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PROTOZOOLOGY
PROTOZOOLOGY
A MANUAL
FOR MEDICAL MEN, VETERINARIANS
AND ZOOLOGISTS
BY
C. M. WENYON
C.M.G.. C.B.E., M.B., B.S., B.Sc. (Lond.)
DIRECTOR-IN-CHIEF OF THE WELLCOME BUREAU OF SCIENTIFIC RESEARCH
FORMERLY PROTOZOOLOGIST TO THE LONDON SCHOOL OF TROPICAL MEDICINE
IN TWO VOLUMES
WITH 565 ILLUSTRATIONS AND !20 COLOURED PLATES
VOL. I
NEW YORK
WILLIAM WOOD AND COMPANY
MDCCCCXXVI
Printed in Great Britain
FELIX MESNIL
MY FORMER TEACHER
THIS BOOK IS DEDICATED AS A TOKEN OF
PERSONAL INDEBTEDNESS AND RESPECT FOR
HIS MANY VALUABLE CONTRIBUTIONS
TO OUR KNOWLEDGE OF
PARASITIC PROTOZOA
CORRIGENDA
3 41, Pig. 23, for Cothurina read Cothurinia.
276, Fig. 125, for hirudinella read hirundinella.
323, line 14, for raice read rajce.
474, line 2, for hirudinis read hiriindinis.
475, line 4 from bottom, for hirudinis read hirundinis.
588, line 1, for Sternotherus read Siernothcerus.
604, Fig. 247, for raice read ra/ce.
710, line 19, for Afeliis read Atehs.
722, inscription to Fig. 307, line 7, for sphendosa read sphcerul
806, line 29, for aspzc read aspi«.
983, line 5, for 25-30 read 26-30.
1116, Plate XX., for Sporozoan read Sporozoon.
PREFACE
The subject of Protozoology has, in recent years, shown a tendency to
become divided into two sections. In the one the student's attention
is directed chiefly to the study of free-living Protozoa, in the other to
parasitic forms, more especially those which give rise to disease in man
and domestic animals. Such a division, if it becomes absolute, cannot
lead to a clear understanding of the group as a whole, for it is evident
that without some knowledge of free-living Protozoa, from which they
have been undoubtedly evolved, a wrong conception of parasitic forms
will be obtained. As in other branches of science, specialization appears
to be inevitable if any advance is to be made, but however specialized
a student becomes, it is his duty to keep himself informed of any progress
made outside his particular field. Anyone who wishes to make an intel-
ligent study of parasitic Protozoa must be acquainted with the funda-
mental principles of general Protozoology, and, indeed, with those of
general Zoology, Physiology, and even other sciences. This is merely
another way of stating the well-recognized fact that all sciences are inter-
dependent. On this account the student of the Protozoa which are
pathogenic to man and domestic animals should have a sound knowledge
of other parasitic Protozoa, and at least a good working knowledge of
non-parasitic forms as well. Conversely, those who study free-living
Protozoa should have a clear conception of the parasitic forms, for the
extensive investigations of recent years have contributed so much to our
knowledge that in many respects they are better known than their free-
living relations, particularly as regards the completeness of their life-
histories and the probable course of their evolution.
In this manual the writer has attempted to present the subject of
Protozoology in such a light that it will be of use to the zoologist who
wishes to obtain information regarding the general principles of the subject
and detailed knowledge of parasitic forms, and to medical men and
veterinarians who are chiefly concerned with those Protozoa with which
they have professionally to deal.
The investigations of Smith and Kilborne on the parasite of Texas
fever of cattle and its transmission by ticks; those of Laveran, Grolgi,
Ross, and Grassi on malarial parasites of man and birds, and their
carriage by mosquitoes ; and the researches of Bruce, who demonstrated the
vii
viii PREFACE
trypanosome nature of the African cattle disease, nagana, and its con-
veyance by tsetse flies, opened an entirely new field of enquiry which has
led to a most extensive study of parasitic Protozoa. The thousands of
papers on the subject which have been published during the past twenty
or thirty years are scattered in numerous journals, many of which are
difficult to obtain by any worker, and impossible by those who are
stationed in parts of the world where good libraries are not available.
Many workers have spoken to the writer of the difficulties associated
with this separation from literature, and it has been largely a desire to
remove at least a part of these difficulties that has led him to undertake
the present work on the subject of Protozoology.
The book deals with all groups of parasitic Protozoa, as well as with
free-living forms, though the latter have been dealt with very briefly,
except in the case of those which are coprozoic in habit and may lead
to confusion with parasitic organisms. The part played by invertebrates
in the transmission of certain parasitic Protozoa of vertebrates necessitates
the examination of invertebrates in order to trace the life-history of any
parasite which may develop in them. As knowledge of the parasites
which are peculiar to these invertebrates is essential if errors are to be
avoided, they have accordingly received special attention.
In reviewing the extensive literature on the subject of Protozoology
it has been necessary to criticize many statements and claims which have
been made, but, in expressing his own views, the writer hopes that he
has explained clearly the reasons which have led him to their adoption,
and that he has treated fairly those records which appear to him to be
of doubtful value.
One of the chief difficulties associated with the production of a manual
like the present one is that hardly a week passes without the publication
of some paper of importance; but an earnest endeavour has been made
to incorporate all new and essential data as they appeared, so that as the
book goes to press in its final form a fair claim can be made that it is
as complete as it reasonably can be. Rapid advances are being made
in the elucidation of the methods of transmission of kala azar and Oriental
sore, and there is every prospect that very soon the sand fly will be in-
criminated definitely as the vector of one or both of these diseases. The
treatment of general paralysis by inducing in patients attacks of malaria
is leading indirectly to the discovery of many interesting facts regarding
the development of malarial parasites. The recently described method
of cultivation of intestinal amoebse is assisting in the solution of many
problems connected with the life-history of these organisms. Three
hitherto supposed coccidia of man have been shown to be nothing more
than parasites of edible fish which are passing casually through the human
PREFAGE ix
intestine. Observations such as these are continually producing changes
in our outlook, so that, however quickly a book is produced, it is bound
to be out of date in certain respects when it appears. Nevertheless, the
greater part of the information which will be found on its pages is well
established, and will be lasting, so that it is sincerely hoped that the
two volumes will provide a reliable record of our knowledge up to the
beginning of 1926.
As the study of spirochaetes is intimately related to that of the Pro-
tozoa, especially in connection with blood w^ork, a section is devoted to
their consideration, though it is definitely maintained that they are not
Protozoa.
Many Protozoa which have affinities with those which produce diseases
in man and domestic animals have been found in the blood of other
vertebrates and in the intestines of invertebrates. A worker who dis-
covers such an organism has considerable difficulty in ascertaining if it
has been previously noted. To meet this difficulty a host list of the
blood-inhabiting parasites of vertebrates and one of the flagellates of
invertebrates have been compiled, and it is hoped they will be useful
references.
As difficulties associated with nomenclature, the accuracy of which
is of such importance, are constantly occurring, the International Rules
of Zoological Nomenclature, which many workers have little opportunity
of consulting, have been included.
The practical side of Protozoology has been constantly kept in mind,
as well as the difficulties which beset the path of those engaged in its
study. A special section deals with methods of investigations. This
is not intended to be a complete account, but merely a guide for the use
of those who already have a working knowledge of laboratory technique.
Authorities for all statements made in the text have been given, and
the exact references will be found in the list of publications at the end
of the book. Practically all these have been consulted in the original,
and with very few exceptions every reference has been verified. The
greater part of this laborious work has been carried out by Miss I. M.
Bellis, Librarian to the Wellcome Bureau of Scientific Research, whose
knowledge of languages and scientific publications has been invaluable.
The writer is glad to have this opportunity of acknowledging his in-
debtedness to her for the great care she has taken with this and many
other parts of the work. The writer has constantly had the assistance of
Mr. Cecil Hoare, Protozoologist to the Wellcome Bureau of Scientific
Research. Many intricate questions have been discussed with him, and
his sound judgment, together with his careful and critical reading of
the proofs, has been a great asset. For many of the drawings, both
X PREFACE
black and white and coloured, the writer is much indebted to Mr. B.
Jobling, now on the staff of the Wellcome Bureau of Scientific Research.
His knowledge of biology combined with his artistic skill has enabled
accurate copies and many original drawings from preparations to be
produced. The writer's thanks are also due to his sister, Miss M. G.
Wenyon, Assistant Secretary to the Royal Society of Tropical Medicine
and Hygiene, who has read carefully the final proofs, and has been a
means of detecting errors which otherwise would have marred the pages.
The writer is indebted to Professor Nuttall, F.R.S., Quick Professor
of Biology and Director of the Molteno Institute of Cambridge ; Dr. A. G.
Bagshawe, C.M.G., Director of the Bureau of Hygiene and Tropical
Diseases ; Professor Warrington Yorke of the Liverpool School of Tropical
Medicine; Professor A. E. Boycott, F.R.S., of University College, London;
Lieut.-Col. W. P. MacArthur, D.S.O., O.B.E., of the Royal Army Medical
College; Dr. Keilin of the Molteno Institute, Cambridge; and the Councils
of the Royal Society of Tropical Medicine and Hygiene and the Royal
Institution for the loan of blocks. He is also indebted to Mr. Clifford
Dobell, F.R.S., for the use of his original diagrams of Aggregata eberthi
and his drawing of the cyst of Balantidium coli, and for permission to
reproduce figures from his publications.
Much assistance has been derived from many of the books on Proto-
zoology or one or other of its branches, particularly Doflein's Lehrhuch
der Protistenkunde, Laveran and Mesnil's Trypanosomes et Trypanoso-
miases, Laveran's Leishmanioses, Minchin's An Introduction to the Study
of the Protozoa, Dobell's The Amoehce Living in Man, Dobell and O'Connor's
The Intestinal Protozoa of Man, and many others; but of all the publica-
tions, apart from original articles, the careful reviews by Professor Mesnil
which have appeared regularly in the Bulletin de VInstitut Pasteur since
1902, and those by various writers in the Tropical Diseases Bulletin,
have been most helpful. Any worker who wishes to keep abreast of
the times cannot do better than to read one or both of these excellent
bulletins with regularity.
Finally, the writer wishes to express his thanks to Mr. N. B. Kinnear
of the British Museum, Natural History, for the trouble he has taken
in checking the host list of birds, and to all the many others who have
been ever ready to give him valuable assistance.
C. M. W.
London,
June, 1926.
CONTENTS TO VOL. I
PART I
genp:ral description of the protozoa
Organization and Life-History of the Protozoa
Typical Division of the Metazoan Nucleus:
Mitotic Division ------
Meiotic or Reducing Division - - - -
greneral morphology of the protozoa
Encystment amongst the Protozoa
The Protozoan Nucleus -----
Multiplication amongst the Protozoa
Syngamy amongst the Protozoa - - - -
Nuclear Division amongst the Protozoa -
Behaviour of Chromosomes during Syngamy
Blepharoplasts, Parabasals, and Kinetoplasts -
Physiology op the Protozoa ....
Life-History of Protozoa -----
Immunity in Protozoal Infections - - - -
Action op Drugs in Protozoal Infections
Status op the Protozoa in the Animal Kingdom
PAGE
1
11
15
18
46
49
62
71
90
108
115
123
133
138
150
152
PART II
SYSTEMATIC DESCRIPTION OF THE PROTOZOA, WITH SPECIAL
REFERENCE TO PARASITIC AND COPROZOIC FORMS - 153
Classification of the Protozoa
PHYLUM: PROTOZOA - - - -
A. SUB-PHYLUM . PL ASMODROM A
1. CLASS: RHIZOPODA
1. Order : AMCEBIDA
2. Order: HELIOZOA
3. Order : RADIOLARIA -
4. Order : FORAMINIFERA
5. Order : MYCETOZOA -
- 155
- 156
- 160
- 160
- 165
- 165
- 166
- 168
- 172
XB^T-^
Xll
CONTENTS TO VOL. I
Systematic Description of the Order Amcebida -
1. Family : amcebid.e -
Genus : Amoeba -
Genus : Hartmannella
Genus : Vahlkampfia
Gemis : Sappinia -
Amceb^ of Plants
Gemis : Pelomyxa
Genus : EntamcEba
Genus : Endamoeba
Genus : Endolimax
Genus : lodamoeba
Genus : Dientamoeba
Diagnosis of Intestinal Amceb^ of Man -----
Action of Drugs on Intestinal Amceb^ - . - . -
AmCEB^E cultivated from F^CES COPROZOIC AUCEBJE . - -
Statistics of Intestinal Amceb^ of Man -----
2. Family : paramcebid^
3. Family : dimastigamcebid^
4. Family : rhizomastigid^ -
II. GLASS : MASTIGOPHORA -----
coprozoic mastigophora -------
Invasion of Blood-Stream by Intestinal Mastigophora
Flagellates which may contaminate Blood and Organ Smears
Division op Mastigophora into Sub-Classes and Orders
\. SUB-CLASS : PhyXomsisWgma. -
2. SUB-CLASS : ZoomsisWgmdi - - - -
A. Monozoic Forms :
1. Order : PROTOMONADIDA -
(1) Sub-Onler : Eumonadea
(2) Sub-Order : Craspedomonadea
Systematic Description of Genera and Species of Sub-Order Eumonadea
1. Family : monadid^
A. Monadid;ie with One Flagelluni -
Genus : Oikomonas
PAGE
173
175
175
175
177
180
181
182
182
235
238
242
248
250
254
257
259
260
260
266
268
270
271
272
274
282
285
285
286
288
288
288
289
289
CONTENTS TO VOL. I xiii
PAGE
Blackhead of Tukkeys -_-__-_ 291
Genus : Craigia - - - 294
Genius : Rhizomastix - - 296
Gemis : Proleptomonas - - 297
B. Monadidoe with Two Flagella - 298
Genus : Heteromita - - 298
Genus : Dimastigamoeba - - 302
Genus : Spiromonas - - 302
Genus : Phyllomitus - - 305
Genus : Costia - - - 305
C. Monadidse with Three Flagella - 306
Genus : Enteromonas - - 306
D. Monadidae with Four Flagella - 308
Genus : Tetramitus - - 308
E. Monadidse with More than Four
Flagella - - - 311
2. Family : trtpanosomid^ - - 312
Relation of Various Types to One Another - - - - 312
Orientation and Origin of Different Types - - . . 316
Subdivision into Genera - - - - - - -318
Cytology of Trypanosomes and Allied Flagellates - - - 321
Method of Reproduction -----.. 336
SyNGAMY ------... 339
Encystation -------.. 34X
General Features of the Life-History ----- 342
Classification - - - - -- - - - 344
Systematic Description of Genera and Species - - - . 343
Genus : Leptomonas - - 348
Genus : Crithidia - - - 355
Genus : Herpetomonas - - 363
Other Members of the Genera Lejytomonas, Crithidia, and Herpetomonas - 369
Forms found in Body Cavity and Salivary Glands - - 370
Roubaud's Genus Cercoplasma - - - - - - 370
Roubaud's Genus Cystotrypanosoma - - - - - 372
Patton's Genus Bhynchoidomonas . - . . . 374
Chatton's Observations on the Trypanosomid^ of Drosophila - 377
Genus : Phytomonas - - 382
Inoculation of Insect Trypanosomid^ into Vertebrates - - 392
CONTENTS TO VOL. I
PAGt
Inoculation of Insect Trtpanosomid^ into Invertebrates - - 395
Genus : Leishmania - - 396
Genus : Trypanosoma - - 442
Methods of distinguishing Trypanosomes- . . _ _ 444
Classification of Trypanosomes -.--.. 456
Curative Action of Drugs and Sera in Trypanosomiasis - - - 459
Systematic Description of Species ------ 463
Group A. Trypanosomes which develop in the Posterior Station
IN THE Invertebrate ----- 463
I. Trypanosomes of Eodents, Cheiroptera, Insectivora, Eden-
tata, Carnivora, and Monkeys - - - 463
(fl) Trypanosomes of Rodents - - . - 453
(6) Trypanosomes of Cheiroptera - - - - 479
(c) Trypanosomes of Insectivora - - - - 482
(d) Trypanosomes of Edentata . - - - 482
(e) Trypanosomes of Carnivora - - - . 433
(/) Trypanosomes of Monkeys - - - . 433
Genus : Endotrypanum - - 485
II. Trypanosome of Man in South America - - - 486
III. Non-Pathogenic Trypanosomes transmitted by Species of
Tabanus, Melojyhagns, or Other Blood-Sucking Arthro-
PODA - - - - - - - - 498
Trypanosomes of Cattle ----- 498
Trypanosomes of Sheep . - - - . 502
Trypanosomes of Antelope ----- 507
Group B. Trypanosomes which develop in the Anterior Station
IN the Invertebrate, or have become secondarily
adapted to Direct Passage from Vertebrate to
Vertebrate - - - - - - - 507
I. Pathogenic Trypanosomes transmitted by Blood-Sucking
Arthropoda ------- 507
General Remarks on Pathogenic Trypanosomes - - - 507
Relation to Game - - - - - - - - 508
Mechanism of Infection - - - - - - - 511
Identification of Trypanosomes in Tsetse Flies - - - 515
Experimentally proved Vectors of Pathogenic Trypanosomes of
Africa - - - - - - - - -517
CONTENTS TO VOL. I xv
PAGE
Passage of Trypanosomes from Parent to Offspring - - - 518
Trypanosomes as Filter Passers ------ 520
Classification of Pathogenic Trypanosomes - - - - 521
1. Pathogenic Trypanosomes transmitted by Species of
Glossina - - - - - -524
(a) Trypanosomes which develop in the Stomach,
Proboscis, and Salivary Glands of Tsetse Flies
— Polymorphic Trypanosomes - - - 524
(b) Trypanosomes which develop in the Stomach
AND Proboscis of Tsetse Flies — Monomorphic
Trypanosomes without Flagella - - 552
(c) Trypanosomes which develop only in the
Proboscis of Tsetse Flies — Monomorphic Try-
panosomes PROVIDED WITH FlAGELLA - - 559
2. Pathogenic Trypanosomes transmitted by Species of
Tabanus OR Other Blood-Sucking Arthropoda —
Monomorphic Trypanosomes provided avith Flagella 565
II. Pathogenic Trypanosomes passing directly from Verte-
brates TO Vertebrates - . - - _ 574
III. Trypanosomes of Birds - - - - - - 577
IV. Trypanosomes of Land Keptiles, including Crocodiles - 581
V. Trypanosomes of Aquatic Vertebrates transmitted by
Leeches :
1. Trypanosomes of Aquatic Eeptiles - - - 585
2. Trypanosomes of Amphibia - - . - 538
3. Trypanosomes of Fish - - - . . 599
3. Family : bodonid.e - - 607
4. Family: prowazekellid.e - 611
5. Family : embadomonadid^ - 615
6. Family : ciiilomastigid^ - - 620
7. Family : cercomonadid^ - - 629
A. Cercomonadidse with One An-
terior Flagellum:
Genus : Cercomonas - - 629
B. CercomouadidiE with Two An-
• terior Flagella:
Gemis : Trimitus - - - 633
C. Cercomonadidse witli Three An-
terior Flagella:
Genus : Tricercomonas - - 634
8. Family : cryptobiid^ - - 636
CONTENTS TO VOL I.
9. Family :
TRICHOMONADID^
Genus
; Trichomonas
Genus ,
• Gigantomonas
Genus .
.- Ditrichomonas
Genus .
; Eutrichomastix
Genus
; Janickiella
Genus :
Trichomitus
Genus .
• Devescovina
Gemis .
.• Foaina -
Genus .
• Retortamonas
Genus .
• Protrichomonas
Genus .
■ Polymastix
Genus .
• Hexamastix
Genus :
• Cochlosoma
10. Family :
DINENYMPHID^ ■
PAGE
646
648
670
670
671
675
676
677
677
677
679
680
681
681
2. Order: HYPERMASTIGIDA - - - 682
3. Order : CYSTOFLAGELLATA - - 684
B. Diplozoic Forms :
4. Order : DIPLOMONADIDA - - - 684
Genus : Hexamita - - 684
Genus : Giardia - - - 691
Genus : Trepomonas - - 711
(\ Polyzoic Forms :
5. Order : POLYMONADIDA - - - 714
Frequency or Intestinal Flagellate Infections of Man - - 714
III. Oi:^*S.S'. CNIDOSPORIDIA - - - - - 716
Order ; MYXOSPORIDIIDA - - - 718
Subdivision of the Myxosporidiida - - 724
Detailed Description of Certain Species - 728
Order : MICROSPORIDIIDA - - - 734
Subdivision of the Microsporidiida - ' - 737
Detailed Description of Certain Genera and
Species . . . . . 740
Certain Microsporidiida of Blood-Sucking
Arthropoda and Neinatoda - - 748
Supposed Microsporidiida in Rabies and En-
cephalitis of Rabbits . . . 754
Order : ACTINOMYXIDIIDA - - - 756
Parasites of Undetermined Position . . . . . 750
SARCOSPORIDTA .---... 76O
GLOBIDIUM - - - - - - . . 769
HAPLOSPORIDIA - - - - - - - 773
RHINOSPORIDIUM - - - - - - - 776
PART I
GENERAL DESCRIPTION OF THE PROTOZOA
PROTOZOOLOGY >. ^:,; x
PART I
GENERAL DESCRIPTION OF THE PROTOZOA
ORGANIZATION AND LIFE-HISTORY OF THE PROTOZOA.
During the latter part of the seventeenth century Antoni van
Leeuwenhoek (1632-1723), working with a simple microscope, investigated
free-living Protozoa and studied the parasitic forms in the intestine of
frogs. He also found that he himself was infected with one of these
organisms, which, as Dobell (1920) has pointed out, was probably the
well-known Giardia intestinalis. The great Dutch microscopist thus not
only discovered free-living Protozoa, but was the first to study parasitic
forms, and he can be justly regarded as the father of Protozoology and
of its more specialized branch. Medical Protozoology. Since Leeuwen-
hoek's day an ever-increasing number of investigators, availing themselves
of the experiences of those who had gone before them and of the steady
improvement in the microscope, have brought to light an enormous
assemblage of minute living creatures, many of which, like the bacteria,
were quite beyond the scope of the simple magnifying apparatus used by
Leeuwenhoek and other early workers. These minute organisms absorb
nourishment and grow, and finally, as in higher animals, reproduce by
detaching portions of their bodies to form those of their offspring, while
any remaining portion dies. It may be that the entire body of the parent
is used up in the production of progeny, or only a small portion of it, as
in higher animals, but in either case, extending from parent to offspring,
there is a continuity which entitles all living creatures to be regarded as
immortal in that a j^ortion at least of the living matter is handed on from
one generation to another, unless accidental death prevents reproduction.
The fact that all the complex mechanisms of life are concentrated in
these minute portions of living matter has led observers to seek in them .
an explanation of the phenomena of life in general. A single organism
may be kept under observation for the whole of its individual existence,
and the visible changes undergone by it during its life, which is terminated
by its final production of offspring, may be actually followed under the
microscope. It seems evident that beyond the scope of the microscope
there exist organisms, or stages of development of visible organisms,
3
4 OEGANIZATION AND LIFE-HISTORY OF PROTOZOA
which cannot be seen — the ultra-microscopic viruses. Dark field illumina-
tion has done much to facilitate the study of these forms, but, as yet,
the exact nature of the numerous minute objects which it has revealed
in every fluid, and which are in constant motion (Brownian movement),
has not been satisfactorily determined, so that at present it is in many
cases impossible to decide whether they are actually living organisms or
granules of inanimate material.
The study of microscopic organisms has revealed the fact that, in
their method of nutrition, some of them resemble plants and others
animals. On the basis of this physiological distinction it has been the
custom to regard them as belonging to one of two main groups — the
Protophyta and the Protozoa. The study of the former has been relegated
to the botanist, and that of the latter to the zoologist. Though some of
these organisms show undoubted affinities with the algse and higher
plants and others with animals, there exists a miscellaneous assemblage
of indeterminate forms which cannot be placed legitimately in either
group. Accordingly, it is safer to regard them all as belonging to one
large group, the Protista, the study of which is known as Protistology,
as first suggested by Haeckel (1866). Without being able to define
accurately the limits of either group, it is nevertheless convenient to
regard the Protista as comprising the two subdivisions of the Protozoa
and the Protophyta. In the case of the former, nutrition is effected by
the ingestion of preformed proteid material, either as solid particles or
in solution. The Protophyta, on the other hand, nourish themselves
like plants on comparatively simple chemical compounds, and when
possessing chlorophyll or some similar substance, make use of the carbonic
acid of the liquid in which they live. Very frequently they secrete around
themselves capsules of cellulose. A typical Protist consists of a small
portion of cytoplasm and a nucleus which contains as its most essential
constituent a substance called chromatin. The contents of the nucleus
are separated from the cytoplasm by a nuclear membrane. Other bodies
may be present in the cytoplasm, but these, at least as definite visible
structures, are not essential to life.
Amongst the existing Protista the most primitive forms are possibly
the bacteria, spirochsetes, and allied organisms, which in many cases do
not appear to possess definitely constituted nuclei, though granules of
a substance which some observers have identified with chromatin are
present in the cytoplasm. Alexeieff (1924fl) maintains that it is not
chromatin, and that this substaiice is absent from bacteria. These
forms, however, are in most cases so minute that accurate information
regarding their cytological structure and life-histories is difficult to obtain.
It can, at any rate, be safely affirmed that those Protista which are most
CELL THEOEY 5
highly developed and most complex in structure possess definite nuclei,
and the small particle of cytoplasm with its included nucleus of which
the body of each is composed is regarded by most biologists as a cell on
account of its resemblance to the cells of higher animals and plants.
The term "cell" was first introduced for the cellulose capsule or wall
which encloses the portions of cytoplasm of which the higher plants are
built up. It was later realized that the wall itself was merely a sup-
porting structure, and that the cytoplasm within it was in reality the
living material. Accordingly, the term "cell" was then applied, not to
the cell wall, but to its cytoplasmic contents. The latter consists typically
of a small mass of cytoplasm containing a single nucleus. When it was
discovered that the tissues of higher animals were also built up of similar
elements or units, the term "cell" was applied to them also. It soon
became evident that, in the case of many microscopic organisms, the entire
body consisted of a similar mass of cytoplasm containing a nucleus, and
the resemblance of these to the cells of higher animals and plants gave
rise to the view that these organisms were single cells, and the distinction
between unicellular and multicellular animals was drawn. This con-
ception, which was first clearly expounded by Schwann (1839), has been
generally accepted, though Dobell (1911) considers it erroneous. He
believes that an amoeba, for instance, is as much an entire organism as
one of the higher animals, and that though the latter may be regarded
as being multicellular, as a result of the division of its cytoplasm and
nucleus into cells, the former should be considered as a non-cellular
organism, and not a unicellular one, since it corresponds, not to any
single cell, but to all the cells which compose the body of the multi-
cellular organism.
When it is realized that amongst the numerous cells which compose the
body of one of these higher animals there are many wandering cells, such
as macrophages, which behave in all essential respects like amoebae, in that
they form pseudopodia, ingest solid proteid material of various kinds, and
multiply by fission, it is difficult to resist the conviction that such a cell has
a definite claim to be regarded as an individual organism like an amoeba
itself. Furthermore, it has been clearly demonstrated that very minute
portions of the tissues, consisting of groups of cells, or even single cells of
higher animals, can be artificially cultivated, and that they will live and
multiply indefinitely provided they are given a continuous supply of suit-
able nutriment. From these culture experiments it seems clear that the
cell, which forms but a part of the entire multicellular animal, is capable of
nourishing itself and reproducing as a single organism. Another illustration
of the power of independent existence and multiplication of isolated cells
of multicellular animals is seen in malignant disease. In this condition
6 ORGANIZATION AND LIFE-HISTORY OF PROTOZOA
certain cells acquire the power of continuous and rapid multiplication,
so much so that they become to all intents and purposes parasites, which
bring about the death of their host. These cells can be inoculated from
animal to animal indefinitely, and in them they will continue to multiply,
just as trypanosomes do in successive passages in experimental animals.
An ovum, according to the non-cellular view, is a non-cellular indi-
vidual, which at once becomes cellular when segmentation occurs. The
cells, each of which gives rise to only part of the individual which will
normally develop from the ovum, are nevertheless potential individuals
themselves, as is demonstrated by the fact that if the cells are separated
from one another artificially, as in the well-known experiments with sea-
urchin eggs, each is capable of giving rise to a complete embryo.
It seems evident that the cells of higher animals are capable of
independent life provided the proper environment exists. Under
natural conditions all the cells of the body contribute to the production
of this environment, which is so delicately balanced that separated and
isolated cells invariably die unless the proper environment is present or
is artificially provided, as in the culture experiments just mentioned.
If the environment necessary for the continued life of cells in the body
can be kept constant, the cells will survive and reproduce indefinitely,
but if some of the cells fail to fulfil their part in the production of this
environment, the other cells will suffer and death will result. It may be
said that any single cell of a Metazoon is living in a condition of symbiosis
with all the other cells. Without entering further into the discussion,
for purposes of this work it is sufficient to follow the more orthodox view
and to regard the Protista as unicellular organisms, or single cells which
still lead a completely independent existence, and the multicellular
organisms as groups of cells which work together for a common end. The
latter have become so completely interdependent that their power of
separate existence has been largely lost. Yet in many features, such as
their structure, mode of life and method of reproduction, nuclear division
and syngamy, they retain the unmistakable characteristics of their uni-
cellular ancestors. It must not be supposed that the ancestors of either
the multicellular or unicellular organisms any longer exist. The primitive
forms from which they may be supposed to have originated have probably
long since disappeared in the course of evolution. The Protista of the
present day, as well as the individual cells of higher animals and plants,
have undoubtedly evolved along different lines and acquired certain charac-
teristics which their common ancestors did not possess. Biologists are
nevertheless justified in still regarding the portion of cytoplasm with its
nucleus as a cell, whether it occurs amongst the Protista or the Metazoa
and Metaphyta, in spite of the fact that the cells of each group may now
CELL KEPRODUCTION 7
possess distinctive features of their own. The cell may be justly regarded
as an individual, whether it is one of the Protista or only part of the body
of a multicellular organism. Tn the latter case it must be admitted
that a number of individuals have remained united as a colony to form
a single larger individual. Of the cells of the latter, only certain ones are
destined for reproduction, as in the case of spores of Cnidosporidia,
where a group of cells is formed by division from a single cell, and of
these only one is a reproductive cell, the others dying after fulfilling other
functions. A single soldier or a regiment of soldiers may both be units
in the military sense, but the soldiers composing the regiment, though
sacrificing their individuality to some extent for the good of the individual
regiment, are as much individuals as the single soldier.
It has been clearly demonstrated that a Protozoon quickly dies if
deprived of its nucleus, and there is little doubt that the cells of higher
animals are similarly dependent on their nuclei. A single unicellular
organism may be divided into several portions, but though those which
do not contain the nucleus may exhibit movements and survive for some
time, they ultimately die, whereas any nucleated portion may re-form
itself into an entire individual which is able to continue its existence.
It is evident the nucleus plays a very important part in the life, and
metabolism of the cell. The Protozoan cell does not differ from other
cells in its capacity to absorb and digest food, and grow and increase in
size. It is able to perform spontaneous movements as a result of con-
tractions of its cytoplasm, though these are reduced to a minimum in
some cases. Finally, the cell is able to multiply, usually by a process
of binary fission, but sometimes by a process of multiple fission. In
binary fission the single nucleus divides into two parts, and this is followed
by division of the entire cell into two daughter cells. Usually, these
are approximately equal in size {equal binary fission), but it may happen
that one daughter individual is larger than the other {unequal binary
fission). When the difference in size is marked, it appears as if a small
daughter individual is separated from a much larger parent which retains
its original form, and the process is spoken of as budding or gemmation.
In the case of multi'ple fission or multiple segmentation, after the first
division of the nucleus the body of the organism does not immediately
divide, but the two daughter nuclei again divide to form four nuclei, and
these may again divide to give rise to eight. After a number of nuclei
have been thus produced by repeated divisions, the body of the organism
segments into, or more accurately buds off, a number of portions cor-
responding to the number of nuclei. This method of multiple fission of
cells, which more correctly should be called multiple gemmation, occurs
in higher animals as well as in the Protozoa, amongst which it is seen
8 ORGANIZATIOX AXD LIFE-HISTORY OF PROTOZOA
typically in the parasitic Sporozoa, and is known as schizogony. Usually
there is a residue which does not participate in the formation of the buds:
it is discarded as a residual body which quickly disintegrates.
During the life-history of many cells a sexual process occurs from
time to time. The advantages gained from such a process, which is
called synga7Mj, are far from being clearly understood. In its simplest
form it consists in the complete union of two cells and fusion of their
nuclei. The uniting cells are known as gmnefes, and the single cell resulting
from the union is a zygote. The zygote proceeds to multiply by binary
or multiple fission.
The process of syngamy must be distinguished from another type of
union which sometimes occurs. Two or more cells may fuse to produce
a multinucleate cytoplasmic body known as a plasmodiiitn. In this
/ ■ .- -
■■^..^' ^ \ ,,*: * /
If ^
A B
Fig. 1. — Diagram of Cells. (Original.)
A. Metazoan cell. The cytoplasm contains a centrosome and a nucleus with a nucleolus.
B. Protozoan cell {Entamoeba). The cytoplasm, differentiated into ectoplasm and endoplasm,
contains a nucleus with central karyosome and numerous food vacuoles. No centrosome is
visible.
manner plasmodia containing many hundreds of nuclei may be formed.
The nuclei show no tendency to unite with one another, as they do in
syngamy, and after the plasmodial phase has existed for some time
segmentation into. uninucleate cells takes place.
The typical cell, wherever it occurs, consists of the two essential parts
— cytoplasm and nucleus (Fig. 1). Each of these is a mixture of
substances of highly complex chemical constitution, the reactions of which
produce the phenomena characteristic of living matter. The cytoplasm
appears to be made up of at least two substances, one of which is suspended
in the other in the form of an emulsion. The nucleus, which is limited
by a nuclear membrane, consists of a substance called nuclear sap, which
occupies interstices in a more solid material. The latter, when viewed in
STRUCTURE OF CELL AND NUCLEUS 9
optical section, has the appearance of a network, and is known as the
linin network, of which the nuclear membrane may be regarded as a special
development. Upon this network, and on the nuclear membrane in the
form of granules or larger masses, is arranged another substance, the
chromatin, which has a strong affinity for certain stains. It is generally
regarded as the most important constituent of the nucleus, and this is
borne out by the fact that nuclear division takes place by an elaborate
process known as 7nitosis, which results in an equal sharing of the chromatin
between the daughter nuclei. In the nucleus of the Metazoan cell there
is usually present a conspicuous body known as the nucleolus. It is devoid
of chromatin, and when nuclear division takes place it passes to one of
the daughter nuclei, the other daughter nucleus forming a new nucleolus.
A very similar body exists in the nuclei of certain Protozoa (Opalina),
and it passes to one of the daughter nuclei when division takes place. In
other Protozoa, as, for instance, in Karyolysus and Hepatozoon, a similarly
achromatic body divides at nuclear division, each daughter nucleus re-
ceiving half (Fig. 35). When such a body occupies a central position in
a Protozoan nucleus it is known as a haryosome, and it has been generally
assumed that it is composed largely of chromatin. It is becoming in-
creasingly evident, however, that the karyosome may be actually devoid
of chromatin, and the supposition that in certain nuclei the entire chro-
matin may be concentrated in the karyosome is a very doubtful one. The
nucleus is often regarded as consisting of two substances — the achromatic
and the chromatic material. The achromatic material, including the
nuclear membrane, linin network, nuclear sap, and other bodies (karyo-
some, nucleolus) which are sometimes present, undoubtedly comprise
several distinct substances, some of which, at any rate, are able to give
rise to chromatin, for the quantity of chromatin in the nucleus varies from
time to time, and increases with its growth. Ahother important constituent
of the cell, which as a rule only becomes visible during nuclear division,
is the centrosome (Fig. 1, A). It is commonly present in the cells of
Metazoa, but it is not so frequently seen in the Protozoan cell. Repro-
duction of a cell by binary fission or multiple segmentation is always
preceded by division of the centrosome, if one is present, followed by
division of the nucleus, which in most cases takes place by mitosis. It
is during nuclear division that the nature of many of the constituents of
the nucleus first comes to light, and for this reason it will be necessary to
consider mitosis, as it occurs typically in the Metazoan cell. During
mitosis there are formed, mainly out of the chromatin, certain bodies
known as chromosomes, which are constant in number for each species of
animal, the same number appearing at each succeeding nuclear division.
There is some evidence that in the resting, or more accurately the non-
10 ORGAXIZATIOX AND LIFE-HISTORY OF PROTOZOA
dividing nucleus, though the chromosomes are no longer visible as
individual units, they still exist as separate entities. During syngamy,
when two gametes unite and their nuclei fuse, the chromosomes of the two
uniting nuclei enter the zygote nucleus, so that, unless a reduction is made
in the number of chromosomes, at each succeeding union the chromosome
number would be doubled. Usually the number of chromosomes in the
gamete nuclei is only half that of the nuclei of other cells of the body, and
the process by which this reduction is brought about is known as the
reducing division, or meiosis.
Though in the vast majority of cases it is recognized that the nuclei
of daughter cells are the products of division of the nucleus of a parent
cell, it is supposed that occasionally amongst the Protozoa nuclei may be
formed from extra-nuclear chromatin granules which appear in the
A B
Fig. 2. — Formation of Nuclei from the Chromidial Body in Arcella vulgaris
( X ca. 300). (After E. Hertwig, 1899.)
A. Normal individual with two nuclei and mass of chromidial substance.
B. The chromidial substance is breaking up and nuclei are being formed from the fragments.
cytoplasm (Fig. 2). It seems to be an undoubted fact that chromatin
material in the form of granules may leave the nucleus and take up a
position in the cytoplasm. This has been described as taking place, not
only in Metazoan cells, but also in the Protozoa. Such granules of
chromatin, which occur in the cytoplasm, are known as cTiromidia. It
is not, however, an easy matter to determine the true nature of granules
which occur in the cytoplasm, and it has not infrequently happened that
identical granules or material have been described as chromatin by one
observer, and as some other substance by another. There seems little
doubt that both in the case of Metazoan cells and Protozoa, chromidia
do not occur so frequently as some have supposed. When the question
of the origin of nuclei from these chromidia is considered there is still
CHROMIDIA 11
greater uncertainty. Some observers believe that the chromatin granules
or chromidia in the cytoplasm may, under certain conditions, arrange
themselves in groups, each of which becomes transformed into a nucleus.
It is difficult to avoid the impression that most, if not all, of the records
of nuclei arising, as it were, by crystallization of chromidia are the result
of misinterpretations, and that the appearances on which the conclusions
have been based might be accounted for in another and more probable
manner. In all cases in which accurate and continuous observation of
reproducing cells has been possible, daughter nuclei have been found to
arise only by division of pre-existing parent nuclei. A classical instance
of this kind is seen in Arcella vulgaris, a binucleate shelled amcBba
(Figs. 2 and 79). Like many other shelled amoebae, in addition to the
true nuclei, Arcella vulgaris contains a mass of material which, on account
of its affinity for certain chromatin stains, is supposed to be of chromidia!
nature, and is called the chromidial body. It was claimed by Richard
Hertwig (1899) and other observers that at certain phases of development
the two existing nuclei degenerate and disappear, and that numerous
secondary nuclei are formed from the chromidial body. Schirch (1914) has,
however, shown that in some cases, at least, the numerous nuclei which
are present result from repeated divisions of the two which occur in the
normal individual. It seems not improbable that the so-called chromidial
body of Arcella and its allies is not really of chromatin nature, but consists
of a special material which may be concerned with the development of
the shell, which is a characteristic feature of these shelled amoebae.
TYPICAL DIVISION OF THE METAZOAN NUCLEUS.
1. Mitotic Division.
The Protozoan nuclei divide in a variety of ways, and it is probable that
amongst them the more primitive types of nuclear division will be found.
There is every transition between what is little more than a simple con-
striction of the nucleus into two parts {amitotic division) and the elaborate
method of division known as mitosis or haryokiyiesis, in which chromosomes
are formed and divided in such a manner that the chromatin of the nucleus
is equally distributed to the daughter nuclei. The division of nuclei by
mitosis occurs most typically in the cells of higher animals and plants,
and it was in their cells that the details of the process were first elucidated.
The terms employed for the different structures and the various stages
which occur were first applied to their nuclei, and were used subsequently
for the corresponding stages which occur during the division of Protozoan
nuclei. Mitosis in its typical form is characterized by the formation
from the chromatin and achromatic material of the nucleus of a number
12 DIVISION OF METAZOAN NUCLEUS
of usually elongate structures called chromosomes, each of which splits
longitudinally into two daughter chromosomes, one of which passes into
each daughter nucleus. This division and separation of chromosomes is
associated with the formation of the achromatic figure which arises in con-
nection with a structure called the centrosome situated in the cytoplasm
outside the nucleus. The whole process can be regarded as talcing place
in a number of stages known as the prophase, metaphase, anaphase, and
telophase (Fig. 3).
PROPHASE. — The centrosome, which is a spherical structure at the
centre of which is a deeply staining granule, the centriole, divides into two
parts which separate from one another. As they separate, the two
daughter centrosomes remain connected by fibres which are arranged as
a spindle, the spindle fibres, while similar fibres radiate into the cytoplasm
from the centrosomes (Fig. 3, B and C). Each centrosome with its
radiating fibres constitutes the aster. Within the nucleus the linin net-
work becomes arranged in what has been supposed to be a long coiled
thread in which the chromatin granules are embedded. This thread is
known as the spireme. Structures such as nucleoli and karyosomes may
break up and disappear, and any chromatin they contain becomes arranged
in granular form with the rest of the chromatin of the nucleus upon the
spireme. Finally, the nuclear membrane disappears, while the spireme
segments into a number of chromosomes (Fig. 3, C). It seems probable
that the conception of the spireme as a single long coiled thread is not
correct, and that from its first appearance it consists of a number of long,
intercoiled, separate segments which become distinct as they contract to
form the chromosomes, the name given to the separate parts into which the
spireme was supposed to divide. With disappearance of the nuclear
membrane the separate chromosomes, each of which can often be seen to
consist of two closely united parallel threads, arrange themselves in a
looped fashion round the equator of the spindle, and in the plane of this
equator in such a manner that the bend of each loop is directed towards
the centre and the two ends away from it (Fig. 3, D and E). The chromo-
somes, which have become shorter and thicker at the equator of the spindle,
form the equatorial plate.
METAPHASE — The chromosomes, which are now arranged as the
equatorial plate, and each of which may consist of two closely apposed
parallel structures, divide longitudinally into daughter chromosomes,
which commence to move towards the pole of the spindle (Fig. 3, E).
ANAPHASE.^The daughter chromosomes now separate completely
into two groups at the poles of the spindle. The natural interpretation
that they are drawn there by the action of the fibres of the spindle to which
MITOSIS
13
they are attached does not appear to be a satisfactory explanation of their
movements (Fig. 3, F).
Fig. 3. — Diagram of Nuclear Division by Mitosis. (After Agar, 1920.
A. Resting nucleus with centrosome.
B. Early prophase with dividing centrosome.
C. Middle stage of projjhase: appearance of spindle and dividing chromosomes.
D. Late prophase.
E. Metaphase with divided chromosomes as equatorial plate.
F. Anaphase: separation of daughter chromosomes.
G. Telojihase: aggregation of chromosomes.
H. Completion of nuclear and cell division and reconstructed daughter nuclei.
TELOPHASE. — The spindle fibres gradually disappear, the nuclear
membrane re-forms around the chromosomes, which gradually become
transformed into the linin network and chromatin characteristic of the
14 DIVISION OF METAZOAX NUCLEUS
original nucleus (Fig. 3, G and H). The centrosome remains outside the
nucleus, the fibres of the aster becoming no longer visible.
The centrosome appears to be the ruling factor in the process, and
the appearance of the aster and spindle fibres can be interpreted as visible
indications of some force which is being exerted. It must be remembered,
however, that in the mitotic division of the nuclei of the higher plants,
as also that of many Protozoa, though all the stages of mitosis seen in the
animal cell occur, definite visible centrosomes are not present. The
fibres of the aster and spindle radiate from an apparently structureless
area, which may be regarded as a potential centrosome. An important
fact to be noted is that for any particular species the number of chromo-
somes present in the nucleus of any cell of the body is constant. In the
much studied cells of Ascaris ntegalocephala, of which there are two
varieties, the number of chromosomes is two or four respectively. In
man it is twenty-two, while in other animals it may be much higher
than this. Each species of animal has thus a definite chromosome
number.
The chromosomes which are formed in any nucleus are not necessarily
all alike in size or form. It is often found that they can be grouped in
pairs, the members of each pair resembling one another more closely than
those of other pairs. The members of each pair are known as homologous
chromosotnes. During the progress of mitotic division the chromosomes
are at first elongate structures, but there is a tendency for them to shorten,
so that at the stage when the equatorial plate is formed they may be
roughly spherical. Though these alterations in size take place, all the
chromosomes are similarly aft'ected. Their relative size and shape remain
the same, so that the homologous pairs can still be recognized.
During the telophase, when the chromosomes of the daughter nuclei
are becoming transformed to reproduce the structure of the resting nucleus,
it can sometimes be seen that the chromatin and achromatic material
of each chromosome is occupied in reconstructing a j^articular portion
of the nucleus. When chromosomes are re-formed at the next nuclear
division, the material in each portion concentrates again into a chromo-
some. In these cases it appears as if there is a permanent separation of
the constituents of each chromosome, even when the nucleus is in the
resting condition. This has given rise to the doctrine of the continuity
of chromosomes, which supposes that each chromosome is a permanent
structure, which, though changing its form, is present as an individual
unit even during the period when the nucleus is not dividing. The proof
of this, however, is exceedingly difficult to obtain, and it must be regarded
at present as little more than a plausible theory.
The chromosomes themselves are not homogeneous bodies, but consist
MEIOSIS 15
of a number of small granules of chromatin of varying size, tlie chromo-
meres, embedded in an achromatic matrix. Very frequently homologous
chromosomes resemble one another very closely as regards the arrange-
ment and variations in size of the chromomeres which they contain.
2. Meiotic or Reducing Division.
A sexual process or syngamy, which consists in the union of two cells
together with fusion of their nuclei, occurs in higher animals and plants,
and it was amongst them that the nuclear changes associated with the
process were first studied. Attention has been drawn to the fact that
the chromosome number for each individual species is constant, so that
it must be evident that, if the nuclei of two cells unite, the number of
chromosomes in the resulting zygote nucleus, which is known as the
synkarion, would be double the usual number. This increase in number
does not actually occur, for the nuclei of the uniting cells or gametes
contain only half the number of chromosomes possessed by other cells.
The reduction is brought about by a special type of mitotic division of
the nucleus during the formation of the gametes (Fig. 4). When the
chromosomes arrange themselves on the spindle fibres as the equatorial
plate, instead of splitting into daughter chromosomes as in ordinary
mitosis, they become separated into two groups, one of each pair of
homologous chromosomes passing to each group (Fig. 4, C and D). In
this way the daughter nuclei contain half the number of chromosomes
possessed by the parent nucleus. The reduction in the number of chromo-
somes in the nuclei of the gametes is effected either at the last cell division
which gives rise to gametes, or at the one immediately preceding it. The
process is known as meiosis, and the nuclear division the meiotic division
or reducing division. When the gametes unite and their nuclei fuse, the
synkarion therefore contains the usual number of chromosomes. The
gamete with half the number of chromosomes is said to be haploid as
regards its chromosomes, while the original cell from which the gametes
were derived and the zygote resulting from their union, which contain
both chromosomes of each homologous pair, are said to be diploid.
Amongst the higher animals, as also frequently amongst the Protozoa,
the gametes can be distinguished as male and female. The former, in
the vast majority of cases, are smaller than the latter, so that the gametes
can be distinguished as 7nicroga?netes and macrogametes. The micro-
gamete of a Metazoon is known as a spermatozoon and the macrogamete
as an ovum. The microgametes are derived from a large number of cells
called spermatogonia, which, like all the other cells of the body, contain
the normal or diploid number of chromosomes. One of these cells in-
16
DIVISION OF METAZOAN NUCLEUS
creases in size and becomes the prmiary spermatocyte. By division two
secondary spermatocytes are produced, and each of these again divides,
giving rise to four spermatids, which become directly transformed into
microgametes or spermatozoa. It is during the first or second of these
two divisions that meiosis occurs and the number of chromosomes is
reduced. When it occurs it is seen that, as the chromosomes arrange
Fig. 4. — Diagram of Meiosis or Reducing Division of a Nucleus with Four
Chromosomes. (Original.)
A. Showing two pairs (dotted and lined) of homologous chromosomes and commencing formation
of spindle.
B and C. Syndesisor conjugation of homologous chromosomes.
D and E. Separation of the conjugated homologous chromosomes.
F. Formation of nuclei, each with half the original number of chromosomes; one of each pair of
homologous chromosomes has entered each nucleus.
In ordinary mitosis the chromosomes at C, instead of separating, would divide, so that two pairs
of homologous chromosomes would pass to each daughter nucleus.
themselves at the equator of the spindle, the individuals of each pair
of homologous chromosomes are closely applied to one another, so that
at first inspection it might be thought that only half the number were
present. This approximation of the chromosomes of each pair is known
as the conjugation of the chromosomes or syndesis, and it is supposed that
MEIOSIS 17
exchange of material takes place between them. As division of the nucleus
proceeds, separation of the conjugating chromosomes occurs, and the
two chromosomes of each pair pass to opposite poles of the spindle. It
will be seen, therefore, that in this division there has been no splitting of
the individual chromosomes as occurs in ordinary mitosis, but merely
a separation of two chromosomes which have come together temporarily
in syndesis. The number of chromosomes in the daughter nuclei are
thus half the original number. If the reduction division occurs at the
division of the primary spermatocyte, then the division of the nucleus
of the secondary spermatocyte is not a reducing one, the chromosomes
splitting longitudinally in the usual manner, so that the number is main-
tained. If the reduction occurs at the division of the secondary sperma-
tocyte, then the division of the nucleus of the primary spermatocyte is
of the ordinary type. In any case, the spermatids which become sper-
matozoa or gametes have half or the haploid number of chromosomes.
In the case of the female cell similar changes occur. Cells called
oogonia grow into primary oocytes. A primary oocyte divides to give
rise to two cells, which are, however, unequal in size. The large one is
the secondary oocyte, and the small one the^^rs^ polar body. The secondary
oocyte divides into two cells, which are again unequal in size. One of
these is the ovum, and the other the second polar body. The first polar
body, which corresponds to a secondary oocyte, may itself divide into
two cells. The nuclear changes which occur in these divisions are similar
to those which occur during the divisions of the spermatocytes described
above, so that the number of chromosomes in the ovum is half the number
present in the o5gonia. There is this difference, however: Whereas each
primary spermatocyte gives rise to four spermatozoa, each primary oocyte
gives rise to one large ovum and two small polar bodies, or three if the
first polar body divides. By this arrangement the cytoplasmic part of
the ovum is increased at the expense of that of the polar bodies, which do
not proceed further to develop. Another difference between the ovum
and spermatozoon is that the centrosome of the ovum has disappeared,
though that of the latter has persisted.
When conjugation or syngamy occurs, the nucleus of the microgamete
or spermatozoon unites with that of the macrogamete or ovum by a
process known as karyogamy to produce the nucleus or synkarion of the
zygote, which again has the diploid number of chromosomes arranged
in homologous pairs. The centrosome of the microgamete becomes the
centrosome of the zygote.
The pairs of homologous chromosomes of the zygote can be recognized
through all the subsequent divisions of the cell down to the moment
when the new adult individual again produces spermatocytes or oocytes.
I 2
18 MORPHOLOGY OF PROTOZOA
One chromosome of each pair was originally derived from the spermato-
zoon and the other from the ovum, and the two, or at least their descen-
dants, have remained distinct during all the subsequent divisions of the
nuclei. When the reducing division or meiosis occurs, the conjugation
of the individuals of each pair of chromosomes takes place, and it is
supposed that at this moment there is interchange of material between
them, and that transmission of hereditary characters is accomplished.
It will be shown below that amongst the Protozoa the production of
gametes may be associated with similar changes in the nuclei, the gametes
possessing half or the haploid number of chromosomes. On the other
hand, cases are known in which no reduction in the number of chromo-
somes takes place during gamete formation. It results that the zygote
contains double or the diploid number of chromosomes. In these cases
the first division of the zygote nucleus is a reducing division, the two
daughter nuclei again having the haploid number. In the one case the
reduction affects the gametes and occurs before syngamy, while in the
other it affects the two daughter cells, resulting from division of the
zygote, and occurs after syngamy.
GENERAL MORPHOLOGY OF THE PROTOZOA.
Of the Protozoa there are a very large number of genera and species,
some of which are free-living forms, while others lead a protected existence
within the bodies of higher animals. The latter have undoubtedly been
derived from the former, and have become modified to such an extent in
adaptation to their hosts that, generally speaking, they are no longer able
to live apart from them. As practically every higher animal is liable to
harbour in its body one or more Protozoa, it is evident that the number
of parasitic species is very large indeed. It should be remembered,
however, that to understand properly the parasitic forms the study of the
free-living Protozoa should not be neglected.
It is customary to regard parasites in general as degenerate organisms,
but though it is true they may have lost many of the organs possessed by
their free-living ancestors, they may have developed others in their place,
and reveal the same degree of adaptation to their environment as free-
living forms. Though a parasite may have lost certain structures which
it no longer requires, it digests its food, grows, and reproduces with all
the complexity exhibited by those which still possess them. It seems
incorrect to regard as in any sense degenerate an organism which is so
completely adapted to its environment as are the majority of parasites.
In fact it might be legitimately argued that if an organism retained
structures for which it had no further use, this would indicate a loss of
SHAPE AND SIZE OF BODY 19
adaptability to environment which in itself should be regarded as a sign
of degeneration.
SHAPE AND SIZE OF THE BODY.— The Protozoa vary considerably
in size, some of them being easily detected with the naked eye. Many
of the ciliates and gregarines can be seen as white specks or elongate
filaments, while certain multinucleate amoeboid organisms may be several
centimetres in diameter. The majority of Protozoa, however, are so small
that they cannot be seen without magnification. The adult individuals
of any species may vary considerably in size amongst themselves, and
there may be marked differences in size between the mature and immature
stages of development.
Protozoa may be of almost any conceivable shape, and the exact form
of the body may be regarded as a direct adaptation to their mode of life
and environment. When living in fluid media, unless the shape is deter-
mined by a relatively tough outer membrane or a skeletal support, there
is a tendency for the organism to assume the spherical form. Amoebae,
in which an outer membrane is entirely absent or represented by an
exceedingly fine pellicle, are spherical unless temporary contractions of
the cytoplasm or pressure of any body against which they come in contact
or over which they are moving overcomes the physical forces to which
they are subject (Fig. 5). So soon as relaxation occurs the spherical
form is resumed. In the majority of Protozoa the body is definitely
elongated even in a condition of repose, and it is evident that this form
is retained without any effort on the part of the organism itself. This
is due in most cases to the development of an elastic outer layer of cyto-
plasm, which retains its shape unless this is temporarily altered by pressure
or the contractions of the cytoplasm (Fig. 6). This outer layer of the
cytoplasm or periplast may attain a high degree of complexity. It may
be so tough, as in many of the Mastigophora and Ciliata, that the shape
of the body is practically constant.
In the case of certain Mastigophora, like Cercomonos, which are adapted
to a creeping mode of existence as well as a swimming one, the body is
constantly changing its shape when it is moving over a surface, with a
tendency to the assumption of an elongate form during progression
through a fluid (Fig. 7). In the majority of Mastigophora and Ciliata
which swim through liquids the body is elongated, and may even have
a spiral form, when movement is associated with revolution about the
longitudinal axis (Figs. 143 and 509). Certain Mastigophora and Ciliata
which lead a swimming existence as well as a creeping one upon the
surface of various objects are frequently flattened dorso-ventrally. In the
swimming forms there is a tendency for one end of the organism to be
more pointed than the other. Certain Protozoa become permanently
20
MORPHOLOGY OF PROTOZOA
attached to objects by means of filaments, and in such cases a cone-
shape is developed, the filament of attachment arising from the apex of
the cone (Fig. 19). Amongst truly parasitic Protozoa the body may be
a motionless'sphere, as in the growing phases of coccidia within the cyto-
plasm of cells; on the other hand, those which live in fluids in the body
spaces and are endowed with powers of
active movement, like free-living forms,
vary considerably in shape.
Amongst the Rhizopoda the body is
usually either globular or irregular in
-Cfi
Fig. 5. — Amceba proteus ( x 200).
(After Leidy, 1879.)
Fig. 6.
1,000).
—Eiiglena viridis ( x ca.
(After Doflein, 1916.]
C, Contractile vacuole ; Ch, chromato-
phores; R, reservoir; S, stigma.
shape, and there is no differentiation between an anterior and posterior
end or a dorsal or ventral surface (Fig. 5). In certain forms, however,
the body is protected by a shell, through an aperture in which pseudopodia
are extruded for purposes of locomotion and capture of food. In such
forms, of which Arcella and Difflugia are examples, it is possible to con-
SHAPE AND SIZE OF BODY
21
sider the aperture which is applied to the surface over whicli the organism
is moving as ventral in position, so that a dorsal and a ventral surface
can be distinguished (Fig. 8). Many Mastigophora are definitely elongate,
Fig. 7. — Cercomonas longicauda ( x 2000) : Changes in Shape undergone by
A Single Individual during Twenty Minutes' Observation. (Original.)
and locomotion takes place in the direction of the flagellate end (Fig. 6).
In these it is evidently possible to distinguish an anterior from a posterior
end. When a mouth aperture or cytostome is present, it is usually near
the anterior end, but slightly to one
side of the terminal flagella. The
surface nearest to which the cytostome
lies may be regarded as the ventral
surface, in which case it becomes
possible definitely to orientate the
organism. In the case of such a
flagellate as Trichomonas (Fig. 26) it
is legitimate to speak of the anterior
flagellated extremity of the body, the
posterior extremity through which the
axostyle protrudes, the ventral surface
^near which the cytostome is placed,
and the dorsal surface which is provided with the undulating membrane
and its basal fibre. This orientation becomes complicated to a certain
extent by the fact that a torsion or twisting of the body towards a spiral
Pig. 8. — Difflugia constricta : A Shelled
Rhizopod from Pond Water
( X 660). (Original.)
The shell is strengthened by adherent grain
of sand.
22 MORPHOLOGY OF PROTOZOA
form may occur. Thus in Trichomonas itself the undulating membrane
takes a slightly spiral course round the body, though its general tendency
is to be on the dorsal surface. Amongst the Ciliata this differentiation may
be carried to a high degree of complexity. In a few forms such as Prorodon
teres (Fig. 24) the cytostome is at the extreme anterior end of the body, and
the cilia pass in longitudinal rows from it to the posterior end. Though it
is possible in these cases to distinguish an anterior and posterior end, there
is actually no dorsal or ventral surface. In other forms the cytostome
has moved from its terminal position, and it at once becomes possible to
regard the surface on which the cvtostome is situated as the ventral one
VJJ.
m^f-^^
m
Fig. 9. — Stylonychia mytilus : Side and Ventral Views ( x ca. 250).
(From Lang, 1901, Slightly Modified.)
The side view shows the ciliate resting on a surface by means of the foot-like cirri formed by
fusion of groups of cilia. The dorsal cilia are few in number. There is a central contractile
vacuole with two excretory canals leading to it.
The ventral view shows the macronucleus in division and two daughter micronuclei. The
V-shaped peristome is bordered on its outer edge by a row of membranes passing round the
anterior end of the ciliate and leading to the cytostome at the apex of the V. A row of cilia
borders the other edge of the peristome, within which is a longitudinal membrane. The
contractile vacuole and parts of the canal are seen as clear areas.
(Fig. 14). In the majority of the free-living Ciliata there are definite
dorsal and ventral surfaces. These are most conspicuous in those forms
which lead a creeping mode of life, owing to the loss of cilia on the
dorsal surface, and the development of cirri and membranelles, through
the fusion of groups of cilia, on the ventral surface (Fig. 9). The cyto-
stome is on the ventral surface; it is not median in position, but displaced
to one side. In the case of attached forms such as Vorticella (Fig. 19)
POLYMORPHISM 23
the ciliated area in which the cytostome lies may be regarded as the ventral
surface, and the filament of attachment as a development from the apex
of the cone-shaped dorsal surface.
POLYMORPHISM.— It has to be recognized that amongst the Protozoa
variations in the shape and form of the body occur at different
stages of development. Such a variation is not a characteristic feature
of the Rhizopoda, for the smallest individuals of any species have essen-
tially the same body form as the fully-grown larger ones. Amongst the
Dimastigamoebidse at certain stages of development one or more flagella are
formed. Though it is purely an arbitrary matter whether the flagellate
stage is considered to be the adult form or not, these amoebae are definitely
polymorphic (Figs. 119 and 120). As all Rhizopoda, however, are able
to encyst under certain conditions, the encysted stage has to be recognized
as another form in which any particular amoeba may occur. Amongst
the free-swimming Ciliata, again, the smallest individuals differ little
except in size from the fully -grown largest forms.
Protozoa which only show a slight degree of variation in body form
during their life-cycle are termed monomorphic, to distinguish them from
polymorphic forms, which show this to a marked degree. This poly-
morphism is well illustrated by the development of the Suctoria (Fig. 532).
Amongst these Protozoa th e attached adult buds off a small ciliated embryo,
which, after leading a free-swimming existence for a time, finally attaches
itself, loses its cilia, and grows into the adult, which is provided with
sucking tentacles. As the ciliated stage is only of a temporary nature,
and is small when compared with the tentacled stage, it is regarded as the
embryo. Amongst the Sporozoa there is a high degree of polymorphism
associated with their complicated cycles of development. In the case of
the malarial parasites, for instance, the organism passes through a constant
series of changes of form (Fig. 391). The minute amoeboid organism
within the red cell grows into the schizont, which breaks up into elongate
merozoites, which again become amoeboid forms in other cells. Some
merozoites develop into gametocytes of two types, which can be distin-
guished from the schizonts. In the mosquito the gametocytes change
in character and produce elongate vermicular zygotes, which pass through
the stomach wall and develop into oocysts, which again produce a large
number of minute sickle-shaped sporozoites, which differ in character from
the merozoites. In this case, as in other Sporozoa, there is a high degree
of polymorphism, as exhibited by a constant series of changes in the size
and form of the body. Very frequently there is a polymorphism associated
with the occurrence of a sexual process and the formation of gametes.
In the gregarines the gametes which unite may be exactly alike, in which
case the process is known as one of isogamy. On the other hand, in certain
24 MORPHOLOGY OF PROTOZOA
gregarines the uniting cells difier from one another (anisogamy), so that
there is a degree of polymorphism as regards the character of the gametes
(Fig. 482). It may happen that the individual which gives rise to gametes
of one type differs from that which gives rise to gametes of the other type.
This differentiation may extend further back in the life-history, so that
it is possible to recognize two distinct types of reproducing individual,
each with its particular characters (Fig. 341). The individuals of one
series may eventually, after a period of multiplication, give rise to gametes
of one type, while those of the other series give rise to gametes of another
type. In such cases it might be supposed that one was dealing with two
distinct organisms, each reproducing its kind. The fact that the gametes
produced by the one unite with those produced by the other proves that
the two series belong to one polymorphic species. This condition is known
as one of sexual dimorphistn, a term which is also employed in a more
general sense to indicate the occurrence of individuals of any species
which can be distinguished as male and female.
Though all these variations in form, which occur as a result of growth,
complicated life-cycles or the sexual process, are examples of polymorphism,
the term is often employed in a more restricted sense. When it has been
decided which stage of the organism is to be regarded as the adult form,
it may be found that the adults resemble one another very closeh^, in which
case the organism is said to be monomorphic. Thus, in the case of
trypanosomes the commonly observed forms in the blood of an animal
may vary very slightly. In these cases the organism is termed a mono-
morphic trypanosome, examples of which are Trypanosoyna evansi and
T. congolense (Figs. 227 and 234). In other cases, as, for instance, Try-
panosoma brucei, it may be possible to distinguish in the blood of an animal
several distinct types — long thin, intermediate, and stumpy trypanosomes
— and forms with or without free flagella (Fig. 225). On this account
T. brucei is regarded as a polymorphic trypanosome. If, however, the
whole life-cycle in the vertebrate and invertebrate hosts of such a form as
T. lewisi, which at certain phases appears monomorphic, is taken into
consideration, it will be found to exhibit as great a degree of variation as
in the polymorphic trypanosomes (Fig. 197).
It seems clear, therefore, that the term " polymorphism " is incapable of
exact definition. Strictly speaking, no Protozoan is monomorphic, while
all are polymorphic. Those which are considered monomorphic show
only a slight degree of variation, while those which are polymorphic show
the variations, but to a greater extent. Any organism may be regarded
as polymorphic because it differs at different stages of its growth and life-
history, or it may be considered as polymorphic because the individuals
which have all reached any particular stage do not resemble one another
RACES 25
very closely. Human beings may be regarded as polymorphic because
the child differs from the adult, or they may be considered polymorphic
because the adults differ amongst themselves. It is in the latter sense that
the term is commonly employed in connection with trypanosomes. It
must be recognized, however, that the trypanosomes which are regarded
as being polymorphic may not all be in the same stage of development.
There is evidence which points to the fact that the shorter stumpy forms
of T. brucei or T. gambiense are the result of growth from the long slender
forms which are present in the blood at the same time (Figs. 222 and 225).
RACES. — Amongst Protozoa, as amongst human beings, there occur
different races of one and the same species. The individuals of one race
differ from those of another in size, shape, rate of multiplication, and other
characters. Each race breeds true to its type to a large extent, so that
even after long periods of multiplication the same differences are observed
in the resulting progeny. On this account it often becomes a matter of
difficulty to decide whether two different forms are merely races of one
species or are actually different species. Thus, in the case of Entarnoeha
histolytica there appear to be several races which can be distinguished from
one another by the average size of the cysts they produce (Fig. 10).
Many researches have been conducted on the race question in species
oi Paramecium, Difflugia, and other Protozoa, especially by Jennings. It
has been observed that the characters of any particular race tend to remain
constant, so that there is considerable difficulty in understanding how these
races arose in the first instance. Evidence has, however, been obtained by
Jennings (1916) in the case of Difflugia corona and by Middleton (1915)
for Stylony cilia, which proves that after long periods of multiplication
definite inheritable variations do occur in the descendants of a single
individual, and this quite apart from any sexual process. It therefore
seems probable that if the observations were continued for a sufficient length
of time, it would be possible to separate from the descendants of a single
individual various races which would be as distinct from one another as
the naturally occurring races. If this were not so, it would be difficult to
understand how evolution could take place at all.
A practical point which arises from the knowledge which has been
acquired regarding races of Protozoa is that the separation of species,
on account of comparatively slight variations in size, is a very questionable
procedure. The literature dealing with parasitic Protozoa contains
numerous instances of the establishment of new species merely because
the dimensions differed slightly from those of a form previously described.
Another type of race peculiarity occurs amongst the Ciliata. It was
shown by Dawson (1919) that Oxytricha kymenostoma, which normally has
both a macronucleus and a micronucleus, may occasionally have the
26
MORPHOLOGY OF PROTOZOA
macronucleus alone. Such an amicronucleate race was cultivated by him
for several years, during which regular multiplication by fission took place.
Abortive attempts at conjugation appeared to be made, but the process
was never completed. Landis (1920) has studied a similar race oi Para-
mecium caudatum, and Patten (1921) one of Didinium nasutum, while
Woodruff (1921a) has described amicronucleate races of OxytricJia fallax
and TJrostyla gra?idis.
P^IG.
10. — Cysts of E. histolytica from Three Distincx Races ( x 2,200).
(After Wenyon and O'Connor, 1917.)
1-3. Race with exceptionally large cysts
7-9. Race with small cvsts
4-6. Race with usual type of cyst.
CYTOPLASM.— The cytoplasm of the Protozoan cell does not differ
in any essential respect from that of cells of multicellular animals. As to
the nature of its minute structure many theories have been advanced.
That which seems to be most satisfactory is Biitschli's view that cytoplasm
is of the nature of an emulsion consisting of at least two substances,
one of which in the form of minute globules is suspended in the other,
which forms the septa between the globules. In optical section, the
CYTOPLASM 27
substance between the globules has the appearance of a network of fibres.
Embedded in these apparent fibres or septa are granules of various kinds
and sizes. The cytoplasm commonly contains vacuoles, which are
spherical spaces containing a material which is more fluid than the con-
stituents of the cytoplasm itself. Very frequently within the vacuoles
are food particles which the organism has ingested. In such cases the
vacuoles are known as food vacuoles or digestive vacuoles, and into them
are secreted acid ferments capable of transforming the food into substances
suitable for assimilation by the cytoplasm. The products of digestion are
gradually absorbed into the cytoplasm, and any residue is got rid of by the
vacuole approaching the surface of the body and discharging its contents
into the medium in which the organism is living. The vacuole is then
no longer visible. In the majority of free-living Protozoa there are one
or more vacuoles, which are known as contractile vacuoles or pulsating
vacuoles. Such a vacuole is near the surface of the body, and when fully
formed contains a clear fluid. By a sudden contraction the contents of
the vacuole are discharged through the surface of the body, and the vacuole
disappears. Very soon, however, a minute vacuole reappears at the same
spot. It gradually increases in size owing to the flow of liquid into it,
sometimes along definite channels. When it has attained its full size,
expulsion of the contents again takes place. These vacuoles appear to be of
an excretory nature, and the intervals between the contractions vary with
the temperature and other conditions. For some reason not clearly under-
stood, contractile vacuoles are frequently absent in parasitic Protozoa.
Within the cytoplasm of many Protozoa there occur various structures
which are to be regarded as secretions of a skeletal nature. In the
Heliozoa, for instance, radially arranged rod-like supports for the pseudo-
podia are formed (Fig. 75), while in many of the Radiolaria complicated
fenestrated shells of a spherical or other shape are secreted in the cyto-
plasm (Fig. 78). These internal structures are not to be regarded as part
of the cytoplasm itself, but bear the same relation to it as the external
shells and coverings, which are sometimes formed around the organism
for protective purposes of a permanent or temporary nature.
A very noticeable feature of the cytoplasm of Protozoa is its differ-
entiation into an ectoplasm and an endoplasm. The former is of tougher
consistency and more hyaline than the endoplasm, and forms a superficial
layer of varying thickness enclosing the more liquid and granular endo-
plasm. The endoplasm, even when the organism is at rest, appears to be
constantly streaming in various directions. The different vacuoles and
bodies, and even the nucleus itself, are constantly changing position as a
result of the currents in the endoplasm. It is in the endoplasm that the
various vacuoles and internal skeletal structures occur, while the ectoplasm
28 MORPHOLOGY OF PROTOZOA
may become highly differentiated. A tough covering to the body, which
may be elaborately marked, is often developed from the ectoplasm, while
it is from this layer that the various permanent organs of locomotion such
as flagella and cilia originate. The ectoplasm also secretes the various
external coverings, such as shells and cysts. In the simpler Protozoa,
like the amoeba? and flagellates, the ectoplasm is merely a thin layer of
clear cytoplasm surrounding the endoplasm. It appears to be only
slightly more resistant than the endoplasm. In the more highly organized
ciliates and gregarines the ectoplasm is highly developed, and itself consists
of several distinct layers. It is a resistant membrane which enables the
organism to retain its shape. In any case, the most superficial layer of the
ectoplasm forms a delicate limiting membrane, the periplast. The surface
of the ectoplasm may be perfectly smooth, or it may be raised into a series
of longitudinal ridges. In other cases it is roughened, or may even develop
a series of symmetrical markings. In the amcebse, many of the simpler
flagellates, and many parasitic protozoa, the ectoplasm forms a complete
layer over the surface of the body, and when solid food is ingested this is
taken in at any part of the body. A particle comes in contact with the
ectoplasm which is gradually raised up round it, and finally closes over
it, so that the object, together with a certain quantity of liquid, is included
in a vacuole which sinks into the endoplasm. In other cases the solid food
particles are ingested in a similar manner at one particular spot on the
body surface. This occurs typically in certain flagellates, where solid food
appears to be ingested only at the base of the flagellum. In other flagel-
lates at this point there is a small excavation or pit in the ectoplasm into
which solid food is taken (Figs. 26 and 33). At the bottom of this pit the
food particle sinks into the endoplasm, and is included in a vacuole. This
depression is frequently of a permanent nature. In association with it
there may be special developments of the organs of locomotion which
create currents in the medium, so that food particles are directed into it.
In Chilomastix one of the flagella lies in a groove, at the posterior end of
which food particles enter the cytoplasm (Fig. 69). The opening in the
ectoplasm, which sometimes is capable of being opened and closed, is known
as the cytostome, while the funnel-shaped pit or tube leading from it to the
endoplasm is the oesophagus or cytopharynx.
As already pointed out, the residue from the digestion of food material
within the food vacuoles is discharged through the surface of the body.
This may occur at any point on the body surface, but in the Ciliata there
may be a permanent opening in the ectoplasm, the cytopyge, which, how-
ever, is usually only visible when a food vacuole discharges its contents
at the posterior end of the body (Fig. 512).
In some ciliates the cytostome is a simple opening on the surface of the
CYTOPLASMIC INCLUSIONS 29
body, but the region round the cytostome (peristome) may be modified
in various ways. There may be a ciliated groove leading to the cytostome
(Fig. 70), or a disc-like area upon which cilia are arranged in a spiral manner
(adoral zone of cilia) may be developed. These cilia are often continuous
with others within the cytopharynx. In the Peritrichida, like Vorticella
and Carchesium, the area round the cytostome is sunk in the form of a
funnel-shaped depression, the vestibulum, the opening of which may be
completely closed by contractions of the cytoplasm. Within the ves-
tibulum is found the cytostome itself, while the food vacuoles and con-
tractile vacuole also discharge their contents into it (Fig. 528).
CYTOPLASMIC INCLUSIONS.— In association with the ingestion of food
and metabolism, granules, globules, and crystals of various kinds may
appear in the endoplasm. These are quite distinct from the partially
digested food in the food vacuoles, though they result from food meta-
bolism. Many Protozoa having affinities with the plants and possessing
chlorophyll are able to form starch, which occurs in the cytoplasm as
characteristic starch granules. They are commonly present in Euglena
and other similar forms. Another substance allied to starch is known as
paramylum. Fat globules are seen especially amongst the Radiolaria
within the inner capsule. They also occur in the marine flagellate
Noctiluca, and it has been suggested that they assist these organisms to
float. Doflein (1910) has noted that, in old cultures of Trypanosoma
rotatorium the flagellates may contain droplets of fat. Another substance
which is of common occurrence in the cytoplasm is glycogen, or a closely
allied substance which was called paraglycogen by Biitschli. These have
a strong affinity for iodine, which colours them an intense brown. Glycogen
is present in gregarines, certain ciliates, and very commonly in the encysted
forms of amoebae and flagellates (Plate II., p, 250). The iodophilic body
which occurs in the encysted stage of lodamceha hiUschlii has given rise to
its generic name. A substance which is of wide distribution amongst
the Protozoa is volutin. It is usually seen in living organisms as globules
of a greenish refractile material which takes a yellow colour in iodine.
Owing to the fact that it may stain deeply with chromatin stains, it has
often been regarded as chromatin. Some observers maintain that it is
actually a forerunner of chromatin. Volutin is often present in the
cytoplasm of trypanosomes and other flagellates, and appears as dark
red granules when they are stained with Romanowsky stains. It commonly
occurs in hsemogregarines and many Sporozoa, as also in amoebae and
ciliates. A substance which may be allied to volutin is seen in the
chromatoid bodies which are present in the cysts of some intestinal amcebae.
They occur so frequently in the encysted forms of Entamoeba histolytica
in the form of bars that they are highly characteristic of this species
30 MORPHOLOGY OF PROTOZOA
(Fig. 96). They are less often seen in the encysted stages of Entamoeba
coli. Like the glycogenic or iodophilic body in the encysted form of
lodamoeba hutschlii, they disappear in the course of a few weeks after
escape of the cysts from the intestine, apparently serving as a supply of
nourishment for the enclosed amoebae.
Another type of cytoplasmic inclusion is the chromatophore, which is
characteristic of many plant-like flagellates grouped amongst the Phyto-
mastigina (Fig, 130). These are bodies which contain various pigments
known as chromatophyll. When green it is called chlorophyll, and when
red haematochrome. As in plants, these bodies enable the organism to
utilize the carbonic acid of the medium in which they live. It has been
show^n that the chromatophores multiply by fission in the cytoplasm, as
also do certain granules known as pyrenoids which may be present in the
chromatophores. It has been surmised that the chromatophores may be
symbiotic organisms living in the cytoplasm.
In the process of ingesting solid food many Protozoa actually ingest
other forms, or even their own species, either in the free or encysted
condition (Fig. 99). The writer has seen a large vacuole in Entamoeba
muris of the mouse filled with actively motile Triclioynonas. The in-
testinal amoebae of man frequently ingest the encysted forms of other
intestinal Protozoa. In many cases the ingested organisms are killed
and digested, but this is not always the case. Instances are known in which
amoebae and even ciliates may have their cytoplasm riddled with vacuoles
in which smaller amoebae or flagellates occur. These may eventually
escape apparently unharmed by their stay in the cytoplasm of another
organism. Protozoa are also liable to invasion by bacteria. Such a
condition approaches, and may actually be, one of parasitism. Instances
of true parasitism are seen in the invasion of the body of Parameciimi by
the Suctorian Sphcerophrya pusilla (Fig. 534), and of various intestinal
flagellates and amoebae by Sphcerita, a vegetable organism which often
resembles a cluster of large cocci (Fig. Ill, 4). The inclusion of smaller
organisms within the cytoplasm of larger ones has always to be remembered,
especially when a process of multiplication by the production of daughter
individuals within the cytoplasm of a parent is considered. The nuclei
of such forms may be mistaken for nuclei belonging to the host. A method
of reproduction of Pelomyxa palustris, a large multinucleated amoeba,
by the production of flagellated forms within vacuoles in its own cytoplasm
has been described by Schirch (1914). It seems not improbable that this
was an instance in which an amoeba had ingested, but failed to kill, a number
of flagellates which were present in the medium. Doflein (1916) mentions
an instance in which the cytoplasm of a ciliate, Stentor ca'rulevs, included
numerous small flagellate organisms.
LOCOMOTOR AND PREHENSILE ORGANS 31
ORGANS EMPLOYED IN LOCOMOTION AND CAPTURE OF FOOD. -The
simplest organs which are used for purposes of locomotion are the
pseudopodia, characteristic of the Rhizopoda or amoebae (Fig. 5). They
are simply processes of cytoplasm which are formed from the surface of
the body. A small elevation of the ectoplasm occurs at any point, and
this gradually increases in size till the endoplasm also takes part in its
formation. When it has reached a certain size it may be withdrawn
gradually, and another formed in some other direction. On the other hand,
it may increase steadily in size till the whole body of the organism flows
into it. In this manner, by the regular production of pseudopodia, an
amceba may move from one spot to another. It is by means of pseudo-
podia passed around any object that food particles are ingested. The
movements and changes in shape associated with the formation and with-
drawal of pseudopodia are termed amoeboid movements, w^hich are exhibited
typically by the amoebae. Certain flagellates as well as Sporozoa, such as
the malarial parasites, may also move in this manner. The pseudopodia
may be blunt finger-like processes of a lobose type, or they may be relatively
long, thin, and tapering, and of a filose type. The long narrow filose
pseudopodia may remain separate from one another, or they may become
united by lateral anastomoses, so that an organism possessing many of
them appears to be surrounded by a fine network of cytoplasm, as in the
Foraminifera (Fig. 72). In the case of the Heliozoa and Radiolaria, the
filose pseudopodia are more permanent structures, known as axopodia,
and are supported by radially arranged axial rods secreted by the endo-
plasm, or formed as outgrowths from the central granule (Fig. 51).
Flagella and cilia are more permanent organs of locomotion. The
former are characteristic of the Mastigophora, and the latter of the
Ciliophora. They are long, narrow, whip-like processes which are capable
of performing vindulating or lashing movements, which cause currents in
the medium and enable the organism to progress through it. A single
flagellum has essentially the same structure as a cilium, though it is usually
larger, and is capable of more violent lashing movements. Generally
speaking, the small number of flagella possessed by a flagellate fulfils
the functions of the large number of cilia possessed by a ciliate. A
flagellum, as pointed out by Alexeieff (191 le), consists of an axial filament,
for which the term axoneme, suggested to the writer by Colonel A. Alcock,
will be employed, and a thin sheath of cytoplasm (Fig. 157). The axoneme
itself takes origin in a minute granule, the blepharoplast, which is situated
in the cytoplasm, and sometimes upon the surface of the nuclear mem-
brane. The axoneme passes to the surface of the body, and there, acquiring
a thin sheath of cytoplasm, becomes the flagellum. There can thus be
distinguished an intracytoplasmic portion of the axoneme and a flagellar
32
MORPHOLOGY OF PROTOZOA
portion. For the former the name rhizoplast is often employed. When
an organism is developing a flagellum, a blepharoplast first becomes
apparent in the cytoplasm, and an axoneme is formed as an outgrowth
from it. When the surface of the body is reached, increase in length still
takes place, the axoneme pushing out a thin covering of cytoplasm. It is
probable that the axial rod of an axopodium is a homologue of the axoneme
of a flagellum.
The flagella of the Mastigophora vary in number. In the typical forms
they are not numerous. There may be only a single one, or as many as
eight. They arise most usually from the anterior end of the body, and
are directed forwards. By their lashing movements they propel the
organism through the medium. In some instances certain flagella arise
from the posterior end of the body, and are directed backwards. Thus,
in Hexamita two of the eight flagella are posterior in position, but their
axonemes can be traced through the cytoplasm to the anteriorly situated
blepharoplasts (Fig. 288). In other
cases, as in Tricercomonas and Cerco-
monas, the axonemes of the posterior
flagellum can be traced over the surface
of the body to the anterior end, where it
enters the cytoplasm and passes to the
blepharoplast (Figs. 259 and 261). In
the flagellates of the genera Trypano-
plasma and Trichomonas such a back-
wardly directed axoneme is adherent to,
or embedded in, the margin of a thin
band of cytoplasm, the undulating mem-
brane (Figs. 26 and 151). In other cases, such as Bodo, one flagellum is
directed backwards, and acts as a trailing flagellum without being attached
to the surface of the body (Figs. 21 and 33). In the case of the trypano-
somes, the blepharoplast occupies an unusual position at the posterior
end of the body. The axoneme arising from it is directed forwards, and
passes over the surface of the body or along the margin of an undulating
membrane as far as the anterior end of the body, where it either terminates
or becomes a flagellum (Fig. 28, B).
All the flagella possessed by a flagellate may be uniform as regards
length and thickness when they fulfil the same function. Frequently,
however, variations occur. In the case of E^nbadomorias, one of the two
flagella, which organisms of this genus possess, is associated with the
cytostome, and is much thicker and shorter, and performs more regular
undulating movements than the anteriorly directed one (Fig. 11).
Flagella are employed not only for purposes of progression, but also for
Fig. \\.~Enibadomonas sp. from
Culture of Intestinal Con-
tents OF Testudo argentina
( X ca. 1,500). (Original.)
LOCOMOTOR AND PREHENSILE ORGANS
33
the capture of food. A cytostome, when present, is always near the point
of origin of the flagella, one of which may be specially modified in con-
nection with the cytostome. Thus, in Chilomastix one flagellum is
permanently within the cytostomal groove, where it functions by creating
currents which assist in the capture of food (Fig. 69). The thicker of the
two flagella possessed by Emhadomonas has a similar function (Fig. 11).
As already remarked, in typical flagellates the flagella are few in
number, but there occur certain forms which possess many flagella.
Fig.
12. — Parajcenia grassii ( x 1,500).
(After Janicki, 1915.)
Fig. 13. — Holomastigoldes hertwigi
( X 320). (After Hartmann,
1910.)
These are the Hypermastigida, which occur chiefly as intestinal parasites
of white ants (Figs. 12 and 13), They stand in this respect as a connecting
link between the Mastigophora and the Ciliophora, with both of which
groups observers have classed them. Though the possession of flagella is a
characteristic feature of the Mastigophora, it must be remembered that
these organs of locomotion are not peculiar to this group. Certain forms
which are classed with the Rhizopoda, and which are amoeboid organisms,
may have flagella at certain stages of development. Similarly, amongst
the Sporozoa the microgametes are commonly supplied with one or two
I. 3
34
MORPHOLOGY OF PROTOZOA
flagella, which enable them to move about in search of the macrogametes
(Fig. 337).
As noted above, the cilia which characterize the tiliophora resemble
small flagella. They have a similar structure, and their axial fibres take
origin in minute granules situated in the ectoplasm. It seems reasonable
to suppose that the axial fibres and the basal granules of cilia are homo-
logous with the axonemes and blepharoplasts of flagella. A single ciliate
possesses a large number of cilia, which exhibit more regularity and
co-ordination in their movements than the flagella of one of the Mastigo-
phora. In some ciliates the body is covered uniformly with cilia, which,
however, are usually arranged in longitudinal
rows (Fig. 14). In other cases the cilia are
limited to special regions of the body. The
cilia may be fairly uniform in length, but fre-
quently those on the extremities of the body
and those which surround the cytostome are
slightly longer than the others. Cilia are often
continued into the cytopharynx. Sometimes,
as in Cyclidium and other forms, one posterior
cilium is much larger than the others, and forms
a kind of tail or caudal process which has very
much the same size and structure as a flagellum
(Fig. 500). Several adjacent cilia may fuse
together to form stout processes known as cirri.
These are seen typically on the ventral surface
of those ciliates (Hypotrichida) which lead a
creeping mode of existence (Fig. 9). They
function as supporting structures or legs. In
some cases, again, rows of cilia may unite to
form membranes. This occurs frequently in
the cytopharynx of certain ciliates, such as
Paramecium, Pleuionema, and others (Fig. 70).
These membranes, or membranelles as the smp.ll
ones are often named, are distinct from the undulating membranes
of Mastigophora (trypanosomes), which are thin ridges of ectoplasm,
and are not formed by the fusion of rows of cilia. The cilia on the
peristome region near the cytostome may differ little from those on other
parts of the body. On the other hand, they may be considerably
modified in character and arrangement. In many forms they are
arranged as a spiral to form the adoral zone of cilia, which are continuous
with those in the cytopharynx. The spiral may be a left-handed spiral
or a right-handed one. It may consist of only a single turn or part of one.
FiG. 14. — BaJaritidiuni ento-
ZOOn FROM THE KeOTUM
OF THE Frog ( x 650).
(Original.)
The longitudinal rows of cilia on
the surface of the body are
represented by dots.
LOCOMOTOR AND PREHENSILE ORGANS
35
or there may be as many as five complete turns. The spiral may be com-
pared with a portion of a fiat watch-spring, the cytostome being situated
at the outer end of the spiral, which lies on the peristome area in front of
the cytostome. The cilia composing the spiral generally consist of several
Fig. 15. — Various Species (jf Suctoria. (After Saville Kent. 1880-1882.)
(a) Sp^cerophrija magim feeding on ciliates ( x .300).
(c) Tokophrijalemnarum {X 100).
(h) Acinetagmndis (X 100).
(d) Discophri/a elongata (X 1.30).
parallel rows, and those of adjacent rows may unite in such a way as to
form a series of spirally arranged, fiat, tongue-like processes or mem-
branelles (Fig. 511). Within the cytostome the cilia may fuse to form one
or more membranes parallel to the axis of the cytopharynx. The general
36
MORPHOLOGY OF PROTOZOA
structure and arrangement of the cilia on the body of ciliates and the
modifications undergone by those associated with the cytostome are
features of importance in the classification and determination of the
species and genera of the Ciliata, just as the number and characters of
the flagella are of importance in the classification of the Mastigophora.
Amongst the Suctoria, which in their youngest stages are provided
with cilia, special organs for use in nutrition are developed in the adults
Pid. 16. Monosiga coiisociatum from Hay Infusion ( x 2,000). (Original.)
1-7. Free and attached individuals of varying size. 8 and 9. Encysted forms.
CFW. 15). These are known as tentacles, and each is a tubular process
of cytoplasm terminating in a disc-like sucker. The latter is applied to
food material, which is taken into the body by a sucking process. It is the
possession of these sucking tentacles which has given rise to the names
Suctoria and Tentaculifera, by which these forms are known.
Another type of structure which probably has to do with the capture
of food is the thin collar which is developed in certain Mastigophora
(Fig. 16). The cytoplasm at the anterior region of the body is raised
LOCOMOTOR AND PREHENSILE ORGANS 37
into a thin cylindrical collar or cuff round the flagelliim. The collared
forms frequently possess attachment filaments, simple or branched, and
often cup-like loricge. The collared forms are generally known as the
Choanoflagellata. Similar flagellated collar cells are found in the group
of Metazoa to which the sponges belong. In many cases it appears that
the collar is not a cylinder, but a cuff with overlapping edges.
A peculiar modification of the ectoplasm which facilitates locomotion
occurs in gregarines. These organisms are able to glide over a surface
without exhibiting any movements of contraction of the body by reason
of longitudinal ridges of ectoplasm between which a quantity of mucoid
material can be rapidly excreted. The excretion of this tenacious material
Fig. 17. — Codonosiga allioides : A Colony of Collared Flagellates on a
Branched Filament ( x 320). (From Lang, 1901, after Kent.)
causes the organism to be pushed forwards without any apparent move-
ments of the body. Similar gliding movements are often exhibited by the
merozoites or sporozoites of the Sporozoa. In the case of certain amoebae
such a gliding movement appears to be the result of constant streaming
of the cytoplasm from behind forwards, w^hile the ectoplasm in contact
with the surface remains stationary, very much as a bag of water can
be pushed along the surface of a table.
ORGANS OF ATTACHMENT.— Though the majority of the Protozoa
are free-living organisms, certain forms are able to attach themselves
temporarily or permanently to objects.
Amongst the Mastigophora there are many pedunculated forms. The
posterior extremity of the body is developed into a filament, by means
38
MORPHOLOGY OF PROTOZOA
of which fixation to various objects is brought about (Fig. 18). Such
forms are more or less permanently attached. By longitudinal division
of the attached flagellate and the continued development of the filament
Fic;. 18. — Various Attached Flagellates. (1, From Lang, 1901, after Kent;
2, From Lemmermann, 1914, after Kent; 3, After Doflein, 1916.)
1. PoUceca dickotoma {X 1,000). 2. Codonosiga botrijtis {x 1,200).
3. Amphimonas (jlohosa ( x 1,500).
from the posterior end of the body complicated branched filaments are
developed (Fig. 17). Sometimes the end of the branch is continued
round the organism as a cup-like expansion or lorica, in which it lives
ORGANS OF ATTACHMENT
39
(Fig. 18, i). Similarly, amongst the Ciliata filaments of attachment, either
simple or branched, may be developed. In some cases, as, for example,
Vorticella, the filament contains a contractile thread, by means of which
it can be suddenly coiled up in a spiral manner and the ciliate withdrawn
when it is subject to adverse stimuli (Fig. 19).
Amongst parasitic Protozoa, many gregarines are provided with
special organs of attachment. The young organism which develops from
the sporozoite is at first intracellular, but as growth occurs it leaves the
host cell, to which, however, it remains attached by a process known as
the epunerite (Fig. 20). This structure is developed in various ways,
and may be compared to the organ of attachment of tape-worms. It
Fig. 19. — VoHicella nehulifera : A Group of
Stalked Ciliates attached to an Object
( X 200). (From Lang. 1901, after
d'Udekem.)
1. Contractile vacuole; 2, daughter individual with
circlet of cilia; .3, dividing form; 4, conjugation.
m
Fig. 20. — A Cephaijne Gre-
GARINE {Corycella armata)
( X ca. 300), showing
Epimerite, Protomerite,
AND DeUTOMERITE. (AfTER
Leger, 1892.)
may be a simple swollen body embedded in the cytoplasm of the cell,
and connected with the parasite by a kind of neck, or there may be de-
veloped from it a series of filaments or roots which anchor the parasite
to the cell. In some cases a large sucker-like process is applied to the
surface of cells, and from it a series of filaments pass into the cells or
between adjacent cells. In other cases the epimerite is supplied with a
40 MORPHOLOGY OF PROTOZOA
series of small spines. After growth of the gregarine is complete, the
epimerite is detached (Figs. 481 and 485).
Many Mastigophora are able to attach themselves temporarily to
objects. This is generally effected by a flagellum, as in species of Bodo
(Fig. 21), but some forms, like Oiko-
monas, can become fixed by a pseudo-
podium-like process developed from the
posterior end of the body. In the case
of trypanosomes and their allies attach-
ment to cells is an important feature of
development in the invertebrate host.
In the intestine, proboscis, or salivary
gland of insects in which development
is taking place, large numbers of the
flagellates may be attached to the sur-
face of the cells, and as longitudinal
division may take place while they are
Fig. 21. — Bodo saltans: A number of In-
dividuals ATTACHED TO A MaSS OF
Debris by the Trailing Flagella
(x 1,000). (Original.)
Fig. 22. — Stentor cceruleus ( x 146).
(Original Drawing from Life
by b. jobling.)
attached, the surface of the cells may become completely covered with
attached organisms. In this process, what usually happens is that the
flagellum disappears, attachment being effected by the tip of the axoneme
(Fig. 150).
In some Protozoa there is a sucker-like development of the surface
SKELETAL OR SUPPORTING STRUCTURES
41
of the body which enables the organism to attach itself temporarily.
In the case of Giardia {Lamblia) the ventral surface develops a large
sucking disc, by means of which the flagellate is able to attach itself to
the surface of the intestinal cells (Fig. 291). Amongst the Ciliata Stentor,
which is conical in shape, is able to fix itself to objects by pseudopodium-
like processes at its tapering posterior end (Fig. 22).
SKELETAL OR SUPPORTING STRUCTURES.— It has already been
pointed out that some Protozoa are able to build for themselves pro-
tective external coverings. Amongst
the Rhizopoda these are seen typically
amongst the Foraminifera and Radio-
laria. The shells may be strengthened
by the adhesion of granules of sand,
spicules, or other material. In the
Foraminifera the shells are external
coverings, the pseudopodia being pro-
FlG. 23. — CiLIATES WITH LORIC.E AND
Opercula which Close the Orifice
WHEN Retraction Occurs ( x 250).
(From Lankester, 1903, after Kent
AND Wright.)
1. CotJiurinaaffinis. 2. Cothurinavalvala.
Fig. 24.
(From
teres ( x 660).
1912, after
Schewiakoff, 1896.)
N, Macronucleus ; n, micronucleus ; o,
mouth; ces., oesophagus with rod-like
supports; f.v., food vacuoles; c.v., con-
tractile vacuole; al, alveolar layer; st,
meridional rows of cilia; «., anal opening.
truded through an opening as a snail emerges from its shell (Fig. 8).
In the Radiolaria the skeletal supports are more complicated, and
consist of spherical or asymmetrically formed fenestrated shells,
strengthened by various radially or tangentially arranged spicules
embedded in the cytoplasm (Fig. 78). The cup-like loricae found
amongst the Mastigophora (Fig. 18, i) and Ciliata (Fig. 23) may be
42
M0RPH0L0C4Y OF PROTOZOA
regarded as external skeletons or supports. These various structures
are secreted by the cytoplasm, from which they are separate. In all
Protozoa which have a distinctive body form it is the rigidity of the
ectoplasm which enables the organism to retain its shape. In certain
cases, what may be regarded as
modifications of the cytoplasm are
developed for purposes of support.
Thus, in certain Ciliata, as, for
instance, Prorodon, the anteriorly
placed cytostome leads to a cyto-
pharynx which is supported by a
series of longitudinally arranged
rods (Figs. 24 and 25). These rods,
or trichites, can be drawn apart and
the cytostome opened by radially
arranged contractile fibres attached
to each rod. In connection with the
cytostome of certain Mastigophora,
such as Chilomastix, the margin of
the cytostomal groove which leads
to the cytostome is supported and
rendered rigid by special fibres
(Fig. 69). In Trichomonas, again.
Fig. 25. — Section through Cyto-
stome OF Prorodon teres, showing
Supporting Rods ( x ca. 600). (From
MiNCHiN, 1912, after Maier.)
N, Nucleus; R, rods; ;«.?-. and m.r.',
myonemes.
Fig. 26. — Trichomonas {Pentatricho-
monas) from the Human intestine
( X 3,200). (After Kofoid and
SwEZY, 1924.)
the line of attachment of the undulating membrane is supported by a
special basal fibre which takes origin in a blepharoplast, and appears to
function by keeping the membrane stretched to its full extent (Fig. 26).
Another structure which also occurs in Trichomonaf! and allied forms
is the axostyle. This is a stift rod which commences in the blepharoplasts,
.SKELETAL OR SUPPORTIXG STRUCTURES
43
and passes through the centre of the body to protrude with a sharp
point at the posterior end. It is a structure which has little affinity for
stains, and its function and origin are not properly understood. Some-
times the flagellates are seen attached to debris by the pointed extremity
of the axostyle, but this is possibly only accidental. It is, perhaps, best
to regard the organ as skeletal in nature. Not infrequently, as explained
above, some of the axonemes which arise from the blepharoplasts at the
(:
Fig. 27. — Trichomonas vaginalis, showing tendency of Axostyle to Split into
A Series of Fibrils ( x ca. 2,000). (After Reuling, 1921.)
anterior end of the body, instead of becoming free flagella at the anterior
end, pass backwards through the cytoplasm to become free flagella at
other parts of the body surface. This condition is well seen in Hexamita
and Giardia (Figs. 288 and 291). It is customary to speak of the intra-
cytoplasmic portions of the axoneme in these flagellates as axostyles,
but this is clearly a misapplication of the term, for there is no evidence
that the axostyle of Trichomonas has any real homology with an axoneme,
though Kofoid and Swezy (19L5) have suggested that it represents an
44
MORPHOLOGY OF PROTOZOA
intracytoplasmic flagellum. The axostyle usually appears as a clear
homogeneous structure, but sometimes a fibre has been described as
passing along its central axis, while Reuling (1921) has noted that the
axostyle of Trichomonas vaginalis may sometimes split into four separate
fibrils which originate in the blepharoplasts. He regards the axostyle as
composed of four united fibres (Fig. 27).
MYONEMES.— It may be accepted that one of the characteristics of
cytoplasm is its power of spontaneous movement. In many Rhizopoda
and Mastigophora there are no visible structures which will account for
this movement, which involves a relatively large expenditure of energy.
In many Protozoa, however, special con-
tractile fibres are developed. An instance
in point is the axoneme of a flagellum, which
by its contractions causes the flagellum to per-
form its lashing movements. Similarly, the
A B
Fig. 28. — Myonemes in Gregaeine and Trypanosome. (From Minchin, 1912,
AFTER Schneider and Minchin.)
A. Clepsidrina nmnieri. B. Trypamsoma perccB ( x 2,000).
contractile fibres in the filaments of attachment of certain ciliates, like
Vorticella, enable the organisms to withdraw themselves suddenly (Fig. 19).
In many of the larger trypanosomes, gregarines, and ciliates there are
developed in the ectoplasm a series of fibres of a contractile nature known
as myonemes (Fig. 28). These run in various directions, and by their
contractions the organisms are able to perform movements of flexion and
extension. They not infrequently give rise to a longitudinal marking of the
surface of the body. A common type of movement seen typically in gre-
garines and merozoites of Sporozoa is the formation of rings of constriction,
which pass as peristaltic waves along the body. In certain ciliates para-
MYONEMES— EXTRUSION FILAMENTS
45
sitic in the rumen of cattle, such as some of the complicated forms like
Diplodinium (Fig. 520), the anterior region of the body is highly developed,
while in association with this there is a complicated system of contractile
fibres which enables the organisms to withdraw the whole anterior ciliated
region of the body into a def)ression, which becomes closed over it. In
a similar manner the ciliated peristomal region of Vorticella and its allies
can be suddenly retracted or withdrawn. The curious elongate ciliate
Spirostomum is well supplied with
longitudinal myonemes, which enable
it to retract suddenly to the globular
form when stimulated (Fig. 509).
The presence of these myonemes
often renders it extremely difficult to
obtain satisfactorily fixed specimens
in the fully expanded condition, as
stimulation of the fixing fluid causes
immediate contraction of the myonemes,
and consequent rounding up of the body.
EXTRUSION FILAMENTS.— In some
Protozoa special structures occur which,
on stimulation, have the property of
discharging filaments of varying length.
These may be protective or aggressive in
function or serve the purpose of fixation.
As organs of protection they are
known as trichocysts, and are found
amongst the Ciliata such as Parame-
cium, Prorodon, Dileptus, and many
other forms. They appear as minute
ovoid bodies embedded in the ectoplasm
(Fig. 29). From the blunt end there arises a fine process which extends
as far as the pellicle or outer layer of the ectoplasm. When stimulated,
the fine process is ejected as a tapering filament. Several explanations
of the sudden formation of the filament have been suggested. One is
that a very rapidly coagulating fluid is discharged. Whether this is the
correct explanation or not, it does not appear that the filament as such
exists in the trichocyst before it is visible externally. A larger organ
with a similar function is the Nessel's capsule or nematocyst. It is
present in Epistylis, and is arranged in pairs (Fig. 529).
Another type of filament which can be suddenly discharged occurs
in the Cnidosporidia (Fig. 30). In this group the resistant cysts or spores
are provided with one or more polar capsules from which long filaments.
Fig. 29. — Trichocysts as seen in
Sections of Paramecium cauda-
tum ( X ca. 1,500). (From Minchin,
1912, AFTER MaIER.)
A. Body Surface.
B. Mouth and oesophagus.
T. Trichocysts; /.t'.,food vacuoles; M.m.,
undulating membrane formed of fused
cilia in the oesophagus .
4G ENCYSTMEXT OF THE PROTOZOA
sometimes fifty or a hundred times as long as the spore itself, can be
extruded. The spores of the Cnidosporida are developed in a complicated
manner from a group of cells, some of which form the polar capsules.
These possess a tough outer covering within which the coiled-up filament
Fig. 30. — Dark Field View of Spore of Nosema apis with Extruded Polar
Filament ( x 1,200). (After Kudo, 1921.)
can be seen. It is supposed that the filament is inverted in the capsule,
and that it is discharged by pressure from within, just as an inverted
finger can be everted by blowing into a glove (Fig. 312).
ENCYSTMENT AMONGST THE PROTOZOA.
The majority of Protozoa under certain conditions which are generally
adverse to their continued existence, or in anticipation of such conditions,
are able to enclose themselves in resistant capsules of varying degrees of
impermeability. Encystment is effected by the secretion from the surface
of the body of a substance which quickly hardens in the medium. In
the majority of instances, while this is taking place, the organism, which
has become contracted to a spherical form, revolves slowly, so that fresh
secreted material is applied regularly to the layer already formed. These
capsules are known as cysts, and are generally composed of a clear, hyaline,
transparent substance. In free-living Protozoa which live in water
encystment takes place when the medium is drying up, and there is danger
of desiccation. In this manner complete drying is prevented, and survival
for long periods may occur in conditions under which it would be im-
possible for the exposed organism to live. It has been shown that the
sand of the desert exposed to the tropical sun contains encysted Protozoa,
which emerge from their cysts when brought into more favourable sur-
roundings. The cysts are usually perfectly smooth on their outer surface,
but sometimes they are roughened by the formation of tubercles or other
markings. Very frequently, after the resistant cyst has been formed
there is secreted a membranous inner lining to the cyst. In such cases
one can distinguish a resistant r/>/r//.s7 from a more delicate cndocyst.
Sometimes cysts are pfovided with })ores, several of which are present
PROTECTIVE AND REPRODUCTIVE CYSTS 47
in those of Dimastig amoeba gruberi (Fig. 120). To prevent drying of the
contents of the cyst, such pores are closed by plugs of some material
formed by the cytoplasm. They probably facilitate emergence from the
cyst.
Those Protozoa which are able to contract during life to the spherical
form produce spherical cysts, but others become encysted without changing
their shape to any extent. Thus, the species of Giardia produces ovoid
cysts, while species of Chilomastix and Embadomonas cysts which are pear-
shaped (Figs. 293, 255 and 256). The cysts (oocysts) which are formed
round the zygotes of various species of Eimeria are frequently ovoid in
shape, while those which form round the zygotes of the Gregarinina are
generally spindle-shaped (Figs. 350 and 483). Though the majority of
Protozoa form cysts at some stage of their development, there are some
forms in which cysts have never been observed.
The behaviour of the organism within the cyst varies considerably.
In many cases the cysts are purely protective in nature, the organism
remaining unchanged in the cyst till circumstances again become favour-
able to a free existence. The encysted organism escapes from the cyst
by its gradual dissolution, or through special pores when these are present.
In other cases multiplication takes place within the cyst. In the case of
Entamoeba coli, for instance, the nucleus of the encysted amoeba divides
repeatedly to produce eight nuclei (Fig. 101). Within the oocysts of
coccidia and gregarines there are produced a varying number of daughter
individuals known as sporozoites (Fig. 337). Similarly, within the oocysts
of the malarial parasites on the stomach of the mosquito there are de-
veloped very large numbers of sporozoites (Fig. 391). Within the cyst
of Giardia there are produced two daughter flagellates, while in that of
Prowazekella lacertw as many as sixty-four daughter flagellates are formed
(Fig. 253). Amongst the ciliates, when cysts are formed, they are usually
purely protective in nature, but in some cases, at least, reproduction
within the cyst takes place. Thus, the various species of Colpoda appear
to reproduce only in the encysted condition. The ciliate becomes spherical,
and by constant rotation forms a spherical cyst. Within it division into
two and then into four daughter ciliates occurs. The cyst is then ruptured
and the four young ciliates emerge. They then grow into the adult form,
when the process is repeated (Fig. 38).
Cyst formation is a very characteristic feature of parasitic Protozoa.
Having adapted themselves to life within another organism, their powers
of survival under external conditions have been largely lost, and it is b^
means of their encysted stages that they are able to pass from one host
to another. It thus arises that whenever an organism passes from one
host to another in such a manner that exposure to external conditions
48 ENCYSTMENT OF THE PEOTOZOA
must occur, it is the encysted forms which render such a transference to
a new host possible. In the case of the intestinal amoebse, though both
free and encysted forms escape from the body, it is only the encysted
stages which are able to carry infection to a new host. Even if direct
transference of unencysted stages occurred, these would, in all probability,
be killed by the digestive fluids of the stomach, which the encysted stages
can withstand. In the large group of insect flagellates (Trypanosomidse),
again, it is by means of minute encysted stages passed in the faeces that
another insect is infected (Fig. 164).
When encystment is about to occur, very frequently changes take
place in the encysting organism. The cytoplasm is freed from all food
residues, and in consequence becomes much clearer. Not infrequently
the cytoplasm becomes charged with food reserve material, such as
glycogen. Sometimes, as in the case of Entamoeba coli and Entamoeba
histolytica, in preparation for encystment special forms of the amoeba
which are smaller than the ordinary free individuals are produced (Figs.
96 and 100). These forms, which are preparing for encystment, are known
as precystic forms. Amongst the Sporozoa encystment is associated
with a sexual process. In the case of the gregarines two individuals
become enclosed in a cyst (gametocyst), within which each gives rise to a
number of gametes (Fig. 465). The gametes unite in pairs, and the
zygote thus produced itself becomes encysted in the oocyst, within which
it divides into a number of sporozoites. In the case of the coccidia, the
zygote is encysted in the oocyst which is formed either before or after
syngamy has taken place (Fig. 337). Within the oocyst the zygote divides
into a number of sporoblasts, which in their turn become encysted in
sporocysts. Inside the sporocysts the sporoblasts divide into sporozoites.
A special type of cyst is produced by the Cnidosporidia. These are
provided with one or more polar capsules from which long filaments can
be rapidly extruded. They serve the purpose of anchoring the cysts in
the intestine, while the wall is opened for the liberation of the enclosed
organism (Fig. 311).
For a long time the resistant encysted stages of the Cnidosporidia,
coccidia, and gregarines were known as psorosperms, a name introduced
by Johannes Miiller (1841) for the spores of Myxosporidiida. The spindle-
shaped oocysts of gregarines were frequently referred to as pseudo-
navicellse, a name first used by von Siebold (1839).
The production of secondary cysts within the primary one occurs
occasionally in other groups, as in the ciliates of the genus Colpoda. A
ciliate may become encysted and undergo a diminution in size within the
cyst, and then form a second cyst. The process may even be repeated
again, giving rise to three concentric cysts. As stated above, the different
PROTECTIVE AND REPRODUCTIVE CYSTS 49
species of Colpoda multiply within cysts. The four daughter ciliates
usually rupture the cyst and escape (Fig. 38). On the other hand, they
may each become encysted within the primary cyst. The process of
encystment was probably first developed for purely protective purposes,
but various reproductive processes have become associated with it. It
must be remembered, however, that encystment is not essential to either
of these processes, as they frequently occur quite apart from any encyst-
ment whatever.
In the majority of cases, when once formed, a cyst undergoes no change
in size or shape, though it may gradually increase in thickness. The
cysts of parasitic forms are ruptured or dissolved by the action of the
digestive fluids of a new host. In some cysts, however, there are formed
special pores which are kept closed by a plug of material which is more
easily dissolved than the rest of the cyst. Amongst the Sporozoa such
a pore in the oocyst is termed a tnicropyle, and through it the daughter
individuals which have been formed within the cyst emerge (Fig. 337).
It sometimes happens that with growth of its contents the cyst in-
creases in size after it is first formed. This process is well illustrated by
the oocyst of the malarial parasite, which increases enormously in size on
the stomach wall of the mosquito (Fig. 391). A similar growth also
occurs in the case of the oocysts of the hsemogregarines {Hejpatozoon), and
the cysts of the flagellate (Prowazekella lacertw), which is parasitic in the
intestine of lizards (Figs. 253 and 254). It will be evident that if the
thickness of the cyst is to be maintained, there must be constant addition
to it of fresh material secreted by the enclosed organism.
The cysts produced by any particular species of Protozoon are usually
fairly uniform in size, and possess distinctive features. On this account
their characters are of great importance for purposes of identification and
classification.
THE PROTOZOAN NUCLEUS.
GENERAL FEATURES. — The nucleus, which is an organized structure
containing chromatin, is the most important constituent of any Protozoan
cell, as, indeed, it is of all cells. It has been shown that the nucleus is
essential to the continuation of life, for individuals which have been
deprived of their nuclei, though they may survive and move about for
some time, quickly degenerate and die, while portions of the cytoplasm,
if they contain nuclei, often survive, regenerate, and continue their
existence. It seems probable that all the activities of the cell are governed
and regulated by the nucleus, which also appears to be mainly responsible
for the transmission of hereditary character.
I. 4
50 PROTOZOAN NUCLEUS
In some Protista, as, for instance, the bacteria and spiroclisetes, it
appears that there is no definite structure which can be called a nucleus,
though a granular material usually identified with chromatin is presumed
to fulfil the functions of the organized nucleus. AlexeiefE (1924a), however,
maintains that the granules are not chromatin, but mitochondria. It is
an exceedingly difficult matter to give a precise definition of the term
nucleus, though every biologist knows that it is a definite circumscribed
structure containing chromatin, and that it behaves in a well-recognized
manner. The fact that it divides when cell division takes place is one
of its most important features, but there are other structures in the
cytoplasm which behave in a similar manner. The one feature which is
not shared by other bodies in the cell is that during the sexual process
the nucleus, or one of its descendants, is able to unite with another
nucleus. In other words, a nucleus is potentially capable of karyogamy.
The majority of Protozoa possess but a single nucleus, except during
the process of multiplication, when two or more may be present. Some
forms, however, possess two nuclei during- the greater part of their life-
history, and are therefore binucleate, while others, again, have many
nuclei and are multinucleate. Such binucleate and multinucleate forms
may be regarded as individuals in which the nucleus has divided prepara-
tory to division of the body, which for some reason or another has been
delayed. In the binucleate and multinucleate individuals the nuclei are
easily recognized as being of one type. It sometimes happens that when
active multiplication by binary fission is taking place, the rate of division
of the nucleus exceeds that of division of the cytoplasm, so that temporary
multinucleate stages occur. In the case of Trypanoso7na brucei and other
pathogenic trypanosomes in laboratory animals, when active multiplication
is proceeding, individuals with four or even a larger number of nuclei
may be seen (Fig. 160). In such cases, however, the condition is quickly
rectified by repeated divisions of the cytoplasm without further division
of the nucleus. In most, if not in all, cases there arrives a period in the
life-history of multinucleate forms when division of the body takes place
and uninucleate individuals are produced. This is well seen in Opalina
ranarum of the intestine of the frog, where the organism is multinucleate
during the greater part of its life-history, but in the spring uninucleate
individuals are produced (Figs. 448 and 449).
Amongst Euciliata there exists a special type of binuclearity- These
Protozoa usually possess two nuclei, which differ from one another not
only in size and structure, but also in function. The larger one of the two
is known as the macronucleus, and the other as the micronvcleus (Fig. 70).
In ordinary division of the organism both nuclei divide, and when the
body is split into two parts the two daughter individuals each have two
BINUCLEARITY 51
nuclei. At certain stages in the life-history the macronucleus disin-
tegrates and disappears, while the micronucleus divides into two parts,
one of which becomes a new macronucleus. This process of regeneration
of the macronucleus occurs most usually in association with the process
of conjugation, but may also occur during the course of the ordinary
asexual multiplication, when it is known as endomixis. The fact that the
macronucleus is formed from one of the products of division of the micro-
nucleus is the primary reason why the macronucleus is regarded as
a nucleus at all. Furthermore, since the macronucleus disappears during
conjugation, and takes no part in the process, it is assumed that the micro-
nucleus is essentially the sexual nucleus, and that the macronucleus is
vegetative in function, and governs the metabolism and activities of the
cell at other times. Though this may be the case, the absolute proof is
difficult to obtain. Apart from the fact that it is small in relation to the
size of the body, the micronucleus behaves in every way during the whole
life of the ciliate as does the nucleus of an organism, such as a flagellate
or an amoeba, which possesses no macronucleus. There is little direct
evidence that the micronucleus of a ciliate is controlling the metabolism
and activities of the cell to a less extent than is the single nucleus of such
an organism as an amoeba.
It is clear that the macronucleus plays an important part in the economy
of the cell, and it is equally clear that it is of nuclear origin, but it does
not seem clear that because of its existence the functions of the micronucleus
are suppressed or supplanted while it is present. The view which main-
tains that the micronucleus is purely passive during the asexual life of the
organism, and only, so to speak, wakes up to activity during conjugation,
while the metabolism of the cell at other times is controlled by the macro-
nucleus, has given rise to the conception of two kinds of chromatin, the
one sexual or generative in function and the other vegetative. Amongst
the Euciliata the two kinds of chromatin are presumed to be separated in
different nuclei, while in other cases the same two elements are supposed
to coexist in the single nucleus. It is thought that dviring the sexual pro-
cess it is the generative chromatin that functions, the vegetative chromatin
having been largely got rid of by so-called reduction or maturation pro-
cesses. At other times it is the vegetative chromatin which is active,
while the generative chromatin, though still present in the nucleus, is
passive.
In this connection it is necessary to recall the fact that in the Mastigo-
phora the flagella take origin from a structure called the blepharoplast. ■ In
its simplest form this consists of a minute homogeneous granule, which
appears to be little more than a thickening of that end of the axoneme
which is in the cytoplasm. In certain stages of development of some
52 PROTOZOAX NUCLEUS
flagellates the flagella are lost, and a non-flagellate stage is developed.
When the flagellate stage is resumed, a new axoneme is developed as an
outgrowth from the blepharoplast, which may or may not have persisted.
fe'
f
..J
1 t ;V
¥
O '
"^■M'i
t
)
o
.P:
■i S 6
Fig. 31. — Parypolytoyna satura, to snow the Origin of the Blepharoplast
FROM THE KaRYOSOME OF THE NUCLEUS ( X 2,600). (AfTER JaMESON, 1914.)
1. Adult flagellate shortly before division.
2. First division completed: two daughter individuals with the old blepharoplasts and flagella.
3. Second division: reconstruction of nuclei and division of body into four.
4. Second division completed: new blepharoplasts are budding off from the karj^osome in the
upper and left-hand individuals, while the right-hand individual retains the old blepharo-
plasts.
5. New blepharoplasts on outer surface of nuclear membi'ane in three of the individuals, while the
left-hand individual retains the old blepharoplasts.
0. Stage shortly before release of four daughter flagellates: the left-hand individual has the old
blepharoplasts and flagella, while the others have new blepharoplasts and young flagella.
Some evidence has been produced by Jameson (1914) in the case of a
^SigeWa^te, Pai'apolytoma satura, and by Entz (1918) in the case oiPolytoma
uvella, that when the non-flagellated stages are about to develop flagella new
BLEPHAROPLAST AND PARABASAL
53
basal granules or blepharoplasts are developed from the nucleus or from its
karyosome (Fig. 31). In the case of Dimastigammha gruberi (Fig. 120), the
amoeboid phase of which develops fiagella under certain conditions, it was
stated by AlexeiefE {I9l2g) and Wilson (1916) that when this took place the
blepharoplasts of the two fiagella migrated into the cytoplasm from the
karyosome of the nucleus, with which they remained connected by a fibre.
As explained below (p. 263), the writer has been quite unable to observe
the origin of the blepharoplasts in this manner. It seems more probable
that the blepharoplasts are present in the cytoplasm, possibly on the outer
surface of the nuclear membrane, during the whole of the amoeboid phase
Fig. 32. — Devescovina striata ( x ca. 1,900).
A. Ordinary flagellate showiiag coiled paraba"^al.
(After Janicki, 1915.)
B. Dividing form.
of tlie organism, and that they move to the surface of the body when
fiagella are commencing to form. In many Mastigophora, in association
with the blepharoplast, is another structure to which Janicki (1911) has
given the name parabasal ; it stains intensely with certain stains (Figs. 32,
33, 67). In some cases — as, for instance, in trypanosomes and their
allies — it seems to be in actual union with the blepharoplast, and to form
with it a composite body, the hinetoplast (Fig. 157). There is no conclusive
evidence that the parabasal body is of nuclear origin, as some have supposed.
It is a well-established fact, however, that division of the organism is
preceded not only by division of the nucleus, but also by division of the
blepharoplast and parabasal as well, and it becomes a tempting hypothesis
54
PROTOZOAN NUCLEUS
to suppose that the nucleus and the kinetoplast of a flagellate represent
two nuclei, as do the micronucleus and the macronucleus of a ciliate.
Such an assumption has been made by Hartmann (1907) and others,
who regard the kinetoplasts as true nuclei, and the flagellates which possess
them as constituting a special group of the Mastigophora, the Binucleata.
As pointed out by Alexeieff (19176), there is no ground for this assumption,
and in order to avoid confusion he proposed the name kinetoplast in place
of kinetonucleus and other terms which implied a nuclear nature. It is
safer to regard the kinetoplast as a
distinct structure concerned with the
activities of the flagellum, even though
it divides when cell division occurs,
and possibly may have originated from
the nucleus in the first place. It might
equally be argued that though the
macronucleus of a ciliate has originated
from the micronucleus, and though it
multiplies by division during reproduc-
tion, it has ceased to be a nucleus in the
true meaning of the term, and has be-
come modified to serve some other pur-
pose, possibly in connection with the
development of large numbers of cilia -
It is worthy of note that the macro-
nvicleus does not divide like a true
nucleus, which in most cases, at least,
shows some indication of mitosis.
Against this view, however, can be
raised the argument that there occur
certain races of ciliates which possess
no micronuclei, though other races of
the same species have both micro- and
(p. :^5), Dawson (1919) discovered an
amicronucleate race of Oxytricha hyiiienostoma, a ciliate which normally
possesses both nuclei. The ciliate was kept in culture for several years, and
though possessing only a macronucleus, it reproduced regularly by fission.
ENDOMIXIS. — As already remarked, the macronucleus of a ciliate
degenerates during conjugation, and a new macronucleus is developed
from one of the products of division of the micronucleus. This re-
placement of the macronucleus from the micronucleus may occur at
times other than during conjugation. Wlien Paramecium aurelia
reproduces repeatedly by simple division over long periods at certain
\^-
Fig. 33. — Bodo caadatus Coprozoic
IN Human F.^ces ( x ca. 1.500).
(Original.)
I and 2. Forms showmg two blepharo-
plasts with associated parabasal.
3. Small individual. 4. Encysted form.
macronuclei. As noted above
ENDOMIXIS
55
intervals, the macronucleus degenerates, and is replaced from the
microniicleus as at conjugation. Woodruft' and Erdmann (1914) described
the process in Paramecium aurelia, and named it endomixis. P. aurelia
contains normally two micronuclei and one macronucleus (Fig. 34). When
endomixis occurs, the macronucleus disintegrates and is eventually
absorbed. The two micronuclei divide to form four, and these again to
form eight micronuclei. Of the four derived from each original micro-
<g5Vb6?Vb> C
Fig. 34. — Endomixis in Paramecium aurelia: Diagrammatic Representation
OF Nuclear Changes as described by Woodruff and Erdmann ,
1914. (After Jennings, 1920.)
A-B. Degeneration of macronucleus and first division of two micronuclei.
C-D. Second division of micronuclei and degeneration of .six of the daughter micronuclei.
E. Division of ciliate to produce two daughter individuals each with a single micronucleus.
F-G. Two divisions of micronuclei to give rise to four, two of which increase in size to become
macronuclei. H. Further division of micronuclei.
I. Division of ciliate to give rise to the normal type as at A.
nucleus, three degenerate, so that two micronuclei are left. The ciliate
now divides, giving rise to two ciliates, each with a single micronucleus. In
each of these daughter ciliates the micronucleus divides to form two, and
again to form four micronuclei. Two of these increase in size and become
macronuclei, while the other two divide to form four micronuclei. The
ciliate now has two macronuclei and four micronuclei. It now divides to
form two ciliates, each of which has two micronuclei and one macronucleus
as in the original form.
56 PROTOZOAN NUCLEUS
Endomixis has been demonstrated in several races of Paramecium
aurelia by Woodrufi and Erdmann, as well as in another species of the
same genus. Fermor (1913) claimed to have seen the same process in
Stylonychia, while Calkins (1915 and 1916) described it in Didinium and
Uroleptus. The meaning of endomixis is not clear. That it takes place
quite apart from unfavourable conditions has been noted by Woodruff
(1925), who also proved that certain races of Paramecium in which it did
not occur died out. All that can be stated is that for the satisfactory con-
tinuation of the functions of the macronucleus, whatever these may be, it
seems necessary in many cases that this structure be renewed from time
to time. Though this usually takes place during conjugation, it may
occur at other times. An exception to this rule is afforded by the
behaviour of the ciliate Spafhidium spathula (p. 132).
STRUCTURE OF THE NUCLEUS.— The nucleus of a Protozoon possesses
a nuclear membrane, which may be regarded as a special develop-
ment of the linin network of fibres or septa which traverse the enclosed
space (Fig. 1). The meshes of the network or spaces between the septa
are filled with a fluid substance known as nuclear sap. Distributed upon
the membrane or network as distinct granules or in one or more larger
masses is the chromatin material, while in most cases, somewhere on the
network, and most usually at or near the centre of the nucleus, is a body
known as the karyosome, which, on account of its affinity for certain stains,
has generally been regarded as consisting partly of chromatin and partly of
an achromatic substance {plastin). In some Protozoan nuclei the
karyosome does not seem to be present, but it appears in the nuclei of the
majority of forms. From what takes place in nuclear division, it appears that
the karyosome is composed mostly, if not entirely, of plastin material, and
that the chromatin of the nucleus is represented by the granules outside the
karyosome, for it is from them that the chromosomes are formed. Doflein
(1922), from a study of the nucleus of the flagellate Ochromonas granulans,
was led to believe that a true karyosome was devoid of chromatin, and
that during nuclear division it gave rise to the achromatic part of the
spindle, while the chromosomes were derived from the peripheral chromatin
which was situated outside the karyosome. On the other hand, Stern
(1924), from a study of the nuclear division of the Heliozoon Acanthocystis
aculeata, arrives at a conclusion which is the exact opposite of this. He
believes that the karyosome breaks up and gives rise to the chromosomes,
while the spindle is formed from the part of the nucleus between the
karyosome and the nuclear membrane.
Sometimes several masses of plastin occur in a single nucleus, but it
seems doubtful if these should all be regarded as karyosomes. It is often
assumed that an intranuclear centrosome, the centriole, is present in the
STRUCTURE OF NUCLEUS 57
nuclei of Protozoa. It is supposed to be embedded in the karyosome,
and only to become recognizable as centrosomic in nature during nuclear
division, when it divides into two parts which separate from one another,
though remaining connected for some time by a fibril, the centrodesmose.
As the daughter centrioles move apart they take up positions at the ends
of the elongating nucleus, while spindle fibres surrounding the centro-
desmose may form between them (Fig. 59). The chromatin of the nucleus
may form definite chromosomes, which arrange themselves as an equatorial
plate at the equator of the spindle. Two daughter plates are formed,
and these travel towards the centrioles at opposite poles of the nucleus.
When the nuclear membrane divides, the spindle fibres and centrodesmose
disappear, a new karyosome is formed around the centriole, and the
chromosomes break up into granules, which are distributed on the
nuclear membranes or linin network. Observers are, however, by no
means convinced that such a granule is a true centrosome, for in many
Protozoa undoubted centrosomes exist in the cytoplasm outside the
nuclei.
The size of the karyosome in proportion to that of the entire nucleus
varies considerably. In many nuclei, especially those of small size, it
has been the rule to regard the bulk of the chromatin as being aggregated
in the relatively large karyosome, and to suppose that little, if any, is
distributed upon the nuclear membrane or linin network. It is becoming
increasingly evident, however, that all nuclei contain some chromatin
on the linin network or membrane (peripheral chromatin). In other cases
the karyosome is relatively small, while definite chromatin granules occur
upon the nuclear membrane or the linin network. The nuclei of the first
type are often spoken of as of the karyosome type, but every transition
between the two types of nuclei occurs. This question as to whether a
centriole is always present or not is a very difficult one to decide, for the
statements regarding it are most conflicting. Some observers are able
to find centrioles in nearly every nucleus they examine, while other
equally competent observers fail to detect them. The difference of opinion
is to be accounted for by the fact that the centriole is merely a minute
granule, the nature of which can only be determined by its behaviour
during actual division of the nucleus. While division is taking place, there
are always numbers of granules in the nucleus. Many of these are chro-
matin granules, and as spindle fibres are often present while the nucleus
is dividing, it is easy to interpret any two granules and a connecting fibre
as centrioles and centrodesmose. It thus happens that it is more than
doubtful if most of the structures which have been described as centrioles
are actually of this nature. The mere presence of a central granule in
a karyosome of a resting nucleus appears to some observers to be sufficient
58 PROTOZOAN NUCLEUS
ground for calling it a centriole. Karyosomes do not always stain homo-
geneously, and may have a granular structure, while the appearance of
a central granule may be merely the result of irregular extraction of stain.
Before deciding as to the centriole nature of a granule, it is necessary to
trace its division and the separation of the two daughter centrioles, and
to observe the actual centrodesmose uniting them. Furthermore, when
spindle fibres are developed for mitotic division, the daughter centrioles.
will occupy the poles of the spindle. Such appearances must not be of
rare occurrence, but must be detected in the majority of dividing nuclei.
When a large number of dividing nuclei of any Protozoon such as an
amoeba are examined, it is a relatively easy matter to find isolated examples
of spindles which have granules at their poles, though the majority of
spindles may not show them. The occasional presence of such apical
granules does not justify the assumption that they are actually centrioles.
It is an undoubted fact that definite spindles may be formed within the
nuclei of Protozoa without there being any evidence of centrioles at the
poles, though it is not difficult for those who desire to see such structures
to convince themselves that granules, which they interpret as centrioles,
are present. The fact that during the mitotic nuclear division of the cells
of higher animals centrosomes are almost always present, and that un-
doubted centrosomes occur in some Protozoa, has undoubtedly led to many
structures being described as centrioles or centrosomes which have quite
another nature. In the present state of our knowledge it is impossible
to state that a centrosome or centriole is an essential constituent of all
Protozoan nuclei. Nevertheless, it cannot be supposed that all the
descriptions which have been given are erroneous. In the division of the
nucleus of Dimastig amoeba gruberi, which has a large central karyosome,
the latter structure elongates and becomes dumb-bell-shaped, and finally
divided into two parts. As these separate, they remain connected by
a fibre which can be shown in many cases to unite two granules which occur
in the two daughter karyosomes (Figs. 61, 4, and 120, 12). AVhether such
granules are to be regarded as intranuclear centrosomes (centrioles) is a
question more difficult to decide.
The true structure of a nucleus is that which it possesses in the normal
living cell. Fixing fluids and other reagents may considerably alter its
appearance, so that the greatest care has to be exercised in the interpreta-
tion of the structures seen in fixed material. Experience has shown that
certain fixing fluids, stains, and reagents produce better results than
others, and are reliable in giving accurate pictures of the true structure.
Nevertheless, the literature dealing with the Protozoa, especially the blood
parasites which have been largely studied in dried films, is full of erroneous
descriptions of nuclei. The dry blood film stained by Romanowsky stains,
STRUCTURE OF NUCLEUS 59
though it may give useful information as to the type of cell or parasite
present, is completely misleading when it comes to a consideration of the
minute nuclear structure. Descriptions of the characters of nuclei which
are based on preparations of this kind are not only worthless but misleading.
From the above description it will be realized that the nuclei of Protozoa
may be roughly divided into two classes: those in which there is a central
karyosome, and those in which no such karyosome is present. Compared
with the size of the nucleus, the karyosome may be a relatively small
structure, or it may occupy a large part of its bulk. As a type of nucleus
with small karyosome, that of Entamoeba histolytica will serve as an illus-
tration (Fig. 95). There is a definite nuclear membrane, on the inner
surface of which practically all the chromatin is arranged in the form of
small granules. At the centre of the nucleus is a small granule, the
karyosome, which presumably consists of plastin material and possibly
some chromatin. Surrounding the karyosome is a clear area, the limits of
which form a sphere (or ring in optical section) of fine granules. These do
not contain chromatin, but represent the inner limit of the linin network
which connects the sphere with the nuclear membrane. The linin network
appears to be free from chromatin. Dobell (1919), who has studied the
nuclear division in this amoeba, could obtain no evidence of the existence
of a centriole in the karyosome, though such a structure has been described
by Hartmann (1908-1913). During division of the nuclei within the cysts
the writer has seen forms which suggest the presence of a central granule
which divides (Fig. 57).
A type of nucleus in which a definite and relatively large karyosome is
present is of frequent occurrence. It is seen typically in trypanosomes,
many free-living amoebae, and other Protozoa (Figs. 48, 89, 224). These
karyosomes are comparatively large structures which are connected with
the nuclear membrane by the linin network. It is possible, though by
no means certain, that some of the chromatin of the nucleus may be con-
centrated in the karyosome, which stains intensely with certain nuclear
stains. The nuclear membrane and the linin network may have com-
paratively little chromatin, which in small nuclei, such as those of trypano-
somes, is difficult to detect. In many cases nuclei of this type are
described as possessing centrioles within the karyosomes. The large
karyosome may appear perfectly uniform and homogeneous, or it may
show indications in stained specimens of being composed of a varying
number of deeply staining bodies embedded in a more faintly staining
l)lastin matrix. The karyosome is often spherical in form, but it may be
irregular in shape. In some cases on the surface of the karyosome there
occur deeply staining granules, which may be chromatin, while the central
part consists of })lastin. Sometimes one or more vacuoles are present.
60 PROTOZOAN NUCLEUS
Between these two types of nuclei many intermediate forms are found,
and individual variations are of common occurrence. These variations
may affect the nuclear membrane, which may be exceedingly fine in some
forms and comparatively thick and dense in others. The arrangement
of the chromatin upon the membrane may be in the form of uniformly
distributed fine granules, or there may be coarse granules more irregularly
distributed, or most of the chromatin may be aggregated into a semi-
lunar mass planted on one side of the membrane.
The linin netw^ork itself may be in the form of a uniform mesh, or it
may consist of radially arranged strands. The meshes of the network
may contain granules other than chromatin or globules of an undetermined
nature. The minute structure of the nuclei is of considerable importance
in the differentiation of species.
Though it is not possible to draw a hard-and-fast line between those
nuclei which possess karyosomes and those which do not, there never-
theless exist certain nuclei in which there appears to be no tendency
towards the formation of a central structure. Amongst the gregarines,
for instance, certain individuals of a particular species may show a single
deeply staining body in the nucleus, or more than one, while in some
Protozoa there are a series of deeply staining bodies upon the nuclear
membrane, while the interior of the nucleus is occupied by a uniform
meshwork of fibrils. It appears impossible to speak of several bodies
in the nucleus as karyosomes, a term which is undoubtedly used by the
vast majority of zoologists, for the single more or less centrally placed
structure described above. Nuclei of the type which has no definite
karyosome may, however, contain a body which may or may not be
central in position, and which is regarded as devoid of chromatin, owing
to the fact that it does not stain intensely with chromatin stains. Such
a structure occurs, according to Metcalf (1909), in the nuclei of species
of Opalina, in which a deeply staining karyosome is not present. It
resembles. the nucleolus which is commonly found in the nuclei of the
cells of higher animals. Bodies of this type have also been described by
Reichenow (1921) in the nuclei of various stages of development of the
hsemogregarines of the genus Karyolysus. They are also present in the
nuclei of Hepatozoon balfouri, and, as in the case of Karyolysus, they divide
during nuclear division (Fig. 35). They are commonly present in the
nuclei of coccidia. They take no part in the formation of the spindle or
the chromosomes.
Another type of nucleus which has to be mentioned is the macro-
nucleus of the Euciliata. It has already been explained that these
Protozoa typically possess two nuclei — the micronucleus and the macro-
nucleus. The former is usually of the type which contains a central
STRUCTURE OF NUCLEUS , 61
karyosome, and when it divides it does so by mitosis, while the latter,
though developed from a nucleus like the micronucleus, has so changed
in appearance and structure that it seems doubtful if it should still be
regarded as a true nucleus (Fig. 37). It is sometimes spherical in form,
but it is more usually slightly elongated. It may be many times as long
as it is broad, and in such cases may have a beaded appearance, as in
Stentor and Spirostomvm (Figs. 22 and 509). It may even be irregularly
branched, as in certain Suctoria (Fig. 531). It consists of a dense material
impregnated with granules which become more evident during division.
Vacuoles are often present, while sometimes, as in the species of Colfoda,
the elongated macronucleus contains within it one or more deeply staining
bodies (Fig. 498). It is evident that the macronucleus differs in many
ways from the micronucleus and the nuclei of other Protozoa. During
division it does not behave as true nuclei do, and there seems to be
little change in its appearance, except for the greater clearness of its
#1 '0^ ^#^ ^^
Fig. 35. — Stages in the First Nuclear Division in the Schizont of Hepatozoon
balfouri, showing Division of the Karyosome, which appears to be entirely
devoid of Chromatin ( x 6,000). (Original.)
granules. It is generally assumed that the granules in the macronucleus
are chromatin, but, if this be so, there must have taken place a remarkable
increase in the chromatin during its formation and growth from the micro-
nucleus from which it was originally derived. It seems not impossible
that this material is not actually chromatin, but some other substance
which has been elaborated to fulfil a special function.
In this connection it will be necessary to refer again to a theory which
was suggested by Schaudinn, and subsequently elaborated by Goldschmidt
and others. According to this theory the Protozoan nucleus is constructed
of two fundamentally different parts, which in the Euciliata are separated
in two distinct nuclei. The one part consists of vegetative material which
controls nutrition, movement, and other vegetative functions, while the
other is composed of generative material which takes part in the syn-
gamic process. This theory has been extended to the chromatin itself,
which is supposed to be of two kinds, the one idiochromatin, which takes
part in syngamy, and is responsible for the transmission of hereditary
62 MULTIPLICATION OF PROTOZOA
characters, and the other vegetative chromatin, which has to do with
the vegetative functions. Amongst the Plasmodroma and the Proto-
ciliata both kinds of chromatin are contained in one nucleus, and it is
supposed that the extrusion of chromatin material from the nuclei of
gametes, which has been described as taking place in certain instances, is
an expulsion of the vegetative chromatin in preparation for syngamy.
If this explanation is the correct one, it has to be admitted that after
syngamy the vegetative chromatin can be re-formed from the generative
chromatin, as illustrated by the formation of new macronuclei from the
micronuclei after syngamy in the Euciliata. The theory depends very
largely on an exact definition of what is and what is not chromatin, and
a correct interpretation of the various parts of the nucleus, about which
at the present time there is considerable difference of opinion. Dobell (1 925)
has described a condition of binuclearity in Aggregata (see p. 873).
MULTIPLICATION AMONGST THE PROTOZOA.
Multiplication takes place by a process of binary fission or gemmation
in which an organism divides into two daughter organisms after division
of the nucleus, or by a process of multiple segmentation, which is generally
known as schizogony amongst the Sporozoa, where it occurs most typically,
after a number of nuclei have been formed by repeated divisions.
BINARY FISSION. — The process of binary fission may give rise to
daughter forms which are equal in size (equal binary fissions), or to forms
w^hich are unequal in size (unequal binary fission). When there is a
marked difference in size between the two, the process is known as budding
or gemmation, a method of multiplication which is seen typically amongst
the attached Euciliata (Peritrichida and Suctoria), where a large form buds
off a small ciliated embryo which does not itself reproduce till it has grown
to the adult form.
In the case of amoebae which have globular bodies, binary fission is
effected by the body becoming elongated and a constriction forming
around the middle of the body (Fig. 36). This deepens till the amoeba
is divided into two parts. The daughter forms may not divide again till
they have grown to the size of the parent. On the other hand, they may
divide before growth is complete, with the result that increasingly small
individuals are produced. If they divide only after they have grown to
a size larger than that of the parent, then larger forms are gradually
produced. In the case of the amoebae it is evidently impossible to state
that division takes place in any one plane, except that it occurs in a plane
at right angles to the axis occupied by the elongate dividing nucleus.
Directly it becomes possible to orientate an organism, and state that
it possesses an anterior and posterior end and a dorsal and a ventral
BINARY FISSION
63
surface, it is found that the plane of division is uniform in the different
groups. Thus amongst the Mastigophora, which have an anterior flagel-
lated end of the body, it is found that in binary fission the body splits
longitudinally from before backwards. In those forms in which a cyto-
stome is present, as in Chilomastix, in which a dorsal and ventral surface
can be distinguished, it is found that a new cytostome is formed near the
original one, and if this is also regarded as being on the ventral surface
then the body splits longitudinally from before backwards and in a dorso-
ventral plane which passes between the cytostomes. In actual division,
however, the body often becomes so distorted that it may be difficult to
Fig. 36. — Successive Stages in Binary Fission of Amceba jtoli/ podia ( x 250).
(From Lang, 1901. after Sciiulze, 1875, modified.)
distinguish the dorsal and ventral surface, though the plane of division
may still be regarded as in this direction. In flagellates such as Tricho-
monas and the trypanosomes, which possess undulating membranes,
division is more complicated (Figs. 160 and 271). A new axoneme grows
out from the free half of the divided blepharoplast and passes along the
border of the membrane. The membrane then divides between the two
axonemes, but the point up to which the membrane has divided at any
stage is always a short distance behind the end of the new axoneme.
When the new axoneme has reached the end of the membrane the division
of the membrane is completed, and the two undulating membranes, each
64
MULTIPLICATION OF PROTOZOA
with its axoneme, are formed. The body of the flagellate then divides
longitudinally from before backwards in a dorso-ventral plane between
the two dorsal membranes. Owing to the blepharoplasts being situated
at the anterior end of the body in Trichomonas and some other flagellates,
the membrane divides from before backwards in these forms. In the
trypanosomes, however, the blepharoplast is situated at the posterior end
of the flagellate, and the membrane
divides from behind forwards. In
either case, the body itself divides
from before backwards after the mem-
brane has completed its division.
Multiplication by binary fission
occurs also amongst the Opalinata
and C-iliata, but division is trans-
verse and not longitudinal, as in the
Mastigophora (Fig. 37). A ciliate
may develop a new cytostome at
some distance behind the first one,
and after division of both the macro-
nucleus and micronucleus the body
divides transversely or at right angles
to the longitudinal axis. It often
happens that, as a result of this
division, the character of the daughter
forms differs from the parent in the
relative size of the cytostome. As
the body of the new individual is
developed from the post-cytostomal
region of the parent, it follows that
the daughter forms will have a cyto-
stome which is relatively longer when
compared with the total length of the
body. In Paramecium the cytostome
of the parent takes up a position at
the centre of the body, and is divided
into two cytostomes of equal or unequal length, after which the body
divides transversely between the two.
Binary fission, when it occurs amongst the Rhizopoda, Mastigophora,
or Ciliophora, usually gives rise to individuals which are roughly equal in
size; but not infrequently, as, for instance, in Trypanosoma lewisi, a large
trypanosome will divide in such a manner as to give rise to one large form
and one which is very much smaller (Fig. 197, 5). The process is repeated
Fig. 37. — Binary Fission of Para-
mecium aurelia ( x ca. 500). (After
Lang, 1901.)
1 and 4, New contractile vacuole; 2 and 6,
dividing macronucleus ; 3 and 5, anterior
and posterior contractile vacuoles, which
will become the posterior vacuoles of the
daughter forms ; 7. new cytostome formed
as bud from original cytostome; 8 and 10,
mitotic division of two micronuclei.
SCHIZOGONY
65
by the large form, which apparently has ceased to grow, so that eventually
its entire cytoplasm is used up in the production of a number of small
forms. At each division there is a nearer approach to equal binary
fission. It is evident that such a method of division approaches a budding
process.
Binary fission usually occurs in the free-living state, and as the division
is taking place the organism may be actively motile. Amongst the
Rhizopoda, the amoebae are frequently perfectly quiescent while binary
fission is proceeding. In some cases, binary fission takes place in the
encysted condition. This appears to be the normal method of multiplica-
tion of species of Colpoda. The organism secretes a cyst in which it
Fic. 38. — Colpoda steini : Multiplication of a Single Indivibual during a
Period of Seven Hours' Observation ( x 650). (Original.)
A. Ciliate about to encyst. B. Encysted ciliate.
C. Division into two completed: commencing division into four.
D. Four daughter ciliates in cyst.
E-G. Escape of ciliates through rupture in cyst wall.
H. Crumpled cyst after escape of ciliates.
divides into two, each of which again divides (Fig. 38). The four daughter
ciliates then rupture the cyst and swim away. Similar divisions within
cysts occur amongst the Rhizopoda and Mastigophora (Fig. 143).
SCHIZOGONY. — By this term is understood a method of multiplica-
tion which occurs typically amongst the Sporozoa (Fig. 39). As the
organism is growing, repeated divisions of the nucleus and daughter
nuclei take place, till finally there may be present a large number of nuclei
in a single mass of cytoplasm. The number of nuclei produced varies
considerably, and may be as few as four or as many as a hundred or more.
The nuclei arrange themselves on the surface of the cytoplasm, which
becomes raised into a series of elevations, into each of which a nucleus
I. 5
66
MULTIPLICATION OF PKOTOZOA
passes. When the requisite quantity of cytoplasm has been raised into
the elevation this is divided off by a constriction, and the daughter forms,
termed merozoites, are produced. These grow into adults, which may
again reproduce by schizogony. It is often supposed that the multi-
nucleate adult, which is termed a schizont, suddenly segments into a
Fig. 39. — Hepatozoon cams : Developmental Stages in the Spleen of a
Bagdad Dog ( x 2,000). (Original.)
1. Young schizont in mononuclear cell. 2. Slightly older schizont.
3. Section of an older schizont \\ith anumber of nuclei.
4. Section of a schizont in w Inch merozoites are commencing to form by a budding process.
.5. More advanced stage of budding process as seen in section of mature schizont.
G. Merozoites and residual body after schizogony : the merozoites are gametocytes which enter
the mononuclear cells of the blood-stream.
7. Section of stage with eight large merozoites which are probably destined to become schizonts
again. 8. Stage similar to that depicted at 7.
number of merozoites, as in the case of malarial parasites. It appears,
however, that in all cases the merozoites, whether few in number or more
numerous, are formed as small buds at the surface of the schizont, as
described above. A variable quantity of the cytoplasm is unused in the
formation of the merozoites. This residual cytoplasm, within which may
be found a certain number of unused degenerate nuclei of varying size,
SCHIZOGONY AND SPOROGONY 67
and any other material to be discarded, such as pigment, is known as the
residual body. It takes no further part in the life of the organism, and
after separation of the merozoites quickly disintegrates (Fig. 39, 6).
Amongst the Sporozoa, after syngamy has taken place, the zygote
divides by a process which is essentially the same as schizogony. This is
termed sporogony, and it gives rise to sporozoites, which differ in size and
shape from the merozoites. The sporozoites arise from the multinucleate
zygote, which may have increased considerably in size and is called the
sporont, by a budding process which is very similar to that by which the
merozoites are formed (Fig. 455). The term " sporogony " is generally
extended to include the whole phase of the developmental cycle from the
beginning of the production of gametes or gametocytes to the formation
of sporozoites from the sporont after syngamy has taken place. To
distinguish the other phase of development during which schizogony
occurs repeatedly without the intervention of a sexual process, it has
been termed agamogony, and the various stages (merozoites and schizonts)
agamonts. The growing agamont is often termed a trophozoite.
During the formation of merozoites and sporozoites it not infrequently
happens that the number of nuclei present is so large that the surface
of the cytoplasm is insufficient to accommodate them all. By a process
of vacuolation of the cytoplasm the available surface is increased. The
vacuoles may open into one another, so that the cytoplasm is reduced to
the condition of a coarse network. In this way the available surface
upon which nuclei can take up their position is increased, so that the
merozoites or sporozoites can be budded off in the usual manner. A
typical instance of this increase in surface occurs during the formation of
sporozoites in the oocysts of the malarial parasites on the stomach of
mosquitoes, as also during schizogony of Aggregata eherthi and other
Sporozoa (Figs. 377 and 391).
A method of schizogony which occurs amongst the piroplasmata must
be mentioned. In these parasites the number of daughter forms produced
are two or four, which are described as arising from the parent by a budding
process, in contrast to the supposed segmentation of the schizont of the
malarial parasites. As already explained, the merozoites of malarial
parasites are not produced from the parent by a sudden splitting of the
body between the nuclei, but by the formation of buds from its surface,
as occurs generally amongst the Sporozoa. The piroplasmata are no
exception to this rule. In some species {Babesia canis) the buds are
usually two in number, but may be four (Fig. 417). In others (B. equi)
there are usually four buds, as in Plasmodium minasense (Fig. 416 and
Plate XVII., 6-15, p. 982). The bud commences as a small cytoplasmic
elevation on the surface of a rounded parasite. It gradually increases in
68 MULTIPLICATION OF PROTOZOA
size at the expense of the cytoplasm of the parent. It is difficult to
understand why an organism which is to produce only two daughter forms
should do so by a budding process instead of by a simple binary fission
into two parts. It seems possible that it is a condition which has evolved
from one in which a larger number of merozoites were originally produced,
as in typical schizogony.
When a schizont is in process of producing merozoites or a sporont
sporozoites, the schizont or sporont may first divide into a number of
intermediate bodies which actually produce the merozoites or sporozoites.
In the case of the coccidium Caryotropha mesnili, when about sixteen
nuclei are present in the schizont, it divides into sixteen portions which
have been called cytomeres or agametoblasts (Fig. 375). The nuclei of
these undergo further divisions, and finally merozoites are budded from
their surfaces. A similar method of multiplication occurs in Klossiella
cobayce and other forms (Fig. 449). Similarly, during sporogony the zygote,
instead of dividing directly into sporozoites, may first produce a number
of sporoblasts, which give rise to the sporozoites. In the coccidia sporogony
takes place within the oocyst which has formed around the zygote, and
it frequently happens that the sporoblasts secrete around themselves
secondary cysts or sporocysts, within which the sporozoites are finally
produced (Fig. 337).
Attention has already been called to the fact that occasionally, amongst
flagellates which normally multiply by binary fission, the rate of division
of the nuclei may exceed that of the cytoplasm during very rapid multi-
plication, so that stages are reached in which an abnormal number of
nuclei are present (Fig. 142). The excessive nuclear multiplication,
however, comes to an end, and the body divides repeatedly till a number
of normal uninucleate forms are produced. In some cases such multi-
nucleate stages occur normally in the developmental process. Thus, in
the course of the development of Trypanosoma lewisi in the flea, the
trypanosomes taken up from the rat enter the cells lining the stomach,
and there grow into large bodies which possess as many as sixteen nuclei,
kinetoplasts, and flagella (Fig. 200). The " sphere," as it is called, then
divides into a corresponding number of trypanosomes. Such a method
of multiplication is really one of delayed division of the cytoplasm,
and must be distinguished from true schizogony. It seems probable
that the final division of the " sphere " takes place by repeated binary
fissions.
During the process of schizogony the merozoites produced by any
particular organism vary as regards size and numbers. In certain cases
the variations are at a minimum, as, for instance, amongst the human
malarial parasites. Plasmodium malaricB of quartan malarial fever pro-
SCHIZOGONY AND SPOROGONY 69
duces nearly always eight merozoites, and these vary little in size (Plate
XIII., p. 934). ^ijnilsiTly, Plasmodium vivax of benign tertian fever pro-
duces, as a rule, sixteen, but departures from this number are not uncom-
mon (Plate XII,, 16-18, p. 926). Amongst other Sporozoa, however, greater
variations occur, as will be described below. In some cases it has been
supposed that the schizogony was of two types- — the one giving rise to a
small number of large merozoites, and the other to a large number of
smaller ones (Fig. 39, 6 and 7). It was supposed that this represented
a sexual dimorphism, one line ending in gametocytes of the female sex,
and the other in gametocytes of the male sex. More careful study of such
cases has thrown doubt on these conclusions, and has tended to show that
every transition, both as regards numbers and size, occurs between the two
types. Thus, in the case of Adelina dimidiata, a coccidium of the centipede,
the merozoites produced by a schizont vary in number from four to
sixteen, as demonstrated by Schellack (1913). As a rule, when the number
is large the merozoites are small, and vice versa. In Hepatozoon canis
(Fig. 39) the number of merozoites produced may be only four, or it may
exceed a hundred. In this case it appears that with successive schizogony
the number of merozoites produced increases, while their size diminishes,
till finally there are formed a large number of small ones which enter the
leucocytes and become gametocytes. It has thus to be remembered that
in any individual species the merozoites produced at schizogony may vary
considerably, both in number and size.
In the case of sporozoites which are produced from the zygote by a
process similar to schizogony, the number and size is much more constant.
Thus the zygotes of coccidia belonging to the genus Eimeria invariably
produce eight sporozoites which are contained in pairs in four sporocysts
(Fig. 337). In other cases, as, for instance, in the genera Barrouxia and
Aggregata, though the number of sporocysts produced by the zygotes of
any particular species may vary considerably, the number of sporozoites
in the sporocysts is constant (Fig. 376). On account of its uniformity
the type of sporogony is of greater value for purposes of identification
and classification than are the forms observed at schizogony.
GEMMATION OR BUDDING.— By this method of reproduction is to
be understood one in which an organism, after its nucleus has divided,
instead of splitting into two equal or nearly equal parts, divides very
unequally, so that one daughter form is very much smaller than the other.
The condition is one of extreme unequal binary fission. It is usual to
regard the large form as a parent individual, and the small one as a daughter.
The process has been described as occurring in free-living amoebae, and the
unequal divisions seen in Tnjpanosoma lewisi, which has already been
referred to, may be regarded as an instance of gemmation (Fig. 197). It
70
MULTIPLICATION OF PROTOZOA
occurs, however, most typically amongst the Euciliata in the attached
Peritrichida like Vorticella, and amongst the Siictoria. In many species of
Vorticella and allied forms the body divides into two equal parts, so that
two equal-sized individuals are attached to the end of a single stalk. One
of these may escape and, attaching itself, develop a new stalk, or it may
remain attached, and the two individuals may form new stalks, so that
eventually a complicated system of dichotomous branches is produced.
The division, though apparently longitudinal, is really transverse, as will
be evident if it is remembered that the organisms are attached to the
stalks by their dorsal surfaces. In some cases the division of the body is
unequal, so that a very small individual is separated from a large one.
These small forms are provided with circlets of cilia, by means of which
Fig. 40. — Ephelota gemrnipara in Gemmation {xca. 350). (After Collin, 1912.)
1. Section of an entire organism, showing method of budding of the macronucleus to form nuclei
of buds. 2. Surface view of budding individual.
they swim away, and ultimately conjugate with one of the larger attached
forms (Fig. 44).
In the Suctoria buds are formed, either from the surface of the body
or in cup-like depressions. In Ephelota, studied by Hertw^g (1876), the
nucleus becomes much branched, and as buds are formed on the surface of
the body, portions of the macronucleus enter each bud. The buds are finally
separated as ciliated embryos (Fig. 40). In other cases, as in Tokophrya
and Choanophrya, there occurs a process of internal budding (Fig. 532).
A depression is formed in the cytoplasm, and the margins of this close to
include a space which communicates with the exterior by a pore. A bud
is formed from the surface of the cytoplasm within this space. A ciliated
embryo is detached, and eventually escapes through the pore.
Though the daughter individuals formed at binary fission may be so
GEMMATION 71
unequal in size that the process is regarded as one of budding or gemmation,
the nucleus of the bud arises by equal division of the nucleus of the parent,
so that the large and small daughter forms have their nuclei of equal size.
A method of formation of the nuclei of buds from chromidia has been
described as occurring in certain Protozoa. Thus, in the case of Entamoeba
histolytica, Schaudinn (1903) supposed that granules of chromatin occurred
in the cytoplasm outside the nucleus. These granules were supposed to
collect in groups at the surface of the organism, become organized into
nuclei, and enter the buds which were forming. Such a process certainly
does not occur in E. histolytica. Another instance in which nuclei have
been described as arising in this manner is that of Arcella vulgaris re-
ferred to above (Fig. 2).
SYNGAMY AMONGST THE PROTOZOA.
As m the higher animals and plants, at certain phases of development,
two cells unite and their nuclei fuse, so amongst the Protozoa a similar
process may occur. This is generally known as a sexual process, or
syngamy. It may take place in one of two ways: either two individuals,
which are known as gatnetes, unite by fusion of their cytoplasm, followed
by union, or haryogamy, of their nuclei; or two individuals become incom-
pletely united, and part of the nucleus of each passes over into the other
individual to unite with its nucleus. After this transference of nuclear
material the individuals separate. The process in which two individuals
unite completely is known as copulation, while that in which interchange
of nuclear material between two temporarily associated individuals takes
place is called conjugation. The two processes are not essentially different
from one another, for it may be considered that in conjugation each of
the two associated individuals really produces two gametes, one of which
is large and contains all the cytoplasm and a nucleus, while the other is
small and consists of a nucleus only. The small gamete produced by one
individual unites with the large gamete produced by the other. It is,
however, convenient to distinguish the process of copulation from that of
conjugation, as the latter is the characteristic method of syngamy amongst
the Euciliata.
COPULATION. — This process consists in the union of two cells with
fusion of their nuclei. The cells are known as gametes, while the single
uninucleated cell resulting from the union is called a zygote, and the nucleus
of the zygote, which is the product of the union of two gamete nuclei, is
the synkarion. The uniting gametes may be the ordinary individuals
which have ceased multiplying, or an ordinary individual, by a special
process of multiplication, may give rise to a number of smaller gametes
72
SYNGAMY IN PROTOZOA
which unite in pairs. In the latter case, the individual which gives rise
to the gametes is known as a gametocyfe, and the process by which it gives
rise to the gametes as gametogony.
The gametes which unite may be alike in size and shape, in which
case they are known as isogametes, and the process of union as isogamy.
On the other hand, they may be recognizably different from one another
in size or structure, and are known as anisogametes. The process is then
called anisogamy or heterogamy. If the gametes differ in size, the large
Fig. 41. — Syngamt of Cercomonas longicauda { x ca. 2,000).
1916.)
(After Woodcock,
1. Two individuals uniting by their posterior ends.
3. Still later stage after nuclei have fused.
4. Stage in which fiagella are lost and body rounded.
2. Later stage in the union.
5. Encysted zygote.
one is called the macrogamete and the small one the microgamete. It
usually happens that the small gamete or microgamete is actively motile,
on which account it is regarded as the male gamete, as it corresponds in
function with a spermatozoon of higher animals. The larger macrogamete,
which is usually a passive body heavily charged with food reserve material,
corresponds with the ovum. There is every transition between the process
of isogamy and anisogamy. Thus, in some cases the gametes are equal in
size, but differ from one another only in the size of their nuclei. In other
COPULATION
73
cases one gamete is only slightly larger than the other, and there is every
gradation towards forms like coccidia or malarial parasites, in which the
macrogamete is a comparatively large cell and the microgamete a very
minute one.
As an illustration of syngamy in which two ordinary individuals unite,
the case of Cercomonas longicauda, as described by Woodcock (1916), may
be considered (Fig. 41). Two flagellates of the ordinary type come
together and unite by their posterior ends, the union gradually extending
forwards. After the two flagellates are completely fused their nuclei
unite to form a synkarion. The zygote which is produced may commence
multiplying by binary fission in the usual manner, or it may encyst.
A similar process occurs in Polytoma uvella, but is modified as a result of
the protective covering of the body (Fig. 42). Two flagellates unite by
their anterior ends, and the cytoplasmic contents of one flow into the other,
Fig. 42. — Polytoma uvella: The Process of Isogamy as observed during thS;
Course of Three Hours ( x 1,500). (Original.)
The contents of one flagellate flow into the other, which gradually becomes spherical. Finally, a
cyst is formed after complete fusion has occurred. The nuclei were no longer visible in the
later stages. The dark rods are the stigmata.
giving rise to a spherical zygote which becomes encysted. In the case of
Copromonas suhtilis two individuals fuse completely, and Dobell (19086)
has stated that each nucleus before union gives off two reduction bodies
(Fig. 48). All these instances are cases of isogamy, in which the gametes
differ little, if at all, from the ordinary adult individuals. Isogamy has
been described by Woodcock (1916) for Sj)iromonas angtista and Co'pro7no7ias
fuminantium.
In most cases, however, certain individuals termed gametocytes, which
may differ from the ordinary reproducing forms, by a special type of
multiplication {(jametogony) give rise to a number of gametes, which then
unite. Syngamy of this type occurs amongst non-parasitic Protozoa, and
has been described, in the case of Foraminifera [TrichosphcEriii'm), Radio-
laria {Thalassicola), and other forms, but the best-known instances occur
amongst parasitic Sporozoa. In the reproduction of Monocystis magna,
74 SYNGAMY IX PROTOZOA
a gregarine of the earth-worm, Cuenot (1901) described the process of
syngamy. Two individuals (gametocytes) encyst together in a common
cyst (gametocyst), and each gives rise to a large number of gametes which
appear to be completely alike (isogamy). The gametes produced by one
individual unite with those produced by the other. The zygotes thus
formed become encysted in secondary cysts (oocysts). In his description
of syngamy in Monocystis rostrata, another gregarine of the earth-worm,
Muslow (1911) also found that there was complete isogamy.
From this condition of complete isogamy, various transition stages
leading to marked anisogamy are known amongst gregarines. Thus, in
the case of Lanlcesteria ascidice, Siedlecki (1899), and in the allied form
Lankesteria culicis of A'edes argenteus, studied by the writer (1911a), the
gametes produced by each gregarine are alike in size, but differ from one
another in that those produced by one gregarine have smaller nuclei than
those produced by the other (Fig. 465). A gamete with a small nucleus
unites with one which has a large one. In the case of Stylorhynchus longi-
collis, heger, L. (1904/^) noted that the gametes produced by one gregarine
were spherical bodies, while those produced by the other were spindle-
shaped structures, each provided with a flagellum. The spindle-shaped
motile gametes were actually larger than the spherical ones, so that if
the former are to be regarded as the male gametes, this instance affords
an exception to the general rule that the male gametes are smaller than
the female (Fig. 482). In the case of the gTeganne Pterocephalus nobiJis,
Leger, L. and Duboscq (1903a) describe the gametes which are formed from
one individual as sniall curved structures (microgametes), and those from
the other as large elongate bodies (macrogametes). In this instance there
is an approach to the condition which is characteristic of the coccidia.
Amongst the coccidia, female gametes or macrogametes are spherical or
ovoid bodies filled with food reserve material in the form of globules, while
the male gametes or microgametes are minute, elongate, sickle-shaped
bodies usually provided with two flagella. The microgametes, which are
provided with two flagella, are composed of chromatin covered by a thin
layer of cytoplasm, and in many respects resemble the spermatozoa of
higher animals (Fig. 337). A similar difference in size exists between
the gametes of the pigmented blood parasites of the genera Plasmodium
and Hwmoproteus and the non-pigmented Leucocytozoon (Figs. 383 and 391 ).
Where a special type of individual, the gametocyte, produces a number
of gametes, the actual number produced by each varies considerably in
different groups. Amongst the gregarines, where two gametocytes are
enclosed in a gametocyst, it is evident that the chance of gametes going
astray is reduced to a minimum, so that both gametocytes produce approx-
imately the same number of gametes. In the majority of gregarines there
COPULATION— C0NJUGATI(3N 75
are many gametes (Fig. 465); there may be not more than a dozen, as in
Schizocystis (Fig. 469), while in the case of Ophnjocystis each gametocyte
produces only a single gamete (Fig. 468).
Amongst the true coccidia or Eimeriidea, the male and female game-
tocytes are not associated, but develop in separate cells of the intestine or
other organ. The number of gametes produced by each individual may be
very unequal in number (Fig. 337). The gametocyte (macrogametocyte)
which gives rise to the macrogamete becomes directly transformed into
a single macrogamete, while the microgametocyte produces a large number
of microgametes. The latter are provided with flagella, and swim away
in search of a macrogamete, which is not itself, endowed with the powers
of movement. It seems evident that the production of large numbers of
microgametes is correlated with the greater uncertainty of the micro- and
macro-gametes coming together. In the case of the malarial parasites
and allied organisms (Hsemosporidiidea), in which fertilization takes place
in the stomach of a blood-sucking insect, the macrogametocyte produces
a single macrogamete, while the microgametocyte gives rise to from
six to ten microgametes (Fig. 391). In the coccidia belonging to the
Adeleidea, the macro- and micro-gametocytes develop in actual contact
with one another. The result of this close association is that, though the
macrogametocyte gives rise to a single macrogamete, the microgametocyte
produces only four microgametes (Fig. 338). In the hsemogregarines of
the genus Karyolysus, in which a similar association of the gametocytes
occurs, Eeichenow (1921) has shown that the microgametocyte produces
only two microgametes (Fig. 457). The marked difference in size betw^een
the microgametes and macrogametes in these cases is associated with the
conditions under which future development will take place. The macro-
gamete is provided with a large amount of cytoplasm heavily loaded with
food reserve material to enable it to survive and develop without nourish-
ment in the encysted condition. As a result of this provision, as in the case
of the ovum, its power of movement has been lost. The male gamete
merely functions as a fertilizing agent, for which its nucleus alone is required,
and for the fulfilment of which a high degree of motility is an advantage.
CONJUGATION. — In the type of syngamy which has just been
described, the two gametes unite completely and their nuclei fuse. This
process is know^n as copulation, to distinguish it from conjugation, which
occurs amongst the Euciliata. In typical conjugation two individuals
associate, and one of the two nuclei, which each then possesses, passes into
the other individual and unites w^ith the nucleus which has remained
stationary. As pointed out above, it is possible to regard the two ciliates
as each producing two gametes, the small gamete (migratory nucleus)
produced by one individual uniting with the large gamete produced by
SYNGAMY IN PROTOZOA
M N U P
Fig. 43. — Syngamy in Paramecium putrmum. (After Doilein, 1916, Slightly
Modified.)
[For description see opposite page-
CONJUGATION 77
the other. The process, which is an exceedingly complicated one, has
been studied in detail, especially in species of Parameciu7n. In the case
of Paramecium putrinum,, for instance, each individual possesses a macro-
nucleus and a micronucleus. When two individuals associate in con-
jugation, they become closely united by their peristomes and the side of
their bodies behind this (Fig. 43). The macronuclei become elongated
and undergo a series of divisions till a large number of fragments are
produced. All these ultimately degenerate and disappear. Meanwhile,
the micronuclei have divided by mitosis, and the two nuclei thus formed
in each ciliate again divide by mitosis. At this stage each ciliate or
conjugant, as it is called, contains four nuclei and a number of degenerating
bodies derived from the macronucleus. Three of the nuclei in each now
degenerate, so that each conjugant is left with only one. This one now
divides again, and of the two resulting nuclei in each conjugant, which as
far as can be seen are exactly alike, one is a stationary nucleus and the
other a migratory one. The migratory nucleus of each conjugant now
passes over and fuses with the stationary nucleus of the opposite conjugant.
The resulting nucleus, which is a zygote nucleus, now divides to give rise
to two, these two to give four, and the four to produce eight nuclei. At
this stage the ciliates, each of which has eight nuclei and still the remains
of the degenerating nuclei, separate from one another and swim away.
Of the eight nuclei, four increase in size and become macronuclei, three
degenerate, while the remaining one retains its character as a micronucleus.
The latter divides to form two micronuclei, and this is followed by division
of the ciliate itself in such a manner that two of the macronuclei and one
of the micronuclei pass to each daughter ciliate. At the next division of
these daughter ciliates the micronucleus divides, and each resulting ciliate
receives one of the two macronuclei and one of the tw^o micronuclei.
Thus, the nuclear condition of the original ciliate is regained. At all
subsequent divisions of the ciliate, both the macro- and micro-nuclei divide.
A. Two associated conjugants with intact macronuclei and commencing division of micronuclei.
B. Macronuclei and micronuclei dividing.
C. Bivided-up macronuclei and two dividing micronuclei in each conjugant.
D. Three of the four micronuclei in each conjugant have degenerated, while the remainmg one is
commencmg to divide.
E. The micronucleus of each conjugant is drawn out into a long spindle.
F. Four resulting micronuclei near the point of union of the two conjugants.
G. Union of the micronuclei in jDairs.
H-L. Progressive division of the micronuclei till each conjugant has eight. The conjugants
finally separate (L.)
M-N. Three micronuclei degenerate, four become macronuclei, while one remains and divides.
The ciliate divides.
0. One product of division of the form with four macronuclei and two micronuclei. It contains
two macronuclei and one dividing micronucleus. The ciliate divides.
P. One product i )f the division of the form with two macronuclei and two micronuclei. It contains
one macronucleus and one micronucleus, and thus resembles the ciliates before they com-
menced conjugation.
78
SYNGAMY IX PROTOZOA
Fig. 44. — Diagrammatic Representation of Nuclear Changes during
Syngamy in YortieeUa nelmlifem. (After Maupas, 1889.)
[For de-^criplion see opposite page.
CONJUGATION 79
This complicated process is best comprehended by reference to the diagram
(Fig. 43). Except for variations in detail, the conjugation of other ciliates
in which the process has been studied takes place in a similar manner. In
Paramecium 'putrinurn the two conjugants are equal in size. In other
ciliates a large individual conjugates with a smaller one, while the most
extreme condition is reached in Vorticella and its allies, in which a small
free-swimming ciliate budded of! from a large pedunculate individual
conjugates with one of the large forms (Fig. 44). The macronuclei in both
degenerate, and the micronuclei undergo a number of divisions, as in
Paramecium. All these degenerate except one which divides to give rise
to a stationary and a migratory nucleus. Each individual, one a large
and the other a small one, now contains two nuclei. Exchange of nuclei
then occurs, as in Paramecium, but the small individual, instead of pro-
ceeding to further development, shrinks and dies, while the large individual
alone survives. The single nucleus of the large surviving individual divides
repeatedly, and a number of macronuclei and one micronucleus are
produced. By successive divisions of the ciliate, similar to those occurring
in Paramecium 'putrinutn, the original condition is regained. In the case
of Paramecium caudatum, the process of syngamy is similar to that of
P. putrinutn (Fig. 45), but in the case of P. aurelia, owing to the fact that
the ciliate possesses two micronuclei instead of one, it is modified in certain
respects. When conjugation occurs, the two micronuclei of each conjugant
divide twice, so that eight are formed. Of these seven degenerate, leaving
in each conjugant one micronucleus and one degenerating macronucleus.
The single micronucleus divides and exchange of nuclei occurs, as in P. jnitri-
num and P. caudatum. After union of the two nuclei the single nucleus
divides twice till four are present, and of these two become macronuclei
and two remain as micronuclei. Each of the latter divides once, so that in
each ciliate there are now two macronuclei and four micronuclei. The ciliate
A. Union of the small free-swimming conjugant with the large attached one.
B. Fragmentation of the macronuclei and division of the micronuclei.
C. D. E. Further divisions of the micronuclei leading to four in the large conjugant and eight
in the small one.
F. All the daughter micronuclei have degenerated except one in each conjugant.
G. The two micronuclei are dividing with the axis of division across the plane of union of the two
conjugants.
H. The two micronuclei in the large conjugant are uniting, while those in the small one remain
separate.
I. The two micronuclei m the large conjugant have united, while those in the small one are
degenerating.
J. The micronucleus of the large conjugant is dividing, while the small conjugant is shrinkmg.
K. The small conjugant has disappeared, while the micronuclei of the large one are dividing.
L, 31. Further divisions of the micronuclei to give rise to eight.
X. Transformation of seven micronuclei into macronuclei and division of remaining micronucleus.
0, P. Division of the body has taken place, giving rise to an individual with four macronuclei
and one micronucleus (O), and one with three macronuclei and one micronucleus (P). By
further divisions the original condition is reached in which the micronucleus and one macro -
nucleus are present.
80
SYNGAMY IN PROTOZOA
Fig. 45. — Diagrammatic Representation of the Nuclear Changes during
Conjugation of Paramecium caudatum. (After Jennings, 1920.)
A. Two associated conjugants.
B. T)egeneration of macronucleus and first division of niicionucleus.
C. Second division of micronuclei to give rise to four, of which three degenerate.
D. Division of remaining micronuclei to produce the gamete nuclei.
E-F. Union of gamete nuclei. G. Separation of the conjugants.
H-J. Division of the nuclei to give rise to eight, of which four increase in size to become macro-
nuclei, while three degenerate.
K. After division of the single micronucleus the ciliate itself divides.
L. After a further division of the micronucleus the daughter ciliates again divide to give rise t(3 the
normal type.
CONJUGATION 81
whicli has separated from its partner divides into two daughter ciliates,
each of which has a single macronucleus and two micronuclei, as in the
original conjugants.
In the conjugation of Collinia branchiarum described below, the two
ciliates unite as inParameciu )ii , and exchange of nuclei takes place(Fig. 495).
The macronuclei, however, behave in a remarkable manner. Each becomes
much elongated, and when exchange of micronuclei is taking place, the
two long macronuclei arrange themselves side by side across the point of
union of the ciliates in such a manner that half of each macronucleus is in
each ciliate. When the ciliates separate the macronuclei divide, so that
Fig. 46. — Conjugation of the Ciliate Cycloposthium bipalmatum, showing
Differentiation of Conjugating Nuclei into Male (^J) and Female ( 2 )
( X ca. 300). (After Dogiel, 1923.)
M., Macronucleus ; Sk., skeletal plate ; An., anus; Ph., pharynx ; My., myonemes ; D., degenerating
micronuclei; Sp., remains of central part of spindle.
each ciliate receives half of each macronucleus. Though this occurs, the
macronuclei ultimately degenerate, and a new macronucleus is formed
from the micronucleus. It will thus be seen that in the Euciliata each of
the two conjugants ultimately contains two nuclei which are exactly alike,
except that one is a migratory or male nucleus, and the other a stationary
or female nucleus. This difference in behaviour is the only indication of sex
differentiation. In the case of Cycloposthium hipalmatum, a ciliate parasitic
in the intestine of the horse, Dogiel (1923, 1925) has noted that, though
conjugation between two individuals takes place in the usual manner, the
two nuclei which take part in the syngamic process differ in that the
migratory one assumes the characters of a male gamete in becoming a
I. ■ 6
82 SYNGAMY IN PROTOZOA
filament provided with a head, while the stationary one retains its original
form (Fig. 46). This observation is a confirmation of the view that the
migratory nuclei in other ciliates are actually male nuclei.
GONOMERY. — A remarkable process of syngamy was described by
Hartmann and Nagler (1908) for Sappinia diploidea, an amoeba isolated
from lizards' faeces. The amoeba is peculiar in being binucleate, the
two nuclei lying close together (Fig. 47). When encystment occurs, two
individuals enter a common cyst. The two nuclei of each individual now^
fuse and then undergo reduction divisions, the reduction bodies degenerat-
ing. After this the two amcebse unite, the nuclei approach one another,
but do not fuse. The amrrbn then leaves the cyst and commences to
d e f
Fig. 47. — Sapjyinia dij)loidea : Fkee and Encysted Stages ( x ca. 1.500). (After
Hartmann and Nagler, 1908.)
a. Usual form with two nuclei. I. Form with dividing nuclei.
c. Dividing form producing two binucleated daughter amcebse.
d. Two amcebse in common cyst. e. The two nuclei in each amoeba have united.
/. The bodies of the two amoebae have fused, giving rise to a binucleated amoeba which escapes
from the cyst and reproduces by binary fission, as at n, h, and c.
multiply by binary fission, the two nuclei dividing by mitosis side by side.
These nuclei are regarded as gamete nuclei, which, however, do not actually
unite, though dividing many times during asexual reproduction till
encystment again occurs. This condition is one of delayed union of gamete
nuclei, a process which is known to occur in higher animals, and which has
been teTmed gono7nery.
METHOD OF UNION OF GAMETES.— The actual union of gametes
during syngamy takes place in a variety of ways, which are dependent on
the structure of the gametes themselves. In the case oi Polytoma uveUa,
Copromonas subtilis, and other forms, the two flagellates approach one
GONOMENY— UNION OF GAMETE8
83
anotlier, and unite first by their anterior ends near the flagellar origin
(Figs. 42 and 48). During this process the flageUates are actively motile.
Their nuclei approach one another and come into contact, and the nuclear
membrane disappears at the line of contact till a common membrane is
formed. In the case of Cercomonas longicauda, Woodcock (1916) observed
union to take place first near the posterior end (Fig. 41).
Union of gametes within the gametocysts of gregarines takes place
in a similar manner. As already explained, sometimes the gametes are
Fig. 48. — Syngamy of Copromonas suhUlis ( x ea.
1. Individual flagellate as seen in living condition.
3. Nuclei dividing to form tirst reduction body.
4. Nuclei dividing t(i fmin sccdud ivdurtion body.
5. Union of nuclei and formation nfcNst
2.000). (After Dobell, 1908.)
2. Early stage in union of gametes.
6. Fully formed zygote in cyst.
alike, and are merely spherical bodies which, coming into contact with
one another, gradually fuse, while their nuclei unite. In other cases the
gametes produced by one gregarine are elongate and provided with
flagella, as in StylorhijncJms, while those produced by the other are spherical
bodies (Fig. 482). Union takes place by one of the elongate flagellated
gametes attaching itself to one of the spherical forms by its pointed
anterior extremity, after which fusion takes place, while the flagellum
disappears. Amongst the coccidia the minute flagellated microgamete
swims activelv and comes in contact with one of the larger immobile
84 SYNGAMY IX PKOTOZOA
macrogametes. Sometimes this occurs before the oocyst has formed; at
other times after its formation, in which case a pore, the micropyle, is
present at one end of the cyst, and through it the microgamete makes its
way. The microgamete enters the cytoplasm of the macrogamete, which
immediately commences to secrete a substance which closes the micropyle.
Though several microgametes may be attracted towards one macro-
gamete, immediately one has entered its cytoplasm this attraction ceases.
The nucleus of the. macrogamete has meanwhile been drawn out into a
long spindle, the fertilization spindle, on the fibres of which the chromatin
granules are distributed. The microgamete nucleus breaks up into
granules, which gradually become distributed upon the fertilization
spindle. The spindle now retracts, and a spherical nucleus containing
chromatin from both the macrogamete and microgamete is again formed
(Fig. 337).
Amongst the pigmented blood parasites of the genera Plasmodium
and Hcemojjroteus a similar type of union occurs. The nucleus of the
macrogamete moves towards the surface of the body, which is raised up
at this point into a small elevation. The elongate motile microgamete
enters this elevation, and its nucleus unites with that of the macrogamete
(Figs. 383 and 391).
Amongst the Euciliata, when conjugation occurs amongst free-swim-
ming forms, it is usually by the peristomes that they become attached
to one another. Actual continuity of cytoplasm appears to take place
just behind the peristomes, to allow of the interchange of nuclei, as
described above. In the attached forms, such as Vorticella, conjugation,
as already noted, takes place between a large attached individual and
a small free-swimming ciliated form which has been budded of! from
another individual. The small free-swimming form attaches itself to the
larger one at a point near the insertion of its stalk, and wdien exchange
of gamete nuclei has occurred it degenerates (Fig. 44).
METHOD OF FORMATION OF GAMETES.— The actual method by which
gametes are formed from gametocytes varies to some extent. Amongst
the gregarines, the nucleus of the gametocyte multiplies by a series
of divisions till the requisite number of nuclei are present (Fig. 465).
These are then arranged upon the surface of the gametocyte, and little
elevations of the cytoplasm are formed. Into each of these there passes
a nucleus. Each small cytoplasmic elevation or l)ud, which has acquired
the characteristic form of the gamete, is now separated by a constriction.
A large amount of the cytoplasm is usually left over as a residual body.
In the case of the coccidia and allied forms, where there is an extreme
condition of anisogamy, one gametocyte, the macrogametocyte, gives rise
to a single macrogamete. It is supposed that this transformation takes
FORMATION OF GAMETES— AUTOGAMY 85
place by the extrusion of one or more reduction bodies. In the case of
the microgametocyte, nuclear multiplication takes place till numbers of
nuclei are formed (Fig. 337). These nuclei at first appear as minute
aggregations of chromatin granules. They change their form on the
surface of the cytoplasm till they appear as dense comma-shaped struc-
tures. Each is then separated with a small amount of cytoplasm, which
contributes to the formation of flagella. In the blood parasites belonging
to the genera Plasmodium, Hcemojjroteiis, and Leucocytozoon, the macro-
gametocyte produces a single macrogamete, as in the coccidia, by the
rapid extrusion of reduction bodies. The microgametocyte gives rise
in the course of a few minutes to six or ten microgametes by a violent
process known as exflageUation, which occurs normally in the stomach of
the invertebrate host, but which may be observed in an ordinary moist
preparation of blood under the microscope (Fig. 381). The details of the
process will be described below in the section devoted to these parasites,
but it may be noted here that the function of the reduction bodies referred
to above is far from clear, and the assumption that the process is com-
parable with the formation of polar bodies during maturation of the
ovum of higher animals does not appear to be correct.
AUTOGAMY. — A process of syngamy which may be defined as self-
fertilization has been described for certain Protozoa under the name of
autogamy. In its most complete form the nucleus of a single individual
divides to form two daughter nuclei. Each of these undergoes reduc-
tion divisions, after which the two surviving nuclei unite. In the
case of Entamoeba coli, Schaudinn (1903) described autogamy in the
encysted stages. The single nucleus of the encysted form divides to give
rise to two nuclei. Each of these gives off two reduction bodies, after
which they divide to form four nuclei, wliich are arranged in pairs at
opposite sides of the cyst. One of each pair is a stationary nucleus and
one a migratory nucleus. The migratory nuclei move to opposite sides
of the cyst, where they unite with the stationary nuclei. The cyst again
has two nuclei, which proceed to divide till the characteristic eight nuclear
stage is reached. The writer (1907) saw certain stages in the development
of the cysts of Entamoeba miiris, which appeared to supply a confirmation '
of Schaudinn's account of E. coli, but there is little doubt that the appear-
ances were capable of another interpretation. All evidence goes to
show that no such process actually occurs in the cysts of E. coli or any
other anio-ba. A somewhat similar process was described by Prowazek
(1904f/) in the cysts of Prowazekella laceyta', while Schilling (1910) recorded
its occurrence in Trypanosofna lewisi. It seems perfectly clear that in
none of these cases was there sufficient evidence to justify the conclusions
whicli were made.
86 SYNGAMY IN PR(3T0Z0A
Hartmann (1909) gave a general account of autogamy amongst Pro-
tista, but a perusal of his paper shows that most, if not all, of the alleged
instances are based on very slender evidence.
PEDOGAMY.— There is another type of self-fertilization which differs
from true autogamy in that a single individual first divides into two
daughter forms after division of its nucleus. When the nucleus of each
has undergone maturation or reduction divisions, the two daughter cells
which are gametes unite. The process which is known as jjedogarny has
W
^^,
.-^^^..^.^
F
Fig. 49. — Pedogamy in Actinosphceriuni eicMomi ( x 80). (From Lang, 1901,
AFTER Richard Hertwig, 1898.)
A. A single multinucleated individual in a primary cyst.
B. Division into a number of uninucleated individuals which become enclosed in secondary cysts.
(!. The contents of each secondary cyst divide into two.
D. The division completed, after which each nucleus undergoes two reduction divisions.
E. The two gametes in each secondary cyst imite.
F. Secondary cysts containing zygotes resulting from the union of the gametes.
been studied by Richard Hertwig (1898) and others in the multinucleated
Heliozoon Actinosphcerium eichkorni (Fig. 49). An individual encysts
and divides into a number of uninucleate forms, which become enclosed
in secondary cysts. Within each secondary cyst a further division into
two individuals takes place. The nucleus of each of these undergoes
two reducing divisions, after which union takes place. In this case the.
PEDOGAMY
87
:-',1^.
w
3
'WW
// ^^
Fig. 50. — Pedogamy in Actiriophri/s sol, Ehrenberg ( x ca. 800). (After
Belak, 1923.)
1 . Loss of pseudi)])odia and their axial filaments.
2. First nuclear division and commencing formation of gelatinous cnvelii])e.
3. Two gamete nuclei in early division stage in the dividing cyti|iiasin within the cyst membrane.
4. Two sejiarate gametes each with a nucleus in process of first niatuiation division (reduction
division).
5 . Later stage of reduction division of the gamete nuclei: the chromosomes in conjugation.
0 . Two later stages in the reduction division of the gamete nuclei.
7. Completion of the reduction division: each gamete has a nucleus and a reduction body (de-
generate nucleus).
[Continued on j)- 88.
88 SYNGAMY IN PROTOZOA
two gametes are formed from a single individual in the secondary cyst.
In such an example there is an extreme instance of inbreeding. More
recently Belaf (19216, 1923) has described in detail a similar process of
pedogamy for another Heliozoon, Actinophrys sol (Fig. 50). A single
uninucleate individual encysts and divides into two daughter forms, which
become gametes. The nuclei undergo two divisions, one of which is
a reducing division in that the number of chromosomes is halved. One
of the products of each nuclear division degenerates. The two gametes
within the cyst then unite. The development is comparable with that
which occurs in the secondary cysts of Actinosphcerium. In the case of
Actinophrys sol, Schaudinn (1896a) stated that two individuals entered
the cyst, but doubt was thrown upon this by Distaso (1908) and Prowazek
(19136), who stated that the two gametes were derived from one individual.
Belaf has finally confirmed the statements of the latter observers. He
has also noted that occasionally two individuals encyst together, and that
each divides to form two gametes, so that four gametes occur within the
cyst. After the maturation divisions have taken place, the gametes
unite in such a way that those formed from one individual unite with
those from the other. In some cases, of the two gametes formed from one
individual, one is motile and the other not, so that a distinction between
male and female gametes can be drawn (Fig 50, lo).
PARTHENOGENESIS. — Amongst higher animals it sometimes happens
that the ovum, which usually develops only after fertilization, does so
without this having taken place. It is evident from what has already been
explained that in such a case the nucleus will only possess half the number
of chromosomes that it would have had if fertilization had occurred. It
has been found that during the parthenogenetic development of the
ovum the nucleus behaves in a variety of ways, by which the double
number of chromosomes is regained. Another feature of parthenogenesis
is that, though the ovum which develops without fertilization may give
rise to the same type of individual as it does when fertilized, this is not
necessarily the case. Thus, the ova of the honey-bee if fertilized develop
into females, if unfertilized into males. Amongst the Protozoa, several
observers have attempted to establish the occurrence of parthenogenesis.
The most notable instance is that described by Schaudinn (1902tt) for
the malarial parasite, Plasmodium vivax of man. This observer supposed
that the female macrogamete, which usually develops only after fertiliza-
8. Two stages in second maturation division of the gamete nuclei.
9. Completion of second maturation division and formation of second i eduction body: the two
reduction bodies are still present in later gamete.
10. Mature gametes, showing sexual dimorphism: the male gamete has pseudopodia.
11. Union of two gametes and commencing fusion of their nuclei.
12. Zj'gotc within its cyst.
PARTHENOGENESIS 89
tion in the mosquito's stomach, is sometimes able to do so in the human
blood-stream without fertilization. The nucleus is described as dividing
into two parts, one of which is cast oli with a portion of cytoplasm and
degenerates. The remaining nucleus multiplies, and reproduction by
schizogony occurs. In this manner it is supposed that the asexual or
schizogony cycle is started again, and it was claimed that this afforded
an explanation of the occurrence of relapses in malaria. The writer has
long held and taught that the parthenogenetic forms depicted by
Schaudinn were instances of red blood-corpuscles doubly infected with
a gametocyte and a schizont (Plate XII, 19, p. 926). Thomson, J. D. (1917),
also came to this conclusion, and showed conclusively that Schaud inn's
figures purporting to represent a parthenogenetic process were really
instances of doubly infected cells.
The cases of parthenogenesis recorded by Prowazek (1904) for Herpe-
tomonas muscarum and by Gonder (1910a, 19116) for Theileria jmrva have
even less evidence to support them than the instance described above.
The various methods by which syngamy is accomplished amongst the
Protozoa may be grouped as follows:
1. Copulation. — Complete union of two individuals.
(1) Two individuals having the characters of the ordinary repro-
ducing forms unite.
((/) The uniting forms are equal in size (isogamy).
(h) The uniting forms are unequal in size (anisogamy).
(2) Two individuals (gametocytes) give rise to a number of smaller
forms (gametes) which unite in pairs,
(a) The gametes produced by the gametocytes are equal in
size and characters (isogamy).
(6) The gametes produced by one individual are unlike
those produced by the other (anisogamy).
(i.) The number of gametes produced by the gameto-
cytes are equal, or approximately equal, in
number.
(ii.) One gametocyte (macrogametocyte) gives rise to
one large gamete (macrogamete), while the other
(microgametocyte) gives rise to a variable
number of small motile gametes (microgametes).
2. Conjugation. — Two individuals (conjugants) associate, their nuclei
divide, and exchange of daughter nuclei takes jjlace, after which the
conjugants separate.
(1) The conjugants are equal in size.
(2) The conjugants are unequal in size, one, a small one (micro-
conjugant), associating with a large one (macroconjugant).
In some cases, after interchange of nuclei the microconjugant
degenerates.
90 NUCLEAR DIVISION IN PROTOZOA
3. Autogamy.- — The nucleus of a single individual divides into two.
Each of these daughter nuclei undergoes reduction divisions, after which
they unite. It is extremely doubtful if this process ever occurs.
4. Pedogamy. — A single individual divides into two. Reduction
divisions of the nuclei of these two daughter individuals which are gametes
take place, after which the gametes unite and their nuclei fuse.
5. Parthenogenesis. — Part of the nucleus of a gamete, which normally
develops only after union wdth another gamete, is extruded, after which
multiplication occurs. There appear to be no convincing records of such
a process amongst the Protozoa.
NUCLEAR DIVISION AMONGST THE PROTOZOA.
The division of a nucleus which takes place by simple constriction
into two parts without formation of chromosomes is known as amitotic
division, to distinguish it from mitotic division, in which definite chromo-
somes and a spindle, associated with the presence of centrosomes, occur
as described above for the nuclear divisions of the cells of higher animals.
Between what appears to be true amitosis and mitosis there occur many
gradations. In some cases the appearances are in every way comparable
with what has been described above as typical mitosis in the cells of
higher animals. In other instances the nuclear membrane persists, and
the whole process of mitosis occurs within the nuclear membrane. In
other cases, again, there appear to be no centrosomes associated with
mitosis within the nuclear membrane, though many observers describe an
intranuclear structure, called the centriole, which is supposed to function
as a centrosome. As regards the nature of this body and its actual
existence there is much diiierence of opinion. That the formation of
a spindle may occur without definite centrosomes being identifiable has
long been recognized in higher plants, so there is no reason to suppose
that this may not happen amongst the Protozoa. When mitosis occurs
within the nuclear membrane, definite chromosomes may be formed at
the equator of the spindle, and these divide into daughter chromosomes
in the usual manner. In other cases, though a spindle is formed, the
chromatin granules become arranged irregularly upon the spindle fibres
without uniting into definite chromosomes. No equatorial plate is formed,
and the nucleus merely constricts into two parts. It is possible that in
some of these instances of irregularly arranged chromatin granules there
are produced a very large number of minute chromosomes which actually
divide. In order to distinguish these intermediate types of mitosis from
typical mytosis, the term promitosis has been introduced by Nagler (1909).
A good illustration of complete mitosis is afforded by the nuclear
MITOSIS
91
%
.v:'^^v.
I ^
'^Vf^n^V^
r7-/-r
S>^J.
^yf0^
i'.
'^-.
^-^^f
Fig. 51. — Stages in the Nuclear Division of Acanthocijstis avideata in which
THE Central Granule Functions as a Centrosome ( x ca. 690). (After
SCHAUDINN, 1896.)
1 . Ordinary individual, showing nucleus and central granule, from which radiate the axial fibres
of the pseudopodia (axopodia).
2, 3. Changes in nucleus nt ci mi men cement of division.
4. Division of central L;iauiile ami iiu •leu- in s|i;reine stage.
5, 6. Formation <>i eludmosoines in nucleus, a- it takes up a central position on the spindle
which forms between the two granules.
7. Disapijearance of nuclear membrane : formation of equatorial plate.
8. Sejiaration of daughter plates of chromosomes.
9. Cytoplasm dividing and two nuclei and central granules returning to the condition of individual
at 1.
92 NUCLEAR DIVISION IN PROTOZOA
division of Acanthocyslis aculeata, one of the Heliozoa, as described by
Scliaudinn (189G6), (Fig. 51). In the ordinary individual the centre of
the body is occupied by a granule, from which radiate the axial fibres
supporting the fine pseudopodia. The nucleus, which has a membrane
and large central karyosome, lies at one side of the central granule. When
division is to take place, the nucleus increases in size and the karyosome
becomes loculated, broken into a number of separate parts, and finally
disintegrated as minute granules which arrange themselves in the form
of a spireme or coiled thread. Meanwhile the supporting fibres of the
pseudopodia have disappeared, while radiating fibres develop in the
cytoplasm in connection w^ith the central granule, which, on account of
the part it plays in nuclear division, must be regarded as the centrosome.
The latter structure divides, and as the two daughter centrosomes separate
a spindle is formed between them, while radiating fibres form two asters.
The nucleus, within which the spireme has segmented into a number of
separate parts, now moves to the equator of the spindle. The nuclear
membrane disappears, and a number of small chromosomes take up a posi-
tion on the spindle as an equatorial plate. The individual chromosomes
divide, and there are formed two daughter plates which move towards
opposite poles of the spindle. At this stage the body of the Heliozoon,
which has become elongated, begins to show a constriction around its
centre. The spindle is finally divided at its centre, and the daughter
chromosomes of each plate become transformed into a karyosome, while
a new nuclear membrane is developed. The centrosome remains as the
central granule of the daughter individual which has been formed, and
new axial fibres are developed. In this division, practically all the stages
of mitosis as seen in the Metazoan cell occur.
Typical examples of mitosis occur also in the case of gregarines, the
nuclei of which divide repeatedly to form the gamete nuclei. Muslow
(1911) has described the process as it occurs in Monocystis rostrata, one
of the species of Monocystis which inhabit the vesicula seminalis of the
earth-worm. The resting nucleus consists of a nuclear membrane and
large central karyosome. When the first nuclear division is to take place
after two gregarines have become encysted together in the gametocyst,
the large karyosome breaks up, while a long twisted thread of chromatin
granules appears at one side of the nucleus (spireme stage). Meanwhile,
from two small areas on the surface of the nuclear membrane, radiations
appear in the cytoplasm to form the two asters. Between these, spindle
fibres develop, and with the disappearance of the nuclear membrane the
chromatin thread becomes segmented into eight looped chromosomes,
which arrange themselves at the equator of the spindle. Each chromo-
some becomes divided longitudinally, and the two groups of eight daughter
MITOSIS
93
4
*i?.^l^
.^1
Fig. 52. — Various Stages in the Late Mitotic Division of the Nucleus of
Monocystis rostrata (1-3x2,000; 4-8x1,700). (After Muslow, 1911.)
1. Resting nucleus. 2, 3. Formation of eight chromosomes.
4. Commencing splitting of the chromosomes.
5. Daughter chromosomes separating at equator of spindle, which is devoid of eentrosomes and
asters.
6. Daughter chromosomes moving towards the poles of the spindle.
7. Chromosomes breaking up into gametes.
8. Reconstitution of the nuclei
94 NUCLEAR DIVISION IN PROTOZOA
chromosomes move to opposite poles of the spindle. The central part
of the spindle disappears, the chromosomes break up into granules, and
with the formation of a nuclear membrane the nucleus is reconstructed.
In subsequent divisions the process is very similar, except that a spindle
is formed without definite centrosomes or asters (Fig. 52).
Very similar mitotic divisions of the nucleus were described by Brasil
(1905) also in the case of a species of Monocystis of the earth-worm (Fig. 53).
In both these instances the nuclear membrane disappears during division,
but in other cases the nuclear membrane persists during the whole mitotic
division of the nucleus.
In the case of Actinosjjhcprium eichhonii, the life-history of which has
been described in detail by Richard Hertwig (1898) in a classic memoir,
• -%
^/
\v
1 2
Fig. 53. — Nuclear Divisions in Associated Monocystid Gregaeines
(Monocystis sp.) of the Earth-Worm (x 900). (After Brasil. 1905.)
1. First nuclear division, showing centrosomes, spindles, and elongate daughter chromosomes.
2. Later nuclear divisions in various stages of mitosis.
very clear examples of mitosis occur. The multinucleate organism, as
mentioned above, becomes encysted in a large primary cyst, within which
it divides into a number of daughter individuals round which secondary
cysts are formed (Fig. 49). Within the secondary cyst a further division
into two individuals takes place. The nucleus of each of these divides
by mitosis to form two nuclei, one of which degenerates. A second
division of the surviving nucleus takes place, and again one of the
resulting nuclei degenerates. After this, the two individuals or gametes
in the secondary cyst unite and their nuclei fuse. The various nuclear
MITOSIS 95
divisions take place by mitosis. When the nucleus of a gamete in the
secondary cyst is about to divide for the first reduction division, there
appears at one side of the nucleus an area of clear cytoplasm towards
which the linin network of the nucleus with its chromatin granules is
drawn (Fig. 54). Into this clear cytoplasm some of the chromatin granules
of the nucleus are attracted, and by their aggregation give rise to the
centrosome. It is possible the centrosome was already present, either
in the nucleus or outside it, and that the commencement of its activities
results in the concentration of the nuclear elements at this pole of the
nucleus, and even the escape of some of the chromatin into the cytoplasm.
Whether Hert wig's account of the origin of the centrosome is correct or
not, when it becomes apparent it is situated at some distance from the
nuclear membrane, and is surrounded by radiations, the bulk of which
are directed towards the nuclear membrane. Division of the centrosome
takes place, and one of the resulting pair takes up a position at the oppo-
site pole of the nucleus. There are now two asters between which spindle
fibres appear. The nucleus occupies a position between the two centro-
somes, and the spindle fibres extend through the nuclear membrane and
the substance of the nucleus, so that there is both an extranuclear and
an intranuclear portion of the spindle. The chromatin granules of the
nucleus now form a series of chromosomes which become arranged in the
form of a plate across the equator of the spindle within the nuclear
membrane. Each chromosome divides, and there result two daughter
plates which, just behind the ends of the elongating nuclear membrane,
move towards the centrosomes. The nuclear membrane, which has
divided, now closes round the chromosomes, which gradually disintegrate,
so that daughter nuclei are formed. One of the nuclei now degenerates.
As already remarked, the nuclei of the two individuals in the secondary
cysts undergo two such divisions, the description just given applying to
the first of these. The second division is of a similar type, and again one
of the daughter nuclei degenerates. Richard Hertwig regards both these
divisions as reduction divisions, though he believed that the chromosomes
actually divide in each instance. It seems reasonable to suppose, from
wdiat is now known to occur in other Protozoa, that in one of the two
divisions splitting of the chromosomes does not take place, but that they
separate into two groups, so that the number of chromosomes in the
daughter or final gamete nucleus is halved. This is all the more probable
since, in Actinophnjs sol, an allied form which has a similar syngamic
process, Bela' (19216, 1923) has noted that in the first of the divisions
the chromosome number of forty-four is reduced to twenty-two (Fig. 50).
To return to AcfinnspluBnuni eichhorni, the many nuclei which an adult
contains become the nuclei of the daughter individuals which form the
96
NUCLEAR DIVISION IN PROTOZOA
■.-tj'^,1 .
^■7^
^Ov
^ J
^.^0'
c„
t->
0>*
,3u-^"*"7~7"1'i
;u>^
^OV'',!M.I J lil ||
S'»jr,i??i?'ri
^^HJ^infiV;
•^^o:,:;^^ '^
'^05^^" 6
v-'V^J'';:./- '//"^!-.>
■^ .it* iis!^^ii'l/4'
%vV/^ 7
••'/'/,
Fig. 54. — First Eeduction Division of the Nucleus of One of the Two
Oametes in the Secondary Cyst of Actinosjyhcerium eichhorni ( x ca. 1,200).
(After E. Hertwig, 1898.)
[For descriplion see opposite page.
MITOSIS 97
secondary cysts. These nuclei are the result of repeated mitotic divisions
of the nucleus of the parent. These divisions differ from those described
as taking place in the gametes in the secondary cysts, in that definite
centrosomes do not occur. Similarly, when the daughter individual in
the secondary cyst divides to form the two gametes, its nucleus divides
without the formation of centrosomes (Fig. 55). Indications of longi-
tudinally arranged fibres can, however, be detected within the nuclear
membrane, and also in a cone-shaped portion of cytoplasm which occupies
huh
^
Fig. 55. — First Nuclear Division in the Secondary Cyst of Aciinospluerium
ciclihorni ( x ca. 1,200). (After E. Hertwig, 1898.)
1. Chromosomes forming in the nucleus. 2. Chromosomes arrtanged as an equatorial plate.
.3. Daughter chromosomes separating as two j^lates.
No definite centrosomes appear at any stage.
the poles of the elongating nucleus. Chromosomes are formed, become
arranged as an equatorial plate, and divide into daughter chromosomes
in the usual manner.
As an illustration of another type of mitosis in which a definite spindle
and chromosomes are formed associated with disappearance of the nuclear
membrane and complete absence of centrosomes, Amoeba glebcB {Hart-
mannella glebce), a soil amoeba described by Dobell (1914c/), may be con-
1. Centrosome with radiations.
2. Two centrosomes at opposite ]iolcs of nucleus in which chromosomes are commencing to form.
3. The spindle has formed between the centrosomes. and chi'omosomes have taken u]) a position
as an equatorial plate. 4. Commencing division of the chromosomes.
5. The chromosomes have divided and two equatorial ]i!ates are foimcd.
6. Passage of the daughter chromosomes towards the ctntrdsomes.
7. Later stage, in whiiji the nuclear membrane is closinu round the chromatin granules.
8. Two daughter uiulci have formed, though the n'liiiiiiis of the spindle and the radiations from
the centrosome, which has itself disappeared, are still to be distinguished.
98
NUCLEAR DIVISION IN PROTOZOA
/5«v^"'
M
i'm)
;\v
#
•»,.—• ^.
/
'•Jt-..»..-,
,„:y
^:
/<9
^:;^
'#>'i
^^J
X'
//
■'' 9
,\
12
Fig. 5G. — Ilartmannella glebce : Binary Fission to show Various Phases of
Nuclear Division (x 2,000). (After Dobell, 1914.)
[ For dcscriplion see opposite pfuje]
MITOSIS 99
sidered (Fig. 56). The resting nucleus consists of a fairly thick membrane
and a large central karyosome round which are arranged a series of
granules. When nuclear division, preparatory to division of the amoeba,
commences, the nuclear membrane becomes thin and the karyosome
fragments into a number of fine granules, while those which surround
the karyosome disappear. Those originating from the karyosome run
together to form larger granules, which become arranged as a long-coiled
chain of beads which, decreasing in length, finally occupies the equator
of the nucleus as a ring. The linin network of the nucleus now shows
indications of spindle-fibre formation and the nuclear membrane dis-
appears. The spindle, which has rounded ends and no centrosomes or
asters, becomes slightly elongated, while the chromosomes, sixteen in
number, which are arranged as a ring round the equator of the spindle,
divide so that two rings of daughter chromosomes are formed. These
separate from one another as the spindle itself becomes greatly drawn
out. Finally, each ring of daughter chromosomes which has moved to
the end of the spindle is broken up and a nuclear membrane is formed.
The daughter nucleus is at first flattened, but gradually increases in size,
and, with reconstruction of the karyosome, assumes the characters of
the original parent nucleus. Before this stage is reached the amoeba,
which has become elongated, is divided by constriction into two parts.
In this division there are no granules which could be interpreted as
centrioles at the apices of the spindle, nor was it possible to discover any
indications of a centrodesmose, so that it would appear that centrosomes
and centrioles are completely absent.
The division of the nucleus of Entmnoeha histolytica, as seen in the
encysted forms, is of a similar type, but the nuclear membrane remains
throughout the process (Fig. 57). The earliest stage appears to be the
division of the minute central karyosome. The two daughter karyosomes
separate, while a spindle forms between them. On the equator of the
spindle, which is surrounded by the elongating nuclear membrane, appear
a ring of chromosomes in an equatorial plate. These divide to form
daughter chromosomes, which pass towards the poles of the elongating
spindle in an irregular manner. According to Kofoid and Swezy (1924o,
1 925) the chromosome number is six. As the spindle elongates the daughter
1. Usual type of amoeba : nucleus with large central karj'osome surrounded by granules.
2, 3. Karyosome breaking up into granules.
4. Chromatin arranged as irregular loop.
."). iJi.sappearance of nuclear membrane : spindle with equatorial plate of chromosomes.
()-9. Division of chromosomes to form daughter plates, which pa.ss to the poles of the elongating
spindle.
1(1. Ciiinmencing division of amoeba.
II. Disappearance of spindle, reconstruction of nuclear membrane, and commencing reconstrue-
tion of karyosome. 12. Encystr d amoeba.
100
NUCLEAR DIVISION IN PROTOZOA
/:
1
<y
§:
%
^^<^^
<'~\
^(y
Fig. 57. — Divisions of the Nuclei in the Cysts of Entamfeha histoli/tim ( x 2,000).
(Original.)
The deeply stained chromatoid bodies are shaded, while the limits of the cytoplasm and vacuoles
are shown in outline.
MITOSIS 101
karyosomes disappear, and there cannot be detected any structures like
centrosomes at the apices of the spindle. When the nuclear membrane
commences to divide, karyosomes of the daughter nuclei reappear.
A modification of the preceding type of division is seen in an amoeba
described by Dobell (1914a) under the name Amoeba lacertce. The amceba
is a common parasite of the intestine of Lacerta muralis and other lizards.
In the resting condition the nucleus consists of a nuclear membrane and
large central karyosome, in which all the chromatin of the nucleus is said
to be aggregated (Fig. 58). When nuclear division commences, coarse
granules of chromatin can be distinguished in the karyosome. These
•^- *:
.^
■•^^
Fig. 58. — Nuclear Division in Valilkampfia dobelli {Amceba lacertm, Dobell, 1914)
(x 2,000). (After Dobell, 1914.)
1. Ordinary form with nucleus containing large karyosome.
2. Karyosome breaking into granules.
3. Elongation of karyosome and arrangement of chromatin granules in meridional lines.
4 7. Elongation and constriction of karyosome.
8-9. Comijletion of nuclear division and commencing division of cytoplasm.
10. Encysted form.
become finer and arranged in meridional lines on the surface of the karyo-
some, which now becomes elongated, as does also the nuclear membrane.
On the surface of the elongated karyosome granules of chromatin are
arranged in longitudinal rows, and some indication of fibres can be de-
tected. The granules gradually collect at the two poles of the karyosome,
which itself becomes constricted at its centre and finally divided into
two parts. This is followed by constriction and division of the nuclear
membrane, which has persisted throughout the division process. The
daughter karyosomes contract to the spherical form, while the granules
of chromatin unite to form larger granules. During this division the
102 NUCLEAR DIVISION IN PROTOZOA
essential features are the appearance in the karyosome of granules which
become irregularly arranged in longitudinal rows on the fibres which
appear in the elongating karyosome. Spindle fibres are thus produced,
but the chromatin granules do not unite to form chromosomes, nor is
an equatorial plate developed. Nothing in the nature of a centrosome
is present.
The stages in the division of Amoeba hyalina (Hartmannella hyalina),
described by Hartmann and Chagas (19106), illustrate the type of division
in which a centriole is supposed to be present (Fig. 59). The resting
nucleus is described as having a centriole within the karyosome. When
0)- ■^- -# ■"^•
'^^M^
5
r*vsi.
)' iw^.
)V
Fig. 59. — Stages in Mitotic Division of Nucleus of Hartmannella hyalina, in
WHICH it is supposed THAT A CeNTRIOLE FUNCTIONING AS A CeNTROSOME IS
PRESENT (x 3,700). (After Hartmann and Chagas, 1910.)
division commences the centriole divides, and as the two halves separate
they are connected by a centrodesmose. The chromatin of the karyosome
breaks up into granules, which become arranged as chromosomes in an
equatorial plate. Each daughter centriole has now taken up a position
at the apex of the spindle-shaped nuclear membrane, within w^hich is
a system of exceedingly fine spindle fibres. The daughter plates of
chromosomes are formed, and these move towards the poles of the spindle.
Finally, the chromosomes at each end run together to form the karyosome,
in which the centriole is included, while the intermediate part of the
spindle disappears. It is by no means clear that the above is an accurate
description. Other observers who have investigated the nuclear division
of this or similar amoebse have failed to detect the centrioles (Fig. 89).
Arndt (1924), in describing the nuclear division of HartmanneUa
MITOSIS
103
klitzkei, which, according to him, takes place by typical mitosis with
extranuclear centrosomes, states that he has been able to demonstrate
simihxr centrosomes in four species of Hartmannella. The centrosome,
which does not originate from an intranuclear centriole, is easily overlooked,
and requires very special technique for its demonstration (Fig. 60). In
the resting nucleus it lies against the outer surface of the nuclear mem-
brane, and when division commences it divides into two daughter centro-
somes, which take up positions at the poles of the spindle. It is evident,
in the light of these observations, that the granule described as a centriole
a ^.
J I
I !
Fig. 60. — Stages in the Nuclear Division of Hartmannella Iditzlcei to snow
THE Presence of the Extranuclear Centrosome as revealed by Mann's
Stain ( x 2,500). (After Arndt, 1924.)
by Hartmann and Chagas cannot be a centrosome, and that the cases of
mitosis which have been recorded as taking place without centrosomes
require reinvestigation.
Another type of nuclear division which is distinct from those described
above occurs in amoebae belonging to the genera Vahlkampfia and Dima-
stigamoeha (Fig. 61). The resting nucleus has a large central karyosome
and peripheral chromatin in the form of fine granules within the nuclear
membrane. The nuclear membrane persists throughout nuclear division,
during which the karyosome becomes elongate and then dumb-bell-shaped,
and finally constricted into two daughter karyosomes. These may
remain connected by a fibre or centrodesmose, which in some cases can
be seen to unite two granules which are embedded in the dense daughter
karyosomes. Between the two karyosomes and surrounding the centro-
104 NUCLEAR DIVISION IN PROTOZOA
desmose are spindle fibres, at the equator of which chromosomes become
arranged. These are formed from the peripheral chromatin granules
of the nucleus, and possibly some which have separated from the karyo-
some. The chromosomes split to form two plates, which move towards
the daughter karyosomes. The centrodesmose and the spindle fibres
disappear, while the nuclear membrane is divided by constriction. Two
daughter nuclei, each with a larger central karyosome and peripheral
chromatin granules, are reconstructed.
In many cases, as, for instance, in trypanosomes and allied flagellates,
in which the nucleus consists of a nuclear membrane containing a large
central karyosome, all that can be detected in nuclear division is the
elongation of the nuclear membrane, within which the karyosome becomes
drawn out and finally dumb-bell-shaped. The narrow intermediate
portion may be quite short, or it may be very much drawn out. In
either case it finally disappears, leaving two daughter karyosomes. By
constriction and division of the nuclear membrane two daughter nuclei
are formed (Fig. 156). It is maintained by the advocates of the centriole
theory that the narrow intermediate portion in the dumb-bell stage
represents a centrodesmose connecting two daughter centrioles which are
lodged in the daughter karyosomes. Certain appearances which are some-
times seen might lend support to this view. Occasionally, a dividing
nucleus may be seen, in which a small granule is situated at each end of
the elongated nuclear membrane. These are connected by a fine fibre,
at the centre of which the still intact karyosome lies. Such an arrange-
ment might be interpreted on the supposition that the centriole within
the karyosome has divided prematurely, and that the two daughter
centrioles have passed out of the karyosome, which has not yet shown
any sign of division. At a later stage the karyosome divides, and the
two daughter karyosomes pass to the ends of the nucleus and again enclose
the centrioles. Such appearances, however, are unusual, and may be
merely accidental arrangements of chromatin granules. Hartmann and
Noller (1918) have given another account of the nuclear division in
Trypanosoma theileri (Fig. 156). They maintain that the apparently
elongated karyosome is really a spindle, at each apex of which is a centriole,
and that fine granules of peripheral chromatin form chromosomes which
become arranged as an equatorial plate. Mitotic divisions of trypanosome
nuclei have been described also by Chagas (1909) and Nieschulz (19226).
As already intimated, the Protozoan nucleus sometimes contains a body
which is entirely devoid of chromatin, and appears to consist of plastin
material alone. In nuclear division it may disintegrate and disappear,
to be re-formed again in the daughter nuclei. In some cases, however, it
divides into two parts, which pass to the poles of the spindle with the
MITOSIS 105
daughter chromosomes, and finally enter the daughter nuclei. Bodies
of this kind have been described by Reichenow (1921) in Karyolysus, and
the writer has seen them in Hepatozoovi baJfouri (Fig. 35). These plastin
bodies are not essentially different from karyosomes, which consist mainly
%
•!»
<^*.
'9
Fig. 61. — Amgeboid Phase of Dimastigamceha gruberi from Culture on Agar
Plate showing Method of Nuclear Division ( x ca. 1,350). (Original.)
1. Usual type of amoeba.
2. Commencing nuclear division. The karyosome has become elongate and granular.
3. The karyosome has become dumb-bell-shaped and the nucleus is filled with granules.
•4. There is an equatorial plate of dividing chromosomes, and the dividing karyosome has formed
the pole caps, which are still united by a fibre (centrodesmose).
5. The daughter chromosomes have become aggregated, and are passing towards the pole caps,
which have lost the connecting fibre. Each pole cajj has a central granule.
6. The nucleus has divided and each half is retracting.
7. Slightly later stage witli tlaughtcr nuclei still further retracted.
8. Form with two reconstituted nuclei.
9. Form with two nuclei in division: equatorial plate stage.
10. Form with two nuclei in different stages of division.
of achromatic material. In nuclear division the plastin substance,
whether it be regarded as a karyosome or not, may divide into two parts,
one of which goes to each daughter nucleus, as in Dimastigamoeba (Fig. 61),
or it may break up and disappear as a single body, to re-form in the
daughter nuclei, as in HartmanneUa (Fig. 56).
106 NUCLEAR DIVISION IN PROTOZOA
When reproduction by binary fission occurs, division of the nucleus
is followed by division of the body of the organism into two parts. When
multiplication by schizogony takes place, or when a number of gametes
are produced, the nucleus divides into two, these into four, and so on,
till the requisite number is reached. The multinucleated organism then
buds from its surface a number of daughter individuals. The repeated
divisions of the nuclei frequently take place by mitosis, especially when
they are multiplying to form gamete nuclei, as in the case of gregarines
or coccidia. It sometimes happens that before the spindle of one division
f/
^
if..
■A
Fig. 62. — First Nuclear Division in One of a Pair of Associated
MONOCTST'ID G-REGARINES {MonocysUs SP.) OF THE EaRTH-WORM. (AfTER
Brasil, 1905.)
1, 2. Two centrosomes are present, the nucleolus is breaking up, while the chromatin has collected
at the centre of the nucleus ( X 900).
3. The spindle has formed, the nucleus has been extruded, and chromosomes are found at the
equator of the spindle { X 9C0) .
•1. The chromosomes have divided and are passing to the poles of the spindle, where the centro-
somes have already divided for the succeeding division ( X 900).
5. Though the nuclei have not been definitely reconstituted, the spindles for the next division
have formed ( X 800).
has disappeared the two asters and the centrosomes, if these be present,
divide again, so that two asters are formed at each end of the spindle.
These may separate and form a new spindle between them, so that when
the daughter chromosomes reach the pole of the original spindle they are
already at the equator of a new one. In this manner very complicated
poly-aster figures may arise. Precocious formation of daughter asters
while the original spindle is still present has been shown to occur in a
gregarine (Monocystis) of the earth-worm by Brasil (1905) (Fig. 62).
Very complicated poly-aster figures similarly occur during nuclear
MITOSIS
107
multiplication in species of Aggregata, as described by Moroff (1908)
and other observers (Fig. 63).
The main types of nuclear divisions of Protozoa may be thus
classified :
1. Mitotic division with centrosomes, asters, achromatic spindle,
chromosomes, equatorial plates, and all the stages seen in the typical
nuclear division of higher animals. The nuclear membrane may or may
not persist during division. A nucleolus or plastin body, if present, may
be divided into two parts, one of which goes to each daughter nucleus,
or it may break up and disappear, the daughter nuclei re-forming their
nucleoli when division is approaching completion.
Fig. 63. — Poly-aster Figure resulting from Successive Nuclear Divisions
IN Male Gametocyte of Aggregata jacquemeti ( x 750). (From Minchin, 1912,
AFTER MOROFF.)
2. Mitotic division of the above type, except that centrosomes and
asters have not been detected.
3. Division in which there is formed within the nuclear membrane
a spindle upon which chromatin granules are irregularly arranged. There
are no asters or centrosomes. It is possible that the granules of chromatin,
though not arranged as an equatorial plate, are actually chromosomes,
which divide into daughter chromosomes as they do in the preceding
types of division. The karyosome may divide into daughter karyosomes,
or break up to be re-formed in the daughter nuclei,
4. Division in which the large central karyosome elongates and becomes
constricted. The two halves move to the ends of the elongating nuclear
membrane to form the pole caps between which a spindle is formed.
The peripheral chromatin becomes arranged as chromosomes in an
108 CHROMOSOMES DURING SYNGAMY IN PROTOZOA
equatorial plate. The pole caps become the karyosomes of the daughter
nuclei.
5. Division in which the karyosome becomes elongated and divided
within the nuclear membrane without development of spindle fibres
or chromosomes. This type of division is seen in the nuclei of small
organisms, and it is probable that it is actually similar to type 4, the
spindle fibres and chromosomes escaping detection owing to their
minuteness.
In those cases in which a centrosome is not present, some observers
claim that its place is taken by an intranuclear centriole.
BEHAVIOUR OF CHROMOSOMES DURING SYNGAMY.
Reference has already been made to the nuclear changes which occur
during the development of the ovum and the spermatozoon, and it has
been pointed out that the chromosome number of the zygote nucleus is
not doubled as a result of syngamy owing to the fact that after meiosis
the nuclei of the uniting gametes contain half the normal number of
chromosomes. Several instances of similar reduction divisions of Pro-
tozoan nuclei, whereby the number of chromosomes is halved, have been
recorded.
Muslow (1911) gives a clear account of a supposed reduction division
in Monocystis rostrata (Fig, 64). The nuclei of the two gregarines which
enter the gametocyst multiply by repeated mitotic divisions in which
eight chromosomes are present, as noted on p. 92. Eventually, after
nuclear division has ceased, a number of gametes are budded off from
each gregarine, and these unite in pairs and their nuclei fuse. During
the last nuclear division, whereby the gamete nuclei are formed, though
eight chromosomes appear on the equatorial plate, when the daughter
plates are formed, there is no splitting of the chromosomes, as has occurred
in previous divisions, but the eight chromosomes are separated into two
groups of four, which move towards the poles of the spindle. It was noted
also that the eight chromosomes composing the equatorial plate consisted
of four pairs of homologous chromosomes, the members of each pair
differing from those of other pairs, and that one of each pair entered each
daughter plate of chromosomes. This last division, which gives rise to
the nuclei of the gametes, is thus a true reduction division or meiosis, like
that which occurs in the production of gametes in higher animals. When
the gametes unite, the nucleus of the zygote, receiving four chromosomes
from each gamete nucleus, again has eight chromosomes or four pairs of
homologous chromosomes.
In the case of Diplocystis schneideri, a gregarine of the cockroach,
MEIOSIS 109
Dobell and Jameson (1915) have given a description of a reduction division
which differs from that of Muslow. According to these observers, during
all the division stages of the nuclei, including the last division which gives
rise to the gamete nuclei, there are three chromosomes which divide to
form the chromosomes of the daughter nuclei (Fig. 65). The nuclei of
the gametes thus have three chromosomes, as do the nuclei of the preceding
stages. When the gametes unite and their nuclei fuse, the zygote nucleus
has six, or double the number of chromosomes found at other stages.
The zygote nucleus now proceeds to division, and it is in this division that
the reduction occurs, three of the six chromosomes passing to each daughter
nucleus. At all subsequent division stages of the nuclei the three chromo-
somes are divided. In Muslow's account of Monocystis rostrata it was
? ^
1 2
Fig. 64. — Last Nuclear Division in One of Two Associated Gregarines,
Monocystis rostrata, to show the Reduction of the Chromosome Number
FROM Eight to Four in the Gamete Nuclei ( x 5,000). (After Muslow, 1911.)
1. Eight chromosomes in nucleus. 2. Eight chromosomes arranging themselves in pairs.
.•{. Separation of the individual chromosomes of each pair.
4. Four chromosomes moving to each pole of the spindle to form the gamete nuclei.
during the last nuclear division in the production of gamete nuclei that
the number of chromosomes was halved, whereas in Dobell and Jameson's
account of Diplocystis schneideri the reduction does not occur at this
stage, but at the first division after the zygote nucleus has been formed.
According to Muslow, the haploid number of chromosomes of Monocystis
rostrata is four, and occurs in the gametes, while all other nuclei have the
diploid number of eight chromosomes; on the other hand Dobell and
Jameson in Diplocystis schneideri find that the diploid number six
occurs only in the zygote, all other stages showing the haploid number
three. The latter observers have noted the same condition in the case
of the coccidium Aggregata eberthi (Fig. 66). In this parasite, during
schizogony nuclear divisions occur in which six chromosomes appear in
the equatorial plate, and they all divide so that the daughter nuclei have
no CHROMOSOMES DURING SYNGAMY IN PROTOZOA
1
iX
m^
r>
/2
'^
r of
/J
Fig. 65. — Nuclear Division in the Gregarine, Di])locysUs sclmeideri, to illus-
trate THE EeDUCTION IN THE CHROMOSOMES IN THE FiRST NuCLEAR DIVISION
IN THE Zygote ( x 2,500). (1-3 after Dobell and Jameson, 1916; 4-14 after
Jameson, 1920.)
1-3. First division in the cregarine, showinfr division and sojjaiation of the three chromosomes.
4-0. Third division in the gregarine, in a\ hich tlucc cliniiiKiscjnics auain divide.
7. Last nuclear division to form the gamete niu^lei: three chroniosumes again divide; there is
no reduction. 8-9. Zygote nucleus with six chromosomes.
10. The six chromosomes arranged in three pairs.
11, 12. Separation of the chromosomes in two groups of three (reduction).
13, 14. Rcconstitution of two nuclei, each with three chromosomes.
MEIOSIS 111
each six chromosomes. Finally, male and female gametocytes which give
rise to male and female gametes are formed. The nucleus of the male
or microgametocyte multiplies by repeated divisions in which the series
of six chromosomes are present (Fig. 66, A to D). They are filamentous
except when arranged as the equatorial plate, when they are contracted
and more or less spherical, though maintaining the same relations as
regards size. At the equator of the spindle the chromosomes divide by
constriction, and the two groups of six daughter chromosomes separate
and. become filamentous again. By repeated divisions of this kind, in
which the daughter asters divide before actual nuclei are formed, very
complicated poly-aster figures are produced. Eventually, as in the schizont,
nuclei which lie on the surface are constituted, and from them the micro-
gametes are formed. The latter are elongate bodies provided with two
flagella at the anterior end (Fig. 376). Meanwhile, certain merozoites of the
female line have become female- or macro-gametocytes. A complicated
series of changes takes place in the nucleus. The nucleolus or karyosome
is thrown out, the nuclear membrane disappears, and a series of six long
chromosomes appears (Fig. 66, E). Finally, a fertilization spindle is formed,
on w^hich the chromatin of the female nucleus is arranged in the form of
granules (see p. 873). The chromatin of the male nucleus, derived from
the microgamete, now enters the spindle, which retracts to form the zygote
nucleus (synkarion). This nucleus now proceeds to division by mitosis,
and the chromosomes are reconstituted (Fig. 66, F to K). It is found
that there are twelve of these — a series of six pairs, the two constituting
each pair being equal in size. Undoubtedly one chromosome of each
pair is derived from the microgamete nucleus and one from the macro-
gamete nucleus. The twelve chromosomes now pass to the equator of
the spindle and become globular in form, and the two constituents of
each pair now unite, giving a stage in which there are only six double
chromosomes (Fig. 66, G). The union, however, is not permanent, for
separation takes place, and one chromosome of each pair passes to one
pole of the spindle, while the other goes to the opposite pole (Fig. 66, H).
In this process there has been no division of the chromosomes, so that
in each daughter group there are only six chromosomes, whereas in the
zygote nucleus (synkarion) there were twelve. The first division of the
synkarion is thus a true reduction division, whereby the original number
of six is regained. It will thus be seen that in every stage of development
of this parasite the nuclei have six chromosomes, except in the synkarion
formed by union of the male and female nuclei, in which there are twelve.
The daughter groups of six chromosomes resulting from the division of
the synkarion now proceed to division again, but, as in the case of the
nuclear multiplication in the schizont and microgametocyte, at each
112 CHROMOSOMES DURING SYNGAMY IN PROTOZOA
M
(3
:<
bb'
cc G
Fig. 66. — Chromosomes of Aggregate eberthi ( x 2,000). (After Dobell
AND Jameson, 1915.)
A . Nucleus of male, showing six long chromosomes at prophase stage of first division .
B. Later stage of first division of nucleus of male: the chromosomes have become compact and
are arranged as an equatorial plate.
C. Later stage: each chromosome has divided to give rise to two groups of six daughter chromo-
somes.
D. One of the groups of six dumlitcr cliroinnsonics arisiim from first division of male nucleus
elongating to form the chroinnsdiiics of one of tlio dauuhtcr nuc-k'i.
E. Nucleus of female before fertilization, sliow ini: six limu chromosomes.
F. Chromosomes in zygote nucleus: early sta^c of first division, showing twelve chromosomes,
six (a-/) derived from the male, and six (■'-/') fnun the female.
G. Chromosomes in zygote nuolens: equatorial ])late stage of first division: the twelve chromo-
somes have contracted and Ixcome associated as six double chromosomes.
H. Chromosomes in dividinu zyt:ote nucleus: the individual chromo,somes of each pair have
separated, giving rise to two groups of six.
K. End of first division of the zygote nucleus : one of the groups of .six chromosomes, which have
elongated, entering the daughter nucleus.
L. Group of six daughter chromosomes on sjiindJe of a later nuclear division of the zygote.
M. Group of six chromosomes forming equatorial plate at second division of the spore
nucleus.
MEIOSIS 113
division the six chromosomes divide, so that each daughter nucleus has
six chromosomes (Fig. 66, L and M). Eventually, a large number of
nuclei are formed. These arrange themselves on the surface of the cyto-
plasm, which segments into a number of sporoblasts.
An exactly comparable process has been described by Reichenow
(192 1) in the case of haemogregarines of the genus Karyolysus (Fig. 457).
Here the haploid number of chromosomes is four, and these occur in nuclei
of all stages except those of the zygotes, which have the diploid number
of eight. When the zygote nucleus divides, four closely united pairs of
chromosomes occur at the equator of the spindle. One chromosome of
each pair then passes towards the pole of the spindle, so that the resulting
daughter nuclei have again only four (see p. 1098). These accounts agree
in that the reduction division occurs at the division of the zygote nucleus,
and not, as Muslow maintains, in the last division which gives rise to
the gamete nuclei. It seems highly improbable that Monocystis rostrata
would differ from other gregarines or coccidia in this respect, and Dobell
and Jameson have suggested that possibly Muslow was dealing with a
mixed infection of two gregarines, one of which has a chromosome number
of four and the other of eight, and that what he considered to be the
reduction division of the form with eight chromosomes was in reality the
ordinary division of the form with four chromosomes.
The nuclear division during the vegetative reproduction by binary
fission, the formation of gametes, and their maturation in the Heliozoon
Actinophrys sol has been the subject of detailed study by Belaf (1923),
as mentioned above. The organism reproduces by simple division.
Finally, encystment occurs and the uninucleated individual within the
cyst divides to form two gametes (Fig. 50). The nucleus of each gamete
divides and one of these degenerates. The remaining nucleus then
divides, and one of the resulting nuclei degenerates. There have thus
been two maturation divisions of the gamete nuclei. Conjugation of
gametes then occurs.. During vegetative reproduction the nucleus
divides without centrosomes by mitosis, while retaining its nuclear mem-
brane. When the chromosomes, which number forty-four, first appear
during nuclear division they are thread-like, but as the equatorial plate
stage is reached they become much shortened, and finally roughly spheri-
cal, in which condition they divide to form daughter chromosomes.
When the encysted individual divides to form the two gametes, the nuclear
division is of the same type as that occurring during the ordinary vegeta-
tive reproduction. The forty-four long chromosomes become arranged
in twenty-two pairs, the members of each pair being closely applied to
one another. Finally, when the equatorial plate stage is reached, there
are present at the equator of the spindle twenty-two pairs of more or
114 CHROMOSOMES DURING SYNGAMY IN PROTOZOA
less rounded cliromosomes. Each chromosome splits into two, so that
the daughter plates and finally the daughter nuclei also contain twenty-
two pairs of chromosomes. Each resulting nucleus then undergoes two
maturation divisions. In the first of these at the equatorial plate stage
there are twenty-two pairs of rounded chromosomes, but when the
daughter plates form the chromosomes do not split, as in the preceding
nuclear division. One chromosome of each pair passes to each daughter
plate, which thus contains only twenty-two chromosomes instead of
twenty-two pairs. The process is similar to that shown at Fig. 4, except
that in the place of the four chromosomes there are forty-four. Of the
resulting nuclei, one degenerates and the survivor divides by mitosis as
before. During this division twenty-two chromosomes appear at the
equator of the spindle, and each divides, so that each resulting nucleus
has twenty-two chromosomes. After union of the gametes, the zygote
nucleus has forty-four chromosomes. During all these divisions the
chromosomes are long filaments at the commencement of nuclear division,
but they gradually retract and finally become roughly spherical, in which
form they are arranged as the equatorial plate.
In connection with the conjugation of ciliates, similar reduction
processes have been described. In these Protozoa, as explained above,
it is only the micronucleus which takes part in syngamy, the macro-
nucleus degenerating. The micronucleus in one individual divides to
form two nuclei, and these again to form four. Of these four, three
degenerate. The remaining one divides again, so that each of the two
associated ciliates contains two nuclei. One of the nuclei in each indi-
vidual now passes over to the other and unites with the stationary nucleus,
after which the ciliates separate. Here, again, if the number of chromo-
somes in the uniting nuclei has not been reduced, it is evident the zygote
nuclei will have double this number. Several observers have maintained
that the first of the three divisions of the micronucleus is really a reducing
division. Hertwig (1889) noted that in Paramecium aurelia, the nucleus
of which has a large number of chromosomes during division, the nuclei
which unite have approximately half the number of chromosomes seen
in the ordinary divisions of the micronucleus during reproduction by
fission. Calkins and Cull (1907), in the case of Paramecium caudatum,
noted that the number of chromosomes in the ordinary dividing nucleus
is about 165. During the first two divisions of the micronucleus during
conjugation there is a reduction in the number to about half this. On
account of their large number it is difficult to count the chromosomes
accurately. Prandtl (1906) found that in Didinium nasutum the first
division of the micronuclei during conjugation was associated with the
reduction of the chromosomes from sixteen to eight. In CoUinia
MEIOSIS 115
branchiarum, Collin (1909) described a reduction of from six to three
(Fig. 495, 5 and 6), while Enriques (1908a) in Chilodon uncinatus saw
a reduction of four to two, and (1907) in Opercularia coarcta a reduction
of sixteen to eight (p. 1174), In all these cases the conjugating or gamete
nuclei possess half or the haploid number of chromosomes, while the nuclei
resulting from the union of the gamete nuclei have the full or diploid
number, which is maintained at all subsequent divisions. This is the
reverse of what occurs in the gregarines and coccidia, as described by
Dobell and Jameson, and Reichenow.
In connection with the process of union of gametes many so-called
reduction or maturation processes have been described. In Eimeria
schubergi, Schaudinn (1900), for instance, described as a maturation pro-
cess the breaking up and extrusion from the nucleus of the macrogamete
of the large karyosome (Fig. 337, ii). From what has been said above of
the reduction division of the nuclei of coccidia, gregarines, and ciliates, it
seems highly improbable that such a process is a reduction at all. In
the case of Cyclosjpora caryohjtica, another coccidium, Schaudinn (1902)
described the macrogamete nucleus as dividing twice, one of the products
of each division degenerating (Fig. 341). This again is explained as a
maturation process for the macrogamete nucleus before it is fertilized by
the microgamete. A similar process is said to take place in the case of
the parasites of malaria. The macrogamete, before fertilization in the
mosquito's stomach, is supposed to extrude one or two polar bodies which
contain some of the chromatin of the nucleus (Fig. 391, i6). In the case
of the conjugation of the flagellate Copromonas subtilis described by Dobell
(19086), where two individuals fuse, before the union of the nuclei each
nucleus is said to divide twice to form two reduction bodies which de-
generate (Fig. 48). After this, the nuclei of the conjugating individuals
unite. From what has been discovered during the past few years regarding
the methods of reduction of the number of chromosomes in connection
with the union of gametes in the Protozoa, it is evident that many
of the processes previously interpreted as reduction or maturation
divisions of the nuclei need to be re-examined in the light of what
is now known. Till this has been done it is useless to speculate as to
their meaning. \
BLEPHAROPLASTS, PARABASALS, AND KINETOPLASTS.
It has been explained above that amongst the Mastigophora the axis
of the flagellum is a filament (axoneme) which arises from a granule called
the blepharoplast. When there are two or more flagella, there arc a
corresponding number of axonemes and blepharoplasts. The several
IIG BASAL GRANULE OF FLAGELLUM
blepliaroplasts, when more than one is present, are often so closely packed
together that it may be difficult to distinguish them individually.
The blepharopjast may be situated upon the nuclear membrane, as
in Cercomonas, or quite separate from it, as in the majority of other
flagellates. It has already been shown above that certain observations
tend to indicate that the blepharoplast is of nuclear origin. In certain
stages a flagellate m.ay lose its flagellum or flagella and become a rou.nded
body with a single nucleus. When the flagellum is about to be re-formed,
it is claimed that a granule separates from the karyosome of the nucleus
and passes out into the cytoplasm through the nuclear membrane (Fig. 31).
Fig. 67. — Trichomonas atufiistd, showing the Spiral Parabasal Body immediately
ANTERIOR TO THE NUCLEUS ( X CU. 2,5()0). (AfTER AlEXEIEFF, 1924.)
An axoneme is then formed from it as an outgrowth, and when the surface
of the body is reached it takes with it a sheath of cytoplasm and becomes
a flagellum.
In association with the blepharoplast, whether it is on the nuclear
membrane or separate from it, there may occur one or more masses of
a substance which stains deeply with many chromatin stains. To such
bodies Janicki (1911) has given the name parabasal (see p. 53). The
name kinetoplast is employed here to designate the compound structure
consisting of a united parabasal and blepharoplast. Kinetoplasts are
typically seen in trypanosomes and allied flagellates. Parabasal bodies
have been described as occurring in Trichomonas by Janicki (1915),
Wenrich (1921), and Alexeieff (1924), but they are only detected after
special fixation — e.g., osmic acid (Figs. 67 and 275).
BLEPHAROPLAST PARABASAL KINET0PLA8T 117
AVhen a flagellate is about to divide, the blepharoplast is usually the
first structure to show any indication of division. It becomes elongated
and constricted into two parts. Very often the two daughter blepharo-
plasts (or two groups of daughter blepharoplasts when several are present)
remain connected by a fibre which may be called the paradesmose, as
suggested by Kofoid and Swezy (1915), to distinguish it from the centro-
desmose which unites the daughter karyosomes, or centrioles which are
supposed by some observers to occur within the karyosome, during division
(Fig. 272). As the blepharoplast elongates and divides and the daughter
blepharoplasts separate, the parabasal also becomes elongated and divides.
If several parabasals are present, without dividing individually, they
separate into two approximately equal groups. The blepharoplast thus
leads the way in division of the parabasal. It sometimes happens that
the blepharoplast divides before the parabasal shows any signs of division.
A figure may be produced in w^hicli the two daughter blepharoplasts are
connected by a paradesmose, at the centre of which the still undivided
parabasal lies. The parabasal now divides, and the two halves move
towards the daughter blepharoplasts. There is some resemblance to
mitosis in this type of division, which has been employed as an argument
in support of the view that the blepharoplasts are centrosomes and
that the kinetoplast is actually a nucleus. The parabasal, however,
does not form chromosomes, nor are spindle fibres developed between
the blepharoplasts, though some claim to have observed these structures
during the division of the kinetoplast of trypanosomes. After the
blepharoplast and parabasal have commenced to divide, the nucleus
itself begins to show signs of division.
In flagellates like Heteromita uncinata and Cerco-monas longicavda, in
which the blepharoplast is on the nuclear membrane, a condition is
seen in which the blepharoplast appears to function as a centrosome
(Fig. 68). The blepharoplast upon the membrane divides, and the two
halves separate. They finally take up positions at opposite poles of the
nucleus, and a definite spindle is formed between them. The karyosome
breaks up, and chromosomes appear at the 'equator of the spindle. The
chromosome plate divides into two daughter plates which move towards
the blepharoplasts. Finally, the nuclear membrane is divided, the
chromosomes disappear, and with the formation of the karyosomes the
nuclei are reconstructed.
It seems difficult to resist the conviction that in such a division the
blepharoplast has fulfilled the function of a centrosome. Its behaviour,
however, may be merely due to its position on the nuclear membrane,
for in flagellates like Parajpolytoma satura, described by Jameson (1914),
in which the blepharoplast is separated from the nuclear membrane,
1].
BASAL GRANULE OF FLAGELLUM
8 9
Fig. 68. — Binary Fission in Ileteromita uncinata ( x 4,000). (Original.)
1 . Normal flcagcllate with blepharoplast on surface of nuclear membrane.
2. The flagellate has become rounded and its blepharoplast divided, while two new flagella have
foiiiKMl. The kaiyosonie has hioken \\\, into granules.
3. The lilc|ili;irnplastsnccu])y the poles of a s|iin(llc whitli has an ecpiatorial plate of chromosomes.
4. The elu-omusonics have divided to form two daughter plates.
5. End view of the equatorial plate. " G. The daughter plates are separating.
7. Formation of two nuclei and the reconstruction of karyosome.
8. Commencing division of the flagellate.
9. The karyosome has re-formed and the flagellate is about to divide.
BLEPHAROPLAST PARABASAL KINETOPLAST 119
mitotic division of the nucleus occurs without any centrosomes at the
poles of the spindle. Instances are known, however, in which the blepharo-
plasts which are separate from the nucleus occupy during nuclear division
positions upon the spindle which centrosomes would be expected to
occupy. Such an example is seen in the division of Oikomonas termo
described by Martin (1912) (Fig. 135).
In the case of Proivazekella lacertce, which has an axoneme originating
in a blepharoplast on the nuclear membrane, the nucleus has one or more
parabasals surrounding it. When division of the nucleus takes place,
the daughter blepharoplasts occupy the poles of the spindle and mitotic
division takes place, as in Heteromita and Cercomonas. The parabasal, if
there is a single one outside the nucleus, becomes elongated and divided
into two parts, one of which passes to each daughter nucleus. When there
are several parabasals they separate into two groups without dividing
individually, very much like the behaviour of mitochondria during division
of spermocytes in the process of spermatogenesis (Fig. 254, s-x).
The function of the centriole in nuclear division has been discussed
above. It will be seen that in Heteromita uncinata, Cercomonas longicauda,
and other forms in which the blepharoplast occurs on the nuclear mem-
brane, and in certain cases where it is separated from the membrane,
the daughter blepharoplasts occupy during nuclear division the same
positions that the daughter centrioles are said to occupy. It is claimed
that as the centriole is functionally a centrosome, the blepharoplasts of
flagellates must also be centrosomes. It is further assumed that, in
those cases in which the blepharoplast occupies a position in the cyto-
plasm apart from the nucleus, it represents a centriole or centrosome
which has left the nucleus or is the result of division of the centrosome
into two parts, one of which remains in the nucleus and still functions
as a centrosome during its division, while the other has left the nucleus
to become a blepharoplast.
The whole subject of the relation of blepharoplasts to centrosomes
is a very complex one, and depends largely on the exact definition of a
centrosome. Some observers definitely assert that the blepharoplast is
a centrosome. Minchin (1914), for instance, stated that in his opinion it
was a well-established fact that in a great many cases blepharoplast and
centrosome were one and the same body. It seems difficult to doubt
this in view of the fact that in the developing spermatozoon of higher
animals the axial filament of the tail which corresponds with an axoneme
is known to be formed as an outgrowth from the centrosome. In fact, the
tail with its axial filament arising from the centrosome is exactly com-
parable with the flagellum with its axoneme and blepharoplast.
The question of the nature of the numerous blepharoplasts possessed
120 BASAL GRANULE OF FLAGELLUM
by the Hypermastigida and the basal granules of the cilia of Ciliophora,
which are to all intents and purposes blepharoplasts, is still more difficult
to answer.
Another point in connection with the blej)haroplasts of flagellates
must be mentioned. Many observers have described fibres which connect
the blepharoplasts with the karyosome of the nucleus, and they suppose
that these fibres represent centrodesmoses which were formed when the
supposed intranuclear centriole divided off the blepharoplasts. As
already remarked, when several blepharoplasts are present, they are
usually packed so closely together that they cannot be distinguished
individually. It not infrequently happens, however, that in certain
individuals of any species of flagellate the blepharoplasts are more dis-
persed, so that it is possible to recognize the actual nvimber present.
Kofoid and Swezy (1920) have described a very complicated system of
fibrillar connections between the various blepharoplasts of Chilomastix,
and they introduce into their scheme a definite centrosome which they
state is present upon the nuclear membrane and is connected by a fibre
with one of the blepharoplasts (Fig. 69). If such a centrosome and
system of fibres is present in this flagellate, it has at any rate escaped
detection by most observers. The complicated system of fibres which
they describe as being present, together with the karyosome, centrosomes,
blepharoplasts, flagella, and other motor organs and marginal filaments
of the cytostomal groove, they name the neuromotor syste?n. This term
has been extended by them to include the fibrillar structures which occur
in other flagellates, such as the complex organisms parasitic in termites,
while Sharp (1914) employs it for the fibrillar apparatus of the ciliate
Diplodinium ecaudatum (Fig. 520). It is quite possible that some of the
fibres have a motor function, but others appear to be merely supporting
rods, while there is at present no direct evidence to prove that they are
comparable to nerve fibrils which the name neuromotor suggests. In
using the term " neuromotor system," groups of structures which are not
necessarily homologous in different organisms have been united under
one name. Kofoid and Swezy, for instance, homologized one of the
fibres which support the margin of the cytostome of Chilomastix, the
basal fibre of the undulating membrane in Trichomonas, and the two
structures of unknown function which commonly occur in the posterior
region of Giardia as parabasals. There seems to be no real evidence that
these are in any way homologous with the true parabasals of other flagel-
lates, and it is worthy of note that several observers have described what
are probably true parabasals in certain species of Trichomonas.
The growth and the formation of new flagella are intimately bound
up with the activities of the blepharoplast. When the blepharoplast of
BLEPHAROPLAST PARABASAL KIXET0PLA8T
121
a flagellate divides, the axoiieme which arose from it remains, as a rule,
attached to one daughter blepharoplast, while a new axoneme grows out
Fig. 69. — Chilomastix mesnili : Free and Encysted Forms, to illustrate the
Structures described by Kofoid and Swezy ( x 6,370). (After Kofoid
AND Swezy, 1920.)
A. Normal flagellate viewed from the ventral or oral side., and showing all the structures of the
body. B. Cyst viewed from the ventral or oral side.
Cent., Centro.3ome; cent.k.. central karyosome; r//s<, cyst wall; cy<.,cytostome; cyt.fl., cytostomal
flagellum or undulatinu; mcnildanc; i iil.iltiz., intranuclear rhizoi^last; l.a.fl., left anterior
flagella; nuc, nucleus; inir.rlii-... iiudrai' rhizoplast; par.b., parabasal body; parast., para-
style; per/s<./.. peristomal liluc; /iriiii.h/i j>li ., primary blejiharojDlast; r.a.fl., right anterior
flagellum; secMeph., secondary blepharoijlast; sp/r.jrr., spiral groove; tert.bleph., tertiary
blepharoplast; tr.rhiz., transverse rhizoplast.
from the other to form a new flagellum. When a group of blepharoplasts,
in flagellates with more than one flagellum, divides into two groups, some
122 BASAL GRANULE OF FLAGELLUM
of the axonemes and flagella remain with one group and some with the
other. There seems to be no regularity in their distribution. Those
blepharoplasts which have no axonemes then form new ones. Very
frequently, before the blepharoplast has actually divided, a new axoneme
grows out from the part of the elongating blepharoplast which will become
one of the daughter blepharoplasts. It may happen that the new axoneme
actually passes into the cytoplasmic sheath of the old flagellum, so that
finally longitudinal splitting of the flagellum occurs. In such a case
division of the sheath of the flagellum alone takes place. It seems highly
probable that in no case does an axoneme itself divide longitudinally. A
new axoneme is invariably formed as a result of the outgrowth from the
daughter blepharoplast. In cultures of Leishmania the flagellum is formed
by outgrowth of the axoneme, which can usually be detected in properly
stained specimens of the parasites as they occur in tissues (Fig. 192).
Certain structures other than axonemes take origin in granules, which
are usually regarded as blepharoplasts. Thus, the two fibres which border
the cytostomal groove in CJiiloynastix arise each from a granule or blepharo-
plast (Fig. 69). Similarly, the basal fibre of the undulating membrane in
Trichomonas originates in a blepharoplast, and when division occurs a
second basal fibre grows out from one of the daughter blepharoplasts into
which the original one has divided. The axostyle of Trichoynonas likewise
arises from the blepharoplasts (Fig. 26). The writer (1907), as well as
Kofoid and Swezy (1915, 1915a), describes the axostyle as splitting
longitudinally during division of Trichomonas muris and other species.
Dobell (1909) stated that the new axostyles in T. batrachorum are formed
from the two halves of the divided paradesmose, which connects the
daughter blepharoplasts during division. Kuczynski (1914, 1918) claims
that the old axostyle degenerates, and that new ones are formed as out-
growths from the daughter blepharoplasts, while the paradesmose dis-
appears. Wenrich (1921) has described a similar origin for the new
axostyles in Trichomonas 7nuris (Figs. 271 and 272).
A great variety of fibres directly or indirectly connected with the
blepharoplasts have been stated to occur in flagellates. Thus, Schaudinn
(1904) describes numerous structures of this kind in Trypanosotna noctucc,
Prowazek (1903, 1904) in Trypanosoma lewisi and in Herpetomonas
tnuscarum,, while McCulloch (1915) figures a very complicated system of
fibres in Crithidia leptocoridis (Fig. 154). It seems that the majority,
if not all, of these are accidental structures, which cannot be considered
as definite organs of the normal flagellates. Whether the marginal fibres
of the cytostomal groove of Chilomastix, the basal fibre of the undulating
membrane, and the axostyle of TricAomowas, and other similar structures
which are connected with blepharoplasts, are to be homologized with
NUTRITION 123
flagella cannot be considered as definitely established. It may also be
open to question if the granules in which they originate, and which are
generally styled blepharoplasts, are actually of this nature.
PHYSIOLOGY OF THE PROTOZOA.
Many of the physiological processes which regulate the life of Protozoa
have been referred to above. It will only be necessary to review these in
a general manner under the headings Nutrition, Movement, Reaction to
Stimuli, Influence of Environment, Influence of Syngamy.
NUTRITION.— The essential food requirements of Protozoa are those of
living matter in general. There is a constant expenditure of energy,
necessitating a continuous supply of nourishment, which includes oxygen,
simple chemical compounds, more complex organic substances, or highly
organized proteid materials. Oxygen is an essential requirement, as it is
of all living matter, but the method by which it is obtained varies, as it
does between the vegetable and animal kingdoms. There are no special
organs of respiration, so that absorption of oxygen and discharge of carbon
dioxide takes place by a process of diffusion through the surface of the
body. Certain Protozoa, like plants, possess chromatophores, and by
means of their pigments or chromophyll are able, in the presence of sun-
light, to obtain oxygen from the carbon dioxide which is in solution in
the liquids in which they live, or which is formed by the organism itself.
The chromatophores, which are green when they contain chlorophyll or
red when the pigment is haematochrome, multiply by binary fission, as do
also certain refringent granules called pyrenoids which they contain.
They behave in many respects as independent organisms, and this has
given rise to the view that they may be actually organisms living in a
condition of symbiosis with the cells in which they occur. This method of
nutrition is described as being holophytic, in contrast to the holozoic type,
which is characteristic of Protozoa, which are devoid of chromatophores,
and which must of necessity absorb oxygen directly from the liquid in
which they live. In either case the organisms require oxygen, so that the
two types of nutrition, the holophytic and holozoic, do not imply any
essential difference in the character of the protoplasm of which their bodies
are composed. This is well illustrated by certain species of Euglena, which
normally have chromatophores, and lead a holophytic mode of existence
(Fig. 6). Under certain conditions, as when cultivated in the dark with
consequent loss of the pigment, they behave as organisms devoid of
chromatophores. The holophytic forms nourish themselves like plants,
and, in addition to the power conferred on them by the coloured pigments
of being able to utilize carbon dioxide for the purpose of acquiring a supply
124 PHYSIOLOGY OF PROTOZOA
of oxygen, tliey are able to elaborate relatively simple chemical compounds
into the protein materials necessary for their existence. Such forms may
be cultivated in solutions of various salts, and, like plants, commonly
elaborate starch or other amyloid substances as one of the products of
assimilation, and not infrequently build for themselves capsules composed
of cellulose. Between these and the completely holozoic forms, which
require, in addition to oxygen, ready-formed proteid materials, either solid
or in solution, there exists a group of organisms known as saprophytes.
These do not possess chromatophores, but are able to live in fluids con-
taining oxygen and complex organic compounds, which nevertheless are
simpler than the proteid materials required by the truly holozoic types.
Amongst the holozoic Protozoa two methods of obtaining proteid
material occur. In the one the organism ingests solid proteid material,
mostly in the form of other living organisms, such as bacteria and other
Protozoa, or, as in the case of parasitic forms like Entamoeba histolytica,
the cells of the host's body (Fig. 95). This solid matter is ingested either
through a definite mouth opening or cytostome, or, when such is not present,
through any part of the body surface by means of pseudopodia which
surround it, or by a movement of the cytoplasm over the object, which
appears to sink into its substance. In the other method, the proteid which
is in solution is absorbed in liquid form. There is no mouth opening, the
material merely passing into the body by osmosis. The latter method is
characteristic of many parasitic Protozoa, such as trypanosomes, malarial
parasites, coccidia, and gregarines. Other parasitic forms, such as the
amoebse, Trichomonas and Balantidium, ingest solid matter either by means
of pseudopodia or definite cytostomes (Figs. 26 and 14).
In the case of Suctoria, which obtain their food by means of sucking
tentacles, these are applied to solid objects, from which the proteid is
extracted in a liquid form, probably as a result of ferments acting at the
points of contact (Fig. 15).
As regards the proteid material ingested, two conditions result. When
it is absorbed in solid form it is enclosed in food vacuoles, in which the par-
ticles are found in various stages of digestion (Fig. 70). When the proteid is
absorbed in a state of solution no such food vacuoles are formed. From
a study of the changes which occur during digestion in food vacuoles it
has been found that when a living organism is ingested it is at first killed
and then gradually digested, leaving finally a residuum of fsecal matter
which is got rid of by the vacuole approaching the surface of the body
and discharging its contents. In the ciliates there frequently exists
a definite anal opening or cytopyge, usually at the posterior end of the
organism, through which the residue is discharged (Fig. 512). The process
of digestion is evidently the result of ferments which are secreted by the
NUTRITION 125
cytoplasm, as various ferments have been extracted from Protozoa.
Generally speaking, the reaction of a food vacuole is at first acid when the
ingested organism is killed. The reaction then becomes alkaline. It is
probable that during the acid phase a peptic ferment is active, while a
tryptic ferment is present during the alkaline phase. Fats also are
capable of being digested. It sometimes happens that the contents of
a food vacuole are alkaline from the commencement, and it appears that
the cytoplasm has some power of varying its response to different types
of food.
The proteid material absorbed from the food vacuoles, or from the
medium in which the organism is living, enters the cytoplasm, and is
immediately elaborated into the constituents of the cell or leads to the
formation of various intermediate bodies. The latter may be regarded as
food-reserve materials which are merely accumulations resulting from the
intake of excess of nourishment, or definite reserves intended for a period
of excessive activity, such as occurs during the sporogony process of
coccidia and gregarines, or the continued development when access to
nourishment is prevented, as when a cyst wall is present.
In the organisms which have a holophytic method of nutrition the
food reserve is stored largely as starch or allied substances of an amyloid
nature. In gregarines preparing for sporogony in the gametocysts the
cytoplasm becomes charged with refractile globules of a substance called
paraglycogen. The macrogaraetocytes of coccidia, which are to continue
development in an oocyst, likewise become loaded with refractile globules
of an albuminous substance. Similarly in the encysted stages of Entaynoeha
histolytica, lodamoeha biltscMii, and other forms, a large amount of a
glycogenic substance is present. It is gradually used up during the period
passed by the encysted form in awaiting a suitable opportunity for emerging
from the cyst. Another substance which is often present is volutin, which
appears in the fresh condition as greenish refractile globules. It stains
deeply with many nuclear stains, and has been supposed to be a forerunner
of chromatin, but of this there is no direct evidence. Many of the granules
which have been described as chromidia are probably of this nature. It
commonly occurs in flagellates, and is often abundant in trypanosomes,
appearing as deep red granules in specimens stained with Romanowsky
stains. Fat globules also occur in Protozoa, and are commonly present
in Radiolaria. The identification of the various granules and reserve
substances is a very difficult matter, dependent on microchemical tests,
solubility in various fluids, and reaction to different stains.
The residue from food digestion, as pointed out above, is discharged
from the body. This may occur immediately after digestion is completed,
or it may be deferred. The substances may assume different forms.
126 PHYSIOLOGY OF PROTOZOA
They may become crystalline excretory crystals, or remain as amorphous
masses. Amongst the Sporozoa, when reproduction by schizogony takes
place, a certain amount of cytoplasm is usually left over as a residual body,
which takes no part in the formation of merozoites. In it is got rid of
a certain amount of excretory substance. Malarial parasites thus dis-
charge the pigment granules which accumulate as a result of digestion
of haemoglobin.
In addition to the substances which have been referred to, and which
may be regarded as steps in the formation of protoplasm or the waste
products from the food, there occur other substances which are elaborated
to fulfil some special function. The conspicuous so-called chromidial
body of shelled amoebae may have to do with the formation of the shell.
The various skeletal structures which occur in the cytoplasm of Radiolaria,
the supporting rods which form the axes of the pseudopodia of many
Heliozoa, and, indeed, the external coverings like the shells of Foraminifera
and the cyst walls themselves, are to be regarded as products of metabolism.
It is evident that the Protozoa which produce such structures must absorb
special substances for the purpose.
Quite apart from the excretion of substances no longer required by the
organism by the rupture of vacuoles containing them at the surface of
the body, there is another method of excretion, which is carried out by a
rhythmically contracting vacuole which is situated near the surface of the
body. Such a contractile vacuole, when fully formed, suddenly contracts,
so that the clear liquid contents are discharged through the surface of the
body. In a short time the vacuole re-forms, and, gradually increasing in
size, reaches its maximum, when it again contracts. In some cases definite
channels in the cytoplasm conduct fluid to the vacuole. The rate of
pulsation varies with temperature and the presence of substances which
affect the density of the medium. It is supposed that the vacuole is a
means of discharging carbon dioxide and other soluble excretory substances,
but the fact that contractile vacuoles are absent in marine Protozoa
and. many parasitic forms, and that fresh- water forms lose the contractile
vacuole when made to live in salt water, suggests that such a vacuole may
be a means of accommodating the organism to the medium in which it lives,
rather than an organ primarily excretory in function. It can hardly be
supposed that marine or parasitic forms are less dependent on excretion
for their existence than those which live in fresh water. It has been con-
jectured that the contractile vacuole may counteract the tendency of the
cytoplasm to become overcharged with water due to the greater absorption
in fresh than in saline water.
On the method of nutrition of any particular organism depends the
character of the medium in which it can be cultivated. Forms like
MOVEMENT 127
Euglena, whicli possess chromatopliores and behave like plants, can be
grown in distilled water in which certain inorganic salts are dissolved.
Saprophytic forms require more complex substances, while holozoic ones
will grow only in media in which proteid material is present. This is
usually in the form of bacteria, which form the staple food of amoebge,
flagellates, and ciliates, when grown on the surface of agar plates or in
liquid media. In other cases, as in the cultures of trypanosomes and
leishmania, bacteria are absent, the proteid materials being derived from
blood-serum.
MOVEMENT. — The power of movement is one of the properti<^s of
cytoplasm in general, and amongst the Protozoa it is seen in its simplest
form in organisms like amoebse, and is most highly developed when special
motile organs are present, such as flagella, cilia, the contractile filaments
in the stalks of the attached Protozoa, and the myonemes of gregarines
and other forms. The cytoplasm is in constant movement wathin the
organism. This streaming of the cytoplasm is undoubtedly the result of
chemico-physical changes which are taking place. In highly-organized
Protozoa, like the ciliates, the currents in the cytoplasm are constant in
their direction, and the various food vacuoles which move with them
perform a definite circuit. In the amoebae, which do not have definitely
orientated bodies, there is more irregularity. It is as a result of this
streaming of the cytoplasm that organisms like amcBbge are able to move
and form pseudopodia. When resting on a surface, the portion of cyto-
plasm in contact with the surface is prevented from movement, while the
streaming of the internal cytoplasm in one direction leads to a forward
movement, which is best illustrated by the roHing movement of a bag of
fluid on an inclined plane. In this manner the whole amoeba may progress
in one direction, or, when the streaming of the cytoplasm is limited, only
portions will move forwards, with the result that pseudopodia are formed.
By changes in the direction of the stream the pseudopodia are withdrawn
and others protruded. Certain pseudopodia, like those of Heliozoa, are
supported by axial fibres, which render them more permanent structures.
They are, nevertheless, capable of performing swinging or bending move-
ments. Whether these are the result of movements of the cytoplasmic
covering or of the axial fibre has not been satisfactorily determined.
As, however, fine pseudopodia devoid of axial fibres can perform such
movements, it would seem that the axial fibre may be purely elastic
in nature, with the function of bringing the pseudopodium back to its
original extended position when the movements of the cytoplasmic covering
cease. The more actively motile flagella and cilia of the Mastigophora and
Ciliophora have essentially the same structure as the axopodia of Heliozoa.
There is an axial fibre (axoneme) covered by a thin sheath of cytoplasm.
128 PHYSIOLOGY OF PROTOZOA
and it may be supposed that tlieir movement is brought about in a similar
manner by changes which occur in the thin cytoplasmic covering, the
axial fibre acting as an elastic support. Similarly, the myonemes which
occur in gregarines and other Protozoa may not in themselves be contractile,
though they may limit the contraction of the cytoplasm itself to definite
channels. It is generally supposed, however, that the filaments themselves
are contractile. In the case of attached forms like Vorticella the stalk
is composed of an axial fibre and a sheath of cytoplasm; when retraction
takes place, the axial fibre assumes the form of a compressed spiral. During
extension it appears that the elasticity of the axial fibre, which returns to
its original condition, is responsible for the extension of the stalk, and it is
possible that the sheath of cytoplasm is the sole cause of the retraction.
That cytoplasm itself, quite apart from the presence of myonemes or other
filaments, is able to perform sudden and rapid movements of contraction
is illustrated by the behaviour of contractile vacuoles.
Another series of internal movements which are common to all cells
provided with nuclei are those associated with nuclear division. The
complicated process of mitosis, with the formation of the spindle and
chromosomes, and the subsequent separation of daughter chromosomes,
is in many cases carried out under the influence of the centrosome. In
many Protozoa, however, no centrosome is visible, but in neither case
has a satisfactory explanation of the phenomenon been given. AVhen a
centrosome is present, it appears to be the centre of activity, for it is
towards it that the rays of the aster and the spindle fibres are directed.
For those who regard the blepharoplasts of flagella as centrosomic in nature,
the action of the flagella is supposed to be another illustration of the motor
activities of the centrosome.
The movements of the cytoplasm which have been considered are
distinct from the locomotion of the Protozoa themselves. An organism
which is in a resting condition and undergoing no changes in shape may
still show the streaming movement of the cytoplasm, but it is nevertheless
these movements of the cytoplasm which bring about the changes in
shape and actual locomotion when these occur. Progressive formation
of pseudopodia and changes in shape in amoebse are the result of continued
streaming movements in one direction, as explained above. In the case
of Mastigophora and Ciliophora it is the result of the continuous action
of the special organs of locomotion, which are so arranged that when
they are in activity the organism is propelled through the liquid medium.
The peculiar gliding or slug-like progression of gregarines has been sup-
posed to be due to the rapid secretion of a tenacious fluid from numerous
pores in the longitudinal grooves of that portion of the ectoplasm which
is in contact with the surface on which the organism is resting. It is
REACTION TO STIMULI— INFLUENCE OF ENVIRONMENT 129
possible that the gliding movements performed by the small gregarine-like
merozoites or sporozoites may be explained in a similar manner.
REACTION TO STIMULI. — The actual direction of progression is the
direct result of external stimuli acting on the organism. Practically all
Protozoa react to stimuli, whether mechanical, chemical, thermal, electric,
or photic. The response to such stimuli has been chiefly studied in the
case of ciliates, in which it has been frequently found that the region of
the cytostome is the most sensitive part of the body. It is evident that
for any Protozoon there is an optimum condition of the medium in which
it lives, and if, during progression, it reaches an environment which is
less favourable to its existence than that which it has just left, there will
be a stimulation of the sensitive area of the body. This stimulation will
result in an altered action of the organs of locomotion, with a consequent
withdrawal from the unfavourable stimulus. The movements of ciliates
when subject to adverse stimuli are very precise, and have been the
subject of extensive investigations. The attraction and repulsion are
known as positive and negative taxis respectively. Generally speaking,
positive taxis indicates a movement towards and a negative taxis a
movement from any particular environment.
INFLUENCE OF ENVIRONMENT.— The actual condition of the environ-
ment in which a Protozoon finds itself is a very important factor in its
development. As already remarked, for each there is an optimum
condition which suits it best. Departures from this are followed by
conditions of depression resulting in degeneration or even death. Lack
of food or excess of it, leading to starvation or overfeeding, also brings
about degenerative changes which are seen in alterations in the structure
of the nuclei, which frequently become enlarged. In certain cases the
nuclei break up entirely, leading to the final death of the organism. To
a certain extent Protozoa can be gradually adapted to changes in environ-
ment, provided these are not brought about too suddenly. It is possible
by gradually raising the temperature of cultures to obtain a race of
organisms which can live at a temperature which would have quickly
killed if applied suddenly. Provided that degeneration has not proceeded
too far, recovery is possible if the conditions are improved. Regeneration
of the degenerate parts takes place. Similarly, Protozoa which have been
mutilated or deprived of portions of their bodies are able to regenerate
themselves, provided the nucleus remains intact.
The majority of Protozoa are able to protect themselves against adverse
conditions by the process of encystment. The tough resistant capsule
which is secreted shuts them off from their environment, so that they are
able to survive unharmed till conditions favourable to a free-living
I. 9
130 PHYSIOLOGY OF PROTOZOA
existence recur. Within the cysts the organism either undergoes no
change or it may continue to multiply. In parasitic forms the cyst
protects the organism during its passage from one host to another, the
encysted form being known as the infective stage. Amongst the Sporozoa
it usually happens that a period of asexual reproduction is followed by
one in which sexual forms are developed. The appearance of these is
generally supposed to be an indication that unfavourable changes are
taking place in the environment, and that encystment, which occurs in
association with conjugation and the production of the zygote, is necessary.
Another feature characteristic of many parasitic forms is the difference
in environment associated with different stages of development. Thus,
in the case of malarial parasites the human blood supplies the conditions
necessary for asexual reproduction and the production of gametocytes.
In the body of the mosquito all asexual stages quickly perish, while the
gametocytes continue their development, which was arrested in the human
blood. The sporozoites ultimately produced will develop no further in
the mosquito, but with the change brought about by their injection into
man further progress occurs. Similarly in the case of trypanosomes the
forms taken up from the blood by the transmitting host quickly lose
their power of developing in the blood, though they do so in the body
of the invertebrate. The metacyclic trypanosomes which are eventually
produced have regained the power of development in the blood.
As a result of abundance of nourishment in the medium the cytoplasm
may become charged with globules of food-reserve material which appear
to be far in excess of that actually required. Thus, the ciVmie Balantidium
coli may be packed with such substances. In many cases this has
apparently little effect on the vitality of the organism, though it has
been shown that in certain forms degenerative changes result.
A feature of this over-nourishment may be seen in certain cases of
gigantism. Thus Tricliomonas vaginalis is often very much larger than
Trichomonas hominis of the intestine. If, however, both these organisms
are cultivated in the same medium, the forms which appear are exactly
alike, so that it would seem that the large size of T. vaginalis is merely
an indication of overgrowth. Similarly the giant forms of Herpetotnonas
mirabilis, which occur in the Malpighian tubes of certain flies, can probably
be accounted for in similar manner.
INFLUENCE OF SYNGAMY.— As already remarked. Protozoa which
become degenerate or pass into a state of depression may recover if
conditions of life become favourable. It is supposed that a similar
recovery may result from the process of syngamy. In the majority of
Protozoa, however, syngamy is not known to occur. In many cases
this is undoubtedly due to the fact that the complete life-history has not
INFLUENCE OF SYNGAMY 131
been elucidated. In some instances, however, unless it is assumed that
syngamy must of necessity take place from time to time, it appears that
reproduction by simple binary fission is continued indefinitely. Such
an organism divides into two daughter individuals, and when these have
become fully grown, division again takes place. A simple life-cycle of this
kind is characteristic of the amoebae, and it is only interrupted by the
amoebae becoming encysted under certain circumstances.
Within these cysts, which are purely protective in function, the
amoebae may or may not continue to multiply by fission. When condi-
tions again become favourable, the cyst is ruptured and the amoebae
escape to continue their multiplicative existence. Similarly, many
trypanosomes can be handed on indefinitely from one animal to another
by simple inoculation of infected blood. There appears to be a continuous
process of reproduction by binary fission without the intervention of
either syngamy or encystment. Under natural conditions, however,
direct transference from vertebrate to vertebrate, except in the case of
Trypanosoma equiperdu?n, does not occur, the life-history being varied
by alternate multiplication in a vertebrate and an invertebrate. As far
as is known at present, multiplication in both hosts is by continuous
binary fission, though some authorities assume that a syngamic process
will be found to occur in the invertebrate. When such a change of hosts
is obligatory, the parasite is said to require an alternation of hosts for the
continuance of its life-cycle. In the case of certain blood-inhabiting
Sporozoa (malarial parasites) the alternation of hosts is characterized
by the occurrence of asexual multiplication in the vertebrate and syngamy
followed by the production of sporozoites in the invertebrate.
Until recently it was considered that the periodic occurrence of
syngamy was essentia] for the continued existence of the race. This view
was the outcome of researches conducted on ciliates by Maupas and
Eichard Hertwig. Thus it was fdiown that Paramecium caudatum, after
a varying period of multiplication by fission, proceeded to conjugate.
Calkins (1904) found that, if conjugation was prevented, the ciliates,
though they continued to reproduce, gradually weakened and died.
Similar results had previously been obtained by Maupas (1888, 1889)
in the case of Stylonychia pustulata and other forms. It was believed
that these experiments proved that a race would invariably die out if
conjugation did not occur. Enriques (1903), working with Glaticoma
scintillans and G. pyriformis, and Woodruf? (1917) with Paramecium
aurelia, proved that this was not the case. The latter observer (1925),
having commenced with a single individual, has carried on the culture
by separating the daughter individuals produced at each division for a
period of fifteen years, during which over 10,000 divisions have taken
132 PHYSIOLOGY OF PROTOZOA
place. Great care was taken to keep the culture medium favourable,
and it was found that the ciliates were just as vigorous at the end
of this period as was the original parent. It is thus evident that,
even in the case of an organism which under natural conditions conjugates
from time to time, the race may survive and still remain in vigorous
condition when this is prevented. Woodruf! (1921) showed that during
this period of repeated binary fission the process of renewal of the macro-
nucleus from the micronucleus, known as endomixis, took place at intervals
(see p. 54). In the case of the ciliate Spathidium spathula, Woodrui?
and Moore (1924) have demonstrated that reproduction can be continued
indefinitely without recourse to endomixis or conjugation when suitable
environmental conditions are supplied.
From the work of Richard Hertwig and Maupas, who considered that
conjugation was essential to survival of the race, arose the theory of
rejuvenescence, which supposes that any race of ciliates dies out through
loss of vigour if conjugation does not take place. It has generally been
assumed that both these observers thought that the rejuvenating process
showed itself in an increase in the rate of multiplication. According to
Jennings (1920) this is a misrepresentation of their views, for it was
definitely stated that the rate of fission before and after conjugation was
not altered. Their view of the change which takes place in conjugation
is that the ciliates which would otherwise have died now continue to live,
and this continued existence itself is a sign of rejuvenescence. Calkins
(1919a), however, has definitely asserted that the failing energy and rate
of multiplication of the pre-conjugation period is abolished by conjugation,
and that in the post-conjugation period the rate of multiplication is
increased. Quite recently Woodruff and Spencer (1924), working with
Spathidium spathula, have clearly shoAvn that conjugation actually does
increase the rate of multiplication, and, furthermore, that on an average
cultures made from forms which have conjugated outlive those from forms
which have not, so that the chances of any particular line surviving are
increased. Careful experiments have not only shown that conjugation
is not necessary to continued existence, but appear to have demonstrated,
in many cases, that following it there is actual depression as regards rate
of division, likelihood of death, and in other respects. If conjugation does
not lead to some change of this kind, it is extremely difficult to account
for the process of syngamy at all. It appears to be unnecessary, yet it
takes place in nature. Minchin (1912) expressed the opinion that it
tends to level down individual variations and keeps the species true to
type. The true explanation may, however, be the reverse of this, as
Jennings (1920) has pointed out.
It has been explained above that there occurs a reduction in the number
INFLUENCE OF SYNGAMY 133
of chromosomes in the nuclei of gametes, or in the two nuclei into which
the zygote nucleus divides. In this reduction division the individuals
of each pair of homologous chromosomes are separated, one of each pair
going to each daughter nucleus. If, for instance, there are four pairs of
homologous chromosomes grouped as ka, B6, Cc, Dr/, at reduction division
one of each pair passes to a daughter nucleus, so that the daughter nucleus
may receive chromosomes in many possible combinations — -ABCD, ABCc?,
ABcrf, khcd, abed, A6CD, etc. In all, there may be sixteen different
combinations. When syngamy occurs, any one of these groups in one
gamete will unite with any one in the other gamete, so that the zygote
nucleus containing eight chromosomes will have a still larger number of
possible combinations, the actual number being eighty-one.
It has been abundantly demonstrated in the higher animals and plants
that the hereditary characters are intimately bound up with the various
chromosomes occurring in the nuclei of the gametes, so that it is clear
that union of gametes with four chromosomes will give rise to eighty-one
different combinations of hereditary characters. In ordinary division
without conjugation all the chromosomes split longitudinally, and half of
each chromosome passes to each daughter nucleus, so that the hereditary
characters are more equally distributed to the daughter nuclei. On this
account, Jennings (1920) sees that the progeny resulting from conjugation
show a greater diversity of hereditary combinations than do the progeny
arising from multiplication by fission. From the point of view of survival
of the race, the diverse individuals resulting from conjugation will be
more likely to provide at least some forms which will tolerate any new
condition of the environment than are the more uniform individuals
which result from continued asexual reproduction alone. The group of
organisms which result from conjugation will be at a distinct advantage
when compared with others when changes in environment take place.
LIFE-HISTORY OF PROTOZOA.
The life-history of a Protozoon is one of continued growth and repro-
duction, which may or may not be interrupted at intervals by a process
of syngamy. When syngamy occurs, two ordinary individuals which do
not appear to differ from those which have been dividing may copulate,
as in Copromonas, or conjugate, as in Paramecium, after which reproduction
is resumed. On the other hand, it may happen that certain young
individuals which arise in the usual manner, and which do not appear to
differ from others which are destined to develop into forms like the parent,
become transformed into individuals of a special type. They are known
as gametocytes, which, when fully grown, produce a number of gametes.
134 LIFE-HLSTORY OF PROTOZOA
The latter unite in pairs to form the zygotes, which give rise to typical
daughter individuals, known as sporozoites. These grow into adults,
which reproduce repeatedly in the manner characteristic of the repro-
ductive phase till certain of the progeny again become gametocytes.
The various forms which occur during the multiplicative phase, which is
known as agamogom/, belong to the asexual generation, while the indi-
viduals themselves are agamonts. In contrast to these, the gametocytes
and the gametes to which they give rise, the zygotes, and the sporozoites
which are ultimately formed, belong to the sexual generation. The
process of development from gametocyte to sporozoite is known as
sporogonij, while the gametocytes themselves are sporonfs. These two
phases of development alternate in that, after reproduction has been
repeated a number of times (agamogony), the sexual method of multiplica-
tion (sporogony) supervenes. The sequence of the two phases is known
as alternation of generations, which is a characteristic of the majority of
the Sporozoa. Amongst the typical gregarines, however, the asexual
generation and agamogony does not occur, the sporozoites into which the
zygotes divide growing directly into gametocytes, which again produce
gametes. The whole life-cycle of a typical gregarine is thus one of
sporogony.
As already indicated, the life-cycle of a Protozoon may at any time
be interrupted by the formation of protective cysts secreted from the
ectoplasm. Some organisms cease multiplying when they become en-
cysted, others continue to multiply within the cyst, while others again
never reproduce except in the encysted condition. Sometimes, as in the
case of parasitic amoebse, special individuals (precystic amoebse) alone
are capable of forming cysts. Amongst the Sporozoa, encystment only
occurs in association with sporogony. A cyst may be formed around
two gametocytes, as in the case of gregarines. It is then distinguished
as a gametocgst. After syngamy has taken place, the resulting zygote
may secrete a cyst known as an oocyst. The zygote may divide into a
number of sporoblasts, and these, either within the oocyst or after their
escape from it, become enclosed in secondary cysts called sporocysts.
Oocysts and sporocysts occur typically amongst the Sporozoa. The cysts
usually have very tough and resistant walls ; at other times they are little
more than thin membranes.
Protozoa may be free-living organisms which spend the whole of their
life in water or in moist situations, or they may be more or less intimately
associated with other animals. According to the degree of this dependence
three classes are usually recognized. There are commensals, which live
in or upon another organism, and, though deriving benefit from this
association, do not injure the host in any way. They deprive it of an
PARASITISM 135
inappreciable amount of material wliicli it might use itself, or feed upon
the waste products. Others are regarded as symbionts, which, living in
similar circumstances, not only derive benefit themselves, but contribute
to the well-being of the host. Thus, Cleveland (1923) has shown that
termites, which feed upon wood, do so by virtue of their intestinal Protozoal
fauna, which actually digest the wood to form substances on which the
life of the termites depends. Other forms are parasites, which deprive
their hosts of their own fluids or tissues, and damage them by destruction
of tissues either directly or indirectly through the formation of toxins.
The line of demarcation between these various types is very indefinite,
so that it is often impossible to decide to which group any particvdar
organism belongs. The numerous discussions which have arisen as to
the pathogenicity of the intestinal flagellates of man is a case in point.
When true parasitism is considered, it must be remembered that the
degree of harm inflicted on the host has a direct bearing on the continued
existence of the parasite. A parasite is an organism which has become
adapted to an existence in another, and has lost at the same time the
power of living outside this host. At some period of its existence it must
be transferred to a new host if it is to survive. This transference may
take place by the production of encysted forms which escape from the
body and are taken up casually by a new host, or an invertebrate
may take up the parasites from the blood and later introduce
them to new hosts. In the first case the parasite does not appear
to be able to produce the encysted stages till some time after infec-
tion of a new host has taken place, and in the second a period must
elapse before the appearance in the blood of the forms capable of infecting
the invertebrate. In any case, the chance of a parasite gaining access to
a new host is a precarious one, and it is evident that the longer a parasite
can survive in one host, the better is its chance of bringing about infection
of another. If, then, a parasite is so virulent that it very quickly destroys
its host, its chances of continued existence are definitely diminished. It
is found in nature that there is such an adaptation of parasite to host,
and vice versa that in all cases of parasitism the parasite damages its host
to the least extent compatible with its own continued existence. When-
ever a parasite is discovered which brings about the death of its host in
a short time, it may safely be assumed that the host is not the natural
one, or that it is a natural one which is in some unnatural condition. In
the case of the pathogenic trypanosomes of Africa, the natural hosts are
the antelopes, to which they do comparatively little harm, while human
beings and domestic animals are unnatural hosts, as they are much more
seriously affected. After a time adaptation may occur, and a host which
was at first an unnatural one may gradually become a natural host.
136 LIFE-HISTORY OF PROTOZOA
Man seems already to have become a natural host to Trypanosoina
gambiense, but to be only in process of becoming so for T. hrucei {T.
rhodesiense).
An important feature of parasitism is the specificity of any particular
parasite for its host. It is found in nature that some parasites are unable
to live in any other host than the one in which they naturally occur.
This undoubtedly depends upon the peculiar character of the body fluids
of these animals. Some parasites have become so specialized that they
cannot survive in any other fluid than the one to which they have become
accustomed. Very frequently, however, a particular parasite is able to
live in hosts which are nearly related, the fluids of which may be presumed
to differ only slightly from one another. Thus Plasmodiuin vivax, which
causes benign tertian malaria, cannot survive in any other vertebrate
host than man, though Mesnil and Roubaud (1920) have shown that it
may multiply for a short period in the chimpanzee. Other parasites are
much less specific, for many of the pathogenic trypanosomes can develop
in small rodents, which under natural conditions are never infected by
them. In such cases it seems probable that, quite apart from the suita-
bility of the fluid of a host, the rapidity with which a host can develop
antibodies is the determining factor as to whether a parasite can establish
itself or not. Instances are known in which it is only after many attempts
to introduce a parasite into a host that success is at last attained. An
instance of this is quoted below (p. 576), where Watson, attempting to isolate
a strain of Trypanosoma equiperdum from horses in laboratory animals,
only succeeded in one after inoculating over 600 animals. The infection,
once established, was then readily inoculated from one animal to another.
It is evident that here the fluids of the animal which gave a successful
result differed from those in which inoculation had failed, or that amongst
the organisms injected on the successful occasion there happened to be
a few which found the environment congenial and were able to resist
the antibodies developed. The fact that subsequent subinoculations were
easily carried out seems to suggest that the explanation is to be found
in the parasites themselves. Not infrequently an animal which has
acquired an infection will free itself, after which it is found to be immune
to further inoculations. On the other hand, it has been shown that in
some cases, when an infection has disappeared or has been much reduced,
further inoculations of the same organism may bring about a super-
imposed infection which may be more severe than that first produced.
Such an instance has been described by Ndller (1917) in the case of frogs
infected with Trypanosoma rotatorium.
It may be stated as a general rule that the specificity of parasitic
Protozoa for their particular hosts is much more marked than is the case
PARASITISM 137
with vegetable parasites, such as bacteria, yeasts, and allied organisms.
It often happens that a parasite in one host may be morphologically
indistinguishable from one in another, yet experimentally it is impossible
to produce cross-infections. Whether such biological races are to be
regarded as distinct species or not is a problem which still requires solu-
tion. From the strictly zoological point of view they should be regarded
as belonging to one. This highly developed specificity of Protozoan
parasites may be kept in mind when organisms of a doubtful nature are
being dealt with. The group of parasites known as Toxoplasma, which
most observers regard as Protozoa, may actually be vegetable organisms,
for it has been found that they are inoculable into a variety of different
hosts.
Another feature exhibited by parasites is one which is termed increase
in virulence. Here, again, illustrations occur amongst the trypanosomes.
T. gatnbiense can be inoculated from man to laboratory animals. In the
first passage the infection may be of slow development, but with successive
passages through these animals a strain wall develop which in its behaviour
differs from that originally introduced. Whereas at first it may have
taken a year to kill the animal in which the trypanosomes were always
scanty, finally it brings about a fatal issue in two or three weeks, the
trypanosomes reproducing rapidly till the blood of the animal is teeming
with them. It is evident that during successive passages the trypano-
somes have gradually adapted themselves to these animals. In the case
of naturally occurring infections, wdiich are characterized normally by
a balance between host and parasite, occasionally infections occur in
which such a balance does not exist. In naturally occurring malarial
infections amongst native children exposed to the bites of infected
mosquitoes there is a balance between the host and parasite, so that
the host appears to be little inconvenienced. Sometimes, however,
severe and fatal cases occur, either because the natural resistance of the
host is low or because the parasites have become peculiarly virulent.
These severe infections are of more frequent occurrence amongst human
beings who have come from non-malarial countries and are suddenly
exposed to infection. It is often claimed that these cases result from
a specially virulent strain of parasite, but it seems more probable that
the host is at fault, and that the fluids of the body differ from those of
the natural hosts. Another illustration is seen in the case of Entamoeba
histolytica. In the majority of cases of infection with this amcBba, the
organism produces a minimum of inconvenience to its host, which is
known as a carrier, but in a small percentage of cases the balance is
broken down and acute symptoms of amoebic dysentery reveal themselves.
It is found that the reaction of a host varies with the strain or race of
138 IMMUNITY IN PROTOZOAL INFECTIONS
any particular parasite emj^loyed. Two strains of the same sj^ecies of
trypanosome may produce very different results. An animal inoculated
with one strain may acquire an infection from which it will recover.
It may have developed an immunity and be no longer inoculable with
this particular strain, though it is still susceptible to inoculation with
another strain of the same species. On this account it is exceedingly
difficult to differentiate species of trypanosome by what have been termed
immunity experiments.
The mechanism of these various phenomena are far from being properly
understood, and it appears that a real explanation will never be obtained
till the biochemist has obtained more information regarding the chemistry
of the living cell and the fluids to which it gives rise.
IMMUNIIY IN PROTOZOAL INFECTIONS.
Immunity in connection with parasitism amongst the Protozoa will
be referred to below in connection with individual parasites, but it will
be necessary to discuss more fully some of the general features which
have just been mentioned above.
NATURAL IMMUNITY.— As remarked above, each parasite has its
own particular host or group of hosts in which it can live, and outside
these limits it is impossible for it to establish itself. This specificity, as
it is called, is well illustrated by the malarial parasites of man. Exactly
how infections are prevented in one host while they take place readily
in another is not properly understood, but, as a result of extensive re-
searches, it is evident that cells and fluids of the body of refractory
animals are of such a nature that parasites introduced cannot develop
and are finally killed. That the serum of the blood is largely responsible
for this natural resistance is proved by the experiments of Laveran (1904ft),
who showed that the blood-serum of baboons, which are usually refractory
to inoculation with Trypanosorna gam.biense, when injected into mice will
cause the disappearance of T. gambiense from their blood, or even prevent
infection if injected forty-eight hours before inoculation with tlie trypano-
some. Such an immunity against infection is a natural immunity. It
is possible, however, in some cases to overcome the natural resistance.
This may be effected either by lowering the resistance of the inoculated
animal, an illustration of the well-known fact that a person in good health
is less liable to disease than one who is in poor condition, or by increasing
the virulence of the parasite. As a rule mice and guinea-pigs are quite
refractory to inoculations with Trypanosoma hwisi of the rat, but Eoudsky
(1910ft, 1911), as will be mentioned below, was able to increase the viru-
lence of the trypanosome, so that mice and guinea-pigs were susceptible.
NATURAL IMMUNITY 139
It is thus evident that in a study of the interrehitions of a host and the
parasite both the condition of the host and that of the parasite have to
be taken into account. The increase in virulence of Tryjmnosoma lewisi
produced by Roudsky was artificial, and it is probable that under natural
methods of transmission such a change would rarely, if ever, take place.
Nevertheless, the observation is an important one, for it demonstrates
that a trypanosome may become modified to such an extent that it will
produce infections in animals in which normally it fails to develop. It
is a generally accepted fact that the animal trypanosome, Tnj'panosoma
hrucei, does not as a rule infect man who is constantly exposed to the
bites of infected tsetse flies, yet there occurs in man in Rhodesia a
trypanosome which has been given the name Tryjpanosoma rhodesiense,
which in all respects appears to be identical with T. hrucei. It is main-
tained by some that it is distinct from T. hrucei, and by others that it is
identical with it. It has, however, to be recognized that it is quite within
the bounds of possibility that the animal trypanosome T. hrucei may
occasionally change, for reasons not yet discovered, so that it becomes
capable of infecting man, or that man may occasionally be in a condition
which will permit infection with the unaltered trypanosome. Duke
(1923, 1923a) believes that an outbreak of trypanosomiasis amongst
human beings in the Mwanza district of Africa, in which the trypano-
some was of the T. rhodesiense type, was due to the inoculation of
the animal trypanosome T. hrucei as a result of the lowered resistance
of the population after a period of famine and heavy ankylostome
infection.
There are many examples of variation in virulence of parasitic Protozoa.
It is well known that if Trypanosoma gamhiense is inoculated from the
blood of man into a rat, the type of infection produced is a chronic one,
very few trypanosomes being present in the blood of the rat at any one
time, the inoculated animal often surviving for many months. In suc-
cessive passages in rats the virulence increases, till finally a strain is
produced which multiplies very rapidly, so that the blood is soon swarming
with parasites, which bring about the death of the host in about ten days.
By passage of the strain through a different host such as the guinea-pig
this virulence for rats may be largely lost. It is regained, however, by
further passage through the rat. Duke maintains that in the spread of
sleeping sickness the epidemic outbursts of this disease are due to direct
passage of the trypanosome from man to man by mechanical transmission
in which some biting insect merely conveys blood from an infected to
a healthy person, just as in laboratory experiments the syringe conveys
blood from an infected to a healthy animal. It is supposed that in this
way the virulence of the trypanosome, which is kept relatively avirulent
140 IMMUNITY IN PROTOZOAL INFECTIONS
under ordinary conditions by a definite cyclical development in the tsetse
fly, is greatly increased.
It is noteworthy that Blanchard and Blatin (1907) have shown that
the marmot during hibernation at a temperature of 6° C. becomes resistant
to trypanosomes, with which it can readily be inoculated when it is in
an active condition. Brumpt (1908a) found that the dormouse showed
a similar immunity during hibernation, though it was observed that the
trypanosome (T. blanchardi) with which it may be naturally infected per-
sists in its blood during the hibernation period. The natural susceptibility
or the resistance of animals to infection with parasites has been advocated
as a means of differentiating species. The method has been mostly
used in the case of trypanosomes, but it has been also applied to other
parasitic Protozoa. As an example may be quoted the effect of inoculating
into rats the two trypanosomes T. congolense and T. nanum, which in
their natural hosts are morphologically indistinguishable from one another.
When inoculated into rats T. congolense gives rise to an infection, while
T. nanum does not, and it is claimed by the advocates of the specific
value of this test that the dift'erence justifies the separation of the two
species. That the test is not as straightforward as at first it might appear
is illustrated by the fact that if T. congolense is inoculated into a goat,
it will be found to have lost its power of infecting rats. It follows, there-
fore, that distinction of species based solely on the ground of resistance
of certain animals is zoologically unsound. Another application of the
same test was made by Adler (1924), who discovered a coccidium in the
intestine of the civet cat in West Africa. Morphologically it resembled
Isospora rivolta, a parasite of dogs and cats. Attempts to infect dogs
and cats with the parasite of the civet cat having failed, it was thought
justifiable to establish a new species. Looking at the question from the
reverse point of view, the susceptibility of a number of different hosts
to a parasite derived from one host is strongly suggestive of the identity
of the parasites which may occur naturally in a variety of hosts. Thus,
birds are very liable to natural infection with a malarial parasite, Plas-
7nodium prcecox. The demonstration that the parasites from one bird
can be inoculated into birds belonging to other species is a valuable
indication that the one parasite may, under natural conditions, occur in
a variety of hosts. The converse is not necessarily true, for development
in one host may bring about such a change in the parasite that it is no
longer able to infect a host which was originally susceptible to it. The
example of passage of Trypanosofna congolense through the goat, referred
to above, is a case in point.
In connection with natural immunity it has to be remembered that
much depends upon the number of parasites — the dose of virus — intro-
RECOVERY FROM INFECTION 141
duced. Theoretically it would be expected that in the case of susceptible
hosts the introduction of a single parasite would bring about infection.
This has actually been demonstrated in the case of Trypanosoma brucei
and mice by Oehler (1913), who showed that the introduction into the
peritoneal cavity of a single trypanosome gave rise to infection. In
other cases, as, for instance, in the inoculation of Leishmania donovarii
to animals, no infection can be detected unless comparatively large doses
are employed. In animals with absolute immunity no infection occurs
even after the use of massive doses. Experiments such as these have been
conducted with animals which are not natural hosts of the parasites
concerned, but there is evidence that even in the case of natural hosts
infection does not always follow exposure, a result which may depend
on the dose of the virus.
Even when a host is a natural one there are always certain individuals
which resist infection. It is well known that, though human beings are
very susceptible to malaria, there are certain individuals who appear to
have a natural immunity, and never show any evidence of infection,
though constantly exposed to the bites of infective mosquitoes. Miihlens
and Kirschbaum (1924), during the inoculation of human beings with
malaria, observed one case which proved resistant to four inoculations,
but became infected after a fifth.
RECOVERY FROM INFECTIONS. — It is a general rule that when once
a parasite has established itself in a host it multiplies actively for some
time, so that the intensity of the infection rises to a maximum, after which
it gradually subsides till finally there may be every reason to suppose
that the infection has completely died out. This recovery may be due
to two causes. Firstly, the fluids of the host may gradually change with
the production of substances injurious to the parasite, or possibly by the
loss of substances which are necessary to the continued development of
the parasite; secondly, the parasite itself may become exhausted and
no longer capable of multiplication unless some change takes place. In
the case of coccidial infections of animals, during the early stages there
is active multiplication by schizogony in the intestinal epithelium.
Gradually this multiplication subsides, and there are produced an increasing
number of male and female gametocytes, which lead to syngamy and the
formation of oocysts, which leave the body. Eventually the sexually
differentiated forms alone can be found, and finally the infection ceases
when all these have been eliminated. In this case it is possible that the
host produces substances which act deleteriously on the parasite, and lead
to the production of the sexual stages, which are bound up, in the case
of the coccidia and other forms, with the distribution of the parasite to
other hosts. On the other hand, it may be that each sporozoite freshly
142 IMMUNITY IN PROTOZOAL INFECTIONS
introduced is only capable of reproducing asexually a certain number of
times, and that when this is completed sexual forms are produced. It
seems clear that the production of substances Vvdiich are generally termed
antibodies in the blood of the host plays some part, for when once a host
has passed through an acute infection it is rarely possible to produce as
intense infection again, while in many cases a complete immunity to
further infection is developed. But the second factor also comes into
play, for it has been shown that as one infection is subsiding it may be
possible to reinoculate the host with the same organism, so as to produce
a superimposed infection. Noller (1917) has shown that frogs which
have passed through the acute stage of an infection with Trypanosoma
rotatorium may be reinfected, though trypanosomes remaining from the
first infection are still present in the blood in small numbers. Such a
superimposed infection may become as intense as the first one, and even
bring about the death of the host. Similarly in the case of piroplasmosis
of cattle, Ed. Sergent and his co-workers (1924) have demonstrated that
superimposed infections are possible. They found that the appearance
of parasites in the blood after the second inoculation was not accompanied
by any of the symptoms which followed the first infection. The animals
had been rer^^ered partially immune, so that the injurious effects of the
parasite were resisted, though its development was not prevented. In
order to distinguish this partial immunity or tolerance immunity from
an absolute or true immunity they have introduced the term " premuni-
tion." It occurs in the infections with Babesia bigemina. The term is
not applicable to infections with Babesia nmtafis, which can also be super-
imposed on an already existing infection, for the first infection is not
accompanied by any recognizable symptoms. This parasite appears to
produce no immunity whatever. Hoare (1923) found that sheep, when
constantly infested with keds, always harbour Trijixinosoma melo'pliagium ,
but if the animals are freed from keds the infection in the sheep gradually
subsides, till after two or three months it can no longer be detected. It
is evident that the batch of parasites introduced by the keds on one
occasion have only a limited term of existence in the sheep, and it wovdd
appear that this is dependent rather on what may be termed an exhaustion
of the parasite than on changes in the sheep, for infection may at any
time be re-established by further introduction of trypanosomes from the
keds. This exhaustion, however, may be the result of continued action
of the antibodies producing a gradual weakening of the parasite.
It seems clear that in the case of many human Protozoal infections,
such as malaria, trypanosomiasis, and amoebiasis, in localities in which
these diseases are prevalent, individuals are constantly being infected
with fresh batches of parasites, and a condition resembling that in the
EECOVERY FROM INFECTION 143
sheep, just mentioned, occurs. In malarious countries, from their birth
children are constantly being bitten by infected mosquitoes, and it is
not unreasonable to suppose that the long duration of malarial infection
in cliildren in these countries is due to continuous reinfection. It has
been demonstrated by Miihlens and Kirschbaum (1924) that human
beings can be reinoculated with malaria when apparent recovery from
a first infection has taken place. They can even be inoculated a third
time, but the successive infections are of decreasing intensity. In view
of the difficulty in determining the complete elimination of parasites
from infected individuals, it is possible that some of these cases were
illustrations of superimposed infections. Recently Van Loon and
Kirschner (1924) in the Dutch East Indies have noted that the native is
relatively immune to inoculation of malarial parasites. In certain cases
it was found to be impossible to produce infection, though large doses of
blood heavily infected with Plasmodium, vivax were injected four or five
times. In other persons who had not experienced a lifelong exposure
infection was readily produced. Sergent, Et. and Ed. (1921c), have,
however, shown that birds in the chronic phase of a malarial infection
do not respond, or respond very slightly, to inoculations with a further
infective dose of parasites. A very striking illustration of the effect of
repeated doses of a virus was an observation made by Miller (1908) on
the haemogregarine Hepatozoon miiris of rats. As a rule these animals
which are infected by the ingestion of mites, acquire an infection which
does not appear to disturb the host in any way. Miller, however, found
that a batch of rats, which were so heavily infested with mites that con-
stant infection with large doses of virus was occurring, were very heavily
infected with the parasite, and that a definite pathological condition
resulted. When recovery from an infection is considered, a distinction
has to be drawn between the cases which have had a single dose of virus
and those which are repeatedly inoculated. Though recovery in a com-
paratively short time appears to be characteristic of many Protozoal
infections, this is not invariably the case. Animals such as cattle, horses
and dogs, which are liable to piroplasmosis, pass through an acute phase
when parasites are exceedingly numerous in the blood. Afterwards the
infection subsides, so that finally the organisms can no longer be detected
by microscopical examination of the blood. Nevertheless, it can be
demonstrated that they are still present and persist for years, bv the
inoculation of large quantities of blood into animals which have never
had the infection. In many cases of infection with Entamceba histolytica
the aniffibse persist in the intestine indefinitely. In these cases a balance
between the host and parasite has been reached, so that the former is
injured to a minimal extent, while the parasite can reproduce sufficiently
144 IMMUNITY IN PROTOZOAL INFECTIONS
to maintain itself. Hosts in this condition are usually termed carriers.
The practical difficulty associated with this type of infection is the im-
possibility of being absolutely certain that any infection has entirely
vanished. In the treatment of trypanosomiasis, leishmaniasis, malaria,
amoebic dysentery, and other infections, this difficulty is constantly being
encountered.
Another feature of recovery from infection has to be noted, and that
is that frequently during the period of abatement of the infection, when
the host may be said to be obtaining a mastery over the parasite, a relapse
occurs in which a fresh outburst of activity on the part of the parasite
leads again to an intense infection. It must be supposed that under
these conditions the control of the host over the parasite has broken down,
and anything which leads to this may bring about a relapse. It is well
known that in malarial infections of man a sudden exposure to cold,
shock resulting from accident, or the intercurrence of some other infection,
may lead to the appearance of large numbers of parasites in the blood.
Such periodic variations in the intensity of infections may, however,
be a feature of the development of the parasite. This periodicity is quite
distinct from the periodicity which results from the developmental cycle,
like that of parasites of malaria, which reproduce only at regular intervals.
In human trypanosomiasis, and also in animals experimentally infected,
it has been frequently noted that the number of parasites in the blood
is not constant. The trypanosomes may be comparatively numerous
on one day and absent on another. This is probably due to variations
in the rate of multiplication, but it is possible that it is also dependent
on variations in the rate of mortality of the trypanosomes resulting from
irregularities in the antibody content of the body fluids of the host. No
satisfactory explanation of this type of periodicity has been discovered.
ACQUIRED IMMUNITY.— Under this heading will be considered the
immunity to infection which a host acquires as a result of an infection.
It has already been shown that in some cases infection may persist for
many years in a latent form, and though there may be considerable
difficulty in determining the complete elimination of an infection, there
is reason to suppose that sometimes a host becomes completely free.
After recovery of this kind the host may be absolutely immune to further
infection, the type of immunity being known as active immunity. The
observations of Van Loon and Kirschner, who failed to produce malarial
infections in natives of the Dutch East Indies, have been referred to above.
In human infections with Leishmania tropica the disease oriental sore, if
allowed to run a natural course, will produce in most cases an absolute
immunity to further infection, so much so that artificial production of
oriental sore by inoculation on an unexposed part of the body has been
ACQUIRED IMMUNITY 145
employed as a means of avoiding the risk of the disfiguring natural
infection on an exposed part such as the face. Another illustration of
absolute immunity conferred by a single infection occurs in the case of
East Coast fever of cattle due to infection with Theileria parva. Animals
which have recovered from one attack are immune for the rest of life.
The same remark applies to rats which have recovered from an infection
with Trypanosoma lewisi. Again, in the case of many of the disease-
producing trypanosomes it has been found that certain animals, such as
the goat and sheep, though acquiring an infection, eventually recover to
such an extent that trypanosomes can no longer be detected. In this
condition they are immune to further inoculations with the same trypano-
some. As in the case of naturally immune animals, these actively im-
munized hosts have been employed as a means of differentiating species.
If it is desired to distinguish two trypanosomes which resemble one
another morphologically, one of them is inoculated into a goat. When
the animal has recovered and is no longer susceptible to inoculation with
this trypanosome, it is inoculated with the other. If infection occurs, it
is assumed that the trypanosomes are different. Though the experiment
undoubtedly indicates a physiological difference between the trypano-
somes, it is far from clear that they belong to distinct species. The test
has been applied by Laveran and Mesnil and others to a group of trypano-
somes which resemble Trypanosoyna evansi, with the result that a number
of species of very doubtful value has been created. Similarly, in the
case of piroplasmosis the test has again been applied. Animals which
recover from an acute attack pass into a chronic phase, during which the
parasites show a gradual diminution in their numbers, till finally they can
no longer be detected except by the inoculation of comparatively large
quantities of blood into a susceptible animal. It has been shown by
Ed. Sergent and his co-workers (1924) that in the case of Babesia bigemina
it is possible to produce a superimposed infection in wdiich parasites
appear in the blood, but this is unaccompanied by symptoms. The infec-
tion, moreover, is less intense than the original one, the parasites quickly
disappearing again. Stockmann and Wragg (1914) showed that cattle
which had recovered from an infection with B. bigetnina, and were
immune to further inoculations with this parasite, were nevertheless
susceptible to Babesia bovis, and behaved, as regards symptoms and
intensity of infection, as animals at their first infection. In this instance
there were morphological differences which justified the separation of
the two parasites as distinct species. On the other hand, a form of piro-
plasmosis in cattle in South America is due to a parasite resembling
B. bovis. Brumpt (1920) showed that cattle which had recovered from
the infection with this parasite were still susceptible to inoculation with
I. 10
146 IMMUNITY IN PROTOZOAL INFECTIONS
the one from South America. There appear to be slight morphological
differences between the two, but whether these are sufficiently distinct
to justify the recognition of the South American form as a distinct species,
Babesia argentina, apart from the cross-immunity test, is open to question.
In connection with piroplasmosis of horses, Nuttall and Strickland (1910)
and du Toit (1919) showed that animals recovered from infections with
Babesia caballi were still liable to infection with Babesia equi. Here again
morphological characters enable the species to be distinguished. The
difficulty of accepting the test as a means of distinguishing species is
illustrated by the experiments of Laveran and Nattan-Larrier (1913) on
canine piroplasmosis. The disease occurs in dogs both in France and
North Africa, and on morphological grounds appears to be due to the same
parasite, Babesia canis, in both places. Yet dogs which have recovered
from infection with the French virus and are completely immune to
further inoculations are susceptible to the North African virus. It would
appear imj)ossible on these grounds alone to recognize two species of
parasite.
As in the case of natural immunity, acquired immunity is dependent
on antibodies which appear in the blood, for the serum of the animals
which have recovered or have been infected for a length of time sufficient
to allow of the production of these substances can be employed as a
curative agent in the case of infected animals. Furthermore, the serum,
when injected into an animal before it is exposed to infection, may entirely
prevent an infection. In this case the immunity is known as passive
immunity, because the host itself has taken no part in the production
of the antibodies, which are merely introduced from another animal.
The extensive investigations of Rabinowitsch and Kempner (1899), and
of Laveran and Mesnil (1901a), on infections of rats due to TryjKinosoma
lewisi threw considerable light on this subject. Infected rats pass through
an acute phase followed by a chronic one, from which ultimate recovery
takes place. The animals are completely immune from reinfection. A small
quantity of the serum (0-5 c.c.) of a recovered animal, if inoculated into
the peritoneal cavity of a rat, will entirely prevent infection when trypano-
somes are inoculated twenty-four hours later. This property is possessed,
though to a less extent, by the serum of animals, such as goats and sheep,
which have recovered from infections with the pathogenic trypanosomes,
and animals, such as cattle, which are in a very chronic stage of infection.
Taliaferro has shown, in the case of T. leivisi, that this is due to the
appearance in the blood of the rat of a substance which inhibits the repro-
duction of the trypanosomes (see p. 467).
Many attempts to produce an active immunity by other means than
actual infection and natural recovery have been made. So-called attenu-
ACQUIRED IMMUNITY 147
ated strains, such as trypanosomes which, as a result of exposure to heat
or other adverse conditions, have lost their power of producing actual
infection, have been injected into animals. In a similar manner killed
trypanosomes, trypanosomes which have been broken up by immersion
in fluids which bring about cytolysis, dried trypanosomes, as well as
cultural forms of trypanosomes, which often have ceased to be infective
to animals, have been tried, but in none of these cases was satisfactory
evidence obtained that the animals inoculated with these altered trypano-
somes had acquired any immunity to inoculation with a virulent strain,
though the application of certain serological tests, such as that of the
complement fixation, has demonstrated that a specific change may have
taken place in the serum of the animals. The response as regards pro-
duction of immunity cannot be compared with that which occurs in the
case of bacteria. Ponselle (1923a) has found that by keeping the heart-
blood of a mouse containing Trypanosoma brucei for twenty-four hours in
a medium of dihydrogen potassium phosphate and hydrogen disodium
phosphate it loses its power of infecting mice, but if injected will render
mice immune to infection with unaltered Trypanosoma brucei (see p. 454).
The bulk of work in connection with the production of immunity in
Protozoal infections has been carried out with trypanosomes, but certain
investigations have been made with other Protozoa. Thus, the Sergents,
Et. and Ed. (19216), have produced a certain degree of immunity in the
case of the parasite of bird malaria, Plasmodium, prcecox. Normal canaries
were very easily infected with this parasite, only 0-72 per cent, resisting
infection out of 965 birds inoculated. If canaries are inoculated with
the sporozoites of the parasite which have been rendered non-infective by
keeping them for twelve to forty-eight hours after removal from the
mosquito, a certain degree of immunity results. It was found that
29*5 per cent, of twenty-four canaries thus treated resisted subsequent
inoculation with the parasite. Similarly, it was found that if the blood
of a canary was drawn off after it had been inoculated with the parasite,
and before the infection had established itself by the appearance of
parasites in the blood, this blood, if injected into healthy birds, produced
an immunity which protected from subsequent inoculation 21 "3 per cent,
of sixty-one canaries.
Many observers have attempted to produce immunity in cattle against
infection with Babesia bigemina and Theileria parva. From both these
infections animals may recover naturally, and possess an absolute immunity
to further infection, but the death-rate is ahvays high, especially in the
case of East Coast fever. No means of producing an immunity apart from
actual infection are known, though in the case of piroplasmosis it is
possible to inoculate the animals at a time when they are best able to
148 IMMUNITY IN PROTOZOAL INFECTIONS
withstand the disease. It is known that young animals recover more
easily than older ones, and that the disease is less severe at a certain
season. It has been shown by a number of observers that by inoculating
young animals with Babesia bigefnina at this particular season it is possible
to obtain a higher percentage of recoveries, and hence of permanently
immune animals, than if they had been exposed to natural infection.
In the case of East Coast fever also young animals are less seriously
affected than older ones, and it would be expected that a similar method
of protection could be applied. As will be shown below, it is not as.
a rule possible to transmit this disease by the inoculation of the blood
of an infected animal, but Meyer (1909) found that this could be effected
by inoculating the macerated spleen and lymphatic glands in which the
reproducing forms occur. By the inoculation of young animals with
emulsions of these organs Theiler (1911a, 19126) noted that though a
number acquired a severe and fatal disease, a much larger number survived
and recovered completely. As many as 50 per cent, of those which
survived proved resistant when exposed to infection by ticks under
natural conditions. Somewhat similar results were obtained by Wolfel
(1912) and Spreull (1914). In the production of immunity by these
methods it is important, as demonstrated by Theiler (1908) and Lignieres
(1903), to employ the particular strain of virus to which subsequent
exposure will occur. A previous infection with Babesia bigemina of
European origin w^ill not produce immunity against the parasite of South
Africa.
Mechanism of lynvnunity. — During the development of an immunity
the blood of the animal acquires certain properties which it did not pre-
viously have, but which are possessed by the blood of naturally immune
animals. It has already been pointed out that the serum of such an
animal will produce a degree of passive immunity when injected into a
healthy animal, which is thereby protected against inoculation with the
organism. Such passive immunity is usually of much shorter duration
than active immunity, which is due to the production of antibodies by the
host itself as a result of an actual infection, or the introduction of modified
or dead parasites, or the products of their dissolution, which stimulate the
host to produce the antibodies without actually giving rise to an infection.
Where active immunity is produced without infection, the substance
introduced is termed a vaccine. It is evident that the immunity produced
is dependent upon the presence of several distinct substances, each of
which has its special action. It was first shown by Laveran and Mesnil
(1901a) that during the course of an infection with Trypanosoma lewisi
the leucocytes of the rat's blood are constantly ingesting trypanosomes,
which are ultimately destroyed. It appears that the serum of an immune
MECHANISM OF IMMUNITY 149
animal actually stimulates this phagocytosis, for Laveran and Mesnil
found that if the serum of such an animal was mixed with trypanosomes
and injected into the peritoneal cavity of a rat, there appeared in the
peritoneal fluid numerous leucocytes which devoured the trypanosomes
with avidity. If the trypanosomes were injected alone, this phenomenon
was not observed to anything like the same extent. Levaditi and Mutter-
milch (1911) showed that the serum affected the trypanosomes in such a
way that they attached themselves to the leucocytes. This was inde-
pendent of the actual process of phagocytosis, for it was found that
attachment to killed leucocytes also occurred. It was shown by Mesnil
and Brimont (1909) that if immune serum were allowed to act upon
Trypanosoma lewisi a change took place, so that the trypanosomes were no
longer able to infect rats even if they were carefully washed free of serum.
It would thus appear that the protective action of the serum is a result
of its power of causing the trypanosomes to attach themselves to the leuco-
cytes which then engulf them. The serum of animals which are immune
to Trypanosoma lewisi also has the property of causing trypanosomes
to become agglutinated into clumps when blood containing them is
mixed with the serum (see p. 452). The presence of agglutinins in the
serum has been shown to occur in the case of other trypanosome
infections.
Another property which the serum may acquire is that of producing
cytolysis, or the gradual swelling up and dissolution of trypanosomes
exposed to its action. It was shown to occur in the case of infections
of animals with the pathogenic trypanosomes by Levaditi and Mutter-
milch (1909), amongst other observers. They also demonstrated that the
serum acquired the property of deviating the complement, a reaction
which has found a practical application in the diagnosis of trypanosome
infections (see p. 452). It seems evident that recovery from any infection
is dependent on the development of antibodies in the blood, which act
upon the particular parasites in various ways. This action of the serum
of an immune animal is specific for the parasite which stimulated its
production. On this account serological tests, which are similar to the
inoculation tests referred to above, have been employed as a means of
differentiating parasites. If the serum of an immunized animal behaves
towards an unidentified trypanosome as it does towards the one which
caused the immunity, then, provided that there is morphological similarity,
it is concluded that they are identical. On the other hand, it is main-
tained by some that, in spite of morphological identity, if the serum fails
to act it is proof of a specific distinction. It is possible that a natural
recovery would never take place unless antibodies were produced, and
that a parasite would continue to multiply continuously till the host
150 ACTION OF DRUGS IN PROTOZOAL INFECTIONS
was destroyed. Certain strains of pathogenic trypanosomes can be
handed on indefinitely from mouse to mouse by direct inoculation of
blood without there being any evidence that the rate of multiplication
by binary fission slackens in any way. In these cases the trypanosomes
multiply so rapidly that the host is overcome by the parasite before any
degree of immunity capable of checking the infection has been developed.
At each inoculation the trypanosomes are introduced into a new host
which has no immune bodies, and multiplication is continued with the
same result. For the development of immunity it is essential that the
rate of multiplication of a parasite shall not be so great as to bring about
destruction of the host before it has time to respond to the infection by
the production of sufficient antibodies to check the development of the
parasite. From the point of view of the parasite this is the condition
most favourable to its survival, and it appears to be the one which obtains
in most, if not all, natural infections.
A parasite may acquire the power of resisting the antibodies in the
serum. Jacoby (1909a) obtained a strain of Trypanosoma brucei which
was resistant to human serum, which normally will cause the disappear-
ance of the trypanosomes from the blood of mice. By repeatedly injecting
small quantities of normal human serum into an infected mouse and con-
tinuing the process in subinoculated mice, a strain of trypanosomes was
eventually secured which, as regards its development in mice, was un-
influenced by as large a dose (2 c.c.) of human serum as the mouse could
tolerate. Leboeuf (1911) in a similar manner obtained races of T. brucei
which were resistant to the serum of baboons.
ACTION OF DRUGS IN PROTOZOAL INFECTIONS.
It is possible that the disappearance of parasites as a result of the
administration of drugs is, in many cases at least, not the result of a
direct poisonous action of the drug upon the parasite. It would seem
natural to suppose that the good effects observed in amoebic infections
which result from the use of emetine and those following the ingestion
of quinine in malaria are due to the direct effect of the drugs upon the
parasites concerned. It appears that the action may be a much more
complicated one, and that drugs may act indirectly by stimulating the
tissues of the host to produce substances which may be regarded as anti-
bodies which are directly responsible for the suppression of the infection.
In support of this contention may be urged the fact that drugs such as
emetine, which are therapeutically active, are not more toxic to the
organisms when tested in vitro than other drugs which have no thera-
peutic properties. The investigations of Dale and Dobell (1917) on the
DRUG-FAST STRAINS 151
action of emetine are discussed below (p. 255). Morgenroth (1918)
believes that quinine combines with the red blood-corpuscles, and thus
prevents the entry into them of the merozoites of the malarial parasites.
Quite recently Yorke and Macfie (1924a) have suggested that in malaria
quinine acts by causing a destruction of a certain number of parasites,
the broken-down parasites then acting as a vaccine in stimulating the
host to produce antibodies, which finally rid the host of all remaining
parasites. So far the presence of the antibodies has not been demonstrated.
Another illustration of what may be the indirect action of a drug is seen
in " Bayer 205." This medicament is remarkably trypanocidal when in-
jected into animals infected with certain trypanosomes. Animals which
have recovered as a result of treatment or uninfected animals which have
received a dose of the drug remain immune from infection for compara-
tively long periods. It is possible that this resistance is due to the pro-
duction by the host of antibodies as a result of the action of the drug
upon its cells. On the other hand, it has to be remembered that when
a drug is administered to an animal it does not follow that the drug
remains unaltered. The fluids of the body act upon it chemically, and
may in this way produce other substances which are definitely toxic to
the parasites. It is known that arsenic compounds in which the arsenic
is in the trivalent form are toxic to trypanosomes in vitro, and are also
therapeutically active, whereas when the arsenic is in the pentavalent
form there is no action in vitro, though there is a therapeutic action which,
however, requires some time to develop. This difference has been
explained by the fact that in the body of an animal the pentavalent
arsenic radical is transformed into a trivalent one.
Another feature of the action of drugs on Protozoa is the development
of drug-fast strains. In the case of mice, for instance, infected with
pathogenic trypanosomes, the repeated treatment of the infection with
such a drug as atoxyl in doses which are insufficient to prevent a sub-
sequent relapse will finally result in a strain of trypanosome which is
quite unaffected by the drug administered to the animals. This strain
maintains its resistance when passed through a series of new mice, but
as Mesnil and Brimont (1908 b) discovered, it is susceptible to the drug
when inoculated into rats, and is still resistant when again passed into
mice. Such a fact appears to be explicable only on the assumption that
the trypanosomes have not become resistant to atoxyl itself, but to a
substance resulting from the action of the drug on the tissues of the
mouse, but not of the rat. Furthermore, it has been demonstrated that
trypanosomes can be rendered arsenic resistant by the inoculation of
infected mice with substances which contain no arsenic. Many writers
refer to quinine-fast strains of malarial parasites and emetine-fast strains
152 STATUS OF PROTOZOA
of Entamoeba histolytica, but at present there is no reliable evidence that
these actually exist. A drug which fails to act on a parasite may do so
because of some peculiarity on the part of the host. The whole subject
of the method of action of drugs in the treatment of Protozoal infections
is exceedingly complicated, and opens a field for extensive investigations.
A very instructive resume of the subject has been made by Dale (1924).
STATUS OF THE PROTOZOA IN THE ANIMAL KINGDOM.
It is usual to regard the Protozoa as constituting a Phylum wdiich
corresponds in status to one of the various Phyla, such as the Mollusca,
Arthropoda, Vertebrata, etc., into which the rest of the animal kingdom
is divided. This is the view adopted by most zoologists, but Dobell and
O'Connor (1921) have recently expressed the view that the Protozoa
constitute a group of organisms which has a status equal to the rest of
the animal kingdom. According to Dobell's contention, discussed earlier
in this work, the Protozoa are non-cellular animals, while the rest of the
animal kingdom includes all cellular animals. On this account he
divides the animal kingdom into two sub-kingdoms — the Protozoa and
the Metozoa. Such a distinction may still be admitted, though there
would be less reason for its recognition if the generally accepted view were
held that the Protozoa are unicellular, and not merely non-cellular animals.
Dobell, having raised the Protozoa to the rank of sub-kingdom, raises
to the status of Phyla the various classes in which they are divided. For
purposes of this work, however, it is unnecessary to discuss this very
intricate subject, and, following the more orthodox view, the Protozoa
will be still regarded as constituting a Phylum.
PART II
SYSTEMATIC DESCRIPTION OF THE PROTOZOA
WITH SPECIAL REFERENCE TO PARASITIC AND COPROZOIC FORMS
CLASSIFICATION OF THE PROTOZOA.
SUB PHYLUM :
PLASMODROMA
CLASS: RHIZOPODA
Order: AM(EBIDA
HELIOZOA
RADIO LARIA
FORAMINIFERA
MYCETOZOA
C'i.-4.s.s. MASTIGOPHORA
SUB-CLASS: Phytomastlgina
Order: GHRYSOMONADIDA
CRYPTOMONADIDA
DINOFLAGELLATA
EUGLENOIDIDA
PHYTOMONADIDA
SUB-CLASS: Zoomastigina
Monozoic Forms
Order: PROTOMONADIDA
HYPERMASTIGIDA
CYSTOFLAGELLATA
Diplozoic Forms
Order: DIPLOMONADIDA
Polyzoic Forms
Order: POLYMONADIDA
CLASS: CNIDOSPORIDIA
Order : MYXOSPORIDIIDA
Suh-Order : Eurysporea
,, Sphaerosporea
„ Platysporea
Order: MICROSPORIDIIDA
Suh-Order: Monocnidea
„ Dicnidea
Order: ACTINOMYXIDIIDA
UNDETERMINED
SARCOSPORIDIA
GLOBIDIUM
HAPLOSPORIDIA
155
PHYLUM: PROTOZOA
CLASS: SPOROZOA
SUB-CLASS: Coccidiomorpha
Order: COCCIDIIDA
Suh Order: Eimeriidea
,, Haemosporidiidea
„ Piroplasmidea
Order: ADELEIDA
Suh-Order: Adeleidea
„ Haemogregarinidea
SUB-CLASS: Gregarinina
Order: SCHIZOGREGARINIDA
EUGREGARINIDA
Sub Order: Acephalinidea
„ Cephalinidea
SUB-PHYLUM: GILIOPHORA
GROUP 1: PROTOCILIATA
CLASS: OPALINATA
GEO UP 2: EUCILIATA
CLASS: CI LI ATA
SUB-CLASS: Aspirigera
Order: HOLOTRICHIDA
Sub-Order .
Astomatea
Stomatea
Section 1 : Gymnostomata
Section 2 : Trichostomata
SUB-CLASS: Spirigera
Order: HETEROTRICHIDA
OLIGOTRICHIDA
HYPOTRICHIDA
PERITRICHIDA
CLASS: SUCTORIA
156 PHYLUM: PROTOZOA
PHYLUM: PROTOZOA GOLDFUSS, 1817.
The phylum Protozoa^ as defined above, is the subdivision of the animal
kingdom in which all unicellular animals are grouped. It may be divided
into two sub-phyla, as suggested by Doflein (1901). The first of these
is the PLASMODROMA, which includes the forms which have
pseudopodia or flagella, and in which syngamy, where it is known to
occur, consists in the complete fusion of two gametes. The second sub-
phylum is the GILIOPHORA, which comprises those Protozoa
which have numerous cilia as motile organs, a special type of binuclearity
(macronucleus and micronucleus), and a process of syngamy in which two
individuals temporarily associate, undergo exchange of nuclei, and then
separate. The class Opalinata, in which syngamy is of the type seen
amongst the Plasmodroma while the binuclearity characteristic of the
other classes of the Ciliophora is wanting, forms a connecting link between
the two sub-phyla.
A. SUB-PHYLUM: PLASMODROMA DOFLEIN, 1901.
This, the first of the sub-phyla into which Doflein divides the Protozoa,
includes forms which have either pseudopodia or flagella as organs of
locomotion, and the parasitic Sporozoa which, owing to their mode of life,
have been modified in various ways. There is either a single vesicular
nucleus or more than one are present. Syngamy takes place by the
complete fusion of gametes, which may be alike (isogamy) or different
(anisogamy). In many forms, after a period of asexual reproduction,
syngamy, followed by a different method of reproduction, occurs (alterna-
tion of generations).
The sub-phylum contains four classes of Protozoa, two of which include
mainly free-living forms, while two contain forms which are exclusively
parasitic. One class is characterized by the amoeboid form of the body
which produces pseudopodia as organs of locomotion, while in another,
though the body may be amoeboid, it possesses one or more flagella. The
Protozoa of the first type belong to the class RHIZOPODA, and those
of the second to the class M ASTIGOPHORA. The separation of these
two classes is rendered difficult by the fact that certain organisms which
are amoeboid and devoid of flagella for the greater part of their existence
may at certain stages develop flagella, while, conversely, forms which
usually possess flagella may have a purely amoeboid phase.
As regards the parasitic types, many observers have grouped them
together in the one class Sporozoa, which was divided by Schaudinn
PLASMODKOMA AND CILIOPHORA 157
(1900) into the Telosporidia and Neosporidia. It appears, however, that
these two groups are so fundamentally different that it is better to follow
Hartmann (1907) and place the Neosporidia in a separate class, for which
Doflein's name Cnidosporidia may be employed, and to reserve the
Sporozoa for the forms included in Schaudinn's group Telosporidia. The
class CNIDOSPORIDIA includes parasitic Protozoa, which are either
amoeboid or almost, if not entirely, motionless. They produce, by a com-
plicated process of development in which several cells take part, very
characteristic encysted stages or spores which are peculiar in possessing
special bodies called polar capsules, from which long filaments can be ex-
truded. The class SPOROZOA also comprises parasitic forms, which
reproduce characteristically by schizogony. After syngamy the zygote
gives rise to a number of sickle-shaped sporozoites. These are either free
within the oocyst which forms around the zygote, or they are enclosed in
a number of secondary cysts, the sporocysts, which are formed inside
the oocyst. Schaudinn included the Sarcosporidia in his group Neosporidia.
These parasites, however, have little in common with the true Cnidosporidia,
and though they produce bodies which are called spores, these are structur-
ally quite different from those of the Cnidosporidia. In fact, very
liUle is known about the true nature of the Sarcosporidia, which will be
considered with certain other parasitic forms (Haplosporidia, Globidium)
of undetermined affinities.
B. SUB-PHYLUM: CILIOPHORA DOFLEIN, 1901.
Ciliophora is the name given by Dofiein to the second of the two sub-
divisions into which he divides the Protozoa. The organisms included in
this group have a comparatively complex structure, and in this respect
may be considered to be the most highly specialized of the Protozoa
(Fig. 70). The body is not, as a rule, subject to changes of shape, unless
as a result of external pressure, there being a definite body form for each
individual. The most characteristic feature is the possession of numerous
hair-like processes, the cilia, which cover either the whole or only part
of the body surface. The cilia are used as organs of locomotion, or for
producing currents in the water for the intake of food. They may also
serve as organs of special sense, such as taste or touch.
The cytoplasm is differentiated into an endoplasm, which contains the
nuclei, contractile vacuoles, and food vacuoles, and a highly-organized
ectoplasm. The latter consists of a superficial membrane, the pellicle,
within which is a layer containing myonemes or contractile fibres, spaces
and canals of an excretory system, basal granules from which the cilia
arise, and sometimes trichocysts, which are small bodies from which
158
PHYLUM: PROTOZOA
Fig. 70. — Diagrammatic Figure of Parame-
cium caudatum ( x ca. 500). (From Minciiin,
1912, AFTER Lang.)
P., Peristome groove; o, mouth; ces., oesophagus with
undulating membrane; f.v.', food vacuole forming
at end of oesophagus; /.r., other food vacuoles; c.v.,
contractile vacuole with surrounding channels lead-
ing to it; ex., excretory crystals; N, macronucleus;
74, micronucleus ; tm, trichocj'sts ; al., alveolar layer;
p., pellicle; um, undulating membrare
threads are discharged. A de-
finite mouth opening or cyto-
stome may, or may not, be
present.
Though the Ciliophora agree
with one another in the posses-
sion of cilia, they differ funda-
mentally as regards their nuclei.
In what may be regarded as the
more primitive forms (Opali-
nata) there are present in each
individual two or more nuclei
which are all of one type, in
which respect an approach to
the Plasmodroma is made.
When syngamy occurs uninu-
cleated forms are produced, and
these, which are gametes, unite
in pairs, with complete fusion of
the bodies and nuclei. In other
forms there are typically two
morphologically distinct nuclei,
one of which is a macronucleus
and the other a micronucleus.
During syngamy the macro-
nucleus disintegrates and takes
no part in the process, while
the micronucleus divides. Two
individuals associate, and one
of the daughter micronuclei of
each individual migrates into
the other and unites with its
remaining daughter micronu-
cleus. When this has taken
place, the associated or con-
jugating individuals separate
and continue to lead an inde-
pendent existence. On the basis
of this distinction Metcalf (1918)
recognizes two groups, the
PROTOCILIATA and the
EUCILIATA. The members
PLASMODROMA AND CILIOPHORA 159
of the group Protociliata (OPALINATA) possess cilia during the whole of
their existence, whereas amongst the Euciliata certain forms (CI LI ATA)
constantly have cilia, while others (SUCTORIA) have them only in their
youngest free-swimming stages, which, however, soon attach themselves
to objects, lose their cilia, and develop suctorial tentacles.
Multiplication amongst the Ciliophora is by binary fission or bud
formation. Amongst the multinucleated Protociliata nuclear division
proceeds somewhat irregularly, and division of the body leads to the
production of two daughter multinucleated individuals, which may, or
may not, possess an equal number of nuclei. In the case of the Euciliata,
which typically possess one macronucleus and one micronucleus, both
these nuclei divide, so that each daughter individual possesses a pair of
nuclei similar to that of the parent.
From the foregoing remarks it will be seen that the phylum Protozoa
may be subdivided as follows:
A. SUB-PHYLUM: PLASMODROMA DOFLEIN, 1901.—
Movement is effected by pseudopodia or flagella, and syngamy, where it
is known, takes place by the complete fusion of gametes.
I. CLASS: RHIZOPODA VON SiEBOLD, 1845.— The predominating
phase is amoeboid, locomotion being effected by means of pseudopodia.
II. CLASS: MASTIGOPHORA Diesing, 1865.— The predominat-
ing phase is flagellate, locomotion being effected by means of flagella.
III. CLASS: CNIDOSPORIDIA Doflein, 1901.— Parasitic forms
which are frequently amoeboid, but which produce characteristic spores
provided with polar capsules from which long filaments can be extruded.
IV. CLASS: SPOROZOA Leuckart, 1879.— Parasitic forms which
reproduce typically by schizogony, and which give rise to sporozoites
enclosed in resistant oocysts after syngamy has occurred.
B. SUB-PHYLUM: CILIOPHORA DOFLEIN, 1901.— Move-
ment is effected by means of cilia.
GROUP 1: PROTOCILIATA Metcalf, 1918.— There are two or more
nuclei, which are all of one type. Syngamy is effected by the complete
fusion of uninucleated gametes.
I. CLASS: OPALINATA.— With the characters of the group.
GROUP "1: EUCILIATA Metcalf, 1918.— There is a definite nuclear
dimorphism, the nuclei being of two types (macronuclei and micro-
nuclei). When syngamy takes place the macronuclei disintegrate, the
micronuclei alone taking part in the process, which is characterized
by the exchange of the products of division of the micronuclei between
two temporarily associated individuals.
160
CLASS: RHIZOPODA
Cilia are present throughout
I. CLASS: CI LI ATA Perty, 1852
the life of the organism.
II. CLASS: SUCTORIA Claparede and Laghmann, 1858.— Cilia
are present only during the young stages, which usually attach themselves
to objects, lose their cilia, and develop suctorial tentacles,
A. SUB-PHYLUM: PLASMODROMA.
I. CLASS: RHIZOPODA V. SlEBOLD, 1845.
CLASSIFICATION.
CLASS: RHIZOPODA
Order: AMCEBIDA
Family: AMCEBID.^:.
Genibs : Amoeba.
,, Hartmannella.
,, Vahlkampfia.
„ Sappinia.
„ Pelomyxa.
„ Entamoeba.
„ Endamoeba.
„ Endolimax.
„ lodamoeba.
„ Dientamoeba.
Family: PARAMCEBID^.
Genus : Paramoeba.
Family: DIMASTIGAMCEBID^.
Genus : Dimastigamoeba.
Family : RHIZOMASTIGID^.
Genus : Mastigamoeba.
Mastigella.
,, Mastigina.
Order: HELIOZOA
RADIOLA.RIA
FORAMINIFERA
Genus : Chlamydophrys.
Order : MYCETOZOA
The Protozoa belonging to the class Hhizopoda (= Sarcodina Hertwig
and Lesser, 1874) are typically organisms which move, and ingest food
by means of pseudopodia. These are cytoplasmic processes of varying
form which are protruded from the surface of the body, and which,
after fulfilling their function, are withdrawn. They may be merely
short, stumpy elevations, or more elongate finger-like processes (Fig. 5).
Sometimes they are very fine, and give the organism a radial appear-
ance. Such radiating pseudopodia, seen typically amongst the Heliozoa,
may be supported by stiff axial fibres, which cause them to be more
permanent structures (Fig. 71). There may be but a single pseudo-
podium, another one being protruded only when the first has been
withdrawn; several may be developed at one time, or large numbers are
produced simultaneously from the whole surface of the body. In the
latter case, anastomoses may be formed between adjacent pseudopodia,
so that the organism has the appearance of being surrounded by a loose
network of cytoplasm. They may be shorter than the diameter of the body,
or many times this length. The cytoplasm may be differentiated into a
ORGANIZATION OF RHIZOPODA
161
superficial clear hyaline layer, the ectoplasm, and a more granular fluid,
endoplasm. A pseudopodium may be formed of ectoplasm alone, or it
may have a core of endoplasm. Within the endoplasm are to be found
the nuclei, food vacuoles, and various granules, while contractile vacuoles
are present in the forms which are not parasitic.
In some Rhizopoda (Foraminifera) the ectoplasm secretes a protective
shell known as a theca, which covers the
body almost entirely (Fig. 72). A pore is left,
through which pseudopodia are protruded,
to enable the organism to move about and
secure its food. In addition to the main open-
ing, the shell may be perforated by niimerous
minute pores. Shells of this kind may be
formed when the organism is only partially
grown, and with increase in size a new and
larger shell is made. With further growth
others still larger are produced, and these,
remaining attached to one another, give rise
to many chambered shells, the separate
sections of which are variously arranged
according to the particular species. The
Radiolaria have a perforated membranous
central capsule, which divides the cytoplasm
into a central mass in which the nucleus
lies, and an extracapsular portion or mantle.
In the latter siliceous skeletal structures
of various kinds are developed. These take
the form of shells or spicules, which are often
conspicuous for the beauty of their design.
Whatever may be the character of the organ-
ism, the predominating phase in develop-
ment is one which produces pseudopodia,
and in the majority no other phase is
known to exist. In some, however, a
transitory flagellate phase occurs, during
which the organism resembles in every
respect a member of the class Mastigophora. On this account it is
exceedingly difficult to define accurately the limits between the two
classes Rhizopoda and Mastigophora. In the latter the flagellate phase
is the predominating one, while in the former it is the pseudopodial or
amoeboid phase. It has been demonstrated in the case of certain organ-
isms (DiniasdtfdiHdha) that the ama'boid or flagellate phase can be pro-
I. 11
Fig. 71. — Actinosphceyiuni eich-
horni : An Entire Individual
( X 90) AND Portion of An-
other ( X 360). (From Lan-
kester's Treatise on Zoology,
after Leidy, 1879.)
c.y.i,Contractile vacuole ;c.«.o, position
of another contractile vacuole which
has just collapsed; cr.fndd vacuole;
r., rotifer just engulfed; y/.v , pscudd-
Tiodium ; psa.,axis of pscutlupodiuni ;
N., nucleus.
162
CLASS: RHIZOPODA
diiced by altering tlie character of the medium in which the organisms
are growing.
I '/w n\MX wv'■
Fig. 72. — Types of Shelled Riiizopoda. (From Lang, 1901, A and B, after
Max Schultze; C, after R. Hertwig.
A. Gromia oviformis with ingested Navicula and seven nuclei ( x ca. 50). The
pseudopodia round the shell should be three times as long as represented.
B. Rotalia freyeri, showing spirally arranged chambers (x ca. 90).
C. Spiroloculina sp., showing four chambers and nuclei (x ca. 30),
In some instances {Mastig amoeba, Mastigina, Mastigella) the body
of the organism resembles an amoeba, in that pseudopodia are formed for
ORGANIZATION OF RHIZOPODA
163
the purpose of locomotion and ingestion of food, while a flagellum is present
as a permanent structure (Fig. 73). Such organisms, though usually placed
amongst the Rhizopoda, might with equal justification be classed with
the Mastigophora.
The majority of the Rhizopoda possess a single nucleus, which divides
only when multiplication occurs. In some cases, however, two nuclei are
present, while in others the organisms are multinucleate. Some of the
multinucleate forms (Mycetozoa) are relatively large, each consisting of
a sheet of cytoplasm (plasmodium) easily visible to the naked eye. Re-
\
m
- o
I.
M
'P
m 9j
m
m^
Fig. 73. — Mastigina hylw : Free and Encysted Stages ( x 1,
(After Collin, 1913
1. Free amoeboid form with four nuclei, to one of which a flagelkim is attached.
2. Encysted form with two nuclei. 3. Encysted form with four nuclei.
production amongst Rhizopoda usually takes place by binary fission, or
simple division into two more or less equal parts. In association with
encystment, when a protective capsule is formed around the organism,
the single nucleus, by repeated divisions, may give rise to a number of
nuclei, and the multinucleate cytoplasmic body within the cyst then
segments into a corresponding number of daughter individuals. The
latter may be amoeboid organisms, like the adults from which they were
derived, or they may be flagellated bodies which swim about for some
time before losing their flagella and again becoming amoebae. In the case
of some of the Foraminifera, there is a complicated life-cycle involving
164 CLASS: RHIZOPODA
an alternation of generations (Fig. 74). Thus, in Polystomella crispa,
a many-chambered shelled form studied by Lister (1895) and Schaudinn
u ^
^
>1
Fig. 74. — Stages in the Life-Cycle of Polystomella crispa ( x ca. 70). (After
Lang, 190L)
A. Young megalospheric individual with three chambers.
B. Fully-grown megalospheric individual.
C. Megalospheric individual in process of formation of flagellated spores.
D. Flcigellated spore more highly magnified. E. Fully -grown microspheric individual.
F. Microspheric individual in jirocess of formation of daughter amoeboid forms which become
megalospheric forms.
1-3, nuclei of various sizes; 4, fragmenting nucleus; 5, chromatin granules.
(1895), the individual (microspheric form) becomes multinucleate, and
then gives rise to a number of daughter amoeboid forms which escape
from the shell (Fig. 74, E and F). Each of these forms a relatively large
ORDERS: AMCEBIDA AND HELIOZOA
165
shell, and grows into a many-chambered individual of another type
(megalospheric form), while the cytoplasm within the shell again gives
rise, by multiple segmentation, to daughter individuals (Fig. 74, A to C).
In this case, each daughter form which escapes from the shell is provided
with two flagella, by means of which it swims about till it meets another
similar form which has been produced by another individual. Conjuga-
tion takes place, and the zygote, losing the flagella, becomes an amoeba,
which forms a small shell and grows into a many-chambered individual
of the first type (microspheric form). In the great majority of the
Rhizopoda, however, no sexual process has been observed.
The class Rhizopoda may be sub-divided into the five orders:
AMCEBIDA, HELIOZOA, RADIOLARIA, FOEAMINIFERA, AND MYCETOZOA.
1. Order: AMCEBIDA Calkins, 1902.
The body consists of cytoplasm unprotected by any shell or skeletal
structure, while movement is effected by the formation of pseudopodia
from any part of the body sur-
face. There is usually a single
nucleus, but some forms have
two and others many nuclei.
The cytoplasm is generally dif-
ferentiated into a softer and
vacuolated inner portion, the
endoplasm, in which the nucleus
and food materials lie, and an
outer, more hyaline, and clearer
layer, the ectoplasm. This order
includes the organisms which
are generally known as amoebae,
and to it belong the various
parasitic forms which occur in
the intestinal canal of man and
animals.
2. Order: HELIOZOA Haeckel,
1866.
Fig. lo.^Actifiophrys sol ( x ca. 600). (From
MiNCHIN, 1912, AFTER GRENACHER.)
N., Nucleus from which radiate the axial fibres of
the pseudopodia; ps., pseudopodia; ax., axial
; C.V., contractile vacuole;/.?;., food vacuole.
The forms included in this
order have a characteristic radial
appearance, the result of fine spiky pseudopodia (axopodia), which are
stiffened and rendered permanent by axial fibres. The latter may radiate
from a granule, probably centrosomic in nature, situated at the centre of
the organism, while the nucleus lies to one side of this (Fig. 51). The
166
CLASS: RHIZOPODA
Heliozoa are popularly known as sun animalcules, and are mostly found
in fresh water. Two common forms are Actinosphcerhwi eichhorni (Fig. 71),
which is multinucleated, and Actinophrys sol (Fig. 75), which has a single
nucleus. Both have been much studied from the point of their nuclear
divisions and pedogamy, as described above (p. 86). Members of the genus
Fig. 76. — Vampyrella later itia : A Single Individual at Different Stages of its
Attack on an Alga ( x 300). (After Cash, 1905.)
1. The free individual. 2. The same applied to the surface of the filament.
3. The filament has been broken, and one segment evacuated.
4. Later stage with four segments detached, two of which are evacuated.
VampyreUa are parasitic forms which bore their way into the cells of alga),
in which they live and multiply (Fig. 76). Another genus, Nuclearia,
parasitizes not only algse, but also other Protozoa.
3. Order: RADIOLARIA Haeckel, 1861.
The members of this order, like those of the preceding one, show
a tendency towards a radial arrangement of the pseudopodia, but
morphologically they are more complicated than the Heliozoa. Various
skeletal structures are commonly produced, while a perforated mem-
branous structure, the capsule, divides the cytoplasm into a central
intracapsular portion, which contains the nucleus, and an extra-
ORDER: RADIOLARIA
CK
167
„, /^vV/iV|||fi|i,,||\.\^.^\vxv^ p
Fig. 77. — Thalassicola x>elagica : An Inhabitant of the Ocean Surface Waters
( X 25). (From Gamble's Article in Lankester's Treatise on Zoology, 1909.)
CK, Central caj^sule; EP, extracai^sular cytoplasm; al, carbonic acid filled vacuoles (alveoli);
2>s., ji.seudopodia.
^S,
f
I
I
Fu;. 78. — Aeanthometra elastica ( x ea. 150). (From Minchin's Protozoa, 1912.)
'<}) , Radiating spines; ps., pseudopodia; c, calymma; ex., central capsule; N., nuclei; x, yellow
cells; 7111/., myophrisks (rod-like bodies).
168
CLASS: RHIZOPODA
capsular portion (Fig. 77). The skeleton may be in the form of radiating
spines, tangentially arranged rods, or definite fenestrated shells (Fig. 78).
The latter may be spherical, with perforations, and several such shells
may be formed concentrically, one within the other, as the animal in-
creases in size, or they may have a definite axis, and be shaped like a cone
or bottle. In many forms the cytoplasm contains peculiar yellow cells
about 15 microns in diameter. These are known as zooxanthellse, and
each is an independent vegetable organism possessing a cellulose wall and
containing a nucleus and chloroplasts. It is probable that they live in
a condition of symbiosis with the host. The Radiolaria are marine
organisms which are found floating on the surface of the ocean. Their
shells are found in large numbers in the deposits of the ocean bed.
4. Order: FORAMINIFERA D'Orbigny, 1826.
These Rhizopoda ( = Testacea Schultze, 1854) may be regarded as amoebae
which have the body protected by an external shell or theca. In the simplest
forms the shell has a single
opening, through which the or- \ V\^ ^ tYxXv
ganism protrudes pseudopodia ^ ^\Wv
for locomotion purposes and
the capture of food, very much
Fig. 79. — Arcella vulgaris, show-
ing Outline of Shell, Side
View of the Circulak Chro-
MiDiAL Body, and Two
Nuclei ( x 1.000). (Original.)
Fig. 80. — Glohigerina buUoides from Ocean
Surface Waters ( x 70). The Shells form
the Main Constituent of the "Globiger-
iNA Ooze " of the Ocean Bed. (After
Ehumbler, from Lister's Article in Lan-
kester's Treatise on Zoology, 1903.)
as a snail emerges from its shell (Imperforata Carpenter, 1862). Such
forms {Arcella, Difflugia, etc.) are very commonly found in stagnant water
(Fig, 79). The shells may be strengthened by adherent sand grains or other
material (Fig. 8). When reproduction is to take place the nucleus divides,
a portion of cytoplasm with one of the daughter nuclei is protruded through
the opening, a new shell is formed around this, and another shelled individual
OKDER: FORAMINIFERA
169
separated by division of the cytoplasm. In other cases, with growth of the
organism, a new and larger shell, which remains adherent to the original one,
is formed to accommodate it. A succession of new shells may be produced,
and these remain attached to one another in such a way as to give rise to
complicated compound shells which are constant in arrangement for any
particular species. In addition to the main aperture the shells may have
numerous minute pores, through which filose pseudopodia may be pro-
truded (Perforata Carpenter, 1862) (Fig. 80). Reproduction of the simpler
forms is by binary fission, while the more complicated types may show an
alternation of generations with the production of flagellated gametes, as
described above (p. 164). The Foraminifera occur either in fresh water or
in the sea. The simpler ones occur in
the former situation, while the more
complicated types are marine forms.
Chalk deposits are composed largely
of shells of marine Foraminifera (Fig.
81). Those which occur in fresh water
are often placed in a separate order,
Thecamoebida (Delage and Herouard.
1896), but there is no sharp line of de-
marcation between these and the true
marine Foraminifera. Some forms,
such as Chlamydojphrys, may pass
through the intestine of an animal in
the encysted condition, and emerge
from the cyst and develop their
characteristic thecse in faeces after
they have left the body.
Chlamydophrys stercorea Cien-
kowsky, 1875.— This shelled amoeba
is of interest, as it is commonly present in fseces of such animals as horses
and pigs, as well as frogs and toads. In the freshly passed faeces, it occurs
in the encysted condition which has passed through the intestine. If
the faeces are kept moist for a few days or planted on agar plates, the
amoebae emerge from their cysts and secrete a thin, egg-shaped, trans-
parent shell, which has a pore at its narrower end, through which the
organism protrudes pseudopodia (Fig. 82). There is a single nucleus with
a large central karyosome. Dobell (1909) gave the measurements of an
average-sized individual as 20 by 14 microns. The writer, who has
obtained cultures from frogs' faeces as well as from dirty water, has
observed forms which are much smaller than this, some of them being
barely 15 microns in length. The organisms readily encyst. If they
Pig. 81. — Shell of Nummulites cum-
mingii ( x 20). Portion of Wall
removed to show the chambers.
(From Lang, 1901, after Brady.)
170
CLASS: RHIZOPODA
have no shell, they merely become spherical and form a cyst; if they are
shelled, they escape from the cyst first. The cysts vary from 6 to 17
microns in diameter. Multiplication takes place by division of the
nucleus, followed by the extrusion, through the pore, of half the cytoplasm
into which one of the nuclei passes. A new shell is secreted round this
portion with its pore directed towards that of the original shell. Finally,
division of the narrow neck of cytoplasm uniting the two shelled individuals
takes place.
Schaudinn (1903) stated that the cysts of Chlamydophrys stercorea
passed through the human intestine, and that sometimes the amwbse
escaped from their cysts and multiplied while still in the intestine. He
Fig. 82. — Chlamydophrys stercorea feom Pigs' F^ces ( x 1,000). (Original.)
A. Ordinary individual. Clear area round nucleus is the chromidial body.
B. Process of binary fission: daughter individual being formed as a bud.
also made the statement that a supposed amoeba, Leydenia gemmipara,
which Ley den and Schaudinn (1896) had found in human ascitic fluid,
was no other than the free amoeboid stage of Chlamydophrys stercorea
which had wandered from the intestine to the peritoneal cavity. There
seems to be no evidence of this whatever, and as Schaudinn was unaware
of the existence of such parasitic forms as Endolimax nana, it is highly
probable that the amoebae he saw in the human intestine and regarded
as C. stercorea were in reality E. nana. As to the nature of Leydenia
gemmipara, there is no reason to suppose that it was anything more than
body cells in a degenerate condition in the peritoneal exudate.
Belaf (1921) has reviewed the genius Chlamydophrys, and concludes
there are six distinct species, which differ from one another in size, method
CHLAMYDOPHRYS STERCOREA
171
of nuclear division, and other details. C. stercorea, according to liim,
measures from 30 to 40 microns in length.
Noller, Krosz, and Arndt (1921) have cultivated from horse and pig
dung a number of thecamoebee belonging to the genera Chlcmiydophrys,
X,
Fio. 83. — Trinema acinus: A Shelled Rhizopod from Pond AVater ( x 2,000).
(Original.)
Fig. 84. — Cochliopodium bilimbosuni ( x 1,000). (After Leidy, 1879.)
Plagiophrys, Trinema (Fig. 83), Groniia, and Cochliopodium (Fig. 84).
Many of these forms multiply readily on agar plates. If pigs' faeces are
172
CLASS: KHIZOPODA
kept moist in a Petri dish for some days, many of these forms appear along
with other coprozoic Protozoa.
Fig. 85. — A PortioiN ui- a LAitGii Plasmodium, possuily a Spkciks of Badhamia,
WHICH WAS GROWN ON AN AGAR PlATE. (ORIGINAL.)
A. General appearance under low magnification ( X 16).
B. Small portion more highly magnified, showing numerous nuclei and vacuoles with
inclusions ( x 1,000).
5. Order: MYCETOZOA De Bary, 1859.
The forms included in this order are characterized by a plasmodial
adult phase. The plasmodium is a large sheet of multinucleated cyto-
plasm which exhibits peculiar streaming movements associated with the
ORDERS: MYCETOZOA AND AMCEBIDA
173
production of branching and anastomosing pseudopodia (Fig. 85). . At
certain stages, portions of the cytoplasm become encysted in resistant
capsules (sporangia), which may be arranged on stalks (Fig. 86). In this
respect there is a striking resemblance to fungi, to which group the
Mycetozoa were originally thought to belong. The sporangia eventually
rupture, and may liberate flagellated organisms which, after a free-living
existence, assume the amoeboid form. By growth, accompanied by
nuclear multiplication, the large plasmodia are produced. The Mycetozoa
are terrestrial in habit, and are commonly found
on the moist surfaces of decaying wood and leaves,
or in similar situations. Some of them may be
grown on the surface of agar plates.
SYSTEMATIC DESCRIPTION OF THE ORDER
AMCEBIDA.
From the point of view of parasitology it is
chiefly members of the order Amoebida which
have to be considered. The vast majority of the
Rhizopoda are free-living organisms, and only a
comparatively small number are truly parasitic
and adapted to their hosts in such a way that
a free extra-corporeal existence does not occur.
The fact that many of the free-living non-para-
sitic forms are able to produce protective cysts
of a resistant nature to enable them to withstand
desiccation has led to some confusion
encysted forms are frequently eaten accidentally
by human beings or animals, and may pass un-
harmed through the intestinal canal. After
escape from the body in the dejecta, they may find themselves in an
environment which is favourable for further development. The amoebae
emerge from the cysts, and by active multiplication increase enormously
in numbers in a comparatively short time. In this way, erroneous
impressions as to their parasitic nature may be obtained. Care must
always be exercised to guard against the possibility of confusing
these coprozoic forms with true parasites. In the case of true
parasites, the only forms which survive outside the body are, as a rule,
the encysted forms, which remain quite passive and unchanged till they
are ingested by another host. The unencysted stages are present in the
freshly passed stool, and show a degeneration which becomes more marked
as the interval since their escape from the body increases. The non-
FiG. 86. — Badhaniia utri-
cularis. (After Lis-
ter, IN Lankester's
Treatise on Zoology,
1909.)
Such «• Group of sporangia ( X 12).
b. Cluster of spores (x 170).
c. Single sjjore.
d. Part of capillitium in in-
terior of sporangium (x 170).
174
FAMILIES OF THE AMCEBIDA
parasitic forms, which have passed through the alimentary canal in the
encysted state, are at the height of their free-living existence some time
after the escape of the cysts from the body.
In the order Amoebida are included a number of well-known free-
living amoebae, such as Amoeba proteus (Fig. 5) and Amoeba verrucosa
(Fig. 87). The majority are uninucleated, but some have tw^o nuclei
(A. binucleata), while others have many nuclei (Pelomyxa). In addition
to these larger forms, there are others which are smaller, and which are
of interest in that some of them are readily cultivated from the fseces of
man and animals, owing to the fact that their cysts are able to pass un-
harmed through the intestine. Such forms are known as coprozoic amoebae.
They have frequently been
referred to as Amoeba Umax, a
name given to an amoeba by
Dujardin (1841) which, accord-
ing to Dobell and O'Connor
(1921), is not now identifiable.
Some of the amoebae ascribed
to this species have been
shown to be the amoeboid
phase of the flagellated organ-
ism Dirnastigamoeba gruberi
mentioned below. Others
appear to be true amoeba?
which have no flagellate
stage. There are many species
which are difficult to identify
on account of their resemblance to one another. They differ in the character
of the cysts they produce, the method of nuclear division, and other details.
The order Amoebida may be considered as comprising the following
families:
1. Family: AMCEBIDA Bronn, 1859. — Amoebae which are not able to
form flagella.
2. Family: PARAMCEBiD^ Poche, 1913. — Amoebae which, in addition
to a nucleus of the usual type, possess an accessory body (Nebenkorper)
which, during division, divides with the nucleus.
3. Family: dimastigamcebidtE. — Amoebae which in the adult form are
able under certain conditions to form two or more flagella, by means of
which they progress as flagellates.
4. Family: RHIZOMASTIGID.E Calkins, 1902. — Amoebae which are pro-
vided with a single flagellum during the greater part of the free-living
existence.
Fig. 87. — Amoeba verrucosa ( x 300)
Cash, 1905.)
(After
GENERA: AMCEBA AND HARTMANNELLA 175
Family: AMCEBID^ Bronil, 1859.
In this family are included a number of free-living amoebao, and most
of the parasitic forms which occur in the intestine of man and animals.
Not many years ago all amoebae, including the parasitic forms, were
placed in the genus Amceha. It is now recognized that several distinct
genera are represented, but the group has not been sufficiently studied
to enable precise definitions to be given. Many of the smaller free-living
forms which were grouped under the name Amceha Umax have been placed
in the genera Hartmannella, Sappinia, Vahlkampfia, which can be identi-
fied by the type of nuclear division and other details, while the parasitic
amoebae have been separated into the genera Entamceha, Endamceha,
lodamceba, Endolimax, and Dientamceha. The exact limits of the genus
Amoeba are doubtful, but the majority of the large free-living uninucleated
forms, such as Amoeba proteus and Amoeba verrucosa, which may have
a diameter of 500 microns or more, are regarded as belonging to it. Much
more information regarding the complete life-histories, the methods of
reproduction and encystment, and the details of nuclear division, are
required before the group can be satisfactorily defined.
Oemis : Amoeba Bory, 1822.
In this genus are included the vast majority of free-living amoebae.
In most cases they are placed in the genus because detailed informa-
tion regarding their structure and development is wanting. It seems
probable that future investigators will show that the only ones which
actually belong to it are the large free-living forms like Amoeba proteus,
Amoeba verrucosa. Amoeba vespertilionis, and Amoeba hydroxena described
by Entz (1912) as parasitic on Hydra oligactis.
Genus: Hartmannella Alexeieff, 1912.
The amoebae belonging to this genus are recognized by the character
of their nuclei and method of nuclear division. The nucleus is spherical,
has a large central karyosome, and peripheral chromatin in the form of
granules either on the inner surface of the nuclear membrane or in the
space between the membrane and karyosome. During division the
karyosome disintegrates, and a spindle is formed upon which definite
chromosomes become arranged as an equatorial plate (Fig. 88). The
nuclear membrane usually disappears at some stage of the division. The
cysts are spherical structures. The numerous species belonging to this
genus are distinguished by the details of nuclear division and the character
of the cysts.
Hartmannella hyalina (Dangeard, 1900).^ — This amoeba, which is often
found in stale fa>ces or in agar plate cultures made from dirty water,
176
FAMILY: AMCEBID^
faeces, or other material, has been referred to by various observers as
Amoeba hyaUna, a name given to it by Dangeard (1900). The generic
name Hartmannella was created by AlexeiefE (1912cf). The amceba
which was cultivated from human faeces by Musgrave and Clegg (1904)
in the Philippines, the one described by Liston and Martin (1911) in
m
0- 0 A
(S^
Fig.
-Stages in the Nuclear Division of a Species of Hartmnnnella isolated
FROM Pigs' F.^ces {xca. 3,400). (Original.)
India as occurring in culture media inoculated with liver abscess pus,
and water, and the form growing on plates after exposure to the air,
as noted by Wells (1911), are probably this species.
The amoeba, when spherical, has a diameter of 9 to 17 microns. It
has a contractile vacuole, while the nucleus consists of a nuclear membrane
■■*■■■ . ' ■ . .^ -
Fig. 89. — Hartmannella hyalina ( x 2,000). (After Dobell and O'Connor, 1921.)
L Ordinary amoeba.
2. Division stage, showing pointed spindle with equatorial plate of chromosome.s.
3. Cyst with crinkled wall.
and large central karyosome (Fig. 89). Peripheral chromatin granules
occur on the nuclear membrane, and in the clear zone between it and the
karyosome. At the time of division the karyosome disintegrates, and
a spindle is formed, at the equator of which the chromatin, in the form
GENUS: VAHLKAMPFIA 177
of a ring of spherical chromosomes, is arranged. The nuclear membrane
disappears during the process, leaving a sharp, pointed spindle in the
cytoplasm. The spherical cysts measure from 10 to 14 microns in dia-
meter. They have a smooth inner wall and a much wrinkled outer one.
The amoeba does not multiply within the cyst, nor does its nucleus undergo
division. When grown on the surface of agar, it not infrequently happens
that amoebae with two or more nuclei encyst, in which case a correspond-
ing number of nuclei occur within the cyst.
There are other amoebse of relatively small size belonging to the genus
Hartmannella, which differ from one another in the details of their nuclear
divisions. Thus H. glehce-, described by Dobell (1914 a), is very similar
to H. hyalina (Fig. 56). The spindle formed during nuclear division has,
however, rounded ends instead of pointed ones. The cyst, moreover, has
a smooth outer surface. This form, or one closely allied to it, often occurs
coprozoically in faeces, and can be cultivated on agar plates (Fig. 56).
At the present time it is impossible to identify many of these coprozoic
amoeba?, but it appears that two fairly well-defined types commonly
occur — the one corresponding to H. hyalina, and the other to H. glebcp.
Genus: Vahlkampfia Chatton and Lalung-Bonnaire, 1912.
Vahlkampf (1905) studied the development of an amoeba, which he
designated Amceba Umax. The nucleus possessed a large central karyo-
some, and during multiplication the nucleus divided with the formation of
pole caps, as in the case of Dimastig amoeba gruberi (Figs. 61 and 90). A.
similar form was named Amoeba 'punctata by Dangeard (1910). Chatton
and Lalung-Bonnaire (1912) created the genus Vahlkampfia for amoebae
showing this type of nuclear division and possessing pores in the cyst wall.
Flagellate forms of the amoebae were not observed, but the conditions
necessary for the production of the flagellate forms were not provided
by them. Several other observers have described amoebae which show
nuclear division of the same type, and which have not been noted to give
rise to flagellate forms. It does not seem improbable that most, if not all,
of these forms would produce flagellate stages if the necessary conditions
existed. Dimastig amoeba gruberi remains as an amceba on agar plates, or
in cultures in egg-albumen water and other media, and does not become
a flagellate unless a sudden change occurs in the medium, as, for instance,
that produced by the addition of tap water. If this is done, flagellates
appear in three or four hours, but they revert to the amoeboid form again
in about a day (Fig. 120). It is probable that many of the amoebae which
have been placed in the genus Vahlkampfia would be capable of transforma-
tion into flagellate forms if they were similarly treated. It is possible, how-
ever, that some of them would not. Hogue (1921), for example, obtained a
I. 12
178
FAMILY: AMCEBID^E
culture of an amcBba, which she named Vahlkamjpfia patuxent, from the
stomach of oysters. Though its method of nuclear division resembled
that of Dimastigamoeba gruberi, she failed entirely to obtain flagellate forms,
though all the methods which cause Dimastigamoeba gruberi to develop
flagella were tried.
Calkins (1913) separated the amcebse which have this particular type
of nuclear division into two genera — viz., the genus VahUamjjfia, to include
the forms which do not develop a flagellate stage, and the genus Ncegleria
(created by Alexeieff, 1912) for those which have such a stage. The
latter forms, as pointed out by Alexeieff (1912a) really belong to
the genus Dimastigamoeba of Blochmann (1894), and will be considered
mm
• 6
Fig.
90.
7 8
Vahlkamiyfia punctata. (After Vaiilkampf, 1905.)
1, 2. Appearance of living amoeba and encysted form ( x 1,500 ?). 3-8. Stages in nuclear
division ( x 3,000 V).
below (p. 260), and as Chatton and Lalung-Bonnaire actually observed
markings which were undoubtedly pores on the cyst wall, it is probable
they were dealing with an organism belonging to the same genus. In
this case, both the names Ncegleria and Vahlkampfia are really synonyms
of Dimastigamoeba.
As already remarked, it is still doubtful if any of the ama>bfe having
the type of nuclear division of Dimastigamoeba gruberi are really incapable
of developing the flagellate stage. The majority, at any rate, have not
been investigated from this point of view. Most of these forms are free-
living amoebae, occurring commonly in damp soil or decomposing vegetable
material, but some of them have been found in the intestines of cold-
blooded animals. Others are to be regarded as coprozoic amwba?, as they
GENUS: VAHLKAMPFIA 179
appear in stale faeces, and have been cultivated from stools on agar plates.
A large number have been named, but it is very doubtful if these are
all distinct species.
Dangeard (1910) described as Atnceba punctata a form of this type
which had cysts with punctate markings. It was studied by Chatton
and Lalung-Bonnaire (1912), who obtained it from human faeces. They
placed it in a new genus as Vahlkampfia 'punctata. The punctate markings
strongly suggest the pores in the cysts of Diynastig amoeba gruberi.
Hartmann (1907a) gave the name Amceba froschi to an amoeba
showing the same type of nuclear division which he had seen in the faeces
of frogs, and the name Amoeba lacertce to a similar form in the intestinal
contents of lizards of the genus Lacerta. Both these forms were studied
by Nagler (1909). The form described by Dobell (1914a) as Amoeba
lacertce, which also occurred in the intestinal contents of lizards, differed
as regards the details of its nuclear division from the form studied by
Hartmann and Nagler. Hartmann (1914) accordingly renamed the form
studied by Dobell Amoeba {V ahlkaynpjia) dobelli. Caullery (1906) gave
the name Amoeba padophthora to an amoeba which parasitized the eggs
of the marine crustacean Peltogaster curvatus, while Chatton (1909)
described, under the name Amoeba mucicola, an amoeba which was parasitic
on the gills of a marine fish. Epstein and Ilovaisky (1914) gave the name
Vahlkampfia ranarum to a large amoeba, reaching 50 microns in diameter,
which they found in the intestine of frogs. Mackinnon (1914) saw an
amoeba, which she referred to as Vahlkampfia sp., in the intestine of the
larva3 of the crane-fly, Tipula sp. An amoeba, which was cultivated by
Whitmore (1911a) from human faeces, liver-abscess pus, and tap water
in Manila, and referred to as Amoeba liynax, was placed in the genus
Vahlkampfia as V. whitmorei by Hartmann and Schilling (1917). The
amoeba described by Porter (1909a) as Amoeba chironomi, from chiro-
nomous larvae, is possibly of the same type, though the nuclear division
was not described. Hogue (1921) recorded 7. patuxent from the stomach
of oysters in America.
In addition to the above-mentioned forms, which have a certain
association with higher animals, a number of free-living species have been
named. Nagler (1909) described Amoeba spinifera, A. lacustris, and
A. albida ; Aragao (1909), A. diplomitotica ; Glaser (1912), A. tachypodia ;
Belaf (1915), A. diplogena ; Jollos (1917), Vahlkampfia magna, V. debelis,
and F. sp.; and Hogue (1914), Vahlkampfia calkensi. Glaser (1912)
described the nuclear division of Ehrenberg's Avnoeba verrucosa as being of
the Vahlkampfia type. An amoeba first seen by Molisch (1903), and later
by Zacharias (1909), is parasitic on Volvox, while another. Amoeba bloch-
manni (Doflein, 1901), first noted by Blochmann (1886), is parasitic on
180
FAMILY: AMCEBID^E
Hwmatococcus. It is possible that both these forms, as well as the others
named above, should be included in the genus Vahlkampfia.
These various amreba? all agree with one another in that the nuclear
division, where it has been studied, is of the type first described by
Vahlkampf (1905), and it is highly probable that further investigations
will demonstrate, in some of them at least, the presence of pores in the
cyst wall and the occurrence of flagellate stages, in which case they wall
have to be transferred to the genus Diynastigamoeha. Meanwhile, however,
till more accurate data are forthcoming, it seems advisable to group these
amoebae under the name Vahlkampfia, which, as pointed out above, may
be a synonym of Dimastigamoeba, rather than to establish a new genus,
which will be necessary if they are finally proved to have no flagellate
stage.
Oeniis : Sappinia Dangeard, 189(5.
The amo'ba? belonging to this genus are peculiar in possessing two
nuclei, which are closely applied to one another. During division, both
Fig. 91. — Sax^pinia diploidea ( x 2,000). (After Dobelt. and O'Connor, 1921.)
1. Ordinary individual with two nuclei in apposition.
2. Cyst containing two individuals.
nuclei divide. When encystment takes place, two amoebae, each with
two nuclei, are enclosed in a common cyst. In the form S. pedata, studied
by Dangeard (1896 a), the free amoebae have the characteristic two nuclei.
The cyst, however, is peculiar in having a pedicle or stalk attaching it to
objects.
Sappinia diploidea (Hartmann and Niigler, 1908). — This is an amoeba
which was isolated by Hartmann and Niigler from lizards' faeces. Accord-
ing to Dobell and O'Connor (1921), it occurs rarely in human faeces, but more
GENUS: SAPPINIA— AMCEBiE OF PLANTS 181
commonly in that of animals, such as the ox and lizard. Hartmann and
Nagler (1908) gave it the name Amoeba diploidea, while Alexeieff (1912a)
placed it in Dangeard's genus Sappinia. The amoeba varies in size from
10 to 30 microns, possesses a contractile vacuole, and has a characteristic
thick pellicle, which is sometimes wrinkled (Fig. 91). It possesses two
nuclei which lie side by side in a central position. They are spherical, and
have large central karyosomes. The amoeba multiplies by binary fission,
the two nuclei dividing and producing two parallel spindles. The daughter
individuals thus have two nuclei. When the amoebse encyst, two in-
dividuals form round themselves a common cyst. According to Hartmann
and Nagler, the two nuclei of each amoeba now fuse. Each nucleus is then
said to give off reduction bodies, which degenerate, after which the cyto-
plasms of the two uninucleate ama?ba? unite. Their nuclei, however,
come into contact with one another, but do not fuse (Fig. 47). The amoeba
emerges from its cyst, and commences to multiply by binary fission as
before (see p. 82).
AMCEB^E OF PLANTS.
Franchini (1922 g, h, j, k, I) in a series of papers stated that he had found amoebse
in the latex of various plants. They occurred either alone or in association with
flagellates of the leptomouas or tryj)anosome type. The plants found infected were
Euphorbias, figs, and allied forms, as well as the lettuce, and were as follows:
Eiqihorbia vertirillatd,, Eiiphorhia nereifolia, Chlorocodon Whitei, Cryptostegia grandi-
Jhra. Stroph(nitliiis I!i<itili ;;iid N. s<-<ni<h-)iti, Acolcantliera venenata, Thevetias-p., Cerbera
Odollam,Fici(s IU'Uja))i'ni(i, Ficiix J'ierrei, Ficus Tholloni, Ficus carica, Ficusparietalis,
Antiaris toxicaria, Lakoocha artocarpus, Chrisophyllon sp., Labramia Bojeri, Tregtdia
Africana, Mimusops schimperi, Sideroxylon inerme, Lactuca sativa, Plumeria alba.
Cultures of some of the amcebse wore obtained by inoculation of blood-agar
plates (Noller's medium) with the latex of the plants, and in this medium most of the
amoebae were found to ingest red blood-corpuscles. In this way cultures were made
from Ficus carica, Chlorocodon Whitei, Cryptostegia grandiflora, Acokanthera venenata,
Plumeria alba, and the lettuce, Lactuca sativa. Three of these amoebae were named
Amoeba chlorocodonis. Amoeba cryptostegice, and Amoeba lactuca;. The descriptions of
the amoebse and the figures are such that it is impossible to form an opinion as to
their nature. It seems not improbable that the cultures obtained may have been
derived from amoebse or their cysts on the cuticle of the plants.
Further remarkable assertions are made by the author (Franchini, 1922 n) in
connection with the inoculation of kittens with cultures of amoebae from the plants
Acokanthera venenata and Plumeria alba. Kittens injected per rectum with cultures
of these amoebae were said to remain well for six to ten days, when they suddenly
became ill with dysentery, which persisted for about ten days. During this period
amoebse, some of which included red blood-corpuscles, were constantly present.
The animals recovered. The figures of these amoebae, again, are unrecognizable,
and cannot be distinguished from cells. It is further claimed that mice are suscep-
tible to inoculation with the amoebae cultivated from latex of Euphorbias, and that,
when these cultures contain trypanosomes and leishmania, as well as the amoebae,
a general infection is produced, and that all these organisms can be recovered by
culture from the heart blood. In another paper Franchini (1923) asserts that
182 FAMILY: AM(EBID^
mice fed ou the latex of the plants or injected intrapeiitoneally with cultures of the
amoebae and flagellates acquired liver abscess in which amcebse with included red
corpuscles occurred. These amoebae are said to reproduce by schizogony, and give
rise to forms like anaplasma, leishmania, and trypanosomes. The figures purporting
to show these forms are quite unrecognizable, and it is impossible to understand the
ai;thor"s conception of these Protozoa.
Genus: Pelomyxa Greef, 1874.
This genus includes certain large free-living multinucleate amoebae,
which may reach a diameter of 2 millimetres. They occur commonly in
stagnant water, and are easily visible to the naked eye. Each individual,
which may contain several hundred nuclei, moves slowly as it throws out
blunt pseudopodia (Fig. 92). The character of the cytoplasm varies with
the medium in which the organism is growing. It is usually much vacuo-
lated, and may be packed with small globules of a refringent substance.
Fig. 92. — Pelomyxa lyalustris : Ordinary Condition during Active Movement
(x 1,000). (After Cash, 1905.)
in addition to sand grains, diatoms, bacteria, and other objects, which
cause it to be peculiarly opaque. At other times the globules are absent,
and the cytoplasm is much clearer. Reproduction takes place by fission,
while gamete formation has also been described by Bott (1906). It is
supposed that uninucleated individuals are divided off, and that these
conjugate in pairs to give rise to zygotes, which grow into the multinucleated
adults (see p. 30).
Genus : Entamoeba Casagrandi and Barbagallo, 1895.
The members of this genus, which inhabit the intestine of higher
animals, vary in diameter from 5 to 40 microns. The pseudopodia are
usually blunt processes, and a new one is rarely formed before the previously
existing one is withdrawn. A central endoplasm can be distinguished
from a peripheral ectoplasm, while a contractile vacuole is not present.
GENERA: PELOMYXA AND ENTAMCEBA 183
The nucleus is spherical, and consists of a definite nuclear membrane, on
the inner surface of which the bulk of the chromatin of the nucleus is
distributed in the form of granules. There is a linin network, upon which
fine granules of chromatin may or may not occur, while a comparatively
small karyosome is present. Reproduction in the vegetative phase is by
simple binary fission, while transmission from host to host is effected by
means of encysted forms. The cyst is a transparent and smooth structure,
and the nucleus of the enclosed parasite, by repeated divisions, gives rise
to a number of daughter nuclei, which vary from four to multiples of four.
The encysted forms are passed out of the body of the host, and undergo
no change till they enter the intestine of another host, where the cyst wall
ruptures, and there is liberated either the multinucleated cytoplasmic
body, which then divides into a number of amoebae, or a number of amoebse
which have been formed before rupture of the cyst. It is not clear which
of these processes actually occurs. Each species of the genus tends to
produce a cyst which, when fully developed, contains a definite number
of nuclei. Thus, E. coli, E. muris, and other forms have eight, while
E. histolytica and E. rananim have only four. Occasionally, the nuclei
are in excess of the usual number. There may be sixteen or more in
E. coli, and more rarely eight in E. histolytica. The cysts of E. ranarum
may have a still larger number. This tendency to nuclear excess may occur
less commonly in the unencysted stages. In the case of E. coli unencysted
forms with eight nuclei have been described, but it is probable that these
were really irregularly-shaped encysted forms, the cyst walls of which,
in stained preparations, were not actually visible. Multinucleate free
forms of E. ranarum were described by Collin (1913), and similar s^^ages
were seen by Keilin (1917) in the case of E. ynesnili, and by the writer
in E. histolytica. It has been supposed by Mathis and Mercier (1917)
that the cysts with an abnormally large number of nuclei represent a
special type of multiplication by schizogony, while those with the normal
number are destined to give rise to gametes. They produced no con-
vincing evidence in support of this view. It is more probable that for
some reason the nuclear multiplication has continued beyond the usual
limits, possibly owing to excess of nutriment, or sometimes to an amoeba
having encysted just as it was about to divide in the free state.
This genus includes Entamoeba coli, the harmless amoeba of the human
intestine; E. histolytica, the pathogenic form producing amoebic dysentery
and liver abscess in man; E. gingivalis, an inhabitant of the human mouth;
and various species which occur as intestinal parasites of animals, such as
E. muris of rats and mice, E. pitheci and E. nuttalli of monkeys, E. bonis
of cattle, E. ovis of sheep, E. testudinis of the tortoise, E. ranarmn of frogs,
E. minchini of the larvae of Tipulid flies, and many other species. It is
184 FAMILY: AMCEBIDiE
probably safe to assume that practically every vertebrate animal, as well
as many invertebrates, will be found to harbour amoebae belonging to this
genus. In the great majority of cases they are of the non-pathogenic
variety, and in this respect resemble E. coli. In a few instances, forms
associated with dysenteric symptoms have been described from animals.
The various species resemble one another very closely, so much so that
in many cases they could not possibly have been regarded as distinct
species, apart from the fact that they occurred in different hosts.
There is some doubt as to the correct spelling of the name Entamceba.
The generic title was created in this form by Casagrandi and Barbagallo
(1895) for E. coli of the human intestine. Leidy (1879), however, had
given the name Endantceba blattcB to the amoeba of the cockroach. If this
form should prove to belong to the same genus as the human amoeba,
then Leidy 's name will have priority. As one of the cockroach amoebae
presents some peculiar features, it is better to regard it at present as
belonging to a distinct genus, Endamoeba (see p. 235).
entam(eb;e of man.
(a) Pathogenic Form.
Entamoeba histolytica Schaudinn, 1903. — Chief synonyms : "Amoeba coli"
Losch, 1875; '"Amoeba dysenteriae" Councilman and Lafleur, 1891; Amoeba coli
(Loscli) Kovacs, 1892; Amceba dysenterice (Councilman and Lafleur) Kovacs, 1892;
Entamceba dysenterice (Councilman and Lafleur) Craig, 1905; Entamoeba coli var. tetra-
gena Viereck, 1 907 ; Entamoeba africana Hartmann, 1907 ; Entamoeba tetragena ( Viereck)
Hartmann, 1908; Poneramoeba histolytica hiihe 1909; Eiitamoeba minuta JL\n\assia,n,
1909; Entamceba nipponica Koidznmi, 1909; Entamceba hartmanni Prowazck, 1912;
Loschia (Viereckia) tetrajsna Cliatton and Laluno-Bonnaire, 1912; Entamceba
brasiliensis AragXo, 1912; Liischia histolytica (Schaudinn) Mathis, 1913; Entamoeba
venaticum Darling, 1915 ; Entamoeba minuta Woodcock and Penfold, 1916 ; Enda-
moeba coli (Losch) Aragao, 1917 ; Endamoeba dysenterice (Councilman and Lafleur)
Pestana, 1917; Entamoeba tenuis Kuenen and Swellengrebel, 1917; Entamoeba
minutissima Bvng, 1917; Endamoeba histolytica (Schaudinn) Craig, 1917; Entamceba
coli communis Knowles and Cole, 1917; Entamceba paradysenterica Chaterjee, 1920;
Caudamceba sinensis Faust, 1923; Karyamoebina falcata Kofoid (and Swezy, 1924);
Entamceba disj^ar Brumpt, 1925.
Everyone who has studied the question is agreed that E. Jii.sfolytiea
was first seen and described by Losch (1875), and named by him " Amoeba
coli.'" Though this name was not correctly written in the original descrip-
tion given by Losch, it was employed for a long time for the amoebae of
the human intestine before it was fully realized that more than one species
existed. Similarly, Councilman and Lafleur (1891) proposed to call the
amoeba "Amoeba dysenteriae," another name which was not correctly
presented. Quincke and Roos (1893) and Roos (1894) were the first to
conclude that two types of amoeba occurred in man, the one an active
ENTAMCEBA HISTOLYTICA 185
form which produced cysts 10 to 12 microns in diameter and was pathogenic
to cats, and the other a less active form which produced cysts 16 to 17
microns in diameter and which did not give rise to infection in cats.
Kruse and Pasquale (1894) similarly described two forms, the one patho-
genic to cats and the other not.
Though Schaudinn (1903), in his account of the amoebse of the human
intestine, made many erroneous statements, he was the first observer to
appreciate clearly the fact that two distinct species exist, the one patho-
genic and the other harmless. Before this, the descriptions referred
sometimes to the one form and sometimes to the other, and often to
a mixture of both. In many cases it is only the association of the amoebae
with pathological conditions, and their occurrence in lesions of the intestine
and abscess of the liver, which are now known to be due only to invasion
of tissues by E. histolytica, that make it almost certain that some of the
earlier writers were actually dealing with this form. If recent investi-
gations had shown that both E. coli and E. histolytica were liable to invade
the tissues, then there would be practically no data whatever to enable
a decision to be made as to which of the forms the earlier writers were
referring. The experiences of the past few years have demonstrated
clearly that E. histolytica alone is responsible for the production of patho-
logical conditions, so that it is perfectly clear that the amoebae described
in the lesions of the intestine, liver, and brain by the earlier writers were
actually E. histolytica, though the descriptions of the amoebse themselves
were in most cases so imperfect that it would be impossible to identify
them. Though Losch, in his original description, expressed a doubt as
to the part played by the amoebae in the production of dysentery, his
really excellent figure depicts an organism which can hardly be any other
than that now known as Entamoeba histolytica.
If Schaudinn had recognized the fact that the amoeba, which Losch
called "Amoeba coli,'' was the pathogenic amoeba, and had given it the
name Entamoeba coli, endless confusion would have been avoided, but as the
matter stands at present there seems to be no alternative, unless further
confusion is to be caused, but to retain Schaudinn's name E. histolytica for
the pathogenic form and E. coli for the non-pathogenic one. The .whole
question of the nomenclature of the intestinal amoebae of man has been
reviewed very thoroughly by Dobell (1919), and readers are referred to his
book for more detailed information on this very intricate subject.
LIFE-HISTORY. — E. histolytica is to be regarded as a tissue parasite of
man, as first demonstrated by Koch and Gaffky (1887), and more clearly
by Kartulis (1885 and 1886). Infection is brought about by the ingestion
of encysted forms, first seen by Quincke and Roos (1893), which have been
passed in the faeces of some other infected person. Under the action of
186 FAMILY: AMCEBID^
the digestive fluids the cyst ruptures. From the work of Chatton (1917a),
who fed cats with material containing cysts, and that of Penfold, Woodcock,
and Drew (1916), who treated cysts with liquor pancreaticus, it appears
that it is the secretions in the small intestine which cause the cyst wall
to dissolve.
Chatton stated that the cyst liberated a four-nucleated amoeba, while
the other observers merely noted that a single amoeba escaped from the
cyst. Whether this happens in the human intestine or not cannot be
stated. Dobell and Stevenson (1918) and the writer have failed to bring
about any escape of amoebae from cysts by means of liquor ]Kmcreaticiis.
From experiments on cats, there can be no doubt that human beings
are infected by the ingestion of cysts. Whatever may be the exact method
of escape of the encysted amoebae and their development after this, it is
a fact that invasion of the intestinal wall by the amoebae quickly takes
place. In the earliest condition the amoebae make their way into the
glands of the large intestine, and crawl to the bottom of these. Here they
multiply, and partly by pressure, and possibly by the secretion of a toxin,
the gland cells degenerate and separate from one another. By this time
the tubule of the gland has probably become blocked, and if the adjacent
glands over a small area of surface are all similarly involved, as is usually
the case, a slightly raised yellowish nodule is produced. Meanwhile, the
amoebae have made their way into the interglandular connective tissue,
and a certain amount of necrotic material from broken-down cells has
collected. In this condition the yellow nodule is in reality a small
amoebic abscess of the mucosa. Very soon this abscess bursts into the
lumen of the intestine, and the contents are discharged, with the result
that a small undermined ulcer is formed (Figs. 93, 94). The amoebae which
thus escape invade other glands, causing the condition to spread, or they
are passed in the faeces with a certain amount of blood and mucus, which
represents the discharge from the abscess. The infected portions of the
intestine may be very limited, so that only a few scattered nodules are
formed, or there may be a more or less continuous infection of all the
glands. After rupture of the primary abscess the ulcer so formed becomes
gradually larger, the amoebae multiplying in the base of the ulcer and
extending over a wider area. They break through the muscularis mucosae,
and extend into the submucous tissues, producing eventually ulcers which
may reach an inch or more in diameter. These ulcers, like the small ones
originally formed, have undermined edges, and become filled with mucoid
material, debris of cells, and amoebae. It is probable that the plugs of
mucus admixed with blood, which occur in the stools of amoebic cases,
represent the evacuations from these ulcers. These masses of mucus
may contain enormous numbers of amoebae.
ENTAMCEBA HISTOLYTICA
187
At any time the discharged amoebae may infect fresh areas, so that
the large intestines of these cases show every stage in the formation of the
ulcers, from the smallest yellow nodules to large, undermined ulcers.
Ks,^
M
G
■uc~-
^
U
A
Fig. 93. — Amcebic Ulceration: Section of a Small Ulcer in a Human Large
Intestine ( x 30). (Original.)
The area marked by a square is shown under higher magnification at Fig. 94.
The best pictures from a histological point of view are obtained by
cutting sections of the small nodules which have not yet become subject
to secondary bacterial invasion, while at post-mortem examinations the
contents of one of these small nodules will often show still live and active
188 FAMILY: AM(EBID.E
amoebae which cannot be obtained so readily from the larger ulcers.
After the amoebae have multiplied and caused an extension of the ulcer
for some varying period a healing process sets in, the amoebae disappear,
and the site of the ulcer is finally represented by a puckered scar of fibrous
tissue, while the peritoneal surface opposite it may be considerably
thickened. In other situations, however, the process of invasion and
ulceration is still continuing, and this affords an explanation of the
persistence of infections with E. histoJytica, which are known to last for
^
3
o ♦
O
''1
Fig. 94. — Eniamceba histolytica in Tissue of Human Large Intestine ( x 500)
(Original.)
The area is shown in the square at Fig. 93.
many years, if not a lifetime, unless eradicated by suitable treatment.
In their extension through the tissues of the intestinal wall the amoebae
not infrequently make their way into blood-vessels, and are carried as
emboli to the liver, spleen, brain, or other organ, where they continue to
multiply and give rise to the well-known amoebic abscesses.
In an infected individual, if the amoebae are multiplying rapidly and
invading one portion of the large intestine after another in quick succes-
sion, the discharge from the ulcers is considerable, and much blood and
mucus will appear in the stool, which becomes of the characteristic
ENTAMCEBA HISTOLYTICA 189
dysenteric type. If the extension is not rapid, then only occasional plugs
of mucus, which may or may not be contaminated with blood, are passed,
and the individual may be quite unaware of his condition. The cases
of rapid extension are regarded as the acute ones, and the amoebae are
all of the large tissue-invading form, many of which contain red blood-
corpuscles. In other cases, where there is not rapid extension, though
a considerable area of the wall must be involved owing to the enormous
number of amoebae or their cysts which are passed in the faeces, a state
known as the " carrier condition " occurs. Exactly what happens in this
condition is not properly understood, for it is difficult to obtain perfectly
fresh post-mortem material from these cases. It is not possible to repro-
duce the carrier condition in animals, which always acquire an acute
infection which either terminates fatally or disappears. From what can
be observed in the stool, it is found that a smaller type of amceba occurs
in the faeces of carrier cases. These are in reality encysting forms (pre-
cystic amoebae), for in association with them are to be found cysts showing
one, two, or four nuclei. In some cases the precystic amoebae and the
encysted forms are passed together in the stool, while in others only the
amoebae or only the cysts are passed. This is probably dependent upon
the varying rate at which the large intestine evacuates itself. It seems
probable that the small amoebae arise in the ulcers by division from the
larger tissue-invading forms under certain conditions which may be
supposed to hinder their free and easy development. As a general state-
ment it can be accepted that Protozoa encyst when the conditions of life
are becoming unfavourable. The small precystic amoebae are formed
from the large ones which have become more superficial in position, and
it might be surmised that if the large amoebae which have escaped from
the tissues into the debris which fills the ulcer remain there for some time,
as they may be supposed to do in the slowly extending cases, no increase
in size occurs through lack of proper food, though they multiply and give
rise at each division to increasingly small forms. These amoebae, deprived
of their proper food, which is to be found only in the tissues in the deeper
parts of the ulcer, become encysted, and escape into the lumen of the
intestine when the ulcer discharges its contents. This discharge may
take place before actual encystment is complete, in which case the small
precystic amoebae will be found in the stool. In certain cases enormous
numbers of cysts are passed in the stool, and it must be supposed that
the process described occurs simultaneously at many parts of the intestine,
not necessarily in large evident ulcers, but in the very small superficial
ones which are not readily detected by the naked eye. The lesions in
these cases may be merely superficial, and, not being of an acute nature,
it is not surprising that certain individuals may be passing extraordinarily
190 FAMILY: AMCEBIDiE
large numbers of cysts without showing any symptoms whatever. In
view of the recent successful culture of E. histolytica by Boeck and
Drbohlav (1925) in egg media, it appears possible that the amoebse may
actually live and multiply on the surface of the intestine without giving
rise to any lesions. It may be that the infection of many symptomless
carrier cases is of this type, and that the precystic amoebae and cysts are
produced by amoebae living on the surface of the mucosa.
As regards the fate of encysted amoebae, there are two views. The
one which maintains that an amoeba which has once encysted in the gut
is unable to leave its cyst in the large intestine of the same host appears
to be in accord with the behaviour of parasitic Protozoa generally.
According to this view an encysted amoeba, in order to develop further,
must pass out of the intestine and be ingested by another or the same
host, so that the cyst may come under the influence of the digestive fluids
of the small intestine. A corollary to this is that if all the amoebae in
an individual could be induced to encyst, an automatic cure would result,
for all the cysts would have to be passed from the body. It is evident,
therefore, that it is just as incorrect to suppose that any case is resisting
treatment because of the impermeable cysts in the intestine as it would
be to conclude that a case of ankylostomiasis was not cured because the
eggs of the worm were too resistant. In the one case cure is effected
by killing the amoebae which produce the cysts, and in the other by killing
the worms which produce the eggs. In either case the presence of cysts
or eggs in the stool is an indication that the organisms producing them are
still present in the intestine, and that treatment has so far failed to kill
the organisms, and not that treatment has failed to kill the cysts or eggs.
According to the second view, though the majority of cysts must neces-
sarily escape from the intestine, some hatch in the large intestine before
they escape, so that the encysted stage can be regarded as a resistant one.
There is no evidence that this actually takes place in the large intestine
of man, though Sellards and Theiler (1924) have succeeded in infecting
kittens by injecting them per rectutn with material which they claim
contained only encysted forms of E. histolytica. Dr. Drbohlav informs
the writer that he has been able to confirm this observation, which has
been repeated by Hoare (1926). The writer has observed in stained
preparations cysts of E. histolytica which appeared to have ruptured and
to have developed hernia-like protrusions. It is just possible that this may
be a natural process, and represents the escape of amoebae from the cyst.
An individual who is in the carrier condition may at any time revert
to one of acute amoebic dysentery. An infected person frequently suffers
from periodic attacks of acute amoebic dysentery when only the large
tissue-invading forms are present in the stool. Between the attacks,
ENTAMCEBA HISTOLYTICA 191
when the acute symptoms have abated, the carrier condition maintains,
when precystic amoebae and cysts are passed. Certain individuals become
infected without suffering from acute dysentery, the infection being
detected only as a result of microscopic examination of the faeces. Those
who become carriers after acute attacks have been termed convalescent
carriers by Walker and Sellards (1913), and the others contact carriers.
Such carriers may remain infected for many years, probably for the rest
of their lives, without at any time being seriously troubled by their
infection. In this respect the infections with E. histolytica are very
similar to those produced by pathogenic bacteria.
As would be expected from the above account, the signs of an infection
with E. histolytica vary considerably. In the acute condition, if there
is extensive ulceration, the quantity of mucus and blood and the number
of amoebse passed in the stool may be considerable. The mucus is
generally of a brownish colour and the blood of a dark red tint. If
ordinary food has been continued, as is often the case, and the large
intestine has not been emptied of faecal matter, this will be present, and
mixed with the blood and mucus to a varying extent. It is not surprising
that the amoebae are found in largest numbers in the mucus which has
been discharged from the ulcers or from the irritated surface in their
immediate neighbourhood. In some cases where active multiplication
of amoebae is in progress over a large surface of the bowel, and food is
continually taken, the stool may be of a soft brown consistency, which
on first inspection appears to differ little from the normal. It will be
found, however, that there is an intimate mixture of faecal matter and
mucus in which large numbers of amoebae occur. Sometimes the stool
is more liquid and of diarrhoeic nature (amoebic diarrhoea), when careful
inspection will reveal small flakes of mucus in which numerous amoebae
may be found. Such cases may be due to superficial invasion of extensive
areas. In many cases it is impossible to decide whether the symptoms
noted are due entirely to the amoebae, or whether they are partly the
result of secondary bacterial infection of the already damaged tissues.
It would be expected that an ulcerated intestine, though producing no
symptoms, would be more liable than a healthy one to be irritated by
food or bacteria, and if diarrhoea results from such irritation it is difficult
to affirm that it is due to the amoebae, though many may appear in the
stool. It not infrequently happens that individuals who are undoubtedly
infected with E. histolytica rarely pass amoebae in the stools, so that many
examinations have to be undertaken before an absolutely certain diagnosis
can be made. In these cases inspection of the mucosa of the lower bowel
by means of the sigmoidoscope has yielded valuable information. Not
only can the ulcerated areas be seen, but scrapings from them will imme-
192 FAMILY: AMCEBIDiE
diately reveal amoebae even in cases which have proved negative after
many examinations of the faeces.
Some observers have attempted to discover a means of diagnosis in
the microscopic appearance of the cells in the stools of amoebic dysentery
cases, but apart from the amoebse there is nothing characteristic of the
condition. As a rule there occur a certain number of cells, including
degenerating epithelial cells, macrophages which have been discharged
from the ulcers, and some pus cells. They are usually present in com-
paratively small numbers, and it is only rarely that the stool contains
the enormous number of cells usually seen in acute bacillary dysentery.
Thomson, J. G. (1918), and Acton (1918) drew attention to the frequent
presence of Charcot- Ley den crystals in the stools of amoebic dysentery
cases, and the latter observer concluded that their presence was pathog-
nomonic of an infection with E. histolytica. Thomson, J. G., and Kobert-
son (1921 and 1921a) have published an account of observations which
tend to confirm the earlier conclusions. It is possible that Charcot-
Leyden crystals appear in any chronic ulcerative condition of the large
intestine, and that their association with E. histolytica is a result of the
amoeba being the most frequent cause of such a condition.
It has been noted above that E. histolytica may find its way to the
liver, spleen, or even the brain, and there give rise to abscesses. In these
situations the process of development is like that in the deeper tissues
of the intestinal lesions. Only the large amoebae are found, and there
seems to be no tendency to the production of the small, precystic amoebse
or cysts, which have never been demonstrated in these situations.
Wherever E. histolytica occurs in the tissues there is no tendency for
the area of invasion to be limited by the formation of fibrous tissue. On
this account the abscesses of the liver are not limited by a fibrotic wall,
as occurs in the case of chronic bacillary abscesses. If a section of the
wall of an amoebic abscess is examined, it will be seen that there is a
gradual transition from normal tissue to the completely necrotic area on
the surface of the abscess wall. The amoebse are found to be most
numerous in what may be called the intermediate zone. On this account
the examination of the pus which first discharges from an amoebic abscess
of the liver may reveal no amoebse. After a day or two, when apparently
the surface of the abscess is breaking away and being discharged, amoebse
may appear in the discharge in large numbers. These amoebse have the
same character as the larger forms found in the intestinal ulcers.
A number of records of the presence of amoebse in the urine have been
published. In the majority of cases these are more than doubtful, but
in one or two instances, as in the cases recorded by Walton (1915) and
Petzetakis (1923), it seems safe to suppose that the observers were actually
PLATE I.
Entamoeba histolytica (x looo) as seen in living condition in a portion of mucus from the
stool of a case of amcEbic dysentery. The mucus, in addition to the amoebae, contains
leucocytes and red blood corpuscles. Many of the amoebae show ingested red blood
corpuscles, from some of which the haemoglobin is diffusing into the cytoplasm, giving
a browinish tinge to the amcebae.
(Original.
[To fa
ENTAMCEBA HISTOLYTICA 193
dealing with amoebae which were of the E. histolytica type, and not with
tissue cells, which frequently lead observers astray. How the amoebse
gain access to the urine is not known, but it may be surmised that a
secondary infection of the urinary tract has taken place, and that amoebse
are discharged from the lesions into the urine, where, however, they
undergo degeneration more rapidly than after their discharge into the
lumen of the bowel from the intestinal ulcers. There is no reason for
regarding the urinary form as a species distinct from E. histolytica, though
Baelz (1883), who was the first observer to see amoebae in the urine,
proposed the name Amoeba urogenitalis.
Warthin (1922) observed E. histolytica in the vas deferens and the
lumen of the dilated tubules of the epididymis and testis. The patient,
a typical case of amoebic dysentery, died in spite of treatment which had
cleared the intestine of its infection. The amoebae were seen in section
of the tissues. They were mostly in clots of blood and fibrin in the lumen
of the dilated tubes, but in some places were invading the walls. They
were remarkable in that they had phagocyted not only red blood-corpuscles,
but also spermatozoa. Hines (1923) noted that a case of amoebic dysen-
tery suffered from enlarged and extremely tender seminal vesicles.
Seminal fluid expressed from the vesicles revealed typical active amoebae
with included red blood-corpuscles.
Petzetakis (1923 and 19236) in Alexandria describes amoebic bronchitis
in which, without actual abscess formation, the lungs appear to be in a
broncho-pneumonic state. There was no evidence of liver abscess, and
only certain cases gave a history of dysentery. The expectoration was
said to contain active amoebae, which in their movements, size, structure,
and included red blood-corpuscles resembled E. histolytica. Those cases
which were free from intestinal infection responded very readily to emetin
treatment. It is evident that these claims require confirmation. Libert
(1924) states that he obtained active forms of E. histolytica in a case of
hepatitis by means of the duodenal tube, an observation confirmed by
Boyers, Kofoid and Swezy (1925).
Several observers have recorded amoebic infections of the skin, but
in most cases there is little evidence that the structures described were
amoebae at all. Maxwell (1912) observed amoebae in fistulae about the
buttocks of cases in Formosa. In this instance it is not improbable that
amoebae had passed into the fistulae from the intestine. Engnian and
Heithaus (1919) gave a description and figures of what they regarded
as E. histolytica from ulcers on the skin of a case which was said
to have an intestinal infection. Judging from the figures and description
it is impossible to recognize the bodies as amoeba?, and it is evident the
authors have had little experience of these organisms. Kofoid and Swezy
I. 13
194 FAMILY: AMCEBIDiE
(1924 a) state, however, that they have examined the material from this
case, and can confirm the occurrence of E. histolytica in the skin lesions.
Furthermore, they claim to have seen another case showing the same
infection.
Smith, S. (1924) states that he has seen amoebse in pus from a knee-
joint, while Sharp and Morrison (1925) claim to have found them in pus
from abscesses in muscles.
MORPHOLOGY. — The morphology of E. histohjtica may be considered
under three headings corresponding with the three phases of. development
— namely, the tissue-invading form, the precystic form, and the cyst.
1. Tissue-Invading Forms. — These may be regarded as representing
the most active phase of development (Plate I., p. 192). They occur
normally in the walls of the intestinal ulcers and of the secondary lesions
produced in other parts of the body. They are to be found in the faeces
after discharge from the ulcers, in the pus draining from abscesses of the
liver and other organs, or in material coughed up after rupture of an
abscess into the lung. As has been pointed out by Dobell (1919), the
amoebae begin to degenerate soon after they have left the intestine, an
explanation of the many discrepancies which characterize the accounts
of the morphology of E. histolytica and the attempts at the establishment
of new species. Even when the amoebae are seen in perfectly fresh stools
within a few minutes of their escape from the body, changes may already
have occurred during their passage down the large intestine. It thus
happens that in most cases in actual medical practice a diagnosis has
to be made from forms which are abnormal, and which do not show the
true structure of the nucleus and cytoplasm of the amoebae as they appear
in the living tissues. Such alterations in character, however, do not
necessarily lead to the death of the amoebae, for kittens may be infected
by injection of material which was passed many hours before.
The tissue-invading form of E. histolytica as a rule varies in diameter
from 20 to 30 microns, but larger or smaller forms may occur (Fig. 95, 1-4).
A very characteristic feature of the amoeba is its activity, large,
blunt pseudopodia being formed and withdrawn in rapid succession.
Progression in one direction is effected by the formation of a pseudopodium
and the flowing of the entire cytoplasmic body into it. The pseudopodia
are often formed quite suddenly with almost explosive violence. The
remarkable activity of a group of these amoebae when seen in a freshly
passed and still warm portion of mucus can only be appreciated when
seen; no description can give a satisfactory picture of this really extra-
ordinary phenomenon. Not infrequently the amoebae become elongated
and glide in a slug-like manner over the surface of the slide without
noticeable change in shape. In so doing the posterior end may have
ENTAMCEBA HISTOLYTICA
195
/
\
s
'»
■■©;
Oi
Fig. 95. — Eniamceha histoJijtiea : Vegetative Forms (x 2,000). (Original.)
1. Large form with single nucleus, three included red blood-eorpiisclcs and two other bodies of
doubtful nature. 2. Large form with dividing nucleus.
3. Large form with two nuclei and food inclusions, possibly altered and swollen red blood-
corpuscles. 4. Large form with clear cytoplasm.
5. Form with spicular chromatoid bodies. 6. Four-nuclear forms from liver abscess pus.
7. Precystic form with large chromatoid body. 8. Precystic form with included bacteria.
9. Precystic form with clear cytoplasm. 10-11. Precy.stic forms of small race.
196 FAMILY: AMCEBIDiE
a ragged appearance, and to it bacteria and other debris may adhere.
This type of movement is common in certain free-living amoebas, and
from it the name " Umax " is derived. Though occasionally E. coli will
be seen to move with an activity almost, if not quite, equal to that of
E. histolytica, this is rarely the case, and the energetic movements of
E. histolytica serve as one of its most important distinguishing features.
When degeneration is advanced the movements become less evident, and
finally cease altogether, though very often evidently degenerate amoebae
will commence moving with remarkable activity when warmed on the
warm stage. In the formation of the pseudopodia the first indication is
a slight elevation of the ectoplasm, but as this increases in size the endo-
plasm quickly flows into it. A characteristic appearance of E. histolytica,
as seen in the stool, is that of an amoeba with a clear, broad, hyaline
ectoplasm sharply marked off from a granular endoplasm. Such forms
may be producing pseudopodia with great activity, and these frequently
consist entirely of ectoplasm. This extreme condition is probably the
result of degeneration. As E. coli does not produce appearances of this
kind, the marked ectoplasm of these altered E. histolytica serves as a dis-
tinguishing feature. In the perfectly fresh and normal individuals the
distinction between ectoplasm and endoplasm is much less marked. The
writer has examined portions of infected mucosa removed through the
sigmoidoscope. Though not more than one minute had elapsed after
removal, the amoebae could be seen actively motile within the pieces of
mucosa and forming ectoplasmic pseudopodia, as in the freshly passed
dysenteric stool. It hardly seems possible to regard the amoebae under
these conditions as being in any way degenerate. On the other hand,
the appearance of an amoeba with a relatively thick ectoplasm surrounding
a globular mass of granular endoplasm is undoubtedly due to a degenera-
tive change. The endoplasm has a ground-glass appearance, and, apart
from the nucleus and food vacuoles, contains, according to Dobell (1919),
numerous small granules which stain intra vita^n with neutral red. The
food vacuoles include red blood-corpuscles (Fig. 95, i), and sometimes
leucocytes or other cells in various stages of degeneration (Fig. 95, i).
Sometimes the whole endoplasm appears packed with red cells, and in
many cases, as these become dehsemoglobinized, the cytoplasm assumes
a yellowish tint. It is only rarely that other objects are ingested by the
amoebae. The number of amoebae in any particular specimen containing
red blood-corpuscles varies considerably. Sometimes as many as 25 per
cent, will show them, while in other cases a long search will reveal only
a single one, or none at all. Amoebae containing red cells may be found
in stools which do not show any blood or other abnormality on naked-eye
inspection.
ENTAMCEBA HISTOLYTICA 197
The writer and O'Connor (1917) noted the occasional inclusion of the
spores of a large bacillus, and on one occasion a large yeast-like organism.
E. histolytica is very fastidious about the kind of food it takes up, and in
the cases just mentioned, though many other structures were present in
the surrounding medium apart from the spores or yeasts, all the amcebse
had selected these particular objects for ingestion. The question arises
as to whether these were taken up by the amoebae before they were
discharged from the ulcers, as probably happens in the case of the included
red blood-corpuscles and other cells, or whether they were ingested during
the passage of the amoebae down the large intestine. E. coli, on the other
hand, ingests indiscriminately all kinds of objects in the intestine, but
apparently not red blood-cells, so that the presence of the latter in an
amoeba is strong presumptive evidence of its being E. histolytica. Occa-
sionally, however, undoubted E. histolytica, as seen in the stool, possess
vacuoles with included bacteria. These organisms are sometimes seen
in amoebae in sections of ulcers from human beings and cats when the
ulcer is invaded by intestinal organisms. This condition is only seen in
the superficial layers. Amoebae, both in faeces and in sections of intestinal
ulcers and liver abscess, especially in cats, may contain numerous irre-
gularly-shaped bodies which appear to be chromatoid in nature. Some-
times they bear some resemblance to Charcot-Leyden crystals (Fig. 95, 5).
In the cultures of E. histolytica, as described by Boeck and Drbohlav
(1925), the amoebae, when grown on egg media, feed largely on bacteria.
In blood media they ingest red cells also.
The nucleus of E. histolytica is a spherical structure 4 to 7 microns in
diameter. It consists of a fine membrane enclosing an alveolar substance
which in fixed material assumes the form of a network of linin threads,
some of which may be radial. At the centre of the nucleus is a small
karyosome surrounded by a clear area, the outer limits of which represent
the inner limits of the alveolar material. In fixed specimens, again, the
clear area appears in optical section to be limited by a ring of fine granules.
The small karyosome is homogeneous, and is said to consist entirely of
chromatin. Hartmann and others claim to have detected a centriole in
the karyosome, but it is very doubtful if such a structure exists. Chromatin
granules are arranged uniformly over the inner surface of the nuclear
membrane. In amoebae which have partially degenerated the nuclei
may have a very different appearance. The karyosome may appear larger
and be definitely excentric in position, while the chromatin on the
membrane may be distributed more irregularly in the form of several larger
masses. Amoebae with nuclei which do not conform to the type are
frequently encountered in perfectly fresh stools. The position of the
nucleus in the endoplasm varies considerably, and is subject to constant
198 FAMILY: AMCEBIDiE
change, as can easily be noted by observing living amoebse in which the
nucleus can be seen. Owing to the density of the cytoplasm and its high
refractive index, the delicate nucleus of E. histolytica is often difficult to
detect in the living amoebae. The nucleus of E. coli, on account of the less
dense cytoplasm, is more readily seen, while structurally it is very different
from that of E. histolytica.
E. histolytica mulitiplies in the tissues by binary fission. There is
first a division of the nucleus, the details of which have been described by
Dobell (1919), and this is followed by division of the cytoplasm into two
more or less equal parts. Kofoid and Swezy (1924a, 1925) state that there
are six chromosomes which appear during nuclear division (Fig. 57).
Reproduction by bud formation, as described by Schaudinn (1903), and
by a process of schizogony, as recorded by Job and Hirtzmann (1918),
are undoubtedly the result of observations on degenerate amoebse, or even
tissue cells. On one occasion the writer has observed amoebae with two
and four nuclei in liver-abscess pus (Fig. 95, 6).
2. Precystic Forms. — As already explained above, under certain con-
ditions E. histolytica becomes encysted, and as the cysts are smaller than
the tissue-invading amoebae, it is evident that before encystment smaller
amoebae are produced. These are probably developed from the large
amoebae by division, while the daughter amoebae, instead of increasing in
size as they do in the tissues, divide again, so that increasingly small forms
are produced. It is possible that the large amoebae, which have become
more superficial in position in the intestinal lesions, suffer from a lack of
fresh tissue or fluid nutriment on which to feed, so that after division
growth does not take place. This shortage of food may be the stimulus
which leads to encystment. The size of the amoebae which actually
encyst varies considerably, and evidence has been brought forward by
the writer and O'Connor (1917), and by Dobell and Jepps (1917, 1918),
that there exist definite races of E. histolytica which can be distinguished
from one another by the average size of the cysts. In the races with small
cysts these may have an average diameter of 7 microns only, while in
those with larger cysts it may be as much as 18 microns. It follows,
therefore, that the precystic amoebae may vary in diameter from 7 microns
upwards (Fig. 95, 7-11). There does not appear to be any evidence to
support the view that in those races with small precystic amoebae the
corresponding tissue-invading forms are smaller than in those producing
larger precystic amoebae.
The precystic amoebae have the same general structure as the tissue-
invading forms, but the cytoplasm is devoid of food vacuoles, the amoebae
having ceased to ingest red blood-corpuscles or other cells, and having
got rid of the remains of those taken in previously.
ENTAMCEBA HISTOLYTICA 199
The precystic forms of E. histolytica were first seen by Elmassian
(1909). He did not realize their nature, and, thinking he was dealing with
a new amoeba, gave it the name E. minuta. The name was employed
subsequently by Woodcock and Penfold (1916) for the smallest races of
E. histolytica, but Elmassian did not use it for the small race, of the
existence of which he was not aware, but for the one of average size which
everyone now admits is undoubtedly E. histolytica. Walker (1911), and
Walker and Sellards (1913), appear to have been the first to realize that
the small amoebae with clear cytoplasm were the precystic forms of the
large tissue-invading amoebae. This has been amply confirmed by many
observers. Shortly before encystment takes place the amoeba often
develops a vacuole containing glycogen, which colours brown with iodine,
as well as one or more refractile bodies. The latter, which often have the
form of rods with rounded ends, were named chroynatoid bodies by Dobell.
They show no marked affinity for iodine, but stain black with iron
hsematoxylin. It is very improbable that they are chromatic in nature.
They have well-defined edges, and are readily seen as greenish refractile
bars in the living amoebae or cysts. The margin of the glycogenic vacuole,
as stained with iodine, is not sharply defined, for it gradually shades off
into the surrounding cytoplasm. In the case of the cysts of lodatnoeba
butschlii, the substance in the vacuole is much denser than that in the
vacuole of the cysts of E. histolytica, and in iodine-stained specimens the
limits of the vacuole, or more correctly those of the glycogenic body
within it, are very sharply defined, the brown colour of the included
substance ceasing abruptly at the margin of the vacuole (Plate II., 5, 6, 9,
and 11-14, p. 250).
The nuclei of the precystic amoebae resemble those of the tissue-
invading forms, except that the chromatin on the membrane often occurs
in larger masses. In some cases the nuclei possess a single large crescentic
mass in addition to smaller ones. Dobell (1919) states that chromatin
granules occur also on the linin network, a condition which he does not
find in the normal nuclei of the tissue-invading forms.
The precystic amoebae are not so active as the tissue-invading forms,
and on account of the larger chromatin gratiules of the nuclei they may
be difficult to distinguish from the corresponding stages of E. coli. In these
cases it will be necessary to discover the characteristic cysts. The smaller
races are still more difficult to distinguish, as they may be confused with
Endolimax nana. The structure of the nucleus, as seen in stained pre-
parations, is important, and a final diagnosis may not be possible till
cysts have been found, it may be after repeated examinations on different
days.
The precystic amoebae and the cysts of E. histolytica were first
200 FAMILY: AMCEBID^E
accurately studied by Huber (1903), though the cysts had previously been
seen and figured by Quincke and Roos (1893), and Rods (1894), They
were again seen by Viereck (1907), and by Hartmann and Prowazek (1907),
who regarded them as belonging to distinct species of amoebae, which
were named E. africana and E. tetragena respectively. This supposed
difference, however, was the outcome of Schaudinn's erroneous account
of the development of E. histolytica, which was almost entirely based on
the appearances seen in degenerating amoebse. There can be no doubt that
E. africana, E. tetrageyia, and E. ttmiuta are merely forms of E. histolytica.
The small amceba described by Prowazek (1912a) as E. hartmanni, and
by Kuenen and Swellengrebel (1917) as E. tenuis, is undoubtedly a small
race of E. histolytica, producing cysts 6 to 8 microns in diameter.
3. Cyst. — The cyst which is formed round a precystic amoeba seems
to be composed at first of a soft material which quickly shrinks and
hardens to a resistant, colourless, smooth, transparent capsule. It is
completely filled by the cytoplasm of the amoeba (Fig. 96). The cyst
wall is about 0*5 micron in thickness, its inner and outer margin being
visible in optical section. When first formed, it encloses the amoeba
and the structures it contains. Thus, the newly-formed cyst contains
the cytoplasm and nucleus, and also the vacuole and chromatoid bodies
if these happened to be present in the amoeba. The cysts are generally
spherical, but they may be elongated or even dumb-bell-shaped. Within
the cyst the single nucleus divides to form two nuclei, and these divide
again, so that in the mature cyst four nuclei are present (Fig. 57). Very
frequently the four nuclei are arranged in pairs at opposite sides of the cyst.
On Very rare occasions eight nuclei may be found. According to Dobell
(1919) the vacuole, if present, gradually disappears as the cyst develops.
It appears as if the glycogen of the vacuole is used up during the nuclear
divisions. The chromatoid bodies are similarly absorbed while the cyst
is waiting outside the body to be ingested by a new host. The chromatoid
bodies usually have the form of rods with rounded ends, and very com-
monly one, two, or three are present in the cyst. They vary in length
from 5 to 10 microns, but longer or shorter forms may occur. Sometimes
they ^e of a different shape, and may be more rounded or irregular in
outline. On other occasions they are filamentous structures, or a large
number of small, irregularly-shaped bodies may be present. When seen
in the living cyst they appear as homogeneous structures which have
a refractive index higher than that of the rest of the cyst. On this
account they are readily distinguished. The chromatoid bodies are of
great diagnostic value, for they occur much more rarely in the cysts of
E. coli, in which case the eight nuclei characteristic of the mature cysts
of this amoeba will be noted.
ENTAMCEBA HISTOLYTICA
201
^><8A
■7
iTk
^
/4
i5
fZ
/J
'#
Fig. 96. — Entamoeba histolytica: Encysted Forms ( x 2,000). (Original.)
1. Form with one nucleus and vacuole. 2. Form with one nucleus and chromatoid bodies.
.3. Form with one nucleus, large vacuole, and chromatoid bodies.
4. Irregularly shaped form with single nucleus, large vacuole, and chromatoid bodies.
5. Form with dividing nucleus.
6. Binucleated form without vacuole or chromatoid bodies.
7. Binucleated form with chromatoid bodies.
8. Binucleated form with numerous chromatoid bodies.
9. Form with four nuclei and chromatoid bodies.
10. Form with four nuclei, chromatoid bodies, and vacuoles with inclusions.
11. Form with four nuclei and two chromatoid bodies. 12. Form with four nuclei alone.
13. Form with six nuclei, two of the original four having divided. Similarly, forms with eight
nuclei occasionally occur. 14-16. Forms belonging to a small race.
202 FAMILY: AM(EBID.E
The cyst of E. histolytica, when seen in fresh material, has a greenish
refractile appearance. Owing to its refractiveness, which is much more
marked than that of the cysts of E. coli, it is sometimes very difficult to
distinguish the nuclei, though the chromatoid bodies may be easily seen.
In iodine solution, however, all the contents can be clearly distinguished
(Plate II., 5-IO, p. 250).
The cysts of E. histolytica vary in diameter from 5 to 20 microns
according to the particular race, but all the cysts of any one race are not
of the same size. Thus, in six cases studied by the writer and O'Connor
(1917) the diameter of the cysts varied as follows: 7 to 9 microns, 7 to 11
microns, 10 to 13 microns, 10 to 14 microns, 11 to 15 microns, 12 to 18
microns. The cysts remained constant in their average size during the
observation, which in some cases extended over several months, so that
it would appear that true races are represented (Fig. 10). Some observers,
however, believe that the small cysts belong to a distinct species of
amoeba. Prowazek (1912 a) gave the name E. hartmanni to these forms,
Kuenen and Swellengrebel (1917) the name E. tenuis, and Brug (1917)
the name E. minutissima. Though the writer has repeatedly observed
the appearance of cysts in the stools of cases in which the acute symptoms
of amoebic dysentery were subsiding, these have always been of the average
size, or larger than this. In no case has he seen the small cysts appear
under these circumstances. It cannot be regarded as finally established
that the races of E. histolytica which produce the small cysts are able
to give rise to amoebic dysentery. Drbohlav (19256) has cultivated a
small race. In the cultures the amoebae resembled the typical
E. histolytica. They did not, however, ingest red blood-corpuscles, and
though producing infection in kittens, failed to give rise to dysentery and
ulceration of the large intestine. The precystic amoebae as seen in faeces
correspond in size with the cysts, so that they are smallest in those
races which produce the smallest cysts. There are no data, however,
to show whether a corresponding variation in average size of the
tissue-invading forms occurs. Shimura (1918) described a race of
E. histolytica with small cysts as a non-pathogenic race, but it has to be
remembered that the majority of carriers who show no symptoms are
passing cysts of the average size. If carriers alone were examined, the
average-sized cysts might with equal justification be regarded as belonging
to non-pathogenic races. In the case of the smaller-sized cysts, diagnosis
from living specimens may be very difficult unless the characteristic
rod-like chromatoid bodies are present. In iodine the details are much
clearer, but it is often necessary to prepare stained films before making
a final diagnosis.
The cysts passed from the body may contain one, two, or four nuclei.
ENTAMCEBA HISTOLYTICA 203
It sometimes happens that the majority of the cysts seen in a specimen
of fseces are in the uninucleate condition, while two and four nuclear
specimens may be very difficult to find. In other cases the majority of cysts
have four nuclei. There seems to be no regularity regarding the stage
of development in which cysts are passed, and this is not surprising when
it is remembered that the evacuation of the large intestine depends upon
the host, and may occur either before or after encystment has commenced.
The cysts of E. histolytica will remain alive for a considerable time after
leaving the body in faecal matter, but if brought into clean water they
will survive a much longer period. During this time the chromatoid
bodies gradually disappear. When the cysts die, various degenerative
appearances become evident, and it is found that the cysts will stain
immediately if brought into eosin solution. It appears that eosin solution
may be used as a test of the life of a cyst, as also of the free amoebae them-
selves. The live cysts or amoebae will not stain immediately, whereas
the dead ones will become red at once. This can readily be demonstrated
by watching the effect of eosin on cysts before and after heating to a
temperature sufficient to kill them.
In his description of the development of E. histolytica, Schaudinn
(1903) described a method of reproduction by bud formation. The
nucleus was supposed to give off chromatin material into the cytoplasm
in the form of granules, which collected in groups on the surface of the
amoebae. Small cytoplasmic buds, each containing a group of chromatin
granules, were formed. These buds were described as becoming enclosed
in very resistant capsules, forming spores, which were much smaller than
the cysts of E. histolytica, as they are now known. Schaudinn claimed
to have produced infection in cats by means of these spores after complete
drying, a procedure which is known to kill immediately the cysts of
E. histolytica. Eecent investigations have failed entirely to confirm
Schaudinn's statements, so that it is safe to conclude that the budding
process and spore formation as described by him do not take place.
PATHOGENICITY.— It has usually been assumed that any individual
who is harbouring E. histolytica must have definite lesions of the intestinal
wall, in the tissue of which the amoebae are living, but though this may
be true to a very large extent, the successful culture of the amoebae in
tissue-free media suggests that they may sometimes live on the surface
of the intestine without giving rise to actual lesions. In some cases the
lesions give rise to large quantities of blood and mucus, so that the acute
condition of amoebic dysentery results. In the great majority of cases,
however, very few, if any, symptoms are noted, so that the infection can
only be detected by microscopic examination of the faeces. The fact
that in some individuals the symptoms of acute dysentery occur, while
204 FAMILY: AMCEBID^
in others the infection is of a mild nature, may be comparable with what
is known to occur in bacterial infections. Many individuals harbour
pathogenic bacilli in their throats without having symptoms of the
disease which may be caused by these organisms. Invasion of the tissues
may occur because the resistance of the host is lowered or because the
virulence of the organism is increased. The former seems to be the most
rational explanation, and in the case of E. histolytica infections it would
seem that the resistance of the intestine is lowered from time to time,
with the result that active multiplication of the amoebae with their exten-
sion into the tissues takes place, so that acute dysentery supervenes. On
the other hand, it has to be remembered that the virulence of protozoa
may vary considerably. In the case of trypanosomes it is well known
that passage of a strain from one animal to another may so change it
that it will bring about death in a few days instead of a few months.
It is possible that the virulence of E. histolytica may vary in a similar
manner, but there is no evidence that this occurs. By quick passage
of a strain from one man to another the virulence might be so increased
that eventually every individual infected would acquire an acute and
fatal amoebic dysentery. Whether this actually happens in nature is not
known, but judging from the results of experiments on kittens the writer
can find no reason to suppose that the amoebae from carrier cases with few
or no symptoms are less virulent than those from acute cases. Brumpt
(1925), however, suggests that there exist two types of amoeba included
under the name E. histolytica, the one infective to kittens and the other
not. To the latter he gives the name Entamoeba dispar, and suggests that it
accounts for many of the carrier cases in countries where amoebic dysentery
is uncommon. The writer does not believe that physiological data of this
kind aiiord a means of distinguishing species.
SUSCEPTIBILITY OF ANIMALS.— Losch (1875) succeeded in infecting
a dog with E. histolytica, and this experiment was repeated by Hlava
(1887), Kruse and Pasquale (1894), Harris (1901), and Dale and Dobell
(1917). Young cats, however, are more easily infected, and it is with
them that most experimental work has been done. Hlava (1887) was
the first to produce infection in these animals, and he was followed by
Kartulis (1891), Kovacs (1892), Quincke and Roos (1893), Kruse and
Pasquale (1894), Marchoux (1899), the writer (1912(?), and many others.
Kruse and Pasquale also infected cats with the amoebae obtained from
liver abscess. The infection has generally been produced by injections
of dysenteric stools per anum, and this is the most reliable method for
infecting these animals. Unless cysts are present the animals cannot
be infected by feeding, a fact first demonstrated by Quincke and Roos
(1893), the first observers to describe the cysts of E. histolytica. Huber
ENTAMCEBA HISTOLYTICA 205
(1903), who rediscovered the cysts, confirmed this observation, which was
repeated by Kuenen and Swellengrebel (1913), the writer and O'Connor
(1917), and Dobell (1917), and others. The infection in kittens is, as a
rule, of a very severe type, the whole of the surface of the large intestine
being infected with amoebae. The infection usually commences at the
lower part of the large intestine, and it is here that the changes in the
mucosa are most marked. Sellards and Leiva (1923a) have shown that
this is probably due to the natural stasis which occurs at this point. By
ligaturing the large intestine of cats at various levels and inoculating
infective material directly into the csecum they have demonstrated that
the infection commences and is most marked just above the ligature.
They see in this an explanation of the fact that in human beings amoebic
ulceration is most marked at the point where stasis occurs. If the animals
live long enough, definite ulcers occur as in human beings, but frequently
all the glands are infected over the whole gut wall and death results from
the general necrosis of the mucosa which is set up. Sellards and Leiva
(1923a) have shown that bacterial invasion of the blood also plays a
part, for they have cultivated various intestinal organisms from the
blood of infected cats. As a method of diagnosis of amoebic infection
in the cat they have employed a daily saline enema. The fluid is quickly
returned, and the flakes of blood-stained mucus which it carries with it
can be examined for amoebae. Infected cats frequently pass -per anum
a whitish fluid containing many broken-down cells and enormous numbers
of amoebae. In less acute cases the stools resemble those of amoebic
dysentery in man, there being faecal material containing masses of mucus
stained with dark red blood. Eecoyery rarely takes place in cats, and
when it does the infection dies out, there being no carrier condition
corresponding to that in human beings. The cysts of E. histolytica are
never formed in cats. As in man, secondary infection of the liver may
take place, leading to the formation of liver abscess, an observation
Avhich was first made by Marchoux (1899), and subsequently by Craig
(1905), Werner (1908), "^Huber (1909), the writer {I9l2d), Dale and
Dobell (1917), Mayer (1919), and Sellards and Leiva (1923ff). Harris
(1901) noted a similar condition in an experimentally infected dog.
The infection in cats may be maintained indefinitely by injecting the
intestinal contents per rectum from one animal to another. The cats
must be only a few weeks old, as large, full-grown animals are more
resistant to infection. As the cysts of E. histolytica never occur in cats,
the infection cannot be handed on from cat to cat by feeding with intestinal
contents. The writer has never succeeded in infecting kittens by means
of material from liver abscess, in spite of the presence of active amoebae.
Guinea-pigs have been infected by Baetjer and Sellards (1914), and
206 FAMILY: AMCEBIDtE
by Chatton (1917, 1918rf). This may be accomplished by injections
per anum or by feeding with cysts per os. In these animals dysenteric
symptoms do not appear, but large tumours develop about the caecum,
and these are found to consist of overgrowths of the tissues due to the
amoebae, which grow and multiply within them. Huber (1909) claims to
have produced a chronic ulceration of the caecum in rabbits by feeding
them with cysts, while Lynch (1915) and Brug (1919a) claim to have
infected rats. Kessel (1923a) states that he has infected rats and mice
with E. histolytica. He finds (1923) that natural amoebic infections of
these animals can be excluded by the examination on two successive days
of faeces obtained after the administration of a purge in the form of stale
bread soaked in magnesium sulphate solution. To such animals cysts
of E. histolytica were given. The infections produced are of a chronic
nature, and persist for months. Free forms, as well as characteristic
cysts, could be obtained in the faeces of the animals after giving them
magnesium sulphate. The infection was handed on from rat to rat.
Chiang (1925) has also infected rats. The amoebae from the experimental
rats, as well as a naturally occurring rat strain {E. histolytica var. murina),
gave rise to typical infections when inoculated to kittens. Clean rats
kept with infected ones contracted an E. histolytica infection.
Attempts which have been made to infect monkeys have been incon-
clusive, owing to the fact that these animals are liable to natural amoebic
infections due to two species of amoebae which are very similar to E. histo-
lytica and E. coli. These animals suffer from amoebic dysentery, and
even amoebic abscess of the liver, as pointed out by Eichhorn and Gallagher
(1916) and others (see p. 226).
CULTIVATION. — Many attempts have been made to cultivate E. histo-
lytica in artificial media, but the only successful results are those of Cutler
(1918) and Boeck and Drbohlav (1925). Other observers have cultivated
only coprozoic amoebae. Cutler used two media.
The first was made as follows: The entire contents of an egg were
broken up by shaking in a glass bottle with beads. To the broken-up
egg 300 c.c. of distilled water were added, and mixture was effected by
shaking. The fluid was then brought gradually to the boiling-point in
a water bath, and kept at this temperature for half an hour. During the
heating the mixture was shaken, so that a fluid was obtained in which
minute egg particles were suspended. It was then distributed in quanti-
ties of 5 c.c. in test-tubes and autoclaved. Before use a few drops of
blood were added to each tube.
The second medium was prepared by boiling 500 c.c. of human blood-
clot for an hour in a litre of water. To the filtrate was added 0-5 per
cent, sodium chloride and 1 per cent, peptone. The fluid was then tubed
ENTAMCEBA HISTOLYTICA 207
and sterilized by steaming for twenty minutes on three successive days.
As in the case of the egg medium, a few drops of blood are added before
inoculation.
Attempts were made to cultivate amoebae from forty-five samples of
fseces containing E. histolytica, and amoebas were grown from six which
contained blood and mucus. Bacteria grew in the media as well as the
amoebae, and it was necessary to subculture every twenty-four to seventy-
two hours on account of the quantity of acid produced by the bacteria.
A temperature of 28° to 30° C. was better than a higher one, as bacterial
growth was reduced. Subculture was effected by transfer of 0-5 to 1 c.c.
of the culture. By this means cultures were maintained for over three
months, and not only did multiplication of the amoebae take place, but
encystment also occurred. Cats were infected by inoculation per rectum
with cultures of more than two and a half months' standing, and typical
dysenteric symptoms with amoebae resulted, while post-mortem examina-
tion showed the characteristic amoebic lesions, from which fresh culture
was obtained. Other animals were infected by feeding them on cultures
containing cysts. Dobell (1919) stated that he attempted without
success to cultivate E. histolytica by this method, and concluded that there
must have been some fallacy in Cutler's work. The writer also failed to
repeat Cutler's experiments. Barret and Smith (1923, 1924), however,
obtained cultures of another amoeba, Entamoeba barreti of the turtle,
Chelydra serpentina. The medium used was a mixture of human blood-
serum 1 part and 0-5 per cent, sodium chloride solution 9 parts. In
each tube 5 c.c. of the mixture was used. A small quantity of mucus
obtained from the intestinal wall was inoculated at the bottom of the
tubes, which were kept at 10° to 15° C, or at room temperature. At first
it was necessary to subculture every twenty-four or forty-eight hours,
but when a culture was established a weekly transfer was sufficient. Two
strains were kept for nine months, during which thirty subcultures were
made. The amoeba? multiplied actively, and corresponded in every way
with those seen in the intestine of the turtles. No cysts were found,
however. Cultures of E. ranarum of the frog have also been obtained.
These results, which were obtained with amoebae of cold-blooded hosts,
led Barret and Smith to suggest that Cutler may have been more successful
with E. histolytica than some had supposed.
Quite recently Boeck and Drbohlav (1925) have cultivated E. histolytica
on solid egg and blood agar slopes covered with Locke's solution containing
serum or egg albumin. From two human cases E. histolytica was isolated
and maintained in subculture for many generations, in one case for more
than eight months, during which 150 subcultures were made. Sub-
culture was made every two or three days, and the tubes were kept at
208
FAMILY: AMCEBID^
30° to 37° C. Bacteria were constantly present in the amoebse. In the
blood medium red blood-corpuscles were frequently ingested by the
amoebae, which structurally corresponded with E. histolytica. Even after
as many as ninety-three subcultures kittens could be infected with the
cultural forms, and a condition exactly like that arising from the injection
of material from cases of amoebic dysentery resulted. In a few instances
the animals developed amoebic abscess of the liver. Cultures were also
obtained from the infected kittens. On one occasion cysts were observed
in the culture tubes. Drbohlav (1925rt) has repeated these experiments,
which have also been confirmed by Thomson, J. G. and Robertson (1925).
ABERRANT FORMS OF E. HISTOLYTICA.— Working in North China,
Faust (1923) has observed in four cases of dysentery a peculiar type of
amoeba which ingests not only red blood-corpuscles, but also bacteria
(Fig. 97). The characteristic
feature of the organism,
which has a diameter of 16 to
17 microns when quiescent
and globular, is its posterior
end. When active it is
definitely elongated, with a
rounded anterior end and a
tapering posterior end which
terminates in a pointed pro-
toplasmic structure (caudo-
style), surrounding which are
sometimes several smaller
..,.r.. ,A -r, ,^-.o X K----K— — proiections.
000). (After Faust, 192.3.) ^ ^ ^ ^
Debris tends to become
adherent to the region of the caudostyle. The nucleus, measuring 3 to 4*5
microns in diameter, is always situated in the rounded anterior end of
the organism. On the inner surface of the nuclear membrane are minute
chromatin granules. The karyosome is a star-shaped structure which may
have a central vacuole. The rays consist of chromatin granules. In two
of the cases examined the infection was a pure one, while in the other two
cases E. histolytica occurred in one and E. coll in the other. Faust states
that there was no difficulty in distinguishing these amoeba? from other
species. Though the cases were followed for some time, no encysted stages
of the organism were seen. The amoeba appears to fix and stain badly,
as compared with E. histolytica or E. coli, which sometimes occurred in
the same sample of faeces. Owing to the fea'.ures described above, Faust
places the amoeba in a new genus as Caudamaeba sinensis. He believes
that it is a cause of amoebic dysentery. As regards the validity of this
Fig. 97. — " Candamceba sinensis " from the Human , ,
•2.000^. fAFTER Faust. 192.3.^ protoplasmic
Debris tends
Intestine (
ENTAMOEBA HISTOLYTICA 209
species it is difficult to form an opinion, as the encysted stages were not
seen. In any case there seems to be little ground for the creation of a
new genus. It has to be remembered, however, that undoubted E. histo-
lytica often move in a slug-like manner, as noted by Dobell and O'Connor
(1921), and that many free-living amoebae, as well as E. histolytica, may
develop the slug-like form with tapering posterior end to which debris
adheres, while other amoebae in the same pure culture move in the more
normal amoeboid manner. Whether Candamoeha sinensis is actually
distinct from E. histolytica future investigations alone will show, but it
seems to the writer that sufficient evidence to justify the distinction has not
yet been produced. Eecently the writer has had an opportunity of observing
E. histolytica in cultures. The assumption of a slug-like form with tapering
posterior end to which debris adheres is quite common. The fact that bacteria
as well as red blood-corpuscles occurred in vacuoles is a feature which may
be met with in undoubted E. histolytica. Schubotz (1905) has figured an
elongated form of E. hlattcB of the cockroach which bears some resemblance
to C. sinensis, while Jepps (1923) has described a somewhat similar form
of E. gingivalis, and Keilin (1917) one in E. mesnili (Fig. 109).
Chaterjee (1920) gave the name Entamoeba jmradysenterica to amoebae
which he found post-mortem in dysenteric lesions, and which he regarded
as a distinct species on account of certain peculiarities of nuclear structure.
The writer has seen preparations of this amoeba, which is unquestionably
a degenerate E. histolytica.
Kofoid and Swezy (19246) gave the name Karyamceha falcata to
an amoeba of the human intestine. As the generic name was preoccupied,
they (1925a) changed it to Karyamoebina (Fig. 98). The amoeba was
first described from three cases. The first harboured, in addition, E.
histolytica, E. coli, Endolimax nana, Dientamceba fragilis, as well as the
form described as CounciUnania lafieuri ; the second E. histolytica ; and
the third E. histolytica, E. coli, and C. lafieuri. Three further cases were
reported in their second paper. The chief distinguishing feature is the
nucleus and the method of nuclear division. The nucleus has a definite
membrane, upon which the chromatin is massed in one or two, rarely more,
crescentic clumps. There is an excentric karyosome round which is a halo.
In division the nucleus elongates, and there is formed at each end a deeply
staining pole cap. On this account the amoeba is supposed to be allied to
members of the genus Vahlkampfia (see p. 177). In Vahlkampfia, however,
the pole caps are formed from the divided karyosome, and it is definitely
stated that in K. falcata the karyosome does not divide. In this form the
pole caps are merely terminal aggregations of the large chromatin masses on
the nuclear membrane. On this account the amoeba cannot be allied with
Vahlkampfia. It is said that in K. falcata about twenty chromosomes occur
at the equator of the elongating nucleus. Cysts have not been observed.
I. ' 14
210
FAMILY: AMCEBID^
As pointed out by the writer (1925), from the fact that the cases from
which K. falcata was first recorded harboured E. histolytica also, w^hile two
of them had other amoebae as well, it seems that definite proof that the
so-called K. falcata is a distinct entity has not been produced. It is
known that in E. histolytica the nucleus not infrequently shows chromatin
arranged in crescentic masses, and it has yet to be demonstrated that
in nuclear division such nuclei never assume the form supposed to be
characteristic of K. falcata.
Of quite another nature are the supposed amoebae which Kofoid and
Swezy (1922) and Kofoid, Boyers and Swezy (1922) have described from the
r
'-■^f
.^--
-- - ■:-■:/
\
Fig.
98. — Free Forms of " Kanjamcebina falcata '" ( x 2,000).
Swezy, 1924, Slightly Reduced.)
(After Kofoid and
1. Form with clear pseudopodium and single crescentic body on nuclear membrane.
2. Nucleus with two crescentic bodies united by a fibre.
3. Dividing form: nucleus with pole caps, centrioles united by centrodesmose, and equatorial
plate of about twenty dividing chromosomes.
bone marrow of cases of arthritis deformans, and from the hypertrophied
lymphatic glands in Hodgkin's disease. Because of a particular type of
division exhibited by the nuclei of certain cells, it is concluded tliat they
are not only amoebae, but actually E. histolytica. It must be apparent to
most protozoologists that far more convincing evidence is required before
this view can be accepted.
(b) Non-Pathogenic Forms.
Entamoeba coli (Grassi, 1879) Casagrandi and Barbagallo, 1895. —
Chief synonyms: "Amcebse" Lewis, 1870; "Anioebse" Cunningham, 1871; Amoeba
coli Crassi, 1879; "Amoeba coli mitis" Quincke and Eoos, 1893; "Amoeba intestini
vulgaris" Quincke and Roos, 1893; Entamceba coli, Casagrandi and Barbagallo, 1895;
ENTAMCEBA COLI
211
Entamceba hominis Casagrandi and Barbagallo, 1897; Entamoeba coli Scliaudinn,
1903; Amoeba coli Brumpt, 1910; Entamoeba williamsi Prowazek, 1911; Entamceba
hartmanni Prowazek, 1912 (pro parte); Entamoeba brasiliensis Aragao, 1912 (pro
parte); Loschia coli Chatton and Lalung-Bonnaire, 1912 ; Entamoeba coli communis
Knowles and Cole, 1917 (^^ro parte); Endameba intestinivulgaris Aragao, 1917 ;
Endameba coli Craig, 1917; Endameba hominis Pestana, 1917 ; Councilmania lafleuri
Kofoid and Swezy, 1921.
This amoeba is a harmless commensal of the digestive tract of man,
and is in no sense a tissue-invading amoeba like E. histolytica. According
to Dobell, it was first seen by Lewis (1870) in India, and was described
more accurately by Cunningham (1871). Grassi (1879-1888) gave various
descriptions of the organism, and erroneously believing it to be identical
with the form originally studied in dysenteric cases by Losch (1875),
gave it the name Amoeba coli, a name which shovdd have been employed
for the pathogenic form only. As has been explained above, Schaudinn
again committed this error, and though,
according to the strict laws of nomen-
clature, E. coli should be the name of
the pathogenic amoeba, its employment
in this sense would lead to endless
confusion, so that it is better to retain
the name E. coli for the harmless amoeba.
Grassi realized that the amoeba was a
harmless inhabitant of the human diges-
tive tract, for he found it not only
in sick, but also in healthy people.
Quincke and Eoos (1893) gave a good
description of E. coli, which they dis-
tinguished from E. histolytica, while
Casagrandi and Barbagallo (1895, 1897)
studied the same organism, which they
named Efitamoeba coli. They took a retrograde step in assuming that
this was the only form which occurred in healthy as well as in dysenteric
subjects. Schaudinn (1903) clearly stated that there were two amoebse,
the one a tissue-invading form and the other a harmless commensal, and
his reputation as a protozoologist resulted in a universal acceptance of
this view, which had been previously put forward by Quincke and Roos.
Since Schaudinn's time numerous names have been given to amoebse which
are undoubtedly merely forms of E. coli. These have been fully discussed
by Dobell (1919), and it is unnecessary to enter into the matter here.
Entamoeba coli is a very common parasite of the human intestine. In
tropical lands, or in other countries where sanitary arrangements are not
satisfactory, it is probable that no person escapes infection. Like E. histo-
FiG. 99. — Entamoeba coli with In-
gested Cyst of E. histolytica
(x ca. 2,000). (After Wenyon
AND O'Connor, 1917.)
212 FAMILY: AMCEBID^
lytica it lives in the large intestine, but it does not invade the tissues.
It develops in the intestinal contents, especially on the surface of the
mucosa, where it feeds on bacteria, yeasts, and other material. It will
ingest cysts of other Protozoa, such as those of Giardia and Isosiwra,
and even the cysts of E. histolytica (Fig. 99). It does not appear to ingest
red blood-corpuscles in its natural habitat. In cases of bacillary dysen-
tery, when enormous numbers of red cells occur in the stool, E. coli may
sometimes be seen moving about amongst them, and showing no inclina-
tion to take them in. The writer has seen red blood-corpuscles adhering
to the surface of motile E. coli, which, however, showed no tendency to
engulf them. Lynch (1924) has, however, been able to induce E. coli
to ingest red cells by incubating them with blood in a test-tube. In the
writer's experience this never occurs in the intestine, and, if it does, it
must be such a rare phenomenon that the general rule given above, that
an amoeba with included red cells is almost certainly E. histolytica, still
holds for all practical purposes. Like E. histolytica, E. coli becomes
encysted in transparent resistant cysts, and it is these forms which
spread infection from one individual to another.
MORPHOLOGY. — E. coli may be considered in three stages: the adult
form, the precystic form, and the cyst.
1. Adult Form. — The fully-groAvn E. coli (Fig. 100) is on an average
larger than E. histolytica, and as usually seen it has a diameter of 15 to
30 microns. Occasionally very much smaller forms, under 10 microns
in diameter, occur. Generally, the amoebae are much less active than
E. histolytica, the movements being very sluggish. Occasionally, however,
the writer has seen undoubted forms of E. coli moving with a rapidity
comparable with that of E. histolytica. The ectoplasm is not so clearly
defined as in E. histolytica, and in the normal individual there is merely
a superficial layer which is clearer than the endoplasm into which it
merges. The degenerating forms of E. coli do not show the exaggerated
extension of ectoplasm which is such a characteristic feature of the
abnormal forms of E. histolytica. The endoplasm of E. coli is often
extensively vacuolated, and the vacuoles contain a great variety of
objects which are chiefly bacteria. The general appearance of the amoeba
is that of a slightly greyish object, which contrasts with the greenish tint
resulting from the high refractive index of the denser E. histolytica.
E. coli is much more fluid in consistency than E. histolytica. Sometimes
the amoebae show various fissures or rectangular vacuoles, which are
probably the result of degenerative changes.
1-3. Forms with vacuolated cytoplasm, including bacteria. 4. Binucleated form.
5-6. Forms with irregularly shaped nuclei and very coarse chromatin masses.
7. Form which has ingested a small binucleated cyst.
8. Form showing excentric position of karyosome.
9-10. Small individuals. 11. Large precystic form with clear cytoplasm.
ENTAMCEBA COLI
213
10 ^1
Fig. 100. — Entamceha coli : Vegetative Forms ( x 2,000). (Original.)
[For di' ■■script inn see opposite page.
214 FAMILY: AMCEBID^
The nucleus of E. coli is a larger and coarser structure than that of
E. histolytica, and is readily distinguished in the living amoeba on account
of the low refractive index of the cytoplasm. In stained specimens it is
seen to have a thicker membrane than the nucleus of E. histolytica. The
chromatin granules are coarser and the karyosome, when it is a single
compact granule, is larger, as also is the clear area around the karyosome.
Dobell (1919) states that the karyosome is nearly always excentric, and that
chromatin granules occur on the linin network between the clear area and
the nuclear membrane. The nucleus of E. coli thus differs from that of
E. histolytica chiefly in its coarseness, and as the nucleus of E. histolytica
quickly changes in character as a result of degeneration, it is very frequently
impossible to distinguish the two amoebae as they occur in the stool from
the appearance of their nuclei alone. The presence of a larger number of
food vacuoles containing bacteria and other objects is a more reliable means
of recognizing E. coli. It must be admitted, however, that it is very often
impossible to distinguish between E. coli and E. histolytica in the free con-
dition. In such cases search must be made for the characteristic cysts.
E. coli reproduces by binary fission, like E. histolytica. The details of
nuclear division have not been followed completely in the free forms; they
are very similar to those of E. histolytica. During the division of nuclei in
the cysts Swezy (1922) states that there are probably six chromosomes.
Several observers, including Schaudinn (1903), Casagrandi and Barbagallo
(1897), and Mathis and Mercier (1917), have described a process of schizo-
gony of E. coli. In stained films it is often very difficult to detect the
wall of a cyst, which becomes highly transparent in cleared preparations.
If such a cyst has an irregular shape, as is not infrequent in prepara-
tions, the appearance of an amoeba with eight nuclei is produced.
The writer has seen and marked such forms as possible schizogony or
multinucleate stages, but in all cases it has appeared more probable that
they were distorted or irregularly shaped forms which were really encysted.
There seems no reason to suppose that E. coli in the free condition repro-
duces in any other way than by binary fission.
2. Precystic Forms. — As in the case of E. histolytica, prior to encyst-
ment there are produced amoebae which are smaller than the adult forms
and have a cytoplasm cleared of all food materials (Fig. 100, ii). The
precystic forms of E. coli are very similar to those of E. histolytica, but
as the average size of the cyst of E. coli is greater than that of E. histo-
lytica, so the precystic amoebae are correspondingly larger. These pre-
cystic forms are probably formed by division of the larger individuals.
3. Cyst. — A cyst wall is secreted round a precystic amoeba which has
become spherical. The nucleus divides to form two nuclei, these divide
to form four, and the four divide again to give the eight nuclei charac-
ENTAMCEBA COLI 215
teristic of the mature cyst (Fig. 101). Occasionally, a further division
will take place, giving rise to sixteen nuclei. The cysts with sixteen
nuclei, though uncommon, are much more frequently encountered than
the eight-nuclear cysts of E. histolytica. Very rarely, cysts with a larger
number of nuclei occur. During the process of nuclear multiplication some
of the nuclei may cease to divide, so that an irregular number of nuclei
of unequal size may result. According to Dobell (1919), soon after encyst-
ment, a glycogen vacuole forms in the cytoplasm, and this reaches its
maximum development at the two-nuclear stage. After this it gradually
shrinks till at the eight-nuclear stage it has disappeared. In the writer's
experience the precystic amoebse themselves may possess a large vacuole
or a series of vacuoles which run together after encystment has occurred.
This vacuole, however, is not always present.
The cysts of E. coli vary in diameter from 10 to 30 microns. They
usually measure from 15 to 20 microns, but larger ones may occur, as
recorded by the writer and O'Connor (1917), who saw one measuring
38 by 34 microns. The commonest type of cyst met with in the stool is
one containing a clear cytoplasm in which are embedded the eight nuclei
(Fig. 101, 4 and 8).
In most cases there occur also a smaller number of cysts of a different
type. These are usually larger than the ones just mentioned, and have
a large central glycogen vacuole which reduces the cytoplasm to a thin
layer lining the cyst wall (Fig. 101, 10-12). There are usually two
nuclei, which generally lie at opposite poles of the cyst. They often
appear as if flattened against the cyst wall by pressure of the vacuole.
In other cases the vacuole is smaller, and there is a thicker layer of cyto-
plasm. The vacuole contains glycogen, which stains brown with iodine
(Plate II., 2, p. 250). More rarely cysts of this type may be seen with
four nuclei, and still more rarely with eight nuclei. A modification is
occasionally seen in which a series of vacuoles occurs round the periphery
of the cyst (Fig. 101, 14), while the cytoplasm with the two, four, or eight
nuclei may occupy its centre. In optical section such cysts have a cart-
wheel appearance. Dobell (1919) considers that the vacuole occurs in the
normal course of development, and reaches its maximum size at the two-
nuclear stage, and that it then disappears. It seems to the writer, how-
ever, that these two-nuclear cysts with the large vacuole have an abnormal
appearance, and it is difficult for him to believe that the eight-nuclear
cysts with their particularly clear cytoplasm have been developed from
the coarsely vacuolated binucleated cysts, which, moreover, are usually
larger than the eight-nuclear cysts present at the same time. On a few
occasions the writer has examined material containing precystic amoebse
with perfectly clear cytoplasm, and has seen in the same material cysts
216
FAMILY: AMCEBID^
m
•'&'
r.)
/^i
t
■'^,
0/
•f / > ^
Fig. 101. — Eniamceha coli : Encysted Forms (x 2,000). (Original.)
\^F<yr description see opposite page.
ENTAMCEBA COLI 217
with similar cytoplasm containing one, two, four, and eight nuclei in
which no indication of vacuole formation has been evident. He has
regarded these as representing the normal encystment process of E. coli
(Fig. 101, 1-4).
The binucleate cysts with large vacuole appear to be derived from
very vacuolated and abnormal-looking precystic amoebse. It is possible
that the real explanation is that in some cases no vacuole is formed at
all, in others that one of moderate size occurs and is ultimately absorbed,
and that in others again there is an excessively large vacuole formed as
an abnormality, and that this prevents the subsequent development of
the nuclei. In the case of E. histolytica the cysts frequently, though not
invariably, contain a vacuole of moderate size which does not impede
nuclear division.
It was suggested by the writer and O'Connor (1917) that there probably
occur races of E. coli in which the average size of the cyst differs, as in
E. histolytica. Matthews (1919), by measurement of a large number of
cysts, demonstrated that this was actually the case.
The nuclei within the cysts have the same structure as that of the
adult amoeba?. According to Dobell (1919), the karyosomes are invariably
excentric in position, and he believes that it is usually possible to determine
with certainty whether the cyst is one of E. coli or E. histolytica from the
nuclear structure alone, provided the cysts have been properly fixed and
stained. In the nucleus of E. histolytica the karyosome is central.
The arrangement of the nuclei within the cyst is subject to variations.
Usually, they are distributed irregularly through the cytoplasm, and careful
focussing at different levels is necessary in order to see and count them.
At other times they are grouped together, sometimes closely, at the centre
of the cyst, where the cytoplasm may be denser than at the periphery.
Chromatoid bodies, first seen by Grassi (1879), and later by Casagrandi
and Barbagallo (1897), are occasionally seen in cysts of E. coli (Fig. 101, 8).
They are usually not so definitely rod-like as those in cysts of E. histolytica,
and may be in the form of one or more lobulated bodies or numerous,
small, irregularly-shaped fragments. Sometimes they are filamentous in
form, and the cytoplasm may be traversed by a kind of network of these
structures (Fig. 101, 9). The writer has seen cysts in which acicular
1-4. Normal method of encystment, showing one to eight nuclei and absence of vacuole.
5. Large form with sixteen nuclei. 6. Form with two nuclei in division.
7. Four-nucleated stage with included bacillus.
8. Form with eight nuclei and chromatoid bodies.
9. Form with eight nuclei and filamentous structures.
10-12. Forms with large central glycogenic vacuole and two nuclei.
13. Form with large central glycogenic vacuole and four nuclei.
14. Form with two nuclei and large peripheral vacuoles.
15. Ruptured eight-nucleated stage with hemia-like protrusion.
218 FAMILY: AMCEBID^
bodies are arranged at the periphery of the cyst in a tangential manner,
while leaving the central cytoplasm, which contains the nuclei, clear.
These acicular bodies were similar to certain bacteria which occurred in
the stool, and in shape resembled Charcot-Leyden crystals. The possi-
bility of their being parasitic in nature has to be considered,
Schaudinn (1903) described a process of autogamy in the cyst of
E. coli. The nucleus of the encysted amoeba divided into two nuclei,
which took up positions at opposite poles of the cyst. Each of these
nuclei then gave ofi chromatin material into the cytoplasm, and then
divided to form two pairs of nuclei, one of each pair being a migrating
nucleus and the other a stationary one. The migrating nucleus of each
pair then passed across the cyst and united with the stationary nucleus
of the opposite pair. In this way a two-nuclear stage was again reached.
Each nucleus then divided, and the daughter nuclei repeated the division
so that a total of eight nuclei resulted. The writer (1907) observed
certain changes in the cysts of E. muris of mice which seemed capable
of a similar interpretation. The observations of Schaudinn have never
been confirmed, and it is abundantly evident that no such autogamy
process occurs in the development of the cysts of any entamoeba. The
eight nuclei undoubtedly result from straightforward repeated divisions.
Mathis and Mercier (1917) expressed the opinion that the usual type of
cyst with eight nuclei were gamete-producing cysts, which in the next host
liberated eight amoebae which conjugated in pairs. The cysts with a
larger number of nuclei were regarded as schizogonic cysts, which were
presumed to give rise to sixteen daughter amoebae which grew into adults
without conjugation. The figures they give are quite unconvincing, and
it is evident from their account that they have not produced sufficient
evidence in support of their view.
PATHOGENICITY. — There is no evidence that E. coli can be pathogenic
to man. That infection is brought about by the ingestion of cysts was
demonstrated by Walker and Sellards (1913), who succeeded in infecting
seventeen of twenty men on whom experiments were conducted. The
infection gave rise to no symptoms, but cysts appeared in the stools in
one to eleven days.
The many attempts made by the writer to infect animals with E. coli
have failed. In conducting such experiments it must be remembered that
many animals harbour amoebae of the E. coli type, and that they produce
cysts which cannot be distinguished from those of the human amoeba.
Kessel (1924a) reports the successful infection of monkeys with E. coli.
Casagrandi and Barbagallo (1897) claimed to have seen the emergence
of amoebae from cysts which had been fed to cats. They supposed that
the cyst wall ruptured, and that eight amoebae escaped from the cyst.
ENTAMOEBA COLI 219
No other observer has been able to repeat this observation, and though
it is clear that the cyst must liberate an eight-nucleate amoeba or eight
uninucleate small amoebae, this has not been conclusively demonstrated.
Kessel (1923a) believes that he has succeeded in infecting rats with E. coli.
CULTIVATION. — Though several observers claim to have cultivated
E. coli on the surface of solid agar media in all cases the amoebae have proved
to be coprozoic organisms. Boeck and Drbohlav (1925) succeeded in
maintaining E. coli for three days in the medium devised for the culture
of E. histolytica. Drbohlav {I92bd) and Thomson, J. G. and Robertson
(1925) have been more successful and have kept strains growing for two
or three months.
ABERRANT FORM OF E. COLL— Mention must be made of an amoeba
to which Kofoid and Swezy (1921, 1921a) have given the name
"^^
Av
1^.
/■':'/
^.,/^'^'
3
Fig. 102. — Free and Encysted Forms of " Comicilrnania lajleuri'' (x 2,000).
(After Kofoid and Swezy, 1921, Slightly Eeduced.)
1. Free form with characteristic nucleus and clear pseudopodium.
2. Encysted form with eight nuclei and chromojihile ridge.
3. Cyst producing first bud through the pore.
Councilmania lafleuri (Fig. 102). It is claimed that this is a distinct
amoeba which has been confused hitherto with Entamoeba coli. It is
supposed to show in its free stage some of the characters of E. Jiistolytica,
such as activity, development of ectoplasm, formation of clear pseudo-
podia, ingestion of red blood-corpuscles, and other features which are
those of E. coli, as distinct nucleus, vacuolation, ingestion of bacteria.
220 FAMILY: AMCEBID^
and the production of an eight-nucleated cyst. The nucleus differs from
that of E. coli in that the karyosome is dispersed instead of being a com-
pact granule. During mitotic division of the nucleus it is claimed that
eight chromosomes are present in place of the six which E. coli is said to
possess. The most characteristic feature, however, and the one on which
the new genus is based, is that in the host in which the cysts are formed
the encysted amoeba buds off, through a pore in the cyst, eight small
amoebje. Associated with the pore is a deeply staining band termed
the chromophile ridge. As has been explained above, the exact method
of exit of E. coli from its cyst is not known, so that the budding process
cannot be held to distinguish the new genus Councilmania from Entamoeba.
There would be more reason to place Entamoeba gingivalis in another genus,
because encysted forms have never been discovered. It appears, how-
ever, from the description and figures, that the supposed budding process
through a pore is most reasonably explained as a result of rupture of the
cyst and the consequent extrusion, by pressure or collapse of the cyst, of
hernia-like portions of the cytoplasm together with the nuclei. The
writer has seen exactly comparable appearances in cysts of E. coli which
have been ruptured by pressure of the cover-glass. As the liquid beneath
the cover-glass evaporates the pressure on the cysts is increased, so that
rupture takes place and portions of cytoplasm with nuclei can be seen
to escape. Similar ruptured cysts are often encountered in the ordinary
stained preparations made by the smear method. The writer has pre-
parations containing ruptured cysts which may be in the two, four, or
eight nucleated stage (Fig. 101, 15). They contain chromatoid bodies,
and bear a striking resemblance to the budding cysts described by Kofoid
and Swezy (1921rt). Werner (1912) gave a figure of a similarly ruptured
cyst showing a hernial protrusion including two nuclei. Casagrandi and
Barbagallo (1897) figure a cyst which is supposed to illustrate the natural
emergence of amoebae, but it is not improbable that they were observing
an artificially ruptured cyst. The writer has seen in stained preparations
similar ruptured cysts of E. histolytica with bud-like extrusions containing
one of the four nuclei. In view of the work of Sellards and Theiler (1924),
who have shown that kittens may be infected with E. histolytica by
injecting material containing cysts only, it is possible that E. coli may
sometimes emerge from its cyst while in the large intestine, and that
some of the appearances of budding may be due to this. It is nevertheless
a fact that artificial rupture of cysts of E. coli will give rise to forms
which are said to be characteristic of C. lafleuri. The deeply staining
band called the chromophile ridge, which is supposed to have some con-
nection with the development of the pore, is probably an artifact in
many cases, the result of irregularities in staining produced by folds or
ENTAMOEBA COLI 221
creases in the cyst wall, or disturbance of the cytoplasm. In some cysts
the structures called chromophile ridges are undoubtedly chromatoid
bodies. Such statements as " chromatoidal body exhausted in the forma-
tion of chromophile ridge," made in connection with the cyst reproduced
at Fig. 102, 2, are quite incomprehensible. It is probable that Kofoid
and Swezy were dealing in some cases with mixed infections of E. coli and
E. histolytica. The writer (1922a, 1925) stated his reasons for regarding
the name Councilmania lafleuri as a synonym of Entamoeba coli. In a
later communication Kofoid, Swezy, and Kessel (1924) reaffirm their
belief in Councilmania lafleuri, and bring forward a number of further
observations which they consider establish the validity of the species.
After carefully reading their paper, the writer still believes that there is
no justification for the genus Councilmania, and that the characters which
distinguish C. lafleuri from E. coli fall within the range of variation of
E. coli itself. Gunn (1922) has examined some of the cases from which
Kofoid and Swezy described C. lafleuri. He has found that the amoebse
present were actually E. coli.
Having discovered similar " budding cysts " in rats and mice, E. muris
and E. decumani are placed in the genus Councilmania as C. muris and
C. decumani by Kofoid, Swezy and Kessel (1923), while it is also claimed
by Kessel (1923a) that rats and mice can be infected with C. lafleuri, and
that the amoeba retains its characters in these animals. Apart from the
" budding " through a pore in the cyst, which the writer believes is a rupture,
the main points which, it is claimed, distinguish the genera Entamoeba and
Councilmania are the character of the cytoplasm and its inclusions, the
clear pseudopodia, the type of movement, and finally the dispersed karyo-
some. There is very great difficulty associated with the identification and
counting of chromosomes in nuclei of the type possessed by these amoebae,
so that the chromosome number quoted for C. lafleuri, C. muris, C. decumani,
E. coli, and E. histolytica (8, 6, 4, 6, 6) cannot be accepted as finally estab-
lished. Kofoid and Swezy (1921a) state that they have encountered jE. 7nuris
in man. E. muris of rats and mice so closely resembles E. coli of man that
the writer is at a loss to know how they arrived at their diagnosis, especially
as Kofoid, Swezy and Kessel (1923) adopt the view that the amoeba belongs
to the genus Councihnania.
Entamoeba gingivalis (Gros, 1849) Brumpt, 1910.— This amoeba, which
is parasitic in the human mouth, was first seen by Gros (1849) in Russia.
He gave it the name Amoeba gingivalis, which was emended by Brumpt
(1910a) to Eutanujeba gingivalis. The organism was seen by Steinberg
(1862), who gave it the name Amiba buccalis, and by Grassi (1879), who
named it Amoeba dentalis. Doflein (1901) referred to it as Amoeba hartu-
lisi, and Kartulis (1906) as Entamoeba maxillaris. It has been described
222 FAMILY: AMCEBIDM
under various names by different observers from the material obtained
from carious teeth or abscesses in the oral and pharyngeal regions. Smith
and Barrett (1915), and in the same year Bass and Johns (1915), studied
this amoeba, and concluded that it was probably the cause of pyorrhoea
alveolaris, and that it invaded the tissues like E. histolytica. There is no
conclusive proof that E. gingivalis is pathogenic in any way or actually
invades the tissues, so that it is safer to regard it as a saprophitic organism
which lives in the mouth, especially in any pockets which may form in
suppurative conditions, along with the numerous spirochsetes, bacteria,
and trichomonas. The observation of Lynch (19156) that E. gingivalis
may occur in material obtained from the interstices of sets of false teeth
worn by individuals with no natural teeth at all and perfectly healthy
gums seems difficult to reconcile with the view that the amoeba is, like
E. histolytica, a tissue parasite. More recently, under the name of
E. macrohyalina, Tibaldi (1920) has described an amoeba obtained from
the tonsil. This again is probably no other than E. gingivalis, which has
been shown by Smith, Middleton, and Barrett (1914) to invade the crypts
of the tonsil under suitable conditions, just as trichomonas and the other
organisms of the mouth may do. The bodies which Artault (1898) dis-
covered in a cavity of the lung, and which he named Amceha pulmoiialis,
are probably the same as those referred to as Entamoeba pulmonalis by
Brumpt (1913c). If they are amoebae, which is by no means clear, they
may be identical with the oral form. It has been suggested that E. histo-
lytica may invade the mouth, and that E. gingivalis is in reality that
species. There seems to be no ground whatever for this conclusion, nor
is there any reason to suppose that more than one species of amoeba
inhabits the mouth. The many names that have been given are the
result of observations on degenerate amoebae, just as has occurred in the
case of E. histolytica and E. coli. Petzetakis (1923 and 19236) claims
to have observed E. histolytica in material coughed up from the lungs in
a type of broncho-pneumonia (see p. 193).
E. gingivalis is a fairly active amoeba when observed on the warm
stage, and possesses an ectoplasm which is even clearer than that of
E. histolytica. Kofoid and Swezy (1924a) note that sometimes in
apparently normal amoebae there is no distinction between ectoplasm and
endoplasm. They state that a definite superficial pellicle is always present.
As regards its activity, E. gingivalis is perhaps intermediate between
E. histolytica and E. coli, and there is a greater tendency to the formation
of several pseudopodia at one time. These are smaller in comparison
with the size of the amoeba than are those of E. coli and E. histolytica,
and according to Jepps (1923a), who has studied the organism in
Malaya, they are never formed in the eruptive manner so characteristic
ENTAMCEBA GINGIVALIS 223
of those of E. histolytica. They are clear, and appear to consist of ecto-
plasm alone when this layer is sharply defined. Jepps, as well as Kofoid
and Swezy (1924a), note that during progression the amoeba may become
elongated, while the hindermost portion becomes drawn out into a tail-
like process to which adhere collections of bacteria, leucocytes, and debris.
The amoebae vary in diameter from 10 microns upwards, but they are
rarely seen with a diameter above 20 microns (Fig. 103). Forms up to
40 microns in diameter have, however, been described. There is dis-
tinguishable a clear, narrow ectoplasmic layer and a highly vacuolated
granulated endoplasm. The many food vacuoles contain a variety of
structures, some of which stain black with iron hsematoxylin, and are
.\::
#
Fig. 103. — Entamceha gingiralis from ScrapinCxS from a Carious Tooth ( x 2,000).
(OrICxINAL.)
In three of the amoebfle are seen the large ingested bodies of doubtful nature.
probably the nuclei of degenerate pus or tissue cells. Smith and Barrett
(1&15) and others state that red cells are sometimes ingested, but the
majority of observers, including the writer, have obtained no evidence
of this. The food vacuoles also contain bacteria of various kinds. The
nucleus is distinctly smaller than that of E. coli, which it resembles,
however, in general features. The nuclear membrane is generally lined
with closely packed granules of chromatin, which in stained specimens
appear as a uniform black ring. There is a karyosome which may be
surrounded by a clear area, as in the nuclei of E. coli and E. histolytica.
According to Dobell (1919), the karyosome is either central or excentric
in position, and there is no chromatin upon the linin network. As in the
case of the nuclei of E. coli and E. histolytica, some observers have main-
tained that there is a centriole within the karyosome.
224 FAMILY: AM(EBID.E
Kofoid and Swezy (1924a) maintain that all descriptions of the nucleus
hitherto given are inaccurate, and that definite differences not previously
noted distinguish it from the nucleus of E. histolytica. They state that
the karyosome is not always a single granule, as in E. histolytica, but is
often composed of a group of granules, and that the halo round the karyo-
some is granular and large, in contrast with the clear and relatively smaller
halo of E. histolytica ; furthermore, the intermediate zone between the
nuclear membrane and halo is clear in E. gingivalis and granular in
E. histolytica, while in the former the chromatin is less regularly arranged
and more liable to clumping on the nuclear membrane. Whether such
minute differences are sufficiently constant to justify the determination of
species future investigations alone will show.
Reproduction of E. gingivalis probably takes place by binary fission,
and the binucleate forms occasionally seen must represent a stage in this
process. The division has never been followed in detail.
Craig (1916) has recorded the finding of cysts, but it is evident from
his figures that the structures described were not cysts at all. Similarly,
Smith and Barrett (1915), and Nowlin (1917), described as cysts structures
which were more than doubtful. The writer has examined E. gingivalis
on many occasions, but was never able to discover encysted forms. This
has been the experience of Dobell (1919), Kofoid and Swezy (1924a),
and other workers. It is probable that cysts occur, but if they have
ever been seen, no convincing description has yet been given.
E. gingivalis can easily be studied in material obtained from carious
teeth or in pus squeezed from pyorrhoeal pockets. In the writer's ex-
perience the amoebae sometimes appear to be absent in particularly foul
mouths when they might be expected to be present, while on other occa-
sions they have been found in the mouths of people who are very particular
as to their dental toilet.
Attempts to infect animals with E. gingivalis have not been successful.
Goodrich and Moseley (1916) have noted that an organism indistinguish-
able from E. gingivalis may be found in pyorrhoeic conditions in dogs,
while Nieschulz (1924c) has described as E. gingivalis var. equi a similar
form from the accumulations round the teeth of horses,
Tibaldi (1920) has recorded the discovery of E. gingivalis in the human
tonsil. He has also described as E. macrohyalina an amoeba of
another type which he has found in two cases of tonsillitis. This amoeba
is considerably larger than E. gingivalis, and may reach a diameter of
40 microns. It has, moreover, a well-marked ectoplasm and a different
type of nucleus, though it must be admitted the figures given suggest
a faulty fixation. It is possible, as noted by the writer (1922a), that
E. gingivalis, which usually lives as a saprophyte, may become modified
ENTAMCEB^ OF MONKEYS
225
in appearance when it inhabits an inflamed tonsil. This is probably the
explanation of the curious amoebae which have been described from abscesses
in the jaw and mouth. Though Tibaldi has drawn attention to an amoeba
which differs from the usual form of E. gingivalis, he has not produced any
evidence, apart from its size, to justify its separation as a distinct species.
Drbohlav (1925c), Howitt (1925) and Dobell (1926) have cultivated
E. gingivalu-. Drbohlav failed to infect kittens with the cultured forms
(see p. 1297).
ENTAMCEB^ OF MONKEYS.
Musgrave and Clegg (1904) stated that they had occasionally observed
natural amebic infections of moid^eys in the Philippines, and the writer
Fig 1U4 — Lntamceb.e from the Intestine of Monkeys {xca. 1,300).
(After Mathis, 1915.)
].. Free form of E. pithed. 2-4. Encysted forms of E. pitheci.
5. Free form of E. nnttalli. 6-8. Encysted form of E. nuttalli.
(1909) observed cysts which were indistinguishable from those of E. coll
in a monkey in Khartoum. Brumpt (1909a) observed similar cysts and
free amoebae in Macacus smicus, while Noc (1909) observed cysts 10 to
12 microns in diameter in three monkeys in Saigon. Castellani (1908)
observed an amoebic abscess of the liver in a Macacus pileatus in Colombo,
and proposed the name Enta?noeba nuttalli for the amoeba. Mathis (1913)
published an account of an amoeba observed by him in Macacus rhesus
and Macacus tcheliensis of Tonkin. He found two distinct types, one re-
sembling E. coll of man in that it produced eight-nucleated cysts (Fig. 104,
I. 15
226 FAMILY: AMGEBIDvE
1-4), and the other like E. histolytica, with cysts containing four nuclei
and chromatoid bodies (Fig. 104, 5-8). Employing the generic name
Loschia proposed for the entamoebse of man by Chatton and Lalung-
Bonnaire (1912), he named these forms L. legeri and L. duboscqi respec-
tively. Prowazek (1912a), however, had previously described and named
E. iiitheci, a form which he had seen in an orang-outang and which
resembled E. coli, though, according to Dobell (1919), he was probably
dealing with more than one species. Swellengrebel (1914) gave the name
E. chattoni to an amoeba seen by him in Macacus rhesus. It was of the
E. histolytica type. Behrend (1914) observed cysts in the faeces of a
Macacus rhesus. They varied in diameter from 8 to 25 microns, some
having four and others eight nuclei. Macfie (1915a) also saw amoebae
in a monkey {Cercopithecus petaurista) of West Africa. It was associated
with dysentery, of which Macfie judged it to be the cause. He named it
Entamoeba cercopitheci. Eichhorn and Gallagher (1916) recorded an out-
break of amoebic dysentery amongst spider monkeys [Ateles ater) in
America. The amoeba is referred to as Amoeba ateles by these authors
and as Entamoeba ateles by Suldey (1924).
McCarrison (1919) stated that monkeys employed by him in nutrition
experiments in India were very liable to attacks of amoebic dysentery.
Bach (1923) described the cysts and free forms of an amoeba of the E. his-
tolytica type which he discovered in a Macacus rhesus which had been
in captivity in Germany for sixteen years. Suldey (1924) has described
a case of spontaneous amoebic dysentery in the chimpanzee. The amoeba
had all the characters of E. histolytica. Amoebae of this type have been
seen by Kessel (1924a) in monkeys in China.
Most of the observations on the amoebae of monkeys have been casual
ones, so that the descriptions given do not necessarily represent the normal
appearance of the healthy amoebae. It is evident that monkeys may
harbour two forms — one, E. pitheci Prowazek, 1912 {=E. legeri Mathis
and Mercier, 1917), which resembles E. coli; and the other, E. nuttalli
Castellani, 1908 {= Loschia duboscqi Mathis, 1913= £'. chattoni Swel-
lengrebel, 1914:=E. cercopitheci Macfie, 1918 =£". ateles Suldey, 1924),
which resembles E. histolytica. The latter is liable to produce amoebic
dysentery and abscess of the liver. It is open to question if these forms
are really distinct from E. coli and E. histolytica.
Mello (1923) in Italy has found that species of Macacus harbour either
E. pitheci or E. nuttalli. The latter is often associated with dysentery, and
the injection of its cysts per rectmn produced dysentery in three kittens,
which passed large numbers of amoebae. In a young orang-outang an
amoeba of another type is described. It measured 25 to 35 microns, and
its mature cyst had eight nuclei. It differed from E. pitheci chiefly in
ENTAMCEBiE OF OTHER MAMMALS 227
the fact that cysts and free forms with over twenty nuclei occiirred.
The multinucleated free forms are regarded as schizonts, and a figure
shows what the author regards as division into daughter amoebae. It
is far from clear that these free forms are not cysts, and the figure illus-
trating the escape of the daughter amoebae from an enclosing membrane
which he says is present might well be interpreted as a ruptured cyst
from which the nuclei are being extruded by pressure. Though the
author refers to the amoeba as a new species, E. multinudeata, it
is evident that he may have been dealing with multinucleated formes of
E. ■pitheci, and that this amoeba comes into line with E. coli, in which
similar stages are by no means uncommon. In stained and cleared
preparations, as pointed out above, it is often exceedingly difficult to
decide whether a form is actually encysted or not. The writer has seen
free forms and cysts of an amoeba resembling E. jyitlieci in Cercopithecus sp.
of West Africa, and with Dr. G. C. Low the cysts alone in the faeces of a
gorilla.
Dobell (1926) has cultivated from monkeys four species of amoeba
including E. nuttaUi. With the last, the complete history of which, in-
cluding excystation, has been studied in cultures, he has produced in
kittens a dysentery which difiers in certain respects from that resulting
from inoculation of E. histolytica.
Brug (1923) has discovered in Macacus cynomolgus a small race of an
amoeba corresponding with the small race of E. histolytica in man. On
the assumption that these human amoebae represent a distinct species {E.
tenuis), he gives the name E. cynonwlgi to the form in the monkey. It is
possible, however, that it is merely a small race of E. nuttaUi.
ENTAMCEB^ OF OTHER ANIMALS.
Entamoebae are of common occurrence in the intestine of animals,
while occasionally they occur in the mouth of the dog and horse, as noted
above (p. 224). Spontaneous amoebic dysentery in dogs has been described
by Kartulis (1891, 1913) in Egypt, Darling (1915) in Panama, and Ware
(1916) in India, Fischer (1918) in China, and Bauche and Motais (1920)
in Cochin-China. In one case noted by Kartulis the dysentery was
associated with abscess of the liver. As the dog is known to be infectible
with E. histolytica, it seems probable that it was actually this species
which was producing the disease. Darling (1915), without differentiating
it from E. histolytica, proposed the name E. roiaticiini for the amoeba
producing canine amoebic dysentery. Franchini, F. (1920 1923), recorded
a case of spontaneous amoeboid dysentery in a cat in Italy. It was con-
cluded that the amoeba was E. histolytica.
228
FAMILY: AMCEBIDiE
Rats and mice are very commonly infected with amoebse [E. muris
(Grassi, 1879)] which closely resemble E. coli both in the free and encysted
stages (Fig. 105). Briig (1919a) has noted that while E. nmris, regarded
by Rudovsky (1921) as a variety E. muris-decummii, is the common
^
-^^9
o
Fig. 105. — Entamoeba maris from the Intestine of a White Mouse ( x 3,000).
(Original.)
One unencysted form and three encysted forms with two, four, and eight nuclei.
form seen in rats, occasionally amoeba? of the E. histolytica type are met
with. Lynch (1915) and Chiang (1925), who also found amoebae. of this
type in rats, believe that rats may actually act as carriers of E, histolytica
(see p. 200).
ENTAMCEB^ OF OTHER MAMMALS 229
Kofoid, Swezy and Kessel (1923), in their conception of a genus
Councilmania, have stated that the common amoebae of rats and
mice belong to this genus, in that they possess clear pseudopodia and
dispersed karyosomes, and are able in the encysted stage to form buds
through a pore, associated with which there may be a problematic struc-
ture called the chromophile ridge. They accordingly transfer E. muris
decumani and E. muris to the genus Councilmania as C. decumani and
C. muris. Furthermore, Kessel (1924) states that in the rat there occurs an
amoeba which differs from C. decumani in that it forms granular pseudo-
podia, has a compact karyosome, and does not form buds, in which
respects it resembles E. coli. It is given the name E. ratti. As explained
by the writer (1922rt, 1925) there are no grounds for retaining the genus
CouncUmania (see p. 219), so that if Kessel's claims regarding these
amoeba? are correct there are to be recognized in rats E. decumani and
E. ratti, and in mice E. muris. According to him, E. decumani and
E. muris may be transferred to both rats and mice. The writer (1925)
feels convinced that much more work will have to be done before the
claims regarding these three species can be accepted. It is possible that
the amoeba of the rat differs from that of the mouse, but at present it
seems safer to regard the amoebse of both rats and mice as E. muris.
An amoeba with free and encysted stages of the E. coli type was
discovered by Brug (1918 a) in rabbits. He named the organism E. cuni-
culi. Rudovsky (1923) saw an Entamoeba in hares. A similar form in
guinea-pigs was named E. cobayce by Walker (1908), and was again referred
to by Chatton (1918c) as E. cavice. It has been seen by the writer in
guinea-pigs on several occasions, while the free and encysted forms of an
amoeba resembling E. muris were met with in the jerboa in the Sudan.
Leger, M. (1918), has also recorded an amoeba of guinea-pigs. He noted
that encysted forms of the four-nuclear type were present, while Holmes
(1923) has observed cysts like those of E. coli.
Theobald Smith (1910) discovered amoebse in sections of intestinal
ulcers in the large intestine of pigs in America. He did not consider
them as pathogenic, but believed they had invaded ulcers which were
due to some other cause. They varied in diameter from 8 to 10 microns,
and each had a single nucleus with a small central karyosome. Prowazek
(1912) described as E. polecki an amoeba found by him in pigs in
Saipan. He claimed to have seen the same amoeba in a child also, but
this was probably a precystic form of E. histolytica. Prowazek's pig
amoeba varied in diameter from 10 to 12 microns, and had a single nucleus
which, in some of his figures, is evidently of the entamoeba type. Hart-
mann (1913), after examining some of Smith's sections, proposed to name
the amoeba E. suis, though admitting its possible identity with
230
FAMILY: AM(EBID^
E. polecki. Noller (1921) states that Feibel in Germany had seen this
amoeba in pigs, while Cauchemez (1922 a) describes it from pigs in France.
The writer has seen it in pigs in England, and it was also met with by
O'Connor in the Ellice Islands. According to Cauchemez, the amoeba
is nearly always uninucleated, and varies in diameter from 5 to 12
microns when round. When elongate, it measures 15 by 5 microns.
Rarely binucleate forms were seen. The amoebae resemble the precystic
forms of E. histolytica. Noller (1922), who emphasizes its resemblance to
E. histolytica, states that the amoeba varies in diameter from 12 to 25
7 8 9
Fig. 106. — Intestinal Amceb^ of Pigs ( x 2,500). (After Niesciiulz, 1923.)
1-2. Enlammha polecki, free forms. ,3-.'5. E. dehliecki. free forms.
G. E. dehliecki, encysted form. 7-8. lodamceba bi'itscMii, free forms.
9. /. biUschlii, encysted form.
microns, and that uninucleate cysts 12 to 15 microns in diameter occur.
The latter may contain numerous splinter-like chromatoid bodies. Douwes
(1921) described four- nucleated cysts with a diameter of 5 to 8 microns.
Whether these are the mature cysts of a small race of the entamoeba
of the pig or some other form is not clear. The correct name for this
amoeba is evidently E. polecH Prowazek, 1912, though some of the forms
figured by Prowazek undoubtedly do not belong to this ama?ba. The
name E. suis becomes a synonym. The amoeba cultivated from pig
ENTAM(EB.E OF BIRDS AND AMPHIBIA 231
faeces by Walker (1908), and named by him Amoeba intestinalis, is not a
parasitic form at all, but a coprozoic organism.
Nieschiilz (19"23f/, 19246) found that pigs harbour two species of Enta-
mwba. There is the large form referred to above, and a smaller one not
more than 5 to 9 microns in diameter, which he proposes to name E. de-
bliecki. Uninucleate cysts with chromatoid bodies are described (Fig. 106.)
Liebetanz (1905) described E. bovis from the stomach of cattle. It
was redescribed by him (1910) and by Braune (1913), and was said to
be 20 microns in diameter. Nieschulz (19226) has met with a smaller,
though possibly the same form in cattle in Germany. The amoebae,
which varied from 5 to 10 microns in diameter, had nuclei of the entamoeba
type. He also saw uninucleated cysts 5 to 12 microns in diameter in the
faeces. He was unable to determine with certainty that these cysts were
derived from the amoebae in the rumen.
Fantham (1920, 1921) refers to an amoeba called by him E. intes-
tinalis {Amoeba intestinalis Gedoelst, 1911), which occurs in the
colon and caecum of horses in South Africa. No details of the structure
are given. He states (1921) that in the faeces he has seen another
form which he names E. equi. It may contain red blood-corpuscles, and
when round has a diameter of 28 to 35 microns. Four- nucleated cysts
containing chromatoid bodies and measuring 15 to 20 microns in diameter
are also mentioned. It is assumed that it is a pathogenic species.
Swellengrebel (1914) discovered free amoebae and uninucleated cysts
in the intestine of sheep, and proposed the name E. avis. The writer
has seen eight-nucleated cysts of the E. coli type in goats' faeces. Fantham
(1923) gave the name E. cajprce to an amoeba of the goat. Very little is
known about these forms. Nieschulz (19236) has found in goats an amoeba
which appears to be identical with the small £^. debliecki of pigs.
Fantham (19106) described as E. lagopodis an amoeba found by him
in the intestine of the grouse, Lagopus scoticus. Cysts with four nuclei
were noted. According to Hartmann (1913), Kuczynski saw a similar
form in fowls, but the encysted stages had eight nuclei. Tyzzer (1920)
saw the same amoeba in American fowls. He noted tha^t in the free
and encysted stages it closely resembled E. coli. To a form in the duck
in S. Africa Fantham (1924) gave the name E. anatis. Cysts with one
or four nuclei are described.
Frogs harbour amoebae (E. ranarum Grassi, 1879) which Dobell has
shown to resemble E. histolytica very closely in the free and encysted
stages (Fig. 107). So similar were these forms that Dobell (1918)
attempted to infect tadpoles by causing them to ingest cysts of E. histo-
lytica. The cysts showed no signs of hatching in the intestines of the
tadpoles, and were passed unaltered in the faeces. E. ranarum was
232
FAMILY: AMCEBID^
studied in tadpoles by Collin (1913), who found that the free amoebsG
sometimes had as many as thirty nuclei (Fig. 107, 7). He regarded these
as schizonts. The amoBba was again studied by Mercier and Mathis
(1918), who described two types of cysts. The usual form had four nuclei,
like the cysts of E. histolytica, while the other had as many as sixteen
nuclei (Fig. 107, 8). As in the case of E. coli, it was conjectured that the
mm
Fig. 107. — Ev.tamoeha ranarum from the Intestine of the Frog. (1-6 after
DoBELL, 1909; 7 after Collin, 1913; 8 after Mercier and Mathis, 1918.)
1. Free form ( x 2,000). 2-6. Encysted forms ( x 2,000).
7. Multinucleated free form (x 1,000). 8. Multinucleated encysted form (x 1,400).
cysts with a small number of nuclei were gamete-producing cysts, while
those with a larger number represented schizogony cysts. No proof in
support of this view was obtained (see p. 218).
Ilowaisky (1922) has described spontaneous amoebic abscess of the
liver in frogs. The amoebse present resembled E. ranarum, which occurred
in the intestine of the same animals. The amoeba seen by Chatton
ENTAMOEBA OF REPTILES AND FISH
233
(1910c) in the rectum of the newt. Triton palmatus, and by Alexeiefi
(1912) in Triton tcBniatus, is very possibly E. ranarum.
Hartmann (19106) described as E. testudinis an amoeba of the tor-
toise, Testudo grcBca. It was also seen by Alexeieff (1912c) in Nicoria
trijuga, a tortoise of Ceylon, while the writer has met with it in Testudo
argentina and T. calcarata. An Entamoeba of the turtle, Chelydra serpentina,
of America, which was cultivated by Barret and Smith (1923), has been
named Entamoeba barreti by Hegner and Taliaferro (1924) (see p. 207).
/
n
Fig.
108. — Entamceha mincliini of Tipulid Larv.e: Free and Encysted Forms
(x 3,200). (After MACKINNON, 1914.)
Dobell (1914a) gave a figure of an entamoeba seen by him in the
wall lizard, Lacerta tnuralis. The writer (1921) encountered a similar
amoeba in Egyptian lizards {Lacerta agilis and Agama stellio). The free
forms were very like those of E. coli, while eight-nuclear cysts occurred
which were indistinguishable from those of the human parasite. What
is probably the same amoeba was seen in Lacerta ocellata by Franchini
(1921a). Cunha and Fonseca (1917) described as E. serpentis an amoeba
seen by them in the snake Drimobius bifossatus of S. America.
Leger and Duboscq (1904) observed an amoeba in the intestine of the
marine fish Box boops and B. saljm. It was studied by Alexeieff (1912),
who placed it in a new genus as Proctamoeba salpcp. According to him,
234
FAMILY: AMCEBID^
four-nucleated cysts with chromatoid bodies are produced. It clearly
belongs to the genus Entamoeba.
Amoebae belonging to this genus occur also in invertebrates. Noller
(19r2a) discovered a form resembling E. histolytica in the vagina of the
horse leech. The free forms varied from 4 to 35 microns in diameter, and
the cysts, which measure 7 to 11 microns, had four nuclei and chromatoid
bodies. Noller gave the name of E. aulastomi to this amoeba, which has
been cultivated by Drbohlav (1925e). Another invertebrate form is
E. minchini, described from the larvae of Tipulids by Mackinnon (1914).
The free amoebae were 5 to 30 microns in diameter, while the encysted
forms contained
a
maximum of ten
nuclei (Fig. 108).
Keilin (1917) de-
scribed as E. mesnili
an amoeba which lives
jjl' in the intestine of the
larvae of the Diptera
Tiichocera hiemalis
and T. annulata. The
amoebae vary inlength
from 6 to 24 microns,
and in breadth from
4 to 8 microns (Fig.
109). There is a clear
ectoplasm and a finely
granular endoplasm
which is free from
food vacuoles. A
curious feature of the
Fig. 109. — Entamceba mesnili from the Intestine of
Larv.e of Trichocera hiemalis and T. annulata
(x 2,000). (After Keilin, 1917.)
1. Uninucleated form.
2. Multinucleatsd form with trailing p.5eiulopodium. to whicl
are adherent bacteria and other debris.
.3. Multinucleated form in division.
4. Encysted form with three nuclei.
amoeba is that many forms are multinucleate and contain from four
to fourteen nuclei according to their size. These multinucleate amoebae
divide to give rise to daughter forms, which are also multinucleate.
Sometimes uninucleate forms are budded off, and these apparently
increase in size and become multinucleate. Encysted forms measuring
8 to 11 microns in diameter are found. They have two to four nuclei.
The nucleus of this amoeba contains a large central karyosome, and in
this respect differs from the typical nucleus of Entamoeha, so that it is
possible that the amoeba belongs to another genus.
Brug (1922) describes as E. helostomce a large amoeba from the intestine
of the water bug Belostoyna sp. of Java. The amoeba was said to be of
the E. histolytica type. Cysts, however, were not seen.
GENUS ENDAMCEBA 235
Fantham and Porter (1911) saw an amceba which they named
E. apis in the bee, Apis mellifica. It resembled E. coli.
Genus Endamoeba Leidy, 1879.
This genus was created by Leidy (1879) for an amoeba of the cockroach
(Periplaneta orientalis). It was named Amwha blattw by Biitschli (1878),
while Leidy gave it the name Endamoeba blattce. If it should be proved
that this amoeba of the cockroach belongs to the same genus as the human
forms to which Casagrandi and Barbagallo (1895) gave the name Entamoeba
coli, then the correct generic name for the human and other forms will be
Endamoeba, as many American writers maintain. Cockroaches, however,
harbour at least two amoebse, one of which undoubtedly belongs to the
genus Entamoeba. The other is Endamoeba blattce, which, according to
Mercier, has such a characteristic nucleus that a distinct genus is justified.
Thomson and Lucas (1926) have recently redescribed the amoeba. Their
description conforms entirely with Mercier's account of the morphology
of E. blatter.
Endamoeba blattae (Biitschli, 1878). — This amoeba was studied by
Schubotz (1905), Janicki (1908, 1909), and later by Mercier (1910), who
has given the most detailed account of its structure and life-history.
E. blattcB lives in the intestine amongst the various nematodes, vegetable
and other protozoal organisms which are found there. It varies very
much in size, ranging from 10 to 120 microns in diameter (Fig. 110). The
average-sized forms measure about 50 microns. The general appearance
varies considerably with the quantity and nature of the food inclusions with
which the endoplasm may be packed. There is no marked distinction
between ectoplasm and endoplasm, but the cytoplasm, which is highly
vacuolated, contains smaller vacuoles near the surface than at the centre.
The movements are sluggish, and one or two blunt pseudopodia are
formed at a time. Sometimes the cytoplasm streams internally in a
peculiar manner, which gives the amoeba a striated appearance. There
is no contractile vacuole.
The nucleus, which differs in many respects from the typical nuclei
of members of the genus Entamoeba, is an ovoid structure measuring
10 to 15 or even 20 microns in its longest diameter. It is limited by
a remarkably thick nuclear membrane, within which, even in the living
amoebae, can be distinguished two zones — a peripheral one consisting of
refringent granules, and a central one of an alveolar nature — the two
zones being separated by a layer of large chromatin granules. The
majority of the amoeba? have a single nucleus, but there occur forms
which are multinucleate.
236 FAMILY: AMCEBID^
Multiplication takes place by binary fission (Fig. 110, 2 and 3). In
nuclear division the large chromatin granules separating the two nuclear
zones are replaced by smaller granules, and these arrange themselves
in the form of a band across the nucleus, which becomes elongated. The
band then divides into two clusters of chromatin granules, which pass
to opposite poles of the nucleus. The latter now becomes hour-glass-
shaped, and finally divided into two. The chromatin of each daughter
nucleus then arranges itself as granules between the two zones, as occurred
in the parent nucleus. Division of the cytoplasm then takes place.
After a number of divisions of this kind, according to Mercier (1910)
a sexual phase is initiated. The amoebse which are to enter on this stage
of development are 40 to 50 microns in diameter, and though the cyto-
plasm is highly vacuolated there are no food inclusions. The nucleus,
which at first has the structure described above, changes its character.
Some of the chromatin in the intermediate zone is extruded from the
nucleus, while the nuclear membrane becomes much thinner. At the
centre there appears a large karyosome made up of an achromatic material
impregnated with chromatin granules, while a centriole can be detected
at the centre. The nucleus elongates, and from the karyosome there is
formed an intranuclear spindle with a centriole at each pole. The
chromatin granules upon the spindle fibres become separated into two
groups, which collect at each pole of the spindle. The nucleus then
divides by constriction. By repeated divisions of this kind, eight nuclei
are ultimately formed (Fig. 110, 4-7). The cytoplasm now becomes
separated into an outer clear alveolar layer and a central granular portion
which contains the nuclei, and this is followed by the formation of a cyst
wall. The cyst has a diameter of 30 to 50 .microns. After encystment,
a second period of nuclear multiplication occurs, with the result that as
many as sixty nuclei may be formed. The number of nuclei varies con-
siderably (Fig. 110, 7-9). The cysts at this stage escape from the
intestine and are taken up by other cockroaches.
In the crop the cyst wall becomes thin, and the centrally arranged
nuclei now take up a position at the periphery. The cyst then passes
into the mid-gut, where it ruptures and liberates the multinucleate cyto-
plasmic body (Fig. 110, 10). A process of budding then occurs, by which
small uninucleate amoebse are separated. These buds are supposed to
be gametes which unite in pairs, giving rise to zygotes, which gradually
increase in size and grow into the free-living adult forms (Fig. 110, 11-23).
The cycle of development is essentially the same as that of Entamoeba
coli. A phase of multiplication by binary fission in the gut is succeeded
by encystment. In the case of E. hlattce, nuclear multiplication commences
before encystment actually takes place, and is continued after the cyst
ENDAM(EBA BLATT^
237
wall has formed till large numbers of nuclei occur, while in E. coli nuclear
multiplication begins only after encystment, and there are rarely more
than eight nuclei. In the case of E. coli the fate of the amoebae which
emerge from the cyst in the newhost is not known, and it is possible that
m-i
rT>:^
■^hM^
^v.
.* <*%i
>-«^l^
^
%%^
,jtf^^^.
■^■?
^^
v^''
tl
S)0
'l^S^.
^«^;?v>'
o© o
'W^/lf 'Wy
;^'
io
f^. .IP
.*
25'
Fig. 110. — Endamceha hlathe from the Intestine of the ('ockkoacu ( x 600).
(After Mercier, 1910.)
1. Amoeba with characteristic nucleus and many vacuolic inclusions.
•1. Amoeba after division of nucleus. 3. Amoeba showing final stage of division.
4-(). Multiplication of nuclei in preparation for encystment.
7-9. Encysted forms; nuclei multiplying.
10. Escape of multinucleated amoeba from cyst.
11-12. Production of gametes from Tuultinuclcated amoeba. l.''-lo. Free gametes.
10-18. Stage.s in unif)n of gametes. iy--3. Stages in growth of zygote.
238 FAMILY: AMCEBIDzE
eight small amoebse are formed, and that these conjugate as in E. blattce,
but there is as yet no evidence that this occurs. It is unfortunate that
Mercier's observations have not been confirmed, and till this has been done
some caution must be shown in accepting his account as absolutely
correct.
Genus: Endolimax Kuenen and Swellengrebel, 1917.
The genus includes parasitic amoebge of small size, each of which has
a single nucleus with a relatively large karyosome of irregular shape.
The cysts are spherical, ovoid, or more irregular in shape, and possess
one, two, four, and more rarely eight nuclei. The genus was created
by Kuenen and Swellengrebel for a small amoeba of the human intestine,
which was named Entamceba nana by the waiter and O'Connor (1917).
Boeck and Stiles (1923) believe that the genus is not sufficiently defined,
and that it might be better to suppress it as a synonym of Entamoeba, or
to regard it as a sub-genus of Entamoeba.
ENDOLIMAX OF MAN.
Endolimax nana (Wenyon and O'Connor, 1917). — Synonymy. — The writer
and O'Connor (1917), who first described this amoeba, named it Entamceba nana, while
Later in the year Kuenen and Swellengrebel (1917), who also discovered it, employed
the name Endolimax iniestinalis. Brug (1917) pointed out that it could not be
included in the genus Entamceba, chiefly because of its distinctive nuclear structure.
and placed it in the genus V aMkam/pfia. Finally Brug (1918) realized that it did
not belong to the genus Valilliampfia, and, accepting Kuenen and Swellengrebel' s
generic title, named it Endolimax nana, by which name it is now generally known,
though Brumpt (1922) refers to it as Endolimax pJiagocytoides, assuming that an
amoeba cultivated from human faeces by Gauducheau (1907. 1998), and named
Entamceba jytiagocytoides, was actually E. nana.
Endolimax ?iana is one of the commonest protozoa of the human
intestine. It was seen by the writer in 1912, and by other observers
before and after this, but its true nature was not recognized. The writer
and O'Connor (1917) found it to be very common in persons in Egypt.
In the free condition E. nana measures from 6 to 12 microns in
diameter (Fig. 111). As usually seen, it moves in a sluggish manner,
but it may be quite active when observed on the warm stage. When at
rest a superficial layer of clear cytoplasm can be distinguished from a
vacuolated endoplasm, but when it performs amoeboid movements little,
clear, blunt pseudopodia are formed. The food vacuoles contain bacteria.
The nucleus is detected with difficulty in the living organism, so that if
the characteristic cysts cannot be found in any specimen of faeces it is
often necessary to prepare stained films in order to distinguish the amceba
from the small precystic form of E. histolytica. The nucleus is a vesicular
structure, and has a diameter of 2 to 3 microns. There is a definite
GENUS ENDOLIMAX 239
nuclear membrane which appears to be free from chromatin, all of which
seems to. be concentrated in the karyosome. The latter varies very much
in shape, and may consist of an aggregation of several distinct granules.
There may be a single angular or irregular mass more or less central in
position; or lying against the nuclear membrane on one side there may
be a large mass connected by a fibre with a smaller mass at the opposite
side. In other nuclei a single large mass may be connected with two or
i
/
/
/
• •••
/#
^
\i
Fig. 111. — Endolimax nana ( x 3,000). (Original.)
1-3. Vegetative forms. 4. Vegetative forms parasitized by Spliasn'ia.
5-6. Uninucleated cysts, one with glycogenic vacuole. 7. Cyst with two nuclei.
8. Cyst with two nuclei in division. 9-12. Cysts with four nuclei.
more smaller granules. The karyosome in its typical form is not a
spherical body, like the karyosomes of the small amoebae (HartmanneUa),
which frequently develop in old material from cysts which have passed
through the intestine. It is the marked irregularity in the shape and
structure of the karyosome which is such a characteristic feature of
E. nana, and enables it to be distinguished from the small forms of E. his-
tolytica (Fig. 95, lo and ii).
The cyst of E. nana was first figured by the writer (1915e), who then
thought it might be a cyst of Chilomastix mesyiili. This error was sub-
240 FAMILY: AMCEBIDyE
sequently corrected by the writer and O'Connor (1917). The cyst, which
has four nuclei in the mature condition, is typically ovoid in shape, and
measures from 8 to 10 microns in length and about half this in breadth
(Fig. Ill, 5-12). As a rule, one side is less convex than the other, so
that the outline is not quite symmetrical. Sometimes the cysts are
spherical, and between these and the typically ovoid forms various
gradations occur. The nuclei in the cysts are constituted similarly to
those of the amoebse, but in the four-nuclear cysts they are very minute.
A characteristic picture is that of a large chromatin body on one side
of the nucleus connected to a smaller body at the other side, as sometimes
occurs in the free forms.
Attention has been drawn by Dobell (1919) to the occasional presence
in the cytoplasm of a glycogen vacuole (Plate II., 22, p. 250). In some
batches of these amoebse a large proportion of the uninucleate cysts
possess such a vacuole, while in others it is not so apparent. As noted
by Swellengrebel and Winoto (1917), the glycogen gradually disappears
in cysts kept for some days outside the body. Dobell has noted the
occasional presence of eight instead of the usual four nuclei within the
cyst. The writer has also seen these form •. The cysts may contain certain
filamentous bodies the nature of which is not clear. Dobell suggests they
may be parasitic or symbiotic bacteria, or possibly chromatoid bodies.
In fresh saline preparations the cysts appear as perfectly clear homo-
geneous structures. The nuclei can rarely be detected, and even in
iodine solution they are often difficult to see (Plate II., 17-22, p. 250).
In specimens stained with iron haematoxylin they are generally quite
evident, but it is difficult to gauge the exact degree of differentiation on
account of the small size of the cysts. The nuclei may occupy any position
in the cyst, but not infrequently they are grouped at one end.
E. nana is an inhabitant of the large intestine, and the writer (1920),
in sections of the large intestine, has noted the presence of these amoebae
in the lumen of the glands. There was no evidence that they could invade
the tissues. Whether the amoebae can also live in the small intestine, as
Dobell (1919) conjectures, is not known. There is no indication that E. nana
is in any way pathogenic, and in this respect it resembles the harmless E. coli.
Attempts at cultivation of E. nana on solid medir. have not met with
success. If faeces containing them are smeared on the surface of suitable
agar medium, cultures of small amoebae of the same size may be obtained,
but these are merely developed from cysts of free-living forms.
Several observers have undoubtedly seen E. nana in human faeces,
gllld have thought they have obtained cultures of it in agar plates. It
is probable that as the E. nana perished the cysts of free-living amoebae
gave rise to a culture, and produced an erroneous impression of culture
GENUS: ENDOLIMAX 241
of E. nana. The amoeba cultivated by Gauducheau (1907, 1908), and named
by him (1907) E. phaf/ocytoides, was probably E. nana in the fresh stool,
but a free-living ama?ba in the culture. A further paper published by
him (1922) tends to confirm this opinion. As his description undoubtedly
applied chiefly to the cultivated form, it seems inadmissible to employ
his specific name phagocytoides for the human parasite, as Brumpt (1922)
and others have done. Thomson and Robertson (1925) have maintained
a strain of E. nana in Boeck and Drbohlav's L.E.A. medium for nineteen
days, during which fifteen subcultures were made.
Kessel (1923rt, 1924f/) states that he has succeeded in infecting rats
and monkeys with E. nana. Chiang (1925) was unable to confirm these
observations on rats.
ENDOLIMAX OF ANIMALS.
Minchin (1910a) described as Malpighiella refrinyens a parasite he
had encountered in the Malpighian tubes of rat fleas {Ceratophyllus
fasciatus). It had an amoeboid phase, and also produced cysts which
Fig. 112. — Malpighiella refringens from the Malpighian Tubes of the Rat Flea,
Ceratophyllus fasciatus : Am(EB0id and Three Encysted Forms ( x ca. 3,000).
(After Minchin, 1910, from Doflein, 1916.)
resemble both in size and appearance those of E. nana (Fig. 112),
The cyst wall, however, is much thicker than that of E. nana. Noller
(1914) observed the organism in the Malpighian tubes of about 90 per cent,
of the dog fleas {Ctenocephahis canis) in Germany. From the fact that
I. 16
242 FAMILY: AMCEBID^E
the unencysted stages do not ingest solid food, he doubts if the parasite
is in reality an amoeba at all. A very similar parasite was seen by Alexeief?
(1913) in the vagina of the leech {Hirudo medicinalis). Dobell (1919)
thinks that if MalpighieUa refringens ultimately proves to be an amoeba,
E. nana may have to be placed in the genus MalpighieUa. An amoeba,
described by Epstein and Ilovaiski (1914) as Ncegleria ranarum, from
the frog probably belongs to the genus Endolimax on account of the
structure of the nuclei, and the encysted forms which resemble those of
E. nana. The free amcebse reached a diameter of 25 microns (Fig. 113).
Tyzzer (1920) described a small amoeba which he found in the intestine
of fowls in America. In the free state it resembled E. nana, but the central
karyosome of the nucleus was more compact. Cysts with a single nucleus
y ^
\ '^"- '
M
ex
Fig. 113. — Endolimax ranarum from the Eectum of the FROG(xt«. 1,200).
(After Epstein and Ilovaiski, 1914.)
a. Free form. b. Encysted form with four nuclei,
c. Encysted form with eight nuclei.
were seen. Though the name Pygolimax gregariniformis was given to the
amoeba on account of its peculiar gregariniform movements, it possibly
belongs to the genus Endolimax.
Brug (1923), in Sumatra, has seen in the monkey, Macacus cynomolgus,
an amoeba which in its free and encysted stages corresponds with E. nana.
He names the amoeba Endolimax cynom.olgi. Chiang (1925) has given the
name Endolimax ratti to an amoeba of the white rat. It is morphologically
identical with E. nana, with which he was unable to infect rats.
Genus: lodamceba Dobell, 1919.
This genus was founded by Dobell for an amoeba of the human intestine
which produces a uninucleated cyst. The latter contains a very distinct,
sharply-defined iodophilic body of glycogen nature which stains a dark
reddish-brown in iodine solution. Because of the presence of this idio-
GENUS: lODAMCEBA 243
philic body the writer (1915e, 1916), who first described it, called it an
" iodine cyst," as its exact nature was not clear. Both the cysts and amoebae
were found in a case by Kuenen and Swellengrebel (1917), and Brug (1919)
came to the conclusion that the amoebae seen by Kuenen and Swellengrebel,
and which they had called " Pseudolimax," were in reality amoebae of which
the "iodine cyst" represented the encysted stage. Kofoid, Kornhauser
and Swezy (1919), and Brug (1921), believe that /. butschlii is a large
race of E. nana, and express the opinion that lodamceha is a synonym of
EndoUmax, and Boeck and Stiles (1923) support them in this conclusion.
It seems, however, that the genus lodamceha is much better defined than
these observers maintain.
IODAM(EBA OF MAN.
lodamoeba butschlii (Prowazek, l9l'2)—Synonijmjj. — Tlicre seems to be con-
siderable doubt as to the correct name of this amoeba. Prowazek (1912«) gave a very
brief and incomplete description of an amoeba which he saw in a child in the Caroline
Islands. He gave it the name Entamceba butschlii. A single cyst is figured, and if
it represents one of the " iodine cysts " it is evidently deformed or degenerate. D obeli
(1919) comes to the conclusion that Prowazek was actually describing the "iodine
cyst " and its amoeboid stage, and that the human parasite should therefore be known
as lodamoeba butschlii. It is quite evident that the figures given by Prowazek cannot
represent either E. coli or E. histolytica. The size of the amoebfe excludes the possi-
bility of its being EndoUmax, nana, and from what is now known of the intestinal
amoebae of man the only amoeba which Prowazek could have observed is the one now
under discussion. On the other hand, Brug (1921) believes that another amoeba,
previously described by Prowazek (1911, 1912) as Entamceba williamsi, was a mixture
of the "iodine cyst " and Entamoeba coli. In support of this contention he states that
he has examined Prowazek's original preparations, and has seen in them the iodine
cysts and the amoeba, an observation which has also been made by Noller (1921).
There can be no doubt, however, that Prowazek's description and figures were based
chiefly on Entamceba coli, and though some of the forms described by him may have
been other amoebae, the name E. williamsi must become a synonym of E. coli. The
fact that Brug and Xoller have found the " iodine cyst " and its amoeba in the original
preparations does not prove that Prowazek actually described them. Taliaferro
and Becker (1922) support Brug and Noller in their contention that the correct
specific name must be williamsi. Brug further considers that the amoebae belong
to the same genus as EndoUmax nana, while Kofoid, Kornhauser, and Swezy (1919)
concluded that they are merely large races of EndoUmax nana. Eodenhuis (1919)
also expressed the opinion that the amoeba belonged to the genus EndoUmax, and
proposed to name it EndoUmax jjileonucleatus. Cauchemez (1921) has studied this
organism, and, in agreement with Byumpt, comes to the conclusion that it cannot be
identified with either of Prowazek's amoebae, E. williamsi or E. biitschUi, and proposes
to name it lodamoeba wenyoni, Brumpt, 1921. This is undoubtedly incorrect, for
if it is necessary to reject both of Prowazek's names, the correct name will be
lodamoeba pileonacleata. It seems, therefore, best to consider the organism as
identical with Prowazek's E. butschlii, and to name it lodamoeba butschlii, as Dobell
(1919) has done. Kuenen and Swellengrebel (1917) used the name "Pseudolimax,"
but not as a generic title, though Brumpt (1922) has adopted it as the generic name
for this amoeba, to which he refers as Pseudolimax wenyoni.
244 FAMILY: AMCEBID^
The free forms of /. hutschlii are intermediate in size between those
of Entamoeba coli and Endolimax nana (Fig. 114, 1-4). They are 9
to 13 microns in diameter, but larger forms up to 20 microns and
smaller ones down to 5 microns in diameter have been seen. Kuenen
and Swellengrebel (1917), who first described the amoeba, gave 10 to
12 microns as the measurement, while Brug (1921) gives 7 to 20 microns.
Taliaferro and Becker (1922) state that the largest form seen by them
measured 20 by 15 microns. There is no marked distinction of ecto-
plasm and endoplasm, and the movements are sluggish, like those of
E. coli. The endoplasm contains numerous food vacuoles, which include
various bacteria. According to Brug, the amoeba feeds only on very
small particles, and does not ingest large bodies, as E. coli often does. In
the living amoeba the nucleus can hardly be detected, a feature which
serves to distinguish it from E. coli, the nucleus of which is nearly always
distinct. As first pointed out by Dobell (1919), and later by Taliaferro
and Becker (1922), it is the structure of the nucleus which is the most
characteristic feature of the free forms. As seen in stained specimens,
it is a vesicular structure with a diameter of 2 to 3-5 microns. There is
a large karyosome, which has a diameter of about a third to a half of that
of the nucleus itself. The membrane of the nucleus is well developed,
while the karyosome is surrounded by a layer of globules composed of
a substance which does not retain the stain as long as the karyosome, and
is thus probably not of chromatin nature. These globules sometimes
indent the karyosome, and give it a stellate appearance, while the septa
between the globules may produce the impression of a series of radiating
fibres connecting the karyosome to the nuclear membrane. Multiplication
by binary fission has been noted by Eodenhuis (1919), but the details of
the process have not been described. Amoebse with cytoplasm devoid of
good vacuoles, and with or without a glycogenic body, are probably pre-
cystic forms (Fig. 114, 5).
The cysts of /. hiitsclilii were first seen by the writer in 1906 in the
Sudan, and were not seen again till 1915, when a description was given.
They appear to be much more frequently encountered in stools than the
free amoebae, and as very heavy infections sometimes occur without it being
possible to discover any amoebae, the writer considered that the cysts might
be vegetable organisms. This view seemed*to receive support from the fact
that filaments grew out from certain cysts when kept under observation in
saline solution. It seems clear from what is known now that those cysts
which produced filaments were really of another nature, and not the cysts
of the amoebaj. The writer has on several occasions kept cysts which were
not identifiable, and has seen them produce long branching filaments across
the preparations, a clear indication that they were spores of fungi.
lODAMCEBA BUTSCHLII
245
^
1
/
>^
\
»
' "I-,.-*'-"'" "
J
•• - (■©•'
s
6
^^c3k
10
/J
/^
/i-
Fig. 114. — lodamoeba hutschUi from the Human Intestine ( x 3,000). (Original.)
!-4. Ordinary type of free form. o. Pi-ecystic form.
()- 1:5. Ordinary type of oncy.stocl form. 1 t-l.l. Encysted forms witli two nneloi.
246 FAMILY: AMCEBID^
The cysts of 7. biUschlii, when spherical, vary in diameter from 7 to
15 microns, but very marked irregularities in shape occur (Fig. 114, 6-15).
There is a definite cyst wall, and in the cytoplasm within the cyst is found
a more or less rounded refractile body and a number of small refractile
granules which are possibly composed of volutin. The single nucleus can
usually be detected in the thickest portion of the cytoplasm, between the
refractile body and the cyst wall. In iodine solution the refractile body
assumes a dark brown colour, and is seen to have a sharply-defined margin,
thus contrasting with the ill-defined limits of the glycogenic vacuoles in
cysts of Entamoeba coli and E. histolytica (Plate II., 11-16, p. 250). The
" iodophilic body " is rarely absent from the cysts. It may be quite
small, but usually has a diameter of a quarter to a third of that of the cyst.
Occasionally two or three separate iodophilic bodies are present. In the
process of staining with iron hsematoxylin and mounting in balsam in the
ordinary manner they are dissolved, the vacuoles alone remaining. As
pointed out by Dobell, the iodophilic body is gradually absorbed in
living cysts kept outside the host. Usually there is a single nucleus
in the cysts, though cysts with two nuclei are not uncommon in some
infections (Fig. 114, 14 and 15). Taliaferro and Becker (1922) found only
four cysts with two nuclei amongst 2,000 consecutive cysts examined.
The nucleus of the encysted form differs from that of the free amoebae in
that the karyosome, instead of occupying a central position, conies to lie
against the nuclear membrane, while the rest of the space within the
membrane is filled with the globules which surrounded the karyosome
in the free amoeba. These may retain the stain irregularly, and give rise
to the appearance of secondary karyosomes in the nucleus. Brug (1919
and 1921) describes the karyosome as being applied to the nuclear
membrane, while between it and the opposite side of the nucleus is a body
which is semilunar in outline in side view, or watch-glass in shape, with
the karyosome at the centre, when viewed in a direction at right angles to
this. It is possible that this appearance is a result of shrinkage of the
globular material filling the space within the nuclear membrane, so that
it forms a more compact body separated from the karyosome and nuclear
membrane by a clear space. The karyosome does not always stain
uniformly, as often a more deeply staining portion can be distinguished
from another staining less intensely.
Dobell (1919) believes that there occur races of /. butschlii which can be
distinguished by the average size of the cysts.
I. bUtscJilii is a fairly common inhabitant of the human intestine. A
remarkable feature of the infections is that often enormous numbers of
cysts are passed without there being any indication of the free forms.
There seems to be no evidence that the amoeba has any pathogenic
lODAMCEBA OF ANIMALS
247
properties. Kessel (1923a, 1924ft) states that he has infected rats and
monkeys with /. hutschlii.
On two occasions, by inoculating Boeck and Drbohlov's medium with
faeces containing cysts of /. hutschlii, Thomson and Robertson (1925)
obtained cultures of amoebae which appeared to belong to this species.
One strain was maintained for forty-six days with forty subcultures.
No cysts were found in the cultures.
lODAMCEBA OF ANIMALS.
O'Connor (1920) describes an amoeba which he found in pigs in the
Ellice Islands. Both free and encysted forms occurred, and save for the
presence of numerous irregular bodies of chromatoid or volutin nature,
Fig. 115. — Cysts of lodamceha of tue Pig {xca. 1,400), drawn from an Iodine
Preparation, showing Marked Variation in Shape and Absence of Iodo-
PHiLic Body in Some Cysts. (Original.)
they bore a striking resemblance to 7. hutschlii. The name /. suis was
suggested, though no data for distinguishing it from the human parasite
were given. Noller (1921), who has studied several cases of infection with
this amoeba in men in Hamburg, stated that Feibel has noted that at
least 20 per cent, of the pigs slaughtered in Hamburg abattoirs had
/. hutschlii in their intestines. In the same year Cauchemez discovered the
organism in pigs in France. He concludes that these animals are probably
the reservoirs from which human beings become infected. Feibel (1922)
has given an account of the observations referred to by Noller. The
writer has on several occasions seen the cysts of this parasite in faeces of
pigs in England (Fig. 115).
248 FAMILY: AM(EBID^
Brug (1920a) described as Endolimax lueneni an amoeba lie met with
in the monkey, Macacus cynomolgus. The amoebae were 7 to 12 microns
and cysts 7 to 10 microns in diameter. The latter closely resembled the
cysts of /. butschlii, and it is evident this monkey amoeba belongs to the
same genus, its name becoming /. keuneni (Brug, 1920). Hegner and
Taliaferro (1924) state that they have seen what appears to be the same
parasite in the Brazilian monkey, Cebus variegatus, while the writer has
seen the cysts of a similar form in the faeces of a gorilla.
Genus: Dientamoeba Jepps and Dobell, 1918.
The genus includes small, delicate, actively motile, parasitic amoebae,
which show a tendency to remain in a binucleate condition. The nucleus
r\)t-
%
2 3
Fig. 116. — Dientamceha frag His ( x 3,000). (Original.)
1. Vegetative form with two nuclei.
2. Vegetative form with two nuclei, one of which has the chromatin on the nuclear membrane.
3-5. Forms with two nuclei. 6-9. Forms with one nucleus.
has a characteristic structure, and encysted forms have been once re-
corded. The single known species of this genus was described by Jepps
and Dobell (1918) as a parasite of man. They noted eight cases,
while Jepps (1921) mentioned ten others in England. The writer saw
this form in 1909, but at that time formed no opinion as to its nature.
It has been recorded in America by Kofoid, Kornhauser, and Plate (1919),
and by Taliaferro and Becker (1922a, 1924); in Manila by Haughwout
and Horrilleno (1920), both in children and adults; by Bijlsma (1919) in
Holland; by Noller (1921) in Hamburg; and by Thomson, J. G. and Robert-
son (1923), and Robertson (1923) in England. Reichenow (1923) examined
the stools of 100 patients in Germany, and found the amoeba in five of these.
GENUS: DIENTAM(EBA 249
Dientamoeba fragilis, Jepps and Dobell, 1918. — This is a small amcBba
which has been seen chiefly in the unencysted stage (Fig. 116, 1-9).
It measures 3'5 to 1'2 microns in diameter. The amoebae are actively
motile, and have a well-marked ectoplasm and endoplasm. The pseudo-
podia are composed almost entirely of ectoplasm, and these are often
flattened or lobed. The endoplasm contains numerous food vacuoles in
which bacteria occur. The most characteristic feature of this amoeba is
its binucleate condition. The nuclei vary in size from 0*8 to 23 microns,
and are exceedingly difficult to detect in the living organism. In stained
specimens the nuclei are seen to be spherical, while there is a central
karyosome consisting of a group of granules embedded in a plastin matrix.
One granule is generally larger than the others. Surrounding this
karyosome is a clear area limited by a fine nuclear membrane, which is
connected with the karyosome by exceedingly delicate threads. At the
point of union of the latter with the nuclear membrane there are certain
granules, which may or may not be chromatin. Apart from these, all
the chromatin of the nucleus is aggregated in the karyosome. Though
the nuclear structure described above is characteristic of the majority of
amoeba? in any one case, the writer has noted that very frequently there is
a different arrangement of the chromatin. In some forms it appears
to be distributed on the inner surface of the nuclear membrane, while a
minute central karyosome can be detected. In other cases, chromatin
granules are separated from the membrane, but lie at some distance from
the karyosome; while in others they are concentrated at the centre of the
nucleus, as Jepps and Dobell state, so that the karyosome may be obscured.
Thomson and Robertson (1923) have called attention to the presence of
the central karyosome round which the chromatin granules are arranged.
It seems doubtful, therefore, if the aggregation of granules at the centre
of the nucleus should be regarded as the karyosome, which appears
to be represented by a minute granule, as in members of the genus
Entamoeba.
Most individuals are binucleate, but a certain number of uninucleate
forms can generally be found. Jepps and Dobell believe that the adult
binucleate amoeba divides to give rise to uninucleate forms, and that as
these grow the nucleus divides in preparation for the division of the
cytopolasm, which does not take place till much later. D. fragilis is a
very delicate organism, and quickly degenerates outside the body. In
so doing, a large central vacuole often appears, reducing the amoeba to
a ring of cytoplasm in which the two nuclei remain. A striking resem-
blance to Blastocystis is thus produced (Fig. 118).
No encysted forms were discovered by Jepps and Dobell, and the
writer and others have similarly failed to find any indication of encystment
250 FAMILY: AMCEBID^.
in cases studied by them. Kofoid (1923), however, describes spherical
cysts of this amoeba containing one or two nuclei and one or more vacuoles.
In a case seen by the writer, in which E. histolytica as well as
D.ffagilis occurred, both infections disappeared after a course of emetine.
Thomson and Robertson (1925) report the successful culture of
D. fragilis in the medium used by Boeck and Drbohlav for the culture
of E. histolytica.
DIAGNOSIS OF THE INTESTINAL AMCEBiE OF MAN.
In order to arrive at a conclusion regarding the nature of the amoebae
or their cysts which are found in human faeces, very careful, and sometimes
prolonged, examinations are necessary. It must always be remembered
that mixed infections are common, so that it is never possible to be
absolutely certain that all the amoebae present have been diagnosed. In
the case of malaria there may be found a large infection of ring forms
about the nature of which there may be considerable doubt. Search may
reveal a few crescents, and though it will then be known that Plasmodiiitn
falciparii?n is present, it will still be impossible to assert that all the rings
belong to this species. Similarly, with the amoebae there may be a mixture
of free forms of E. coli and E. histolytica, and the discovery of the charac-
teristic cyst of one species does not exclude the possibility of some of the
free forms belonging to the other (Fig. 117).
Examinations repeated on several different occasions reduce this error
to a minimum, but cannot entirely eliminate it. It was demonstrated
by the writer and O'Connor (1917) in a series of cases that the positive
findings which result from the examination of the first specimen yield
only one-third of the positive results obtained by examinations repeated
on a number of successive days. Very often it may be impossible to
determine the nature of the free amoebae in any specimen. The large
forms may be E. coli or E. histolytica. The intermediate forms may
be either of these or /. hutschlii, while the small forms may be any
of these or E. nana or D. fragilis. The precystic forms of E. coli
and E. histolytica may be difficult to distinguish from one another.
Diagnosis is most easily made by finding the cysts in saline or iodine
preparations, if not on one day, then on another. If cases are examined
repeatedly, encysted forms will generally be found (Plate II., p. 250).
As regards the large amoebae from 15 to 20 microns or more, if they
occur in dysenteric stools and are very active, they are probably E. histo-
lytica. If so, the nucleus should be difficult to see, and search may reveal
forms including red blood-corpuscles. In the latter case the amoebae
are certainly E. histolytica. E. coli may, however, be present in dysenteric
PLATE II.
Cysts of various human intestinal Protozoa, as seen in iodine solution (x 2000).
1-4. Entamoeba coli. 17-22. Endolimax nana.
5-10. Entamceha histolytica. 23. Giardia intestinalis .
1 1- 1 6. lodamaeba butschlii. 24. Chilomastix mesnili.
(Original.) [To face p. 350.
DIFFERENTIATION OF HUMAN AMCEB^E
251
conditions of a bacillary nature. The amoebae are less active, the nuclei
are clearly visible, while food vacuoles containing bacteria and other
objects are present. In cases of doubt it is necessary to wait till formed
Fig. 117. — The Intestinal Amceb.e of Man (x 1,250). (After Wenyon, 1922.)
A-C. Entamoeba histolytica.
A. Tissue-invading f(n-ni with one nucleus and six ingested red blood-corpuscles.
B. Precystic amoeba. C. Cyst with four nuclei and chroniatoid bodies.
D-F. Entammha coli.
D. Large amoeba with one nucleus and various ingested food bodies.
E. Precystic amoeba. F. Cyst with eight nuclei.
G-I. lodamoeba biiischlii.
G. Free amoeba. H. Precystic am ceba.
I. Cyst with a single nucleus and glycogenic vacuole.
•J-L. EndoUmax imna.
J. Free amoeba. K. Precystic amoeba, L. Cyst with four nuclei.
M-N. Dienfamceba fragilis.
M-N. Forms with one and two nuclei.
stools are being passed, when the characteristic cysts of E. coli or E. his-
tolytica will probably appear. The former are seen either with eight
nuclei or with two nuclei and large central vacuole; while the latter have
one, two, or four nuclei, and not infrequently chromatoid bodies and
252 FAMILY: AMCEBID^
vacuoles. Examined in iodine solution, the details are more readily seen,
for the nuclei of the cysts of E. histolytica are not easily distinguished in
saline solution. Very active amoebae with very marked ectoplasm and
pseudopodia being formed entirely of ectoplasm, are most probably
E. histolytica. If the amoebae are about 10 to 15 microns in diameter,
then diagnosis is difficult, and a careful search in saline and iodine solution
must be made for cysts of E. coli, E. histolytica, or I. butschlii. The
last is distinguished by its deeply staining iodophilic body and single
nucleus; the others by the number of nuclei and presence or absence of
chromatoid bodies. If no cysts can be found, it will be necessary to
make stained films, when /. butschlii can be recognized by its large
central karyosome. If its nucleus has a small karyosome and chromatin
granules on the membrane, an amoeba may be E. histolytica or E. coli.
Attention to details of the nuclear structure, as described above, may
assist in diagnosis, but it must be admitted that there is difficulty in
distinguishing the precystic forms of these amoebae.
If precystic amoebae occur in any specimen, then it is very unusual for
cysts not to be present also, and if they are not found at the first examina-
tion, later examinations will almost certainly reveal them.
If the amoebae are quite small and vary in size from 5 to 10 microns,
or a little over this, they may be free forms of E. nana, precystic forms of
small races of E. histolytica, I. biitschlii, or D. fragilis. Here, again,
the discovery of cysts will enable a diagnosis of the first three to be
readily made. If cysts cannot be found, then films must be stained.
The small forms of E. histolytica will show their characteristic nuclei, and
D. fragilis the two nuclei characteristic of this amoeba. Amoebae
with a single nucleus, showing a large, irregularly-shaped karyosome, are
almost certainly E. nana, though it is just possible they may be difficult
to distinguish from /. biitschlii, which, however, is rarely seen in
the unencysted condition. If /. biitschlii is present, its cysts will almost
certainly be found and recognized in iodine solution. The cysts of
E. nana are typically of an ovoid shape, while the small cysts of E. histo-
lytica are usually spherical or nearly so. The small cysts of E. histolytica
often show chromatoid bodies, and the details of the cysts and those of
E. nana should be quite clear in properly stained films.
It must be remembered that in the great majority of cases a diagnosis
can be arrived at by the careful examination of thin saline and iodine
preparations, and that stained films are only necessary in exceptional cases
or for confirmatory purposes.
The presence in large amoebae of food vacuoles containing bacteria,
yeasts, or other objects, amongst which may be cysts of the intestinal
Protozoa, such as those of E. histolytica, Giardia intestinalis, Isospora
DIFFERENTIATION OF HUMAN AMCEBiE 253
belli, is almost conclusive evidence of the amojbae being E. coli. It
should be remembered, however, that dying or dead E. histolytica may be
invaded by bacteria of all kinds, while occasionally a particular type of
body, such as the spores of a bacillus, may be taken up by apparently
healthy E. histolytica. All the intestinal amoebse of man, as well as the
free-living amoebae, are liable to invasion by the vegetable organism
Sjphcerita, a name given to it by Dangeard (1886), who saw it in a Heliozoan.
It was seen by the writer (1907) in E. muris of mice. It has the
appearance of spherical masses of coccus-like bodies which are highly
refringent in the
living condition. In
films stained by iron
haematoxylin they are
black. They occur in
vacuoles in the cyto-
plasm (Fig. Ill, 4).
A less common para-
site of similar appear-
ance is one which
occurs within the
nuclear membrane.
It was named Nucleo-
fhaga by Dangeard O^
(1896), who saw it
in the nuclei of free- ^ _
living amoebae. Noller
(19^1) has described Yig. 118. — Btastocystis hominis from Human Faeces fixed
its occurrence in the in Sciiaudinn's Fluid and stained with Iron H.ema-
nuclei of E. nana and toxylin ( x 2,000). (Original.)
/. biltschlii. ^^ addition to the nuclei the thin layer of cytoplasm surrounding the
. largecentral vacuole contains dark staining granules of volutin.
It IS difficult to 1-7, Ordinary forms; 8, dividing form.
give any rules for the
separation of amoebae or their cysts from other structures in faeces. Blasto-
cystis hominis Brumpt, 1912, is very commonly present, and varies con-
siderably in size (Fig. 118). It has a large central vacuole, while the
cytoplasm is reduced to a thin layer in which one or two small nuclei lie at
each pole of the cyst. Refractile globules of volutin which may be present
in the cytoplasm must not be mistaken for the nuclei, which are much less
distinct. The organism has a much more delicate appearance, and is
generally less refractile than the amoebic cysts. Usually the central vacuole
contains non-refractile material; at other times it contains a highly refrac-
tile body which may have a yellowish or brownish tint. Blastocystis may
254 FAMILY: AMCEBID^
be no more than 5 microns in diameter, or 20 microns or more. In varying
number it can be found in practically every stool examined. It reproduces
as a rule by binary fission, and multiplies rapidly in certain media,
such as that used for the cultivation of E. histolytica. Occasionally,
as pointed out by Alexeieff (1911rf), forms with numerous nuclei are
seen, and it appears that the cytoplasm concentrates round these
nuclei, producing eventually a number of daughter forms within the
original cyst membrane. Such a form was seen in human fseces by
the writer and O'Connor (1917) in Egypt. Blastocystis is a vegetable
organism, but not infrequently it may be simulated by cells, flagellates,
or amoebae, which in a degenerating condition develop a large central
vacuole. Cysts of amoebae may be confused with fat globules (castor oil),
or globules of semi-digested muscle fibres. The latter may be perfectly
spherical and homogeneous. They are usually of a yellow-brown tint.
In iodine solution they stain a pale brown colour, or, as sometimes happens,
they become definitely pink. They are highly refractile, and show no
internal structure either in saline or iodine solutions. When once an
observer has become familiar with the actual appearance of the cysts
of the intestinal Protozoa, it is hardly possible to confuse them with
other objects, and this familiarity can only be obtained by practical
experience with the microscope. Intestinal epithelial cells swollen as a
result of degeneration, and the large macrophages which are sometimes
seen with included red blood-corpuscles, have been frequently mistaken
for amoebae. These cells, however, never exhibit active movements,
while their nuclei have an appearance which is quite different from that
of the nuclei of amoebae. As dead and immobile amoebae may easily be
confused with large cells, and vice versa, it is safest to regard no cell
as an amoeba unless definite amoeboid movements are seen. Polynuclear
leucocytes, in which the nucleus has separated into four parts, may be
mistaken for four-nucleated cysts. Occasionally, cysts of free-living
Protozoa which have been swallowed in food or water may be met with
in perfectly fresh stools. It cannot be too strongly emphasized that
specimens examined should be as fresh as possible.
ACTION OF DRUGS ON INTESTINAL AMCEBiE.
There is only one drug which can claim to luive any marked specific
action on the intestinal amoebae of man, and this is emetine. Curiously
enough, it affects only two of these — namely, E. histolytica and /.
butschlii. The former is known to be a tissue parasite, while there is no
evidence that I. butschlii is anything more than a harmless commensal
which lives in the intestinal contents. On E. coli and the other forms
there is no evidence that emetine has any action whatever.
ACTION OF DRUGS 255
It has long been known that ipecacuanha is a specific for amoebic
dysentery, but Vedder (1912) was the first to show that this action of
ipecacuanha depended upon the alkaloid emetine. Rogers (1913), in
India, was the first observer to introduce this alkaloid in the routine
treatment of amoebic dysentery, a course which had been previously
recommended by Vedder (1912). The latter observer believed that it
acted directly on the amoebae and poisoned them, and Rogers made
similar claims. It appears, however, as has been demonstrated by the
writer and others, that active E. histolytica, either in faeces or liver- abscess
pus, can be mixed with relatively strong solutions of emetine, and that
the amoebae will remain as perfectly active as those in control preparations.
Unless it is assumed that the medium in which the amoebae happen to
be — namely, the faecal matter or the pus — absorbs or fixes the emetine,
so that it never actually comes in contact with the amoebae, it must be
concluded that the alkaloid has no immediate toxic action on the amoebae.
That such an explanation of the failure of emetine to kill amoebae in
these experiments may have something in its favour is borne out by
certain tests made by Pyman and the writer (1917) on cultures of free-
living amoebae on agar plates. The agar was made up with varying
strengths of different salts of emetin, and it was found that the amoebae
did not grow on the medium which contained the salts, which are known
to be specifics for amoebic dysentery, though the bacterial growth upon
which the amoebae feed was little altered in character. Furthermore, it
has been shown by Brown (1922) that if the emetine solution which is to
be introduced into the agar is first mixed with pus for a few minutes, the
liquid portion separated by centrifugation has lost its power of arresting
growth of amoebae on the plate. It would seem that in this experiment
the dead cells and debris in the pus had absorbed the emetine from the
solution, so that there may be some reason for suspecting that when
material such as faeces or pus containing E. histolytica is mixed with
solutions of emetine, the failure of the drug to kill the amoebae may be due,
in part at least, to its absorption by the dead material. It has also to be
remembered that even if emetine has no direct action on E. histolytica
exposed to it for a comparatively short time, it may still have such an
action over a longer period in preventing growth and multiplication.'
It should be possible to test this point on cultures of E. histolytica.
Dale and Dobell (1917) investigated the action of emetine on experi-
mentally infected cats, and came to the conclusion that the drug only in-
directly kills £'. histolytica by acting primarily on the host. In the case of
cats they stated that it neither acts as a prophylactic when given before
infection is attempted, nor as a curative agent after E. histolytica has estab-
lished itself in the large intestine. Mayer (1919) had similar experiences,
256 FAMILY: AMCEBIDiE
but Sellards and Leiva (1923) have shown that, as a rule, amoebic dysentery
in kittens is so much more acute than it is in human beings that the action
of emetine in the two hosts is hardly comparable. By employing large
animals, in which the dysentery arising from injection of E. histolytica is
less acute than it is in kittens, and by treating the animals with emetine
solutions 'per rectum in a dose of 10 milligrams per kilogram of body-weight,
they have demonstrated a definite therapeutic action of the drug. Further-
more, by employing the same method of treatment in kittens immediately
infection has taken place, similar results were sometimes obtained.
Ware (1916) reported an outbreak of what appeared to be amoebic
dysentery in a pack of fox-hounds in India. Seven of the animals, some
of which had been obstinately ill for several months, were given injections
of from I to 1 grain of emetine. There was an immediate response with
cessation of symptoms. All the animals recovered completely except
one, which relapsed. Whatever may be the mechanism of its action, it
is certain that in man emetine has a remarkable effect. Attacks of
amoebic dysentery are in most cases cut short by the hypodermic injection
of 1 grain of the drug on a few successive days, while the introduction
of the drug has diminished the number of secondary complications, such
as liver abscess, which formerly were of common occurrence.
As in the treatment of so many protozoal diseases (malaria, trypano-
somiasis), though it is comparatively easy to suppress the parasites to
the extent that acute symptoms disappear, it is extremely difficult to
rid the host of E. histolytica entirely, and relapses are therefore prone to
occur. Very frequently, after the treatment of acute amoebic dysentery,
with the disappearance of symptoms the patient passes into the carrier
condition. In a certain number of cases it is possible by intensive treat-
ment to rid a patient entirely of an E. histolytica infection, but to obtain
evidence that this has happened it is necessary to continue the examina-
tions of the stools over a period of many months.
It was shown by the writer and O'Connor (1917) that the administra-
tion of 1-5 grains of emetine hydrochloride daily (1 grain subcutaneously
each morning and | grain by the mouth each night) for a period of twelve
days would in a certain number of cases eradicate E. histolytica infections.
Another method of giving emetine is in the form of the powder of bismuth
emetine iodide in cachets. This drug was introduced during the war by Low
and Dobell (1916), and has been extensively used. A cachet containing
3 grains of the drug, corresponding to 1 grain of emetine hydrochloride,
is given each day for twelve days by the mouth.
Emetine has a remarkable action in cases of threatened amoebic abscess
(hepatitis), though it is not quite clear if the drug alone, without operative
treatment, will cause an amoebic abscess which has already formed to
COPROZOIC AMCEB^ 257
disappear, though some observers, such as Rogers, chiini that it will.
The writer with O'Connor observed a case in Egypt of a liver abscess
which was draining after operation. Active amoebae were constantly
present in the pus in spite of the administration of large doses of emetine
both subcutaneously, by the mouth, and by injection into the abscess
cavity. Here it would seem that failure of the emetine to reach the tissues
in which the amoebse were actually living, possibly as a result of defective
circulation, would account for the residt.
The action of emetine in getting rid of infection of 7. butschlii
was first noted by the writer and O'Connor (1917) in Egypt. The same
result was obtained by others, as recorded by Dobell, Gettings, Jepps,
and Stephens (1918). If it is correct that emetine only acts on E. histo-
lytica indirectly by its influence on the tissues of the host, and has no
action on E. coli and other intestinal amoebae, it is difficult to understand
how it affects I. butschlii, which, as far as we know% is similar in habits to
E. coli.
In some cases of E. histolytica infections emetine fails to act, and in
these it can only be supposed that the intestine is in such a condition that
the tissues in which the amoebae are living are not reached by the emetine,
possibly as the result of defective circulation in certain portions of semi-
necrotic mucosa. The view that emetine resistant strains of E. histolytica
exist requires definite proof, of which at present there is none, before
it can be accepted. There also does not seem to be any evidence that
the administration of emetine wall cause a sudden encystment of amoebae
in the gut, as has been claimed. When it is understood that encystment
is not a simple process, but depends first of all on the production of pre-
cystic amoebae, it is difficult to see how this can be brought about in a
short time. It is possible that, if precystic amoebae are present at the
time of administration of emetine, this might accelerate their encystment.
There is no evidence that the cases which resist emetine do so on
account of the presence of cysts. Certain resistant cases, as noted by
the writer and O'Connor (1917), appear never to pass cysts, free active
amoebae alone being found in the stools whenever these are examined.
AMCEB^ CULTIVATED FROM FAECES— COPROZOIC AMCEB^.
The fact that amoebae develop in faeces after they have been passed,
and on the surface of agar plates inoculated with f^ces, has misled ob-
servers into believing that they had been able to cultivate the intestinal
amoebae. Kartulis (1891) claimed to have cultivated amoebae of the
human intestine, but it was pointed out by Celli and Fiocca (1894, 1895),
and Casagrandi and Barbagallo (1895, 1897), that the cultures contained
I. 17
258 FAMILY: AMCEBID^
only free-living non-parasitic forms. Miisgrave and Clegg (1904, 1906)
made extensive observations in Manila. They thought they had culti-
vated the amoebae of the human intestine and isolated them from the
water supply. They also stated that it was possible to produce dysentery
in monkeys by injecting cultures of these amoebae. The w^riter (1907),
using the same medium, attempted to obtain cultures of E. muris of mice
and E. coli of man, but succeeded in growing only free-living amoebae.
He pointed out that the amoebae obtained in culture by Musgrave and
Clegg in no way resembled E. coli, which they claimed to have cultivated.
It appeared that what actually happened was that cysts of free-living
amoebae were constantly passing through the intestine of man and animals,
and that it was these which were responsible for the cultures obtained.
Walker and Sellards (1913) again investigated the claims made by Mus-
grave and Clegg, and showed that the writer's explanation was undoubtedly
correct. By causing individuals to ingest the cysts of the amoebae which
appeared on agar plates, they were able to isolate the same amoebae a
few days later by smearing agar plates with the faeces. Fantham (1911a)
gave a description of E. coli based entirely on agar cultures of free-living
amoebae.
The amoebae which appear on agar plates after smearing them with
the faeces of man or animals are usually small forms which are rarely
more than 10 to 20 microns in diameter. They are actively amoeboid,
and live by ingestion of bacteria which grow at the same time. It is
necessary, if good cultures are to be obtained, to have a medium which
is not too rich in nutrient material, so that the bacteria do not overgrow
the amoebae. The medium used by Musgrave and Clegg is very suitable,
and consists of agar 20 grams, sodium chloride 0-5 gram, extract of beef
(Liebig) 0-5 gram, water 1 litre. The solution is then made 1-5 per cent,
alkaline to phenolphthalein. About 10 c.c. of the medium is warmed till
liquid, and poured into a Petri dish, where it is allowed to set. On this
medium with a low power of the microscope the amoebae may be seen
spreading across the surface beyond the edge of the bacterial growth.
Multiplication is rapid at laboratory temperature, and in a few days a
plate will contain thousands of amoebae. In the central and older parts
of the culture the amoebae encyst in spherical cysts. Subculture is readily
effected by transferring small portions to fresh plates. It is possible, by
using a finely-drawn-out glass filament with a rounded bead at the end,
under a low power of the microscope, to transfer a single isolated amoeba
to a new plate, and thus to obtain a perfectly pure culture of a single
species.
It has already been mentioned that cultures of amoebae often appear
in stale stools, and care must be taken not to confuse them with Endolimax
STATISTICS OF HUMAN INTESTINAL AM(EB^
259
nana, to which they bear some resemblance. Though these cultural
amoebse resemble one another superficially, they belong to several distinct
species. They can be differentiated fropi one another by a careful study
of the nuclear division and the encysted stage. The amoebse isolated in
this way are usually species of HartmayineUa and Dimastig amoeba. The
shelled form Chlamydophnjs stercorea and its allies may also occur on agar
plate cultures of faeces of animals.
STATISTICS OF INTESTINAL AMCEB^ OF MAN.
As in the case of most of the parasitic infections of the intestine, the
incidence of amoebic infections in any community is directly related to
the efficiency or otherwise of the sanitary arrangements. Where there
is every possibility of food and water becoming contaminated with fsecal
material, either directly, or indirectly by the agency of flies, there the
percentage of individuals harbouring intestinal Protozoa will be high.
It was shown by the writer and O'Connor (1917) that flies in Egypt are
constantly feeding on fsecal material, and that the cysts of intestinal
Protozoa, and even the unencysted forms, may quickly pass undamaged
through the intestine, and in this way be deposited on food.
It is probably correct to state that all the intestinal amcebde of man
are world-wide in their distribution, the number of individuals actually
infected varying with the locality. In tropical countries, where sanita-
tion is generally bad, the incidence is high, while in England it is relatively
low, though even here the figures are higher than might be expected.
It has already been pointed out that the percentage of infections resulting
from a single examination of each case is fallacious, and that repeated
examinations usually yield a figure which is at least three times as great
as that obtained by a single examination. In Alexandria and London
during the war the writer and O'Connor (1917) found the following
percentages of infection amongst different groups of men:
5-
•^
1 .
li
328
11
M
o
556
1.-
P
961
Gabarri
Prison
{British).
Iladra Prisi
(Natives).
11
87^
^1
CO g
-S.g
Total examined
1,979
168
524
48
Entamosha histolytica . .
5-3
6-4
10-8
3-2
1-8
13-7
11-5
41
Entamceba coli
20-0
31-7
39-0
10-4
120
48-6
20-7
18-7
Entamoeba sp. (?)
1-3
1-8
—
2-0
17-2
0-57
M
—
lodamoeba biitschlii
3-0
2-0
5-2
()-3
—
14-8
7-0
4-1
Endolimax nana
0-5
—
1-0
3-0
12-0
"
~
~
260 FAMILY: PARAMCEBID^
The figures given for Hadra Prison show the results obtained by
single examinations of natives of the country. In the case of E. nana,
the low figure is explained by the fact that the examinations were largely
made before it was recognized that E. nana was a parasitic amoeba.
During the war a large number of examinations were made of healthy
persons in the British Isles, and though isolated cases of E. histolytica
infection in individuals who had never left the country had already been
recorded by Marshall, D. G. (1912), the writer (1916) and others, it was
Yorke and his collaborators (1917) who first showed that amoebic infections
were quite common amongst the indigenous population. Dobell (1921)
has examined the records of several observers, and after allowing for
the errors of the single examination concludes that the percentages
of infections to be found amongst the artisan population are as follows :
E. histolytica, 7 to 10; E. coli, 36 to 54; E. nana, 9 to 13; /. biitschlii,
0-5 to 0-75.
Boeck (1921) has published the results of examination of eighty-three
industrial school children in America. Each case was examined, on an
average, 5*3 times. He gives the following figures of percentages: E. histo-
lytica, 10-8; E. coli, 49-3; E. nana, 6-0; /. biitschlii, 1*2. Similar records
have been published from other parts of the world.
2. Family: PARAMCEBiDiE Poche, 1913.
This family includes the single genus Paratnosba, which was created
by Schaudinn (1896) for a marine amoeba, Paramceba eilhardi, which
possessed, in addition to its nucleus, an accessory body (Nebenkorper).
Both the nucleus and the " Nebenkorper " divided during division of the
amoeba. Janicki (1912) pointed out that two amoebae {A. pigmentifera
and A. chcBtognathi) which Grassi (1882) had discovered in the body cavity
of the small marine worms of the genera Spadilla and Sagitta belonged to
this genus (Fig. 119). During division of the amoeba the nucleus divides
by mitosis, while the " Nebenkorper " divides by simple elongation and
constriction. Small elongate flagellates, each with a single flagellum, are
produced. These, after multiplying by division, conjugate and give rise
to zygotes which become the amoebae.
3. Family: dimastigamcebidte.
This family includes amoebae which are able, under certain conditions,
to develop flagella and behave as flagellates. When they occur in faeces
or are cultivated on the surface of agar plates they live and repro-
duce as amoebae, but if brought into liquid media they quickly grow
flagella and swim about for some hours, after which the flagella are lost
and the amoeboid phase is resumed. One of these amoebae was isolated
FAMILY: DIMASTIGAMCEBIDvE 261
from human faeces by Schardinger (1899), who named it Amoeba gruberi.
Wasielewski and Hirschfeld (1910) showed that an amoeba which they
called A. Umax at certain stages developed two fiagella. A similar
observation was made by AlexeiefE (1912^) on an amoeba referred to as
A. punctata (Dangeard). Martin and Lewin (1914) showed that a
soil amoeba, which they called Vahlkampfia soli, readily developed two
flagella when an agar plate containing the encysted forms was flooded
with tap water containing 025 per cent. NaCl and 0-05 per cent. MgS04.
Wherry (1913), working with a similar amoeba, could produce the trans-
^'^i0^^
\
...f^T-.
M '--faK
I ■■■■ -^
.' ■^■^ ,^^ "
-•■■'•; ":■'•. '•':■•"'
m0
y ■:■
\ p;i^#^^::
'om |i-,'o.
o ' ■ ■
M' ■
"''"'\';-'\'-
v.'« .' '? ' , . '
■■■ ■:.• * \ V ■
n •::•
X^^Si>^^-J
J-.. '.;•; TTiuiv!^ '^-f !••;'•
v^
1 ■^^"9>^.a:;"1?^^' ■' , .* ...i
Sr5»:.Sr>-^-'
4
Fig. 119. — Parasitic Amceb.e of the Genus Paramceba. (After Janicki, 1912.)
1. Free form of P. pigmentifera with two nuclei (X 1,800).
2. Dividing form of P. chcstognathi ( x 2,700).
3. Flagellate stage of P. pigmentifera ( X 3,650).
4. Dividing flagellate form of P. pigmentifera (x 3,650).
formation by merely diluting a loopful of liquid egg-medium culture of
the amoebse with several loopfuls of distilled water, observations which
were confirmed by Wilson (1916). In all these cases the flagellates had
two flagella of approximately equal length, and traceable to two blepharo-
plasts in the cytoplasm or to the nuclear membrane. It is probable that
all these amoebae belong to the species which was first isolated from human
faeces by Schardinger (1899), who called it A. gruberi. On account of
its flagellate stage, it was placed in a new genus, Noegleria, by Alexeieff
(1912), who later (1912a) came to the conclusion that it belonged to the
262 FAMILY: DIMASTIGAMCEBIDiE
genus Dimastigamceba founded by Bloclimann (1894), its correct name
being Dimastigamceba gruberi. According to Boeck and Stiles (1923),
the name Dimastigamceba of Blochmann (1894, 1895) refers to another
amoeba, as also Alexeieff's name Ncegleria. They adopt the name Wasie-
lewskia, proposed by Hartmann and Schiissler (1913), and employed by
Zulueta (1917), for the form described here, which they refer to as Wasie-
leivskia gruberi. As, however, there is little doubt that the organism
described by Whitmore (19116) as Trimastigamoeba philippinensis is the
same amceba, his generic name has priority over all names except Dima-
stigamceba, which nevertheless appears to be the correct name for the
genus.
It is evident that Dimastigamoeha gruheri might be classed with the
Mastigophora, instead of with the Rhizopoda. It illustrates very clearly
the close relationship of the two groups,
Dimastigamceba gruberi (Schardinger, 1899). — This amoeba, which
occurs commonly as a coprozoic organism in faeces of human beings and
animals, has been studied by the writer in cultures made from dirty water
and old faeces. Both on agar plates and in liquid media the organism
remains in its amoeboid phase, but if sudden changes are made the
flagellate phase appears in two to three hours, and lasts up to twenty-four
hours or longer than this under exceptional circumstances. Thus, if some
of the growth on agar plates is scraped off and mixed with two or three
drops of tap water, in two or three hours, according to the temperature,
enormous numbers of flagellates are developed. In twenty-four hours
they have all reverted to the amoeboid form again. A further addition of
tap water brings about the reappearance of the flagellate forms. It is
quite easy to watch under the microscope the transformation of one of the
amoebae into the flagellate. The amoeba becomes rounded, and two
flagella commence to grow from the surface of the body. They can be
seen to be connected with two small granules, the blepharoplasts, which
lie close together on the surface of the body. The nucleus, which is
readily seen on account of its large retractile karyosome, may remain near
this point, or it may be at some other part of the amoeba, or its position
may be constantly changing. It has not been possible to observe the
origin of the blepharoplasts from the nucleus or its karyosome. They are
first detected as such after the flagella have commenced to form. The
flagella gradually increase in length, and become more violent in their
action. The organism now elongates and becomes pear-shaped, the more
pointed end being the flagellar end. The nucleus, if it has not remained
near the blepharoplasts during the growth of the flagella, approaches
this end of the body. At this stage the typical flagellate is formed. The
posterior region of the body is swollen, the anterior being narrow. At one
DIMASTIGAMCEBA GRUBERI 263
side of the anterior end can, not infrequently, be made out a slight de-
pression, having the appearance of a small cytostome. The two blepharo-
plasts, which were first clearly described by Alexeieff (19125f), lie one in
front of the other on the surface of the cytoplasm within this depression,
and the flagella arising from them pass through the opening of the depres-
sion. In many individuals a short fibre can be traced from each blepharo-
plast as far as the nucleus, where it ends in a small thickening or elevation
of the nuclear membrane. In some forms the nucleus may be near the
centre of the body, or even at the posterior end, and in such cases it may
or may not be possible to trace fibres from the blepharoplasts to the
nucleus. The nucleus has a large central karyosome, which is connected
with the nuclear membrane by radiating filaments. On the inner surface
of the membrane are granules of chromatin. The nucleus of the amoeboid
form is spherical, but in the flagellate phase, in which a connection between
the blepharoplasts and nuclear membrane can be made out, the latter
structure may be slightly drawn out towards the blepharoplasts. A con-
tractile vacuole is present. In the flagellated forms it is behind the nucleus
in the thicker portions of the body. The flagellates, which are typically
pear-shaped, vary in length from 10 to 30 microns. The relation of the
blepharoplasts to the nucleus are of considerable interest, Alexeieff
(1912^) stated that when the flagellate phase was to appear, two granules
separated from the karyosome and migrated to the surface of the body,
retaining in some forms a connection with the karyosome. Wilson (1916)
also described the separation from the karyosome of a granule, which
migrated into the cytoplasm and became the blepharoplasts. The writer,
after examining many thousands of amoebae at all stages of flagellum
formation, has failed entirely to trace the origin of the blepharoplasts from
the karyosome. Appearances suggestive of such an origin are occasionally
seen, but they are too inconstant to justify the conclusions that the
blepharoplasts arise in this manner.
On agar plates there occur amoebse with one, two, or four nuclei
(Fig. 61). Those with one nucleus develop, as a rule, a single pair of
flagella (Fig. 1 20, 1-7) ; those with two nuclei two pairs (Fig. 120, 13 and 14) ;
and those with four nuclei four pairs (Fig. 120, 15). It is evident, therefore,
that each nucleus has associated with it a pair of blepharoplasts. If an
amoeba has a nucleus in process of division, it will still develop flagella,
but in this case two pairs appear, as in the forms with two nuclei (Fig. 120,
II and 12). It seems evident that with nuclear division the blepharoplasts
have divided. In some cases an amoeba, with a single nucleus showing no
sign of division, will develop two pairs of flagella (Fig. 120, 8-10). It
would seem justifiable to conclude that the single pair of blepharoplasts
has divided preparatory to nuclear division, which has not as yet visibly
264
FAMILY: DIMASTIGAMCEBIDvE
. »r
/ il
ts
-1^
#
#
^^i
.%
• ^
/f
/?
Xf
Fig. 120. — ^Flagellate Fokms of Dimastigamoeba gruberi developed a Few Hours
AFTER PLACING THE Am(EB^ IN TaP WATER ( X ca. 1,400). (ORIGINAL.)
[For description see opposite page.
DIMA8TIGAM(EBA GRUBERI 265
commenced. This would be in agreement with what is known to occur
in other flagellates, in which the first stage of division of the organism
is division of the blepharoplasts. These appearances suggest that the
blepharoplasts are present in the cytoplasm even during the amoeboid
phase of the organism. They are so minute that, unless their connection
with the flagella can be detected, it is impossible to distinguish them from
other granules which occur in the cytoplasm. It seems probable that in
the amoeboid phase they are adjacent to, or actually upon, the nuclear
membrane, and that when flagella are to be formed they move towards
the surface of the body, retaining in many cases a connection with the
nuclear membrane. There seems to be no real evidence that they are
derived from the karyosome of the nucleus. Exceptionally only one
flagellum is developed by uninucleated amoebse, and three by binucleated
or even uninucleated amoebse.
The amoebse themselves, judging from cultures commenced from a
single individual, vary in size from about 5 to 20 microns (Figs. 61 and 121).
They are fairly actively motile, and usually form blunt pseudopodia, but
sometimes fine hair-like or radiating pseudopodia are produced, giving the
organism the appearance of a Heliozoan. The nucleus contains a large
central karyosome connected with the nuclear membrane by radiating
septa which traverse the clear space. On the inner surface of the nuclear
membrane, and just internal to it, is a layer of granules of chromatin. The
amoeba3 are difficult to recognize from others of the genus Harlmannella,
unless the stages of nuclear division, the cysts, or the production of
flagellates can be observed. The nuclear division has been described
above (p. 103). The cysts are spherical structures, which in uninucleated
forms vary in size from 5 to 12 microns. Their most characteristic feature
is the presence of a number of pores in the cyst wall, which is composed of
a double membrane (Fig. 121). On agar plates not only do the uninucleated
amoebse encyst, but also the multinucleated forms, which produce corre-
spondingly larger cysts with an increased number of pores. Cysts from
12 to 18 microns in diameter have usually two nuclei, while larger ones
have more. Thus a cyst 31 microns in diameter had three nuclei and
twenty to thirty pores, while another was 21 microns in diameter, had
six nuclei, and fifteen to twenty pores. The cytoplasm of encysted forms
contains a number of conspicuous refractile bodies which may be larger
than the nuclei. They stain black with iron hsematoxylin, and are probably
of a volutin nature.
1-7. Various forms of biflagcllate type.
8-10. Forms with single nucleus, four flagella, and tAvo pairs of blepharoplasts.
11-12. Forms with dividing nuclei, four flagella, and two pairs of blepharoplasts.
13-14. Forms with two nuclei, four flagella, and two pairs of blepharoplasts.
15. Form with four nuclei, eight flagella, and corresponding blepharoplasts.
266
FAMILY: EHIZOMASTIGIDiE
Whitmore (19116), working in the Philippines with cultures of amoebae
isolated from water on agar plates, noted that a certain amoeba developed
a stage with three flagella. His figures also show forms with two and four
flagella. He gave it the name Tritnastigamceba philippinensis. The
amoeboid phase was 16 to 18 microns in diameter, while the cyst was
oval, and measured 13 to 14 microns by 8 to 12. In the flagellate phase,
with flagella slightly shorter than the body, the organism was elongated,
and measured 16 to 22 microns by 6*5 to 8 microns. As Dimastigamceba
gruberi may sometimes develop three, four, or more flagella, it is clear
Fig. 121.^ — Amceboid Phase of Dimastigamceba gruberi off Agar Plate ( x 3,000).
(Original.)
1. Amceba showing nucleus with large central karyosome and peripheral granules and contractile
vacuole (cv.).
2. Encysted form showing double membrane of cyiist wall and three pores.
3. Encysted form in which inner membrane has separated from the outer membrane excejit at the
pores.
that Whitmore was actually observing this species, his name Trimastig-
amceba becoming a synonym. From the work of Bunting (1922) it appears
that flagellates of the genus Tetramitus may also have an amoeboid phase
(see p. 310).
4. Family: RHizoMASTiGiDyE Calkins, 1902.
This family includes certain free-living amoeba?, and possibly some
parasitic forms which possess a single flagellum. They are usually classed
MASTIGAMCEBA—MASTIGELLA— MASTIGINA 267
with the Rhizopoda, and not with the Mastigophora, because they live
mostly as amoebae and crawl about by means of pseudopodia, instead of
swimming by means of their flagella. Unlike the members of the family
DimastigamoebidsB, they retain the flagellum throughout the amoeboid
phase. There are three genera. The genus Mastigamoeha includes large
amoebae with a flagellum as long as, or longer than, the body. The
axoneme of the flagellum arises from the nuclear membrane. The genus
Mastigella includes similar forms, in which the flagellum is unconnected
with the nucleus. The genus Mastigina comprises amoebae which have
a short flagellum, the axoneme of which arises from the nuclear membrane.
The majority of these forms are free living, and for one of these Goldschmidt
(1907) described a complicated life-cycle, which, however, has not received
confirmation. A few parasitic forms have been described. Frenzel (1892)
described as Tricholimax hylce a flagellated amoeba from the intestine of
tadpoles of the genus Hijla in the Argentine (Fig. 73). Goldschmidt (1907)
placed it in the genus Mastigina. Collin (1913) studied this organism,
Mastigina hylcB, in tadpoles of newts and Bufo calamita in Europe.
Spherical cysts, 25 to 28 microns in diameter, were produced, and these
contained two or four nuclei. What was possibly the same organism
was seen by Hoare, working in the writer's laboratory, in the intestine
of Triton vulgaris in England. Becker (1925) has seen it in tadpoles of
Rana clamata and R. cateshiana in America. He notes that the single
short inactive flagellum arises from a blepharoplast situated on the nuclear
membrane, and at one end of a cap-like structure which partially covers
the nucleus. From the whole surface of the cap radiating fibres pass into
the cytoplasm. From the blepharoplast a deeply staining curved rod
passes into the cytoplasm. It is homologized with the basal fibre (rhizo-
style) of the membrane of Trichomonas. Becker sees in the structure of
this organism a ground plan of the morphology of such flagellates as
Trichomonas, Chilomastix, and even Giardia.
Another parasitic form is that described by Liebetanz (1910) as
Mastigamoeha bovis from the rumen of cattle. It measures about
25 microns in longest diameter, and is provided with a flagellum about
twice as long as the body. The cytoplasm is dift'erentiated into a well-
marked ectoplasm and endoplasm, and there is a large central nucleus.
268 CLASS: MASTIGOPHORA
II. CLASS: MASTIGOPHORA Diesing, 1865.
CLASSIFICATION.
CLASS: MASTIGOPHORA
SUB-CLASS: Phytomastigina
Order: CHRYSOMONADIDA
CRYPTOMONADIDA
DINOFLAGELLATA
EUGLENOIDIDA
PHYTOMONADIDA
SUB-CLASS : Zoomastigina
Monozoic Forms
Order: PROTOMONADIDA
Sub-Order: Eumonadea
Family: monadid^
TRYPANOSOMID^
BODONID^
,, PROWAZEKELLID^
EMBADOMONADID^
Family : CHILOMASTIGID^
CERCOMONADIDiE
CRYPTOBIID.^
TRICHOMONADID^
DINENYMPHID^
Sub-Order: Craspedomonadt
Order: HYPERMASTIGIDA
„ CYSTOFLAGELLATA
Diplozoic Forms
Order : DIPLOMONADIDA
Genus : Hexamita
,, Giardia
Trepomonas
Polyzoic Forms
Order: POLYMONADIDA
Family : CALONYMPHID^
The Protozoa which are included in the class Mastigophora ( = Flagellata
Cohn, 1853) are commonly known as flagellates, and comprise a very varied
assemblage of organisms which have one feature in common — namely, the
possession of one or more flagella. In the case of Protozoa belonging to
other classes, flagella may be temporarily present at certain stages of
development, as, for instance, in the case of the microgametes of coccidia;
but in the Mastigophora the flagella are present during the greater part of
the life of the individual, and occur in the active, fully-grown, motile stage
of the organisms. The majority of the Mastigophora are free-swimming
creatures which move about in liquid media by the lashings of their flagella.
Some of them resemble amoebae more than flagellates, for, in addition
to swimming, they may crawl over surfaces by means of pseudopodia.
Others, again, secrete filaments or stalks, by means of which they are
attached to objects. In some cases there is developed at the end of the
filament a cup-like receptacle (lorica) in which the flagellate is lodged
(Fig. 18). In other cases, groups of flagellates are held together by a
common gelatinous matrix, the whole colony moving about as a single
unit as a result of the joint action of the flagella of the several individual
flagellates.
In what may be regarded as the most primitive forms the body con-
sists of a portion of cytoplasm showing no differentiation into ectoplasm
GENERAL ORGANIZATION 269
or endoplasm. Superficially, it is covered by an exceedingly delicate
membrane or periplast, which does not prevent amoeboid movements,
formation of pseudopodia, or the ingestion of food particles at any point
of the body surface. There is a single nucleus, while one or more basal
granules or blepharoplasts, which lie upon the nuclear membrane or
free in the cytoplasm, are also present. From each blepharoplast there
arises an axoneme, which may be traced to the surface of the body, and
thence into a flagellum, which consists of the axoneme covered by a sheath
formed by the periplast. In the non-parasitic forms contractile vacuoles
are present. Flagellates of this relatively simple type are usually free-
swimming organisms, but some of them are able to attach themselves
temporarily to objects by a process like a pseudopodium or, with or with-
out losing their fiagella, to crawl about like amoebae for a time. The fila-
ments and cup-like receptacles mentioned above are formed as secretions
from the surface of the body, as also is the gelatinous matrix which binds
together the colonial forms. These structures are not actually parts of
the organism, and are not to be regarded as modifications of the superficial
layer of the cytoplasm. The more highly developed flagellates may be
considered to have arisen from the simpler forms by changes in the peri-
plast or by the development of internal structures. With a thicker
periplast there is still the possibility of change in body form, though this
is limited, while the power of ingesting food at any part of the body
surface is lost. A definite cytostome is developed, usually near the origin
of the fiagella. With further development of the periplast, the body
becomes rigid and a definite body shape is acquired. In many cases this
thickened rigid periplast is of a high degree of complexity, and may be
elaborately marked. The fiagella, when more than one are present,
usually arise near together at the anterior end of the b'ody. Sometimes,
however, they are spread over a wider area. In certain forms some of the
fiagella arise from the anterior end of the body and one or two from the
posterior end. In the case of the latter the axonemes may pass directly
backwards through the cytoplasm from their respective blepharoplasts
{Giardia, Hexamita), or they may pass forwards through the cytoplasm
to the anterior end of the body and, turning backwards, pass over the
surface of the body to enter the fiagella when they reach the posterior end.
When an axoneme passes over the surface of the body, the periplast may
be raised into a ridge or membrane (undulating membrane), along the edge
of which the axoneme passes (Trichomonas, Trypanosoma). In certain
flagellates the periplast at the anterior end of the body becomes raised
into a collar or cuff, which surrounds the fiagella (Choanoflagellata, or
collared flagellates).
In addition to the complexities in organization which are the result of
270 COPROZOIC MASTIGOPHORA
elaborations of tlie superficial layer of the body, there occur others which
arise from the formation of internal structures. In Trichomonas there is
developed an organ called the axostyle, which is traceable through the
body from the region of the blepharoplasts to the posterior end, through
which it projects as a pointed rod. It is supposed by some to be a modified
axoneme (see p. 42). Similarly, in this flagellate, which possesses an
undulating membrane, a stiff fibre is developed along the base of the
membrane. In Chilomastix the edge of the cytostomal groove is rendered
rigid by two fibres which pass along its margins. The blepharoplast may
be a simple granule in which the axoneme originates, or associated with it
there may be another body of variable size and shape — the parabasal.
The parabasal and the blepharoplast may be intimately connected, as in
the trypanosomes and allied flagellates, to form a compound organ — the
kinetoplast.
The majority of Mastigophora are uninucleated, and possess one or,
at most, a small number of flagella with a corresponding number of ble-
pharoplasts, which are usually closely grouped together, so that the indi-
vidual blepharoplasts may be difficult to detect. The order Hypermas-
tigida, however, includes flagellates which, though uninucleated, possess
a large number of flagella and blepharoplasts.
The members of the order Diplomonadida {Giardia and Hexamita) have
two nuclei and eight flagella and blepharoplasts, while the members of the
order Polymonadida are multinucleate, and have a large number of flagella
and blepharoplasts.
Reproduction amongst the Mastigophora is usually by binary fission,
the division being a longitudinal one, which commences as a rule at the
flagellated end of the organism after the blepharoplast and nucleus have
divided. This division may take place in the free-swimming condition,
or after the flagellate has lost its flagella and become an amoeboid or
rounded form, or in some cases after encystment has taken place. Cyst
formation as a means of protection against desiccation commonly occurs.
COPROZOIC MASTIGOPHORA.
As in the case of free-living amoebae, the encysted forms of many free-
living flagellates are able to withstand the action of the digestive fluids
of an animal's intestine. They pass unchanged through the intestine,
and liberate the flagellates in the faeces. There are thus coprozoic flagel-
lates as there are coprozoic amoebae. Some flagellates which live in
stagnant water and infusions are able to live in the intestine, especially
of cold-blooded animals. It is possible that the Hexamita of the frog's
intestine is identical with a similar form which lives in water. Berliner
INVASION OF BLOOD BY MASTIGOPHORA 271
(1909) noted that Copromonas major occurred coprozoically in lizard's
faeces, and that occasionally it occurred in the unencysted stage in the
lizard's intestine. Some flagellates which are more truly parasitic, such
as Trichomonas, are not only readily culturable in artificial media, but may
survive for long periods in faeces outside the body, while others, such as
Giardia, quickly die after leaving the body. Other forms, such as Bodo
and Cerco7nonas, rarely if ever occur in the intestine in any but the en-
cysted stages, but they are the commonest forms to develop coprozoically
in stale faeces.
INVASION OF BLOOD-STREAM BY INTESTINAL MASTIGOPHORA.
Between the forms which are more specially adapted to life in the
intestine, like Giardia and Trichomonas, and the true parasitic flagellates
belonging to the Trypanosomidae and Cryptobiidae various gradations
occur. Several observers have found that intestinal flagellates may
occasionally invade the blood-stream. Danilewsky (1889) noted that
the intestinal Hexamita sometimes invaded the blood vessels of the
edible frog and tortoise. Labbe (1894), and more recently Ponselle (1919),
made a similar observation in the case of the frog. The latter was able
to produce a blood infection of Rana temporaria by inoculating blood
containing Hexamita from an infected edible frog. Labbe (1894) also
stated that he had seen a Bodo and Hexamita in the blood of a lizard
{Lacerta sp.), while Hexamita has been seen in the blood of the toad
{Bufo calamita) in large numbers by Lavier and Galliard (1925). Lan-
franchi (1908) saw Trichomonas in the blood of a pigeon. Gonder (19106)
observed Giardia in the blood of a falcon (Elamus coeruleus) which had
been shot. It is possible that in this case the blood was contaminated
from a wounded intestine. Martoglio (1917) saw a Tetratrichomonas in the
blood of a fowl, while Chatton (1918a) observed a Eutrichomastix in the
blood of the gecko {Tarentola mauritanica) . Reichenow (1918) observed
the same flagellate in the blood of Lacerta muralis and L. viridis. He
noted that the mites which fed on the lizards also became infected, and
that young lizards were able to acquire an intestinal infection by eating
infected mites. During the examination of the blood of animals which
had died in the Zoological Gardens in London, Plimmer (1912a) on
several occasions found intestinal flagellates in the blood-films. He
observed Hexamita in the blood of tortoises {Cyclemys trifasciata, Cistudo
Carolina, Testudo angulata), and Trichomonas in the blood of snakes
(Coluber leopardinus, Naia tripudians, Heterodon simus, Python sebce).
It is possible that in some of these cases the flagellates appeared in the
blood-films as a result of damage to the intestine at the post-mortem
examination. Sangiorgi (1922) states that he observed a Trichomonas
272 CONTAMINATION OF FILMS BY MASTIGOPHORA
in the heart blood of a dead mouse, and considered the flagellate had passed
from the intestine into the blood-stream; while Knowles, Napier, and Das
Gupta (1923) saw the flagellate in the liver and spleen of a rat during the
course of kala-azar investigations. In the case of human beings, the
writer (1920) found that the whole mucosa of the large intestine of a
case was invaded by Trichomonas, while Pentimalli (1923) saw the same
organism in the blood of a patient on two occasions at ten hours'
interval. Several instances of the occurrence of flagellates of the lepto-
monas type in the blood and intestine of lizards are mentioned below. In
the majority of these cases the invasion of the blood by intestinal flagel-
lates was discovered post-mortem, so that it is not improbable that it had
occurred either after or shortly before the death of the animal. In other
cases the animals were evidently ill and in such a condition that the
natural resistance to such invasion may have been absent. The fact,
however, that such an invasion may take place is some indication of the
possibility of intestinal flagellates acquiring the habits of blood parasites.
FLAGELLATES WHICH MAY CONTAMINATE BLOOD AND ORGAN
SMEARS.
In this place may be mentioned a number of flagellates which have
been encountered in smears made from the blood and organs of various
animals. These forms have been regarded as parasites, but this is more
than doubtful, for a source of error which arises from time to time in blood
work has not been excluded. The distilled water used in laboratories
for E-omanowsky staining may become contaminated with free-living
flagellates in the bottle or even in the pipette used in the manipulations.
When the water is added to the film, either for the dehsemoglobinizing of
a thick film or for the dilution of the alcoholic stain on the slide, the
flagellates adhere to the film, and are found as blue-staining bodies with red
nuclei and one or more flagella according to the nature of the flagellate
in the water. The assumption that the flagellates occur in the blood is
easily made if their possible source is not recognized. Henry (1917) drew
attention to this fallacy in connection with Dymond's supposed h?emogre-
garine of trench fever. The writer has encountered this fallacy on several
occasions. In one case in particular he was asked to look at a remarkable
flagellate in a malarial blood-film. The organism appeared with a blue
vacuolated cytoplasm with red nucleus and flagellum. Red-staining
bacteria were also present. The possible origin of these was recognized,
and an examination of the distilled water which had been used for diluting
the Leishman stain revealed their presence.
Franchini (1913) in Italy described a flagellate from the blood and liver
of a patient who had been in Brazil. The organism was also examined and
FALLACIES RESULTING FROM CONTAMINATION 273
reported upon by Brumpt (19136). In the films it occurred as blue-staining
cytoplasmic bodies, sometimes pigmented and with one or two red-staining
masses. In two cases short flagella were noted. Furthermore, encapsuied
forms described as cysts were found. Brumpt noted that the films
contained the structures described by Franchini, as well as many bacteria,
some of which were actually within the cytoplasm of the flagellates.
The organism was placed in a new genus by Franchini as Hcemocystozoon
braziliense. The presence of encysted forms and the occurrence of
bacteria within the flagellates, as well as in other parts of the films, are quite
inconsistent with the assumption that the flagellate originated from the
Fig. 122. — Trypanopsis maligmis, as seen in Dried Smears of the Liver stained
BY Eomanowsky Stain ( x ca. 4,000). (After Leger, M., 1920.)
One form appears to be ingested by a leucocyte, but the association is probably accidental.
Similar forms were found in blood-films.
blood. The use of a distilled water infected with flagellates and bacteria
could easily give rise to the appearances described, and this seems the
probable explanation of their origin. Franchini attempted culture from
the blood in N.N.N, medium, and no growth was obtained, a further
confirmation of the view expressed here of their extraneous origin.
In the same category probably must be placed the flagellates described
by Leger, M. (1920), from a fatal case of pyrexia in a human being in French
Guiana. As figured by Leger, the organism appears as elongate flagellates
with single flagellum, rounded forms with one to three flagella, and rounded
forms without flagella (Fig. 122). Two chromatin masses were present,
and the flagellum arose from one of them. In some of the rounded, non-
i. 18
274 CLASS: MASTIGOPHORA
flagellate forms numerous chromatin bodies occurred. The organism was
never plentiful in the films. It was named Trypanopsis malignus by its dis-
coverer. The figures resemble very closely the forms which appear in films
as described above, and the writer feels that the possibility of the flagellate
having arisen from a slightly contaminated distilled water was not excluded.
Another fallacy which may occur is the result of contamination of
exposed blood-films or smears by house-flies, which are very commonly
infected with Herpetomonas muscarmn. The flagellates are frequently
passed in large numbers in the fseces of flies, and such faeces deposited on a
film may be smeared over it by the fly itself or in some other way. When
stained, the presence of flagellates in the film will be liable to cause
confusion.
When an animal which has died is opened for examination, smears
made from the liver, spleen, or other organs are very readily contaminated
with the intestinal contents if the intestine has been opened even very
slightly. Yeasts or even flagellates may thus contaminate the smears and
lead to a wrong diagnosis. In practically all these cases it will be found
that, in addition to the flagellates, the films contain a varied assemblage of
bacteria, the presence of which should always give rise to suspicion. It
is a common practice to open up animals which have been shot with the
object of making films from the heart-blood and organs. A slight wound-
ing of the intestine has often led to the passage of intestinal contents
into the peritoneal cavity, and consequent contamination of blood-films.
DIVISION OF MASTIGOPHORA INTO SUB-CLASSES AND ORDERS.
Certain Mastigophora resemble plants in that they are provided with
chromatophores containing chlorophyll, by means of which they lead a
holophytic existence. They may secrete capsules composed of cellulose,
while many of them possess red pigmented stigmata. These forms,
which are very closely allied to the unicellular algte, have been placed
by Doflein (1916) in the sub-class Phytomastigina, to distinguish
them from the Zoomastigina, which includes the flagellates which
have a holozoic method of nutrition, and are evidently animal in nature.
The latter ingest, solid food at all parts of the body surface like amoebae by
means of pseudopodia or through a special opening, the cytostome, or
they absorb by osmosis only preformed proteid matter in solution.
The members of the sub-class Phytomastigina are mostly free-living
organisms which in many cases are closely related to the algae. Many of
them possess chlorophyll and have a holophytic mode of life. Reproduc-
tion is by binary fission, while syngamy, which is either isogamous or
anisogamous, commonly occurs. Certain Euglenoidida are parasitic in
SUB-CLASS: PHYTOMASTIGINA
275
the intestine of tadpoles, while members of the genus Copromonas are
commonly found in stale fa3ces. Following Doflein (1916) the sub-class
Phytomastigina is divided into five orders:
1. Order: CHRYSOMONADIDA.
Simple forms of small size which possess chromatophores mostly
coloured with a brown pigment. There are one or two flagella. Cysts
Fig. 123. — Various Chrysomonadida. (From Oltmann, 1922, after
Various Authors.)
1-2. Chri/siinnilia. ni<1i<ins {x 1,000). 5. Cyst oi Chromulina favicans formed endo-
3. Och'roiiKuias .^uiip/f.rix ca. 400). genously ( x ca. 1,500).
4. Chronmlnid pnsrhcrl {x ca. 1,500). 6. S;/iicri/'pta volvox, a, co\onia.\iorm {x ca.650).
7. Pontos'phoerahaeckeli{x 1,600).
with siliceous walls are formed endogenously within the cytoplasm. The
surface of the body may be limited by a rigid membrane, or such a structure
276
CLASS: MASTIGOPHORA
may be absent, the organism being capable of amoeboid movements. Some
forms develop cup-like loricse in which they live. Numerous individuals
may be held together by a gelatinous matrix to form colonies. The order
(=Chrysomonadina Stein, 1878) includes Chrysamopba, Ochromonas, Chro-
mulina, Pontosphcera, and other genera i^
(Fig. 123). ^\V
2. Order: CRYPTOMONADIDA.
Small forms with two flagella and a
thick, rigid periplast, which gives them a
characteristic ovoid shape. The body is
often flattened, while a longitudinal groove
is frequently present on one surface. This
asymmetry permits of a definite orientation.
Chromatophores of varying colour are usu-
ally present. Included in the order {= Cryp-
tomonadina Stein, 1878) are Cryptomonas,
Chilomonas and other genera (Fig. 124).
Fig. 124. — ^A, Crijj)toriionas ovata; B, Chilomonas
Paramecium (x 1,000). (After Doflein,
1916.)
Chr, Chromatophore; B, blepharoplast ; N, nucleus;
8, oesophagus; Rh, rhizoplast.
Fig. 125. — CeraUumJiirudinella :
Optical Section (Length
100-700 Microns). (From
Doflein, 1916, after Lau-
terborn.)
N, Nucleus ; Rf, equatorial groove with
flagellum; ^gr, long free flagellum.
3. Order: DINOFLAGELLATA Butsciili, 1885.
These organisms, known also as Peridinians, which are mostly marine
forms, have a thick, rigid covering to the body, which is variously shaped.
SUB-CLASS: PHYTOMASTIGINA
277
There are two flagella, one of which usually lies in a groove in the thick
covering of the body. Chromatophores may or may not be present. There
are a large number of genera, of which Ceratium, Gymnodinium, Diplo-
dinium, and Goniodoma are representatives (Figs. 125, 126).
Fig. 126. — Gymnodinium rhomboides (1 and 2), and G. spirale (3) (xca. 1,000).
(From Oltmann, 1922, after Schutt.)
qf. Equatorial groove ; If, longitudinal groove.
4. Order: EUGLENOIDIDA.
Large forms covered with a definite periplast often longitudinally
marked. The shape of the body may be permanent or it may change
according to the rigidity of the periplast. At the anterior end of the
body is a depression, in which the flagellum arises. Sometimes there
are two flagella. In some forms a cytostome leading to an oesophagus
occurs in the anterior depression. There is a characteristic system of
excretory vacuoles, consisting of a reservoir into which discharge one or
more contractile vacuoles. A red pigment spot, the stigma, is often found
at the anterior end of the body, while green chromatophores are fre-
quently seen in the cytoplasm. The order ( = Euglenoidina Biitschli, 1884)
includes well-known genera such as Euglena (Figs. 6 and 128, B), Astasia
(Fig. 127), and Phacus (Fig. 128, A), and the coprozoic Copromonas (Fig. 133).
5. Order: PHYTOMONADIDA.
These forms, which are often considered to be unicellular algae, possess
definite cellulose walls and are devoid of cytostome. There are usually two
flagella, which emerge through a pore in the cell wall. Green chromato-
phores often occur, while some are coloured red by a pigment known
as hsematochrome. Eed-pigmented stigmata are not infrequently present.
Colonial grouping of a varying number of individuals is a common feature,
while there may be a complicated life-history, in which syngamy is
associated with the production of differentiated male and female gametes
278
CLASS: MASTIGOPHORA
as in Volvox (Fig. 129). Included in the order ( = Phytomonadina Bloch-
man, 1895), amongst other genera, are Chlafnydomonas (Fig. 130), Hmmato-
coccus (Fig. 131), Polytoma (Fig. 42), Parapolytoma (Fig. 31), and colonial
forms like Gonium (Fig. 132), Pandorina, and Volvox.
In the sub-class Zoomastigina are found numerous free-living forms as
well as the various parasitic or saprophytic flagellates which occur in man
Fig. 127. — Astasia ienax
(X 650). (After Stein, 1878.)
Two individuals, showing changes
in form due to peristaltic waves
of contraction; each possesses
a nucleus, two flagella, and
oesophagus, at base of which is
a contractile vacuole.
Par
Fig. 128. — Fhacus lotigicandus (A) ( x 650) and
Euglena oxyuris (B) ( x 450). (From Doflein,
1916, after Stein.)
Par, Paramylum; P, pyrenoid; N, nucleus; T', contractile
vacuole; st, stigma.
and animals. The sub-class is usually divided into several orders as
follows: Order Protomonadina, including simple forms with few flagella;
the Polymastigina, more complex forms with several flagella and possibly
other organs; the Hypermastigina, forms which are mostly parasitic in
SUB-CLASS: ZOOMASTIGINA
279
white ants, and which have a very complex structure and large numbers
of flagella ; and the Cystoflagellata, marine flagellates of peculiar organiza-
Fig. 129. — Portion of a Spherical Colony of Volvox globator, in which Sexually
Differentiated Gametes have developed ( x ca. 1,000). (From Lang,
1901, after CiENKOWSKY and BiJTSCHLI.)
S, Male gametes; 0, female gametes.
Fig. 130. — Chlamydomonas angulosa (l-i) and C. longisUgma (5-8), showing Method
OF Multiplication ( x ca. 1,000). (From Oltmann, 1922, after Dill.)
a. Stigma; chr, chromatophores; g, flagella; k\ nucleus; pi/, pyrenoids; v, contractile vacuole.
tion. Doflein separates from the Polymastigina, in the order Distoma-
tina, certain flagellates {Hexamita, Giardia) which have a bilateral
symmetry associated with the presence of two nuclei and two sets of
280
CLASS: MASTIGOPHORA
organs. The three last-named orders are fairly well defined, but there is
more difficulty in connection with the Protomonadina and the Polymasti-
FiG. 131. — Hcematocoecus liluvialis ( x ca. 2,500). (After Reichenow, 1910).
A. Individual from a culture in a special medium, giving rise to forms without hsematochrome,
The nucleus, three pyrenoids, and the stigma, as a dark rod near the right-hand margin,
are clearly visible.
B. Usual form with structure obscured by hamatochrome. S, stigma scarcely visible.
Fig. \^2.—Qonium pectorale : Colony of Sixteen Individuals, each with Two
Flagella (X ca. 480). (From Minchin, 1912, after Stein.)
^ , In surf ace view ; iJ, in side view; A'^, nuclei; cv., contractile vacuole; s<, stigmata.
SUB-CLASS: ZOOMASTIGINA 281
gina. The two merge into one another, and certain forms which are
usually placed in the one order might with equal justification be trans-
ferred to the other. It would seem better, therefore, to consider most
of the flagellates usually included in these two orders as belonging to
one order, Protomonadida, and to reserve an order, Polymonadida, for
the flagellates belonging to the family Calonymphidse, which includes
parasitic forms possessing many nuclei and blepharoplasts from which
arise a large number of flagella.
Hartmann and Chagas (1910a) divide their Protomonadina into two
sub-orders — the Monozoa, including forms in which there is only a single
nucleus and set of organs; and the Diplozoa, those which have a bilateral
symmetry and double set of organs. It seems better, however, as Doflein
has done, to separate the Diplozoic forms in another order entirely, for
which the name Diplomonadida may be employed. The Zoomastigina
can be considered from the point of view of the number of nuclei the
adult forms possess, and this is perhaps the best basis for their primary
subdivision. The majority of forms possess a single nucleus, and these can
be regarded as Monozoic forms; others (Giardia) possess two nuclei, and
are therefore Diplozoic; while others again (Calonymphidse Grassi, 1911)
have many nuclei, and are therefore Polyzoic (Figs. 291, 301).
The sub-class Zoomastigina may, therefore, be subdivided as
follows:
A. Monozoic Forms.
There is a single nucleus and a varying number of flagella and ble-
pharoplasts.
1. Order: PKOTOMONADIDA.— The flagella are few in number (rarely
more than six).
2. Order: HYPEEMASTIGIDA.— The flagella are very numerous.
3. Order: CYSTOFLAGELLATA Haeckel, 1873.— The body is large and
globular, and possesses a peculiar tentacle as well as a single flagellum.
B. Diplozoic Forms.
There are two nuclei, while the flagella, blepharoplasts, and other
structures are similarly duplicated, giving rise to a bilateral symmetry.
4. Order: DIPLOMONADIDA.— With the characters of the Diplozoic
forms.
C. Polyzoic Forms.
There are more than two nuclei and numerous flagella and ble-
pharoplasts.
5. Order: POLYMONADIDA.— With the characters of the Polyzoic
forms.
282
SUB-CLASS: PHYTOMASTIGINA
1. SUB-CLASS: Phytomastigina Doflein, 1916.
The majority of flagellates belonging to this sub-class are free-living
organisms. Certain Euglenoidida of the genus Copromonas commonly
occur in stale faeces, while others are parasitic in the intestine of tadpoles.
Copromonas subtilis Dobell, 1908. — ^This organism, for which Dobell
19086) established the genus, has an elongate body covered by a rigid
Fig. 133. — Copromonas subtilis : A Coprozoic Flagellate from F^ces
(1-6, X 2,600; 7-16, x 4,000). (Original.)
1-2. Typical flagellates. 3-6. Stages in division.
7-15. Successive stages in division of nucleus. The decolorized karyosome appears to have a
central granule which divides. The two halves remain connected by a fibre.
16. Connecting fibre of two halves of dividing central granule.
periplast. It is ovoid in outline and distinctly flattened, and possesses
a cytostome leading to a long, narrow oesophagus. There is a single
flagellum, which arises from the wall of the oesophagus. The nucleus is
central in position, while a blepharoplast lies anterior to it. It is possible
COPKOZOIC EUGLENOIDIDA 283
that the form which was named Copromonas subtilis by Dobell is identical
with Scytomonas pusilla Stein, 1878.
Copromonas subtilis was first described by Dobell (19086) from the
faeces of frogs and toads. Dobell and O'Connor (1921) report its occurrence
once in human fseces, not as a parasite in the freshly passed stool, but as a
coprozoic organism which had evidently developed from cysts after the
stool had been passed. The writer has seen this flagellate in cultures
of pig's fseces. It is an elongate organism with an average length of
15 microns (Fig. 133). Longer forms up to 20 microns and smaller ones
of 4-5 microns also occur. The body is covered with a thick, rigid pellicle,
so that there is little change of shape. The anterior end is somewhat
pointed, and there is here a cytostome leading to an oesophagus which
extends through half the length of the body, the posterior end of which
is rounded. There is a single flagellum, which arises from a blepharoplast
situated in the wall of the oesophagus near the nucleus. During forward
progression the tapering flagellum projects as a rigid filament, the move-
ments being confined to the distal third or half. According to Dobell, near
the blepharoplast is a clear vesicle, the reservoir, into which the contents
of a minute contractile vacuole are periodically discharged. The nucleus
is centrally placed, and consists of a spherical membrane and a large
central karyosome. Multiplication is by longitudinal division from before
backwards, after division of the nucleus and blepharoplast. The flagellum
is discarded, and after division of the blepharoplast two new flagella are
developed as outgrowths from the two daughter blepharoplasts, which,
during division, remain connected by a long fibre which lies transversely
across the body and parallel to the spindle of the dividing nucleus. Syn-
gamy occurs, as first described by Dobell (19086). Two flagellates unite
by their anterior ends, the union extending backwards till their two bodies
are completely fused (Fig. 48). Each nucleus is described as undergoing
a reduction of its chromatin, after which union takes place. During the
conjugation one flagellum is withdrawn, so that the zygote has a single
flagellum, by means of which it moves about actively. The zygote may
commence dividing after leading a free existence for some time, or it may
encyst. Encystment may also occur without conjugation. The cysts
are ovoid or spherical structures with thin walls and clear contents. They
measure 7 to 8 microns in length. Berliner (1909) gave the name Copro-
monas major to a form which he cultivated on agar plates from the faeces
of lizards. Like the form cultivated from goat's fseces by Woodcock
(1916), which he named Copromonas ruminantium, it is slightly larger than
C. subtilis. Both these may be merely races of the smaller flagellate.
Berliner stated that the flagellates were sometimes present in the free-
swimming stage in the intestine of lizards.
284
SUB-CLASS: PHYTOMASTIGINA
In addition to the forms just considered, which are coprozoic in habit,
certain Eiiglenoidida are definitely parasitic.
Tadpoles appear to be commonly infected with certain chlorophyll-
bearing flagellates allied to the free-living Euglena. Alexeieff (1912/")
Fig. 134. — Euglenoid Flagellates from the Intestine of Tadpoles of Bana
pipiens and Other Species. (After Hegner, 1923.)
1-3. Euglenamorpha hegner i (x 1,600): (1) Living specimen showing three flagella, reservoir,
stigma, chromatoi^hores, and nucleus; (2) specimen fixed in Schaudinn's fluid and stained
with iron hsematoxylin ; (3) specimen stained with iodine.
4. Livmg specimen of Phacus ( x 1,600). 5. Euglena spirogyra ? ( X 780).
noted them in large numbers in the rectum, and states that Brumpt had
made a similar observation. He placed the organism in the genus Euglena
without giving it a specific name. He also observed a species of Phacus
in the same host. He regarded the flagellates as accidentally present in
PARASITIC EUGLENOIDIDA 285
the intestine. He points out that the allied Astasia captiva described by
Beauchamp (1911) from a turbellarian Catenula lemnce was more truly
parasitic, as it perished after removal from its host. AlexeiefE (1912/)
further records the presence of Astasia mobilis in a species of Cyclops. It
occurred not only in the intestine, but also in the developing embryos in
the egg sac, a fact which led Alexeiei? to express the view that it might
be transmitted hereditarily from host to host, and to restate Biitschli's
theory that Sporozoa may have evolved from these or allied flagellates.
Hegner (1923c) has given a description of Euglenoids studied by him in
tadpoles in America (Fig. 134). One form had a single flagellum like the
common free-living type Euglena spirogyra, while another possessed three
flagella. To the latter Wenrich (1923) has given the name Euglenamorpha
hegneri. It appears to be as truly parasitic as other Protozoa in the
intestine. It does not survive when removed from its host for any
length of time, but is readily passed from tadpole to tadpole by feeding.
Hegner also noted the presence of a species of Phacus. The three
types agreed with one another in the possession of green chromatophores
and bright red stigmata. Another form discovered in tadpoles of Lepto-
dactylus ocellatus of Brazil has been placed in a new genus Hegneria by
Brumpt and Lavier (1924). The single species, H. leptodactyli, varies in
length from 40 to 50 microns and has seven flagella. There is a large
anterior vacuole across which the intracytoplasmic portions of the seven
axonemes pass to end in seven blepharoplasts on the posterior wall of the
vacuole.
2. SUB-GLASS: Zoomastigina Doflein, 1916.
A. Monozoic Forms.
1. Order: PROTOMONADIDA.
As already remarked, the flagellates included in this order (=Proto-
monadina Blochmann, 1895) are forms of relatively uncomplicated
structure. They are monozoic, and possess a single nucleus and one or
more flagella, each of which has an axoneme arising from a blepharoplast
situated upon the nuclear membrane or separated from it. In the latter
case there is often a complex structure, the kinetoplast, made up of a
body called the parabasal and one or more blepharoplasts. The axoneme
forms the central core of the flagellum. It arises from the blepharoplast,
and usually takes a straight course to the surface of the body, whence it
enters the flagellum. Sometimes, however, when the surface of the body
is reached, it passes along the surface for some distance before entering the
flagellum, and the line of attachment may be raised into a thin membrane.
In some forms the cytoplasm at the anterior end of the body is raised
286 ORDER: PROTOMONADIDA
into a cylindrical collar or cuff around the base of the flagelluni. The
majority of flagellates belonging to the Protomonadida are free-swimming,
but some of them develop attachment filaments, and it is in these forms
that cup-like sheaths (loricse) and collars commonly occur.
This order includes a large number of free, non-parasitic, and coprozoic
forms, as well as certain parasites such as the trypanosomes and some of the
intestinal flagellates of man and animals.
Many of the simpler Protomonadida are able to ingest solid food at any
part of the body surface by means of pseudopodia, just as amoebae do.
These forms are sometimes known as the Pantostomatina.. Others,
however, only ingest food near the base of the flagellum, where a permanent
cytostome may or may not be present. In the case of the parasitic
blood-inhabiting Trypanosomidae and the Cryptobiidse there is no
cytostome, and nutrition is effected by the absorption of nutrient material
from the blood in solution. In some saprophytic forms it is probable that
both solid food is ingested as well as nutriment in a soluble form. The
Protomonadida do not, as a rule, possess any accessory internal organs,
but in some of them axostyles, parabasals, supporting filaments and other
structures are developed.
The order PROTOMONADIDA may be subdivided into two sub-orders, the
Eumonadea, which are free-swimming forms, and the Craspedomonadea,
which possess attachment organs, and which may or may not have collars
or loricse.
(1). Stib-Order : Eumonadea.
The members of this sub-order are flagellates of relatively simple
structure which have one or a small number of flagella. Each flagellum
arises from a blepharoplast, which may be on the nuclear membrane or
separate from it. When more than one flagellum is present, one may
function as a trailing flagellum. Accessory structures such as axostyles
are sometimes present. The following families may be recognized:
1. Family: MONADID^ Kent, 1880. — Flagellates of simple structure with
one or more free flagella, the axonemes of which originate in blepharo-
plasts which are either upon the nuclear membrane or removed from it.
When there is more than one flagellum, one may function as a trailing
flagellum. The body, which is very metabolic, may or may not be pro-
vided with a cytostome.
2. Family: TRYPANOSOMiD.'E Doflein, 1901. — Flagellates which have a
single flagellum and are parasitic in vertebrates, invertebrates, or plants.
The body is usually elongate, and the axoneme of the flagellum in its course
from the blepharoplast to the point of origin of the flagellum may, if the
blepharoplast be near the nucleus or posterior to it, pass along the border
SUB-ORDERS: EUMONADEA AND CRASPEDOMONADEA 287
of an undulating membrane. There is no cytostome. The flagellates
frequently assume a rounded leishmania form devoid of flagella.
3. Family: bodonid^. Doflein, 1901. — Flagellates which have two
flagella, which arise near a laterally placed cytostome. One of the flagella is
directed backwards as a trailing flagellum. A parabasal body is associated
with the two blepharoplasts, which are separated from the nuclear mem-
brane. The encysted forms are ovoid structures containing a single flagellate.
4. Family: PROWAZEKELLID^ Doflein, 1916. — Parasitic flagellates
which have two flagella, one directed forwards and the other backwards
as a trailing flagellum. The blepharoplasts are on the nuclear membrane.
The cysts are spherical structures, which increase in size after they are first
formed and produce within them a large number of daughter flagellates.
5. Family: embadomonadid^e Alexeieff, 1917. — Flagellates with two
flagella, one directed forwards and the other backwards or laterally
through a large cytostome. The blepharoplasts, which lie near the nuclear
membrane, are not associated with a parabasal. The cysts are ovoid or
pear-shaped structures containing a single flagellate.
6. Family: CHILOMASTIGID^. — Flagellates with four or more flagella,
one of which lies in a large cytostomal groove, while the others are directed
forwards. The blepharoplasts of the flagella are closely grouped together
near the nucleus, and there is no parabasal. The margins of the cytostomal
groove are supported by fibres. The cysts are ovoid or pear-shaped
structures containing a single flagellate.
7. Family: CERCOMONADID^ Kent, 1880. — Flagellates which have one
or more flagella, the axoneme of one of which passes backwards over
the surface of the body, to which it is adherent, without development
of an undulating membrane. The blepharoplasts are upon the nuclear
membrane. The cysts are simple ovoid or spherical structures containing
a single flagellate.
8. Family: CRYPTOBIID.E Poche, 1913. — Flagellates which have two
flagella, one of which is directed forwards while the other passes backwards
and is attached to the surface of the body, which may be raised into an
undulating membrane. The two blepharoplasts are separate from the
nucleus, and there is a parabasal associated with them. Cysts may or
may not be produced.
9. Family: TRiCHOMONADiD^. — Flagellates which have three or more
flagella; one axoneme may pass backwards along the margin of an undu-
lating membrane. The blepharoplasts form a group near the nucleus, and
there may or may not be a parabasal. A pointed rod-like structure, the
axostyle, passes through the cytoplasm from the anterior to the posterior
end of the body, through which it protrudes. The cysts are ovoid or
spherical, and contain a single flagellate.
288 FAMILY: MONADID^
10. Family: DINENYMPHID.E Grassi, 1911. — Flagellates which have
several flagella, the axonemes of which are directed backwards and
attached to the borders of a series of undulating membranes. There is an
axostyle, as in the Trichomonadidae.
(2). Suh-Order : Craspedomonadea,
The sub-order Craspedomonadea includes flagellates which are more
or less permanently attached to objects (Figs. 16, 17, 18). The point of
attachment is the posterior end of the body, and from this a filament may
be secreted, at the end of which the flagellates wave about. In some cases
the filament becomes a complex, tree-like system with a flagellate at the
extremity of each branch. Each attached flagellate may develop around
itself a gelatinous or chitinous cup-like sheath or lorica. The latter is
formed both by attached flagellates, which have no filaments, as well as by
those which possess them. Another modification undergone by some of
these attached flagellates is the development of a cytoplasmic cylindrical
collar or cuff with overlapping margins round the base of the flagellum
at the anterior end of the body. The collared forms may or may not have
loricae as well. The Craspedomonadea are not parasitic forms, and they
often appear in fluids containing decomposing vegetable matter such as
hay infusion. They need not be considered any further here.
SYSTEMATIC DESCRIPTION OF THE GENERA AND SPECIES IN
THE FAMILIES OF THE SUB-ORDER EUMONADEA.
The flagellates in this sub-order are unattached, free-swimming forms,
some of which are parasitic, though the majority are not. They include
types with a single flagellum and very simple structure, and a series of
transition forms leading to more complicated flagellates with at least
six flagella.
1. Family: MONADID^ Kent, 1880.
The flagellates belonging to this family include the simplest of the
Mastigophora. They possess one or more flagella, the axonemes of
which take origin in blepharoplasts which are situated either upon
the nuclear membrane or separate from it. When there is more
than one flagellum all may be directed forwards, or one may be
differentiated as a trailing flagellum. A cytostome may or may not be
present, while the body is often liable to marked amoeboid changes of
form. Apart from the nucleus, blepharoplasts, and axonemes there are
no internal structures except the food vacuoles and the contractile
vacuoles in the non-parasitic forms. The Monadidse could be subdivided
GENUS: OIKOMONAS
289
into a number of sub-families according to the number of flagella present,
but it seems unnecessary to give these names. The following groups can
be recognized:
A. MONADID^ WITH ONE FLAGELLUM.
A number of minute flagellates which possess a single flagellum have
been described from stagnant water and infusions. Some of these are
exceedingly minute, and many of them may be the " swarm spores " of
plants or other Protozoa. Undoubtedly, many of the forms which are
included with the Phytomastigina might be classed with the Monadidae,
but for the purpose of this work they have been omitted.
Genus: Oikomonas Kent, 1880.
A typical member of this genus, as defined by Kent, has an ovoid or
spherical body and a single flagellum, while the posterior end of the body
Fig. 135. — Oikomonas termo : Free and Encysted Forms ( x ca. 2,000). (After
Martin. 1912.
1-2. Usual type.
6. Encysting zygote.
3-4. Dividing forms.
5. Stage in conjugation.
7. Mature cyst.
may form a pseudopodium by means of which temporary attachment to
objects can be effected. Several species were described by Kent as
occurring in stagnant water.
Oikomonas termo (Ehrenberg, 1838). — This flagellate, which is possibly
identical with the flagellate described by Miiller (1773) and Ehrenberg
(1838) as Monas termo, and by Stein (1878) as Cercomonas termo, was
studied by Martin (1912), who recovered it from soil (Fig. 135). The body,
I. 19
290
FAMILY: MONADIDiE
when spherical, has a diameter of about 4-5 microns. It possesses a
spherical nucleus with a large central karyosome. Near the surface of the
anterior end of the body is a blepharoplast, from which arises a single
flagellum which is as long as, or longer than, the body. Reproduction is
by binary fission, and spherical resistant cysts are produced.
From the intestine of man and animals, several observers have de-
scribed flagellates of this type. Liebetanz (1910), who examined the
contents of the rumen of cattle, encountered several types of uniflagellate
organism. A form which had an egg-
shaped body and long flagellum spring-
ing from its narrow anterior end he
placed in Kent's genus Oikomonas, while
he created the genus Sph(Eromonas for a
type with a spherical body, and the
genus Pirotnonas for one with a pear-
shaped body and a flagellum arising at
a point a short distance behind its
narrow anterior end. He further dis-
tinguished two species of Oikomonas
{0. communis and 0. minifna), three of
S-phceromonas {S. communis, S. rninima,
and S. maxima), and three oi Pirojnonas
{P. communis, P. minima, and P.
maxirna). The members of the genus
Oikomonas varied in length from 4 to
11 microns, those of the genus Sphwro-
monas from 3 to 14 microns, and those
of the genus Piromonas from 4 to 18
microns. It is clearly an error to
establish these species on size alone.
In fact, Braune (1913) united the
species of Sphceromonas in the one
species S. cotmnuiiis, while Fonseca
(1916) believes that the genus Piromonas is identical with Sphceromonas,
and that the difference in the shape of the body described by Liebetanz
is only an indication of change in body form. He, nevertheless, records
two species of Sphceromonas from cattle which differ from one another only
very slightly (Fig. 136). He also records the finding of S. communis in
the goat and guinea-pig {Cavia porcelliis), as well as in cattle in Brazil.
It is undoubtedly fallacious to separate these uniflagellate organisms
in different genera, as Liebetanz has done. It is not improbable that they
all belong to the genus Oikomonas.
1
Fig. 136. — Sphwromonas communis
(1) AND S. liebetansi (2) (x ca.
2,000). (Aftek Fonseca, 1916.)
GENUS: OIKOMONAS 291
Under the name of Oikomonas granulata YakimofE, Solowzoff, and
Wassilewsky (1921) describe a small flagellate isolated by them by inocu-
lating agar plates with the stools of two cases of diarrhoea in Petrograd.
They distinguish the organism from the free-living form 0. termo on
account of the presence of certain granules in the cytoplasm. Yakimoff,
Wassilewsky, KornilofT, and Zwietkof? (1921) state that they have isolated
0. termo from the fa?ces of guinea-pigs and mice by employing the same
technique. Another form isolated from guinea-pig faeces is described
as Sphceromonas rossica, and one from rabbit faeces as Piromonas rossica.
The description of these forms is most unsatisfactory, and there are no
grounds whatever for the assumption made that the flagellates were
actual parasites of man or animals. They were undoubtedly dealing
with free-living forms which had passed through the intestine in the
encysted state, or, what is more probable, with flagellates in water con-
taminating the vessels in which the samples of faeces were collected.
The writer has seen an organism of the Oikomonas type in the tortoise
Testudo calcarata. The body is spherical or ovoid, and possesses a single
long flagellum. When spherical, the body varies in diameter from 5 to 16
microns. There is a nucleus with large central karyosome, while the
axoneme of the flagellum arises from a blejjharoplast near the surface of
the body.
The organisms discovered in human faeces by Kofoid and Swezy
(19216), which they regard as representing two species of Craigia (see
p. 29-1), not improbably belong to the genus Oikomonas.
Blackhead of Turkeys.
This disease, which takes the form of an entero-hepatitis associated
with black discoloration of the head, especially in young turkeys, may
be considered here on account of its association with a flagellate infection.
Theobald Smith (1895) described as Amoeba meleagris certain structures
which he found in the intestinal and liver lesions. Cole and Hadley (1910)
believed that the amoebae were really the schizogony stages of a coccidium
which it was supposed had been acquired from sparrows. Theobald
Smith and Smillie (1917), however, showed that the coccidium of the
sparrow was an Isospora, while that of the turkey was an Eimeria.
Hadley and Amison (1911) came to the conclusion that the lesions were
not due to a coccidium, but to TricJiomonas which had invaded the tissues
and become mostly aflagellate amoeboid bodies. Jowett (1911a), working
in South Africa, came to the same conclusion. Hadley (1916, 1917),
after further investigations, stated that he had actually seen flagella
on some of the tissue forms, and was still further convinced of their
292 FAMILY: MONADID^E
Trichomonas nature. Tyzzer (1919), however, refutes these statements,
and returns to Theobald Smith's original view that the invading
organism is actually an amoeba, and that the disease is comparable to
amoebic dysentery in man. Further investigations by Tyzzer (1920a)
showed that the amoeboid bodies which invaded the tissues exhibited
peculiar jerky movement when seen alive, and this fact, combined with
(0> . •:
Fig 137. — llistomonas meleagris from the Intestine of Turkeys affected with
Blackhead ( x 1,400). (After Tyzzer, 1919.)
a. Section of large intestine, showing parasites in mucosa. b. Dividing form.
c-d. Forms showing nuclei and blepharoplasts, with attached fibres.
the presence of a blepharoplast from which axonemes appeared to pass to
the surface of the body, and the formation of a fibril between daughter
blepharoplasts when division occurs, strongly suggested flagellate affinities
(Fig. 137). Tyzzer, however, did not believe that the parasites were
Trichoynonas which had lost their flagella after invasion of the tissues.
He regarded them as aberrant flagellates, for which he proposed the name
IIisto?nonas meleagris, recognizing in them the bodies which Theobald
BLACKHEAD OF TURKEYS
293
Smith originally called Amoeba meleagris. Tyzzer and Fabyan (1920),
and Tyzzer, Fabyan, and Foot (1921) showed that the disease could be
produced in young turkeys by the subcutaneous inoculation of diseased
tissues. Local lesions followed by generalized infection in the form of
nodules occurred. The same result occasionally followed the inoculation
of pigeons, whereas chickens only developed a local skin lesion. It had
been pointed out by Smith and Graybill (1920) that blackhead could be
produced in turkeys by feeding them with ova of Heterakis jjapillosa.
Tyzzer, Fabyan, and Foot (1921) confirmed these observations, but
concluded that the helminth was not the actual cause of the disease, but
that it was merely one of the causes of the condition favourable to invasion
of the body by Histomonas
meleagris. In support of the
conclusion that this organism is
not simply the common Tricho-
monas of the intestine which has
invaded the tissues, Tyzzer and
Fabyan (1920) point out that
blackhead may occur in young
birds, which appear on examina-
tion of the intestine to be quite
free from flagellates, and that
feeding newly hatched turkeys
with infected tissues does not
lead to the appearance of flagel-
lates in the gut.
Tyzzer (1924) and Drbohlav
(1924) have now found that
young chickens contract the
disease when fed upon liver tissue
of diseased turkeys. They are
less susceptible to the disease than turkeys, and usually recover from
the acute symptoms. When the acute symptoms abate, the intestine is
found to harbour an organism which in many respects resembles an amoeba,
except that it is provided with one to four short flagella, which impart to
the living organism a peculiar jerky movement, as noted by Tyzzer (1920)
in the case of the tissue forms (Fig. 138). This infection occurs in chickens
which have been carefully isolated and fed on sterile food, and the par-
ticular organism is the only one present apart from bacteria. Control
chickens not fed upon liver tissue have no such infection. The recovered
chickens with the intestinal infection are regarded as carriers. The
organism, which, it is believed, is the same as the one which occurs in the
Fig. 138. — Flagellates from the F/eces
OF Young Chickens infected from
Turkeys suffering from Blackhead
( X 2,000). (Original from Giemsa
Stained Film prepared by Drbohlav.)
294 FAMILY: MONADID^
diseased tissues, can be cultivated from chicken fseces on egg medium,
and the cultures fed to young chickens produce the same condition as that
resulting from ingestion of liver material. It has not, however, been
possible to obtain cultures directly from the tissues. From these
observations it would appear that the organism named H. meleagris is
actually a flagellate which has one to four fiagella, the axonemes of which
arise from a blepharoj^last or group of blepharoplasts; that it lives in the
intestine as a flagellate and is able to invade the tissues. There is no trace
of axostyle, undulating membrane, or basal fibre, so that its relation to
Trichomonas cannot be upheld. It seems possible that, as the majority
of the flagellate forms have but one flagellum, this is the normal condition,
and that the rarer forms with more than one flagellum are the result of
precocious division of the blepharoplast. In many respects the organism
resembles a member of the genus Oikomonas.
Genus: Craigia Calkins, 1913.
Calkins (1913) founded the genus Craigia for an organism said to be
parasitic in the human intestine, and which was first described by Craig
(1906) from the Philippines as Paramoeba hominis. Barlow (1915) stated
that he had discovered a similar but smaller organism in Honduras and
named it Craigia migrans. He claimed to have seen over 150 cases of
infection, and attributed to the presence of the organism the numerous
symptoms, including fever, dysentery, and even abscess of the liver, from
which his cases suffered. Such assertions it is manifestly impossible to
accept. C. hominis, described by Craig, is said to live in the intestine of
man, and to have both an amoeboid and a flagellate stage. The amoeboid
form is described as resembling E. coU, and measuring in diameter 10 to 25
microns. It was said to form uninucleated cysts, from which, after further
development, numbers of flagellates escape. The latter grow and attain
a diameter of 10 to 20 microns. Each flagellate is depicted as consisting
of a rounded body and a long tapering process, which, though described
as a flagellum, certainly does not appear like one in the figures accompany-
ing Craig's descrij)tion. After many futile attempts to discover such an
organism, the writer was very kindly given some preparations by its
discoverer. In these, which, unfortunately, were poorly stained, the
writer could find only typical free forms of E. coli and Chilomastix mesnili,
a flagellate which was hitherto unrecorded from the Philippines, where the
films were made. The three anterior flagella which this latter organism
possesses were very difficult to detect on account of the imperfect staining.
The posterior extremity of the organism, however, was drawn out in
many cases into a tapering process which resembled the structures which
were called flagella by the original discoverer of C. hominis. Both
GENUS: CRAIGIA 295
the amrebso and the flagellates were of the dimensions given by this
observer for the corresponding stages of C. hominis. In these pre-
parations no other Protozoa were present, so it seems probable that these
had been regarded as C. hominis. When Barlow's description of Craigia
appeared, the writer asked him for preparations, but was informed that none
was available, and the films he had prepared were so poorly stained that he
had not been able to recognize the nature of the organism, but that Craig,
to whom he had sent the films, had been able to convince himself that
Craigia was present. At the writer's request Dr. Newham, during a recent
visit to Honduras, made films from a number of cases showing intestinal
flagellates. The writer has examined these, and could find only the well-
/.^
^^K
\
1 Z 3
Fio. 139.—Craigia migrans (x ca. 3,000). (After Kofotd and Swezt, 1921.)
1. Rounded type of flagellate. 2. Elongate type of flagellate. 3. Encysted form.
known forms. In several of the films Chilomastix was present. This flagel-
late, which is evidently quite common in Honduras, was not identified by
Barlow, so it is not improbable that he mistook this organism for C. hominis.
The extensive investigations made during the war have cleared up
many doubtful points in connection with the intestinal Protozoa of man,
but neither C. hominis nor C. ynigrans has been rediscovered. The
writer has long held the opinion that no such parasites of the human
intestine exist, and in this conclusion he is in agreement with Dobell
(1919). Kofoid and Swezy (19216), as stated above, have claimed to have
observed cases of infection with both species of Craigia. The parasite
seen by these observers does not in its amoeboid phase resemble E. coli in
any way, while in the flagellated stage the flagellum is an exceedingly fine
structure which is difficult to detect, and does not show the least resem-
blance to the tail -like processes figured by Craig. The organism corre-
296
FAMILY: MONADIDiE
spends in every way with Sphceromonas communis described by Liebetanz
from the rumen of cattle (Fig. 139). There seems, therefore, to be no
doubt that Kofoid and Swezy have discovered a small uniflagellated
organism in human fseces. The fiagellum cannot be detected in every
one of the organisms, but when it is present it arises from a granule
near the surface of the parasite, while a fibre is depicted as connecting
.^ this granule with the centrally
r
/
A
^
^
^i-?^
o
Fig. 140. — BhizomasUx gracilis Alexeieff,
1911, FROM Intestine of Larva of Tipula
sp. ( X 4,000). (After MACKINNON, 1913.)
A. Flagellate form.
B. Cyst with one nucleus.
C. Cyst showing nuclear division.
D. Cyst with two nuclei.
placed karyosome of the nu-
cleus. In its course it passes
through the nuclear membrane.
Spherical cysts which contain a
uninucleated cytoplasmic body
and resemble the cysts of some
free-living amoebae also occur.
The discovery of such a form
does not in any way establish
the authenticity of the genus
Craigia. Kofoid and Swezy
state that they have seen their
organism in six cases. It is
possible that, owing to the ex-
treme fineness of the fiagellum
and the difficulty of detecting it
exceptin well-stained specimens,
this organism has sometimes
been mistaken for Endolimax
nana, to which it bears a super-
ficial resemblance. On the other
hand, th e possibility of its bei ng a
coprozoic flagellate of the genus
Oikomonas which has developed
in the fseces after they have left
the body has to be considered.
Gemis
Rhizomastix Alexeieff,
1911.
The flagellates of this genus
have rounded or pear-shaped
bodies and a central nucleus.
There is a long flagellum arising from the anterior end of the body, and
its axoneme is continued into the cytoplasm in the form of a long fibre
which terminates in a blepharoplast behind the nucleus.
GENERA RHIZOMASTIX AND PROLEPTOMONAS 297
Rhizomastix gracilis AlexeiefE, 1911.^Tliis flagellate (Fig. 140), wMch
varies in length from 6 to 11 microns, has been described from the intestine
of axolotls by Alexeief? (1911), and by Mackinnon (1913) from tipulid
larvse. It has the structure described above, and produces spherical
cysts, within which nuclear division occurs.
YakimofE and Kolpakoff (1921) described as Pararhizomastix hominis a
flagellate isolated by them from human faeces planted on agar media.
The organism closely resembles Alexeief?'s Rhizomastix agilis of the
axolotl. The authors do not
state the grounds on which
they create the new genus,
nor why they regard the
flagellate as a human parasite,
and not a coprozoic organism,
which it undoubtedly is.
Genus: Proleptomonas Woodcock, 1916.
This genus was founded by Woodcock
(1916) for a flagellate which he discovered in
cultures from faeces of goats (Fig. 141). On
account of its resemblance to the leptomonas
of insects, it was placed by him in a new
genus, P)'oleptomonas, of which there is one
species.
Proleptomonas fsecicola Woodcock, 1916. —
This flagellate measures from 7 to 8 5 microns
in length by 1-25 to 1-75 microns in breadth
(Fig. 141). There is a long anterior flagellum
16 to 21 microns in length and a central
nucleus, in front of which is a blepharo-
plast from which arises the axoneme of the
flagellum. Woodcock thinks it possible that P. fcecicola may be the
present-day representative of the ancestral type from which the para-
sitic flagellates of the genus Leptomonas were derived. Fantham (1922)
has seen a similar flagellate in decomposing cabbage, and, owing to
the fact that a definite kinetoplast was present, he regards it as differing
from Woodcock's Proleptomonas. He gives it the name Herpetomonas
hrassicce. Another form found in soil he names H. terricolce. These
flagellates, however, do not belong to the genus Herpetomonas, and
it is not improbable that they are identical with Proleptomonas fcecicola.
Fig. 141. — Proleptomonas
fcecicola : A Coprozoic
Flagellate from F.eces
of Goats ( x 3,000).
(After Woodcock, 1916.)
298 FAMILY: MONADIDvE
B. MONADIDiE WITH TWO FLAGELLA.
Many free-living flagellates provided with two flagella have been
described. Such are the various flagellates placed by Stein in the genus
Monas. These are minute organisms which occur in stagnant water.
They have ovoid or elongate amoeboid bodies and two flagella, the thinner
one of which is about twice the length of the other. There is no cytostome,
but a contractile vacuole is present. They produce minute spherical
cysts.
Yakimoff and Solowzofl^ (1921r/) identified as Monas vulgaris a flagellate
they obtained by inoculation of agar plates with human faeces in Russia.
Yakimoff and his co-workers seem to believe that this affords sufficient
evidence of parasitism in the human intestine. The organism is un-
doubtedly a free-living form which in the encysted condition contami-
nated the stool after it had been passed.
Genus: Heteromita Dujardin, 1841.
This genus was established by Dujardin for certain flagellates which
had hitherto been included in the genera Monas or Bodo, and which pos-
sessed pear-shaped bodies provided with two anterior flagella, one of which
was two or three times as long as the other. The longer flagellum, which
was finer than the shorter one, could function as a trailing flagellum. It
seems not improbable that the flagellate for w^hich Krassilstschik (1886)
created the genus Cercobodo and that for which Klebs (1892) proposed
the name Dimorpha really belong to the genus Heteromita. Several
flagellates of this genus were studied by Dallinger and Drysdale, for one
of which Kent (1880-1882) proposed the name Heterotnita uncinata.
The genus Heteroinita can be defined as including minute flagellates
which have pear-shaped bodies from the more pointed anterior end of
which arise two flagella of unequal length. The shorter one, which may be
thicker than the other, is from once to twice the length of the body and is
directed forwards. The finer and longer flagellum may be two to four
times the length of the body. It performs lashing movements, and when
in contact with a surface may act as a trailing flagellum. The axonemes of
the flagella, which commence in blepharoplasts on the nuclear membrane,
pass to the anterior end of the body, and thence directly into the flagella.
There is no cytostome, and a contractile vacuole is present in the posterior
region of the body. The body is exceedingly amoeboid when in contact
with a surface. In this condition the flagella may be lost, the flagellate
then moving about like a small amoeba.
Reproduction is by longitudinal fission of free-swimming forms, or
GENUS: HETEROMITA 299
rounded individuals with or without flagella. Encystment in spherical
cysts occurs. The genus Cercomonas (see p. 629) contains flagellates of
Fig. 142. — Heteromita uncinata from Culture on Agar ( x 4,000).
(Original from Life.)
1-7. Vari(nis types of flagellate with single nucleus, and contractile vacuole.
S. BinucleatVd dividing form. " 9. Trinucleated form with three contractile vacuoles.
1(». Multinucliatcd form with many contractile vacuoles.
11. Form without llagella (five nuclei).
very similar structure, except that the axoneme of the trailing flagellum
passes over the surface of the body from the anterior to the posterior end
300 FAMILY: MONADID^
before entering the flagellum. The genus Bodo includes flagellates which
also have two flagella, but the axonenies of these arise from two blepharo-
plasts which are separated from the nuclear membrane, and which have a
parabasal body associated with them. There seems to be no justification
whatever for Ndller's (1922) inclusion in the same genus of flagellates of
the Cercomonas type with adherent axoneme and those of the Heteromita
(Cercobodo) type, much less for his assumption that they all belong to the
Rhizojjoda.
Heteromita uncinata Kent, 1880. — This was one of the flagellates
studied by Dallinger and Drysdale (1873), who accurately described the
main features of its life-cycle. What appears to be this organism was
seen by the writer as a coprozoic flagellate in old human faeces. It is
pear-shaped, with a rounded posterior end and somewhat pointed anterior
end (Fig. 142). It varies in length as a rule from about 3 to 8 microns,
but exceptionally large forms up to 10 microns in length occur, A con-
tractile vacuole is present in the hinder region of the body, and there is no
cytostome. Arising from the pointed anterior end are two flagella of
unequal length. The shorter, which is slightly thicker than the other, is
approximately as long as the body, and directed forwards during progres-
sion. The longer flagellum, two to four times the length of the shorter
one, performs wide sweeping movements in front of the flagellate when it is
swimming freely. If the long flagellum comes in contact with the glass,
the flagellate still moves forwards by the action of the shorter flagellum,
while the long one trails behind over the surface. In stained individuals,
the axonenies of the two flagella can be traced to the surface of the nuclear
membrane, which may be drawn out into a cone at the point of union
(Fig. 68). In some individuals, two blepharoplasts can be distinguished
at the apex of the cone. The centre of the nucleus is occupied by a large
karyosome. In the free-swimming condition the body of the flagellate
retains its pear shape, but if it comes in contact with a surface it exhibits
amoeboid changes of shape. In pure cultures reared from a single
flagellate there occur amoeboid forms devoid of flagella, so that a definite
amoeboid phase has to be recognized. When grown on agar plates there
occur much larger multinucleated forms, with a corresponding number of
contractile vacuoles and pairs of flagella. Dallinger and Drysdale de-
scribed the fusion of numerous flagellates to form a multinucleated body.
That the multinucleated forms which occur on agar plates do not always
arise in this way is shown by the fact that, after staining, they may have
all their nuclei in process of division, the body containing a number of
spindles. These multinucleated forms, as they occur on agar plates, are to
be regarded as instances of delayed division of the cytoplasm.
The flagellate reproduces by longitudinal division in the free-living
HETEROMITA UNCINATA 301
condition, but most usually after having become spherical and quiescent.
The amoeboid forms without flagella also multiply by binary fission. The
blepharoplasts on the nuclear membrane divide, and the two pairs of
daughter blepharoplasts take up positions at opposite poles of the elongat-
ing nuclear membrane (Fig. 68). Two new axonemes grow out from two
of the daughter blepharoplasts, and these form flagella at the surface of
the body as division is proceeding. The karyosome of the nucleus breaks
up, and a small number of chromosomes appears at the equator of the
spindle which forms within the nuclear membrane between the blepharo-
plasts. Daughter plates are formed by division of the chromosomes, and
a long spindle stretches across the elongated body of the flagellate. The
daughter plates approach the blepharoplasts, the intermediate part of the
spindle disappears, and the nuclear membrane closes round the daughter
chromosomes, which concentrate into the characteristic karyosomes. The
cytoplasm now becomes constricted and divided into two parts, and two
flagellates result. Division of the amoeboid forms takes place in the same
manner except for the absence of flagella.
Under adverse conditions the flagellate loses its flagella, becomes
spherical and encysts in spherical cysts 3 to 6 microns in diameter. The
cyst wall appears perfectly smooth and shows no indication of pores.
On agar plates larger spherical, ovoid, or more irregularly shaped cysts up
to 10 microns in diameter occur. As the included cytoplasm may contain
as many as sixteen nuclei, it is probable they are formed by the encystment
of the multinucleated forms which occur in these cultures. The emergence
from the cysts of large numbers of minute flagellates and the conjugation of
two individuals, as described by Dallinger and Drysdale, were not observed.
The life-history and structure of this flagellate is of interest in that it
closely resembles Cercomonas longicauda, another coprozoic organism
(Fig. 259). It differs chiefly in the fact that both flagella arise at the
anterior end of the body, there being no tendency for one of the axonemes
to pass along the surface of the body before entering a flagellum. The
amoeboid forms of C. longicauda retain the two flagella, while those of
H. uncinata usually discard them.
It seems not improbable that the flagellate which Sangiorgi (1922a)
cultivated from human faeces, and which he named Pirobodo intestinalis,
belongs to the genus Heteromita. As described, it had a pear-shaped
body with two long flagella arising from the pointed anterior end. The
dimensions given are 12-8 to 16-6 microns for the length and 9-6 to 14-4
for the breadth. The description is, however, so inadequate that it is
impossible to identify the organism with any degree of accuracy.
302 FAMILY: MONADIDiE
Genus : Dimastigamoeba Blochmann, 1894.
There is some question as to whether the organisms included in this
genus shoukl be regarded as Mastigophora or E-hizopoda (Figs. 61, 120,
121). It appears that the great part of their existence is spent as amoebae,
in damp soil or similar situations, but that at certain times, when excess
of fluid is suddenly added to the medium, they temporarily assume a
flagellated condition. Two fiagella are developed, the body becomes
elongated, and the organism has the characters of a typical member of the
Mastigophora. After leading a free-swimming existence for about a day
the fiagella are lost, and the amoeboid condition is again assumed. The
axonemes of the two fiagella appear to be connected with the nuclear
membrane, and in this respect the flagellates resemble those of the genus
Heteromita. Division, however, takes place only in the amoeboid phase,
during which the nucleus divides in a characteristic manner, differing in
this respect from the method of nuclear division of Heteromita. Further-
more, the spherical cysts are provided with a number of pores which render
them easily recognizable. This genus has been considered more fully in
the group of flagellated amoebae (p. 262).
Genus: Spiromonas Perty, 1852,
This genus includes flagellates which in the adult condition have narrow
elongate bodies which are spirally twisted. There are two fiagella, which
arise from the anterior end of the body near a small cytostome. Two
blepharoplasts lie near the insertion of the fiagella and the nucleus is
centrally placed. When reproduction takes place, the body becomes
spherical and enclosed in a cyst, within which division into daughter
fiagellates takes j)lace.
Spiromonas angusta (Dujardin, 1841). — This organism, which in the
adult stage has an elongate spiral body 12 to 13 microns long by 1-75
to 2 microns broad, was referred to by Dujardin (1841) as Heteromita
angusta and Stein (1878) as Bodo gracilis. Kent placed it in the genus
Spirofnonas (Fig. 143). It was studied by Woodcock (1916) in cultures
made from goat's fseces. The smallest forms have ovoid bodies measuring
2-5 by 1 micron, and provided with two backwardly directed fiagella,
which may be more than three times the length of the body. As growth
takes place the body becomes definitely spiral, while with further develop-
ment it becomes bean-shaped. In the largest forms the fiagella are about
as long as the body, which undergoes no change in shape and appears to
be quite rigid. The nucleus is in the anterior half of the body, while the
axonemes of the fiagella arise from two blepharoplasts in front of the
nucleus. Multiplication occurs only in the encysted condition, which is
GENUS: SPIROMONAS
303
brought about by the flagellate losing its flagella and becoming spherical.
Within the cyst it divides usually into three, but sometimes into two or
four small forms, which develop flagella and escape from the cyst, Syn-
FiG. 143. — Spiromonas angnsta : A Coprozoic Flagellatk from Pig's F.eces
( X 3,000). (Original.)
1-2. Narrow fornis showing spiral groove.
3-4. Thicker fornis in which spiral groove is not evident.
5-6. Forms witiuiut tiagella retracting for encystment; one has a large vacuole.
7-8. Encysted forms with one nucleus; one has a vacuole.
9-10. Similar forms with two nuclei. 11. Encysted form with four nuclei.
12. Encysted form after division into four; each develops two flagella and escapes from
the cy.st. 13-14. Encysted forms after division in two.
gamy was also observed to take place. Two individuals may form a
common cyst, within which they unite, or they may first unite and form a
cyst afterwards. A coprozoic Sjnromonas has been seen by the writer in
304
FAMILY: MONADID^
pig's fseces. Though certain individuals reached a length of 15 microns,
the flagellate is probably identical with that studied by Woodcock. In
the younger forms the body is distinctly flattened, and resembles a blade
of grass twisted into a spiral. There is a small but definite cytostome,
though Woodcock stated that no cytostome was present in the form
studied by him. The two flagella arise from the region of the cytostome,
one apparently from its anterior lip and the other from a point within it.
In some individuals a thread, which may be the axoneme, can be traced to
the membrane of the spherical nucleus which occupies a central position
in the body. In some individuals there appears to be a granule at the
base of each flagellum near or on the surface of the body. The older
individuals become more cylindrical in form, though they may still show
Fig. 144. — Phyllomitus undulans : A Coprozoic Flagellate in Goat's F.eces
( X 3,000). (After Woodcock, 1916.)
1-2. Ordinary type of flagellate. 3-4. Dividing forms.
indications of a spiral twist. In preparation for division the body
gradually retracts to a spherical form and encysts. The nucleus divides
to form two nuclei, and these again to give rise to four. The body then
divides into four daughter flagellates. In some cases two and in others
three daughter flagellates are formed.
Sangiorgi (1917) described as Toxobodo intestinalis a small flagellate he
had cultivated from human faeces. Its measurements were 8 to 9- 6 microns
by 3-2 to 4-8 microns. From the figures, it appears that the flagellate
was probably a Spiromonas. Both it and the one named T. sangiorgii,
and cultivated from mouse fasces by YakimofE (1925), are probably
S. angusta. Similarly, the coprozoic flagellate seen by Alexeieff (1918) in the
faeces of the horse and tortoise, and which he named Alphamonas coprocola,
is probably the same spiral organism, as pointed out by Woodcock (1921).
GENERA PHYLLOMITUS AND COSTIA
305
Genus: Phyllomitus Stein, 1878.
This genus includes Phyllomitus undulans, which was originally de-
scribed by Stein. Woodcock (1916) obtained it in culture from goat's
faeces. It has an ovoid body, and varies in size from 6 to 13 microns by
3 to 8 microns (Fig. U-t). There are two flagella, one about twice the
length of the body and the other less than half this. The two flagella
are united by a membrane. Multiplication is by binary fission.
Fig. 145. — Costia necatrix from Skin of Fish. (After Moroff, 1903.)
a. Side view of flagellate ( X 2,000).
b-c. Probable division forms with two new flagella developing ( X 2,000).
d. Section of skin with attached flagellates. e. Encysted form ( X 2,000).
Genus: Costia Leclerq, 1890.
This genus was founded by Leclerq (1890) for a flagellate which is
parasitic on the skin of fish. The organism is pear-shaped, and has two
or four flagella arising in a groove.
Costia necatrix (Henneguy, 1883).— This flagellate, the only member of
the genus, was discovered by Henneguy (1883, 1884). He placed it in the
genus Bodo as Bodo necator, while Leclerq (1890) created for it the new genus
I. 20
306 FAMILY: MONADID^
Costia. The body, which is somewhat flattened, is pear-shaped in outline
(Fig. 145). At the anterior pointed end is a funnel-like depression, from the
bottom of w^hich arise the flagella. According to Morof? (1903), there are
two long flagella which extend beyond the body and two short ones con-
fined to the interior of the funnel. It seems possible that the two short
flagella are new ones forming preparatory to division, and that the organism
has really only two long flagella. The body measures from 10 to 20
microns in length by 5 to 10 microns in breadth. The parts of the flagella
beyond the body have a length slightly shorter than that of the body itself.
There is a spherical nucleus at the middle of the body, while behind it is
a contractile vacuole. Reproduction is by longitudinal division, while
spherical cysts 7 to 10 microns in diameter are formed. The flagellates
are parasitic on the skin of fish, to which they are attached by their
flagella. They sometimes occur in enormous numbers on young fish
artificially reared, and have been suspected of causing a high rate of
mortality.
C. MONADID^E WITH THREE FLAGELLA.
Genus: Enteromonas Fonseca, 1915.
This genus was founded by Fonseca for a flagellate named by him
Enteromonas ho?ninis which he found in human faeces in Brazil (Fig. 146).
The various descriptions he has given of the organism are not in agreement.
The last account given by him (1920) describes the flagellate as having a
splierical body 5 to 6 microns in diameter. There was a nucleus near the
anterior end 1 micron in diameter. Running from the nucleus to the
anterior end of the body was an axoneme which terminated in a blepharo
plast, from which arose three flagella. There was no cytostome. The
cytoplasm contained food vacuoles, but no other structures. Encysted
forms were not encountered. Chalmers and Pekkola (1917a, 1918) re-
corded the finding of an organism in human faeces which they believed to
be identical with Fonseca's E. hominis, while Chatterjee (1917) erro-
neously ascribed to the genus Monocercomonas an organism with similar
structure from human beings in India. Later in the same year (1917a)
he gave an account of a new organism which he named Trichomastix
hominis, on account of the fact that some of the individuals had four free
flagella. It is very probable that the forms with four flagella were pro-
ducing new flagella in process of division, for, of the thirty-five individuals
figured, only four are shown with four flagella. All the others have three,
with the exception of one with two. It seems clear, therefore, that the
organism is not a Trichomastix (Eutrichomastix) at all, and that it is the
same as the form previously described by him as Monocercomonas, which
again appears to be identical with the forms first seen by Fonseca. The
GENUS: ENTEROMONAS
307
flagellate described by him (1919) as Enteromonas Bengalensis is possibly
the same organism, though some of the figures suggest Embadomonas
intestinalis. Leger, M. (1918a), described a similar form from man in
Guiana, and regarded it as E. hominis. These various accounts agree
in describing E. hominis as a flagellate with rounded body, three flagella,
and no cytostome. Yakimoft" (1925) gave the name Enteromonas fonsecai
to a form in the guinea-pig.
There seems to be considerable doubt as to the accuracy of the descrip-
tions of the genus Enteromonas. Dobell and O'Connor (1921) suggest that
the various observers were actually dealing with Tricercotnonas intestinalis,
and that the fourth posterior flagellum had been overlooked (Fig. 261). It
appears, however, that another and more probable explanation can be
found. As described by the writer (19106), Chilomastix mesnili may occur
as a small spherical flagellate with three anterior flagella (Fig. 256, 7, 8).
In these forms the cytostomal groove
and its enclosed flagellum may be
difficult to detect or quite invisible, so
that flagellates appear to have the
structure ascribed to Enteromonas
hominis. This mistake appears to
have been made by Chalmers and
Pekkola, for the writer has been able
to examine their original films. There
can be no doubt that their E. hominis
is merely a small rounded form of
C. mesnili. Though they state that
the latter flagellate was never found in association with E. hominis, the
writer has found typical forms in the films. In their description of C. mesnili,
these observers (1918) draw attention to the small round forms with ob-
scured cytostomal groove, but it did not occur to them that their E. hominis,
previously described, might be the same forms. It is not improbable that
Fonseca and other observers were also describing as Enteromonas the small
forms of C. mesnili. If this be so, then the name Enteromonas becomes a
synonym of Chilomastix. Fonseca (1918) described as E. intestinalis of the
rabbit a flagellate which was said to have the same structure as his E. hominis
of man. This form, as well as the human one, was seen by da Cunha and
Pacheco (1923), who also saw another in the viscacha in Brazil. If the
human form is a Chilomastix, it is not improbable that the rabbit one is
also, as it is liable to infection with a species of Chilomastix. Lynch (1922a)
has, however, obtained from the guinea-pig a culture of a flagellate having
the structure ascribed to Enteromonas. It apparently showed no tendency
to develop into Chilomastix, with which the guinea-pig may also be infected,
Fig. 146. — Enieromonafi hominis {x ca
2,000). (After Fonseca, 1910.)
308 FAMILY: MONADID^
but remained as a small rounded organism with three flagella, one of which
sometimes functioned as a trailing fiagellum. Whether this flagellate should
be regarded as belonging to the genus Enteromonas depends on whether
Fonseca's human Enteromonas was or was not a Chilomastix. Brug (1923)
gave a description and figures of E. Jiominis in Sumatra. Some of the
figures are suggestive of Tricercomonas intestinalis, and as Jepps (1923)
had encountered this flagellate in Malaya, it seemed possible that Brug
was actually dealing with it. Brug has kindly allowed the writer to see
preparations of his flagellate, which is undoubtedly T. intestinalis.
The determination of the structure of small flagellates in faeces is an ex-
ceedingly difficult procedure. A careful observation of living individuals, or
those which have been killed by exposure to osmic vapour or iodine solution,
will often yield more information than the study of fixed and stained films.
D. monadid;e with four flagella.
A number of coprozoic or intestinal flagellates have been described
which possess four anterior flagella and no accessory structures in the
cytoplasm beyond the nucleus and blej^haroplasts. These have been
placed in various genera, but it is very doubtful if many of these are valid.
Their classification is rendered difficult by members of the genus Eutricho-
mastix (p. 671), which correspond in structure except for the possession
of an axostyle. Other flagellates having a similar structure, but possess-
ing a fibre in the place of a true axostyle, belong to the genus Retortamonas
(often called Monocercomonas, p. 677). The axostyle or the fibre may
not be visible, in which case the flagellates resemble Monadidae with four
flagella. It is evident that when an organism is seen with four flagella and
no axostyle or fibre, it may be a Eutrichomastix or Retortamonas in which
these structures are not visible, or a true Monad with four flagella.
Genus: Tetramitus Perty, 1852.
This genus was created by Perty (1852) for certain free-living, pear-
shaped flagellates, which, possess a cytostome and four flagella, one of
which might be a trailing flagellum. There are several species recorded
by Perty, Klebs, and others. It is possible that some of the flagellates
with four flagella which occur in faeces belong to this genus. The form
described by Dobell (1908c) as Monocercomonas bufonis from the toad is a
pear-shaped organism 12 to 15 microns in length and 3 to 6 microns in
breadth. There is no cytostome. The nucleus lies near the blunt end
of the body, and in front of it are four blepharoplasts from which arise
four flagella, all of 'which are directed forwards. There- was no axostyle
or fibre in the body. This flagellate evidently does not belong to the genus
Monocercomonas (Retortamonas), as the axial fibre is absent. It may belong
GENUS: TETRAMITUS
309
to the genus Tetramitus, as Dobell suspected, though the absence of cyto-
' stoma is against this view.
As noted above, the Monocercomonas described by Chatter jee (1917)
from the human intestine probably has only three flagella, and not four,
and should not be included in this genus.
Aragao (1916) established the new genus Coj^ro/z^rtA'/ia? for a flagellate with
four anterior flagella, which ap-
peared in cultures of human and
rat faeces in egg-albumen water
(Fig. 148). The organism is pear-
shaped, with a blunt anterior
end, at one side of which is a
cytostome. The length of the
body is 16 to 18 microns and the
breadth 7 to 9 microns. Smaller
forms, however, occur. The
Fig. 147. — GMlomitus cavice from
THE C^CUM OF THE GuiNEA-PiG
(x ea. 2,000). (After Fonseca,
1916.)
Fig. 148. — Copromastix prowazelci : A Copro-
zoic Flagellate appearing in Cultures
of Human and Rat F^.ces (x ca. 3,000).
(After Aragao, 1916.)
1. Usual type. 2. Dividing form.
flagellates multiply by binary flssion. The blepharoj^last from which the
four flagella take origin first divides into two, some of the four flagella re-
maining with one portion and the others with the other portion. The nucleus
divides by mitosis, and this is followed by division of the body. New flagella
are formed from the blepharoplast till each daughter individual has four.
Aragao names the flagellate Cojxromastix prowdzeki, but it corresponds very
closely with Tetramitus rostratus, a free-living form first seen by Perty
(1852). It is probable that it as well as Copromastix aragaoi cultivated
from human faeces by Yakimoff (1925) are actually this species.
Bunting (1922) obtained by culture from the csecal contents of rats
an amoeba which after reproduction in this form became transformed into
a flagellate of the Tetramitus type, with four flagella, a lateral cytostome,
and contractile vacuole. After reproduction in the flagellate stage had
taken place, reversion to the amoeboid phase occurred. Spherical cysts
6 to 18 microns in diameter were produced by the amoebse. Rats and mice
310 FAMILY: MONADID^
were fed on the cultures. When examined jpost morteyn one to five days
later, no amoebae or flagellates could be found in the intestine, but cysts
were present. Culture from the intestinal contents again gave cultures
of amoebae and flagellates. It is evident that the flagellate and the
amoeba are coprozoic, and that they represent different phases of develop-
ment of one organism. The flagellate phase as figured by Bunting has a
striking resemblance to Cojjrotnastix jrvowazeki. It is possible that these
organisms are related to Dimastigamceha, which also has both a flagellate
and an amoeboid phase (see p. 262).
Fonseca (1916) created the genus Chilo7nitus for a flagellate of the
caecum of the guinea-pig {Cavia porcellus and C. a/perea) in Brazil. It is
said to vary in size between two extremes. The large form is 12 to 17
microns in length by 4 microns in breadth, and the small one 8 to 10 microns
in length by 4 to 5 microns in breadth (Fig. 147). The anterior end is
rounded and the posterior end tapering, while the body itself is very rigid
owing to a well-developed ectoplasmic layer. There are four anterior
flagella which arise from a blepharoplast and a cytostome which is much
shorter than that of Chilomastix. There is no flagellum within the cyto-
stome. The nucleus near the flagellar origin is not vesicular, and appears
to consist of a mass of granules. The margins of the cytostome are not
stiffened by marginal filaments as in Chilomastix.
Lavier informs the writer that he has seen this flagellate, which was
named Chilomitus cavics by Fonseca, in the rodent Viscacia viscacia of
the Argentine. In addition to the structures noted by Fonseca he has
seen an axial fibre passing longitudinally through the body.
Chalmers and Pekkola (1918) created the genus Protetramitus for a
flagellate which was described as having a spherical body and four flagella
arising from blepharoplasts, near which was the single nucleus. The
flagellate Protetramitus testudinis was found in the tortoise {Testudo
calcarata). The writer has examined the films, and finds that, in addition to
Entamoeba testudinis and Balantidium testudinis, a number of flagellates are
present — Hexafnita, Trichomonas, Eutrichomastix — and a large organism
with a single flagellum which appears to belong to the genus Oikomonas.
Unfortunately, owing to faulty technique, the majority of the organisms
are imperfectly fixed, and many have actually dried in the film. The
result is that many of the Trichomonas and Eulrichimastix have become
rounded, while the axostyles, supporting filament and undulating mem-
brane, are not visible. It is easy, however, to trace every degree of this
change between the flagellates which were named Protetramitus by
Chalmers and Pekkola and the typical Trichomonas and Eutrichomastix.
The flagellate named Protetramitus testudinis is thus nothing more than a
rounded and altered Trichomonas or Eutrichomastix.
GENERA: CALLIMASTIX AND SELENOMONAS
311
E. MONADID^ WITH MORE THAN FOUR FLAGELLA.
Of flagellates with more than four flagella, the only genus which should
be mentioned is CaUimastix Weissenberg, 1912. The genus was created
by Weissenberg for a flagellate parasitic in the body cavity fluid of a
species of Cyclops. He gave it the
name CaUimastix cyclopis.
A very similar form discovered by
Braune (1913) in the rumen of cattle
was named by him C. frontalis
(Fig. 149, A). The body, which is
spherical or ovoid, has a diameter of
about 12 microns, and possesses a
single nucleus with large central ka-
ryosome. The characteristic feature
of the flagellate is its possession of a
number of flagella which spring from
a row of blepharoplasts. The flagella
are arranged in one plane, and appear
as if united laterally to form a band
about 30 microns in length. This
organism was seen by Fonseca (1916)
in cattle, sheep, and goats in South
America. He established a new
family, Callimastigidse.
An organism which may be re-
lated to CaUimastix frontalis is Seleno-
monas j^alfitans, which was described
by Simons (1921) from the caecum of
guinea-pigs (Fig. 149, B). It seems
probable that Ancyromonas ruminan-
tium, which Certes (1889) found in the
rumen of cattle, is the same organism.
Prowazek (1913a), as Kerandel (1909)
had done before, saw it in the blood-
films from African antelopes, and
concluded that these had been con-
taminated from the intestine. He
created the genus Selenomonas. Soon
after Woodcock and Lapage (1913)
observed it in the stomach of goats,
and placed it in a new genus as
Fig. 149.
(After Fonseca, 1916.)
A. CalUmastix frontalis from rumen of ox,
sheep, and goat ( x ca. 4 ,000).
B. Selenomonas rumimintium from caecum of
wild guinea-pig ( X ca. 4,000).
312 FAMILY: TRYPANOSOMIDiE
Selenomastix ruminantium, realizing that it was the same as the organism
described by Certes. Prowazek's name evidently has priority, as the
organism certainly does not belong to Kent's genus Ancyromonas. Da
Cunha (1915) noted the organism in the caecum of guinea-pigs, as did
also Fonseca (1916) and Simons (1920, 1921). The form in the guinea-pig
was described in detail by Boskamp (1922). The body of the organism
is a rigid crescent measuring 6-8 to 9-1 by 1-8 to 2-3 microns. A bunch
of flagella springs from the hollow of the crescent, near which is a
deeply staining mass. Reproduction is by transverse fission through the
flagellar region. Half the flagella pass to each daughter individual.
Boskamp believes that the organism is not a Protozoon, but is related to
the Spirilla. The writer has seen the organism in large numbers in the
caecum of a guinea-pig in England. It seems quite possible that Fonseca's
Callimastix is a rounded form of the same or a similar organism.
2. Family: TRYPANOSOMID^ Doflein, 1901.
In this family are grouped a number of closely related flagellates. They
are the true trypanosomes typically seen in the blood of vertebrates or
their invertebrate hosts; the leptomonas, crithidia, and herpetomonas,
which have only an invertebrate host, in which they live mostly as intestinal
parasites; the leishmania, which, like the trypanosomes, have both a verte-
brate and an invertebrate host, though the latter is not definitely known;
and the phytomonas, which have both an invertebrate and plant host.
RELATION OF VARIOUS TYPES TO ONE ANOTHER.
All the members of the family resemble one another in the possession
of a nucleus and a single flagellum which arises from a composite structure,
the kinetoplast (Fig. 150). The latter is made up of a posterior deeply
staining body, the parabasal, and an anterior blepharoplast in which the
axoneme of the flagellum has its origin. The kinetoplast, or the parabasal
alone, is often termed the kinetonucleus, a name proposed by Woodcock
(1906), while Laveran and Mesnil, in their writings, refer to it as the small
nucleus or centrosome. It is also called the micronucleus, a name which
should not be employed, for it is used to designate one of the highly
specialized nuclei of the Ciliata. The term kinetonucleus implies that it is
equivalent to a nucleus, and in this sense is misleading. On this account
the term kinetoplast, first employed by AlexeiefE (19176) for the corre-
sponding structure in Bodo caudatus, will be used here. The portion of the
axoneme or axial filament of the flagellum between the blepharoplast and
the surface of the body where the flagellum commences is often called
the rhizoplast, but this term has been used for many different fibrils. The
body of one of these flagellates usually consists of an elongated, flattened,
ORGANISATION
313
Fig. 150.— Various Forms assumed by Flagellates of the Genera Lepto-
monas (1-16), Crithidia (17-26), and Trypanosoma (27-50). (Original.)
The lines represent the surface of epithelium to which flagellates become attached.
1-4 and 5-7. Evolution of round form (cyst) into leptomonas form 8.
9-11. Attached forms in which the flagella are lost.
12. Overgrown cercoplasma form.
13-16. Retrogression of leptomonas form into round form (cyst).
17-20. Evolution of round form (cyst) into crithidia form.
21-23. Attached forms in which flagella are lost.
24-26. Retnigrc'ssiou of crithidia furm into round form (cyst).
27-29. Evolution nf round form (cyst) into trypanosome form.
30-39. VarinustyiK'.s of trypanosome form. _ .
40-42. Attached forms in which Hagella are lost, but undulatmg membrane is still
present.
43-45. Attached forms in which flagella are lost and no undulating membrane present
(RhjTichoidomonas forms).
46-47 and 48-50. Retrogression of trypanosome form to round form (cyst).
314 FAMILY: TRYPANOSOMID^
and curved blade of cytoplasm which is more or less tapering at each end.
The surface of the body is covered by a very fine but denser layer of
cytoplasm, the periplast. That such a fairly strong and resistant mem-
brane is present Minchin (1909a), from a study of the cytology of Tnjpano-
soma lewisi, considers obvious from the manner in which the trypanosomes
retain their body form under trying circumstances. The cytoplasm has
a finely alveolar structure, and very frequently a distinct vacuole occurs
near the kinetoplast.
The simplest flagellate type is the leptomonas, which has an elongated
and sometimes slightly curved blade-like body, and the various structures
described above (Fig. 150, 8). All the other flagellates of this family may
be regarded as having arisen from the leptomonas form. The first modi-
fication is the displacement backwards of the kinetoplast, which takes up
a position near, but still anterior to, the nucleus. There is a considerable
lengthening of the axoneme, which now, instead of passing through the
body of the flagellate, takes a lateral course to the convex margin, and
then passes along the surface of the body or on the edge of a cytoplasmic
ridge — the undulating membrane — to the anterior end of the body, and
thence into the flagellum. The free margin of the membrane, when one is
present, is longer than the attached margin; hence it is thrown into folds,
and has an undulatory movement when in action. Flagellates of this type
are distinguished as crithidia (Fig. 150, 20). A further change occurs with
continued displacement of the kinetoplast, which passes the nucleus and
ultimately occupies a position near the posterior end of the flagellate.
The axoneme then passes along the surface of the body for almost its
entire length or along the margin of an undulating membrane, so that the
trypanosome form is reached (Fig. 150, 30). These three flagellate types —
leptomonas, crithidia, and trypanosome — may all of them transform in a
converse manner to produce finally shorter and more rounded individuals
till the leishmania form arises (Fig. 150, i, 17, 27). The latter has a small
round or ovoid body containing the nucleus and kinetoplast. There
is no flagellum, but the axoneme can often be detected as extending from
the kinetoplast to the surface of the body. These leishmania forms,
under suitable conditions, will transform again into any one of the flagel-
late types from which they were originally derived ; or, if they arise in the
intestine of an invertebrate, as in the case of those flagellates which are
limited to an invertebrate host, they may encyst and escape in the faeces.
Since purely insect flagellates are transmitted from one host to another
by small encysted leishmania forms, it follows that the leishmania forms
which escape from the cysts after they are ingested are much smaller than
the fully-grown flagellate stages. In the intestine of the host these small
leishmania forms increase in size, develop flagella, and change their shape
ORGANISATION 315
till the fully-developed flagellate stage is reached. Multiplication may-
take place at any period of this development. All the intermediate forms
between the cyst and the elongate flagellates have been termed by Patton
(1909) pre-flagellates. Conversely, in the hind-gut the flagellate forms, by
a reverse process, become leishmania forms again. These eventually
encyst and escape with the dejecta of the insect. The forms between the
adult flagellates and the cyst have been styled 2^ost-flagellates. It is
doubtful, however, if the cycle, as, for instance, that of Crithidia gerridis
(Fig. 166), is as definite or simple as this nomenclature implies. If
nutrition is lacking in the intestine, it may happen that flagellate forms
become shorter and attach themselves to the gut wall. They have then
become post-flagellates, but a fresh supply of nutriment may lead them
to develop again into fully-formed flagellates. The terminology, however,
is not inconvenient, since the fully-formed flagellates in an insect, whether
of the leptomonas, crithidia, or trypanosome form, represent the height
of an infection. The rounded or short forms occurring before this stage
is reached are found in the stomach and are pre-flagellates, while those
developed after it in the hind-gut are post-flagellates.
At certain stages of their development in the invertebrate the flagel-
lates may show a tendency to become attached to the lining cells of the
organs (gut, Malpighian tubes, salivary glands) in which they live (Fig.
150, 9-11, 21-23, 40-45)- This attachment, which takes place by the
flagellar end of the body, is associated with a change in morphology.
There is a loss of the flagellum, though the portion of the axoneme between
the kinetoplast and the anterior extremity of the body persists. In this
condition the flagellates may still retain the trypanosome, crithidia, or
leptomonas structure as far as arrangement of the kinetoplast, nucleus, and
undulating membrane is concerned. The attached flagellates are usually
subject to a shortening of the body, so that every transition between the
elongate forms and ovoid leishmania forms may be seen attached to the
surface of the cells. On the other hand, the posterior portion of the body
of the attached flagellate may undergo an overgrowth (Fig. 150, 12, 45).
An extreme type of this condition is seen in the case of the Cercoplasma
forms described by Roubaud (1908a, 19086, 1911a) for Herpetomofias
mirabUis and H. mesnili (Fig. 172), Similar forms were seen by Swingle
(1911) in the case of H. lineata, in which the post-nuclear part of the
body may reach a length of 300 microns. It seems to the writer that
the Rhynchoidomonas forms of Patton (1910a), in which the axoneme
terminates at the anterior end of the body, are probably attachment forms
of a flagellate of the trypanosome type (Fig. 150, 45, Fig. 174). In these
there is a certain degree of overgrowth of the posterior portion of the body,
though nothing comparable with that which occurs in the Cercoplasma
316
FAMILY: TRYPAXOSOMIDiE
forms noted by Roubaud and Swingle. At any time, probably with the
advent of fresh nutriment into the intestine, these attached forms may
become free, and, as a result of increase in length of the axoneme, develop
flagella for a swimming mode of life.
ORIENTATION AND ORIGIN OF THE DIFFERENT TYPES.
It will be seen that in the simplest flagellate type, the leptomonas, the
flagellar end is undoubtedly anterior, for the living organism progresses
in this direction, and in the more
highly constructed trypanosomes
with undulating membranes the
flagellar end, according to the
view expressed above, is still
anterior. In the blood of certain
fish there occurs another type
of flagellate, the trypanoplasm,
which possesses two flagella
(Fig. 151). During progression,
one is directed forwards and the
other, attached to an undulating
membrane, backwards. There is
little doubt that the anterior end
of this organism is the one from
which the f orwardly directed free
flagellum arises, and it has been
supposed by some that the try-
panosome type has been derived
from these forms by the suppres-
sion of the free flagellum. It
will be evident that, if this is the
case, the flagellar end of a try-
panosome must be regarded as
posterior. The evidence in favour
of trypanosomes as they occur
in vertebrates having originated
from the leptomonas and crithidia forms of invertebrates seems almost con-
clusive, while the trypanoplasms undoubtedly belong to quite another group
of flagellates (see p. 637). Leger, L. (190%), expressed the opinion that
some trypanosomes had been derived from a trypanoplasm ancestor
(trypanosomes with flagellum posterior), while others had originated from
leptomonas ancestors (trypanosomes with flagellum anterior). Liihe (1906)
Fig.. 151. — Trypanoplasms from Fish
(x 2,000). (After MiNCiiiN, 1909.)
1. Trj/panoplasma keysselitzi of the tench.
2. Trypanoplasma ahramidis of the bream.
3. Trypanoplasma borreli of the rudd.
OKGANISATION
317
expressed similar views, and considered the two types of trypanosome
generically distinct. Woodcock (1906), in discussing the phylogeny of the
trypanosomes, arrived at the conclusion that two distinct families are
represented (Trypanosomatidse and Trypanomorphidse), one including
heteromastigine forms evolved from trypanoplasm ancestors originally
parasitic in the vertebrate intestine, and the other including herpetomona-
dine forms evolved from insect or invertebrate flagellates. In the former
the flagellar end is posterior, while in the latter it is anterior. There
seems to be, however, no sound argument to support this view. Repre-
sentatives of both Woodcock's groups are culturable, and in these cultures
they all tend to revert to flagellates of the insect type— viz., leishmania,
leptoraonas, and crithidia forms — a fact which speaks strongly in favour of
B
Fig. 152. — Agglomeration of Trypanosomes and Trypanoplasms by their
Posterior Ends as a Kesult of the Action of Sera ( x ca. 1,300). (A,
AFTER LaVERAN AND MeSNIL, 1912; B, AFTER SCHINDERA, 1922.)
A. Trypanosoma lewisi. B. Crijplohia helicis.
the similarity of their origin. In his genus Trypanomorpha, Woodcock
places Trypanosoma noduce and probably some of the avian and mamma-
lian trypanosomes, while the genus Trypanosoma includes all other forms.
The phenomenon of agglomeration first demonstrated by Laveran
and Mesnil (1900a) in the case of Trypanosoma lewisi may be regarded as
throwing some light on this question. When acted upon by certain sera
the trypanosomes become clustered in rosettes, with their flagellar or
anterior ends directed outwards and their non-flagellar ends united at
the centre of the cluster (Fig. 152, A). Schindera (1922) has shown that
Cryptobia helicis of the snail similarly becomes agglomerated under the
influence of sera. In this case, again, it is the anterior end with the
free flagellum which is directed outwards, while the posterior end with
the posterior flagellum is at the centre (Fig. 152, B).
318 FAMILY: TRYPANOSOMID^
The most generally accepted view is that the trypanosomes of verte-
brates were originally purely insect flagellates which gradually became
adapted to the blood medium when the insects became blood-suckers.
The flagellates then passed into the vertebrate, and became adapted to
life in the blood-stream. Minchin (1908), however, held the opinion that
trypanosomes were originally intestinal flagellates of vertebrates which
thence passed into the blood-stream, and secondarily became parasites
of blood-sucking insects. In a later paper (1914) he appears to have
relinquished this view, and writes of the ancestral forms of the trypano-
somes as insect flagellates. Mesnil (1918), however, expresses himself in
favour of the view that trypanosomes originated from leptomonas forms
parasitic in the intestine of the vertebrate. It seems to the writer that the
evidence available points to the evolution of trypanosomes from purely
insect flagellates, as explained above.
SUBDIVISION INTO GENERA.
The grouping into different genera of the flagellates belonging to the
family under consideration is, in our present state of knowledge, exceed-
ingly difficult, and for the want of some definite scheme great confusion
in nomenclature has resulted. Some of the flagellates have only an in-
vertebrate host; others have two hosts, a vertebrate and an invertebrate;
while others, again, are known only in the vertebrate. According as to
whether they are limited to an invertebrate, or have both a vertebrate
and an invertebrate host, or whether, in their highest stage of develop-
ment, they reach the leptomonas, crithidia, or trypanosome form, it is
possible to group them in general in the following provisional manner,
always remembering that the flagellates which have no vertebrate host,
and which pass directly from insect to insect, do so in an encysted stage,
which does not occur in those flagellates which pass from insect to verte-
brate or vice versa. Other flagellates of the leptomonas type have an
invertebrate and a plant host.
1. Flagellates of the genus Leptomonas are those which never develop
beyond the leptomonas stage. In the course of their life-history they
show only the leishmania and leptomonas forms. They are confined to
invertebrate hosts, and pass from one to another by means of cysts voided
with the dejecta.
2. Flagellates of the genus Crithidia show, in the course of their
development, leishmania, leptomonas, and crithidia forms. They are
limited to invertebrate hosts, as in the members of the genus Leptomonas,
and are transmitted in a similar manner by means of cysts.
3. Flagellates of the genus Herpetomonas are, again, purely invertebrate
SUBDIVISION INTO GENERA
319
parasites, but they attain a higher degree of development. In their cycle
are found all four types — leishmania, leptomonas, crithidia, and trypano-
sonie. Here, again, infection is passed on by encysted forms.
4. Flagellates of the genus Leishmania resemble those of the genus
Leptomonas in having only the leishmania and leptomonas forms, but they
differ in having both a vertebrate and an invertebrate host. Both the
leishmania and leptomonas forms may occur in either host. It is presumed
GENERIC TITLES
33
m
O
m
-o
-I
O
z
s>
C/)
FLAGELLATE TYPES
c^rz!:^
:^
TRYPANOSOME
TRYPANOSOME
GRITHIDIA
LEPTOMONAS
LEISHMANIA
(2
LEISHMANIA
Fig. 153. — Diagram of Classification of the Trypanosomes and Allied
Flagellates. (After Wenton, 1913, 1921.)
that infection passes from invertebrate to vertebrate or from vertebrate
to invertebrate, but the exact mechanism of this is not yet known.
5. Flagellates of the genus Phytomonas, which resemble those of the
genus Leptomonas, but differ in having both an invertebrate and plant
host.
6. Flagellates of the genus Trypanosoma have also a vertebrate and
invertebrate host. They resemble those of the genus Herpetomonas in
320 FAMILY: TRYPANOSOMID.E
having at various stages of their development all four types of flagellate.
They are passed from invertebrate to vertebrate or from vertebrate to
invertebrate.
It will be seen that to complete the series there should be a form with
two hosts corresponding with the genus Crithidia, but no such flagellate
is known at present.
This grouping of these nearly related organisms in the genera defined
above may be represented in tabular form (Fig. 153).
As will be explained below, the type species of the genus Leptomonas
is a flagellate of the nematode worm Trilobus gracilis. Unfortunately,
this flagellate has never been re-examined in the light of present-day
knowledge, so that there is some doubt as to its true nature. For the
present purpose it is regarded as having the structure of the members of
the genus as described above. The type species of the genus Herpeto-
monas is H. muscarum, a common flagellate of the house-fly. It is assumed
here that the trypanosome forms which may occur in association with
it are actually stages in its development, and recent work supports this
view, though some observers believe they belong to a distinct parasite
of flies. If this latter view should prove correct, then a new name would
have to be found for the genus Herj^etomonas as here defined. It would,
however, in no way invalidate the scheme of classification. Knowledge
concerning many of these flagellates is still very imperfect. It is probable
that some of those which are only known in an invertebrate host will
ultimately be found to have a vertebrate one also. On the other hand,
those which are only known in a vertebrate host have undoubtedly an
invertebrate host as well, though it is at present unknown. There is only
one exception to this rule in Trypanosoma equiperdum, the cause of
dourine of horses, which passes from vertebrate to vertebrate without the
intermediary of an invertebrate, though the possibility of an alternative
method of transmission through an invertebrate cannot be excluded
entirely.
The matter is still further complicated by the claims made by some
observers that certain of these flagellates, which in nature appear to be
purely insect parasites, are experimentally inoculable into vertebrates,
and produce in them a condition somewhat resembling that produced
by flagellates which naturally have both hosts. It will be realized how
closely all the forms are related to one another. Moreover, some of them
appear to be actually in a process of transition from one genus to
another. This relationship is further illustrated by their behaviour in
culture media. For instance, a flagellate which is only known in the
trypanosome stage in the blood of a vertebrate will in such a culture
develop into a multiplicity of crithidia, leptomonas, and leishmania forms.
CYTOLOGY 321
Similarly, it was first shown by Rogers (1904) that the leishmania forms
of the parasites of kala-azar were in reality flagellates by their develop-
ment into leptomonas forms in culture.
It is probable that the transformation which occurs in culture is an
imitation of that which takes place in the invertebrate host.
In this description, wherever the words leishmania, leptomonas, cri-
thidia, or trypanosome are employed in an adjectival sense, they refer
to stages in the development of any of the flagellates which show these
particular forms, and are not used in a generic sense. When they are
employed as nouns, they refer to a member of the particular genus. Thus
one may speak of a leishmania form of a trypanosome, a trypanosome
form of a herpetomonas, or a leptomonas form of a leishmania.
CYTOLOGY OF TRYPANOSOMES AND THE ALLIED FLAGELLATES.
Before describing the individual genera, it will be necessary to consider
the structure and method of multiplication in greater detail. The whole
group shows a marked uniformity of minute structure, though, as will be
seen, considerable variation occurs in the actual size and shape of the body,
especially amongst the trypanosomes.
CYTOPLASM.— The body is covered by the periplast, and within it
the cytoplasm is generally perfectly clear and of a very fine alveolar struc-
ture. Vacuoles may be present, especially the one already referred to,
which is near the kinetoplast. It has been described as contractile in
Herpetomonas tnuscarum, but this is probably incorrect. Occasionally,
forms with a highly vacuolated cytoplasm are seen, but these are abnormal
or degenerating individuals.
Apart from the periplast, the cytoplasm is of uniform consistency,
and shows no differentiation into ectoplasm and endoplasm. Immediately
below the periplast longitudinal fibres can sometimes be made out,
especially in some of the larger trypanosomes (Fig. 28, B). These are
generally supposed to be myonemes or contractile fibres. Minchin (1909,
1909a), who had observed them in the trypanosomes of the perch and eel,
was unable to distinguish them in the smaller Trypanosoma lewisi.
GRANULES.— Various kinds of granule may be present in the cyto-
plasm, especially anterior to the nucleus. Some of these may take a red
or purple tint with Romanowsky staining. In T. lewisi, Minchin (1909a)
refers to them as " chromatoid granules," and many writers have con-
sidered them to be volutin. On account of their affinity for certain
chromatin stains, they have sometimes been mistaken for nuclei.
Flagellates of this group are sometimes packed with these granules,
but their presence seems to depend upon the rate of metabolism. The
21
322
FAMILY: TRYPAKOSOMIDiE
Fig. 154. — Various Internal Structures which have been described im
TrYPANOSOMID^. (1-5, AFTER ChATTON AND LeGER, 1911; 6, AFTER Mc-
CULLOCH, 1915; 7, AFTER PROWAZEK, 1904: 8, AFTER MlNCHlN, 1909; 9-10,
AFTER NiESCIlUI.Z, 1922.)
[For descriplion s&e opposUe page.
CYTOLOGY 323
flagellates which occur in cultures of trypanosonies and leishniania are
sometimes perfectly free from granules of this kind, while at other times
many are present. Their exact nature is doubtful, but there is no evidence
that they have originated from the chromatin of the nucleus, as some have
supposed. Doflein (1910) has noted the presence of fat globules in the
flagellates occurring in old cultures of Trypanosoma rotatorium of the
frog. In Herpetomonas muscarmn in the posterior region of the body
there sometimes occur rod-shaped structures which show bipolar staining
(Fig. 159). The writer (1913a) regarded them as bacteria which had
entered the cytoplasm, but similar structures in other flagellates have
been interpreted as evidence of a process of internal budding (see p. 338).
Granules other than metaplastic ones which have arisen as a result of
metabolism have been occasionally described in the cytoplasm. Thus, in
Trypanosoma raice (Fig. 247, 12, 13) a granule surrounded by a clear area and
lying near the nucleus was described by Robertson (1909a). Minchin (1909a)
described a refringent granule lying behind the kinetoplast in Trypano-
soma lewisi. It was present in specimens killed by osmic acid vapour and
examined wet without further treatment, but was not detected in stained
specimens (Fig. 154, 8). The writer recently noted that in an ordinary
dried and stained film of T. lewisi nearly every trypanosome possessed a
fairly deeply staining granule surrounded by a clear halo (Fig. 197, 16).
It was of uniform appearance and adjacent to the nucleus. No fibre or
filament could be detected in connection with it, and no suggestion can
be offered as to its nature or function. In some individuals it was rod-
shaped and in others double. A similar rod-shaped body was described
by Nieschulz (1922a) in the cultural forms of bird trypanosonies (Fig.
154, 10).
AXIAL AND OTHER FILAMENTS.— Another structure which has been
described is the axial filament. Prowazek (1905) depicted a complicated
fibrillar system in T. lewisi. A filament connected the karyosome of the
nucleus with the parabasal body, from which another filament ran through
the cytoplasm to another granule situated posteriorly to the nucleus, while
from it another passed to the anterior end of the body. None of these
structures were detected by Minchin (1909a) in his careful study of the
cytology of T. lewisi. Prowazek (1904) described an axial filament in
Herpetomonas muscarum as extending from a centrosome associated with
1-5. Herpitomonas drosophilcs in division, showing formation of axoplast from the dividing
kinetoplast (x c.a. 4,000).
6. Crithidia leptocoridis, showing complicated system of fibrils (x .3, .500).
7. Herpetomonas muscarum, showing fibrils ( x m. 4.000).
8. Trypanosoma lewisi, showing retractile granule, which is present in wet osmic killed trypano-
somes (x 3,000).
9-10. Cultural forms of bird trypanosome, showing granule within nuclear membrane and rod-
shaped structure in the cytoplasm ( x 3,000).
324 FAMILY: TRYPANOSOMIDiE
the kinetoplast to another centrosomic body near the posterior end of the
flagellate (Fig. 154, 7). The writer (1913a) was unable to detect such a
filament in this flagellate. A similar filament (axoplast) was described
by Chatton and Leger, M. (1911), in Leptomonas (Herpetomonas) droso-
philce (Fig. 154, 1-5). It extended from the kinetoplast to the posterior
end of the body. When division was taking place it degenerated, and a
new one was formed between the two daughter kinetoplasts. At first
straight, it soon became U-shaped. The limbs of the U increased in length
till finally each was as long as the body, which meanwhile had commenced
dividing from its anterior end. When division of the body was complete,
the limbs of the U formed the new " axoplasts " of the daughter flagellates.
In Crithidia leptocoridis parasitic in the gut of the box-elder bug, Mc-
Culloch (1915) has described a system of fibres still more complicated
(Fig. 154, 6). In this case the " axostyle " commencing in the blepharo-
plast runs to the posterior end of the body, where it terminates in a granule
called the " chromatin granule." In addition, there is a fibre connecting
the blepharoplast or the kinetoplast with the karyosome of the nucleus,
and another (" myoneme ") running from the posterior end of the body
to terminate in the flagellum. In a subsequent work, however, the same
writer (1919) gives a diagram of C. lejJtocoridis which only shows one of
these fibres — namely, that connecting the blepharoplast with the karyo-
some of the nucleus. The majority of observers have not detected or
described these fibres in the flagellates they have examined. The writer
(1913a) has examined many forms which have been carefully fixed and
stained, and has seen no such structures present with the constancy which
would be expected if they were essential parts of the anatomy. If large
numbers of individuals of any species are examined, occasionally fibres
resembling those described by the various writers may be seen, but other
explanations of their presence can be given. Folds or creases in the peri-
plast or abnormally developed flagella may give rise to these appearances.
The whole group is such a homogeneous one that it is highly improbable
that structures so complicated would be present in one species and com-
pletely absent in another.
A fibre connecting the blepharoplast or parabasal with the karyosome
of the nucleus has been more frequently described. McCulloch (1915)
refers to it as the rhizoplast, and not only mentions its occurrence in
Crithidia leptocoridis (Fig. 154, 6), but also (1917) in C. euryophthalmi
(Fig. 168), while Kofoid and McCulloch (1916) note it in Trypanosoma
{Herpetomonas) triatomw, a flagellate of the bug Neotoma fuscipes, Chagas
(1909) described a similar connecting fibril in the case of the developmental
forms of Trypanosoma cruzi in the bug Triatoma megista.
Here, again, it may be said that this fibre has not been seen with any
CYTOLOGY
325
constancy, and, if present, it has been overlooked by the majority of
competent observers. The complicated system of fibres associated with
the nuclear changes and divisions of Trypanosomu noctuce and its develop-
ment in the owl and mosquito, as described by Schaudinn (1904), are
only of historic interest.
An axial filament was described in Herpetomonas muscarum from the
fly Calliphora erythrocephala by Alexeieff (1911e). He also observed in
this fly the peculiar Rhynchoidomonas forms first described by Patton
Fig. 155. — Herpetomonas muscarum from Intestine of Calliphora erythrocephala
(x 1,500), ARRANGED TO SHOW AlEXEIEFF'S ViEW OF THE ORIENTATION OF
THE Rhynchoidomonas Forms. (After Alexeieff, 1911.)
1. Typical leptomonas. 2. Leptomonas with rhizostyle.
3-4. Axoneme of flagellum still visible, while rhizostyle is more marked.
5-6. Axoneme no longer visible, while rhizostyle is well developed.
7. Dividing form.
It appears j/robable. however, that the forms at 3-7 should be reversed, and that the structure
called the rhizostyle is in reality the attached axoneme, which is longer owing to the back-
ward migration of the kinetoplast.
(1910a) in Lucilia (Fig. 155). In the latter there is a deeply staining
line running from the kinetoplast along the surface of the body past
the nucleus to the pointed extremity of the body. The other extremity
is drawn out into a tapering process. In some of these forms there
is to be detected a faintly staining line extending from the kinetoplast
to the end of this process. The natural interpretation of this appear-
ance would be that the deeply staining band is the axoneme, and
that the flagellates have the trypanosome structure, though a some-
what remarkable one. Alexeieff, however, interprets them differently.
326 FAMILY: TRYPANOSOMID^
He regards the deeply staining band as the axial filament, here well
developed, while the faintly staining line passing to the opposite extremity,
and not always present, as representing the axoneme. Of the two views,
it seems to the writer that the first one is correct, otherwise one must
assume that this particular form is totally different from all other flagel-
lates of the group. The writer (1913a) studied these forms in the house-
fly, and could find no evidence to support Alexeiefl's view.
NUCLEUS. — The nucleus, which can only be satisfactorily studied in
specimens which have been prepared without drying, generally consists of
a nuclear membrane enclosing a clear space, at the centre of which is a
karyosome which stains deeply with chromatin stains. In some of the
larger trypanosomes, radiating fibres — the nuclear meshwork — can be
seen connecting the karyosome with the nuclear membrane (Fig. 247).
The latter is clearly seen as a definite structure in the largest trypano-
somes. In the smaller forms it may be difficult to distinguish from the
surrounding cytoplasm, the nucleus appearing as a clear space in the
cytoplasm with the karyosome at its centre. In such cases it is probably
safe to assume that a very fine membrane is present, for the clear space
persists in nuclear division, during which it becomes elongated and finally
divided. In the nuclei of the larger trypanosomes, in addition to the
central karyosome, smaller granules, apparently of a chromatin nature,
are sometimes present on the inner surface of the nuclear membrane or
even distributed upon the nuclear meshwork. Occasionally, in place of
the single karyosome, several comparatively large chromatic bodies are
present. The karyosome usually stains uniformly and intensely, but in the
larger forms several more deeply staining areas may be present, suggesting,
as Robertson (1909a) has pointed out for Trypanosoma raicB, that it may be
made up of two substances, the chromatin proper and a plastin material.
Sometimes a more deeply staining granule has been distinguished at the
centre of the karyosome, and from what occurs in nuclear division this
granule has been interpreted as a centriole or intranuclear centrosome.
Nieschulz (1922a) has noted the presence of a small granule on the nuclear
membrane of the crithidia forms which appear in cultures of bird trypano-
somes (Fig. 154, 9). It was not present in the trypanosome forms. It
miist be remembered, however, that the appearance of the karyosome
after such stains as iron hsematoxylin varies considerably with the degree
of extraction of the stain.
In nuclear division, as usually seen in wet fixed films, there is an elonga-
tion of the nucleus associated with an elongation of the karyosome (Fig.
156, i). The latter finally becomes constricted at its centre and divided into
two daughter karyosomes. The nuclear membrane then becomes constricted
between these, and two nuclei similar to the original one are produced.
CYTOLOGY 327
The division is generally equal, so that the two daughter nuclei are approxi-
mately of the same size. Not infrequently during division the nucleus
becomes much elongated, while the halves of the divided karyosome occupy
the poles and are connected by a fine line, which is usually referred to as
the centrodesmose, and is supposed to connect the two daughter centrioles
which may be presumed to lie embedded in the daughter karyosomes. This
is all the more striking in cases which have sometimes been noted where the
chromatin of the nucleus, instead of being concentrated into a single karyo-
some, is in the form of two or more granules lying on the inner surface of
the nuclear membrane. At nuclear division, a small central granule first
divides, and the two separating halves are connected by a fibre. The
chromatin granules themselves then divide into two groups, and two
daughter nuclei are ultimately formed. In these cases the central
granule, which may or may not be a true centrosome, is not obscured by
concentration of the chromatin around it in the form of a karyosome.
In other cases it would seem that the central granule divides, and the
halves, still connected by a fibre, separate, and eventually pass out of
the karyosome, the division of which is retarded. There is then produced
an elongated nucleus with a granule at each end, and a centrodesmose
connecting them. The karyosome, still undivided, lies at the centre of
the centrodesmose. Though the gradual elongation and constriction of
a uniformly staining karyosome is the usual appearance in nuclear division,
a number of observers have maintained that after appropriate staining
the elongating karyosome can be resolved into a spindle, upon the fibres
of which the chromatin is distributed in the form of granules or chromo-
somes. Schaudinn (1904) described a mitotic division of the nucleus in
Trijpanosoma noctucB ; Rosenbusch (1908-09) in T. lewisi and T. brucei ;
Hindle (1909) in T. dim.orphon ; Chagas (1909) in T. cruzi ; Alexeieff (1912e)
in T. lewisi and T. brucei; Kuhn and Schuckmann (1912), Kuczynski
(1917), and Schuurmans-Stekhoven (1919) in T. brucei; Hartmann and
Noller (1918) in T. theileri ; and Nieschulz (1922a) in bird trypanosomes.
Hartmann and Noller state that in the case of T. theileri there is formed
an intranuclear spindle which appears to originate in the achromatic
material of the karyosome, while the chromatin substance in the form of
granules is first distributed irregularly on the spindle (Fig. 156, 5-8). The
spindle elongates, as also does the nuclear membrane, so that there results
a long oval nucleus enclosing a sharp-pointed spindle, the ends of which
may rest on the nuclear membrane. At this stage the chromatin granules
collect at the equator of the spindle to form an equatorial plate, which
quickly divides into daughter plates. The latter move towards the poles
of the spindle, wdiich divides at its centre. Each half, with the chromatin
granules of the daughter plate, which have again become irregularly dis-
328
FAMILY: TRYPANOSOMID.E
tributed on the spindle, concentrates into the daughter karyosome.
Nuclear division is completed by the division of the nuclear membrane.
During the whole of this process the nuclear membrane has persisted and
enclosed the spindle. Nieschuiz (1922a) has described a similar method
^ (^
i
Fig. 156. — Nuclear Division ik Trypanosomid.e. (1, Original; 2-3, after
NiESCHULZ, 1922; 4, after Citagas, 1909; 5-8, after Hartmann and Noller,
1918.)
1. Usual apiiearance in wet fixed films stained by iron hajmatoxylin.
2-3. Two stages in division of the cultural form of the tryimnosome of the ling-ousel [Turdus
torquatvs), showing mitosis of the nucleus ( x 3,000).
4. Division of the nucleus of Trypanosoma cruzi by mitosis (x ca. 3,0C0).
5-8. Division of cultural forms of Trypanosoma thcilcri, showing mitotic division of the nuclei
(X ca. 2,600).
of division of the nuclei of cultural forms of bird trypanosomes (Fig. 156,
2-4). He noted the formation of an equatorial plate which divided to
form daughter plates, but was unable to distinguish individual chromo-
somes, the plates appearing as dark bands across the spindle. Schulz
CYTOLOGY 329
(1924) has found that the nucleus of Leishmania donovani divides by
mitosis, as also that of Leptomonas fasciculata in cultures from the intestine
of Culex pipiens and Theobaldia annulata. It seems probable, therefore,
that the nucleus of the trypanosomes and the allied flagellates divides by
a form of mitosis, but the process is difficult to detect in such minute
objects, and can only be demonstrated by special staining. The ordinary
appearance at nuclear division is that of an elongating, deeply staining
karyosome which becomes constricted at the centre, as described above.
An unusual type of nucleus has been described in the broad, leaf-like
forms of Trypanosoma rotatorium (Fig. 150, 38). It was first noted by
Franga and Athias (1906) in the trypanosome of Hyla, and then by Martin
(1907) in T. boueti. It was subsequently observed by other writers in
T. rotatorium of frogs (see p. 592). Instead of being spherical, the nucleus
is in the form of a long spindle, which is frequently curved. One end is
near the kinetoplast, while the other may be near the posterior extremity
of the flagellate. It has been shown by Noller (19136) that the large
trypanosomes of this type in the frog are the survivors of a tadpole
infection with flagellates of the more normal type, and are to be re-
garded as abnormal or overgrowth forms. The long drawn-out nucleus
may represent a division process which has been arrested at this stage.
KINETOPLAST.— The kinetoplast consists of the blepharoplast and
parabasal body. The two appear to be united. This union has been
described as a system of fibres forming a cone, with the parabasal at its
base and the blepharoplast at the apex, or as a definite membrane enclosing
a space with the parabasal and blepharoplast at opposite poles. That
there is such a membrane is borne out by certain appearances seen in
degenerating trypanosomes, which the writer (1913a) studied in smears
made from heavily infected animals some hours after death. It was noted
that many trypanosomes were in various stages of disintegration, and
that frequently the cytoplasm had disappeared, leaving only the nucleus
and the kinetoplast, with the axoneme and flagellum still attached
(Fig. 157). The axoneme will be seen to terminate in the blepharoplast,
which appears to be lying on the surface of a membrane connecting
it with the parabasal. If the parabasal were a free and independent
structure, it would be expected that in disintegration or cytolysis the
parabasals would not remain united, as they appear to do. Dividing or
already divided blepharoplasts and parabasals still show this connection
with one another and with the axoneme of the flagellum. It is interesting
to note that in these degenerating forms there is no evidence of a fibre
connecting the kinetoplast with the karyosome of the nucleus, as several
observers have described.
The first indications of division are seen in the kinetoplast. The
330
FAMILY: TRYPANOSOMIDiE
blepharoplast becomes transversely elongated, narrowed at its centre,
and finally divided into two parts, the axoneme remaining attached to
one half, Coincidently with this the parabasal, if a rounded body, also
becomes transversely elongated, constricted, and divided into two, and
finally two kinetoplasts are produced, each constituted as the original one.
In flagellates like Herptotnonas muscarum, the kinetoplast is an elongated
structure with the blepharoplast at its anterior end and the parabasal
at its centre. The blepharoplast first divides, and the two halves separate
(Pig. 158). The parabasal becomes dumb-bell shaped and also divides.
At a certain stage of the process the dividing kinetoplast is in the form
of a Y, wdth a blepharoplast at the end of each of the two anteriorly
Fig. 157. — Kinetoplasts and Attached Axonemes of Degenerate Trypanosoma
rhodesiense, as seen in Dried Films stained by Leishman Stain. (After
Wenyon, 1913.)
1. Blepharoplast united to parabasal; the part of the axoneme which borders the membrane
appears thicker than the intracytoplasmic portion, owing to a sheath of cytoplasm.
2. Early division with dividing blepharoplast.
3. Division of blepharoplast and parabasal.
4. Completed division of parabasal and blepharoplast and outgrowth of new axoneme.
.5 and (3. New axonemes forming before division of parabasal is complete.
directed limbs. The various stages of this division process give the
impression that the blepharoplast is leading the way in division, and it
appears as if the stress exerted by the separating daughter blepharoplasts
causes the parabasal and the entire kinetoplast to divide. The writer
(1913a) compared this division with that of the nucleus of Cercomonas
longicauda, which in the resting condition is composed of a spherical
nuclear membrane with large central karyosome. The anterior part
of the membrane is really cone-shaped, and at the apex of the cone is
the blepharoplast, from which two flagella arise. In nuclear division
the blepharoplast first divides. As the two halves separate a spindle
CYTOLOGY 331
is formed between them, while the karyosome breaks up into chromo-
somes, which arrange themselves upon the spindle as an equatorial plate.
In this case the blepharoplast is undoubtedly functioning as a true centro-
some. In many respects the division of the nucleus of Cercomonas longi-
cauda, as also that of Heteromita uncinata, resembles that of the kinetoplast
of Herpetomonas muscarum, in which, however, the parabasal does not
form chromosomes, but merely splits into two equal parts (Figs. 68 and 259).
Kobertson (1913) has shown that in Trypanosoma gambiense division of
the kinetoplast may take place in such a manner that the two daughter
blepharoplasts are connected by a fibre, at the central point of which the
parabasal is situated. When the daughter blepharoplasts still further sepa-
rate, the parabasal divides and two kinetoplasts are thus formed. Such
a method of division has a striking resemblance to that frequently seen
A 1 /\ /\ An
Fig. 158. — Dividing Kinetoplasts of Rerpetomonas muscarum. (After Wenyon,
1913.)
Upper row after fixation in Hermann's fluid.
Lower row after fixation in Schaiidinn's fluid.
in division of the nucleus itself. This raises the question of the nuclear
nature of the kinetoplast. Hartmann (1907), assuming this to be the
case, regards the flagellates of this group as possessing two nuclei, and
places them in a special order, the Binucleata. The evidence of the true
nuclear nature of the kinetoplast in the sense that it is a second nucleus
is, however, wanting. The mitotic divisions of the kinetoplast with
formation of chromosomes, wdiich some observers have recorded, can
hardly be taken seriously.
If, however, such a mitotic division actually occurred, it would be
strong presumptive evidence of the nuclear nature of the kinetoplast.
From analogy with other flagellates, the terminology here employed
appears to be the safest one. In many flagellates, axonemes arise from a
blepharoplast in close association with which is a parabasal body which
332 FAMILY: TRYPANOSOMID^
assumes various shapes and forms. In division, both the blepharoplast
and parabasal body divide (Fig. 32).
Schaudinn (1904), in his description of what he regarded as the origin
of the " binucleate " trypanosome from the uninucleate halteridium of
the little owl, supposed the kinetoplast to arise by an unequal division
(heteropolar mitosis) of the nucleus, the smaller portion becoming the
kinetoplast. The origin of the flagellar blepharoplast from the nucleus
was described by Jameson (1914) in Parapolytoma satura, and by Entz
(1913, 1918) in Polytoma uvella (see p. 52). Though there may be some
evidence that in other flagellates the blepharoplast arises by division of
an intranuclear centrosome (P. uvella), the proof that the flagellates of
the trypanosome group have a stage devoid of a kinetoplast is still wanting.
At certain stages of development of these flagellates, especially when they
assume the leishmania form, the kinetoplast approximates to the nucleus
to such an extent that the two frequently lie in complete apposition, and
by some observers they have been supposed to fuse. Roubaud (19116,
1911c) described such a fusion in the leishmania forms of Herpetomonas
(Leptomonas) sudanensis, a flagellate of an African fly (Pycnosoma), but that
actual union had taken place seems very doubtful. In this connection,
mention may be made of the remarkable process of union of kinetoplast and
nucleus, which was first described by Moore and Breinl (1907) for Trypano-
soma gambiense, and later by the same observers (1908) for T. equip er dum ;
by Moore, Breinl, and Hindle (1908) for T. lewisi ; and by Fantham (1911)
for T. ga?nbiense and T. rhodesiense. The process is described as taking
place in the blood and organs of heavily infected experimental animals.
A periodic variation in the number of trypanosomes in the blood occurs,
and at the height of the wave there appear in the blood forms which possess
an axial filament extending from the kinetoplast to the nucleus. The
filament, which is supposed to consist of chromatin material, breaks up
into granules which fuse with the nucleus. The cytoplasm of the trypano-
some is then cast of!, leaving a small rounded body ("latent body"),
consisting of little more than the nucleus with which the chromatin of
the kinetoplast is supposed to have united. The " latent bodies " are
to be found in the smears of the lung, spleen, and other organs. They are
said to give rise to trypanosomes again by formation of cytoplasm around
them, while by division of the intranuclear centrosome, one half of which
separates, the new kinetoplast is formed. In the work of Moore, Breinl,
and Hindle, the transformation of the trypanosome into the "latent
body," and the converse process of the growth of the latter into the try-
panosome, was studied in stained films in which a series of forms supposed
to illustrate the process were depicted. That leishmania forms do actually
occur during the course of development of many trypanosomes, and that
CYTOLOGY 333
these may again grow into trypanosomes, there is no question, but it
must not be forgotten that in the body of heavily infected animals many
trypanosomes undergo degenerative changes. It seems to the writer that
the whole process as described by Moore and Breinl is the result of the
combination of these involution forms in a hypothetical cycle. Fantham
(1911), however, maintains that he actually observed the growth of these
latent bodies in vitro, and gives a figure purporting to show the trans-
formation of a small "latent body " of the leishmania type into a fully-
formed trypanosome possessing flagellum and undulating membrane. The
resulting organism was many times the bulk of the original body, and the
whole of this remarkable metamorphosis is stated to have taken place
in about one hour. It is known that the leishmania forms of the parasites
of oriental sore, or kala-azar, require at least forty-eight hours to become
fully-formed flagellates, so that it is impossible to accept the statement
regarding such a rapid development without confirmation.
It seems, therefore, that no reliable evidence of the origin of the
kinetoplast of trypanosomes and the allied flagellates from the nucleus
has yet been produced, and though in some respects the kinetoplast
behaves like a nucleus during division, this does not justify an assertion
that it is a true nucleus. The whole question is a very difficult one, and
involves the more general one of the definition of the nucleus itself.
FLAGELLUM. — The flagellum arises from the anterior end of the body,
and, like that of other flagellates, consists of a cytoplasmic sheath enclosing
an axial filament, the axoneme, which can be traced through the cytoplasm
to the blepharoplast. In flagellates of the crithidia and trypanosome
'type the axoneme passes to one side of the body, and then along the
surface of the body or on the edge of a ridge of cytoplasm — the undulating
membrane — to enter the flagellum at the anterior end of the flagellate.
The simplest arrangement occurs in organisms of the leptomonas type.
It can be seen that the axoneme is much finer than the flagellum, and
this is an indication that the flagellum is composed of an axial filament,
the actual continuation of the axoneme, and a sheath of the periplast.
In the degenerated and cytolized trypanosomes already referred to
(Fig. 157), it will be noted that the flagellum is still attached to the
blepharoplast by a very fine line which represents the intracytoplasmic
portion of the axoneme.
Furthermore, after division of the blepharoplast, the half which has
no attached axoneme commences to form a new one as an outgrowth,
which grows parallel to the original axoneme and close to it. When the
new axoneme reaches the surface of the body, it may continue its growth
within the sheath of the old flagellum, so that when the sheath divides
longitudinally, an appearance of longitudinal division of the flagellum
334
FAMILY: TRYPANOSOMIDiE
may result. As regards the origin of the new flagelliim, there has been
some difference of opinion. Some observers, as, for instance, Laveran and
Mesnil (1904, 1912), Minchin (1908), Woodcock (1909), and others, have
described longitudinal splitting of the flagellum, but the writer (1913a),
from observations on Herpetofnonas tnuscarum and trypanosomes, came
to the conclusion that a longitudinal division never occurs, and that the
new flagellum is the result of the growth of a new axoneme from the newly-
formed blepharoplast, a view previously upheld by Schaudinn (1904),
MacNeal (1904), and Prowazek (1905). Hartmann and Noller (1918), in
a study of the cytology of Tryfanosoma theileri, arrive at the same con-
FiG. 159. — Dividing Forms of Herpetomonas muscarum (x 2,000). (After
Wenton, 1913.)
The outgrowth of a new axoneme from the divided blephaioplast produces the appearance of an
organism with two flagella.
elusion, as also did Rosenbusch (1909a), though he gave an erroneous
description of the division of the kinetoplast. Mackinnon (1910), in the
case of H. homolomyicB and H. scatophagce, describes the intracytoplasmic
portion of the axoneme as dividing, the new flagellum being then formed
as an outgrowth of the new axoneme. There seems little doubt that the
flagellum is entirely formed as a result of the outgrowth of a new axoneme
from the new blepharoplast, a process which may even commence before
division of the blepharoplast is complete. In some cases the new flagel-
lum attains a considerable length before the kinetoplast is actually
divided, so that the organisms appear to have two flagella. //. muscarum
CYTOLOGY 335
is a case in point, for in certain infections this precocious flagellum for-
mation may have taken place to such an extent that nearly every indi-
vidual has two flagella (Fig. 159). It was this appearance which led
Prowazek (1904) to regard this organism as a biflagellate, and to state
that the type species of the genus Herpetomonas necessarily possessed two
flagella. It was proved, however, by the work of Patton (19086), Porter
(19096), Mackinnon (1910), and the writer (1911a) that the forms with
two flagella were in reality dividing forms in which precocious flagellum
formation had taken place in anticipation of coming division of the
flagellate.
Fran9a (1920ff) maintains that in H. muscarmn there is actually a
division of the kinetoplast, intracytoplasmic portion of the axoneme
(rhizoplast), and the flagellum, while in flagellates of the genus Lepto-
monas, after division of the kinetoplast, a new axoneme grows out from
the daughter blepharoplast to form a new rhizoplast and flagellum. It
appears to the writer that such a distinction cannot be drawn between
the genera Herpetomonas and Leptomonas.
UNDULATING MEMBRANE.— As already explained, in the crithidia
and trypanosome forms the axoneme passes along the surface of the body
to its anterior end, where it may or may not be continued into a flagellum.
In the majority of crithidia and trypanosomes the line of attachment of
the axoneme is raised into a thin ridge, the undulating membrane, which
varies in length with the position of the kinetoplast. It is attached to
the convex edge of the curved, blade-like body of the flagellate. The free
border is longer than the attached one, hence it is thrown into folds. The
axoneme runs along the free border, and the constant undulating move-
ments of the membrane are probably the result of contractions of the
axoneme, though some observers believe that they are due to certain
myoneme fibres which they claim to have detected. The membrane
consists of little more than the periplast, while the axoneme runs in a
canal at its margin. When the axoneme leaves the body of the flagellate,
the periplast is continued as the sheath of the flagellum. In some trypano -
somes the undulating membrane is broad and well developed {Trypanosoma
rotatorium), while in others it is very narrow (T. congolense). On the other
hand, in the trypanosome stages which occur in the development of
purely insect flagellates (Herpetomonas), well-developed undulating mem-
branes may be present in some cases, while in others there is no definite
membrane, the axoneme merely passing along the surface of the body
(Fig. 150, 40-45). This condition is well seen in the trypanosome stages
of H. mirabilis (Fig. 172).
336 FAMILY: TRYPANOSOMIDiE
METHOD OF REPRODUCTION.
Reproduction takes place most usually by longitudinal fission (Fig. 160).
After division of the kinetoplast and nucleus, and formation of a new
flagellum, as described above, the cytoplasm divides by a fission com-
mencing at the anterior end between the flagella and extending backwards
till two flagellates result. The resulting organisms remain attached by
their posterior ends for some time till final separation takes place. The
newly-formed flagellates are usually equal in size, but quite frequently they
are unequal, so that a small form may be separated from one many times
its size. In these cases, though the cytoplasm of the two daughter
forms may differ in amount, the nuclei and kinetoplasts are equally divided.
In the crithidia and trypanosome forms the division is more complicated,
owing to the presence of the undulating membrane (Fig. 160, 9 i6). In
both these, after division of the blepharoplast has commenced, a new
axoneme begins to form from what will be the daughter blepharoplast.
This grows parallel to the original axoneme along the border of the un-
dulating membrane, and as the extremity of the new axoneme is closely
applied to the original one, the impression of longitudinal splitting may be
given. After the new axoneme has grown to some extent, the undulating
membrane commences to split from behind forwards and between the two
axonemes. The point up to which the membrane is split at any stage of
the process is always a little behind the end of the new axoneme, so that
beyond the point to which the membrane has split the new and old axonemes
still lie close together. The stage at which complete separation of the
axonemes takes place or the extent of division of the membrane depends
on the size of the daughter individual. If division into two equal flagellates
is taking place, then the process of new axoneme formation and splitting
of the membrane extends right up to the anterior end of the flagellate. If,
however, the division is into unequal flagellates, then there is a shorter
axoneme formed and a shorter membrane is split off. In either case there
results a flagellate with two axonemes, flagella, and undulating membranes,
and as division of the kinetoplast and nucleus will then be complete, with
two kinetoplasts and two nuclei. At this stage the body of the flagellate
divides, the fission commencing at the anterior end between the two flagella
and membranes. It extends backwards till two flagellates are formed,
each with a nucleus, kinetoplast, undulating membrane, and flagellum.
In the division of a trypanosome, therefore, the splitting of the membrane
and growth of the axoneme takes place from behind forwards, while
the body divides from before backwards (Fig. 160, 13-16). Division of
the non-flagellate leishmania forms also takes place after division of the
kinetoplast and nucleus. In these cases, in which the axoneme is still
REPRODUCTION
337
present, it will be seen that at division a new axoneme is formed from the
blepharoplast, but it does not extend beyond the surface of the body into
Fig. 160. — Diagrammatic Representation of Method of Division of the
Various Forms of the Trypanosomid.e. (Original.)
1-4. Division of leishmania form. 5-8. Division of leptomonas form.
9-12. Division of crithidia form. 13-16. Division of trypanosorae form.
17-20. Delayed division of cytoplasm resulting in appearance of multiple division of trypanosome
form.
a flagellum. The more or less rounded body then divides between the
axonemes.
It not infrequently happens that, after division of the kinetoplast
I.
22
338
FAMILY: TRYPANOSOMID^
and nucleus, and new flagellum formation, the division of the body is
delayed for some reason (Fig. 160, 17-20). The daughter kinetoplasts
and nuclei may proceed to a further division, giving rise to forms with
four sets of these structures. Such forms are sometimes seen in actively
multiplying trypanosomes like T. brucei in the blood of a rat, where in-
dividuals with four nuclei, four kinetoplasts, and four undulating mem-
branes and flagella arise. By a further division of one or more of the
nuclei and kinetoplasts, still more complicated forms are produced.
Fig. 161. — Crithidia enryoplithalmi from Fig. 162. — Crithidia euryophthalmi
Intestine of the Bug, EuryopJithalmus
convinus (x ca. 2,400). (After McCul-
LOCH, 1919.)
Multiple division forms (spheres) in the epithelial
cells of the crop.
FROM Intestine of the Bug,
Euryophthalmus convinus ( x 3,500).
(After McCuleocii, 1919.)
Forms showing what is described as in-
ternal budding.
During the development of T. lewisi in the flea, the intracellular phase
results in the formation of multinucleate forms, in which as many as
sixteen trypanosomes are represented before cytoplasmic division occurs
(Fig, 200, 5-11). A similar form has been described by McCulloch (1917)
as an intracellular phase of the development of Crithidia euryophthalmi
in the crop of the bug, Euryophthalmus convivus (Fig. 161). Multiple
segmentation forms of T. lewisi also occur in the blood of the rat
in the early phases of an infection (Fig. 197). The process has been
SYNGAMY 339
described for Leishmanin donovani by Mackie (1915ffl), and for T. cruzi
by Hartmann (1917), but in both these cases the descriptions are not
convincing, for it would appear from the figures produced that the
so-called schizonts are merely the broken-of! portions of the cytoplasm
of large cells enclosing leishmania which, as a result of degeneration or
feeble staining, have their outlines imperfectly defined, so that the appear-
ance of cytoplasmic bodies containing many pairs of nuclei and kineto-
plasts is produced. It must be remembered that simple binary fission
is the normal method of reproduction, and the multiple segmentation
often termed schizogony merely represents retarded division of the cyto-
plasm, and is not to be compared with the true schizogony which occurs
in the Sporozoa as the normal method of multiplication.
Mention must be made of another type of reproduction which has been
described in only one instance. This is the so-called internal budding
noted by McCulloch (1919) as occurring in Crithidia euryophthalmi (Figs.
162 and 168). In this process, repeated division of the nucleus is supposed
to take place till the posterior region of the body contains a varying
number of separate nuclei. Around each a portion of cytoplasm becomes
concentrated, while a kinetoplast is formed from the nucleus. The
original flagellum, together with the axoneme and kinetoplast, degenerates.
There is thus formed an elongated cytoplasmic body in which are em-
bedded a number of leishmania forms. Presumably by rupture of the
cytoplasm of the original parent, these escape and produce the crithidia
forms again. This is a remarkable process which has not hitherto been
observed. The figures, which are supposed to illustrate the process,
are far from convincing, and suggest the possibility that yeasts or
other structures, or even leishmania forms of the flagellate itself, may
have been adherent to the surface of the organisms. Minchin and
Thomson, J. D. (1915), however, mention certain structures in the cyto-
plasm of Lejptomonas pattoni as evidence of a possible endogenous bud
formation, a process which they also consider may occur in T. lewisi.
They, however, never actually observed the formation of buds, and did
not feel justified in describing the process.
SYNGAMY.
The possibility of a sexual process occurring in trypanosomes and their
allies has attracted considerable attention. Schaudinn (1904) described
syngamy in the case of Trypanosoma noctuce, and Prowazek (1904, 1905)
in T. lewisi and Herpetomonas muscarum. Various processes of syngamy,
parthenogenesis (development of the female gamete without fertilization),
and ethiogenesis (development of the male gamete without fertilization),
340 FAMILY: TRYPANOSOMID/E
are included in the complicated descriptions of the latter writer. The
observations, or more correctly the deductions, made by him were un-
doubtedly the outcome of a theoretical bias, and cannot be accepted.
Influenced by these statements, numerous observers, without any evidence
whatever, have described as male and female flagellates the narrow and
broad forms which occur in almost every infection. The figured stages
of conjugation can always be interpreted as the final stages of a division
in which a narrow form, the supposed male, is separating from a broader
one, the supposed female. The instance recently described by Fran9a
(1920cf) for Phytomonas davidi is unconvincing, as the forms figured can
easily be explained as dividing individuals or the casual association of
two flagellates.
As regards the pathogenic trypanosomes. Woodcock and Lapage (1915)
go to the other extreme, and suggest the complete loss of syngamy in this
group. It is quite possible that a sexual process takes place in association
with the development of trypanosomes in their invertebrate hosts, but the
most careful observations, such as those of Robertson (1913) on the develop-
ment of T. gambiense in tsetse flies, and of Minchin and Thomson (1915)
on T. lewisi in the flea, have failed to reveal one, though the possibility
of its having escaped detection is admitted. In order to demonstrate a
sexual process, something more than a casual association of two unequal
forms in stained films is necessary. The formation of "latent bodies,"
as described by Breinl and Moore, and referred to above, was supposed by
them, without any real evidence, to depend on a fusion of the kinetoplast
and nucleus, and to represent a process of self-fertilization or autogamy.
Roubaud (1911c, 19126) noted that in certain insect flagellates, when
they became leishmania forms in the hind-gut in preparation for encyst-
ment, the kinetoplast approached the nucleus. He concluded that actual
fusion of the two sometimes took place, and that a process of autogamy
was represented. It is very doubtful if any fusion occurs, as the dis-
turbing effect of drying the parasites on films so obscures the true nuclear
structure that it is impossible to be certain that any apparent fusion of
two closely applied bodies is not merely artificial. Quite apart from the
defects due to drying, it often happens that the kinetoplast lies over the
nucleus, and it becomes impossible to distinguish the two as separate
bodies in such minute organisms. Even if such a fusion as that described
takes place, there is no evidence that it represents syngamy in any form.
It is clearly evident that, of the numerous statements w^hich have been
made regarding the occurrence of sexual difierentiation and syngamy
amongst the Trypanosomidse, not one has any evidence to support it.
CYST FOKMATION 341
ENCYSTATION.
In those Trypanosomidse which are confined to insect hosts, infection
is spread from one to another by encysted forms passed in the fseces. In
the trypanosomes, however, cysts do not occur, as infection takes place
either by the inoculation of free forms through the proboscis, or by the
ingestion by the vertebrate of free forms passed in the faeces of the insect.
The cyst, in the case of the purely insect flagellates, is formed in the hind-
gut or rectum around small leishmania forms which are produced there
by the gradual shortening of the elongated forms (Fig. 150, 13-16, 24-26,
46-50). They are described as differing from the unencysted leishmania
forms in that they have a much more definite and deeply stained outline.
In some cases the cyst wall is depicted as quite thick, and even radially
striated. It may be very difficult to distinguish the kinetoplast from
the nucleus, the two structures often lying very close together or in com-
plete apposition. The process of encystment in Herpetofnonas muscarutn
was described by Prowazek (1904), and by Minchin (1908) in H. grayi
(Fig. 173, 10-16). In the latter the cysts, which were called " Schleim-
cysten " by Prowazek, commence to form around the blunt posterior
end of the flagellate, which is still in the elongate flagellate state. As
the cyst forms, the organism becomes more and more retracted and the
flagellum withdrawn, till finally it becomes a pear-shaped structure in
which the flagellum is represented only by the short axoneme in the cyto-
plasm. The cyst then closes round the more pointed anterior end. It
is at first of a gelatinous nature, and encloses a cytoplasm in which the
nucleus and kinetoplast can be distinguished. The axoneme finally
disappears, leaving in its place an area which stains red with Giemsa stain,
and this in its turn vanishes also. The nucleus and kinetoplast become
broken up into separate granules, so that their identity is difficult to make
out unless the more deeply staining ones are derived from the kinetoplast.
The cyst, at first pear-shaped, becomes more circular in outline, the more
or less spherical condition being the final one. There seems, however,
to be some doubt as to the nature of the structures called cysts in the
case of H. grayi. Koch (1906) suggested that the flagellate was possibly
the developmental form of the crocodile trypanosome, while Minchin
(1908) thought it possibly represented a bird trypanosome. Kleine
(1919a) claims that the flagellate is actually derived from the crocodile
trypanosome, as tsetse flies bred in the laboratory acquire an infection
when fed upon crocodiles harbouring this trypanosome; while Lloyd,
Johnson, Young, and Morrison (1924) have produced evidence that it
may be derived from either the crocodile, monitor, or toad. If, then,
the so-called cysts are actually true cysts, and not merely leishmania
342 FAMILY: TRYPANOSOMID^
forms — which, owing to deposit round them on the film, have taken
on the appearance of cysts — it has to be assumed that the flagellate of
the tsetse fly not only passes from fly to crocodile, but also from fly to fly
by means of cysts; or that the crocodile becomes infected by ingesting the
cysts passed in the faeces of the fly. The discovery by Lloyd (1924) of
a typical Leptomonas in the labial cavity of the proboscis and the mid-gut
of Glossina morsitans still further complicates the question of the nature
of the so-called cysts of these flagellates.
In the process of encystment of H. muscarum, the flagellate first
retracts itself to an ovoid body around which the cyst forms. The retrac-
tion, however, may take place in three different ways, as will be described
below (Fig. 171). That the cysts of insect flagellates are actually re-
sistant bodies has been proved by the writer (1912c) in the case of the
leptomonas of the flea, Pulex irritans (p. 351). It must be remarked,
however, that it is exceedingly difficult in most cases to form a definite
opinion as to whether a minute leishmania form, as seen in the hind-gut
of an insect, is actually encysted or not, and the mere fact that in
dried films stained by Romanowsky stain a red line surrounds them can
hardly be regarded as evidence of the presence of a cyst wall. The thick
envelopes which have been figured as cyst walls by numerous observers
are probably artefacts due to the staining of granular material which has
become heaped round the leishmania forms in the process of drying the
films. In other cases, the structures described as cysts have probably
been yeasts, or even spores of microsporidia. That a membrane actually
exists seems to be proved by the resistance of these forms to drying, but it
must be admitted that the detection of a cyst wall is a far more difficult
matter than many have supposed. Hoare (1923), in his work on the
development of the trypanosome of the sheep in the ked, draws attention
to the possibility of mistaking yeasts or other artefacts for cysts, and has
shown that the supposed cysts of the flagellate of the ked, which w^as at
one time thought to be a parasite peculiar to this insect, are merely
rounded forms, with deposits of stained granular material round them,
or yeasts. The cysts of H. grayi may be capable of a similar interpreta-
tion (Fig. 173). The changes in nuclear structure which occur during
the alleged encystation of this flagellate may be merely evidence of
degeneration.
GENERAL FEATURES OF THE LIFE-HISTORY.
In the description of genera given above it has been explained
that the flagellates belonging to the genera Herpetomonas, Crithidia, and
Leptomonas have only an invertebrate host, while those belonging to the
genera Trypanosoma and Leishmania have both vertebrate and inverte-
LIFE-HISTOKY 343
brate hosts. As regards the forms which occur in vertebrates, it will be
found that the organisms may assume any of the forms between the try-
panosome and the leishmania types. In the blood or other fluids of the
body they are usually provided with flagella, and have the trypanosome
structure, but occasionally, as in the case of Trypanosoma lewisi, free-
swimming forms of the crithidia or leptomonas type occur also. If the
flagellates are intracellular, they tend to be of the leishmania type, as in
the case of Leishmania donovani, L. tropica, and T. cruzi. In the case of
T. cruzi, however, after a number of intracellular leishmania forms have
been produced, they gradually become transformed through a crithidia
phase into flagellates having the trypanosome structure, while maintaining
their intracellular position. Any of these various forms, whether free-
swimming in the fluids of the body or in the cytoplasm of cells, can
reproduce by binary fission.
In the invertebrate host, the members of the genera Trypanosoma and
Herpetomonas may occur in any form between the leishmania and the
trypanosome. The members of the genus Crithidia never pass beyond
the crithidia stage, while those of the genus Leptomonas never pass beyond
the leptomonas stage. Multiplication of all these forms by binary fission
takes place as in the vertebrate. During the development in the inverte-
brate, the flagellates may be provided with flagella, by means of which
they swim freely in the lumen of the gut, proboscis, salivary gland, or in
other situations. These free forms were termed nectomonads by Minchin
and Thomson (1915). On the other hand they may attach themselves
to the lining cells by their anterior extremities, in which case the
flagella are lost, but in the arrangement of the nucleus, kinetoplast, and
axoneme they may have the trypanosome, crithidia, leptomonas, or leish-
mania structure. Such attached forms have been called haptomonads by
Woodcock (1914). The attached forms may retain their elongate character
in the anterior portions of the intestine. In the hind-gut there is a general
tendency for the elongate flagellates to become much shortened, though
the nuclei and kinetoplasts may retain their relative positions. The
haptomonad forms occur very commonly in the case of those flagellates
which are limited entirely to invertebrates, but they are also found in the
invertebrate phase of development of trypanosomes. Thus, they occur
in the case of T. lewisi in the hind-gut of fleas, T. vivax in the proboscis of
Glossina morsitans, and T. gambiense in the salivary glands of G. palpalis.
The flagellates belonging to the genera Herpetomonas, Crithidia, and
Leptomonas have but a single invertebrate host, infection being spread
by means of encysted leishmania forms passed in the dejecta. When
such an encysted form is eaten by a new host, the liberated leishmania
form gradually grows to the adult flagellate form. During this period of
344 FAMILY: TRYPANOSOMID^
growth, multiplication by binary fission may occur. The various forms
which occur before the adult flagellate stage is reached have been called
jne-fageUates by Patton (19086). When the fully-formed flagellate stage
has jjersisted and reproduced for some time, there occurs a gradual retrac-
tion of the body towards the leishmania form in preparation for encystment.
The forms leading to encystment have been called post-flagellates. These
are usually attached to the surface of the cells lining the hind-gut, and are
thus haptomonad forms. In the case of the development of trypanosomes
in the invertebrate, the final infective forms which pass back to the verte-
brate have the trypanosome structure, and have been developed from
attached or haptomonad forms of the crithidia type. These infective
forms have been termed metacyclic trypanosomes by Brumpt (1913). In
the case of some trypanosomes {T. lewisi, T. cruzi, T. melophagium), they
are produced in the hind-gut of the invertebrate and escape in the faeces,
which are ingested by the vertebrate (development in the posterior station) ;
while in others (T. gambiense, T, vivax, T. granulosum) they develop
in the anterior part of the alimentary tract, in the salivary glands (tsetse
flies), or proboscis sheath (leeches), and enter the vertebrate during the
biting act (development in the anterior station).
CLASSIFICATION.
The classification of the members of this family is a difficult one on
account of the many gaps in knowledge and the contradictory statements
made by different observers. The flagellates, which are limited entirely
to invertebrate hosts, are handed on from one to the other by encysted
forms in the faeces. Those which have both a vertebrate and invertebrate
host, as far as is known, always pass from the latter to the former in the
unencysted condition. In the case of flagellates of the genus Trypano-
soma, the infective forms are of the trypanosome type (metacyclic trypano-
somes). The exact form which is infective in the case of members of
the genus Leishmania is not known, but it may be assumed that encysted
forms are, at any rate, unnecessary. Assuming this to be the case, it is
possible to divide the members of the family into two groups — those
limited entirely to invertebrates, in which infection is contaminative
through one insect ingesting cysts passed by another {Leptomonas, Cri-
thidia, Herpetomonas), and those occurring in both vertebrate and in-
vertebrate hosts, in which infection is passed from the invertebrate to the
vertebrate by the former inoculating unencysted flagellates during the
act of feeding or passing unencysted flagellates in its faeces, which either
contaminate the puncture wound or are eaten by the vertebrate {Leish-
mania, Trypanosoma). The flagellates with two hosts can be divided into
two groups according as the highest stage of development is the leptomonas
CLASSIFICATION 345
form {Leishmania) or the trypanosome form {Trypanosoma). The latter,
again, can be subdivided into those forms, such as T. leivisi, which are
carried by fleas; T. cruzi, conveyed by reduviid bugs; T. melophagiutn,
transmitted by the sheep ked; and possibly T. theileri and its allies, the
invertebrate hosts of which are probably tabanid flies and the trypano-
somes of land reptiles, including crocodiles, in which development in the
invertebrate leads to the formation of metacyclic trypanosomes in the
hind-gut, and passage of these in the faeces (development in the posterior
station); or into those like T. gambiense, T. vivax, and T. congolense, and
the trypanosomes of some cold-blooded vertebrates, the development of
which in the invertebrate results in the formation of metacyclic trypano-
somes in the region of the proboscis and their inoculation during the biting
act (development in the anterior station). The trypanosomes which
develop in the anterior station, a term first proposed by Duke (1913), can
further be grouped into those developing in biting flies (the pathogenic
trypanosomes of mammals) and those which develop in leeches (the try-
panosomes of aquatic reptiles, amphibia, and fish). The trypanosomes
of birds are difficult to place, for some have claimed that development
takes place in the mosquito, and that they are inoculated at the time the
mosquito bites. On the other hand, it seems very probable that the true
host of the bird trypanosomes will be found amongst the ectoparasites
which infest the young in the nest, and it is possible that infection may be
contaminative, as in T. lewisi. For this reason, in the scheme of classifica-
tion given below, the trypanosomes of birds have been placed in both
groups with a note of interrogation. Similarly, the trypanosomes of
land reptiles, including crocodiles, are placed in both groups, for it is not
definitely known whether the development is in the anterior or posterior
station, though the latter is probable in the case of the trypanosomes of
the crocodile and the monitor which develop in tsetse. flies.
The trypanosomes which develop in biting flies in the anterior station
include the pathogenic forms of tropical Africa, which are conveyed by
species of Glossina (tsetse flies), and possibly the pathogenic forms of the
T. evansi type, including similar forms in many parts of the world, which
are conveyed by Tabanidse and their allies. It has not been actually demon-
strated that T. evansi develops in the anterior station in tabanid flies,
though its similarity to T. hrucei renders this not improbable. A develop-
ment in the posterior station is, however, possible.
As regards those which develop in tsetse flies, it will be shown below
that three types of development occur, as pointed out by Duke (1913) and
Bruce (1914). In one type the process commences in the stomach, but
the infection spreads forwards to the proboscis and ultimately to the
salivary glands, in which infective metacyclic trypanosomes are produced.
346 FAMILY: TRYPANOSOMID^
In the second the stomach phase occurs, and is followed by invasion of the
proboscis, but not the salivary glands. In the third the whole develop-
ment occurs in the proboscis, there being no stomach phase. As far as
present knowledge goes, the trypanosomes of cold-blooded vertebrates,
with the exception of those of land reptiles and crocodiles, develop in
leeches. There is a stomach phase leading to invasion of the proboscis
and proboscis sheath, from which trypanosomes escape into the wound as
the leech feeds. Finally, there is T. equiperdimi, in which an invertebrate
host is at all events unnecessary, the infection being handed directly from
vertebrate to vertebrate. This trypanosome is undoubtedly allied to
those transmitted by biting flies, and evidence has been produced that
infection can be sometimes spread by the agency of these insects. The
flagellates of the leptomonas type parasitic in euphorbias, which have
both an insect and plant host, have been separated under the generic
name Phytomonas.
The classification outlined above and arranged in tabular form below
has the advantage of convenience, if nothing more. It, however, recognizes
what is definitely known about these flagellates, and probably indicates
their phylogenetic history. The leishmania are probably derived from
insect leptomonas, and the trypanosomes from a crithidia or herpeto-
monas. Those which have a development in the anterior station may
have arisen by direct inoculation into the blood, while those with a
posterior station may have infected the vertebrate in the first instance
by way of the alimentary canal. It is known that certain lizards harbour
leptomonas in the intestine. It is very probable that this infection is
acquired from the insects on which the lizards feed. Similar flagellates
occur in the blood of lizards, and the natural inference is that they have
invaded the blood-stream from the intestine. If the insects, which were
responsible for the intestinal infection, were accustomed to suck the blood
of lizards, it would be possible for them to become infected from the blood,
in which case they might or might not lose the power of becoming infected
by ingesting the faeces of infected insects of their own kind.
TABULAR CLASSIFICATION OF THE FLAGELLATES OF THE
FAMILY TRYPANOSOMID^E.
A. Flagellates with only an invertebrate host. Infection is contamina-
tive by means of cysts.
(a) Leptomonas.
(6) Crithidia.
(c) Herpetomonas.
CLASSIFICATION 347
B. Flagellates with both a vertebrate and an invertebrate host.
Infection is contaminative or inoculative. No cysts occur.
(a) Flagellates in which the highest development is the lepto-
monas type. Infection of the vertebrate is either inocu-
lative or contaminative. {Leishmania.)
(b) Flagellates in which the highest development is the try-
panosome type. (Trypanosoma.)
1, Trypanosomes which in the invertebrate develop in
the posterior station. Infection of the verte-
brate is contaminative.
(a) T. lewisi and other similar forms in small mammals.
(6) T. cruzi.
(c) Non-pathogenic trypanosomes transmitted by
keds, species of Tabanus, or other biting flies.
T. melophagium, T. theileri, T. ingens.
(d) Trypanosomes of birds (?).
(e) Trypanosomes of land reptiles (?).
2. Trypanosomes which in the invertebrate develop in
the anterior station. Infection of the verte-
brate is inoculative,
(a) Trypanosomes transmitted by blood - sucking
arthropoda.
(1) Pathogenic trypanosomes transmitted by
species of Glossina.
(a) Development in the stomach, probos-
cis, and salivary glands. T. gam-
biense, T. brucei {T. rhodesiense).
(6) Development in the stomach and
proboscis. T. congolense, T.
simice.
(c) Development in the proboscis only.
T. vivax, T. uniforme, T. caprce.
(2) Pathogenic trypanosomes transmitted by
species of Tabamis or alHed flies, and
possibly by ticks. T. evansi, T. equinum.
(3) Trypanosomes of birds (i).
(4) Trypanosomes of land reptiles (?).
(6) Trypanosomes transmitted by leeches.
(1) Trypanosomes of aquatic reptiles.
(2) Trypanosomes of amphibia.
(3) Trypanosomes of fish.
C. Pathogenic trypanosomes usually passing directly from vertebrate
to vertebrate (T. equiperdum). (As these have undoubtedly
become secondarily adapted to this mode of transmission, it might
be more logical to group them with the pathogenic forms trans-
mitted by Jbiting flies.)
D. Flagellates with both an invertebrate and a plant host {Phyto-
monas). P. davidi and similar forms.
348 FAMILY: TRYPANOSOMID.^
SYSTEMATIC DESCRIPTION OF GENERA AND SPECIES.
Genus: Leptomonas Kent, 1880.
The genus Lejjtoinonas, as defined above, includes flagellates which in
their life-cycles exhibit both leishmania and leptomonas forms, and which
are confined to invertebrate hosts.
Biitschli (1878) described a flagellate which he found in the gut of a
nematode {Trilobus gracilis), and Kent (1880) named it Leptomonas
butschlii as the type of the genus. Unfortunately, this flagellate has not
been studied in the light of present knowledge, so that it is still uncertain
if it conforms with the definition of the genus Leptomonas given above.
A very large number of species have been discovered in invertebrate
hosts, mostly arthropods, and of these anything like a complete life-history
is known only in a few instances. The form seen by Biitschli in the
nematode T. gracilis has already been mentioned. Chatton (1924)
records one seen by him in a marine nematode. Amongst the Mollusca,
Porter (1914) described L. patellce from the limpet Patella vulgaris, and
Mello (1921) L. jmchylabrcE from another mollusc, Pachylabra moesta.
Leptomonas ctenocephali (Fantham, 1912). — Though Patton (1908c)
had seen a leptomonas in the Indian flea, Ctenocephalus felis, and its larvae,
the flagellate of the dog flea, C. canis, was first seen by Basile (1910a), who
mistook it for developmental forms of Leishmania donovani. The same
error was made by Basile and Visentini (1911), Sangiorgi (1911), Mar-
zocchi (1911), and Alvarez and da Silva (1911). The rounded leishmania
stages of the parasite were seen by Swellengrebel and Strickland (1910).
Noller (1912c^, 1914) discovered the flagellate in dog fleas and their larvae
in Germany, and concluded that it was a specific parasite distinct from
L. donovani. He studied the infection in fleas, and noted that it was
usually confined to the hind-gut, which was often completely lined with
attached forms. Fantham (1912) proposed the name Herpetomonas
ctenocephali for the flagellate, and Brumpt (1913) the name H. pseudo-
leishmania. The writer (1913a) observed the flagellate in dog fleas in
England, and later (1914a) in Malta, while da Silva (1913) studied it in
connection with attempts to transmit kala-azar in Portugal. Laveran
and Franchini (1919), Chatton (1919), Tyzzer and Walker (1919), Shortt
(1923), and Drbohlav (1925) studied cultures of the flagellate, and noted
that they differed from those of L. donovani.
Though the flagellate of the dog flea is named Leptomonas ctenocephali,
it must be recognized that morphologically indistinguishable forms have
been previously described and named from other fleas, and if these should
be proved to be identical with that in the dog flea, the name given to the
GENUS: LEPTOMONAS
349
form in the dog flea will become a synonym. The first-named form is one
which Mackinnon (1909) described in Ctenophthahnus agyrtes, and which
she named Herpetomonas ctenophthalmi. Swingle (1911) gave the name
H. pattoni to one which he found in species of Ceratophyllus and Pulex,
while Chatton and Delanoe (1912a) identified as this species a form in
the larv8B and adults of C. fasciatus. Brumpt (1913) gave the name
H. debreuli to a flagellate of C. sciurorum, and Laveran and Franchini
(1915) the name H. ctenopsyllce to one in Ctenopsylla musculi. Patton
and Rao (1921) gave the name H. pulicis to the form in the human flea,
P. irritans, but it is a synonym of Crithidia pulicis. This form, again,
was first seen by Basile (191 1«), who regarded it as L. donovani. Similar
^%
g^|J"#
Fig. 163. — Longitudinal Section of the Intestine and Transverse Section
OF A Malpigiuan Tube of the Dog Flea, showing Leptomonas cienocephali
LINING THE HiND-GUT AND THE MaLPIGHIAN TuBE (x Ca. 170). (ORIGINAL.)
flagellates have been seen in other fleas, but as far as is known they
correspond very closely with L. ctenocephali, and it is not improbable
that they may be identical with it. Cross-infection experiments with
bred fleas will have to be undertaken before this is finally settled. All
these forms belong to the genus Leptomonas, as here defined.
In the dog flea the infection is limited to the intestinal tract and the
Malpighian tubes which open into it just behind the stomach (Fig. 163).
Most usually, flagellates do not occur in the stomach, but when the infec-
tion is exceptionally heavy, it may extend forwards to this portion of the
intestine. As a rule, the infection stops abruptly at the pyloric opening,
where a large cluster of free and attached organisms often occurs. The
condition in which the flagellates are found in the gut depends to some
350 FAMILY: TRYPANOSOMID^
extent on the amount of blood present. The flagellates have a marked
tendency to attach themselves to the lining epithelium, which may be
completely covered with a mosaic of flagellates, mostly of a stumpy type.
It is by the flagellar end that attachment is made, and the flagellum
becomes much reduced in length till it is represented only by the axoneme;
the anterior end of the organism then lies in contact with the epithelial
cell. The majority of flagellates are attached to the epithelium, and this
is probably a result of the behaviour of the gut when the flea feeds.
During this act, by means of transmitted light, the gut can be seen to be
in a state of violent peristalsis, the waves passing first in one direction
and then the other. The result is that the first droplet of liquid ejected
from the rectum by the flea contains pure unaltered blood, and if the flea
has been feeding on a rat infected, for instance, with Trypanosoma lewisi,
the living trypanosomes may be found in the first droplet passed. It is
clear that if all the leptomonas were free in the gut cavity, the majority
would be voided with the dejecta. Only those forms which are free or
have become detached escape in the ejected blood, and in this all the
various stages of the flagellate which occur in the gut can be found.
When there is little nourishment in the hind-gut, practically all the flagel-
lates are in the attached condition, but after a meal of blood, many active
flagellates can be seen in the gut contents, the long flagellate forms being
developed from the shorter non-flagellate attached ones. The infection
may spread into the Malpighian tubes, where the same series of free and
attached forms are to be found. Towards the posterior end of the intes-
tine the attached flagellates, and also those free in the cavity, become
smaller, till finally little ovoid leishmania forms are produced. These,
together with all the larger forms up to the longest flagellates, are found in
large numbers in the faeces of the flea, which consist of droplets of digested,
semi-digested, or pure blood. The flea has such a voracious appetite that
it will continue to feed for a long time, filling its stomach again and again
with fresh blood, while it repeatedly voids what is apparently pure blood
from its rectum. The general rule is that the largest flagellates are found
in the fore part of the hind-gut, either free or attached, and the smallest
forms in the rectum, but this rule is not absolutely constant. Sometimes
the whole gut is lined with short stumpy forms with very few long forms,
at others there is a larger number of long forms. In attachment there is a
tendency for groups of flagellates to be arranged as a disc, with the flagella
directed towards the centre or in a hemispherical mass, the so-called
rosette, which has its base on the epithelium, the flagella of the individual
flagellates being directed centrally. Such groups increase in number till
the whole gut is covered. In these groups all individuals may be long or
short forms, or a single group may show every transition from the largest
LEPTOMONAS CTENOCEPHALI 351
flagellate forms to the smallest rounded ones which have no free flagellum.
The clusters or rosettes of attached forms increase in size by multiplication
of the individual flagellates, which are able to divide longitudinally in
whatever form they occur.
The small leishmania forms which arise in the hind-gut appear to
develop a cyst wall. The absolute proof of the existence of a cyst m
such minute forms is, of course, difficult to obtain, though the writer
(1914r/) has shown that these supposed encysted forms are protected in
some way against desiccation. The fseces of an infected flea, which were
passed while feeding, were received on to a sterile cover-glass held a short
distance behind it. The droplet was spread into a thin film with a sterile
needle and allowed to dry. The cover-glass was then placed in a dry
sterile test-tube for twenty-four hours, after which it was transferred to
N.N.N, medium, in which a culture of the flagellates was obtained. A
similar experiment was made by the writer (1912c) with the flagellate of
the human flea, Pulex irritans. This is sufficient evidence to show that in
the fgeces there occur forms which can withstand complete drying, and in
all probability these are the small apparently encysted leishmania bodies.
It is assumed that the flagellates and unencysted forms must be killed m
the process of drying.
It is well known that the larvae of these fleas feed largely on the fseces
of the adults, and, as demonstrated by Noller (1914), they take up the small
cysts, for the same flagellates can be found in their intestine. Here, also,
both elongated and shorter forms occur, but the writer has never seen the
gut covered with attached flagellates, as in the adult. Drbohlav (1925)
has shown that the flagellate infection of the larvse survives in the pupse,
and appears as an intestinal infection in the newly-emerged adults.
It will be seen that the infection is a simple one, which passes from
one insect to another by means of encysted forms voided in the faeces.
The various types of organism from the flea's intestine are shown in
Fig. 164. The longest forms have a body 18 microns in length. There is
a distinct tendency to curvature like the blade of a curved sword. Very
narrow forms occur, as also much broader ones, and between the long
flagellate forms and the minute leishmania ones every stage can be traced.
The small encysted bodies which are finally produced are barely 3 microns
in diameter. Reproduction takes place by binary fission, and this is not
confined to any particular stage, flagellates of all sizes and shapes taking
part in the process. No stage of intracellular reproduction corresponding
with that of Trypanosoma lewisi in the epithelial cells of the flea's gut
has been seen in this flagellate or in any other leptomonas.
There is no evidence that L. ctenocephali has any vertebrate host, in
spite of the claims, which appear somewhat dubious, of Laveran and
35:
FAMILY: TRYPANOSOMID^
Franchini (1913a) that they were able to infect mice. These experiments-
will be considered more fully below. The presence of L. ctenocephali in
fleas led Basile and others to the view that Leishmania donovani undergoes
a development in the flea, the natural flea flagellate being mistaken for
developmental forms of the parasite of kala-azar.
As will be seen in the lists of hosts, leptomonas have been found in a
number of fleas, and some of these have been given specific names without
Fig. 164. — Lejytomonas ctenocepliali from Intestines of Dog Flea {Ctenocephahis
canis) {x 2,000). (Original.)
At bottom right-hand comer are the presumably encysted forms which occur in faeces.
there being any real justification for this procedure. The form in the human
flea, Pulex irritans, was studied by the writer (1912c), and was named L.
pulicis by Patton and Eao (1921). They found fleas naturally infected, and
also succeeded in infecting fleas experimentally by feeding them on cultures
of the flagellate. Larvae kept with infected fleas became themselves in-
fected. They ingest the rounded forms passed in the faeces of the adult,
and acquire an infection of the stomach in which the flagellates live and
LEPTOMONAS CTENOCEPHALI 353
multiply. The flagellates survive the pupal stage, and in the adult flea
appear in the hind-gut and Malpighian tubes. The various forms found
in the adult fleas are described as pre-flagellates, flagellates, and post-
flagellates. Certain round forms, called pre-flagellates, are described
from the Malpighian tubes of the flea, and to account for their presence
the improbable assumption is made that they have been carried there
by adhering to flagellate forms which have migrated from the gut. It
would be expected that the pre-flagellates would only exist in the larvse,
as it is admitted that the flagellates develop in the stomach of the larvse.
The pre-flagellates, it will be remembered, are the rounded forms which
result from the ingested encysted stages, and which develop into the full-
grown flagellates. If they occur in the adult flea, one must suppose that
they have not completed their development in the larvae which ingest
them, as some of them are admitted to do, and that they have passed
through the pupal stage of the flea. The occurrence of these forms in
the adult flea rather suggests that the forms pre-flagellate, flagellate, and
post-flagellate, which Patton describes in this and other insect flagellates,
do not follow one another in succession so regularly as he supposes. It
seems more probable that the flagellates may become rounded leishmania
forms, which may again develop into flagellates in the same host, without
necessarily passing on to the encysted stage, to be voided in the faeces.
As already remarked, cultures of L. ctenocephali and the flagellates
of other fleas can readily be obtained on N.N.N, medium by receiving
the voided droplets of liquid faeces of fleas on sterile cover-glasses and
transferring them to the culture fluid. The writer (1914a) obtained such
cultures from dog fleas in Malta. Laveran and Franchini (1919), Chatton
(1919), Tyzzer and Walker (1919), and Shortt (1923) have obtained
cultures by washing the fleas in sterilizing fluids and dissecting them under
aseptic conditions. These cultures grow readily, can be maintained by
subculture for any length of time, and show all the forms which occur in
the insect gut. There is never any tendency towards the formation of
crithidia or trypanosome forms. Growth is very rapid, much more so
than in the case of the allied pathogenic leishmania. The cultures remain
alive for long periods, and enormous numbers of flagellates are produced.
In one instance in the writer's experience, active flagellates were still
present six months after the tube of N.N.N, medium had been inoculated,
and a subculture was obtained from it two months later, when active
flagellates had disappeared, though leishmania forms were still present.
In old cultures, many abnormal and evidently degenerating forms occur.
Tyzzer and Walker (1919) made a careful comparative study of cultures
of Leishmania donovani (Mediterranean strain) and Leptomonas cteno-
cephali. The flea flagellate grew more rapidly at 21° C. than L. donovani,
I. 23
354 FAMILY: TRYPANOSOMID^
while it still multiplied at 10° C, whereas L. donovani did not. L. cteno-
cephali showed a greater tendency to grow in clumps with the fiagella
internally directed, and it was generally more active than L. donovani.
The fully-grown flagellates of L. donovani varied in length from 9 to 12-5
microns, with fiagella 7-5 to 15-3 microns long. The corresponding stages
of L. ctenocephali measured 11 to 16-8 microns, with fiagella 7-3 to 21
microns in length. In the case of L. ctenocephali, the long forms fre-
quently had the aflagellar end extremely attenuated or ribbon-like, while
spiral twisting of this part of the body was common. In L. donovani the
nucleus was centrally situated, with the kinetoplast near the anterior end
Fig. 16.5. — Leptomonas pulicis of the Human Flea [Fulex irritans). (Original).
A cluster of flagellates from a culture in N.N.N, medium ( x 2,000).
of the body, while in L, ctenocephali the nucleus was definitely in front
of the middle of the body and the kinetoplast was near it. Chatton (1919)
had already drawn attention to the long, acicular forms which occurred
in cultures of L. ctenocephali, and which were absent from cultures of
L. donovani. Drbohlav (1925) hasshownthat dog fleas may be infected with
L. ctenocephali by injecting them per rectum with cultures, or by allowing
them to feed on cultures through a membrane. The cultures of L. pulicis
of the human flea studied by the writer (1912c) are very similar (Fig. 165).
Laveran and Franchini (1919, 1920) claim to have produced a
generalized infection of mice and guinea-pigs by inoculating them with
GENUS: CRITHIDIA 355
cultures of L. ctenocephali. They state that a local infection occurred in
one guinea-pig inoculated in the testis. Noller {I9l2d) failed to infect
a dog with L. ctenocephali, and Chatton (1919) was equally unsuccessful
with mice. Shortt (1923a) attempted to infect dogs, monkeys, cats, mice,
pigeons, and frogs. The animals were examined by the smear and culture
method, but no evidence of infection was obtained. Yamasaki (1924)
also failed to infect mice and dogs, and noted that the flagellate differed
morphologically from Leishmania donovani. Drbohlav (1925) has failed
completely to produce any infection in a series of about 150 animals,
including one monkey, dogs, guinea-pigs, rats, and mice. In the light of
these failures the claims of Laveran and Franchini that practically every
animal inoculated acquired an infection are difficult to explain.
Laveran and Franchini (1920a) also claim to have infected Euphorbia
plants {E. sauliana and E. pilosa) by inoculating them with cultures
of L. ctenocephali. The flagellates were said to be present in the plants
for at least thirty-five days. Shortt (1923) introduced cultures of this
flagellate into small pockets in E. royleana in India, where they survived
for six days. Some of the flagellates became elongated, and showed the
peculiar twisting of the posterior end of the body so characteristic of the
natural Euphorbia flagellate.
By feeding bed bugs on cultures of the leptomonas of Pulex irritans
and cultivating from the intestine, Patton, La Frenais, and Rao (1921) have
shown that the flagellates can survive in the bug at least thirty-seven
days. Shortt (1923) has also shown that active multiplication of L. cteno-
cephali takes place in the stomach of bed bugs fed on cultures. Up to
forty-eight hours there may be a very heavy infection of the stomach,
after which it subsides, till in eight days very few flagellates occur.
Flagellates may, however, still be present in the hind-gut. Multiplica-
tion of the flagellate will also take place for a few days in bugs which have
died after feeding. The effect of giving the bugs feeds of blood after
ingestion of the culture has not been tried.
Genus: Crithidia Leger, 1902.
This genus was first created by Leger, L. (1902a), for a flagellate
{Crithidia fasciculata) which he had found in Anopheles maculipeyinis.
The name was based on the short, stumpy, leishmania forms which Leger
considered characteristic of this genus. As, however, these forms occur
in flagellates of the genera Leptomonas and Herpetomonas, this character
cannot be considered of generic value. Leger's genus Crithidia was
emended by Patton (1908a) in accordance with the definition given above.
The flagellates of this genus are purely parasites of invertebrates, and
in their most highly developed form are elongated organisms with a
356
FAMILY: TRYPANOSOMID^
rounded posterior and tapering anterior end. The kinetoplast lies close
to, but still in front of, the nucleus, while the axoneme, before entering
^!'->y
/J
28
25 /'
m ^ ^
Fig. 166. — Crithidia gerridis from the Water Bugs, Gerris fossarum and
Microvelia sp. (No. 9x460; others x 8')0). (After Patton, 1908.)
1. Pre-fiagellate forms from mid -gut of nymph.
2. Early stage of development of axoneme.
3. Development of flagellum and commencing division.
4 and .5. Dividing forms with developing flagella.
6 and 7. Forms evolving towards the crithidia type.
8. Cluster of rounded fmins with developing flagella.
9. Cluster of critliidia forms adherent to a particle by their flagellar ends.
10-12. Elongate crithidia form. 13. Club-shaped crithidia forms.
14. Dividing crithidia forms. 15-20. Short and narrow crithidia forms.
21-26. Stages in development of post-flagellate forms which escape m faeces and lead to infection
of young nymphs.
CRITHIDIA GEEIRDIS 357
the flagellum, passes along an undulating membrane to the drawn-out
tapering anterior end of the body. These long forms become shorter
and finally converted into round leishmania forms, which appear to encyst
and escape in the faeces of the invertebrate. As in the case of leptomonas,
the cysts lead to infection of a new host.
Crithidia gerridis Patton, 1908. — This flagellate is an intestinal parasite
of the water bug, Gerris fossarum, where it was first seen by Patton. It
is also found in a species of Microvelia, and another water bug related
to Perittopus (Fig. 166). It was chiefly studied by Patton in Microvelia.
The alimentary canal of the Microvelia consists of a narrow oesophagus
opening into a sacculated crop. The latter opens into the short, dilated
mid-gut, which nearly always contains a greenish-yellow fluid. The mid-
gut is followed by the small intestine, at the anterior end of which open
four long, narrow Malpighian tubes. The small intestine is followed by
the dilated colon continuous with the short, straight rectum. The eggs
hatch into nymphs, which by five moults attain the adult condition. In
the crop of the nymphs are found the encysted forms which have been
ingested with water (Fig. 166, i). Very shortly after their ingestion,
these round forms produce flagella and begin to multiply. The smallest
round forms are 4 to 6 microns in length by 3 to 4 microns in breadth.
At first, these round forms possess only nucleus and kinetoplast. Very
soon, from the latter the axoneme is formed, but when it reaches the
surface of the body, instead of immediately entering the flagellum, it
passes along the edge of a narrow undulating membrane, the rudiment
of the structure which is seen fully developed in the adult crithidia forms
(Fig. 166, 2-5). These forms, having increased in size, now measure
6 to 10 microns by 4 to 8 microns. Multiplication takes place at this stage
by binary fission. Patton states that the flagellum actually divides
longitudinally, but this is certainly incorrect. By active division rosettes
of rounded flagellates are produced, with the flagella directed outwards
(Fig. 166, 8). These rosettes are attached in masses to the lining epi-
thelium. They gradually break up, and the individual flagellates swim
away. The pole opposite that to which the flagellum is attached elon-
gates, while the flagellated pole becomes drawn out with the flagellum.
In this manner the typical crithidia forms arise. They vary from 15 to
45 microns in length and 2 to 4 microns in breadth (Fig. 166, 10-12).
The anterior end of the body is drawn out to a fine point where the axoneme
enters the flagellum. There is an undulating membrane on the part of
the body anterior to the nucleus, and the axoneme passes along its margin.
The posterior end of the body is rounded. The nucleus is spherical and
situated at the centre of the parasite, while the kinetoplast is 1 to 1-5
microns in front of it. These long forms are often agglomerated together
358 FAMILY: TRYPANOSOMID^
by their flagellar ends, or attached to cells or debris (Fig. 166, 9). Multi-
plication of these forms again takes place.
Flagellates of all sizes and shapes are found not only in the crop, but also
in the other parts of the intestinal tract. In the rectum there is a gradual
production of short forms, by a process the reverse of that which occurred
in the crops of the nymphs, by the drawing in of the anterior and posterior
ends (Fig. 166, 21-26). Oval or round forms measuring 4 to 6 microns
by 3 to 4 microns are thus produced. The flagellum is lost or absorbed,
the axoneme alone remaining. These forms become enclosed in cysts of
various sizes. Not only are these cysts voided with the faeces of the bug,
but any other forms which may be present in the rectum also escape,
so that it is possible the nymphs may become infected, not only by ingestion
of the cysts, but of the unencysted forms also. Patton has noted that
the bugs have cannibalistic habits, and often kill and feed on one another,
so that infection may take place in this manner. The flagellates were
never found in any other organ than the intestine, and there was no
evidence that infection of offspring through the eggs could take place.
The life-history of Crithidia gerridis is very similar to that of Lepto-
monas ctenocephali, there being direct infection from host to host by
means of cysts voided in the faeces. The difference is that the flagellates
develop further towards the trypanosome type. Becker (1923, 19236),
in a study of C. gerridis in Gerris remiges in North America, actually
noted that trypanosome forms occasionally appear. He has also seen the
flagellate in Microvelia americana, G. marginatus, and G. rufoscutellatus.
Fantham and Porter (1916) stated that they had infected vertebrates
by inoculating them with C. gerridis. Becker (1923«) has failed entirely
to confirm these observations.
A cycle of development similar to that of C. gerridis has been described
by Patton (1909) for C. tahani of Tahanus hilarius and Tahanus sp., and
by Porter (1911) for a parasite of the human flea, Pulex irritans. The
latter flagellate was given the name C. pidicis, which had previously been
used by Balfour (1909a) for a similar, though not necessarily identical,
form discovered by him (1906a) in the flea, Loemopsylla cleopatrce, in the
Sudan. From the observations of Noller (1916), it seems probable that
the Crithidia of tabanid flies are really developmental stages of Trypano-
soma theileri (see p. 501).
Crithidia hyalommae O'Farrell, 1913. — This flagellate is worthy of
special consideration, not only because it infects the body cavity fluid
of its host, Hyalomma cegyptium, but also because actual infection of the
ova is described as leading to infection of the hatched offspring (Fig. 167).
The parasite was found by O'Farrell in ticks living on cattle in the
Anglo-Egyptian Sudan. In the first place, it might be suggested that
CRITHIDIA HYALOMM^
359
Fig. 167. — Grithidia liyalommce from Body Cavity of Hyalomma wgyptium of tii]
Sudan (x 2,000). (After O'Farrell, 1913.)
1-2. Leishmania forms in hsemocoele fluid, (described a.s pre -flagellate forms).
3. Plasmodia! phase from hsemocoele fluid.
4-10. Development of flagella and transformafon into crithidia forms.
11-12. Fully-formed crithidia.
13-15. Retrogression form in haemoccele fluid (described as post-flagellate form).
16. Ovarian form (described as post-flagellate stage).
17. Plasmodia! form in ovary. 18. Form in salivary gland
19. Stages in ovarian cell.
360 FAMILY: TRYPANOSOMIDyE
the flagellates represented developmental forms of a cattle trypanosome,
but this was considered to be negatived from the fact that only a few
ticks from any single animal were found infected. No other ticks on the
animals than this particular species showed infection. The cattle, more-
over, were invariably healthy. A remarkable feature of the infection is
that it is not an intestinal one, but is confined to the body cavity or
hsemocoele. At the height of an infection, which occurs just before the
tick oviposits, the smallest drop of fluid obtained by cutting off one of
the legs is found to be swarming with flagellates. In the early stages
of an infection, only round leishmania forms occur, but these gradually
develop into the adult crithidia forms. Multiplication takes place in
the usual way, all stages of the flagellate participating in this. After ovi-
position, and just before the death of the tick, round leishmania forms
(post-flagellate forms) may appear in the fluid. The intestinal diver-
ticula and Malpighian tubes were not found to harbour the parasite, though
as non-flagellate forms they were sometimes found in the salivary glands,
but this was exceptional. Infection of the ovaries is described as taking
place by the flagellates piercing the walls of the oviducts, and then entering
the ova. Some of the flagellates remain in the cells of the oviducts,
where they become transformed into leishmania forms. Those that
enter the eggs likewise become of the leishmania type, and here they
may be seen in process of division. It is unfortunate that, in his account
of this developmental process, O'Farrell does not make any reference to
the examination of the newly-hatched young. Examination of the ovaries
by the section rather than the smear method would have given more
trustworthy results as regards the supposed invasion of the eggs. As
the author says, the hsemocoele fluid became " a felted mass of crithidial
bodies and waving flagella," and it must be difficult in such a case to
exclude the contamination of the interior of an egg with hsemocoele fluid
when smears are made.
Several other instances of infection of ova by flagellates are on record,
but in all cases the smear method was used, though Porter (19096) claims
to have actually observed penetration of the egg of Nepa cinerea by the
living flagellate forms of Leptomonas jaculum. Flu (1908), Swingle
(1909), and Porter (1910) have described invasion of the ova of Melophagus
ovinus, the sheep ked, by the flagellates of these insects. Porter (19096,
1909c), though claiming to have observed L. jaculum and C. gerridis
within the eggs of their hosts, considers that they degenerate without
infecting the egg. This condition is supposed to lead up to that in M.
ovinus, where invasion of the ova is said to be followed by multiplication,
so that hereditary infection occurs. The statements regarding M. ovinus
can hardly be accepted in view of the fact that it is now known that the
CRITHIDIA HYALOMM/E 361
flagellate, which was supposed to be peculiar to the ked, is the develop-
mental form of the sheep trypanosome. Hoare (1921a), working in the
writer's laboratory, could find no evidence of invasion of the eggs of either
M. ovinus or N. cinerea, but noted the accumulation around the ova
of spermatozoa, which produced an appearance of discarded flagella.
It is possible that these were mistaken for the flagella of flagellates.
Prowazek (19126) described infection of the egg of Sarcophaga by
L. sarcophagce, but it is not clear that intestinal contamination was
avoided. It is evident the question of transmission of flagellates through
the ova requires to be studied by the more accurate method of sectioning
the ovaries and eggs. In the case of such a host as Hyalomma cegyptium,
which, apparently, only sucks blood, it would appear that ovarian infec-
tion would be the only method of transference from host to host if the
possibility of a cattle trypanosome is excluded. In this connection it must
not be forgotten that the apparently harmless Trypanosoma theileri is
often present in cattle in such small numbers that it can only be demon-
strated by culture methods. This source of the infection in the tick has
not been considered, nor is mention made of any infection in the newl}'-
hatched nymphs. Only ticks which had been feeding on the cattle were
found infected. It is evident that the infection of the eggs, and the supposed
hereditary infection of offspring hatching from the eggs, has not been demon-
strated for C. hyalommcB nor any of the other flagellates mentioned above.
Crithidia euryophthalmi McCulloch, 1917.— This flagellate is parasitic
in the gut of the bug Euryophthahnus convivus, which feeds on the plant
Lupinus arboreus, growing in sand dunes near San Francisco (Fig. 168).
It was discovered by McCulloch (1917), who has given an account of its
life history, which is of interest in that two phases of development not
hitherto recorded in the life-history of these flagellates are described.
These are multiple segmentation and internal budding. The alimentary
tract of the bug consists of fore-, mid-, and hind-gut (Fig. 168). The
fore-gut is made up of the mouth, pharynx, oesophagus, and proven-
triculus; the mid-gut of the crop, mid-stomach, pyloric expansion, and
intestine; and the hind-gut of the colon, into which open the Malpighian
tubes and the rectum. The type of flagellate found varies with
the position in the gut. The oesophagus and proventriculus have
always been found free from infection, the hind-gut has shown a
slight infection in the rectum in some instances, while it was in the
mid-gut that the heavy infections occurred. The stages which usually
occur in the hindgut of insects are in this bug found in the pyloric
expansion.
The forms which occur in the stomach (crop, mid-stomach, and pyloric
expansion) are:
362
FAMILY: TRYPANOSOMID^
f^-ii i i \
33
\n
Fig. 168. — Crithidia euvyo'pliiliclmi from Intestine of Euryophthahnus convivus,
arranged so as to show the various forms which occur in different
Parts of the Alimentary Canal (x 1,750), (After McCulloch, 1917.)
1. Diagram of alimentary canal: res., oesophagus; cr., crop; mid.stom., mid-stomach; pyl.ex.,
pyloric expansion; int., intestine; int.gl.,Lntestina,\ glandular epithelium ; ?/ii., Malpighian
tubes; c, colon; r., rectum. 2-35. Various flagellate types explained in text.
1. Ovoid forms, which are presumably those taken up casually in the
food. They are about 3-2 microns in length, and occur in the crop
(Fig. 168, 2).
2. Every stage in growth of the ovoid forms uj) to the elongate flagellates
10 to 30 microns in length. As growth takes place, they migrate back-
CRITHIDIA EURYOPHTHALMI 363
wards from the crop to the mid-stomach and pyloric expansion (Fig. 168,
2-7 and 13-18).
3. Multiple division forms, which resemble the intracellular stages of
development of Trypanosoma lewisi in the flea (Fig. 200, 9). They occur in
the crop, and are presumably produced by growth associated with nuclear
multiplication of flagellates which have entered the lining cells, and which
by segmentation give rise to a number of flagellates corresponding with
the number of nuclei (Fig. 168, 8-9).
4. Forms which are supposed to show a process of internal budding
(Fig. 168, 12).
5. Binary fission forms of the usual type occurring in the crop and
pyloric expansion (Fig. 168, 7).
6. Crithidia stages from the crop, which become free forms (necto-
monads) in the mid-stomach and pyloric expansion (Fig. 168, 15-18).
7. Crithidia stages from the crop, which become attached forms
(haptomonads) in the mid-stomach and pyloric expansion (Fig. 168,
13-14)-
8. Final ovoid stages, which occur both in the mid-stomach and pyloric
expansion. They are formed by a process the converse of that which
occurred in the crop when the ovoid forms grew into the elongate crithidia
forms. They become encysted, and pass back to the rectum as infective
forms, to be passed in the faeces (Fig. 168, 19).
The interesting feature of this infection is that the cycle takes place
comparatively far forwards in the gut, the final stages occurring in the
pyloric expansion of the stomach, and not in the hind-gut. The ovoid
encysted forms taken into the crop grow into the crithidia forms, which
reproduce in the usual manner by longitudinal division, by an intracellular
multiple segmentation, and by the curious internal bud formation. The
latter is quite unique, and has not been described by any other observer
(see p. 338). The crithidia forms produced in the crop pass back to the
mid-stomach and pyloric expansion, where they may attach themselves
to the lining epithelium or remain free. In either case, they retrogress
to form the small ovoid encysting bodies. The various stages are illus-
trated in the diagram given by McCulloch, but confirmation of the intra-
cellular stages and the internal budding process is required before they
can be finally accepted.
Genus: Herpetomonas Kent, 1880.
The genus Herpetomonas was created by Kent (1880) for a flagellate
of the house fly {Musca domestica), which was first mentioned by Burnett
(1851, 1852) under the name Bodo. The next record of the fly flagellate is
364 FAMILY: TRYPANOSOMID.E
that of Leidy (1856), who said he had frequently found Bodo muscarum
in the intestine of the house fly in immense quantity. Later Stein (1878)
referred to it as Cereomonas muscce domesticce, and gives Bodo 7nuscce
domesticce (Burnett) as a synonym, though, as noted above, Burnett
referred to it only as Bodo. Finally, Kent (1880) referred it to his genus
Herpetomonas, and called it H. muscce domesticce (Burnett), though this
specific name was really Stein's. It would seem, therefore, as pointed
out by Hoare (1924), that the correct name should be H. muscarum
Leidy, 1856, as there is no doubt that Leidy and Burnett were both
observing this flagellate, in spite of the fact that Becker (1923c) considers
it a nomen nudum. Grassi (1879a) referred to the flagellate as
Schedoacercomonas muscce domesticce, and in 1882 as Monomita muscarum.
The majority of observers refer to the organism as Herpetomonas muscce
domesticce.
The members of this genus, as defined in this work, have not only
leptomonas and crithidia forms in their cycle of development, but also
trypanosomes forms. They are, nevertheless, purely invertebrate para-
sites, which pass from host to host in the encysted stage. In the writer's
opinion, the bulk of evidence is in favour of the view that the flagellate
of the house fly has a trypanosome stage occasionally, though it is most
usually seen in the leptomonas form. If it should be demonstrated that
the trypanosome forms which occur in the house fly in reality belong to
a distinct species of flagellate, then the generic name Herpetomonas
cannot be employed for the genus as here defined, and it will become
a synonym of Leptomonas. In this case, probably Patton's name Rhyn-
choidomonas (p. 374) would have to be employed. Whether the name
stands or not, it is an undoubted fact that there are many insect flagellates
which conform to the definition of the genus Herpetomotias as given here,
and which was emended in this sense by the writer (1913). The recent
work of Drbohlav (1925), who has obtained cultures of the flagellate of
Lucilia ccesar, aiiords a direct confirmation of the conclusions reached
here. He informs the writer that cultures commenced with a single
organism showed not only leptomonas, but also trypanosome forms. With
these cultures specially bred Musca domestica, as well as Fannia regina
and L. sericata, were infected. It may be concluded, therefore, that the
flagellate of the house fly is identical with that of L. ccesar, and has both
leptomonas and trypanosome stages in its life-history.
Herpetomonas muscarum (Leidy, 1856). — This flagellate is very com-
mon in the intestine of the house fly, Musca domestica, in all parts of the
world (Figs. 159 and 169). In some localities, especially in the tropics,
practically every fly examined is found to be infected. It has been
described from a variety of hosts other than Musca domestica, but from
GENUS: HERPETOMONAS
365
the work of Chatton and his collaborators (1911-1913) on the parasites of
species of Drosophila, and that of Patton (19126) on Musca nebulo and
Lucilia serenissima, it appeared at one time that the specificity of the
Fig. 169. — Eerpetomonas muscariim from the Intestine of the House Fly,
FIXED IN SCHAUDINN'S FlUID AND STAINED WITH IrON H.EMATOXYLIN
(x 2,000). (Original.)
1-3. Short forms with single ilagellum.
•4-9. Dividing forms, showing formation of new iiagella as new outgrowth and division of kineto-
plast and nucleus.
insect flagellates for their hosts was greater than has been supposed, and
that it was possible that many of the flagellates which had been regarded
as H. muscarum were in reality distinct species. Patton (1921), however,
366 FAMILY: TRYPANOSOMID^
as a result of further observations, states that he has found this flagellate
in Madras in the following hosts: if. nebulo, M. humilis, Famiia canicu-
laris, Borborus sp., Drosophila sp., Lucilia argyricephala, L. craggi ; while
Becker {1923d) has shown by actual cross-infection experiments that in
North America it may infect Phormia regina, Lucilia sericata, Calliphora
erythrocephala, Cochliomyia {Clirysomyia) macellaria, Musca domestica,
and Sarcophaga bullata. He believes that H. ?nuscarum, H. lucilice,
H. callipliorcE, H. sarcophagce, and the Herpetomonas which occurs
naturally in P. regina and C. macellaria, belong to one species,
H. muscarum. The similar results obtained by Drbohlav (1925) have
been referred to above (p. 364).
As regards the distribution of H. muscarmti in the fly, it may
occur in any part of the gut up to the opening of the proventriculus.
In the fully-grown leptomonas form it has a pointed, blade-like body
up to 30 microns in length and 2 to 3 microns in breadth. The flagellum
is often three times the length of the body. The nucleus is central in
position, and the elongated kinetoplast is near the anterior end, and con-
sists of the usual parabasal body, and the blepharoplast from which the
axoneme of the flagellum arises. The anterior end of the body is often
truncated or cut off, and a clear area may sometimes be seen to run
into the cytoplasm towards the kinetoplast. In some cases this clear,
funnel-like area appears to be continued past the kinetoplast, where it
terminates indefinitely in the cytoplasm. The fact that in some indi-
viduals structures like bacteria were seen at the posterior end of the body
led the writer (1913a) to suggest that this structure might be of the
nature of a cytostome. This, however, seems very doubtful, for Becker
(1923c) could detect no cytostome. It is found not only in the adult
leptomonas forms, but also in the shorter and broader types on the way
to encystment.
A feature of this flagellate, which has given rise to some controversy,
is the frequent occurrence of two flagella (Fig. 169). This fact led
Prowazek (1904) to define the genus Herpetomonas as including biflagellate
organisms. The observations of Patton (19086), Porter (19096), Mackinnon
(1910), and the writer (1911a) have clearly shown that the biflagellate
individuals, which may comprise the majority of forms seen in an infection,
are in reality dividing forms (Fig. 159). When division is proceeding,
the blepharoplast elongates transversely, and a new axoneme growing
out of a new flagellum appears even before division of the blepharoplast
is completed. By the time the blepharoplast has divided, the new flagellum
may be as long, or nearly as long, as the original one. The daughter
blepharoplasts may proceed to division again, with a new axoneme forming
from each one, and this may occur before the parabasal or the nucleus
HERPETOMONAS MUSCARUM
367
has completed the first division. In this manner, organisms with four
flagella and a single dividing nucleus may appear, and give the impression
of a dividing biflagellate organism. A similar condition is sometimes
seen in trypanosomes dividing actively in the blood of inoculated rats,
where large forms may occur with four nuclei, four kinetoplasts, and four
membranes and flagella. That the explanation given of the biflagellate
appearance is the correct one is borne out by the fact that in flies, where
active multiplication is not in progress, the flagellate has only a single
flagellum.
It is in the leptomonas form that the flagellate is most commonly seen
in flies. As pointed out by the writer (1913a), the kinetoplast may change
Fig. 170. — Herpetomonas muscarum of House Fly (x ca. 2,000) : Transformation
OF Leptomonas into Trypanosome Forms, (After Wenyon, 1913.)
its anterior position for one near the nucleus, in which case the axoneme
passes along the surface of the body (Fig. 170). Such forms have the
crithidia structure, though an undulating membrane, as a definite band
of cytoplasm, is not actually present. With further migration backwards
of the kinetoplast, trypanosome forms are produced. The conditions
under which this takes place are not known. The occurrence of these
three phases has been noted in many allied flagellates. Some observers
believe they represent distinct species, but the bulk of evidence is in
favour of regarding all the forms as belonging to the cycle of the one
flagellate. Fig. 170 shows the various transition forms in an infection
where the leptomonas and the trypanosome types both occur (see also
Fig. 155). Rosenbusch (1909) noted these different forms in the flagellate
of the house fly, which, on this account, he termed Crithidia musccB
domesticoe. Becker (1923c) has confirmed these observations, while the
368
FAMILY: TRYPANOSOMID^
culture experiments of Drbohlav (1925), referred to above (p. 364), appear
to be conclusive.
By a gradual shortening of the body of both the leptomonas and
trypanosome forms, smaller stumpy individuals are produced, and
these become attached to the lining epithelium of the hind-gut. Repro-
duction of all these free and attached forms takes place by longitudinal
fission, often producing enormous infections of the gut. Encystment
takes place by a gradual shrinkage of the body, or in some of the
trypanosome individuals by the doubling of the body into a U, the space
between the limbs of which gradually fill in, so that the axoneme follows
a characteristic curved course in the cytoplasm. There are three methods
Fig. 171. — Three Methods by which the rounding-up (Encystment) of Ilerpe-
tomonas muscarum takes place in the Hind-Gut of the House Fly. (After
Wen YON, 1913.)
1-3. Retraction of leptomonas form.
4-8. Rounding up of trypanosome form by looping of the body.
9-11. Rounding -ujj of trypanosome form by retraction of body.
by which retraction of the body and encystment may take place (Fig.
171). Becker (1923c) thinks that encystment of H. muscarum always
takes place after the trypanosome type has been developed. The cysts
appear to have a definite cyst wall. Their function is undoubtedly the
transmission of infection from fly to fly, but, as Patton (19106) and Becker
(1923c) have shown, flies may be infected by ingestion of adult flagellates
or the pre-encysting forms passed in the faeces, as well as by cysts. The
cysts would probably ensure protection against a period of desiccation.
From his earlier work, Patton concluded that, though the larvae of
Musca nehulo might be infected with flagellates, these did not appear to
survive the pupal stage, as flies hatched from infected larvae were free
from infection. In a later paper (1921) he appears to have modified this
view, for he states that infections will pass through the pupae to the
adults. Becker (1923c) was unable to detect larval infections. As
GENERA: LEPTOMONAS, CRITHIDIA, HERPETOMONAS 369
already pointed out, H. muscarum, like the flagellates considered above,
passes from insect to insect in cysts voided in the fseces by infected
individuals. It differs from L. ctenocephali and C. gerridis in that the
flagellate may assume the trypanosome form in the course of its develop-
mental cycle.
Patton (1921) records the successful culture of H. muscarum in N.N.N.
medium. The strain was obtained by dissecting out the peritrophic
membrane of a Lucilia argyricephala, and inoculating the medium with
some of the contained flagellates. Glaser (1922), who also succeeded in
cultivating the organism, has shown that grasshoppers can be infected by
inoculation into the body cavity. The cultures obtained by Drbohlav
have been noted above (p. 3G4).
Franchini and Mantovani (1915), and Fantham (1922), claim to have
infected rats and mice with H. ynuscarum. Glaser (1922) and Becker
(1923a) have been unable to confirm this observation.
OTHER MEMBERS OF THE GENERA LEPTOMONAS, CRITHIDIA,
HERPETOMONAS.
As will be seen from the list (p. 14:02), the number of invertebrate
flagellates is very great, but in the majority of cases nothing like a com-
plete cycle has been observed. In some of those where it is known, as,
for example, L. ctenocephali, L. culicis, L. jaculum, C. pulicis, C. gerridis,
etc., the cycle of development is a comparatively simple one, the encysted
forms ingested growing through the pre-flagellate form into the adult
flagellate, and then retrogressing through a post-flagellate form into the
cyst, which escapes in the faeces and is ingested by a new host. In most
cases the feeding habits of the adults, as the house fly, are such that
infection by the ingestion of cysts is possible. In other cases, as, for
instance, fleas and sand flies, the adults are blood feeders, which have
no opportunity of ingesting cysts. There are, however, larval stages,
which are omnivorous feeders, and the adults become infected during
metamorphosis from the infected larvae. Other blood suckers have no
stage capable of ingesting cysts, and it would appear that infection can
be derived only from the blood. The crithidial infection of Hyalomma
cegyptium, mentioned above, is of this type. The recent discovery of a
Leptomonas in the proboscis and intestine of Glossina morsitanshy Lloyd
(1924) afiords another instance of the same type. The origin of this Lepto-
monas is not known, but if it conforms with other flagellates of tsetse flies,
it must have originated from the blood of some vertebrate. It may be
connected with the leishmania infections of man which occur in Nigeria, or
be derived from the blood of a reptile on which these flies readily feed.
I. 24
370 FAMILY: TRYPANOSOMID^
Forms found in the Body Cavity and Salivary Glands.
The infections are in most cases purely intestinal ones, thongh the
flagellates may sometimes find their Avay into the Malpighian tubes.
Occasionally, however, the infection extends from the gut to the body
cavity. Zotta (1912 and 1921) described a flagellate infection of Pyrrho-
coris aptera, a plant bug. The organism {L. jjyrrhocoris) occurred, not
only in the gut, but also in the body cavity and salivary glands, as again
noted by Franchini (19226). C. hyalommce, described above, is peculiar
in that it occurs in the body cavity of the tick, whence it infects all the
tissues of the body, including the salivary glands, but is absent from the
intestine.
Robertson (1912) found flagellates of the leptomonas type in the
salivary glands of a plant bug {Leptoglossus memhranaceus), and HoUande
(1912) found a form, which was named by him L. emphyti, in the hsemocoele
fluid of a hymenopteran larva {Emphytus cinctus), a mere puncture of the
cuticle yielding a fluid teeming with flagellates. Glasgow (1914) noted
a salivary gland infection in another bug {Peribalus litnbolarius). Working
with pentatomid bugs {Pentatoma ornata and P. juniperina), Franchini
(19226) stated that not infrequently the crithidia, which inhabited the
intestine, invaded the salivary glands. Poisson (1925) has noted that
L. naucoridis of the water bug Naucoris macnlatus, though usually con-
fined to the intestine, may, in the case of heavy infections, invade the
body cavity and internal organs, including the salivary glands. While
dissecting a species of Culex in Tonkin, Mathis (1914) noted infection of
the salivary glands with a flagellate of the crithidia type. In the case
of the plant bugs, it is possible that the flagellates may be developmental
stages of some plant parasite, while those in the tick and mosquito may
have been derived from vertebrate trypanosomes.
Roubaud's Genus Cercoplasma.
While in the Congo, Roubaud discovered a remarkable flagellate in
the intestine of Pycnosoma putorium. He (1908) named it L. mirabilis,
but subsequently (1911) created the new genus Cercoplasma, in which
he placed it, together with other similar forms he had found. It will be
seen that, apart from certain large giant individuals, the flagellate shows
the usual types of the genus Herpetomonas, and in view of the fact that
some of the flagellates ascribed by Roubaud to his genus Cercoplasma
lack these giant forms, they will be regarded as belonging to the genus
Herpetomonas. Roubaud's flagellate then becomes H. mirabilis (Roubaud,
1908). The main features of the flagellate are shown in Fig. 172. The
giant forms may exceed 200 microns in length, with a maximum breadth
GENUS: CERCOPLASMA
371
of 3-5 to 5 microns. The leptomonas and trypanosome forms are of the
usual dim.ensions, being 18 to 20 microns in length, with a flagellum up
to twice the length of the body. All intermediate stages between these
small forms and the giants occurred. In the rectum, small forms, 4 to 10
microns in length, were found. These were evidently encysting forms.
It is difficult to account for the giant forms, which have been seen only
in this and the allied flagellates, H. mesnili and H. lineata, though smaller
forms of the same type occur in Chatton's H. roubaudi described below
(Fig. 176). It appears to be not improbable that they are merely abnormal
overgrowth forms, in which, for some reason, nuclear division has been
delayed, allowing a great increase in the cytoplasm to take place, as occurs
Fig. 172. — H&rpetomonas mirabilis from Pycnosoma initorium, showing Various
Trypanosome, Leptomonas, Elongate Cercoplasma, and Eounded
Forms (x 900). (After Roubaud, 1909.)
in the case of Trypanosoma rotatorium of frogs, Trichomonas vaginalis,
and other flagellates.
Herpetomonas mesnili (Roubaud, 1908) was first called Leptomonas
mesnili by Roubaud (19086), and later included in his genus Cercoplasma
(1911a). It is a parasite of Lucilia latifrons, and Lucilia sp. of the Congo.
Both this parasite and H. mirabilis were later found by Roubaud in a species
of Pycnosoma and Lucilia in the French Sudan. In H. mesnili, the giants
are not more than 70 microns in length, while the small forms vary from
7 to 8 microns and upwards, and have a flagellum from 12 to 14 microns
in length. Round and encysting forms are not described. The flagellate
was only seen twice in the Congo — once in a fly in pure culture, and
once in association with two other flagellates, one morphologically a
crithidia and the other a leptomonas.
372 FAMILY: TRYPANOSOMIDiE
Patton (1921) reports having found H. mirahilis in various flies in
Madras. He has seen it in Lucilia argyricephala, L. craggii, Chrysomyia
(Pycnosoma) megalocephala, and C. albiceps {Pycnosoma putorium). The
cycle of development in L. argyricephala is described as follows. In the
larvse the growth of rounded leishmania forms into leptomonas forms
and finally into the elongate cercoplasma forms was noted. The flagel-
lates persist in the pupse, and appear in the adult flies. Here in the
hind-gut the leptomonas forms become transformed into flagellates
of the trypanosome type. The nucleus becomes elongated, while the
kinetoplast passes backwards to a point near the posterior end of the body.
The trypanosome stage having been reached, the flagellates become
shorter, and, finally, rounded leishmania or post-flagellate forms are
produced. It is by ingestion of these that the larvae and, presumably,
the adults become infected. It is possible that this flagellate represents
one of the phases of development of H. muscarum.
Another flagellate, which Roubaud places in his genus Cercoplasma,
is Herpetomo.nas caulleryi (Roubaud, 1911). This was found in Auchme-
romyia luteola in the French Sudan. It agrees with the two forms
H. mirahilis and H. mesnili as regards the various small forms, but the
giant forms were not seen. The flagellate discovered by Roubaud (1912c)
in a species of Drosophila in the French Sudan, and named by him Cerco-
plasma droso])hilw, is probably identical with one of the species of Herpeto-
monas described by Chatton from D. confusa (see below). All these
flagellates were limited to the intestinal tract of the flies.
Swingle (1911) has described large giant forms of Herpetomonas lineata
of Sacrophaga sarracenice in North America. In this case the longest forms
may even reach a length of 385 microns, and, like Roubaud's giants, there
is a swollen anterior end containing nucleus and kinetoplast, and a very
long drawn-out post-nuclear region. The flagellum is short, a fact which
would suggest that they are normally attached to the gut epithelium. As
in H. mirahilis, the giant leptomonas forms were associated with flagellates
of the trypanosome and other types. This form, again, may be a phase
of H. muscarum.
Roubaud's Genus Cystotrypanosoma.
The genus Cystotryjxinosoma was proposed by Roubaud (1911) for
flagellates which have the trypanosome structure, and which produce
cysts in the rectum of the flies. Of this type is a flagellate he found in the
intestine of a species of Lucilia, probably L. sericata, at Bamako in the
French Sudan. Ordinary trypanosome forms occur which in the rectum
become smaller and doubled as a U, and by a fusion of the limbs there is
produced an ovoid body which encysts. Larger forms of the rhyn-
GENUS: CYSTOTRYPANOSOMA 373
choidomonas (see below) type of Patton were also found. According to
Roubaud, it has been the custom to include in the genus Trypanosoma
the typical forms parasitic in the blood of vertebrates, and those which
are purely insect flagellates. The latter produce cysts, as noted above,
while the former do not. Accordingly, he proposes to divide the genus
Trypanosoma into two sub-genera: Trypanosoma for the blood parasites
' .f ." #" ^^ «^
Fig. 173. — Trypanosoma (jrayi in Intestine of Glossina 2)alpalis (x 2,000).
(After Minchin, 1908.)
1-7. Division of typical crithidia forms. 8. Trypanosome form.
9-16. Stages in the supposed formation of the cyst.
of vertebrates, and Cystotrypanosoma for the cyst-producing forms of
invertebrates. Another flagellate, which Roubaud includes in the latter
genus, is Herpetomonas grayi Novy, 1906 (called by Novy, Minchin, and
others Trypanosoma grayi, and by Roubaud Crithidia grayi). This organism
(Fig. 173) occurs in the digestive tract of Glossina palpalis, G. brevipalpis,
374 FAMILY: TRYPANOSOMIDyE
G. fusca, and G. tachinoides, where it may give rise to confusion with de-
velopmental stages of T. gamhiense and other trypanosomes. It occurs in
the trypanosome form as well as others, and cysts are said to be produced in
the rectum. Kleine and Taute (1911), and Kleine (1919a), working with bred
flies which were fed on a known infected crocodile, have demonstrated that
H. grayi is in reality the developmental form of T. l-ochi, the crocodile
trypanosome; while Lloyd, Johnson, Young, and Morrison (1924) have
shown that laboratory bred G. tachinoides become infected with H. grayi,
or flagellates indistinguishable from it, after feeding on monitors {Varanus
exanthematicus) harbouring T. varani, or on toads {Bufo regularis) harbour-
ing a trypanosome resembling T. varani, as well as on crocodiles. It
appears, therefore, that H. grayi represents the invertebrate phase of a
trypanosome, so that the alleged presence of encysted forms in the rectum
of the flies requires an explanation, as these stages are not known to occur
in the case of any other trypanosome. Fraser and Duke (19126) failed to
cause laboratory bred flies to infect themselves from the faeces of infected
flies. As explained above (p. 342), it seems probable that the supposed
cysts of H. grayi are not actually of this nature. Minchin, Gray, and
Tulloch (1906), and Minchin (1908) suggested that H. grayi might be a bird
trypanosome. Of this there is at present no direct evidence.
The genus Cystotrypanosoma, as defined by Roubaud, corresponds with
the genus Herpetomonas as it is interpreted in this work. As members of
Roubaud's genus Cercoplasma produce cysts, the distinction between it
and the genus Cystotrypa?iosoma is not very clear.
Patton's Genus Rhynchoidomonas.
Pattou (1910a) described a flagellate which he had found in the Mal-
pighian tubes of Lucilia serenissitna of Madras, and which appeared to difler
from the well-recognized types. He described it under the generic name
of Bhynchotnonas, but as this was pre-occupied, later in the same year he
substituted the name Rhynchoidomonas (Fig. 174). The flagellates were
only seen in a single fly. Flagellates of the same type were seen by the
writer (1911) in the gut and Malpighian tubes of house flies in Bagdad.
These flagellates were also seen by Alexeieff (1911) in species of CaUiphora
and Lucilia in Europe (Fig. 155), and by Patton (1921) in another fly in
Madras.
In the writer's experience (1911a) they occurred in association with
H. muscarum, and it was concluded that they represented developmental
stages of this common house-fly flagellate. Later, in Aleppo, the question
was again studied by the writer, and the view was adopted that the try-
panosome forms actually belonged to the cycle of H. muscarum, as every
stage in the migration backwards of the nucleus could be traced.
GENUS: RHYNCHOIDOMONAS 375
Diinkerly (1911) and Alexeieff (1911/, 1912e) also regarded these forms
as representing developmental stages of H. muscarum, which, however, is
most usually met with in the leptomonas form. Chatton (1913) expressed
the opinion that the flagellate, H. sp. (1) referred to below (p. 378), of the
Malpighian tubes of the adult Drosophila confusa had been evolved from
the intestinal species. Hence, he suggests that these Malpighian tube
forms should be placed in a distinct genus for which Patton's name has
priority. The type species of this genus, according to Chatton, will be
g § r^ ^<:% (
9
Fig. 174. — Life-Cycle of BhynchoUlomonas siphunculUue in Intestine of
Siphunculina funlcola (x ca. 2,000). (After Patton, 1921.)
1-2. Pre-flagellate forms from stomach of fly.
3-5. Growth of flagellate and formation of flagellum in Malpighian tubes.
6-9. Development of fully-formed flagellate of Rhynchoidomonas type in Malpighian tubes.
10-13. Retraction of flagellate forms towards the post-flagellate stage in Malpighian tubes.
14. Post-flagellate stages which escape from the Malpighian tubes into the intestine and are
passed in the faeces.
7?. drosophilcB [H. sp. (1)], which is said to occur only in the Malpighian
tubes of D. confusa. As many insect flagellates are known to occur in the
intestine, and occasionally in the Malpighian tubes {e.g., L. cteiiocephali
of the flea), it cannot be considered as finally established that the Mal-
pighian tube forms are distinct from the intestinal ones.
Patton (1921) has given an account of what he regards as the complete
life-cycle of one of these forms, which he names R. siphunculince, and
376 FAMILY: TRYPANOSOMID^
which occurs in the Malpighian tubes of the eye fly, Sijiliunculina funicola,
of Madras (Fig. 174). The life-cycle which he describes follows closely
those of other arthropod flagellates studied by him (Fig. 166). The pre-
flagellate stage resulting from ingested cysts occurs in the stomach of the
fly, but, unlike the pre-flagellate forms of species of leptomonas and
crithidia, these do not reproduce, but merely increase in size, while the
axoneme becomes evident. Further growth takes place only in the
Malpighian tubes, where the typical rhynchoidomonas forms are produced.
When fully formed, these may measure 55 microns in length. The nucleus
lies nearer the anterior than the posterior end, and the kinetoplast lies
near but posterior to the nucleus. From the kinetoplast the axoneme
passes to the anterior end along the surface of the body. A definite
undulating membrane is not developed, nor is the axoneme continued
beyond the anterior extremity of the body. The part of the flagellate
behind the nucleus varies considerably in length according to the stage
of development. At first it is quite short, but in the fully-formed flagel-
lates it may be drawn out into a long, tapering, cytoplasmic process three
or four times as long as the portion of the body anterior to the nucleus.
Multiplication takes place by longitudinal division after division of the
kinetoplast and nucleus, but, contrary to what usually occurs in other
Trypanosomidse, the body commences to divide at the posterior ex-
tremity. After the flagellate stage has been reached, development
towards the post-flagellate takes place. The long posterior portion of
the body is withdrawn, and forms very much like pre-flagellate stages
arise. In some of these the kinetoplast is near the posterior extremity
of the body, and the nucleus nearer the anterior end. These forms are
attached to the cells of the Malpighian tubes in clusters. Eventually,
small rounded or oat-shaped forms are developed, and these escape into
the gut and are excreted with the faeces. Patton was able to demonstrate
that this flagellate never developed in the larvae of the fly which were fed
on the dead bodies of adult flies harbouring this parasite. On the other
hand, another form (Herpetotnonas siphunculincB), which occurred in the
intestine of the fly, readily infected the larvae and appeared in the intestine
of the adult. The fact that typical trypanosome forms with free flagella
occurred in the cycle of the H. siphunculincB in the intestine of the fly,
and that this flagellate was never found in the Malpighian tubes, raises the
question of its relationship to the rhynchoidomonas form, which may be
a stage of evolution of H. siphunculincB. The peculiar features of its
morphology may be due to the fact that it has invaded the Malpighian
tubes, which is not its usual habitat. The writer cannot agree with
Patton that the rhynchoidomonas forms are not of the trypanosome type.
It is known that in typical trypanosomes, as, for instance, T. lewisi
FLAGELLATES OF DROSOPHILA 377
the post-nuclear region of the body may be extremely long. A similar
though more marked hypertrophy occurs in Roubaud's H. tnirabilis.
Furthermore, in typical trypanosomes the width of the undulating
membrane varies considerably, so that in some forms the axoneme appears
to pass along the surface of the body, as in the rhynchoidomonas forms
here under discussion, while it is well known that in many forms of try-
panosome no flagellum exists. If these variations were combined in one
individual, and the kinetoplast brought nearer the nucleus, then the
characteristic rhynchoidomonas form would be produced. As a matter
of fact, in some of the forms depicted by Patton the kinetoplast is far
behind the nucleus, so that in all essential respects the rhynchoidomonas
forms are of the trypanosome type, and the flagellate will be considered
here as belonging to the genus Herpetotnonas. The fact that the axoneme
does not extend beyond the anterior end of the body probably indicates
that these rhynchoidomonas forms are really attached forms. Further-
more, this fact may explain the commencement of division at the posterior
unattached end of the body instead of at the attached anterior end, where
it usually occurs.
Chatton's Observations on the Trypanosomidae of Drosophila.
lu certain eases, as appears chiefly from the work of Chatton and his colleagues
on the flagellate parasites of various species of DrosopMla, the cycle of develop-
ment may not be so simple as in the forms described above. In Drosojjhila confusa
he has been able to identify four, or possibly five, distinct species as a result of
extensive breeding experiments extending over several years. He has succeeded
in separating the flagellates, and has obtained them in pure culture in different
batches of the fly.
In order to comprehend properly Chatton's views, it wiU be necessary to describe
a structure which occurs both in the larvae and adults of the Drosophila (Fig. 175).
This is the peritrophic membrane which arises at the oesophageal opening of i^he
stomach as a cylinder and passes back through the stomach to end in the hind-gut.
The anterior end of this membrane is attached as the diagram shows, but the pos-
terior end is lying free in the gut cavity. It is a membranous structure, possibly of
a chitinous nature, and, as far as can be seen, is not perforated, so that organisms
cannot pass through it. The lumen of the cylinder is the endotrophic space, while
that between it and the gut lining epith-elium is the peritrophic space. The function
of the membrane is not properly understood, but it naturally suggests a filtration
process in connection with nutrition.
Of the flagellates of 1). confusa, Leptomonas roubaudi Chatton, 1912, is j)erhaps
the simplest (Fig. 176). It has only been found in the Malpighian tubes of the larva
and the adult, where it occurs in the various forms depicted. It will be seen that the
elongate forms are leptomonas in type, and these gradually merge into trypanosome
forms, which become round and finally encysted. According to the definition of
genera adopted here, this parasite will be known as Herpetomonas roubaiuli.
The second flagellate is Trjjjianosoma drosopJiilce Chatton and AlUaire, 1908.
It occurs in the larva?, pupse and adults of D. confusa. In the larvse and pupae
it occurs only in the peritrophic space, while in the adult it occurs only in the Mai-
378
FAMILY: TRYPANOSOMID.E
pighian tubes or in the peritropliic space near their openings. The flagellate occiirs
in the trypanosome form, which, still maintaining this structure, becomes a smaller
trypauosome form. This becomes doubled into a U form in which the two limbs
fuse, and the resulting body then encysts. This process is similar to that described
above for H. muscarum. Here, again, the cycle is a simple one (monophasic), and
as in the first-mentioned flagellate, by simple reduction in size and retraction of
the body, the cyst is produced (Fig. 177). This flagellate, showing the trypano-
FiG. 175. — Akrangement of the Peritrophic Membrane in the Intestine of
DrosopMla confusa. (After Chatton and Leger, 1912.)
m.p, Peritrophic membrane; ces, oesophagus; Lend, flagellates iii the endotrophic position; I. per,
flagellates in the peritrophic position .
some form in its cycle, becomes Herpeiomonas sp. (1). Chatton (1913) notes that
the trypanosome forms which occur in the Malpighian tubes are related to the
Khynchoidomonas described by Patton. The third and fourth flagellates of this fly
are closely related. They are described as L. drosoplillce by Chatton and Alilaire
(1908), and Leptomonas sp. by Chatton and Leger, M. (1912rt). The former occurs
as an endotrophic infection in the larva and as a peritrophic infection in the adult,
while the latter is only found in the adult in the endotrophic space. The first of
these, L. droso-philcc, which will be called here //. drosojMlce, occurs in the adult fly in
FLAGELLATES OF DROSOPHILA
379
various forms— trypaiiosome. crithidia, leptomoxias, leishmania and cyst (Fig. 178).
The k'ptoinonas lorius are regarded a^ the flatjellates ^\hich develop diiectly from
the cysts. V>\ l)ack^^ul■d migration of the kimaophx^^t, tlie ciithidia lonn^, and
Fig. 170. — llei-pdontona-s roiibaudi from Malpigiiian Tubes or D,-oh.ophila confasa
(x ca. 2,000). (After Chatton, 1912.)
1. Malpighian tube packed with flagellates.
3-4. Large attached forms of the cercoplasraa type (gregarinien).
5-9. Transitions from the leptomonas (monadien) to the trypanosome form (spermoide).
10-11. Stages of encystment.
finally the trypanosome forms, are evolved. As the latter pass to tlie liind-giit, the
kinetoplast comes forward again, and there are again produced leptomonas forms
Fig. 177. — Herpetomonas sp. of Dmsophila phalerafa (x ca. 2,000). (After
Chatton and Leger, 1912.)
This flagellate is similar to H. sp. (1) of D. confusa.
1-2. Rhynchoidomonas forms. 3. Tryimnosome forms.
■i-5. Encystment after looping of body.
which attach themselves to the epithelial lining of the gut. Here they become still
further retracted, till the round leishmania forms which produce the cysts result.
Reproduction takes place in all these stages, and the reduction in size, leading to
380
FAMILY: TRYPANOSOMID^
cyst formation, is rather the result of successive divisions unassociated with growth
than to actual retraction. In this cycle it will be seen that the leptomonas forms
appear in two phases, so that, to use Chattou's term, the developmental cycle is
diphasic in contrast to that of R. roubaudi and H. sp. (1) described above, in which it
is monophasic.
There is a modified cycle of development exhibited by another peritrophic form
often associated with H. drosophilce which is monophasic. The free leptomonas
forms, instead of becoming free trypanosome forms, as in H. drosophilce, pass to the
hind-gut as leptomonas forms, where they become smaller and attach themselves
to the gut wall. At the same time the kinetoplast migrates backwards, so that the
attached forms really have the trypanosome structure. This cycle corresponds
closely with that of Ilerpetomonas sp. (1), the second flagellate mentioned above,
and can be considered as a condensed cycle by the loss of the active trypanosome
stage, Avhich is only revealed after the leptomonas forms have attached themselves.
Fig. 178. — Herpetomonas drosophilce fkom In-
testine OF Drosojihila confusa ( x 2,000).
(After Ciiatton and Legek, 1911.)
1-2. Leptomonas forms (monadien).
3-8. Transformation of leptomonas into trypanosome
forms (trypanoide).
9-13. Transformation of leptomonas forms (monadien)
into small ovoid bodies (spermoide).
14. Attached forms (gregarinien) in rectum which
become encysted after becoming trypanosome
forms (spermoide).
Fig. 179. — Herpetomonas sp. of
DrosopliUa p>lialerata ( x ca.
2,000), Free Leptomonas
AND Transformation of
attached leptomonas
Forms (Gregarinien) into
Trypanosome Form (Sper-
moide) BEFORE EnCYST-
MENT. (After Ciiatton
AND Leger, 1912.)
Chatton and Leger, however, speak of this flagellate as Leptomonas p., as they have
not sufficient evidence, in the shape of pure infections in the fly, to justify separating
it entirely from the diphasic form, H. drosophilce. A flagellate of D. phalerata is,
however, very simUar to it (Fig. 179).
The fifth flagellate is Leptomonas sp. Chatton and Leger, 1912. It, again, is a
Herpetomonas [H. sp. (2)], according to the definition adopted here, and differs from
H. drosophilce in being endotrophic and not peritrophic in the adult fly. It occurs
only as an intestinal parasite of the adult fly. The forms met with are similar to
those of //. drosophilce, with the exception that reduction in size of the body takes
place to a certain extent and then ceases, so that cysts are not formed. From
FLAGELLATES OF DROSOPHILA
381
observations on the allied flagellates of D. ampelophila it would appear that these
small forms, still provided with flagella,, are found in the fseces, where they can
readily he seen. In this endotrophic parasite, which has a diphasic cycle, cyst
formation has so far not been discovered.
The flagellates described by Chatton and his co-workers from D. confusa can be
tabulated as follows:
1. //. rotibaudl { = L. roabandi Chatton, 1912).^Malpighian tubes of larva and
adult: monophasic cycle.
2. H. SI). (1) i = T. drosophilw Chatton and Alilaire, 1908 = Bhynchoidomonas
drosophilw Chatton, 1913).— Larva (peritrophic), adult (Malpighian tubes): mono-
phasic cycle.
3. H. drosopliilcB ( = L. drosopMlre
Chatton and Alilaire, 1908). — Larva
(endotrophic), adult (peritrophic): di-
IJhasic cj'cle.
4. H. p. (=£. p. Chatton and liCger,
1912). — Larva (endotrophic), adult (peri-
trophic): monoi:>hasic cycle. It appears
that this may represent an alternative
cycle of //. drosophilce, a view which re-
ceives support from the later observa-
tions of Chatton and Aubertot (1924),
mentioned below.
5. E. sp. (2) (=£. sp. Chatton and
Leger, 1912). — Adult (endotrophic):
diphasic cycle.
As many of the tryptanosome forms
of insect flagellates do not appear to
have a well-developed membrane, the
axonemo running either through the
cytoplasm or attached directly to the
surface of the body, Chatton employed
the term lepiotnjpanosome to distinguish
them from the trypanosomes [eutrij-
panosome), which are the typical verte-
brate forms with a well-developed mem-
brane. In later writings he employed
the name trijpanoide for the trypanosome
forms of the insect flagellates. Accord-
ing to Chatton' s nomenclature, the series
of forms through which a flagellate may
pass are these: stade monadien (leptomonas forms), which by backward migration
of the kinetoplast becomes the stade trypanoide (trypanosome form).
The latter may revert to the monadien phase again. The monadien forms by
shrinkage of the body become the short leptomonas forms, which attach themselves
to the hind-gut epithelium. These Chatton terms stades gregariniens, and they by
migration of the kinetoplast may assume the trypanosome arrangement, when they
are known as stades spermoides. These latter forms become encysted. So that, in
the diphasic cycle of H. drosopMlce and the allied H. rubrostriatw, the following
stages are passed through : monadien, trypanoide, monadien, gregarinien, spermoide,
cyst (Fig. 178). The monophasic cycle of //. roubaudi (Fig. 176) is simpler: mona-
dien, gregarinien, spermoide, cyst.
Fig. 180. — Herpetomonas ruhrostriatw
OF Drosophila rubrostriata ( >■ ca. 2,000).
(After Chatton and Leger, 1912.)
1. Leptomonas forms (monadien).
•2. Forms approaching the trypanosome type
(trypanoide), which again become
leptomonas forms (monadien).
3. Retracting forms attached to cells of hind-
gut (gregarinien).
4-5. Encystment.
382 FAMILY: TRYPAXOSOMID^
Chatton and liis co-workers have devoted much time and trouble to the experi-
mental side of this question, and though sucli a multiplicity of flagellates from a single
host is somewhat disconcerting, his published results are difficult to explain on any
other basis. Some of the flagellates have been kept in pure culture in a batch of
flies for over two years, and, according to Chatton, the infections have always
remained the same. Another, perhaps unexpected, result of his earlier work is that
flagellates of nearly allied species seem to be specific to their hosts. Thus, working
with three other species of Drosophila — viz., D. ruhrostriata, D. 'plialeraia, and I),
ampelophila — it was found that when bred in captivity with infected I>. confnsa
they did not acquire infection, though they themselves at other times are found to
harbour flagellates which are difficult to distinguish from those of D. confusa. The
flagellate of each host appeared to be specific for that host. As a result of his
experiments, Chatton has named two of the flagellates, which become H. rubro-
striatcB Chatton and Leger, 1911, and H. ampdopMlce Chatton and Leger, 1911
(Fig. 180). The flagellate of B. ruhrostriata remained a pure iDcritrophic infection
in a batch of flies from June, 1910, to March, 1911, during which time over 200 flies
were examined. In these flagellates, both the diphasic and monophasic forms, like
H. drosophilce and H. p., occurred. On the other hand, a batch of D. ampelopliila
bred from June to December, 1910, always showed H. ampelopMlce as an endotrophic
form, which corresponds to H. sp. (2) of D. confusa.
In a later publication Chatton and Aubertot (1924) modify the view regarding
the specificity of the flagellate H. drosophilw ( =L. drosophilce Chatton and Alilaire,
1908). In B. confusa it is always endotrophic in the larva, and both endotrophic
and peritrophic in the adult. It has now been found that both larvfe and adults of
B. ruhrostriata can be infected with this flagellate. In both larvse and adults the
infection commences as an endotrophic one, but in the adult it may become peri-
trophic after a few days, owing to migration of the flagellates round the posterior
free end of the peritrophic membrane. It follows that the flagellate H. r\ihro-
striatce may be identical with H. drosophilw.
Genus: Phytomonas Donovan, 1909.
As explained above, the flagellates which are included in this genus
have only the leptomonas and leishmania forms. A very good case for
retaining them in the genus Leptomonas can be made, but as they occur
in both plants and invertebrates, and sometimes in vertebrates also, if
Strong's observations receive confirmation, they are conveniently placed
in a separate genus like the forms included in Leishmania (Fig. 181).
The name Phytomonas, suggested by Donovan (1909), will be employed.
Lafont (1909) described a flagellate of the leptomonas type as occurring
in the latex of a plant, Euphorbia pilulifera, in the island of Mauritius. He
named it Leptomonas davidi, and later rediscovered the organism in two
other plants, E. thymifolia and E. hypericifolia. It is now known to occur
in various parts of the world, as the table shows (p. 390). Various species
of Euphorbia are involved, and it was supposed that flagellate infections
were limited to plants of this family till Migone (1916) described an in-
fection of Araujia angustifolia {Funastrum boneoriensis) in South America.
Migone proposed the name Leptomonas elmassiani for the flagellate of
GENUS: PHYTOMONAS
383
A. angustifoUa, while Franga (1921) has given the name L. bordasi to
another flagellate which, Migone informed him, he had found in a plant
(Morreira odorata) belonging to the same family. Franchini (1922c,/, A)
claims to have found flagellates of various kinds, not only in plants
belonging to the Euphorbiacese, but in many others. Franga (1920a) has
Fig. 181. — Phijtomonas davidi in the Juice of an Indian Euphorbia. (From
Drawings presented to the Writer by Dr. E. Row of Bombay.)
Two plant cells are shown ( X 2,000).
shown that the bug Stenocejjhalus acjilis is responsible for the spread of
the infection from plant to plant in Portugal, while Strong (1924) has
incriminated another bug {Chariesterus cuspidatus) in Central America.
Strong, moreover, claims to have shown that lizards, which devour these
bugs, acquire an intestinal infection with the flagellate, which, when
384 FAMILY: TRYPANOSOMIDiE
inoculated from the lizard's intestine into the skin of the monkey,
produces a lesion resembling oriental sore, in which leishmania forms
of the parasite occur. The evidence that the flagellate of the lizard
is actually that of the bug is not quite convincing.
Phytomonas davidi (Lafont, 1909). — The flagellate has been studied
most fully in Portugal by Franga. He has discovered the invertebrate
host of the flagellate, and has described what he regards as its cycle of
development. As observed in the latex, P. davidi has the usual lepto-
monas structure (Fig. 181). The body measures 16-5 to 19-5 microns
in length by 1-5 in breadth. The extremities are tapering, and the
flagellum measures from 10-5 to 16 microns. A peculiar feature seen in
some of the organisms is a twisting or folding of the posterior portion of the
flat, blade-like body of the parasite two or three times round its longitudinal
axis (Fig. 184 E). That this twisting is merely the result of the medium
in which the flagellate is growing is demonstrated by an observation of
Shortt (1923) that if Leptonionas ctenocephali of the dog flea is inoculated
into a small fissure made in a Euphorbia, the flagellates persist there for
six days, during which some of them become longer, and show the same
twisting of the posterior part of the body. The nucleus usually lies at
the junction of the anterior and middle thirds of the body, with the kineto-
plast about 3 microns anterior to it. Shorter flagellates are also seen,
and even round leishmania forms. Multiplication is by the usual method
of longitudinal division.
Culture of the flagellate was attempted by Fran9a (1914) without
success, but Nieschulz (1924rf) has successfully cultivated and maintained
on blood agar a strain from Euphorbia cereiformis received from Franchini.
He refers to the flagellate as Herpetomonas euphorhiw.
Inoculation from plant to plant was attempted by Noc and Stevenel
(1911), who claimed to have transmitted the infection to healthy plants
by injecting material with a glass pipette. As the local inoculation
seems to have produced a generalized infection in forty-eight hours, there
would appear to be some doubt as to the accuracy of the result. Fran9a
(1914), trying the same experiment, after over a hundred failures, only
succeeded twice in producing a localized infection of the plant. As a rule,
the natural infection is found only in certain parts of the plant, and it
spreads gradually from twig to twig. Instead of its usual white appear-
ance, due to the presence of starch and other granules, the latex becomes
a clear liquid, in which these substances are not found. In sections of
the plant, the flagellates occur in enormous numbers, sometimes as
veritable emboli, in the lactiferous tubes, in which the latex has been
completely changed in character. This alteration not only brings about
the death of the infected part of the plant, but eventually causes degenera-
GENUS: PHYTOMONAS
385
tion and abnormal growth of the parasite, apparently as a result of ex-
haustion of nutriment. Amongst the degenerated parasites are some
forms of large dimensions. The latter may reach a length of 30 microns
and a breadth of 6 microns. The kinetoplast either entirely disappears
or becomes hypertrophied. This abnormal increase in size may be com-
parable with that of the giant forms of Herpetomonas fnirabilis and
H. tnesnili described above, and it may be that the presence of giant forms
in the fly and latex can be attributed to similar; disturbances of nutrition.
The effect of the infection on the latex has been mentioned. In a
section of a healthy leaf the lactiferous tubes are seen to be filled with
starch and other grains, whereas in an infected leaf the tubes are com-
FiG. 182. — Plujtomonas davidi in a
Lactiferous Tube, as seen in a
Section of a Twig of Ewphorhia
segetalis. (After Franca, 1914.)
Fig. 183. — Stenocephalus agilis ( $ ), the
Transmitter of Phytomonas davidi
in Portugal ( x 3). (After Franca,
1920, Modified.)
pletely devoid of these (Fig. 182). Furthermore, the chlorophyll gradually
diminishes, and the plant finally withers and dies. Occasionally, however,
an infected twig will recover.
Fran§a has also noted the flagellates in the sheath of the fruit, while
in the fruit itself he has seen minute bodies which, however, he cannot
certainly identify with the flagellates. He suggests the possibility of
their being forms destined to infect the seeds and bring about infection
of the young plants, a kind of hereditary infection analogous to the
supposed infection of the ova of insects. Strong (1924) has noted that
all parts of the plant, including the roots, may be infected.
A transmitting host of the flagellate has been sought by several
observers. Lafont (1909) noted that the plants (E. hypericifolia) haj^-
I. 25
386 FAMILY: TRYPANOSOMID.E
boured hemiptera, and in one of these, Nysius ewphorhice, he found a
flagellate of the leptomonas type. He succeeded (1911) in infecting
healthy plants by means of these bugs, but failed to infect E. peplus,
which is never found naturally infected in Mauritius, Bouet and
Roubaud (1911), employing eighty specimens of the bug Dieuches humilis,
also succeeded in carrying infection from one plant {E. pilulifera) to
another. Rodhain and Bequaert (1911) observed flagellates in the intestine
of an hemipteran larva taken off infected Euphorbia indica in the Congo.
Franga (1919 and 1920a), working in Portugal with E. segetalis, has
succeeded in transmitting the infection by the agency of a bug, Steno-
cephalus agilis (Fig. 183). The bug is chiefly nocturnal in its habits,
and, when feeding, punctures the leaf in many places. The points of
puncture — the primary lesions — when examined, are found to contain
minute rounded or slightly elongate forms of the flagellate, which are
very similar to those which occur in the salivary glands of the bug. It
is later that the infection extends from the primary lesion to the latex,
and becomes general. Franga has traced the development of the flagellate
in the bug up to an invasion of the salivary glands (Fig. 184). The forms
ingested by the bug when feeding on infected latex multiply rapidly in
the gut up to the fourth day. It is supposed that there then occurs a
process of syngamy, in which two flagellates, after losing their flagella
and kinetoplasts, fuse completely. Unfortunately, this appears to have
been deduced from stained films only, so that it cannot be accepted as
reliable. From the fourth day onwards there appear large giant forms
up to 50 microns in length, and rounded multinucleate bodies. After
this period, only small forms 4-5 to 7 microns are found. These are,
presumably, the infective forms, for they occur, not only in the gut, but
also in the salivary glands. Small round leishmania forms, some of which
appeared to be encysted, were found occasionally in the hind-gut, and
once in the proboscis. Invasion of the salivary glands seems to take
place by a forward migration of the intestinal forms, which make their
way to the proboscis and thence up the salivary duct, as in the cycle of
development of Trypanosoma gambiense in tsetse flies. Flagellates were
not found in the hsemocoele fluid, though a dipterous larva (one of the Ocyp-
terincB or G^^/mnosominoe), inhabiting the body cavity, was found infected.
Galli-Valerio (1921), working in Switzerland, has found Euphorbia
gerardiana infected at a height of 1,300 metres above sea-level. The plants
provided one specimen of Stenocephalus, and in this bug he claims to have
found the intestinal flagellates and the small metacyclic forms in the
salivary glands described by Fran9a. Franchini (19226) collected the insects
and bugs from a large number of infected Euphorbias near Bologna. In no
case was Stenocephalus found, and it is concluded that other arthropods
GENUS: PHYTOMONAS
387
Fig. 184. — Life-Cycle of Phytomonas davidl as described by Franca (x ca.
1,000). (After Franca, 1920.)
A. Infective forms in salivary gland of bug (Stenocephalns agilis).
B. Forms in primary lesion on cuticle of plant, which results from the bite of the bug.
C. Forms in latex when infection becomes generalized.
D. Forms in fruit.
E. Forms in intestine of bug. F. Resistant forms in faeces of bug.
388 FAMILY: TRYPANOSOMID^
probably play a part in the transmission of the flagellates, a view with which
Fran9a (1922) disagrees. In a later paper Franchini (1922^) states that he
has found the flagellate in flies {Anthomyia maculata) taken off the plants.
Strong (1924) has published an account of experiments conducted
with the flagellates of Euphorbias in Central America. He has shown
that the coreid bug Chariesterus cuspidatus infects itself from the Euphor-
bias, on the juices of which it feeds. It was also noted that certain lizards
{Cnemidophorus letnniscatus) which fed upon insects harboured in the
posterior portion of the intestine a flagellate indistinguishable from
that of the bugs. It was evidently of interest to investigate the con-
nection between these flagellates and those of cutaneous leishmaniasis
which occurred in the district. Monkeys, dogs, guinea-pigs and mice
were inoculated intraperitoneally and subcutaneously with the flagellates
from the plants, bugs, and lizards. All these experiments were entirely
negative as regards the production of generalized or local infections,
except in one monkey inoculated subcutaneously on the abdomen with
flagellates from the lizard, in which a papule appeared on the eighth day.
It increased in size, and finally ulcerated. On the sixteenth day, definite
leishmania were discovered in the lesion, and these were found to be
numerous in sections of the ulcer, which was removed when the animal
was killed on the twenty-fourth day. As no similar lesions resulted from
inoculation of the flagellates from the plants or the bugs, it is concluded
that the flagellates had become capable of infecting the skin of the monkey
as a result of their modification in the intestine of the lizard. As pointed
out above, the proof that the flagellate of the bug is identical with that
of the lizard was not obtained. In the light of the observations of Franya,
Galli-Valerio, and Strong, it is interesting to recall the fact that several
observers, as noted above (p. 370), have recorded the presence of flagellates
in the salivary glands of plant bugs.
Of these plant flagellates, Fran9a (1921) recognizes three species,
which are said to differ as regards the dimensions of the fully-grown
leptomonas forms. He notes that the Euphorbia flagellate of Portugal
may be distinct from Lafont's original form from Mauritius. Should this
prove correct, he suggests the name Leptomonas lafonti. The dimensions
in microns of the leptomonas forms of the three species of Phytomonas,
as given by Fran9a (1921) are shown in the table below (p. 389). It must
be admitted, however, that much more extensive observations will have to
be made before they can be accepted as indicating specific distinctions.
Fantham (1925) proposes the name Herpetomonas ficuurn for a flagellate
of Ficu$,edulis.
It has been noted above (p. 335), that Fran9a (1920a) believes that
flagellates, «!f the genera Herpetomonas and Leptomonas can be distin-
GENUS: PHYTOMONAS
389
guished by their method of division. In the case of the former, it is
claimed that the kinetoplast, rhizoplast, and entire flagellum divide;
while in the latter, a new axoneme grows out from the daughter blepharo-
plast to form a new rhizoplast and flagellum. Nieschulz (1924(f) appar-
ently interprets Franga as making the claim that in flagellates of the
genus Leptomonas no rhizoplast is present, though this structure is clearly
shown in Franga's figures. Having found that in the cultural forms of the
flagellate of Euphorbia cereiformis a rhizoplast occurs, he gives it the new
name Herpetomonas euphorbice, as Franga groups the Euphorbia flagellates
studied by him in the genus Leptomonas. Actually, there is no difference
between the flagellate studied by Nieschulz and those studied by Franga.
P. elmassiani.
P. davidi.
P. bordesi.
Length of body
12 to 15
16-5 to 19-5
24 to 27
Length of flaa;elhim . .
4-5 to 7-5
10-5 to 16
7-5 to 9
Distance of kinetoplast from
anterior end of body
1-5
1-5
2-2 to 3
Distance between the kineto-
plast and nucleus . .
1-5
3
3
Leno-th of nucleus
1-5
2 to 3
2-2 to 3
Distance between nucleus and
posterior end of body
7-5 to 10-5
10-5 to 12
16-6 to 18
Laveran and Franchini (1920a) have discovered leptomonas in a number of
Euphorbias in Italy as follows: (Bologna) E. peplus, E. diilcis, E. falcata, E. nerei-
folia, E. virosa ; (Florence) E. humifusca ; (Ferrara) E. peplus ; (Syracuse) E. peplus ;
(Catania) E. grandis. In two of these, only rounded non-flagellate forms were found,
and they think it possible a distinct species is represented. Euphorbias in Pans
were not found infected, but an attempt was made to inoculate plants {E. snuliana
and E. pilosn) with cultures of Leptomonas ctenocephali of fleas. As long as two
months after, the inoculated twigs were not growing so well as the control ones,
while smears from the latex showed typical flagellates. The examination of the
controls was entirely negative. In the same paper, these observers claim to have
produced a mild infection in mice by inoculating them with the flagellate of the
Euphorbias. Franchini (1921b, 1922c') stated that he had found four members
of the family Apocynaceae (Acolcanthera spectabilis, A. venenata, Funiumia elastica,
and Thevetia nereifolia) infected, and that in Euphorbia nereifolia and E. ccerulescens
he had found flagellates which had the trypanosome arrangement of the kinetoplast
and nucleus. These had a length up to 12 microns. The undulating membrane,
when visible, was poorly developed. Eounded forms also occurred, and these
appeared to be produced by the flagellate first becoming looped and the space
between the limbs gradually filling up with cytoplasm. Franchini has given the
name Trypanosoma euplwrbiw to this flagellate. Even if his statement is to be
relied upon, it has yet to be demonstrated that he was not dealing with a hitherto
undetected form of development of Phytomonas davidi.
The same observer (1922/) described a flagellate infection of cabbages, which
were infested with various species of pentatomid bugs (Pentatomaornatum, P. ornatum
var. pedorale, P. oleraceum). These bugs commonly have an intestinal crithidia
infection, and it is claimed that the flagellates sometimes invade the salivary glands.
390 FAMILY: TRYPANOSOMID.E
The cabbage leaves, which are heavily infested with bugs, become yellow and
unhealthy. In these, Franchini claims to have found the flagellates and leishmania
forms. In a discussion which took place after the announcement, Roubaud stated
that he had frequently observed the intestinal infection of the bugs, but, though he
had specially looked for them, he had failed entirely to find flagellates in the salivary
glands of the bugs or in the tissues of the cabbages. In another paper Franchini
{ld22d) describes as Critliidia oxycareni an intestinal crithidia of the bug Oxycarenus
lavaterce, which lives in bushes of the species Altea syriaca. He states that he found
leishmania forms of the flagellate in the ffecal deposits of the hug on the surface of
the leaves, and that these forms occurred also in the tissues of the leaves. Franchini
(1922(7) states that he examined a number of latex-producing plants in the Botanical
Gardens in Paris, with the following results: Flagellates of the trypanosome
type were seen in five species of the family Euphorbiacese {Euphorbia calyculata, E.
nereifoUa, E. viroso, Elteophorbia drupifera, Exocwria emmarginaia), and leishmania
forms in one (Manihot dichotoma). Crithidia were seen in one of the Asclepiadacese
{Cryptosteiga grandiflora). Of two Apocynacese, leptomouas were present in Cerbera
odollam, and a large trypanosome with membrane but no flagellum in Caudronia
javanensis. Amongst the Urticaceae, trypanosome forms were found in Ficus
benjamina and leishmania forms in Ficus tliolloni. Of the Sapotacese examined,
Sideroxylon inerme contained a herpetomonas (leptomonas) form, Clirysojjhyllum
glabrum and C. sp. a large trypanosome with undulating membrane and no flageUum.
The statement is made that mice were inoculated, and that trypanosomes were seen
in the blood. It will be noted that in an earlier paper, Laveran and Franchini stated
that the Euphorbias of Paris were not infected, and that they were successfully
inoculated with L. ctenocepholi. Franchini (1922A;) has described the presence of
flagellates of the leptomonas, crithidia, and trypanosome type in the juice of the
fruit and the latex of F. parietalis. They have been cultivated, and with the
cultures mice were inoculated. Leishmania forms were found in the blood of the
animals. In a later paper (1922m) an account is given of attempts to infect Euphor-
bias with other flagellates. With cultures of Leishmania tropica, E. segetalis was
infected ; with L. donovani, E. ipecacuanha ; and with Herpetomonas muscarum , E.
geniculata. The infected plants were constantly in poor condition compared with
the controls, while leishmania forms occurred regularly in the plant juices for as long
as three months. Franchini (1923a) again claims to have successfully infected
Euphorbias with the intestinal flagellates of Musca dGmestica, Sarcophaga 7 {vriior-
rhoidalis, Calliphora erythrocephala, and Pentatoma ornatum. Shortt (1923) has noted
the persistence of Lejytomonas ctenocephali for six days after being introduced into
a small excavation on a bough of a Euphorbia plant. As will be seen below, many
of the statements contained in papers to which Franchini' s name is attached, and
which describe successful inoculations of insect flagellates to vertebrates, are of
such a nature that it seems impossible to estimate their real value. It is evident
that many of them cannot be accepted till reliable confirmation is forthcoming.
RECORDED PHYTOMONAS INFECTIONS OF PLANTS.
Euphorbiacese.
E. brasiliensis, Noguchi, 1924, Honduras.
E. callitrichoides. Strong, 1924, Central America.
E. caproni, Monti (quoted by Visentini, 1914), Sardinia.
E. cereiformis, Franchini, 1923, France,
E. cyparissias, Aubertot, 1923, Alsace. Bruni, 1925, France.
E. dulcis, Laveran and Franchini, 1920, Italy.
GENUS: PHYTOMONAS 391
B. esula var. mosana, Zotta, 1921, France.
E.falcata, Laveran and Franchini, 1920, Italy.
E. gemrdiana, Galll-Valerio, 1921 and 1923, Switzerland.
E. grandidens, Franchini, 1923, Italy.
E. grcmdis, Laveran and Franchini, 1920, Italy.
E. Jielioscopia, Franchini. 1923, France; Avibertot, 1923, Alsace.
E. humifusca, Laveran and Franchini, 1920, Italy.
E. hijperici folia, Lafont, 1909, Mauritius; Vincent, 1910, Reunion; Noc and Stevenel,
1911, "Martinique and Antilles; Iturbe, 1918, Venezuela; Strong, 1924, Central
America.
E. indica, Rodhain and Bequaert, 1911, Belgian Congo.
E. nereifoUa, Laveran and Franchini, 1920, Italy; Franchini, 1923, Italy.
E. neruri, Row, 1915, Bombay.
E. officinarum, Franchini, 1923, Italy.
E. peploides, Sergent, Et., 1921, Algeria.
E. peplus, Franga, 1911, Portugal; Tjaveran and Franchini, 1920, Italy.
E. pilulifera, Lafont, 1909, Mauritius, and 1911, Madagascar, Mayotte, and Zanzibar;
Donovan, 1909, Madras; Vincent, 1910, Reunion; Carougeau and le Fera, 1910,
Madagascar; Bouet and Roubaud, 1911, Dahomey; Leger, 1911, Upper Senegal
and Niger; Noc and Stevenel, 1911, Martinique; Leboeuf and JaveUy (v.
Laveran and Mesnil. 1912, Franca, 1914), New Caledonia; Row, 1915, Bombay;
Tejera, 1919, Venezuela; Strong, 1924, Central America; Noguchi, 1924,
Honduras.
E. scliimperiana, Monti (quoted by Visentini, 1914), Sardinia.
E. secretalis {segetalis f), Tejera, 1919, Venezuela.
E. segetalis. Franca, 1911, Portugal; Visentini, 1914, Italy.
E. splendens, Franchini, 1923, Italy.
E. striata, Fanthani, 1925, S. Africa.
E. thymifolia, Lafont, 1909, Mauritius, and 1911, Madagascar and Mayotte; Vincent,
1910, Reunion; Carougeau and le Fera, 1910, Madagascar; Row, 1915, Bombay;
Tejera, 1919, Venezuela.
E. virosa, Laveran and Franchini, 1920, Italy; Franchini, 1923, Italy.
Asclepiadacese.
Araujia angiistifolia, Migone, 1916, Paraguay; Cordero (quoted by Franca, 1921),
Uruguay; Franchini, 1923.
Cynachum aciitum, Zotta, 1923, Roumania.
Morreira odorata, Migone, 1921, Paraguay.
Aselepias curassavica, Hegner, 1924, and Noguchi, 1924, Honduras.
Asclepias syriaca, Holmes, 1924, Baltimore; Noguchi, 1924, New York.
Apocynacese.
Acolcantliera spectabilis, Franchini, 1922, Italy.
Acolcanihera venenata, Franchini, 1922, Italy.
Cerbera odollam, Franchini, 1922, Paris.
Funtumia elastiea, Franchini, 1922, Italy.
Thevetia nereifolia, Franchini, 1922, Italy.
Sapotacese.
Sideroxylon inerme, Franchini, 1922, Paris.
Urticacese.
Ficus parietalis, Fisknchim, 1922, France.
Ficus benjamina, Franchini, 1923, Italy.
Fieiis ediilis, Fantham, 1925, S. Africa.
392 FAMILY: TRYPANOSOMID^
INOCULATION OF INSECT TRYPANOSOMID^ INTO VERTEBRATES.
A number of investigators, particularly Laveran and Franchini, and
Fantham and Porter, have claimed that vertebrates, particularly mice,
may be infected easily with insect flagellates by inoculation or feeding.
In some cases it is stated that a definite disease condition resembling
kala azar has resulted, and that the infection can be handed on from
animal to animal by inoculating emulsions of the infected organs. The
infection is associated with the presence of leishmania forms in smears of
the organs, while sometimes actual leptomonas forms occur in the blood.
The experiments of these investigators have been repeated by a number
of competent observers, who have failed entirely to substantiate their
claims. It would seem probable that some fallacy, such as the interpre-
tation as leishmania of structures which are of another nature, has been
responsible for the very high percentage of positive results claimed. The
only reliable test of an infection is the discovery of undoubted parasites in
smears of the blood or organs, or the development of flagellates in cultures
made from the blood or organs on N.N.N, or other suitable medium.
After Basile's claim that Mediterranean kala azar was transmitted from dog to
man by the dog and human fleas, Ctenocephalus canis and Pulex irritans, had become
known, the relation of the naturally occurring flea flagellates to Leishmania donovani
became the subject of many investigations. The question was raised as to whether
the insect flageUates could give rise to infections when inoculated into vertebrates.
Laveran and Franchini (1913) published an account of the infection of mice with
Leptomonas ctenocejjhaU. After inoculation by the intraperitoneal route, the para-
sites were found by direct examination in the peritoneal exudate and in the blood
for as long as sixty days. In the blood, both leishmania and leptomonas forms
occurred, while after death leishmania forms were found in the smears of liver and
spleen. Mice inoculated with peritoneal exudate of inoculated mice also acquired
an infection. Later (1914a, 1919, 1920) su.ccessful infections of mice, rats, guinea-
pigs, dogs, and monkeys {Macacus cynomolgus) were reported. Mice were readily
infected by inoculation of emulsions of the organs of infected mice, while rats were
infected by inocidation with heart blood, and dogs with spleen emulsion of infected
mice. Again, in other papers (1914&, 1914c, 1919a) it is recorded that, working
with L.pattoni of Ceratophyllus fasciaius, rats and mice were found to be susceptible
to inoculation and feeding. Eats and mice placed in jars with infected fleas for
forty-eight hours became infected with L. pattoni, and it was shown that mice could
be infected by contaminating their food with infected fleas. Mice were also infected
by the oral administration of Orithidia melopliagia {Trypanosoma melopliagium).
Experiments (1913a, 1914a) were also carried out with rats and mice and Crithidia
fasciculata of Ano2^1ieles maculipennis with similar residts. This flagellate was also
inoculated from one mouse to another, and an interesting result was obtained by
cutaneous injection. A local sore developed, in which leishmania forms were said
to occur.' There was also a general infection at the same time. Galli- Valeric (1923)
also states that more than two months after inoculation of a rat with the flagellates
from MeJophagus ovinus the animal died, and leishmania forms were found in its organs.
INOCULATION OF VERTEBRATES FROM INSECTS 393
FrancMni and Mantovani (1915) also claim to have infected rats with Herpeto-
monas muscarum of house flies. They state that they obtained a culture from the
heart blood of an inoculated rat in N.N.X. medium. The only organisms seen in the
cultures had the appearance of anaplasma, and they claim that mice were successfully
inoculated by means of the cultures. The mice showed leishmania forms in their
organs. It is impossible to understand what the authors mean by the small ana-
plasma forms, which were apparently the only ones seen in the culture. It is
difficult to conceive of a culture of H. muscarum which would not show the usual
leptomonas forms. Laveran and Franchini (1919rt, 1920) report that mice and
guinea-pigs were infected by inoculation of cultures of L. ctenocephali, which was
again recovered by culture from the blood. Similarly (1919a), cultures of L. jaculum
of the water bug Nepa cinerea were obtained by inoculating mice intraperitoneally
with the intestinal contents of the bugs and cultivating from the heart blood, and
mice were infected by inoculation of cultures of C. melophagia. The infections were
carried on to other mice by injections of liver and spleen material. Laveran and
Franchini (1920, 19206) gave accounts of successful experiments with cultures of
the leptomonas of Phlebotomus. Two dogs were inoculated in the skin of the thigh.
One developed a local lesion in which large cells containing numerous leishmania
occurred, while the other acquired a general infection (see p. 436). Guinea-pigs and
mice were also infected, and leishmania and other forms found in the organs. Kou-
baud and Franchini (1922) state that several mice, which were placed in jars in
which fleas {Ctenopsylla muscuU) were breeding, acquired infections, and that
leishmania forms in which the kinetoplast was not clear were found in the organs-
From the spleen of one of these mice another mouse was infected. They also claim
(1922o) that mice inoculated snbcutaneously with dried faeces of fleas became
infected. In a later paper these authors (192.3) state that a culture was made from
the heart blood of one of the mice two and a half months after its inoculation.
Nothing appeared in the culture for some time, but over three months later the tube,
which had been put aside, was examined and flagellates were found. Subcultures
were successfully obtained. It is evident that if flagellates took such a long time to
appear in the cultures, they must have been exceedingly scanty in the heart blood of
the mouse. Laveran and Franchini (1923) give an account of experiments con-
ducted with the flagellates of the bug Pentatoma ornatum. These were inoculated
to mice and passed through other mice in series. In all cases infection resulted,
though it is admitted that the organisms were present in small numbers only. These
were said to be of the leishmania, piroplasma, or anaplasma type. Cultures were
repeatedly made from the heart blood or organs of the experimental animals, but in
only one case was a positive result obtained. In this culture only round forms were
present, no flagellates being seen. The figures accompanying the description serve
a useful purpose in that they illustrate what the author is willing to accept as evidence
of infection in animals.
The organisms discovered in infected animals by Franchini and those who have •
associated themselves with him were usually of the leishmania type, though the
elongated flagellates were often said to be present in the blood-stream and occa-
sionally in the organs. As a rule, the parasites were scanty in number, the animals
not showing the intense infection which sometimes occurs in mice inoculated with
Leishmania tropica or L. donovani. Some of the figures, or rather diagrams, produced
by these observers, however, show large cells of the macrophage type packed with
parasites, as seen in oriental sore and kala azar. Experiments of a similar kind
have been recorded by Fantham and Porter (1915rt). ^Vorking with L. jaculum
of the water bug Nepa cinerea, they claim to have successfully infected mice
by inoculation or feeding with the intestinal contents of the bugs. A puppy, like-
394 FAMILY: TRYPANOSOMID.E
wise, is described as becoming infected after being made to ingest lleas, some of wliich
harboured L. ctenocephali. A more extensive series of experiments was published
later (1915&). In these, four flagellates were used [L. jaculum, L. stratiomyice,
L. pedicuU, and C. gerridis), and various vertebrates as follows: the stickleback
(Gasterosieus aciileatus), newt {3Iolge vulgaris), frog (Bana temporaria), toad {Bufo
vulgaris), lizard {Lacerta vivipara), snake {Tropidonotus natrix), and mice {Mus
musculus). These animals were infected with one or more of the flagellates, either
by inoculation or feeding. In many cases, Fantham and Porter believe that the
organisms acted as pathogenic agents, and brought about the death of the animals.
Still another series of experiments is recorded by these observers (1915c). On this
occasion, they claim to have infected birds (canaries, martins, sparrows) by feeding
them with L. jaculum or L. culicis of Cnlex pipiens. In these experiments they
claim to have found leishmania and flagellate forms of the parasites in the blood
and various organs, and state that the birds became ill from the infections induced.
It is suggested that it is possible that in nature these infections may be one of the
causes of mortality amongst birds.
The remarkable feature of all these experiments is the apparent ease with which
infections were produced. Other workers, as, for instance, Noller (1912(Z), failed
to infect a young dog with L. ctenocepliali. Chatton (1919) failed entirely to infect
mice with the same flagellate, and the writer has had a similar experience with the
cultures of the leptomonas of Pulex irritans. Tyzzer and Walker (1919) conducted
very careful experiments with L. ctenocephali. Though they inoculated mice, some of
which were newly-born, by various routes, they never succeeded in producing an
infection. Patton (1921) has stated that he has failed entirely to infect mice with
several species of insect flagellate, while Glaser (1922) attempted without success
to repeat Franchini and Mantovani's experiments with H. muscarum. Hoare
(1921rt) made a very careful study of the question, and carried out a series of experi-
ments with the flagellates of GalUphora sp., ISlepa cinerea, and Meloplmgus ovinus,
The vertebrates inoculated or fed with one or other of these flagellates were mice,
newts, frogs, and sticklebacks. Though very searching observations were made,
involving not only the examination of smears, but also cultures from the heart blood
and organs, in no single instance was an infection demonstrated. Hoare' s experi-
ments indicate, at any rate, that infections cannot easily be produced, and that the
claim that purely insect flagellates may take on pathogenic properties seems very
doubtful indeed. As Hoare points out, in conducting experiments of this kind, only
undoubted leishmania forms should be accepted as evidence of infection. In the
successful experiments recorded above, the observers have undoubtedly been willing
to accept as leishmania forms bodies of a doubtful nature. This is clearly shown by
the frequent references to leishmania forms with a single nucleus and the anaplasma
forms in cultures. Roubaud and Franchini (1922), for instance, state that the
parasites in the infected mice mostly had a single nucleus, and that the kinetoplast
. was very difficult to distinguish. In the absence of a kinetoplast, it is not easy to
comprehend the reasons for regarding the bodies as flagellates at all. They might
equally well be yeasts, the organism which has been named Encephalitozoon, or the
merozoites of some Sporozoon such as Klosiella, which may infect the endothelial
cells of the bloodvessels.
Glaser (1922) made unsuccessful attempts to infect six mice, a rat, and a guinea-
pig with //. muscarum, while Shortt (1923rt) conducted a series of experiments
with L. ctenocephali of the dog flea, and L. lucilice of Lucilia craggii, and rats, mice,
monkeys, dogs, pigeons, and frogs. The animals were either fed or inoculated
in various ways, and were subsequently examined by the smear and culture
method. Over fifty experiments were made, and in not a single instance was
PLATE III.
Various Vegetable Organisms which simulate Protozoa when they occur in Dried
Blood-Films or Smears of Organs stained with Romanowsky Stains. (1 and
2, X 2000: 3-6, x 1000)
1. Histoflasma capsvlatum in macrophage from smear of human lymphatic gland. Note resem-
blance to Leishmania.
2. Cryptococcus farcinimosus, the cause of lymphangitis of horses, from smear of lymphatic
gland. Note resemblance to Leishmania.
3. Group of large vegetable cells in a blood- film contaminated with intestinal contents of a
rabbit. They bear some resemblance to haemogregarines.
4-6. Groups of yeast-like organisms in blood- films contaminated from cultures. They may be
confused with Leishmania, merozoites of Sporozoa, or spores of Microsporidia.
(1 AND 2, AFTER ROCHA-LIMA; 3-6, ORIGINAL.)
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[ To face p. 394
INOCULATION OF INVERTEBRATES FROM INSECTS 395
an infection noted. Yamasaki (1924) also failed to infect mice and dogs with
L. ctenocephali.
Becker (1923a), using C. gerridis, H. muscarum, and the flagellates of the sheep
ked, failed entirely to produce infection in seventeen rats, three mice, one rabbit, and
one guinea-pig. As in the case of Iloare's and Shortt's experiments, cultures on
X.N.X. medium were attempted. Strong (1924) likewise failed to infect animals
with the flagellates of the bugs found on Euphorbias, while Drbohlav (1925) failed
to produce any infection with L. ctenocephali in about 150 animals examined by
the smear and culture method after inoculation.
There is no longer any doubt that the C. melophagia of the sheep ked is in reality
T. melophagium of the sheep, yet, by inoculation of this trypanosome to mice,
Laveran and Franchini claim to have obtained infections of the leishmauia type.
As pointed out by Hoare (1921a), if an infection had occurred, it would almost
certainly have been of the ordinary trypanosome type. Buchner (1922), who also
faUed to infect mice with the ked flagellate, likewise points out this fallacy in the
experiments of Laveran and Franchini. It seems impossible to accept the remark-
able statements regarding the successful inoculation of insect flagellates to verte-
brates till definite confirmation is forthcoming.
The negative results obtained by so many com-
petent observers suggest that the positive results
reported may have been due to misinterpretations.
It is possible, however, that occasionally isolated
parasites may survive in the organs and give rise
to cultures, but it is doubtful if such a condition
can be regarded as an infection. There is cer- _ ^ ^^^
tainly no reliable evidence that, even if such a ;'^i'^'^j<8'cf^^6^^^^E0
survival of the parasites occurs, they give rise to '^^iP'^^^^^jQ^'0^"''
serioixs and fatal disease. Though Fantham and '^'^*£?^*i^ \ ® ^^
Porter claimed that sticklebacks were killed by ^O^, ^}m
the infections induced by feeding them on the
intestines of the water bug, Hoare found that the ^IG. 185. -Vnjptoroccm muris
fish thrived on this diet. {^ ««• ^'^7„^-, ^^^^^^"^
There occur in the organs of rats and mice J^angiorgi, 19-...)
structures which can readily be mistaken for
leishmauia. Thus, Sangiorgi (1913) has described as Toxoplasma musculi certain
minute bodies found by him in the spleen of a mouse, and as T. ratti similar
forms from the rats. Sangiorgi (1922&) also recorded a Cryptococcus from mice, and
it is this organism which was named C. muris by Shortt (1923a), who discovered
it in mice in India (Fig. 185). Whether the structures described by Sangiorgi are
Toxoplasmata or not, it is evident that they and the cryptococcus could be easily
mistaken for leishmauia. The same remarks apply to the parasite of rabbits
described as Enceplialitosoon cuniculi and the similar form in mice, both of which
occur fairly commonly in the organs of laboratory animals (p. 754).
INOCULATION OF INSECT TRYPANOSOMIDiE INTO INVERTEBRATES.
As trypanosomes can be inoculated from one vertebrate to another,
so can invertebrates be inoculated with flagellates obtained from, other
invertebrates. Zotta (1912) observed a leptomonas in Pyrrhocoris aptera,
a plant bug. The infection occurred, not only in the intestine, but also
396 FAMILY: TRYPANOSOMID/E
in the body cavity, whence all the organs of the body were invaded. He
(1921) succeeded in obtaining a culture of the organism L. pyrrJiocoris in
N.N.N, medium. In the same year (1921a) he investigated the effect of
these cultures on other arthropods by inoculating them in the body cavity.
He found that active multiplication occurred, some of the experimental
arthropods becoming overrun with flagellates. In this manner he suc-
ceeded in infecting Notonecta glauca (water boatman), Naucoris cunicoides
(aquatic bug), Galleria mellonella (caterpillar of bee-hive moth), CaUiphora
sp. (larva of blow-fly), Tenebrio molitor (larva of meal-worm). The most
intense infections were produced in the larva of the meal-worm and the
caterpillar. Glaser (1922) has similarly succeeded in infecting Melanoplus
femurrubrum (grasshopper) and Amblycorypha oblongifolia (locust) with
H. muscarum of the house fly.
By feeding bed bugs on cultures of Leptotnonas pidicis, Crithidia cteno-
cephali, and Herpetomonas muscarum, Patton, La Frenais, and Eao (1921)
have shown, by making cultures from the intestine in N.N.N, medium
at varying intervals after feeding, that the flagellates can survive for
thirty-seven, eight, and forty-five days respectively.
Genus: Leishmania Ross, 1903.
The flagellates included in this genus are characterized by the possession
of both a vertebrate and an invertebrate host, as in members of the genus
Tryjjanosotna, from which they differ in that only leishmania and lepto-
monas forms occur in the cycle of development. In no case, how^ever,
has an invertebrate host actually been demonstrated, but the evidence
that such a host exists is so convincing that this feature has been included
in a definition of the genus.
From the purely morphological point of view there are at present no
data which afford a means of distinguishing members of the genus Leish-
mania from those of the genus Leptomonas. In both there occur only the
leishmania and leptomonas forms. The members of the genus Leptomonas
are handed on from one invertebrate to another by the contaminative
method by means of encysted forms passed in the fseces. No such stages
are known in the case of Leishynania, though they may occur. It would
thus be quite logical to include Leishynania in the genus Leptomonas.
Nevertheless, on account of the existence of two hosts in the former and
a single one in the latter, the retention of the separate genera is a con-
venience. The inclusion of Leishmania in the genus Herpetomonas, as
Patton and others have done, cannot be admitted, as the members of the
genus Herpetomonas have definite trypanosome stages which do not occur
in Leishmania.
The first observer to see one of the parasites which are now regarded
GENUS: LEISHMANIA 397
as belonging to the genus Leishmania was Cunningham (1885) in India,
who described " Peculiar Parasitic Organisms in the Tissue of a Specimen
of Delhi Boil." The parasitic organisms referred to were the large macro-
phages which were supposed to be amoebae, while the leishmania within
them were regarded as spores. Firth (1891), who made similar observa-
tions, proposed the name Sporozoa furunculosa for the large cells containing
the spores. As the name was given primarily to the supposed amoeboid
forms which are now known to be tissue cells. Firth's name cannot be
employed for the parasites.
Leishmania were next seen and recorded by Marchand (1904) in the
spleen of a Chinaman who had died in Germany. A demonstration was
given before the Leipzig Medical Society on February 3, 1903. This
observer inclined to the view that the bodies within the large cells were
degeneration products of nuclei. On May 30, 1903, appeared Leishman's
paper on " The Possibility of the Occurrence of Trypanosomiasis in
India," wherein he described the parasites which he had found three
years before in cases of dum-dum fever. He recognized their resem-
blance to the round forms which occurred in trypanosome infections. On
July 11 of the same year Donovan (1903) recorded the presence of the
same parasites in this disease. Laveran and Mesnil (1903, 1903a) examined
some of Donovan's films, and, owing to the scarcity of the parasites
and the fact that many appeared adherent to red blood-corpuscles, they
regarded them as piroplasmata, and proposed the name Piroplasma dono-
vani (November 3, 1903). Koss came to the conclusion that the organism
was a Sporozoon, and suggested the name Leishmania (November 14 and
28, 1903). The name for the organism of kala azar is, therefore, Leishmania
donovani (Laveran and Mesnil, 1903). In March, 1904, appeared Bentley's
announcement of the discovery of the organism in cases of kala azar.
Nicolle (1908) gave the name Leishmania infantum to the parasite causing
kala azar in the Mediterranean area.
Wright (December, 1903) described a similar organism from a case of
oriental sore in an Armenian child wdio had been brought to Boston. He
proposed the name Helcosoma tropicum for the parasite, which he con-
sidered to be a Protozoon allied to the Microsporidia. Marzinowsky and
BogrofE (1904) in Russia discovered the organism in a sore on a boy who
had resided in Persia, and proposed the name Ovoplasma orientale. Sub-
sequent investigations have shown that the organism is morphologically
indistinguishable from that of kala azar, and must be included in the same
genus. The correct name for the parasite of oriental sore is Leishmania
tropica (Wright, 1903). Eogers (1904) made the important discovery
that flagellates developed in sodium citrate solution to which spleen pulp
containing L. donovani had been added. At first he regarded them as
398 FAMILY: TRYPANOSOMID^
trypanosomes, but later came to the conclusion that they were herpeto-
monas (leptomonas) developed by growth of the leishmania. Leishman's
original view as to the flagellate nature of the bodies was thus fully estab-
lished. It was not till four years later that Nicolle (19086) and Nicolle
and Sicre (1908) obtained a similar culture from the leishmania of oriental
sore, an observation which demonstrated more clearly the close relation-
ship of the two parasites. Subsequent work established the fact that the
parasites were the actual causes of the two diseases, which were shown to
have a wide distribution in the Old World, while cutaneous leishmaniasis
was found to occur also in South and Central America, where Vianna (1911)
gave the name L. hrasiliensis to the parasite. It was further demonstrated
that dogs are liable to the same two diseases.
The organisms belonging to the genus Leishmania, which infect human
beings, are thus to be regarded as flagellates of the leptomonas type, which
in man and the dog are almost invariably in the leishmania stage, though
very rarely the leptomonas form has been observed. There occur,
however, certain flagellate infections of other vertebrates in which the
predominating forms are of the leptomonas type. These organisms also
will be considered as belonging to the genus Leishmania. Button and
Todd (1903) stated they had seen a flagellate of the leptomonas type in
Gambian house mice, but a later examination of stained films led Todd
(1914) to the view that the flagellate was really a trypanosome (T. acomys).
Balfour (1916) called attention to the fact that he and Archibald some years
earlier had seen such a flagellate in the gerbil in the Sudan, but in neither
of these cases was the structure of the organism accurately determined.
The Sergents, Ed. and Et. (1907), observed flagellates of the lepto-
monas type in a stained blood-film of a pigeon in Algiers. The body of
the organism was 17 to 20 microns in length, while the flagellum measured
19 to 35 microns. The figures show an organism very similar to Herpeto-
monas muscarum. It was only found in a film made on one occasion, and
has never been rediscovered. Knuth (1909a) found similar forms in
smears of the heart blood of a roebuck in Africa, but the animal had been
dead some time, was partly devoured and decomposed, and was infested
with fly larvae, so that the origin of the flagellates was doubtful. They
may have been deposited by flies. Fantham and Porter (1915) gave a
figure and description of a similar form observed by them in the living
condition in a mouse in England. As the flagellate was seen only in the
fresh blood, and was described as very active, it is difficult to understand
their statement that the drawings were made with the camera lucida.
A nucleus and kinetoplast, which are exceedingly difficult to detect
without staining, are clearly shown.
The Sergents, Lemaire, and Senevet (1915) demonstrated the presence
GENUS: LEISHMANIA 399
of flagellates of the leptomonas type in the North African gecko {Tarentola
tnauritanica) by making cultures from the heart blood. Bayon (1915)
discovered flagellates of this type in the cloaca of the chameleon {Chamce-
leon pumilus) of Robben Island, an observation confirmed by the writer
(1921) for Chamwleon vulgaris of Egypt. Another form was found by
Leger, M. (19186), in the blood of a lizard {Anolis sp.) of Martinique.
Fantham and Porter (1920) have described and figured a leptomonas
from the blood of a South African fish {Dentex argyrozona), while Laveran
and Franchini (1921), under the name of Herpetotnonas myoxi, record a
similar form from the dormouse {Myoxus glis) of Italy (see p. 442). Strong
(1924) has seen flagellates of the leptomonas type in the intestine of the
lizard {Cnemidophorus lemniscatus) of Central America.
In the case of the flagellates which are only seen in the blood in the
living condition, it is always possible that they were in reality trypano-
somes or crithidia stages of these. This possibly applies to the forms seen
by Balfour in the gerbil, by Fantham and Porter in the mouse, and by
Laveran and Franchini in the dormouse. Quite recently the writer saw
very active flagellates in the urine of a rat. At first they were thought
to be leptomonas, but more careful study of the shape and movements
produced the impression that they were crithidia. Stained films, however,
proved that only trypanosomes of the T. lewisi type were present, and as
the rat was infected with this trypanosome, it was evident the trypano-
somes had passed into the urine from a wound made at the autopsy.
Richardson (1925, 1926) found numerous leishmania in the spleen of
a horse which died in Uganda. The writer saw the films, which resembled
those from cases of kala azar. Curson (1926) has given the name Leish-
mania caprcB to supposed leishmania seen in films made from the ear of a
goat in S. Africa.
As regards the various species of Leishmania described from man,
it is generally admitted that they are morphologically indistinguishable
from one another. Little assistance has been obtained from animal inocu-
lations, for it has been found that L. donovani, which produces a general-
ized infection in man, may give rise to purely cutaneous lesions in animals,
as also occasionally in man; while L. tropica, which causes local cutaneous
lesions in man, may produce generalized infections in animals. Attempts
have been made to differentiate the species by serological tests, the use of
which for the separation of true species is of very doubtful value. The
most precise statements are those of Noguchi (1924). He employed
strains of L. donovani, L. infantum, L. tropica, and L. brasiliensis. Rabbits
were inoculated intravenously on four occasions at five to seven day
intervals. The sera from these animals were then used on cultures to
test their agglutinating power. It was found that in dilutions of ^\, or
400 FAMILY: TRYPANOSOMID^
even yi^^, the serum of the animals inoculated with L. donovani agglut-
inated this organism and L. infantum, but not the two others. Similarly,
the serum from an animal inoculated with L. tropica agglutinated this
organism alone, and the same was true of the serum of an animal inocu-
lated with L. brasiliensis. From these reactions it appears that sero-
logically the organisms tested fall into three groups, in conformity with
the clinical types of disease produced. If the sera were added to the
culture media, they were similarly specific in changing the character of
the growth of the homologous organisms.
LEISHMANIA IN MAN.
The Parasite of Kala Azar.
Leishmania donovani (Laveran and Mesnil, 1903). — This organism,
which is often referred to as the Leishman-Donovan body, is a rounded,
non-flagellate stage of a flagellate which infects the vascular endothelium
and wandering macrophages of human beings, and produces the disease
known as kala azar.
DISTRIBUTION. — Kala azar occurs in India, in Madras and in the
district north of the Bay of Bengal, in Calcutta and along the Ganges
and Brahmaputra, in Bengal and Assam. Cunningham and Pundit
(1925) have recently discovered the disease in the extreme South of
India opposite Ceylon. In China it occurs north of the Yang-tse in
a district between the coast and a line joining Pekin and Hankow. It
has also been recorded from Sumatra by Smits (1916), but from informa-
tion the writer has received there appears to be considerable doubt
regarding this observation. In Southern Russia it is found both west
and east of the Caspian Sea, in Transcaucasia and Turkestan, while Kiilz
(1916) has found it to be endemic in Mesopotamia. The whole of the
Mediterranean littoral and many of the islands are homes of the disease,
as also an area on the Blue Nile west of Abyssinia extending as far as
Khartoum in the north and towards Kodok in the south. The writer has
received information that the disease has been discovered in Kenya Colony.
A case in a child has been recorded by Bouilliez (1916) near Lake
Chad, and another by Tournier (1920) in the Gaboon, both in West Africa.
SYMPTOMOLOGY. — The disease occurs most usually in children or
young adults, and is due to invasion of the endothelial cells of the capil-
laries by the parasites, which are mostly concentrated in the spleen, bone
marrow, and liver, though the lymphatic glands, or, indeed, any organ,
may be found infected. The symptoms produced are chiefly enlargement
of the spleen and liver, progressive emaciation, anaemia, and an irregular
type of fever. Other symptoms may occur, such as enlargement of the
LEISHMANIA DONOVANI 401
lymphatic glands, pigmentation and dryness of the skin, and oedema.
These may be ascribed to the general malnutrition, while dysentery,
cancrum oris, and pneumonia are complications due to secondary bacterial
infections. Left untreated, the disease nearly always ends fatally, though
a small percentage of recoveries may take place. Recovery has also been
noted after certain bacterial infections, which seem to act adversely on
the leishmania. The duration of untreated cases may be only a few
months in acute forms of the disease, or several years in the more chronic
type.
RELATION OF INDIAN KALA AZAR TO THE SIMILAR DISEASE IN
OTHER LOCALITIES. — Kala azar was first recognized as a distinct disease
in India, but after the discovery of the characteristic leishmania as its cause,
it was soon found to have a much wider distribution. The discovery of
kala azar in the Mediterranean area as a disease which afiected chiefly very
young children at once raised the question of its identity with that of
India. The parasite causing the Mediterranean type of the disease known
as infantile kala azar was named Leishrnania infantum by Nicolle (1908).
The discovery of the disease in the Caspian region and in the Sudan,
where children and young adults are mostly affected, and the realization
that in India it is by no means limited to adults, as at one time was
supposed, have raised doubts as to the validity of the species L. infantum.
Morphologically, there is no distinction between the leishmania from
the various areas in which the disease occurs, nor is there any marked
difference in which animals respond to inoculation. In the Mediterranean
and Caspian areas, the disease is associated with a similar one in dogs,
whereas in India kala azar in dogs has not been discovered, though it has
been very carefully looked for. It is known, however, that dogs can be
infected with the Indian parasite. There seems no r-eason, therefore,
to separate the Mediterranean leishmania from that of India, and the
parasite of kala azar, wherever it occurs, wall be designated Leishmania
donovani. Noguchi (1924) has found that serologically L. donovani and
L. infantiim are identical.
PATHOLOGY. — The chief histological change in the organs of kala azar
cases is an increase in the large macrophages, which are presumably
derived from the endothelial cells of the capillaries. Correlated with
this is an increase in the proportion of mononuclears in the blood, though
the general leucocyte picture is usually one of leucopenia. The very much
enlarged spleen shows an increase in fibrous tissue, and a multiplication
of the macrophages, which are often loaded with parasites (Fig. 186).
Similar changes occur in the liver, where the fibrotic change may be very
marked, while the bone marrow shows a great increase in these large cells
I. 26
402 FAMILY: TRYPANOSOMIDyE
(Fig. 187). In whatever part of the body parasites are found — and they
may occur in any organ or tissue — they are practically always within
the cytoplasm of large cells of the endothelial type. It was Christophers
(1904) who first showed that, pathologically, kala azar was essentially an
infection of the endothelial cells of the blood-vessels. It must be remem-
bered that in smears of organs or in blood-films, the parasites are often
seen extracellularly, but, though such forms must occur in the passage
of parasites from cell to cell, the extracellular position as usually seen
is due to the breaking-up of the large cells in preparation of the films.
si's . 4'«'<^««'vv ?'
Fig. 186. — Section of Human Spleen (x 750): Leishmania donovani within
Macrophages. (After Nattan-Larrier, 1913.)
Not infrequently, portions of the cytoplasm, fragmentation bodies, of
these large cells are broken off in the process of film-making, and if found
to harbour parasites, they may produce an appearance of multiple seg-
mentation, especially when the outlines of the organisms are imperfectly
stained. In sections of tissues where artificial rupture of the cells has
not taken place, the parasites are practically always found to be intra-
cellular. Furthermore, in films, parasites are sometimes seen lying over
red blood corpuscles, and have been described as actually within these
cells, like the malaria parasite and piroplasmata. This is merely an
LEISHMANIA DONOVANI 403
appearance artificially produced in preparation. The organism may be
found in films of the peripheral blood, either in cells of the mononuclear
or polynuclear variety.
The parasites, as already remarked, may occur in any tissue of the
body within the macrophages. They were demonstrated by Christophers
(1904) in intestinal ulcers, and in the papules which sometimes occur in
the skin of cases of kala azar. Bramachari (1922) has noted that cases
^f^- «i^''a
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Fig. 187. — Section of Human Liver (x750): Leishmania donovani within
Macrophages and the Glandular Cells. (After Nattan-Larrier, 1913.)
of the disease which have apparently recovered after antimony treatment
may develop nodules on the skin, which in one case were distributed over
the body, and resembled a form of nodular leprosy. Leishmania w^ere
present in all these lesions, though they had apparently disappeared from
the internal organs. Another similar case has been described by Shortt
and Bramachari (1925). Perry (1922) has found that in cases of kala azar
the subepithelial tissues of the wall of the intestine may be much swollen,
owing to the presence of enormous numbers of macrophages packed with
404 FAMILY: TRYPANOSOMID^
leishmania (Fig. 188). This condition has led him to suggest the possi-
bility of the spread of infection by the escape of parasites from the body
in the dejecta. Shortt (1923c), and Shortt, Swaminath, and Sen (1923),
have demonstrated the escape of L. donovani in the urine.
©
f'
■9
@
!
^ .or •:•■-.- ;V:vV-:'. ^
Fig. 188. — Section of Disorganized Villus of Small Intestine, with Leish-
mania donovani in Macrophages (x ca. 750). (After Perry, 1922.)
DIAGNOSIS BY DISCOVERY OF THE PARASITE.— Diagnosis of the
disease is established by the discovery of the parasite. This is usually
accomplished by making films of material obtained by puncture of the
spleen, and staining by Romanowsky stain. The operation is not entirely
free from danger, as in some cases, especially when the spleen is very soft
and large, the wound has continued to bleed and death has resulted. AVhen
due care has been taken, however, there is little risk of haemorrhage. An
ordinary hypodermic syringe may be employed, and it should be perfectly
dry. The best result is obtained when very little blood is abstracted, on
which account suction should be discontinued as soon as blood appears
above the needle. If this be done, there will be a greater number of spleen
cells, which are the ones required for finding the parasites. The less danger-
ous operation of liver puncture will often reveal the organism, and some
observers maintain that it is as reliable for diagnosis as puncture of the
spleen. Examination of ordinary blood-films will sometimes reveal the
parasite as first pointed out by Christophers (1904), but many films may
have to be examined before a single parasite is seen. In some cases,
however, they seem to have been easily found in the peripheral blood.
LEISHMANIA DONOVANI 405
Thus, Donovan (1905, 1909a) states that he had found parasites in the
finger blood of over 93 per cent, of the cases in Madras, while Patton
(1907, 1912a), in the same place, had positive results in thirty-eight out
of forty-five cases. Nicolle and Comte (1908a) in Tunis demonstrated
leishmania in the peripheral blood of a case of the Mediterranean disease,
and Cannata (1913-1914) in Italy found them in fifteen out of sixteen
cases after examination of many films from each, an observation which
was confirmed by Vaglio (1914), who was successful in eleven cases.
Knowles (1920) has examined cases from this point of view at Shillong in
Assam, with the following results: Seventy-three cases were examined,
and parasites discovered in the blood of thirty-three. From the seventy-
three cases, 682 films were scrutinized, and sixty-seven of these were
positive, revealing 2,839 parasites. It will thus be seen that diagnosis by
direct examination of the peripheral blood is not always a simple matter,
but usually necessitates the careful and prolonged study of many films.
Knowles and Das Gupta (1924a) have demonstrated parasites in 67 per
cent, of seventy cases by the use of thick films of the peripheral blood.
Mayer and Werner (1914) and the writer (1914) demonstrated the
possibility of diagnosis by culture of the peripheral blood. Blood taken
from the finger with due care to avoid bacterial contamination is
inoculated to a series of tubes of N.N.N, medium, a few drops being added
to each tube. Flagellates develop in the tubes after a variable period of
two or three weeks. This observation has been confirmed by Row (1914),
Giugni (1914, 1914a), Cannata and Caronia (1914), Cornwall and La
Frenais (1916), and Knowles (1920). Though it is a method of diagnosis
worthy of trial, a negative result cannot be held to exclude an infection.
Culture of material from spleen or liver puncture should be carried out
at the time of film-making, for, when the infection is a slight one, the
parasites may be missed in the smears, though sufficiently numerous to
develop in the culture.
DIAGNOSIS BY SEROLOGICAL TESTS.— Attempts to obtain a method
of diagnosis based on the principle of complement fixation has yielded
only discordant results.
Napier (1921, 1922) noted that if a drop of commercial formalin
be added to 1 c.c. of the serum of a case of kala azar, the serum solidifies
in a few minutes, and very quickly becomes opaque, like the coagulated
white of an egg. This reaction, which is called theformol gel test or aldehyde
reaction, is fairly constant in kala azar. It occurs only partially in
tuberculosis, leprosy, and heavy malarial infections, and disappears
progressively during the course of treatment oi cases of kala azar by
means of tartar emetic.
Bramachari (1920) described as the globulin precipitation test a reaction
406 FAMILY: TKYPANOSOMID^
which occurs with the serum of kala azar cases. If one part of serum
is mixed with two parts of distilled water, an opacity is produced. If
the water is poured on the surface of the serum, a ring effect is obtained.
The test has been elaborated into a quantitive one by Bramachari and
Sen (1923).
Wagener (1923) has shown that the injection of alkaline extracts of
Leishmania from cultures into the skin of rabbits previously rendered
sensitive by injections of cultural forms of Leishmania produces a local
reaction in the form of an erythematous papule, which reaches its height
in forty-eight hours, and persists from three to five days. The antigen
can be prepared from both L. tropica and L. donovani, as it is not specific
for either parasite. If these results are confirmed, the reaction may be
of use for diagnostic purposes. The serological observations made by
Noguchi (1924) have been referred to above (p. 399).
MORPHOLOGY. — The parasite Leishmania donovani, which is morpho-
logically indistinguishable from L. tropica, is a small organism usually
circular or oval in outline (Plate IV., 7-10, p. 406). It consists of a mass of
cytoplasm covered by a definite membrane. The cytoplasm contains
two very characteristic structures, the recognition of which is essential
to the identification of the organism. One is the nucleus, and the other
the kinetoplast. The former is a more or less spherical body, with a
diameter about one-third to a half of the shortest diameter of the organism.
It usually lies against the membrane, and is somewhat flattened on this
side. The flattening may be so marked that its form is reduced to that
of a hemisphere, or even of a thin disc, so that in optical section it is seen
as a semicircle or merely a narrow structure lying along one side of the
parasite. The extreme flattening of the nucleus often appears to be
intensified by the presence of one or more vacuoles in the cytoplasm,
which may be so large as to reduce the parasite to the condition of a thin-
walled sac. The second structure of importance is the kinetoplast, which
is usually seen as a rod lying with its long axis directed towards the nucleus.
In preparations it may appear as a small spherical body, but in most cases
this is due to its long axis being perpendicular to the slide. In ordinary
dried films stained by the Romanowsky method, the nucleus appears as a
mass of bright red granules, while the kinetoplast, which is a more solid com-
pact body, takes a deep reddish-purple tint. In deeply stained parasites
a red line, first described by Christophers (1904), can be traced from the
blepharoplast, which lies near the centre of the kinetoplast, to the surface
of the parasite. This is the axoneme, which gives rise to the flagellum
of the leptomonas forms which develop in cultures (Plate IV., 6, p. 406).
The size of the parasite varies considerably. When spherical, it measures
from 1 to 3 microns in diameter. More usually it is ovoid, with the long
PLATE; IV.
Leishmania tropica and L. donovani from Cases of Oriental Sore and Kala-Azar:
Dried Films Stained with Romanowsky stains. ( x 2,000).
1. Portion of a field in a smear from an oriental sore, showing L. Ircpica scattered as a result of
rupture of an endothelial cell.
2. Detached portion of cytoplasm of endothelial cell showing L. tropica. The outlines of the
parasites are not visible. Such bodies have been interpreted as schizonts.
3. Red cell with superimposed L. tropica.
i. Detached portion of cytoplasm of endothelial cell with L. tropica.
5. Large endothelial cell packed with L. tropica.
6. Three parasites (L. tropica) showing axonemes.
7. Portion of a spleen smear showing L. donovani.
8. Detached portion of cytoplasm of endothelial cell in peripheral blood-film of kala-azar case
showing L. donovani.
9. Large endothelial cellin peripheral blood- film of kala-azar case with a single parasite (L. dono-
vani) in the cytoplasm.
10. Group of nine parasites {L. donovani) in smear from cervical lymphatic gland of kala-azar case.
(Original.)
in .iluiot a
,-;^;j^ cu^i -a Cjiiu'i^ .01
PLATE IV.
^
§
■* *^ .Al
^
;*^
[To /ai;« p. 406
LEISHMANIA DONOVANI 407
diameter from 2 to 5 microns and the shorter 1-5 to 2-5 microns. In these
forms one end is often more rounded than the other. Occasionally, more
elongated forms somewhat resembling a cigar or torpedo in shape occur,
and in these the kinetoplast may be so closely applied to the nucleus as
to escape recognition (Fig. 192). In the smear of a child's spleen made
by the writer in Malta, the majority of the parasites were of this type.
Occasionally, larger parasites occur, especially in inoculated animals,
where they may attain a diameter of 8 or 9 microns. In a spleen smear
from a typical case of kala azar there occur numbers of large cells, the
macrophages, some of which are packed with parasites. Many of these
cells will have broken down in preparation of the smear, and the liberated
parasites will be scattered amongst the debris. Detached portions of the
cytoplasm of these cells containing groups of leishmania have been called
" gangues" by French writers (Plate IV., 2, 4, 8, p. 406). In less heavily
infected cases a careful examination of the films will have to be made, as
Fig. 189. — Flagellate Forms of Leishmania donovani in the Tissues of an
Experimentally Infected Dog ( x ca. 2,000). (After Wenyon, 1915.)
the parasites may be present in very small numbers. A group of two
or three parasites, or even single ones, will be found in the cytoplasm of
a small percentage of the cells. In such cases, careful attention must be
paid to the morphology, and no structure should be called a leishmania
unless the sharp outline, the deeply staining rod-like kinetoplast and
the more palely staining and larger nucleus have been clearly seen. The
crucial test in any doubtful case is the development of the flagellate lepto-
mouas form in culture. In every film, in addition to the parasites which
show the typical structure, there occur abnormal or degenerate types,
about the nature of which it is often impossible to form an opinion.
As seen in dried smears stained by the usual Romanowsky methods,
the nucleus appears as an aggregation of red-staining granules. This is
an artificial picture, for in films which have been fixed, without drying,
in a suitable fixative, and stained by the iron-haematoxylin method, the
nucleus is seen to have a membrane enclosing a clear space, at the centre
408 FAMILY: TRYPANOSOMID.E
of which is a spherical karyosome (Fig. 192, 7-8). The kiuetoplast
is a compound body consisting of a rod-shaped parabasal and a blepharo-
plast from which the axoneme arises. In dried films the axoneme is often
seen as a red line after deep staining with Romanowsky stains.
There is no evidence that the leishmania exist in any other than the
typical form in the infected host, with the single exception recorded by
the writer (1915a) of the occurrence of leptomonas forms, such as appear
in cultures, in the spleen of a dog infected with leishmania from a case of
Indian kala azar (Fig. 189). In this animal the leishmania were of a
particularly large size and varied shape.
Maitra (1924), in India, found in a peripheral blood-film, made from a
case which was clinically one of kala azar, flagellates which appeared to be
of the leptomonas type. Subsequent examinations of the blood did not
reveal any flagellates, so that it is not improbable that the film had been
contaminated. The writer knows of an instance in which similar flagellates
were deposited on a blood-film by a fly. There is nothing in Maitra's
account to suggest that such a contamination took place, but, from
information the writer has received, such a fallacy was not excluded,
for the flagellates were only found in one of several films made at the
same time.
MULTIPLICATION. — The only method by which Leishmania donovani
multiplies is by binary fission. Dividing forms with two nuclei and two
kinetoplasts, and these structures actually in process of division, can easily
be found in stained films. The minute details of the division process can
only be followed in properly fixed films. In dried films stained by Roman-
owsky stains, the red mass representing the nucleus elongates, becomes
dumb-bell-shaped, and then divides into two parts. The kinetoplast
divides by elongation and division of the blepharoplast, followed by a
similar process in the parabasal. After division of the blepharoplast, a
new axoneme is formed from that daughter blej)haroplast, which is not
attached to the old axoneme. In dividing leishmania, it is sometimes
possible to distinguish two parallel axonemes arising from an incompletely
divided kinetoplast.
Multiple segmentation has been described by several observers
(Mackie, 1914, Yakimoff, 1915a). The evidence rests on the appearance
in films of cytoplasmic bodies within which are arranged a varying number
of nuclei and kinetoplasts, without any outlines to indicate separate
organisms. In the writer's experience, these bodies probably represent
detached portions of cytoplasm of the large cells containing leishmania,
of which the outlines are not clearly visible, either as a result of imperfect
staining or degenerative changes undergone by the parasites (Plate IV., 2,
p. 406). Similar appearances are often seen when the large cells are still
LEISHMANIA DONOVANI
409
intact, and where the cytoplasm is dotted over with pairs of nuclei and
kinetoplasts, just as they are in the supposed multiple segmentation forms.
Still more doubtful are the forms which Archibald (1913, 1914), Smallman
(1913), and Statham and Butler (1913) have described. These are more
or less spherical portions of cytoplasm containing granules, which have
no such definite arrangement as the nuclei and kinetoplasts in the bodies
just discussed. They bear some faint resemblance to schizogony stages
of malarial parasites or other organisms as seen in dried smears. Here,
again, the origin of these structures is in the cytoplasm of large cells with
granular cytoplasm, portions of which have been broken off. They are
merely fragmentation bodies, and have no relation to the leishmania.
CULTURE. — The greatest interest attaches to the culture of leish-
mania. Rogers (1904) demonstrated that flagellates of the leptomonas
Fig. 190. — Culture Forms of Leishmania donovani fixed with Schaudinn's
Fluid and stained with Iron H.^matoxylin (x 2,000). (Original.)
type appeared in citrate solution, to which material from spleen puncture
of cases of kala azar had been added, an observation which proved con-
clusively the flagellate nature of the puzzling Leishman-Donovan body
(Fig. 190). Though flagellates developed and multiplied in the medium
employed at the ordinary laboratory temperature, this was not always
the case, and subculture Was not satisfactorily obtained. Rogers obtained
better results with citrated human blood acidified with citric acid, but it
was Nicolle (1908c) who demonstrated the possibility of culture at a tem-
perature of 22° C. in the water oi condensation in tubes of Novy and
410 FAMILY: TRYPANOSOMID^
McNeal's rabbit blood-agar, and in a simplified medium now known as
the N.N.N. (Novy, McNeal, Nicolle) medium. Furthermore, in this
medium subculture was readily obtained, so that the flagellate could be
maintained as easily as any bacterial organism. Since that time the
culture method has been universally adopted, and is a recognized aid to
diagnosis. A strain of L. donovani isolated in 1910 was reported by
Nicolle (1925) to be still growing in N.N.N, medium. During these years
it has been subcultured 395 times.
The change undergone by the leishmania when introduced into the
medium can be studied in heavy infections by examination of the inocu-
lated material at frequent intervals. According to Leishman and Statham
(1905), who studied the development, the first change is an increase in
size of the leishmania, a growth of the nucleus, and an increased vacuo-
lization of the cytoplasm. In many cases the leishmania become pyriform
in shape. After forty-eight hours, growth of the flagellum commences.
It is a rapid process, and takes place from an eosin staining vacuole or
body which lies in front of the kinetoplast. This body moves to that end
of the organism which will be its anterior end in subsequent development.
Its contents are extruded as a series of fine filaments, which unite and
form the rudiments of the flagellum. As this process was studied only
in dried films, it is probable that the appearances are artefacts due to a
rupture of the vacuole. It is far more probable — and this is supported
by what is known of the formation of flagella in other organisms — that
the axoneme, which is sometimes visible in the leishmania, continues its
growth, and extends through the surface of the parasite to form the
flagellum. Active flagellate forms may be seen in cultures at any time
between forty-eight and seventy-two hours after the medium has been
inoculated. Very soon many of the organisms become still more elon-
gated till the body measures from 10 to 20 microns in length, while the
flagellum is often longer than the body. The fully-formed flagellate is
flattened like a blade of grass. Sometimes one edge of the organism is
convex and the other slightly concave, giving it the shape of a curved
sword-blade. In a culture of four or five days' growth various types of
flagellates are present, including round forms 4 to 5 microns in diameter
with long flagella, broad pyriform individuals with rounded anterior and
tapering posterior ends, and measuring about 10 microns in length and
4 to 5 microns in breadth, and the longer sickle-shaped forms already
mentioned (Fig. 190). The various types are connected by intermediate
forms. Reproduction by longitudinal division takes place rapidly till the
culture at the end of a week to ten days may be swarming with flagellates.
At division the blepharoplast divides, and a new axoneme is formed by
outgrowth from the blepharoplast. The parabasal then becomes constricted
LEISHMANIA DONOVANI 411
and divides, and at about the same time nuclear division commences. In
properly fixed material the nucleus is seen to elongate, while the spherical
karyosome at its centre elongates also, and is finally divided into two
parts, after which the entire nucleus becomes constricted at its equator,
and finally two result. Schulz (1924) maintains that the nucleus divides
by mitosis. Meanwhile, the new axoneme has continued its growth, and
a new flagellum is formed which gradually increases in length. Though
multiplication of the flagellum by longitudinal division has been described,
it is extremely doubtful if such a process ever occurs. After the new
flagellum has developed, splitting of the body, whether in the rounded
or elongated form, commences between the two flagella, and extends in a
posterior direction till two flagellates result. A characteristic feature
of the cultures is that the flagellates tend to remain clustered in groups,
with their flagella directed towards one another, so that rosettes or spheres
of organisms are formed with the flagella entangled at the centre. As the
cultures become old, elongate forms become less numerous, and many
rounded, non-flagellate bodies appear which resemble in many respects
the original leishmania. Many of these are, undoubtedly, degenerate or
dead forms, but the fact that subculture can often be obtained from
cultures of this type proves that some of them, at any rate, are living.
Cultures can be obtained from the spleen, as first shown by Rogers (1904),
or any other organ in which the parasites occur. They were cultivated
from the blood by Mayer and Werner (1914), and by the writer (1914),
while Shortt (1923c), and Shortt, Swaminath, and Sen (1923), have suc-
ceeded in growing them from the centrifuged deposit from the urine of
three cases of kala azar.
Noguchi has found that L. donovani in culture can be differentiated
from other species of Leishmania by serological tests (p. 399).
NATURAL INFECTIONS OF ANIMALS.— The only animals which have
been found naturally infected with L. donovani are dogs and cats, and the
latter only on one occasion, when Sergent Ed. and Et., Lombard, and
Quilichini (1912) published an account of a case of kala azar on a farm
near Algiers. The infected child was associated with a dog and a kitten
about four months old, both of which were infected.
The dog has been frequently found infected, especially in the Mediter-
ranean region, and often in association with infected human beings. This
has given rise to the view of the canine origin of kala azar.
The natural disease in dogs may run an acute or chronic course, and
the symptoms, as in man, are loss of weight, fever, anaemia, enlargement
of the liver and spleen. The dogs appear in bad condition, and are mangy
and often die of intercurrent infections. Recovery takes place more
frequently than in human beings.
412 FAMILY: TRYPANOSOMID^
The first observation of canine kala azar was made by Nicolle and Comte (1908)
in Tunis, an endemic centre of the human disease. A large number of examinations
were subsequently made in Tunis and other parts of Lybia by NicoUe, the YakimoiSs
(1911c), Gray (1913), and others, with the result that the ordinary street dogs were
found infected to the extent of about 1-6 per cent. Examining a series of dogs which
were evidently in bad condition, Nicolle (1914) found a percentage of 5-5 infected.
Similar observations were made by Sergent Ed. and Et. (1910), Seuevet (1912), and
Lemaire, Sergent, and Lheritier (1913) in Algiers, where the human disease also exists.
The canine disease has also been seen in parts of Africa where the disease in man is
rare or unknown. Thus it has been found in Morocco by Delanoe and Denis (1916),
and at Dakar (Senegal) by Lafont and Heckenroth (1915). In the Sudan, Bousfield
(1911) found bodies somewhat resembling leishmania in a dog, but Archibald (1914)
examined many dogs in the endemic area without encountering a single case of the
canine disease. In endemic centres in Europe the disease in dogs has been frequently
encountered. In Malta, Critien (1910 and 1911) found three out of thirty dogs
infected, and tlie writer (1914a) six out of forty-six. Alvarez and Pereira da SUva
(1910, 1911) examined 300 dogs in Lisbon and found eight infected, and in a later
seriesfour out of 109. Martinez (1915) discovered the first canine case inSpain, while
Pittaluga (1914) observed three infected dogs in Tortosa and Beninar. In Italy and
Sicily, where infantUe kala azar exists, the canine disease has also been found. At
Bordonaro in SicUy, for instance, Basile (1910) claims to have found infection in as
many as twenty-seven out of thirty-three dogs examined. In Palermo itself Jemma
(1910 and 1912) found no case amongst 227 dogs examined, but in the environs of
the town discovered two infected animals, one of which was in close association with
a human case. In the same town Caronia and di Giorgio (1914) examined with
negative results 1,005 dogs, while in Catania Pulvirenti (1911) saw three infections
in a series of 275 dogs. These places in Italy and Sicily are endemic centres of the
disease, but canine kala azar has also been found in Rome, which is not an endemic
centre, though a single case in a child has been recorded here. The human disease
has, however, been recorded from Nice by Labbe, Targhetta, and Ameuille (1918).
In Greece, Cardamatis (1912) found eighty -one dogs infected amongst 589 examined
in Athens, and Lignos (1913), in the Isle of Hydra, found a percentage of infections
of 16-66 from May to October, while later (1916) another series examined during
the winter (October to April) gave a percentage of 8-77. In the Trans-Caspian
region Dschunkowsky and Luhs (1909?>) observed cases of canine kala azar, while in
Turkestan Kohl-Yakimofl, Yakimofl and Schokhor (1913), Yakimofl and Schokhor
(1914), and Yakimoff (1915«) found dogs to be infected in a percentage varying from
25 to 35, according to the season. Adelheim (1924) has reported kala azar in a child
and a dog in Eiga. Both contracted the disease in Tashkent, where the family had
been living. Cesari ( 1 925) reports the canine disease at Grasse in the South of France.
In India the results have been very different. Donovan (1909b), working in
Madras, examined 1,150 dogs, 256 of which came from the kala azar quarter of the
city, without finding a single infection. Donovan (1913) and Patton (1913) recorded
the same result after a further examination of 2,000 dogs, also in Madras. Mackie
(1914) failed to find an infection amongst ninety-three dogs examined in the villages
of Nowgong (Assam), where the human disease is endemic. On the other hand,
Castellani (1912) claims to have observed the disease in several dogs in Colombo,
which is not an endemic centre of kala azar in man. Such an anomalous statement
can hardly be accepted till confirmatory evidence is forthcoming. Mr. Burgess, of
the Bacteriological Institute of Colombo, at the writer's request, kindly made
spleen smears from 250 dogs in Colombo. In none of these was the writer able to
find leishmania.
LEISHMANIA DONOVANI 413
RELATION OF HUMAN TO CANINE KALA AZAR. — The important
question arises as to whether the naturally occurring disease of dogs is due
to Leishmania donovani or to some other species. The frequent associa-
tion of the disease in dogs with human cases in the Mediterranean area,
and the morphological identity of the parasites, are facts which make it
impossible to regard the organism from dogs as other than L. donovani.
Furthermore, the disease produced in dogs by inoculation with the parasite
from human sources is identical with the natural canine disease, while
the organism from the canine disease is inoculable to animals, with results
similar to those which result from inoculation of the human virus.
The apparent absence of the canine disease in endemic areas in India
has been urged as evidence that the Indian disease is distinct from the
Mediterranean. The Indian disease is, however, inoculable to dogs, so
that the freedom of the Indian dog from infection probably depends on
some factor not at present understood. In the present state of know-
ledge, and lack of absolute proof of the method of transmission of the
disease, it is better to consider all the various systemic diseases in man
and dogs as due to one parasite, L. donovani. It is hardly necessary to
again remark that morphologically (in smears and cultures) the parasites
from the various sources are identical.
Though it is admitted that the human and canine diseases are caused
by the same organism, this does not mean that the dog is to be regarded
as a reservoir of the virus. Some have maintained that in Italy the
disease necessarily passes from dog to man, but so many cases occur which
cannot be associated with any infected dog that it would appear that the
infection of the animal is as much an accident as the infection of the human
being. Areas occur in which, apparently, only dogs have the disease,
while in others only human cases are known. It is claimed, however, by
Basile (1916) that in Bordonaro in Sicily, where a high percentage of
naturally infected dogs occurred, the extermination of these has led to an
almost complete disappearance of the human disease.
SUSCEPTIBILITY OF ANIMALS.— Nicolle (1908a, 1909o), and Nicolle,
Comte, and Manceaux (1908), were the first to show that L. donovani of
Mediterranean origin was inoculable to dogs and monkeys. The failure
to produce infection in animals by observers in India was advanced as
a proof of the existence of two species of leishmania in kala azar. It is
now known that the Indian virus, if injected in sufficiently large doses,
will give rise to infections as often as the Mediterranean virus. Infection is
produced most readily by intraperitoneal inoculation of large doses of the
material obtained by crushing an infected spleen, liver, or bone marrow
in normal saline solution. In larger animals, inoculation can be made
intrahepatically or intravenously. Subcutaneous inoculation does not
414 FAMILY: TRYPANOSOMID^E
produce infection so readily. Animals may be infected by injection of
large doses of the cultural forms, but infection is less likely to take place
than after a dose of the virus from the organs of man or another animal.
Organisms which have been maintained by subculture for long periods
are less liable to infect than those more recently isolated.
The infected animals often recover, and, as has been clearly demon-
strated by Laveran, on the passage of the virus from animal to animal it
loses its virulence to such an extent that finally infection does not occur.
This is equally true of the mouse, dog, and monkey — the animals which
have been used to the largest extent — though from the recent observations
of Young, Smyly, and Brown (1924) in North China, the hamster appears
to be more susceptible. The majority of animals, if young when inoculated,
continue to increase in weight in spite of their infection, though subject
to minor disturbances of health such as slight attacks of fever. In some
cases the infection is more acute, and after a rapid loss of weight death
occurs.
The infection produced in experimental animals is of slow develop-
ment, and cannot be compared with that resulting from the inoculation
of pathogenic trypanosomes. The lack of a suitable experimental animal
has been a great handicap to investigation work.
In some cases, by the inoculation of the skin, observers have been able
to produce with L. donovani local cutaneous lesions resembling oriental
sore.
Shortt (19236) inoculated a number of caterpillars and other inverte-
brates with cultures of L. donovani. In the case of one caterpillar, active
single and dividing flagellates were found in the body cavity fluid a week
later.
The Mediterranean virus has been successfully inoculated into dogs by several
observers since Nicolle's first success in 1908. Jemma, di Cristina, and Cannata
(1910) were successful in Italy, and Novy (1908) in America with a culture which
had been sent to him. NicoUe and Blaizot (1912) proved that the jackal was also
susceptible. Yakimoff (1915rt), working in Turkestan, succeeded in infecting dogs
and mice with the local virus. The most extensive series of experiments with dogs
has been made by Laveran (1917), who employed a virus obtained in Tunis. Of
thirty dogs inoculated with material from the organs of infected animals, twenty-six
became infected. In five dogs which died of the disease, the average duration was
257 days. Two dogs killed on the 454th and 456th days were still found infected,
though the condition of the organs showed them to be on the road to recovery.
Nicolle and Laveran have both noted keratitis in infected dogs. The Indian virus
was first inoculated to dogs by Donovan (1913). At about the same time Patton
(1913) was also successful with the dog and jackal. The writer (1914rt, 1915a) in
London infected a dog from a case of kala azar from India. Subsequently the virus
was passed through four successive dogs, when the inoculations were discontinued.
Mackie (19156) also succeeded in infecting dogs with the Indian virus. Laveran
(1913, 1917), commencing with a culture of L. donovani obtained from Row in India,
LEISHMANIA DONOVANI 415
inoculated twelve dogs either intravenously or intrahepatically. Five of these
became infected. Two others were infected by inoculation with both cultures and
spleen material from a heavily infected monkey. The conrse of the disease resembled
that produced by the Mediterranean virus. In two cases keratitis was observed.
Xicolle (1909) succeeded in infecting monkeys {Macacus sinicus and M. cyno-
molgus) with the Mediterranean virus in Tunis, while Laveran (1917), with the same
virus, inoculated fourteen monkeys {31. sinicus, M. cynomolgus, and M. rhesus),
of which two acquired a fatal infection, seven only a slight one, while five did not
become infected. Marshall (1911), working in the Sudan, succeeded with five
monkeys {Cereopithicus sabceus) out of seven inoculated with the Sudan virus.
Archibald (1914) also infected a monkey of the same species. Monkeys infected may
die in a couple of months, or the disease in them may run a chronic course terminating
in recovery. The infection shows the same irregularities as in the dog. With the
Indian virus. Row (1912) produced a general infection in M. sinicus and 31. cyno-
molgus. Of especial interest are the results obtained by this observer after local
inoculation of the skin. A 31. sinicus was inoculated with material from the spleen
of a case of kala azar by scarification of the skin, and another by subcutaneous
injection of a culture. In both cases local nodules appeared at the sites of inocula-
tion several months later. Leishmania were present in these nodules, one of which
was excised and used for further inoculations. With the material thus obtained
another monkey was inoculated in the skin, with the production of a local infection,
while two mice and a monkey injected intraperitoneally acquired a general infection.
Exjjeriments of a similar kind were carried out by Korke (1914). He noted, how-
ever, that subcutaneous inoculation sometimes gave rise to a local skin lesion, and
at others to a generalized infection. In some cases where a local lesion was produced,
a generalized infection occurred at the same time. Tyzzer and Walker (1919)
produced a purely local lesion by inoculating a monkey cutaneously with cultures of
the Mediterranean virus. In this case, there was an incubation period of four
mouths. Laveran (1913, 1917), working with the Indian virus, infected monkeys
in Paris, and found the course of the infection similar to that produced by the Medi-
terranean virus. Shortt ( 1923&) inocu^lated thirteen monkeys with virus from man or
other monkeys, and obtained infection in ten. Laveran and Pettit (1909a) were the
first to produce an infection in mice with the IMediterranean virus. Successful results
were also obtained by Yakimofi and Kohl-Yakimoff (1912) and Rutelli (1914) in
Italy. Continuing his experiments, Laveran (1920) noted that the virus could be
handed on from mouse to mouse, but that the depreciation in virulence was very
marked. He gives the following results of the passage of a strain of L. donovani
of canine origin through mice:
Numher of
Mice Infected.
1st inoculation . . . . 17
2nd ,, . . . . 13
3rd ,, .. .. 16
4th ,, . . . . 19
5th ,, .. .. 14
6th ,, .. .. 6
It th\is appears that with successive passages through mice, though infection usually
takes place, the percentage of natural recoveries increases. The infected mice
became anaemic, showed marked enlargement of the spleen, and degeneration of the
testicle. Mice examined over a year after inoculation were in some cases still
infected, while in others, though no parasites could be found, the characteristic
lesions were still present.
Not. Infected.
Becovered.
1
7
2
8
3
8
0
15
1
9
0
5
416 FAMILY: TRYPANOSOMID^
With the Indian virus, Row (1912, 1913) was successful in inoculating mice. The
virus employed was obtained either from a local cutaneous nodule in a monkey, the
spleen of infected monkeys, or cultures of the organism. Maokie (1914, 1915/))
infected mice from material from human cases, and Laveran (1917) was also success-
ful. Shortt (19236) produced a heavy infection in a mouse with the Indian virus.
Adelheim (1924) produced heavy infections in mice with a virus he obtained from a
dog which had been brought to Riga from Tashkent. Subcutaneous injection pro-
duced, not only a generalized infection, but also a local sore in which the parasites
tended to persist longer than in the internal organs.
Rats have also been infected with the Mediterranean virus by Laveran (1912c)
and Yakimoff and Kohl-Yakimoff (1912rt). Patton (1912a) infected a rat with the
Indian virus, while Cornwall and La Frenais (1916) infected one by injection of
cultures and another by feeding it on bread soaked in culture. The organisms
were only demonstrated in the infected animals by the culture method. The writer
(1915c) was also successful in infecting white rats directly from a human case.
Guinea-pigs were first shown to be susceptible to the Mediterranean virus by
Laveran and Pettit (1909/>). Franchini (1911) claimed to have produced a general
infection in a young guinea-pig by injection of cultures. With the Indian virus
guinea-pigs have not yet been infected. The only general infection in a rabbit was
recorded by Mantovani (1912). Volpino (1911) infected the cornea of a rabbit by
scarification with material from the spleen of an infected dog. About three months
after the cornea showed a lesion which resembled those produced by the virus of
syphilis. Numerous leishmania were present in the lesion. Rabbits do not appear
to have been infected with the Indian virus. Rabbits and guinea-pigs are evidently
difficult to infect, as many failures have been recorded. Cats also have never been
infected, though on one occasion a naturally infected kitten was discovered in Algiers.
With the Sudan virus Archibald (1914) infected the jerboa and the gerbil, while
with the Indian virus Mackie (1914) infected the flying fox {Pteropus edwardsi).
In this connection it is of interest to note that Archibald (1914), working with the
Sudan virus, was successful in infecting two monkeys by feeding them with crushed
infected spleen of man or experimental monkey. He faUed to infect a young dog
by this method.
As regards infections in animals, it is rarely that leishmania can be foimd in
the peripheral blood. Liver puncture can generally be carried out, but parasites
are not numerous in this organ. Spleen puncture is difficult to perform, though on
one occasion the writer diagnosed a case of infection in a dog by this method in
Malta. Bone marrow can be obtained by trephining a rib or one of the long bones
of the leg under an anaesthetic. In dogs, at any r.ate, this method gives the most
reUable information. Cultures from the blood have also been obtained.
Dogs and monkeys which have recovered from infections have been shown by
NicoUe (1910) and NicoUe and Comte (1910) to be immune to further inoculations.
Laveran (1914a) notes that a monkey which had recovered from infection with the
Mediterranean virus was immune to inoculation with the Indian one.
From the foregoing summary it wdl appear that many successful inoculations
of animals have been effected. The infections, however, cannot be compared with
those produced by pathogenic trypanosomes, for they are nearly always of slow
development, and the number of organisms found is generally small. The want of
an easily inoculable and susceptible host for L. donovani has been a great obstacle
to the carrying out of experimental work on the method of transmission of kala azar.
Recently, however, Smyly and Young (1924), and Young, Smyly, and Brown
(1924), have shown that in Xorth China the hamster {Cricetulus griseus) is more
susceptible than other laboratory animals. Successive passages of a virus were
LEISHMANIA DONOVANI 417
effected, and as it showed no signs of becoming attenuated, it is possible that this
animal may prove useful for experimental purposes, and lead to important results
on the aetiology and transmission of kala azar. Meleney (1925) has shown that the
infection progresses steadily in its intensity till, at the end of fifteen months, the
tissues of the spleen, liver, bone marrow, lymphatic glands, and intestinal mucosa
have been largely replaced by macrophages packed with parasites. The parasites
are found also in other organs, including the meninges, where the macrophages occur.
They were also demonstrated in th-) glandular cells of the liver.
Franchini (1922m) claims to have produced infection of the plant Eupliorhia
ipecacuanha by inocidating it with cultures of L. donovani.
TRANSMISSION. — Since Eogers's demonstration of the development of
flagellates of the leptomonas type from leishmania, and the recognition
of the close resemblance of these to natural insect flagellates, it has been
generally assumed that Leishmania donovani has an invertebrate host.
Though many attempts have been made to discover such a host and the
method of transmission of kala azar, the problem still remains unsolved.
Many different invertebrates, chiefly bugs and fleas, have been considered,
and some observers claim to have effected transmission of infection by the
agency of fleas. As leishmania are present in the peripheral blood of cases
of kala azar, they are readily ingested by blood-sucking insects, while
the flagellate forms which appear in cultures undoubtedly represent an
insect developmental phase, as they do in cultures of trypanosomes, the
invertebrate hosts of which are known in many cases. It was suggested
by the writer (1914o) that oriental sore and kala azar may be caused
by insect flagellates which only accidentally infect man. Normally, the
flagellates would pass from insect to insect, as do all naturally occurring
insect flagellates. Occasionally, they would infect human beings, and
give rise to the diseases mentioned. According to this view, the virus
could be maintained indefinitely in the insects, which would be infected
from one another, though an insect would be capable of infecting itself
by sucking the blood of an infected human being.
As first demonstrated by Patton (1912a), it is well known that L.
donovani will develop into the leptomonas form in the stomach of the bed
bug. The flagellates can be recovered from the intestine of the bug by
the culture method as long as six weeks after parasites were first ingested.
Mice can be infected with the forms in the intestine of the bug nine days
after the feed on kala azar cases. An enormous amount of time and
energy has been spent in investigating the claims of the bed bug, but
no actual proof that it is the transmitter of kala azar has been obtained.
Kecently, Knowles, Napier, and Smith (1924), Christophers, Shortt and
Barraud (1925, 1925a), and Shortt, Barraud and Craighead (1926) have
found that female Phlebotomus argentipes acquire a heavy leptomonas
infection of the intestine and pharynx after feeding on kala azar cases.
I. ' 27
418 FAMILY: TRYPANOSOMID^
The flagellates may even extend into the buccal cavity. This fact, com-
bined with Sinton's observation that the distribution of the disease in
India corresponds with that of P. argenti])es, leaves only a definite trans-
mission experiment to prove that kala azar is conveyed by the bite of the
sand fly. The claim that the flea is the transmitter of kala azar in the
Mediterranean area has not been substantiated. If it be assumed that
an insect vector exists, then there are two possibilities as to the mode of
infection. The organism may either be injected by the insect by way
of the proboscis (inoculative), or it may be voided in the faces of the insect
in some form, and thus infect the wound or be ingested (contaminative).
It has been suggested that L. donovani may escape in the faeces of
patients. Manson and Low (1904) demonstrated its presence in the ulcers
of the intestine, while Perry (1922) has seen the villi heavily infected with
parasites. Mackie (1914c) saw bodies resembling leishmania in mucus
from the intestine, but Knowles (1920) examined mucus very carefully,
and though he saw bodies more closely resembling leishmania than those
noted by Mackie, he pronounced no opinion as to their nature. It seems
probable that these bodies are yeasts, which frequently show a striking
resemblance to leishmania in stained films. Shortt (1923) has cultivated
leishmania from the urine of kala azar cases, so that the possibility of
spread of infection by water has to be considered, but experience has
shown that the parasites quickly degenerate in water. The nature of the
parasite is not in favour of such a method of transmission, though Adel-
heim (1924) has noted that a healthy mouse kept in a jar for five months
with an infected mouse contracted the disease. As the infected mice
commonly had ulcers in the intestine in which parasites could be demon-
strated, it was thought that oral contamination was responsible for this
contact infection.
The common association of ankylostomiasis with kala azar has
suggested the possibility of the ankylostomes being a source of infection.
Knowles (1920) investigated the worms taken from kala azar cases, and
even the eggs and embryos hatching from them, without finding anything
to support this view.
The following experimental work with insects has been carried out
with a view to the discovery of a transmitting host:
Bugs. — The bed bug {Cimex roiundaius) was suspected by Kogers as a possible
carrier of kala azar on account of its frequent presence in houses where cases of
the disease occurred. Patton (1912a), working in Madras, also favoured this view,
and conducted a series of experiments by which he claimed to have proved the
correctness of the theory. By feeding bugs on cases of kala azar, in which the
leishmania were numerous in the perijiheral blood, and dissecting them at varying
intervals, he found that the leishmania had developed into flagellates of the lepto-
monas form as they do in cultures, and that some multiplication had taken place.
LEISHMANIA DONOVANI 419
In bugs dissected eight to ten days after feeding, tlie flagellate forms had given rise
to rounded bodies again. The various stages were compared with the similar
natural flagellates of insects, and were described as pre-flagellates, flagellates, and
post-flagellates. It was found, however, that the flagellates did not persist in the
bug, and, furthermore, it was noted that a second feed of blood often caused the
organism to disappear more quickly. The writer (1912c and 19156) criticized the
conclusions drawn from these experiments, and pointed out that the development
which took place in the bug was probably due to the large quantity of blood in the
stomach, and that it represented merely a temporary culture as occurred in the test-
tube. This view received support from the fact that a development of Tryiyanosoma
lewisi would take place in the bug, which cannot be considered to be a host for this
flagellate. Patton was never able to demonstrate actual transmission by bed bugs.
Mackie (1914 and 1915) publislied an account of further investigations with the
bed bug. He dissected over 1,500 bugs from kala azar areas without finding a
single one infected. Two monkeys were inoculated with 209 and 606 crushed bugs
without becoming infected. Young bugs born in the laboratory to the number of
131 were fed on kala azar cases. In only two dissected twenty four hours after
feeding were leishmania seen. On another occasion, 191 young bugs gave a negative
result. Cornwall and La Frenais (1916) succeeded in causing bugs to ingest cultural
forms of L. donovani. The bugs were then fed on rabbits. It was found that in
some cases the flagellates multiplied and persisted up to twenty-nine days. Attem]3ts
were made to infect citrated rabbit's blood by causing these bugs to bite through
skin. The blood was then distributed in N.N.N, medium. In no case was a culture
of flagellates obtained. The faeces of the bugs never contained encysted forms of
the flagellate such as are found in the fseces of insects with a natural flagellate
infection. Rounded forms and flageUates were, however, seen in the rectum, but
these appeared to be in process of degeneration. A peciiliar type of organism, called
the " thick-tailed form," was seen in the biigs. This consists of a rounded body of
the usual leishmania structure measuring 5 to 6 microns in diameter, and provided
with a long flageUum which is very much thicker than that of the ordinary flagel-
lated forms. It is thus apparent that the bed-bug hypothesis has not been
established, and no proof has yet been given that the development which takes place
in the bug is other than a temporary culture of the organism. This is all the more
probable from an account of investigations made by Patton, La Frenais, and Rao
(1921) in Madras. By feeding bugs on material containing Leptomonas pulicis,
Crithidia ctenocephali, and Ilerpeiomonas muscarum, and making cultures in N.N.N.
medium from the alimentary tracts of the bugs at varying intervals, it was shown
that these flagellates persisted for twenty-four, eight, and forty-five days respectively.
By similar experiments made by feeding bugs with cultures of Leishmania tropica and
L. donovani, these authors (1921) obtained cultures from bugs after forty-four and
forty-one days respectively. Shortt (1923) has also obtained active multiplication
of Leptomonas ctenocephali in the intestine of bugs fed on cultures.
A series of experiments with bugs was conducted by Adie (1921) in India. Many
attempts were made to obtain a satisfactory develojunent of L. donovani in the bed
bug, but without result. Finally, some bugs which had died after being fed on spleen
puncture material from a case of kala azar were placed in saline solution in the
incubator at 27^ C. These were examined about thirty-six hours later, and in one
tliere were found numerous developmental forms of leishmania. These not only
occurred in the lumen of the gut, but also in the intestinal cells in clusters, which are
compared with the intracellular stages of development of T. lewisi in the flea.
Apparently, similar stages were not found in the numerous live bugs dissected, so
that it would seem that here, again, the development was of the cultural type, and
420 FAMILY: TRYPANOSOMIDyE
had taken place within the cytoplasm of dead cells, just as it does in a culture
medium. The figures purporting to illustrate the development are not convincing,
and suggest the possibility of a mixed infection of leishmania and some other parasite,
such as a Sporozoon.
Patton (1922) states that by a special technique he has confirmed Adie's
observations. He does not describe the technique, but, presumably, it is the culture
of leishmania in the bug's intestine after removal from the body. Neither Patton
nor Adie was able to obtain the intracellular development in living bugs. Because
multiplication takes place in dead or dying cells, it is not legitimate to conclude
that it will also occur in living ones.
Cornwall and La Frenais (1922) repeated these experiments with living bugs.
They fixed the intestines entire and examined them in serial section, so as to retain
the normal relations of the cells. Though developmental forms of leishmania
occurred in the lumen of the intestine, sometimes in enormous numbers, there was
no indication of any intracelhdar development.
The bodies which Adie (1922, 1922a) saw in the salivary glands of bugs, and
which were regarded as leishmania, have proved to be spores of a microsporidian.
Shortt and Swaminath (1924) have tested the infectivity of the developmental forms
of L. donovani in bed bugs Avhich had fed on a case of kala azar with parasites in
the jDeripheral blood. Nine days after feeding on the case the bugs were dissected,
and emulsion of their intestines injected intraperitoneally into mice. In the case
of one of these animals a culture was obtained from the spleen 123 days after the
injection. No parasites could be discovered in smears of the organs. Thus the
forms in the bugs on the ninth day were infective to mice, so that, as tliese intestinal
forms are presumably passed in the bug's dejecta, it is possible they might be
ingested or contaminate the puncture wound during or after feeding. Xicolle and
Anderson (1925) using over 2,000 bugs in Tunis failed to transmit L. donovani
from dog to dog, while Shortt and Swaminath (1925) were equally unsuccessful in
similar experiments with monkeys in India.
Conorhinus mbrofasciatus. — Donovan (1909a) suggested this bug as a possible
vector of L. doyiovani, but no evidence of the development of the parasite in this bug
could be obtained by Patton (1912a).
Fleas. — The occurrence of kala azar in dogs naturally turned the attention of
investigators to the possibility of fleas acting as transmitters. This view was first
exj^ressed by NicoUe (1908d), and was investigated by Basile in Italy (1910a, 1911,
1911a), who published the results of a series of observations by which he claimed
to prove that fleas were the true hosts of L. donovani. It has already been remarked
that at Bordouaro in Sicily, an endemic centre of the human disease, a high per-
centage of dogs was found to be infected. According to Basile, the only ectopara-
sites common to dog and man are the fleas Pulex irritans and Ctenoceplialus canis.
Attention has been drawn above to the fairly frequent association of infected human
beings and dogs in the same house.
Basile' s investigations were conducted on two lines — namely, attempts at
infection of healthy dogs by fleas and the study of the leishmania in the flea. In the
first place (1911), he claimed that fleas which fed on spleen juice of cases of kala azar
became infected with cultural forms of leishmania, and that these produced infection
when injected into dogs. Three dogs were then said to have acquired the disease
by causing them to live with an mfected dog in Bordonaro. In another experiment
four dogs were infected in Rome by placing on them fleas taken from infected dogs
in Bordonaro. Experiments of this kind were rei>eated, and from what is now
known of the difficulties attending the inoculation of animals it is remarkable with
what apparent ease positive results were obtained. Sangiorgi (1911) relates that he
LEISHMANIA DONOVANI 421
received iu Turin an infected dog from Tunis. This dog was placed in a kennel with
another dog, unfortunately not examined, which was afterwards found infected.
The brothers Sergent, Lheritier, and Lemaire (1912) allowed a pup, previously
examined and found uninfected, to be bitten eighty-two times by fleas fed one to
eight days previously on an infected dog. The pup was later found infected. Care
had been taken to keep the pup free froin ectoparasites. The experiments of Basile,
apart from any doubt one may have on account of his uniformly successful results,
may be criticized from the point of view of the difficulty of excluding with any degree
of certainty a previous infection in these animals, though the author claims to have
done this. During the long incubation period of a leishmaniasis it is almost im-
possible to exclude other sources of infection. The same remark may apply to the
experiment of Sergent and his co-workers. In many cases it requires exhaustive
study and examination to detect a small infection in an animal when it has been
killed, but the difficulty is increased a hundredfold when the animal is alive, and
reliance has to be placed on puncture of the organs. It is almost impossible to
perform a spleen puncture on a dog during life, while liver puncture is most unreliable
as a means of revealing an infection.
Working in Malta {1914rt), the writer carried out a careful exjDeriment. Four
young dogs were sent from England by sea, and, on arrival in Malta, two were placed
in a flea-proof cage and two in an exposed cage near the other. Over 400 fleas were
then transferred from an infected dog kept in another part of the town to the flea-
proof cage. Within three weeks the dogs appeared evidently ill, and between five
and six weeks after exposure to infection they both died within a day of one another.
They were very emaciated and ansemic. No trace of leishmania infection could be
detected, and the jjost-mortem appearances were quite unlike those of kala azar in
dogs. The organs were very pale, and the spleens reduced in size and almost white.
The animals had died of aniiemia through abstraction of blood by the fleas, which had
multiplied to an enormous extent. The two control dogs remained perfectly healthy.
Basilo (1911) first announced that the dog flea fed upon spleen juice containing
leishmania became infected with cultiiral forms of the parasite. It may be remarked
that it is extraordinarily difficult to induce fleas to feed on such material. The
writer has always failed after many attempts. Basile then claimed to have found
flagellate forms of leishmania in the human flea. In criticism of these statements,
it was pointed out that fleas were liable to natural leptomonas infections, and Basile
then qualified his statements by claiming to be able to distinguish the natural
leptomonas of fleas from those forms derived from the leishmania. It was evident
he had not excluded the natural infections of the fleas before placing them in contact
with spleen juice containing leishmania. For the study of flea infections, NoUer
(1912rf) had introduced a method of controlling fleas by fixing them on fine wire.
The writer has employed this method with very good results. Basile stated that he
had used this method in one exiieriment, and that in two days 200 fleas from an
infected dog were fixed on wire in this manner. On the third day they were all fed
on a newly-born pup, and the faeces they passed while feeding were examined. Three
of the fleas were found infected with leishmania. The two more heavily infected
fleas were then dissected and the gut contents injected into two mice, one of which
was found infected fifty-six days later. The fixing of a flea on wire is a delicate
operation which requires much experience, and may take as long as half an hour.
The feeding of the flea till it passes faeces on to a cover-glass, which is then m.ade into
a film and stained, may occupy ten minutes or as much as an hour. It is incon-
ceivable that Basile tethered 200 fleas and examined them in so short a time.
Furthermore, if his object was to discover infected fleas, this could have been more
quickly done by simple dissection. The object of Poller's method is to enable
422 FAMILY: TRYPANOSOMID^
repeated examination of the faeces of fleas to be made, so that natural intestinal
infections can be excluded before employing them for feeding experiments. On
account of these and other incomprehensible statements, it seems impossible to
accept Basile's claim that he has demonstrated the flea transmission of Mediter-
ranean kala azar. Pereira da Silva (1913, 1915) conducted a very careful series of
experiments, employing Noller's method amongst others, with a view to deter-
mining the possibility of L. donovani developing in the human and dog flea. He
could obtain no evidence whatever of such a development, and came to the con-
clusion that the flea is not the transmitting agent of kala azar. Basile (1914,
1914rt) attempted to explain the negative results obtained by other workers by
assuming that certain meteorological conditions existed during his experiments
which were absent during those of other observers. It is of interest to note here
that the writer (1912c), experimenting with fleas by Noller's method, could obtain
no evidence of the development of L. tropica in these insects (see below). Xicolle
and Anderson (192.3, 1924) have published an account of most careful attempts to
transmit kala azar to ten dogs by means of numerous dog fleas which had fed upon
known infected animals. The exposure to the fleas lasted from three weeks to seven
months, but in no case did an infection result. Two of the dogs were made to
swallow 510 and 410 fleas. Every source of fallacy was excluded, and it is rightly
concluded that the experiments lend no support to the flea transmission hypothesis.
Mosquitoes. — Franchini (1911a, 1912) allowed Anopheles macuUpennis to feed on
cultures of L. donovani. The parasites persisted in the gut up to twenty-four hours,
and leishmania forms were passed in the faeces. The same mosquitoes were fed on
spleen puncture material from cases of kala azar. Tieishmania were ingested, and
persisted up to forty-eight hours. After thirty hours large round forms were present,
while at the end of forty-eight hours a flagellate leptomonas form Avas found. He
claims to have controlled his experiments by numerous dissections of mosquitoes
not fed on leishmania material. Flagellate infections of A. macuUpennis are, how-
ever, very common. No evidence of the possibility of transmission was produced.
Patton (1907 and 1912fl.), in India, could obtain no evidence of any development
of leishmania in Culex pipiens, A. Stephens i, and Stegomyia sugens, but natural
flagellates were found in some of these. Similarly, Mackie (1915) was completely
unsuccessful with 266 culex and eighteen anopheles.
Lice. — Patton (1907-1912rt) obtained negative results after feeding lice {Pedicnlus
capitis and P. vestimenti) on kala azar cases in the blood of which leishmania
occurred. Mackie (1915) fed large numbers of lice on kala azar patients, and,
furthermore, dissected larger numbers collected from cases without finding any
trace of leishmania in them. In all, over 3,000 lice were thus examined.
House Flies. — Patton noted that L. donovani degenerated very rapidly in the
intestine of the house fly.
Ticks. — Patton, working with Ornithodorus savignyi, could obtain no evidence
of development of L. donovani. Basile (1910a) and Marshall (1912) likewise had
negative results with ticks.
Sand Flies. — The recent experiments of Knowles, Napier, and Smith (1925) have
directed attention to sand flies of the genus PhJebotomus. They point out that Sin ton
has informed them that in India the distribution of Phlebotomus argentipes coincides
with that of kala azar. An investigation of this fly in Calcutta has shown that
twenty-five out of fifty-six female flies bred in the laboratory contracted a leptomonas
infection after feeding on kala azar cases. Bred flies, forty-six in number, fed on
control cases acquired no such infection, while 407 wild flies (317 ? and 90 (^) also
showed no infection. Similarly, 210 wild P. minutas were uninfected. Experimenting
with P. minutus, it was found that this flv would not feed on man. One hundred
LEISHMANIA TROPICA 423
and three unidentified wiJd sand flies also gave a negative result, bringing the total
of the controls to 857. Christoi)her8, Shortt, and Barraud (1925, 1925fl), and Shortt,
Barraud and Craighead (1926), in Assam find the flagellates in massive numbers in
the pharynx and extending to the buccal cavity. They state that this seems to be
all that is required, short of a final proof of a transmission experiment, to demon-
strate that kala azar is transmitted by the bite of the sand fly. With Culicoides
macrostoma the first-named observers (\^2ob) obtained no development in forty-eight
flies fed on cases.
ACTION OF DRUGS ON LEISHMANIA DONOVANI.— As regards the action
of drugs on the parasites, a great advance was made by the introduction
of tartar emetic treatment in kala azar by Di Cristina and Caronia (1913),
after the success obtained by Vianna earlier in the same year with this
remedy in dermal leishmaniasis of South America. Rogers (1915) intro-
duced the treatment into India, and the drug is now widely used. It is
recognized as a specific against the leishmania, and has so far influenced
the mortality that, whereas formerly the majority of cases died, now the
great majority recover.
The drug (or the corresponding sodium salt, which is supposed by some
to be less toxic) is given intravenously. A dose of 10 c.c. of a 1 per cent,
solution {i.e., 1-5 grain) can be given once or twice a week during two to
three months till a total of about 2 grams (30 grains) has been adminis-
tered. Improvement, as shown by loss of fever, reduction in the size of
the spleen, and a better general condition, takes place rapidly, but it is
found that parasites still persist in the spleen, and are culturable even up
to two months after the commencement of treatment, which must there-
fore be continued beyond this period.
Knowles (1920) found by culture that living leishmania might still be
present in the spleen after 174 centigrams of tartar emetic had been
administered intravenously. Stibacetin, or stibamine, first used in the
treatment of kala azar by Caronia (1916), is an organic antimony com-
pound which appears to be equally efficacious, but liable to a decomposition
which renders it toxic. It can be administered intramuscularly. Urea
stibamine, introduced by Bramachari (1922), stibamine glucoside and " von
Heyden 471 " have given good results, even in cases which were resistant
to tartar emetic.
The Parasite of Oriental Sore.
Leishmania tropica (Wright, 1903). — This organism, which is morpho-
logically indistinguishable from L. donovani, is the cause of the cutaneous
infections known as oriental sore (Plate IV., p. 406). The disease occurs in
the New as well as the Old AVorld. In the latter, in the vast majority of cases
the lesions are limited to the skin of the exposed part of the body, but as a
very rare exception they may extend to the mucous lining of the mouth,
nose, and pharynx. In the New World, though in most cases the lesions
424 FAMILY: TRYPANOSOMID.E
are confined to the skin, they appear to be of a more chronic character
than those of the Old World, while in about 10 per cent, of cases the
mucosae are involved. This latter condition produces in the mouth,
pharynx, and nose extensive ulcerations and necroses, which may last
for years, and reduce the victim to a condition of profound cachexia. In
the Old World type, occasionally, ulceration may extend from the skin to
the inner surface of the lips or nose, as recorded by Cardamatis and
Melissidis (1911), in Greece by Pulvirenti (1913), and La Cava (1912 and
1914) in Italy, and by Christopherson (1914) in the Sudan. In these cases
there is not the extensive involvement of the post-pharyngeal region so
characteristic of the disease in South America. Castellani (1913), however,
claims to have discovered leishmania in the pharyngeal ulcerations of a case
in Ceylon. There had been no previous skin lesion, and, as Laveran (1917)
remarks, if this was a case of infection with L. tropica, a fact which was not
demonstrated, the case is quite abnormal and of an exceedingly rare type.
DISTRIBUTION. — The distribution of oriental sore is a very wide one.
It occurs in Spain, Italy, and Greece, and more recently a case has been
described from France by Ravaut (1920). It is fairly common in North
Africa, and has been found in various localities along the northern coast
area, especially at Biskra. It is found in Egypt and the Sudan, and also
in the French Congo, in the district of Lake Chad, and on the Niger. Asia
Minor, Arabia, Mesopotamia, Persia, and the southern parts of Russia
are endemic centres. In India it is common along the north-west frontier
districts, and even farther south, as at Cambay near Bombay. It does
not, however, extend to the kala azar areas in the east. In America it
occurs chiefly in Brazil and Peru, but also frequently in Guiana, Paraguay,
Panama, Yucatan. Cases have also been recorded from the Argentine,
Uruguay, Bolivia, Equador, Colombia, and Venezuela. The disease in
the New World is known under various names, such as espundia, uta, buba,
pian-bois, forest yaws, bosch yaws, and has been known for many years,
though it was not till 1909 that Lindenberg, Carini, and Paranhos demon-
strated leishmania in the ulcer of Bauru in Brazil. Splendore (1911) and
Carini (1911) were the first to show that the disease of the naso-pharyngeal
region was due to infection with leishmania.
It is thus quite clear that, though in most cases the disease in South
America is limited to the skin, and in this respect resembles the disease of
the Old World, in a certain percentage of the cases secondary lesions appear
in the naso-pharyngeal mucosa and lead to a very chronic type of ulcera-
tion. Furthermore, the purely cutaneous type appears to be more severe
and of longer duration than the oriental sore of the East, which rarely
lasts for more than a year or eighteen months. It is possible, therefore,
that the parasites causing the two diseases are not identical. Vianna
LEISHMANIA TROPICA 425
(1911) proposed the name Leishmania brasiliensis for the American form,
on account of a filament lie had observed in the parasites. This is un-
doubtedly the axoneme which is often demonstrable in L. tropica of the
Old World. Escomel (1911, 1913a, 1914) noted elongate forms of the
parasite provided with short flagella. He again (1922) refers to them,
and gives a figure showing typical leptomonas. Accordingly, he pro-
posed to name the organism L. americana var. flagellata. Rebagliati
(1914) and Monge (1914) also claim to have observed flagellate forms of
leishmania in the South American ulcers. La Cava (1912) has recorded
similar forms in infections of L. tropica in Italy. Laveran and Nattan-
Larrier (1912a) observed unusually large forms of the parasite in smears
from a South American sore. There was a large central vacuole, and the
nucleus was flattened out against one side of the parasite. On account
of these peculiarities, they suggested the name L. tropica var. americana
for the parasite. Exactly similar forms, however, are met with in the
L. tropica of the East. Velez (1913), who discovered the disease in Peru,
proposed to name the local parasite L. peruviana. It is evident that, of
the various names proposed for the South American parasite, L. brasili-
ensis has priority over the others. No one, however, has been able to
establish any morphological difference between this parasite and L. tropica,
either as it occurs in the tissues or in cultures. The organisms cannot be
distinguished from L. tropica except by the serological tests devised by
Noguchi as described above (p. 399). Thomson and Balfour (1910) described
a curious type of cutaneous leishmaniasis in the Sudan, in which the
lesions were nodular and showed no tendency to ulceration. Here, again,
the organism was morphologically indistinguishable from L. tropica, but
Brumpt (1913c), regarding the disease as distinct from oriental sore,
proposed to name the parasite L. nilotica.
It seems better to retain the name L. tropica for both forms till
more reliable proof of specific difference is forthcoming. On the other
hand, the close resemblance of L. tropica to L. donovani led to Manson's
suggestion that oriental sore is a local manifestation of an infection with
the same organism that causes kala azar in much the same way as vaccinia
may be supposed to be a local manifestation of smallpox. In this con-
nection it is of interest to note that Nicolle and Manceaux (1910a) found
that in experimental monkeys and dogs an animal recovered from oriental
sore was immune to this disease but not to kala azar, while one re-
covered from kala azar was immune to both. Patton (1922) records an
instance of a patient who contracted kala azar after having recovered
from oriental sore. Laveran's experiments with mice show that they
react differently to L. tropica and L. donovani. Furthermore, the dis-
tribution of the diseases is against the view of the identity of the two
426 FAMILY: TRYPANOSOMIDiE
organisms, though undoubtedly many arguments could be raised in support
of their inclusion in a single species.
SYMPTOMOLOGY. — ^The cutaneous lesion due to L. tropica commences
as a small, red papule, which is usually supposed to be the result of an
insect bite. Instead of disappearing, however, it persists and increases
in size, and may eventually give rise to a nodule an inch or more in diameter.
After persisting for about a year, shrinking commences. The nodule
finally dries into a scab, which eventually falls off, leaving a thin depressed
scar. More usually, however, after a variable period of growth, the surface
breaks down, and an ulcer with round edges is formed. Secondary
bacterial infection takes place, and the ulcer may become as large as the
palm of the hand. In the non-ulcerating variety, fluid obtained by punc-
ture is found to contain large numbers of parasites, but in the ulcerating
form these may be more difficult to detect, as scrapings from the granu-
lating surface contain many pus cells and extraneous organisms. In such
cases the best procedure to adopt in order to discover the parasites is to
puncture the surrounding red margin of skin and run in a fine glass pipette,
so as to obtain the tissue below the contaminated surface. Without the
finding of the parasite, certain diagnosis is impossible, for the lesions often
appear in remarkably atypical form, and even when they appear typical
they resemble certain tropical ulcerations of quite another nature. If
parasites cannot be demonstrated by direct examination, the culture
method may be of assistance. In one case seen by the writer, an undiag-
nosed lesion on the ear contracted in South America had been treated
unsuccessfully for several years. Though scrapings from the sore and
puncture of the margin failed to reveal leishmania in stained films, yet
flagellates grew in cultures inoculated with material obtained by puncture
after sterilization of the skin. The organisms must have been very scanty,
for it was not till after the lapse of three weeks that the characteristic
organisms had multiplied sufficiently to be detected.
The lesions in oriental sore are usually confined to exposed surfaces
of the body — e.g., hands, wrists, feet, legs, and face. They are often single,
but two or three sores are quite common. More rarely a larger number
are present, and these may be scattered over the surface of the body.
Cardamatis and Melissidis (1911) record a case in Greece in which there
were thirty-five sores distributed about the hands, arms, and face, while
Torres (1920) in South America observed one in which 248 distinct lesions
occurred on various parts of the body. As a rule there is no constitutional
disturbance, except in those South American cases in which naso-
pharyngeal involvement occurs, when the patient is often very much
reduced in health. Lymphangitis in the lymphatics and glands draining
the infected region is not uncommon, and organisms have been obtained
LEISHMANIA TROPICA 427
by puncture of the enlarged glands. Neumann (19096) on two occasions
discovered leishmania in the peripheral blood of a case of oriental sore, an
observation confirmed later by Patton (1912) in India. The presence of
parasites in the peripheral blood in oriental sore is a very rare occurrence.
The writer has searched in vain for them on many occasions, and attempts
at culture from finger blood have given only negative results.
■^^■^N^^-'^^'-
%
te
'J
%^
B
Fig. 191. — (Sections of Oriental Sore. (After Nattan-Larrier, 1913.)
A. General view, shi I wiiiLC clcvatcil nature of sore, absence of epithelium on surface, and dark areas
consisting of acciiiiiulatiiinM if inacro])hages { x 7).
B. Tissue of sore, showing ma' ro|iliai:cs coiitaining Leishmania tropica ( x 1,000).
PATHOLOGY. — In sections of oriental sore, especially the non-ulcerat-
ing variety, the new growth is found to consist of a fine reticulum of
connective tissue, in the meshes of which are numbers of large cells often
packed with parasites (Fig. 191). The cells resemble those met with in
428
FAMILY: TRYPANOSOMIDiE
cases of kala azar, and probably have a similar origin in the endothelial
lining of the vessels. The epidermis over the new tissue is often very
thin and degenerate, and in certain cases islets of epithelium are found
more deeply. In the ulcerating form the deeper tissue is composed of large
cells, but on the surface large numbers of pus cells are present also, and
the structure resembles that of an ordinary granulation tissue.
MORPHOLOGY.— There is very little to add to the description given
above for L. donovani, a parasite which cannot be distinguished morpho-
logically from L. tropica. In any individual smear from a case of
oriental sore, however, the parasites appear to have a greater range of
form and size than they have in a smear from a case of kala-azar. The
elongate, cigar-shaped forms are
more frequently encountered in
oriental sore, as also the larger
types of parasite (Fig. 192). They
occur in the cytoplasm of the large
macrophages, and their appear-
ance extracellularly in smears, as
in the case of L. donovani, is due
to rupture of cells in film-making.
In the ulcerating variety of sore,
wdiere there has been secondary
bacterial infection, and where pus
cells are abundant, there is a ten-
dency for degenerate parasites to
appear, and in various stages of
disintegration they may be found
in these cells. In such cases it
may be exceedingly difficult to
identify them, and when yeast-like
organisms are present also, con-
Yeasts often stain in a manner
closely resembling leishmania, and frequently exhibit a red area and a
deeply-staining granule. Rocha Lima (1912) drew attention to this
source of error, and pointed out that the organisms described by Darling
in Panama as Protozoa under the name of Histoplasma capsulation were
in reality yeast-like bodies from a case of blastomycosis. Yeasts, however,
have a much more distinct capsule, and the red area is more irregular in
shape than the nucleus of leishmania, while the deeply-staining dot does
not take on the characteristic rod-like form. Furthermore, reproduction
takes place by budding, and evidence of this can generally be found.
Another yeast-like organism, at one time considered to be a Protozoon
Fig. 192. — Leishmania tropica from
Oriental Sore ( x ca. 5. ()(»()). (After
Wenyon, 1911.)
1-3. Elongate torpedo- or cigar-shaped forms.
4-5. Abnormal forms in which kinetoplast alone
is visible.
6. More rounded form with dividing nucleus.
7-8. Appearance after fixation with Schaudinn's
fluid and staining with iron-hsematoxylin.
fusion with these is easily made.
LEISHMANIA TROPICA 429
allied to leishmania, is C ry ptococcus farcinimosus , the cause of lymphangitis
of horses (Plate III., p. 394).
The occurrence of flagellate forms of Leishmania tropica in oriental sore
has been referred to above (p. 425). The writer has never seen such forms,
though not infrequently fibres and filaments amongst the debris, especially
if associated with parasites, may give the appearance of flagella.
CULTURE. — Leishmania tropica, which grows in artificial media as readily
as L. donovani, was first successfully cultivated by Nicolle (19086) in Tunis,
Row (1909), in India, was also successful, as also were Markham Carter
(1909), Marzinowsky (1909), and the writer (1911a). Pedroso (1910)
appears to have first obtained culture of L. tropica from the South American
disease. The writer (19126) also cultivated the organism from a case of
South American origin. The cultures of L. tropica behave like those of
L. donovani, the most suitable temperature for growth varying from
22° to 25° C. It is possible that L. tropica grows more vigorously than
L. donovani, but variation in individual strains is often considerable. In
the writer's experience, certain strains of L. tropica grow very readily and
others with greater difficulty, especially in the first subculture. Similarly,
it sometimes appears that a slightly higher temperature is more favourable,
but there is no uniformity in this. Giugni (1914a), for instance, claims
that the optimum temperature for L. tropica is 28° to 29° C, while that
for L. donovani is 21° to 22° C. The writer has kept many strains of both
L. tropica and L. donovani growing for long periods at a temperature of 24°C.
Nicolle (1925) reports having maintained a strain of L. tropica in culture
for over fifteen years, during which it has passed through 384 sub-cultures.
Morphologically, the forms which appear in cultures of L. tropica are
indistinguishable from those of L. donovani (Fig. 190). Some have main-
tained, as Row (1909) has done, that L. tropica produces larger forms
than L. donovani, but the size of the flagellates varies with the age of the
culture, and also with different batches of medium, which can never be
prepared with absolutely uniform composition. Such a variety of forms
occurs in the cultures, and these in such varying proportions, that com-
parison between different cultures is exceedingly difficult to make.
To obtain cultures from an oriental sore, it is necessary to secure
material free from bacteria. In the ulcerating varieties, this can only be
done by carefully sterilizing the skin at the edge of the ulcer with iodine
or other antiseptic, making a puncture with a needle or sharp knife, and
drawing off material by means of a sterile pipette. The material in the
pipette is then blown into the liquid at the bottom of a tube of N.N.N.
medium. Flagellates are to be detected in the tubes in from three days
to three weeks, according to the number of organisms introduced.
430 FAMILY: TRYPANOSOMID^
As noted above (p. 399), Noguchi finds that the cultural forms of
L. tropica can be distinguished from other species of Leishmania by
serological tests.
NATURAL INFECTION OF ANIMALS.— Before the discovery of L. tropica
in oriental sore, several observers had already noted that in localities
in which the human disease occurred dogs were liable to develop similar
ulcers, especially on the nose and ears. Neligan (1913), working in
Teheran in Persia, where oriental sore is endemic, discovered leishmania
in the cutaneous lesions of a dog. Not only were the parasites present
in the skin lesions, but they were also found in the spleen, liver, and
bone marrow. YakimofE and Schokhor (1914) found leishmania in the
cutaneous lesions of dogs in Turkestan, and they suggested the name
L. tropica var. canina for this parasite. They produced no evidence, how-
ever, that it was different from the human parasite, which occurred in the
same locality. Gachet (1915) examined twenty-one dogs in Teheran, and
found skin lesions due to leishmania infection in fifteen of them. Dschun-
kowsky and Luhs (19096), in Transcaucasia, discovered a dog with leish-
mania in the spleen, liver, and bone marrow. Avari and Mackie (1924)
have discovered leishmania in ulcers on the ears of a dog in Bombay,
and mention another similar infection of a dog in the Punjab which was
brought to their notice by Eow, who (1925) has described the case.
Thus, in Teheran and farther west in Transcaucasia, leishmania are
found in dogs, not only in skin lesions, but also in the organs. The question
arises as to whether here the two diseases, canine kala azar and canine
oriental sore, exist side by side, or whether L. tropica in dogs leads to a
general as well as a cutaneous infection. In the Mediterranean region,
the naturally occurring canine kala azar is not associated with skin lesions,
though a case of spontaneous cutaneous leishmaniasis of the dog has been
noted by Sergent, Gueidon, Bouguet, and Catanei (1924) in Algeria,
where canine kala azar occurs. In Transcaucasia, both oriental sore and
kala azar exist in human beings, and it is not improbable that both occur
in dogs. In Teheran, on the other hand, human kala azar is not known.
It must be remembered that experimentally the virus of oriental sore
may produce a general infection in inoculated animals, while that of kala
azar can produce local skin lesions. The subject of canine leishmaniasis
in these areas requires further investigation.
As regards the South American disease, Pedroso (1913) noted ulcers
on the skin of two dogs which were associated with a man infected
with L. tropica. In one of the ulcers the author claims to have found
leishmania, but there seems to be some doubt as to the accuracy of this
observation.
LEISHMANIA TROPICA 431
DIRECT INOCULATION FROM MAN TO MAN.— Before the discovery
of L. tropica, it was well known that oriental sore could be handed on from
man to man by inoculation of the skin with the material from a sore. In
some places, such as Bagdad, Mosul, etc., it was the custom to inoculate
on the arm or some covered part of the body, with a view to developing
an immunity which would prevent the disfigurement of a natural infection
on the face. One attack of the disease as a rule confers an immunity
which lasts for the rest of life.
Definite evidence of the transference of the parasite in this way was
first produced by Marzinowsky (1909), who inoculated himself. Parasites
were demonstrated in the sore, which was first visible seventy days after
inoculation. Nicolle and Manceaux (1910a) obtained a positive result
by inoculation of cultures by scarification of the skin. The writer (1912a)
inoculated himself in a similar manner with material from a sore in
Aleppo. After a preliminary suppuration the wound healed, and it was
not till nearly seven months later that a minute red speck appeared at
the site of inoculation. This increased in size, and L. tropica was con-
stantly present for one and a half years, during which it persisted.
Patton (1912) inoculated himself and developed a sore after sixteen
days. Bouilliez (1917) inoculated himself accidentally. Material from a
syringe entered the conjunctival sac, and about four months later a papule
appeared on the internal surface of the lower lid. It increased in size to
that of an almond, and a second papule appeared. L. tropica was demon-
strated in the lesions. It is possible, therefore, for the parasite to infect
a healthy mucous membrane. It does not appear to be able to pass
the healthy skin, as was demonstrated by the writer. Material from a
sore was placed on the healthy skin and allowed to dry naturally, but no
sore developed at this spot, though at another spot where the skin was
scarified a typical lesion resulted.
These experiments of direct inoculation have their parallels in natural
infections. Numerous records occur of individuals who have developed
oriental sore at the site of some accidental wound or abrasion of the skin.
It is also well known that a person with one sore may auto-infect himself
by scratching on other parts of the body.
SUSCEPTIBILITY OF ANIMALS.— It was first demonstrated by Nicolle
and Manceaux (1910) tliat dogs could be inoculated in the skin with
L. tropica, and that local cutaneous lesions containing the parasites resulted.
Since then a number of observers have shown that dogs, cats, monkeys,
rats, mice, and guinea-pigs can be similarly inoculated. In the case of
small mammals such as mice, intraperitoneal inoculation has resulted in
generalized infections, resembling in many respects those produced by the
inoculation of L. donovani.
432 FAMILY: TRYPANOSOMIDiE
That the dog is susceptible to inoculation with L. tropica was first proved by
Nicolle and Manceaux (1910). These animals developed sores after inoculation
of virus from human cases or from cultures. The virus was handed on from dog to
dog. Laveran (1915rf, 19 IG) produced local lesions in dogs by inoculating material
from the organs of mice which, as will be shown below, are liable to a generalized
infection of L. tro2)ica. Dogs which had recovered from a first infection are found
to be reinoculable, but a second attack conferred an immunity against further
infection. Attempts at the production of a general infection in dogs like that in
kala azar, by injection of virus intraperitoneally or intravenously, by Nicolle and
I\Iauceaux, Laveran and the writer, have given only negative results. The duration
of the inoculated disease in dogs is much shorter than in man.
With the South American virus the writer (1913) succeeded in inoculating a dog
oil the ear directly from a human case. A cat was also infected. Strong and his
co-workers (1913) also infected a dog with the South American virus.
Monkeys were first inoculated by Nicolle and Sicre (1908a). Since then, Nicolle
has extended his observations, and successful results have also been obtained by
Row (1910), Patton (1912), Bouilliez (1917a), and Laveran (1912fZ, 1917). Various
species of Macacus and Cercopithecus and the mandrill {Cynocephalus mormon) are
found to be susceptible. The lesions produced resemble more closely those in man,
but they are of shorter duration. With the South American virus the writer (1913)
produced cutaneous lesions in a baboon. Sant' Anna (1913) successfully inoculated
a species of Cercopithecus.
The observation made first by Row in India that local skin lesions could be
produced in monkeys by inoculating L. donovani has been referred to above (p. 415).
Mice were first shown to bo susceptible to L. tropica by Gonder (1913). The
animals inoculated intraperitoneally with large doses of culture developed not only
a general infection, but also swelling and cutaneous lesions of the legs and tail.
Leishmania were present in all these lesions, and there was marked enlargement of
the liver and spleen. General infections m mice were also produced by Row ( 1 914a),
Sergent, Ed. (1915), and Pavoni (1915), and especially by Laveran (1914?), 1915/?, c),
who has studied the question in detail. As a result of numerous experiments, it
appears that in Laveran' s hands the animals were easily infected by intraperitoneal
injection of cultures or virus from other animals. In most cases, the first signs of
infection in male mice, with which Laveran chiefly worked, is an infiltration of the
peritesticular connective tissue, which becomes much thickened and oedematous,
and is found to contain large numbers of parasites. Subsequent to this infection, a
general infection of the internal organs takes place, associated with oedema of the
limbs and tail. Fluid from these parts is found to contain parasites. The skin over
the swollen testicular region, tail, and limbs breaks down and ulcers result. In
female mice local skin lesions alone often appear.
Of a series of sixty-seven mice which were infected, forty-three showed only the
local lesions without a general infection, fifteen had both local lesions and a slight
general infection, while nine had local lesions and a fairly intense general infection.
Mice as a rule do not show signs of infection for about a month.
The disease progresses for several months, and the animals may die or recover.
The results of inoculation of mice with L. tropica thus appear to differ in a very
striking manner from those obtained with L. donovani. Row (1914a, 1924), on the
other hand, working in India, has produced general infections in mice with L. tropica.
but has never noted the involvement of the skin or testes. The animals have reacted
in every way as they do towards L. donovani, the parasites being numerous in
the spleen, liver, and bone marrow. In some cases localized infections of the mucosa
of the small intestine were noted in regions where lymphoid tissue occurred.
LEISHMANIA TROPICA
433
Laveran (1917) has shown that rats respond in a similar manner, especially if
inoculated in the testicle. BouiUiez has also infected rats, and has noted local
lesions in these animals. He was working in the district of Lake Chad (Chari Kivcr),
and employed Mus concha and another rodent, which was probably Arricanthus
niloticus richardi. Another small rodent {Golunda campance) of this district was
also infected by him. Laveran had similar results also with Meriones shawi and
Myoxus glis. A guinea-pig inoculated in the testicle by Laveran with material
from an infected mouse became locally infected, and a gerbil responded in the same
manner as mice. Infection in animals has not always been a simple matter, for
many observers have failed to produce infections, possibly because the dose of virus
had been too small.
Franchini (1922m.) states that he has infected the plant Euiihorbia segctalis by
inoculating it with cultures of L. tropica
TRANSMISSION. — At the present time it is generally believed that the
sand flies of the genus Phlebotomus are responsible for the spread of oriental
sore (Fig. 193). These flies
were first suggested as possi-
ble vectors by Pressat (1905),
and Sergent, Ed. and Et.
(1905a), while the experi-
ments conducted by Sergent,
Ed. and Et., Parrot, Donatien,
and Beguet (1921) in Algiers,
by Aragao (1922) in South
America, and Adler and
Theodor (1925a) in Palestine,
as also those of Laveran
and Franchini (1920) in
France, lend support to this
view without, however, sup-
plying the absolute proof.
The last-named observers in-
oculated dogs in the skin with
cultures of the leptomonas of
Phlebototnus, and produced lesions resembling oriental sore, in which para-
sites were found. Similarly, Sergent and his co-workers (1921) produced
a characteristic oriental sore in a man by inoculating crushed-up PJilebo-
tomus papatasi, Aragao a similar sore on a dog by inoculating crushed-up
Phlebotomus which had previously fed on a sore, and Adler and Theodor a
papule containing leishmania on the skin of a man by inoculation of lepto-
monas from P. papatasi. It is possible that the leptomonas discovered by
the writer (1911) in Phlebotomus of Aleppo was actually Leishmania tropica.
It was discovered by the Sergents, Lemaire, and Senevet (1914), and
later by Chatton and Blanc (19186), and Nicolle, Blanc, and Langeron
I. 28
Fig. 193. — Phlebotomus papatasi ( $ ), the Prob-
able Transmitter of Leishmania tropica
( X ca. 13). (After Whittingham AND Rook,
1923.)
434 FAMILY: TRYPANOSOMID^
(1920), that the blood of the gecko {Tarentola mauritanica) harboured a
flagellate of the leptomonas type, which was only demonstrable by culture
of the heart blood. This gave rise to the view, first enunciated by the
original discoverers of this organism, that the gecko, on which sand
flies were known to feed, probably acted as a reservoir for the virus of
oriental sore. Nicolle, Blanc, and Langeron (1920), by careful examina-
tion of the cultural forms of the gecko flagellate, concluded that they were
distinguishable from the cultural forms of L. tropica. Moreover, injection
of cultures of the gecko flagellate into the skin of man and monkeys failed
to give rise to oriental sore. They conclude, with ample justification,
that there is no real evidence that the gecko flagellate has any connection
with L. tropica.
Strong (1924) has produced in the monkey a lesion resembling oriental
sore in which leishmania occurred by subcutaneous inoculation of the skin
with a flagellate of the leptomonas type, which occurs in the intestine of
the lizard, Cnemidophorus lemniscatus, of Central America, where cutaneous
leishmaniasis is endemic. The lizard, it is assumed, acquires its infection
by feeding on plant bugs, which in their turn obtain the flagellates from
the juices of Euphorbias (pp. 383, 442). Further investigations will be
required before it can be accepted that the flagellate in this lesion is
identical with that causing the naturally occurring human disease, or that
the sequence of events described by Strong has any setiological significance
in connection with the natural method of its transmission.
It has been shown that L. tropica will develop in the bed bug like
L. donovani, but there appears to be little reason for suspecting it to be a
vector of oriental sore. Lloyd (1924) has found a typical Leptomonas in
the proboscis and intestine of Glossina morsitans in Nigeria. As this fly
feeds only on blood, it would appear that the flagellate must have been
derived from the blood of some animal or man. As human leishmaniasis
occurs in Nigeria, the flagellate of the tsetse fly may represent a Leishmania.
Experiments on the possibility of L. tropica developing in insects have
been made by several observers.
Bugs. — Tlie writer (1911«) observed that when the bed bug fed on an oriental
sore before ulceration had set in, it took up leishmania, and a development similar to
that described previously by Patton for L. donovani, took place. Pattou (1912)
published a more extensive series of experiments with bed bugs, and obtained
resiilts similar to those he had obtained with the parasite of kala azar. Working
later in England, the writer (1912c) again found that L. tropica developed into
flagellates in the bed bug. In no cases did active multiplication occur such as
would be expected in the true invertebrate host, and, as with L. donovani, it appeared
that the blood in the stomach of the bug had acted merely as a culture medium.
By a series of ingenious arguments similar to those employed in support of his
claim of the transmission of kala azar by bed bugs, Patton attempted to prove
that this insect also transmitted oriental sore. The bed bug was supposed to bite
LEISHMANIA TROPICA 435.
exposed surfaces of the body more commonly than any other part. Whatever may
be said in favour of the bed bug being a possible vector of L. donovani, no sound
arguments, epidemiological or other, can be adduced in support of the claim that it
is the cause of oriental sore.
Patton (1922) stated that he has been able to obtain a development of L. tropica
in the bed bug similar to that obtained by Adie with L. donovani (see p. 419).
Presumably, intracellular stages were seen, but as these occurred in the cells of
the gut only after its removal from the body and incubation at a suitable
temperature, they can hardly be recognized as representing a normal process of
development, and still less as proving conclusively that the bed bug is the true host
of L. twjyica in Cambay in India, as Patton maintains. No host can be regarded as
being conclusively incriminated in the transmission of L. tropica or any other
parasite till the infection has been actually transmitted by it.
Fleas. — Working with fleas {Pulex irritans and Ctenocephalus cants), which the
writer (1912c) fed on an oriental sore resulting from his inoculation in Aleppo, no
evidence of development of L. tropica could be obtained. In these experiments the
fleas were attached to wire according to Noller's method, and before feeding on the
sore were proved, by examination of the fseces ejected during feeding, to be free
from flagellate infection. When fed on the sore, it was noted that leishmania were
ejected with the faeces even in the first portion passed, proving that the fleas had
actually ingested parasites. The fleas were then incubated at 22° C, the optimum
temperature for culture. They were fed from time to time on the wrist, but no
evidence of flagellates which might have developed from the leishmania could be
found in the ejected faeces. Fleas found naturally infected with leptomonas con-
stantly passed flagellates in the faeces. The fleas which had given negative results
for leishmania were then fed on a rat harbouring Trypanosoma lewisi, and afterwards
on the wrist as before. On the sixth day infective forms appeared, and continued in
the faeces, thus proving that the conditions of the experiment were suitable for the
development of a natural flagellate of fleas.
Laveran (1917) describes attempts to transmit L. tropica from mouse to mouse by
means of fleas. Four healthy mice, together with others heavily infected with
L. tropica, were placed in a glass jar which was serving as a flea breeding-place. There
were so many fleas present that the mice had eventually to be removed for fear
of their being killed by continued abstraction of blood. Three of the mice were
examined after five months, and the fourth after eight months, but no infection had
taken place.
Lice. — Patton could obtain no evidence of the development of L. tropica in lice.
Mosquitoes. — The writer (1911a), working in Bagdad, fed thirty-one Culex
fatigans on oriental sore. It had been proved by dissection immediately after
feeding that mosquitoes readily took up leishmania from a sore of the non-idcerating
variety. The mosquitoes dissected twenty-four, forty-eight, and seventy-two hours
after feeding showed no trace of flagellates. In a few out of a large number of JEdes
argenteus {Stegomyia fasc lata) which had fed daily on the sore, rounded bodies possibly
derived from the leishmania were found on dissection twenty-four or forty-eight
hours after feeding. An attempt at transmission by means of twenty-six of these
mosquitoes which had fed repeatedly on the sore and then on the arm gave no result.
Phlebotomus. — The writer (1911 ) first recorded the existence of a natural Lepto-
moitds of the sand fly in Aleppo, an endemic centre of oriental sore. W^hat is
probably the same flagellate has been found in P. papatasi in Palestine by Adler
and Theodor (1925a). It is possible that the flagellate was actually Leishmania
tropica. Mackie ( 1 914b) then gave the name Herpetomonas 2)hlebotomi to a flagellate
436 FAMILY: TRYPANOSOMID^
found in Phleboiomus minutiis in Assam. Sliortt (1925) has examined the original
preparations, and finds that it is actually a Bodo, the name of which is therefore
Bodo phlebotomi.
Later Mackie again encountered flagellates in sand flies in the same locality. On
this occasion elongate forms definitely crithidial in type were present in the films which
were seen by the writer, so that it is evident that the flagellate was not a leptomonas.
It may represent a trypanosom.e of a lizard, on which these flies are known to feed.
Laveran and Fran chini (1920, 1920b) state that they found a flagellate which they call
H. iMehotomi in P. papatasi in Italy. In this case, again, the figures of the organism
might be interpreted as representing crithidia. Cultures were obtained, and with
them two dogs were inoculated in the skin of the thigh. One developed a local
lesion resembling oriental sore, and the other a generaUzed infection like kala azar.
In both cases leishmania forms of the flagellate were said to occur in the lesions.
Patton (1919, 1920) refers to H. pHebotomi in connection with remarks on the
probable transmission of oriental sore in Mesopotamia by P. papatasi and P. minutiis.
He has informed the writer that he did not actually see the flagellates in these flies.
Sergent, Ed. and Et., Parrot, Donatien, and Beguet (1921) had sand flies sent
from Biskra to Algiers, a three days' journey. On one occasion seven P.jjapatasi
received were crushed in saline and inoculated into the skin of a human being by
scarification. Two months and twenty-four days later a papule which changed
into a typical sore containing leishmania appeared, though flagellates had not been
seen in the inoculated material. Aragao (1922) in South America fed P. inter-
medius on sores, and three days later crushed them in saline. This material was
applied to a scarification on the nose of a dog, which developed a sore in which
leishmania were found. These experiments prove that the sand fiy can carry the
virus in a virulent form for at least three days, for in the case of the flies employed
by the French observers it is possible that they had just fed on a sore in the military
hospital at Biskra, where they were caught. The experiments of Adler and Theodor
(1925«) are more conclusive. In a single P. papatasi in Palestine numerous lepto-
monas were found in the whole extent of the alimentary canal, including the oeso-
phagus and its diverticulum. The flagellates were inoculated into the skin of a
human being on June 26. On July 31a small papule had formed, and in it leish-
mania were found. Adler informs the writer that another positive inoculation
from a naturally infected fly has been made, while flies have been infected with
flagellates by allowing them to feed on oriental sores.
Hippoboscidse. — Gachet (1915) noted that the dogs of Teheran were heavily
infested with Ilippobosca canina. Examining a fly which had just gorged itself on
a sore on the face of a dog, leishmania were found in the blood in its stomach . Gachet
thinks that the frequence of cutaneous leishmaniasis of dogs in Teheran may be due
to the prevalence of this fly.
Stomoxys. — The writer (191 la) showed that Stomoxys were capable of taking up
leishmania from a sore, but no development took place.
House Flies.— Laveran (18806) first suggested that the oriental sore of Biskra
might be due to fly transmission. The writer ( 191 la) and Patton (1912) experimented
with house flies, but found that the leishmania degenerated after being ingested.
Cardamatis and Melissidis (1911rt) claim that L. tropica persists in flies up to six days,
but they were undoubtedly observing the natural flagellates of the fly. It is, how-
ever, highly probable that the house fly, which swarms around the exposed sores,
especially in children, may sometimes carry the virus on its feet or proboscis to
abrasions on the skin of another person. The leishmania may also pass rapidly
through the gut of the fly and be deposited with the dejecta, as occurs with other
LEISHMANIA TKOPICA 437
organisms. Thus, trichomonas in faeces will appear quite unaltered in the dejecta
of the fly five minutes after being taken up.
With reference to the cutaneous leishmaniasis of South America, there has been
much speculation as to the transmitting host. Biting flies and ticks of various
kinds have been blamed, but little definite observation has been carried out. Town-
send (1915) inoculated a guinea-pig in the skin with flagellates he found in a Chiro-
nomid {Forcipomyia). A i^apule developed at the site of inoculation, and bodies
supposed to be of the nature of leishmania were found in it. As the flagellates occur
neither in the proboscis nor salivary glands of the fly, he believes that transmission
is effected by deposition of fly dejecta in the skin, and subsequent contamination of
the puncture wound inflicted. No proof was produced that the organism, if an
organism at all, in the papule was in reality L. tropica. The experiments conducted
by Aragao (1922), which have been noted above, suggest the possibility of Phleboto-
miis intermedius being the vector of the South American cutaneous leishmaniasis,
while the observations of Strong (1924) suggest a possible connection with the
flagellates of Euphorbias.
ACTION OF DRUGS ON LEISHMANIA TROPICA.— As in the case of
L. do))ova)i L tartar emetic and the corresponding sodinm salt have a specific
action on the parasites. Cures may be eiTected by scraping, excision,
and tlie use of strong reagents, which not only destroy the parasites, but
the tissues as well. Such are crystals of permanganate of potash, carbolic
and nitric acids, solid carbon dioxide, and methylene blue. Tartar emetic
may be used as in the case of kala azar, or in the form of an ointment
locally. Emetin, as first pointed out by Photinos (1920), brings about
death of the parasite and a cure of the disease w^hen injected into the lesion.
Possibility of Confusing Leishmania with other Organisms.
Huntemliller (1914) described under the name of Plasmosoma jeri-
choense an organism he had found in sections of tissue removed from
a " Jericho boil." He considered it to be an entirely new Protozoon.
The wTiter was able to examine the sections, which showed the organism
to be badly-stained Leishmania tropica, which is often very difficult to
stain in tissues, especially when unsuitably fixed. Similarly, Chalmers
and Kamar (1920) described as Toxoplasma pyrogenes certain structures
obtained from the spleen of a fatal case of splenomegaly in the Sudan.
From information the writer has received, there is no doubt that the
supposed toxoplasma was merely degenerating or badly-fixed Leishmania.
Similarly, the yeast-like organism Cryptococcus farcinimosus, which
was discovered and named by Rivolta (1873), was regarded by many
observers as a Protozoon, though its original discoverer had recognized
its true nature. Pocha Lima (1912) drew attention to the fact that yeasts,
as seen in stained smears, often simulated leishmania (Plate IIL, p. 394).
Such a fallacy has always to be borne in mind when the organs of animals,
especially those which have died and the tissues of which may have been
invaded by bacteria or yeasts, are examined for Leishmania.
438 FAMILY: TEYPANOSOMID.E
LEISHMANIA IN ANIMALS.
The definition of the genus Leishmania, which has been adopted here,
is such that it includes all flagellates which attain the leptomonas form,
and which have both a vertebrate and an invertebrate host. The latter
feature is in the nature of an assumption, for, as shown above, the actual
invertebrate hosts of L. donovani and L. trojnca have not been demon-
strated, though the probability of such hosts existing is so great as to
amount almost to a certainty. In addition to the two forms already
considered as producing diseases in man, there exist certain other lepto-
monas forms, which have been described as natural infections of verte-
brates, and which must be included in this genus, though here also
the invertebrate host has yet to be demonstrated (see p. 398). These
naturally occurring infections are not to be compared with the artificial
ones which Laveran and Franchini, and Fantham and Porter, claim to
have produced in animals by the injection into them of purely insect
flagellates. The latter have been considered in the section devoted to
the insect flagellates (see p. 392). It seems probable that the naturally
occurring infections in lizards result from their feeding on infected insects.
Leishmania tarentolse Wenyon, 1921. — Sergent, Ed. and Et., Lemaire,
and Senevet (1914), while searching for a host of L. trojnca in North Africa,
discovered that cultures of a typical leptomonas could be obtained from
the heart blood and organs of the gecko, Tarentola mauritanica. The
cultures closely resembled those of L. tropica, and led to the view that
the gecko was a possible reservoir host of the human parasite, especially
as the sand fly Phlebotomus, the supposed vector of oriental sore, frequently
feeds on the lizard. The observation was confirmed by Chatton and
Blanc (19186), and by Nicolle, Blanc, and Langeron (1920) at Tamerza.
The latter observers studied the cultures carefully, and came to the con-
clusion that the flagellates could be distinguished from those in cultures
of L. tropica. The organisms must be present in the heart blood of the
lizard in very small numbers, for in two positive cases out of twelve geckos
examined, flagellates were not to be detected in the cultures till twenty-
four to thirty-six days had elapsed. Nicolle and his co-workers believe
that the organism is probably of intestinal origin, and is only accidentally
present in the blood. Laveran (1915) could obtain no infection in geckos
by inoculating them with L. tropica. Chatton and Blanc (19186) inocu-
lated geckos with cultures of L. tarentolce, and were able to recover the
flagellate from heart blood by culture in 50 per cent, of the cases after
one to two months. In nature, 35 per cent, of geckos were found infected.
Cultures of trypanosomes (T. platydactyli) were also obtained, but these
could be readily distinguished. They also inoculated geckos with cultures
LEISHMANIA IN ANIMALS 439
of L. tropica, and were able to recover the organism by cultures of heart
blood even after the expiry of twelve days. Pittaluga and Buen (1917)
in Spain, and Laveran and Franchini (1921a) in Italy, have examined
specimens of T. mauritanica by the culture method, and have found
them infected with L. tarentolce. Laveran and Franchini state that the
living flagellates were actually observed in the blood, but in most cases
the presence of the organism was demonstrated by the culture method
only. Cultures of the trypanosomes were also obtained. Franchini
(192k/) states that a further examination of these lizards has shown
that the flagellate may occur in the rectum and cloaca in the lepto-
monas and leishmania form.
By feeding bed bugs on geckos, Chatton and Blanc (1918a) obtained
a temporary development of L. tarentolce in the stomachs of the bugs.
Leishmania henrici (Leger, 1918).— This organism, discovered by M.
Leger (19186), and named by him Leptomonas henrici, was present in the
blood of two out of thirty lizards (Genus Anolis) examined in Martinique.
The body of the flagellate measured 15 to 16 microns in length and 4 to 5
microns in breadth. The flagellum was longer than the body. Leish-
mania forms were also seen, but more rarely. Leger subsequently found
that over half the lizards harboured what was apparently the same organism
in the rectum, so that there had probably been an invasion of the blood
and organs from the intestine. The flagellate probably originates from
some insect upon which the lizards feed, a fact which indicates how a
leishmania infection may arise from an insect flagellate first becoming
established in the intestine of the vertebrate. It opens up the possibility
of L. donovani infecting man by way of the intestinal tract.
Leishmania chamaeleonis Wenyon, 1921. --A typical leptomonas flagel-
late was observed by Bayon (1915) in the cloaca of Chamceleon pumilus
of Robben Island. What was undoubtedly the same organism was
discovered by the writer (1921) in Egypt in C. vulgaris. The flagellate
was present in the cloaca in enormous numbers, where they lived in the
mucus or invaded the lumen of the glands (Fig. 194). Intracellular forms
were not seen, nor were cultures obtained from the heart blood. The
measurements given by Bayon are incorrect, as the writer, who saw his
preparations, can testify. The flagellate has a body about 15 microns
in length, and the flagellum is slightly longer than this (Fig. 195). The
width of the body in the long forms w^as about 3 microns. From these
long flagellates may be traced a series of gradually diminishing individuals
of varying size and shape, till minute round forms barely 2 microns in
diameter are produced.
The last have relatively long flagella. Others are devoid of flagella
and have the leishmania form, and some oval bodies with deeply staining
440
FAMILY: TEYPANOSOMID^
outline appeared to be encysted (Fig. 195, d). Some experiments con-
ducted with house flies showed that a temporary infection of the gut
resulted from feeding them on the cloacal mucus. It is in this material,
rather than in the actual faeces that the flagellate occurs.
Tnierons
Fig. 194. — Leishmania chamccleonis in the Lumen of a Gland of the Cloaca of
Chamceleon vulgaris ( x 1,700). (After Wenyon, 1920; from Parasiiologtj, vol. xii.)
Franchini (1921a) has examined two specimens of C. vulgaris, and
has noted that the flagellates may occur in small numbers in the upper
parts of the intestine. Leishmania forms are said to occur in the stomach.
LEISHMANIA IN ANIMALS 441
No infection of the blood or other organs could be detected. Two mice
which were fed on cloacal contents were said to have become infected.
Free leishmania forms are described as occurring in the heart blood and
bone marrow, and both these and leptomonas forms in the liver and spleen.
By employing N5ller's blood-agar plate method, cultures of the flagellate
were obtained from the cloacal contents of a chameleon. The flagellate
of the chameleon is of interest when compared with L. henrici, which
occurred, not only in the intestine of its host, but also in the blood.
Further investigation of the flagellate of the chameleon will probably
show that it also may occur in the blood-stream.
Fig. 195. — Various Types of Leishmania chamceleonis found in Cloaca of
Chamwleon vulgaris (x 2,200). (After Wenyon, 1921; from Parasitology, vol xii.).
Leishmania hemidactyli (Mackie, Gupta, and Swaminath, 1923). —
This parasite appears to be very similar to L. tarentolce. It was discovered
by the authors, who named it, in cultures made from the blood of the
Indian gecko, Hemidactylus gleadovii. Direct examination of the blood
failed to reveal any flagellates, and no mention is made of a concurrent
intestinal infection. A trypanosome named Trypanosoma hemidactyli
was also present in the blood.
Franchini (1921a) records the presence of leptomonas and leishmania
forms of a flagellate in the rectum and cloaca, and also other parts of the
gut, of Lacerta ocellata. They were also said to be present in the leish-
mania form in the heart blood and liver.
442 FAMILY: TRYPANOSOMID^
Another flagellate of the leptomonas type has been found by Strong
(1924) in the hind-gut of the lizard, Cnemido'phorus lemniscatus. He
suggests that the infection is acquired by the lizards eating certain plant
bugs which harbour what he assumes, on morphological grounds, to be
the same organism. The bugs become infected by feeding on the latex
of Euphorbias, which are also infected with the same flagellate. These
observations, combined with the fact that Strong has succeeded in inocu-
lating the lizard flagellate into the skin of the monkey, where a lesion
resembling oriental sore is produced, serves to indicate the close relation-
ship of all the flagellates of the leptomonas type.
Leishmania denticis (Fantham and Porter, 1919). — This flagellate was
found in four out of forty-one silver fish [Dentex argyrozona) examined by
Fantham (1919) and Fantham and Porter (1920) in South Africa. It was
called by them Herpetomonas denticis, but is a flagellate of the typical
leptomonas form, while leishmania stages also occur. The body measures
5 to 24 microns in length and 1-5 to 2-5 microns in breadth. The flagellum
is often longer than the body, and is relatively longer in the shorter forms.
Non-flagellate leishmania forms measured 2-5 to 4-5 microns by 1-5 to 2-5
microns. The organism was found most frequently in the heart blood,
and also in smears of the liver, spleen, and kidney. It was not abundant
in any fish examined, nor was it present in the intestinal tract.
Fantham (1922) records as H. xenopi a flagellate from the rectum of
the South African clawed toad, Xenopis Icevis. No details of the infection
or of the flagellate are given.
Leishmania myoxi (Laveran and Franchini, 1921). — Three out of seven
dormice {Myoxus glis) captured near Bologna were found infected. The
organism, named Herpetomonas myoxi, was found only in stained smears
of the blood, spleen, and liver. It occurred mostly as leishmania forms,
which measured 1-8 to 3-6 microns in length by 1-2 microns in breadth.
They were either free or within mononuclear cells. In addition, a certain
number of elongate non-flagellate forms were seen. These measured from
5 to 20 microns in length by 1-2 microns in breadth. Flagellate lepto-
monas forms 12 to 20 microns in length were also encountered. Though the
figures depict an organism of the leptomonas type, the writer feels that
confirmation is necessary before the statements regarding it are accepted.
Genus : Trypanosoma Gruby, 1843.
The flagellates of the genus Trypanosoma attain the trypanosome
structure at some stage of their development, and occur as parasites in
the blood and tissues of vertebrate animals. For many of them there
have been demonstrated invertebrate hosts, which transmit them from
one vertebrate to another either by direct inoculation through the mouth
GENUS: TRYPANOSOMA 443
parts in the act of feeding, or indirectly by the vertebrate accidentally
ingesting the infective fseces. The vast majority of trypanosomes are
known only as they occur in the blood of the vertebrate, but it is safe to
assume that an invertebrate host exists for every one, with the possible
exception of Trypanosoma equijjerdum, the cause of dourine, which is
handed on directly from horse to horse during the sexual act. In some
instances the trypanosome is only known in the invertebrate, but that a
vertebrate host also exists is rendered probable by the fact that typical
infections can be produced in laboratory animals by inoculating them
with these insect flagellates. Such infections differ from the transitory
infections which may result from the inoculation of purely insect lepto-
monas, crithidia, or herpetomonas, which, as explained above, cannot be
regarded as having vertebrate hosts.
Trypanosomes have been found in every class of vertebrate, and it is
because some of them produce disease in man and domestic animals that
these flagellates have attained considerable importance and have been
the subject of many investigations, the literature dealing with which is
now very extensive.
According to Laveran and Mesnil, whose excellent treatise on trypano-
somes and trypanosomiasis summarizes our knowledge of these flagellates
up to the year 1912, the first observer to see a member of the genus was
Valentin of Berne, who discovered a trypanosome in the blood of the
trout, Salmofario, in 1841. In the two succeeding years Gluge of Brussels,
Mayer of Bonne, and Gruby of Paris published three papers on the try-
panosomes of the frog. To these organisms Gruby gave the name Try-
panosoma. From 1843 to 1880 little advance was made in our knowledge
of trypanosomes except for their discovery in various amphibia, the black
rat, the field mouse, and the mole. Timothy Lewis (1878, 1879) published
accounts of the trypanosome of the rat in India, but these flagellates were
first recognized as of great importance on the announcement in India of
the discovery of a trypanosome in the blood of horses and camels suffering
from surra by Griffith Evans (1880), and in the disease nagana of horses
and cattle in Africa by Bruce (1895). Discovery of various other trypano-
somes in domestic and other animals followed these observations, which
led up to the discovery in the blood of a man in the Gambia by Forde of
an organism which was recognized and described as a trypanosome
{T. gambiense) by Button (1902). The next observer to see a trypanosome
in man was Castellani, who (1903) announced his discovery of a trypano-
some in the cerebro-spinal fluid of a case of sleeping sickness in Uganda.
This observation was confirmed immediately afterwards by Bruce and
Nabarro (1903), who demonstrated the causal relationship between the
trypanosome and the disease.
444 FAMILY: TRYPANOSOMID^
Though it was long known that the diseases caused by trypanosomes in
man and domestic animals in Africa were transmitted by flies belonging
to the genus Glossina, the role of these blood-sucking diptera was not
properly understood till Kleine (1909, 1909a) proved that a period of about
twenty days was required for development of the trypanosome in the fly
before the latter was able to bring about infection. Before Kleine's
discovery in 1909, the repeated failure to transmit infections by tsetse
flies had led observers to hold the view that they acted merely in a
mechanical manner in carrying infective blood from one animal to
another, with a lapse of a minimal interval of time between the two bites.
The work of Rabinowitsch and Kempner, Swingle, Noller, the writer,
Minchin and Thompson, and others established the role of the flea in
transmitting T. lewisi from rat to rat, and proved that a development
took place in the flea, leading to the appearance of infective forms of the
trypanosome in the flea fseces which were eaten by other rats.
Brumpt, Robertson, Noller, and others demonstrated the develop-
ment of trypanosomes of fish and frogs in leeches, and the part they play
in handing on the infection from one animal to another. The work of
Chagas, Brumpt, and others has proved the role of Triatoma megista and
other reduviid bugs in the transmission of the human trypanosome, Try-
panosoina cruzi, of South America, while, finally, the observations of
Noller, Kleine, and Hoare have proved the transmission of T. melophagium
of sheep by the sheep ked {Melophagus ovinus).
METHODS OF DISTINGUISHING TRYPANOSOMES.
The number of trypanosomes which have been named is very great,
and the list is constantly being extended. It becomes of importance,
therefore, to be able to distinguish one from another, and it has resulted
that, quite apart from morphological details, various methods of separating
the species have been devised. In many quarters, the discovery of a
trypanosome in a new host has been taken as sufficient ground for the
creation of a new species. Though this procedure is not in accordance
with the rules of nomenclature, there is something to be said in favour of
it. for the trypanosome of a particular host will be referred to by its name
till it has been definitely proved to be identical with some other previously
named type. This is likely to lead to less confusion in the literature than
if workers had to deal with a large number of unnamed trypanosomes,
or trypanosomes which had received already existing names because of
certain resemblances they might have to these. Scientifically, it is just
as incorrect to group together under one name without sufficient evidence
what may eventually prove to be distinct species as to give dift'erent
names to forms which ultimately may be found identical.
GENUS: TRYPANOSOMA 445
The characteristics which are of use in distinguishing trypanosomes
are the following:
1. MOVEMENT. — When viewed alive under the cover-glass, trypano-
somes vary very much in the movements they perform. Some are
sluggish and do little more than wriggle and twist about in a limited area.
This is true of many of the larger trypanosomes, like those of fish and frogs.
On the other hand, amongst smaller types there is a similar variation.
T. gamhiense is moderately motile, and may travel some distance across
the field of the microscope. T. lewisi is more active, while T. vivax takes
its name from its remarkable motility. It darts about amongst the red
blood-corpuscles and quickly passes out of the field.
2, MORPHOLOGY. — The morphological features of trypanosomes
depend on the size and shape of the body, variations in the size and posi-
tion of the nucleus and kinetoplast, and the degree of development of the
undulating membrane and flagellum. All these features, as also others,
have to be taken into consideration in describing the characters of any
trypanosome. There may, however, be considerable difficulty in doing
this, as they vary at different stages of development, and anything like a
complete cycle is known only in a few instances. Thus, T. lewisi varies
remarkably at different stages of development in the rat and the flea
(Fig. 197). Similar variations occur in the case of T. cruzi of man (Figs.
207, 209), T. rotatorium of the frog (Fig. 237), and indeed, in all trypano-
somes in wdiich anything approaching a complete life-history is known.
As in most cases only one stage in the development has been seen, and
that in the blood of the vertebrate, knowledge of the exact morphology
of these forms is very incomplete, and has often led to different stages of
one and the same trypanosome being described as distinct species.
The general shape of the body of a trypanosome is that of a curved,
flattened blade (Fig. 150). One margin of the body is generally convex
and the other concave. The ends are tapering. The nucleus lies most
usually near the centre of the body, and the kinetoplast near the posterior
end. The axoneme commences at the blepharoplast, and after traversing
the cytoplasm for a short distance passes along the border of the undulat-
ing membrane, which arises from the convex edge of the body as a thin ridge
of cytoplasm. At the posterior end of the body, the membrane terminates
and the axoneme may or may not be continued into a flagellum. Though
on first appearance many trypanosomes seem to differ structurally from
this type, they are, however, all traceable to it. The variations which
occur may be considered as arising in one of two ways. Firstly, there may
be an increase in the length of the convex border, giving rise to forms which
are more and more curved till a complete spiral may be reached (Fig. 236, 2).
Secondly, there may be a great increase in the width of the flagellate,
446 FAMILY: TIIYPANOSOMID.E
leading to forms which are remarkably broad (Fig. 150, 37). In some
cases, both these modifications occur, with the result that there arise the
very remarkable leaf-like trypanosomes which are seen particularly in
amphibia (Fig. 150, 37).
In other cases increase in thickness as well as breadth occurs,
and solid ovoid forms arise which are again typically seen in frogs
(Fig. 150, 39, and 238).
It must be remembered that in ordinary stained films of blood these
complicated forms are generally distorted to such an extent that their
actual shape is obscured. The true form of the body can only be satis-
factorily seen in the living condition or in specimens fixed without drying.
Furthermore, during life the trypanosome is constantly altering its shape
by contractions of its body, but in relaxation it returns to one or other of
the types indicated in the diagram (Fig. 150).
The length of the body behind the kinetoplast is also subject to
variation. In some forms the kinetoplast is actually at the posterior
extremity, or very near it, as in T. vivax, T. congolense, and the meta-
cyclic or infective forms of T. lewisi in the flea (Fig. 197, 20-23). In other
trypanosomes this region of the body may be greatly prolonged, as in
certain forms of T. lewisi in the rat (Fig. 197, 1-3) and the trypanosomes
which occur in toads (Fig. 238).
The shape of the posterior end of the body is of some diagnostic
importance, though it must not be forgotten that the extremity is subject
to changes brought about by contractions of the living cytoplasm. Some
trypanosomes, like T. lewisi and T. cruzi, have habitually a very sharply
pointed posterior end, while others like T. vivax and T. congolense, have
this extremity rounded (Plate V., p. 456). Many pathogenic trypano-
somes not infrequently have the posterior end sharply cut off or flattened.
The swollen condition of the posterior half of the body in T. vivax is highly
characteristic of this species (Fig. 231).
The undulating membrane naturally has its attached border shorter
than the free one, so that it is thrown into folds. The degree of undulation
varies in dift'erent trypanosomes, and consequently the length of the
axoneme. The degree of undulation is usually judged by the appearance
of the axoneme. In T. lewisi, in the forms which occur late in the infection
of a rat, the attached flagellum is only slightly undulating, whereas in
T. gambiense it is much more so (Plate V. a and l, p. 456). In other
trypanosomes the degree of undulation may be still more marked.
As already noted, the axoneme may terminate at the anterior extremity
of the body, as in T. congolense and the stumpy forms of T. brucei and
T. gambiense, or it may be extended as a flagellum (free flagellum) for a
varying distance, as in the majority of forms (Plate V., p. 456).
GENUS: TRYPANOSOMA 447
So far reference lias been made only to the flagellates of the trypano-
some type, but it must be remembered that during the evolution of
trypanosomes, either in the vertebrate or invertebrate host, other types
appear — viz., crithidia, leptomonas, or leishmania forms. Thus, during
the early stages of infection of the rat with T. lewisi, a great variety of
forms may be found in the blood and organs, as also in its other host,
the flea (Fig. 197). During the development of T. gambiense and other
trypanosomes in tsetse flies, crithidia and other forms are found (Fig. 223).
T. cruzi, though appearing in the blood of the vertebrate as small flagel-
lates of the trypanosome type, reproduces intracellularly in the organs
as leishmania forms, and presents a still greater v^ariation in structure in
its invertebrate host, the reduviid bug, Triatotna tnegista (Figs. 206, 207,
209). It will thus be apparent that, before accurate knowledge of the
morphology of any trypanosome can be claimed, it is necessary to study
every stage of its development, both in the vertebrate and invertebrate
hosts.
The cytoplasm of a trypanosome is usually clear and homogeneous or
finely alveolar, but there is frequently present a vacuole near the kineto-
plast. Sometimes the cytoplasm contains granules which are greenish
and refractile in life, and stain deeply purple with Romanowsky stains.
They are most frequently seen in the anterior region of the body, and
probably consist of volutin. According to Doflein (1916), the cytoplasm
of cultural forms of T. rotatorium of frogs may contain droplets of a fatty
substance. In some of the larger trypanosomes longitudinal markings
of the surface of the body have been described, and these are generally
regarded as contractile fibres or myonemes lying in the outer layer of
the cytoplasm (Fig. 28, B). There is no definite ectoplasm layer as
distinct from an endoplasm, but the surface of the body is limited by fine
membrane or periplast representing a concentration of the superficial
cytoplasm. The undulating membrane may be regarded as a lateral
extension of this limiting layer of denser cytoplasm.
The nucleus is typically spherical, and consists of a nuclear mem-
brane enclosing a clear material at the centre of which lies a karyosome
(Fig. 156). It is situated usually at the centre of the flagellate. In some
cases, as in the posterior nuclear forms of T. brucei, it may lie near the
posterior end of the body and sometimes actually behind the kinetoplast
(Fig. 224). In others, as for instance, in T. lewisi, it has moved in the
reverse direction, and is typically found anterior to the central point of
the body (Fig. 197, 19).
The kinetoplast, consisting of the blepharoplast and parabasal, as ex-
plained above (p. 329), lies at a short distance from the posterior end of
the body. It may actually be situated at the extreme posterior end, as in
448
FAMILY: TRYPANOSOMID^E
T. congolense. The parabasal part of the kinetoplast varies considerably.
It is a comparatively large body, often slightly elongated, or egg-shaped in
T. cruzi. It is smaller and spherical in most pathogenic forms, while in
T. equinutyi of the South American disease of horses (mal de Caderas), it
is apparently absent (Plate V., i, p. 456).
In a single blood-film from an infected animal it will be found that the
trypanosomes are not all of the same size, and in the case of some patho-
genic forms {T. gambiense, T. brucei) it has been the custom to describe
those present as belonging either to the " short stumpy," " intermediate,"
or " long thin " forms (Figs. 221, 224). The measurement of the try-
panosomes and the relative positions of the various structures may be
roughly made by the micrometer eye-piece, but the flagellates are often
13
lA
15
le
17
18
19
20
21
22
Microns
23 24 25
26
27
28
29
30
31
32
33
34
35
16
14
r
^
12
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's^
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/
\
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y
\
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.\
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y
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V
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r-
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T.evansi
T. brucei
Fig. 196. — Curves representing the Distribution, by Percentages, in
Respect to Length of Trypanosoma evansi and Trypanosoma brucei. (After
Bruce, 1911.)
so twisted in the stained film that this is a matter of difficulty. A more
accurate method is to draw the trypanosomes to a fairly large scale with
the aid of a camera lucida, and to project on to the paper the micrometer
scale through the same system of lenses. The trypanosomes may then
be measured by setting the measuring dividers according to the scale on
the paper. A large number of individuals can thus be measured, and the
average dimensions taken, while a curve can be plotted showing the
percentage of trypanosomes of any length between the maximum and
minimum (Fig. 196). A feature of these curves is that very frequently
they show a notch which has been interpreted as an indication that in such
infections two particular types of trypanosomes are present — not neces-
sarily two species, but that the one species tends to produce in its develop-
GENUS: TRYPANOSOMA 449
ment two main groups of organisms. Some have suggested that it
indicates a tendency towards the production of male and female indi-
viduals, but, at present, there is no evidence to support this view.
3. PATHOGENICITY. — Trypanosomes are often grouped as patho-
genic and non-pathogenic forms. The former are those which give rise
to disease in man or domestic animals, and they do so, not because these
are their natural hosts, but because the man or animal is susceptible to
inoculation with a trypanosome, which in its natural host is non-patho-
genic. As a general statement, it is safe to regard all trypanosomes as
non-pathogenic to their natural hosts. The vast majority resemble
T. lewisi of the rat, which, under ordinary circumstances, cannot infect
any other host than the rat. A small number of trypanosomes are,
however, inoculable into man, the domestic animals, and experimental
laboratory animals, and in these unnatural hosts they often produce
serious symptoms of disease. To their natural hosts, which in many
cases are the big game of Africa, they are apparently harmless. The
various pathogenic trypanosomes, however, vary in the effect they
produce on laboratory animals, and these variations are of some assistance
in the identification of the species. T. brucei, for instance, is readily
inoculated into the rat, mouse, and guinea-pig, whereas, with T. pecorum,
these animals cannot be infected. The human strain of T. brucei
{T. rJiodesiense) inoculated from man into the rat rapidly produces a
very heavy infection which quickly kills the animal, whereas T. gambiense
under these circumstances may fail to infect the rat, though it usually
does so, leading to a chronic type of infection characterized by the
presence of a small number of trypanosomes in the blood at any time,
the animals surviving for even a year or more. By passage from rat
to rat the virulence of such a strain may be increased, till finally the
infection may become as intense and as rapidly fatal as that produced
by T. brucei.
The animals most frequently employed for these tests are rats, mice,
guinea-pigs, dogs, monkeys, goats, and even the larger domestic animals,
such as donkeys, horses, mules, and cattle.
It sometimes happens that in the inoculated animals there appear
particular forms of trypanosome which were not present in the original
host. Thus, in man, T. brucei (T. rhodesiense) closely resembles T. gam-
biense, but in the inoculated rat there appear a certain number of
posterior-nuclear forms which enable the trypanosomes to be distinguished
from T. gambiense (Plate V. a and b, p. 456).
In these experiments it is of importance to note the period of incuba-
tion before trypanosomes appear in the blood of the inoculated animals,
the intensity of the infection produced, and the duration of the infection.
I. " 29
450 FAMILY: TRYPANOSOMID^
In some animals, particularly the goat, only transitory infections are
produced, the animals quickly ridding themselves of their trypanosomes.
The incubation period and the subsequent course of the infection varies
with the dose or number of trypanosomes injected, and also with the type
of injection, the intraperitoneal route leading to more certain and rapid
infection than the subcutaneous. Naturally, any conditions which lower
the vitality of the experimental animals at the same time lower their
resistance, and lead to a more intense infection.
4. IMMUNITY. — As noted above, some animals, though acquiring
an infection from inoculation, recover after a lapse of time. This is
particularly true of the goat, sheep, and ox. Such recovered animals
are found to be resistant to reinoculation with the same trypanosome,
but are still susceptible to another. Laveran and Mesnil have employed
this method extensively in differentiating trypanosomes. Thus, a goat
rendered immune to T. brucei (T. rhodesiense), as proved by reinoculations,
was susceptible to T. gatnbiense. Though this method will undoubtedly
distinguish trypanosomes of distinct species, it is possible that mere races
of one and the same species may give similar results. Furthermore,
animals which have recovered from an infection with one species may
sometimes be reinfected with the same species. Thus, Noller (19136)
has shown that frogs may be infected twice or even three times with the
trypanosome, which occurs naturally in these animals (T. rotatorium).
Such superimposed infections demonstrate that in many cases the
immunity acquired against any particular trypanosome is very inconstant,
and that great caution has to be exercised in making deductions from such
cross-infection experiments. Martin and Darre (1912) gave an account
of a trypanosome which had been acquired accidentally by Lanfranchi
when working with T. evansi in the laboratory. The strain of trypano-
some recovered from his blood, and also that with which he supposed he
had infected himself, were investigated by Mesnil and Blanchard (1914).
Lanfranchi stated it was T. evansi, but Mesnil and Blanchard, by use of
immunity tests, concluded that the two strains were different. They
were, however, unable to identify the human strain with any known
trypanosome, and decided to refer to it as " Trypanosoma Lanfranchi.'''
Such a result is a direct indication of the unreliability of the immunity
test. In his work on T. melophagium of sheep referred to below, Hoare
(1923) has shown that so long as sheep are infested with keds, trypano-
somes are present in the blood. If the keds are removed, the infection
disappears in two or three months, only to reappear again when exposure
to keds again takes place. In this case it would seem that any batch of
trypanosomes introduced by a ked are able to multiply in the sheep and
survive for a limited period. If their disappearance is due to an acquired
GENUS: TRYPANOSOMA 451
immunity on the part of the sheep, this at any rate is insufficient to
prevent a fresh infection.
5. SEROLOGY.— Rabinowitsch and Kempner (1899) were the first
to demonstrate the protective property of the serum of animals recovered
from trypanosome infections. They showed that 0-5 c.c. of serum
from a rat recovered from an infection due to T. lewisi was sufficient to
protect a normal rat against infection when the serum and blood containing
trypanosomes were inoculated at the same time. Laveran and Mesnil
(1901a) extended this observation, and demonstrated that if sufficient
serum from an animal, such as the sheep or goat, rendered immune to any
particular trypanosome was mixed with infective blood from another
animal containing the same trypanosome, normal animals inoculated
with the mixture did not become infected, whereas the same serum mixed
with another trypanosome did not protect against infection with the
latter. This property of the serum for destroying trypanosomes and
preventing infection may be retained for long periods. The serum of a
chronic case of sleeping sickness in man has a similar action in the case of
T. gamhiense, whereas the serum of a normal individual is not protective.
Furthermore, Laveran (19026, 1903) was the first to demonstrate that
normal human serum had a marked protective action when inoculated to
mice at the same time as either T. brucei, T. evansi, or T. equinum. The
normal human serum was in some cases even curative when injected into
animals already infected.
Mesnil and Ringenbach (1911) showed that it had a similar action on
the human strain of T. brucei {T. rhodesiense). Laveran and Nattan-
Larrier (1912c) discovered that this reaction was far from constant, as
different human strains of this trypanosome behaved differently towards
human serum. Freshly isolated strains tended to be killed by human
serum, while this ceased to be the case after the strain had been subjected
to many passages through laboratory animals. Laveran (1915a) showed
that one particular strain of T. gamhiense, even after being kept for
twelve years in laboratory animals, still resisted normal human serum.
Mesnil and Blanchard (1916), however, proved that other strains of
T. gamhiense may lose this resistance after long periods. Conversely,
T. brucei may acquire a resistance not previously possessed by it after
many passages, as proved by Jacoby (1909). Laveran (1904a) also
demonstrated that the serum of the higher apes, especially the baboons
(Cynocephalus), behaved like human serum. Mice injected with serum
at the same time as T. brucei did not become infected, whereas, with
T. gamhiense, they were not protected.
The serum of animals recovered from infections may have a trypano-
lytic or disintegrating action on the tryj^anosomes in vitro, as first shown
452 FAMILY: TRYPANOSOMID^
by Franke (1905) in the case of the serum of cattle recovered from
T. brucei infections. Laveran and Mesnil (1900a, 1901a) first demonstrated
the agglutinating action of the serum of recovered rats on T. leivisi.
When the serum is allowed to act upon the tryj3anosomes in vitro, they
become arranged in clusters or rosettes with their flagellar ends directed
outwards (Fig. 152, A). Though the trypanosomes are attached to one
another by their posterior ends, there is no loss of activity, as evidenced
by the continued movement of the flagella. After some time the cluster
breaks up, and the individual trypanosomes swim away. The pheno-
menon is often termed " agglomeration " to distinguish it from bacterial
agglutination, which involves loss of vitality of the individual bacteria,
there being no tendency for the clumps to break up.
Laveran and Mesnil (1901a) also found that if T. lewisi were injected
into the peritoneal cavity of rats which had recovered from an infection,
the trypanosomes quickly became attached to leucocytes, while no such
attachment occurred in the case of rats not previously infected. This
observation was extended to other trypanosomes by Mesnil and Brimont
(1908, 1909), while Levaditi and Mutermilch (1910) studied it in detail.
The last observers found that the reaction depended on immune sub-
stances in the blood of recovered animals, for the addition of immune
serum from these recovered animals to a mixture of trypanosomes and
cells obtained from artificially produced peritoneal exudate caused the
trypanosomes to become attached to the cells. The serum still retained
this power even after heating. This reaction, though in certain cases
quite specific, is too inconstant to be relied upon as a means of differentiat-
ing trypanosomes.
Many experiments have been made to test the deviation or fixation
of complement in trypanosome infections with a view to diagnosis, but
the results so far obtained are very discordant. Sometimes, however,
definite and uniform results are obtained, as in the work of Watson (1915),
and Woods and Morris (1918) on infections of horses with T. equiperdmn.
Woods and Morris, working with dogs, found that, using as antigen a
salt solution of the spleen of a heavily infected animal, complement
fixation usually followed, but sometimes occurred before, the appear-
ance of trypanosomes in the blood of the dog. The reaction, however,
always appeared before clinical manifestations of disease. Therapeutic
injections of arsenobenzol into infected dogs not only caused the try-
panosomes and clinical symptoms to disappear, but so altered the
serum that the complement fixation test became negative, as in normal
dogs.
The complement fixation test has been employed on a large scale for
diagnostic purposes in the case of dourine of horses in Canada. The
GENUS: TRYPANOSOMA 453
technique devised by Watson (1915 and 1920) was used. As antigen,
trypanosomes from heavily infected rat's blood were employed. They
were separated by repeated centrifugation in saline solutions from the
blood-cells and sera.
The tests were carried out as in the Wassermann test for syphilis.
Between the years 1912 and 1919 it was applied to 40,000 horses in Canada,
with the result that infection was detected in many animals which clinically
were not suspected of suffering from dourine. By adopting the practice
of slaughtering all animals giving a positive reaction, the disease has not
only been prevented from spreading, but has been almost, if not entirely,
stamped out. Writing of these results, Watson (1920) points out that the
test is absolutely reliable from a diagnostic point of view. Clinically,
the incubation period of the disease may vary from two weeks to three
months, but from the results obtained by the serological test it became
apparent that the animals show signs of infection in from ten to twenty
days.
Schoening (1924), using the dourine antigen, applied the test to camels
to be imported into the United States, and was able to demonstrate that
a trypanosome infection was present. The organism in these animals
was probably T. evansi, so that the positive result obtained proved that
the test is not specific for any particular species of trypanosome. It is
evidently what is termed a group reaction.
Very interesting serological studies with cultures of frog trypanosomes
(T. rotatorium) have been made by Noller (1917). He employed the
flagellates grown on horse blood-agar plates, so that the cultural forms
could be removed with a minimal amount of admixture with the ingre-
dients of the culture medium. Horse serum produced sedimentation in
emulsions of the flagellates in a dilution of 1 in 20, and a macroscopic
agglutination in 1 in 40 to 1 in 80. The flagellates were all killed by the
undiluted serum in one hour, while in a dilution of 1 in 10 the majority
were killed in this time, and none were alive on the following day. With
horse serum inactivated by heating to 56° C, sedimentation alone was
obtained, and this only when the undiluted serum was used, whereas the
agglutination up to 1 in 80 occurred with the active serum. The inacti-
vated serum, moreover, had no trypanocidal action. Guinea-pig serum
produced sedimentation in dilutions of 1 in 10 to 1 in 20, but appeared
to have little agglutinating power. Its trypanocidal action, however,
was marked, but ceased at a dilution of about 1 in 160. As Noller remarks,
this result is directly the opposite of that obtained by Mendeleeff-
Goldberg (1913) in experiments conducted with cultures in the liquid of
N.N.N, medium. The serum of infected frogs (Rmia esculenta) gave sedi-
mentation in dilution of 1 in 80 and agglutination in 1 in 40. The undiluted
454 ' FAMILY: TRYPANOSOMID^E
serum killed most of the flagellates in a short time, a percentage of 1 or 2
surviving, whereas in dilutions of 1 in 10 only about 90 per cent, were
killed. With higher dilutions the number of surviving flagellates increased.
The inactivated serum (56° C.) had no trypanocidal action. The agglu-
tinin was also destroyed, a result which shows it to be thermolabile, and
thus different from the agglutinin which occurs in horse serum. Working
with serum of uninfected frogs, similar results were obtained. It thus
appears that the agglutinating action of the serum is no indication of
the power of the animal to resist infection, for not only were uninfected
frogs infected by injection of cultures, but superimposed fatal infections
were produced in already infected frogs. These results appear to be
analogous to those obtained by Mesnil and Blanchard (1916), who
proved that human sera had a marked trypanocidal action on both
T. gambiefise and T. hrucei [T. rJiodesiense), both of which may infect
human beings.
Though serological tests may serve to distinguish strains of trypano-
somes, it does not follow that the trypanosomes thus differentiated are
true species in the zoological sense. As in the case of the immunity test
referred to above, different races of one and the same species may show
differences in serological reaction. Ponselle (1923a) has produced a certain
degree of immunity in mice with a vaccine of T. hrucei. A solution con-
sisting of dihydrogen potassium phosphate (H2KPO4) 1'8 grams, hydrogen
disodium phosphate (HNa2P04,2H20) 0-2 grams, and distilled water
100 c.c. is prepared and sterilized at 115° C. for twenty minutes. To
2-5 c.c. of this solution is added with sterile precautions 0*5 c.c. of
heart blood of a mouse at the end stage of its infection with T. hrucei.
After twenty-four hours at 20° C. the mixture is inoculated intra-
peritoneally to mice in a dose of 0"1 c.c. It was found that in four
or five days the serum of the mice had acquired definite agglutinating
properties against T. hrucei, and that in many cases after eight to
ten days the mice were immune to inoculations with doses of trypano-
somes which produced the usual rapid and fatal infections in control
animals.
6. CULTURE. — The culture method, though it has been mostly
employed to determine infections which are not evident on microscopical
examination of the blood, as in the case of T. theileri of cattle and
T. melophagium of sheep, has also been used for purposes of identification.
Thus, Noller, working with cultures of Crithidia suhulata of Tahafius
glaucopis, claims to have proved that this is merely the insect phase of
Trypanosoma theileri on account of the exact similarity of the culture
forms of each. He also demonstrated the similarity between cultures
of the sheep trypanosome and the flagellate of the sheep ked {Melophagus
GENUS: TRYPANOSOMA 455
ovinus), and came to the conclusion that they were merely stages of one
organism {T. melo-phagium), a fact which has been conclusively demon-
strated by Hoare (1923).
The course of development of trypanosomes in cultures is of considerable
interest, for, undoubtedly, it is an imitation of the development which
takes place normally in the invertebrate host. Most observers have
noted that the trypanosomes introduced into culture media commenced
multiplying and became transformed into flagellates of the crithidia type.
Thomson, J.D. (1908), first noted in the case of cultures of the trypanosome
of gold fish that the first division process of the trypanosomes resulted
in the formation of crithidia forms (Fig. 247). Active multiplication of
these takes place for some time, but eventually trypanosome forms again
appear. There seems little reason to doubt that these represent the meta-
cyclic trypanosomes which appear at the end of the development in the
leech. Delanoe (1911) noted that in old cultures of T. lewisi small try-
panosomes appear, and it is evident from his figures that these correspond
with the metacyclic trypanosomes which are developed in the rectum of
fleas, and which are the actual infective forms. Hoare (1922, 1923),
working with cultures of T. melophagium, both from the blood of sheep
and from the intestine of the ked, has noted the same fact. Small trypano-
somes appear in the cultures after the crithidial phase, and these are
identical with the metacyclic trypanosomes which are developed naturally
in the hind-gut of the ked. It seems clear, therefore, that the course of
development of any trypanosome in culture is a parallel of the natural
development in the invertebrate. Noller (1920c), working with cultures
of T. loxicB and T. syrnii of birds, T. theileri of cattle, and T. melophagiimi
of sheep on blood-agar plates, states that at low temperatures the flagellates
remain in the crithidia form, but that elevation of the temperature to
37° C. causes their transformation into trypanosomes. It seems that the
only possible biological explanation of this phenomenon is that the
heightened temperature causes the flagellates to revert to the warm-
blooded vertebrate phase, which is a step in advance of the metacyclic
trypanosomes which appear in the cultures in liquid media at low tem-
peratures. It would seem reasonable to suppose that the transforma-
tion noted by Noller as occurring on agar plates after an elevation of
temperature is not comparable with the appearance of trypanosomes in
liquid media kept at a uniformly low temperature, but rather with the
changes undergone by the metacyclic trypanosomes after they gain
entrance to a vertebrate.
7. INSECT VECTOR. — Finally, the capacity to develop in invertebrate
hosts may be employed as a means of difEerentiating trypanosomes.
T. gambiense is capable of infecting Glossina palpalis, and only rarely
456 FAMILY: TRYPANOSOMIDiE
G. morsitans, whereas the reverse is the case with T. brucei {T. rJiodesiense),
though in the blood of man the two trypanosomes resemble one another
closely. The power to infect invertebrates is not always as specific as
this, for T. lewisi can undergo its development, not only in the rat fleas,
but also in the dog and human fleas. This method of diagnosis has been
named by Brumpt (1914a) xenodiagnosis. He found during his investiga-
tions into the development of trypanosomes of fish, frogs, and snakes
that leeches often developed infections after feeding on animals in which
no trypanosome had been found. Similarly, with T. cruzi various species
of reduviid bugs may acquire infections when the trypanosomes are too
scanty to be found by microscopical examination in the animals on which
they fed. Bruce et al. (1913a, 1914^), in Nyasaland, employed the te.st
in a reverse manner by feeding batches of tsetse flies on susceptible animals
in order to determine the nature of the infection of the flies, the develop-
mental forms of the trypanosomes in the flies being more difficult to
identify than those in the blood of a vertebrate.
CLASSIFICATION OF TRYPANOSOMES.
At the present time our knowledge of the life-histories of the majority
of described trypanosomes is so imperfect that it is impossible to classify
them accurately in any system. Attempts have been made to divide the
group into separate genera. For instance, Llihe (1906) proposed to
separate the mammalian trypanosomes from all others under the generic
name of Trypanozoon. More recently, Chalmers (1918) has attempted a
still more elaborate classification, with the establishment of a number of
genera which are quite indeterminate. Such attempts fail to assist in the
clear understanding of this already complicated group, and only lead to
greater confusion. On morphological grounds alone all the trypanosomes
undoubtedly belong to one genus, Trypanosoma. Where anything like
a complete history is known, they are found to be polymorphic, exhibiting
in their development every type between the leishmania and the try-
panosome form. As a rule, reproduction by fission of any of these forms
may occur. Variations in the shape and size of the body, the relative
positions of the kinetoplast and nucleus, the degree of development of
the undulating membrane and flagellum, cannot be regarded as generic
characters.
From the descriptions which will be given below it will be seen that
there are two main courses of development in the invertebrate. There is
the development which leads to infection of the biting parts of the inver-
tebrate, so that the vertebrate is inoculated during the biting act; and,
secondly, there is the development which leads to a hind-gut phase,
PLATE V.
'^- Various trypanosomes ol man and animals (x 2000). — A. T. gam-
biense. B. 7. brucei (T. rhodesiense). C. T. evansi. D. T. uniforme.
E. T.caprae. F. 7 . vivax. '^G. T. simiac. H. T. congolense. I. T. ^
equinum. J. T.eqmperdum. K. T.cruzi. L. T.lewisi. M. T.theikri.
(After various authors and originals.)
[To /ace /». 45^'
GENUS: TRYPANOSOMA 457
the vertebrate being infected by accidentally ingesting the faeces of the
invertebrate or the invertebrate itself. In the former case the trypano-
somes are described by Duke (1913) as having an anterior station in the
invertebrate. Employing this suggestion, it will be convenient to describe
the others as having a posterior station. In the case of T. gamhiense in
Glossina palpalis, infection of the salivary glands follows an intestinal
phase of development (Fig. 216). In other trypanosomes {T. congolense),
also transmitted by species of Glossina, an intestinal phase of development
leads to infection of the proboscis alone, the salivary glands not being
infected (Fig. 217), while in others (T. vivax) there is no intestinal develop-
ment, the whole cycle taking place in the proboscis of the tsetse fly (Fig.
218). These three variations of development in the tsetse flies may be
used as a basis for classifying some of the pathogenic trypanosomes, as has
been done by Duke (1913) and Bruce (1914). Many of the trypanosomes
of cold-blooded vertebrates, as, for instance, T. inopinatum of frogs, are
transmitted by leeches. In these invertebrates the trypanosomes develop
in the stomach, and finally in the proboscis sheath (anterior station),
whence they gain access to the wound inflicted by the leech when it feeds
(Fig. 243). All these methods of infection by tsetse flies and leeches
may be spoken of as inoculative, since the trypanosomes are inoculated at
the time of biting. The other method of infection is contaminative, for
infection takes place through infected faeces or the invertebrate itself'
being ingested or, possibly in some cases, by faecal contamination of the
wound inflicted by the invertebrate. Thus T. lewisi develops in the flea,
leading to infection of the rectum (Fig. 199). Rats acquire the infection
by eating the faeces of the fleas. If the complete life-histories of all the
trypanosomes were known, it might be possible to group them according
to such data as have been just outlined. In the case of several trypano-
somes of small rodents, it is now known that the invertebrate hosts are
fleas, and that the infection of the vertebrate is contaminative as in T.
lewisi, while the trypanosomes of fish are carried by leeches, as in the case
of T. inopinatmn of the frog. In a certain number of cases, however,
the trypanosome is known only in its invertebrate host, but the existence
of a vertebrate host is rendered highly probable from the fact that these
flagellates are easily inoculable into vertebrates and produce a definite
infection comparable with the infections produced by inoculation of
trypanosomes from vertebrate to vertebrate. Thus, Lafont (1912) dis-
covered a flagellate in the gut of Conorhinus rubrofasciatus. When
inoculated to mice it produced a typical trypanosome infection, and
for this reason he gave it the name Trypanosoma hoylei.
According to the scheme given on p. 346, the trypanosomes can be
grouped in the following manner:
458 FAMILY: TRYPANOSOMIDiE
Group A. — Trypanosomes which develop in the posterior station in
the invertebrate:
I. Trypanosomes of rodents, Cheiroptera, Insectivora, Edentata,
monkeys.
II. The trypanosome of man in South America, T. cruzi.
III. Non-pathogenic trypanosomes transmitted by species of Tabanus,
Melophagus, or other blood-sucking Arthropoda, including the
large forms from cattle, sheep, and antelopes.
Group B. — Trypanosomes which develop in the anterior station in the
invertebrate or have become secondarily adapted to direct passage from
vertebrate to vertebrate:
I. Pathogenic trypanosomes transmitted by blood-sucking Arthro-
poda.
II. Pathogenic trypanosomes secondarily adapted to direct passage
from vertebrate to vertebrate.
III. Trypanosomes of birds (?).
IV. Trypanosomes of land reptiles (?).
V. Trypanosomes of aquatic vertebrates transmitted by leeches:
1. Trypanosomes of aquatic reptiles.
2. Trypanosomes of amphibia.
3. Trypanosomes of fish.
The pathogenic forms are those which produce disease in man and
domestic animals, but these cannot be regarded as the natural hosts.
In Africa, the pathogenic forms are naturally parasitic in the wild game,
where they are relatively non-pathogenic. They only become pathogenic
when inoculated into susceptible animals which have not developed a
relative immunity as a result of exposure for many generations. The
virulence of T. lewisi, which under natural conditions is quite harmless,
may be increased till it becomes definitely pathogenic, and T. inopinatum,
harmless for the African frogs, is pathogenic for those of France. The
pathogenic trypanosomes, however, form a convenient group, and are
transmitted in most cases by species of Glossina in Africa. There are
some pathogenic trypanosomes, however, in the transmission of which the
tsetse fly can play no part, as, for instance, T. evansi of surra and
T. equinum of mal de Caderas, which occur in countries where tsetse flies
are not found. In these cases other biting flies of the genus Tabanus
and its allies fulfil the role. As regards Trypanosotna equiperdum, its
affinities are undoubtedly with the trypanosomes of the pathogenic group.
It appears that its capacity of passing directly from vertebrate to verte-
brate has been secondarily acquired as a result of the situation of its
GENUS: TEYPANOSOMA 459
development in the vertebrate. It may be a form of T. evansi modified
by long passage from vertebrate to vertebrate without an arthropod
intermediary.
CURATIVE ACTION OF DRUGS AND SERA IN TRYPANOSOMIASIS.
The serum of certain normal animals when injected into rats or other
laboratory animals infected with pathogenic trypanosomes will sometimes
cause their temporary disappearance. For instance, a dose of 0-1 to
1 c.c. of human serum injected into a mouse infected with T. brucei
may cause the trypanosomes to disappear entirely from the blood. Some
strains of T. brucei resist such treatment, as also does the human strain
(T. rhodesiense). The serum from animals, such as the goat, which have
recovered from an infection is little more active than a normal serum,
so that at present there seems little possibility of a serum therapy in
trypanosomiasis being devised.
Much more definite results have been obtained with chemical agents.
Ehrlich and Shiga (1904) gave an account of the action of the organic dye
trypanrot on trypanosomes. They showed that a fair proportion of
experimentally infected animals could be permanently cured by its
means. A long series of investigations on allied organic compounds was
carried out by Nicolle and Mesnil (1906), and it was found that a definite
relationship existed between the structure of the molecule and the thera-
peutic action.
Thomas (1905) announced the fact that the organic arsenic compound
atoxyl had a specific action on trypanosomes, and was very much less
toxic than arsenious acid, which had previously been employed in the
treatment of sleeping sickness. The introduction of atoxyl led to a
series of investigations under the direction of Ehrlich, which resulted in
the elucidation of the chemical nature of atoxyl and the preparation of
other organic arsenic compounds, notably arsenophenylglycine, and
finally salvarsan. Many other allied drugs were produced, and it is
chiefly in one or other of these forms that arsenic is now employed in the
treatment of trypanosomiasis.
Antimony in the form of sodium or potassium antimony tartarate
(tartar emetic) has a marked action on trypanosomes, which disappear
rapidly from the blood of animals after intravenous injection. As they
disappear the trypanosomes show evident signs of degeneration, while
smears from the spleen show quantities of debris from the broken-down
organisms. Though the trypanosomes may disappear entirely from the
blood after a single injection, they almost invariably reappear after a
number of days. It seems apparent that it is rarely possible to give at a
460 FAMILY: TRYPANOSOMID^
single injection a dose sufficiently large to kill all the parasites and yet not
to kill the host. Accordingly, in treating trypanosomiasis it is necessary
to continue the treatment with small doses over long periods in the hope
of ultimately killing all the trypanosomes or assisting the body to do so.
There is a danger in prolonged treatment that drug-fast strains may
be created. It was noted that after treatment by a single dose of a drug
trypanosomes reappeared after varying intervals. Further treatment
caused them to disappear again. Eventually, after several relapses, the
drug frequently became incapable of causing the organisms to disappear
from the blood. This phenomenon was studied by Ehrlich and his co-
workers. It was discovered that there was a real resistance on the part
of the trypanosomes, for it persisted even when the trypanosomes had been
subjected to many passages through animals which had had no previous
injections of drugs. Strains resistant to various arsenic and antimony
compounds were obtained. It was further demonstrated that certain
arsenic-free substances, as, for instance, pyronine and acridine, were able
to produce strains resistant to atoxyl. In some cases arsenic resistant
strains could be made resistant to tartar emetic by injecting other arsenic
compounds. These facts are of great importance from the point of view
of treatment of trypanosome diseases. It may be that in this process a
kind of natural selection occurs, the more resistant survivors always
producing larger numbers of resistant forms after the susceptible ones
have been killed by the drug. Mesnil and Brimont (19086) have showni,
however, that a race which had become resistant to atoxyl, and had main-
tained this resistance when passed through mice, lost it when transferred
to the rat, only to regain it w^hen again passed into the mouse. It is
evident that the tissues of the host play a part in the therapeutic process.
A curious action of certain drugs, such as pyronine and others of the
oxazin series, on trypanosomes was noticed first by Werbitzki (1910). If
animals infected with T. hrucei are treated with these drugs, it will be
found that an increasing number of the trypanosomes lose the parabasal
body in the kinetoplast. In some cases the strain becomes normal again
after several passages through animals, but occasionally all the trypano-
somes present show this peculiarity, which persists through many passages.
The exact meaning of this alteration is not understood, but it is interesting
to note that in T. equinum of horses of South America the parabasal is
normally absent.
Voegtlin et al. (1920) have studied the action of various arsenic and anti-
mony compounds on trypanosome infections. They note that the trivalent
arsenic and antimony are markedly toxic for animals and also for trypano-
somes, which disappear very rapidly after intravenous injections. The
substances have a marked trypanocidal action in vitro. On the other
GENUS: TRYPANOSOMA 461
hand, the pentavalent arsenic and antimony compounds are much less
toxic, do not cause the trypanosomes to disappear at once, and have no
trypanocidal action. This indicates that if the arsenic or antimony
compounds are in the form of R.As = 0, symptoms of toxicity appear
at once and trypanosomes disappear rapidly. If they are in the form of
/OH
R.As = 0 a much longer time is required. It is concluded that during
\0H
the interval or latent period the pentavalent compounds are being reduced
in the body to trivalent ones. In the case of the arsenobenzol derivatives
(salvarsan, etc.), which act slowly and have no trypanocidal action in
vitro, it is believed that an oxidation to the trivalent forms takes place
{R.As=:As.R becomes R.As:=0). In the case of atoxyl, which again
shows a latent period before it acts, and which has no trypanocidal action
in vitro, the process seems to be one of reduction to the trivalent form.
Terry (1915) showed that if atoxyl and blood were incubated together, the
mixture acquired marked trypanocidal properties.
In animals such as rats and mice the various salvarsan and neo-
salvarsan compounds vary in their toxicity and in their therapeutic
efficiency. The toxic doJ^e for these animals varies from about 0-2 to
0-6 gram per kilogram of body weight, while a dose which is approx-
imately one-tenth of this will clear the blood of pathogenic trypano-
somes in about twenty-four hours. The toxic dose of tartar emetic is
about 0-04: gram per kilogram of body weight, and a dose of 0-02 gram
per kilogram will clear the blood of trypanosomes in about fifteen to thirty
minutes.
A drug (Bayer 205), which was first introduced in Germany by
Haendel and Joetten (1920), and Mayer and Zeiss (1920), appears to
have an action on trypanosomes which is more specific than that of any
drug hitherto employed. It is claimed that cures can be uniformly brought
about in small animals, and also in horses suffering from dourine. Further-
more, in small animals the single dose (0-003 gram per kilogram of body
weight) necessary to bring about a cure is only one-sixtieth of that which
can with safety be given to the animals. The ratio between the minimal
therapeutic dose and the maximum tolerated dose is thus 1 : 60. In
this respect, again, the drug is superior to any trypanocide which has
been used before. The writer (19216) tried the drug in the case of mice
infected with a very virulent strain of T. equvperdum, and was able to
confirm the statement of the German investigators.
Kleine and Fischer (1922) and Kleine (1924) find that the drug is
efficacious in the case of human trypanosomiasis, and also gives promising
therapeutic and prophylactic results in the disease of domestic animals.
462 FAMILY: TRYPANOSOMIDiE
Low and Manson-Bahr (1923) have also obtained apparent cures in a
large percentage of human cases treated by them. It appears that the
drug gives a fair promise of cure only in the cases which have no involve-
ment of the central nervous system. As regards the action of the drug
on trypanosomiasis of domestic animals, Kleine and Fischer (1923) find
that its action is less marked than in the case of human beings, while in
the animals T. brucei is more responsive than T. vivax or the closely allied
T. cajjroe. It appears that the more nearly the infected host resembles the
natural reservoir, the less active is the drug. Thus, T. brucei is more
readily eradicated from man than from cattle, for the latter are more
closely related to the buffalo, which is one of the natural reservoirs of this
trypanosome.
Another drug which has a marked trypanocidal action in the case of
experimentally infected laboratory animals is tryparsamide, the sodium
salt of N. phenylglycineamide-j9-arsonic acid. Its action in sleeping
sickness has been the subject of an investigation by Pearce (1921) in the
Belgian Congo. Van den Branden and Van Hoof (1923) have followed up
some of the cases treated by Pearce, and report that a cure can be effected
in 100 per cent, of early cases of human trypanosomiasis in the Belgian
Congo when the cerebro-spinal fluid is still normal, and that in a large
percentage of more advanced cases a similarly successful result can be
obtained.
In the treatment of human beings suffering from trypanosomiasis, the
drugs hitherto most usually employed are atoxyl or soamin and tartar
emetic. Injections of one or both of these must be continued over long
periods, and cure may be effected in a certain number of cases. It must be
remembered, however, that some cases tend towards a natural recovery,
and appear to respond very well to treatment, while others get progressively
worse in spite of the remedies used. On this account, great caution has
to be exercised in ascribing good results to any particular line of treatment,
while a cure cannot be said to have certainly taken place unless there
have been no signs of the disease for some years.
In the treatment of trypanosomiasis of domestic animals, the above-
mentioned compounds, as well as liquor arsenicalis, have been tried with
varying success. Tartar emetic administered intravenously seems to give
the best results. Hornby (1919), working in Rhodesia, noted that horses
and other equidse were more liable to infection with T. brucei than with
T. congolense and T. vivax, while the reverse was the case for cattle.
Hornby found that tartar emetic had little effect in saving horses infected
with T. brucei, but was of great value for cattle harbouring T. congolense
or T. vivax. He has informed the writer that as many as 80 per cent, of
the cattle may be saved by the use of this drug if treatment is commenced
TRYPANOSOMA LEWISI 463
early. His practice is to give with a syringe 1 gram of the drug intra-
venously every five days till six doses have been injected. Though the
animals may not be entirely cleared of infection, they are saved from
death, improve clinically, and get into good condition again. If relapse
or reinfection occurs, the treatment is repeated.
SYSTEMATIC DESCRIPTION OF SPECIES.
Group A. Trypanosomes which Develop in the Posterior Station
in the Invertebrate.
I. TRYPANOSOMES OF RODENTS, CHEIROPTERA, INSECTIVORA, EDENTATA,
CARNIVORA, AND MONKEYS.
(a) Trypanosomes of Rodents.
The best-known trypanosome of this group, Tryjmnosoma lewisioi the
rat, will be considered as a representative of the group.
Trypanosoma lewisi (Kent, 1880). — Synonyms: Herpetomonas lewisi Kent,
1880; Trinmnomonas lewisi (Labbe, 1881); Tryjmnosoma rattonim Borner, 1881;
Trichomonas lewisi (Crookshank, 1886); Trypanosoma sanguinis ^s.saxt'kak, Durham,
and Blandford, 1898; Tr?//»anomo«as mttri Mm Danilewski, 1889; Trypanozoon lewisi
(Liihe, 1906); Trypanosoma longocandense Lingard, 1906.
According to Laveran and Mesnil (1912), the first person to see this
trypanosome was Chaussat, who discovered it in the blood of Rattus rattus.
He mistook it for a nematode embryo, and it was Lewis in 1877 who
recognized as a flagellate the organism he saw in the blood of R. decumanm
and R. rufescens in India. In his manual on Infusoria, Kent (1880) re-
ferred it to the genus Herpetomonas, as did also Biitschli (1881). Laveran
and Mesnil (1901(^) showed that this flagellate did not differ in any essential
respect from the type of the genus Trypanosoma created by Gruby (1843)
for the parasite of the frog, and that therefore the parasite of the blood of
rats should be known as T. lewisi, which name it has retained, though several
observers have needlessly attemjited to create new genera for its reception.
Distribution. — T. lewisi is very common in R. rattus and R. decumanus
in all parts of the world where these rats occur. In India it is found
in R. rufescens and R. niveiventer, in Africa in R. maiirus, in Christmas
Island in R. macleari, in Tunis in R. alexandrinus . It has been recorded
from other small rodents, though in many cases it is probable that the
trypanosomes were not T. lewisi. A trypanosome of the S. African
gerbil {Tatera lobengula) is regarded by Fantham (1925) as a race of
T. lewisi.
Course of Infection in the Rat. — T. lewisi, which is readily inoculated
from rat to rat, can be conveniently studied in the white rat. The try-
464 FAMILY: TEYPAXOSOMID^
panosomes appear in the peripheral blood from four to six days after
intraperitoneal inoculation of infected blood from another rat, and the
resulting infection may be divided into two phases. In the first, a great
variety of forms occurs in the blood, most of which are in process of
division (Fig. 197). This is the multiplication phase, but it gradually
subsides, giving place to a phase in which the trypanosomes are much more
uniform in character, and are the forms generally recognized as T. lewisi.
The first phase is of short duration, and multiplying forms are rarely
seen in the peripheral blood after the eighth or ninth day, when the only
forms to be found are those of the second phase, which lasts from one to
four months. When inoculation has been made intraperitoneally — and
this is the readiest method of bringing about infection — it is stated by
Laveran and Mesnil (1912) that multiplication first commences in the
peritoneal cavity, and that these stages are much more numerous in the
peritoneal exudate than in the blood. Before their appearance in the
peripheral blood after intraperitoneal inoculation, it appears, from still
unpublished observations by A. C. Stevenson, that active multiplication
has been taking place in the small vessels of the internal organs, especially
the kidneys. He was unable to demonstrate the active multiplication in
the peritoneal cavity, though in the later stages of an infection trypano-
somes occurred in the exudate. AYithin two days of peritoneal inocula-
tion, multiplying forms can be demonstrated in sections of the organs.
In the ordinary course of events, T. lewisi does not seriously injure the
rat, which recovers from its infection and nearly always has an immunity
to reinfection. Miss M. Robertson, however, informs the writer that if
only a slight infection occurs after a first inoculation, the rats may be re-
infected. In some cases, rats are reported to have died as a result of
heavy infections.
Roudsky (1910-1911), by rapid passage from rat to rat of the whole
blood of an animal when the trypanosomes were at the multiplication
phase, was able to raise the virulence of T. lewisi till it became definitely
pathogenic to rats, and not only infected mice, which are seldom susceptible
to the ordinary strains, but sometimes killed them. Further, the infection
in mice was transmissible from mouse to mouse. This strain of heightened
virulence was also inoculable to rabbits, guinea-pigs, and other rodents,
which are rarely susceptible or entirely resistant to T. lewisi. It was
suggested by Reichenow (1917) that the numerous trypanosomes of mice
and other rodents, which morphologically resemble T. lewisi, and even
a trypanosome which he found in African apes, might actually be
T. lewisi. Yamasaki (1924) attempted without success to infect mice
and monkeys by means of fleas which had become infective after feeding
on rats.
TRYPANOSOMA LEWISI 465
Morphology.— The trypanosome form which is present in the blood
of the rat for the longest time is the one which occurs after the multipli-
cation phase (Fig. 197, 18-19, and Plate V., l, p. 456), and is generally
spoken of as T. lewisi, though this name applies to all stages of its develop-
ment in the rat and flea. The trypanosome as seen in the later stages
of an infection is a very characteristic organism. Very similar forms
occur in the blood of other small mammals, and they are often referred
to as being of the T. leivisi type. These forms (Plate V., l, p. 456) are
about 25 microns in length, and have a distinctly curved body which is
sharply pointed at its posterior end. There is a well- developed kineto-
plast situated at some distance from the pointed posterior extremity.
The nucleus is definitely anterior to the central point of the body. The
undulating membrane is not markedly convoluted, the axoneme along
its border running a fairly straight course. There is a well-developed
flagellum beyond the anterior extremity of the organism. The curved
body, the sharp posterior end, and the excentric position of the nucleus
give these forms of T. leivisi a very characteristic appearance. Apart
from the nucleus and kinetoplast, the cytoplasm of the trypanosome is
usually free from granules, but certain structures not always visible have
been described as of occasional occurrence (see p. 323).
During the multiplication phase of T. lewisi, which commences shortly
after inoculation, the trypanosomes which occur in the blood-vessels
exhibit an extreme degree of polymorphism. There are large broad
trypanosomes with prolonged and pointed posterior ends with their
kinetoplasts adjacent to the nucleus, very much smaller forms of the same
type, and small round forms provided with flagella. Types intermediate
between all these also occur. These variations are best comprehended
by reference to the figure (Fig. 197, 1-15).
In the living condition the typical trypanosomes are exceedingly active,
and dash about with great energy amongst the red blood-corpuscles
with flagellar end in front, quickly passing from one microscopic field to
another. The large multiplication forms and others seen in the early
stages of an infection are much less motile.
Multiplication. — As already noted, the multiplication phase is of short
duration, and is characterized by the marked polymorphism of the try-
panosomes. It may be said to commence with the large broad trypano-
somes, which measure at least 35 microns in length and have the kineto-
plast near the nucleus (Fig. 197, 1-3). Division of the kinetoplast takes
place, followed by that of the nucleus. From the daughter kinetoplast
is formed a new axoneme, which does not grow to the length of the original
one, so that a short undulating membrane is formed. The cytoplasm then
divides between the flagella, and a small daughter individual is separated.
I. 30
466
FAMILY: TRYPANOSOMIDyE
21
%%
25
Fig. 197. — Trypanosoma lewlsi (x 2,000). (Original.)
1-15. Forms which occur in the blood of the rat during the reproducing pha
16-19. Forms which occur in the blood in the later stages of an infection.
20-23. Metacyclic trypanosomes which occur in the faeces of infective fleas.
TRYPANOSOMA LEWISI 467
Before it has become completely detached, division may again commence
in the parent form, and the process may be repeated several times, so that
a large individual, now, however, much reduced in breadth, with several
small ones not completely separated, may occur (Fig. 197, 4-9). These
small forms detach themselves, and may in turn divide more or less
equally (Fig. 197, lo-ii). On the other hand, the small forms may
become round, and, while increasing in size, the kinetoplast divides
repeatedly, together with the nucleus, the division of the latter being
always a little behind that of the former, while new axonemes grow out
from the newly-formed kinetoplasts. Cytoplasmic bodies are in this way
produced which have 2, 4, 8, or 16 nuclei and kinetoplasts, and a corre-
sponding number of axonemes and flagella (Fig. 197, 6-7). The nuclei
are peripherally arranged, and the body becomes indented between the
nuclei, and finally segmented into a number of organisms, which resemble
the round parent form from which they were derived. Eventually, these
small individuals elongate and become transformed into the trypanosome
forms. The multiplication forms gradually disappear from the blood, and
are replaced by the typical trypanosomes, which appear no longer to
multiply. During the multiplication phase the various forms met with
are referable to the types described, but all intermediate stages between
these are met with, and a blood-film made at this period shows a wonderful
series of organisms belonging to the various leishmania, leptomonas,
crithidia, and trypanosome types described above. The origin of the
large trypanosomes which commence the reproductive phase is doubtful.
It is probable that they are the result of growth of the inoculated forms,
which are those which occur in the late phase of an infection.
Reaction to Sera. — Laveran and Mesnil (1901a) first demonstrated
that the serum of rats which had acquired immunity to T. lewisi after
recovery from an infection had a marked agglutinating effect on the
trypanosomes if blood containing them were mixed with the immune
serum. In a few minutes the trypanosomes attached themselves to one
another by their posterior ends, producing finally clumps of organisms
(Fig. 152). The trypanosomes in these clumps are quite active, and the
condition of agglutination may pass off, the individual trypanosomes
swimming away. In other cases, if the agglutination persists, the trypano-
somes eventually cease their movements and degenerate. There is
evidence which indicates that two distinct substances are involved — an
agglutinin and a trypanolysin. In some instances an auto-agglutination
has been observed in the blood of infected animals. According to Talia-
ferro (1923, 1924), the serum of rats in the late stages of an infection
contains a substance which inhibits the development of the trypanosomes.
If 2 c.c. of such a serum is mixed with washed trypanosomes and injected
468
FAMILY: TRYPANOSOMIDiE
intravenously into healthy rats, no multiplication of the trypanosomes
occurs, and no infection results, whereas, if the same experiment is con-
ducted with the serum of a normal rat, the trypanosomes multiply and
infection results in the usual manner. Coventry (1925) could not detect
this substance in the blood of rats before the fifth day of an infection,
though it is undoubtedly present, as reproduction is declining before this.
There is a rapid increase in the quantity present in the blood between the
fifth and sixth days, and a more gradual one up to the thirty-fifth day,
after which it decreases up to the time when the infection ends.
Culture. — T. leivisi is readily cultivated in blood-agar media, and
can be maintained for indefinite periods by subculture. In these cultures,
the trypanosome forms disappear till every type of organism between
leishmania and crithidia forms
are met with, and large clus-
ters of flagellates of all kinds
are formed in which the organ-
isms are arranged with their
flagella directed inwards to-
wards the centre of the mass.
Delanoe (1911) showed that
in old cultures trypanosome
forms tend to reappear. They
differ in structure from those
originally introduced, and bear
a striking resemblance to the
infective metacyclic trypano-
somes which are produced in
the rectum of fleas. This
observation lends support to the view that the type of development
which occurs in the culture tube is an imitation of the invertebrate cycle
of the trypanosome.
The cultural form of T. lewisi will infect rats, though after long
maintenance by subculture its power of doing so becomes diminished.
Pathology. — In its normal condition T. lewisi is not pathogenic to
rats, which naturally recover from their infections. In accordance with
this, practically no change is produced in the organs. In the strains of
heightened virulence studied by Roudsky (1910-1911) degenerative
changes with enlargement of the organ and lymphoid infiltrations occur
in the liver and spleen.
Transmission." — That T. leivisi was transmissible from rat to rat by
fleas was first proved by Eabinowitsch and Kempner (1899), who infected
Fig. 198. — Ceratophyllns fasciatus, the Trans-
mitter OF Trypanosoma lewisi ( x 20).
(Original.)
TRYPANOSOMA LEWISI 461)
rats by transferring to them fleas {Ceratojjhylhis fasciatus) taken from
infected animals (Fig. 198). Swingle (1911) also conveyed infection by
means of fleas (C lucifer and Pulex hrasiliensis), but the exact mechanism
of infection was first definitely established by the work of Noller (1912(7),
the writer (19136), and Minchin and Thomson (1915), though Swellen-
grebel and Strickland (1910) had previously proved that infection was not
conveyed by the flea in the act of biting, and had described the course of
development in the flea which terminated in the production of the small
metacyclic trypanosomes in the rectum. It is now known that infection
takes place by uninfected rats eating the dejecta of fleas, or the fleas
themselves, which have previously fed on infected rats (Fig. 199). Fleas
do not become infective till after the lapse of about six days from the time
of their feed on infected blood, during which interval a definite cycle of
development takes place in the intestine. Yamasaki (1924) claims that
the dog flea is able to infect by its bite as a result of regurgitation of
trypanosomes which occur in the stomach and proventriculus, and that
this method is as effective as the fsecal method of transmission.
Minchin and Thomson's method of experiment was to introduce clean
rats into a cage of fleas which had previously had an opportunity of feeding
on an infected rat. After remaining in the cage for about three days the
rats were removed and exposed to chloroform vapour for a short time to
immobilize the fleas upon them. The fleas were removed from the rats
and returned to the cage. The course of the infection in the rats was then
studied. The development in the fleas was traced by exposing clean
fleas to infection from an infected rat, and examining them after various
intervals.
Cycle in the Flea. — As already remarked, the main outlines of the
developmental cycle in the flea culminating in the production of meta-
cyclic trypanosomes in the rectum was first described by Swellengrebel
and Strickland (1910), and Swingle (1911), while the mechanism of infection
was established by Noller (1912(7) and the writer (19136). Minchin and
Thomson (1911) discovered the intracellular stage in the stomach of the
flea, an observation confirmed by Noller (1912(7). Minchin and Thomson
(1915) published a detailed account of the complete developmental cycle
in the flea, and the experiments which led them to accept the view that
infection of the rat was brought about by its ingesting the excreta of the
flea. Further experiments on the mechanism of transmission with the
dog flea, in which he claims that infection may be brought about by the
bite, have been conducted by Yamasaki (1924).
The trypanosomes, which are of the type seen in the late phase of an
infection in the rat, are taken into the stomach of the flea, where during
the first six hours they undergo a change, which, however, is chiefly a
470
FAMILY: TRYPANOSOMID^E
physiological one, in that they cease to bring about infection if injected
into rats (Fig. 200, 1-4). They appear to become more rigid in character,
and possibly more violent in their movements. At about the end of this
period invasion of the lining cells of the stomach takes place (Fig. 20,
4-12). Actual penetration was observed by Noller (1912^), who saw a
-^'c^ ^^f^
^p^y?
Fig.
199. — Diagram of Trypanosoma lewisi in the Blood of the Eat and
IN THE Flea. (After Wen yon, 1922.)
A. Trypanosomes as seen in the rat at late iDhase of infection (X 1,500).
S. Trypanosomes in stomach of flea.
B. Intracellular phase of development in stomach (X 1,500). R. Rectal phase.
C. Attached flagellates in rectum : evolution of crithidia into metacyclic trvpanosome
form ( X 1,500).
D. Free metacyclic trypanosomes which bring about infection Mhen ingested bv rat
(X 1,500).
trypanosome enter the cell by its posterior end. Within the cell a vacuole
forms, in which the trypanosome may be seen to exhibit active movements.
It becomes doubled on itself, the two limbs of the U thus formed merging
into one another to form a pear-shaped body. The volume of this pear-
shaped body appears to be less than that of the trypanosome that entered
TRYPANOSOMA LEWISI 471
the cell, so that a reduction in size seems to have taken place. The pear-
shaped body now grows in size, while the kinetoplast and nucleus multij)ly
by repeated divisions. New fiagella are formed from axonemes which
develop from the daughter kinetoplasts, while the original flagellum still
persists with its axoneme attached to one of the kinetoplasts. The bodies
produced were described by Minchin and Thompson as " spheres." They
may be spherical, with the fiagella arranged irregularly about the surface
of the " sphere," or the flagellar end of the original parasite may still
survive, while the new fiagella are arranged parallel to it, forming a tuft
of bunched fiagella. In the living condition the " spheres " are in constant
motion. The number of nuclei and kinetoplasts produced is generally
eight to ten, but there may be as many as fourteen. The diameter of
the fully-developed "sphere" is 8 to 10 microns, and it finally divides
into a number of trypanosomes which bear a striking resemblance to the
original forms taken up from the rat's blood. The invaded cell is often
reduced to a mere membrane enclosing the activ'ely moving trypanosomes.
It is suggested that the periplast of the original trypanosome contributes
to the formation of this membrane. By rupture of the cell the trypano-
somes escape into the stomach of the flea (Fig. 200, 11-12). Sometimes
several " spheres " are developed in a single cell. The intracellular phase
of development occurs in all parts of the stomach, and, commencing about
six hours after the feed, it may cease as early as eighteen hours or persist
as long as four or five days. The trypanosomes which escape by rupture
of the cell may again enter other cells and repeat the process, but how
many times this may occur is not known. It is probably very variable.
The next stage is the migration backwards of the trypanosomes to the
hind-gut and rectum (Fig. 200, 12). These forms, which have pointed
posterior ends and the kinetoplasts near but still posterior to the nuclei,
are evidently approaching the crithidia form. Minchin and Thompson
distinguish them as crithidiomorphic forms. Change in structure, which
may have commenced before the trypanosomes actually leave the stomach,
now takes place. This consists in a loss of activity, shortening of the body
with rounding of the posterior end, diminution in length of the flagellum, •
and transposition of the nucleus and kinetoplast to give the true crithidia
structure. Multiplication by fission of these crithidia forms takes place, and
there then ensues the established rectal phase, in which a great variety of
forms occurs (Fig. 200, 13-19). There are the typical short attached
(haptomonad) forms, the free-swimming (nectomonad) crithidia forms, and
finally the trypanosome forms. The small attached or haptomonad forms
are derived from the trypanosomes which migrated from the stomach,
and they give rise to the small infective trypanosomes (Figs. 197, 20-23,
and 200, 19, T). The attached forms multiply, as do also the free-swimming
472
FAMILY: TRYPANOSOMIDiE
Fig. 200. — Diagram of Life-Cycle of Trypanosoma lewisi in the Flea ( x 2,000).
(After Minchin and Thomson, 1915.)
1. Trypanosome from rat's blood.
2. Slightly modified trypanosome after few hours in flea's stomach.
3-12. Stages in intracellular multiplication.
13-18. Two ways in which established rectal phase may arise from the stomach trypanosomes.
19. Established rectal phase, showing haptomonads (/t),nectomonads (?i), transitional crithidia
types (t.r.). and metacyclic trypanosomes (T).
20. Secondary infection of the pyl(3ric region of the hind-gut, showing forms similar to those
which occur in the rectum.
TRYPANOSOMA LEWISI 473
crithidia forms, till the whole of the rectum may be covered with organisms.
All these forms appear in the faeces of the fleas, but it is probable that
it is only the small trypanosomes which bring about infection. Though
the main development and attachment takes place in the rectum, this
may also occur, but to a smaller extent, at the anterior end of the hind-
gut near the pyloric opening.
Though Swellengrebel and Strickland (1910) had established the fact
that rats could not be infected by the bites of the flea, the exact mechanism
of infection was not understood till Noller {I9l2d) published the results
of his experiments. This observer employed the convenient method of
handling individual fleas by tethering them on fine wire, a jirocedure
adopted by showmen. By its use the movements of a flea can be com-
pletely controlled. Noller's results were confirmed by the writer (19136),
using the wire method, and later by Minchin and Thompson (1915) with
untethered fleas. Noller found that the fleas repeatedly passed faeces or
blood during the act of feeding, and that this could be collected and
examined. About six days after the flea had fed on an infected rat, the
small infective trypanosomes, as well as other forms, appeared in its
faeces. Fleas in this condition were allowed to feed on uninfected rats,
care being taken to prevent the voided faeces contaminating the skin.
The faeces ejected were received on a cover-glass held behind the flea while
feeding, and were transferred at once to the mouth of another rat. This
experiment, repeated many times, always resulted in infection of the
second rat and never the one bitten. Observing rats on which free fleas
were placed, it was noted that the latter had the habit of congregating
about the root of the tail, where they would feed when the rat was asleep.
Aroused by their bites, the rat turns its head to allay the irritation or
dislodge the fleas, which, startled by its movements, eject their faeces and
escape into the fur. The freshly-passed faeces are then easily licked up
by the rat. In this manner, by fleas passing from infected to uninfected
rats, T. lewisi is transmitted in nature. Minchin and Thompson, working
with Ceratojphyllus fasciatus in the free condition on rats, noted that only
a small percentage actually became infected. In the case of the fleas
used by the writer, all became infected after feeding on an infected rat.
Yamasaki (1924) obtained similar results.
This mode of transmission was demonstrated by Noller (1912(^) in the
case of the dog flea, Ctenocephalus canis, and by the writer (19136) for this
flea, as well as the human flea, Pulex irritans, and the Indian plague flea,
Xenopsylla chcBopis. Minchin and Thompson (1915) proved it for the
European rat flea, Ceratophyllus fasciatus, so that it is clear that many
species of flea may act as vectors of T. leivisi. Furthermore, the complete
development may take place in fleas which in nature rarely, if ever, have
474 FAMILY: TRYPANOSOMID.E
an opportunity of feeding on rats. For instance, Brunipt (1913) showed
that the swallow flea, C. hinulinis, might serve as a host for T. Jewisi,
and that the faeces of the fleas were infective to rats in the usual manner,
while Noller {\^12d) showed that the development could take place in
Ctenopsylla musculi.
It is highly probable tliat in nature infection may take place by rats
actually devouring the infected fleas themselves.
From the above description it will be seen that the development in
the flea consists of an intracellular multiplication phase in the stomach,
followed by the transformation of the trypanosomes into crithidia forms
and their migration to tlie rectum, where the attached phase results.
Eventually, after the expiry of six days from the time of feeding, small
metacyclic trypanosomes are voided in the faeces and ingested by the
rats. It is important to note that at no stage was a sexual process
encountered. Yamasaki (1924), who claims that the dog flea can transmit
the trypanosome by its proboscis, also states that the intracellular stage
is not essential to complete development in the flea.
Possible Transmission by Other Arthropods. — In addition to the experi-
mental work with fleas, a good deal of attention has been paid to
the rat louse, Hcematopinus spinulosus. Prowazek (1905) described a
developmental process, including syngamy, in the louse, leading to infec-
tion, not only of the gut, but also the body cavity fluid. The trypano-
somes were supposed to be inoculated to the rat by the bite of the louse.
Subsequent observation has not confirmed the developmental cycle,
though it has been definitely shown by McNeal (1904), Nuttall (1909), and
Baldrey (1909) that infection can be conveyed to rats by transferring lice
from infected animals. Noller (1914) studied the question of louse
transmission, and was unable to find any evidence in favour of Prowazek's
sexual phase, nor of the invasion of the body cavity fluid or biting organs.
According to him, T. lewisi undergoes changes in the intestine of the
louse, which are comparable to the culture of the trypanosome in artificial
media. No established infection is produced in them as in the case of the
flea, which, once infected, remains so for the rest of its life owing to con-
tinued m\dtiplication of the attached forms in the rectum. The faeces of
lice which have ingested infected blood will produce infection if eaten by
the rat, as also will the louse itself, and Noller thinks that in nature the
louse may convey the trypanosome in a mechanical manner by being
devoured while it still has trypanosomes within it, though as a vector it is
of little importance compared with fleas. Several observers have shown
that rats can be infected by feeding them with the blood or organs of
infected rats.
It has been shown by the writer (19r2c) and otliers that T. lewisi will
TRYPANOSOMES OF RODENTS 475
uiulergo clianges comparable to those seen in artificial culture in blood
media in the stomach of bed bugs and other arthropods, where a compara-
tively large quantity of blood is taken in and only slowly digested. This
condition must not be mistaken for true infection. It has been a constant
source of errors in experimental work with flagellates and biting arthropods.
In many cases it may be difficult to decide between cultural developments
and true infections, but in the latter the parasites tend to persist for long
periods in spite of constant feeding, whereas, in the former, a second feed
of blood often causes the flagellates to vanish. The experiments of
Patton, La Frenais, and Rao (1921), referred to on p. 355, are of interest
in this connection.
(a) Other Trypanosomes of Rodents,
The trypanosomes of rodents include the majority of forms known
to occur in small mammals. The best known is T. lewisi of tlie rat,
which has been dealt with above in some detail, and all the trypanosomes
of this group resemble it closely. Species have, however, been created on
slight differences in size, the failure of rats to become infected after
inoculation, and the immunity of the hosts to infection with T. lewisi.
In the few cases where it has been possible to study the complete develop-
ment in the vertebrate, the resemblance to T. lewisi is very marked. The
course of development in fleas in those cases which have been investigated
is also identical with that of T. lewisi. It is possible that most, if not all,
of these forms represent races of T. lewisi which have become adapted to
particular hosts. It is evidently impossible to place reliance on differential
characters which are based on slight morphological variations, especially
when it is remembered that in the case of T. lewisi what were merely
difi'erent stages of development of this trypanosome have been given
special specific names.
Trypanosoma duttoni Thiroux, 1900. — This is a trypanosome which
occurs in mice {Mus morio and M. tnusculus) in various parts of the world.
According to Laveran and Mesnil (1912), in dimensions and method of
multiplication in the mouse it closely resembles T. lewisi (Fig. 201, 9).
Though easily inoculable from mouse to mouse, rats and guinea-pigs are
not infected. Roudsky (1912), however, was able to increase its virulence
till it was inoculable to rats, just as he raised the virulence of T. lewisi,
as shown above, till mice became susceptible. Brumpt (1913) w^as able
to demonstrate that T. duttoni had a cycle of development in the swallow
flea, Ceratophyllus hirudinis, like that of T. lewisi. In the faeces of the
fleas were found the small infective trypanosomes, and seven mice which
were fed with the faeces became infected. The swallow flea can hardly
be the natural host of the mouse trypanosome, yet in this flea its
476 FAMILY: TRYPANOSOMIDiE
development is apparently completed. T . musculi Kendall, 1906, is prob-
ably the same trypanosome.
T. avicularis Wenyon, 1909, from the . zebra mouse {ArvicantJnis
zebrce), is of the T. lewisi type (Fig. 20 1, n). It was discovered in the
Sudan.
T. acomys Wenyon, 1909, of the spiny mouse {Acomys sp.), was de-
scribed by the writer in the Sudan (Fig. 201, 12-13). I^ resembles
T. diittoni, but is somewhat larger. The complete development was not
studied.
T. grosi Laveran and Pettit, 1909.- — This is a parasite of the field
mouse, Mus sylvaticus. It was probably first seen by Gros in Russia in
1845. It is of the T. lewisi type, but is not inoculable to other animals.
Laveran and Mesnil (1912) state that mice which had recovered from an
infection were found to be sometimes inoculable with Roudsky's virulent
strain of T. lewisi. The multiplication forms have not been seen.
T. microti Laveran and Pettit, 1909.- — The host of this trypanosome
is the field vole, Microtus arvalis. It is very active and of the T. Uwisi
type. The reproductive stages have not been described (Fig. 201, 10).
T. blanchardi Brumpt, 1905.^ — This trypanosome was discovered by
Brumpt in the dormouse, Myoxus nitela. Its dimensions and develop-
ment in the dormouse closely resemble those of T. lewisi in the rat.
Brumpt (1913) was able to transmit it by means of the flea, Ceratophyllus
laverani, the faeces of which contained infective trypanosomes. The
trypanosomes seen by Galli-Valerio (1903) in the blood of M. avellanarius,
and named by Blanchard T. myoxi, is possibly this species. T. eliomys
Fran9a, 1909, is certainly identical with T. blanchardi.
T. evotomys Hadwen, 1912. — This trypanosome was discovered by
Hadwen in the field mouse, Evototnys saturatus, in Canada. It resembles
T. lewisi, but developmental stages were not described.
T. peromysci Watson, 1912. — This is another trypanosome of the
T. lewisi type which occurs in the Canadian deer mice, Peromyscus mani-
culatus, P. nebracensis, and other species. The multiplication was not
studied.
T. rabinowit£chi Brumpt, 1906. — This form was discovered by AVittich
(1881) in the hamster, Cricetus frumentarius . It closely resembles T. lewisi,
1. T. vesper'Alionis of the bat (Pipistrellus pipistrellvs).
2. T. megadcrmce of the Sudan bat (Megaderina frons).
3-4. T. hcybergi of the Congo bat (Nycteris hispida).
5-0). T. ialpce of the mole (Talpa europcea).
7-8. T. nahiasi oi the rabbit. 9. T. duttoni of the mouse.
10. T. microti of the field vole (Microtus arvalis).
11. T. avicularis of the zebra mouse (Lemniscomys zebra).
12-13. T. acomys of the spiny mouse (Acomys sp.).
14-15. T. legeri of the sloth (Tamandua tridactyla).
TRYPANOSOMES OF RODENTS
477
Fig. 201. — Various Trypanosomes of Small Mammals (x 2,000). (1, Original;
2, 11, 12, 13, AFTER Wenyon, 1909; 3 and 4, after Eodhain, 1923; 5 and
G, AFTER Coles, 1914; 7 and 8, after Laveran and Mesnil, 1912; 19, after
TiiiROUx, 1905; 10, after Laveran and Pettit, 1909; 14 and 15, after
Mesnil and Brimont, 1910.)
[For description see op2)osile page .
478 FAMILY: TEYPANOSOMIDiE
but does not infect the rat. It is identical with T. criceti Liihe, 1906.
Noller (1912c) studied its development, and found it was morphologically
identical with T. lewisi, but not inoculable to rats, mice, or guinea-pigs.
Small trypanosomes, like the infective forms of T. lewisi, were found in
the rectum of fleas, Typhlopsylla assitnilis, Ceratophyllus fasciatus, and
Ctenocephalus canis, which presumably are able to transmit the infection.
T. nabiasi Railliet, 1895. — This trypanosome, which Blanchard
(1904) referred to as Trypanosoma cuniculi, occurs in rabbits, Lepus domes-
ticus and L. cuniculus (Fig. 201, 7-8). It was first seen by Jolyet and
Nabias (1891), and has been found by numerous observers in various
parts of Europe. It is of the T. lewisi type and is not inoculable to rats
and mice, but can be maintained in rabbits. The multiplication phase
has not been properly studied. Brumpt (1913) proved its development
in and transmission by the rabbit flea, Spilopsyllus cuniculi.
A trypanosome of the guinea-pig was described and figured by Kunstler
(1898). Judging from the figure, it would seem that the organism was not
a trypanosome at all.
Cazalbou (1913) claimed that he had discovered a large trypanosome in
rabbits in France. It was 80 microns in length, the free flagellum being
10 to 12 microns long. There was a w^ell-developed membrane. Though
only one trypanosome was seen in one of a series of rabbits which died,
the trypanosome was assumed to have been the cause of death. Cazalbou
suggested the name T. gigas for this trypanosome. There seems to be
considerable doubt as to the accuracy of this observation.
T. acouchii Brimont, 1909. — This is a trypanosome of the agouti
{Myoprocta acouchy) of French Guiana, and is of the T. lewisi type. Two
rats and two guinea-pigs were inoculated, with negative results. Multipli-
cation forms are not known.
T. indicum Liihe, 1906. — This form occurs in the Indian palm squirrel
(Sciurus pahnarum). It resembles T. lewisi, but is distinctly smaller.
Multiplication forms have not been seen.
T. spermophili Laveran, 1911. — This is a small trypanosome of the
T. lewisi type, and is found in Spermophilus musicus, S. guttatus, and
S. everstnanni of Russia and Siberia. The Canadian trypanosome
T. citelli Watson, 1912, occurring in the squirrel, Citellus richardsoni, is
possibly the same species.
T. otospermophili Wellman and Wherry, 1910. — This trypanosome
is very similar to T. spermophili^ and occurs in the Californian ground
squirrel, Otospermophilus beecheyi. Neither this nor the last-named species
has been fully studied.
T. bandicotti Lingard, 1904. — This trypanosome was discovered by
Lingard in 1893 and named by him (1904). It occurs in the bandicoot
TRYPANOSOMES OF CHEIROPTERA 479
{Nesokia gigantea) of India, and closely resembles T. lewisi, from whicli
it differs in that it is inoculable to guinea-pigs, in which it gives rise to
fatal infections. The naturally infected animals are always young, a fact
which suggests that an immunity is developed, as in the case of T. lewisi
in the rat.
T. akodoni Carini and Maciel, 1915, in the South American rat,
Akodon fuliginosus ; T. eburneensce Delanoe, 1915, of the West African rat,
Rattus couchar ; T. guist'hani Delanoe, 1915, of the Savannah rat, and
T. crocidurce Brumpt, 1923, of the shrew, Crocidura russulus, of France, are
all of the T. lewisi type, but in no case is the complete development known.
(b) Trypanosomes of Cheiroptera.
A trypanosome of the bat was first noted by Dionisi (1899a) in Italy
in Miniopterus schreibersii. Donovan (quoted by Laveran and Mesnil,
1904) found trypanosomes in the large Indian bat, Pteropus tnedius.
Battaglia (1904) gave the name T. vespertilionis to a trypanosome of
Vesperugo noctula, while Ed. and Et. Sergent (1905) described T. nicol-
leorum and T. vespertilionis from the North African bats, Myotis rnurinus
and Vespertilio kuhli. In the same year Petrie (1905) saw a trypanosome
in the English bat, Vesperugo pipistrellus. It was found later in the same
bat in other parts of Europe, while Bettencourt and Franga (1905) in
Portugal found it in three species of Vesperugo (F. pipistrellus, V. serotinus,
and V. nattereri), and named it T. dionisii. Cartaya (1910) described,
under the name of T. phyllostomcp, a trypanosome of the American bat,
Phyllostoma perspicillatuyn .
Laveran and Mesnil (1912) state that in their opinion all these various
forms belong to T. vespertilionis Battaglia, 1904, which has a striking
resemblance to T. cruzi (Fig. 201, i). Nicolle and Comte (19086), in
Tunis, found Vespertilio kuhli to be commonly infected with the large and
small trypanosomes described as separate species by Ed. and Et. Sergent
(1905). They expressed the opinion that they both belonged to the one
species, T. vespertilionis. Cultures on blood-agar medium were obtained,
and these were easily carried on by subculture. Nicolle and Comte
(1909) attempted to infect three young bats by means of the cultural
forms, but no infections resulted. Laveran and Mesnil (1912) state that
these observers succeeded in infecting one out of twenty bats inoculated.
The writer (1909) described a larger trypanosome from the Sudan bat,
Megaderma frons, under the name of T. inegaderynce (Fig. 201, 2). It has
a length of 40 microns, and is distinctly larger than the largest known
forms of T. vespertilionis, which varies in length from 14 to 24 microns
and in breadth 1 to 2 microns. Iturbe and Gonzalez (1916) described as
T. lineatus a trypanosome seen by them in the Venezuelan bat, Vampirops
480
FAMILY: TRYPANOSOMID.E
lineatus. It measured 19-5 microns in length, had a well-developed
membrane and a central nucleus. According to them, it resembled
*
1?^
Fig. 202. — Trypanosoma vespertilionis [=Schizo-
trypanum jyipistrelli Chatton and Courrier,
1921) OF THE Bat, Vesperugo plpistrellus.
(After Chatton and Courrier, 1921.)
1. Two cysts in the mucosa of the intestine ( x ca. 100).
2. Two cysts in the stroma of the ovary, one of which
(rt) contains crithidia forms, and the other (b)
trypanosomes (x ca. 500).
3. a and b, Trypanosomes from the blood; c, crithidia
form from the tissues ; d, cultural form of trypano-
some (x ca. 2,000).
T. brucei rather than T,
Leger and Baury (1923) de-
scribe as T. morinorum a
trypanosome of the bat [Hip-
posiderus tridens) of Senegal.
It is broader than T. vesper-
tilionis, and measures 30 by
7 to 8 microns. The part of
the body behind the kineto-
plast represents about half
the length of the body. The
kinetoplast is close to the
nucleus, which is centrally
placed. There is a free flagel-
lum of 7 to 15 microns in
length. A closely allied form
is T. heybergi, w^hich was dis-
covered by Rodhain (1923)
in the insectivorous bat,
Nycteris hispida, of the
Belgian Congo (Fig. 201, ;,-4)-
It is also a broad trypano-
some, but differs from
T. morinorum in some of its
dimensions.
As regards the various
trypanosomes mentioned
above, it is at present im-
possible to decide whether
those that have been given
specific names are good
species or not. In no case
has the complete develop-
ment been studied, and no-
thing is known of the range
of variation of the blood
forms of any one of them.
The work of Chatton and
Courrier (1921) shows that the life-history may be a very complicated one.
These observers have described, under the name Schizotrypaniim pipistrelli,
TRYPANOSOMES OF CHEIROPTERA 481
a trypanosome of Vesperugo pipistrellus of Alsace. As they admit, they
have little evidence to indicate that they were not dealing with T. vesper-
tilionis, except that in this instance they discovered a somewhat remarkable
developmental process which had not been previously observed (Fig. 202).
By cutting sections of various organs of infected bats they noted that the
trypanosome reproduces within cysts which may reach a diameter of 200
microns. In this respect it resembles T. cruzi, and is placed by them in
the genus Schizotrypaymm . As will be shown below, there is no actual re-
production by schizogony of T. cruzi, which multiplies by binary fission
like all other trypanosomes, so that there is no valid ground for placing it in
a separate genus. For the same reason the form described by Chatton and
Courrier will be included in the genus Trypanosoma. The cysts referred
to above were found in various situations — mucosa and submucosa of the
stomach and intestine, the gall bladder, kidney, bladder, spleen, ovary,
uterus, epididymis, and peritoneum. Within the cysts there occurred flagel-
lates of various forms, but in any individual cyst all the flagellates were of
the same type. The simplest forms seen were short stumpy crithidia forms.
It appears as if multiplication occurs within the cysts by repeated division
of these forms. When the cyst is mature the short forms increase in
length, and finally become the typical trypanosomes, which escape into the
blood by rupture of the cyst. It will be noted that in this trypanosome
the reproducing forms are of the short crithidia type, whereas in the cysts
of T. cruzi, to be described below, the multiplying forms are of the leish-
niania type. The trypanosomes which appear in the blood of the bat do
not differ from T. vespertilionis, as described by other observers, so that it
seems highly probable that Chatton and Courrier have observed the
reproductive process in T. vespertilionis for the first time. Coles (1914)
gave a description of T. vespertilionis of the English bat. He noted that
in the heart blood there occurred, beside the typical trypanosomes,
immature forms which from his microphotographs appear to have a
close resemblance to the stumpy crithidia forms seen by Chatton and
Courrier within the cysts. Very similar forms have been seen in smears
of the liver and lung by Franchini (1921).
As regards the method of transmission of the trypanosomes of bats
very little is known. Gonder (1910) discovered trypanosomes in the
stomach of mites (Liponyssus arcnatus) taken off bats. He believed that
the mite would be found to be thevector of T. vespertilionis. Nicolle and
Comte (1909), however, suspected the bug, Cimex pipistrelli, which is
frequently found on young bats in Tunis, and Pringault (1914) claims to
have transmitted the trypanosome to four out of five bats by the bite of
this bug. Bats were also infected by inoculating them with crushed bugs.
Sergent, Et. and Ed. (1921a), have noted the occurrence of flagellates of the
I. 31
482 FAMILY: TRYPANOSOMIDtE
leptomonas and leishmania types in this bug, and raise the question of
their being developmental forms of the bat trypanosome. What are
probably developmental stages of the trypanosome were seen by Franchini
(1921) in the mite, Leiognathus laverani. Rodhain (1923) found that mites
(Leiognathus) taken from infected bats harboured crithidia and trypano-
somes, so that it seems probable that this mite is the vector of the try-
panosome named T. heybergi by Rodhain.
Battaglia (1914) has claimed that T. vespertilionis is pathogenic to
rabbits. He makes a similar claim for T. lewisi. No other observer has
succeeded in confirming these statements, attempts at infecting laboratory
animals with the trypanosomes of bats having invariably failed.
(c) Trypanosomes of Insectivora.
Trypanosoma talpae Nabarro, 1907. — Petrie (1905) discovered a
trypanosome in the English mole, Talpa europcBci. The trypanosome
was again seen by Thomson, J. D. (1906), and by Franga (1911a) in
Portugal in T. europcea and T. cceca. Though resembling Try]m7iosoma
lewisi in some respects, it is not inoculable to rats (Fig. 201, 5-6). Nabarro
(1907) gave it the name T. talpce. Laveran and Franchini (19136) found
developmental forms of the trypanosome in the mole flea {Paloeopsylla
gracilis).
T. soricis Hadwen, 1912. — This is a trypanosome of the wandering
shrew {Sorex vagrans) in Canada. It is of the T. lewisi type, but reaches
a total length of only 17*5 microns.
T. brcdeni Rodhain, Pons, Vandenbranden and Bequa^rt, 1913. —
This form, again, is of the T. lewisi type, and occurs in Petrodromiis tetra-
dactylus of the Belgian Congo.
T. denysi Rodhain, Pons, Vandenbranden and Bequsert, 1913. —
This trypanosome, which is larger than the preceding one, was discovered
in Pteromys volans. It had a total length of 37 to 48 microns, of which
8 to 10 microns represented the flagellum.
T. xeri Leger and Baury, 1922. — This form occurs in the fossorial
squirrel {Xerus erythropus) of Senegal, and is very similar to T. denysi.
(d) Trypanosomes of Edentata.
A trypanosome, named T. legeri by Mesnil and Brimont (1910), was
discovered by Brimont in an ant-eater, Tamandua tridactyla, in French
Guiana (Fig. 201, 14-15). The body of the trypanosome is 30 to 35 microns
in length, and the flagellum 10 to 13 microns. In breadth it varies on
either side of 5 microns. The posterior extremity extends for about
14 to 16 microns beyond the kinetoplast. Besides these large forms
there occurred others which were smaller, and resembled Trypanosoma
TRYPANOSOMES OF INSECTIVORA, EDENTATA, ETC. 483
lewisi in shape and dimensions. The undulations of the membrane are
more marked than in the rat trypanosome. Mesnil and Brimont (1908a)
described a trypanosome in another edentate {Choloepus didactylus) in
the same locality which may be identical with T. legeri. It occurred in
the blood in association with Endotryjmnum schaudinni (p. 485).
(e) Trypanosomes of Carnivora.
Trypanosoma pestanai Bettencourt and Franga, 1906. — This trypano-
some occurs in the badger, Meles taxus, of Portugal. It has a breadth of
5 to 6 microns and a total length of 30 to 32 microns. The posterior ex-
tremity is prolonged beyond the kinetoplast for a considerable distance,
and there is a flagellum 4-3 microns in length. The membrane is well
developed.
A trypanosome was seen by Fehlandt (1911) in an otter in Tanganyika,
and one in a lion by Week (1914) in East Africa. In both these cases it is
supposed the trypanosomes were of the pathogenic forms of Africa.
(/) Trypanosomes of Monkeys.
Trypanosoma prowazeki Berenberg-Gossler, 1908. — This trypanosome
was discovered by Berenberg-Gossler in a monkey {Brachyurus calvus)
from the Amazon district (Fig. 203, i). It measured (flagellum included)
21 microns in length by 2 microns in breadth. The flagellum was 7 microns
long. Laveran and Mesnil (1912) regard it as allied to T. cruzi.
T. minasense Chagas, 1909. — This trypanosoma, first seen by Chagas,
appears to be a common parasite of marmosets, Hapale penicillata and
H.jacchus, of South America (Fig. 203, 2). The body of the trypanosome
measures 30 to 35 microns in length, and there is a free flagellum 8 to 10
microns long. The breadth is 4 to 6 microns.
T. vickersae Brumpt, 1909. — This form was discovered by Brumpt
(19196) in Macacus cynomolgus (Fig. 203, 4-5). Its length is 20 to 22
microns, of which the flagellum occupies about 8 microns. In general
structure and pathogenicity it resembles T. cruzi. It was inoculable to
M. cynomolgus and to other monkeys, M. rhesus and M. sinicus, as also
to rats, mice, guinea-pigs, dogs, and marmosets. The same trypanosome
appears to have been discovered in a M. rhesus at the Rockefeller Institute
by Terry (1911), who proposed to name it T. rhesi.
A very similar, if not identical, trypanosome which bears some resem-
blance to the established forms of T. lewisi was found in M. sinicus in
Algiers by Et. Sergent (1921). The trypanosome was not seen on direct
blood examination, but was obtained in culture in N.N.N, medium, in
which it grew very readily.
T. lesourdi Leger and Porry, 1918. — This trypanosome occurs in the
484
FAMILY: TRYPANOSOMIDiE
monkey, Ateles pentadactylus, of French Guiana, It is a small trypano-
some with a body 14 microns in length and a flagellum 5 microns long.
The kinetoplast is large and round, and situated 3 microns from the
posterior extremity. There is a well-developed membrane.
T. devei Leger and Porry, 1918. — This form was found in Midas midas
in French Guiana. It is a long, thin trypanosome, the body of which
measures 37 microns and the flagellum 7 microns. The breadth is 2 to
2-5 microns. The kinetoplast is some distance from the posterior end of the
body, and there is a well-developed membrane. It is of the T. leivisi type.
Brimont (1909) discovered a trypanosome in a howler monkey {Alouatta
senicula) captured in French Guiana (Fig. 203,3). Oiily a single trypano-
FiG. 203. — Trypanosomes of Monkeys (x 2,000). (1, after Berenberg,
GossLER, 1908; 2, after Carini, 1909; 3, after Brimont, 1912; 4 and 5,
AFTER LaVERAN AND MeSNIL, 1912.)
1. T. prowazeki of the Ouakasi monkey (Ouakasi calvus).
2. T. minasense of the marmoset (Hapale penicillata).
3. T. sp. of the howler monkey {Alonatta senicula).
4-5. T. vickerSfB (Maraca fascicularis= M . cynomolgus).
some was seen, and it had a length of 28 microns, of which the flagellum
occupied 9 to 10 microns.
In Africa, in endemic centres of sleeping sickness, trypanosomes have
been noted by several observers in monkeys. They have generally been
regarded as-T. gambiense. Ziemann (1902a) recorded a trypanosome in
a chimpanzee in the French Congo, Kudicke (1906) a large trypanosome
in Cercopithecus sp, in German East Africa, and Button, Todd, and
Tobey (1906) one from C. schmidti of the Belgian Congo, which measured
about 25 by 2-5 microns. Martin, Leboeuf, and Roubaud (1909) saw a
trypanosome in a lemur (Galago demidojffi) of the French Congo, while
Koch, Beck, and Kleine (1909) observed a trypanosome in a captured
monkey, and regarded it as T. gambiense, as also did Bruce et al. (1911<^).
GENUS: ENDOTRYPANUM
485
Reiclienow (1917, 1920c) observed trypanosomes of the T. lewisi type in
both chimpanzees and gorillas, as also in a lemur (Perodictus) and in
C. cephus in the Cameroons. He proposed to name the trypanosome
T. lewisi var. primatum, as on morphological grounds he regards it as a
variety of T. lewisi. Yamasaki (1924), as already noted, failed to infect
monkeys by means of fleas which had become infective after feeding on
rats harbouring T. lewisi. Direct inoculation of blood from infected rats
into monkeys has also failed to infect them with T. lewisi. Chagas
(1924), in Brazil, found monkeys {Chrysothrix sciureus) naturally infected
with trypanosomes. These were studied in inoculated guinea-pigs and
dogs, with the result that he arrived at the conclusion that the trypano-
some was T. cruzi, with which it agreed in its morphology and method of
multiplication.
Genus: Endotrypanum Mesnil and Brimont, 1908.
Mesnil and Brimont (1908a) described under the name Endotrypanum
schaudinni a curious parasite which occurred in the red cells of Choloepus
y
Fig. 204. — Endotrypanum schaudinni in the Blood of the Sloth, Cholceinis
didactylus. (1-3, after Mesnil and Brimont, 1908; 4-6, after Darling,
1914).
1-3. Parasites in the red blood-corpuscles (x ca. 1,800).
4-6. Two intracorpuscular forms and one free form ( x ca. 3,000).
didactylus, the two-toed sloth of Guiana (Fig. 204). As it is undoubtedly
related to trypanosomes, it is considered here. It was elongated and
piriform in shape, one end being blunt or rounded and the other fine and
486 FAMILY: TRYPANOSOMID^
tapering. It was longer than the diameter of the corpuscle, and either
pushed this out at one point or was curved to adapt itself to the space
available. In the stained films it consisted of blue staining cytoplasm,
and possessed a large, round, red nucleus, by the side of which was a rod-
like body. As a trypanosome occurred in the blood at the same time, the
possibility of these bodies being intracorpuscular stages of the trypano-
some naturally occurred to the observers. The parasite did not possess a
fiagellum, and no axoneme was visible. It measured 8 to 11 microns in
length by 2-5 to 4 microns in breadth. No free forms were discovered,
and there did not occur any which could be considered as intermediate
between the trypanosomes and the intracorpuscular parasites.
This curious organism was again seen by Darling (1914) in Panama.
He had an opportunity of studying it in the living condition. The
parasite was within the red cells immediately after the blood was taken.
It showed active movements, and eventually liberated itself from the cell.
One end was rounded and the other tapering, and in some there was a
definite undulating membrane extending towards the pointed extremity.
In stained specimens the nucleus and kinetoplast described by Mesnil
and Brimont were seen, and in addition a filament running along one side
of the organism. This was undoubtedly the axoneme. The general
appearance of the parasite was that of a crithidia or cultural form of a
trypanosome, to which it seems to be nearly related. Labernadie and
Hubac (1923) also discovered the organism in Guiana. They noted both
intracellular as well as free forms. In some there was a free fiagellum
4 to 6 microns in length, while occasionally the kinetoplast was at the
posterior end of the organism, giving the parasites a definite trypanosome
structure. The organism was seen by the writer and Scott (1925.7) in
Brazilian sloths (C. didactylus) which had died in London.
II. THE TRYPANOSOME OF MAN IN SOUTH AMERICA.
Trypanosoma cruzi Chagas, 1909. — Synonyms: Schizotrypanum criizi
(Chagas, 1909); T. escomeli Yorke, 1920. This trypanosome, which pro-
duces a disease in man in South America, will be considered here, as it
appears to be more nearly related to T. lewisi than to the other pathogenic
trypanosomes of man and animals (Plate V., l, p. 456).
T. cruzi was first discovered by Chagas in 1907, and described by him
(1909) as a parasite of the reduviid bug, Triatoma tnegista. The bugs
were known to attack man in certain parts of Brazil, and Chagas discovered
crithidia forms of a flagellate in the hind-gut of specimens of the bug
collected at Minas. Some of these were allowed to feed on a marmoset,
Hapale penicillata, which three weeks later showed trypanosomes in its
TRYPANOSOMA CRUZI
487
blood. The trypanosomes were found to be inoculable to dogs, guinea-
pigs, and rabbits. Extending his observations, Chagas ultimately found
the organism in a cat, and later in children who suffered from a wasting
disease which had long been known in the country. Chagas first placed
the trypanosome in the genus Trypanosoma, but later, on account of its
Fig. 205. — Diagram of Trypanosoma cruzi in the Blood and Tissues of Man
AND IN THE BuG (Triatoma megista). (After Wen yon, 1922.)
A. Leishraania forms in muscle fibre of heart. B. Trypanosome forms in muscle fibre.
C. Trypanosome forms in blood. S. Trypanosomes in stomach of bug.
R. Rectal phase of development. D. Multiplying crithidia forms in rectum of bug.
E. Metacyclic trypanosome forms which produce infection. These forms usually have no flagella.
peculiar intracellular mode of development as leishmania forms, created
the new genus, Schizotrypanutn, for its reception. This name was chosen
because it was believed that reproduction took place by schizogony, but it
is now known that multiplication, though occurring within cells in the
leishmania stage, is by the usual method of binary fission, so that it is
preferable to retain the trypanosome in the genus Trypanosoma.
488 FAMILY: TRYPANOSOMID^
As already remarked, T. cruzi was first discovered in children at Minas
in Brazil. Later it was shown to occur in other parts of Brazil also, and
by Tejera (1919a) in the States of Zulia and Trujillo in Venezuela, and
by Escomel (1919a) in Peru. As will be shown below, the infection in the
reduviid bugs is much more widespread in South America than is the
disease in human beings which is often termed Chagas' disease.
Symptomatology. — The disease has been described in detail by Chagas
and other observers. It occurs in children of all ages, but assumes
an acute form in the first year of life. In these cases the incubation
period varies between ten days to a month. There is fever, wasting
anaemia, enlargement of the liver, spleen, and lymphatic glands, and
especially of the thyroid, producing a puffy condition of the face and body.
A more chronic condition exists in older children, in which the above
symptoms develop more slowly, while the involvement of the thyroid
gland produces a pseudo-myxoedematous or a well-defined myxoedematous
condition. The chronic form occurs in children up to fifteen years of age,
and is associated with retarded development of mind and body. In any
of these cases there may occur special symptoms attributable to involve-
ment of the heart, meninges, or brain. The disease, though most com-
monly occurring in children, also attacks adults. T. cruzi does not occur
in great numbers in the blood of infected individuals. As a rule there
is a scanty infection, the parasite being found with difficulty on direct
examination. It is more readily demonstrated by inoculation of blood
into a susceptible animal like the marmoset or guinea-pig. It has also
been found in the cerebro-spinal fluid. The reproducing forms occur in
ceils of various organs which are histologically altered by the parasites.
Pathology. — The pathological changes caused by the trypanosomes
consist in the degeneration of the invaded cells, and a leucocyte invasion
of the affected tissue in which numerous leishmania and other forms of the
parasite occur (Fig. 206). There is an increase of fibrous tissue, often
leading to definite sclerosis. This is especially well seen in the thyroid
and ovaries. The changes are most marked in those organs most heavily
invaded by the parasite, and neither in man nor animals is it possible to
predict which part of the body will be most affected.
Morphology. — The trypanosome itself is a curved, stumpy organism
with a sharp posterior end (Fig. 209, 1-3, and Plate V., k, p. 45G). Its
length, including the flagellum, varies on either side of 20 microns, but not
to any great extent. Some individuals are broad and others narrow, and,
as has been suggested in the case of other trypanosomes, this variation
was supposed by Chagas to represent a distinction between female and
male trypanosomes. The proof of this, however, is lacking. Brumpt
TRYPANOSOMA CRUZI
489
<(^
^
% .A
ti«
i;iN
■~^
d*^
s
^ %
.. i,'' »•••
^^':: V^^^'^
N^^^
.-> <-•
w -••'■■' :■.
,.^c«
.-%
,T-fe «a*» "
J^
/-:**
<l!>
" >»:
.„ ...1
Fig. 20G.^Tnjpanosoma cruzi : Leisiimania Forms in Sections of Tissues of
Human Case ( x ca. 1,000). (After Ciiagas, 1916.)
1. Heart muscle. 2. Brain. 3. Thyroid.
490 FAMILY: TRYPANOSOMID^
(1912) believes tliat the narrow forms are the young ones escaped from the
cysts, and that they gradually grow into the broader individuals. The
nucleus is central in position, while the kinetoplast is a relatively large
ovoid or egg-shaped body close to the pointed posterior end. The undulat-
ing membrane is narrow and only slightly convoluted. The flagellum
represents about a third of the total length of the organism. The curved
character of the short broad body with the large "egg-shaped" kineto-
plast and comparatively straight membrane gives T. cruzi at this stage
of its development a very characteristic appearance. Chagas described
certain forms within the red blood-corpuscles, but this observation has
not been confirmed, and it is probable he was merely dealing with super-
imposed trypanosomes or other structures. T. cruzi appears to be a
peculiarly fragile organism, for in the process of making blood-films from
infected animals many of the trypanosomes are damaged.
Escomel (1919a) described what he believed to be the first case of
T. cruzi infection to be noted in Peru. In his description of the trypano-
some he gave the length as 20 to 40 microns, and stated that the kinetoplast
was not well developed. From the description, it appeared to Yorke
(1920a) that Escomel must have been dealing with some trypanosome
other than T. cruzi. He accordingly proposed to name it T. escomeli.
In the following year Escomel (1920) gave a more detailed account of the
trypanosome. He corrected his previous measurements, while from the
figures he gave there is little doubt that he was actually dealing with
T. cruzi, so that the name T. escomeli becomes a synonym.
Multiplication. — Longitudinally dividing forms of T. cruzi, such as
are found in the blood in the case of other trypanosome infections, do
not occur, and this is explained by the type of reproduction which was
specially studied by Vianna (1911), and which bears a striking resemblance
to the method of multiplication of the trypanosome of the bat, Vesperugo
pipistrellus, as described by Chatton and Courrier (see p. 480). The
multiplication of T. cruzi takes place within the cells of nearly every
organ of the body — not only the endothelial cells of the capillaries and
lymphatics, but also the organ cells themselves. In some cases one
organ is more involved than another, a feature which accounts for the
special symptoms seen in certain cases. The heart and voluntary
muscles, the nervous system, thyroid, lymphatic glands, bone marrow,
suprarenal capsules, ovaries, and testis have all been found invaded by
the multiplying forms. The process can be readily studied in sections of
the heart muscle and other organs of mice, rats, and guinea-pigs, or in
smears made from these organs (Fig. 207). Multiplication appears to
commence after the invasion of a cell by a single trypanosome which,
losing its membrane and flagellum, becomes a leishmania form measuring
TRYPANOSOMA CRUZI
491
about 4 microns in diameter. This commences to divide by simple fission
after division of its nucleus and kinetoplast (Fig. 207, 17-20). By repeated
fissions in this manner intracellular cysts are produced which contain large
numbers of leishmania forms. The cyst is more of the nature of a vacuole,
as a definite wall is not present, the cell being enlarged and reduced to a
Fig. 207. — Trypanosoma cruzi in Smear of Heart of a Mouse (x 2,000).
(Original from Preparation made by Dr. Tejera.)
1-9. Stages in development of a trypanosome from rounded flagellated stage.
10-15. Stages in development of a trypanosome from elongate flagellated stage.
16. Posterior nuclear trypanosome form. 17-20. Division of leishmania form.
21-23. Growth of flagellum in leishmania form. 24-28. Division of flagellated forms.
mere enclosing membrane with its nucleus flattened and pushed to one
side. At a certain stage each of the leishmania forms develops a flagellum,
and by gradual changes in the arrangements of its parts becomes trans-
formed into a crithidia form, and finally into a trypanosome of the blood
type (Fig. 207, 1-20). In any single group of organisms the change affects
492 FAMILY: TRYPANOSOMIDiE
all the individuals at the same time and at approximately the same rate,
so they all arrive at maturity together. During the development of
flagella and the transformation into trypanosomes division may still take
place (Fig. 207, 24-28). Rupture of the cell liberates the trypanosomes,
which escape into the blood-stream. According to Brumpt (1912), when
they first enter the blood-stream they are very narrow, active trypano-
somes which grow into the broader forms. A point which does not appear
to be definitely decided is whether the infection of fresh cells is brought
about by the blood trypanosomes entering new cells, and there becoming
again transformed into leishmania forms, which recommence the division
process, or whether new cells are infected by leishmania forms escaping
from ruptured cells. In sections of the organs of infected mice or guinea-
pigs, the writer has often seen ruptured cysts containing the leishmania
forms, and isolated leishmania forms scattered amongst the cells, so that it
does not seem improbable that they might continue the process of multipli-
cation if taken into the cytoplasm of other cells. It might be supposed
that the blood type is only capable of development in the invertebrate
host, but this does not seem to be the case, for all the phases of reproduction
of leishmania forms within the cells will commence after inoculation of an
animal with blood containing the mature trypanosomes.
Chagas (1909) described a peculiar form of pulmonary reproduction
in which small cysts are produced by the looping of a trypanosome into a
ij-shape and its concentration into an ovoid body. The kinetoplast is
supposed to be thrown out, and the nucleus divided into eight small nuclei.
Finally, the contents of the cyst divide into eight small merozoites, which
are presumed to enter the red blood-corpuscles and develop into mature
trypanosomes of the male and female type. This method of reproduction
has not been confirmed, and there is little doubt that Chagas was dealing
with another organism, probably Pneumocystis carinii (Fig, 450).
Hartmann (1910, 1917) has described a process of schizogony which
commences by a single trypanosome becoming a leishmania form within
a cell. Nuclear and kinetoplast divisions take place repeatedly, and by
growth a large cytoplasmic body is produced containing many nuclei
and kinetoplasts. Segmentation into separate leishmania forms then
occurs. Hartmann also describes a schizogony stage in which the nucleus
alone is present, the kinetoplast being absent. The figures given by Hart-
mann are far from convincing, and suggest the presence in a cell of numerous
leishmania forms which have lost their outlines through degeneration.
The schizonts appear to be portions of the cytoplasm of cells containing
the nuclear remains of degenerating or badly fixed parasites. Similar
appearances have led to the view that Leishmania donovani also repro-
duces by schizogony (p. 408).
TRYPANOSOMA CRUZI 493
Culture. — Tryjpanosoma cruzi cultivated in N.N.N, medium produces
the various crithidia and trypanosome types of organism seen in the
development of the trypanosome in Triatoma megista. Animals may be
infected with the cultural forms. In the writer's experience, it is very
difficult to obtain subcultures. Noguchi (1924a), working at yellow fever
in Brazil, on one occasion cultivated from a patient's blood, not only the
leptospira of yellow fever, but also a trypanosome, the presence of which
had not been suspected. The trypanosome, which was probably T. cruzi,
remained alive in the leptospira medium for many weeks. No statement
regarding subculture was made.
Susceptibility of Animals. — T. cruzi is readily inoculable into laboratory
animals, though there is a marked tendency for it to change its virulence.
Guinea-pigs infected by inoculation of the intestinal contents of the bug,
Triatoma megista, frequently die in a couple of weeks. On the other hand,
passage through guinea-pigs for some time may lead to such a decrease
of virulence that the animals only acquire a temporary infection, from
which they recover. For this reason a strain is best kept up by changing
the animal host from time to time. Mice, rats, rabbits, dogs, and cats can
all be infected, as also monkeys {Macacus and Cercointhecus) and mar-
mosets. As will be seen below, the armadillo also acquires an infection.
The virulence of the strain may be so low that it can be kept only in very
young animals, which are more susceptible than older ones.
Transmission. — As already stated above, Chagas (1909) first showed
that Trypanosoma cruzi could be transmitted to animals by allowing
infected bugs {Triatoma megista) to feed on them (Fig. 208), an observation
which he later (1912) extended to two other species {T. infestans and
T. sordida). Larvae hatched in the laboratory became infective in ten to
twenty-five days after feeding on infected animals, and this, according to
Chagas, was associated with the appearance of small trypanosomes in the
body cavity fluid and in the salivary glands. The details of the develop-
ment are, however, not as well understood as that of Trypanosoma gam-
biense in Glossina palpalis. Working with imported bugs in France, Brumpt
(1912) found that the trypanosomes of the blood type ingested by the larvae
quickly became changed into stumpy crithidia forms, which reproduce
rapidly (Fig. 209, i-8). The daughter individuals become elongated, and
transform themselves into flagellates of the long crithidia type, till the
posterior part of the mid-gut contains large numbers of these forms in
varying stages of division (Fig. 209, 9-12). After about twenty days
amongst the multiplying crithidia forms, there appear smaller trypano-
some forms which have been evolved from the former by migration of the
kinetoplast towards the posterior end (Fig. 209, 13-16). As the larva?
become older, the small metacyclic trypanosomes appear to be the
494
FAMILY: TRYPANOSOMID^
dominant type present in the intestinal infection, which was still found
to persist five months after the feed on infected blood. The fseces of the
infected bug contain numerous metacyclic trypanosomes, and are infective
to animals (Fig. 205). The infectivity of the fgeces commences with the
appearance of the trypanosome forms. Chagas (1909), as noted above,
described flagellates of the trypanosome type in the body cavity fluid
of the bugs, and stated that
he had also seen them in the
smears of the salivary glands.
This infection is supposed to
spread from the gut by way
of the Malpighian tubes.
According to Brumpt, this
phase cannot be of constant
occurrence, as he was unable
to demonstrate it, even
when he examined bugs with
a heavy intestinal infection.
Torres (1915) failed to de-
monstrate flagellates in the
body cavity fluid of infected
reduviids {T. megista), though.
he succeeded in infecting
animals by allowing the bugs
to bite through gauze, which
prevented fsecal contamina-
tion of the skin. Extending
his observations, Brumpt
(1912) was able to demon-
strate that, in addition to
T. megista, other allied species
are easily infected — T. infes-
tans, T. chagasi, and T. sordida
— while Brumpt and Gon-
zales-Lugo (1913) proved this
Brumpt also obtained develop-
C. rotundatus, and C. boueti,
Mayer and Eocha Lima
of T. 7negista, which was
Fig. 208. — Triatoma megista ( $ ), One of the
Transmitting Hosts of Trypanosoma cruzi
( X 3). (After Chagas, 1909.)
Dorsal view and side view of head, showing recurved
proboscis.
for Rhodnius prolixus, another reduviid.
ment in the bed bugs, Cimex lectularius
and even in the tick, Ornithodorus moubata.
(1914) found that infection of the intestine
associated with a penetration of the epithelial cells by the trypano-
somes, persisted for at least two years. They also showed that
T. cruzi would undergo development in the bed bug, C. lectularius, and the
TRYPANOSOMA CRUZI
495
tick, 0. moubata, but that infection was not transmitted by their bites.
Mayer (1918) found that specimens of 0. moubata still contained infective
flagellates in the intestine five years after feeding on an infected animal.
Neiva (1913a), experimenting on the transmission of canine piro-
plasmosis by means of Rhipicephalus sanguineus, infected dogs with Try-
panosoma cruzi. In the bed bug Brumpt found that the infection persisted
for over two months, and that the same forms occurred as in the true host,
Triatoma ^negista. Moreover, the faeces of the bed bug were infective to
animals. In one instance, Blacklock (1914) was able to transmit T. cruzi
by allowing infected Cimex lectularius to feed on an animal. Yamasaki
(1924) experimented with the dog flea, but found that in this insect a
rapid degeneration of the trypanosomes took place. Brumpt (1914)
Fig. 209. — Development of Trypanosoma cruzi in Gut of Bhodnius prolixus
(x 2,000) from a Film of the Intestinal Contents. (Original from
Preparation made by Dr. Tejera).
1-3. Trypanosomes of the blood type which are ingested by the bug.
4-9. Various crithidia forms. 10-\'I. Dividing crithidia forms.
13-16. Metacyclic trypanosomes which escape in the fseces of the bug.
noted that reduviid bugs had the habit of attacking each other, and also
of ingesting the liquid fseces passed by themselves or other bugs. That
infection may be acquired in this manner was proved by feeding bed bugs
on diluted fseces containing crithidia forms of T. cruzi. Some of the
bugs became infected, and the flagellates persisted in them for over two
months. Though admitting the cannibalistic habits of the reduviid bugs,
Torres (1915) does not think they can infect one another, as they only suck
the clear body cavity fluid, in which he could find no evidence of trypano-
somes. Though he found that bugs feed on one another's faeces when in
captivity, he believes that under natural conditions this does not occur.
496 FAMILY: TRYPANOSOMID^
Hoffmann (1922) again calls attention to this habit in the case of Rhodnhts
prolixus. The larvse were able to continue their development by sucking
blood from the recently fed parent bugs or other larvae. The possibility
of their becoming infected in this way is evident.
In Venezuela, Tejera (19196) found naturally infected with T. cruzi,
not only R. prolixus, which is the natural vector, but also another reduviid
bug, which he informs the writer has been since identified as Erathyrus
cuspidatiis. Neiva and Pinto, quoted by Pinto (1923, 1924), have effected
transmission by means of R. pictipes.
It will be seen from the above account that active development of
T. cruzi takes place in the mid- and hind-gut of the reduviid bugs, and that
crithidia and finally metacyclic trypanosome forms appear in the feeces.
Chagas believes that the latter gain access to the salivary glands, and
that the bugs produce infection by their bites. In most cases, however,
this salivary gland infection does not take place, and as the faeces of the
bugs are infective when injected into animals, natural infection may occur
by the wound inflicted by the bug becoming contaminated with faeces
passed by the bug while feeding or by the faeces being ingested, as in the
case of T. lewisi. Brumpt (1913a) and Mayer and Rocha Lima (1914)
have shown that mice may be infected by placing infective blood on the
buccal mucous membrane, so that oral infection by means of faeces of an
infected bug may occur. Brumpt (1912) showed that T. cruzi could
penetrate the healthy conjuctiva, and subsequently (1913a) showed that
infection could take place through the healthy skin of young mice.
Reduviid bugs are found naturally infected, not only in the districts
in which the human disease is endemic, but also in other localities. Thus,
Neiva (1914) in the State of Rio noted that Triatoma vitticejjs, and in the
State of San Salvador, T. sanguisuga, T. dimidiata, and R. prolixus,
might be infected with Trypanosoma cruzi, while Maggio and Rosenbusch
(1915) described the infection of T. infestans in the Argentine. Brumpt
and Gomes (1914) have found T. chagasi naturally infected far from human
habitations. This seems to suggest that the bug infection is dependent
on some other host than man, in whom infection occurs only in certain
localities. Pinto (1923) states that Triatoma hrasiliensis has been found
infected in various parts of Brazil, and that dogs may be infected with
the trypanosomes they harbour. A natural infection with T. cruzi has
been demonstrated in the following reduvid bugs: Triatoma megista,
T. infestans, T. sordida, T. dimidiata, T. chagasi, T. geniculata, T. viiticeps,
T. sanguisuga, R. prolixus, and R. pictipes, though they have not all been
incriminated as transmitting the disease to man.
Reservoir Hosts. — Chagas (1912) noted that T. genicidata harboured
a flagellate in its intestine which was indistinguishable from the develop-
FLAGELLATES ALLIED TO T. CRUZI 497
mental forms of T. cruzi in the intestine of Triatoma megista. T. geni-
culata lives in the burrows of the armadillo {Dasypus novemcinctus), which
is commonly infected with a trypanosome. Both this trypanosome and
the flagellate of the bug were inoculable to guinea-pigs. The trypanosome
which appeared in each case resembled T. cruzi, and Chagas concluded that
the armadillo was a reservoir host. Torres (1915) showed that in the
endemic centres of the disease three species of armadillo (D. novemcinc-
tus, D. sexcinctus, and D. unicinctus) were often naturally infected with
T. cruzi. In the burrows in which these animals lived, a reduviid bug,
Triatoma geniculata, fed upon them. Chagas (1918) found that as many
as 46 to 50 per cent, of armadillos {D. novemcinctus) harboured the try-
panosome, as did also the bugs, T. megista, living in their burrows. The
armadillos were frequently found infected far from human habitations.
It would appear, therefore, that the armadillo is the natural host of a
trypanosome which occasionally infects man. Crowell (1923) examined
the organs of a naturally infected armadillo captured in Brazil, and found
the usual developmental form of T. cruzi in the muscle fibres of the heart.
A cat was found by Chagas (1909) to be naturally infected. Chagas (1924)
has found that in Brazil in the Para district monkeys {Chrysothrix sciureus)
may be naturally infected with T. cruzi. The trypanosome was inoculable
to guinea-pigs and young dogs. The latter animals died of the infection,
and were found to have the characteristic reproduction forms in the
heart muscles.
OTHER FLAGELLATES RELATED TO TRYPANOSOMA CRUZI.
It is probable that certain flagellates which have been found in the
gut of blood-sucking reduviid bugs are closely related to T. cruzi. Thus,
Lafont (1912) described a form seen by him in the gut of Triatoma
rubrofasciata in Mauritius. In the gut of the bug there occurred trypano-
some, crithidia, and leishmania forms, which resemble very closely the
stages of T. cruzi in its invertebrate host. Encysted leishmania forms were
also described as occurring in the rectum, but from the figures given there
is no evidence that a cyst wall actually exists. It is of interest to note that
Lafont was able to infect mice by intraperitoneal injection of the gut
contents of the bug. Trypanosomes appeared in the blood of the mice,
and remained there up to a maximum of eight days. A transitory
infection was also produced in the rat and the monkey {Macacus cijno-
molgus). It seems probable that this flagellate, which Lafont named
T. boylei, will be found to be a trypanosome of some vertebrate. Cri-
thidia conorhincB, described by Donovan (1909a) from Triatoma rubro-
fasciata in India, is also possibly a vertebrate trypanosome. The same
I. ^2
498 FAMILY: TRYPANOSOMID^
remark applies to T. triatomce, described by Kofoid and McCiilloch (1916)
from the bug, Triatoma jprotracta, which lives in the nest of the wood rat,
Neotoma fuscipes, of California. Hefpetomonas rangeli Tejera, 1920,
horn. Rhodnius prolixus, and Crithidia vacuolata Rodhain, Pons, Vanden-
branden, and BequaBrt, 1913, from Rhinocoris albopilosus, may also
represent the invertebrate phases of trypanosomes.
III. NON-PATHOGENIC TRYPANOSOMES TRANSMITTED BY SPECIES OF
TABANUS, MELOPHAGUS, OR OTHER BLOOD-SUCKING ARTHROPODA.
Trypanosomes of Cattle.
Trypanosoma theileri Laveran, 1902. — Synonyms: T. transvaaliense
Laveran, 1902; T. lingardi Blanchard, 1904; T. Mmalayanum Lingard, 1906;
T. indicum Lingard, 1907; T. muktesari Lingard, 1907; Trypanozoon theileri Liilie,
1906; Trypanosoma wriiUewsMi Wladimirofl and Yakimoff, 1908; T. americanum
Crawley, 1909; T. frcmJcFiosch, 1909; T. falshawi Kmith, 1909; T.sclieini Knuth,
1909; T. rutherfordi Hadwen, 1912; T. schonebecM Mayer, 1913.
Theiler (1903) described a large trypanosome which he had found in
cattle in South Africa. He had sent blood-films to Laveran, who (1902a)
named it T. theileri. Since that date similar forms have been discovered
in various parts of the world, and have received different names. Lingard
(1903-1907) described three species from Indian cattle — T. himalayanum,
T. indicum, and T. fnuktesari. Frosch (1909) described as T. franJc a
trypanosome of cattle in Germany, while Knuth (1909) recorded T . falshawi
and T. scheini from Singapore and Annam. Watson and Hadwen (1912)
saw a similar form, named T. rutherfordi, in Canada. Crawley (1909)
gave the name T. americanum to a trypanosome of American cattle.
A large form was described from the Lithuanian bison by Wrublewski
(1908), and named T. wrublewskii by Wladimiroff and Yakimoff (1908).
The last observer (1915) came to the conclusion that the trypanosome
was in reality T. theileri. T. transvaaliense was described by Laveran
(1902a) from blood-films from South African cattle sent him by Theiler.
He regarded it as a distinct species, because the kinetoplast was midway
between the nucleus and posterior end of the body instead of being near
the posterior end. For the same reason Croveri (1920) suggested that the
form in cattle in Somaliland was a variety, T. theileri var. somalensis.
It is now known that this degree of variation in the position of the kineto-
plast occurs in T. theileri.
T. theileri is a large trypanosome measuring 60 to 70 microns in length
and 4 to 5 microns in breadth, and frequently shows well-marked myonemes
(Fig. 210, 3-4, Plate V., m, p. 456). Smaller forms also occur of a minimum
length of 25 to 30 microns. It seems reasonable to suppose that the
TRYPANOSOMA THEILERI
499
Fig. 210. — Large Trypanosomes of Mammals (x 2,000). (1 and 5, after Bruce
et al, 1915 and 1910; 2, after Kinghorn and Yorke, 1913; 3 and 4, after
LtJHE, 1906.)
1. Trypanosoma cephnlophi of the duiker (Cephalophus grimmi).
2. T. trngelaplii from the blood of Tragdaphu-s spekei.
3-4. T. theileri from the blood of cattle.
5. T. ingens from the blood of the reed buck, bush buck, and ox.
500 FAMILY: TRYPANOSOMID^
forms described under the various names mentioned above belong to this
species. The trypanosomes are never very numerous in the blood of
adult cattle, and it is highly probable that the variations in size on which
the different species are based merely indicate different developmental
stages. By injecting the forms which were named T. transvaaliense by
Laveran into an ox, Theiler, according to Laveran and Mesnil (1912),
produced an infection showing the typical T. theileri forms. Similar
results were obtained by Behn (1910a) in Germany. By inoculating
calves with the blood of a cow in which trypanosomes had been demon-
strated by the cultural method, a comparatively large infection was pro-
duced. At first the trypanosomes were small and numerous, but after
five days they became scanty and assumed the large form characteristic
of T. theileri.
T. theileri var. somalensis, described from cattle in Somaliland by
Croveri (1920), does not differ in any essential respects from T. theileri.
It is commonly seen in animals used for the preparation of rinderpest
serum, and is said to become pathogenic during the course of this disease.
T. theileri has frequently been demonstrated in the blood of cattle by
the cultural method when direct blood examination has been negative.
The first experience of this kind was that of Miyajima (1907), who was
attempting to cultivate a cattle piroplasm in Japan. In the cultures
flagellates appeared, and he supposed he had demonstrated a flagellate
stage in the development of the piroplasm. Miyajima's experiments were
repeated by Martini (1909) in the Philippines. He was able to demon-
strate that the flagellates had no connection with the piroplasm. These
results were confirmed by various observers in Europe, Africa, and America,
and it was shown that the flagellates in the cultures were derived from
T. theileri, which was present in very small numbers in the blood. The
culture is made by abstracting sterile blood from the jugular vein, and
adding it to twice its volume of ordinary nutrient bouillon. The mixture
is kept at a temperature of about 25° C, and flagellates of various forms
begin to appear towards the end of a week, and attain their maximum in
a fortnight. Subculture may be carried out in the same medium or in
blood-agar media. In the cultures every variety of form between small
round bodies of the leishmania type having a diameter of 2 to 3 microns
up to large crithidia forms occur. The largest forms Avhich may have the
trypanosome structure are 60 to 70 microns in length, and resemble T. thei-
leri as seen in the blood. Herds of cattle examined by the culture method
have shown a percentage of infected individuals varying from 10 to 70 per
cent. As far as is known, the infections in no way inconvenience the host.
Theiler (1903) claims to have transmitted the trypanosome through the
agency of Hippobosca rvfjies and H. wacidata. Flies fed on infected
TRYPANOSOMA THEILERI 501
cattle were at once transferred to uninfected animals, and in two cases
out of four an infection resulted. Such a transmission, if it actually took
place, is evidently a purely mechanical one, which might be accomplished
by any biting insect. The difficulty of excluding an infection in the cattle
apart from the culture method, which was not employed by Theiler,
raises doubt as to whether the experimental animals were really free from
infection before exposure to the flies.
Noller (1916) succeeded in obtaining a culture on blood-agar of Cri-
tJiidia suhulata, a flagellate first described by Leger, L. (1904c), from the
gut of Tabanus glancopis, and, owing to the resemblance of the cultural
forms to those of T. theileri, he came to the conclusion that C. suhulata is
Fjg. 211. — Tahatms Uvniola (T. soeius) (?) of the Sudan, with Wings extended
(x2-5). (After King, 1911.)
This species very commonly harbours a crithidia, which is probably a developmental form of
Tryfanosoma theileri.
really the developmental form of T. theileri in the tabanid fly, which is to
be regarded as the true insect host of this trypanosome. It has been
suggested above (p. 358) that C. hyalommce, which occurs in the tick, may
possibly be a developmental form of this trypanosome. If C. suhulata
is merely the insect phase of T. theileri, it seems probable that this applies
also to other similar flagellates of Tabanidse and their allies, such as those
seen by the writer (1909) in the Sudan. They were especially common in
Tahanus tceniola {T. soeius), which was a voracious blood-sucker (Fig. 211).
Noller (1925) appears to have established this identity in the case of the
crithidia of Hcematojpota pluvialis. He injected clean calves with cultures
of the flagellate of the flies and recovered trypanosomes from the blood
by culture on the fifth, sixth, and tenth days.
502 FAMILY: TRYPANOSOMID^E
Trypanosomes of Sheep.
Trypanosoma melophagium (Flu, 1908). — Synonyms: CritMdia melophagia
Flu, 1908; Leptomonas Roubaud, 1909; L. melophagi Mesnil, 1909; C. melophagi
Swingle, 1909; Sheep-trypanosome Woodcock, 1910; CritMdia Wenyon, 1913;
L. melophagia Brumpt, 1913; T. woodcoclci Brumpt, 1913; Herpetomonas melopliagia
Doflein, 1916; Trypanosoma {Cystotrypianosoma) melophagia Bnimpt, 1922.
This trypanosome, the developmental stages of which in the sheep ked
{Melo])hagus ovinus) were the first forms to be discovered, was seen by
PfeifEer (1905), w^ho referred to it as a " trypanosome-like flagellate." Flu
(1908) described the ked flagellate as Critliidia tnelophagia, and, like its
original discoverer and many subsequent observers, including Roubaud
(1909), Porter (1910), Swingle (1911a), Dunkerley (1913), regarded it as an
Fig. 212. — The Sheep Ked, Melophagus ovinus (9), and its Pupa, the Trans-
mitter OF Tryjmnosoma melophagium ( x 8). (After Hoare, 1923.)
The scale shows the natural size of the fly.
organism peculiar to the ked. Woodcock (1910), however, observed a
trypanosome in the blood of an English sheep, and suggested the possibility
of the ked flagellate being merely the invertebrate phase of this parasite.
The trypanosome of sheep was again seen by Behn (191 1, 1912) in Germany,
and its relation to the ked flagellate was investigated by Noller (1917)
and Kleine (1919a). Noller obtained cultures of both the sheep trypano-
some and the ked flagellate, and showed that the cultural forms were
identical. He noted that flocks of sheep which were most heavily infested
with keds were likewise most heavily infected with trypanosomes, and he
concluded that the ked flagellate was actually the developmental form of
TRYPANOSOMA MELOPHAGIUM
503
the sheep trypanosome, as Woodcock had suggested. Noller pointed out
that its correct name was T. ^nelophagium. Kleine (1919a) also studied
the trypanosome, and came to the conclusion that the ked inoculated it
to sheep from its salivary glands. The whole question has been the subject
Fig. 213. — Life-Cycle of Trypanosoma melopliagium in the Blood of the Sheep
AND IN the Ked [Melophagus ovinus) ( x 1,560). (After Hoare, 1923; from
Parasitology, vol. xv., p. 395.)
1. Trypanosome in blood of sheep; form ingested by ked.
2. Trypanomorphic crithidia form which leads to typical crithidia (4, 5) by division (3).
3. Dividing form. 4-5. Typical crithidia forms in mid-gut.
G-8. Development of small crithidia forms in hind-gut.
9 9«. Two methods of division of crithidia forms, giving rise either to small jiyriform crithidia
(10) or metacyclic trypanosomes (10a). By migration of the kinetojilast the crithidia
may become a metacyclic trypanosome (10, 10a).
106. Leishmania forms taking no part in cycle.
of exhaustive investigation by Hoare (1922, 1923) in England. He has
shown conclusively that uninfected lambs can be infected by feeding them
with the hind-gut of infected keds, and, furthermore, that the bite of the
ked is unable to bring about infection. A study of the trypanosome in the
ked has shown that the flagellate produces metacyclic trypanosomes in
504 FAMILY: TRYPANOSOMID^
the hind-gut, and that the development is one in the posterior station, as
in the case of T. lewisi in the flea (Fig. 213). The many observers who
regarded the ked flagellate as peculiar to the insect have described encysted
forms in the rectum, and it was supposed that these were ingested by other
keds, which consequently became infected. That such an infection did not
take place was proved by Kleine (1919a), who found that uninfected keds
hatched from pupse in the laboratory did not become infected when kept
with keds already infected. He showed, furthermore, that uninfected
keds did not become infected when fed on goats which did not harbour
trypanosomes. It is evident, therefore, that the bodies described as
cysts in the faeces of the keds by various observers who have investigated
>
® ® ^ §,. - -^
'mm
Fig. 214. — Structures in the Hind-Gut of the Ked, which might be Inter-
preted AS Cysts of Flagellates (x 2,000). (After Hoare, 1923.)
1. Accumulation of staining material round a flagellate producing appearance of a
homogeneous cyst wall. 2-5. Stained granular debris round leishmania forms.
G. Deposit round short flagellate form. 7-8. Yeasts of the Cryptococcus type.
9. Metacyclic trypanosome superimirosed on a yeast. 10-12. Yeasts in various stages.
this flagellate were not of this nature. They were in many cases leish-
mania forms round which deposits of stain had taken place, or even other
organisms, such as yeasts (Fig. 214). It is possible that the cysts Avhich
have been described in the case of H. grayi of tsetse flies may be of a
similar nature. The cycle of development of the ked flagellate, as de-
scribed by Porter (1910), in which the various phases (pre-flagellate,
flagellate, and post-flagellate) occur are quite erroneous. The work of
Hoare has finally established the identity of the ked flagellate and the
trypanosome of sheep, and, furthermore, shows that many of the Crithidia
of blood-sucking arthropods require reinvestigation from the point of view
of their possible relationship to vertebrate trypanosomes. T. melophagiimi
TRYPANOSOMA MELOPHAGIUM 505
is usually present in small numbers in the blood of infected sheep, and,
as in the case of T. theileri, its presence is best detected, as first shown
by Behn (1911), by the use of thick films or, as N5ller (1920c) demon-
strated, by abstracting blood from a vein and diluting it in culture tubes
under sterile conditions with an equal quantity of bouillon. The mixture
is incubated at 30° C. for a week or more, after which time the scanty
trypanosomes w^ll have multiplied sufficiently to be readily detected.
By the culture method Hoare was able to demonstrate that the sheep in
a ked-infested flock were infected Lo the extent of 80 per cent. In lambs
which were experimentally infected by feeding them with the hind-gut
of keds, the trypanosomes are for a short time sufficiently numerous to be
detected in the blood by the examination of a few wet films. The infection,
however, subsides in the course of one to three months, and if the animals
are kept free from keds it will disappear entirely. The sheep, however,
can be readily reinfected, and it seems probable that there is only a very
slight degree of immunity, and that flocks of sheep are kept infected by
constant reinfection. The trypanosome appears to have no harmful effect
on the sheep.
The trypanosome in the blood of the sheep is of large size, like T. thei-
leri (Fig. 215). It is from 50 to 60 microns in length, and the portion of
the body behind the kinetoplast is pointed and represents about one-
third the length of the entire body. The nucleus is central in position,
and the kinetoplast is a short distance behind it and about 9-6 microns
from the posterior end. There is a short free flagellum about 5-6 microns
in length. No multiplication forms have been seen in the blood.
The early stages of development in the ked have not been followed,
but these insects are practically invariably infected when taken oft" sheep.
The predominating type is a crithidia which appears to be confined to the
Stomach (Fig. 215, 2-3). It multiplies rapidly by longitudinal fission, and
becomes attached in large numbers to the wall of the hind-gut, especially
round the pyloric opening of the stomach. In this attached condition
many of the crithidia forms by repeated divisions unassociated with growth
become smaller forms, which by migration backwards of the kinetoplast
to the posterior extremity of the body are transferred into short stumpy
metacyclic trypanosomes (Fig. 215, 4-5). The latter are presumably
those which lead to infection of the sheep. They resemble in many respects
the small metacyclic trypanosomes of T. lewisi.
Cultures of the trypanosome, whether commenced from the blood of
sheep or from the intestine of the ked, can be maintained at 30° C. in
Noller's medium, which consists of N.N.N, medium to which glucose has
been added. In the cultures from the sheep's blood large trypanosomes
at first occur, but these quickly become crithidia forms like those in cultures
506
FAMILY: TRYPANOSOMID^
from the ked's gut. In older cultures of both kinds there appear numbers
of small trypanosomes, which are like the small metacyclic forms developed
in the hind-gut of the ked. In fact, the behaviour of the trypanosome in
cultures appears to be directly comparable with its development in the ked.
%,^jr
Fig. 215. — Trypanosoma meloph%gium of the Sheep and Sheep Ked, Melophngm
ovinus (x 2,000). (After Hoare, 1923.)
1 . Trypanosome from blood of sheep .
2. Three crithidia forms from mid-gut of ked.
3. Small crithidia and leishmania forms from mid-gut of ked.
4. Epithelium of hind-gut of ked with various attached flagellates.
5. Metacyclic trypanosomes attached to epithelium of hind-gut.
Attempts to inoculate mice, rats, and guinea-pigs with the flagellates
from the ked and with cultures have been invariably unsuccessful except
in the case of Laveran and Franchini (1914, 1919), who claim to have
infected mice by feeding them or inoculating them intraperitoneally
with the flagellates from the ked. The infection, however, was said to be
TRYPANOSOMES OF ANTELOPES 507
of the leishmania type. Galli-Valerio (1923) claims to have produced a
similar infection in a rat. If these results are accurate, this is the only
known instance of a trypanosome producing a leishmania infection without
the occurrence of trypanosomes at the same time. Hoare (1921cr) in the
case of rats, mice, and guinea-pigs, and Buchner (1922) with mice, failed
entirely to produce any infection with these flagellates.
Examining ticks {Ixodes ricinus) from sheep. Bishop (1911) claims to
have seen a single crithidia form in the tick. It is possible this was a
cultural form of the sheep trypanosome.
Trypanosomes of Antelope.
Button, Todd, and Tobey (1906) described as T. tragelajjhi a large
trypanosome from the blood of a West African bush buck, Trogelaphus
sylvaticus (Fig. 210, 2). Kleine and Fischer (1911) found a similar form
in the reed buck, Cervicapra arundinum, near Tanganyika, and Rodhain,
Pons, Vandenbranden, and Bequsert (1913a) one in Cephalopus grimmi
and Cohus vardoni in the Congo. It does not seem improbable that
these forms are actually Trypanoso7na theileri. Bruce, Hamerton, Bate-
man, and Mackie (1909a) discovered a much larger form in the reed buck
{Cervicapra arundinum), in the bush buck {Tragelaphus sylvaticus), and
in an ox in Uganda. On account of its large size it was named Trypano-
soma ingens (Fig. 210, 5). It measures from 72 to 122 microns in length
and 7 to 10 microns in breadth. The trypanosome was also seen by
Eraser and Duke (1912a) in the blood of a bush buck in Uganda. From
the dimensions given, it will be seen that it is distinctly larger than any
known form of T. theileri, and on this account is possibly a distinct species.
Nothing is known of its life-cycle. A trypanosome of the same type was
seen by Dodd (1912) in the blood of two mouse deer {Tragulus javanicus)
which had died in the Zoological Gardens of Sydney. Bruce et al. (1913c)
gave the name Trypanosoma cephalophi to a large form seen by them in
the blood of the duiker, Cephalophus grimmi (Fig. 210, i).
Group B. Trypanosomes which Develop in the Anterior Station in the
Invertebrate or have become Secondarily Adapted to Direct Passage
from Vertebrate to Vertebrate.
1. PATHOGENIC TRYPANOSOMES TRANSMITTED BY BLOOD-SUCKING
ARTHROPODA.
General Remarks on the Pathogenic Trypanosomes.
Under this heading are included certain trypanosomes which produce
disease in man and domestic animals. As stated above, the true verte-
brate hosts of these trypanosomes, in tsetse fly areas of Africa at least,
508 FAMILY: TEYPANOSOMIDiE
are not those in which disease is produced, but rather the wild animals of
the country, which harbour them without suffering in any serious manner,
just as Trypanosoma lewisi occurs in the rat. In other parts of the world,
with the exception of South America, where the capibara is said to be the
reservoir for T. equinum, the pathogenic trypanosomes, which are of the
T. evansi type, appear to be transmitted amongst the domestic animals
alone. This is undoubtedly accounted for by the fact that it is only in
Africa that domestic animals come into close contact with the game.
It is on account of the importance of these trypanosomes from an economic
standpoint that they have attracted so much attention.
In tsetse-fly areas of Africa the domestic animals have been found
infected as follows:
Horse, Mule, and Donkey: T. brucei, T. vivax, T. congolense.
Ox: T. gamhiense{1), T. brucei, T. vivax, T. congolense, T. uniforme,
T. montgoyneryi.
Pig and Camel: T. brucei, T. congolense.
Sheep and Goat: T. gambiense (1), T. brucei, T. vivax, T. congolense,
T. caprcB.
Dog: T. gambiense, T. brucei, T. congolense, T. montgomeryi.
Relation to Game. — In Nyasaland in the fly country below Kasu Hill,
the Royal Society's Commission under Bruce (1913e) found that the
wild game harboured trypanosomes to the extent of 31-7 per cent. The
species found were T. brucei (7-8 per cent.), T. pecorum {T. congolense)
(14-4 per cent.), T. simice (1-7 per cent.), T. caprcB (11-1 per cent.), and
T. ingens (1-7 per cent.). As regards the wdld tsetse flies {Glossina morsi-
tans), of 1,060 examined by Bruce et al. (1914/) T. brucei was found once,
T. pecorum six times, T. simice twelve times, and T. caprce fourteen times.
Similar results had previously been obtained by Bruce (1895) in Zululand,
though at that time all the pathogenic trypanosomes were considered to
belong to the species T. brucei.
Domestic animals living in the area were found infected to a limited
extent, but their numbers were so small as to constitute little danger.
Of 140 goats examined, five showed T. pecorum and one T. caprce ; and of
twenty-two dogs, six harboured T. pecorum and ten T. brucei.
Kinghorn and Yorke (1912a) found that trypanosomes were of frequent
occurrence in the domestic stock of North-East Rhodesia. As regards
the big game, a conservative estimate placed the percentage of those
infected at about 50 per cent, in the Luangwa Valley, and 35 per cent, in
the Zambesi-Congo basin. The trypanosomes found were T. brucei
{T. rhodesiense), T. vivax, T. congolense {T. nanum and T. pecorum),
T. montgomeryi, T. multiforme {T. brucei or T. gambiense, or a mixed
PATHOGENIC TRYPANOSOMES AND GAME
509
infection), and T. tragelaphi. The animals harbouring trypanosomes
included bush buck, water buck, puku, impala, sitatunga, eland, and
duiker. Duke (1913a) also found that a considerable percentage of the
wild game in West Uganda is infected with trypanosomes (T. hrucei,
T. congolense, T. vivax, T. uniforme, and trypanosomes having a " sus-
picious resemblance to T. gambiense'^). Similar results were obtained by
Kleine and Fischer (1911), R-odhain, Pons, Vandenbranden, and Bequa?rt
(1912, 1913a), Taute (1913), Week (1914), and others.
The following table given by Bruce and his co-workers (1913e) shows
the results of the examination of wild animals in Nyasaland:
Eland
Sable
Water buck
Koodoo
Bush buck
Hartebeest
Eeed buck
Oribi
Duiker
Buffalo
Lion
Hyjena
Elephant
Wart hog
WUd cat
Porcupine
Total
10
6
5
0
13
9
3
2
10
7
35
6
19
12
26
4
7
2
9
2
1
0
3
2
2
0
33
7
3
0
1
0
180
59
14
26
20
The possibility of the existence of a reservoir of T. gambiense in game
and other animals will be discussed below. The evidence that any such
reservoir exists is not at all clear. As regards the other trypanosome of
man in Africa, which appears to be merely a strain of T. brucei, but which
is usually referred to as T. rhodesiense, the position is a difficult one. In
areas where the disease nagana of domestic animals is common, and the
human disease due to this strain of T. brucei is absent, all observers are
agreed that the trypanosome of this type in the game is T. brucei. In
areas in which the human disease occurs opinions differ. In Nyasaland
the Royal Society's Commission under Bruce (1913 to 1914) concluded
that the trypanosome in man, domestic animals, and game was identical,
and called it T. brucei vel rhodesiense. Kinghorn and Yorke (1912
to 1913) in North Rhodesia referred to the trypanosome in man as
510 FAMILY: TRYPANOSOMID^
T. rhodesiense, and concluded that the similar form in the game was also
T. rhodesiense. Kleine and Taute, however, in Tanganyika referred to the
human form as T. rhodesiense, but believed that that which occurred in
domestic animals and game was another species— namely, T. brucei.
According to them, a reservoir host of T. rhodesiense has not been dis-
covered. This subject will be referred to in more detail below.
There seems to be little evidence that T. evansi (including several
named species of trypanosome which appear to be merely races of
T. evansi), which has a wide distribution in tsetse-free areas of the Old and
New World, and which infects cattle, horses, mules, donkeys, camels, and
elephants, has any reservoir comparable with the game reservoirs in Central
Africa. It has been supposed that the buffalo or pig may act in this
capacity in India, while in South America it has been stated that one form
(T. venezuelense) occurs naturally in the dog, monkey, and capibara, and
another {T. equinutn) in the last-named animal.
Game Reservoirs of Trypanosomes of Men and Domestic Animals in Africa.
Buffalo {Bos coffer): T. brucei, Bruce et al., 1897. T. vivax, Duke, 1913.
T. ^iniforme, Duke, 1913. T. congolense, Duke, 1913; Bruce et al., 1913.
Bush Buck {Tragelaphus scriptus): T. gamhiense {T. multiforme), Kinghorn and
Yorke, 1912. T. brucei, Bruce et al., 1897; Kleme and Fischer, 1911; Kiugliorn
and Yorke, 1912; Taute, 1913. T. caprce, Bruce et al., 1913. T. vivax, Bruce et al.,
1911 ; Kleine and Fischer, 1911. T. cazalboui ( = T. vivax), Rodhain, Pons, Vanden-
branden, and Bequsert, 1913. T. uniforme, Duke, 1912; Fraser and Duke, 1912.
T. congolense, Kinghorn and Yorke, 1912; Kleine and Eckard, 1913; Rodhain,
Pons, Vandenbranden, and Bequsert, 1913; Bruce, 1913. T. dimorpJion ( = T. con-
golense), Dutton, Todd and Kinghorn, 1907; Montgomery and Kinghorn, 1908;
Johnson, 1920. T. theileri {T. tragelapM ?), Dutton, Todd, and Tobey, 1906.
T. ingens, Bruce et al, 1909; Fraser and Duke, 1912 ; Rodhain, Pons, Vandenbranden,
and Bequsert, 1913. Undetermined, Montgomerj and Kinghorn, 1908; Kleine and
Fischer, 1911; Week, 1914; Dutton, Todd, and Kinghorn, 1907.
Chimpanzee: T. gambiense [?], Ziemann, 1902.
Duiker {CejjJialophus grimmi): T. brucei, Bruce et al., 1913; Taute, 1913.
T. vivax, Kinghorn and Yorke, 1912. T. congolense, Kinghorn and Yorke, 1912.
T. theileri, Rodhain, Pons, Vandenbranden. and Bequtert, 1912. T. ingens, Bruce
et al., 1912; Rodhain, Pons, Vandenbranden, and Bequsert, 1912.
Eland {Taurotragus oryx): T. brucei, Taute, 1913; Davey, 1916. T. caprce,
Bruce et al, 1913. T. congolense, Kinghorn and Yorke, 1912; Bruce et al, 1913;
Davey, 1916. Undetermined, Week, 1914.
Elephant: T. brucei {T. elephantis), Bruce et al, 1909.
Hartebeest (Bubalis lichtensteini): T. brucei, Kingliorn and Yorke, 1912;
Bruce et al, 1913; Taute, 1913. T. congolense, Kleine and Fischer, 1911 (Pferde-
antelopen); Bruce et al, 1913. Undetermined, Montgomery and Kinghorn, 1908.
Hippopotamus: Undetermined, Kleine and Taute, 1911.
Hy-ENa {Hycena crocuta): T. brucei, Bruce et al., 1897. T. congolense, Bruce,
1913, Undetermined (? T. gambiense), Duke, 1913.
PATHOGENIC TRYPANOSOMES AND GAME 511
Koodoo (Sfrepsiceros capensis): T. brucei, Bruce e^ oZ., 1897. T. caprce, Brnce
et ol., 1914. T. cazalhoui ( = T. vivax), Rodliain, Pons, Vandenbranden, and Bequsert,
1913. T. congolense, Kinghorn and Yorke, 1912; Bruce et al., 1913.
Lemur {Galago demidoffi): T. gambiensi (?), Martin, Lel>oeuf,and Roubaud, 1909.
Lion (Felis leo): Undetermined, Week, 1914.
Monkey: T. gambiense (?), Kudicke, 1906; Button, Todd. and Tobey, 1906; Koch.
Beck, and Kleine, 1909; Bruce et al, 1911.
Mpala {Mpyceros melampus): T. bnicei, Kinghorn and Yorke, 1912. T. caprce,
Bruce et al., 1914. T. congolense, Kinghorn and Yorke, 1912; Bruce, 1914.
Oeibi {Oribia seoparia !): T. brucei, Bruce, et al. 1913. T. caprce, Bruce et al.,
1913. T. congolense, Bruce et al., 1913. T. ingens, Bruce et al., 1913.
Otter (Liitra capensis ?): Undetermined, Fehlandt, 1911.
PuKU {Cobus vardoni): T. vivax, Kinghorn and Yorke, 1912. T. cazalhoui
{ = T. vivax), Rodhain, Pons, Vandenbranden, and Bequsert, 1913. T. ingens,
Rodhain, Pons, Vandenbranden, and Bequaert, 1913.
Reed Buck {Cervicapra arimdinum): T. gambiense (? T. brucei), Simpson, 1918.
T. brucei, Bruce et al, 1903 and 1913; Taute, 1913. T. caprte, Bruce et al, 1913.
T. vivax, Connal, 1917; Simpson, 1918. T. cazalhoui {=T. t'ii;oa;), Rodhain, Pons,
Vandenbranden, and Bequsert, 1913. T. congolense, Kleine and Fischer, 1911;
Bruce et al, 1913. T. theileri, Kleine and Fischer, 1911. T. ingens, Bruce et al,
1909 and 1913. Undetermined, Kleine and Fischer, 1911; Week, 1914.
Roan {Hiptpotragus equinus): T. vivax, Duke, 1923. T. cazalhoui { = T. vivax),
Rodhain, Pons, Vandenbranden, and Bequsert, 1913. T. congolense, Kinghorn and
Yorke, 1912; Davey, 1916.
Sable {Hipj^otragus niger): Undetermined, Week, 1914.
Serval {Felis serval ?): Undetermined, Week, 1914.
SiTATUNGA {Tragelaphus spelcei): T. gambiense, Duke, 1912. T. brucei, Duke,
1921. T. vivax, Duke, 1912. T. uniforme, Duke, 1912 and 1923. T. tragelaplii,
Kinghorn and Yorke, 1912; Duke, 1912. T. ingens, 1912.
Steinbock {Baphiceros campestris): T. brucei, Bruce et al, 1903.
Wart Hog {Phacoceros cethiopicus) : T. brucei, Kinghorn, and Yorke, 1912; Bruce
ct al, 1913. T. congolense, Bruce et al, 1913; Simpson, 1918. T. simice. Bruce et
al, 1913.
Water Buck {Cobus ellipsipriimnns): T. fc)-»cej, Kleine and Fischer, 1911; King-
horn and Yorke, 1912; Bruce et'al., 1913; Taute, 1913; Stohr, 1913; Duke, 1923.
T. caprce, Bruce et al, 1913. T. vivax, Kleine and Fischer, 1911; Kinghorn and
Yorke, 1912; Duke, 1913; Johnson, 1920. T. uniforme, Duke, 1913. T. congolense,
Kinghorn and Yorke, 1912; Bruce et cd., 1913. T. ingens, Bruce et al, 1914.
Undetermined, Kleine and Fischer, 1911; Week, 1914.
Wildebeest {Connochcetes gnu ?): T. brucei, Bruce et cd., 1897. Undetermined,
Week, 1914.
Mechanism of Infection. — Under natural conditions tlie pathogenic
trypanosomes are transmitted to man and domestic animals by blood-
sucking arthropods. In the tsetse-fly areas of Africa those flies which
belong to the genus Glossina are chiefly responsible, though it is possible
that other biting flies may occasionally play a part (Fig. 216). From the
table (p. 517) it will be seen that one species of tsetse fly is able to transmit
512
FAMILY: TRYPANOSOMIDiE
several species of trypanosome, and this fact led Kleine and Fischer (1912)
to express the view that any species of tsetse fly would probably be able
to transmit any of the pathogenic trypanosomes with which it was in
contact. The flies, which inject the trypanosomes when they bite, become
infective after the trypanosome has passed through a definite cycle of
development, terminating in the production of metacyclic trypanosomes.
The cycle requires about twenty days for its completion. In an ingenious
experiment, Rodhain, Pons,
Vandenbranden, and Bequa^rt
(1912c), induced G. morsitans
infected with T. brucei to feed
through a membrane covering
a tube in which citrated blood
was contained. After a fly had
fed, the number of trypanosomes
in a portion of the fluid were
counted, and it was estimated
that a single infected fly was able
to inject 1,562 metacyclic try-
panosomes while feeding.
It has been clearly demon-
strated that a purely mechanical
transmission may also occur by
the fly contaminating the wound
it inflicts with infective blood
which it has recently taken into
its proboscis from another host.
Duke (1919) believes that the
ejndemic of sleeping sickness
which swept over Uganda was
largely due to mechanical
transmission of infection from
man to man by Glossina pal-
palis. This view is further
developed by Duke (1921, 1923,
1923r/.), who concludes that wherever human trypanosomiasis occurs in
epidemic form in Africa, the transmission is a mechanical one. Certain
experiments made by him (1923a,) are held to prove that when the human
trypanosome is passed directly from monkey to monkey by direct
inoculation of blood, it eventually loses its power of passing through the
complete cycle in the tsetse fly, and he assumes that a similar change may
occur after prolonged mechanical transmission from man to man.
Fig. 216. — Glossina morsitans ( 9 ) Dorsal
AND Side Views (x 4-5). (After
Xewstead, 1924.)
TRANSMISSION OF PATHOGENIC TRYPANOSOMES 513
Hornby (1921) found that mechanical transmission of trypanosomes
amongst domestic stock in Rhodesia is by no means uncommon. A few
animals which have acquired infection in tsetse-fly areas, if brought into
close contact with animals in a tsetse-free district, may lead to the infection
spreading through the stock. In such cases infection is spread by flies
other than tsetse flies, and presumably in a mechanical manner. All the
pathogenic trypanosomes which are transmitted by tsetse flies have been
shown by various observers to be capable of mechanical transmission by
mosquitoes or species of Stomoxys and Tabanus. In the case of T. evansi
and the forms allied to it both in the Old World and America, this is the
only method of transmission which has been demonstrated, unless the
claim made by Cross and Patel (1921) regarding the transmission of
T. evansi by ticks in India indicates a cycle of development com-
parable with that in tsetse flies in Africa. Mechanical transmission
of T. evansi {T. hippicum) by the house fly was proved to be possible
by Darling (1912).
Direct inoculation of blood from an infected to a healthy animal will
bring about infection, and it is by this means that the various laboratory
strains of trypanosomes have been maintained for experimental work.
Many strains have been kept in rats or guinea-pigs for numbers of years,
but it must always be remembered that such artificially maintained strains
may acquire peculiarities which they did not originally possess in the
normal host. There is a variation in the animals inoculable with any one
trypanosome, and, furthermore, after successive passages the virulence
may become much increased. Intraperitoneal and intravenous inocula-
tions lead to infections more readily than subcutaneous ones. It is
highly probable that after long maintenance in animals like rats in the
unnatural conditions of direct passage, without any fly intervention as
occurs in nature, trypanosomes become profoundly altered, not only
morphologically, but also physiologically, so that care has to be exercised
in comparing such forms with those recently isolated from their natural
hosts. Bruce et al. (19136) expressed the opinion that " it is absurd to
expect to arrive at any classification at all approaching a true one by the
study of strains of trypanosomes kept for many years and undergoing
many vicissitudes in our European laboratories."
Bruce (1897) noted that a dog which had eaten a piece of the congealed
heart blood of a heifer which had died of nagana contracted the disease,
while many instances are on record of animals becoming infected after
eating the organs of infected animals. Experimental work has demon-
strated the infective power of blood introduced into the mouth, stomach,
conjunctival sac, and vagina. Under natural conditions it is known that
T. equiperdum is transmitted through mucous membranes, while rats
I. 33
514
FAMILY: TRYPANOSOMID^
become infected with T. leivisi by eating the faeces of infected fleas, a
method of infection which is probably applicable to other trypanosomes
also.
Attempts have been made to infect invertebrates with the pathogenic
trypanosome by inoculating blood from infected vertebrates. Wendel-
stadt and Felmer (1909) proved that T. brucei could survive in the tissues
Salivary glands. Second phase of multipli-
cation. Free-flagellated fusiform critliidia,
producing infective trypanosomes.
Hypopharynx.
Used as passage
only.
Labial cavity. U.<-ed
as passage only.
loventriculus. End
of first phase. Long
slender free • flagel-
lated trypanosomes.
Mid gut. First phase of multipli-
cation. Free-flagellated fusiform
trypanosomes. No crithidia.
Fig. 217. — Diagnostic Characters of Trypanosoma bmcei and Trypanosoma
gambiense in the Tsetse Fly. (After Lloyd and Johnson, 1925.)
Z
Preinfective and
.infective in Lumen
of hypopharynx.
Salivary glands. No phase.
Hypopharynx. Preinfective forms
enter and become infective In-
fective forms F, cuinulate.
Labial cavity. Second phase of
multiplication. Loosely fixed
colonies of ribbon-shaped cri-
thidia, aflagellate or almost
so, producing free - flagellated
slender posterior-nuclear prc-
infected forms.
Froventriculus. End
of first phase. Long
slender free-flagc'.lated
trypanosomes.
Mid gut. First phase of multi-
plication. Ribbon-shaped try-
panosomes, aflagellate or almost
so. No crithidia.
Fig. 218. — Diagnostic Characters of Trypanosoma congolense in the Tsetse
Fly. (After Lloyd and Johnson, 1925.)
of beetles for at least seven days. More recently Iwanow (1925) has found
that T. equiperdum will live for eleven days in caterpillars {Galleria mel-
lonella) kept at laboratory temperature. Active trypanosomes were seen
up to the ninth day, while mice could still be infected by inoculation of
the tissues of the caterpillars up to the eleventh day.
PATHOGENIC TRYPANOSOMES IN TSETSE FLIES
515
Identification of Trypanosomes in Tsetse Flies. — As various pathogenic
trypanosomes undergo development in tsetse flies, it is of importance
to be able to identify them. Most observers have adopted the method
of identifying the trypanosomes which appear in animals after the flies
have been allowed to feed upon them. This is a laborious method which
entails considerable delay. Though it has been possible in many cases to
Salivary glands. No phase.
Hypopharynx. Preinfective forms
enter and become infective- In-
fective forms accumulate.
Labial cavity. Only phase of
mill tiplicati on . Compact
colonies of fusiform crithidia,
long free flagellum, produc-
ing free-flagelljte posterior-
nuclear preinfective forms.
Mid gut. No phase. Blood forms
which enter disintegrate.
Fig. 219.
-Diagnostic Characters of Tnipanosoma vivax in the Tsetse Fly.
(After Lloyd and Johnson, 1925.)
Salivary glands. No phase.
Labial cavity
No phase.
Mid gut. Only phase of multiplica-
tion. Large free-flagellated undu-
lant crithidia, long slender crithidia,
short - flagellated trypanosomes.
Complete life history not known.
Fig. 220.— Diagnostic Characters of Trypanosoma grayi in the Tsetse Fly.
(After Lloyd and Johnson, 1925.)
make a shrewd guess as to the species from what is known of the site of
development in the fly, there has been no certainty about the identification
apart from the trypanosomes of the polymorphic type (T. brucei, T. gam-
ble nse), which are known to be the only ones which invade the salivary
glands. Lloyd and Johnson (1924). however, after a careful study of the
516 FAMILY: TRYPANOSOMID^
developmental forms of various trypanosomes in the fly, have reached
the conclusion that it is possible to identify the trypanosomes from their
morphology alone.
It is known that T. brucei and T, gambiense develop in the stomach
into long thin trypanosomes, which then make their way to the proboscis,
enter the hypopharynx, and travel to the salivary glands, where crithidia
forms and eventually metacyclic trypanosomes are produced (Figs. 217 and
224). They pass down the hypopharynx with the salivary secretion, and
are injected into the vertebrate when the fly feeds. It is evident that in
the case of these trypanosomes they may occur in the stomach, proboscis,
and salivary glands. Those which may be found in the proboscis are
merely travelling forms, either on their way to the salivary glands from
the stomach, or from the salivary gland to the vertebrate host. At all
stages the trypanosomes have fiagella except the metacyclic forms, which
resemble the short stumpy trypanosomes occurring in the blood.
In the case of T. congolense, development takes place in the stomach,
with the production of long slender trypanosomes, which migrate to the
proboscis (Figs. 218 and 228). In the labial cavity crithidia forms are pro-
duced, and these make their way into the hypopharynx, where the crithidia
forms give rise to metacyclic trypanosomes which resemble the blood
forms. As in the vertebrate host, all these stages are devoid of fiagella,
so that they can be distinguished by this character from most of the
stages of T. brucei and T. gatnbiense. The metacyclic trypanosomes of
T. brucei and T. gambiense, though devoid of fiagella, differ from the
metacyclic trypanosomes of T. congolense in size and other respects.
The development of trypanosomes of the T. vivax group in tsetse flies
is limited to the proboscis (Figs. 219 and 233). Trypanosomes are taken
into the stomach, but these quickly degenerate. Before they do so they
can be distinguished from other trypanosomes by their characteristic
swollen posterior ends and fiagella. The trypanosomes in the proboscis
become quickly transformed in the labial cavity into crithidia forms with
flagella. These pass into the hypopharynx, where metacyclic trypano-
somes of the blood type are evolved. All these stages have fiagella.
As regards the trypanosomes which may occur in the stomach, a
difficulty is introduced in that another trypanosome, T. grayi, commonly
occurs in this region (Figs. 220 and 173). It probably represents develop-
mental stages of the trypanosome of the crocodile or the monitor. The
characteristic type is a broad crithidia form. Trypanosome forms also
occur, but these have very short flagella, and differ in other respects from
T. brucei and T. gambiense. It will thus be seen that the following forms
can be recognized in the different regions of the body of tsetse flies in
which development occurs:
PATHOGENIC TRYPANOSOMES IN TSETSE FLIES 517
Stomach.— T. brucei and T. gambiense : Trypanosomes of the blood
types which multiply and become transformed into long slender trypano-
somes with flagella.
T. congolense : Trypanosomes of the blood type which multiply and
become transformed into long trypanosomes without flagella.
T. vivax : Trypanosomes of the blood type which quickly degenerate.
T. grmji : Long crithidia forms and long trypanosome forms with very
short flagella.
Labial Cavity. — T. brucei and T. gambiense : Trypanosomes of the
blood type on their way to the stomach; long slender trypanosomes with
flagella which are passing from the stomach to the hypopharynx.
T. congolense : Trypanosomes of the blood type on their way to the
stomach; long trypanosomes without flagella from the stomach; crithidia
forms without flagella.
T. vivax : Trypanosomes of the blood type; crithidia forms with flagella.
Hypopharynx. — T. brucei and T. gambiense : Long trypanosomes
with flagella on their way from the stomach and labial cavity to the salivary
glands; short stumpy metacyclic trypanosomes without flagella passing
from the salivary gland to the vertebrate.
T. congolense : Crithidia forms without flagella from the labial cavity ;
metacyclic trypanosomes of the blood type.
T. vivax : Crithidia forms from the hypopharynx; metacyclic trypano-
somes of the blood type.
Salivary Gland. — T. brucei and T. gambiense : long slender trypano-
somes with flagella from the stomach; crithidia forms; short stumpy
metacyclic trypanosomes without flagella.
There seems to be some doubt as to the path taken by trypanosomes
in their passage from the labial cavity to the hypopharynx. In the
diagrams given by Lloyd and Johnson (Figs. 217-220) they are represented
as passing through a slit in the wall of the hypopharynx. Authorities
are by no means agreed that such an opening exists. If the hypopharynx,
which is really a continuation of the salivary duct, is a closed tube, then
it must be supposed that the trypanosomes enter it at its open end at the
extremity of the proboscis.
Experimentally Proved Vectors of Pathogenic Trypanosomes of Africa.
Glossina brevipalpis: T. brucei, Bruce et al., 1914; Braun and Teichmann,
1914. T. congolense, Bruce et al., 1914; Braun and Teichmann, 1914. T. caprce,
Bruce et al, 1914. T. simice, Bruce et al, 1914 (dissection of flies only).
Glossina fusca: T. gambiense, Ross (P. H.), 1908. TJndetermiyied, Greig, 1905
(? which fly).
518 FAMILY: TRYPANOSOMIDiE
Glossina longipalpis: T. pecaudi, Bouet and Roubaiid, 1910. T. dimorphon,
Bouet and Roubaud, 1910. T. casalboui, Bouet and Roubaud, 1910.
Glossina longipennis: T. dimorphon, Ross (P. H.), 1913. Undetermined,
Greig 1905 (? which fly).
Glossina morsitanS: T. gambiense, Taiite, 1911; Rodhain, Pons, Vanden-
branden, and Bequsert, 1912; Kleine and Fischer, 1912; Bruce, 1915. T. brucei,
Taute, 1909 (quoted by Kleine); Bruce et al., 1913; Duke, 1916. T. brucei {G. palli-
dipes"!}, Bruce et cd., 1895. T. brucei (T. dimorjihon), Fehlandt, 1911. T. brucei
(T. rhodesiense), Kinghorn and Yorke, 1912; Bruce et (d., 1913. T. pecaudi, Bouet
and Roubaud, 1911; Rodhain, Pons, Vandenbranden, and Bequaert, 1912. T. con-
golense, Fehlandt, 1911; Rodhain, Pons, Vandenbranden, and Bequaert, 1912;
Bruce et al., 1913; Kinghorn and Yorke, 1912; Duke, 1916. T. dimorpilion, Bouet
and Roubaud, 1912. T. vivax, Duke, 1916. T. cazalboui, Bouet and Roiibaud,
1911; Rodhain, Pons, Vandenbranden, and Bequsert, 1912; Roubaud, 1915. T. caprce,
Fehlandt, 1911; Bruce et al, 1913. T. uniforme, Duke, 1916. T. simiw, Bruce
et al, 1912. T. simiw {T. ignotum), Kinghorn and Yorke, 1912.
Glossina pallidipes: T. gambiense, Ross (P. H.), 1907. T. brucei, Duke, 1916.
T. brucei {G. morsitans ?), Bruce et al., 1895 (?). T. congolense, Croveri, 1919. Un-
determined, Bruce, 1895; Greig, 1905 ("? which fly).
Glossina palpalis: T. gambiense, Dutton, Todd, and Hannington, 1907; Kleine,
1909; Bruce et al, 1909; Robertson, 1912; Fraser and Duke, "1912; Kleine and
Fischer, 1913. T. brucei, Minchin, Gray, and Tulloch, 1906; Miuchin, 1907; Martin,
Lebceiif, and Roubaud, 1908; Kleine, 1909; Fischer, 1911; Fraser and Duke, 1912;
Eckard, 1913. T. j>ecaudi, Cazalbou, 1906; Bouet and Roubaud, 1910. T. congo-
lense, Bruce, 1910; Fehlandt, 1911; Duke, 1912. T. dimorphon, Dutton, Todd,
and Hannington, 1907; Roubaud, 1907; Bouet, 1907; Bouet and Roubaud, 1910.
T. vivax, Bruce et al., 1909. T. cazalboui, Bouet, 1907; Boufiard, 1909; Bouet and
Roubaud, 1910. T. uniforme, Fraser and Duke, 1912. Undetermined, Bruce and
Nabarro, 1903; Bruce, Nabarro, and Greig, 1903; Nabarro and Greig, 1905; Greig
and Gray, 1905.
Glossina swynnertoni: T. brucei {T. rhodesiense), Duke, 1923.
Glossina tachinoides: T. gambiense, Lloyd and Johnson, 1924. T. brucei,
Lloyd and Johnson, 1924. T. brucei {T. pecaudi), Macfie, 1914. T. pecaudi, Bouet
and Roubaud, 1910. T. congolense, Macfie, 1914; Lloyd and Johnson, 1924.
T. dimorphon, Bouet and Roubaud, 1910. T. vivax, Macfie, 1914; Lloyd and
Johnson, 1924. T. cazalboui, Boiiffard, 1910; Bouet and Roubaud, 1910.
Stomoxys: T. gambiense, Schuberg and Kuhn, 1911. T. brucei, Minchin, Gray,
and Tulloch, 1906; Martin, Leboeuf, and Roubaud, 1908; Schuberg and Kuhn. 1911.
T. pecaudi, Bouet and Roubaud, 1912. T. dimorjyhon, Bouet and Roubaud, 1912.
T. cazalboui, Bouffard, 1907; Bouet and Roubaud, 1912.
Tabanus: T. brucei, Sergent (Ed. and Et.), 1906.
CuLEX: T. gambiense, Roubaud and Lafont, 1914. T. brucei {T. rhodesiense),
Roubaud and Lafont, 1914.
Mansonia: T. brucei, Martin, Lebceuf, and Roubaud, 1908; Heckenrotli and
Blanchard, 1913.
A'edes (Stegomyia): T. gambiense, Roubaud and Lafont, 1914. T. brucei,
FilUeborn and Meyer, 1907; Roubaud and Lafont, 1914.
Passage of Trypanosomes from Parent to Offspring. — As a general rule,
it may be said that the young born of an infected parent are not
INFECTION THROUGH PLACENTA AND FILTER PASSERS 519
themselves infected, and that they are just as susceptible to inoculation
as the parent was in the first place. An infected animal often gives birth
to still-born young, and in some instances the young born alive have been
found infected. The first observation of intra-uterine infection was made
by Sivori and Lecler (1902), who noted that a guinea-pig infected with
T. equinum of mal de Caderas gave birth to an infected young one.
Sergent, Ed., Et., and Lheritier (1919) showed that the blood of still-
born offspring of camels infected with T. herherum was infective to dogs.
Sergent, Ed., Et., and Donatien (1920) further reported the finding
of trypanosomes in the organs of still-born camels, and noted that if the
infection in the parent is in the acute condition, the foetus becomes
infected, while no infection takes place if the parent has clinically
recovered, though its blood is still infective to laboratory animals.
Bassett-Smith (1919) and Stevenson (1919) showed that trypano-
somes occurred in the organs of the foetuses of rats which were infected
with T. rhodesiense. Bassett-Smith (1921) also noted that young guinea-
pigs born of a parent infected with T. gmnbiense showed trypanosomes
in the blood about a month after birth. In this case, the infection
may have occurred through the milk. A guinea-pig born of an infected
parent and another born of a healthy parent were exchanged. The
healthy mother, suckling the infected young one, did not acquire
an infection, nor did the healthy young one become infected,
though suckled by the infected parent. Nattan-Larrier (1921) showed
that T. cruzi sometimes passed through the placenta to the foetus in
guinea-pigs.
The question of transmission of trypanosomes from parent to offspring
by way of the milk has been studied by Lanfranchi (1915, 1916, 1918,
1918a). Infected dogs, cats, guinea-pigs, and rats were used. Milk from
infected animals was inoculated into susceptible animals, and infections
were produced with T. brucei, T. rhodesiense, T. gambiense, T. evansi, and
" T. lanfranchi " (T. evansi). Offspring suckled by infected mothers
became infected with T. brucei, T. gambiense, and " T. lanfranchi."
Nattan-Larrier (1913) noted that T. cruzi was frequently present in the
milk of infected animals and T. equiperdmn occasionally. Velu and
Eyraud (1916) noted that one pup of a litter suckled by a bitch infected
with the horse trypanosome of Morocco, T. moroccanum (T. evansi),
acquired the infection. Evans (1880) noted that a pup which was suckled
by a bitch infected with T. evansi acquired the infection. Kellesberger
(1925) has seen a woman and her ten-day-old infant both with trypano-
somiasis in the Congo. It would seem probable that this was an instance
of intra-uterine infection. The degree of enlargement of the spleen and
the number of trypanosomes in the blood of the infant would seem to
520 FAMILY: TRYPANOSOMID^
exclude the possibility of infection after birtli from the milk or as a result
of mechanical transmission by insects.
Trypanosomes as Filter Passers. — Various observers have tested the
capacity of trypanosomes to pass through porous filters which will
not allow the passage of bacteria. In the case of relapsing fever spiro-
cheetes it is known that the entire organism, probably on account of its
peculiar movements, is able to pass through such filters. In the case of
trypanosomes it is the opinion of some observers that this factor cannot
account for the passage through the pores of the filter, and that some
stage which is smaller than the usual form must exist. Novy and
MacNeal (1904a) first showed that the passage through a Berkefeld
filter of diluted blood containing T. lewisi yielded a filtrate which was
infective to rats. Experiments with T. hrucei gave negative results.
In these experiments, according to Wolbach, Chapman, and Stevens (1915),
the filters had been " thinned down " and were not shown to be impervious
to bacteria. Bruce and Bateman (1908) used filters which were proved
to prevent the passage of Micrococcus melitensis, and found that T. evansi
from the blood and organ juices of normal animals, and those which had
been treated with antimony and the cultural forms from blood-agar
medium, could not pass through. Bruce et al. (1911ji') again made
similar experiments with the developmental forms of T. gambiense in
Glossina palpalis, but obtained only negative results. Wolbach, Chapman,
and Stevens (1915) conducted a very careful series of experiments under
varying pressures in which care was taken to prevent the clogging of the
pores of the filters. Three trypanosomes were used- — T. gambiense, T.
brucei, and T. lewisi. The conclusion arrived at is that trypanosomes from
cultures and animal tissues are not filterable through bacteria-proof filters.
More recently Reich and Beckwith (1922) and Reich (1924) have repeated
the experiment. They used the macerated organs of guinea-pigs which
had died of T. brucei infections. The fluid was filtered after the addition
of a loopf ul of culture of Bacillus prodigiosus. The filtrate was immediately
inoculated into guinea-pigs, and a control culture was made on glucose
agar medium to determine the presence or absence of bacteria. In a series
of seventy-two experiments in which the filtrate was free from bacteria,
guinea-pigs became infected with T. brucei on twenty occasions. A series
of seven experiments made with highly infected blood taken from the
animals during life gave only negative results. It appears, therefore, that
the filterable form is to be found in greatest number in the organs, espe-
cially the liver and spleen, of animals which have died of an infection. It
does not follow from these experiments that invisible or ultra-microscopic
stages of trypanosomes exist. The plasticity of the body compared with
that of rigid bacteria would enable an organism to pass through narrow
CLASSIFICATION OF PATHOGENIC TRYPANOSOMES 521
passages and round corners in which bacteria would become impacted.
It has been shown that filters which are impermeable to bacteria on
filtration will, nevertheless, allow bacteria to grow through them if
sufficient time for multiplication is allowed. It is quite possible that in
the experiments of Reich and Beckwith the positive results depend upon
the altered trypanosomes in the tissues of dead animals being more
plastic and even smaller than those in living animals. Such forms are
probably in a degenerate condition, though capable of revival if brought
into a favourable environment such as occurs when they are inoculated
into a living animal.
Classification of the Pathogenic Trypanosomes. — Many attempts have been
made to separate the pathogenic trypanosomes from one another
on purely morphological grounds. To a certain extent this can be
done, but there are many named species which cannot be recognized
from their morphological features alone. Thus, there exist many trypano-
somes which are structurally like T. evansi, but have been separated by
cross-immunity tests and differences in the susceptibility of laboratory
animals. It seems to the writer that it has not yet been proved that these
tests are of specific value. It would be a remarkable circumstance if
T. evansi, which in its natural home in India produces surra in horses,
had not spread to other countries in view of the extent to which horses
have been moved from one part of the world to another during the last
two or three hundred years. Many of the trypanosomes of North Africa
and South America, for instance, resemble T. evansi in their morphology,
but have been separated from it as distinct species by the methods men-
tioned above. It seems more probable that these are merely races of
T. evansi which have been slightly modified by local conditions after long
separation from the parent stock. The same remark applies to the various
trypanosomes of the T. congolense group. If this view is adopted, then the
pathogenic trypanosomes can be grouped in species according to their
morphological characters, and the very similar forms which reveal dif-
ferences in regard to immunity and virulence for laboratory animals only
may be considered as races of these. On this basis it is possible to recognize
in the tsetse-fly areas of Africa the following forms: T. brucei (including
T. rhodesiense) in man and animals, T. gafubiense in man and occasionally
in domestic animals, T. congolense and T. simice in animals, T. vivax in
animals (once found in man), T. cajproe and T. uniforme in animals. In
tsetse-free areas of Africa, as also in other parts of the world, there is
T. evansi or its races and T. equiperdmn in animals.
The pathogenic trypanosomes are often spoken of as being either
polymorphic or monomorphic. In this connection the terms apply purely
to the appearance of the trypanosomes in the vertebrate host or in inocu-
522 FAMILY: TRYPANOSOMID^
lated animals. As a matter of fact, when the whole life-cycle is taken into
account, all trypanosomes are markedly polymorphic. If it is understood
that only the blood stage in the vertebrate is referred to, the term has its
application. Thus T. gambiense, T. brucei, and T. rhodesiense are under
these circumstances polymorphic. There are two extreme types of try-
panosome. One is long and narrow, and has a well-developed fiagellum,
while the other is shorter and broader, and has no fiagellum. A form
intermediate between these two types occurs in which there is a short
fiagellum. The three types are spoken of as " the short stumpy," " the
long thin," and " intermediate " trypanosomes. T. evansi and its allies —
T. congolense, T. simiw, T. vivax, T. unifortne, T. cajprce, T. equiperdum,
and T. equinum — on the other hand, are monomorphic. In the case of
T. evansi, T. equiperdutn, and T. equinum all the trypanosomes have
flagella, and, unless they are dividing forms, they are of more uniform
dimensions than the polymorphic forms, and are very similar to the long
narrow forms of T. brucei. In T. vivax, T. unifor?ne, and T. caprce all
forms have flagella and a characteristic swollen posterior end of the body.
They differ from one another in size. In the case of T. congolense and
T. simicB, again, the trypanosomes are miich smaller than the members
of the other groups, and there is no fiagellum.
In accordance with these morphological distinctions, there are dif-
ferences in the developmental cycles in the invertebrate host. Thus the
polymorphic forms [T. ga?tibiense and T. brucei) develop in tsetse flies
in the stomach, proboscis, and salivary glands; the T. congolense group in
tsetse flies in the stomach and proboscis ; and the T. vivax group in tsetse
flies in the proboscis only. T. evansi and its allies are not carried by tsetse
flies, but are transmitted by tabanids or other biting flies. No develop-
mental cycle has been detected in them. T. equiperdmn, which is possibly
derived from T. evansi, is in an anomalous position in that it does not
require an invertebrate host.
When the size of any species of trypanosome is referred to, it must be
remembered that this is the average size obtained by the measurement of
several hundred or a thousand individuals. On the basis of the foregoing
facts, the classification of the pathogenic trypanosomes as given on p. 458
(Group B, I. and II.) may be extended as follows:
I. Pathogenic Teypanosomes transmitted by Blood-Sucking
Arthropoda.
1. Pathogenic Trypanosomes transmiUed by Species of Glossina.
(a) Development in stomach, proboscis, and salivary glands of
the tsetse flies. Polymorphic trypanosomes (short stumpy forms
CLASSIFICATION OF PATHOGENIC TRYPANOSOMES 523
without flagellum, long thin forms with flagellum, and intermediate
forms).
T. gainbiense. — Does not show posterior nucdear forms in small labora-
tory animals.
Size: Length 13 to 32 microns (average 22-5 microns); breadth 1*5
to 3 microns.
Pathogenicity: When established in laboratory animals, very virulent.
Much less virulent at first inoculation from man, but monkey most sus-
ceptible.
T. brucei {T. rhodesiense, T. pecaudi). — Shows posterior nuclear forms
in small laboratory animals.
Size: Length 12 to 35 microns (average 21 to 23 microns); breadth
1-5 to 3-5 microns.
Pathogenicity: Very virulent for laboratory animals, even at first
inoculation from man or other naturally infected hosts.
(6) Development in the stomach and proboscis of tsetse flies. Mono-
morphic trypanosomes with no flagellum.
T. congolense {T. nanum, T. pecormn). — Size: Length 9 to 18 microns
(average 14 microns); breadth 2 to 3 microns.
Pathogenicity: Virulent for all laboratory animals. After passage
through goat loses virulence for laboratory animals, and then resembles
the natural strain, T. naniwi, which is not inoculable to laboratory
animals.
T. smiicB. — Size: Length 14 to 24 microns (average 18 microns); breadth
1 to 2-75 microns.
Pathogenicity: Not inoculable to small laboratory animals, but highly
virulent for monkeys and goats.
(c) Development in the proboscis only of tsetse flies.
Monomorphic trypanosomes with flagellum and characteristic swollen
posterior end.
T. vivax (T. cazalboui). — Size: Length 15-5 to 30-5 microns (average
25 microns); breadth 2 to 3 microns.
Pathogenicity: Not inoculable to laboratory animals as a rule, though
rabbits have been infected.
T. unif or 7ne.— Size: Length 12 to 19 microns (average 16 microns);
breadth 1-5 to 2-5 microns.
Pathogenicity: Not inoculable to laboratory animals.
T. caprcB.—^ize: Length 18 to 32 microns (average 25-5 microns);
breadth 1-75 to 4-25 microns.
Pathogenicity: Not inoculable to laboratory animals.
524 FAMILY: TRYPANOSOMIDJ^
2. Pathogenic Trypanosomes transmitted by Species of Tabanus or
Other Blood-Sucking Arthropoda.
(a) Monomorphic trypanosomes with flagelliim. Kinetoplast well
developed.
T. evansi and many allied forms which are probably races of T. evansi.
Size: Length 18 to 34 microns (average 24-9 microns); breadth 1-5 to
2*5 microns.
Pathogenicity: Virulent for all laboratory animals.
(6) Monomorphic trypanosomes with flagellum. Kinetoplast rudi-
mentary.
T. equinum. — Size: Same as T. evansi.
Pathogenicity: Virulent for all laboratory animals.
IL Pathogenic Trypanosomes Secondarily adapted to Direct
Passage from Vertebrate to Vertebrate.
(a) Monomorphic trypanosomes with flagellum.
T. equiperdum. — Size: Length 16 to 35 microns (average 24 microns);
breadth 1-5 to 2-5 microns.
Pathogenicity: When established, virulent for all laboratory animals,
but very difficult to inoculate from horse in the first place. Dog most
susceptible.
According to this scheme, the above trypanosomes can be separated
from one another on morphological grounds, with the possible exception of
T. equiperdum, which structurally is closely related to T. evansi.
1. PATHOGENIC TRYPANOSOMES TRANSMITTED BY SPECIES OF
GLOSSINA.
( /) Trypanosomes which Develop in the Stomach, Proboscis, and Salivary Glands of
Tsetse Flies. — Polymorphic Trypanosomes
Trypanosoma gambiense Dutton, 1902.— Synonyms : T . ugandense Ciis.iQ\-
lani, 1903; T. castellanii Kruse, 1903; T. hominis Manson, 1903; T.fonlii Maxwell-
Adams, 1903; T. gamhice Maxwell-Adams, 1903; Trypanosoon gambiense (Liilie, 1906);
Trypanosoma rovumense Beck and Week, 1913; T. tullochii Minchin, 1907; T. nige-
riense Macfie, 1913 ; Castellanella gambiense (Chalmers, 1918); C. castellanii
(Chalmers, 1918).
T. gambiense was first seen by Ford in the blood of a man in the Gambia,
and was recognized as a trypanosome and named by Dutton (1902). In
the following year, Castellani described as T. ugandense a trypanosome
he found in the cerebro-spinal fluid of a case of sleeping sickness in Uganda,
an observation which was soon confirmed by Bruce and Nabarro (1903).
Following these discoveries, it was definitely established that the disease
known as sleeping sickness was merely the final stage of human trypano-
TRYPANOSOMA GAMBIENSE 525
somiasis, and that the trypanosomes of Button and Castellani were
identical. Hence, T. gcunbiense stands as the correct name, while the
other names become synonyms.
Distribution. — T. gambiense occurs only in Africa. On the West
Coast it is limited to a district between 15° N. and 15° S. of the ec^uator.
Further east, it is restricted to the area between 10° N. and 10° S., its
eastern limits being Lakes Victoria and Tanganyika. In these regions
it occurs in greatest intensity along the rivers and shores of the great
lakes, and, wherever it is found, there the tsetse fly, Glossina palpalis^
also occurs. This association of the fly with the infection in man led to it
being suspected as the carrier of the disease, but the actual part played
by the fly was never properly explained till Kleine made his observations
on the behaviour of trypanosomes in tsetse flies, the first account of which
was published in 1909.
Symptomatology. — In the blood of man the trypanosome never occurs
in great numbers, and long search may be required to discover it. Some-
times it can be more readily found by examination of fluid obtained by
puncture of an enlarged lymphatic gland, and later in the disease in the
cerebro-spinal fluid. At other times its presence has only been demon-
strated by inoculation of fairly large quantities of blood (20 c.c. or more)
into some susceptible animal like the monkey. As a rule, however, careful
search of one or more ordinary blood-films will reveal its presence.
This may have to be repeated on several occasions, for, as is usual in
these infections, the number of trypanosomes in the blood is subject to
definite fluctuations. Age and sex do not appear to influence infection to
any extent unless, owing to habits of occupation, any particular age or sex
is more liable to exposure than another.
It has been noted in certain districts of Africa that natives may be
found to harbour the trypanosome, though apparently perfectly healthy.
On this account it may be difficult to give a definite incubation period,
which is said to vary between two or three weeks and seven years.
Generally, the first symptom noted is irregular fever, which is uncontrolled
by quinine. This is followed by enlargement of the lymphatic glands
and spleen, anaemia, and wasting. A cutaneous eruption in the form of
circular red patches may occur. This stage is due to the invasion of the
blood and lymphatic system by the trypanosomes. A second stage is
due to the extension of the infection to the central nervous system, during
which various nervous symptoms become manifest, leading finally to that
lethargic condition which has given rise to the name sleeping sickness.
Recovery frequently takes place as a result of treatment in the first stage
of the disease, but more rarely when the second stage is reached (see p. 459).
526 FAMILY: TKYPANOSOMID^
Pathology. — The lesions produced in man by T. gamhiense consist
of a hyperplasia of the lymphatic tissue of the body. There is enlargement
of the lymphatic glands and spleen. In the later stages the meninges
are affected, while there is an increase in the cerebro-spinal fluid. Most
marked are the changes about the arteries of the brain and cord, leading
to a thickening of the arterial coat, together with invasion of the area
around the vessel by round cells, which give rise to the characteristic
round-celled infiltration (Fig. 221, A). Mott (1899, 1906) pointed out that
Fig. 221.— Section of Brain of Fatal Cases of Sleeping Sickness. (After
Stevenson, 1922 and 1923; from Tmns. Boy. Soc. Trop. Med. and Tlyg., vol. xi.)
A. Perivascular infiltration by round cells (x200).
B. Frontal lobe, showing two''trypanosomes in grey matter (x ca. 1,000).
the lesions in sleeping sickness resembled those of general paralysis. As
a rule, in animals which do not live long after inoculation, these lesions
are not apparent, but in monkeys with long-standing infections, and even
in smaller animals if they survive for several months, the same round-celled
infiltration about the cerebral vessels can sometimes be demonstrated.
In sections of the tissues of man, trypanosomes are with difficulty found,
but their distribution in guinea-pigs infected with a strain of T. gambiense
from Nigeria has been studied by Stevenson (1917, 1918). Though present
TRYPANOSOMA GAMBIENSE 527
in the blood-stream in very small numbers, they may be found in the
lymph channels in any part of the body in greater numbers. They do not
appear to be intracellular, but can be found between the cells of the brain,
heart, stomach, kidney, and, in fact, any organ of the body where small
patches, probably of an oedematous nature, occur in which the trypano-
somes are considerably more numerous than elsewhere (Fig. 221, B).
Similar results were previously obtained by Wolbach and Binger (1912),
who studied the distribution of T. gavnhiense in rats, guinea-pigs,
and monkeys, and by Yorke (1911) in the case of T. brucei (T. rhodesiense)
in goats.
Stevenson (1922) examined the brain of a fatal case of sleeping sickness
in which trypanosomes had not been seen in the blood for many months,
though careful search had been made. There was a high degree of round-
celled infiltration of the vessels of the brain, and trypanosomes could be
demonstrated in the intercellular spaces of the brain substance (Fig. 221).
These observations seem to indicate that the site of election of the try-
panosomes is not the actual blood-stream itself, but rather the lymphatic
channels. In this connection it is of interest to note that abstraction of
fluid from lymphatic glands as a means of discovering trypanosomes more
readily than in the blood was first advocated by Greig and Gray (1904) as
a means of diagnosing the disease in man. Clapier (1921) observed try-
panosomes in large numbers in the fluid abstracted from hydroceles and
Newham (1919) in peritoneal exudate.
In the case of heavy infections in experimental animals, smears from the
spleen, bone marrow, or other organs may show trypanosomes in an intra-
cellular position. This is a result of phagocytosis, and the trypanosomes
are quickly destroyed. Phagocytosis of this kind occurs in the case of
other trypanosomes, and the process as it occurs m vitro in the case of
T. lewisi was described by Laveran and Mesnil (1904). In the process of
digestion the trypanosomes assume various forms which must not be
mistaken for developmental stages. The trypanosomes which occur in
the cerebro-spinal fluid of human beings in sleeping sickness often have a
curiously abnormal shape.
There appear to be no data to indicate how early in the disease trypano-
somes enter the cerebro-spinal fluid. Broden and Rodhain (1908) state
that the degree of involvement of the central nervous system can be
gauged by the cell content of the cerebro-spinal fluid, A normal fluid
contains not more than three lymphocytes per cubic millimetre. In the
earliest stages of involvement there is an increase in the lymphocytes.
Later there appear medium-sized mononuclear cells, and still later large
vacuolated mononuclear cells. In a series of cases which were clinically
in an advanced stage Pearce (1921) found the number of cells in 1 c.mm.
528 FAMILY: TRYPAXOSOMID^
of cerebro-spinal fluid to vary from 15 to 467. The blood leucocytes in
these cases varied from 4,500 to 12,000 per c.mm.
Keratitis is common in animals infected with pathogenic trypanosomes,
and it occurs less commonly in man. In the case of animals, Morax
(1906, 1907) and Yorke (1911) showed that the condition was due to the
presence of large numbers of trypanosomes in the lymph spaces, w^hich
were often swollen and oedematous.
Morphology. — T. gambiense, after its inoculation into man by Glossina
palpalis, presumably invades the blood and lymphatic channels, and there
multiplies by repeated longitudinal division. On account of its scarcity
in the blood of man, its morphology has been studied chiefly in the blood of
susceptible animals. In the blood of such an animal as the rat or guinea-
pig the trypanosome varies in length between 15 and 30 microns, and, as
pointed out by Minchin (1908), occurs in three types, for which reason it
is regarded as a polymorphic tryjianosome (Plate V., a, p. 456). There is
a short and broad form w^hich has no flagellum, a long thin form with a
flagellum, and an intermediate form (Fig. 222, A-C). These three types
are not sharply defined, as they merge into one another by inappreciable
gradations. Robertson (19126) has shown that the short forms are the
result of division of the long ones, and that they grow into long forms
which divide. The majority of trypanosomes in any infection come
within the dimensions given above, but longer forms are sometimes seen
nearly 40 microns in length. These are generally dividing forms. On the
other hand, trypanosomes less than 15 microns in length are seen,
especially in inoculated rats and guinea-pigs. The occurrence of peculiar
short stumpy forms in the blood of a guinea-pig inoculated with the
South Nigerian human trypanosome combined with its low virulence for
human beings, especially in children, led Macfie (1913a) to suggest its
separation as a distinct species, T. nigeriense (Fig. 222, D). As these
very short forms occur in rats inoculated from human beings with un-
doubted T. gambiense, and as the virulence of this trypanosome for man
varies considerably in other parts of Africa, it is highly probable that
Macfie's trypanosome is merely a strain of T. gambiense of exceptionally
low virulence.
In T. gambiense the nucleus, occupies a central position and the kine-
toplast a point a short distance from the posterior end of the body. As
regards the undulating membrane, it is of moderate width and not markedly
convoluted, being more so than in T. lewisi and less so than in some of the
other pathogenic trypanosomes. Granules of volutin may or may not
be present in the cytoplasm. Multiplication is by longitudinal division in
the usual manner, and calls for no special remark. The supposed sexual
process associated with the production of " latent bodies " described by
TRYPANOSOMA GAMBIENSE
529
Moore and Breinl (1907) has already been considered (p. 332), It may be
noted that the occurrence of short broad forms and long thin trypanosomes
has been supposed to indicate a sexual dimorphism, of which at present
there is no evidence. Robertson (19126) considers that the short broad
Fig. 222. — Trypanosoma gambiense (x 2,000). (A, B, and C, after Bruce, 1912;
D, Original.)
A. Long thin form with fiagellum. B. Intermediate form with short flagellum.
C. Broad stumpy form without flagellum.
D. Small forms "from a rat inoculated with the Nigerian strain.
trypanosome 13 to 20 microns in length is the adult blood form. When
proceeding to divide, growth takes place till the long forms are produced.
These are the dividing individuals, which give rise to the short broad forms
again. Robertson (19126) believes that the short broad forms alone are
capable of development in the tsetse fly.
I. 34
530 FAMILY: TRYPANOSOMIDiE
As already remarked, trypanosomes occur in the cerebro-spinal fluid.
Here they may be found in the later stages of the disease, and are peculiar
in that they exhibit a marked want of uniformity in size and shape-
Curious rounded, stumpy, or pear-shaped forms are often encountered.
These are to be regarded as abnormal or involution forms, and are of no
special significance in the life-history of the trypanosome.
Susceptibility of Animals. — It is possible to inoculate T. gaynhiense
into all the ordinary laboratory animals. Monkeys are readily infected,
but baboons (Cynocephalus) are refractory. Those of the genera Macacus,
Cyno7nolgus, and Cercopithecus (especially C. ruber) are very susceptible.
The smaller animals such as rats, mice, guinea-pigs, and rabbits are not so
readily infected as monkeys when the inoculations are made directly from
man. After a strain has passed through a monkey, it becomes more virulent
for the smaller animals. The dog and the cat are susceptible, as also
goats, sheep, horses, and cattle. Fowls are inoculated with difficulty. The
course of infection in these animals varies considerably. The virulence
of any particular strain increases with passage from animal to animal till
it causes death in two to three weeks in rats and guinea-pigs, and even in
monkeys. The first passage from man may, in small animals, give rise
to an infection lasting several months, or even a year or more, during which
time trypanosomes are with difficulty discovered in the blood. In the
larger domestic animals the infection is of a mild and chronic type,
trypanosomes often being demonstrable only by inoculation of their blood
into more susceptible animals.
Animal inoculations are of service as an aid to diagnosis. One or two
cubic centimetres of blood from a man may be inoculated intraperitoneally
into a rat or guinea-pig, or larger quantities into a monkey. It must be
remembered that a failure to produce infection does not prove that
trypanosomes are absent. In the writer's experience, inoculation
of fairly large quantities of blood known to contain trypanosomes
into rats has not infrequently failed to give rise to any recognizable
infection.
Culture. — In the usual blood media, as, for instance, N.N.N, medium,
which answers well for many flagellates of this group, T. gamhiense may
survive for a fortnight or more, and show changes of structure to the
crithidia type, but it does not multiply to any extent, so that the main-
tenance of a culture by sub-inoculation of fresh tubes does not succeed,
though the flagellates transferred may linger for a week or more before
finally disappearing. Media which contain a comparatively large propor-
tion of human blood give better results than others, but the satisfactory
maintenance of T. gamhiense outside the body has not yet been accom-
plished.
TRYPANOSOMA GAMBIENSE
531
Transmission. — T. gambiense is conveyed from man to man by the
tsetse fly, Glossina palpalis, which has a distribution in Africa corre-
sponding fairly closely with that of the trypanosome (Fig. 223). After
Bruce (1895) had demonstrated the carriage of T. brucei by G. 7norsitans,
many prophetic utterances as to the probable transmission of T. gatnbiense
by tsetse flies were made. The first of these was that of Brault (1898), who,
Fig. 223. — Diagram of Trypanosoma gambiense in the Blood of Man and the
Tsetse Fly {Glossina palpalis). (After Wen yon, 1922.)
A. Ordinary forms in the blood of man. T>. Dividing forms in the blood of man.
P. Trypanosomes passing through proboscis of fly.
S. Trypanosomes in stomach of fly. I. Trypanosomes in salivary gland of fly.
E. Long trj^anosomes which make their way from the stom.ach to the salivary glands via the
proboscis.
B. Crithidia forms developed from the long trypanosome forms in the salivary glands.
C Metacyclic trypanosomes developed from the crithidia forms in the salivary glands. These
forms produce infection when injected with the salivary secretion of the fly.
before the actual discovery of T. gambiense, predicted that sleeping sickness
would probably be found to be a disease caused by trypanosomes and
transmitted by tsetse flies. Bruce, Nabarro, and Greig (1903), working
in Uganda with wild G. palpalis caught off animals, showed that batches
of flies fed on sleeping sickness cases were able to transmit infection to
monkeys up to forty-eight hours after feeding. Combined with the fact
532 FAMILY: TRYPANOSOMID^
that trypanosomes {T. hrucei) had been seen by Bruce (1897) in the
proboscis up to forty-six hours after feeding, the conclusion was arrived at
that the transmission was a mechanical one. The experiments carried out
by Bruce, Nabarro, and Greig (1903), however, indicated that a cyclic
development in the fly was not excluded, for freshly caught flies were
shown to infect monkeys. It was shown by Minchin (1908) and others
that in mechanical transmission it was not possible for a fly to infect more
than one animal, the proboscis being apparently purged of trypanosomes
at the first feed. Minchin, Gray, and Tulloch (1906), and Minchin (1908),
working with single flies {G. palpalis), transmitted T. gamhiense nine times
out of ten by the method of interrupted feeding, by which the flies were
allowed to commence their feed on an infected animal and to complete it
on a healthy one. Bruce et at. (1910/), by using laboratory bred flies,
proved that mechanical transmission of T. gatnbiense by G. palpalis could
take place within two hours of feeding. It was recognized that in the
earlier experiments noted above, when wild flies were used, what had been
regarded as mechanical transmission after forty-eight hours was probably
due to the flies having already been infected in nature.
Much confusion regarding the behaviour of trypanosomes in the flies
was at flrst caused by Herpetotnonas grayi, which was not distinguished
from T. gamhiense (Figs. 173 and 220). Minchin, Gray, and Tulloch (1906)
first showed that this flagellate was distinct from T. gamhiense, a fact
which was recognized later by Novy (1906), who examined films sent him
by Gray. After H. grayi had been recognized, the behaviour of T. gam-
hiense in G. palpalis was studied by Minchin, Gray, and Tulloch (1906),
who found that the ingested trypanosomes disappeared entirely from the
gut of the fly in four days. This led Minchin (1908) to express the view
that T. gamhiense in Uganda was transmitted by G. palpalis in a purely
mechanical manner; though influenced by the work of Koch (1905) and
Stuhlmann (1907), chiefly on T. hrucei, he still held that, given the proper
conditions and the proper fly, a true cyclic development would be found
to take place. Koch (1905) noted that a fluid free from red blood-corpuscles
and containing large numbers of trypanosomes could be expressed from
the proboscis of wihl tsetse flies, G. fusca (? G. hrevipalpis), G. morsitans,
and G. palpalis. From what is now known, these trypanosomes, which
were of the vertebrate blood type, were undoubtedly the metacyclic
infective forms. It was probably these forms which Gray and Tulloch
(1905) found in the salivary glands of a fly. Stuhlmann (1907) confirmed
Koch's observations, and also noted long narrow forms in the proven-
triculus. He found that from 3 to 14 per cent, of wild tsetse flies, G. fusca
(? G. hrevipalpis), had trypanosomes in the proboscis. Roubaud (1908)
also obtained trypanosomes from the proboscis of wild flies. Bruce
TRYPANOSOMA GAMBIENSE 533
(1903) had shown in ZuluUmd that the trypanosomes which develop in
the stomach of the tsetse flies are not infective to animals, an observation
which was confirmed by Koch (1905), Gray and Tulloch (1905), Minchin,
Gray, and Tulloch (1906), and Bouet (1907). Minchin (1908) remarks
that Manson made the suggestion that this lack of infectivity was due to
the trypanosomes being in a developmental stage, and was in favour of a
developmental cycle in the fly. Other observers, notably Cazalbou (1906),
Dutton, Todd, and Hannington (1907), Bouet (1907), Eoubaud (1907),
Ross, P. H. (1908), and others, made contributions to the subject without,
however, solving the problem. They all effected transmission of trypano-
somes by means of tsetse flies fed first on infected animals and shortly
after on healthy ones. As wild flies were used, it is highly probable that
some of the flies were already infected when caught. Ross, P. H. (1907),
succeeded in infecting a monkey with what he regarded as T. gambiense
by means of wild G. pallidipes, and in the following year (1908) a monkey
with the same trypanosome by G. fusca feeding alternately on an infected
and the healthy animal. There is, however, considerable doubt as to
the species of trypanosome used in these experiments. Kleine (1909a),
working in German East Africa with T. brucei, discovered that laboratory
bred flies do not become infective till after the expiry of about twenty
days from their infecting feed. This important observation proved con-
clusively that a definite cycle of development took place in the fly, and
explained the failure of the earlier observers, who did not extend their
experiments over a sufficiently long period after feeding the flies on infected
animals. Kleine's experiments were conducted with T. brucei and G.
palpalis, but his results were quickly confirmed by Bruce et al. (1909,
1910a, 191 1(^), working in Uganda with T. gambiense and G. palpahs.
The important fact of the necessary incubation period in tsetse flies
having been established, it was soon demonstrated by Bruce et al. (191 Irf)
that T. gambiense went through a cycle of development terminating in
infection of the salivary glands, where infective metacyclic trypanosomes
appeared (Fig. 223). Bruce et al. (1911c) showed that after ingestion by
the fly, T. gambiense in the stomach remained infective to inoculated
animals for two days, after which the infectivity was lost. The forms
which appear in the salivary glands eventually become capable of infecting
animals, the period of non-infectivity of the fly forms corresponding with
that during which the fly is unable to transmit the infection by its bite.
Though G. palpalis is the host of T. gambiense, not every fly which
feeds on an infected animal becomes infective. The percentage of flies
in which the developmental cycle completes itself varies, but it is well
under 10 per cent. As regards the transmission of T. gatnbiense by other
species of tsetse flies there is some experimental evidence. Rodhain,
534 FAMILY: TRYPANOSOMID^
Pons, Vandenbranden, and Bequa^rt (19126) succeeded in transmitting
T. gambiense by means of laboratory bred G. morsitans fed on infected
monkeys. Kleine and Fischer (1912) also succeeded in a similar experi-
ment, as did Bruce et al. (1915). Taute (1911) fed G. morsitans on a
monkey which had been infected from a man. These flies kter infected
healthy monkeys. Lloyd and Johnson (1924), working in North Nigeria,
have transmitted T. gambiense by means of G. tachinoides , in which a
complete development of the trypanosomes occurred. It appears that
in certain areas this fly is responsible for the spread of sleeping sickness.
These instances can only be regarded as exceptions to the general rule
that T. gambiense is transmitted in nature by G. 'palpalis.
T. gambiense may also be transmitted in a mechanical manner by
mosquitoes and other biting flies. Thus, Heckenroth and Blanchard
(1913) showed that mosquitoes {Mansonia uniforinis) could infect guinea-
pigs within twenty-four hours of feeding on an infected animal, while
Minchin, Gray, and Tulloch (1906) were successful with Stomoxys which
had partially fed on an infected animal and had completed its feed on a
healthy one. In the latter case, the trypanosome transmitted was possibly
T. brucei, and not T. gambiense. Duke (1919, 1923) has come to the
conclusion that mechanical transmission of a virulent strain of T. gam-
biense from man to man was largely responsible for the spread of sleeping
sickness through Uganda from 1900 to 1910. He believes that when
human trypanosomiasis occurs in epidemic form, mechanical transmission
is responsible for the rapid spread of the disease, while transmission
associated with the cycle of development in the fly maintains the disease
in endemic form.
Cycle in the Tsetse Fly.— The main outline of the development of
T. gambiense in G. palpalis (Fig. 223) was described by Bruce et al. (1911f7).
It was studied in greater detail by Robertson (1913), whose account is
follov/ed here.
When a fly hatched from the pupa ingests blood and trypanosomes
from an infected animal, one of several alternatives may occur.
1. The trypanosomes may be destroyed and disappear during the
fifty to seventy-two hours during which the blood is being digested.
1. Trypanosome of the blood-stream. 2. Division of blood form.
3. Tryijanosome in mid-gut thirty-six to forty-eight hours after feeding.
4-6. Tryimnosome in hind-gut third or fourth day of cycle.
7. Trypanosome in mid-gut on fifth day.
8. Large multiple form (delayed division) sixth day.
9-11. Trypanosomes in gut twelfth to twentieth day.
12-13. Slender proventricular types — final form of gut development.
14-15. Form newly arrived in salivary glands.
16-20. Typical salivary gland— crithidia forms.
21-23. Final trypanosome types in salivary glands (metacj'clic trypanosomes).
TRYPANOSOMA GAMBIENSE
535
Fig. 224. — Development of Trypanosoma gamblense in Glossina palpalis (x 3,000)
(After Robertson, 1913.)
[ For description see opposite page.
536 FAMILY: TRYPANOSOMID^.
2. Trypanosomes may not entirely disappear with digestion of the
first feed of blood, but do so at the second.
3. They may survive and multij)ly in the gut, although a second feed
of blood has been superadded.
i. They may survive and multiply in the crop for as long as twelve
days, provided the crop has never been entirely emptied of blood. In such
cases the gut may be entirely free from trypanosomes. Those in the crop
are unable to survive a complete emptying of the organ, and no permanent
infection of the fly results if this takes place.
5, The trypanosomes may persist in greater or less numbers both in
the gut and in the crop of the same fly.
6. The whole of the partially digested blood which survives from the
first feed may be displaced by the fresh blood of the second feed without
the trypanosomes which are present in the stomach disappearing. The
crop in these cases may be either empty or filled with new blood.
Of these various conditions, any one of which may result from a feed
of infected blood, the last appears to be the one which leads to a permanent
infection of the fly. It is thus evident that only a small percentage of the
flies actually fed will acquire an infection. In the flies in which infection
will occur, active multiplication of the trypanosomes in the mid- and hind-
gut commences soon after the infecting feed, and continues progressively.
There is no intracellular stage of the trypanosomes, no stage in which they
are attached to the gut wall, and in no case do they disappear from the
gut to reappear at a later period. Reproducing trypanosomes are thus
constantly present in the lumen of the gut.
Thirty-six to forty-eight hours after ingestion many degenerating
trypanosomes are present, together with dividing healthy forms, which
appear to be all of the short broad type which were present in the blood of
the vertebrate. They differ little at this stage from the blood type
(Fig. 224, 1-2), though the undulating membrane may be a little straighter
and the kinetoplast slightly displaced towards the nucleus (Fig. 224, 3).
The division results in the formation from the parents of daughter indi-
viduals, which exceptionally may have, for a short time only, the crithidia
arrangement of nucleus and kinetoplast. The trypanosome arrangement,
however, is quickly regained. These crithidia forms only occur at the
early divisions, and they are the only indication of a crithidia phase in the
intestinal development. By the tenth day a large number of trypano-
somes remarkable for the variety of their shape and size has been pro-
duced, but the maximum length attained by any one does not exceed
35 microns (Fig. 224, 4-1 1). At this stage there may app r a small
number of characteristic slender individuals. From the tentL to the
fifteenth day these slender forms are developed gradually from the broader
TRYPANOSOMA GAMBIENSE 537
forms in increasing numbers. They are to be regarded as the regular
proventriculus type, and difier from the broad forms which they are
replacing in having a more finely granular cytoplasm, a nuclear karyosome
reduced in size, and a nuclear membrane which stains more deeply (Fig. 224,
12-13). Division of the slender forms may still take place. During this
multiplication period there are produced an enormous number of trypano-
somes, which invade the anterior part of the mid-gut, and finally the
anterior gut and proventriculus. The anterior part of the anterior gut
and the proventriculus contain the long slender trypanosomes which invade
this portion of the intestine between the tenth and twentieth day. Pro-
longed fasting causes the infection to pass back again till it becomes
limited to the middle and hind part of the mid-gut. A fresh feed again
brings the infection forwards to the proventriculus. If, however, new
blood is taken in while the proventriculus infection exists, the trypano-
somes maintain their position. The long slender trypanosomes are the
forms which were seen by Stuhlmann (1907) in the proventriculus.
The further development is brought about by the slender proventriculus
forms passing into the labial cavity and hypopharynx, and thence into the
narrow tubular portion of the salivary gland, which consists of a narrow
tube, a broader cellular part, and finally the still broader terminal glandular
portion (Fig. 224, 14-16). The trypanosomes attach themselves to cells
of the cellular part or commencement of the glandular part. They
gradually transform into broad crithidia forms with rounded posterior
ends (Fig. 224, 16-20). They multiply, and gradually the cavity of the
gland becomes filled with flagellates which vary in shape and size
between tadpole-shaped crithidia forms and trypanosome forms resembling
very closely the vertebrate blood type (Fig. 224, 20-23). Division of all
these forms takes place, the crithidia forms being mostly attached to the
surface of the glandular cells. Fresh slender trypanosomes are constantly
travelling up the duct from the hypopharynx, so that there is a continuous
production of fresh crithidia forms, which in their turn produce the try-
panosomes of the blood type. The flies seem to become infective from
two to five days after the slender forms invade the gland. The whole
development occupies about twenty days. The cytology of the gland
forms calls for no special remarks except that their nuclei appear to be
richer in chromatin than those of the slender invading forms.
It will thus be seen that there is an intestinal multiplication phase of the
trypanosome forms, leading to the formation of slender trypanosome
individuals which invade the proventriculus and hypopharynx. These
pass to the salivary glands by way of the duct, and become flagellates of
the crithidia type, which in turn gives rise to metacyclic trypanosomes,
closely resembling the blood type which commenced the developmental
538 FAMILY: TRYPANOSOMIDiE
cycle in the gut (Figs. 217 and 223). As the glands are not infective
when injected into animals till the final trypanosome stages appear, the
latter are the actual infective metacyclic forms. During the whole of this
development no sexual process was observed. In spite of failure to
observe it, Robertson considers that there is a good deal of circumstantial
evidence that a conjugation or some equivalent process takes place. The
passage through the fly seems to have some biological significance in
playing an " essential role in maintaining the integrity of the species,
quite apart from its being a convenient method of transmission." If a
sexual process occurs, it will probably be at that stage " which is absolutely
essential to the production of a trypanosome viable in the blood of the
vertebrate — namely, the crithidial phase in the salivary gland."
Reservoir Hosts. — Owing to the increase of sleeping sickness along the
shores and on the islands of Lake Victoria Nyanza, the prophylactic
measure of removing the native population was adopted. Though this
was carried out, five years later Duke (1915) found that two fly-boys who
had been bitten by G. palpalis on the lake shore or islands contracted
sleeping sickness, an inadvertent experiment which proved that reservoirs
of T. gambiense still existed in the locality. Bruce et al. (191 le) failed to
find T. gambiense in animals examined on the lake shore. Duke (1912a,
1912c), however, was able to demonstrate that the sitatunga, Tragelaphus
spekei, harboured a trypanosome which he regarded as T. gmnbiense. It
was concluded that this antelope was acting as a reservoir for the virus
in 1915. But whether the flies acquired their infection only from this
source must be doubtful, for more recent observation seems to indicate
that the islands had not been kept so free from human beings as was at
first supposed. It was proved by Gray and Tulloch (1907) that the dogs
of LTganda in endemic areas of sleeping sickness might harbour what was
apparently T. gambiense, an observation which was also made, according
to Koch, Beck, and Kleine (1909), by Van Someren. Bruce and his co-
workers (1910c) showed that cattle might act as a reservoir for T. gam-
biense. Healthy animals could be experimentally infected by G. palpalis,
and in the fly area they found a naturally infected cow. In a similar
manner, antelope (water buck, bush buck, reed buck) could be infected
with T. gambiense, while bred flies fed on these animals became infected.
No antelope, however, were found naturally infected, though trypanosomes
were found in a monkey {Cercopithecus pygerythrus centralis) from the
lake shore. Fraser and Duke (1912) showed that antelope may remain
in perfect health for over a year after experimental infection with T. gam-
biense, and that G. palpalis could be infected from them 315 days after
inoculation. Blood from an antelope 327 days after inoculation was still
capable of producing infection in rats. Kleine and Taute (1911) succeeded
TRYPANOSOMA GAMBIENSE 539
in infecting sheep and goats in the same manner. Prolonged search
amongst antelope by Bruce, Kleine and Fischer, Duke and Fraser, and
other workers for a reservoir host for T. gaynhiense did not meet with any
success, except in the case of the sitatunga noted above. Koch, Beck,
and Kleine (1909), and Bruce et al. (1911e) reported having found monkeys
naturally infected with trypanosomes resembling T. gambiense. Trypano-
somes which were possibly T. gambiense were seen by Kleine and Eckard
(1913) in a cow, a sheep, and a goat in Tanganyika, by Duke (1913a) in a
buli'alo and a hyena in Western Uganda, by Yorke and Blacklock (1915)
in a cow in Sierra Leone, and by Simpson (1918) in a reed buck in the
Gold Coast. In none of these cases can it be taken for granted that the
trypanosome was certainly T. gambiense. Unless studied in small labora-
tory animals, it is impossible to distinguish T. gambiense from T. brucei,
and in most of these instances of supposed infection with T. gambiense this
was not done. Even Duke's observation (1912a, 1912c) on the sitatungas,
which has been generally accepted, is open to doubt, for, reinvestigating
these animals (1921), he found that the trypanosomes with which they
were naturally infected produced posterior nuclear forms when injected
into guinea-pigs, and were more virulent than those jDreviously isolated.
He concludes that T. gambiense, by long residence in the sitatunga, has re-
verted to the T. brucei type, but at the same time admits that during the
earlier investigations the significance of posterior nuclear forms was not
realized, so that they were not specially looked for, and may have been
neglected. It would seem, therefore, that search for a reservoir host for
T. gambiense has shown that occasionally domestic animals living in associa-
tion with human beings amongst whom the disease occurs may acquire an
infection, but there is little or no evidence to incriminate the wild game as
reservoirs of this trypanosome. There does not appear to be such a close
connection between T. gambiense and the wild fauna of Africa as in the case
of T. brucei. Probably, therefore, man himself, and sometimes the domestic
animals near him, are most usually the sources from which G. palpalis
derives its infection. T. gambiense, which undoubtedly originated from a
trypanosome of animals (probably T. brucei) in the first place, has now
become adapted to man to such an extent that there is little tendency for
it to infect the game. In this respect it stands in marked contrast to the
human strain of T. brucei {T. rhodesiense).
Trypanosoma brucei Plimmer and Bradford, 1899. — Synonyms: T. suis
Ochmann 1905; Trypanozoon brucei {Lxihe, 1906); T. suis {L,iihc. 1906); Trypanosoma
pecaudi Laveran, 1907; T. togolense Mesnil and Brimont. 1909; T. elephantis Bruce
et al., 1909; T. rhodesiense Stephens and Fantham, 1910; T. ancepis Bruce et al., 1914
T. Uganda; Stephens and Blacklock, 1913; Castellanella brucei (Chalmers, 1918)
C. rhodesiense (Chalmers. 1918); T. multiforme Kinghorn and Yorke, 1913 (?)
T. cqui Blacklock and Yorke, 1914 ('?); T. dulcei Knuth and du Toit, 1921.
540 FAMILY: TRYPANOSOMIDiE
This trypanosome was discovered by Bruce in 1895, and proved to be
one of the causes of nagana, a disease which had long been known to
attack domestic animals in Zululand. Accounts of his observations were
published in 1897 and 1903. The trypanosome was named Trypanosoma
brucii {T. brucei) by Plimmer and Bradford (1899), from the forms which
occurred in an infected dog which had been sent to England by Bruce.
Stephens and Blacklock (1913) noted that the strain was monomorphic,
and resembled T. evansi rather than the polymorphic form here described
as T. brucei. Plimmer and Bradford (1899) described their trypanosomes
as monomorphic, hence Stephens and Blacklock think that the poly-
morphic Uganda trypanosome, which is now generally called T. brucei,
cannot be the same as the original monomorphic Zululand trypanosome,
the true T. brucei. They therefore suggest the name T. ugandce for the
polymorphic form. Bruce, on the other hand, regards his original Zululand
strain as the same as the polymorphic form now generally known as
T. brucei, and ascribes the discrepancy to a change in type which has
probably taken place owing to long maintenance in laboratory animals.
That some profound change had taken place receives support from
Roubaud's observations (1913) that the Pasteur Institute strain was no
longer capable of infecting Glossina morsitans. The writer has noted on
several occasions that trypanosomes inoculated from the blood of man into
laboratory animals may show posterior nuclear forms at first, and that these
disappear entirely in subsequent passages, the trypanosomes tending to
become more and more monomorphic. The figure given by Bruce (1897)
of the trypanosome in the blood of the dog shows definitely a polymorphic
form, while one of the trypanosomes appears to have the nucleus in a
posterior position.
There can be no doubt that one of the trypanosomes causing nagana is
really a polymorphic trypanosome, whatever Plimmer and Bradford said
about the strain they examined in 1899. It is quite possible that they over-
looked or neglected to describe the forms without flagella, a point which
can only be determined by a re-examination of their original films. It is
worthy of note that, though T. brucei had been studied by many observers,
posterior nuclear forms had not been described till Stephens and Fantham
(1910) noted them in T. rhodesiense, the cause of a disease in man. This
trypanosome is indistinguishable from T. brucei, and will be regarded as
the human strain of this species. The question is more fully discussed
below.
Distribution. — The polymorphic trypanosome which was named by
Plimmer and Bradford (1899) is now known to be of wide distribution in
Africa, extending from the Sudan to Zululand, though it has frequently
been described under different names. It has been recorded from the
TRYPANOSOMA BRUCEI 541
Sudan, Uganda, Rhodesia, East Africa, the territory around Lakes Nyasa
and Tanganyika, and all the districts bordering on the Transvaal except
those to the south. It will be seen that in West Africa this trypanosome
is apparently absent, but a form morphologically indistinguishable from
it was described from Togoland and the surrounding districts by Mesnil
and Brimont (1909), and was named by them T. togolense. It was dis-
tinguished from T. brucei by immunity and inoculation tests. A trypano-
some of much wider distribution in West Africa is T. pecaudi Laveran,
1907, which produces a disease known as baleri. Laveran and Mesnil
describe this trypanosome as differing in certain respects from T. brucei,
especially as regards the presence of certain small forms. The trypano-
some which was described by Balfour (1909) and the writer (1909) in the
Sudan as T. pecaudi is indistinguishable morphologically from T. brucei.
Macfie (1913) described a trypanosome from Northern Nigeria which was
indistinguishable from T. brucei, and the same form was isolated by Macfie
(1914) by feeding wild G. tachinoides on guinea-pigs, so that it would appear
that a trypanosome of the polymorphic type exists in animals all over
tropical Africa. According to Laveran and Mesnil, T. pecaudi and T. togo-
lense can be distinguished from T. brucei by their immunity reactions and
other features. It is extremely doubtful, however, if any real specific
differences exist between these forms. Similarly, the trypanosome which
was isolated from a bush buck in the Luangwa Valley by Kinghorn and
Yorke (1912c), and named by them T. multiforme on account of certain
short forms of the T. congolense type, which were mixed with others of
the T. brucei type, may in reality be T. brucei, or possibly a mixed infection
of T. brucei and T. congolense. T. suis, described by Ochmann (1905)
and Geisler (1912) from pigs in Somaliland, is probably T. brucei, as
also T. elephantis, discovered by Bruce et al. (19096) in an elephant of
Uganda. Bruce et al. (1914^) discovered a trypanosome in three dogs
in Nyasaland, which they regarded as an aberrant form of T. brucei.
It produced a chronic type of infection instead of the usual acute one,
and differed slightly in other respects from the usual T. brucei strains.
It was inoculable to rabbits, dogs, and white rats, but not to oxen, goats,
monkeys, or guinea-pigs. Though it was considered to be a modified
form of T. brucei, the name T. anceps was suggested in case it should be
decided to regard it as a new species.
Duke (1913) isolated a trypanosome of the T. brucei type from donkeys
in East Africa. It was readily inoculable to most laboratory animals,
but of seven guinea-pigs inoculated, only one became infected. The
trypanosome was shown to develop in G. 2J(ilp(ilis with salivary gland
infection. Knuth and Du Toit (1921), for reasons which are not quite
clear, propose to name this trypanosome T. dukei. There seems to be no
542 FAMILY: TRYPANOSOMID^
reason to regard it as other than T. brucei. For all practical and scientific
purposes, the polymorphic trypanosome, which is highly virulent for
small experimental animals, and which produces in these animals a varying
percentage of posterior nuclear forms, may be regarded as T. brucei.
There is, however, one difficulty in connection with T. 'pecaudi. Bouet
and Roubaud (1910) studied the development of a trypanosome which
they regarded as T. pecaudi in G. tachinoides, G. longipalpis, and G. pal-
palis. In the first they claim that the development commenced in
the stomach, and was followed by infection of the proboscis only, the
salivary glands not being involved. If this observation is correct, then
there is a definite departure from what is known to occur in the case of
T. brucei. Bouet and Roubaud's account of the development has not yet
received confirmation, and as there is a possibility that they were not
actually dealing with a polymorphic trypanosome, it is better to ignore
it at present.
Susceptibility of Animals. — T. brucei is undoubtedly the most
virulent of the known pathogenic trypanosomes. It is inoculable into
all mammals, including monkeys, with the exception of the baboon
{Cynocephalus), as noted by Bruce (1903). The latter animals enjoy an
immunity, as do the majority of human beings. In the case of the last
named it has long been known that travellers who have been, constantly
bitten by tsetse flies {G. morsitans), and who have lost all their transport
animals through the ravages of these flies, have themselves remained
perfectly healthy. The question of the immunity of man will be referred
to again in connection w4th the relationship of T. brucei and T. rhodesiense.
Horses, mules, and donkeys are very susceptible, and die in a period
varying from a fortnight to three months. For camels the strain is
equally virulent. In the writer's experience, a convoy of over seventy
camels taken into the Bahr el Ghazal province of the Sudan all died of
the infection within a period of two months. Cattle, on the other hand,
are not so rapidly killed as horses, but recoveries are rare. Sheep and
goats are still less susceptible. Death may occur in four or five months,
or few symptoms may be shown. Ultimately recovery may take place,
with immunity to further infection. Pigs, on the other hand, quickly
succumb. Dogs acquire the infection easily, and die in about a fortnight
from the time of inoculation. Cats are slightly more tolerant. Rats and
mice are easily infected. The former live for about a fortnight after
inoculation, and mice for a shorter period. Sometimes, however, rats may
survive for three weeks or more. Guinea-pigs live for three or four weeks.
Monkeys, with the exception of the baboons, die of an intense infection
in three weeks or a month. Many other animals are inoculable, and acquire
infections similar to those indicated above. As a rule, the trypanosome
TRYPANOSOMA BRUCEI 543
multiplies rapidly in the blood till near the end enormous numbers are
present, and death may take place quite suddenly, suggesting a blockage
of some important part of the circulatory system. It must not be forgotten
that the virulence of any particular strain increases with passage through
animals. Many laboratory strains have acquired a high virulence for
rats, mice, and guinea-pigs after long maintenance in these animals.
Some strains will kill mice in two or three days and rats in less than
a week.
Hornby (1919rt), iii Rhodesia, has noted that if a convoy of horses
and cattle are taken through fly areas, the general rule is for the horses or
other equidse to become infected with T. brucei, while the cattle acquire
T. congolense and T. vivax. With few exceptions this rule seems to apply
fairly regularly throughout the tsetse fly areas of Africa. It is possible
therefore that nagana is a disease caused by several distinct trypanosomes.
As regards the susceptibility of birds, Mesnil and Martin (1906) showed
that T. brucei could survive in geese for as long as three months, as proved
by inoculation of blood into guinea-pigs. Durham (1908) had similar results
in the case of an inoculated falcon {Cerchneis tinnunculus). Wendelstadt
and Felmer (1909) showed that T. brucei could survive in the circulation
of snakes and tortoises for about a week. Trypanosomes inoculated into
the body cavity of two beetles survived seven and two days respectively,
as proved by the inoculation of rats. Similarly in a snail the trypano-
somes survived for two days.
Morphology. — T. brucei, being a polymorphic trypanosome, varies
considerably in size (Plate V., b, p. 456). The measurements of a series
given by Bruce (1911) are shown in the curve compared with T. evansi
(Fig. 196). There occur the same three forms as are found in T. gambiense
— namely, the short broad or stumpy form without a flagellum, the long
slender one with a fairly long flagellum, and the intermediate form
(Fig. 225). The short forms, however, tend to be broader than the corre-
sponding ones of T. gambiense, and there is more variation in the position
of the nucleus. In a certain number of broad forms, especially in infection
in small laboratory animals, the nucleus becomes displaced towards the
posterior end (posterior nuclear forms). Sometimes it may actually lie
posterior to the kinetoplast. These posterior nuclear forms occur, not
only in the undoubted T. brucei, but also in T. pecaudi and other West
African strains, a fact which lends support to the view that they are
identical. As will be seen below, they also occur in the human strain of
T. brucei [T. rhodesiense), and afford a means of distinguishing this try-
panosome from T. ga^nbiense. It must be remembered, however, that it is
only in the large infections seen in rats, mice, and guinea-pigs that the
characteristic posterior nuclear forms are met with to any extent. The
544
FAMILY: TRYPANOSOMID^
Fig. 225. — Trypanosoma briicei (x 2,000). (After Bruce, Harvey, Hamertox,
AND Ladt Bruce, 1914.)
A. Broad stumpy form; two posterior nuclear forms are shown, one with kinetoplast behind
the nucleus, and one with it in front.
B. Intermediate form with short flagellum. 0. Long slender form with flagellum.
TRYPANOSOMA BRUCEI 545
measurements of T. brucei (Ziiliiland strain) in inoculated animals are
given by Bruce and his co-workers (1914) as follows:
Number of
Species of Animal. i Trypanosomes
Measurement in Microns.
Measured. Average Maximum Minimum
Length. Length. Length.
160
21-2
31-0
12-0
260
21-5
32-0
16-0
30
22-9
35-0
17-0
500
20-8
28-0
17-0
Monkey
Dog \ .
Guinea-pig
Rat
A curve showing the percentage of trypanosomes of various lengths
is given in Fig. 196. The percentage of posterior nuclear forms varies
in difEerent strains. In the case of an infection in rats of the Sudan strain,
thirty-six trypanosomes out of 1,138 were found by the writer (1912) to
show this posterior displacement of the nucleus.
Transmission. — The transmission of T. bnicei by the tsetse fly was
demonstrated by Bruce (1897) in Zululand. At that time Bruce con-
sidered the fly to be G. niorsitans, but from observations made later,
Austen (1903) came to the conclusion that the fly with which Bruce
worked was probably G. pallidipes. It was Kleine (1909a) who first
demonstrated that flies did not become infective till a period of eighteen
to twenty days had expired from the time of feeding. The experiments
were made by feeding G. palpalis on sheep and a mule previously infected
with T. bnicei by the bites of G. morsitans. The flies were then fed each
day on a healthy animal, and it was found that the only animals to
become infected were those bitten after the long incubation period.
Bouet and Roubaud (1910), working w^ith the same trypanosome
(T. pecaudi) in West Africa, found that G. longipalpis, G. tachinoides, and
G. palpaJis were all capable of transmitting the trypanosome, though
the former was the most frequent carrier and the latter only rarely so.
They further found that the same trypanosome was carried by G. mor-
sitans. Macfie (1914) and Gallagher (1914) obtained strains of T. brucei by
feeding wild G. tachinoides on guinea-pigs in the Eket district of Nigeria.
Transmission by means of G. tachinoides has also been effected by Lloyd
and Johnson (1924). Bruce et al. (1913, 1913o), working in Nyasaland,
found that G. morsitans was the usual carrier, but they also noted (19146)
that G. brevipalpis could serve as a vector, both with the Nyasaland strain
and that from Zululand.
Cycle in the Tsetse Fly. The developmental cycle of T. brucei in
G. morsitans, as demonstrated by Bruce et al. (1914rt, 1914/?, 1914/), follows
I. 35
546
FAMILY: TRYPANOSOMIDiE
very closely that of T. gambiense in G. palpalis as described above (Figs. 217
and 223). There is an intestinal phase followed by migration forwards of
long thin trypanosomes to the proventriculus, labial cavity, and hypo-
pharynx, and finally to the salivary gland, where, after the production of
free and attached crithidia forms, the infective metacyclic trypanosomes
arise (Fig. 226). The characters of the developmental forms as described
by Lloyd and Johnson (1924) have been referred to above (p. 515). They
Fig. 226.— Developmental Form of Trypanosoma brucei in Glossina tachinoides
(x 2,000). (After Lloyd and Johnson, 1924.)
1-3. Forms in mid-gut.
5-G. Crithidia forms from salivary glands.
4. Form in proventriculus.
7-8. Infective forms from salivary glands.
are so similar to the developmental forms of T. gambiense that it is
impossible to differentiate the two trypanosomes as they occur in the fly.
Lloyd and Johnson give the dimensions in microns of the infective forms
as they occur in the salivary glands of G. tachinoides as follows:
Average Length.
Average Length
of Flagellum.
Average Breadth
at Nucleus.
T. brucei . ,
T. gambiense
15-8 (13-3-18-0)
14-6 (12-1-17-3)
2-1 (M-3-5)
1-7 (0-5-2-8)
2-2 (1-4-3-0)
1-5 (1-0-2-5)
Reservoir Hosts.— In his investigations in Zululand, Bruce (1895)
found that the wild G. morsitans readily infected dogs and other animals.
As there were no domestic animals alive in the district, it was evident that
TRYPANOSOMA RHODESIENSE 547
another source of infection existed. The wikl fauna was examined, and
it was discovered that 24 per cent, harboured trypanosomes. In later
investigations in Nyasaland, Bruce et al. (1913e) found that as many as
31-7 per cent, of the wild game harboured T. brucei or other species
pathogenic for domestic animals. Similar results were obtained by
Kinghorn and Yorke {I9l2a) in North-East Rhodesia, but they wrote of
the trypanosome as T. rhodesiense, which, however, they regarded as
identical with T. brucei. The wild game do not appear to be seriously
afJected by their infections, and it is evident that they form a reservoir
for the virus, which is transmitted to domestic animals by the tsetse flies
(see p. 508). On account of the wild fauna, development of these countries
is handicapped to such an extent that some have advocated the complete
extermination of the game. If, as seems probable, T. rhodesiense is
identical with T. brucei, then the question is a still more important one.
The Human Strain of Trypanosoma brucei.
Trypanosoma rhodesiense Stephens and Fantham, 1910, — This try-
panosome, which produces a disease in man differing in many respects
from that caused by T. gambiense, was first recognized as distinct from the
latter by Stephens and Fantham (1910). The chief feature not shown by
T. gambiense is the presence of posterior nuclear forms in small laboratory
animals inoculated from man. The disease in man is of a more serious
type than that produced by T. gambiense, and runs a course of only a few
months. It is only exceptionally that the symptoms characteristic of
sleeping sickness appear. The disease is too rapidly fatal to allow of the
changes in the central nervous system which occur in the more chronic
infections with T. gambiense.
Distribution. — The disease in man has a very restricted distribution
when compared with sleeping sickness due to T. ga^nbiense. It is limited
to the districts east and west of Lake Nyasa, and occurs in Northern
Rhodesia, Nyasaland, the south-east corner of Tanganyika Territory,
and the north-east part of Mozambique. Cases have also been recorded
from South Rhodesia near Livingstone. Outside this area, in w^hich
T. gambiense infections do not occur, there are only two records of the
occurrence of the infection. Duke (1923) studied an epidemic at Mwanza
in the district bordering the south-east corner of Victoria Nyanza, while
Archibald (1922) isolated from sleeping sickness cases in the Southern
Sudan a trypanosome which corresponded morphologically with T. brucei.
Though T. gambiense infections occur at the north end of Lake Victoria,
they have not been recorded from Mwanza, the only district where the
two infections appear to overlap being in the Southern Sudan.
548 FAMILY: TRYPANOSOMIDiE
Hearsey (1909) was the first to report cases of human trypanosomiasis
from districts in which G. palpalis was not known to occur, and to susj^ect
the existence of a disease distinct from the well-known sleeping sickness.
Relation to T. brucei of Animals and T. gambiense of Man.— Though the
characters of the tryj^anosome as described by Stephens and Fantham
serve to distinguish it from T. gambiense, this is not so for T. brucei in
animals, which it resembles so closely as to be morphologically indis-
tinguishable. The difficulty in dealing with this trypanosome is that there
is a divergence of opinion as to whether it is distinct from T. brucei or not.
Bruce and his co-workers (1913e) in Nyasaland came to the conclusion
that no differences exist between the trypanosomes producing disease
in man and animals, and wrote of it as T. brucei vel rhodesiense. The
trypanosome of Nyasaland was also found to be identical with a strain of
T. brucei from Zululand, from which country the original T. brucei came.
This similarity ap|)lies to all stages of the organism, whether in man, wild
game, experimental animals, or tsetse flies, so that there seems no reason
to regard T. rhodesiense as being distinct from T. brucei. Kinghorn and
Yorke (1912) arrived at the same conclusion in Rhodesia. It is, however,
a well-known fact that in many localities where nagana is widespread
amongst animals, and where human beings are constantly bitten by
G. morsitans which are actually transmitting T. brucei to animals, no
cases of human infection have been recorded. Moreover, in those districts
in which trypanosomes of this type produce disease in both man and
animals the number of human cases is much lower than those in animals.
Some observers, as, for instance, Taute (1913), Kleine (1914), Beck (1914),
believe that the human cases in these areas are due to a distinct trypano-
some, T. rhodesiense, and the animal cases to T. brucei. Kleine (1923)
again makes this assertion, and concludes that an animal reservoir of
T. rhodesiense is unknown. Those who regard the trypanosomes as
identical suppose that man is much less susceptible to infection than
animals. Furthermore, it has been suggested that this type of human
trypanosomiasis is a new disease, and there is some evidence in support of
this view. It is conceivable that it was only in one area that T. brucei
became capable of infecting man, and that, having once acquired this
property, the particular strain has now commenced to extend to other
areas.
Taute believes the two trypanosomes are distinct, not only for the
reasons given above, but from the results of a series of experiments con-
ducted by himself and Huber (1919). A large number of human beings,
natives of Africa belonging to different tribes, to the number of 129,
and also the two observers themselves, were inoculated with T. brucei
derived from four horses and two mules which were discovered naturally
TRYPANOSOMA RHODESIENSE 549
infected. In no case did an infection result. In an earlier experiment
Taute fed infected G. morsitans on animals and on himself. He was
immune, but all the animals acquired the disease. These experiments,
at any rate, prove that man is not easily inoculated with T. brucei, though
they do not conclusively prove that he never can be. Kleine (1923)
further maintains that the only means of distinguishing T. brucei
from T. rJiodesiense in naturally infected flies and animals is to test the
susceptibility of human beings, as was actually done in the experiment
mentioned above. If this view is correct, it becomes practically impossible
to distinguish them. To the writer, however, it seems that the evidence
at present available is in favour of the identity of the human and animal
strains. The animal strain (T. brucei) is not readily inoculable to man,
but once having gained a footing there, it is more easily passed to other
men. In attempting to isolate T. equiperdum from horses, Watson only
succeeded in inoculating the trypanosome to a laboratory animal after
many hundred failures. Directly this was accomplished, the blood of this
animal readily infected other laboratory animals, and even horses, so that
the strain was easily maintained.
It seems probable that T. gamhiense also originated from T. brucei of
animals, it may be centuries ago, and that having passed from man to
man through many passages, has become modified morphologically
(disappearance of posterior nuclear forms) and as regards its virulence
for laboratory animals. The human strain of T. brucei, on the other
hand, represents the animal strain which has only recently infected man,
and, having been subjected to few passages, still maintains its morpho-
logical characters and virulence. T. gambiense is sufficiently distinct to be
regarded as a species, but T. rhodesiense is merely a strain of T. brucei
in man.
Duke (1921, 1923a) has expressed a similar view, but suggests that
T. gambiense and T. brucei are still more nearly related. He points out
that his previous investigations (1912c) of the trypanosomes occurring
in the Sesse Islands of Victoria Nyanza before the population was removed
revealed only T. gambiense in man and a similar trypanosome in the
sitatunga. He reinvestigated the subject over ten years after the islands
were depopulated. He finds that G. palpalis, no longer having human
beings to feed upon, nourishes itself on the sitatunga, which have increased
considerably in numbers. The trypanosome now isolated from these
animals is of the T. brucei type, and Duke believes that the trypano-
somes of the T. gambiense type, which originally were handed on in a
mechanical manner from man to man by the flies, have, since the de-
population, been handed from sitatunga to sitatunga by the same flies,
which have been driven to feed on them exclusively, with a consequent
550 FAMILY: TRYPANOSOMIDiE
reversion of the trypanosome to its antelope type. As pointed out above
(p. 539), the possibility of the occurrence of posterior nuclear forms
in small laboratory animals was not excluded in Duke's investigations
of 1912.
It is well known that G. morsitans, the chief carrier of T. hrucei, lives
a considerable distance from water, while G. palpalis, the vector of
T. gambiense, is found along the water-courses or near the lake shores.
The former is sometimes spoken of as the " dry fly," and the latter as
the " wet one." It may be supposed that when T. hrucei first gained
entrance to human beings, who naturally live near to water, it was
G. palpalis which necessarily passed the infection from man to man.
Antelope, on the other hand, spend the daytime in districts far from
water, where G. morsitans is found, and travel long distances at night
to drink. In this manner it may be supposed that the human strain
gradually became adapted to G. palpalis, while the animal strain remained
in association with G. morsitans. If t.his be the case, there would be a
double chance of the trypanosome becoming modified morphologically.
Duke (1923) described an epidemic of trypanosomiasis east of Mwanza
in the Tanganyika Territory (lat. 5° to 2° 3' S., long. 33° 30' to 34°),
which throws light on the question under discussion. The trypanosome
causing the disease was of the T. hrucei type when inoculated to small
animals. Duke believes the human infection arose in 1919 during a
famine and an influenza outbreak which reduced the resistance of the
already ankylostome-ridden population, and made them susceptible to
infection with T. hrucei, which occurred in the game. He does not think
the outbreak was due to imported infection. The vector in this locality
is the recently discovered G. swynnertoni. Duke believes that the epidemic
was due to mechanical transmission from man to man, in spite of the fact
that wild flies, infected from either game or man, were discovered in this
locality. The cyclically infected flies, including those infected from
man, he regards as responsible for the spread of infection amongst the
game, man not being susceptible even when the flies had a salivary gland
infection derived from man. According to this hypothesis, a fly with a
salivary gland infection acquired from a feed on an infected human being
would not necessarily be capable of infecting another human being, though
it would certainly infect game. In the writer's opinion very substantial
evidence is required before this view can be accepted.
It has still to be mentioned that Laveran and Mesnil (1912) separate
T. rhodesiense as a distinct species from T. gamhiense and T. hrucei, as
a result of cross-immunity and serological tests carried out chiefly by
Mesnil and Ringenbach (1911a, 19126), Laveran (191 1«, 1912, 1912a),
and Laveran and Nattan-Larrier (1912, 19126).
TRYPANOSOMA BRUCEI 551
Morphology, — As already remarked, the human strain of T. brucei
is morphologically indistinguishable from that derived from animals,
so that Fig. 225, illustrating the latter, will apply equally well to the
human strain. In the inoculated small animals the posterior nuclear
forms appear. The number of these, however, varies considerably.
Thus, in three strains isolated from men and investigated by the writer
and Hanschell (1913) the percentages in rats examined on different days
varied between 0 and 9-3, 0 and 7-2, and 13 and 40. These observations
were made in a series of rats, in which 1,000 trypanosomes were counted
every three days. After long passage through rats, the number of posterior
nuclear forms may diminish considerably till they become difficult to find.
Susceptibility of Animals.— In its effect on animals, T. rhodesiense does
not differ in any way from T. brucei. It is readily inoculable from
man to laboratory animals. If a rat is inoculated with even a few drops
of blood from a human case, it quickly acquires a large infection. The
trypanosome is virulent to rats from the start, and in this respect differs
from T. gambiense. A rat inoculated directly from a man with T. gam-
biense acquires a very chronic type of infection, during which trypanosomes
are rarely numerous in the blood. It is only after many passages through
rats that T. gambieyise attains anything like the virulence the human
strain of T. brucei has at its first passage into laboratory animals.
Transmission. — The human strain of T. brucei was proved to be
conveyed by G. morsitans by Kinghorn and Yorke (1912) in Northern
Rhodesia. These observers found that the percentage of wild flies infected
varied with the altitude. In a valley (2,100 feet, temperature 75° to 83°)
1 in 534 flies was found naturally infected, whereas on the plateau (4,400
feet), with a mean temperature 15° to 20° lower than in the valley, only
1 in 1,260 was found infected. By actual feeding experiments in the
valley, about 3 per cent, of flies became infective. Flies fed and kept at
the lower temperature did not become infective, though it was shown that
after a period of sixty days at the lower temperature the flies became
infective when the temperature was raised. The low temperature is thus
compatible with the early stages of development, but the final stage
requires a higher one.
Bruce and his co-workers (1913, 19146) in Nyasaland found that
G. morsitans was the principal agent, but G. brevipalpis was also incrim-
inated. The course of development in the fly is identical with that of
T. gambiense and the animal strains of T. brucei.
Reservoir. — As regards the reservoir hosts, Bruce and his co-workers
found that in Nyasaland a large proportion of the wild game harboured
the trypanosome, and Kinghorn and Yorke (1912) found the same state
of affairs in Northern Rhodesia They, like Bruce, found a natural
552 FAMILY: TRYPANOSOMID^
infection in the dog. From what has been stated above, it is evident
that there are no means of distinguishing the trypanosomes which occur
naturally in game and tsetse flies from the form found in man. Kleine
(1923), who regards the human strain as a species {T. rhodesiense) dis-
tinct from T. brucei, maintains that, in spite of morphological identity,
the forms seen in the wild animals by Bruce and his co-workers and by
Kinghorn and Yorke are T. brucei, and that a true reservoir for the human
trypanosome, T. rhodesiense, has yet to be discovered,
{b) Trypanosomes which Develop in the Stomach and Proboscis of Tsetse
Flies — Monomorphic Trypanosomes without Flagella.
Trypanosoma congolense Broden, 1904. — Synonyms: T.dimor'plion'LsiVGxan
and MesnU, \W4: pro x>arte ; T. wfwmm Laveran, 1905; Trypanozoon dimorphon {Liihe,
1906) 2rro 2)(irte ; T. nanum (Liihe, 1906); T. congolense (Liilie, 1906); Trypanosoma
confusum Montgomery and Kinghorn, 1909; T. pecortim Bruce et al., 1910; T. soma-
liense Martoglio, 1911; T. cellii Martoglio, 1911; T. frobeniusi Weissenborn, 1911;
Duttonella pecorum (Chalmers, 1918); T. montgomeryi Laveran, 1909 (?); T. ruandce
van Saceghem, 1921.
Distribution. — This is a small trypanosome found chiefly in cattle,
but also in horses and sheep. It was first recorded by Broden (1904) in
the Congo. What was probably the same form was discovered by Balfour
and Head in the Sudan, and was named T. nanum by Laveran (1905f/).
Montgomery and Kinghorn (1909) suggested the name T. confusum for
this trypanosome on account of the doubt as to its identity. Finally,
Bruce et al. (19106), because of the same confusion, proposed to start
de novo with the name T. pecormn. There seems little doubt that all these
forms are identical, in spite of certain differences as regards the suscepti-
bility of small laboratory animals. T. nanum, for instance, as noted by
Bruce et al. (19116), is said to be not inoculable into rats and mice, whereas
the form named T. jpecorum could be often transmitted to these animals.
Bruce (1914), however, stated that after passage through the goat,
T. pecorum ceased to infect rats and mice, and came to the conclusion that
T. nanmn was merely a strain of T. pecorum which had lost its virulence.
Aders (1923) in Zanzibar has noted the same loss of virulence after passage
through the goat, sheep, and giant rat. Morphologically, the various
forms are indistinguishable, and it seems safer to regard the small patho-
genic trypanosomes which are widely distributed in Africa as belonging to
one species. There is a greater difficulty, however, with a form which
was named T. dimorphon by Laveran and Mesnil (1904). This trypano-
some was first seen by Button and Todd in the Gambia in 1903, and the
strain brought home was sent to Laveran and Mesnil, who have employed
it in a long series of investigations. They maintain (1912) that it is
distinct from any of the forms mentioned above, not onlv because of cross-
TRYPANOSOMA CONGOLENSE
553
immunity tests, but also on account of the fact that in infected animals a
small percentage of the trypanosomes present measure up to 25 microns
in length, while the remainder are small forms like T. congolense. It must
be admitted, however, that the trypanosome was isolated in the early
days of trypanosome investigations, and that the possibility of mixed
infections was not then considered, Yorke and Blacklock (1911), from
the examination of two naturally infected horses in the Gambia, came to
the conclusion that the original T. dimorphon strain was a mixed one of
'-^
Af^f*^
x^
%x
\^
Fig. 227. — Trypanosoma congolense (x 2,000). (After Bruce, Hamerton,
Bateman, Mackie, and Lady Bruce, 1910 and 1911.)
T. congolense and T. vivax. Whichever view may be correct, it is doubtful
if the exact counterpart of the original strain has been rediscovered since,
though French writers have frequently employed the name T. dimorpJwn for
the trypanosome of the T. congolense type, while others have used it for one of
the T. briicei type. The small pathogenic trypanosome of wide distribution
in Africa should therefore be known by Broden's name, T. congolense.
Morphology.^T. congolense is the smallest of the pathogenic African
trypanosomes, and varies in length from 9 to 18 microns, with an average
554 FAMILY: TRYPANOSOMIDyE
of 14 microns (Fig. 227, Plate V., h, p. 456). Its breadth is under 3 microns.
There are no forms with a fiagellum, though sometimes in certain indi-
viduals there may be difficulty in deciding whether a short one is present
or not. The nucleus is central in position, while the kinetoplast often
projects over the border of the parasite. According to Laveran and
Mesnil (1912), T. dimorphon occasionally shows much larger forms up to
20 or even 25 microns in length, but the majority of the trypanosomes in
any pure infection of T. congolense fall within the dimensions given above.
The trypanosome occurs naturally in horses, donkeys, oxen, goats,
sheep, pigs, and dogs, in which it produces a rather chronic wasting disease
associated with fever and progressive ansemia. Rats may sometimes be
inoculated, but, as pointed out by Bruce and his co-workers (19136), many
strains have no effect on rats.
Working with what was undoubtedly this trypanosome in the Sudan,
the writer produced an infection in tw^o dogs which he had inoculated
from cattle.
Transmission and Reservoir. — The wild game of Nyasaland were shown by
Bruce et al. (1913e) to be reservoirs of T. congolense, as many as 14'4 per
cent, of those examined in the "fly country" being found infected
(see p. 509). It was further shown (1914c) that the strain with which
they worked (T. pecorutn) was conveyed by Glossina morsitans. Bouet
and Roubaud (1910), with the trypanosome they called T. dimorpJion,
found that G. longipalpis was the chief carrier, but that G. tachinoides and
G. palpalis could also act as vectors. Bruce et al. (1910a) and Fraser and
Duke (1912c) in Uganda found that the strain (T. pecorum) was carried
by G. palpalis, while Bruce et al. (19146) found that G. hrevipalpis could
transmit the trypanosome in Nyasaland. Croveri (1919) showed that in
Somaliland G. pallidipes was the vector of this trypanosome, which was
referred to as T. somaliense. Duke (1923c) has again effected the trans-
mission of T. congolense by G. palpalis in Uganda.
Cycle in the Tsetse Fly. — The mode of development in the tsetse
fly, which differs from that of T. gambiense, was studied by Robertson
(1913) and Bruce et al. (1914c). There is at first an intestinal development,
followed by migration forwards of long narrow trypanosomes to the
hypopharynx, where a change into attached crithidia forms and then
into trypanosomes of the blood type takes place, after which the flies
are infective (Fig. 218). There is no invasion of the salivary glands, as
there is with T. gatnbiense. Duke (1912) in Uganda found that the part
of the proboscis which became most heavily infected was the labrum,
while in only one instance were trypanosomes observed in small numbers
in the hypopharynx. After the forward migration of the intestinal forms,
TRYPANOSOMA CONGOLENSE
555
a change into crithidia forms takes place, and these attach themselves to
the inner surface of the labrum, just as they do in the salivary glands in
the case of T. gambiense. It is from these crithidia forms that the final
infective metacyclic trypanosomes are evolved.
Lloyd and Johnson (1924) find that during the early stages of develop-
ment the forms in the gut are short trypanosomes which are feebly
undulant and have no free flagellum (Fig. 228). There are then produced
long trypanosomes with free flagella, which migrate to the proventriculus
and thence to the labial cavity. These are difficult to distinguish" from
Fig. 228. — Developmental Forms of T. congolense in Glossina tachinoides
(x 2,000). (After Lloyd and Johnson, 1924.)
1-3. Forms in mid-gut. 4. Form in proventriculus.
5-6. Forms from fixed colonies in labial cavity.
7-8. Pre-infective forms from labial cavity in hypopharynx.
9-10. Infective forms from hypopharjmx.
11. Infective form from hjrpopharynx of Glossina morsitans.
the corresponding forms of T. gambiense or T. brucei. In the labial
cavity they become crithidia forms without flagella, and are attached in
compact colonies. The corresponding stages of T. vivax have flagella.
Subsequently slender crithidia forms, with the nuclei and kinetoplasts
close together at the posterior end of the body and with flagella, are
produced. These invade the hypopharynx and give rise to infective
forms, which resemble the trypanosomes of the blood in that there are
no flagella. In contrast to T. vivax infections the metacyclic trypano-
somes are numerous in the hypopharynx.
ength.
Breadth at Nucleus
IM
1-6
11-1
1-7
11-2
1-6
556 FAMILY: TRYPANOSOMID^
A series of measurements of the infective forms from the hypopharynx
of three species of tsetse fiy gave the following average dimensions:
Glossina tachinoides . .
Glossina palpalis
Glossina morsitans
By attention to the features detailed above, Lloyd and Johnson claim
that a T. congolense infection can be recognized in tsetse flies (see p. 514).
It is possible that T. 7nontgo7neryi Laveran, 1909, seen once by Mont-
gomery and Kinghorn (1909) in Ehodesian cows, and T. somaliense and
T. cellii, described by Martoglio (1911) as the cause of disease in cattle,
horses, sheep, and camels in Somaliland, belong to the T. congolense group.
The same remark applies to T. frobeniiisi, discovered by Weissenborn
(1911) in Hamburg in the blood of a horse which had been brought there
from Togoland. T. montgoyneryi or a very similar form was again seen
/•\'
O
Fig. 229. — Tryimnosoma montgomeryi from Blood of Ntasaland Dog (x 2,000).
(After Kinghorn and Yorke, 1913.)
by Kinghorn and Yorke (1912a) in a dog in Ehodesia, and, as it is distinctly
broader than T. congolense (1*25 to 6-5 microns), it may be a separate
species (Fig. 229). Lloyd has, however, shown the writer a slide of
undoubted T. congolense from a sheep in which numerous forms apparently
identical with T. montgomeryi occur. The trypanosome found by Eding-
ton (1908) in a horse in Zanzibar is probably T. congolense. Writing of
this trypanosome, Aders (1923) notes that with the importation of cattle
from Africa there are introduced, not only T. congolense, but also T. hrucei,
T. vivax, and a trypanosome resembling T. evansi. Of these, only T. con-
golense has established itself in the island, and this has taken place in the
absence of tsetse flies. Tabanids and possibly other biting flies appear
to be the vectors.
With reference to T. somaliense and T. cellii, Donizio (1921) has reinves-
tigated the trypanosomes of Italian Somaliland, and has found that two
forms exist^one, of the T. hrucei type, affecting chiefly equidae, and the
other, of the T. congolense type, producing disease in cattle. He concludes
TRYPANOSOMA SIMILE
557
with ample justification that Martoglio was probably dealing with one or
both of these trypanosomes in pure or mixed infections.
Stirling (1921) found a trypanosome in large numbers in the blood of
a bullock which had died in the Central Provinces of India. The try-
panosome was quite unlike T. evansi, measured 11 to 18 microns in length,
with an average of 14-5 microns, and had the characters of T. congolense.
Stirling concluded that it was actually T. congolense, of which this is the
first record outside Africa. In such a case there might have been some
grounds for the creation of a new species, but there are none whatever
for the name T. ruandce given by Van Saceghem (1921) to a trypanosome
of the Belgian Congo which is undoubtedly T. congolense.
Trypanosoma simiae Bruce et al. 1912. -Synonyms: T. ignotum Kinghorn
and Yorke, 1912; Didtonella simice (Chalmers, 1918).
This is a trypanosome which was discovered by Bruce et al. (1912) in
Nyasaland. What was probably the same trypanosome was also seen
later by Kinghorn and Yorke (19126) in Rhodesia. The latter observers
^-kJ\^.
/
^^^^jx7 .^S^ f^
,,^-
Fig.
230.^Trt/2mnosoma simice from Blood of Monkey (x 2,000). (After
Bruce, Harvey, Hamerton, and Lady Bruce, 1914.)
isolated it by feeding wild G. morsitans on monkeys in the Luangwa
Valley, and, being unaware that it had already been named, called it
T. ignotutn.
Morphologically T. simice resembles T. congolense, which has been
considered above, except that it is distinctly larger (Fig. 230, Plate V., g,
p. 456). Bruce et al. (1913rf) found that the trypanosome was remarkable
for its virulence to the monkey and domestic pig. Goats and sheep are
also susceptible, but other animals, including rats, mice, and guinea-pigs,
are refractory. It was noted that when a monkey and a goat are exposed
to bites of infected flies, both acquire an infection, but the monkey in such
558
FAMILY: TRYPANOSOMID^
an acute form that death takes place in a few days. The infection in the
goat is of a chronic nature, and the animal may recover. If, however, a
monkey is inoculated from the goat, as a rule no infection takes place,
an experiment which proves
that passage through the goat
may profoundly modify the
virulence of a tryjianosome.
The loss of virulence of
T. peconi7H for the rat after
passage through the goat is
another instance of the same
change. These observations
are of considerable interest in
throwing light on the real value
of inoculation tests as a means
of separating trypanosomes
generally.
Morphology. — T. simicB varies
in length from 14 to 24 mi-
crons; the majority of forms
measure about 18 microns,
and are thus larger than those
of T. congolense, which have
an average length of only 14
microns (Fig. 230). There is
no flagellum, but in some in-
dividuals there is a difficulty,
as occurs also with T. congo-
lense, in deciding whether the
last few microns represent a
flagellum or not. The nucleus
is central, while the kinetoplast
is at the margin of the try-
panosome, about 1-5 microns
from the posterior end, which
is generally more or less
rounded. As suggested by
Hornby (1921), it is possible
that T. siniice is merely a race of T. congolense modified by passage through
the wart hog.
Transmission and Cycle in Tsetse Fly.— The transmission of T. simicB by
Glossina morsitans was demonstrated by Bruce et al. (1912) in Nyasaland,
Fig. 231. — Trypanonoma simUc IN the IjABIUM
(left) and Hypopiiarynx (right) of Glossina
morsitans (x 520). (After Bruce, Harvey,
Hamerton, and Lady Bruce, 1913.)
TRYPANOSOMA VIVAX 559
an observation made independently but shortly after by Kingliorn and
Yorke (19126) in the Luangwa Valley. Bruce et al. (19146) found that
G. hrevipalpis might be naturally infected with this trypanosome. They
also showed (1913cZ) that the wart hog {Phacochcerus cBthiopicus) was the
natural reservoir. They found that the course of development in the fly
was similar to that of T. congolense, the usual period of about twenty days
being required before the fly becomes infective. The development
commences in the stomach. Finally, the labial cavity is infected, where
crithidia forms are evolved. These invade the hypopharynx and develop
into metacyclic trypanosomes (Fig. 231).
(c) Trypanosomes which Develop only in the Proboscis of Tsetse Flies —
Monomorphic Trypanosomes provided with Flagella.
Trypanosoma vivax Ziemann, 1905. — Synonyms: T. cnsalbouiLa,VGran, 1906;
Trijpanozoon vivax (Liihe, 1906); Trypanosoma bovis Kleine, 1910; T. angolense
Broden and Eodhain, 1910; Buttonella vivax (Chalmers, 1916).
As with so many of the pathogenic trypanosomes, there has been considerable
confusion over the correct name of Trypanosoma vivax. Ziemann (1905) described a
very active trypanosome from the blood of cattle, sheep, and goats in the Cameroons.
It was of wide distribution, and was seen many times in the area he investigated.
With blood taken from infected animals a series of inoculations was made. Eight
grey rats which had suffered from T. lewisi infection were inoculated, and died in
eight to eleven days. One white rat was inoculated, but did not become infected,
nor did a cat. A dog showed scanty trypanosomes after ten days, but these quickly
disappeared, and did not recur. Laveran (1906) gave the name T. cazalboiii to a
ver}^ similar trypanosome which Cazalbou had studied in cattle in the Upper Niger
region. This trypanosome, though inoculable to sheep and goats, was not inoculable
to monkeys, dogs, guinea-pigs, rats, and mice.
The question is whether this form is identical with Ziemann's T. vivax. If it
be regarded as identical, it has to be explained how Ziemann infected his eight
rats, for all subsequent workers are agreed that the trypanosome of this type
is not inoculable to these animals, and it was chiefly for this reason that the new
species, T. cazalboui, was created. It is just possible that Ziemann mistook T. lewisi
in the rats for T. vivax, for he states earlier in his paper that when T. brucei and
T. leivisi exist together in the rat, they are easily distinguished, whereas with T. vivax
and T. lewisi this may be very difficult unless stained Alms are examined, and no
details of the infection in the rats are given.
That the eight rats died is again explicable from the fact that wild rats frequently
die in captivity quite apart from any infection. Whether this will explain the dis-
crepancy or not, it may be noted that in the other inoculations — namely, the white
rat, the cat, and the dog — only a slight transitory infection took place in the dog, and
this is in agreement with all later observations on T. vivax. Laveran and Mesnil
(1912) state very emphatically that it is impossible to identify a trypanosome
inoculable to rats (T. vivax) with one which is not thus inoculable (T. cazalboui).
They consider that T. vivax cannot be employed as a name for any known trypano-
some. Bruce et al. (1910e), on the other hand, came to the conclusion that T. vivax
and T. cazalboui are identical. They compared the Uganda strain, which Laveran
560 FAMILY: TRYPANOSOMIDiE
had examined and pronounced to be T. cazalboui, with Ziemaun's original prepara-
tions, and could find no difference between them. If they are not identical, it
means that Zieniann's T. vivax, which was discovered by him in numbers of animals
over a wide area, has not been rediscovered. This is highly improbable. It seems
far more likely that T. vivax is the active trypanosome which has been found in the
blood of cattle, sheep, and goats by many observers in various parts of Africa, and
which is not inoculable into laboratory animals, and that Ziemann and Cazalbou
were working with the same trypanosome. The results of inoculations given by
Ziemann agree with this, apart from the eight grey rats which he states became
infected. In this case he may have been using a specially virulent strain; or, as
seems more probable, he may have been dealing with a mixed infection of two
trypanosomes (T. vivax and a small trypanosome like T. congolense), one of which
was inoculable to rats, as has been suggested by Yorke and Blacklock (1911) and
Blacklock (1912). A similar siiggestion was made by Kleine and Fischer (1912).
and it seems probable that though Ziemann recognized the typical very active form
(T. vivax), in some of his inoculations he injected it along with another one [T. hrucei)
which he did not recognize, and which infected his grey rats. It seems hardly
justifiable to abandon Ziemann's name, T. vivax, for this form because of the single
discrepancy when it conforms in other respects with the trypanosome which has
been studied subsequently, and probably with greater accuracy. There is another
point which must be mentioned. Ziemann gave as the dimensions of his trypano-
some a length of 18 to 26 microns, with some forms reaching 30 microns. Laveran
and Mesnil (1912) give for T. cazalboui an average length of 21 microns. Therefore,
Ziemann's measurements are higher than the latter' s. Bruce' s measiirements for
T. vivax, however, agree with Ziemann's, as do those of Eodhain, Pons, Yanden-
branden, and Bequsert (1913a) for a trypanosome of the T. cazalboui type seen by
them in the Belgian Congo; and, as noted above, Laveran examined Bruce's Uganda
strain, and pronounced it to be T. cazalboui.
Taking all these facts into consideration, there can be little doubt that the
trypanosome generally called T. cazalboui by French workers is the same as the
one seen by Ziemann and named T. vivax, a name which, on account of the trypano-
some's motility, is a particularly suitable one. The trypanosome which Kleine
(1910) named T. bovis, and which he discovered in cattle in the Tanganyika district,
is almost certainly T. vivax, as also that referred to as T. angolense by Brodeu
and Eodhain (1910) in the Congo. Walravens (1924) has given the name T. rod-
haini to a trypanosome found in the pig in the Belgian Congo. It resembles
T. vivax in having a flagellum, but differs in being much less active and in having
a narrow body. As no measurements are given, it is evident further investigations
are required before the validity of the species can be accepted.
Distribution. — T. vivax is widely distributed throughout the tsetse
flv areas of Africa. It has been found most commonly in cattle, sheep, and
goats, but also occurs in equines. The infected animals usually die in
from fifty to ninety days. According to Hornby (1921), T. vivax is
generally less virulent to cattle than T. congolense, and a certain number
of the animals recover naturally. They are, however, easily reinfected.
Goats may recover from their infection, but the other animals rarely do.
As remarked above, monkeys, dogs, guinea-pigs, rats, and mice are not
inoculable.
Blacklock and Yorke (1913a) have shown, however, that rabbits may
TRYPANOSOMA VIVAX 561
sometimes be inoculated and the strain carried on in them. Kleine (1923)
states that on one occasion he produced a transitory infection in a monkey.
Morphology. — T. vivax can be distinguished from other pathogenic
trypanosomes, not only by its activity, which enables it to dash about in
a fresh blood preparation with great energy, but also by its morphological
features (Fig. 232, B, and Plate V., f, p. 456). It measures 18 to 26 microns
and has a definite flagellum. As regards the structure of the body,
the bulk of the cytoplasm lies posterior to the nucleus, giving to this
part of the body, which consists of a clear alveolar cytoplasm, a swollen
and broad appearance. The body narrows at the nucleus and tapers ofE
to the anterior end. The kinetoplast is at or near the posterior extremity,
and is well developed. The nucleus is central, while the undulating
membrane is less developed and the axoneme straighter than in T. brucei
or T. evansi. The flagellum is 3 to 6 microns in length.
Transmission and Reservoir. — T. vivax was found in the blood of a
bush buck by Bruce et al. (1910e) in Uganda. Rodhain, Pons, Vanden-
branden, and Bequaert (1912) recovered the trypanosomes from various
antelopes in the Belgian Congo, as also did Kinghorn and Yorke (1912a)
in North-East Rhodesia (see p. 508).
Several species of tsetse fly are capable of transmitting T. vivax.
Bruce et al. (1910a, 1911 A) found that development took place in about
20 per cent, of Glossina palpalis fed on infected animals. These flies were
also found naturally infected. The researches of Pecaud (1909), Bouffard
(1909, 1910), Bouet and Roubaud (1910, 1911a), and Roubaud (1910)
have shown that the trypanosome with which they worked, and which
they called T. cazalhoui, could be transmitted by G. palpalis, G. tachi-
noides, G. longipalpis, and by G. morsitans, while Rodhain, Pons, Vanden-
branden, and Bequeert (1912) also transmitted it by G. morsitans.
Cycle in the Fly. — The development in the fly as first noted by Bruce
et al. (1910a) illustrates a third type of evolution (Fig. 219). In this case
there is no stomach phase of development, the multiplication of the try-
panosomes taking place in the proboscis only. Crithidia forms are pro-
duced in the labial cavity, and these attach themselves in large numbers
to its walls. The hypopharynx is invaded, and finally there are produced
the infective metacyclic trypanosomes of the blood type.
Lloyd and Johnson (1924) find that the trypanosomes taken into the
gut quickly degenerate, and can thus be distinguished from T. gambiense,
T. brucei, and T. congolense, which develop in this situation (Fig. 233).
In the labial cavity they quickly change into crithidia forms with flagella
and become attached to the walls in compact colonies. When a colony
is small the flagellates are short and boat-shaped, and when it is large
I. 36
562
FAMILY: TRYPANOSOMID.E
A
Fig. 232.— Trypanosomes of the T. vivax Ctroup (x 2,000). (After Bruce,
Harvey, Hamerton, Mackie, and Lady Bruce, 1911 and 1913.)
A. Trypanosonui caprce. B. Trypanosoma vivax. C. Trypanosoma uniforme.
TRYPANOSOMA VIVAX
563
they are long and scroll-like. The posterior end is seldom truncated, as
in the corresponding forms of T. congolense. At a later stage the nucleus
and kinetoplast are close together at the posterior end of the body, and
it is these forms which invade the hypoj)harynx. The nucleus then
moves forward, and the infective metacyclic trypanosomes are produced.
These are slender, markedly undulant trypanosomes with sharply-pointed
Fig.
!33. — Developmental Form of T. vivax in Qlossina tachinoides (x 2,000).
(After Lloyd and Johnson, 1924.)
1-2. Degenerate forms in mid-gut and crop.
3-4. Crithidia forms from fixed colonies in labial cavity.
5-6. Pre-infective forms in labial cavity.
7-9. Infective forms in hypopharjTix (G. morsiians and G. palpalis).
posterior ends and a free flagellum, which is from one-third to one-fourth
the length of the body. Measurements of a number of infective forms
from three species of tsetse fly gave the following average dimensions:
J- .J Length of the Breadth at
ijengm. ^lagellum. Nucleus.
Glossina tachinoides
Glossina palpalis
Glossina morsitans .
14-4
14-9
14-8
3-9
4-3
4-1
1-8
1-9
1-6
By means of the above characters it is possible to recognize a T. vivax
infection in tsetse flies without the necessity of infecting animals (see p. 515).
Mechanical transmission by means of Stomoxys was effected by
Boufiard (1907) and by Bouet and Roubaud (1912a).
564 FAMILY: TRYPANOSOMID^
Possibility of T. vivax Infecting Man. — An observation of Macfie
(19176) is of considerable interest in connection with this trypanosome.
He discovered in two blood-films made on two occasions from a native
of the Gold Coast a trypanosome which morphologically resembled
T. vivax. Of 200 trypanosomes measured, the longest was 24 microns and
the shortest 18 microns, giving an average of 20-7 microns. The organism
was evidently monomorphic, and completely unlike the ordinary T. gam-
biense of this district. Furthermore, it had the swollen and rounded
posterior end of T. vivax, and large terminal or nearly terminal kinetoplast.
As T. vivax is exceedingly common in domestic animals in West Africa,
the author, having demonstrated its presence in 76 per cent, of the hump-
backed cattle of Accra, it is possible that in this case T. vivax, usually not
inoculable to man, has been able to obtain a footing in a human host.
Macfie is inclined to regard the infection as actually one of T. vivax in man.
Should this conclusion be confirmed, it is of interest in the light of the
much-disputed relationship of T. brucei and T. rhodesiense, where again a
trypanosome which is readily inoculable to domestic animals may only infect
human beings under exceptional circumstances. It, furthermore, raises the
question of the possibility of other trypanosomes infecting human beings.
Possibility of T. vivax occurring in South America. — Leger, M. and
Vienne (1919) discovered a trypanosome in cattle in Venezuela, which
they named T. guyanense. As this name was already pre-occupied
(Mesnil, 1912), Lavier (1921) proposed to substitute the name T. viennei.
As regards its morphological characters and the susceptibility of laboratory
animals, it resembled T. vivax of Africa. Tejera (1920rt) studied the
organism, and thought it possible that it was actually T. vivax which had
been introduced from Africa some years before. If this view is correct, it
is remarkable that the trypanosome should have established itself in South
America, where the tsetse fly, the natural vector of T. vivax, does not occur.
Trypanosoma caprae Kleine, 1910. — This trypanosome was first
studied by Kleine (1910) near Tanganyika, and was afterwards investigated
by Bruce et al. (1913/) in Nyasaland. It is a very actively motile trypano-
some, like T. vivax, which it resembles closely (Fig. 232, A, and Plate V., e,
p. 456). It is, however, more heavily built, has a larger and more clumsy
appearance, and varies in length from 18 to 32 microns, with an average
of 25-5 microns. Measured across its broadest part, which, as in T. vivax,
is posterior to the nucleus, it is found to vary in breadth from 1-75 to 4-35
microns, with an average of 3 microns. The undulating membrane is
broader than in T. vivax, and there is a fiagellum 4 to 9-5 (average 6-5)
microns in length. It occurs in cattle, sheep, and goats, which may
recover from their infection. It is not inoculable to small animals in the
laboratory.
TRYPANOSOMA VIVAX 565
As demonstrated by Fehlandt (1911), and by Bruce et al. (1913/, 1914e),
T. cajprcB is transmitted by Glossina morsitans. Bruce et al. (19146) also
effected transmission by means of G. brevipalpis. There is no stomach
phase of development in the fly, the whole cycle taking place in the labial
cavity and hypopharynx. Bruce and his co-workers (1913) found that
IM per cent, of the wild game harboured this trypanosome.
Trypanosoma uniforme Bruce ei al., 1911. — This trypanosome was first
studied and named by Bruce et al. (1911a) in Uganda. It is a small form
of the T. vivax type (Fig. 232, C, and Plate V., d, p. 456). Its movements,
though vigorous, cannot be compared with those of T. vivax. The anterior
part of the body does not show the same degree of narrowing as in T. vivax,
so that there is not so great a contrast between the width of the body
anterior and posterior to the nucleus. The post-nuclear region of the body,
however, is decidedly bulbous and the posterior end rounded. T. uniforme
varies in length from 12 to 19 microns, with an average of 16 microns.
The width is from 1-5 to 2-5 microns. The kinetoplast is well developed
and near the posterior extremity. The membrane is distinct, though
narrow, and there is a fiagellum 2 to 5 microns in length.
Like T. vivax and T. caprce, this trypanosome affects cattle, sheep, and
goats, but is not inoculable to the smaller animals. The animals infected
usually die in about thirty to sixty days.
Glossina palpalis was shown by Fraser and Duke (1912c) in Uganda to
be the carrier of T. uniforme. The development is confined to the proboscis,
as in T. vivax and T. caprce. Flies do not become infective till twenty-
seven to thirty-seven days after feeding. It was also shown that the
trypanosome was harboured by antelope on the lake shore in Uganda.
It was the only trypanosome isolated from wild animals, including thirty-
two lake-shore antelope, though the G. palpalis of the area examined were
known to be infected with T. gambiense and T. vivax. A healthy goat
was fed upon by 1,020 flies collected on the lake shore. The animal first
showed T. uniforfue in its blood, and some days later T. vivax also.
It will be noted that the three trypanosomes, T. vivax, T. caprce, and
T. uniforme resemble one another very closely. They difEer only in their
average dimensions. It is open to question whether they represent
distinct species or should be regarded as merely varieties or races of T. vivax.
2. PATHOGENIC TRYPANOSOMES TRANSMITTED BY SPECIES OF TABANUS
OR OTHER BLOOD-SUCKING ARTHROPODA. MONOMORPHIC TRY-
PANOSOMES PROVIDED WITH FLAGELLA.
The trypanosomes included in this group are placed provisionally
amongst the forms which develop in the anterior station in the invertebrate.
In no case, however, have the details of the development been worked out.
566 FAMILY: TRYPANOSOMID^
As far as present information goes, it appears that the trypanosomes are
transmitted by biting flies in a purely mechanical manner, but it is possible
that a definite developmental cycle will be discovered. Should the method
of transmission prove to be purely mechanical, then a new group would
have to be formed to include them.
Trypanosoma evansi (Steel, 1885). — Synonyms: Spirochceta evansi Steel, 1885
Trichomonas evansi (Crookshank, 1886); Herpetomonas evansi (Crooksliank, 1886)
Trypanosoma evansi var. mborii Laverau, 1903; T. herherum Ed. and Et. Sergeut
1904; Trypanozoon evansi (Liihe, 1906); Trypanosoma soiidanense Laveran, 1907
T. hippicum Darling, 1910; T. venezuelense Mesnil, 1910; T. annamense Laveran
1911; T. marocanum Sergent, Lheritier and Belleval, 1915; Castellanella evansi
(Chalmers, 1918); T. equinum Voges, 1911 ("?).
Under the name of surra, a disease of horses had been known for many
years in India. Evans (1880) described an organism he found in the
blood of horses, camels, and mules suffering from this disease. It was
rediscovered by Steel (1885), who regarded it as a spirocheete, but the work
of Crookshank (1886) and others revealed its true nature. The disease
as it occurs in India was the subject of lengthy reports by Lingard (1893).
Surra is now known to be caused by T. evansi, which is found naturally
in horses, mules, donkeys, cattle, camels, elephants, and dogs. It is,
furthermore, inocalable into most of the laboratory animals.
Distribution. — Owing to the movement of horses about the world,
surra is now a widespread disease. It occurs in India, Burma, Assam,
Ceylon, South China, Siam, Sumatra, Java, Philippines, Mauritius,
Madagascar. Animals afterwards found to be infected have been imported
to Australia and the United States, but precautionary measures have
prevented any extension of the disease. From India it extends into
Persia, South Russia, Mesopotamia, and Arabia.
Susceptibility of Animals. — The disease in horses is nearly always
fatal in a period varying from a week to six months. The infected
animals show fever, loss of appetite, anaemia, wasting, and various oedemas.
Similar symptoms are to be noted in camels, in which, however, the duration
may extend to three years, while spontaneous recovery may take place.
As a rule, the disease in cattle is of a milder type. T. evansi appears to
be much less virulent to cattle than to horses. The animals show few
symptoms as a rule, and nearly always recover naturally, but outbreaks
affecting cattle seriously have been described from Java by Penning
(1899, 1900) and Schat (1902), and in Mauritius by Edington and Coutts
(1907). Elephants are affected very much as camels are. An observation
by Cameron, recorded by Evans (1910) and Evans and Rennie (1910),
is of interest in this connection. Trypanosomiasis was discovered in a
herd of seven to nine elephants at Pyinmana in Burma. The trypanosome
TRYPANOSOMA EVANSI 567
morphologically and in inoculations appeared to be T. evansi. The animals
were in poor condition and suffered from fever. Treatment with liquor
arsenicalis was carried out over a long period, during which the animals
were kept at work, and in two to three years they not only recovered
clinically, but their blood ceased to be infective to rats. Dogs are very
susceptible to T. evansi, and in India hunting packs have sometimes
suffered heavily. Death may occur in a week, or not till three or four
months after infection. Cats can be infected, as also pigs. In experi-
mental inoculations rats and mice develop very large infections, and die
in about a fortnight. In guinea-pigs the infection is not so intense, and
death results in about one month. Rabbits show still milder infections,
but the animals die in about the same period. Monkeys are also sus-
ceptible, and the disease produced terminates fatally in about two months.
According to Laveran (1904a), baboons (Cynocephalus) are immune.
Sheep and goats, though they sometimes contract a fatal infection,
generally recover after six months. During this period the trypanosomes
may be so scanty in the blood that they can only be demonstrated by
inoculation of blood to more susceptible rats or guinea-pigs. Goats
which have recovered from their infection are found to be immune to
reinoculation. Laveran and Mesnil have employed these immune animals
in the differentiation of T. evansi from other nearly allied forms.
The virulence of a strain of T. evansi is greatly increased by successive
passages through small animals. In the first few passages after inoculation
from an infected horse the duration of life in these animals is at least
double what it will be later in sub-inoculations.
Morphology.— T. evansi is a monomorphic trypanosome which always
possesses a fiagellum (Fig. 234 and Plate V., c, p. 456), though Bruce
(1911) states that rarely short stumpy forms without flagella occur. In
this respect it differs from T. brucei and T. gamhiense, which are definitely
polymorphic, in that the short stumpy forms are frequently found.
Measuring 820 individuals, Bruce (1911) found a variation in length of
T. evansi between 18 and 34 microns, with an average of 24-9. The
breadth is given as varying between 1-5 and 2 microns. The curve
(Fig. 196) shows the percentage of trypanosomes of various lengths from a
large number measured as compared with T. brucei.
Transmission.— Surra is transmitted from animal to animal by various
blood-sucking flies, chiefly those belonging to the genus Tabanus (Fig. 211).
Up to the present no evidence of a cycle of development comparable with
that of T. (jambiense and other trypanosomes in tsetse flies has been
demonstrated for T. evansi. Rogers (1901) in India recorded successful
transmission experiments. " Horse flies " were allowed to feed partially
on infected dogs, and then to complete their meal on healthy dogs,
568
FAMILY: TRYPANOSOMID.E
some of which became infected. If the interval between the feeds was
over twenty-four hours, no infection took place, Musgrave and Clegg
(1903) in the Philippines transmitted surra by biting flies. Monkeys,
horses, dogs, rats, and guinea-pigs were thus infected. In one experiment
the house fly carried the infection from an infected to a healthy dog by
feeding successively upon a wound on each. In a similar manner fleas
were shown to be capable of carrying infection between dogs and cats.
Working with a North African strain (T, herherum) Sergent, Ed. and Et.
(19056, 1906a), effected a mechanical transmission with Stomoxys and
Fig. 234. — Trypanosoma evansi from Blood of Various Animals (x 2,000).
(After Bruce, 1911.)
Tabanus nemoralis. Eraser and Symonds (1908), working in the Federated
Malay States, found that four species of Tabanus {T. fumifer, T. jjartitus,
T. vagus, and T. minimus) would convey the trypanosome if not more
than five minutes elapsed between the feeds on the infected and healthy
animals. With species of Stomoxys and Hcematopota they obtained nega-
tive results. Leese (1909) at Mohand in U.P., India, obtained positive
results with Tabanus, Hcematopota, and Stomoxys, and he records an
outbreak of the disease among horses where the only biting fly was
Lyperosia minuta. Baldry (1911) at Muktesar in India inoculated the
TRYPANOSOMA EVANSI 569
intestinal contents of various species of Tahanus (T. orientis, T. tropicus,
T. subcallosus) and Stomoxys calcitrans into guinea-pigs at varying
intervals after feeding on infected horses. Up to twenty-four hours the
animals became infected, but not later. Bouet and Eoubaud (1912a),
working with a Sudan strain {T . soiidatiense), effected transmission with
S. calcitrans. Mitzmain (1913) conducted very careful experiments in
the Philippines with T. striatus, which were bred in the laboratory. In
these experiments the trypanosome was transmitted by the method of
interrupted feeding, where only a short interval intervened between the
two feeds. It was further shown that the contaminated labellum did not
appear to be a factor in the conveyance of the trypanosomes, which were
present in the gut of the fly up to thirty hours after feeding. Transmis-
sion was also effected with S. calcitrans, and in one instance by means of
the louse, Hcematopinus tuberculatus. Sergent and Donatien (1922),
working with the strain known as T. herberum, again obtained a mechanical
transmission with Stomoxys, while Donatien and Lestoquard (1923)
observed that dogs which frequented the stables occupied by infected
dromedaries became infected through the numerous Stoinoxys which were
always present. It will thus be seen that up to the present the only
known method of transmission of Trypanosoma evansi in nature is a
mechanical one, in which various biting insects inoculate healthy animals
within a short time of their having fed on infected ones. It would seem
very probable, however, that this is not the whole of the story, and that
further research will reveal some form of development in the fly, leading to
a permanent infection similar to that which occurs in various species of
Glossina in Africa.
Cross and Patel (1921) in India claim to have transmitted T. evansi
from camels to healthy rabbits by means of ticks. A number of ticks
(Ornithodonis crossi and 0. laborensis) were fed on camels. Some were
allowed to complete their feed, while others were interrupted before this
was finished. Those which had not completed their feed were allowed
to finish it upon healthy animals from one to twenty minutes later. The
others were similarly fed again five to twenty-two days later on healthy
animals. In no case did infection result. After forty-six days the result
was again negative, but after sixty-seven days forty-two of the ticks,
together with two others which had fed on an infected camel twenty-two
days before, produced an infection in a healthy rabbit. After a further
interval of sixteen days thirty-six ticks were fed on a clean rabbit, and
again after eighteen days on another rabbit. Both these animals became
infected. It is concluded that ticks can harbour the virus for long periods
(67 to 101 days), and then produce outbreaks of surra. Trypanosomes
first appeared in the rabbits eight to ten days after the ticks had fed,
570 FAMILY: TRYPANOSOMID^
but no statement is made as to whether the rabbits died of their infections
or not. In a further series of experiments, Cross (1923) confirms his
original findings. The ticks transmitted the trypanosome seventeen days
and one month after feeding on an infected animal. He thinks it probable
that a cyclic development occurs in the ticks. He has also transmitted
the trypanosome mechanically by means of Tabanus albimedius, when the
feeds on the infected and uninfected animals followed one another imme-
diately., Yorke and Macfie (1924) report that they received about 200
0. crossi from Cross in India. Though the ticks had been fed on an
infected dog in India, and after their arrival in Liverpool were found to
contain well preserved, though motionless, trypanosomes, they failed to
infect rabbits on which they were fed. The writer also failed to infect
rabbits and rats with a batch of similar ticks received from Cross.
Though Singh (1925) states that he has confirmed the observations of
Cross and Patel, the subject is one which requires further investigation.
Reservoir. — The question of a reservoir host for Trypanosotna evansi
has been frequently raised. Camels, in which the disease pursues a
chronic course, must act in this way, as also the buffalo, which may carry
the trypanosome without suffering to any great extent. Baldry (1910)
expressed the opinion that the pig was a source of infection for other
animals, and he showed that it was susceptible to inoculation.
Treatment. — As regards treatment, the best results have been obtained
by the use of atoxyl subcutaneously and arsenious acid by the mouth,
as recommended by Holmes (1910) in India. Maya (1912) had good
results with this treatment in Mauritius. Thiroux and Teppaz (1910)
report favourably on the action of orpiment by the mouth associated with
atoxyl or tartar emetic injections, while Cross (1920, 1920a) found that
tartar emetic intravenously gave promise of success.
Other Trypanosomes of the Trypanosoma evansi Type.
Forms in Asia and Africa.
Trypanosoma evansi var. mborii Laveran, 1911. — A disease of drome-
daries known as mbori was first described by Cazalbou (1903) in the
French Sudan. It occurs in the districts of the Niger and Senegal Rivers,
and was first noted by its discoverer at Timbuctoo. It affects horses as
well as dromedaries, and produces a disease similar to surra. Laveran
(1904c) considered the trypanosome which causes the disease to be a variety
of T. evansi, and he (1911) named it T. evansi var. mborii. It is inoculable
into the same animals as T. evansi, but is less virulent. Morphologicallv
it is indistinguishable from the trypanosome of surra.
A trypanosome of the same type has been recorded as producing a
TRYPANOSOMES ALLIED TO T. EVANSI 571
disease in dromedaries in Italian Somaliland by Martoglio (1911), and in
South- West Africa by Reinecke (1911). Tlieiler (19066) met with the
same trypanosome in South Africa in dromedaries which had come from
Somaliland.
T. annamense Laveran, 1911. — Another trypanosome morphologically
indistinguishable from T. evansi is one first noted by Blanchard (1888)
in horses in Tonkin and Annam. It has been studied by various observers,
and found to occur also in cattle. Laveran (1911) studied the trypano-
some, and found that goats which had acquired an immunity to the true
T. evansi of India could still be infected with the Annam strain. Accord-
ingly, he designated the trypanosome T. annamense.
T. soudanense Laveran, 1907. — ^Another disease caused by a trypano-
some, and again in the same animals, is the debab of Algeria and Egypt,
and probably North Africa generally. It extends into the same districts
in which Cazalbou studied the disease mbori. A strain of this trypano-
some, which was isolated from a camel, was studied by Laveran (1907) by
immunity tests in goats. This led him to regard it as a species distinct
from that causing mbori. The trypanosome, which he named T. souda-
nense, is not distinguishable from T. evansi save by its immunity reactions.
It is possibly this trypanosome or the variety of T. evansi causing mbori
which is responsible for the disease of camels in Khordofan and Somaliland.
T. berberum Sergent, Ed. and Et., 1904, and T. marocanum Sergent,
Lheritier, and Belleval, 1915 — These two trypanosomes of the T. evansi
type are also recorded from North Africa. T. berberum produces a disease
of camels and horses similar to debab throughout North Africa, while T.
marocanum was encountered in an outbreak amongst horses at Casablanca.
On the evidence of cross-immunity tests these trypanosomes were stated to
differ from one another and also from T. evansi. Sergent, Ed. and Et., and
Donatien (1920) have shown that T. berberum may, at the height of an
infection, pass through the placenta and bring about infection and death of
the young in utero. Camels which have passed the acute stage of the disease
bear healthy young, which, however, possess no immunity to infection,
Vialatte (1915) and Donatien and Parrot (1922) have reported T. berberum
as occurring naturally in dogs, while similar observations for T. marocanum
have been made by Delanoe (1920) and Velu (1920).
A trypanosome of camels in Russian Turkestan was named T. nince
I'ohl-yakimov by Yakimoft" (1921a), who claims that it differs from T. evansi
in pathogenicity to laboratory animals and serum reactions, tests which
are quite insufficient to justify the creation of a new species.
It will be seen from the above account that these various supposed
species produce diseases in those animals which are known to suffer
from surra. Moreover, they are morphologically indistinguishable from
572 FAMILY: TRYPANOSOMIDJE
T. evansi, from which they have been separated by Laveran and others by
cross-immunity tests alone. They resemble T. evansi in that tabanid
flies are probably responsible for their transmission. It is therefore
a reasonable hypothesis to suppose that they are merely races of T. evansi.
The results of inoculation and immunity tests are merely indications of a
dift'erence in virulence of various strains of the same trypanosome.
Forms in Central and South America.
In America, domestic stock is also liable to infection with trypano-
somes of the T. evansi type, and it seems probable that these also may be
merely races of T. evansi.
Trypanosoma hippicum Darling, 1910. — This trypanosome, which very
closely resembles T. evansi, was first seen by Darling (1910) in mules arriving
in Panama from the United States. It produces in equines a disease which
is very like surra. It is inoculable into laboratory animals, in which it
gives rise to the same types of infection as those caused by T. evansi.
Laveran and Mesnil (1912) state that the large forms sometimes seen in
T. evansi infections do not occur in the case of T. kippicufn, and that it can
be distinguished from the trypanosome of surra by cross-immunity tests.
T. venezuelense Mesnil, 1910. — This form, which was first seen by
Rangel (1905), is very similar to T. hippicion and T. evansi, and causes a
disease of equines and dogs in Venezuela.
Morphologically it is indistinguishable from either, and as no cross-
immunity tests had been carried out at that time, Mesnil (1910), who
examined a strain sent to Paris, considered it safer to give it a new name
provisionally. Leger and Tejera (1920) have recently investigated this
trypanosome, and compared it with T. evansi. They claim that it differs
from T. evansi in dimensions, in virulence for laboratory animals, and
response to various medicaments and blood-sera. Taking these facts
into consideration, together with the results of cross-immunity tests,
they conclude that T. venezuelense is a distinct species. The comparisons
were made, however, with a strain of T. evansi which had long been
maintained in laboratory animals. It is very questionable if the slight
differences noted justify the retention of T. vetiezuelense as a distinct
species. Rangel (1905) stated that the trypanosome occurs naturally
in the domestic dog, the wild dog {Canis azare), capibara {Hydrochcerus
capibara), and howler monkeys {Mycetes vrsinus and M. seniculus).
T. equinum Voges, 1901.— Synonyms : T. equina Yoges, 1901; T. elmassiani
Liguieres, 1902; Trypanosoon equinum (Liilie, 1906).
This is a trypanosome which produces a disease of horses known as
mal de Caderas. It occurs in various parts of South America (Brazil,
TRYPANOSOMES ALLIED TO T. EVANSI 573
Bolivia, Paraguay, Argentine). Mules and donkeys also acquire the
disease, but in them it is less acute than in horses. Cattle, sheep, and
goats, which usually recover, take the disease in a very mild form, trypano-
somes only being demonstrable by inoculation of the more susceptible
smaller animals. The duration of the disease in horses varies from about
one to four or five months. Voges (1901) quotes an instance in which a
regiment received 600 horses, of which 500 died of the disease in the
course of the succeeding five months. Inoculated to the smaller laboratory
animals, an acute infection is produced comparable with those produced
by T. evmisi and T. bnicei.
T. equinmn is remarkable in that it differs from all known pathogenic
trypanosomes in the absence of the kinetoplast, or rather the parabasal
body, for the axoneme can still be seen to originate in a minute blepharo-
plast, as is well illustrated in the figures of detached flagella depicted by
Sivori and Lecler (1902). In length it measures from 22 to 24 microns,
of which about five comprise the flagellum (Plate V., i, p. 456). Dividing
forms may be as much as 30 microns in length. The breadth of the
trypanosome is 3 to 4 microns. The nucleus is central, and there is a
well-developed membrane. T. equinum, apart from the condition of the
kinetoplast, of which the blepharoplast alone is present, closely resembles
T. evansi.
It has been noted that from time to time epidemics occur amongst the
capibaras {Hydrochcprus capibara) in districts in which T. equinum is
endemic. Migone (1910) studied one of these outbreaks, found trypano-
somes resembling T. equinum in the blood, and noted that the animals
died with symptoms which he stated resembled those of mal de Caderas.
The evidence, though not absolutely conclusive, seems to suggest that
these animals may act as a reservoir for the virus, though the fact that
they die of the infection does not support this view. The disease is
probably transmitted by species of Tabanus and Stomoxys. Sivori and
Lecler (1902) claimed to have obtained mechanical transmission by means
of S. calcitrans.
These various South American trypanosomes resemble T. evansi so
closely that it seems more reasonable to regard them as races of T. evansi
rather than distinct species. In connection with the absence of the
parabasal body in T. equinum, it must be remembered that similar forms
in other trypanosomes can be produced experimentally by the action of
certain drugs (p. 460).
574 FAMILY: TRYPAN0S0MID.1
II. PATHOGENIC TRYPANOSOMES PASSED DIRECTLY FROM VERTE-
BRATES TO VERTEBRATES.
Trypanosoma equiperdum Doflein, 1901. — Synonyms: T. rougeti Laveran
and Mesnil, 1901; Trypanozoon equiperdum {Luhe, 1906); Castellanella equiperdum
(Chalmers, 1918).
Unlike other pathogenic trypanosomes, T. equiperdutn is transmitted
directly from animal to animal during the sexual act, as occurs with the
organism of syphilis. It produces in horses and donkeys a disease known
as dourine, which is endemic in various countries of Europe, in India
and probably other parts of Asia, in North Africa, North and South
America, and Canada. It was first named T. equiperdum by Doflein
(1901), and a few days later T. rougeti by Laveran and Mesnil,
Symptomology. — The disease is usually of a chronic nature. The
first symptoms are noted about ten days or a fortnight after infection,
and consist of oedema of the sexual organs. About a month later charac-
teristic lesions in the shape of plaques appear on the skin. These vary in
size from that of a shilling to the palm of the hand. They are raised, and
give the impression of a hard subcutaneous disc. Each plaque may persist
for several days, or it may disappear in a few hours. A period of gradual
weakening and loss of flesh supervenes, accompanied by fever and pro-
gressive anaemia. Finally, paraplegia and various nervous symptoms
appear, and the animal dies in from two months to a year after infection.
The females usually abort during the course of the disease. Very rarely
recovery has taken place, after which the animals are immune to reinfection.
Sergent, Donatien, and Lheritier (1920) have shown that horses which
have entirely recovered as judged by disappearance of clinical symptoms,
either naturally or as a result of treatment, may still transmit the disease.
Stallions which had acquired the disease were treated with atoxyl and
orpiment till complete clinical recovery had taken place. The animals were
then returned to full regimental duty, but the blood was examined from time
to time by inoculating dogs. The following history of four stallions is given :
1. For a month after complete clinical recovery the blood still infected
dogs. During three years 3| litres of blood injected into nineteen dogs
failed to infect any.
2. For a year after recovery 1|^ litres of blood failed to infect eight
dogs. Four months later one of two dogs injected became infected.
During the next two years 2 litres did not infect any of ten dogs.
3. During two years 2-2 litres of blood did not infect eleven dogs.
During the third year, however, dogs were infected.
4. During three and a quarter years 3-64 litres of blood did not infect
nineteen dogs. The blood then infected one of two dogs injected.
TRYPANOSOMA EQUIPERDUM 575
Watson (1920) studied an infected mare, which suffered from three to
four day periods of fever every twenty-four to twenty-eight days asso-
ciated with oedematous swellings of similar duration. These swellings
were examined every few hours by abstraction of serum with a fine needle,
and the trypanosomes were found to pass through a definite cycle. The
first specimens of serum showed few organisms. Later they increase in
number till at the fortieth hour agglomerations were present. At about
the forty-fourth hour all the trypanosomes were found to have been
ingested by the macrophages. At forty-eight hours only debris of try-
panosomes could be recognized in the cells, while on the third day no
trace of them could be found and the swelling disappeared.
Watson found that the virulence of T. equiperdum for horses was
increased after passage through the mouse, and the infection produced
was associated with the constant presence of trypanosomes in the blood-
stream, a condition never observed in the natural disease or in horses
experimentally infected by injection of trypanosomes directly from a
naturally occurring case of dourine.
The discovery of the trypanosome in the naturally infected horses and
donkeys is often very difficult. It occurs in very small numbers in the
blood-stream, but is more numerous in the exudate from the areas of
oedema and in fluid obtained from the plaques. Watson (1920) believes
that the organism is not a blood-parasite at all, and that it only occa-
sionally gains access to the blood-stream from the connective tissue lymph
channels, which constitute its usual habitat. For diagnostic purposes it
is often necessary to inoculate large quantities of blood (100 to 400 c.c.)
intraperitoneally to dogs. If the dogs do not become infected, this does
not exclude infection in the horse. The complement fixation test, as
carried out by Woods and Morris and Watson, has been referred to above
(p. 452).
Susceptibility of Animals. — The trypanosome is inoculable into the
dog and rabbit, and more rarely to rats, mice, guinea-pigs, monkeys,
sheep, and goats. There is, however, a great variation in virulence, so
that with certain strains animals are easily infected, while with others no
infection takes place. Any individual strain is liable to change its
virulence, so that a marked irregularity in the results of inoculations
occurs. The dog seems to be the most susceptible animal, and is usually
employed for purposes of diagnosis when trypanosomes cannot be found
by direct examination of the blood of the horse or donkey. Dogs and
rabbits infected by inoculation are able to transmit the infection during
the sexual act.
Dogs usually die of an infection in two to three months. In rabbits the
disease is of a chronic nature, and reveals the lesions characteristic of the
576 FAMILY: TRYPANOSOMID^
infection in horses. Recovery may take place in about a year, or death
occurs before this. With virulent strains rats and mice survive from one
to three weeks. Guinea-pigs succumb in one to three months. Other
animals which have been inoculated generally recover.
Watson (1920) states that the Canadian strain of T. equiperdum was
transmitted to a white mouse after hundreds of unsuccessful attempts
with dogs, rabbits, guinea-pigs, rats, and mice. When once established
in mice, the trypanosome was readily inoculable to the other animals.
Furthermore, after passing through the horse again for several successive
passages, it was readily recoverable by inoculation of laboratory animals.
Thus its power of infecting laboratory animals was not lost after return to
the original host. In the first instance, when the first successful inoculation
of a mouse occurred, this animal was the eighty-fourth of a series of rats
and mice which had been inoculated during a period of four weeks with
fluid rich in trypanosomes which had been collected from the plaques
appearing on an infected mare. The remaining eighty-three animals did
not become infected. This change in virulence after passage through an
animal is perhaps comparable with Bruce's (1914) observation that T. con-
golense (T. pecormn) lost its virulence for laboratory rats after passage
through the goat (see p. 552). The strain isolated by Watson in mice
after many failures behaved in mice and in horses like T. evansi. Though
it was actually isolated in the first place from the serum from the oedema-
tous swellings, it is just conceivable that the trypanosome which infected
the mice was not T. equiperdum, but T. evansi, a trypanosome, however,
which is not known to occur in Canada. In any case, the change in
character of the trypanosome after passage through mice raises the
question of relationship of these two forms.
Morphology. — T. equiperdum is a trypanosome of the T. evansi type
(Plate v., c, p. -ISO). There is always a fiagellum, and the trypanosome
varies in length from 25 to 28 microns. Blacklock and Yorke (1913)
examined three strains of the trypanosome obtained from various European
laboratories. Two of the strains correspond with T. equiperdum, but one
was polymorphic in nature and resembled T. brucei. It was concluded
that this form was a different type of dourine-producing trypanosome,
and it was named T. equi. The strain was said to have originally :come
from Algeria, in which country dourine is known to be due to T. equiperdum
of the normal type. In the case of a trypanosome so far removed from its
original host, quite apart from the possibility of changes in morphology,
accidents of interchange with other laboratory trypanosomes may have
occurred, a fallacy which certainly cannot be excluded.
Transmission. — As already remarked, dourine is spread from animal
to animal by the direct contact of mucous surfaces. That the disease may
TRYPANOSOMES OF BIRDS 577
be carried in other ways has been proved to occur. Thus, Schuberg and
Kuhn (1911) obtained a mechanical transmission by means of Stomoxys
calcitrans, and Sergent, Ed. and Et. (1906a), with a tabanid fly, Tabanus
nemoralis.
III. TRYPANOSOMES OF BIRDS.
The trypanosomes of birds are considered here amongst the forms
which develop in the anterior station in the invertebrate, though actually
in no case is the vector known, much less the type of development which
occurs. Mosquitoes have been suspected of being the transmitting hosts,
and certain observers have noted flagellates in the salivary glands of these
insects, but there is no evidence that these have been derived from bird
trypanosomes. It is quite possible, when the true intermediate host is
discovered, it will be found that development of the trypanosome takes
place in the posterior station.
The first satisfactory account of the occurrence of trypanosomes in
birds was that of Danilewsky (1888). Since then a large number of
forms has been described from well over a hundred species. In the great
majority of cases, little more has been done than to give an account of the
trypanosomes as they appeared in a single blood-film; no detailed study
has been carried out. In one or two instances, however, more extended
observations have been made. Schaudinn (1904) published a remarkable
account of the development of the trypanosome {T. noctuce) of the little
owl. He stated that an alternation of a trypanosome with an intra-
cellular halteridium phase occurred in the life-cycle. Subsequent observa-
tions, in spite of various attempts by Woodcock (1909) and others to
substantiate Schaudinn's views, have clearly demonstrated the fallacies
of his conclusions.
Trypanosoma paddae Laveran and Mesnil, 1904. — The best account of
a bird trypanosome is that of Thiroux (1905), who described the infection
due to T. paddce Laveran and Mesnil, 1904, in the Java sparrow {Munia
oryzivora). The trypanosome was first seen by Levaditi in birds imported
to France. By means of this strain, Thiroux was able to infect other birds
and to study the course of the infection. Inoculated intraperitoneally,
trypanosomes appeared in the blood of the birds in twelve hours, whereas by
the intramuscular or subcutaneous method the incubation periods were nine
and twelve days respectively. Furthermore, there was marked irregularity
in the results of inoculation. Some birds only became infected after a
second inoculation. After their appearance in the blood, the trypanosomes
increased in number during nine to fifteen days, after which the number
declined day by day till they could only be found with difficulty. In some
cases the infection brought about the death of the birds. The trypano-
I. 37
578
FAMILY: TRYPANOSOMID^
Fig. 235.— Trypanosomes of Birds (x 2,000). (1-10, after Minchin and Wood-
cock, 1911; 11, AFTER Bruce, Hamerton, Bateman, Mackie, and Lady
Bruce, 1911; 12-14, after Thiroux, 1905.)
1-10. Various types of Trypanosoma norivce in the little owl { Athene nodua).
11. Trypanosoma galUnarum in blood of Uganda fowl.
12-14. Trypanosoma paddce in blood of Java sparrow (Munia oryzivora).
TRYPANOSOMES OF BIRDS 579
some was also inoculable to other birds (Serinus serinus, S. canarius,
Lagonosticta minima, Mariposa phcenicotis, Estrilda cinerea). The infection
in canaries was more intense and the mortality higher than in the natural
host. These observations serve to indicate the possibility of one species
of trypanosome having several hosts (Fig. 235, 12-14).
Multiplication of T. paddce takes place by longitudinal division in the
usual manner, but the dividing forms are only seen in the blood during
the early stages of an infection. Later no division forms can be found,
and in this respect the trypanosome resembles T. lewisi of the rat. Exami-
nation of the spleen and bone marrow did not reveal a greater number of
parasites than the blood. In the case of other bird trypanosomes the
bone marrow appears to be the site of election, for they can often be found
there when the blood-examination has been negative. For instance,
Minchin and Woodcock (1911) noted this in the case of T. noctuce of the
little owl, and Woodcock (1910) in T. fringillinarum of the chaffinch.
These observers pointed out that the trypanosomes were absent from the
blood, but were to be found in the bone marrow, especially in winter and
spring.
Thiroux succeeded in cultivating T. paddce in blood-agar media.
Avian Trypanosomes in General.
Though a large number of trypanosomes of birds have been given
specific names, it is evident that the validity of many of these is very
doubtful. Where infections have been studied in any detail, it has been
observed that the trypanosomes are very polymorphic.
Morphology. ^Minchin and Woodcock (1911) noted a great range in
size of T. noctuce, the largest forms being found in the winter and spring
(Fig. 235, I -10). Thus, there are small forms with a total length of 26-5
microns and a breadth of 3-5 microns, intermediate forms measuring
44 to 47-5 microns by 5 to 5-5 microns, and large massive forms 54 to 60
microns by 5-5 to 6 microns. The small forms gradually grow into the
large forms, which are found in winter in the bone marrow. It is supposed
that in the summer the small forms are reproduced from the large ones by
a process of schizogony, but this hypothetical reproductive process was
not observed. The small forms reproduce by longitudinal division, and
also give rise to certain stout trypanosomes which, according to the
authors, are destined to undergo development in the mosquito. The proof
that the mosquito, Culex pipiens, is the transmitting host of T. noctuce,
and that the changes undergone by the trypanosome in the stomach of
the mosquito, as described by Woodcock, (1914), are true developmental
stages, is as yet lacking. It is evident that T. noctuce is markedly poly-
morphic in the owl, a feature which, if of general occurrence, renders the
580 FAMILY: TRYPAXOSOMIDiE
identification of species exceedingly difficult, especially as the majority
of those which have been named have only been seen in one particular
phase in a single blood-film. Furthermore, very little is known as to the
number of hosts any trypanosome may infect. As noted above, Thiroux
(1905) found that T. jJaddce was inoculable to a number of different birds.
Similarly, Noller (1920c) found that T. loxice of Loxia curvirostm was
inoculable to canaries and finches.
Though trypanosomes have been described from many different birds,
they can all be referred to one or other of the types described by Minchin
and Woodcock as occurring in the cycle of development of the trypano-
some of the little owl, Athene noctva, and which are illustrated in Fig. 235.
As regards the details of their morphology, bird trypanosomes conform to
other members of the genus. Nieschulz (1922a) has described a rod-
shaped structure which occurs in the cytoplasm of cultural forms and a
granule which is present on the nuclear membrane. These have already
been referred to above (Fig. 154).
Transmission. — As regards the natural transmission of bird trypano-
somes very little is known. Schaudinn (1904), and later Woodcock
(1914), stated that C. pipiens was the intermediate host of T. noctucB.
Danilewsky observed that young birds in the nest only a few days old
were already infected with trypanosomes. Duke and Robertson (1912)
noted that T. gallinarum (Fig. 235, ii), first described by Bruce et al.
(19in) in Uganda fowls, underwent a development in Glossina palpalis,
resulting in the production of crithidia forms in the stomach. It was
concluded, however, that the tsetse fly was not the true host. What is
possibly the same trypanosome was seen by Mathis and Leger (1911a)
in fowls in Tonkin. It is probable that the transmitting hosts of bird
trypanosomes will have to be sought amongst the blood-sucking arthropods,
which especially infest the nests. There is evidence, however, that bird
trypanosomes will develop in mosquitoes. Woodcock (1914) described
the changes undergone by T. noctuce in C. pipiens. The trypanosomes
taken up by the mosquitoes underwent multiplication and became
crithidia forms, while finally long slender trypanosomes and very much
rmaller stumpy trypanosomes were produced. The latter forms bear a
resemblance to the metacyclic trypanosomes which are developed in the
hind-gut of the flea in the case of T. lewisi. Noller (1920c) also noted
that T. loxice underwent a development in C. pipiens, as also in A'edes
argenteus. This culminated in an accumulation of flagellates in the hind-
gut of the mosquitoes. He noted that when T. loxice and T. syrnii were
cultivated on blood-agar plates at 18° to 20° C, there was rapid multiplica-
tion of crithidia forms, and that a transformation into trypanosomes
took place when the plates were incubated at 37° C. In cultures of T.frin-
TRYPANOSOMES OF LAND REPTILES 581
gillinanim, Woodcock (1914) likewise noted that a trypanosome phase
followed the appearance and multiplication of crithidia forms. It is
evident that the cycle of development of bird trypanosomes in the inverte-
brate will follow the usual lines, in which crithidia forms first appear, to
be followed by metacyclic trypanosomes. No actual transmission by
means of mosquitoes or any other invertebrates has as yet been effected.
An interesting observation made by Mathis (1914) may be urged in support
of the view that the transmitting hosts of bird trypanosomes are mos-
quitoes. In a species of Ciilex in Tonkin he noted an infection of the
salivary glands with flagellates of the crithidia type, and conjectured that
these might have been derived from some bird trypanosome (p. 370).
Culture. — That trypanosomes of birds are relatively easy to cultivate
in blood-agar media was first demonstrated by Novy and McNeal (1905).
Danilewsky (1888), however, had previously observed multiplication of
trypanosomes in hanging-drop preparations of bird's blood. Novy and
McNeal, and Nieschulz (19226) noted that infections could be demonstrated
in birds by the cultural method w^hen blood-examinations were negative.
In these cultures the trypanosomes multiply rapidly, becoming trans-
formed into crithidia and rounded or ovoid forms. In older cultures
trypanosomes again appear. The cultures may be maintained indefinitely
by subculture. Novy and McNeal, and Thiroux (1905) found that birds
were with difficulty infected from the cultural forms.
Noller (1920c) and Nieschulz (19226) have cultivated trypanosomes
from a number of birds on blood-agar plates. The plates kept at room
temperature show mostly crithidia forms. If they are kept at 37° C, the
crithidia forms assume the trypanosome structure, but again revert to
the crithidia form when the temperature is reduced.
IV. TRYPANOSOMES OF LAND REPTILES INCLUDING CROCODILES.
The first definite record of a trypanosome in a reptile was that of
Laveran and Mesnil (1902), who described T. damonice of the tortoise,
Damonia reevesii, though as early as 1883 Kunstler had noted a flagellate
in the blood of a mud tortoise, and considered it to be allied to the try-
panosomes. Since Laveran and Mesnil's discovery, various trypanosomes
have been described from crocodiles, tortoises, snakes, and lizards.
In very few cases is the method of transmission actually known, but
such information as is available appears to indicate that the trypanosomes
of land reptiles, including crocodiles, are transmitted by blood-sucking
arthropoda, while those of aquatic reptiles are transmitted by leeches.
On this account the trypanosomes of reptiles are considered under two
headincrs.
582 ■ FAMILY: TRYPANOSOMID^
As regards the trypanosomes which have blood-sucking arthropods as
their vectors, it is not definitely known whether the development is in the
anterior or posterior station, though the behaviour of T. hoclii of the
crocodile in the tsetse fly is suggestive of a contaminative method of
infection.
T. kochi Laveran and Mesnil, 1912.- — The crocodile trypanosome was
first seen by Minchin, Gray, and Tulloch (1906) in Uganda, but no descrip-
tion was given. Koch (1906) studied it in greater detail, and suggested
the possibility that certain flagellates (T. grayi, Herpetotnonas grayi)
frequently encountered in tsetse flies had their origin in the trypanosome
of the crocodile, on which the flies were noted to feed (see p. 373). As
Laveran and Mesnil (1912) have pointed out, Koch did not suppose that
the flagellates of the flies, which he thought might have developed from
the crocodile trypanosome, were in any way related to T. gambiense,
though writers have wrongly attributed this view to him. Kleine and
Taute (1911) described experiments which gave definite support to Koch's
view of the development of the crocodile trypanosome in tsetse flies.
In one experiment, thirty-two bred flies {Glossina palpalis) were fed on a
crocodile, with the result that eleven were found to harbour H. grayi
when dissected eight to fourteen days later. They believed, however,
that the tsetse flies could acquire the flagellates from other hosts than
the crocodile. Ross, P. H. (1911), found flagellates of this type in
G.fusca, while Bruce et al. (19146) discovered that both G. palpalis and
G. hrevipalpis were liable to be infected with H. grayi. They suggested
that the flagellates were probably derived from the crocodile, iguana, or
some water bird, as both these flies resemble one another in the habit of
living near water. Lloyd and Johnson (1924) have found the flagellate
in G. tacJiinoides in Nigeria. Kleine (1919a) definitely asserts that the
flagellate represents developmental forms of the crocodile trypanosome.
Roubaud (1912), basing his conclusions on a series of negative feeding
experiments and on the fact that Minchin (1907) had described encysted
stages of the flagellate in the rectum of the flies, stated that H. grayi was a
flagellate peculiar to the flies, and was handed on, like other purely insect
flagellates, from fly to fly by means of the cysts (Figs. 173 and 220). It is,
however, far from clear that the bodies described by Minchin were actually
cysts, and it is difficult to understand how tsetse flies, either in the adult
or larval stages, could ingest such cysts. It seems probable that Kleine's
view is the correct one, in which case the name of the crocodile trypano-
some will be T. grayi Novy, 1906, and not T. l-ochi Laveran and Mesnil,
1912. Lloyd and Johnson (1924) and Lloyd, Johnson, Young, and
Morrison (1924), however, produce evidence that the flagellates of the
H. grayi type in G. tachinoides may be derived from monitors {Varanus
TRYPANOSOMES OF LAND REPTILES 583
exanthematicus) as well as crocodiles on which the flies feed. If this be the
case, they would represent developmental forms of T. varani, first seen
by the writer (1909) in the Sudan. It is possible that under the names
T. grmji and H. grayi several reptilian trypanosomes have been grouped,
and that it will not be possible to identify the flagellates first named
H. grayi with any particular trypanosome. The question of the flagellates
of tsetse flies is still further complicated by the recent discovery by Lloyd,
Johnson, Young, and Morrison (1924) that crithidia forms indistinguish-
able from those of T. grayi appear in the intestine of G. tachinoides after
feeding on toads {Bufo regularis), which harbour trypanosomes resembling
T. varani (see p. 374).
The trypanosome (Fig. 236, 3) seen by Bruce et al. (1911/) in the
crocodile {Crocodilus niloticus) had a total length of 87 microns, which
was made up as follows: posterior end to the kinetoplast, 18 microns;
kinetoplast to anterior end, 46 microns; flagellum, 23 microns. The
body showed longitudinal myoneme striations. The trypanosome was
cultivated by Koch (1906), and by Kleine and Taute (1911). A try-
panosome about half the length of the form seen by Bruce was recorded
from C. catejpractus by Button, Todd, and Tobey (1907) in the Congo.
Other Trypanosomes of Land Reptiles.
Martin (1907) described as T. boneti a broad leaf-like trypanosome
from Mahuia raddoni of French Guinea, while Bouet (1909) gave the name
T. martini to a similar form found by him in M. maculilahris and M. perro-
teti of the Ivory Coast. Fran9a (1911a) named the form from the second
of these hosts T. jperroteti. The writer (1909) described as T. mahuicB
a trypanosome of M. quinquetcBniata of the Southern Sudan (Fig. 236, 6-7).
The trypanosome occurred in two forms — one a broad leaf-like trypano-
some resembling T. rotatorium of the frog and measuring 30 to 40 by
8 microns, and the other a smaller trypanosome like T. inopinatutn of the
frog and measuring 20 to 25 by 2 to 2-5 microns. It is possible that the
various species described from the skinks of the genus Mahuia are different
stages of one polymorphic trypanosome like T. rotatorium of frogs, in
which case Martin's name, T. boueti, will have priority. A broad leaf-like
trypanosome was described by the writer (1909) as T. chamceleonis from
Chamceleon vulgaris of the Sudan, and another similar form from the
monitor {Varanus niloticus) as T. varani.
Robertson (1908) recorded trypanosomes from two geckos of Ceylon.
One which occurred in Hemidactylus leschenaultii w^as named T. lesclie-
naultii. It measured 56 to 60 microns in length, and had a flagellum
measuring 17 to 20 microns. The other, named T. pertenue, occurred in
584
FAMILY: TRYPAXOSOMID^
Fig. 236. — Trypanosomes of Reptiles (x 2,000). (1 and 2, after Mathis and
Leger, 1909; 3, after Bruce, Hamerton, Bateman, Mackie, and Lady
Bruce, 1911; 4-7, after Wenyon, 1909.)
1 2. Trypanosoma primeti of the snake, Tropidonotiis piscator, of Tonkin.
3. Trypanosome of the Uganda crocodile {T. kochi ?).
4 5. Trypanosoma erythrolampri of the snake, Erylhrolamprns cesudapii, of South America.
G-7. Trypanosoma mabnice of the lizard, Mabuia quinquitoiniata, of the Sudan.
TRYPANOSOMES OF AQUATIC REPTILES 585
H. tried ri. It was 30 to 35 microns in length, with a flagellum 15 to 20
microns in length. A similar trypanosome from P sylodactylus caudicinctus
was named T. galliji by Bouet (1909), while Mathis and Leger (1911)
mention the occurrence of another in Acanthosaura fruhstorferi in Tonkin.
Catouillard (1909) gave the name T. platydactyli to a trypanosome of
Tarentola mauritanica of Tunis. It was cultivated in N.N.N, medium
by Sergent, Ed. and Et., Lemaire, and Senevet (1914), while Chatton and
Blanc (1918a) showed that it developed readily in bed bugs fed on the
geckos. Todd and Wolbach (1912) mentioned the occurrence of trypano-
somes in Againa colonorum and Lygosoma sp. of the Gambia.
Trypanosomes have been recorded from a number of land snakes.
T. erythrolampri, seen by the writer (1908) in the South American snake,
Erythrolamprus cesculajni, is a long narrow form measuring 30 to 34 by
5 to 7 microns (Fig. 236, 4-5). It was found in the blood of a snake which
had died in the Zoological Gardens in London, and some of the flagellates
had the crithidia structure. It is possible that the crithidia arrangement
of nucleus and kinetoplast was the result of changes occurring after the
death of the host. The writer (1909) gave the name T. najce to a trypano-
some of the Sudan cobra {Naja nigricollis). It measured 50 microns in
length, and was only seen in the living condition. Bouet (1909) described
as T. clozeli a large broad trypanosome of the African snake, T ropidonotus
ferox. The nucleus and kinetoplast were close together near the middle
of the body, which measured about 100 to 106-5 by 10 to 25 microns.
Button, Todd, and Tobey (1907) record a trypanosome from the puff adder,
Bitis arietans, of the Gambia. According to Johnston and Cleland (1910),
Love discovered a trypanosome in the Australian snake, Diemenia textUis.
V. TRYPANOSOMES OF AQUATIC VERTEBRATES TRANSMITTED BY
LEECHES.
1. Trypanosomes of Aquatic Reptiles.
The best-known trypanosomes of this group are those of aquatic
chelonians. The development of T. vittatoe has been studied by
Robertson (1908).
Trypanosoma vittatae Robertson, 1908. — This trypanosome (Fig. 237)
was discovered in the soft tortoise {Emyda vittata) of Ceylon by Robertson
(1908), who studied it not only in the vertebrate host, but also in the
leech {Glossosiphonia sp.), which is probably the invertebrate vector of the
trypanosome. Development, however, was found to take place also in
the horse leech, Pcecilobdella granulosa.
In the blood of the tortoise (Fig. 237, 1-4), the largest trypanosomes
have a body measuring between 60 and 70 microns in length and 8 to 9
586
FAMILY: TRYPANOSOMID^
s^nciccsc^
Fig. 237. — Trypanosoma vittatce, Parasite of the Ceylon Tortoise, Emyda
vittata. AND THE Leech, Poeeilobdella granulosum ( x 1,600). (After Robertson,
1909.)
1-4. T. vittatce in blood of tortoise.
5-6. Division stages of rounded -off trypanosome in intestine of leech.
7. One of the products of division.
8. Elongation of body to form crithidia stage.
9-10. Division of elongating forms.
11-13. Tryi^anosome forms evolved from the crithidia forms
TRYPANOSOMES OF AQUATIC REPTILEsS 587
microns in breadth. The shortest forms were about 25 microns long and
4 to 5 microns broad. Intermediate forms also occurred. The undulating
membrane is markedly frilled. The fiagellum measures up to 25 microns
in the large and small trypanosomes, being relatively longer in the latter.
In the large forms the body is seen to be longitudinally marked by parallel
lines, an indication. of myonemes. When examined in the fresh condition,
the trypanosome is seen to writhe about locally with little progression.
Occasionally there is a slow translatory movement, the trypanosome
revolving spirally on its axis. In the blood of the tortoise division stages
were rarely found, and these only in the case of trypanosomes of the
intermediate size. It is possible that active multiplication only takes
place in the early stages of an infection, or is chiefly confined to the internal
organs.
In the crop of the leech a cycle of development takes place, resulting in
the formation of crithidia forms (Fig. 237, 5-10). The earliest stage of
this cycle consists in the rounding-of! of the large trypanosomes, a process
which can be studied in fresh blood-preparations under the microscope.
The large trypanosomes become retracted in various ways to form globular
masses of cytoplasm. The myonemes cease to be visible, as also the nuclei.
The axonemes become detached from the membranes, and finally disin-
tegrate. These globular bodies then commence to divide. By two
divisions four pyriform bodies are produced from each, and these remain
more or less attached to one another while they form flagella. The latter
first appear as short rods, which increase in length till their full size is
reached. These flagellate bodies, when stained, are found to have the
crithidia structure. By their further multiplication the crop of the leech
becomes populated with a large number of long, slender, and very actively
motile crithidia forms, which eventually give rise to metacyclic trypano-
somes (Fig. 237, 11-13). The development appears to be limited to the
crop, and the exact mechanism of the infecting process was not elucidated.
The leech, Glossosiphonia sp., is peculiarly suited to play the part of an
intermediate host, as it has the habit of wandering from one tortoise to
another.
Other Trypanosomes of Aquatic Reptiles.
Trt/panosoma danwnice, the first trypanosome to be described in a reptile,
has been mentioned above. It was discovered by Laveran and Mesnil (1902).
The length was 32 microns, of which the fiagellum formed about one-third,
and the breadth 4 microns. Trypanosomes have been met with in a
number of other chelonians. Button and Todd (1903) and Button, Todd,
and Tobey (1907) noted the presence of trypanosomes in tortoises of the
Gambia, as did Minchin (1910) in one in Uganda. Bouet (1909) gave the
588 FAMILY: TRYPAXOSOMIDyE
name T. pontyi to a trypanosome of the tortoise, Sternotherus derbianus, of
Africa. T. chelodina was recorded from Chelodina longicollis by Johnson
(1907), and what is probably the same form from Emydura hrefftii by
Johnston and Gleland (1910, 1912), who saw it also in C. longicollis.
In aquatic snakes trypanosomes also occur. Mathis and Leger (1909fl)
gave the name T. primeti to a trypanosome discovered by them in Tropi-
donotus piscator and Hypsirhina chinensis (Fig. 236, 1-2). There occur
large forms measuring 105 by 14 microns and small forms measuring
57 by 7 microns. Brumpt (1914a) saw a trypanosome which he named
T. brazili in the Brazilian water snake, Helicops modestus. He demon-
strated a complete development terminating in metacyclic trypanosomes
in the leeches, Placobdella brasiliensis and P. catenigera (Fig. 452). The
whole of the development was confined to the stomach, no infection of the
proboscis sheath occurring even after several months. Brumpt suggests
the possibility of snakes becoming infected by swallowing the leeches.
In the case of another snake [Rhadmcea merremii), specimens of the leech,
P. brasiliensis, which had been allowed to feed on it were later found to
contain developmental stages of a trypanosome. The snake was then
carefully examined, and found to have a small infection of a trypanosome
resembling T. brazili.
2. Trypanosomes of Amphibia.
(a) Trypanosomes of Anura.
Ghige (1842) appears to have been the first to have seen what was probably a
trypanosome in the blood of the frog-. In the following year Mayer described
various forms of the same organism under the names of Amceba rotatorium. Para-
mecium loricatum, P. costatnm, while later in the year Gruby gave a better descrip-
tion, and suggested for it the new name Trfipanosoma sanguinis. The trypanosome
was seen by other observers, and Lieberkiilni (1870) proposed the name Monas
rotatoria and Eay Lankester (1871) the name TJndulina ranarum. Grassi (1881-
1882) studied the trypanosome in various frogs and toads, and separated from
T. sanquinis the forms which, though possessing a membrane, appeared to have no
flagellum. under the name of Paramecioides costatns. These were evidently the
forms studied by Mayer, and named by him Paramecium costatnm. The trypano-
somes of frogs and toads were then studied by various observers in many parts of
the world, and owing to their extreme polymorphism, numerous names appeared
which are undoubtedly synonyms.
Noller (1913&) has studied the whole question, and has come to the conclusion
that only two certain species are represented amongst the large number of trypano-
somes described from frogs and toads — viz., T. rotatorium (Mayer, 1843), and T. inojii-
natum Sergent, 1904.
Trypanosomes conforming to one or other of the types seen in T. rotatorium
have been described from frogs and toads from various parts of the world, but it is
not possible definitely to assert that they all belong to one species, though in many
cases this is highly probable (Fig. 238). The following names, which may be synonyms
TRYPANOSOMES OF AMPHIBIA
589
Fig. 238.— Trtpanosomes seen in the Blood of Congo Frogs (x 2,000).
(After Dutton, Todd, and Tobey, 1907.)
They were described under the following names: 1-3 and 5, T. loricatum ; 4 and 6,
T. mega ; 7, T. laryoseukton.
590 FAMILY: TRYPANOSOMIDiE
of T. rotatorium, have been used for tryi)anosomes of frogs and toads, apart from
those already given: T. mega Button and Todd, 1903; T. Icaryozeukton Dutton and
Todd, 1903; T. rotatorium var. nana Ed. and Et. Sergent, 1905; T. nelspruitense
Laveran, 1905; T. belli Nabarro, 1907; T. borelli Marchoux and Salimbeni, 1907;
T. hijlce Franga, 1908; T. leptodactyli Cariui, 1907; T. innominatum Pittaluga, 1905;
T. somalienselixuiwyii, 1906; T. bocrt</ei Fran§a, 1911; T.hocageivaiY.imrvaSinA magna
MathisandLeger, 1911 ; T.c7*fl<^oBiMatliisandLeger, 1911 ; TJwmidrtAverinzev, 1918.
Similarly, in the case of T. inojnnatum the following names appear to be
synonyms: T. undulans Franca and Athias, 1906; T. elegans Franga and Athias,
1906; T. hendersoni Patton, 1908.
Laveran and Mesnil (1912) arrange the trypanosomes of frogs and toads in four
groups. They separate from the two species named above T. leptodactyli of Lepto-
dactylus ocellatus of Brazil and all the trypanosomes of toads. Fran9a (1925)
believes that T. mega and T. haryozeulcton of Bufo regularis are good species.
Trypanosoma rotatorium (Mayer, 1843). — As a result of the work of
Noller (19136), it would appear that this trypanosome is primarily a
parasite of the tadpole, and is handed on from one tadpole to another by
the leech, Hemiclepsis marginata. In the tadpole, and also in young frogs,
the flagellate is of the usual narrow trypanosome type (Fig. 239, 1-2). In
older frogs there appear many remarkable forms which are to be regarded
as derived by overgrowth from the more typical trypanosomes of the
tadpole (Fig. 239, 11-12).
Morphology. — The tadpole trypanosome, according to Noller (19136),
has a body measuring from 25 to 35 microns in length. The nucleus lies
at the centre of the body, and is 2 to 2-8 microns in diameter. It is
spherical, and in properly fixed specimens is seen to have a central karyo-
some. The flagellum is 12 to 15 microns in length. The posterior end
of the body is sharply pointed and the undulating membrane is well
developed. Trypanosomes first appear in the tadpole five or six days
after exposure to infection by the leech. The first trypanosomes to
appear are small and narrow, and it is about the tenth clay after exposure
that the infection reaches its height, and the more typical trypanosomes
corresponding with the measurements given above appear. They are,
however, not numerous, as only about twenty occur in a square (18 by
18 mm.) cover-glass preparation of the blood. Reproduction takes
place in the usual manner by longitudinal division. Noller was unable to
discover any intracellular forms or stages of multiple division as described
by Machado (1911), nor was he able to obtain any evidence justifying the
separation of any of these trypanosomes into male and female individuals,
as this observer has done.
The trypanosomes of the adult frog occur in three main types:
1. Long narrow forms with well-developed undulating membrane,
spherical nucleus, compact kinetoplast, sharp-pointed and drawn-out pos-
terior end, and flagellum. The periplast may be longitudinally marked.
TRYPANOSOMES OF AMPHIBIA
591
-^cJ^^'
^0^^^^^^S
ES
Fig. 239. — Tri/panosoma rotatorium Parasitic in the Frog and the Leech
{Hemiclepsis marginata) (x 2,900). (After Noller, 1913.)
1-2. Typical forms in the blood of the tadpole. 3. Crithidia forms from stomach of leech.
4-7. Try]^anosome forms from stomach of leech.
8-10. Nuclear divisions of roimded-ofE forms which develop in cover-glass cultures of the large
striated forms from frog's blood.
11. Flat leaf-like form from frog's blood.
12. Solid striated form from frog's blood.
592 FAMILY: TRYPANOSOMIDiE
2. Large compact individuals (Fig. 239, 12), more or less spherical or
ovoid in shape, with a longitudinally striated periplast, spherical nucleus,
and spherical kinetoj)last which lies near the nucleus. The undulating
membrane is well developed, while the axoneme usually terminates at the
anterior end. In some, however, there is a short flagellum. The posterior
end of the trypanosome is often rounded.
3. Flat leaf-like forms (Fig. 239, 11), with rounded or pointed posterior
end, well-developed undulating membrane, and long flagellum. The
periplast is usually not striated. The nucleus is a long drawn-out structure,
the posterior end of which lies near the kinetoplast at the posterior end of
the trypanosome, while the other end terminates at the middle of the body.
According to Noller, it is in the order given above that these forms
appear in the blood of the frog. Those of type 1 are undoubtedly
developed by growth from the tadpole form, which is of the same shape
but smaller. By a further growth and thickening the large solid forms of
type 2 are produced. Whether the leaf-like forms of type 3, with their
curious elongate nuclei, are developed from the solid forms in some way
or from those of type 1 through an increase in breadth and not in thickness
cannot be stated with certainty, but the latter would seem to be more
probable. The three types are not sharply marked off from one another,
as connecting links occur. It is thus evident that T. rotatorium of the
tadpole and frog exhibits a great variety of shape and form, and it is for
this reason that numerous synonyms have arisen.
Susceptibility of Frogs and Other Animals.— Noller (19136, 1917), working
in Europe, has published accounts of inoculation experiments per-
formed with the trypanosome of frogs. The blood of tadpoles of Rana
esculenta infected with trypanosomes was inoculated into adult frogs,
which developed a larger infection of the forms characteristic of frogs
than they had before. Further inoculations were carried out with large
doses of cultural forms from blood-agar plates. Though the frogs had
already a small infection, they developed an enormous one which killed
them. The blood and organs were swarming with the large trypanosomes,
and this was especially marked in the kidneys, where veritable emboli of
these forms occurred. These infections were undoubtedly superimposed
on old-standing ones. Inoculation of R. temporaria, which is rarely found
naturally infected, with cultures of T. rotatorium derived from R. esculenta
led to a milder blood infection though the kidneys were found heavily
loaded with trypanosomes. The tree frog, Hyla arborea, was also infected,
a fact which suggests that T. hylce of Fran9a (1908c?) is actually T. rota-
torium. Two toads, Bombinator igneus, were inoculated with very large
doses of culture, and no infection took place. This species of toad
has never been found naturally infected with a trypanosome. Similar
TRYPANOSOMES OF AMPHIBIA
593
experiments with the tortoise and goldfish gave only negative results.
It is possible that T. rotatorium. occurs in a number of different hosts.
Transmission.— The intermediate host of T. rotatorium, as first demon-
strated by Fran9a (1908a), and then by Noller (19136), is the leech, Hemi-
clepsis marginafa (Fig. 240), but before discussing the development in this
invertebrate it will be necessary to describe some details of its anatomy.
The mouth opens into the proboscis, a thick-ridged cylinder which is
armed with teeth, and completely retractable into
the proboscis sheath (Fig. 244). The sheath is an ^^
infolding of the anterior end of the body, forming &,
a cavity in which the proboscis lies. Through the g^
anterior opening of the proboscis sheath the proboscis ^\
can be protruded at the time of feeding, while the rM
margin of the proboscis sheath is applied to the skin.
It will thus be seen that in the act of feeding the
contents of the proboscis sheath can gain easy access
to the wound made by the proboscis. An oesophagus
leads from the proboscis to the stomach or crop,
a large structure with lateral diverticula occupying
most of the body cavity. From the stomach an
intestine with diverticula at its anterior end leads to
the anus.
On account of the scanty infection in the tadpole,
it was impossible to observe the earliest stage of
development in the leech. In two or three days after
feeding on the infected tadpole there are present in
the stomach stumpy forms which were described as
having the leptomonas structure, but which may in
reality be crithidia (Fig. 239, 3).
How these actually arise was not determined.
They may have been the result of repeated binary
fissions of the ingested trypanosomes, or, perhaps,
what is more probable, the products of the seg-
mentation of a spherical stage such as Robertson
(1907, 1909, 1909r/) and Brumpt (1905) have described in the development
of fish trypanosomes in leeches (see p. (J03). Noller has observed such
a multiplication of the large thick individuals in cultures made from frog's
blood (Fig. 239, 8-10). From the third day onwards there begin to appear
very active narrow crithidia forms. At the end of a week narrow trypano-
somes occur, and they gradually replace the other forms (Fig. 239, 4-7).
Towards the end of the period of digestion (ten to fourteen days) the
trypanosomes migrate forwards to the proboscis, and pass out of the mouth
T. 38
Fig. 240. — Hemiclep-
sis marginata, A
Transmitter of
Trypanosomes of
Frogs and Fish
( X 3). (After
Harding, 1910.)
594 FAMILY: TRYPANOSOMIDiE
into the proboscis sheath, where they multiply rapidly. Infection takes
place from the proboscis sheath during the sucking act. After feeding, the
leech has emptied its proboscis sheath and multiplication of the trypano-
somes commences again in the stomach, and reinfection of the proboscis
sheath again occurs towards the end of digestion. The development in the
leech takes place in the stomach alone, the intestine being free from
flagellates.
No intracellular stage was observed in the leech, nor did the trypano-
somes invade the body cavity. As Noller points out, it is remarkable how
easily the young leeches infect themselves from tadpoles which have a very
scanty infection of trypanosomes. Over a hundred young leeches were
thus infected. On the other hand, twenty-six leeches had a full feed on an
adult frog, in the blood of which occurred the large solid giant forms as
well as the thin leaf-like ones. Not a single leech was infected, though
they were kept under the same conditions as regards temperature as the
others (10° to 20° C). It might be urged that the trypanosomes of the
adult frogs belonged to a different species from that of tadpoles, but this
view is not tenable, as occasionally the young frogs show the typical
tadpole forms, while the frogs raised from the tadpoles only showed the
larger forms. For these and various other reasons, Noller concludes that
the typical tadpole forms become transformed into those which appear in
the frogs on account of change in the character of the blood associated
with the metamorphosis of the tadpole into the frog.
Culture. — T. rotatorium is readily cultivated in blood-agar medium.
Of special interest is the behaviour of the large trypanosomes in vitro.
The phenomenon was first observed by Danilewsky (1885a, 1889) and
Chalachnikov (1888), and was later observed by Mathis (1906), Franga
and Athias (1907), Button, Todd, and Tobey (1907), LebedefE (1910),
Doflein (1910), and lastly by Ndller (19136). It consists of the rounding-
ofE of the trypanosome and its segmentation into a number of small
individuals. The accounts differ somewhat in details, the following
being based on the work of the last-named author. A drop of blood
from a frog was diluted with a similar quantity of bouillon, and a moist
preparation made and sealed to prevent drying, and examined at ordinary
laboratory temperature (10° to 25° C). If one of the large solid striated
trypanosomes (type 2) is kept under observation, it will be found to lose
its membrane and flagellum. Furthermore, the longitudinal markings
disappear, and, owing to various fibrous structures which appear adherent
to the cytoplasmic mass, it seems as if the striated periplast is thrown off.
Nuclear division can be seen to take place, and finally after about five to
six hours the cytoplasm divides, the two daughter individuals remaining
side by side. Each daughter then divides again, and the process is
TRYPANOSOMES OF AMPHIBIA 595
repeated till a collection of twenty to thirty-two small cytoplasmic bodies
devoid of flagella results. After twenty to twenty-four hours from the
commencement the small bodies begin to exhibit trembling movements,
and careful observation reveals a short flagellum on each. Division of these
forms continues, and they gradually elongate and assume the crithidia
form with a short undulating membrane. After forty-eight hours each
original trypanosome will have given rise to a cluster made up of about
150 small crithidia forms. The clusters then break up, and the individuals
swim away and continue their multiplication. It will be noted that the
original trypanosome loses its flagellum entirely, and those of the daughter
individuals are newly formed. Furthermore, the division is always a
repeated simple binary fission, and not a multiple segmentation. Noller
was able to make a similar observation on the broad leaf-like trypanosomes
(type 3). In the case of these the process proceeds more slowly, and the
large cytoplasmic body formed from the original tryj)anosome extrudes a
number of bud-like processes representing the daughter individuals, which,
however, do not detach themselves. As many as forty-eight may be
present after forty-eight hours. After another twenty-four hours flagella
develop at the pointed extremities of these processes, and finally a mass of
small crithidia forms is produced. They remain grouped together, however,
for a much longer time than in the development of the trypanosome of
type 2. Noller seems to think that the length of time that these daughter
forms remain together in clusters is suggestive of the division having taken
place within the periplast of the original trypanosome. It is probable
that this development, which takes place in vitro, represents the early
development in the leech, Hemiclepsis marginata. Noller believes that
the leech, Piscicola geometra, will also prove to be a vector of T. rotatorium.
Ponselle (19236) has shown that this development of the large trypano-
some is directly dependent upon the reaction of the medium. It will not
take place in blood mixed with simple saline solution, but occurs if broth
is used to dilute the blood. Broth having an acid reaction (about pH 6-3),
he tested saline to which 0-2 per cent. HCl was added, and found that the
development took place. By substituting a more complex mixture such
as Ringer-Locke solution for the simple saline, the development was even
quicker. It was found that cultures of T. rotatorium could easily be
obtained in a mixture of broth and one-tenth its volume of defibrinated
rabbit's blood. No development occurred in a mixture of equal parts of
distilled water and defibrinated rabbit's blood. In the case of T. inopi-
natum development readily occurred in the latter, but not in the
former, so that each mixture appears to be specific for its particular
trypanosome.
Trypanosoma inopinatum Ed. and Et. Sergent, 1904.— This trypano-
596
FAMILY: TRYPANOSOMID^
some, like T. rotatorium, is a parasite of the edible frog, Rana esculenta.
and lias been described under a variety of names owing to the fact that
the blood form changes considerably during the course of an infection.
2 "^-^^^r,.^
9
Fig. 241.
,000).
-TrypiDiosonia inopinatum OF the Frog (;
(After Franca, 1915.)
1. Fully-grown form {T. undulans). 2-3. Forms of intermediate- size {T. clegam).
4-G. Typical small forms {T. inopinatum). 7-10. Cultural forms.
It was originally named T. inopinatum by its discoverers in Algiers, then
other stages were named T. undulans and T. elegans by Franga and Athias
(1906) in Portugal, and what is probably the same form in R. tigrina,
T.hendersoni by Patton (1908c) in India. Observations by Brumpt (1906c)
and Fran9a (1909, 19116, 1915), who followed the infection in frogs from
its beginning, conclusively demonstrated that all these forms are merely
stages of development of one and the same organism (Fig. 241).
TRYPANOSOMES OF AMPHIBIA 597
Morphology. — The first trypanosomes to appear in a frog after inocula-
tion are small trypanosomes with a body 16-5 to 21 microns in length by
1-5 to 2-2 microns in breadth. The posterior end of the body is pointed,
while the margin of the undulating membrane is fairly straight. The
flagellum is 6 to 10-5 microns in length. The nucleus is central and the
kinetoplast well developed. These are the forms seen by the original
observers, who noted that the trypanosome bore a striking resemblance to
the late phase form of T. leivisi of the rat (Fig. 241, 4-6). After a few
days, during which the small forms alone are present in the blood, larger
forms begin to appear by growth of these. They measure 35 to 36 microns
in length and 2-2 to 3-5 microns in wddth. These are the trypanosomes
which were originally described as T. elegans (Fig. 241, 2-3). The flagellum
is only 5 to 6 microns in length. By continued growth they give rise
to still larger forms (T. undulans) 36 to 37 microns by 4-5 microns
(Fig. 241, i). Sometimes much larger forms up to 54 microns in length
occur. About a month after the infection first appeared the large forms
may be the only ones present in the blood.
Apparently none of these various types of trypanosome seen in the
blood is undergoing division. Fran9a (1915), in smears of the lung, has noted
within the cells leishmania forms, many of which are in process of division.
Between these and the small trypanosomes every intermediate stage can
be traced. It would thus appear that reproduction takes place by division
of leishmania forms in the lung or other organs in much the same way as
occurs in T. cruzi. The leishmania forms are apparently derived in the
first place from the flagellates inoculated by the leech.
Transmission. — Billet (1904), who found a variety of flagellates in the
intestine of the leech {Helobdella algira), which fed upon the frog, came to
the conclusion that it was the transmitting host of the trypanosome
(Fig. 242). Accordingly, Brumpt (1906c) in Paris obtained a number of
these leeches from Algiers. He found that Rana esculenta of France was
easily infected by the bites of the leeches, the first trypanosomes appearing
in their blood in eight to ten days. The European frog, moreover, was
very susceptible, for in many cases the infection proved fatal. The
trypanosomes were found to be present in enormous numbers, the heart
of frogs which had died being filled with an embolus of the organisms.
R. temporaria is also susceptible to the infection. Franca (1915, 1920)
notes that the heaviest infections follow inoculation with the young
forms of the trypanosome.
The process of development in the leech has been described by Brumpt
(1906c). The large form of the trypanosome is the one taken up by the
leech, and in the stomach it gives rise to numbers of crithidia forms.
This process does not seem to have been described in detail, but from
598
FAMILY: TRYPANOSOMIDiE
observations under the cover-glass in wet films Franca has noted that the
large trypanosomes became more rounded and that multiplication of the
nuclei and kinetoplasts takes place till several pairs are present. The
cytoplasm segments into a corresponding number of crithidia forms after
axonemes have grown out from the kinetoplasts. It is probable also that
the large forms divide unequally and repeatedly, giving rise to small
crithidia forms with gradual reduction in size of the parent. In whatever
manner the process takes place in the leech, the stomach soon becomes
crowded with large numbers of these crithidia forms, which multiply by
fission in the usual manner. Eventually a
return to the trypanosome type is noted,
and it is these forms which probably, by
migration along the oesophagus to the open-
ing of the proboscis, gain access to the
cavity of the proboscis sheath, w^here they
accumulate, and are transmitted to the
frog when the leech feeds. In the case of
this trypanosome of the frog, as with the
others which have been considered above,
the trypanosomes taken up by the inver-
tebrate become at first crithidia forms,
which later are transformed into try-
panosomes again. These metacyclic try-
panosomes which appear at the end of
the cycle reproduce the infection in the
vertebrate.
In connection with the transmission of
T. inopinatum by the leech, H. algira,
Brumpt (1907) noted what is interpreted
as a definite hereditary transmission in the
leech. The embryos, when they hatch from
the egg, attach themselves to the ventral
surface of the parent leech, and in this position were found by Brumpt
to be infected. He has been able to observe this infection persisting
through five successive generations of leeches. The infected young are
able to infect frogs. Brumpt considers that the egg is infected while still
within the parent, but he does not seem to have excluded the possibility of
the young leeches being infected soon after hatching from flagellates which
escape into the water from the intestine of the parent. He does not state
whether young leeches removed from the egg are already infected or not.
Culture.— Ponselle (1923) has shown that T. inopinatum is readily
culturable in a mixture of equal parts of defibrinated rabbit's blood
Fig. 242. — Helohdella algira
(x5), THE Transmitter of
Trypanosoma inopinatum.
(After Brumpt, 1922.)
Dorsal view and ventral view with
attached eggs.
TRYPANOSOMES OF NEWTS AND FISH 599
inactivated at 56° C. for thirty minutes and distilled water. In this
mixture T. rotatorium will not develop.
According to Brumpt (1914a), T. lejdodactyli of the Brazilian frog,
Leptodactylus ocellatus, undergoes a complete development, terminating in
the appearance of metacyclic trypanosomes in the proboscis sheath, in the
leech, Placobdella hraziliensis.
Lloyd, Johnson, Young, and Morrison (1924) have shown that labora-
tory bred Glossina tachinoides in Nigeria develop a crithidial infection of
the intestine after feeding on toads, Bufo regularis, which harbour trypano-
somes resembling T. varani.
{b) Trypanosomes of Urodeles.
A trypanosome in an American newt {Diemyctyhis viridescens) was
described by Tobey (1906) under the name of T. diemyctyli. The body of
the trypanosome measures 45 to 50 microns, and there is a fiagellum
24 microns in length. The breadth varies from 2 to 5 microns. The undu-
lating membrane is well developed. Hegner (1921) has called attention
to the frequency with which these newts are infected. Ogawa (1913)
described a trypanosome named by him T. tritonis from the Japanese
newt, Triton jpyrrhog aster. It measures 57 to 80 microns in length by
2-4 to 6-4 microns in breadth. The fiagellum is about 15 microns long.
It was readily cultivated in bouillon to which a tenth part of defibrinated
rabbit's blood had been added.
3. Trypanosomes of Fish.
The trypanosomes of fish have attracted attention since Valentin's
discovery of what was either one of these flagellates or a trypanoplasm in
the blood of a trout (Salmofario) in 1841. A large number have since been
seen in both fresh and salt-water fish in various parts of the world, and
many of them have been given specific names. In some cases there is
evidence that one and the same trypanosome may have several hosts.
Morphology. — The trypanosomes of fish usually have long and narrow
bodies (Fig. 243). When observed alive, they wriggle about in a peculiar
snake or worm-like manner, and frequently roll themselves into knots,
only to extend themselves again. The trypanosome of a ray (T. giganteum)
may be as much as 130 microns in length, while that of the pike (T. retnaki)
may have a body only 15 microns long. The majority of forms are about
50 microns in length, with a breadth of 2 to 5 microns. There is a fiagellum
of varying length and a well-developed undulating membrane. The kine-
toplast is generally large, and the nucleus, which is often easily visible
in the living trypanosome, is centrally placed. Sometimes, as in the
case of T. remaJci, two types of trypanosome are present in the blood
600
FAMILY: TRYPANOSOMID.^
Fig. 243. — TpaPANOSOMES of Fresh Water Fish (x 2,000). (After Minchin, 1909.)
1-3. 7'ri/panosoma granulosmn of the eel.
7-8. T. renidki of the pike.
11. T. abramidis of the bream.
4-6. T. perccB of the perch.
9-10. T. tincce of the tench.
12. T. percce, showing niyonemes.
TRYPANOSOMES OF FISH
GOl
(Fig. 243, 7-8), as pointed out by Laveran and Mesnil (1901c) and Minchin
(1909). There are large forms (T. remahi var. magna) measuring 45 to 57
microns in length, of which nearly 20 microns is taken up by the flagellum,
and small forms {T. retnaJd var. parva), which may have a body 10 to 25
microns in length with a flagellum from 10 to 17 microns long. It seems
Fig. 244.
-Diagram of Tkypanosomes in the Blood of a Fish and in
THE Leech. (After Wenyon, 1922.)
A. Trypanosomes in blood of fish. B. Developmental forms in stomach of leech.
S. Trypanosomes in stomach of leech. P. Develoj^mental forms in proboscis sheath.
C. Crithidia forms in proboscis sheath of leech.
D. Metacyclic trypanosomes in proboscis sheath of leech.
hardly probable, however, that the forms seen in the pike belong to two
species, though Minchin (1909) asserts that the two types are sharply
marked off from one another.
Some of the larger trypanosomes of fish, as, for instance, T. perccE
Minchin, 1909, of the perch, may have long longitudinal myonemes w^ell
developed (Fig. 243, 12).
Susceptibility of Fish. — Trypanosomes of fish are directly inoculable
from one to another. Thus, Laveran and Mesnil (1904) state that they
602
FAMILY: TRYPANOSOMID^
had been able to infect pike and eels by injecting blood from infected fish.
Very few attempts, however, have been made to infect fish with trypano-
somes from other species. Lebailly (1906) made some experiments of this
nature without success. Robertson (1911) found that the trypanosome of
the goldfish, perch, and bream could be transmitted to goldfish by the leech.
Transmission. — In nature, the trypanosomes of fish are carried by
leeches (Fig. 244). Some attempts by Minchin (1909) to infect the crus-
tacean Argulus by placing them on fish gave no result.
Fig. 245. — Piscicola geometra, Dorsal and
Ventkal Views (x 3), Transmitter of
Trypanosomes and Trypanoplasms of
Fresh Water Fish. (After Harding,
1910.)
Fig. 246. — PontobdeUa muricata,
the Transmitter of Try-
panosomes OF Marine Fish
(Natural Size). (After
Harding, 1910.)
As long ago as 1857 Leydig had noted the presence of flagellates in the
stomach of the leeches {Piscicola and PontobdeUa) which had fed on fish,
and Doflein (1901) suggested the possibility of these invertebrates being
vectors of the fish trypanosomes. Keysselitz [cited by Hofer (1904)j
was able to transmit the trypanosomes of tench, carp, and pike by means
TRYPANOSOMES OF FISH 603
of the leech, Piscicola geometra (Fig. 245). Brumpt (1904) observed the
development of enormous numbers of trypanosomes in the stomach of
Hemiclepsis marginata which had fed on infected fish, and Leger, L. (1904e),
made a similar observation with species of Piscicola fed on loaches infected
with T. barhatulcB. BrumjJt (1905) succeeded in infecting young carp and
two bull-heads by exposing them to the bites of leeches. Brumpt then
traced the development of T. granulosum of the eel in H. marginata, and
of various trypanosomes of marine fish — T. solce and T. cotti in Trachelob-
della punctata, and T. scyllU and T. rajce in Pontobdella muricata. In the
case of T. granulosum, he noted that after multiplication had taken place
in the stomach of the leech the flagellates migrated forwards and passed
through the proboscis into the proboscis sheath, whence infection of the
wound inflicted by the proboscis took place. Neumann (1908, 1909)
described the development of T. giganteum and T. variabile of the skate in
the leech {Pontobdella), and was able to infect Raja punctata with T. varia-
bile by means of P. muricata (Fig. 246).
Cycle in the Leech.— Robertson (1907) has studied the trypanosomes
in P. murciata, and suggested the possibility of their being derived from
the trypanosome (T. rajce) of the skate (Fig. 247). A further contribution
(1909, 1909a) to the subject was made by this observer, and the flagellates
of the leech were definitely associated with T. rajce. The first stages of
development in the leech, according to Robertson, is a rounding-oft" of the
trypanosome, with loss of undulating membrane and flagellum (Fig. 247,
i-io). The latter is finally cast ofi from the body, and may continue its
movements in this free condition for some time. The rounded cytoplasmic
body resulting from this change then undergoes division. The whole
process can be watched under the microscope in a fresh blood-preparation.
The single nucleus can be seen at the centre of the parasite, and its division
into two can be followed. After division of the nucleus the cytoplasm
divides, and two smaller bodies are produced. These in their turn divide,
and the four daughter individuals repeat the process. In a film thirty-six
hours after preparation there were present still unaltered trypanosomes
actively motile and non-motile individuals in groups of four, six, or
eight. At about this stage in the daughter forms there appear short
stiff rods which by gradual growth become flagella. They seem to take
about twelve hours or more to become motile. The flagellate forms
thus produced are more or less rounded, and by change in shape and
elongation, during which further multiplication occurs, various types of
flagellate, some of which have the crithidia forms, arise (Fig. 247, ii). In
the leech the rounding-ofl process and division into non-flagellate daughter
forms and the early formation of the flagella take place in what Robertson
calls the first stage of digestion. The production of the large number of
604
FAMILY: TRYPANOSOMIDtE
( \
Fig. 247. — Development of Trypanosoma rajw in the Leech, Pontohdella muricata
(2-14, X 4,500; 1, 15, 16, LOWER MAGNIFICATION). (After Eobertson, 1907
AND 1909.) rr^ ^ • -•
[ For description see opposite page.
TRYPANOSOMES OF FISH 605
flagellates of various types occurs during the middle period, when the blood
is being digested into a green-brown fluid (Fig. 247, 12-14). In the third
period of digestion the crop or stomach becomes nearly empty, and long,
slender, very active flagellates of the typical trypanosome type appear.
These forms migrate forwards, and presumably find their way into the
proboscis sheath, though this is not actually mentioned (Fig. 247, 15-16).
The whole developmental process is very similar to that of T. vittatce of the
tortoise described above. It is presumably the long narrow trypanosomes
of the proboscis which bring about infection of the vertebrate.
The development of T. gmnulosum of the eel in Hemiclepsis, as de-
scribed by Brumpt, is very similar to that of T. rajce. At the end of
twenty-four hours after feeding, however, flagellates had vanished from
the stomach, and were undergoing development as leptomonas (? crithidia)
forms in the intestine, whence they eventually migrated to the stomach
and along the oesophagus to the proboscis and its sheath, where the meta-
cyclic trypanosomes were to be found.
According to the observations of Brumpt (1904-1906), the trypanosomes
of fresh-water fish are carried by Hemiclepsis marginata, in which the
development is of two types (Fig. 240).
I. The trypanosomes develop in the stomach alone, and here the
crithidia forms and eventually the metacyclic trypanosomes appear.
There is no infection of the intestine nor of the proboscis sheath. Infection
of the fish takes place by active migration or regurgitation forwards of
the metacyclic trypanosomes while the leech feeds. To this category
belong T. ahramidis, T. remaki, T. barbi, T. percce, T. acerincB, and
T. squalii.
II. The trypanosomes develop in the stomach and then pass into the
intestine, where the flagellates persist. Before the leeches become infective
the intestinal forms reinfect the stomach, from which the proboscis sheath
is infected with metacyclic trypanosomes. To this group belong T. granu-
losum, T. danilewskyi, T. phoxini, and T. carassii.
In the case of other trypanosomes, only part of the cycle was observed.
A development in the stomach was followed, but the subsequent events
were not traced. To this group belong T. barbatulce, T. langeroni, T . scar-
dinii, T. leucisci, and T. elegans. Tanabe (1924) has noted that the
trypanosome of the Japanese loach {Misgurnus anguillicaudatus) multiplies
for a period of three or four days in the intestine of the leech {Hirudo
nipponica). No transmission experiments were carried out.
1. Large form in blood of skate.
2-10. Rounded forms from the alimentary tract of the skate. Some of these are without flagella,
and most of them are in process of division.
11. Crithidia form in crop of leech. 12-14. Trypanosome forms from crop of leech.
15-16. Slender forms from the proboscis of the leech.
606
FAMILY: TRYPANOSOMID^
As regards marine fish, Brumpt (1906) studied the development of
T. cotti and T . solce in Trachelobdella punctata. As was subsequently con-
firmed by Robertson (1909) in the case of T. rajce, the trypanosomes lose
their fiagella, and active multiplication in the non-flagellate condition
takes place. It is only after some days that crithidia and trypanosome
forms reappear. The development is confined to the stomach. In the
case of T. scyllii and T. rajce, the same type of development occurs in
^O
Fig. 248. — The Trypanosome of the G-oldfish in Culture ( x ca. 2,000).
(After Thomson, J. D., 1908.)
1-2. Forms from the blood of the fish. 3-5. Crithidia forms in early cultures.
6. Granular crithidia forms in older cultures.
7-8. Crithidia form and metacyclic trypanosome form from culture on the forty-thiid day.
Pontohdella muricata, but infection of the intestine follows the stomach
phase, whereupon the forms in the stomach disappear. In no case did
Brumpt observe infection of the proboscis sheath. In one instance,
T. cotti was transmitted to a fish (Cottus hubalis) by the bite of an infected
leech.
Culture. — The trypanosomes of fish are easily cultivated in blood-agar
media. Thomson, J. D. (1908), cultivated the form in the goldfish, and
FAMILY: BODONID.^ 607
made the interesting observation that in okl cultures there appeared
trypanosome forms, which were evolved from the crithidia forms which
occurred earlier. This was a clear demonstration that the cultural forms
resemble in type and sequence those which occur in the invertebrate host
(Fig. 248).
3. Family: bodonidte Doflein, 1901.
In this family are included a number of flagellates which have one or
more anteriorly directed fiagella, and one which is often, though not always,
longer and thicker than the others, and which trails behind the organism
during progression as a trailing flagellum. The simplest forms belong to
the genus Bodo, and have been described under various names [Cystomonas
Blanchard, 1885, Prowazekia Hartmann and Chagas, 1910, etc.) as occur-
ring in human faeces and also urine.
Oenus: Bodo (Ehrenberg, 1830) Stein, 1875.
The flagellates belonging to this genus have ovoid bodies, an antero-
lateral cytostome, a central nucleus and a kinetoplast consisting of a
parabasal body, and two blepharoplasts, from which arise the axonemes
of the two fiagella. Species of Bodo occur commonly in stagnant water
and infusions, so their presence in faeces and urine is usually the result of
the development of encysted forms which have gained entrance to the
material from the air, or the receptacle, or have been ingested and passed
through the alimentary canal. In the case of the faecal forms, it seems
clear that in all cases the organisms which have been described as Bodo
or Proivazehia have been purely coprozoic forms which have developed
after the stool has been passed. Thus Porter (1918) describes cases of
human infection with P. cruzi and B. stercoralis in South Africa without
producing any evidence that such extraneous sources of contamination
have been excluded. A number of observers have claimed to have found
Bodo-\ike organisms in urine. None of these accounts is entirely satis-
factory, and having regard to the fact that organisms develop very rapidly
in decomposing urine outside the body, in most cases it is safe to assume
that the flagellates have developed after the urine had been passed. In
other cases the flagellates may have been Trichomonas, which are known
to occur in the urethra, and are quickly changed in appearance by the
action of urine. In one instance, however, Powell and Kohiyar (1920)
have described a case in which flagellates were present in the urine drawn
aseptically from the bladder of a man in India. The case was repeatedly
examined during five years, and the organism was constantly present. It
is described as a " Bodo-like " organism, but the details of its structure
were not accurately made out. The writer has examined some of the
fixed material, and can only say that the flagellate which was present had
608
FAMILY: BODONID^
a round or ovoid body, a nucleus, and two flagella. It seems quite possible
that it is actually a species of Bodo, but more than this cannot be stated.
Knowles and Das Gupta (1924) have seen a species of Bodo in the
saliva from the human mouth. It was seen on three occasions between
August 18 and October 27. Knowles, Napier, and Smith (1924) record
the occurrence of a flagellate belonging to this genus in the rectum of the
sand fly, Phlebotomus minutus, in India. To Alexeieff (1910fl) is due the
credit of first pointing out that the flagellates described as Protvazelxia did
not differ structurally from those of the genus Bodo.
Bodo caudatus (Dujardin, 1841) — The synonymy of this species is given by
Dobell and O'Connor (1921) as follows: Amjihimonas caudata Dujardin, 1841; Bodo
urinarius Hassall, 1859 ; Diplomastix
caudata Kent, 1881 ; B. asiaticus Castellani
and Chalmers, 1910; Prowazelcia cruzi
Hartmann and Cliagas, 1910; P. weinbergi
Mathis and Leger, 1910 ; P. asiatica
(Castellani and Chalmers) Whitraore,
1911; P. javanensis Flu, 1912; P. urinaria
(Hassall) Siuton, 1912; P. i/r/ZiCrt Sangiorgi
and Ugdulena, 1916; and with these must
be included B. stercoralis Porter, 1918,
and P. ninoe IcoM-yaTcimoviYaiMmoff, 1916.
It is probable that B. caudatus
is the commonest coprozoic flagellate
to appear in human fseces. It is in
no sense an inhabitant of the human
intestine, but develops in fseces after
they have left the body, and can
often be obtained by inoculating
fseces on to agar plates.
The forms which develop in decom-
posing urine, as described by Hassall
(1859)andSinton (1912), are probably
the same species (Fig. 249). Accord-
ing to Klebs (1892), the flagellates
vary in length from 11 to 19 microns,
It may be long and slender or more
or less rounded. The posterior end of the body is pointed and the body
is somewhat flattened. At one side of the anterior extremity is the
mouth, which may be considered to be on the ventral surface. Dorsal to
the mouth is a small contractile vacuole, near which is the kinetoplast,
a structure made up of a deeply staining parabasal and two blepharo-
plasts, each of which gives origin to an axoneme which passes to the
surface of the anterior end of the body to form a flagellum. There is a
0 *i' S
Fig. 249. — Bodo caudatus from Human
TJpaNE (x 1,650). (After Sinton,
1912.)
1-2. Two types of flagellate.
3. Encysted form.
4-5. Emergence of flagellate from cyst.
but the shape of the body varies.
GENUS: BODO
609
nucleus at the centre of the body consisting of nuclear membrane and
large central karyosome. The cytoplasm contains various food vacuoles,
especially in the posterior region. The two flagella which arise from the
anterior end of the body are of unequal length. One is anteriorly directed,
and is about the same length as the body, while the posteriorly directed
one, which trails over the body in progression, is about twice this length.
Fig. 250.~Bodo edax (x 1,400). (After Kuhn, 1915.)
1. Flagellate showing nucleus, kinetoplast, large contractile vacuole, and two flagella.
2. Division of blepharoijlasts and formation of two new flagella; separation of nucleus into
two parts.
.3. Commencing division of the nucleus, the karyosome occupying the hollow of the dumb-bell-
shaped structure.
4. One part of the nucleus divided, with karyosome in the middle.
5. More advanced stage with two kinetoplasts. each with two flagella.
6. Nucleus elongated and karyosome now dividing.
7. Division of body and long drawn-out karyosome.
8. One of products of division, nucleus being reconstructed.
9. Encysted form. 10. Escape of flagellate from cyst.
Dobell and O'Connor (1921) state that the trailing flagellum may be
attached to the surface of the body for a short distance. The cysts|^of the
organism are ovoid bodies 5 to 7 microns in length. As a rule, each cyst
contains a single nucleus and kinetoplast, though in some cases, as a result
of division, two of each of these may be present.
I. 39
610
FAMILY: BODONIDiE
The flagellate multiplies by longitudinal fission after division of the
kinetoplast and nucleus. The organism is readily cultivated in hay and
other infusions, and it will also multiply on the surface of agar plates.
According to Sinton (1912), division of the flagellate takes place once in
every four hours, so that in a short time very large numbers are present
in the medium.
Bodo edax Klebs, 1892. — This is another species which may appear in
faeces, though less commonly than B. caudatus (Fig. 250). It is slightly
smaller than B. caudatus, and more stumpy in form. The two flagella are
(1-4, AFTER PaRISI,
Fig. 251. — Bhynchomonas nasuta (1-4, x 1,800; 5-6, x 3,800)
1910; 5-0, AFTER Belar, 1915.)
I. Usual type of flagellate. 2-3. Dividing forms. 4. Encysted form.
5. Stained flagellate, showiiag details of structure.
6. Dividing form with two kinetoplasts and nucleus dividing by mitosis.
approximately equal in length, and both are longer than the body. The
organism was studied by Kiihn (1915). In its method of multiplication
and cyst formation it is very similar to B. caudatus.
Genus: Rhynchomonas Klebs, 1892.
Stokes (1888) in America described as Heteromita tiasuta a flagellate
which was ovoid in shape and provided with one trailing flagellum, over
the point of origin of which there exteiided a digital process. Klebs
(1892) created the genus RhyncJiomonas for this organism. It was seen
as a free-living flagellate in fresh water by Stokes (1888) and Belaf (1915),
and in salt water by Griessmann (1913), while Parisi (1910) obtained it as a
GENUS: RHYNCHOMONAS 611
coprozoic flagellate from the intestinal contents of cockroaches. Belaf,
who obtained a culture of Rhynchomonas nasuta, has studied its structure
and method of division (Fig. 251). The organism is ovoid in shape, and
resembles members of the genus Bodo in the possession of a nucleus and
kinetoplast. From the latter there arise two axonemes, one of which
becomes a flagellum in the notch formed by the digital process, while
the other is continued to the end of the process, but does not become a
flagellum. The single flagellum acts as a trailing flagellum. The flagellate
is evidently closely allied to species of Bodo, and the notch behind the
digital process bears a striking resemblance to the cytostome of the
flagellates of this genus. Not infrequently in such a form as B. caudatus
the portion of the body in front of the cytostome has the appearance of a
digital process, so that it does not seem improbable that the notch formed
by the digital process may be actually a cytostome.
4. Family: prowazekellid^ Doflein, 1916.
This family includes the single genus Prowazekella, established by
Alexeieif (1912), the members of which are parasitic in the intestine of
lizards. The encysted forms are remarkable in that great increase in
size takes place after the cyst wall has been formed.
Prowazekella lacertae (Grassi, 1879). — Grassi(1879rt), who first saw this flagel-
late, iuchided it in liis genus, Monocercomonas. He afterwards (1881a) placed it in
Dujardin's genus, Heteromita, while Prowazek (1904fl) referred it to the genus Bodo.
Alexeieff (1911) described another form from the intestine of newts, salamanders,
and axolotls, and included the two forms in the genus Heteromita. In the following
year (19126) he created the new genus, Prowazelcella, for these flagellates.
P. lacertcB occurs in the intestine of lizards {Lacerta, Tarentola, etc.).
The fully-grown flagellate has an elongate pyriform body, 10 to 30 microns
in length (Fig. 252). It has a tapering posterior end and blunter anterior
end, from which arise two flagella. One of these is directed forwards, and
may be four times the length of the body, while the other is a trailing
flagellum about twice the length of the body. The latter, in its backward
course, is sometimes attached to the surface of the body for a short
distance before becoming free. There is a nucleus near the anterior end,
consisting of nuclear membrane and a central karyosome. Surrounding
the nucleus are one or more bodies, the parabasals, while extending from
the anterior end of the nuclear membrane is an axoneme (rhizoplast),
which is continued into the two flagella. The life-cycle of the flagellate
was described by Chatton (19176) in the case of the form which occurs in
the gecko, Taretitola fnauritanica. The flagellate multiplies by longitudinal
division in the gut of the lizard. Certain forms then lose their flagella,
and, becoming ovoid in shape, produce cysts which have a diameter of
612
FAMILY: PROWAZEKELLID^
about 10 microns (Fig. 253). The writer (1921) has produced some
evidence that two of the ovoid forms become encysted together, and that
syngamy, with complete union of the cytoplasm and nuclei, follows
(Fig. 254, n-s). A vacuole now appears in the cytoplasm, and the single
nucleus begins to divide. The vacuole increases in size till the cytoplasm
Fig. 252. — Prowazelcella lacence ( x 2,300). (After Belak, 1921.)
A-B. Two types of flagellate, showing nucleus, parabasal body, and flagellar connections.
C-F. Stages in division.
is reduced to a thin layer lining the cyst. At the two-nuclear stage the
nuclei lie at opposite poles of the cyst, which has a large central vacuole
and bears a close resemblance to Blastocystis, with which it has been
compared. Repeated divisions of the nuclei take place, while the cyst
increases in size till it may reach a diameter of about 70 microns. At this
PKOWAZEKELLA LACERT^E
613
stage there are about sixty-four nuclei present. The cytoplasm still
lines the cyst, which has a large central vacuole often traversed by thin
strands of cytoplasm. According to Chatton (19176), the cytoplasm then
becomes heaped up round each nucleus, flagella are developed, and finally
a number of small flagellates having the structure of the free forms are
produced (Fig. 253). The cysts are found in large numbers in the hind-
FiG. 253. — Frowazekella lacertw from the Intestine of the North African
Gecko, Tarentola mauritanica, as seen in Living Condition ( x 720), (After
Chatton, 1917.)
1. Free flagellate. 2. Uninucleated cyst.
3-5. Growth of cyst, multiplication of nuclei and segmentation of contents.
G. Cyst containing flagellates.
gut of lizards, and they are passed in the fgeces. Presumably, when the
cysts are eaten by other lizards, the flagellates are liberated in the intestine.
The minute structure of the flagellate stage of P. lacertce has
been described by Belaf (1921rt). The flagella actually rise from two
minute granules, which are at the extreme anterior end of the flagellate
614
FAMILY: PROWAZEKELLID^
microns
Fig. 254. — Trowazelcella lacertce from the Intestine of the Lizard {Laeerta agilis)
( X 1,250). (After Wenyon, 1921; from Parasitology, vol. xii.)
a-h. Various types of flagellate. In some the backwardly directed flagellum is attached to the
surface of the body for a short distance.
n-s. Probable stages in sjngamy and encystment.
t-y. First nuclear division in zygote and formation of vacuole.
m, I, i. Growth of cyst and multiplication of nuclei.
/, k. Stages corresponding with those at o and f.
PROWAZEKELLA LACERT^E 615
(Fig. 252). From each of these there passes backwards a rhizoplast
(axoneme). The two soon merge into one another, and are continued
to the nuclear membrane as a single rhizoplast. In addition to these
structures, there are two rings. One surrounds the rhizoplast a short
distance behind the basal granules or blepharoplasts, and a kind of funnel
connects the ring with the rhizoplast. About half-way between the
blepharoplasts and the nucleus is a second ring, which surrounds the
rhiziplast. As already described, the spherical nucleus is surrounded by
several bodies or a single elongated body which stains deeply. These
may be regarded as parabasals. When the flagellate divides there is, first
of all, division of the blepharoplasts, and two new flagella are formed.
The two pairs of blepharoplasts then s'eparate till they occupy the poles of
the elongating nucleus. The chromatin becomes arranged at the equator
of an intranuclear spindle in a compact mass formed by a group of
chromosomes. The chromatin mass splits into two daughter plates,
which pass to the poles of the nucleus which now divides. The parabasal
body or bodies become arranged outside the nucleus as an elongate mass
parallel to the nuclear spindle, and with nuclear division this divides into
two parts. Finally, division of the flagellate takes place. The parabasal
bodies persist in the cysts also, and at each division of a nucleus they are
divided into two groups, one of which passes to each daughter nucleus.
Prowazek (1904a) claimed to have demonstrated a process of autogamy
within the cyst, but there is no evidence that such a process takes place.
He concluded also that the cysts of the flagellate were identical with
Blastocystis. This also is not correct. Typical Blastocystis, which is a
vegetable organism, occurs in the intestine of lizards, and is easily mis-
taken for the cyst of P. lacertce (Fig. 118).
Alexeiefl (1911) discovered a form in the intestine of newts, sala-
manders, and axolotls. He regarded it as a distinct species, and later
(19126) gave it the name P. longifiUs.
5. Family: EMBADOMONADiD^ Alexeiei!, 1917.
The flagellates belonging to this family have ovoid bodies and an
anterior nucleus. On one side of the anterior end of the body is a
cytostome, and anterior to it, and close to or actually upon the nuclear
membrane, are two granules, the blepharoplasts, which give rise to two
flagella. One flagellum is long and thin, and passes forwards as an
anteriorly directed flagellum. The other flagellum is shorter and thicker,
and passes backwards to protrude through the cytostome. There is a
single genus which has the characters of the family.
616 FAMILY: EMBADOMONADID^
Genus: Embadomonas Mackinnon, 1911.
The genus Embadojnonas was founded by Mackinnon (1911, 1915)
for flagellates found in the intestine of tipulid and trichopteran larvae.
Two species were described in these insects. Later a form was discovered
by the writer and O'Connor (1917) in Egypt in the human intestine, since
when other forms have been found in the intestine of vertebrates and
invertebrates.
EMBADOMONAS IN MAN.
Embadomonas intestinalis (Wenyon and O'Connor, 1917). — This
flagellate was found in man in Egypt in two cases by the writer and
O'Connor (1917). They placed the flagellate in a new genus as Waskia
intestinalis, but it was evident, as first pointed out by Chalmers and
Pekkola (1918), that it really belongs to Mackinnon's genus Embadomonas.
Fonseca (1920) expressed the opinion that the genus Waskia should be
retained, but it is quite clear that the human parasite shows no features
of generic value which will differentiate it from the genus Embadomonas.
Since E. intestinalis was first described in Egypt, it has been discovered
in other localities. It was found by Kofoid, Kornhauser, and Plate
(1919) in New York in four men who had returned from overseas, and in
four who had not been abroad. Hogue (19216) has reported one case from
Baltimore. Broughton-Alcock and Thomson (1922a) have seen a case in
a man who had returned to London from China, while Jepps (1923) reports
cases from Malaya. As will be shown below (p. 633), Chalmers and
Pekkola (1919a) included this flagellate in their Dij)locercomonas suda-
nensis which they described in the Sudan. A form identical in every way
with E. intestinalis was seen by the writer in the caecum of a guinea-pig
which had been sent to Macedonia from Egypt in 1918, while he has
cultivated the flagellate on three occasions from guinea-pigs, and once
from a wild rat in England.
In the cases examined by the writer and O'Connor in Egypt the
flagellates, when present in the diarrhoeic stool, occurred in large numbers.
In fresh material these were very active, and progressed in a peculiar jerky
manner. The long thin anterior flagellum performed lashing movements,
and it was evidently the organ of progression. The shorter and thicker
flagelluni which protruded through the cytostome had a more regular and
slower action. The shape of the body varied considerably (Fig. 255).
Some forms were elongate and about three times as long as they were
broad, while others were almost spherical. Sometimes the posterior end
of the body was drawn out into a tapering process. When seen with tlie
cytostome at the side, the narrow forms often had an outline resembling
that of a bird. In length the flagellates varied from 4 to 9 microns, and
GENUS: EMBADOMONAS
617
Fig. 255. — Embadomonas intestinalis from the Human Intestine. (After
Wenyon and O'Connor, 1917; Faust, 1922; and Jepps, 1923.)
1-0. Appearance of flagellates in living condition (x ca. 3,000).
.5-6. Dividing form.s. 7-8. Cysts in fresh condition (x ca. .3.000)
9-12. Flagellates fixed and stained, showing relation of two blepharoplasts (x ca. 3,500).
13. Dividing form ( x ca. 3,500). 14-17. Encysted forms stained ( X ca. 3,500).
18. Cyst as seen from end (x ca. 3,500). 19. Ovoid cyst (X ca. 3,500).
20-22. Large forms described as Embadomonas sinensis by Faust (X ca. 2,000).
23-2G. Free and encysted forms as depicted by Jepps (X ca. 3,500).
618 FAMILY: EMBADOMONADID^
in breadth from 3 to 4 microns, while the spherical forms were about
9 microns in diameter. The anterior flagellum was as long or longer
than the body, while the thicker cytostomal flagellum was shorter than
this. Many of the spherical forms were evidently dividing flagellates, as
they were seen to possess two cytostomes, one on each side of the anterior
end of the body, and two pairs of flagella.
The encysted forms as seen in fresh material are whitish, opalescent,
pear-shaped bodies (Fig. 255, 7-8). The anterior end is distinctly narrowed,
and often forms a sort of tubercle. In the living condition they vary in
length from 4-5 to 6 or even 7 microns, while the breadth varies from
3 to 4-5 microns. Dobell and O'Connor (1921) have given the dimensions
of the cysts as less than this, but their measurements were taken from
fixed and stained preparations made by the writer and O'Connor in Egypt.
The writer has recently examined further fresh material, and can verify
the measurements previously given in the account by the writer and
O'Connor (1917). In stained films the flagellates are seen to have an
alveolar cytoplasm, within which bacteria may occur in food vacuoles
(Fig. 255, 9-13). Near the anterior end is the spherical nucleus, which
has a central karyosome. On the nuclear membrane occur two granules,
the blepharoplasts, from which the flagella arise. Several stages in the
division process were seen, but the details were not followed. The
spherical forms with two cytostomes were seen to have two nuclei and four
flagella, while other similar forms were seen with an elongated dividing
nucleus with a pair of blepharoplasts and two flagella at each extremity
of the nucleus. It is possible that the margins of the cytostome are
supported by marginal fibres, as in Chilomastix, but the small size of the
organism makes it difficult to determine this point with accuracy.
In stained films the cysts are seen to have a somewhat peculiar internal
structure. There are generally two dark lines marking out an elongate,
oval, or spindle-shaped area within the cyst (Fig. 255, 14-19). It is often
nearly as long as the cyst itself, and within it is what appears to be the
karyosome of the nucleus. In some cysts two dumb-bell-shaped bodies or
karyosomes are seen. The writer and O'Connor (1917) interpreted the
structure as being a much elongated nucleus. A very similar body was
figured by Dobell (1909), and interpreted as the elongated nucleus in cysts
which he identified as being the encysted forms of Trichomonas of the frog,
but which are probably cysts of a species of Efnbadomonas which the writer
has seen in English frogs. Dobell and O'Connor (1921) figure the cyst of
E. intestinalis as having a round, more or less central nucleus, and the
outline of the cytostome at one side towards the anterior end. Jepps
(1923) has figured the cyst as having an elongate central cytostome and a
round nucleus with central karyosome (Fig. 255, 25-26). The cyst thus
EMBADOMONAS INTESTINALIS 619
resembles those of Chilomastix mesnili, but is smaller. Quite recently the
writer has examined cysts of E. intestinalis, which have been fixed in osmic
acid vapour and stained by Leishman stain. In these preparations there
is a central red granule surrounded by a blue ring, which undoubtedly
represents the nucleus and its karyosome. The two lines which extend
the whole length of the cyst probably represent the margins of the cyto-
stome, and not the nuclear membrane. The dumb-bell-shaped structures
mentioned above may have been dividing karyosomes.
In one of the two infections seen by the writer and O'Connor (1917)
in Egypt, E. intestinalis persisted for one and a half months. Though the
flagellates were seen in diarrhoeic conditions, there was no evidence that
they were the actual cause of the trouble.
Hogue (19216) reports the successful culture of E. intestinalis in a
medium made by cooking the white of one egg with 100 c.c. of 0-7 per cent,
solution of sodium chloride. During heating, the mixture is constantly
shaken. It is then filtered and placed in test-tubes, after which it is
autoclaved. By subculture every other day, the flagellate was kept alive
for over eight weeks at a temperature of 35° C. In the cultures the
flagellates multiply by binary fission, and also produce the typical cysts.
The writer (1921a) has succeeded in cultivating E. intestinalis as also
forms from the guinea-pig, rat, tortoise, and frog, not only in Hogue's
egg medium, but also in a soft rabbit blood-agar medium. The cultures
were maintained both at 24° and 30° C.
Faust and Wassell (1921) described as E. sinensis a flagellate seen
by them in nine cases of diarrhoea in North China. A further account
of the organism has been published by Faust (1922). The average size is
given as 14 by 4-2 microns, but longer forms up to 20 microns were seen.
From the description and figures it appears that the two flagella are of
equal length and thickness (Fig. 255, 20-22). The encysted forms, how-
ever, correspond in shape and size with those of E. intestinalis. In a case
of E. intestinalis (that of Broughton-Alcock and Thomson noted above)
which the writer had an opportunity of studying, flagellates up to 17 microns
in length were seen. The larger forms were more frequently encountered
in cultures. The statement made by Faust and Wassell that the flagella
of their species were ec^ual in length and thickness requires confirmation,
for in all the forms examined by the writer (man, guinea-pig, rat, tortoise,
frog) the cytostomal flagellum has been thicker and shorter than the
other. It seems to the writer that it is exceedingly doubtful if E. sinensis
is a distinct species from E. intestinalis, especially as the encysted forms
are alike. The flagellate described as Enteromonas Bengalensis by Chaterjee
(1919) may be E. intestinalis (see p. 307).
620 FAMILY: CHILOMASTIGID^
EMBADOMONAS IN ANIMALS.
Embadomonas wenyoni (Foiiseca, 1917). — This form closely resembles
E. intestinalis of man, with which it may be identical. It was described
by Fonseca (1917) as Waskia wenyoni, and was found in the caecum of
the Brazilian monkey, Cebus carya. The description referred to the
spherical dividing forms, which have two sets of flagella and two cyto-
stomes. They correspond 'u\ every way with the dividing stages of
E. intestinalis.
E. agilis Mackinnon, 1911.- — This flagellate was discovered by Mac-
kinnon (1911, 1915) in tipulid and trichoi^teran larvae. It varies in size
from 4 to 1-5 microns to 11 by 3 microns. The cysts measure 3-5 to 4
by 4 by 3 microns.
E. alexeieffi Mackinnon, 1911. — This form, which is slightly larger than
the preceding one, occurred only in tipulid larvae. It measured 7 to 16
by 5 to 9 microns, while the cysts measured 5 to 6 by 4 to 5 microns. The
cysts of this and E. agilis are described and figured as being ovoid in shape,
with no tendency to a narrowing of the anterior end, as not infrequently
occurs in those of E. intestinalis.
E. belostomae (Brug, 1922). — Brug (1922) records as Waskia belostonicea
an Embadomonas which he found in the water bug, Belostoma sp., in Java,
It actually shows no specific differences from other species which have
been described.
A flagellate of the genus Embadomonas has recently been seen by the
writer in the intestine of a tortoise {Testudo argentina) which died in the
Zoological Gardens in London (Fig. 11). Structurally, it did not differ
from E. intestinalis, but w^as distinctly larger, as it varied in length from
12 to 19 microns. Encysted forms were not seen. It was successfully
cultivated. A form having the same dimensions was discovered by the
writer in the rectum of an English frog. A culture was obtained, and in
this the characteristic cysts resembling those of E. intestinalis were
produced.
6, Family: CHILOMASTIGID^,
This family includes flagellates which have three anteriorly directed
flagella, and one posteriorly directed one which lies in a long cytostomal
cleft. Characteristic oval or pear-shaped cysts are produced, within which
the single nucleus and the cytostomal cleft can be distinguished. It
includes the single genus Chilomastix. The genus Tetrachilomastix, which
was founded by Fonseca (1916) for flagellates having the Chilomastix
structure, except the possession of four anterior flagella instead of three,
is not free from doubt.
GENUS: CHILOMASTIX 621
Genus: Chilomastix Alexeieii", 1910.
Alexeiefl (1909) described as Macrostoma caulleryi a flagellate of this type from
the intestine of tadpoles, and it was in this genus that the writer (19106) placed
the human form as M. niesniU. It was later discovered that the name A[ aero stoma
was not available, so Alexeieff (1910) included the flagellate in Perty's genus
Tetramitus. It was evident, however, that the parasitic forms were not of the
same type as the free-living Tetramitus, so Alexeiefl (19126) established the new
genus, Chilomastix, by which name these forms are now generally known.
The flagellates of this genus have pear-shaped bodies, three anteriorly
directed flagella, and a large cytostomal cleft, within which is a fourth
flagellum. There is a vesicular nucleus near the anterior end of the body,
and between it and the anterior end of the cytostomal cleft is a group of
blepharoplasts which give origin to the four flagella and to two filaments
which pass along the margins or lips of the cytostomal cleft. Reproduc-
tion is by longitudinal division. Characteristic pear-shaped cysts are
produced. In each cyst there is a single flagellate, of which the nucleus,
cytostomal cleft, and blepharoplasts can often be clearly distinguished.
CHILOMASTIX IN MAN.
Chilomastix mesnili (Wenyon, 1910).— As pointed out by Brumpt (1912rt)
and ('Jialmers and Pekkola (1917), Davaino (1854) was the first observer to mention
this flagellate. In 1860 he redescribed and figured it. Though his figures were
imperfect in that only a single anterior flagellum was shown, his statements regarding
the cytostomal cleft render it very probable that he was actually dealing with this
organism. He referred to it as Gercomonas liominis variety A, the variety B being
TrieJiomonas. In the same year Moquin-Tandon (1860), some months before the
ai^pearance of Davaine's work, referred to the latter's two varieties of Gercomonas,
the account of which had not then been published. He must have had some know-
ledge of Davaine's forthcoming work, for, though he did not give any recognizable
description or figures of the flagellates, for the variety " A " he proposed the name
Gercomonas davainei, and for the variety "B" the name Gereomonas obliqua. It
seems clear that if there is no doubt as to the identity of Davaine's flagellates, the
correct name for the human GMlomastix, as pointed out by Kofoid (1920), should
be GMlomastix davainei Moquin-Tandon, 1860, while that of the human intestinal
Trichomonas should be Trichomonas obliqua Moquin-Tandon, 1860. It seems un-
desirable, however, to change the name Ghilomastix mesnili, which is now in general
use, and though it is very jirobable that Davaine was actually observing this flagel-
late, his description would have been quite madequate to establish its identity
were it not for the fact that the human intestine harbours only a limited number
of organisms of distinctive structure. Davaine's description might apply to Emba-
domonas intestinalis, which, however, is a much rarer organism than Ghilomastix
mesnili. Cunningham (1871) in India, Marchand (1875), Leuckart (1879), Grassi
(1881a), Epstein (1893), Roos (1893), and others probably saw this flagellate, but
they did not describe it accurately, and confused it with Trichomonas. It must
have been frequently referred to as Gercomonas, a name which was formerly employed
by medical writers as a general name for any flageUate of the human intestine.
The flagellate named by Prowazek (1911) Fanapepea intestinalis, that by Pro-
wazek and Werner (1914) Gijathomastix liominis, and that by Gabel (1914) Difcimus
622 FAMILY: CHILOMASTIGIDiE
tunensis are almost certainly identical with Chilomastix mesnili, though Gabel failed
to recognize the flagellum within the cytostome. The flagellates belonging to the
genus Enleiomonas established by Fonseca (1915) are probably, in some cases at
least, small rounded forms of CMlomastix (see p. 307).
C. ?nesnili is usually about 10 to 15 microns in length, though very
small spherical forms not more than 3 to 4 microns in diameter may be
met with as well as larger ones up to 20 microns in length (Fig. 256). The
anterior end is rounded or sometimes definitely flattened, while the
posterior end varies considerably. It is sometimes blunt, and at other
times drawn out into a long thin tapering tail. There is a long cytostomal
cleft about half as long as the body itself, and this is obliquely arranged in
such a way that, if a flagellate is observed with the cytostomal cleft
upwards, the rounded anterior end pointing away from the observer and
the posterior end towards him, then the anterior end of the cytostomal
cleft is nearer the left side of the body of the flagellate, while the posterior
end is nearer the right side. The two margins of the cytostomal cleft
often form definite lips, which may even overlap one another. There is
also a groove on the body which varies in development in different indi-
viduals. If the flagellate be regarded as in the position indicated above,
then the groove commences near the anterior end of the body to the left
of the cytostomal cleft, and passes round the body in a spiral manner
parallel to the cleft (Fig. 256, 9-10). It may terminate at the posterior
end of the cytostomal cleft, but is often continued beyond it, and may
make two complete turns of the body. On account of this spiral groove,
many of the flagellates appear to have the posterior region of the body
curiously twisted. In some infections the spiral groove is not evident.
There is a spherical nucleus near the anterior end of the body, and just
anterior to it is a group of blepharoplasts, which, according to Dobell
(Dobell and O'Connor, 1921) are six in number. The cytostomal cleft
commences just behind the group of blepharoplasts. Three of the
blepharoplasts are in front of the others, and from each of these there
arises an axoneme which passes to the anterior surface of the body, there
to enter one of the three anteriorly directed flagella. Each flagellum is
about as long as the body. The three posterior blepharoplasts give rise
to three different structures. The central one gives rise to a flagellum,
which is thicker than the anterior flagella, and which lies in the cytostomal
cleft. Some observers, as, for instance, Boeck (1921a), believe that there is
a membrane within the cytostomal cleft, and that the flagellum is attached
to its margin. This is probably not correct, as sometimes the flagellum
leaves the cytostomal cleft in which it usually lies. If the flagellate is
observed in the position described above, it will be noted that of the three
posterior blepharoplasts that on the left gives rise to a deeply staining fibre,
CHILOMASTIX MESNILI
623
which runs along the left margin of the cytostomal cleft, round its posterior
end, and up the right side for a short distance. From the right blepharo-
plast another fibre passes along the right margin of the cytostomal cleft
for about three-quarters of its length to a point near which the other
Fig. 256. — Chilomastir, mesnili from the Human Intestine (x 2,000). (7-8,
AFTER WeNYON, 1914; 9-10, AFTER WeNYON AND O'CONNOR, 1917.)
1-6. Ordinary forms as seen in stained films.
7-8. Small gfobula-r forms in which the cytostomal groove is not apparent.
9-10. Drawings of living spacimens, showing the twisted appearance of the posterior region of
the body. ' 11-14. Encysted forms in stained films.
marginal fibre terminates. The six blepharoplasts are usually so closely
packed together that they appear as a single deeply staining body. The
mouth or cytostome is at the posterior end of the cytostomal cleft at the
point where the marginal fibre turns round the posterior margin of the
cleft.
624 FAMILY: CHILOMASTIGIDiE
Kofoid and Swezy (1920) have given another interpretation of the
structure of the cytostomal cleft and blepharoplasts (Fig. 69). According
to them, the cytostome is an elongate aperture at the bottom of the cleft.
It is described as having the shape of the outline of a dumb-bell and sup-
ported by a fibre running completely round its margin. This fibre, as
also those found on the margins of the cleft itself, are said to originate
from one of the blepharoplasts. Of the latter, the left-hand one, which
turns round the posterior margin of the cleft, is called the peristomal
fibre and the other one the parabasal. As regards the blepharoplasts,
Kofoid and Swezy describe three which are united with one another by
various fibres called rhizoplasts, and with a granule on the nuclear mem-
brane which they call the centrosome. There seems little ground for
homologizing one of the marginal fibres with the parabasal bodies of other
flagellates, while the interpretation of the group of blepharoplasts is open
to question, especially as Belar (1921a) has published a description of the
structure of Chilomastix aulastomi of the leech {Aulastomum gulo), which
agrees e^ntirely with the account given above as far as the blepharoplasts
and cytostomal apparatus are concerned.
The method of multiplication of C. rnesnili is undoubtedly by longitu-
dinal fission after division of the nucleus. Though the writer has seen
isolated stages of this process, it has not been followed in detail. The
longitudinal fission of C. aulastomi has been described by Belaf (1921o),
and it may be presumed that the division of C. mesnili will be very similar
(Fig. 257). Apparently, the cytostomal cleft and its fibres vanish, and a
single granule appears in place of the group of blepharoplasts, which
may be supposed to have become more closely packed together. This
granule is on the surface of the nuclear membrane, and it divides into two.
The two granules then take up positions at opposite poles of the nucleus,
and an intranuclear spindle is formed between them. The nucleus, which
retains its membrane, then moves to a more central position, and the
chromatin of the nucleus becomes arranged at the equator of the spindle
in the form of a plate of chromosomes. Two daughter plates are formed
by division of the chromosomes, and these move to opposite poles of the
spindle. Meanwhile, new flagella begin to grow out from the two granules,
which then subdivide into the several blepharoplasts. The elongated
nucleus is finally divided at its centre, and the daughter nuclei assume
the characters of the nucleus of the adult flagellate. Two new cyto-
stomal clefts and the other structures associated with them are formed.
The body of the flagellate now divides by constriction, and two flagellates
result.
C. mesnili is often found in the stool in the encysted condition. The
cysts, which were first described and figured by Prowazek and Werner
CHILOMASTIX MESNILI
625
(1914), are pear-shaped structures which vary in length from 7 to 10 microns
(Fig. 256, 11-14). In the majority of cysts one end is narrower than the
other, though occasionally the two ends are more alike. In the fresh
condition practically nothing of the internal structure can be made out,
lf\ ttm
Fig. ■2o7.^Ghllomastix aulastomi ( x 2,300). (After Belar, 1921.)
1. Usual type of flagellate.
2. Comtnencing division with two centrosomes on nuclear membrane.
3. Spindle with centrosomes and chromosomes at equator of spindle; newflagellaare forming.
4. Nuclear division nearly complete. 5. Commencing division of flagellate.
though a few greenish refractile granules are sometimes seen. In iodine
solution the single round nucleus and the cytostomal cleft can be faintly
distinguished (Plate II., 24, p. 250). In stained specimens, however,
practically all the details which can be seen in the flagellates themselves
are visible. The nucleus is near the narrow end of the cyst, and near it can
I. 40
626
FAMILY: CHILOMASTIGID^
be seen the group of blepliaroplasts, which are often more scattered than
in the flagellates themselves.
The cytostomal cleft can be seen extending for the greater part of the
length of the cyst, while the flagellum can often be detected within it.
The cysts most usually remain in this condition, and are passed from the
body sometimes in large numbers, so that several can be seen in every
field of the twelfth objective. Though large numbers of the cysts have
been examined by the writer and other observers, no indication of nuclear
division has been noted. Kofoid and Swezy (1920), however, describe a
division process within the cyst. Division of the blepharoplasts is followed
by mitotic division of the nucleus, while the cytostomal cleft and the
marginal fibres are duplicated. There results a cyst containing a flagellate
Fig. 258. — Cysts of Chilomastix mesnili with Two Nuclei: Six Nuclei in Various
Phases of Mitosis (x 3,500). (After Hegner, 1923.)
with two sets of the various structures possessed by the ordinary cysts.
Division of the cytoplasm into two flagellates would presumably be the
next stage, but this was not observed. Hegner (19236) has also observed
binucleate cysts of C. mesnili, and has noted that the single nucleus divides
by mitosis in which about five chromosomes are present (Fig. 258).
Binucleate cysts are undoubtedly of rare occurrence, as no other observers
have seen them.
G. mesnili is sometimes present in very large numbers in diarrhoeic
stools, both in the free and encysted condition. In formed stools only the
cysts are found. The persistence of the infections is well illustrated by a
case observed by the writer and O'Connor (1917) in Egypt, where C. mesnili
was continually present during an observation period of fifty days. In
another case it was present for ninety days, except for an interval of a
CHILOMASTIX IN ANIMALS 627
month, when it was apparently absent or present in such small numbers
as to escape detection. As in other flagellate infections, the number of
organisms present is subject to marked periodic fluctuations. The evidence
as to pathogenicity is still wanting. The writer (1920) has noted that in
sections of the large intestine the flagellates may be found within the
lumen of the glands, but could find no indication that invasion of the
tissues could take place. Kessel (1924a) reports the successful inoculation
of monkeys with C. mesnili.
The culture of C. mesnili has been successfully carried out by Boeck
(1921a), who used a medium consisting of one part of human serum to
four parts of Locke's solution, to which 0-25 grain of dextrose had been
added. In this medium at 37° C. the flagellates survived and multiplied
for eight or nine days till they were overgrown by the bacteria. By sub-
culture every two or three days the strain was maintained for about five
months. Reichenow (1923) has also cultivated the organism in a medium
prepared by dropping dilute serum into hot saline, so that flocculi are
formed. He has grown it from stools which were microscopically negative,
a fact which demonstrates the value of the culture method for diagnostic
purposes. Boeck and Drbohlav (1925), and Thomson, J. G., and Robertson
(1925), report the culture of C. mesnili in Boeck's L.E.S. medium.
The small round forms of C. mesnili, which have a diameter of 3 to
6 microns, often have the cytostomal groove obscured (Fig. 256, 7-8).
In this condition they resemble the flagellate described as Enteromonas
hominis by Fonseca. From an examination of their films, the writer is
able to state that the cases of E. hominis infections recorded from the Sudan
by Chalmers and Pekkola are in reality ones of C. vnesnili, in which the
majority of the fiagellates are in the small rounded form. This fact raises
the question as to whether the other cases of E. Jiominis infection are not
due to the same organisms. If this be so, the name Enteromonas becomes
a synonym of Chilomastix (see p. 307).
CHILOMASTIX IN ANIMALS.
Flagellates of this genus are fairly common parasites of animals.
Though many of these have been given specific names, it is very doubtful
if they can be distinguished from one another. C. mesnili varies so much
in size, as also do the cysts, that this feature is of little value in the dif-
ferentiation of species. Thus Alexeief! (1914) expressed his belief that the
human flagellate, C. mesnili, is identical with C. caulleryi of frogs.
C. hettencourti Fonseca, 1915, is parasitic in the intestine of rats (Rattus
norvegicus). This form has been seen by the writer on several occasions in
both rats and mice, and he can find no differences between it and the human
form. Fantham (1925) records as C. muris a form in the gerbil {Tatera
628 FAMILY: CHILOMASTIGID^
lohengula) and the rat (Rattns concha) of South Africa. C. caprce Fonseca,
1915, is a very similar form found in the rumen of goats (Capra Jiircvs).
C. cuniculi Fonseca, 1915, occurs in the caecum of rabbits {OryctoJagus
cuniculus). The form named C. cuniculi var. rossica by Yakimoff,
Wassilewsky, Korniloff, and ZwietkofE (1921) is unquestionably identical
with C. cuniculi.
C. rosenbuschi Fonseca, 1916, occurs in the intestine of the viscacha
{Lagostoynus 7naximus), a South American rodent, and C. intestinalis
Kuczynski (1914) in the guinea-pig {Cavia jjorcellus). The latter is fairly
commonly present in guinea-pigs in England. Chalmers and Pekkola
(1918) recorded the occurrence of a Chilomastix in the gerbil {GerhiUus
pygurthus) of the Sudan. Bach (1923) has seen the cysts and free forms
of a Chilomastix in a monkey, Macacus rhesus, and Hegner (1924) the cysts
in another monkey, Cebus apella. Species of Chilomastix occur in other
hosts than mammals. Thus, Alexeieff (1909) described C. caulleryi from
the intestine of tadjjoles, axolotls, and salamanders. A form, probably
C. caulleryi, was seen by Fantham (1922) in the South African clawed toad
{Xenopus IcBvis). Alexeieff (1910) also saw a flagellate of the same type
in the marine fish, Motella tricirrata and M. inn stela. He (19126) gave it
the name C. motellw, while another form which he saw in the|fish. Box
salpa, he identified with the human C. 7nesnili. Brumpt (1912a), however,
regarded it as a distinct species, and gave it the name C. hocis. Martin
and Robertson (1911) mention the occurrence of a species of Chilomastix
in the intestine of the coal fish {Gadus virens). The writer (1921) recorded
Chilomastix sp., a small form from the gut of two Egyptian lizards, Lacerta
agilis and Agamci, stellio, and he has since seen similar forms in films made
by Chalmers and Pekkola of the intestinal contents of the gecko, Tarentola
annulurus of the Sudan. Belar (1921a) has described as C. aulastomi a
species which occurs in the hind-gut of the horse leech, Aulastomum gulo.
It is possibly this form which Alexeieff (1910)-records as having been seen
by Chatton in Hcemopsis sanguisuga. The writer has seen a flagellate,
probably C. caulleryi, in the common English toad. Both free and
encysted stages occurred.
Under the name of C. gallinarum, Martin and Robertson (1911) de-
scribed a flagellate from the caecum of fowls (Fig. 265, A). According to
their description, there were four anterior flagella, but no mention is made
of one within the cytostomal cleft. Fonseca (1916) created the genus
Tetrachilomastix for flagellates of this type, and later (1920) states that
there is a fifth flagellum within the cytostome. The chicken parasite
would then become T. gallifiarum, differing from species of Chilomastix in
having four instead of three anterior flagella. The encysted stages are
similar to those of species of Chilomastix. The writer has studied the
FAMILY: CERCOMONADID^ 629
fowl flagellate, and finds that only four flagella are present, and that it has
the characteristic structure of members of the genus Chilomastix. In the
form figured by Martin and Robertson the cytostomal fiagellum was
evidently in an unusual position outside the cytostomal cleft. Fonseca's
genus, Tetrachilomastix, thus becomes a synonym of Chilomastix. San-
giorgi (1917) placed in this genus as T. intestinalis a flagellate he saw in
human fseces. It had four anterior flagella and was cultivated. There
is no evidence that it was not a Trichomonas.
Chatterjee (1923) has given the name Tetrachilomastix bengalensis to a
flagellate which he says occurs commonly in the human intestine in India.
According to his description, it has the general structure of a Chilomastix,
but differs in that there are four anterior flagella, while a fifth runs along
the border of an undulating membrane situated at one side of the large
cytostomal groove. The attached axoneme may extend posteriorly as
a flagellum. Through the courtesy of the author the writer has been
able to examine preparations of the flagellate, and he is quite unable to
convince himself that the organism differs in any essential respect from
C. mesnili. It appears to him that the undulating membrane is merely
the edge of the fold which occurs in twisted forms. The fixation of the
flagellates and the cysts was not entirely satisfactory.
7. Family: CERCOMONADID^ Kent, 1880.
This family includes flagellates which may be supposed to have
originated from flagellates of the Heteromita type in which the trailing
flagellum has become attached to the surface of the body. In addition
to the attached flagellum, which is posteriorly directed, there are one or
more free anteriorly directed flagella.
A. Cercomonadidse with One Anterior Flagellum.
Genus: Cercomonas Dujardin, 1841.
The members of this genus have two flagella, which arise from the
anterior end of the body. One flagellum is directed forwards as a free
flagellum, while the axoneme of the other turns backwards over the
surface of the body to which it is attached. It becomes a flagellum at the
posterior end of the body. Though the name Cercomonas has been
frequently used to designate intestinal flagellates of man, these have
belonged to other genera, such as Trichomonas and Chilomastix. The
flagellates of this genus are common in infusions, where they were first
seen and named by Dujardin (1841). They also appear in old faeces
as coprozoic organisms, but there is no evidence that they are ever para-
sitic in the human intestine. Some observers, as, for instance. Porter
(1918), record them as occurring in the human stool, but it is probable
630 FAMILY: CEECOMONADID.E
that in all these instances they had developed from cysts of free-living
forms.
Cercomonas longicauda Dujardin, 1841. — The commonest form to
appear in old faeces is probably C. longicauda, which was described by
Dujardin (1841). It was seen by Klebs (1892), who referred to it as
DunorjjJia longicauda. The writer (1910) met with it in stale faeces, and
maintained it in culture in hay infusion, and also on agar plates. The
flagellate has a more or less pear-shaped body, which varies in length from
2 or 3 microns to as much as 18 microns. The shape of the body, which
is metabolic, changes very much according to the kind of medium in
which the flagellate is living. In surface films it becomes definitely
amoeboid. There is no cytostome, and food is ingested in an amoeboid
manner. A contractile vacuole has not been seen. Near the anterior
region of the body is a nucleus consisting of a nuclear membrane and
central karyosome. The membrane is drawn out into a cone-like pro-
longation, and at the apex of the cone there commences a rhizoplast formed
of two closely applied axonemes, which passes to the anterior end of the
body. One axoneme enters a forwardly directed flagellum, which may
be two or three times the length of the body. The other passes backwards
over the surface of the body to the tapering posterior extremity, where it
enters a tail flagellum. At the tip of the nuclear cone, from which the
axonemes arise, there is a granule which represents two minute blepharo-
plasts.
Reproduction is by longitudinal division (Fig 259). The first indica-
tion of this process is the formation of two new flagella from the granules
at the tip of the nuclear cone, while the karyosome takes the form of a
band. The granules now divide into two pairs, each of which gives origin
to two flagella. The daughter granules move apart, and the nuclear
membrane becomes spindle-shaped, while the band of chromatin is
arranged as a plate at the equator of the spindle. The plate is made up of
a number of chromosomes. By their division two plates are formed, and
these pass to opposite poles of the nucleus, which now stretches right
across the body of the flagellate, and has a pair of blepharoplasts at each
end. The nucleus is finally divided at its centre, and this is followed by
division of the flagellate.
The flagellate becomes encysted in spherical cysts, which are 6 or 7
microns in diameter. There is a central nucleus with large karyosome,
while the cytoplasm is often filled with refringent granules which stain
black with iron haematoxylin. The cysts can be dried, and will give rise
to cultures again if placed in suitable medium.
Woodcock (1916) isolated C. longicauda from the faeces of sheep and
goats. He observed the process of syngamy (Fig. 41). Two organisms
GENUS: CERCOMONAS
631
become united by tlieir posterior ends, and gradually fuse. The nuclei
unite, and at this stage the flagellate has the appearance of a uninucleate
^C^% '
^J^
•v;»
Fig. 259. — Cercomonas longiccmda (x ca. 2,000). (After Wen yon, 1910, 1913.)
1-2. Flagellate showing attached flagelluni passing over surface of body.
3-9. Various stages of division; the blejiharoplast functions as a centrosome.
10. Encysted form (stained). 11. Encysted form (living).
organism with two flagella. The flagella are then lost, and encystment
in a spherical cyst takes place.
632
FAMILY: CERCOMONADID^
Liebetanz (1910) described three species of Cercomonas from the
stomach of cattle. These differ from one another only in size, and as no
indication is given of the posterior flagellum, it seems very probable that
they are merely elongate forms of the organisms which he describes as
Sphceromonas, and which have been dealt with above.
Castellani and Chalmers (1910) described as Heteromita zeylcniica a
flagellate seen in human faeces in Ceylon.
It was again recorded by Castellani (1917)
from Macedonia. The statement that a
flagellum existed at each end of the body
shows that the flagellate does not belong
to the genus Heteromita. These authors,
however, state that thev believe Heferoinifa
A
B
Fig. 260.
(A) Species of Helkesimastix Copkozoic in Goat and fSiiEEP Dung (x 2,250).
(After Woodcock, 1921.)
]. H. major ; 2 and 3, //. fcecicola.
(B) Trimitus moteUce from the Eectum of the Marine Fish, Motella tricirrata
(x 2,250). (After Alexeieff, 1910.)
to be a synonym of Cercotnonas, so that it is highly probable that the
flagellate was C. longicanda occurring coprozoically in the faeces.
Genus: Helkesimastix Woodcock and Lapage, 1915.
This genus was established by W^oodcock and Lapage (1915) for a
certain small flagellate which they encountered in cultures of goat's fseces.
The original description was corrected by Woodcock (1921). The flagel-
late resembles Cercotnonas in that it possesses two flagella, the axoneme of
one of which is adherent to the surface of the body as far as its posterior
GENERA: HELKESIMASTIX AND TRIMITUS 633
end (Fig. 260, A). It differs, liowever, in that the anterior flagellum is
exceedingly short, while the posterior flagellum is about the length of
the body. The axonemes appear to arise from the nuclear membrane as
in Gercomonas. Spherical encysted forms occur, and syngamy was observed
to take place by the gradual union of two individuals. Two species are
described, the smaller of which, H.fcvcicola, had an ovoid body measuring
4 to 6 microns in length.
B. Cercomonadidae with Two Anterior Flagella.
Genus: Trimitus Alexeieft", 1910.
This genus was founded by Alexeieff (1910) for a small flagellate which
had two anteriorly directed flagella and one posteriorly directed, the
axoneme of which passed over the surface of the body. There is one
species, Trimitus motellce, which occurs in the intestine of Motella tricirrata,
a marine fish. One of the anterior flagella is about as long as the body
and the other about half this (Eig. 260, B). The posterior flagellum is a
thick one, which is attached to the body as in Gercomonas, and has a length
four or five times that of the body itself. There is a nucleus near the
anterior end of the flagellate, and near it a granule in which the axonemes
of the flagella originate. A similar flagellate was discovered by Duboscq
and Grasse (1923, 1924:) in the termite, Calotermes flavicollis, of France.
It resembled T. motellce of Alexeieff, except in the possession of an axostyle
and a small rod-like parabasal which was attached to the blepharoplast
(Fig. 279). They believe it possible that Alexeieff had overlooked these
structures, and think that the flagellate sometimes has two and at other
times three anterior flagella. In a later paper (1924a) they point out
that the flagellate, in their opinion, is merely a young form of Trichomonas
dogieli (see p. 675).
Chalmers and Pekkola (1919) described as Dicercomonas siidanensis a
flagellate which they found in human faeces. The name was subsequently
(1919a) changed by them to Diplocercomonas sudanensis, as the name
Dicercomonas had been previously used (Diesing, 1865; Grassi, 1879).
According to their description, the flagellate resembles Gercomonas with
the exception that there are two anterior flagella instead of one. The
writer has been able to examine the original films, and finds two flagellates
are actually present. One of these is Tricercotnonas intestinalis, described
below, and the other Embadomonas intestinalis, and it is evidently owing
to the fact that the double nature of the infection was overlooked that the
presence of a new flagellate having the structure described above was
accepted. There were no flagellates present which had the characters of
Diplocercomonas sudanensis, except some examples of Tricercotnonas, in
which only two anterior flagella were visible.
634 FAMILY: CERCOMONADIDyE
C. Cercomonadidae with Three Anterior Flagella.
Genus: Tricercomonas Wenyon and O'Connor, 1917.
This genus was founded by the writer and O'Connor (1917) for a flagel-
late having the general structure of a member of the genus Cercomonas,
except that it possessed three anterior flagella instead of one. There is
only one species.
Tricercomonas intestinalis Wenyon and O'Connor, 1917. — This is a
minute pear-shaped organism which has three anterior flagella and a
fourth posterior one, the axoneme of which is attached to the surface of
the body. The name T. intestinalis was given to this flagellate by the
writer and O'Connor, who discovered it in diarrhoeic stools in Egypt.
The writer saw the same organism later in several cases in Macedonia,
while it was recorded by Kofoid, Kornhauser, and Plate (1919) and Kofoid
(1920) in soldiers who had returned to New York from service abroad.
Lynch (1922a) and Boeck (1924) have seen it in North America, Jepps
(1923) in Malaya, and Da Cunha and Pacheco (1923) in Brazil.
The flagellate, which is an active metabolic organism when seen in
freshly passed stool, is 4 to 8 microns in length (Fig. 261). It is pear-
shaped, but the side along which the axoneme of the posterior flagellum
passes is somewhat flattened. The posterior extremity is often drawn out
into a tail, while the flagellum is continued for a short distance beyond the
end of the tail. In stained specimens a nucleus can be seen near the
anterior end of the body. It has a central karyosome, while the nuclear
membrane is drawn out into a cone, from the apex of which the axonemes
of the flagella originate.
Reproduction takes place by longitudinal division, but only isolated
stages of this process were seen. The accounts of the flagellate given by
Lynch (1922a) and Boeck (1924) agree in the main with that of the writer
and O'Connor (1917). The organisms described by Brug (1923) and
Jepps (1923) as Enteromonas hominis are unquestionably T. intestinalis.
Boeck (1924), who has cultivated the organism in his L.E.S. medium
from a human case, gives the measurements of the flagellate as 4 to 10
by 3 to 6 microns. The nucleus is described as spherical, and two ble-
pharoplasts were noted near the nuclear membrane. In one of these the
axonemes of the three anterior flagella originated, while the other gave
origin to the axoneme of the posterior flagellum. Boeck believes that
the cone-like appearance of the nucleus described by the writer and
O'Connor is not a normal one. A similar cone-like arrangement occurs,
however, in Cercomonas longicauda and Heteromita uncinata. In one of
the cases studied by the writer and O'Connor, encysted forms of T. intes-
tinalis were encountered. Cysts were also seen by Boeck. These are
GENUS: TRICERCOMONAS
635
oval in outline, and measure 6 to 8 microns in length by about half this in
breadth (Fig. 261, 5-8). In stained films it is seen that the cysts have one,
two, or four nuclei. At the four-nuclear stage the nuclei are arranged
in pairs at opposite ends of the cyst. The infections which were studied
in Egypt did not persist for long periods. In one case the flagellate was
seen daily for nine days, when it disappeared.
It should be mentioned here that in their work on the intestinal
Fig.
^61. — Tricercomonas intestinalis from the Human Intestine (x 2,600).
(After Wenyon and O'Connor, 1917.)
1-4. Flagellates as seen in living condition.
9-10. Flagellates in stained films.
5-8. Encysted forms in stained films.
11. Dividing form.
Protozoa, Dobell and O'Connor (1921) came to the conclusion that the
E. hominis described from man by Fonseca, and which has been referred
to above (p. 306), is the same as T. intestinalis. Jepps (1923) refers to the
flagellate seen by her in Malaya as E. Jiominis. It is assumed by Dobell and
O'Connor that Fonseca and other observers have overlooked the posterior
flagellum, and have erroneously supposed that only three anterior flagella
are present. This is, of course, quite possible, as the determination of the
It is, however,
636 FAMILY: CRYPTOBIIDiE
possible that a flagellate of tlie type of E. hotni?iis actually exists as a
human parasite, and further observations are necessary before it is finally
concluded that Tricercomonas is a synonym of Enteromonas. Lynch
(1922a), who has studied a case of infection with T. intestinalis in America,
has also cultivated from the intestine of the guinea-pig a flagellate having
the characters of Fonseca's E. Jiominis, so the possibility of such a form
occurring in man cannot be excluded.
There is another aspect of the question, which has been referred to
above. In cases of infection with Chilomastix tnesnili it is not unusual to
find small rounded forms of this flagellate in which the cytostomal groove
is difficult to detect (Fig. 256, 7-8). These have essentially the structure
ascribed to E. hominis by the various observers who have recorded this
flagellate. Having examined the original films, the writer is in a position
to state that the flagellates described by Chalmers and Pekkola as
E. hominis from the Sudan are actually the small round forms of C. mesniJi.
It is possible, therefore, that Enteroynonas is a synonym of Chilomastix.
Duboscq and Grasse (1924), from observations on the flagellates of
termites, arrive at the conclusion that both Enteromonas and Tricercomonas
are merely young forms of other flagellates. In the case of Enteromonas,
as pointed out above, there is considerable evidence in favour of this
view, but in the case of Tricercomonas of man there is no indication
whatever that it is a young form of any other flagellate. The form in the
termites which Duboscq and Grasse considered as of the Tricercomonas
type possessed both an axostyle and a parabasal (Fig. 279), in which
respects it differed from T. intestinalis from the human intestine.
Boeck (1924), and Thomson, J. G. and Robertson (1925), report the
cultivation of T. intestinalis in Boeck's L.E.S. medium.
8. Family: cryptobiid^ Poche, 1913.
The flagellates which are included in this family are found in three
situations— viz., the blood of fish, the intestinal canal of fish and the
Chsetognathan Sagitta, and the vesicula seminalis and spermatophores of
molluscs and other invertebrates. Structurally, the flagellates from these
three situations are so similar to one another that many observers regard
them as belonging to a single genus. As will be explained below, the
correct name of the genus is Cryptobia Leidy, 1846, and strictly all the
forms should be included in this genus. The blood-inhabiting forms have
so long been known under the name Trypanojplasma, given them by
Laveran and Mesnil (1901c), that it seems better at present to retain them
as a distinct genus.
The body of a typical member of the genus consists of an elongate
flattened portion of cytoplasm in which is a nucleus and a kinetoplast
GENUS: CRYPTOBIA 637
consisting of a parabasal body and two blepliaroplasts. From each
blepharoplast arises an axoneme which passes through the cytoplasm as a
rhizoplast to the anterior end of the body. Here one enters a flagellum,
which is directed forwards, while the other passes backwards along the
border of an undulating membrane to become a flagellum at the posterior
end of the body. The question of the possible origin of trypanosomes
from these forms by the loss of the anteriorly directed flagellum has
been discussed above (p. 316). It seems very improbable that trypano-
somes have originated in this way. The primitive type from which they
have been evolved is presumably a flagellate of the leptomonas form seen
typically in insects, while the forms now being considered seem to have
sprung from Bodo or Cercomonas ancestors. In fact, the members of this
family are structurally very like species of Bodo and Cercomonas. From
the former they differ in the absence of the cytostome, and in the back-
wardly directed axoneme being attached to an undulating membrane,
while from the latter they differ in the possession of a kinetoplast. It is
probable that the forms which occur in the blood of fish have been derived
from intestinal forms which have invaded the blood-stream.
In association with a blood habitat a method of transmission through
the agency of leeches has been evolved, while the purely intestinal forms
are presumably handed on directly from fish to fish by the ingestion of
forms which escape in the fgeces. The molluscan forms, which live in the
vesicula seminalis or spermatophores, are probably transmitted directly
from host to host during copulation.
It will be most convenient to consider these flagellates under the
following headings— Invertebrate Forms, Intestinal Forms of Fish, and
Blood Forms of Fish.
A. Invertebrate Forms.
The first flagellate of this family to be seen was described by Leidy
(184:6) from the sexual organs of various species of snail {Helix) in America.
He named the flagellate Cryptobia helicis, but in the following year (1847)
renamed it Cryptoicus helicis, as the name Cryptohium had been previously
employed for a beetle. Diesing (1851) referred to it as Bodo helicis, while
Leidy (1851 and 1856) accepted Diesing's conclusion that it belonged to
Ehrenberg's genus Bodo. It was found by Keferstein and Ehlers (1860)
in Helix jwmatia in Germany, and was studied in detail by Friedrich (1909).
It was further studied by Jollos (1910), Crawley (1909), and by Delanoe
(quoted by Laveran and Mesnil, 1912) in France in H. pomatia, H. hor-
tensis, and H. nemoralis, and by Belaf (1916). It is evidently a common
parasite of the various species of Helix in America and Europe. The
correct name of this organism is clearly Cryptohia helicis Leidy, 1846,
638 FAMILY: CRYPTOBIIDiE
though most writers have placed it in Laveran and Mesnil's genus, Tnj-
panojjlasma, which was established by them in 1901. According to the rules
of nomenclature, Leidy had no necessity to change the name to Cryptoicus.
Cryptobia helicis Leidy, 1846. — According to Belar (1916), C. helicis is
typically an elongate organism varying in length from 6 to 20 microns
(Fig. 262). The breadth varies considerably, there being comparatively
narrow forms not more than 3 microns in breadth and others which are
much broader. Typically, however, the organism has an elongate form. The
body is distinctly flattened. There is a nucleus consisting of a nuclear
membrane enclosing a space in which there is a central karyosome and a
number of scattered chromatin granules. Nearer the anterior end is the
kinetoplast, consisting of an elongate parabasal body and two blepharo-
plasts, which are often so close together as to appear as one. From each
there arises an axoneme. One of these passes through the cytoplasm to
the anterior end of the body, where it becomes a flagellum, w^hich is about
as long as the body itself. The other passes to the surface of the body
in a lateral or backward direction, and then runs over the surface of the
body, to which it is adherent, as far as the posterior extremity. It is then
continued in a short flagellum. There does not appear to be an undulat-
ing membrane at the line of attachment of the axoneme.
The flagellate multiplies by binary fission. The process has been
described in detail by Belar. The single pair of blepharoplasts divides
to form two pairs, the original axonemes remaining with one pair, while
new axonemes commence to grow out from the other j)air. As the two
pairs of blepharoplasts separate, the parabasal body splits from before
backwards. At the same time changes take place in the nucleus. The
karyosome becomes elongated and dumb-bell-shaped, and its two halves
become more and more separated, though still connected by a fibre, as the
nucleus itself increases in length. The chromatin granules of the nucleus
at a certain stage appear to be collected at the equator of the nucleus as an
irregular plate. This plate is divided into two parts, which travel to
opposite poles of the nucleus. Finally, the nucleus is divided into two.
The nuclear division is thus a modified form of mitosis. By the time
nuclear division is complete, the flagellate has two complete sets of organs.
The body now divides, and two organisms result. Belar described what
he supposed to be conjugation, in which two flagellates unite, their nuclei
and kiuetoplasts fusing, but in a later paper (1924) he admits that this
was an erroneous interpretation of the appearances seen by him.
1-4. Various types of flagellate. 5. Commencing division of nucleus.
6-7. Divided blepharoplast and commencing division of parabasal.
8. Blepharoplast and parabasal completely divided; nucleus dividing.
9-10. Pinal stage of division.
CRYPTOBIA HELICIS
639
Fig. 262. — Cryptobia helicis from the Eecepxaculum Seminis of the Snail,
Helix 2)omatia {x 3,800). (After Belar, 1915.)
[For description see opposite page.
640 FAMILY: CEYPTOBIID^
So far, no observer has noted an encysted stage of this flagellate,
though it appears evident that the flagellate is handed on from snail to
snail during copulation.
Poche (1903) described a very similar flagellate from the stomach of
certain Coelenterates {SiphonojjJiora). The organism was studied by
Keysselitz (1904), who gave it the name Trypmwphis grohheni. The
flagellate measures 65 by 4 microns, has a centrally placed nucleus, and a
small kinetoplast which is near the anterior end of the body. There is a
short anterior flagellum and another posterior one, which is continuous
with an axoneme attached to the body by a narrow undulating membrane.
The flagellate differs from the members of the genus Cryptohia, considered
above, in the shortness of the anterior flagellum and the small size of
the kinetoplast, but it is doubtful if this is sufficient justification for its
inclusion in a distinct genus.
A flagellate, which probably belongs to the genus Cryptohia, has been
described by Hesse (1910) from the vagina of leeches (Hirudo medicinalis
and Aulastomum gulo) as Trypanoplasma vaginalis. Cryptohia carinarice
was recorded by Collin (1914) from the seminal receptacle of the mollusc
Garinaria mediterranea. Another form, which undoubtedly belongs to the
same genus, was recorded by Fantham and Porter (1910) as T. dendrocoeli,
from the intestine of the fresh- water planarian, Dendrocoelutn lacteum.
The organism was studied by Gelei (1913). Its structure and method
of reproduction was very similar to that of G. helicis. Intracellular
forms also occurred, as previously noted by Fantham and Porter, but
Gelei did not observe them in the cells of the ovary, as these authors
maintained. He considered the intracellular forms as being merely an
indication of the phagocytic powers of the host cells. Hamburger (1912)
described as a Trypanoplasma a flagellate of the mole cricket, Gryllotalpa
vulgaris. Structurally, it resembled a Gercomonas in that there was no
separate kinetoplast, and it is possible that it belongs to this genus.
Under the name Trypanoplasma isidorce Fantham (1923) describes a
flagellate of the receptaculum seminis of the pond snail Isidora tropica
in South Africa,
B. Intestinal Forms of Fish.
The first flagellate of this type to be seen in the intestine of marine fish
was one discovered by Dahl in 1887 in Gydopterus lumpus. It was referred
to by Mobius (1888) as Diplomastix dahlii. Keysselitz (1906) named it
Trypanoplasma ventricoli, as Leger, L. (1905), had placed in this genus as
T. intesiinalis a similar flagellate from the marine fish, Box hoops (Fig. 263).
Elmhirst and Martin (1910) gave the name T. congeri to a form from
the stomach of the conger eel. Conger niger. Its method of multiplication
CRYPTOBIA OF FISH
641
by binary fission was described by Martin (1910). Alexeieff (1910)
encountered T. intestinalis, not only in B. boops, but also in Motella tri-
cirrata. Apstein (1910) studied the form in Cyclopterus, and described it
as Heteromita dahlii.
According to these authors, the various flagellates have essentially the
same structure as Cryptohia helicis. Alexeieft' gives the measurements of
T. intestinalis as 14 to 18 microns by 3 to 5 microns. The anterior
flagellum measures about 14 microns, while the posteriorly directed
flagellum, the axoneme of which is attached to the border of an undulating
membrane, measures about 28 mi-
crons.
Martin (1913) published an ac-
count of the flagellates from these
three fish. He was convinced that
the form named T. intestinalis has
three anterior flagella, which are
often twisted together so closely that
observers have erroneously concluded
that only one flagellum is present.
Accordingly, Martin places it in a
new genus as Trypanoplasynoides
intestinalis. Structurally, according
to him, it resembles C. helicis, except
that it possesses three anterior flagella
instead of a single one. As regards
the flagellate of Cyclopterus, first seen
by Dahl, Martin states that a small
cytostome is present at the base of the
flagella, the axoneme of one of which
passes backwards over the surface of
the body, to which it is attached,
without there being any evidence of an undulating membrane. For these
reasons he follows Apstein (1910) in retaining it in the genus Heteromita as
H. dahlii. It certainly does not belong to this genus, the members of
which have two free flagella and axonemes arising from blepharoplasts
on the nuclear membrane (Fig. 142). It is better to retain it in the genus
Cryptohia at present, as also the form in the conger eel, for which Martin
retains the name Trypanoplasma congeri. Woodcock and Lodge (1921)
described as C. trematomi a flagellate which was found in the stomach and
intestine of a fish {Trefnatotnus hernacchii).
Walker (1910) stated that he had obtained a culture of a trypanoplasm
from the intestine of the frog, Bana jyalustris, by inoculating agar plates. It
I. 41
Fig. 263. — Intestinal Trypanoplasms
OF Fish. (After Martin, 1913.)
1 . Cryptohia congeri from the stomach of the
conger eel {Conger niger) ( x ca. 1,800).
2. Cryptohia dahlii from the stomach of
Cyclopterus lumpiis (x ca. 1,200).
3. Cryptohia [Trypanoplasma ides) intestinalis
from stomach of Box hoops ( x ca. 1,800).
642 FAMILY: CEYPTOBIIDiE
seems more probable that he was dealing with some other flagellate, possibly
a Cercomonas. Hovasse (1924) has given the, name Trypanoplasma sagittce
to a form he has found in the intestine of a little marine worm belonging
to the genus Sagitta.
C. Blood Forms of Fish.
Laveran and Mesnil (1901c) created the genus Trypanoplasma for a
flagellate they discovered in the blood of the fresh-water rudd, Leuciscus
enjthrojjhthahnus, which they named T. horreli (Fig. 151). Since that date,
a number of similar forms have been discovered in the blood of fresh-
water fish. Curiously enough, no one has yet described an intestinal
form from fresh-water fish, though such forms have been seen in marine
fish, in which, however, the blood forms do not occur. Structurally, the
blood flagellates are similar to those of snails and the intestine of marine
fish, so that strictly they should be included in the genus Cryptohia.
The blood forms have been so long known by the name Trypaiioplasfna
that it seems better to retain this name for them provisionally. They
are carried from host to host by leeches, while the other forms have a
different method of transmission. In the case of the intestinal flagellates
of fish, and possibly those of snails, it might be expected that encysted
forms would occur. Such, however, have not yet been described. Future
investigation may, however, reveal encysted forms, in which case the re-
tention of the name Trypanoplasma for the blood forms would be justified.
The possibility of a difference in life-cycle was suggested by Woodcock
and Lodge (1921).
The various tryj)anoj)lasms of fish resemble one another very closely,
so much so that Keysselitz (1906), who studied these flagellates in many
species of fresh-water fish, came to the conclusion that they all belonged
to the species T. horreli. Other observers, however, have given specific
names to the forms occurring in different fish.
The following species have been described:
T. horreli Laveran and Mesnil, 1901: Leuciscus eryihrophihahnus (rudd).
T. cypriniVlGhn, 1903: Carassius vulgaris (carp), C. auraiius (goldfish).
T. varium Leger, 1904: Cobitus barbatula (loach).
T. guernei Brumpt, 1905: Coitus gohio (bull-head).
T. barbi Brumpt, 190G: Barbus fluviatilis (barbel).
T. abraviidis Brumj)t, 1906: Abraviis hrama (bream).
T. Initiw Brumpt, 19C6: Salmo fario (trout) = T. valentini Gauthier, 1920:
Salmo fario.
T. sp. Rodliain, 1907: Labeo macrostoma
T. gurneyorum Minchin, 1909: Esox lucius (pike).
T. Clarice Mathis and Leger, 1911: Clarias macrocephnlus.
T. sp. Mathis and Leger, 1911: Monopterus javancnsis.
T. heysselitzi Minchin, 1909: Tinea tinea (tench).
T. sp. Tanabe, 1924: Misgurnus anguillicaudatus.
T. nince koM-yaJcimov Yakimofl, 1925: Silurus glaris.
GENUS: TRYPANOPLASMA 643
Keysselitz (1906) published au account of the trypanoplasmas of fish
which he had seen in Perca fluviatilis, Acerina cernua, Lota vulgaris,
Barbus fluviatilis, Cyprinus carpio, Carassius vulgaris, Tinea tinea, Ahramis
bratna, Leuciscus idus {Idus melanotus), L. cephalus {Squalius cephalus),
L. ergthrophthalmus {Scardinius erytkrophthalttius), L. rutilus, Esox lucius,
and Cobitis barbatida. He regarded them as all bejpnging to the one
species, T. borreli, to which Leger, L. (1904/), had ascribed a form seen
by him in the minnow, PJioxinus Icevis.
The various species described differ from one another merely in their
dimensions, in the position of the nucleus, and other details. The parasites
are usually scanty in the blood of fish, so that in most cases the observa-
tions have been made on only a few individuals. It is impossible to be
sure, therefore, that the forms do not belong to one species, as Keysselitz
maintains.
Trypanoplasma borreli Laveran and Mesnil, 1901. — This flagellate, as
described by Laveran and Mesnil, is a flattened elongate organism with a
rounded anterior end, and usually a somewhat pointed posterior end
(Fig. 151). It is distinctively curved, so that one side of the flattened body is
convex and the other concave. The nucleus lies just behind the anterior
third of the body near the convex border. In properly fixed specimens it is
seen to consist of a nuclear membrane and central karyosome. Opposite
the nucleus, and near the concave border, is the kinetoplast, consisting of
an elongate parabasal, just anterior to which are two closely applied ble-
pharoplasts. From one of these arises an axoneme, which passes forwards
round the anterior end of the body, and thence backwards on the edge of
the undulating membrane along the convex border to the posterior end,
where it becomes a flagellum. From the other blepharoplast there arises
an axoneme, which passes into the anteriorly directed flagellum. The
body of the flagellate is about 20 microns in length by 3 to 4 microns
in breadth. The two flagella are about 15 microns in length. Slightly
larger or smaller forms occur, while in preparations many curious distorted
flagellates result from the metabolic nature of the body. Reproduction
in the blood of the fish is by longitudinal division, and follows very closely
the process as described above for Crgptobia helicis.
The trypanoplasm of the rudd is not only inoculable to uninfected
rudd, but also to minnows, as proved by Laveran (19046). Similarly, the
naturally occurring trypanoplasm of minnows is inoculable to rudd.
Plehn (1903) succeeded in inoculating the trypanoplasm of carp to other
carp.
Ponselle (1913) has succeeded in cultivating the trypanoplasm
{T. varium) of the loach. The medium employed consisted of a 2 per cent,
agar in tap water without the addition of salt, to which one volume of
644 FAMILY: CRYPTOBIID^
defibrinated rabbit's blood was added as in N.N.K. medium. The cul-
tural forms resembled the blood flagellates, except that the undulating
membrane was less developed. In some the posterior flagellum was
detached as a trailing flagellum, giving the organism a likeness to species
of Bodo.
According to Plehn (1903) and Keysselitz (1906), the trypanoplasms are
liable to produce various morbid symptoms in the fish, which appear
paler than normal fish do, and may show oedematous swellings of the body.
In some cases there is marked loss of vitality, terminating in death.
Transmission, ■ — In nature the trypanosplasms are transmitted from
one fish to another by leeches. Leger, L. (1904e), noticed that numerous
trypanoplasms occurred in the intestine of leeches {Hemiclepsis marginata)
which had fed on infected fish (Fig. 240). He also saw small forms in a
species of Piscicola which had fed on infected minnows. Brumpt (19066)
found that T. guernei of the bull-head and T. barbi of the barbel multiplied
in the intestine of Piscicola, while T. ahramidis of the bream developed in
H. marginata. The question of transmission was studied more completely
by Keyssilitz (1906) in P. geometra (Fig. 245). According to him, there is
at first a conjugation in the crop of the leech of forms which he regarded as
gametes. The zygote thus formed has no flagella, but is an ovoid body con-
taining a nucleus and kinetoplast, each of which is supposed to be the result
of fusion of the corresponding structures of the gametes. No confirmation
of this process has yet appeared. In the crop of the leech there is active
multiplication of the flagellates by fission till a large number is present.
These vary very much in size and shape, but there is a tendency towards
the production of small slender forms, which eventually make their way
into the proboscis sheath. It is presumably these slender proboscis
sheath forms which enter the wound inflicted by the leech in the act of
feeding. Multiplication was also noted to take place for a short period in
Hirudo medieinalis.
Robertson (1911) studied the development of the trypanoplasm of gold
fish in England in Hetniclepsis marginata and Piscicola sp. (Fig. 264).
About four to five hours after a young leech had fed, dividing trypano-
plasms could be seen in the crop. These appear to be somewhat broader
than the blood forms which were ingested. Multiplication proceeds till,
on the second day, slender comma-shaped forms make their appearance.
All intermediate types between these and the broad forms are still present.
After some days the slender forms move forwards to the proboscis and
1. Form in blood of goldfish. 2. From crop of leech forty-four hours after feeding.
3. From crop three and a half days after feeding. 4. From crop six days after feeding.
5. From crop six days after feeding. 0. From crop seven days after feeding.
7. From crop ten days after feeding. 8. From crop twenty-five days after feeding.
TRYPANOPLASMA BOERELI
645
Fig. 264. — Trupanoplasyna cyprini from the Blood of the Goldfish and the
Intestine of the Leech, Hemiclepsis marginata (x 4,000). (After Eobert-
SON, 1911.)
[For description see opposite page.
646 FAMILY: TRICHOMONADID^
enter the sheath, in which situation they may be seen in incredible numbers,
either free or attached to the wall of the sheath by their fiagella. By
the end of the tenth day, before which the leech is not ready to feed
again, there are numerous flagellates in the proboscis sheath and anterior
part of the crop, but none farther back. Exceptionally, the fresh feed will
clear the leech of flagellates entirely, but, as a rule, the proboscis sheath,
which is emptied at the feed, becomes filled with the slender forms which
have resulted from multiplication of those left in the sheath or by a
further migration forwards from the crop. A fish, upon which infected
leeches had fed, first showed trypanoplasms in its blood on the seventh
day. According to Brumpt (19066), the trypanoplasms with which he
worked multiply in the gut, but do not invade the proboscis sheath.
Tanabe (1924) has given an account of the development of the trypano-
plasm of the Japanese loach {Misgurnus anguillicaudatiis) in the leech,
Hirudo nipponica. In the intestine active multiplication occurred during
the first three days, so that large numbers of small forms were produced.
After this there was a gradual disapjDearance of the flagellates, though in
some cases they persisted for eight or nine days. No mention is made of
any attempts to transmit the infection by means of these leeches.
9. Family : Trichomonadid^.
The flagellates belonging to this family are characterized by the pos-
session of a variable number of flagella, a definite cytostome, and a rod-
like structure, the axostyle, which arises from the blepharoplasts and passes
through the body to its posterior end, through which it usually protrudes.
In some forms, one of the flagella is directed backwards, and its axoneme
may be attached to the border of an undulating membrane. In such cases
there is usually a stiff basal fibre, which lies along the line of attachment
of the undulating membrane to the body (Fig. 265). The family includes
the following genera:
Genus: Trichomonas Donne, 1837.
Ditrichomonas Cutler, 1919.
Gigantomonas Dogiel, 1916.
,, Eutrichomastix Kofoid and Swezy, 1915.
Janickiella Duboscq and Grassi, 1923.
Trichomitus Kofoid and Swezy, 1919.
Devescovina Foa, 1905.
.» Foaina Janicki, 1915.
Retortamonas Grassi, 1879.
Protrichomonas Alexeiefl, 1911.
Polymastix Biitschli, 1883.
Hexamastix Alexeieff, 1912.
Cochlosoma Kotlan, 1923.
GENUS: TRICHOMONAS
647
It must, however, be mentioned that flagellates of the genus Tricho-
monas are very liable to exhibit changes of structure, so that they appear
to resemble members of another genus. By detachment of the membrane
flagellum a flagellate of genus EutricJiomastix is simulated. The genus
Hexamastix could be accounted for by supposing that the membrane
flagellum of a Trichomonas with five
anterior HngeWn (Pentatrichomonas)
had become detached from the
membrane, so as to give rise to
forms with six anterior flagella and
no undulating membrane. The
axostyle and the supporting fibre of
the undulating membrane may be
difficult to detect, so that forms
with a number of flagella and no
other structures result. It has
been shown above (p. 310) that
the genus Protetramitus was
founded on what were merely
altered forms of Trichomonas or
Eutrichomastix. It is not im-
probable that the genus Tricho-
mitus was similarly established for
flagellates which actually belong
to the genus Trichomonas, and in
which the axostyle was for some
reason invisible, as not infre-
quently occurs with undoubted
Trichomonas. When such forms
are encountered in films, careful
search will usually disclose a
series connecting them with the
typical unaltered Trichomonas.
In practically every film of material
containing Trichomonas there
occur rounded cytoplasmic bodies
possessing a nucleus and blepharo-
plasts, but with no other struc-
tures visible, similar rounded forms in which the flagella can be seen,
others with the axostyle evident, and, finally, the typical Trichomonas
with the undulating membrane and its attached flagellum. Under these
circumstances the determination of genera, which may be regarded
Fig. 265. — Flagellates from the C^cum
OF THE Fowl (x 4,000). (After
Martik and Robertson, 1911.)
A. Ohilomastix galUnarum.
B. Trichomonas eberthi.
C. Trichomonas galUnarum.
D. Eutrichortiastix galUnarum.
648 FAMILY: TEICHOMONADIDiE
as modified Trichomonas, is an exceedingly difficult one, and no definite
decision can be reached unless it is clearly established that the characters
are constant.
Oenus : Trichomonas Donne, 1837.
The flagellates of this genus have more or less pear-shaped bodies,
three to five anterior flagella, and a recurrent axoneme, which is attached
to the border of an undulating membrane. The axoneme may or may
not be continued beyond the membrane as a posterior flagellum. There
is a definite cytostome near the base of the flagella. An axostyle is present,
and also a fibre which runs along the line of attachment of the undulating
membrane. A nucleus is situated at the anterior end of the body, and
anterior to it is a group of blepharoplasts, from which the flagella and
other structures arise. In some forms a parabasal has been described in
the cytoplasm between the nucleus and basal fibre of the undulating
membrane. Reproduction is by binary fission, and encystment also
occurs.
TRICHOMONAS IN MAN.
There is a large number of species of this genus, which differ from one
another in size, shape of the body, and the number of flagella. It appears
that at least three occur in man : T. hominis of the intestine, T. elongata
of the mouth, and T. vaginalis of the vagina. It cannot, however, be
considered as definitely established that these are distinct species.
Trichomonas hominis (Davaine, 1860). — This common intestinal flagel-
late of man was first recorded by Davaine in 1854 as Cercomonas. In 1860
he gave a figure and more detailed description of the organism under the
name Cercomonas Jioininis. It was later placed in Donne's genus, Tricho-
monas, by Leuckart (1879) and others, and is now generally known as
T. hominis. As noted above (p. 621), Moquin-Tandon in the year 1860
had already proposed the name C. obliqua for a flagellate usually regarded
as a Trichomonas, so that, according to rule, the name should be T. obliqua
if there is no doubt as to the identity of the organism named by him.
Grassi (1879a, 1881a) referred to it as Monocercomonas hominis. On
account of these difficulties of nomenclature. Stiles (1902) proposed the
new name Trichomonas confusa.
T. hominis is probably the commonest intestinal flagellate of man
(Fig. 266). As a rule, it is only seen in diarrhoeic stools, but that it is
still present when the stools are formed can be demonstrated by adminis-
tering saline purges, or by the inoculation of faeces into certain media, as,
for instance, Hogue's egg medium, and incubating at 24° C. for a few days,
as advocated by Hegner and Becker (1922) and Reichenow (1923). Its
occurrence in diarrhoea, however, is due most probably to the fact that it
is only when the stools are liquid that the flagellate in its active condition
TRICHOMONAS HOMINIS
649
is swept out of the intestine. Its presence is not necessarily an indication
that it is the cause of any intestinal derangement which may exist. It
is a pear-shaped organism measuring 5 to 15 microns in length. Occa-
sionally longer forms are seen. The shape of the body, which is normally
pear-shaped, changes considerably from time to time, and under certain
conditions pseudopodia are formed. The anterior end of the body is
somewhat bluntly pointed, while the posterior end is more tapering and
terminates in the protruding axostyle. The flagella, which are as long as
Fig. 266. — Trichomonas hominis from the Human Intestine ( xca. 2,000.)
(1-3, AFTER Faust, 1921; 4-6, after Wenyon and O'Connor, 1917.)
1-3. Forms with three anterior flagella (Tritricliomonas) .
■4-6. Forms with four anterior flagella [TricJiomoruts).
or longer than the body, arise from the anterior extremity. These are
usually four in number. They move about from one side of the body to
the other, performing sweeping movements very much like the action of a
whip which is lashed to and fro. Very frequently the proximal portions
of the flagella appear adherent to one another or twisted to form a common
stem. On the ventral side of the base of the flagella is a slit-like cytostome.
There is a well-developed undulating membrane, which passes in a slightly
650 FAMILY: TRICHOMONADID^
spiral manner along the dorsal surface to the posterior end of the body.
The posteriorly directed axoneme is attached to the border of the mem-
brane, and may be continued beyond it posteriorly for a short distance as
a fiagellum. The extension beyond the membrane of a flagellum does not
appear to be a constant feature. In some cases it is exceedingly difficult
to detect such a flagellum, especially if it is a short one, as is usually the
case. Faust (1921) regards the axoneme as terminating at the end of the
membrane (Fig. 266, 1-3). The membrane is in constant motion, while
the anterior flagella are sweeping over the body, first on one side and then
on the other. The flagellate progresses in a jerky manner, revolving con-
tinuously on its longiti^dinal axis owing to the action of its membrane.
When observed for any length of time, many of the flagellates in a specimen
become degenerate. Various changes may take place, all of which may
lead to misconceptions as to the character of the organism. The axoneme
may become detached from the membrane and lash about, so that if the
other finer flagella are overlooked, as is easily done, the organism may be
regarded as having only one long thick flagellum. Such forms have
probably been mistaken for Cercomonas. The flagellate may lose its
membrane flagellum entirely, while the cytoplasm at the anterior part of
the body throws out quite suddenly a long finger-like pseudopodium,
which travels backwards and at the same time becomes shorter. When it
reaches the posterior end of the body it vanishes, while another one is
formed again at the anterior end. These finger-like pseudopodia pass
down the body regularly, and may be regarded as a series of high narrow
waves, resulting from uncontrolled action of the membrane which has been
deprived of its axoneme. The movements are so peculiar that Castellani
(1905) was misled into describing this form as a new amoeba {Entamoeba
undulans). The posterior end of the body may become swollen, so that
the flagellate appears to have a spherical mass attached to it by a narrow
neck. This mass of cytoplasm may be broken off. In normal individuals
it is only the tip of the axostyle that protrudes from the body, but
in many degenerating forms, possibly as a result of retraction of the
cytoplasm or actual separation of portions of it, a greater length of
axostyle is exposed. If the flagellates do not degenerate, they gradually
become rounded and perfectly quiescent. In this condition the membrane
with its attached axoneme passes round the circumference of the now
spherical body, while the axostyle and basal fibre are curved within it.
The anterior flagella may entirely disappear. It is these rounded flagel-
lates which are suspected of proceeding to encystment, but encysted stages
of T. hominis have not been seen. In T. cavicB of the guinea-pig, however,
it is the spherical forms of this type which become encysted, and this
appears to be true also of T. muris of mice. Prowazek (1904fl) and
TKICHOMONAS HOMINIS 651
Bohne and Prowazek (1908) described a Blastocystis as being the cyst of
T. hominis, and sujjposed that this encystment was associated with a
process of autogamy. Several writers, including Bensen (1909), claim to
have confirmed this observation. There is no doubt, however, that Blasto-
cystis, a vegetable organism which can be cultivated, has no connection
whatever with T. hominis (Fig. 118). The cysts described by Lynch (1916)
are so similar to the cysts of Chilomastix mesnili that they cannot be
regarded as cysts of T. hominis till further evidence has been produced.
Lynch (1915c) described, both from faeces and cultures, spherical cysts with
granular contents, but from the description it is impossible to conclude
that he was dealing with encysted Tricho^nonas. They did not show any
of the characters of the encysted forms as seen in animals. In a later
paper this author (1922) admits that cysts of T. hominis have not yet been
discovered.
T. hominis reproduces by longitudinal division. The process has not
been studied in detail in this species, which is usually of small size. As
regards the division of other species, there are many conflicting statements
as to what actually occurs. In stained films, a few further details of the
structure of T. hominis can be made out. The human flagellate is a very
small form which easily shrinks on fixation, so that it is exceedingly
difficult to make satisfactory preparations. In addition to the various
details which can be detected in the living organisms, it can be seen that
there is a spherical or slightly ovoid nucleus near the anterior end of the
body. It consists of a nuclear membrane surrounding a clear space, at
the centre of which is a karyosome. Anterior to the nucleus is a closely
packed group of blepharoplasts. From these arise the axonemes of the
anterior flagella, and also the one which borders the undulating membrane,
as well as a stiff fibre, the basal fibre, which passes through the body just
below the attachment of the undulating membrane. Posteriorly, the
basal fibre tapers to a point. Parallel and close to it can sometimes be
distinguished a row of deeply staining granules.
The axostyle is another structure which commences at the blepharo-
plasts. It has the form of a broad bar which takes a straight course through
the body to the posterior extremity, through which it protrudes as a sharp-
pointed caudal process. The axostyle, unlike the basal fibre, does not stain
black with iron haematoxylin. In the living organism the axostyle is perfectly
passive, and only moves with the contractions of the cytoplasm around it.
T. hominis feeds by ingesting bacteria through its cytostome, and these
can be seen in various food vacuoles. It is possible that it also absorbs
nourishment in solution through the surface of its body. Sometimes red
blood-corpuscles are present in vacuoles. The writer has seen them within
T. hominis in cases of bacillary dysentery when many red cells are jjresent
652 FAMILY: TEICHOMONADID^
in the stool, and also in cultures in media containing rabbit blood. The
presence of included red cells has been advanced as an argument in favour
of the pathogenicity of this flagellate, but there is no reason to suppose
that they have been taken up by the flagellates anywhere than in the
lumen of the gut. In the case of Entamoeba histolytica, it is probable that
the red cells are ingested by the amoebae while they are still in the tissues.
Cultivation. — Several observers have cultivated T. Jioininis. Escomel
(1913) stated that he had obtained a culture in a vegetable medium, but
there was no evidence that he had done anything more than keep the
flagellates alive, as sometimes happens, for many days in the liquid faeces
themselves. Lynch (1915a, 1915c) was able to keep T. hotninis, as well
as the other human species, alive for some days in acid bouillon. In the
case of T. hominis a few subcultures were made, and it seemed evident that
multiplication had taken place- Boyd (1918, 1919) was more successful
by using a saline suspension of fresh faeces in which T. hominis was taken
through seven subcultures during sixty-five days, Ohira and Noguchi
(1917) cultivated T. hotninis (? T. elongata) in a mixture of ascitic fluid and
Ringer's solution. Active multiplication took place in the deeper parts of
this medium and by subculture every forty-eight hours when the tubes
were kept at 23° to 27° C, or every twenty-four hours when, at 37° C,
the flagellates were kept alive indefinitely. Barret, who has successfully
cultivated Balantidium coli and Blastocystis in a 10 per cent, solution of
inactivated human blood-serum in 0-5 per cent, sodium chloride solution,
informs the writer that he has cultivated T. hotninis in this medium and
taken it through thirty subcultures. The writer has successfully cultivated
the flagellate and maintained it by subculture in Hogue's egg medium at a
temperature of 24° C. Bacterial growth takes place rapidly, and to keep
the flagellates alive it is necessary to subculture every week. At higher
temperatures subculture must be made more frequently. The culture
method has been used for diagnosis purposes by Hegner and Becker (1922)
and Reichenow (1923). Boeck and Drbohlav (1925), and Thomson, J. G.
and Robertson(1925),have cultivated T. hominis inBoeck's L.E.S. medium.
Method of Infection. — The question of the method of infection with
T. hotninis presents some difficulties. Definite encysted forms have not
been discovered in man. If infection occurs from man to man, and if it is
true that cysts do not occur, then it must be assumed that infection takes
place by ingestion of the active flagellates themselves, which are known to
survive for a considerable time outside the body. It was shown by the
writer and O'Connor (1917) in Egypt that flies fed on faeces containing
T. hotninis deposited in their dejecta live flagellates five minutes later.
In this manner, food or drink could readily become contaminated with the
living organisms.
TRICHOMONAS HOMINIS 653
Infection of Animals. — Several observers claim to have infected animals
with T. hominis. Thus, Escomel (1913) stated that he had succeeded in
infecting the rabbit, guinea-pig, dog, and cat. Lynch (1915a) thought
he had infected rabbits by injecting them per rectum with cultures, but
in a later paper (1922) he doubts his original claim, owing to the difficulty
he has had in positively excluding a previous infection in animals. Boyd
(1919) stated that he had infected a rat by feeding it with cultures of
Trichomonas. Owing to the doubt which attaches to these experiments,
Hogue (1922) attempted to infect cats, kittens, and rabbits which had,
by repeated examinations, been proved to be free from Trichomonas
infections. The animals were fed and injected per rectum with cultures of
the flagellates, but in no case did an infection result. Kessel (1924a)
states that he has infected monkeys.
Pathogenicity. — The fact that T. hominis occurs most commonly in
diarrhoeic stools has led observers to regard it as a pathogenic organism.
When there is no diarrhoea, the flagellate is still present, though it is only
rarely found in formed stools. As the cysts are not known, its presence
can be recognized only when diarrhoea occurs or after the administration
of a purge. It frequently happens that individuals suffer from a chronic
looseness of the bowel, and that no explanation of this condition can be
discovered. In a certain number of such cases T. hominis is present in
the stools, and many clinicians assume that they are the exciting cause
of the disorder. As there are many cases of this type in which flagel-
lates cannot be discovered, it is quite illogical to assume that the few in
which they are present owe their condition to these organisms.
The writer (1920) examined by section post-mortem material from
five cases of T. hominis infection. In one of these the large intestine
showed the flagellates in the lumen of the glands, actually breaking through
the glandular cells, and distributed in the interglandular connective tissue
(Fig. 267). Whether this invasion of the tissues of the intestinal wall is an
indication of pathogenicity or not cannot be stated. It is possible that
the Trichomonas invaded the tissues shortly before or even after death.
It may be mentioned, however, that guinea-pigs, which are commonly
infected with T. cavice, often show ulceration of the large intestine and
csecum, with definite invasion of the tissues by the flagellate in perfectly
fresh material which has been taken from animals which have been killed.
That invasion can occur at other times is borne out by an observation
made by Pentimalli (1923). Examining fllms made from blood taken from
a patient's vein under aseptic conditions, he found Trichomonas present.
A further examination made ten hours later showed the same organism in
smaller numbers. Further examinations were negative. No information
as to the presence or absence of an intestinal infection was obtained.
654
FAMILY: TRICHOMONADID.E
Kessel (1925) has seen the flagellate in pus from an amoebic abscess of the
liver.
T. liominis infections may be very persistent. In Egypt, the writer
and O'Connor studied a case in which the flagellates were present during
an observation of sixty-two days. Cases are on record, however, in which
they are known to have been present for many years.
«
m
\^
■m m
1 1 \
^Jte
Fig. 267. — Section of Human Large Intestine, showing Invasion of the Wall
BY Trichomonas liominis (x ca. 1,000). (After Wenyon, 1920.)
The flagellates pass through gaps in the lining cells of the intestinal glands into the surrounding
connective tissue.
VARIETIES OF TRICHOMONAS HOMINIS.^As already pointed out,
T. hoininis usually possesses four flagella. In any infection it will be seen
that the great majority of the flagellates have a definite number of flagella,
so that it seems clear that for any particular form the number is constant.
It is known, however, that in certain cases the majority of flagellates have
five flagella, and in other cases three (Figs. 26, 266, 289). Some observers
have given special generic names, according to the number of flagella.
Thus, the four-flagellate type has been called Tetratrichomonas by Parisi
(1910), the five-flagellate type PentatricJiomonas by Mesnil (1914) and
VARIETIES OF TRICHOMONAS HOMINIS
655
Chatterjee (1915), and the three-flagellate type Tritrichomonas by Kofoid
(1920). It seems doubtful if these forms should be placed in different
genera. The type species of Trichomonas is T. vaginalis Donne (1837), and
this form has always been seen to possess four flagella, though it cannot be
affirmed that the three or five flagellate types never occur in the vagina.
Trichomonas hominis, therefore, would be the correct name for the intestinal
form with four flagella, there being no need to employ the generic title
Tetratrichomonas. The generic names Tritrichomonas and Pentatrichomonas
can be employed for the types with three and five flagella, or what appears
to be safer is to regard the various types as varieties of one species, so
that it is possible to distinguish in the human intestine T. hominis var.
Fig. 268. — Trichomonas trijpanoides from Intestine of a Termite, Eeticiilitermes
Jucifugus, SHOWING One, Two, and Four Anterior Flagella (x ca. 2,500).
(After Duboscq and Grasse, 1924.)
tritricJiomonas, T. hominis var. tetratrichomonas, T. hominis var. j)enta-
trichomonas. Duboscq and Grasse (1924) have shown that in the case of
T. trypanoides, described by them from termites, there is a single thick
anterior flagellum which frequently becomes divided longitudinally to
give rise to two, three, or four separate flagella (Fig. 268). The jcommonest
type in man is undoubtedly the one with four flagella. Most observers,
however, fail to record the number of flagella, which, moreover, are very
difficult to count. In an infection in which a certain number of flagellates
have, say, four flagella, it will be found that others have a smaller number,
so that to determine the prevailing type in any infection it is necessary to
count the flagella on a number of flagellates, a procedure which involves
656 FAMILY: TRICHOMONADID^
considerable expenditure of time and labour. It should be clearly under-
stood that before a Trichomonas is reported as having a particular number
of flagella, it is necessary to observe this number in the majority of the
forms present. Some dividing forms will have a larger number of fiagella,
while in others it will be impossible to detect the full number. Neverthe-
less, by careful observation it is not difficult in the case of pure infections
to determine the normal flagellum number for the form present. The
writer has seen the form with four flagella on many occasions, that with
five only a few times, but the one with three only once.
Derrieu and Raynaud (1914) proposed the name Hexamastix ardin
delteili for the human form with five flagella, while Chalmers and Pekkola
(1916) mistook it for a Hexamita, which they named Octo7nitus hominis
(Fig. 289). Kofoid and Swezy (1924) employ the name Pentatrichomonas
ardin delteili for the one with five flagella. One of the five flagella is
described as a trailing flagellum longer than the others (Fig. 20).
Trichomonas elongata Steinberg, 1862.- — Hoffle (1850) appears to have
been the first to observe Trichomonas in the mouth. Steinberg (1862)
studied these oral flagellates and named three distinct species: T. elongata,
T. caudata, and T.flagellata. It is evident that he was dealing with various
forms of one flagellate, so that if the oral Trichomonas be regarded as a
species distinct from that of the intestine, its proper name will be T. elon-
gata, the first of those proposed by Steinberg, and not T. buccalis, the name
suggested by Goodey and Wellings (1917). It is probable that Leeu-
wenhoek saw the flagellate in the tartar of his own and other people's
teeth, but there are no means of identifying it unless it is assumed that
Trichomonas is the only flagellate which can possibly occur in material
taken from the mouth. That other flagellates may occur in the mouth
has recently been demonstrated by Knowles and Das Gupta (1924), who
have found a species of Bodo in this situation. Mliller (1773) also noted
that flagellates developed in the course of four days in water to which
tartar from the teeth has been added. He named the organism Cercaria
tenax, but there is no conclusive evidence that he was actually dealing
with Trichomonas. It has been maintained by Goodey and Wellings
(1917) that, in the oral form, the axoneme does not extend beyond the end
of the membrane as a flagellum, as it does in T. hominis (Fig. 269). The
writer has, however, found no difference in this respect between the
Trichomonas of the mouth and that of the intestine. The writer and
O'Connor (1917) noted that, in a case which constantly showed Tricho-
monas in the month, the flagellates never appeared in the diarrhoeic stools,
though they were specially looked for on many occasions. Lynch (1915o)
could find no Trichomonas \n the faeces of a woman who harboured Tricho-
monas, both in the mouth and vagina. The Trichomonas of the mouth
TRICHOMONAS ELONGATA
G57
possesses four flagella, as noted by Goodey and Wellings (1917), and does
not differ as regards size and structure from that of the intestine.
Jepps (1923a.) believes that the oral form is more actively amoeboid than
that of the intestine, but though it is stated that the organism agrees
with the one described by Goodey and Wellings, only three flagella are
figured.
Trichomonas have been noted by the writer (1920) in pus exuding from
the follicles of the tonsil. Several observers, including Schmidt (1895)
and Artault (1898), have seen the flagellates in sputum coughed up from
the lung, while Strube (1898), Cohnheim (1903), Zabel (1904), Schmidt
Fig. 269. — Trichomonas elongata from the Human Mouth (x 4,100).
(After Goodey and Wellings, 1917.)
(1904), and Eosenfeld (1904) observed them in stomach contents in cases
of carcinoma and other conditions. They have also been recorded as
occurring in pleural exudate by Litten (1886) and Roos (1893). Parisot
and Simonin (1921) observed the flagellates in large numbers in the
expectorations of a case of gangrene of the lung. At post-mortem they
were present in abundance in the gangrenous areas, but not in others.
These forms are certainly identical with the oral species, though the name
T. pulmonalis has been given to the form seen in sputum by Schmidt
(1895). The invasion of the lung is comparable with the spread of spiro-
chsetes and bacteria to this organ from the mouth when conditions become
favourable to their growth.
I. 42
658 FAMILY: TRICHOMONADIDiE
The Tricliomonas of the mouth was maintained in culture for a short
time by Lynch (1915a) by means of the method used by him for culture
of T. liominis. The results with subculture were not entirely satisfactory.
Ohira and Noguchi (1917) were more successful. They employ a mixture
of equal parts of ascitic fluid and Ringer's solution. By making sub-
cultures every day large numbers of organisms were obtained. The usual
forms measured 10 to 15 microns by 4 to 8 microns, and possessed four
flagella. Occasionally, larger forms up to 25 by 12 microns occurred.
Multiple division forms, in which four, six, or eight individuals separated
from the large body, were also seen (see p. 652).
Trichomonas vaginalis Donne, 1837. — This species, which was first
seen by Donne (1837), is of fairly frequent occurrence in cases of vaginitis,
in which the exudate has an acid reaction (Fig. 270). It has been studied
especially by Blochmann (1884), Kunstler (1884), Bensen (1900), Lynch
(1915a), Reuling (1921), and Hegner (1925). Bensen gives its measure-
ments as varying from 18 by 6 microns to 26 by 16 microns. Some forms
are narrow and elongate, while others are almost spherical. Bensen
erroneously concluded that there were three anterior flagella, and also
failed to note the axostyle. In a case studied by Lynch (1915a), Triclio-
monas was present, not only in the vagina, but also in the mouth. The
oral forms are described as possessing four flagella, and what is evidently
an axostyle was seen protruding from the posterior extremity. This
organ was more definitely seen by Kunstler. Lynch states that the
vaginal forms were the same in every respect as those in the mouth, and
he concludes that the two are identical. It is interesting to note that
he found no flagellates in the faeces. Reuling gives the measurements of
T. vaginalis as 10 to 30 microns by 10 to 15 microns, and Hegner 7 to 21
microns by 6 to 18 microns. The undulating membrane, with its sup-
porting fibre and attached axoneme, extended for only a third, or at most
half, the length of the body. A definite axostyle was present, but in
some cases Reuling found in its place four separate fibres (Fig. 27). Both
Reuling and Hegner describe four anterior flagella.
A series measured in the living condition by the writer gave the
following dimensions in microns: 29-5 by 19-2, 21-6 by 18-5, 20-0 by 16-5,
18-0 by 18-0, 18-0 by 12-6, 16-0 by 14-5, 14-5 by 9-0, 12-6 by 9-0, 11-0 by 10-0.
In the infections studied, the majority of forms had four flagella (Fig. 270).
Some, however, had only three, while a few which were not evidently
dividing forms had five. There was a definite axostyle extending from
the region of the nucleus to the posterior end, through which it projected.
In many of the largest forms this structure was completely obscured.
The undulating membrane extended in the larger spherical forms for only
about half the length of the body, but in the smaller ones it was as long as
TRICHOMONAS VAGINALIS
659
the body, while the attached axoneme terminated at the posterior end of
the membrane or was continued for a short distance as a flagellum. In
some individuals a definite cytostome could be detected, while a supporting
rod at the base of the membrane was also present. Certain individuals
which were evidently degenerating gave rise to structures resembling
Blastocystis (Fig. 270, 7-9). There was very little difference in the length
of the membrane in the large and small forms, the larger forms appearing
Fig. 270. — Trichomonas vaginalis (x 1,300).
1-6. Ty|iical forms.
(Original, from Living Specimens.)
7-9. Degenerate forms.
to have been developed from the smaller ones by overgrowth of the pos-
terior part of the body. The smaller forms did not differ in any respect
from T. hominis of the intestine.
From the case investigated by him, Lynch (1915rt) obtained a culture
of both the vaginal and oral Trichomonas, which, however, were maintained
only for a short time.
A Trichomonas has also been found in the male urethra in cases of
urethritis. It was seen in the urine by Dock (1894, 1896), Marchand (1894),
660 FAMILY: TRICHOMONADID.E
Miura (1894), and Fonseca (1916), and is not uncommon in the centrifuged
deposit from urine. It is possibly the same species as T. vaginalis.
T. vaginalis appears to be of common occurrence in cases of vaginitis,
where the exudate is acid in reaction. Thus, Hausmann (quoted by
Blochmann, 1884) found it present in 30 to 40 per cent, of females examined,
while Donne, its original discoverer, found it very common in France.
Brumpt (1913c) obtained 10 per cent, positive examinations in Paris, and
the writer has found it common in England. There appears to be little
reason to suppose that T. vaginalis is in any way a pathogenic organism.
It seems quite possible that the three species of human Trichomonas
really belong to one species, and that the differences which occur are due
to variations in nutrition. The writer has studied the Trichomonas of
the mouth, vagina, and intestine. Those of the mouth and intestine
resemble one another so closely that it is impossible to differentiate them,
and this is also true of the smaller forms which occur in the vagina. The
large vaginal flagellates are probably overgrowth forms. Ohira and
Noguchi (1917), as noted above, observed large forms of the oral Tricho-
monas in their cultures. In cultures made by the writer in Hogue's egg
medium the mouth and intestinal forms were identical, and though the
vaginal form was not maintained in subculture in this medium, those
flagellates which remained active for some days were indistinguishable from
the cultural flagellates of the mouth and intestine. Lynch (1922), who
has cultivated all these forms, states that under the same conditions they
are identical, and that there is no means of differentiating them. Should
this prove to be the case, the name T. vaginalis will have priority over all
others.
TRICHOMONAS IN ANIMALS.
Species of Trichohionas are very common parasites of the intestinal
canals of animals. The csecums and large intestines of guinea-pigs and
rats, for instance, are often swarming with these flagellates, which, on
account of their large size, are more easily studied than the human forms.
They are common in birds, reptiles, and amphibia, and also occur in
invertebrates. Many of these have been given distinctive names, but
whether each host has its own species cannot be stated at present. The
various species described are very uniform in character, and differ from
one another chiefly in size. T. muris of the mouse varies in length from
3 to 20 microns at least, so that dimensions are of little value as specific
characters unless they can be proved to be constant.
Trichomonas muris (Grassi, 1879). — This common flagellate of the intes-
tine of rats and mice, and possibly other rodents, was first noted by Grassi
(1879a), who named it Monocercomonas muris. Later (1881a) he referred
TRICHOMONAS MURIS
G61
Fig. 271. — Trichomonas muris of the Mouse, showing Structure of Flagellate
AND Method of Division (x 2,000). (After Wenricii, 1921.)
1. Form showing parabasal body (pale grey streak) after fixation in Flemming'.s osmic acid
solution.
2. Form in which parabasal body is not evident after fixation in Schaudinn's fluid.
3. Commencing division — formation of new basal fibre as fine outgrowth from blepharoplast.
4. A similar >t,iL;f tn that at 3 — nucleus shows six chromosomes, each with indication of
douhlcn.ituiv.
5. The l)l( |ili:ii(.ij|a>ts have divided, and the daughter blepharoplasts are connected by a para-
dc<ni..M'; til.' iMw lia>.-.l tilnv and new inciiihranc liauclliiiii aiv .(.iinccted with the divided-
ol? lil<'|.liai..|.laMs: m\ (•lir..iii.iM mi-s with nn in.licat icni .>t .Imil.lr nature.
6. Similar ■>taL;c sli-htly nioic advaiK'cd. slniwin;^ (•(miiiK'nciiiM (Icjciicration of axostyle.
7. The six chromosomes have divided into two groups nf >i\ daii-htcr chromosomes, which show
indications of a double nature; the axostyle has disapiKMnd.
8. Nuclear division is complete, and each daughter nutlrus has six chromosomes which again
appear double; the paradesmose is still present; the axostyle has disappeared.
9. The nuclei are reconstructed, and daughter axostyles (clear streaks) are growing out from
the blepharoplasts.
10. Outgrowth of new axostyles complete; paradesmose still present.
662 FAMILY: TRICHOMONADIDiE
to it as CimcB7iomonas muris. As it occurs in mice it was investigated
by the writer (1907). It has been recorded from the field vole {Microtus
arvalis) by Lavier (19216). T. muris varies in length from 3 to 20 microns.
It has the usual pear-shaped body, possesses three anterior flagella, a
terminally protruding axostyle, and a well-developed membrane bordered
by an axoneme which becomes a flagellum at the posterior end of the
organism (Fig. 271). At the base of the flagella is a slit-like cytostome.
In stained films the oval nucleus can be seen near the anterior end of the
flagellate. It has a definite membrane, and the chromatin is distributed in
the form of fine granules throughout its substance, while a central karyo-
some may be present. Anterior to the nucleus can be seen two groups of
closely aggregated blepharoplasts. The anterior of these gives rise to the
three flagella, and the one which borders the undulating membrane.
From the other arises a stiff, deeply staining basal fibre, which passes down
the body parallel to the base of the undulating membrane. Parallel to
the basal fibre, and close to it, is a row of granules, a second row of which
may also be present. The axostyle, the pointed tip of which protrudes
through the body posteriorly, commences at the blepharoplasts and passes
through the body. In the region of the nucleus it seems to pass between
the cytostome and the nucleus, and the latter often appears to be partially
embedded in it. Wenrich (1921) describes another structure which can
sometimes be detected in the cytoplasm, especially after fixation in weak
Flemming's solution without acetic acid. It is a sausage-shaped body
lying between the nucleus and basal fibre of the undulating membrane
(Fig. 271, i). It has a length of a little less than half that of the body.
Wenrich considers it to be of the nature of a parabasal. A similar structure
was seen by Janicki (1915) in T. hatraclwrum of the frog (Fig. 275), and by
Alexeieff (1924) in T. augusta (Fig. 67).
The cytoplasm contains food vacuoles in which bacteria occur. Occa-
sionally, as noted by the writer (1907), large vacuoles filled with bright
refractile coccus-like bodies are seen. It was suggested that they were
possibly parasitic in nature, and it is now generally recognized that they
are spores of a fungus of the genus Sph(srita, which was established by
Dangeard (1886) for a similar parasite of free-living amoebae and flagellates
(see p. 252). Dangeard recognized the organism as belonging to the
Chytridiacese, and gave it the name S. endogena. It was studied in free-
living amoebae by Chatton and Brodsky (1909), who gave the name
S. dangeardi to a form in Euglena. What may be a distinct species was
seen by the writer (1907) in Entamoeba muris and T. muris, by Cragg (1919)
in E. coli, by Dobell (1919) in Endolimax nana, and by Noller (1921) in
E. coli, E. histolytica, lodamoeba biitschlii, and Dientamosba fragilis. Da
Cunha and Muniz ( 1 923) gave the name S. minor to the form in TricJiomonas.
TEICHOMONAS MURIS 663
Lwoff (1925), referring to the form in E. coli and E. histolytica, suggests
that it is distinct from the one which parasitizes free-living amoebse, and
proposes to name it S. normeti. The development of the organism is a
simple one. A spore enters the cytoplasm and grows into a multinucleated
sphere enclosed by a membrane. It breaks up into a number of spores,
which are not provided with flagella (Figs. Ill, 4, and 173, 4). The spores
escape from the cyst, and after entering the cytoplasm of other amoebse or
flagellates, repeat the process of growth and spore formation.
T. muris multiplies by longitudinal fission (Fig. 271). The first step
in the process is the division of the two groups of blepharoplasts, so that
two pairs are formed. These separate from one another, and as they do
so they are seen to be connected by a fibre, the paradesmose, which may
still persist even when the blepharoplasts have reached opposite sides of
the body. From the anterior of the two new blepharoplasts three flagella
arise, while from the posterior one a new basal fibre grows out parallel
to the pre-existing one. At the same time a new membrane forms as a
new axoneme grows out from the anterior blepharoplast. The new
membrane, axoneme, and basal fibre gradually increase in size till they
equal those already existing. Meanwhile, the nucleus has been under-
going changes. The fine chromatin granules run together to form definite
chromosomes. The writer (1907) concluded there were six of these, but
Kofoid and Swezy (1915a) give the number as five and Kuczynski (1918) as
eight. Wenrich (1921) has published a clear account of the division stages of
T. muris, and has shown that there are actually six chromosomes (Fig. 271).
The nucleus becomes elongated, the nuclear membrane persisting during
the whole process of nuclear division. Each chromosome then becomes
constricted and divided into two, so that six pairs of chromosomes can
be seen. The nucleus becomes elongated, and one set of daughter chromo-
somes passes to one end of the nucleus and the other set to the other end.
The nuclear membrane becomes constricted and divided. The chromo-
somes in each daughter nucleus now break up into finer granules, and
the original type of nucleus is reproduced. During the nuclear division
changes have been occurring in the axostyle. Here, again, there is a
difference of opinion as to what actually happens. The writer (1907)
believed that, as the blepharoplasts separate, the axostyle was divided
longitudinally from before backwards. Eventually, the two daughter
axostyles were united only at their posterior extremities. By this time
the flagella, membrane, and other parts of the new flagellate were fully
formed. The cytoplasm became elongated, and had nucleus, blepharo-
plasts, cytostome, and flagella at each end, while stretching between the
blepharoplasts were the axostyles, united by their tips at the middle of the
elongated body. The cytoplasm was then divided between the axostyles,
664
FAMILY: TEICHOMOXADIDiE
and two flagellates formed. They often remained united by the tips of
the axostyles for some time before finally separating. In this process of
division, as the blepharoplasts separate from one another, they are at
Fig. 272. — Trichomonas oiigfttsf a, illustrating the Structure of the Flagellate
AXD the View that the Axostyle Splits Longitudinally during Division
(;< 1,450). (After Kofoid and Swezy, 1915.)
first connected by a paradesmose, as explained above, while passing from
each blepharoplast in another direction is a limb of the dividing axostyle.
As the blepharoplasts take up positions at opposite poles of the elongated
body of the flagellate the axostyle becomes completely divided, and as the
TRICHOMONAS MURIS
665
body of the flagellate elongates the two daughter axostyles finally occupy
a straight line between the two blepharoplasts. This is the line which
would be occupied by the paradesmose if it persisted, and this has given
rise to the view that the axostyles are really derived from the paradesmose,
and that the old axostyle had disappeared. The writer (1907) came to
the conclusion that the paradesmose had disappeared before this stage,
and that the structure uniting the blepharoplasts at the final stage of
division was formed by the longitudinally divided axostyles. This view
was supported by the observations of Kofoid and Swezy (1915a) on
Fig. 273. — Trichomonas anfjiista from the Frog, Bnna hoi/lei : Plasmodium Phase
WITH Eight Nuclei and Four Axostyles (x 2,175). (After Kofoid and
SwEZY, 1915.)
T. inuris, T. augustci, and other species (Fig. 272). Dobell(1909) came to
the conclusion, however, that in T. batrachorum of the frog the axostyles
of the daughter fiagellates arise from the paradesmose. On the other
hand, Kuczynski (1914), from a study of T. niuris and other species,
maintains that neither view is correct, and that the old axostyle disappears,
while new axostyles are formed as outgrowths from the blepharoplasts,
and arise like the new basal fibre. Martin and Robertson (1911), from a
study of T. eherthi of fowls, could arrive at no definite conclusions as to
what happened. Wenrich (1921), from a study of T. vuiris, finds himself
666 FAMILY: TRICHOMONADID^
in agreement with Kuczynski. The method of formation of the axostyle
evidently needs reinvestigation.
Kofoid and Swezy (1915a) have described a remarkable process of
multiple segmentation in T. muris, T. augusta, and other species (Fig. 273).
By repeated divisions of the nuclei and blepharoplasts, and formation of
new flagella and other structures, complex organisms are produced which
may have eight nuclei and sets of organs. By multiple segmentation,
eight daughter individuals are formed. These forms were not seen by the
writer in a prolonged study of many mice infected with T. ynuris, nor have
they been seen by other observers in those species of Trichomonas in
which Kofoid and Swezy claim that the process occurs.
It seems probable that T. muris becomes encysted in spherical
cysts about 6 to 8 microns in diameter. These forms were described by
the writer (1907). Within the cyst can be seen the nucleus, blepharoplast,
axostyle, membrane, and flagella of the flagellate. Kuczynski (1914)
states that both in the case of T. muris and T. cavice of the guinea-pig he
has seen such encysted forms in which the enclosed flagellates have double
sets of organs. It is often difficult to judge whether T. tnuris is encysted
or not. The flagellates have a habit of becoming perfectly spherical and
quiescent in j^assed faeces, but that such forms are not encysted can be
demonstrated by warming them on the warm stage, when they will be
seen to renew their activities and assume their usual form.
Wenrich (1921) believes that two species of Trichomonas occur in
mice. The large form, T. tnuris, varies in length from 8 to 20 microns
with an average of 12-9 microns. Its nucleus in division has six chromo-
somes. The smaller form, which is possibly T. parva of AlexeiefE, varies in
length from 6 to 9 microns. During division its nucleus has only three
chromosomes. The writer has, however, seen forms which have a length
of barely 3 microns. If Wenrich's statement regarding the difference in
the chromosome number is accepted, the two species must be recognized,
but further information is required before his view is finally adopted.
Trichomonas caviae Davaine, 1875. — This flagellate, first mentioned and
named by Davaine (1875), is very similar to T. muris, and often occurs in
large numbers in the caecum and large intestine of guinea-pigs. As already
remarked above, it can sometimes be seen to be invading the intestinal
wall in sections of the intestine fixed immediately after death. Whether
these lesions in which the flagellates occur are caused primarily by the
Trichomonas or not has yet to be determined. Like T. muris, with which,
indeed, it may be identical, T. cavice varies in length from about 3 to 20
microns (Fig. 274). In some infections the csecum is swarming with
large forms alone, while in others every transition in size between the
smallest and largest individuals can be traced.
TRICHOMONAS CAVI^
667
T. cavicB becomes encysted in spherical cysts about 7 microns in
diameter, as first noted by Galli-Valerio (1903), There does not appear to
be any multiplication within the cyst, which is probably purely protective.
Fig. 274. — Trichomonas cavice from Large Intestine of Guinea-Pig ( x 3,000).
(Original.)
1-8. Flagellates from one preparation, showing great variation in size.
9. Encysted form.
668 FAMILY: TRICHOMONADID^
The flagellate has been cultivated by Chatton (1920). He employed
a medium consisting of ordinary bouillon, to which had been added 1 c.c.
of blood to every 10 c.c. of bouillon. In this, T. cavice grew in association
with numerous bacteria. By subculture every three or four weeks the
cultures were maintained for a year, when they were lost owing to acci-
dental contamination with fungi. The culture apparently grew at any
temperature between 20° and 37° C, but they survived longer at the
lower temperature, when multiplication of the bacteria and flagellates
was less rapid than at higher temperatures. Though the cultures were
started from typical Trichomonas with undulating membrane, the flagel-
lates assumed the Eutrichomastix form in culture when the axoneme
bordering the membrane became a free flagellum. In attempts to rid the
cultures of bacteria, guinea-pigs were inoculated intraperitoneally with
culture. Six hours after, when the peritoneum was examined, the flagel-
lates had assumed the Trichomonas form again. Chatton believes that
Eutrichotnastix cavicB, which in natural infections is very frequently found
along with the T. cavice, is merely a form of this flagellate which it assumes
in media of low density.
Faust (1921a) has stated that the Trichomonas which occurs in guinea-
pigs in Pekin differs from those described from this animal elsewhere.
The size of the organism is given as 8 to 14 microns by 6-5 to 10 microns.
The protruding portion of the axostyle is said to be two-thirds the body
length. There are three anterior flagelia, which have a length over half
that of the body and a long posterior flagellum. On account of the
supposed difference from T. cavice, Faust proposes to call this form
T. flagelliphora. From the plate accompanying his description, which
the author says depicts characteristic specimens, the writer can find no
evidence that he is dealing with a species distinct from the ordinary form
which is common in guinea-pigs in other localities.
Other Species of Trichomonas.
A large number of other species of TrichoiHonas have been described,
and these have been studied especially by ITobell (1909), Alexeiefi' (1909-
1911), Kuczynski (1914), and Kofoid and Swezy (1915). They occur in a
variety of hosts, as summarized below, and many specific names have
been given, but it is clear that in most cases the evidence necessary for
the establishment of new species is wanting.
Mammals.— T. suis Gniby and Delafond (1843) (stomach of pig); T. talmi
Fonseca, 1915, three free flagelia (Talus novemcinctus, armadillo); T. ruminantium
Braune, 1913, three free flagelia (rumen of cattle). Fantham (1920) records this
form from the reticulum of the sheep and ox, and (1921) gives the name T. equi to
one in the horse. T. chagasi Haselmann and Fonseca, 1918, three free flagelia
{Cerodon rupestris); T.felis Da Cunha and Muniz, 1 922, four free flagelia (cat) : Brumpt
TRICHOMONAS OF OTHER ANIMALS
6G9
(1925) (cat and dog). Brnnipt (1909rt) noted a form in Macacus sinieus. Fantliam
(1925) records T. mysiromyis from the white-tailed rat {Mystromyft alhicaudatus).
Birds.— T. eberthi Martin and Robertson, 1911, three free flagella; and T.galli-
namm IVfartin and Robertson, 1911, fonr free flagella (caecum of fowls). T. columbo}
Rivolta, 1 878 (pigeons), and T. eolumbarum Prowazek and AragHfo ( 1 9:)9), are possibly
the same as T. columbw. Ratz (1913ff) observed a TrieJiomonas in the liver of a
pigeon, while Kotlan (1923) described Trichomonas eberthi and a new species, Tetra-
iriehomonas anatis, from the ciccum of ducks.
Lizards. — T. Ucertce Prowazek, 1904, three free
flagella [Lneerta sp. and other lizards); T. mabiiiw Dobell,
1910, llnce free flagella [Mabuia carinata); T. sp. Dobell,
1910, three free flagella {Ilemidaciylus leschencmlti); T. sp.
Wenyon, 1921, three free flagella {Agama stellio and
Lacerfa agilis); T. sp. Franchini, 1921 {Lacerta ocellata).
Snakes. — The writer has seen and cultivated a form
with three free flagella from Python molurus of India.
Tortoises. — T. brumpti K\exG'\eR,\Q\2, fonr free flagella
{Nicoris trijuga). It has been seen by the writer in other
tortoises {Testudo radiata, T. calcarata, and T.argentina).
Crocodiles. — A form identified as T. irrowazelci was
seen by Parisi (1910) in Crocodilus palustris.
Amphibia. — T. batrachorum Perty, 1852, three free
flagella (frogs, toads, and newts, etc.) (Fig. 275);
T. augusta Alexeieff, 1911, three free flagella (frogs,
toads, and newts, etc.); T . ptroumzehi Alexeieff, 1909, fonr
free flagella {Salamandra maculosa, Triton cristatus, Alytes
obstricans); T. tritotiis Alexeieff, 1911, three free flagella
(newts); T. mirabilis Kuczynski, 1918, three free flagella
{Bufo sp. of the Congo); Tetratrichomonas batrachorum
Escomel, 1925, four flagella {Telmatobitis gebsM, South
America). Exechlyga acuminata Stokes. 1884, is probably
T. batrachorum.
Fish. — T. legeri Alexeieff, 1910, three free flagella
{Box boops); T. prowazehi Alexeieff, 1910, four free
flagella {Box salpa); T. fp. Fantham, 1919 {Mugil capito).
Leeches. — T. sanguisugce Alexeieff, 1911, three free
flagella {Bccmopis sanguisuga); T. granulosa Alexeieff,
1911, three free flagella {Ecemopis sanguisuga); T. ninw
Icohl-yalimowi Yakimofl, 1917 {Luminatisturkestanensis).
Molluscs. — T. limacis Dujardin, 1841 (land snail, Limax agrestis).
Termites. — T. termitis Dogiol, 1916, four free flagella {Bhinotermes sp.); T. macro-
stoma Dogiel, 1916, four free flagella {Ilodotermes mossamhicus); T. dogieli Duboscq
and Grasse, 1923; three free flagella {('alotermes flavicollis); T. trypanoides Duboscq
and Grasse, 1924, four free flagella {L'd i<uil Hermes lucifugus); T. termopsidis Cleve-
land, 1925, four free flagella {Terniopsis iifradnisis).
Fig. 275. — Trichomonas
bat7'achorum of the
Frog, showing Para-
basal Body after
Fixation in Her-
mann's Fluid
( X 2,400). (After
Janicki, 1915.)
The axostyle is abnormal in
not being pointed at its
posterior end.
Invasion of the Blood-Stream by Trichomonas.
The observation of Pentimalli (1923) of Trichomonas in the human
blood-stream has been mentioned above (p. 653). Lanfranchi (1908)
670 FAMILY: TRICHOMONADID^
discovered a Trichofnonas in the blood of a pigeon. He claimed to have
inoculated it to rabbits and guinea-pigs. Martoglio (1917) discovered a
similar form which had four free flagella in the blood of fowls in Eritrea,
and proposed to place it in a new genus as Hcemotrichomonas gallinarum.
He also places in this genus as H. ophidium the Trichomonas discovered
by Plimmer (1912a) in the blood of snakes which had died in the Zoological
Gardens. Lanfranchi (1917) again refers to the form previously described
by him, and places it in Martoglio's genus as H. colmnbcB. These forms,
which occur in the blood, are almost certainly the result of invasion of the
vessels by intestinal flagellates, so that there is no justification for the
genus Hcemotrichomonas, as indeed Sangiorgi (1922), who saw a Tricho-
monas in the heart blood of a dead mouse, has pointed out. For some
reason which is not quite clear he believes that the flagellates seen by
Lanfranchi and Martoglio in the blood of the fowd and pigeon were not
Tricho?nonas, but Toxoplasma. As pointed out by Plimmer (1912), the
intestinal Trichomonas of amphibia are liable to invade the blood-stream
shortly before death. As noted above, the writer (1920) has seen T. hominis
in the tissues of the intestinal mucosa of human beings (Fig. 267).
Genus: Gigantomonas Dogiel, 1916.
This genus was established by Dogiel (1916) for a flagellate of the
intestine of the termite, Hodotermes mossambicus. The chief characters
are the size and the fact that one of the anterior flagella is thicker and
longer than the others.
Gigantomonas herculea Dogiel, 1916. — This is the only representative
of the genus. It measures from 60 to 75 microns in length and 30 to 35
microns in breadth. In structure it resembles a Trichomonas. It seems
possible that the flagellate represents an overgrown form of T. macrostoma ,
which Dogiel found in the same host.
Genus: Ditrichomonas Cutler, 1919.
This is a genus which was founded by Cutler (1919) to include a flagel-
late of termites which has essentially the same structure as Trichomonas
(Fig. 276). The single species, D. termitis, possesses only two anterior
flagella. It has two blepharoplasts, a nucleus, axostyle, and a basal fibre
running along the line of attachment of the undulating membrane, to
which the backwardly directed flagellum is attached. One of the ble-
pharoplasts, which Cutler terms the membrane granule, gives rise to the
basal fibre and the axoneme of the posterior flagellum. The other gives
origin to the axonemes of the two anterior flagella and the axostyle, as
well as a rod-shaped body called the parabasal. The latter structure may
be half the length of the body, and appears to be homologous with the
GENUS: EUTRICHOMASTIX
671
parabasal described by Janicki (1915) in Devescovina striata (Fig. 32) and
T. hatracJiorum (Fig. 275). Similar though smaller parabasal bodies have
been described in species of Tricliomonas. Thus, they were seen in
T. augusta (Fig. 67) by Alexeieff (1911A) and Kuczynski (1914). The
latter observer (1919) found the parabasal of constant occurrence in
T. mirahilis, which also possessed the basal fibre, so that the view of
Kofoid and Swezy (1915a) that the basal fibre of Trichotnonas is homo-
logous with the parabasal of other flagellates is untenable.
Fig. 216.-~Ditrichomonas termitis (x 940). (After Cutler, 1919.)
a. Usual type of flagellate, showing the deeply staining parabasal body.
b. Dividing form, showing two basal fibres and membranes and new axostyles developing.
c. Later division stage, showing duplication of all the structures.
Duboscq and Grasse (1924) describe as T. trypanoides a flagellate of
termites which has a single thick anterior flagellum. In certain individuals
it is represented by two, three, or four finer flagella (Fig. 268). They
include Cutler's flagellate in the genus Trichomonas, and propose for it
the name T richoftionas immsi, as the name T. termitis was employed by
Dogiel (1916) for another form in white ants.
Genus: Eutrichomastix Kofoid and Swezy, 1915.
This genus includes flagellates, which resemble Trichomonas except
for the absence of an undulating membrane, the posterior flagellum of
Trichomonas being represented by a trailing flagellum (Fig. 265, D). They
672 FAMILY: TEICHOMONADID^
have generally been known by the generic name Trichomastix, but owing
to the fact that Vollenhoevan had previously proposed this name for an
insect, Kofoid and Swezy (1915«) introduced the name Eutrichomastix.
It seems probable that, in some cases at least, the Eutrichotnastix forms are
merely Trichomonas in which the posterior flagellum has become free.
Chatton (1920), as noted above, found that in cultures the TricJiomonas
of the guinea-pig might assume either form. Eeichenow (1918, 19206)
noted that occasionally in lizards (Lacerta muralis and L. viridis) the blood-
stream was invaded by Eutrichoinastix from the intestine. In one case in
which a lizard had died of such an infection, at the time of death the only
forms present in the blood were of the Eutrichomastix type. On the next
day, however, in addition to these there were other flagellates of the
Trichomonas type present. Reichenow considers it possible that the
latter had been derived from the former, and that the two types may be
stages of one organism. In favour of this view is the well-known fact
that where flagellates of the Trichomonas type occur, very frequently
others of the Eutrichomastix form are present at the same time. Thus,
Dobell (1909) noted that T. batrachorum was often associated in the
frog's intestine with E. batrachorum, and a similar association was noted
by Prowazek (1904rt) in the case of lizards, and by Martin and Robertson
(1911) in fowls. On the other hand, it appears that sometimes the
flagellates are found in the Eutrichomastix form when Trichomonas is
absent, as in the case of E. serpentis seen in a snake by Dobell (1907a).
The writer has cultivated a Trichomonas of the tortoise {Testudo radiata),
the python {Python molurus), and the frog, and in these cases there was no
tendency for the flagellates to assume the Eutricho^nastix form. For the
present, therefore, it seems best to regard the flagellates as belonging to
distinct genera.
The flagellates of the genus Eutrichomastix have the same structure as
those of the genus Trichomonas, except that all the flagella, which are
four in number, are free, there being no undulating membrane. One of
the four flagella usually functions as a trailing flagellum.
It is unnecessary to give a detailed description of these flagellates,
which in their life-history and structure correspond very closely with the
various species of Trichomonas.
Haughwout and Horrilleno (1920) state that they saw a flagellate of
the Eutrichomastix type in a human stool in Manila. They refer to it as
Eutrichomastix sp. As only a single flagellate was seen, it is possible that
they were dealing with an altered Trichomonas.
E. lacertce was described by Prowazek from the intestine of species of
Lacerta. What is probably the same form occurs also in other lizards,
as noted by the writer (1921) in the case of L. agilis and Agatna stellio.
GENUS: EUTRICHOMASTIX
G73
\ ^#^
Fig. 277.
-Eutrichomastix lacertce in the Lizard and the Mite (x ca. 1,300).
(After Reichenow, 1920.)
I. Section ni intestine of lizard {Psammodromm hispanicm), sliowing wound of epithelium into
which a flagellate and bacteria have penetrated.
2 6. Flagellates from the blood of the lizard (Lacerta muralis).
7. Lymphocyte in the blood of the lizard with two ingested flagellates.
8. Large mononuclear cell from blood of lizard with ingested flagellates.
9. Large cell from body cavity of lizard with ingested flagellates.
10. Intestinal epithelial cell of the mite [Liponyssus saurarum) with included flagellates.
674 FAMILY: TRICHOMONADID^
E. batrachorum was described in detail by Dobell (1909) and E. serpentis
by Kofoid and Swezy (1915a). In the latter case, multiplication by binary
fission, as also by multiple segmentation with the production of eight
daughter individuals, is described, as noted by these authors in the case of
species of Trichomo?ias (p. 666). Dobell (1909) described the encysted
forms of E. hatrachormn and T. batrachorum as small ovoid bodies measuring
6'5 by 5 microns. They bear a striking resemblance to the cysts of species
of Embadomonas (Fig. 255, 14-19). The writer on one occasion obtained
a culture of an Embadomonas from the rectum of the common English
frog. The encysted forms corresponded very closely with those described
by Dobell, so that it seems very probable that the supposed cysts of
E. batrachorum and T, batrachorum actually belonged to undetected Emba-
domonas. Working with E. lacertce, Eeichenow (1918, 19206) noted that
the flagellate sometimes invaded the intestinal wall, body cavity, and even
the blood-stream of the lizards (Lacerta), and that the mites {Liponyssus
saurarum) which suck their blood become infected with the same flagellate
(Figs. 277, 458). In mites which have a second feed of blood, the flagel-
lates multiply rapidly and increase in size. They occur in numbers in large
vacuoles in the lining cells of the intestine. It was demonstrated by
Reichenow that the mites can remain infected for at least thirteen days,
and he succeeded in infecting a newly hatched Lacerta muralis by feeding it
on infected mites. In this connection it is interesting to note that Chatton
(1918a) obtained a culture of a species of Eutrichotnastix from the heart
blood of the North African gecko, Tarentola mauritanica. These cultures,
which contained bacteria in addition to flagellates, were maintained
indefinitely in subculture.
As in the case of the genus Trichomonas, numbers of species of Eutrichomastix
have been given names. Tricliomastix liominis, described by Chatterjee (1917rO»
is probably a small form of Chilomastix mesnili (see p. 3C6), and it seems probable
that some of the forms ascribed to the genus really belong to Trichomonas, the
posterior flagellum having become detached from the undulating membrane.
E. ruminantium (Braune, 1913) occurs in the rumen of cattle, while in fowls is
found E. galUnarum (Martin and Kobertson, 1911). Kotlan (1923) has described
this species from ducks, while Da Cunha and Muniz (1925) have named three species
from Brazilian birds. E. caviw (Grassi, 1881) is parasitic in the caecum of the
guinea-pig. Yakimoff, Wassilewsky, Korniloff, and Zwietkoff (1921) give the name
E. caviw var. rossica to a form seen by them in the guinea-pig, and which is un-
doubtedly identical with E. cavice. Fonseca (1916) records E. caviw from the wild
guinea-pig {Oavia aperea) and the aguti [Basyprocta aguti) of Brazil. In reptiles
there are several named species, all of which may belong to the form E. lacertce
(Biitschli, 1844), Avhich was redescribed by Prowazek (1904o) from species of Lacerta
and by Franchini (1921«) from Lacerta ocellata. E. vipcrw (Leger, 1904) occurs in
Vipera aspis and E. serpentis (Dobell, 1907) in Boa constrictor. E. mabuiw (Dobell,
1910) occurs in the Ceylon lizards, Hemidactylus leschenaulti and Mahuia carinata,
and E. saurii (Fonseca, 1917) in a Brazilian lizard, AmphisJ)W)ia sp. E. batrachorum
GENUS: JANICKIELLA
675
(Dobell, 1909) occurs in frogs, and probably other amphibia. From fish there have
been recorded E. motellce (AlexeieiS, 1910) from Motella tricerrata and E. snljjce
(Alexeieff, 1910) from Box salpa. In invertebrates are found E. trichopterce (Mackin-
non, 1910) from trichoiiteran larvae. It was recorded also by Mackinnon (1915) from
tipulid larvae (Fig. 278). Mackinnon (1913) discovered a flagellate in tipulid larvtie
which differed from EutricTiomastix trichopterce, which was also present, in that it
possessed four, instead of three, anterior flagella in addition to the trailing flagellum.
For this reason it was placed in a
new genus as Tetratriehomastix
parisii. In a later communication
Mackinnon (1915) described spheri-
cal cysts 4 to 5 microns in diameter.
The nucleus of the single flagellate
within the cyst divided once to form
two nuclei. These cysts belonged
either to T. parisii or E. trichop-
terce.
Genus: Janickiella Duboscq and
Grasse, 1923.
Duboscq and Grasse (1923)
created a new genus, Janickiella,
for a flagellate (J. grassii) which
they found in the intestine of
the termite, Calotermes flavi-
collis. In many respects it re-
sembles members of the genus
Eutrichomastix (Fig. 279, 3). It
is ovoid in shape, with a cyto-
stome and long protruding axo-
style. In front of the anteriorly
situated nucleus are two ble-
pharoplasts. One of these is
large and gives origin to a long,
thick, trailing flagellum and a
rod-like parabasal. The other
is small, and from it arise the
axonemes of three fine anteriorly
directed flagella and two rows
of granules. In addition to this flagellate, the termites harboured other
forms. Two of these were very small flagellates which resembled Trimitus
with two anterior flagella or Tricercomonas with three anterior flagella
(Fig. 279, 1-2). Duboscq and Grasse (1924rt), as a result of further observa-
tions, have reached the conclusion that the small flagellates are young
stages of the Eutrichomastix form, which is itself merely a young form of
D
Fig. 278. — Eutrichomastix trichopterce from
Intestine of Trichoptera Larvae ( x ca-
2,600). (After Mackinnon, 1910.)
A. Flagellate showing four anterior flagella, one of
which is a trailing flagellum ; nucleus is some-
what farther back than usual ; axostyle is showTi ,
but not the cytostome, which is sometimes
clearly visible. B. Dividing form.
C. Encysted form. D. Division within the cyst.
676 GENERA: TRICHOMITUS AND DEVESCOVINA
Trichomonas dogieli (Fig. 279, 4). Is it further suggested that other flagel-
lates, such as Joenia, may enter into the life-cycle of Janickiella grassii, and
that the flagellates belonging to the genera Enterotnonas and Tricercomonas
may be merely young forms of others. It has been pointed out above that
E. hominis is probably a young form of Chilomastix mesnili (see p. 307).
Fig. 279. — Flagellates from Intestine of the Termite, Calotermes flavicoUis,
ILLUSTRATING THE DEVELOPMENT OF Janichielld grassii (x ca. 2,000). (After
DUBOSCQ AND GrASSE, 1924.)
1. " Trimitus " stage with two anterior flagella. 3. " Eutrichomastix " stage.
2. " Tricercomonas " stage with three anterior flagella. 4. " Trichomonas '" stage.
Genus: Trichomitus Swezy, 1915.
This genus was founded by Swezy (1915fl) for a flagellate from am-
phibians. It resembles a member of the genus Trichomonas with three
flagella, but differs in the absence of an axo.style. It was named
Trichomitus parvus. Later Kofoid and Swezy (1919) placed in this genus as
T. termitidis a structurally similar but much larger flagellate found in the
termite, Termopsis angusticollis, of California. It varies in length from
75 to 150 microns. An elaborate system of fibres, called the neuromotor
GENERA: FOAINA AND RETORTAMONAS
677
system, is described in connection with the blepharoplasts and nucleus.
In addition to multiplication by binary fission, a process of multiple
fission is said to occur. As during division the nucleus behaves differently
from that of Trichomitus jyarvus, it is suggested that T. termitidis be
regarded as belonging to a sub-genus, Trichomitopsis.
In connection with this genus, it has to be remembered that the
detection of an axostyle in Trichornonas is not always a simple matter.
In any preparation containing large numbers of Trichomonas, a number
of forms ahvays occur in which an axostyle is not
visible. Furthermore, in the large overgrown
forms of T. vaginalis, the axostyle is frequently
quite obscured, so that it seems possible that
the forms included in the genus Trichomitus may
in reality belong to the genus Trichomonas.
Genus: Devescovina Foa, 1905.
This is a genus which was established by
Foa (1905) to include certain flagellates which
occur in the intestine of termites. The genus
is undoubtedly related to Eutrichomastix.
D. striata has been studied by Janicki (1911).
There are four flagella, three of which are
directed forwards, while one, which is much
longer than the others, acts as a trailing flagel-
lum (Fig. 32). There is a blepharoplast from
which the flagella arise, and behind it is the
nucleus, which appears to be embedded in the
axostyle. In relation to the nucleus and coiled
round the anterior part of the axostyle is an
elongate deeply staining body, the parabasal.
that Kofoid and Swezy (1915a) homologize the basal fibre of Trichojyionas.
Fig. 280.— Poamrt gracilis
FROM Intestine of the
Termite, Calotermes cas-
taneus (x 1,825). (After
Janicki, 1915.)
It is with this structure
Genus: Foaina Janicki, 1915.
This genus was created by Janicki (1915) to include a flagellate of
termites, which resembles Devescovina in many respects (Fig. 280). In
place of the long coiled parabasal there are two small parabasals.
Genus: Retortamonas Grassi, 1879.
Grassi (1879a) created three new genera: Monoeereomonas, Betortamonas, and
Schedoacercomonas. In the first genus he included intestinal flagellates of man,
guinea-pig, snake, frog, mouse, and lizard. It is probable that all these were
Trichomonas, and that Monoeereomonas is a synonym of Tricliomonas. The name
678
FAMILY: TRICHOMONADIDiE
has, however, been generally used for another group of flagellates owing to the fact
that Grassi (1881a) included in the genus a form which he called Monocercomonas
insectorum, a name which he regarded as including two flagellates previously named
by him (1879a) Schedoacercomonas gryllotalpce and S. melolonthct. Neither of these
is a Trichomonas, so that Grassi (188 1«) was quite wrong in placing them in his genus
Monocercomonas, which included a number of undoubted Trichomonas. In 1879,
however, he had given the name Betortomonas gryllotalpce to a flagellate of the mole
cricket, and as this name was placed before Schedoacercomonas gryllotalpw and
S. melolontha', both of which appear to belong to the same genus, the correct generic
name for these flagellates is Betortamonas, and not Monocercomonas. The question
was still further complicated by the fact that Grassi (1881a), without any apparent
reason, altered the name Betorta-
monas gryllotalpce to Plagiomonas
gryllotalpce, which is therefore
merely a synonym.
The flagellates of the
genus Retortmnonas are close-
ly allied to Eutrichomastix.
There are four flagella, one
of which is a trailing flagel-
lum. In the place of the
typical axostyle of Eutri-
chotnastix, there is a fibre
which stains deeply. In
many of the flagellates, how-
ever, such a fibre cannot be
distinguished, and they re-
semble Monadidee with four
flagella (see p. 308). The
first forms to be described
were Retortmnonas gryllotalpce
Grassi, 1879 (syns. Schedoacercomonas gryllotalpce Grassi, 1879; Monocer-
comonas insectorum Grassi, 1881, pp..; Plagiomonas gryllotalpce Grassi,
1881), of the mole cricket, Gryllotalpa sp., and R. jnelolonthce Grassi, 1897
(syns. S. melolonthce Grassi, 1879; M. insectorum, Grassi, 1881, p.p.) of
the cockchafer, Melolontha vulgaris. Parisi (1910) described as Tricho-
mastix orthopteronwi a similar form from the cockroach, while Jollos
(1911) gave the name Monocercomonas cetonice to one from larvse of Cetonia
sp. Hamburger (1912) also studied this flagellate. Mackinnon (1912)
observed a form in tipulid larvse, while Franya (1913) described forms from
Oryctes nasicornis, 0. grypus, and Phyllognatus silenus. Belar (1916) gave
a detailed account of the structure and division of R. orthopterorum. The
organism is pear-shaped as a rule, and measures 3 to 6 microns in length.
There is no cytostome (Fig. 281). Four flagella arise from the blepharo-
FiG.281. — Betortamonas orthopterorum {x 3,800).
(After Belak, 1916.)
1-2. Flagellates showing four flagella (one a trailing
flagellura), axostyle, nucleus, and blepharoplast.
3-5. Division stages.
GENUS: PROTRICHOMONAS 679
plast near the nucleus at the anterior end of the body. One of the flagella
functions as a trailing fiagellum. Arising in the blepharoplast and passing
through the body to its posterior end is an axostyle. This structure
cannot, however, be distinguished in all the forms. Furthermore, the
axostyle appears to differ from the corresponding structure in Trichomonas
and Eutrichomastix in that it stains deeply with iron hsematoxylin. It is
possibly not an axostyle at all in the strict meaning of the term.
Very frequently Retortamonas occurs in association with Polymastix,
which differs chiefly in its peculiarly ridged periplast. Mackinnon (1912)
noted that Polymastix not infrequently cast its periplast, with the result
that flagellates of the Fefortamonas type resulted.
Genus: Protrichomonas Alexeieff, 1911.
Very closely allied to Retortamonas is the genus Protrichomonas, which
was founded by Alexeieff (1911/0 for a flagellate which he discovered
Fig. 282.— (A) Protrichomonas tegeri (Alexeieff, 1910), from CEsophagus of
3IARINE Fish, Box salpa (x 1,500). (B) ProtricJiomonas anatis Kotlan, 1923,
IN Rectum of Duck (x 2,000). (A, after Alexeieff, 1910; B, after
KOTLAN, 1923.)
(1910) in the oesophagus of the marine fish, Box boops (Fig. 282). Alexeieff
(1910) noted that it had three anterior flagella of equal length arising from
a blepharoplast in front of the nucleus. A structure like an axostyle
passed backwards through the body from the blepharoplast. He named
the parasite provisionally Trichomonas (?) legeri, in spite of the fact
that there was no undulating membrane. Later (1911/0 he came to the
conclusion that it did not belong to the genus Trichomonas, and placed it
in a new genus, Protrichomonas.
Kotlan (1923) ascribed to this genus, under the name P. anatis, a
flagellate which he found in the intestine of ducks and other aquatic birds
680
GENUS: POLYMASTIX
{Nyroca ferruginea and FuUca atra). The flagellate has an ovoid body
measuring 10 to 16 microns by 4 to 6 microns. There are three anterior
flagella as long as, or longer than, the body. They arise from an anteriorly
placed blepharoplast. The nucleus is situated near the centre of the body.
Arising from the blepharoplast, and passing backwards through the
cytoplasm, are two fibres. They pass one on each side of the nucleus, and
then run close together to the posterior extremity of the body, through
which they protrude as a pointed body. From the figures, these two fibres
appear as if they might be the margins of an axostyle.
Fig. 283. — Polymastix melolonthce from Gut of Insect Larv.e (x 4,000).
(After Mackinnon, 1913.)
1. Ordinary type of flagellate. 2. Dividing form.
Genus: Polymastix Biitschli, 1884.
Blitschli established this genus for a flagellate to which Grassi (1881a)
has referred as Trichomonas melolonthce from the intestine of the larva of
the cockchafer (Melolontha). Similar forms were discovered by Hamburger
(1911) in larvae of Cetonia sp., Mackinnon (1912, 1913) in larvae of Tipida
sp., and Franca (1913) in larvae of Oryctes nasicornis. The form studied by
GENERA: HEXAMASTIX AND COCHLOSOMA 681
Mackinnon appears to be the same as P. melolonthcB of the cockchafer
(Fig. 283). The body is pear-shaped, with a rounded anterior and pointed
posterior end, which may be forked or otherwise deformed. There are
four flagella arising in pairs from two blepharoplasts at the anterior end
of the flagellate. Between the blepharoplasts, according to Mackinnon,
there is a cytostome. The nucleus lies just behind the blepharoplasts,
and it is spherical or pear-shaped. It contains a large karyosome. A
characteristic feature of the flagellate is the presence of a definite rigid
periplast, which is raised into ridges or folds which run in a more or less
longitudinal direction. An axostyle is present, but is not always well
developed. The flagellate multiplies in a somewhat curious manner.
The karyosome becomes dumb-bell-shaped and then divided, and this is
followed by division of the nucleus. One nucleus, together with one of the
blepharoplasts and its two flagella, and part of certain granules which lie
just anterior to the nucleus, become gradually transferred to the posterior
end of the organism which elongates. The body is then divided by
constriction across the middle. This form is of interest in that it shows
features which characterize some of the highly complicated forms included
in the order Hypermastigida, such as Lojphomonas hlattanmi, a flagellate
which occurs in the intestine of the cockroach (Fig. 286). The mode of
division of L, hlattarum, is very similar to that of Polymastix melolo7ithce,
while the superficial periplast may show longitudinal markings.
Genus: Hexamastix Alexeieff, 1912.
This genus was created by Alexeieff (19126) for a flagellate of the
intestine of the newt, Triton tceniafiis. The flagellate resembles in all
essential respects a member of the genus Eutricho-
iiiastix, except that there are six flagella. It was
first placed by Alexeieff (1911) in the genus Poly-
mastix, from which he removed it in 1912. It may
be related to the forms of Tricliomonas with five
anterior flagella.
Genus: Cochlosoma Kotlan, 1923.
This genus was created by Kotlan (1923) for an ^ ^„, ^ „
., „ „ • 1 • n 11 • • i- Fig. 284. — Cochlosoma
ovoid flagellate with six flagella arising from a a««iw Kotlan 1923
blepharoplast at the anterior end of the body from the Cecum op
(Fig. 284). Behind the blepharoplast was a single the Duck (x 2,000).
nucleus, while two fibres arising from the blepharo- '^ J7^^ otlan,
plast passed backwards through the cytoplasm, one
on each side of the nucleus, to the posterior end of the body, through
which they protruded. The characteristic feature of the flagellate,
682
FAMILY: DINENYMPHID^
however, was the presence on one face of the anterior region of the body
of a circular depressed area, which resembled in some respects the
sucking disc of species of Giardia. There is a single species, Cochlosoma
anatis, which occurs in the intestine of ducks. The large forms measured
10 to 12 microns by 6 to 7 microns, while smaller forms were 5 to 9 microns
by 3 to 6 microns. The flagella, which ap-
peared to vary in number, but of which there
were usually about six, were directed back-
wards over the body.
10. Family: DINENYMPHID^ Grassi, 1911.
Amongst the numerous remarkable para-
sitic flagellates which occur in termites is a
form which was placed in a separate family,
the Dinenymphidse, by Grassi to include
Dinenympha gracilis Leidy, 1877 (Fig. 285).
There is a single nucleus, a structure like
an axostyle, and several flagella. The last
arise from the anterior end of the body, are
all directed backwards, and are attached to
ridges producing an appearance of a series
of undulating membranes which take a spiral
course over the body. This flagellate evidently
has affinities with Trichomonas, and forms
a connecting link with the Polymonadida.
Koidzumi (1921), who has named a number
of new species, believes that the structure
resembling the axostyle is in reality an
elongate blepharoplast for the numerous
flagella, as he could detect no separate
blepharoplasts in the forms he examined. Comes (1912) believes that
D. gracilis reproduces by multiple segmentation. .
2. Order : HYPERMASTIG-IDA.
This order ( = Hypermastigina Grassi, 1911) includes a number of very
complicated flagellates which are parasitic chiefly in the intestine of white
ants (termites). There is a single nucleus and numerous flagella which arise
from as many blepharoplasts. Axostyles and parabasal bodies may be
present. Lophomonas hlattarum Stein, 1860, occurs in the intestine of the
cockroach. It is pear-shaped and possesses a single nucleus, in front of
which are two groups of blepharoplasts, from each of which axonemes, giving
rise to a tuft of flagella, orginate. An axostyle passes backwards from the
Fig. 285. — Dinenympha gra-
cilis FROM THE Intestine
OF Termes lucifugus
(x 1,000). (After
ZULUETA, 1915.)
The flagellate possesses a single
axostyle and nucleus, and a
series of spirally arranged mem -
branes with attached flagella.
ORDER: HYPERMASTIGIDA
683
Fig. 286. — Various Hypermastigida. (1, after Janicki, 1915; 2 and 5, after
GrASSI AND SaNDIAS, 1893; 3, AFTER BuTSCHLI, 1889; 4, AFTER KOIDZUMI, 1921.)
1. Lophomonas blaltarum of the cockroach (X 2,900).
2. Trichonympha agilis of termites ( x ca. 300).
.•?. Joenia annectens of termites (x ca. 300).
4. Teratonympha mirahilis of termites ( x en- 300).
5. Spirotrichonympha flagellata of termites ( x ca. 300).
684 OEDER: DIPLOMONADIDA
blepharoplasts to the posterior end of the body, including in its course the
nucleus, associated with which is a parabasal body. The Hypermastigida
are subdivided into a number of families and genera, including the Tricho-
nymphidse, Leidy, 1877, which have since been studied by Grassi (1917),
Kofoid and Swezy (1919), Koidzumi (1921), and others (Fig. 286).
3. Order : CYSTOFLAGELLATA Haeckel, 1873.
This order includes certain marine Protozoa, of which Noctiluca miliaris,
a phosphorescence-producing organism, is the best known. The body is
spherical, and may reach a diameter of over 1,000 microns. It has a
groove leading to the cytostome, in front of which is a thick tentacle,
with a length equal to half the diameter of the body, and a single
fiagellum. Reproduction is by binary fission or bud formation.
B. Diplozoic Forms.
4. Order : DIPLOMONADIDA.
The flagellates belonging to this order ( = Diplozoa Hartmann and
Chagas, 1910) differ from all others in that the nucleus and other organs
are duplicated, so that the body has a bilateral symmetry. They may
be supposed to have originated from certain uninucleate Protomonadida,
which have commenced a division process that has been arrested before
division of the body has taken place. The order contains the three genera :
Hexamita, Giardia, and Trepo^nonas.
Genus: Hexamita Dujardin, 1841.
The flagellates of this genus have pear-shaped bodies provided with
six anteriorly directed flagella, and two which arise from the posterior end.
There are two nuclei at the anterior end of the body. The genus was
founded by Dujardin (1841) to include three species, two of which occurred
in stagnant water and one in the intestine and pectoral cavity of frogs and
newts. He described the organisms as having pear-shaped bodies with
four anterior and two posterior flagella, hence the name Hexamita. It
appears that H. inflata of stagnant water is the type species of this genus,
though Dujardin placed in the same genus, H. intestinalis, the parasitic
form (Fig. 287). It is now known that the latter, as pointed out by
Grassi (1879) for the form in the frog, in addition to the two posterior
flagella, has six anterior ones, so that Dujardin evidently overlooked two
of the latter. Dobell (1909) points out that there is little doubt that
Dujardin was observing the eight-flagellate parasite, only six of the flagella
of which he was able to count. If he made this error over the intestinal
form, it is evident he was equally liable to make the same mistake as regards
the type species, H. inflata, of stagnant water, for he places them in the
GENUS: HEXAMITA
685
same genus. It is now known that the forms in stagnant water likewise
have two posterior flagella, as well as six anterior ones, so that it seems
evident the name Hexamita must be employed for these flagellates. Dobell
(1909), though admitting that Dujardin overlooked two flagella in the
intestinal form, apparently thinks he may not have done so in the case of
the type species, H. inflata, though both were described at the same time
and were regarded as having the same number of flagella. Dobell therefore
adopts for the parasite of frogs the name Octomitus, proposed by Prowazek
Fig.
587. — Hexamita intestinalis from the Eectum of the Fkog ( x 5,000).
(Original.)
1. Ventral view of living flagellate.
3. Appearance in stained film.
4-5. Arrangement of nuclei and blepharoplasts as
after exposure to osmic acid vapour and drj':
G. Binucleated cyst.
2. Side view of living flagellate.
seen in flagellates stained by Giemsa stain
ng-
7. Cyst after division of two nuclei.
(1904f/). It seems to the writer that if it be accepted that Dujardin
overlooked flagella in the intestinal form, as he undoubtedly did, it must
be assumed he did so in the free-living form also. Klebs (1892), who first
realized that the intestinal form had eight flagella, described as Urophagus
rostratus a free-living form of similar structure, but which was said to
possess a cytostome at the posterior end of the body. As pointed out by
Alexeieff (1910), it seems very doubtful if he was correct in supposing a
686
ORDEE: DIPLOMONADIDA
cytostome to exist in this remarkable position. It seems more probable
that he was observing species of Hexamita, in which had occurred some
deformity of this part of the body, which is known to be very metabolic.
Moroff (1903) proposed to employ Kleb's name, Urophagus, for these
flagellates, owing to the uncertainty as regards the flagellates which
Dujardin named Hexamita. There seems to be no doubt, however, that
Dujardin was actually dealing with forms which are known to possess
Fig. 288. — Hexamitus muris from the Intestine of the Mouse {xca. 3,000).
(After Wen yon, 1907.)
1. Ordinary free form. 2-4. Dividing forms.
5-6. Encysted forms, showing division of nuclei.
eight flagella, so that there is no reason why his name Hexamita should
not be employed.
Hexamita muris (Grassi, 1881). — This species was first seen by Grassi as
a parasite of the intestine of mice and other small rodents. It was named
by him Dicercomonas tnuris. What is probably the same form was seen
by Prowazek (1904a) in rats, and named Octomitus intestinalis. Lavier
(19216) records the flagellate from the field vole, Microtus arvalis. The
organism was studied by the writer (1907). It has a rounded anterior
GENUS: HEXAMITA 687
and a pointed posterior end, the latter being subject to changes in shape.
In the small intestine of mice, the forms seen are 4 to 7 microns in length
by 2 to 3 microns in breath. In the caecum, longer and broader forms
occur, which may measure as much as 10 microns by 5 or 6 microns. The
latter may be adult forms of those found higher up in the intestine. From
the anterior end arise six flagella in two groups of three (Fig. 288, i).
From the posterior end arise two flagella. In stained films it will be seen
that the axonemes of the anterior flagella arise from two closely applied
granules, each of which appears to be a compound structure composed of
four blepharoplasts. From each granule there passes backwards a band-
like structure, the axoneme, which is continued into a posterior flagellum.
The axonemes of Hexamita are often referred to as axostyles, but there
seems no reason to suppose that they are homologous with the axostyle
of Trichomonas. In Hexmnita, the axonemes usually stain deeply, while
in TricJiomonas the axostyle does not readily stain. It has been suggested
by Kofoid and Swezy (1915a) that the axostyle of TricJiomonas represents
the axoneme of a backwardly directed flagellum, as in Hexamita. At the
anterior end of the body of H. muris, and just behind the blepharoplasts,
are two nuclei, between which the axonemes pass. Very frequently the
nuclei, blepharoplasts, and anterior parts of the axoneme stain as a single
compact and lobed mass, so that there is difficulty in distinguishing the
separate parts {cf. Fig. 287).
Multiplication of H. muris takes place by longitudinal division
(Fig. 288, 2-4). There is division of the blepharoplasts and nuclei, and
with it division of the axonemes, so that there are produced rounded
bodies with four nuclei and four axonemes. Presumably, by division
of the body into two parts, two daughter individuals, each with two
nuclei and two axonemes, are formed. Dobell (1909) has expressed it
as his opinion that the division stages of H. muris, figured by Foa (1904)
and by the writer (1907), were degenerate and fused forms which have
nothing whatever to do with division. This is certainly not the case.
Very similar division forms have been seen by Alexeieff (1908) and Swezy
(1915) in species of Hexamita from amphibia (Fig. 290).
The encysted stages of H. muris also occur, and can be found in the
caecum. These are elongate bodies with rounded ends (Fig. 288, 5-6).
They measure 6 to 7 microns in length by 3 to 4 microns in breadth. In
stained films the cyst can be seen to contain a single flagellate. In some
cysts, nuclear division has taken place, so that four nuclei are present.
If faeces of mice which are known to contain H. muris are diluted
with water, cultures of this flagellate may be obtained. This seems to
suggest that the forms which are found in stagnant water may actually be the
same species as those which live in the intestine of amphibia and rodents.
688
ORDER: DIPLOMOXADIDA
Supposed Hexamita of Man.
Chalmers and Pekkola (1916) have recorded as Octomifus hominis a
flagellate found by them in the human intestine in the Sudan (Fig. 289, 6.)
Fig. 289. — ■Trichomonas with Four and Five Flagella from a Film supposed
TO SHOW Hexamita hominis (x 3,600). (1-5, Original; 6, after Chalmers
AND Pekkola, 1916.)
1-5. Trichomoiuis with long drawii-ont axostyle.
6. Chalmers and Pekkola's drawing of Hexamita hominis.
As this form possesses a single nucleus, and does not have the structure of
Octomitus {Hexamita), doubts as to its validity were raised by Kofoid and
GENUS: HEXAMITA 689
Swezy (19216), wlio proposed establishing for it a new genus, Ditrichomastix,
and by Dobell and O'Connor (1921), who suggested that it was possibly a
dividing form of Tncercomonas intestinalis. Prom an examination of the
original film, the writer is able to state that the supposed Hexamita is a
Trichomonas. As is usual in a film, it is not possible to detect the complete
structure in every flagellate, but there is no doubt that the infection is one
of Trichotnonas, and no other flagellate (Fig. 289, 1-5). The majority of
forms in which the anterior flagella can be counted have four, a few have
five, while others have a smaller number. The protruding portion of the
axostyle in many is very long, while the basal fibre in some appears to be
continuous with the posterior flagellum. In no case were six anterior
flagella present, and it seems probable that some at least of the anterior
flagella depicted by Chalmers and Pekkola were merely fibres in the
medium.
Other Species of Hexamita.
According to Dobell (1909), the first observer to see a flagellate belong-
ing to this genus was Ehrenberg (1838), who named a form seen by him in
frogs, Bodo intestinalis. Dujardin (1841) named it Hexainita intestinalis
and described two other species which he saw in stagnant water, H. no-
dulosa and H. inflata. Biitschli (1878) united the two latter forms under
the name H. inflata, and called the parasitic one H. intestinalis. Grassi
(1879) referred to the form in the frog as Monomorphus ranarum. The
form described by Prowazek (1904rt) as Octomitus intestinalis from the
intestine of rats is certainly identical with H. ?nuris of the mouse, while
0. dujardini, described by Dobell (1909), from frogs and toads, is H. intes-
tinalis. Moroff (1903) described a species of Hexamita from the rainbow
trout. He regarded it as identical with the parasite of frogs and toads.
AlexeiefE (1910) observed a form in the fish, Motella tricirrata and M. mus-
tela, and (1911) another in species of Triton and axolotl. These were all
regarded as identical with H. intestinalis of the frog. H. parva is the
name given to a form seen by Alexeiefl (1912c) in the Ceylon tortoise,
Nicoria trijuga. The writer has seen this or a similar form in Testudo
radiata, T. calcarata, and T. argentina, and another in Python molurus.
Mackinnon (1912) saw a form in the intestine of tipulid larvae. Swezy
(1915), who has given the most detailed account of the structure and
division of these flagellates, described two new species {H. ovata and
H. batrachorum), both from the intestine of amphibia (Fig. 290). Escomel
(1925) gave the name H. brmnpti to a form found in the South American
batrachians, Telmatobius escomeli and T.gehshi. Belar (1916) described a
species {H . periplanetce) from the cockroach. Da Cunha and Muniz (1922),
H. avium from Brazilian birds, and Kotlan (1923), H. intestinalis from
the duck. Noller and Buttgereit (1923) recorded H. columbce from the
I. 44
690
OKDER: DIPLOMONADIDA
pigeon, and Da Ciinlia and Miiniz (1925) H. acuminata and H. elongata
from other birds of Brazil. Moore observed a flagellate in large numbers
in the intestine of North American trout. She at first regarded it as
a species of Giardia, and suggested the name G. salmonis. Later, both
YiG. 290. — Hexamita ovata of the Amphibian, Diemyctylus torosus, showing
Structure and Method of Division (x 2,583). (After Swezy, 1915.)
1. Normal flagellate, showing two nuclei, blepharoialasts, and axonemes of jiosterior flagella.
2. Early division stage: each nucleus has apical daughter blepharoplasts connected by a para-
desmose; new axonemes and posterior flagella have developed; the chromatin is in the form
of a spireme.
3. Later division stage: each nucleus has two groups of two daughter chromosomes; the para-
desmose is still present.
4. Still later stage: the daughter nuclei are reconstructed, and the paradesmose has disappeared.
r>. stage just prior to division of the body. 6. Multiple division form.
Moore (1923) and Davis (1923) studied the flagellate in trout {Salvelinus
fontinalis and Sahno shasta), and found that it in reality belonged to the
genus Hexamita. They concluded that it invaded the intestinal cells,
but the figures of the intracellular stages given by Davis are unconvincing.
GENUS: GIARDIA 691
Schmidt (1920) gave the name Octomitus intestinalis truttce to a form from
the intestine and gall bladder of European Salmonidae.
Alexeiefi (1917, 1917o) placed the forms seen by him (1910, 1912c) in
the tortoise and fish in a new genus, Octomastix, as 0. parvus and
0. motelloi. Grasse (1924), who has seen the tortoise flagellate in the
urinary bladder of Emys orbicularis, accepts this genus, the characters of
which differ from those of Hexamita in minor details only.
Invasion of the Blood by Hexamita.
Danilewsky (1889) first pointed out that the Hexamita of frogs was
able to invade the body cavity and even the blood-stream when the hosts
were in poor condition. Plimmer (1914, 1916) observed flagellates of the
Hexamita type in blood-films of tortoises (Nicoria 'punctularia and Cistudo
Carolina) which had died in the Zoological Gardens. Ponselle (1919)
again observed a species of Hexatnita in the blood of the edible frog, Rana
esculenta. The infection was readily transmitted to other frogs {R. tem-
poraria) by intraperitoneal inoculation of blood. Lavier and Galliard
(1925) have also seen the parasite in the blood of frogs, but were unable
to infect other frogs by inoculation.
Genus : Giardia Kunstler, 1882,
The members of this genus, which are all parasites of vertebrates with
the single exception of a form discovered by Thomson, J. G. (1925), in a
parasitic nematode {Vianella sp.), are characterized by the possession
of two nuclei and a bilaterally symmetrical body, which is rounded
anteriorly and tapered posteriorly. There is a dorsal convex surface and
a flattened ventral surface, on which is a well-developed sucking disc
with a raised edge circular in outline except at its posterior end, where
it is indented to form a notch. There are eight flagella, four of which
arise from the margin of the sucking disc, two from the posterior end of the
body, and two from a median position in the notch of the sucking disc.
The axonemes take a complicated course in the body.
A flagellate of this genus was first seen by Leeuwenhoek, who found
himself infected in 1681, as pointed out by Dobell (1920). The human
form was again seen by Lambl (1859), who called it Cercomonas intestinalis.
Grassi (1879a) established the genus Dicercomonas with two sub-genera,
Monomorphus (Hexamita) and Dimorphus (Giardia), but later (1881a) he
replaced Dimorphus by Megastoma. He regarded the form in man as
identical with others found by him in domestic animals. Blanchard (1888a)
proposed the name Lamhlia, which has been in general use for some
years. Kunstler (1882), however, had established the genus Giardia for
the flagellate seen by him in tadpoles, and there is little doubt that this
is the correct generic name for these organisms.
692
ORDER: DIPLOMONADIDA
Fig. 291. — Giardia intestinaUs from the Hvma'^ Intestine (x 5,000). (Original.)
1-4. Variations in size and shape of body. 5. Partial side view,
G-8. Variations in shape as seen in .-ide view.
GENUS: GIARDIA 693
The members of the genus, which vary little in minute structure,
are characterized by having a body which in shape resembles a longi-
tudinally split pear. The dorsal surface is convex, while the ventral one
is flat. The tapering posterior end or tail is a flexible structure which can
be turned up over the convex dorsal surface. The rest of the body is
rigid. On the ventral surface is the sucking disc, which is almost circular
in outline save for a posterior indentation or notch. It has a raised edge,
and by its means a flagellate is able to rest attached to the surface of epi-
thelial cells. The four pairs of flagella, which are symmetrically arranged,
originate in a series of blepharoplasts, the exact distribution of which has
been variously described by different observers. Two nuclei are present,
one lying on each side of the middle line of the body. There is no cyto-
stome, though some observers incorrectly refer to the sucking disc by
this name. Reproduction is by binary fission, which usually takes place
within an ovoid cyst. Occasionally, division occurs in the unencysted
condition. The body of a typical representative of the genus is distinctly
flattened dorso-ventrally, though the degree of convexity varies consider-
ably (Fig. 291). In some, which are probably the products of a recent
division, the body is not more arched than a watch-glass, while in others,
which are fully grown, it is almost hemispherical. When swimming in
fluid media, the flagellate sways from side to side as any flattened object
does when progressing through a liquid. The exact arrangement of the
flagella, blepharoplasts, and axonemes is difficult to elucidate; so much
so that the various observers who have undertaken the study of these
flagellates have given different accounts. The difficulty of interpretation
refers particularly to the region between the nuclei. The writer (1907)
described what he considered to be the arrangement in Giardia muris of
mice, and subsequent observations on G. intestinalis of man and other
forms convince him that his original description was substantially correct.
In the internuclear region the structures are so closely packed that the
separate blepharoplasts cannot be recognized except in specimens which
have been almost completely discoloured after staining with iron haema-
toxylin. Ordinary dried films stained by Giemsa stain not infrequently
show the granules and axonemes distinctly, especially in individuals which
have been flattened or even fortuitously dissected. Thin sections of the
intestine in which the flagellates have been cut often show the structures
more clearly than in flagellates mounted whole. Kofoid and Swezy
(1922), Simon (1922), and Hegner (1922) state that there is only a single
anterior blepharoplast on each side, but it appears from their figures that
the single elongate blepharoplast is really composed of at least two closely
applied blepharoplasts. They suppose that when two are present on each
side this is an indication of commencing division. The following, in the
694 OEDEK: DIPLOMONADIDA
writer's opinion, appears to be the arrangement : All the blepharoplasts and
axonemes have a superficial position on the ventral surface of the body.
Between the two oval nuclei, which are also near the ventral surface,
and slightly anterior to them, are four blepharoplasts arranged in pairs on
each side of the middle line of the body. The lateral blepharoplast of
each pair is slightly posterior to the one which is more centrally situated.
Unless the stain is sufiiciently extracted, each pair appears as a slightly
elongated single blepharoplast. From the lateral ones there arise two
axonemes, which pass forwards and, taking a curved course, cross one
another. They reach the border of the sucking disc, pass along it for some
distance, and finally enter flagella at points on its outer margin. From
the median blepharoplasts there also arise two axonemes, the so-called
axostyles, which pass backwards either on the surface of the body or just
beneath it to the posterior extremity, to be continued into the posterior
flagella. From these anterior central blepharoplasts there also arise two
fibres which pass forwards and towards one another. They unite after a
short course, and are continued as a single fibre, which is lost in the
cytoplasm of the anterior part of the body. The single fibre sometimes
appears as a group of radiating fibres. There is another pair of blepharo-
plasts centrally placed on the surface of the body in the hollow of
the notch in the sucking disc. From them arise two axonemes which
immediately enter flagella, which appear to arise directly from the
blepharoplasts.
It seems probable that there is still another pair of blepharoplasts,
from which the axonemes of the fourth pair of flagella originate. The
axonemes of these can be traced forwards along the margins of the notch
in the sucking disc, and can often be seen to terminate in a pair of
granules at the anterior end of the notch. These are not improbably the
blepharoplasts, which, however, appear to be connected with the anterior
lateral blepharoplasts by fine fibres. If the granules are not the blepharo-
plasts, then it must be assumed that the fibres which connect them with
the anterior lateral blepharoplasts are continuations of the axonemes, and
that they terminate in the blepharoplasts from which originate the
axonemes of the anterior crossed flagella. Not infrequently, granules may
be seen in stained specimens at the point of entry of the axonemes into
the flagella. This is particularly true of the posterior flagella, but these
granules probably indicate a thickening of the superficial layer of cyto-
plasm or periplast, and cannot be regarded as blepharoplasts. In the
arrangement, as just described, there can be distinguished a pair of lateral
crossed flagella, the axonemes of which arise from the anterior lateral
blepharoplasts; a pair of lateral uncrossed flagella with axonemes arising
from the same blepharoplasts, or more probably from others posterior to
GENUS: GIAEDIA 695
them; a pair of posterior flagella, the axonemes of which originate in the
anterior median blepharoplasts; and a pair of central flagella having
axonemes arising from the central blepharoplasts. The last pair of
flagella are thicker than the others, and usnally lie parallel to one another
on the surface of the body. As in the case of the axonemes of Hexamita,
those of the posterior flagella of Giardia are often regarded as axostyles,
but they cannot be homologized with the true axostyle of a TricJwynonas.
They undoubtedly represent the intracytoplasmic portions of the axial
filaments of the flagella, and are thus true axonemes. Simon (1922)
figures them as broad anteriorly at their attachment to the blepharoplasts
and tapering to a point posteriorly. Actually, they are of uniform
thickness throughout. Very frequently there occur two deeply-staining
curved or rounded bodies, which lie side by side just posterior to the
sucking disc and dorsal to the axonemes of the posterior flagella. They
are of largest size in the fully-grown individuals. These bodies have been
homologized by Kofoid and Christiansen (1915, 1915a, 19156) with the
parabasals of other flagellates, but there is little real evidence in support
of this view. The position of the blepharoplasts, as described above,
seems to the writer to be the true arrangement. Other observers have
considered that the axonemes of the eight flagella are all traceable to an
anterior group of four blepharoplasts, while Kofoid and Christiansen
(1915) in the case of G. muris and Kofoid and Swezy (1922) in the case
of G. intestinalis conclude that they all terminate in two, and suppose
that the presence of four in this region indicates the first stage in a division
process, each blepharoplast having divided to give rise to two. This
view is supported by Simon (1922) and Hegner (1922, 1922a). From the
appearances seen in G. intestinalis of man and G. muris of mice, as also
other forms, the writer believes that the undividing flagellate actually has
eight blepharoplasts, each of which gives origin to an axoneme of a
flagellum, as described above (Fig. 290). On a priori grounds alone, it is
highly probable that each flagellum has its own blepharoplast. This is
true of flagellates generally, and the members of the genus Giardia are
unlikely to be exceptions to the general rule. When the blepharoplasts
lie close together they often stain as a single body, so that the individual
blepharoplasts are difficult to detect.
Several observers have described two fibres connecting the anterior
lateral blepharoplasts with granules on the anterior extremity of the
nuclear membrane. Bensen (1908) figures them in G. muris, while Kofoid
and Christiansen (1915, 1915a) describe in G. 'muris and G. microti a
continuation of these fibres to the karyosomes of the nuclei. They are
also figured by Kofoid and Swezy (1922) in G. intestinalis. The writer
has seen in G. intestinalis and other forms actual fibres connecting the
696
ORDER: DIPLOMONADIDA
anterior lateral blepharoplasts with the granules on the nuclear membranes,
but he is not convinced that these fibres are continued to the karyosomcs
of the nuclei (Fig. 290).
The nuclei are two ovoid bodies which lie one on each side of the
middle line of the body near its ventral surface in the region of the sucking
disc. Each consists of a nuclear membrane, within which is a karyosome,
usually elongate in form. In what appear to be older individuals, several
separate chromatin masses united by a meshwork of fibres are present.
Fig. 292. — Giardia intestinalis : Various Stages of Division (x ca. 6,0UO).
(After Wenyon and O'Connor, 1917.)
On the anterior surface of the membrane adjacent to the blepharoplasts
is the granule mentioned above.
The flagellates multiply by a complicated process of longitudinal
division. So seldom are division stages of the free flagellates encountered
that most observers consider that the process occurs usually in the encysted
condition. It seems probable that changes in the nuclei and blepharo-
plasts take place preparatory to division, which is completed within the
cyst. The writer and O'Connor (1917) and Kofoid and Swezy (1922)
obtained preparations of G. intestinalis of man which showed undoubted
GENUS: GIARDIA
697
division forms of the unencysted flagellates, while Kofoid and Chris-
tiansen (1915, 1915a) described a similar process of binary fission in
G. microti and G. muris. It seems evident that binary fission may occur
in the free condition, though most usually it takes place within the cysts,
which are passed in large numbers in the fseces of infected individuals.
The flagellates undoubtedly multiply in the intestine, and unless binary
fission in the unencysted stage takes place more frequently than has beeu
observed, it has to be assumed that the two flagellates which have resulted
from division within the cyst are able to escape from the cyst without it
leaving the host, a condition of affairs which is quite exceptional for
intestinal Protozoa. In the case
of most Protozoa, the encysted
forms are destined to escape from
the host in order to ensure infec-
tion of others.
The stages of division of G. in-
testinalis are shown at Fig. 292.
It will be seen that the nuclei have
divided, and that there has been
duplication of the sucking disc
and various blepharoplasts, axo-
nemes, and flagella. The body
finally divides from before back-
wards. The details of the process
have not been worked out. It is
evident that division within the
cyst takes place in a similar
manner, but here the various du-
plicated structures are so crowded
together that it is impossible to
follow the details with any degree
of accuracy (Fig. 293).
The flagellate encysts in an ovoid cyst which forms first around the
anterior end of the body. It extends backwards and gradually encloses
the tail, which is finally retracted within the cyst. In recently encysted
individuals the flagella and tail may be seen to be moving within the cyst.
In stained specimens it will be noted that the two nuclei move to the
anterior end of the body, where they divide to form four spherical nuclei.
The fibre which forms the margin of the sucking disc becomes duplicated,
and the two are often coiled in various ways. The blepharoplasts have
each divided, and new axonemes and flagella have been developed, so that
the cyst encloses an ovoid mass of cytoplasm containing four nuclei and
Encysted
Intestine
Fig. 293. — Giardia intestinalis :
Forms from the Human
(x 3,000). (Original.)
1. Form with two nuclei.
2-.5. Forms with four terminal nuclei.
6. Form in which two of the nuclei have
migrated to the opposite pole and the
flagellate is dividing within the cyst.
698
ORDER: DIPLOMONADIDA
numerous fibres which are difficult to trace. One pair of nuclei moves to
the opposite end of the cytoplasmic body, which divides longitudinally
to form two flagellates. There is no evidence that two flagellates ever
become encysted in a common cyst, as maintained by Schaudinn (1903),
Bohne and Prowazek (1908), and Woodcock (1915). Hartmann's (1909)
opinion that autogamy occurs in the cyst is likewise unsupported by fact.
In the smaller flagellates, which are probably the youngest forms seen
in any infection, each nucleus has a single central karyosome (Fig. 294).
In the larger or older individuals the karyosome is replaced by a
number of granules distributed upon a meshw^ork. It is supposed by some
observers that the formation of these granules is a preparation for nuclear
division, and that ultimately eight chromosomes are formed. Rodenwaldt
(1912) described the nuclear division of G. intestinalis . He noted that
the nucleus of the free flagellate contained either a central karyosome or
Fig. 294. — Giardia intestinalis of Man, to illustrate the Growth of the Flagel-
late, FROM A Film in which dividing Forms were Present (x ca. 1,500).
(Original.)
eight separate bodies. When a flagellate with a nucleus of the latter
type encysted, the eight masses or chromosomes arranged themselves in
two longitudinal rows, while the granules on the anterior end of the nuclear
membrane divided into two. The nucleus then became constricted at its
centre, and finally divided, each daughter nucleus receiving four of the
chromosomes. If this be correct, it would appear that the division of the
single karyosome in the flagellate stage into eight masses represents the
commencement of division, which is completed after encystment has
taken place. Kofoid and Christiansen (1915, 1915a.) described binary
fission of G. muris and G. microti, and Kofoid and Swezy (1922) that of
G. intestinalis. The process bears a close resemblance to that seen in
G. intestinalis by the writer and O'Connor (1917). The details of the
nuclear division were studied by Kofoid and Christiansen (1915, 1915a),
by Boeck (1917), and by Kofoid and Swezy (1922). The resting nucleus
possesses a central karyosome. The first stage in division is supposed to
be the division of the single pair of anterior blepharoplasts to produce
GENUS: GIAEDIA
699
two pairs. The writer has already expressed it as his opinion that the
flagellate possesses two pairs of anterior blepharoplasts, and he believes
that when division is taking place there actually occur four pairs of
anterior blepharoplasts. Boeck describes the changes in the nucleus as
taking place in the following manner (Fig. 295) : The fibril, which is said
to connect the granule on the anterior end of the nuclear membrane with
the karyosonie, becomes extended to the opposite pole of the nucleus.
The karyosome then becomes more irregular in shape, and finally divided
into eight chromosomes. Meanwhile, the granule on the anterior end of
Fig. 295. — Mitotic Division of Nucleus of Giardia microti { x ca. 7,300). (After
BoECK, 1917.)
1. Ordinary resting nucleus with karyosome cormected with centrosome by a fibril.
2. Karyosome has elongated.
3. Elongated karyosome has split longitudinally, and each half is dividing into four chromosomes.
4. Division of each half of karyosome into four chromosomes is complete. The centrosome has
divided, and the two daughter centrosomes are connected by a fibre (paradesmose).
5. The eight chromosomes have united to form four double chromosomes at the equator of the
spmdle. The paradesmose is no longer visible.
6. The four double chromosomes have divided to form two groups of four which move towards
the poles of the spindle.
7. Division nearly completed; chromosomes fused. 8. Completed division.
the nuclear membrane has divided, and one half migrates over the surface
of the nuclear membrane to the opposite pole of the nucleus. It remains
connected with the other half, which retains its anterior position, by
a centrodesmose which lies on the surface of the nuclear membrane.
Between the two granules, which are functioning as centrosomes, a spindle
is formed within the nuclear membrane, and upon the fibres of the spindle
the eight chromosomes arrange themselves. The chromosomes are
described as four pairs of homologous chromosomes, and the individuals
of each pair become closely associated to form four double chromosomes
at the equator of the spindle. One chromosome of each pair now moves
700 ORDER: DIPLOMONADIDA
towards the anterior centrosome, while the others pass to the posterior
centrosome. The chromosomes of each group then fuse to form a karyo-
some, and this is followed by constriction and division of the nuclear
membrane, so that two nuclei are formed. In the writer's experience,
G. intestinalis of man frequently shows nuclear pictures, which correspond
with all the stages figured by Boeck (1917) for G. microti, with the exception
of the actual division of the nuclei. In such preparations, though very
large numbers of flagellates are present, none of them is actually dividing
in the free stage. If the various nuclear appearances undoubtedly
represent a mitotic division of the nuclei, as first pointed out for G. intes-
tinalis by Rodenwaldt (1912), one would expect to find more frequently a
corresponding number of flagellates with two pairs of nuclei and others
with their bodies actually in process of fission. It is possible, as Roden-
waldt maintains, that usually the nuclei of the free forms prepare for a
division which is completed in the cyst. In one case noted above, the
writer and O'Connor (1917) encountered numbers of free flagellates actually
in process of binary fission (Fig. 292), and similar forms have been de-
scribed by Kofoid and Swezy (1922), so that it has to be admitted that
division in the free state can take place.
Kofoid and Christiansen (1915, 1915cf) have described multinucleate
stages of G. ?nicroti and G. 7nuris. Both the free flagellates and. the
encysted forms are described as dividing by multiple segmentation. It
is remarkable that in some of the figures the two normal nuclei of the
free or encysted flagellates are in the position and possess the characters
they usually have, while the other supposed nuclei are smaller and have
a different appearance. It is difficult to understand how such a multi-
nucleate condition can have arisen if the two normal nuclei are still in
their usual situation, and are iinaltered in size and appearance. The
writer has seen very much vacuolated specimens of G. intestinalis having
at the centre of each vacuole a granule which might be mistaken for a
karyosome. It seems highly probable that it is structures such as these
which have been interpreted as nuclei. Similar multinucleate cysts of
G. intestinalis have been described by Kofoid and Swezy (1922). As many
as sixteen nuclei are said to be present. In no case was division of the
encysted form or daughter flagellates observed. Noc (1909a) gave an
illustrated description of what he considered to be multiple division of
G. intestinalis. It was supposed that after nuclear divisions a number of
minute flagellates were produced, but it is evident from his figures that
some of the forms depicted are not G. iyitestinalis, even if they are living
organisms.
Various species of Giardia have been described from man and animals,
but the specific characters are in most cases very ill-defined. Simon (1922)
GIARDIA INTESTINALIS 701
and Hegner (1922a) maintain that species may be distinguished by the
average dimensions of a number of individuals, and they illustrate in a
graphic manner the measurements which are necessary for identification
(Fig. 296).
Reuling and Rodenwaldt (1921) have attempted to revive the genus
Lamblia by suggesting that G. agilis, described by Kunstler (1882) from
tadpoles, differs sufficiently from the other forms to justify their in-
clusion in a separate genus, Lamblia. The tadpole parasite is a long
narrow organism with a small sucking disc (Fig. 298), while all other
forms rarely have a length as much as twice that of their breadth.
They believe that the generic name Giardia should be retained for the
narrow form, of which there is the one species, G. agilis Kunstler, 1882.
The broader forms, which include all the others, are to be placed in
Blanchard's genus, Lamblia, the type species being the human parasite,
L. intestinalis (Lambl, 1859). The authors seem inclined to this view
rather from a desire to retain the name Lamblia for the human parasite
than from conviction that the differences between the two forms are of a
generic value. It does not seem to the writer that matters are assisted in
any way by splitting into two sub-genera the very compact and uniform
genus, Giardia, merely because certain forms in the tadpole are narrower
than those in other animals. As pointed out by Hegner (1922), the
differences described are certainly not of generic value.
The various forms of Giardia which are known are invariably inhabi-
tants of the small intestine. In mammals they are to be found in the
upper parts of the small intestine and duodenum. When they occur
lower down, it is probable that their appearance is accidental. They can
be studied in sections of the intestine, and are often found in large numbers
in the tubules of the secreting glands, a fact which probably aft'ords an
explanation of the difficulty in getting rid of an infection in human beings
by the administration of intestinal disinfectants.
GIARDIA IN MAN.
Giardia intestinalis (Lambl, 1859). — As noted above, this flagellate was
named Cercomonas intestinaUs by Lambl (1859). Diesing (1851) had. liowever,
given this name to a flagellate which Ehrenberg had previously described as Bodo
intestinalis. As pointed out by Dobell (1909), Diesing was in error in so doing, as
Ehrenberg's flagellate was not a Cercomonas, but probably a Hexamita. Hence,
DobeU concludes that Lambl's specific name is still available for the human Giardia.
Kofoid (1920), believing that Lambl's name was not available, adopted Grassi's
name, and referred to the human form as 6. enterica Grassi, 1881 ; while Simon (1922)
states that Stiles has shown that the name G. enterica, which is in reality a synonym
of G. muris, cannot be employed, and proposes to adopt the name G. lamblia StUes,
which was put forward in a paper by Kofoid and Christiansen (1915). If the rules
of uomenclaturo are strictly adhered to, Lambl's specific name intestinalis cannot
702 ORDEE: DIPLOMONADIDA
be employed for the human Oiardia, since the name Cercomonas intestinalis was
already given to another flagellate (Hexamita of frogs, Diesing, 1851), when Lambl
used it in 1859 for the Giardia of man. As Boeck and Stiles (1923) point out, it
appears that the correct name for the human Giardia will have to be Giardia lamblia
Stiles, 1915. The better-known name, G. intestinalis, will, however, be retained here.
This species is a common intestinal parasite of man, and has a workl-
wide distribution. It lives in the upper parts of the small intestine, thus
differing from the other intestinal Protozoa of man, which are inhabi-
tants of the large intestine, with the possible exception of the coccidia.
Mliller (1889) discovered it in the duodenum of one case at autopsy, an
observation w^hich was repeated by Moritz and Holzl (1892). Cohnheim
(1903, 1909) and Zabel (1901-1910) recorded what was probably G. intes-
tinalis in stomach contents in cases of carcinoma. Boyd (1921) in Canada
obtained large numbers of the flagellates by means of a duodenal tube
passed on a convalescent typhoid case. A similar observation has been
made by McGill (1922), Knighton (1922), Simon (1922), Silverman (1923),
and Libert and Lavier (1923). As the bile obtained by the operation
described as duodeno-biliary drainage contains large numbers of the
organisms, it is concluded that they have actually invaded the bile ducts
and gall bladder. This was confirmed by an observation of Smithies
(quoted by Knighton), wdio found the flagellates in the gall bladder at
surgical operation. Westphal and Georgi (1923) also record the discovery of
Giardia in a gall bladder opened at operation. The writer has seen G. i?ites-
tinalis in sections of the small intestine from fatal cases of typhus fever.
The general shape of the flagellate and arrangement of the various
organs conform with the description given above (Figs. 291, 294). The
length of the body, not including the tail flagella, varies from 10 to 18
microns, though longer and shorter forms sometimes occur. The breadth,
which is a little more than half the length of the body, is subject to
greater variations than the length. Simon (1922) gives the following
measurements in microns for the flagellate: length 9-25 to 20-25 (average
13-7), breadth 5-0 to 10-25 (average 7-46).
Encysted forms are very commonly seen in the stools of infected
individuals. It is only in diarrhoeic conditions that the free forms are
seen. The cysts are ovoid bodies varying in length from 8 to 14 microns
(Fig. 293). Simon (1921) gives for the length 8-0 to 14-0 (average 10-7)
microns, and for the breadth 6-0 to 10-0 (average 7-47) microns. In the
fresh condition the cysts are quite transparent. With careful observa-
tion it is usually possible to distinguish the nuclei, the central axonemes,
and some of the flagella. The nuclei are situated at the anterior end
of the cyst, and each of these may have divided to form a total of
four small spherical nuclei. In iodine solution or in stained films the
GIAEDIA INTESTINALIS 703
various structures are more readily detected (Plate II., 23, p. 250). Within
the cyst the flagellate ultimately divides into two, but the process of
division is an exceedingly complicated one on account of the numerous
structures present. The cysts, like those of other intestinal Protozoa,
vary in their permeability to stains and other reagents. On this account,
good pictures of the cyst content are only obtained in the case of permeable
cysts. As pointed out above, G. intestinalis is sometimes seen dividing
in the free condition. As the flagellate possesses no cytostome and the
cytoplasm is free from food vacuoles, it is evident that nourishment is
effected by the absorption of fluid nutriment through the surface of its
body. Bacteria are sometimes seen in evidently degenerate forms.
Pathogenicity. — The question of the pathogenicity of G. intestinalis,
as that of other intestinal flagellates of man, has given rise to considerable
controversv. It is an undoubted fact that the flagellates are rarely seen,
except in diarrhoeic conditions, but that they are often present in normal
individuals can be demonstrated by the finding of cysts in the formed stool.
The number of cysts present in the stools are subject to fluctuations. They
may be absent from the stool for varying periods, and reappear again later.
Certain individuals are known to have remained infected for many years
without showing any symptoms, but this fact cannot be raised as an
argument against the occasional pathogenicity of the flagellate, as the
same condition frequently occurs in infections with Entxmioeha histolytica.
In some cases of Giardia infection there occur periodic attacks of diarrhoea
associated with the passage of large quantities of clear mucus, in which
enormous numbers of free flagellates occur. It is difficult to avoid the
impression that this mucus has been produced at that part of the intestine
where the flagellates are most numerous, and is the result of irritation set
up by their presence. It is possible that in certain individuals which are
more susceptible than others, the attacks of diarrhoea correspond with
periods of active multiplication of the flagellate.
In the case of animals, as, for instance, the rabbit, which is commonly
infected with a species of Giardia, sections of the small intestine may show
all the glands packed with organisms either free in the lumen of the duct
or applied to the surface of the cells. When such a condition exists in
man, it would not be surprising if the gland cells were irritated by the
presence of such large numbers of flagellates. There does not appear to
be any tendency for the flagellates to cause ulceration or to penetrate the
epithelial surface. The majority of observers believe that G. intestinalis
may give rise to intestinal disorders, but the absolute proof of this is
difiicult to obtain. The diarrhoeic condition associated with an infection
is often spoken of as dysentery, but actually true dysentery does not
result. Though quantities of mucus may be present and the stools
704 ORDEE: DIPLOMONADIDA
diarrhoeic in form, blood never occurs in pure Giardia infections. Westphal
and Georgi (1923) have noted that in certain chronic disorders associated
with jaundice the flagellates were present in large numbers in the duo-
denum, and in one case their presence in the gall bladder was demon-
strated at operation. They believe that a definite inflammatory condition
of the bile duct and gall bladder is set up by their presence.
Animal Experiments. — The fact that rats and mice are often infected
with Giardia led Grassi (1879-1888) to express the opinion that human
beings become infected from these animals. He claimed to have infected
himself by means of the intestinal contents of rats, Perroncito (1901)
stated that he had infected mice by feeding them with material from
human cases, and Fantham and Porter (1916) made similar claims. The
fact that mice are often naturally infected with Giardia renders such
experiments very doubtful. Even prolonged examination of the faeces
of the animals before the experiment may fail to exclude the natural
infection. More recently, Deschiens (1921) has studied the question more
fully. He was convinced that he had succeeded in infecting animals by
means of human material. Thus, two cats were infected from human
beings and two others from mice. All four animals developed a dysen-
teric condition which w^as fatal in three of the cases. Five mice naturally
infected with Giardia failed to react to the human form, whereas five
mice which were not naturally infected developed an infection with
dysenteric symptoms, which proved fatal in three. The flagellates which
appeared in the cats after ingestion of human material were said to be
identical with those occurring after infection from mice. From these
results Deschiens was inclined to regard G. intestinalis of man and G. muris
of rats and mice as identical. Furthermore, he is convinced of the
pathogenic role of these flagellates. It should be remembered, however,
that both cats and mice are often found naturally infected.
Simon (1922) obtained white rats and wild rats which were free from
Giardia infections. Attempts to infect them with G. intestinalis of man
failed entirely, though they were readily infected with Giardia of mice.
For this reason, and on morphological grounds, he concludes that the
human infection is not contracted from rodents, but passes directly from
man to man. The writer has attempted on several occasions to infect
mice with the cysts of the human form, but has never succeeded. Quite
recently he has conducted a carefully controlled experiment with four
kittens. One of them had a natural Giardia infection. About 10 to 20 c.c.
of fluid human stool containing numerous cysts was administered to each
animal by means of an oesophageal tube. All four developed diarrhoea,
and two actually passed blood and mucus. Cysts of Giardia were present
in the faeces for two days, after which they disappeared, except in the case
GIAEDIA IN ANIMALS 705
of the naturally infected animal. Two of the cats died on the fourth day,
when a careful examination of the intestines was made both in the fresh
condition and in stained sections. There was no sign of any infection
with Giardia. The naturally infected cat and one other survived for
three weeks. They completely recovered from the intestinal disturbance
caused by the inoculation. The naturally infected animal alone continued
to pass cysts. The ajiimals were killed, and, as was expected, Giardia was
found only in the one which was already infected before the experiment.
GIARDIA IN ANIMALS.
As already remarked, a number of different species of Giardia have
been described, but, with the exception of G. agilis of the tadpole, the
various forms are very uniform in appearance. Simon (1922) and Hegner
{1922a) have introduced a biometric method for the separation of species
similar to that which has been employed in the case of trypanosomes.
They maintain that if a sufficiently large number of individuals is
measured, species can be separated by constructing curves showing the
percentage of flagellates of any one size. Simon and Hegner claim that in
this wa}^ it is possible to separate the human form from that of rats and
mice, and Hegner those occurring in the dog and rabbit from each other
and from those of human beings and rats (Fig. 296). Hitherto the occur-
rence of Giardia in different hosts has been the chief factor which has
influenced observers in the establishment of species. Grassi (1879o)
gave the name Dimorphus muris to the form in the mouse, while later
(1881a) he noted the occurrence of Giardia in human beings and also in
the cat, dog, rabbit, sheep, rat and mouse — Mus muscidus, Rattus rattus,
R. decumanus, M. sylvestris, Arvicola (Microtus) arvalis — and introduced
the new name Megastoma entericum. Grassi and SchewiakofE (1888)
added to the list of hosts A. (Microtus) amphibius. These observers
regarded the animal forms as belonging to the same species as that
occurring in man. In the writer's experience, Giardia is commonly
present in cats, dogs, rats, mice, and rabbits in England. Kofoid and
Christiansen (1915) have described as G. microti a form which occurs in
Microtus calijornicus of California. They believe it to be distinct from
G. muris, which they found, not only in mice, but also in Peromyscus
maniculatus gambeli. Davaine (1875) described as Hexamita duodenalis
a flagellate from the duodenum of the rabbit. It is undoubtedly a species
of Giardia, so that the correct name for the form in the rabbit is G. duo-
denalis, though Bensen (1908) proposed to name it G. cuniculi (Fig. 296, b).
Fonseca (1916) observed it in both the rabbit and Ccendu viUosus of South
America. A form he saw in the monkey {Cebus caraya) he regarded as
identical with the human G. intestinalis. Splendore (1920) gave the name
I. 46
706
ORDER: DIPLOMONADIDA
G. pitymisi to a torm occurring in the field vole, Pitymis savii. Simon
(1922) has noted in Microtus fennsylvanicus acadicus in Nova Scotia a
Giardia which appears to be identical with the form described by Kofoid
and Christiansen as G. microti (Fig. 296,/).
Hegner (1923a) has recorded as G. cavicB a form found by him in the
guinea-pig in America (Fig. 296, ^r). It is a small form like G. muris, but
Fig. 296. — Diagrammatic Kepresentation of Various Species of Giardia,
SHOWING Specific Differences. (From Hegner and Taliaferro, 1924,
after Simon and Hegner.)
a, 0. intestinalis of man; b, G. diioderuilis of rabbit; c, G. muris of rats and mice; d. G. agilis of
frog tadpoles; e, G. cants of the dog; /, G. microti of field mouse; g, G. cavice of guinea-pig.
broader in proportion to its length, while the deeply staining bodies
behind the sucking disc are represented by two rods which lie trans-
versely and somewhat obliquely across the body. Another named
species is G. sanguinis found by Gonder (19106) in blood-films made
from a falcon {Elanus coeruleus) which had been shot in the Transvaal.
Noller (19206) described as G. ardece a form seen by him in the intestine of
herons {Ardea cinerea and Ardetta minuta), while Kotlan (1922) discovered
similar forms in the shrike {Lanius collurio) and avocet {Recurvirostra
GIARDIA IN ANIMALS
707
avocetta). Later (1923), he recorded as G. ardece flagellates which he found
in Ardea cinerea, A. rubra, Nycticorax griseus, and Pelegadis falcinellus.
Kiinstler (1882), who founded the genus Giardia, described as G. agilis
a form which occurs in the tadpole. Observing flagellates, which appeared
to be of a different type in tadpoles, Kunstler and Gineste (1907) proposed
to name two new species, G. gracilis and G. alata. Alexeieff (1914) expressed
it as his opinion that all the forms belonged to the one species, G. agilis,
a view which is shared by Hegner (1922), who has given a description of
the tadpole flagellate.
As already remarked, apart from G. agilis, which is distinctly elongate,
the various species of Giardia are very much alike in appearance, and the
various morphological differences which have been described are quite
Fig. 297. — Various Species of Giardia of Mammals (
A. G. miiris of the mouse. B. G. duodenalis of the rabbit.
,300). (Original.)
C. G. sp. from the cat.
inconstant and cannot be employed for the separation of species. If,
however, a large number of flagellates from a human case be examined and
compared with those occurring in rats or mice, the impression is gained
that the human form is longer in proportion to its breadth than the mouse
form. By actual measurement this is shown to be the case, and it is not
improbable that a comparison of the average dimensions of the forms from
other animals may show that constant differences in size occur, as Simon
and Hegner maintain. Such a method of identifying species is, however,
a long and tedious process.
Giardia muris (Grassi, 1879). — This species was first seen by Grassi
(1879«, 1881a), who regards it as identical with the human form (Figs. 296,
c, and 297, A). It was described by the writer (1907), and by Bensen (1908)
and Kofoid and Christiansen (1915a). Bensen believed that it could be
distinguished from other forms by certain morphological characters,
especially those of the two deeply-staining bodies which lie dorsal to the
708 ORDER: DIPLOMONADIDA
axonemes of the tail flagella. As pointed out by Simon (1922), none of
these characters is of sufficient constancy to be of any value for separating
species. According to him, it is only by the average dimensions that
species can be recognized. His measurements for G. niuris are: length,
7-25 to 12-75 (average 9-75) microns; breadth, 5-25 to 9-75 (average 7-26)
microns. The dimensions of the cysts are very similar to those of G. intes-
tinalis of man. Simon believes that in one white rat examined by him
there occurred two distinct species. One of these was evidently G. muris.
The other is referred to as G. sp. It was larger than G. muris, and varied
in length from 10-25 to 16-75 microns (average 13-25) and in breadth from
6-25 to 9-25 microns (average 7-49). Both white rats and wild rats known
to be free from natural infections were readily infected with G. 7nuris from
mice by Simon. He was unable to infect these animals with G. intestinalis
or G. microti. Fantham (1925) records G. muris from Rnttus concha and
Tatera lohengula.
White mice and rats are commonly infected with G. muris, which quickly
spreads when introduced to a batch of these animals. There seems little
reason to regard it as in any way pathogenic, though Kofoid and Christi-
ansen (1915) maintain that the intestines of infected animals are altered
to a yellow colour, which is most evident at the site of heaviest infection.
Hegner (1923a) has found G. muris in wild rats and mice in America.
From a study of Rattus norvegicus in Paris, Lavier (1924) concludes that
these rodents harbour two species of Giardia. One of these is G. muris,
while the other appears to be morphologically identical with G. intesti-
nalis of man. As all attempts to infect rats with the human Giardia have
failed, Lavier believes that the form in the rat is a distinct species, for
which he proposes the name Giardia simoni. It is apparently the form
referred to by Simon as G. sp.
Giardia microti Kofoid and Christiansen, 1915. — This is a form which was
discovered by Kofoid and Christiansen (1915) in meadow mice {Microtus
californicus californicus) in California. They supposed that it could be
distinguished from G. muris on morphological grounds, but Simon (1922)
has shown that this is not the case, and that the species can only be distin-
guished by its measurements (Fig. 296). He gives these as: length, 8-25 to
13-75 (average 11-11) microns; breadth, 5-25 to 10-25 (average 7-58) microns.
Simon was unable to infect rats or mice with G. microti obtained from
M. pennsylvanicus acadicus. It is not improbable that the form seen by
Grassi in Arvicola {Microtus) arvalis in Italy, and studied by Lavier (19216)
in France, and that described by Splendore (1920) as G. jpitymisi of
Pitymys savii of Italy are identical with G. microti.
G. viscacise Lavier, 1923. — This species was discovered by Lavier
(1923) in the viscacha {Viscacia viscacia), a rodent of South America.
GIARDIA IN ANIMALS 709
The dimensions of the fixed and stained forms were given as, 13 to 18
microns by 6-5 to 12 microns. The living forms appeared somewhat
longer, and varied in length from 17 to 20 microns and in breadth from
9 to 12 microns. The cysts were 11 to 13 microns in length by 7 to 7-5
microns in breadth. Thomson, J. G. (1925), has made the interesting
observation that intestinal nematodes {VianeUa sp.) from the same rodents
harbour what appears to be the same organism. Two of these rodents
died in the Zoological Gardens in London. On examination, hundreds
of nematodes heavily parasitized with the flagellate were found, though
neither flagellates nor cysts could be discovered in the intestinal contents.
The rodents also harboured numerous nematodes of the genus Tricho-
strongylus, but in none of these was the flagellate found. It seems highly
probable that the flagellate is G. viscacice, which, being ingested by the
worms, had found a habitat suitable for its multiplication. It is of
interest to note that Brumpt (1910a) has observed that certain Ascaridse
parasitic in the colon of horses appear to feed exclusively on the Infusoria
— ciliates and flagellates — which live in this part of the intestine.
G. duodenalis (Davaine, 1875).— This flagellate was first described by
Davaine as Hexamita duodenalis. Grassi (1881a) regarded the rabbit form
as identical with that of man, a view which was held by Metzner (1901).
Bensen (1908) applied to it the name Lamblia cuniculi. The correct name
is undoubtedly G. duodenalis. The rabbit flagellate has been studied by
Hegner (1922a), who gives the measurements as follows: length, 12-7 to
18-7 (average 15-8) microns; breadth, 7-7 to 11-0 (average 9-1) microns.
It is thus both broader and longer than G. intestinalis (Figs. 296, 297, B).
The two deeply-staining bodies at the base of the tail are described as
being often bent and longer than in other species. Fonseca (1915) de-
scribed a form which he regarded as this species in Coendu villosus, as
also in the rabbit of Brazil. Hegner (1922a) believes that possibly he
was dealing with a distinct species,
G. canis Hegner, 1922. — This form, which was first noted by Grassi
(1881a), was again mentioned by Grassi and Schewiakoff (1888) and by
Janowski (1897). The writer has seen it in dogs in England. Hegner
(1922a) states that it has a characteristically broad anterior end
(Fig. 296, e). It varies in length from 11-9 to 17-0 microns (average 13-8)
and in breadth from 7-6 to 10-2 microns (average 8-5).
Giardia cati Deschiens, 1925. — This form was first seen by Grassi
(1881a), and was named by Deschiens (1925). Later, Hegner (1925a)
gave the name G. felis to a parasite of the cat in America. Hegner's
flagellate, which may not be the same as the one studied by Deschiens,
measured from 10-5 to 17-5 microns in length and from 5-25 to 8-75 microns
in breadth. The cysts measured 10-5 by 7-35 microns. In the writer's
710
ORDEE: DIPLOMONADIDA
experience English cats are commonly infected. Hegner (1924) has seen
cysts in the faeces of a wild cat, Lynxruffus. They measured 11-01 to 13-55
microns by 6-57 to 8-47 microns. Fantham (1923) gave the name G. suri-
catcB to a form in the meercat, Suricata tetradactyla. Deschiens (1925a)
has seen cyst of a species of Giardia in the fseces of two lions.
G. bovis Fantham, 1921, and G. equi Fantham, 1921.— These forms
were recorded without details by Fantham, from the ox and horse in
South Africa. Later (1923) he states that G. equi measures 20 by 10
microns, and the cysts 12 to 15 by 9-2 microns, and (1925) that the cysts
of G. bovis measure 11 to 11"5 by 7 microns. Nieschulz (1923) found cysts
of Giardia measuring 10 by 5-2 microns in the faeces of a calf in Holland.
G. caprae Nieschulz, 1923. — This form was discovered by Nieschulz
(19236, 19246') in the goat in Holland. The free forms measured 9 to 17
by 6 to 9 microns. Cysts measur-
ing 12 to 15 by 7 to 9 microns were
also seen.
Hegner (1924) has seen the
cysts of Giardia in the faeces of a
monkey, Atelus geoffroyi. The
measurements given are: length,
11-01 to 14-40 microns; breadth,
6-77 to 9-31 microns. It is stated
that they are obviously different
from the cysts of the human para-
site. The writer has seen cysts
of Giardia in a young monkey
(Cercopithecus) from West Africa.
G. sanguinis (Gonder, 1910).—
As pointed out above, this form
was found by Gonder in the
blood-films of a falcon shot in
the Transvaal. In view of the fact
that blood-films made from birds
which have been shot frequently
show contamination with intes-
tinal organisms, there is little
doubt that Gonder was dealing
with an intestinal form which had contaminated the blood through the
wounded intestine. Noller (19206) described as G. ardew an intestinal
form from the herons, Ardetta miniita and Ardea cinerea. Kotlan (1922)
has recorded Giardia from a shrike {Lanius coUurio) and an avocet
(Recurvirostra avocetta), while Rudovsky (1923) has found one in a buzzard.
Fig. 298. — Giardia agilis OF the Tadpole
( X 4,300). (After Hegner, 1922.)
GENUS: TREPOMONAS 711
He,o;ner (1925a) has seen giardias in the black-crowned night heron
and the great blue heron in America, and Da Ciinha and Muniz (1922) in
Ardea socoi, Gathartis aura, and Nycticorax ncevius in Brazil.
G. agilis Kunstler, 1882. — This form occurs in tadpoles, but the infection
disappears when the metamorphosis into the frog takes place. The tadpole
flagellate differs from all other known species of Giardia in the length of
the body (Figs. 296, d, and 298). Hegner (1922) notes that structurally
it differs in no respect from other species, though Eeuling and Rodenwaldt
(1921) described certain differences on account of which they suggested
the retention of the name Giardia for this form and the name Lamhlia for
others. Hegner gives the measurements of G. agilis as follows : length, 14-4
to 28-9 (average 20-0) microns; breadth, 3-5 to 5-1 (average 4-5) microns.
Encysted forms have not been seen, though Alexeieff (1914) encountered
small spherical cysts about 10 microns in diameter in a recently meta-
morphosed frog. He supposes the flagellates encyst soon after meta-
morphosis of the tadpole, and that the cysts, which he regards as those
of G. agilis, escape into the water and are ingested by tadpoles in the
following spring.
The form described by Fantham (1923) as G. xenojji, from the clawed
frog, Xenopis Icevis, may be the same species. He also records it from
Bufo regularis.
G. denticis Fantham, 1919.— This flagellate was recorded by Fantham
(1919) from the blood and intestine of the South African silver fish {Dentex
argyrozona). It is not clear that the flagellates in the blood were not due
to intestinal contamination. G. salmonis, recorded by Moore (1922) from
trout in America, has been shown by Davis (1923) to be a Hexamita (p. 690).
G. varani Lavier, 1923.— This form was described and named by
Lavier (1923) from the Nile monitor ( Varanus niloticus). The length of the
body varied from 15 to 21 microns and the breadth from 8 to 11 microns.
Genus: Trepomonas Dujardin, 1841.
This genus was established by Dujardin for a flagellate which occurred
in sea-water infusions, and which he named Trepomonas agilis. Klebs
(1892) also studied this organism and named other species. The writer
and Broughton-Alcock (1924) have seen a form, probably T. agilis, on one
occasion as a coprozoic flagellate in the stool of a human being suffering
from mucous colitis (Fig. 299).
The organism is distinctly flattened and is oval in outline. There are
two longitudinal grooves on the posterior half or two-thirds of the body,
one on one surface and the other on the opposite surface. Sometimes they
appear as if they are formed by a folding over of the edge of the body in
712
ORDER: DIPLOMONADIDA
this region in such a way that one edge is folded forwards and the other
backwards. An organism viewed from the anterior or posterior end has
the appearance of an S, the hollows of the letter corresponding with the
grooves. Running round the anterior end of the body is a horseshoe-
shaped structure tapering at its extremities, which lie at the commence-
FiG. 299. — Trepomonas agilis as a Coprozoic Flagellate in Human F,eces
(x 3,000). (After Wenton and Broughton-Alcock, 1924; from Trans.
Boy. Soc. Trop. Med and Hijg., vol. xviii., p. 9).
1. Form with narrow groove.
2. Form with gaping groove with turned-out edges producing impression of lobes.
3. Early division form.
ment of the grooves. Within this structure can usually be distinguished
four deeply-staining granules. Two of these lie near one another at the
anterior end of the body, while the others are nearer its extremities.
Arising from a point near the commencement of each groove are a number
of flagella. One of these is a conspicuous long flagellum directed outwards,
while the others are short and lie in the groove.
There appear to be three short flagella, but it is
not always possible to distinguish this number.
Klebs figured a long flagellum and three short ones.
The nature of the horseshoe structure is doubt-
ful. In dividing forms it splits into two, and one
half moves to the opposite end of the body.
Before it divides, however, division of the granules
within it takes place, so that it is possible that
the two anterior granules which are surrounded by
a clear area are the true nuclei, the other two
granules blepharoplasts, and the structure itself a
parabasal. The organism would appear to be re-
lated to Hexamita, the six short flagella in the grooves corresponding with
the six anterior flagella and the two long ones with the posterior flagella.
Fig. 300. — Trepomonas
sp. from Eectum of
Marine Fish, Box
salpa ( X 2,250).
(After Alexeieff,
1910.)
GENUS: TREPOMONAS
713
The degree of development of the grooves varies considerably. Sometimes
each is a narrow slit, while at other times it is wide and gaping. The margins,
which are folded over, may be turned back, producing the appearance of
Fig. 301. — Calonymjiha grassii (x 1,600) from the Intestine of the Termite,
Calotermes grassii. (After Janicki, 1915.)
two large curved lobes one on each side of the posterior region of the body.
The inner margin of each groove often appears to be strengthened by a fibre
which passes round the
posterior end of the
body, where a notch
sometimes occurs, and
has its ends on the ex-
tremities of the horse-
shoe body. It sometimes
appears as if this fibre is
an actual continuation
of the latter structure.
Alexeieff (1910) dis-
covered a form in the
rectum of the marine
fish. Box saljKi (Fig. 300).
The form he described
was evidently a dividing
flagellate, and only the
two long flagella were
depicted.
Fig. 302. — Htei^hanonym'pha siUestrini (x 1,200) from
Intestine of Termite, Calotermes castaneus.
(After Janicki, 1915.)
tl4 INTESTINAL FLAGELLATES OF MAN
C. Polyzoic Forms.
5. Order: POLYMONADIDA.
The flagellates included in this order are polyzoic, and possess many
nuclei and blepharoplasts, each of which gives origin to one or more
flagella. In association with each nucleus, there may be a parabasal,
while an axostyle is present. The members of this order may be sup-
posed to have been derived from flagellates of the Eutrichomastix type, in
which multiplication of nuclei and organs has taken place without division
of the body. The order includes the single family CalofiymphidcB, founded
by Grassi for certain flagellates of termites which have the above charac-
teristics. The family includes several genera, such as Calonympha (Fig.
301) and Stephanony mpha (Fig. 302).
FREQUENCY OF INTESTINAL FLAGELLATE INFECTIONS OF MAN.
Human beings are commonly liable to infection with the following
five intestinal flagellates : Giardia intestinalis, Chilomastix mesnili, Tricho-
monas hominis, Embadomonas intestinalis, Tricercotnonas intestinahs
(Fig. 303). The flagellated organism described by Kofoid and Swezy as
a species of Craigia, but which is probably a species of Sphceromonas or
Oikomonas, possibly identical with S. conmiunis, described by Liebetanz
(1910) from the rumen of cattle, is undoubtedly of rare occurrence. The
last-named organism has been seen only by Kofoid and Swezy in five
persons resident in America, and in one person who had returned from
India (see p. 295). Tricercomonas intestinalis was seen in about a dozen
cases of diarrhoea by the writer and O'Connor (1917) in Egypt. It is a
small flagellate which is exceedingly difficult to identify. It was seen by
the writer again in several cases of diarrhoea in Macedonia in 1918. Kofoid,
Kornhauser, and Plate (1919) record three cases of infection in soldiers
returned to America from abroad. The possibility of the identity of this
flagellate with the form described as Enteromonas hominis has been dis-
cussed above (p. 653). It is probable that it is of fairly common occur-
rence, as recent observations have extended its known distribution.
The difficulty of identifying it accurately may lead to its being regarded as
a small form of Trichomonas ho?ninis, Chilomastix mesnili, or even Emba-
domonas intestinalis. The last-named flagellate was seen by the writer
and O'Connor (1917) in two cases in Egypt. It was again recorded by
Kofoid, Kornhauser, and Plate (1919) in four patients returned to New
York from overseas, and in four others who had never left the United
States. A case was also seen by Hogue (19216) in the same country, while
another was seen by Broughton-Alcock and Thomson, J. G. (1922a), in a
person who had returned to England from abroad. It has since been
recorded from other localities.
RELATIVE FREQUENCY 715
The species of Giardia, ChUomastix, and T richomonas are more exten-
sively known, and can be considered to be world-wide in their distribution.
As the two former can be recognized by their cysts, they can be detected
in the formed as well as the diarrhoeic stool, whereas the latter is only
rarely seen in the formed stool owing to the absence of the encysted forms.
G. intestinalis was found by the writer and O'Connor (1917) to be
present in 4-1 to
16 per cent, of ..^rSt. . rx B
normal individ-
uals in Egyj)t.
Dobell (1921)
estimates that
18 to 27 per cent,
of the artisan
population of
the British Isles
harbour this
flagellate, while
Boeck (1921)
found it present
in 48-1 per
cent, of eighty-
three industrial
school children
examined in
America.
As regards
C. mesnili, the
writer and
0'Connor(1917)
obtained a per-
centage of 3-2
of infections
amongst nor-
mal individuals
in Egypt. For
the population of the British Isles, Dobell gives 6 to 9 per cent., while
Boeck gives 1-2 for American school children.
T. hominis was seen by the writer and O'Connor in only 3 per cent, of
hospital cases in Egypt, and in a much smaller percentage of healthy
people. Amongst the population of Britain, Dobell mentions that it was
only occasionally seen, while it was not met with at all by Boeck m his
examinations of American school children.
Fig. 303. — The Flagellates of the Human Intestine
(x 2,000). (After Wenyon, 1922.)
A-C. Giardia intestinalis, free and encysted forms.
D-F. ''Iii/oiini^ti.r iiir.^)/l/i. free and encysted forms.
G-I. I'lniiiiiilniiiniiit-i i iilr^il iKiIis. free and encysted forms.
J-L. Tnci n-diuuiris iiitisiiii(i/is. free and encysted forms.
M-0. Trichomonas hominis, forms with three, four, and five flacrella.
16
CLASS: CNIDOSPORIDIA
III. CLASS: CNIDOSPORIDIA DoFLEiN, 1901.
CLASSIFICATION.
CLASS: CNIDOSPORIDIA
Order: MYXOSPOEIDIIDA
Sub-Order: Eurysporea
Famihj : CERATOMYXID^E
Genus : Leptotheca
Ceratomyxa
Myxoproteus
Wardia
»5 Mitraspora
Sub-Order: Sphaerosporea
Family : CHLOROMYXID.5;
Genus : Chloromyxum
»» Agarella
Family : SPH^ROSPORID^
Genus : Sphserospora
»' Sinuolinea
Sub-Order: Platysporea
Family: myxidiid^
Genus : Myxidium
)• Sphaeromyxa
Zschokkella
Family: MYXOSOMATIDJ^;
Genus : Myxosoma
»» Lentospora
Family: myxobolid^
Genus : Myxobolus
»> Henneguya
!» Hoferellus
Order: MICROSPOEIDIIDA
Sub-Order: Monocnidea
Family: glugeid.e
Genus : Glugea
Family : NOSEMATID^
Genus : Nosema
>' Perezia
Gurleya
Thelohania
»> Stempellia
Duboscquia
Plistophora
Family: COCCONEMID^
Germs : Cocconema
Family: mrazekiid^
Genus : Mrazekia
Octosporea
-, Toxonema
Spirillonema
Sub-Order: Dicnidea
Fam ily : T E L O ]\I Y x I D .5:
Genus : Telomyxa
Order: ACTINOMYXIDIIDA
Genus : Tetractinomyxon
»» Hexactinomyxon
5' Triactinomyxon
»5 Synactinomyxon
>> Sphseractinomyxon
Parasites of Undetermined
Position
SARCOSPOEIDIA
GLOBIDIUM
HAPLOSPORIDIA
GENERAL ORGANIZATION 717
The Protozoa belonging to this class are amoeboid organisms during
the growing or trophic phase of development, while dissemination is
effected by means of resistant spores, which are peculiar in being provided
with one or more polar capsules. The latter, under certain conditions of
stimulation, as, for instance, those of the intestinal fluids, extrude long
filaments which are supposed to attach or anchor the spore to the intestinal
wall till the enclosed amoeboid body, the actual infective agent, is able to
escape from the spore and invade the tissues of the new host. Gluge
(1838) was the first observer to see small spores of one of these parasites
in fish, but Johannes Miiller (1841) discovered much larger ones in a
number of fish, and referred to them as psorosperms, a name which was
long used for them and the spores of coccidia and gregarines. The
Cnidosporidia are often grouped with the Sporozoa, which Schaudinn
(1900) divided into two sub-classes, the Telosporidia and the Neosporidia,
the former to include the coccidia and gregarines, and the latter the
Myxosporidiida, Microsporidiida, Actinomyxidiida, and Sarcosporidia
The Telosporidia, however, have little in common with the Neosporidia.
They have definite intracellular stages, reproduction is by schizogony,
while the zygotes resulting from a conjugation of gametes become encysted
in resistant oocysts, within which they give rise to sporozoites. The
Myxosporidiida, Microsporidiida, and Actinomyxidiida, on the other hand,
though sometimes intracellular parasites, reproduce mostly by binary
fission and not by schizogony, while the zygotes do not become encysted,
nor do they give rise to sporozoites. Furthermore, the very characteristic
spores possessing polar capsules are produced. Very little is known about
the affinities of the Sarcosporidia, but it seems clear from their com-
paratively simple spores that they are in no way related to the Cnido-
sporidia, which produce the highly complex spores provided with polar
capsules. In their development the spores of Cnidosporidia difi'er
fundamentally from those of all other Protozoa, the resistant or encysted
stages of which are produced by a cell secreting a capsule round itself.
Subsequently the entire cell or the products of its division survive. In the
case of the Cnidosporidian spore a single cell divides to form several cells,
some of which give rise to the polar capsules, others to the spore mem-
branes, while one or two alone survive. The production of the spore
involves the sacrifice of several cells for protective purposes, while no such
sacrifice is associated with spore formation in other Protozoa. This
difference led Emery (1909) and Ikeda (1912) to suggest that the Cnido-
sporidia are in reality Metazoa. Attention has been again called to this point
by Dunkerly (1925), who sees in this dift"erentiation of cells a process by
which Metazoa may have evolved from Protozoa. It seems, therefore,
best to follow Hartmann (1907), and separate the Myxosporidiida, Micro-
718 CLASS: CNIDOSPOKIDIA
sporidiida, and Actinomyxidiida from the Sporozoa, with which Schaudinn
first grouped them, and to place them in a distinct class for which the name
Cnidosporidia, suggested by Dofiein (1901) for the order, can be employed.
The Sarcosporidia do not appear to be related either to the Sporozoa or the
Cnidosporidia, and will be considered with other forms with doubtful
affinities, as was done by Labbe (1899).
A typical member of the class commences its existence as a small
amoeboid body which has escaped from the spore in the intestine of the
host. It makes its way to the tissue or body space in which its subsequent
development wdll occur. Here it may grow into a multinucleate Plas-
modium through repeated nuclear divisions not being followed by division
of the cytoplasm, or it may multiply by binary fission or possibly by
multiple segmentation or gemmation, so that a large number of uninucleate
forms is produced. In either case spore formation eventually occurs.
In the multinucleate plasmodial forms certain of the nuclei become
separated with a portion of cytoplasm as small round cells which lie in
vacuoles in the plasmodium. These uninucleate cells (pansporoblasts)
in the vacuoles then become transformed into spores, while the plasmodium
continues to increase in size. In the uninucleate forms the spores arise
from one of the uninucleate parasites (pansporoblasts). The process of
development of the spore from the uninucleate body with the production
of the polar capsules is a very complicated one, and the type of spore
produced in the dift'erent genera of Cnidosporidia varies considerably.
The ultimate infective agent within the spore is an amoeboid body which
has one or two nuclei. It has often been referred to as a sporozoite, but
there is no evidence that it is homologous with the typical sporozoites of
Sporozoa, The Cnidosporidia include three orders: Myxosporidiida,
MiCROSPORiDiiDA, and Actinomyxidiida.
A. Order: MYXOSPORIDIIDA.
In these forms the trophic or growing phase is a multinucleate
plasmodium, which resembles an amoeba in that it is motile and forms
pseudopodia (Fig. 304). They are typically parasites of cold-blooded
vertebrates, a large number of species infecting fish, amongst which they
give rise to severe and fatal epidemics. In some cases they live as
harmless amoeboid organisms in the body spaces, such as the gall bladder,
urinary bladder, or tubules of the kidney, where they float about or are
attached to the walls by pseudopodia. It is in these forms that the
complicated process of spore formation has been chiefly studied. In other
cases they are definite tissue parasites, which may give rise to nodules,
sometimes of large size, on the skin and gills or in the muscles and
other organs (Fig. 305). In the tissues the parasite may grow actually
OEDER: MYXOSPORIDIIDA
719
within a cell, which becomes much distended, as in the case of muscle
fibres (Fig. 306, D). Many forms, however, develop in the intercellular
Fig. 304. — Various Cnidosporidia. (After Thelohan, 1894.)
A. Ceratomyxa appendiculata Thelohan, 1894, from gall bladder of Lophius piscatorius and
L. budegassa. Spores 50 x ,5-7 n-
B. Chloromtjxmnleydigi Ming., 1890, from gall bladder of various marine Elasmobranchs. Spores
about 13x10 /J.
C. Several small forms of Leptotheca agilis Thelohan, 1894, attached to a specimen of C. leydigi.
D. Glugea ?»ar(ow/5 Thelohan, 1894, from the gall bladder of marine fish (Wrass), Julis vulgaris
and J . giofredi. Spores 8 x 3 /t.
E. Sphcerospora (ifi'ersrews Thelohan, 1894, from the kidney tubules of Ble.nnius photis and Creni-
lahrus melops. Spores 10 /< in diameter.
F. Leptotheca agilis Thelohan. 1894, from the gall bladder of the ray, Trigon vulgaris. Spores
11-12x6-7/1.
spaces, the tissues of the host and the parasites being closely intermingled
(Fig. 306, A-C). The infected area of tissue is frequently shut off by the
720
ORDER: MYXOSPORIDIIDA
development, on the part of the host, of a fibrous capsule, within which
occur the remains of host cells with their hypertrophied nuclei, and the
multinucleate plasmodia containing a varying number of spores. The
central portion of such an encapsuled area, owing to degeneration of the
central part of the parasite, may consist of granular debris and spores,
while thee apsule itself is lined by the multinucleate cytoplasm of the
parasite, which continues to grow and produce spores. In old nodules
spores and debris alone may be detected, while later still fibrosis or even
calcification may occur, and all trace of the parasites be lost.
Infection is brought about in the first place by the small amoeboid
organism, which frequently has two nuclei, escaping from the spore in the
intestine of the host. It is claimed by some that at this stage syngamy
takes place, but the evidence of this is conflicting. The amoeboid
organism, which is now known as a planont, makes its way to that par-
FiG. 305. — Miixoholus pfeifferi .- Section through the Body of a Barbel, showing
Two Tumours caused bt the Parasite. (After Keysselitz, 1908.)
ticular tissue or body space which the species infects. In some cases it is
evident that a multiplication of these small forms occurs, and this may
take place within the cytoplasm of cells, in the intercellular spaces of the
tissues, or in the lumen of the gall bladder or other body cavities. Finally,
growth into the large multinucleate plasmodia takes place. It was main-
tained by Cohn (1896) that Myxidium lieherhuhni, which infects the
gall bladder of fish, was able, in the multinucleate phase, to bud from its
surface numerous small uninucleate forms. Laveran and Mesnil (1902a)
showed that no such budding takes place in this species, and that the
formation of numerous short pseudopodia, and the fact that young para-
sites often become applied to the surface of older ones, are responsible
for the misconception. They showed that multiplication takes place by
equal or unequal division of the young forms. Kudo (19226) has, however,
described a process of internal budding in the case of Leptotheca ohlmacheri
in the kidney of the frog (Fig. 311).
GENERAL ORGANIZATION
The behaviour of the spores after ingestion by a
721
new host has been
studied by several observers, whose accounts are by no means concordant.
In the case of M. bergense, a parasite of the gall bladder of the saithe,
Gadus virens, Auerbach (1910) noted that after entering the duodenum of
the fish the polar filaments of the spore were extruded and the two valves
of the spore capsule separated. This allowed the binucleate amoeboid
body to escape. The two nuclei then fused, and the resulting uninucleate
amoeboid body made its way to the bile ducts, into one of the cells of which
B
r #6^1?®®^'^^:^!
Fig. 306.
My.robolas pfeifferi in the Tissues of the Barbel. (After
Thelohan, 1894.)
A. Portion of intestinal wall of the barbel infected with 2Iif.roholiis pfeifferi.
B. Connective tissue of kidney of barbel infiltrated with Mi/xnhnJus pfeifferi.
C. Portion of the fibrous tissue shown in A more highly niagnitied.
T>. Muscle fibre of the barbel infected and destroyed by Myxohohis pfeifferi. Spores 14 x 10 /i.
it entered. Later it is described as leaving the cell and multiplying by
binary fission in the lumen of the bile ducts or gall bladder. The uni-
nucleate amoeboid forms then associate in pairs, while the nucleus of one
of each pair divides to form two nuclei, one of which is discharged from the
cytoplasm. The two cells, one of which has a reduced nucleus, now unite
to form a binucleate mass with one large and one small nucleus. Other
observers, as, for example, Davis (1916), Georgewitch (1917), Erdmann
(1917), Schuurmans-Stekhoven (1919), and Kudo (1922), working with
other species, maintain that such a union does not take place, and that
I. 46
722 OEDER: MYXOSPORIDIIDA
the binucleate stage is not produced by union of two uninucleate indi-
viduals, but by the actual division of the nucleus of the uninucleate form.
The order Myxosporidiida is divided by Doflein (1901) into two sub-
orders, Disporea and Polysporea. The members of the former are
parasitic in the body spaces as large amoeboid organisms, which may
measure as much as 85 by 25 microns. Within each individual a single
cell (pansporoblast) is separated in a vacuole. The single uninucleate cell
by further development gives rise to two spores, which remain embedded
in the cytoplasm of the adult. After their formation the parasite dies
and the two spores are liberated. In the Polysporea, which include the
majority of the Myxosporidiida, the adult parasite produces a large number
of spores. It is these forms which invade the tissues, give rise to large
tumours, and often produce an intense infection of the host. In an
infected area of tissue, which can only be satisfactorily examined in
sections, it is frequently difficult to define the limits of a single parasite,
which extends as a reticulum amongst the tissue cells and fibres, producing
the condition known as difiuse infiltration (Fig, 306). In such cases the
spores, when produced, are scattered amongst the tissue elements, which
often show marked hypertrophic changes, though sometimes this does not
occur, the individual cells and fibres being little altered in appearance.
In those cases in which a fibrous capsule is formed, the multinucleate
layer of cytoplasm which lines the capsule appears to be a single parasite.
Kudo (1919) has, however, pointed out that such a division into
Disporea and Polysporea is an artificial one, as the number of spores
produced by any particular species is by no means as constant as such a
classification implies. He maintains that the spore stage is still the only
one which affords constant characters by which various genera and
species can be identified (Fig. 307).
The spore consists of a shell composed of two valves which are united
in a sutural plane like two watch-glasses placed with their rims together.
The sutural line may be straight, as in the case of watch-glasses, or it may
be more irregularly curved, giving the appearance of an S in side view.
The form of the spore varies with the shape of the valves and the presence
1. Leptothera informis. 13. Sinuolinea capsularis.
2. Myxnimili iisrnrnutiis. 14. Mi/.n'di/nit /inirenwi:
3. Wanlni ,>rn,nn,a. IT). M i/xld, ma i ii llatum.
4. Ceni/niiif/.iii sjiinosa. 1<>. Splm rnnn/ni Im'hiami.
5. Ccniluiin/ni Iriiiiral/l. 17. S plui rniini.ni I iir ii mild.
6. Ccnihnn'ii.nt nnirrahi.. IS. Z.-^rlmkl.; Iht a,h, lln,, imilii .
7. Crr,tln„iiix<i sphrrK/om. li). J/y.ro.v„/,„/ ,l,iianli„l.
8. J////v^^•/-u/v/ riiprlni. 2(1. .\h/.n,su„m dnjanlini.
9. Mllnispuni niiidata. 21. f.rj,ln.<i„,ni nnila.
10. Ch!on>,u//.nnn. leydigi. 22. Mi/.ruh,diis nmissii.
11. Ghloromyximh caudatum. 23. Hoferdlus cyprini.
12. Sphcerospora rostraia. 24. Henneguya gurleyi.
25. Henneguya psorospennica.
GENERAL ORGANIZATION
723
Fig. 307. — Spores of Myxosporidiida (x 1,500). (After Kudo, 1919.)
[For description see opposite page.
724 OEDER: MYXOSPORIDIIDA
of accessory appendages (Fig. 307). In Ceratomyxa there are lateral
appendages, in Myxoproteus anterior ones, in Wardia a posterior fringe,
in Mitraspora a posterior filament, in Hoferellus a posterior spine, and in
Henneguya a posterior tail-like process. The surface of the shell or valves
is smooth or marked with ridges. Within the shell are the polar capsules,
which in most cases are situated at the narrow anterior end of the spore.
In the Myxidiidse there is one at each end of the spore, while in a few
species of Wardia the polar capsules are central in position. Each polar
capsule is spherical or pyriform in shape, and opens to the exterior by a
separate pore which is at the anterior end of the spore except in the spores
of the Myxidiidse, in which no distinction between anterior and posterior
ends can be made. There are always two polar capsules in the spore,
except in Myxoholus, which has one, and Chloromyxum and Agarella, which
have four.
Within each polar capsule is a coiled filament, which can be extruded
through its pore. The filament is coiled round the long axis of the spore,
except in Sphceromyxa, in which it is coiled round an axis at right angles to
this. The filament is long and thin in all forms except Sphceromyxa, in
which it is short, thick, and tapering. In addition to the polar capsules,
the spore contains the infecting agent in the form of a cytoplasmic body,
sometimes called the sporoplasm, containing usually two nuclei, and
frequently an iodophilic vacuole filled with glycogenic material.
Subdivision of the Myxosporidiida.
The following classification, taken from Kudo's monograph (1919) on
the Myxosporidiida (=Myxosporidia Biitschli, 1881), is based chiefly on
the characters of the spores (Fig. 306). It difiers in the inclusion of the
genus Agarella in the family Chloromyxidee.
1. Sub-Order: Eurysporea Kudo, 1919.
Largest diameter of the spore at right angles to the sutural plane.
One polar capsule on each side of the plane. Sporoplasm with no iodino-
philous vacuole. Vegetative form found in body cavity (except in two
species). Great majority parasites of marine fish. Monosporous, disporous,
and polysporous.
(1) Family: CERATOMYXiD.tE Doflein, 1899.
With the characters of the sub-order.
Genus: Leptotheca Thelohan, 1895.
Shell valves of spore hemispherical or shortly rounded. Fifteen
species. Disporous (seven unknown). Fourteen species in body cavity,
one in tissue; all in marine fish. Type species: Leptotheca agilis Thelohan.
CLASSIFICATION 725
Genns : Ceratomyxa. Thelohan, 1892.
Shell valves conical and hollow, attached on the bases; free ends
extended, tapering to more or less sharply pointed or rounded ends.
Sporoplasm usually does not fill the cavity, but is located asymmetrically
in it. Thirty-five species, Disporous (twenty-three species), mono-
sporous and disporous (three species), disporous and polysporous (four
species), and unknown (five species). All (except two species in urinary
bladder) in the gall bladder of marine fish. Type species: Ceratomyxa
arcuata Thelohan.
Ge»«s .• Myxoproteus Doflein, 1898, emend. Davis, 1917.
Spores roughly pyramidal; with or without distinct processes from the
base of the pyramid. Three species. Disporous (one species unknown).
All in urinary bladder of marine fish. Type species: Myxoproteus ambiguus
(Thelohan) Doflein.
(Te»«s.- Wardia Kudo, 1919.
Spore form of isosceles triangle with tw^o convex sides. Oval in profile.
Surface of shell with fine ridges which turn into fringe-like processes at
the posterior end. The polar capsules, large and perfectly spherical,
situated at the central portion of the spore, opening at the anterior tip.
Two species. Polysporous (one species unknown). Tissue parasite (one
species) of fresh-water fish and amphibia, both found in Illinois, U.S.A.
Type species: Wardia ovinocua Kudo, 1919.
GewHs.- Mitraspora Fujita, 1912, emend. Kudo, 1919.
Spores spherical or ovoidal. Two polar capsules pyriform, one situated
on each side of the sutural plane. Shell longitudinally striated, with or
without long and fine filaments projecting posteriorly in a row at right
angles to the sutural plane at the posterior side. Three species. Di-
sporous and polysporous. All found in kidney of fresh- water fish. Type
species: MitrasjJora cyprini Fujita.
2. Suh-Order : Sphaerosporea Kudo, 1919.
Spores spherical or subspherical, with two to four polar capsules.
Sporoplasm without iodinophilous vacuole. Vegetative form found in
body cavity and tissue. Monosporous, disporous, and polysporous.
Parasites of marine and fresh-water fish and amphibia.
(1) Family: CHLOROMYXID^ Thelohan, 1892.
Spores with four polar capsules. Monosporous, disporous, and
polysporous.
726 ORDER: MYXOSPORIDIIDA
Gemis : Chloromyxum Mingazzini, 1890.
With the characters of the family. Spores without posterior tail-like
prolongations. Twenty-two species. Eighteen in body cavity, four in
tissue. Seven from marine and twelve from fresh-water fish, two in
amphibia, one in insect. Type species: Chloromyxum leydigi Mingazzini.
Genus : Agarella Dunkerly, 1915.
Spores prolonged at posterior end into two processes. Only species
Agarella gracilis Dunkerly from testis of Lepidosiren
(2) Family: SPH^ROSPORID^ Davis, 1917.
Spores with two polar capsules. Monosporous, disporous, and poly-
sporous.
Genus: Sphaerospora Thelohan, 1892.
Spores with two polar capsules. Monosporous, disporous, and poly-
sporous. Ten species. Body cavity and tissue. Five from fresh-water
and five from marine fish. Type species: S2)ha}rospora dirergens Thelohan.
Genus: Sinuolinea Davis, 1917.
Spores with or without lateral processes. Two polar capsules spherical.
Sutural line sinuous. Five species. Disporous and polysporous. In the
urinary bladder of marine fish. Type species: Sinuolinea dimorjjJia
Davis.
3. Sub-Order: Platysporea Kudo, 1919.
Sutural plane of the spore coincides with or forms an acute angle
with the longest diameter. One or two polar capsules. Sporoplasm with
or without an iodinophilous vacuole.
(1) Family: MYXIDIID^ Thelohan, 1892.
Two polar capsules, one at each end. Sporoplasm without any iodino-
philous vacuole. Spores fusiform.
Gefius : Myxidium Biitschli, 1882.
Spores more or less regularly fusiform, with pointed or rounded ends.
Polar filaments long and fine. Twenty-six species. Monosporous, di-
sporous, and polysporous. Twenty-two in body cavity, four in tissue.
Fifteen in marine and six in fresh-water fish, two in fishes from both
waters, and three in reptilia. Type species: Myxidium lieberl'ilhni
Biitschli.
CLASSIFICATION 727
Ge/i?f.s' ; Sphaeromyxa Thelolian, 1892.
Spores fusiform, with truncated ends. Polar filament short and thick.
Trophozoites large and disc-shaped. Seven species. Polysporous (two
unknown). Six in body cavity, six in marine fish, one in amphibia.
Type species : Sphceromyxa balbianii Thelohan.
G<?n»,s; Zschokkella Auerbach, 1910.
Spores semicircular in front view, pointed at ends. Polar capsules
large and spherical, opening on the fiat edge near the tips. Sutural line
usually curved in S form. Four species. Monosporous, disporous, and
polysporous. Body cavity. Two from marine and two from fresh-water
fish. Type species: ZschokJcella hildce Auerbach.
(2) Family: MYXOSOMATID^ Poche, 1913.
Two polar capsules at the anterior end. Sporoplasm without iodino-
philous vacuole.
Genus: Myxosoma Thelohan, 1892.
Spores ovoidal, flattened, and more or less elongated. Three species.
Polysporous. Tissue parasites. Two in fresh-water and one in marine
fish. Type species: Myxosoina dujardini Thelohan.
Genus: Lentospora Plehn, 1905.
Spores similar to Myxobolus in form. Sporojolasm without any iodino-
philous vacuole. Six species. Disporous and polysporous (two unknown).
One in marine and three in fresh- water fish, two from fishes in both waters.
Type species: Lentospora cerebralis (Hofer) Plehn.
(3) Family: MYXOBOLID.ZE Thelohan, 1892.
Spores with one or two polar capsules at the anterior end, with or
without posterior processes. Sporoplasm with an iodinophilous vacuole.
Majority polysporous in fresh- water fishes.
Genus : Myxobolus Biitschli, 1882.
Spores ovoidal or ellipsoidal; flattened. One or two polar capsules at
the anterior end. Shell without posterior process. Sixty-three .species.
Polysporous (nine species unknown). Fifty-nine species in tissue; four
unknown. Five in marine and fifty-six in fresh- water fish, one in annelid,
and one in amphibia. Type species: Myxobolus miilleri Biitschli.
728 OKDER: MYXOSPORIDIIDA
Gemis : Henneguy a Thelohan, 1892.
Spores more or less globular or ovoidal. Two polar capsules at the
anterior end. Posterior end of the shell valves prolonged into more or
less extended processes, which unite and form a tail in the median line.
Thirty-two species. Polysporous, disporous, and monosporous. Twenty-
eight species in tissue and four in body cavity. In fresh- water fish, except
one. Type species: Henneguya psorospermica Thelohan.
Genus: Hoferellus Berg, 1898.
Spores pyramidal, with two posterior processes from the lateral faces.
One species. Polysporous. Tissue and body cavity of fresh-water fish.
Type and only species: Hoferellus cyprini Doflein.
DETAILED DESCRIPTION OF CERTAIN SPECIES.
Myxobolus pfeifferi Thelohan, 1894. — This organism is a common
parasite of the barbel, Barhus fluviatilis. It infests all the tissues of the
body, including the skin and gills, on which it gives rise to nodules and
tumours, which may be of large dimensions. In the infected tissues the
Plasmodia are closely intermingled with the host cells, so that often there
is difficulty in defining the limits of the parasite. The infected area of
tissue is frequently enclosed by a fibrous capsule formed by the host, and
in this the parasite in the form of a multinucleate plasmodium continues
its development (Fig. 305). Spores are continuously being formed in the
cytoplasm of the parasite. Eventually the parasite dies, and all that
remains is a fibrous nodule which, on section, is seen to contain many
spores in the interstices of the tissue. Spore formation has been studied by
Keysselitz (1908). In the plasmodium, one of the nuclei becomes separated
with some cytoplasm as a cell, which Keysselitz calls the propagative cell
(Fig. 308). After unequal nuclear division a large and small cell are
produced. • Two such couples become associated, and the aggregation of
the two large and two small cells proceeds to the development of two
spores. The two small cells spread over the surface of the two large cells
to form an envelope, while the nuclei of the two large cells multiply till a
total of twelve are present. The cytoplasm of the two large cells may now
unite. Of the twelve nuclei, four, which are gamete nuclei, become
centrally placed, while eight take up a peripheral position. If the cyto-
plasm has united division takes place, so that two bodies are produced,
each with two centrally placed gamete nuclei and four peripheral nuclei.
Each of these cells with six nuclei develops into a spore. Two of the
peripheral nuclei form the two valves of the spore, while two give rise to
MYXOBOLUS PFEIFFERI
29
the two polar capsules. The two gamete nuclei come to lie in a cyto-
plasmic body at the posterior part of the spore. Finally, the two gamete
nuclei fuse. Apparently, the spores escape into the water and are ingested
by fish. The amoeboid body presumably escapes from the spore and
Fig. 308.
-Development of the Spores of Myxobolus pfeifferi from the
Pansporoblast (x 2,500). (After Keysselitz, 1908.)
1. Single propagative cell formed from the multinucleate plasmodium.
2. Division to form one large and one small cell.
3. Association of two pairs to give a group of two large and two small cells.
4-5. Formation of six-cell stage. Each small cell which does not multii^ly tends to spread a
covering over its own sister cells.
6. Stage with fourteen nuclei, two of which are the nuclei of the original small cells.
7. Division into two bodies, each with six nuclei, while the nuclei of the small cells take up a
position at the angles between them.
8. Each body now divides into three cells, two of which, with single nuclei and vacuoles,
form the polar capsules, one with two nuclei the infective agent, while two nuclei
become peripherally arranged and form, together with some cytoplasm, the valves of
the spore.
9. More advanced stage of one of the developing spores shown in 7.
10- U. Fully developed spores.
finds its way to the tissues of the fish, in which it develops into the multi-
nucleate Plasmodium. As is to be expected, the tracing of this part of
the development is beset with many difficulties. Other observers, such as
Schuurmans-Stekhoven (1919), state that there is no syngamy.
730
OEDEE: MYXOSPOEIDIIDA
Sphaeromyxa sabrazesi Laveran and Mesnil, 1900.^ — This parasite
was discovered in tlie gall bladder of the sea-horse, Hijjpocamqnis hrevi-
rostris, by Laveran and Mesnil (1900). Schroder (1907, 1910) studied its
development in the gall bladder of H. guttulatus. It lives in the gall
bladder and larger bile ducts as a more or less circular disc of cytoplasm,
which may reach a diameter of half a centimetre. There is a definite
hyaline ectoplasm and a much vacuolated endoplasm, in which numerous
retractile granules are embedded. It is probable that infection is com-
FiG. 309.
-Spore Formation in Sphceromyxa sabrazesi (x ca. 1,500).
(After Schroder, 1907 and 1910.)
A. Pro i^agative cell with two nuclei. B. Union of two propagative cells.
C. Cell with four nuclei, two small nuclei of envelope cells, and two large nuclei, which contribute
to the formation of two spores .
D. Division of the cell after nuclear multiplication into two spore-forming bodies. Each
contains six nuclei and two commencing polar capsules. At the centre are two residual
nuclei. E. Two sj^ores nearing comjjletion.
F. Complete spore before union of two nuclei in the sporoplasm.
G. Two nuclei of sporoplasm have united.
menced by the small uninucleate amoeboid body which, escaping from
the spore in the intestine of the fish, invades the bile ducts. By nuclear
multiplication and growth of the cytoplasm the large plasmodia are
produced. Spore formation commences by the separation of one of the
nuclei with some cytoplasm as a small cell, which remains in the cytoplasm
of the parent (Fig. 309). The nucleus of this cell divides into a large
and a small nucleus, and, as in the case of Myxoholus j^feiffefi, two such
binucleate cells become fused into a single quadrinucleate cell, which
contains two large and two small nuclei. This cell gives rise by further
SPH.^KOMYXA AND CEEATOMYXA
731
development to two spores. The process of spore formation is very similar
to that of M. pfeifferi, but the spores are very dilrerent in character. The
infective amoeboid body in the fully-formed spore has at first two nuclei,
but these fuse, so that the final infective agent has a single nucleus. During
spore formation the parasite continues to increase in size, while nuclear
multiplication is going on. Each parasite produces a large number of
spores, which in any individual are in various stages of development.
Fig. 310. — Ceratomyxa dreimnopsettce (xca. 700). (After Awerinzew, 1909.)
. Parasite with two vegetative nuclei and a macrogametocyte and microgametocyte.
. Similar form in which the gametocytes have divided into two macrogametes and two
microgametes. C. Similar form after conjugation of the gametes.
. Each zygote has given rise to six cells. E. Each grouji of six cells is producing a spore.
. Single sjjore nearing maturity. G. More advanced stage of development of two spores.
Ceratomyxa drepanopsettae Awerinzew, 1909. — This parasite was
discovered by Awerinzew (1909) in the gall bladder of the plaice {Drepa-
nopsetta platessoides), where it lives as an amoeboid organism. At first
the trophozoite has two nuclei, and it was concluded by Awerinzew that
this stage resulted from nuclear division of a uninucleate form. In view
of the subsequent conjugation process, Minchin (1912) concluded that it
was more probable that union of two uninucleate forms had taken place.
732 OEDEE: MYXOSPOEIDIIDA
Each nucleus now divides unequally, so that two small and two large
nuclei are present (Fig. 310). The small ones are vegetative and the
large ones generative nuclei. The generative nuclei become separated
in the cytoplasm of the parent as a large and a small cell, which may be
regarded as female and male gametocytes. Each of the gametocytes
divides to form two large female gametes and two small male gametes,
while the two vegetative nuclei remain unchanged. Conjugation between
male and female gametes takes place to form two zygotes, so that the
parasite again reaches a quadrinucleate stage in which its cytoplasm con-
tains two vegetative nuclei and two zygotes, each with a single nucleus.
Each zygote now proceeds to the formation of a spore. A number of
cells is produced, two of which give rise to the two valves of the spore,
two to the two polar capsules, and one to the infective amoeboid body.
After each zygote has formed a spore the parasite dies and degenerates,
the spores being liberated. Thus each parasite produces only two spores.
It was Myxosporidiida of this type that Doflein (1901) grouped under the
heading Disporea.
Leptotheca ohlmacheri (Gurley, 1893). — This parasite was first observed
by Ohlmacher (1893) in the kidney tubules of Bufo lentiginosus. It was
studied by Whinery (1893) and Gurley (1893, 1894). The latter observer
named it Chloro?nyxum ohlmacheri, while Thelohan (1895) gave the name
Leptotheca ranee to a form in the kidneys of Rana esculenta and R. tem-
poraria. Labbe (1899) placed Ohlmacher's parasite in the genus Lepto-
theca as L. ohlmacheri, and came to the conclusion that it was identical
with L. rancB. The parasite has more recently been studied by Kudo
(19226) in R. clamitans and R. pipiens in America.
According to Kudo, the spore contains two uninucleate amoeboid
bodies, and when it is placed in gastric juice or weak pepsin hydrochloric
acid, the amoeboid bodies show slow movements. The polar filaments
are extruded from the capsules, and finally the valves of the spore separate
(Fig. 311). By this time the two amoeboid bodies have united into a
single binucleate form, which escapes from the spore. It is probable that
the two nuclei fuse, for the earliest stages found in the kidney tubules
contain a single nucleus. There was no evidence of any intracellular
stage, the whole development appearing to take place in the lumen of the
tubules. It seems probable that multiplication of these forms by binary
fission takes place for some time, after which growth into the adult spore-
forming parasite occurs.
The single nucleus divides into two nuclei of equal size, which, though
at first alike, soon become difi'erent in appearance, so that a vegetative
nucleus can be distinguished from a generative one. The latter quickly
divides again, so that a trinucleate parasite is produced. This contains
LEPTOTHECA OHLMACHEKI
733
11. — Leptotheca ohlmacheri. Parasitic in the Kidney Tubules of
THE Frog (x 2,350). (After Kudo, 1923).
A. SejDaration of values of the spore, extrusion of polar filaments, and escape of binucleate
amrehoid body under the action of pepsin hydrochloric acid.
B. Later staL'r nf cscaiic nf amoeboid body under action of gastric juice.
C. Ama'lxiid infective IhkIv in which the two nuclei have united.
D, E. Multiplieatinii \>y binary fission. F. Fully grown iminucleate form.
G. Nucleus has divided into vegetative and generative nuclei.
H. The generative nucleus has divided to form the typical trinucleated form.
I. Form with two generative nuclei, one vegetative nucleus, and a bud containing also a
smaller vegetative nucleus and two smaller generative nuclei.
J. Form with one vegetative nucleus and two areas, each with six generative nuclei, which will
give rise to two spores.
K. Form with one vegetative nucleus and two spores nearing the completion of their develop-
ment.
734 OEDEE: MICEOSPOEIDIIDA
one vegetative nucleus and two generative nuclei. By a process of
gemmation or budding trinucleate individuals may be separated. In
tbis process the vegetative nucleus divides into two. One of these divides
again to form a vegetative nucleus and a generative nucleus. The latter
again divides to form two generative nuclei. Eound the group of these
three nuclei cytoplasm concentrates and the trinucleate bud is separated.
Apparently this process can be repeated several times, so that the number
of trinucleate parasites in the kidney tubules is greatly increased in number.
Spore formation takes place in the trinucleate forms, each generative
nucleus giving rise to one spore. The generative nucleus divides to form
two nuclei, and these again to form four. Of these four, two are devoted
to the formation of the two valves of the spore, one divides to form two
nuclei which give rise to the two polar capsules, while the fourth divides
to form the nuclei of the two infective amoeboid bodies which occur in the
fully-formed spore. Each generative nucleus of the original trinucleate
individual thus gives rise to six nuclei, so that a parasite in which two
spores are developing simultaneously, which is not always the case, will
have one vegetative nucleus and twelve generative nuclei, each group
of six generative nuclei being destined to form one spore.
B. Order: MICROSPORIDIIDA Labbe. 1899.
The parasites included in this order produce small spores, which are
frequently less than 5 microns in length. The spores often resemble
yeasts or bacilli, but possess one or, exceptionally, two polar capsules
from which, after treatment with certain reagents or under pressure,
exceedingly long filaments are extruded (Fig. 30). The latter may reach
a length of 500 microns or more. The organisms occur as intracellular
amoeboid parasites (Fig. 312). As multiplication takes place, the para-
sitized cells often become hypertrophied in a remarkable manner. In the
case of some hosts, only special organs are attacked, but in others, as in the
silkworm disease, the whole body is overrun by the parasites. The ova
may become infected, with the result that the parasites pass from the
parent to the ofTspring. After multiplication in the amcfiboid phase has
gone on for some time, certain spherical uninucleate forms (pansporo-
blasts or sporonts) undergo a complicated development to produce the
characteristic spores. The spores produced by each pansporoblast vary
in number from one to sixteen or more according to the particular genus
or species.
The Microsporidiida are found commonly in the intestinal epithelium
and other tissues of the aquatic larvse of insects. They occur also in
certain vertebrates such as fish, in which tumour-like nodules may be
GENERAL ORGANIZATION 735
formed in the muscles or skin. The infection can be recognized by the
presence of the numerous spores in the teased-out tissues. In sections it
Fig. 312. — Diagram of Life-Cycle of Nosema homhycis in Intestinal
Epithelium of Silkworm (xc«. 3,000). (After Stempell, 1909.)
can be seen that the infected cells of the intestine or other tissues are
hypertrophied and filled with numerous minute, rounded, or amoeboid
736 ORDEE: MICROSPORIDIIDA
cytoj)lasmic bodies containing one or more nuclei, and multiplying by
binary fission or multiple segmentation. Large numbers of spores also
occur in the cells, and these often have the appearance of yeasts, cocci, or
bacilli, from which they may be difficult to distinguish, unless there is an
opportunity of causing extrusion of the polar filament. The presence of
the polar capsule and the extrusion of its filament can be rendered evident
by treating the spores with irritating fluids such as dilute acid, iodine
solution, or perhydrol, or by pressure between slide and cover-glass. In
the fresh condition, the filaments are best seen by dark ground illumina-
tion (Fig. 30). They may be stained by the silver nitrate methods
employed for demonstrating spirocheetes. Owing to the small size of the
spores of the majority of Microsporidiida, their detailed structure is difficult
to make out, while the varying efiect of different fixatives accounts for
the great diversity of the accounts which have been given. Stempell
(1909) described the spore of Nosema bombycis {¥ig. 312). He believed
that the polar capsule was an elongated body occupying the length of the
spore, and that the infective agent was in the form of an equatorial band
of cytoplasm surrounding the polar capsule in the space between it and
the spore wall. This cytoplasm was described as containing four nuclei.
Many observers have adopted Stempell's views regarding the structure of
the spore. Schuberg (1910), working with Plistophora longifilis, stated
that the polar filament was coiled on the inner surface of the spore wall,
and that the infective agent was in the form of a circular band of cytoplasm
containing a single nucleus. He maintained that a definite polar capsule
did not exist. Leger and Hesse (1916a), in the case of the- spores of
P. macrospora, N. bombycis, and other forms, described the polar capsule
as a large sac-like body occupying the greater part of the interior of the
spore, and the infective agent as a small mass of cytoplasm in a clear space
at the posterior end of the spore. The polar filament was coiled within
the polar capsule. The band of cytoplasm described by other observers
appeared to be nothing more than the retracted and distorted polar
capsule itself, and the supposed nuclei in it optical cross-sections of the
coiled filament. Kudo (1920) found that the spores of Stempellia magna,
which, on account of their large size, were very suitable objects of study,
were constructed as Leger and Hesse maintained (Fig. 313). The spores
of the genus Mrazekia, as described by Leger and Hesse (1916), are formed
on the same plan, with the exception that the proximal part of the polar
filament is thickened as an axial manubrium (Fig. 317). As regards the
minute structure of the smallest spores (Cocconema, Toxonema, Spirillo-
nema), nothing is known (Fig. 318).
It is probably safe to assume that the spores of Microsporidiida have a
large polar capsule occupying the bulk of the interior of the spore, and
CLASSIFICATION
737
that the infective agent, containing one or two nuclei, lies behind the polar
capsule in the clear space at the posterior part of the spore. The envelope
of the spore consists, in some cases at least, of two valves. The develop-
ment of the spore from the single cell (sporoblast) which gives rise to it
appears to be a very complicated one. A number of cells are formed, as
in the development of the spores of Myxosporidiida, and some of these
give rise to the outer covering of the
spore; others form the polar capsule and
infective amoeboid bodv.
Subdivision of the Microsporidiida.
Doflein (1901) classified the Micro-
sporidiida on the basis of the number of
spores produced by each pansporoblast,
but Leger and Hesse (1922a) point out
that many forms are far from constant in
the number of spores produced, a fact
previously noted by Chatton and Krempf
(1911). It is maintained that the only
constant feature on which a classification
can be based is the character of the spore
itself, and, as in Kudo's classification of
the Myxosporidiida, they propose a system
which has the character of the spore as
its basis. They divide the Microsporidiida
into two groups — the Monocnidea, which
have spores with one polar capsule; and
the Dicnidea, with spores with two polar
capsules. The former appear to be of two ^
types. In the one the parasite is a multi-
nucleate cytoplasmic body, which is con-
stantly increasing in size, and when de-
veloping often includes the nuclei of tissue cells
certain uninucleated cells (pansporoblasts or sporonts) become separated in
vacuoles in the parent cytoplasm, and these give rise to spores (Fig. 304, D).
Parasitesof this type are called Polysporogenea (family Glugeidse) by Doflein
to distinguish them from those of the second type, Oligosporogenea (family
Nosematidae), which in the vegetative stage are uninucleate bodies multi-
plying by binary fission or schizogony. Finally, as in the Polysporo-
genea, pansporoblasts are formed, and these give rise to a varying number
of spores (one to sixfeen or more). It must be admitted, however, that
I. 47
Fig. 31.3. — Structure of Micro-
spoRiDiAN Spores. (1 and 2,
AFTER Kudo, 1920; 3, after
Leger and Hesse. 1916.)
1-2. ^Tpore oi Stempellia magna (x 2,360).
1. Extruded filament, polar capsule,
and infective body-
2. Same before extrusion of filament.
3. Spore of Plisto'phora Diacrospora
(x 2,500). Polar capsule with
coiled filament and infective
body with two nuclei.
As in the Myxosporidiida,
738 OKDER: MICROSPORIDIIDA
there is some doubt as to the exact method of spore formation in the Poly-
sporogenea, which in many cases give rise to tumour-like structures in
fish, in which the parasites and the host tissues are so closely intermingled
(diffuse infiltration) that the details of development are difficult to follow.
The Oligosporogenea are more readily studied, as the small uninucleate
parasites are scattered through the cytoplasm of cells.
The following classification of the order Microsporidiida is based on
that suggested by Leger and Hesse:
1. Suh-Onler: Monocnidea Leger and Hesse, 1922.
The spore, which varies in shape, has only a single polar capsule.
(1) Family: GLUGEID^ Gurley, 1893.
The spores, ovoid or pyriform in shape, are developed from pansporo-
blasts formed in vacuolic spaces in the cytoplasm of the parasite, which
continues to grow and produce more nuclei as spore formation is pro-
ceeding. Each pansporoblast gives rise to two sporoblasts, and finally to
two spores.
Genus: Glugea Thelohan, 1891.
(2) Famihj : NOSEMATlDyE Labbe, 1899.
The spores, which are ovoid or pyriform in shape, are developed from
uninucleate rounded bodies which are the products of multiple or binary
fission of the vegetative forms. Each uninucleate body, which is a
pansporoblast, gives rise to a varying number of spores, which may or
may not be enclosed in a capsule.
Genus: Nosema Niigeli, 1857.
Each pansporoblast gives rise to a single spore.
Genus : Perezia Leger and Duboscq, 1909.
Each pansporoblast gives rise to two spores.
Gemis: Gurleya Doflein, 1898.
The spores are elongated, being broad at one end and somewhat
tapering at the other. Each pansporoblast gives rise to four spores.
Genus: Thelohania Henneguy, 1892.
Each pansporoblast typically gives rise to eight spores, but sometimes
only four or as many as sixteen are formed.
CLASSIFICATION 739
Germs: Stempellia Leger and Hesse, 1910.
Each pansporoblast gives rise to one, two, four, or eight spores, which
vary in length from 2 to 6 microns. The smallest spores occur when eight
are formed, and the largest when there is only one.
Genus : Duhoscqia Perez, 1908.
Each pansporoblast gives rise to sixteen spores.
Genus: Plistophora Gurley, 1893.
Each pansporoblast gives rise to many spores (more than sixteen).
(3) Family: coccoNEMiD^ Leger and Hesse, 1921.
The spores are spherical and resemble cocci.
(Te«».s' .• Cocconema Leger and Hesse, 1921.
(4) Family: MRAZEKiiD^ Leger and Hesse, 1922.
The spores are cylindrical, and are either straight, spiral, or curved.
They resemble bacilli, vibrios, or spirilla.
Genus : Mrazekia Leger and Hesse, 1916.
The spores are cylindrical, like bacilli. Each has an axial manubrium,
which can be extruded from one end of the spore. The polar filament is
attached to the end of the manubrium. Each pansporoblast gives rise to
one or more spores.
Genus: Octosporea Flu, 1911.
The spores are slightly arched and cylindrical, like bacilli, but there is
no axial manubrium. Each pansporoblast gives rise to eight or sixteen
spores.
Genus: Toxonema Leger and Hesse, 1922.
The spore is arched and resembles a vibrio. The ^pansporoblast gives
rise to eight spores.
Genus: Spirillonema (=Spironema Leger and Hesse, 1922).
The spores are spiral and resemble spirilla. The pansporoblast gives
rise to eight spores.
2. Sub-Order: Dicnidea Leger and Hesse, 1922.
The spore is oval in outline, and possesses two polar capsules, one
at each end of the spore.
Family: telomyxid.b Leger and Hesse, 1910.
Genus: Telomyxa Leger and Hesse, 1910.
740 ORDER: MICROSPORIDIIDA
DETAILED DESCRIPTION OF CERTAIN GENERA AND SPECIES.
Genus: Glugea Thelohan, 1891.
The members of this genus are typically parasites of fish, but they
occur also in reptiles, frogs, and worms. Their characteristic feature is
that they occur as multinucleate plasmodia, as a rule embedded in or
infiltrating the tissues. The ovoid spores are produced from pansporo-
blasts, which are separated in vacuoles in the multinucleate plasmodium.
Sometimes they occur free in the body-cavity spaces. In this respect
they resemble the Myxosporidiida (Fig. 304, D).
Glugea anomala (Moniez, 1887). — This is a parasite of the tissues and
organs of various fresh-water fish, chiefly the sticklebacks (Gasterosteus),
on the skin of which it gives rise to white nodules. On section such a
nodule is seen to have a fibrous capsule, within which is a multinucleate
cytoplasmic body. The central part of the nodule is occupied by numerous
ovoid spores, and these are also present in vacuoles in the peripheral cyto-
plasmic part. There are also present a number of large nuclei, which appear
to be the nuclei of the tissue cells which have been almost completely
destroyed. The spores are ovoid, and measure, as a rule, from 4 to 4*5
by 3 microns (Fig. 381, i). The polar filament may be 150 microns long.
Germs : Nosema Nageli, 1857.
This genus includes Microsporidiida, which in the vegetative phase
resemble the members of the genus Thelohania. The uninucleate pan-
sporoblast by a complicated process of development gives rise, however,
to a single spore.
Nosema bombycis Nageli, 1857. — This parasite, which is the best-
known member of the genus, gives rise to the notorious silkworm disease.
Its life-history was studied by Stempell (1909). The infection is com-
menced by the small amoeboid body which escapes from the spore after its
ingestion by a silkworm Bomhyx mori (Figs. 312 and 314). It multiplies in
the intestine. The resulting parasites, which are uninucleate, pass between
the epithelial cells into the hsemocoele space, and thence into the various
tissues of the body, including the ovary. These stages were called planonts
by Stempell. They eventually enter the cytoplasm of cells and become
meronts, which multiply by binary fission, gemmation, or schizogony.
The products of this multiplication are often arranged in rows like a string
of beads. After the cytoplasm of the cell is exhausted, the uninucleate
forms become transformed into spores. Four nuclei are formed in each,
and two of these with some of the cytoplasm form the spore capsule, while,
of the two remaining, one takes part in the formation of the terminal
FAMILY: NOSEMATID^
741
polar capsule, and the other becomes the nucleus of the infective amoeboid
form or sporozoite, which escapes from the spore when it is taken into the
intestine of a new host. The spores are so minute and the capsule so
thick that the details of the development are exceedingly difficult to follow.
Another form, Nosema apis Zander, 1909, with spores measuring 1-6 to 6-4
by 2-5 to 3-4 microns, is supposed to be the cause of bee disease (Fig. 30),
y^'^J^3K%^''
'"^^^■'-v-5-'fi»U»''^i'
Fig. 314. — Nosema honibycis : Developmental Stages and Spoke Formation in
Intestinal Epithelial Cells of Silkworm {xca. 2.000). (After Stempell, 1909.)
while N. frenzelincB Leger and Duboscq, 1909, parasitizes a gregarine
{Frenzelina conformis), which is itself parasitic in the crab, Pachygrctjjsus
marmoratus.
Genus: Gurleya Dofiein, 1897.
The members of this genus have elongate spores, which are broader
at one end than at the other.
Gurleya francottei Leger and Duboscq, 1909. — This organism is
parasitic in the epithelium of larvae of Ptychoptera contaminata. The
pansporoblast gives rise to four spores, which are radially arranged
(Fig. 315).
Genus : Thelohania Henneguy, 1892.
The members of this genus occur as minute parasites in the cytoplasm
of cells of aquatic invertebrates. There is little tendency to the produc-
tion of multinucleate plasmodia. Multiplication is usually by binary
742
ORDER: MICROSPORIDIIDA
fission, but sometimes division into uninucleate forms does not take place
till a larger number of nuclei is present. Eventually, a uninucleate form
becomes a pansporoblast, and produces typically eight ovoid spores which
:"\
Fig. 315. — Gurleya francottei. Parasitic in Intestinal Epithelium of Larvae of
Ttyclwptera ( x ca. 2,000); Stages of Development of Individual Parasites
AND Section of Intestinal Epithelium, showing Parasites in situ. (After
Leger, L., and Duboscq, 0., 1909.)
a-f. Stages in multiplication by binary fission. g. Form with two unequal nuclei.
h-iii. Division of nuclei in pansporoblast and formation of four spores.
are enclosed in a thin capsule derived from the superficial layer of the
pansporoblast.
Thelohania varians (Leger, 1897). — This is a common parasite of the
larvse of Simulium reptans and S. ornatnm, which are often heavily
infected. The cells of the body are seen to be filled with uninucleate
FAMILY: NOSEMATIDiE
743
parasites, forms with two nuclei about to divide, and some multinucleate
forms which divide into uninucleate forms. Division may take place in
such a way that rows of small forms are produced. In addition to the
vegetative forms, the cells contain spores in clusters of eight and others
more irregularly arranged. The spores vary in length from 4 to 5 microns.
Another species, T. chcetogastris, described by Schroder (1909), is parasitic
in an oligochsete worm (Fig. 316).
-Thelohania chcetogastris. Parasitic in the Oligoch.ete Worm,
Ghcetog aster diaphanus. (After Schroder, 1909.)
1. Connective tissue cell containing three schizonts and spores ( x ca. 1,500).
2. Muscle cell with reproducing forms ( X ca. 1,500).
3-7. Stages in formation of eight spores from the single pansporoblast ( x ca. 2,500).
Genus: Stempellia Leger and Hesse, 1910.
Sternpellia mutabilis Leger and Hesse, 1910. — This parasite occurs in
the cells of the fat body of the nymph of Ephemera vulgata. It resembles
very closely a Thelohania, except that the pansporoblasts give rise to
one, two, four, or eight spores. The latter vary in length from 2 to 6
microns, the largest spore being formed when the pansporoblast gives rise
to only one spore and the smallest when it gives rise to eight. The only
other member of the genus is S. magna (Kudo, 1920), parasitic in larvae
of Culex j)ipiens and C territans in North America.
Genus: Duboscqia Perez, 1908.
Duboscqia legeri Perez, 1908. — This form is a parasite of the termite
Termes lucifigu.s. It gives rise to white nodules up to 500 microns in
diameter in the body cavity. Each nodule shows a peripheral layer of
multinucleate cytoplasm, within which are a number of large nuclei up
to 60 microns in length. The latter are probably the hypertrophied nuclei
744 ORDER: MICROSPORIDIIDA
of the infected cells. The central part of the nodule is occupied by
pansporoblasts measuring 12 by 7 microns, and groups of sixteen spores
enclosed by a capsule. Each group is developed from one pansporo-
blast. The spores, which are ovoid, measure 5 by 2-5 microns. This
genus is evidently closely related to Thelohania, with which it may be
identical.
Genus: Plistophora Gurley, 1893.
The Microsporidiida belonging to this genus are found in fish. They
produce small white nodules in the tissues. The pansporoblast gives rise
to more than sixteen spores.
Plistophora typicalis Gurley, 1893. — This parasite occurs in the
stickleback and other fresh-water fish, in which it gives rise to whitish
nodules in the muscles. These are 25 to 35 microns in diameter. Each
pansporoblast gives rise to numerous (more than sixteen) ovoid spores,
which eventually fill the nodules. Other forms are P. sttgoynyice of
Stegomyia fasciata {Atdes argenteus), and P. simulii of larvse of Simulium.
Genus: Cocconema Leger and Hesse, 1921.
The Microsporidiida belonging to this genus are characterized by their
spherical spores, which resemble cocci (Fig. 318). Leger and Hesse (1921)
have described four species from aquatic larvse or worms.
C. micrococcus occurs in the fat body of the larvse of Tanyjnis setiger.
The spore has a diameter of 1-8 to 2 microns.
C. polyspora occurs in the same host and in the same situation, but the
spores are larger, varying in diameter from 2 to 3-2 microns.
C. octospora, the spores of which have a diameter of 2-1 microns, is
found in the intestinal epithelium of larvse of Tanytarsus sp.
C. slavinae with spores 3 microns in diameter, occurs in the intestinal
epithelium of the aquatic worm, Slavina appendiculata.
Kudo (19246) recognizes two other species — C. stempelli and C. miyairii.
Genus: Mrazekia Leger and Hesse, 1916.
The members of this genus produce cylindrical spores which, in addition
to a polar filament, possess a manubrium, which may be regarded as the
thickened proximal part of the filament (Fig. 317). The manubrium
occupies the central axis of the spore, and the filament is coiled round it.
Both the manubrium and filament are extruded. When spore formation
takes place, a single uninucleate cell or pansporoblast gives rise to one,
four, eight, or sixteen spores.
M. caudata Leger and Hesse, 1916. — This species is parasitic in the
lymphocytes of aquatic worms of the genera Tubifex and Limnodrilus.
FAMILY: NOSEMATIDtE
745
The spore, which measures 16 to 18 microns in length and 1-3 to 1-4 in
breadth, has the end opposite that from which the filament is extruded
drawn out into a pointed process as long as the spore itself. The pan-
sporoblast gives rise to one spore (Fig. 317, i).
M. brevicauda Leger and Hesse, 1916. — This form occurs in the fat
body of larvae of Chironomus flumosus. The spore, which measures
20 to 30 microns by 1-4 to 1-5 microns, has a short pointed process.
The jjansporoblast gives rise to one spore (Fig. 317, 2).
M. striata Leger and Hesse, 1916. — This form occurs in the lympho-
cytes of the aquatic worm, Lumhriculus variegatus. The spores, which
Fig. 317.-
4
-Spores of Microsporidiida of G-enus Mrazelia ( x 1,750). (After
Leger and Hesse, 1916.)
1. j\I. caudata. 2. M. brevicaudata. 3. M. stricta.
4. M. argoisi. 5. M. argoisi : manubrium extruded.
6. M. argoisi : complete extrusion of manubrium and polar filament.
measure 13 to 14 microns by 1-8 to 2-0 microns, have no process. Each
pansporoblast gives rise to one spore (Fig. 317, 3).
M. argoisi Leger and Hesse, 1916. — This is a parasite of the fat body
of the fresh-water crustacean, Asellus aquaticus. The spores, which have
no pointed process, measure 17 to 23 microns by 3-5 microns. Each
pansporoblast forms a single spore (Fig. 317, 4-6).
M. bacilliformis Leger and Hesse, 1922.— This species is a parasite
of the fat body of larvae of OrtJiodadius sp. The spores measure 5 by 0-8
microns, and each pansporoblast gives rise to eight spores.
M. tetraspora Leger and Hesse, 1922. — This form occurs in the fat
body of larvae of Tanypus sp. The spores are 6-5 to 8 microns in length
746 ORDEK: MICEOSPORIDIIDA
by 0-8 micron in breadth. There is a short hyaline prolongation 1-2 microns
long at one end of the spore. The pansporoblast gives rise to four
spores.
M. niphargi Poisson, 1924. — This is a parasite of the amphipod^
Niphargus stygius. The spores measure 8 to 9 by 2 microns. The
pansporoblast gives rise to eight or sixteen spores.
M. piscicola Cepede, 1924.— This is the first species of the genus to
be described from a vertebrate. It occurs in the pyloric coeca of the
whiting, Gadus merlangus.
A closely allied, if not identical, genus is Myxocystis Mrazek, 1897.
These Microsporidiida give rise to white spheres, often ciliated externally^
which float about in the body-cavity spaces of aquatic worms {LimModrihis),
Mrazek (1897), who first described these forms, later (1910) demonstrated
that the white spheres were agglomerations of wandering cells, the cyto-
plasm of which was infected with uninucleate Microsporidiida, which
multiplied by binary fission. Each uninucleate pansporoblast gave rise
to a single spore. The spores are ovoid, the narrow end being drawn
out into a kind of neck. The spores of M. mrazehi Hesse, 1905, parasitic
in Limnodrilus hoff^neisteri, measure 9 to 10 microns by 1 to 2 microns.
Genus: Octosporea Flu, 1911.
This genus was created by Flu (1911) for a parasite of the intestinal
epithelium of the house fly. He thought it was a schizogregarine, but
Chatton and Krempf (1911) proved that it belonged to the Microsporidiida.
The spores are bacillary and slightly curved. No details of their structure
could be made out in the fresh condition, and there was no indication of
a manubrium. The pansporoblast gives rise to eight or exceptionally
sixteen spores.
Octosporea muscae domesticae Flu, 1911. — This parasite was first seen
by Flu (1911) in various tissues of the house fly. Chatton and Krempf
(1911) saw it in Drosophila confusa and D. plurilineata, and first realized
that it was a microsporidian. The young forms, 3 microns in diameter,
occur in the epithelial cells of the intestine. Reproduction takes place
by schizogony, forms with as many as thirty-two nuclei occurring. The
spores are 5 to 6 microns in length by 1 micron in breadth. The only other
species is 0. ^nonospora Chatton and Krempf, 1911, parasitic in the same
species of Drosophila and Homalomyia scalaris. The spores are 4 to 5
microns in length.
(ze»)»s; Toxonema Leger and Hesse, 1922.
Toxonema vibrio Leger and Hesse, 1922. — This parasite is the only
member of the genus (Fig. 318). The total length of the spore is 3-5
FAMILY: TELOMYXID^ 747
microns, and it is curved so that the distance between its two ends is
2 microns. Each pansporoblast gives rise to eight spores T. vibrio
occurs in the fat body of larvas of species of Ceratopogon.
Genus : Spirillonema.
Leger and Hesse (1922a) suggested the generic name Spironetna for
those Microsporidiida which have spiral spores resembling spirilla (Fig. 318).
As the name Spironetna was given by Klebs (1892) to a flagellate, the
name SpiriUonetna may be used.
Spirillonema octospora (Leger and Hesse, 1922). — This parasite, the
only member of the genus, is found in the fat body of larvae of Ceratopogon.
The spiral spore is 8 to 8-5 microns long and 1 micron wide. Each pan-
sporoblast gives rise to eight spores (Fig. 318).
4{;* ^'-/3 \l^ S^(
'•fi% '-Z^^^ ^1" ^'^*
G
Fig. 318. — Spores of Microsporidiida. (After Leger and Hesse, 1922.)
1-4. Bacterial types of spore deeply stained as they appear in smears or sections of
tissue ( X 1,000).
5-12. Types of spore to show some details of structure ( x 3,000).
1, 5, 6. Cocconenia. 2, 12. Toxonema vibrio.
3. Mrazekiahacilliformis. 4, 11. Spirillonema octospora.
7. Glugea. 8, 9. Telomyxa. living and stained. 10. Mrazekia tetrasporn.
Genus : Telomyxa Leger and Hesse, 1910.
Telomyxa glugeiformis Leger and Hesse, 1910. — This form, which is
the only representative of the genus, occurs as a parasite of the cells of
the fat body of the larvae of Ephemera vulgata (Fig. 318). The spores are
ovoid, and measure 6*5 by 4 microns. There is a polar capsule at each
end of the spore. After the multiplicative phase, certain uninucleate cells
become pansporoblasts, and each gives rise to groups of eight, sixteen, or
more cells, which become transformed into spores. Leger and Hesse
regarded this organism, the spores of which have two polar capsules, as
a connecting link between the Myxosporidiida and Microsporidiida.
748
OKDER: MICROSPORIDIIDA
Microsporidiida of Blood-Sucking Arthropoda and Nematoda.
As already remarked, the Microsj^oridiida are parasites chiefly of Arthro-
poda, and some of the forms which may be encountered in experimental
work will be considered briefly. It is important to remember these
organisms when insect flagellates are being studied. They may be con-
fused very readily with the minute leishmania forms of certain flagellates,
as pointed out by Chatton (1911a) and Shortt (1923), a mistake which
undoubtedly has been made on more than one occasion.
MOSQUITOES.— Hesse (1904, 1904a) found a parasite in the cells of
the fat body of larvse of Anopheles tnaculipennis in France. The spores
_ of this organism, which Hesse named Thelohania
legeri, measured 8 by 4 microns. The filament,
which was extruded from the spores when placed
in iodine water, measured 50 microns in length.
The mosquito larvse seemed to be unaffected by
the presence of the parasite. Another species,
T. illinoisensis, was described by Kudo (1921)
from the larvse of A. punctipennis and A. qua-
drimaculatus of North America. It was very
similar to Hesse's species, T. legeri. The spores,
however, appeared to be smaller (4-75 to 6
microns), while the filament was longer (60 to
97 microns). In a later paper Kudo (1924)
describes this form in detail, and compares it
with T. legeri in films from A. maculipennis
and A. bifurcatus larvae sent to him by Hesse.
He comes to the conclusion that T. illinoisensis,
which occurs in larvse of A. crucians, as well as
the mosquitoes mentioned above, is identical
with T. legeri. It appears to be a parasite
specific to larvse of Anopheles, and develops in
the cells of the fat body. Reproduction takes
place by repeated binary fission. Eventually,
forms with four nuclei are produced (Fig. 319).
These divide into two sporont mother cells, each with two nuclei. The
two nuclei divide, and this is followed by division of the mother cell,
so that again stages with two nuclei are produced. The two nuclei then
fuse, and at the same time fine chromatin granules appear in the cyto-
plasm. The cell with a single nucleus is the sporont, which by successive
nuclear division, the first of which is mitotic, reaches an eight-nuclear stage.
Within it eight sporoblasts are formed, and each of these becomes a spore.
Fig. 319. — Diagram of
Nuclear Changes in
Late Schizogony and
Early Sporogony of
Thelohania legeri. (After
Kudo, 1924.)
The two nuclei in the final i^ro-
ducts of multiplication fuse to
form the nucleus of the sporont,
which eventually produces the
spores.
SPECIES IN ARTHROPODA
749
Kudo (1922a, 1924a) has given the name T. opacita to a parasite of
larvse of Culex testaceus {C. apicalis) and C. territans, also of North America
(Fig. 320). The name was suggested by the effect the parasite has on its
host, which becomes of an opaque white colour. Its developmental
Fig. 320. — Developmental Stages of Thelohania opacita (1-20, x 2,300,
21-23, X 2,360). (After Kudo, 1924.)
1-3. Binary fission. 4-6. Multinucleate forms.
7. Final binucleate product of multiplication.
8-10. Union of nuclei to form sporont.
U-Ki. Division of nuclei 1" U<vm ciulit. 17. Formation of eight sporoblasts
18. l'ans|)ciioM,isl ((inf.iiiiiiiu ciiilit young spores.
19. Pans|i(iiiil.l,i.st (■(iiitiiiiiiiiL.' •■iulit mature spores.
•20. Panspdio blast with four si^ores. 21-23. Normal-sized spores.
cycle is very similar to that of T. legeri. Reproduction is by repeated
binary fission. Finally, binucleate forms are produced. These, by fusion
of the nuclei, give rise to sporonts (pansporoblasts). The rounded
pansporoblast produces, as a rule, eight sporoblasts, which become spores.
750 ORDER: MICROSPORIDIIDA
These are ovoid in shape, and measure 5-5 to 6 by 3-5 to 4 microns.
The polar filament is 110 microns in length. Occasionally, the pansporo-
blast gives rise to only four sporoblasts, which produce correspondingly
larger spores, measuring 8 to 8-5 by 4-5 to 5-5 microns. The polar filament
in these cases reaches a length of 200 microns.
The same author (19246) describes as T. obesa a parasite of the fat body
of an anopheline {A. quadrimaculatus ?) larva. The pansporoblast, which
gives rise to a group of eight spores, is 9 to 10 microns in diameter. The
fixed and stained spore measures 4 to 4-5 by 3 to 3-5 microns.
Another species recorded by this author (19246) is T. pyriformis from
the fat body of larvae of A. crucians or A. quadrimaculatus. The fixed
and stained spore measures 3-5 to 4 by 2 to 2-8 microns. In the fresh
condition it appears considerably larger, and measures 4-8 to 5-4 by 2-7
to 3 microns.
Kudo (1920, 1921) described as T. magna a microsporidian of the
larvae of C. 'pvpiens and C. territans in North America. Later (19246)
he transferred it to the genus Stempellia (Fig. 313). It occurs in the cells
of the adipose tissue, and the larvae were heavily infected. The parasite
multiplies by binary fission or by schizogony. Finally, a division of a
parasite into four cells, which remain connected together, takes place.
A further division of each of these may occur. The resulting cells are
sporoblasts, which develop into spores. The spore measures 12-5 to 16-5
by 4 to 4-6 microns. The extruded filament may reach a length of 350
to 400 microns.
Two other species of Thelohania are recorded by Kudo (19246) from
Culex leprincei of North America. One of these is named T. rotunda.
The spore is broadly ovoid or sub-spherical, and when fixed measures
2-5 to 3 by 2-3 to 2-7 microns. The other, T. minuta, has an ovoid spore
measuring when fresh 3-5 to 3-7 by 2-4 to 2-7 microns, and when fixed
2-5 to 3-3 by 1-5 to 2 microns. Both occur in the adipose tissue of the
larvae, while T. minuta has been found in the pupae also.
The writer has seen a Thelohania which was discovered by MacGregor
in larvae of Aedes (Ochlerotatus) nemorosus in England. The fresh spores
measured 6 to 7 by 4 to 4*5 microns. The parasite occurred chiefly in the fat
body, and appeared to be specific for the larvae of this particular mosquito,
as the larvae of other mosquitoes, including other species of the same genus,
in the pond at the same time were not infected. Attempts to infect larvae
of 0. nemorosus from another locality and larvae hatched in the laboratory
failed, though enormous numbers of the spores were ingested. No infec-
tion of the body cavity took place. In the pond, in which at one time
early in March at least 50 per cent, of the larvae of this species were infected,
the infection gradually died out during the course of one month, though
SPECIES IN ARTHROPODA 751
there appeared to be every chance of its survival in the larvae of 0. nemo-
rosus, which were constantly present. It seems evident that infection
depends on certain conditions not at present known.
Another form described by Kudo (19246) is Nosema anophelis, a
parasite of larvae and adults of A. quadrimaculatus. In the larvae it
occurred in the gastric pouch, and in the adults in the epithelial cells of
the anterior part of the mid-gut and in the neighbouring fat body. The
young forms, which reproduce by binary fission, are 1-5 microns in
diameter. Each pansporoblast produces a single spore, which measures
from 4-7 to 5-8 microns in length by 2-3 microns in breadth. The filament
is 50 to 60 microns in length.
Marchoux, Salimbeni, and Simond (1903) described as N. stegomyiw
a parasite of the larvae and adults of A'edes argenteus (Stegomyia fasciata)
in Brazil. It occurs in the intestine, body cavity, and tissues of the
posterior part of the body, including the ovaries. It is supposed that
two kinds of spore are produced, the one colourless, the other brown.
The colourless spore gives rise to multinucleate plasmodia up to 40 microns
in diameter, and the brown spore to long filaments. There is considerable
doubt regarding the description of the parasite, the microsporidian nature
of which has not actually been demonstrated. Chatton (1911a) placed the
parasite in the genus PlistopJiora.
Bresslau and Buschkiel (1919) recorded as Thelohania sp. a parasite
of larvaB of Theobaldia annulata in Germany. Noller (19206) mentions the
occurrence of a parasite which he supposes to be Thelohania legeri in
larvae of A'edes nemorosus in Germany. As T. legeri, according to Kudo
(1924), is specific for Anopheles, it is probably some other species. Ndller
also mentions Nosema sp. as occurring in A'edes nemorosus and A. cantans.
Bresslau and Buschkiel (1919) gave the name iVosema culicis to a parasite
of larvae of Culex pipiens. The spores measured 4-5 to 5-5 by 1*8 to 2-4
microns. What are possibly spores of Microsporidiida were seen by Pfeiffer
(1895) in larvae of Culex sp. in Germany, by Grassi (1900) in larvae and
adults of Anopheles sp. in Italy, and by Ross (1906) in adults of C . fatigans
and Aedes sp. in India. Kudo (1921), who has reviewed the literature
dealing with Microsporidiida of mosquitoes, doubts if these are, in most
cases at least, true Microsporidiida.
SIMULIUM. — The larvae of various species of Simulium are very liable
to infection with Microsporidiida. Heavy infections occur, so that the
larvae often appear swollen and white in colour, while in some cases actual
nodular tumours are produced. The first form noted was one which
occurred in S. ornatuni, and was named Glugea variants by Leger, L.
(1897). The parasite was studied by Debaisieux (1919a), who found it
also in S. reptans. He transferred it to the genus Thelohania. It occurs
752 ORDER: MICROSPORIDIIDA
in the body cavity and adipose tissue, and produces spores measuring
6-5 to 8 by 4-5 to 5-5 microns. Strickland (1913) in America named
three species which he placed in the genus Glugea. They were transferred
to the genus Thelohania by Debaisieux and Gastaldi (1919), who found
them in Belgium. T. bracteata and T. fibrata were found in S. venustum
and S. ochraceum in South America by Lutz and Splendore (1904, 1908),
in S. bracteatum and S. hirtipes by Strickland (1913) in North America,
and in S. maculata by Debaisieux and Gastaldi (1919) in Belgium. The
parasites occur in the fat body of the larvae. The spores of T. bracteata
measure 3 to 4 by 2-5 to 3 microns, and those of T. fibrata on an average
7 by 3-5 microns. Another species, T. ?nultispora, seen by Strickland
(1913) in S. vittatum and S. bracteatum, and by Debaisieux and Gastaldi
(1919) in S. maculata, also occurs in the fat body, but produces spores of
an intermediate size.
Lutz and Splendore (1904) included in their species Nosema simidii,.
which embraced the forms noted above, a parasite which Debaisieux and
Gastaldi (1919) placed in the genus Plistophora. It was seen in S. venustum
and S. ochraceum by Lutz and Splendore, and in S. tnaculata by Debaisieux
and Gastaldi. It produces regular rounded tumours in the tissues of the
larvse. The spores vary in size from 4-5 to 8-5 by 3-5 to 5-5 microns.
FLEAS. — These arthropods are also liable to infection with Micro-
sporidiida. Noller (1912) found a form which he named Nosema pulicis in
the salivary glands, Malpighian tubes, and fat body of the dog flea (Ctenoce-
phalus canis) in Germany. The oval spores measured 2-5 to 5 microns
in length by 1-5 to 2 microns in breadth. The polar filament was 65 to 85
microns long. Another form was described by Korke (1916) from the dog
flea in India. He suggested the name N . pulicis, but as the spores are
smaller than those of N. pulicis, Kudo (19246) has given it the name
iV". ctenocephali. Shortt (1923), in a study of Leptomonas ctenocephali of
the dog flea, has drawn attention to the care which must be exercised in
distinguishing the spores of N. ctenocephali from leishmania forms of the
flagellate.
BED BUGS.—Certain small ovoid bodies which Adie (1922, 1922a)
found in the salivary glands and other tissues of bed bugs in India, and
which were regarded as stages of Leishmania donovani, are, according to
Christophers (1922), Microsporidiida, for which he proposes the name
Nosema adiei (see p. 420). The parasite has been described by Shortt and
Swaminath (1924a), who have also met with it in bugs in India. The
intestine is most commonly infected. The spores are ovoid or elliptical
bodies measuring 3 by 1-7 microns. Minute small amoebulse 1-6 microns
in diameter occur, as also larger forms measuring 3-2 by 2-7 microns.
SPECIES IN NEMATODA
753
These free forms in dried films stained by Romanowsky stain have blue
protoplasm and one or two red chromatin areas. When there are two of
unequal size, some resemblance to Leishmania donovani may result, but
the smaller of the two red areas is never rod-shaped.
NEMATODES.— Lutz and Splendore (1908) gave the name Nosema mystacis
to a parasite of the intestinal cells and reproductive organs of Ascaris
Fig. 321. — Thelohania reniformis, Parasitic in Intestinal Epithelium
OF Protospirura muris {\, x 1,560; 2-15, x 2,200). (After Kudo, 1923.)
1. Four cells of intestinal epithelium showing various stages of parasite.
2. Fresh spore. 3. Stained spore.
4-8. Growth and nudtiplication by binary fission.
9-14. Growth of pansporoblast and formation of eight spores.
15. Spore with e.xtnidcd filament.
tmjstax of Brazilian cats. The bodies seen by Bischoff (1855) and Kefer-
stein (1861) in the same helminth were possibly spores of this species.
Kudo and Hetherington (1922) have described as Thelohania reniformis
a parasite which they found in the lining cells of the intestine of Proto-
spirura muris, a common helminth of the stomach of mics (Fig. 321). The
I. 48
754 OKDER: MICROSPORIDIIDA
spores are reniform, and measure 3 to 4 by 1-5 to 1-8 microns. The
polar filament is 45 to 55 microns in length. The pansporoblast gives rise
to eight sporoblasts, which become eight spores.
Supposed Microsporidiida in Rabies and Encephalitis of Rabbits
and Mice.
Wright and Craigliead (1922) observed a form of paralysis in young rabbits,
and found that it was due to an organism which they thought might be a Protozoou.
It was found in most of the tissues of the body, but was specially noticed in the
kidneys and urine and in nerve cells of the spinal cord, which were quickly destroyed
a
Fig. 322. — Encephalitozoon cuniculi in Section of Brain of Rabbit (x950).
(After Da Fano, 1924, from Journ. Path, and Bad.)
Parasites in cysts which may be merely vacuoles in macrophages.
by its presence. The same disease of rabbits had been observed by Bull (1917),
Oliver (1922), and Twort (1922). They described the changes in the nervous system
without associating them with any particular organism. In attempts to reproduce
human diseases, encephalitis lethargica and herpes, in rabbits, the naturally occurring
disease of rabbits and the associated organism have given rise to some confusion.
Doerr and Zdansky (1923) described the lesions in the brains of rabbits inoculated
with the virus of encephalitis lethargica, and discovered that similar lesions occurred
in uninoculated animals. They saw in the brain certain small bodies which they
thought were probably parasites resi)onsible for a disease of rabbits which was being
ENCEPHALITOZOON 755
confused with encephalitis lothargica. Later in the year Levaditi, Nicolau and
Schoen (1923) also saw the organism, and recognized it as the cause of an ence-
phalitis of rabbits which had no connection with the human disease. They gave it
the name Encephalitosoon cunieuli, and expressed the opinion that it was a micro-
spordian. Doerr and Zdansky (1923rt, b) then gave a clear description of the
organism. They noted that it occurred in the form of spores, which were either
distributed through the tissues of the brain or collected together in cysts. Levaditi,
Nicolau and Schoen {1924a) have given a complete review of the subject and de-
scribed their own results. They were able to inoculate the organism to rabbits, rats,
mice, and dogs. In rabbits it was found only in the brain and kidneys, though
Wright and Craighead (1922) had observed it also in the spleen, liver, and myo-
cardium, as also in the urine, which suggested to them a possible source of infection.
Levaditi and his co-workers have actually demonstrated the infectivity of the urine.
'7%
Fig. 323. — Encephalitozoon cunieuli in Atrophying Nerve Cell and scattered
THROUGH THE BrAIN SUBSTANCE ( x 1,200). (AfTER Da FaNO, 1924.)
During the course of their experiments they (1924) discovered that mice were liable
to a similar infection. Cowdry and Nicholson (1924) have also observed an organism
in mice, which appears to be morphologically identical with that in rabbits, and gives
rise to similar lesions in the central nervous system. The lesions consist of menin-
gitis of the cortex and septa of the brain, perivascular infiltration of the blood-
vessels, and nodules composed of masses of cells which may be necrotic centrally.
Marked changes occur in the kidneys, especially in the heavily infected epithelium
of the tubules, and in the liver and spleen.
In the infected organs the parasites are either scattered through the tissues or
enclosed in masses in what are called cysts (Figs. 322, 323). It appears more probable
that these are the remains of endothelial or other cells, for often a large flattened
nucleus can be seen on the cyst wall. In fact, Wright and Craighead describe the
infected cells as being reduced to membranes containing the organisms. By the
rupture of the enclosing membrane the spores are scattered through the tissues.
756 OEDEK: ACTINOMYXIDIIDA
The individual parasite is a small ovoid body about 2-5 microns in length by
0-5 to 1 micron in breadth. At the end there are one or two chromatin-like granules.
In many respects it resembles a small yeast, but reproduction by budding has not
been observed. Levaditi and his co-workers believe that they have demonstrated
small cytoplasmic bodies (pansporoblasts), which give rise to the spores, and they
conclude that the parasite is a microsporidian, in spite of the fact that these parasites
have never been found in warm-blooded vertebrates. They have not demonstrated
the presence of a polar capsule and filament, while their account of the development
of the spore requires confirmation. It seems premature to conclude that the
oi-ganism is even a Protozoon. The writer in 1909 saw what was evidently the same
organism in sections of the brain and liver of a rabbit, but was unable to arrive at any
conclusion regarding its nature. Da Fano (1924) has given a good description of
the organism and the lesions it produces in the brain of rabbits in England, and
Smith and Florence (1925) its appearance in the kidneys. Goodpasture (1924) has
seen it in the lungs.
Levaditi, Xicolau and Schoen (1924?>) suggest that the virus of rabies is probably
a microsporidian which enters the body in some invisible stage, and produces
eventually the Negri body. Manouelian and Viala (1924) go even further, and claim
to have demonstrated in the cells of the brain and salivary gland of dogs organisms
which are indistinguishable morphologically from those in the disease of rabbits
described above. They name the organism Encephalltozooii rabiei. Levaditi,
Nicolau and Sclioen (1924c), in a later paper, confirm the observations of Manouelian
and Viala, and claim that the Negri body is the cyst stage of the parasite. Ignoring
the specific name rabiei, they place it in the genus Gliigea as O. lyssce. Here, again,
there is no evidence that the organism is a microsporidian, as indeed Manouelian and
Viala suspect.
The presence of the parasite in mice, whatever its true nature may be, introduces
another fallacy into experiments which involve the discovery of Lslshmania in the
organs of animals inoculated with insect flagellates (see p. .395). The figures of
Encephalitosoon cunieuU and the similar parasite of mice show how easy it would be
for such parasites, when seen in smears stained with Eomanovsky stain, to be mis-
taken for Leishmania.
C. Order: ACTINOMYXIDIIDA.
The parasites included in this order (=Actinomyxi(lia Stole, 1899),
which were first seen and named by Stole (1899), occur in aquatic worms.
The spores are complicated structures consisting of a capsule composed of
three valves, each of which may be drawn out into a long spine which may
be bifurcated, so that there is a definite triradiate arrangement (Fig. 324).
Three polar capsules are present, and the mature spore contains a variable
number of amoeboid infective agents, often referred to as sporozoites.
The Actinomyxidiida have been studied by Stole (1899), Leger, L. (1904a),
Caullery and Mesnil (1905a), Ikeda (1912), and Mackinnon and Adam
(1924), to whose researches most of what is known of these parasites is due.
The development of the spore is a complicated process which resembles
that of the spores of Myxo sporidi da. It has been traced in certain species
by Caullery and Mesnil (1905a), Ikeda (1912), and Mackinnon and Adam
(1924). There are, aosording to Ikeda, the following five genera: Tetracti-
GENERAL ORGANIZATION
757
notnyxon, HexactinoDiyxon, Triactinomyxon, Synactmomyxon, and Sphcerac-
tinomyxon, which differ from one another in the character of the spores
and other details.
According to the observations of Mackinnon and Adam (1924) on
Triactinomyxon legeri in Tubifex tubifex, the life-history is as follows
(Fig. 325) : The pansporocyst, a spherical cyst of about 60 microns in
diameter, contains eight closely packed spores, the three tails of which are
Fig. 324. — Spores of Various Actinomyxidiida. (From Caullery and Mesnil,
1905, AFTER StOLC, LeGER, AND CaULLERY AND MESNIL.)
I. II exact inomyxon psammorocj/stis ( x 450). II. Triactinomyxon ignotum ( x 250).
Ila. Terminal portion of i^ipore of T. ignotum, showing eight " sporozoites " and three jiolar
cap.su les ( x 900).
III. Synactinomyxon tubificis. A, Surfacs view of spore with three polar capsules; B, side
view of spore ( x 900).
IV. Sphceractinotnyxon stole i. A, Side view of spore; B, end view, showing three jjolar capsules
( X 900).
g, Germinal mas.s; sp.., " sporozoite "; ti.e, nucleus of envelope cell; n.ii, nucleus of polar capsule
cell; u, polar capsule.
folded within the cyst membrane. By rupture of the cyst the spores are
liberated when the tails become extended, and the characteristic tri-
radiate arrangement is seen. The individual spore varies in size. Its
length up to the point where the three rays originate varies between
90 and 140 microns, while its breadth varies from 11 to 16 microns. The
rays also vary in length, but on an average this is 150 microns, while the
breadth is 8 to 14 microns. At the end of the spore are three polar capsules,
from which filaments can be extruded, while adjacent to them is a mass
758
ORDER: ACTINOMYXIDIIDA
of cytoplasm (spongioplasm), in which are embedded, in three columns
of eight, twenty-four sporozoites. In addition, the spongioplasm contains
three nuclei. The spongioplasm, either intact or segmented into several
Fig. 325. — Diagram of Life-History of Triactinomyxon ignoUim. (After
MACKINNON AND Adam, 1924, FROM THE Quavt. Jourti. Mic. Set.)
1. Sporozoite.
2-6 Formation of pansiiorocyst (3' to 5' stages in T. legeri given as alternative).
7-11. Formation of gametes and their reduction bodies (black dots).
12. Union of gametes.
13. Zygote and subsequent nuclear divisions (r.n., residual nuclei; g.»., nuclei of sporojjla.smic
mass; p.c.n., nuclei of polar capsule cells; sp.n., nuclei of sporal envelojje).
14. Young sjiore. 1.5. Ripe spore.
masses, moves from the region of the polar capsules towards the point of
origin of the rays, where a pore is probably present. Meanwhile, the
twenty-four sporozoites unite in pairs, giving rise to twelve binucleate
amoeboid bodies. The nuclei of these, though they may come into contact
GENERAL ORGANIZATION 759
with one another, do not fuse. These binucleate amoeboid bodies, which
are about 11 microns in diameter, escape into the intestine of the worm
and proceed to develop, so that auto-infection has to be recognized.
The two nuclei of each amoeboid body divide to form a total of four nuclei,
two of which migrate to the periphery and, together with some of the
cytoplasm, form the capsule which develops, while the two remaining
nuclei increase in size. The cytoplasm within the capsule, which increases
in size, divides to form two cells, while by a further division four cells are
produced, two of which are larger than the other two. By further
divisions, which proceed somewhat irregularly, eventually eight small cells
and eight large cells are formed. These are gametes, and it appears that
certain reduction bodies are discharged from their nuclei. The gametes
unite in pairs (anisogamy), forming eight zygotes within the cyst. Each
zygote, which, in addition to the nucleus, contains a granule of what
appears to be residual chromatin, develops into a three-rayed spore.
By a series of nuclear divisions, two, four, six, and then seven nuclei are
formed. Three of these migrate to one end of the cytoplasmic mass,
where they become the capsulogenous cells from which the polar capsules
are formed, three of them pass to the opposite pole and form the cells
which give rise to the three-rayed spore envelope, while the remaining
nucleus divides many times till twenty-seven nuclei are formed. Of these
nuclei, twenty-four are nuclei of sporozoites and three the residual nuclei
of the spongioplasm, in the substance of which the twenty-four sporozoites
are eventually developed.
In this development it will be seen that the union of the sporozoites is
not a syngamic process, as the nuclei remain distinct. When the nuclei
divide to form four nuclei, two of the nuclei, together with some of the
cytoplasm, form the cyst wall, which encloses the rest of the cytoplasm
containing two nuclei. Eventually sixteen gametes are produced from
these two cells, and it seems probable that eight of these, which are macro-
gametes, are derived from one sporozoite, while the eight microgametes
are formed from the other. Finally, there is a spherical cyst containing
eight zygotes. When each zygote has completed its development, the
spherical cyst contains eight closely packed spores. The worm Tubifex
tubifex appears to harbour at least four species of Triactinornyxon.
T. ignotum. Stole, 1899, has eight sporozoites in each spore; T. legeri
Mackinnon and Adam, 1924, has twenty-four; T. sp. Leger, 1904, has thirty-
two; and T. mrazeki Mackinnon and Adam, 1924, has more than fifty.
The cycles of development of these species resemble one another very
closely, and it appears probable that, apart from the difference in
shape of the spores, the members of the other known genera of
Actinomyxidiida develop in a similar manner. Granata (1925), who
760 SARCOSPORIDIA
recognizes a sixth genus, Neoactinomyxon, has recently published a
memoir on the morphology, development, and systematics of the
group.
PARASITES OF UNDETERMINED POSITION.
There are a number of organisms which are usually grouped with the
Cnidosporidia, though they do not show any of their main characters.
It is doubtful if some of them are Protozoa at all. The chief of these are
the Sarcosporidia, which are parasitic in the muscle fibres of vertebrates,
and have the form of elongate chambered bodies filled with sickle-shaped
spores; the closely related Globidium (cysts of Gilruth), which give rise to
nodules in the mucosa of the stomach and intestine of ruminants and
other animals; the Haplosporidia, which occur chiefly in invertebrates
in the form of uninucleate or multinucleate cytoplasmic bodies and
resistant spores; and the Serumsporidia, which are found in the body-
cavity fluids of aquatic Crustacea and larvae as small round cells which
multiply by binary fission or schizogony. The Rhinosporidia, which
produce nasal polypi, have been usually classed with the Haplosporidia,
but Ashworth (1923) has shown conclusively that they are fungi, and
not Protozoa, and this appears to be true of Globidium and possibly the
Sarcosporidia.
Sarcosporidia Biitschli, 1882.
The parasites included under this heading are regarded as belonging to
the genus Sarcocystis Ray Lankester, 1882, and are usually classed with
the Cnidosporidia; but, as noted above (p. 717), there are no adequate
grounds for this. They are often grouped as a separate order, Sarco-
sporidia Biitschli, 1882. They are found chiefly as parasites of the striated
muscle fibres, and less frequently of the unstriated fibres of mammals; but
a few forms have been recorded from birds and reptiles.
They have been found occasionally in man, and are very common in
sheep, cattle, and horses. A form which occurs in mice and rats has been
studied more than others on account of the fact that experimental infec-
tions can be produced by feeding the animals on infected tissues.
Morphology. — In whatever hosts they occur, the parasites are very uniform
in appearance, though the forms described vary considerably in size.
Since the discovery by Miescher (1843) of a form in muscle fibres of mice,
they have frequently been known as " Miescher's tubes," and the spores
as " Rainey's corpuscles." They are sometimes so small that they can
only be detected with the microscope. At other times they are seen as
tiny white streaks in the muscle fibres, while they may be as much as
5 centimetres in length. When seen entire after removal from the tissues,
GENERAL OEGANIZATION
761
each parasite has a whitish aj^pearance, is cylindrical with somewhat
pointed ends, and has a slightly lobulated surface (Fig. 326). Each is
normally embedded in a muscle fibre, but this ultimately degenerates, so
that the parasite is left in the connective tissue. There is an enclosing
membrane which, in the larger forms, shows a radial striation. It is not
quite clear whether this membrane is formed by the parasite or by the
tissues. Within the membrane a thin multinucleate layer of cytoplasm
is sometimes supposed to be present, and this layer
also contains vacuoles in which uninucleate cells /^/T
occur. In most cases it is impossible to detect
such a multinucleate cytoplasm, and all that can
be seen is a layer of uninucleate cells lying in
spaces in a homogeneous material which may,
however, be cytoplasm. This material gives rise
to a series of septa, which divide the bulk of the
parasite into a number of chambers (Fig. 327).
Those near the membrane contain single spherical
cells, and are smaller than the ones nearer the
centre of the parasite. The more deeply situated
chambers are larger and contain a number of
round cells, while those that are fully developed
contain a variable number of characteristic
sickle or crescent-shaped bodies usually called
spores. These are covered by a delicate mem-
brane, which cannot be compared with the re-
sistant covering of the spores of Microsporidiida.
In old parasites, the central part usually consists
of a space filled with free spores in various stages
of degeneration and granular debris. The central
portion is that which was first formed, and it
ultimately degenerates, while the parasite still
increases in size and produces new septa and
spores peripherally.
The spores are presumably produced by
multiplication of the peripheral cells which
reproduce by binary fission, while the space in which the cell lies dilates
to form a chamber. When a number of such cells is present, they
become transformed into the spores. The latter, which measure
from 10 to 15 microns in length, may be readily stained in dried films
by Romanowsky stains, but the true structure can only be made out
in properly fixed smears. Each is crescentic in shape, and has one
extremity rounded and the other pointed (Fig. 329). Towards the rounded
Fig.
OF
326. — Sarcocystis
THE Pig. (After
Manz, 1867.)
A single cyst from a muscle
fibre, showing the striated
capsule and a rupture
thi-ough which can be seen
the groups of spores in the
chambers.
762 SAECOSPORIDIA
end is an elongate nucleus consisting of a membrane and central karyosome.
The cytoplasm of the spore towards the pointed end is clear and hyaline,
while the rest contains a number of granules of a material which stains
deeply. The clear portion was supposed to be of the nature of a polar
capsule, chiefly as a result of the statements of Van Eecke (1892), who
claimed to have produced an extrusion of filaments as occurs in the spores
of Microsporidiida. This observation has never been confirmed, and it
seems evident that the clear part of the spore contains nothing but hyaline
cytoplasm, and in no way corresponds with the polar capsules of the
Microsporidiida, A fully-grown parasite will contain many thousands of
spores, which escape when rupture takes place.
Fig. 327. — Diagrammatic Representation of Longitudinal Section of a
Sarcocyst in the Muscles of the Ox (xea. 500). (Original.)
Life-History. — The development of the parasite has been studied
chiefly in experimentally infected mice. Erdmann (1910, 1910a, 1914)
states that in the intestine of the mouse the spore membrane ruptures and
liberates a small amoeboid body which enters the intestinal cells. There
follows a period of multiplication. The parasites persist in the gut wall
for a few days only. They then disappear, and are only detected about
forty days later in the muscle fibres. According to Negri (1910), who
studied experimental infections in rats, the youngest forms seen in muscle
fibres have a length of 25 microns, and are cytoplasmic bodies contain-
ing about twelve nuclei (Fig. 328). Segmentation of this multinucleate
Plasmodium then occurs, so that a number of cells are enclosed in a mem-
branous sheath. This development occupies from forty-eight to sixty
days. The sheath may rupture, and the cells wliicli are liberated infect
GENEKAL ORGANIZATION
7G3
@
IN-
other muscle fibres, so that intense infections of all the muscles may occur.
On the other hand, the parasite may increase steadily in size, so that after
seventy days it may be half a centimetre in length and contain numerous
cells which are apparently formed by division of those originally produced.
Towards the centre of the parasite some of the cells are transformed into
the sickle-shaped spores. With increase in size larger numbers of spores
are formed, and finally the characteristic structure, as described above,
is reached.
Alexeiefi (1913a), in a study of the parasite of sheep, came to the
conclusion that the enclosing envelope consisted of three zones, the inner
of which is continued as the septa,
which enclose the uninucleated cells
and spores (Fig. 329). He believes
that the envelope, together with the
septa, are in reality derived from the
host cell, and do not belong to the
parasite, which is represented by the
round uninucleate cells which multi-
ply by division, and the spores into
which they ultimately become trans-
formed. According to this view, each
sarcocyst is not a single parasite
which is producing spores, but a large
number of uninucleate parasites en-
closed by an envelope and septa
derived from the host. Chatton and
Avel (1923), from a study of S.platij-
dactyli of the gecko, come to the con
elusion that the enveloping membrane
belongs to the parasite, and is not de-
veloped from the tissues of the host.
Negri (1908) believes that he has
demonstrated multiplication of the spores themselves by binary fission,
not only in the case of S. muris, but also S. bertrami of the horse, while
Teichmann (1911) claims to have made a similar observation in the case of
S. tenella of sheep.
The parasites in old infections may be as much as 5 centimetres in
length. It is quite evident that the dimensions cannot be employed as a
means of distinguishing species, so that there is very little evidence that
the numerous species of Sarcocystis which have been described are valid.
Theobald Smith (1901, 1905) was the first observer to demonstrate
that mice could be infected by feeding them with spores. Negri (1910)
Fig. 328. — Sareocystis muris in
Muscles of Mice. (After Negri,
1910.)
1. Form 25 microns in length fifty days
after feeding.
2. Form 52 microns in length fifty days
after feeding.
3. Section of portion of a parasite fifty days
after feeding.
4. Section of portion of a parasite sixty-
eight days after feeding.
764
SARCOSPORIDIA
and Darling (1910a) showed that guinea-pigs could be infected with the
parasite of rats, and Darling points out that the forms in the guinea-pig are
morphologically identical with those described by him from man. Erdmann
Q
^31
ch'.spb.
Fig. 329. — Sarcocystis tenella of Sheep: Diagrammatic Representation of
Small Portion of Cyst (x ca. 1,000). (After Alexeieff, 1913.)
E, Envelope of cyst consisting of three zones — external (Ze.), median {Zm.), and internal (Zi.).
The latter is continued as the septa, which divide the cyst into chambers. Outside the
envelope of the cyst is a fibrous zone (Zf.), a connective tissue layer (fc). and a muscular layer
()n.). The chambers near the envelope contain the sporoblasts (ch.spb.), which become
transformed into spores (ch.sp.). According to Alexeieff, the envelope and the septa arc
developed from the tissues of the host, the parasites being the cells which become sporoblasts
and spores.
(1910a) succeeded in infecting mice with the parasite of sheep, while Darling
infected guinea-pigs with the form found by him in the opossum. It
seems clear that these parasites are not specific to any particular host.
GENERAL ORGANIZATION 765
Crawley (1916) gives an account of the early stages of development of
the spore in the intestine of mice. He supposes that the spores first
differentiate in the lumen of the gut into male and female forms. These
enter the epithelial cells and undergo further changes. The nucleus of
the male enlarges and its cytoplasm disappears entirely. The nucleus is
then supposed to produce microgametes, as in coccidia. The female,
however, retains its cytoplasm, and is eventually fertilized by a micro-
gamete. Crawley supposes that the zygote so formed proceeds to multiply
in the cell, as Erdmann has described. The description given by Crawley
seems very unconvincing, and some of his figures might equally well
represent degenerating parasites, while others might conceivably be stages
in the development of Eimeria falciformis, which is a common intestinal
parasite of mice. According to Marullaz (1920), in mice which have been
fed on infected material the spores can be found in the intestinal cells
within two hours. Soon after this they become round and the nucleus
divides by mitosis. Finally, division into two takes place. The daughter
forms may escape into the intestine again and infect other cells. Mul-
tiplication by binary fission goes on for about ten days. Meanwhile,
parasites have been entering the lymphatic spaces of the villi, and thence
make their way to the liver and spleen, where the author found them on
the eleventh day after feeding. These forms measure 3 by 4 microns,
and have a single nucleus. From the forty-fourth to the fifty-fifth day
similar forms were found in the muscle fibres. In addition, certain parasites
in the muscles had two nuclei, and in one case there appeared to be a
division into eight cells. The author regards the last named as an early
stage in the development of a typical sarcocyst. Arai (1925) fed mice with
spores of S. tenella of sheep. He noted that, during the first three hours,
the spores could be found in all parts of the intestine, but only those in the
upper parts of the small intestine were unchanged in appearance. Those
occurring lower down were evidently in a degenerate or dying condition.
The unchanged spores high up in the intestine during the three hours
following the feed applied themselves to the surface of the epithelium,
passed in between the cells, but not into them, and appeared finally in the
subepithelial tissue. Within six hours of feeding all spores had dis-
appeared from the lumen of the intestine, while they could not be detected
in the subepithelial tissues later than four hours after feeding. On one
occasion a spore was found in blood taken from the tail five hours after
feeding, and on two occasions in the heart blood six hours after feeding.
Between this time and the appearance of young parasites in the muscle
fibres thirty-five to fifty days later the spores were not traced. It seems
to be an undoubted fact that in the case of the mouse the spores penetrate
the intestinal wall, but it is an exceedingly difficult matter to trace such
7GG SARCOSPORIDIA
minute objects in their wanderings, and at the same time to exclude every
possibility of confusing them with portions of cell nuclei or other small
bodies in the tissues.
Very little is known about the development of Sarcosporidia in other
animals. Bertram (1892) described young stages of the S. tenella of sheep.
The smallest forms consisted of elongate cytoplasmic bodies 4 to 5 microns
in length with a single nucleus. Older forms possessed a definite mem-
brane, and consisted of round or oval cells lying in spaces in a matrix. By
multiplication of thecells the spaces are enlarged and the matrix between
the spaces becomes the walls or septa of the chambers. When multiplica-
tion has produced the requisite number of cells in a chamber, they become
transferred into typical spores.
Pathogenicity. — In the majority of cases, even when fairly heavy
infections exist, there is little evidence that the host is adversely affected.
Sheep have sometimes died with very heavy infections, and death has been
attributed to the Sarcosporidia. Creech (1922) has described extensive
muscular degeneration in pigs caused by these parasites. In experi-
mental mice the animals sometimes die, apparently as a result of intense
infection. A curious feature of the Sarcosporidia is that they appear to
contain a substance which is highly toxic to animals. Pfeiffer (1890)
showed that the parasites of sheep were highly toxic if injected into mice,
rabbits, and even sheep. Kasparek (1895) also showed that subcutaneous
injection of the sheep parasite killed mice and guinea-pigs. Laveran and
Mesnil (1899) made aqueous or glycerine extracts, and found that the
extract of 0-001 gram of fresh parasite when injected subcutaneously
killed rabbits in five to ten hours. Rats, mice, sheep, and frogs were not
affected. They named the toxin " sarcocystin." Similar experiments
were conducted by Rievel and Behrens (1904) with the Sarcosporidia
obtained from llamas. Teichmann (1910) used a dried extract of the
sheep parasite, which killed rabbits when injected intravenously in a dose
of 0-0002 gram dissolved in saline solution. Rats and guinea-pigs were
refractory. It was shown by Teichmann and Braun (1911) that rabbits
could be immunized against the toxin, and that the serum contained
antibodies which could produce passive immunity in other animals.
Method of Infection.^ — Though it is easy to understand how infection
will spread amongst animals, such as rats and mice, which eat flesh, it is
difficult to see how this happens in the case of cattle and sheep, which are
nearly always infected and are purely herbivorous in diet. It occasionally
happens that in cattle the Sarcosporidia infect the skin, and recently
Sergent, Ed. (1921), has had the experience of finding the spores in blood-
films made from these animals after pricking the skin. He has raised the
HUMAN INFECTIONS 767
question of the possibility of biting flies taking up spores from the skin
Watson (1909) also drew attention to the occurrence of spores in blood
films.
Sarcosporidia in Man.
According to Darling (1909, 1910a), who has recorded two cases of
human sarcosporidiosis, the infection is rare in man. The organism
iy.
1
Fig. 330. — Sarcocystis lindemanni from the Muscle Fibres of the Human
Larynx. (After Baraban and St. Remy, 1893.)
1. Longitudinal section of a muscle with a cyst in situ ( x 300).
2-3. Transverse section of infested muscle fibres ( x 300).
4. Portion of a section of a cyst from which the spores have dropped out, showing the septa
( X 680), ' 5. Single spore ( x 1,600).
768
SARCOSPOKIDIA
described from man by Rivolta (1878) as Gregarina lindemanni is probably
one of these parasites. Rosenberg (1892) saw certain structures, which are
possibly Sarcosporidia, in the heart muscle of a man. He proposed to
name the parasite S. hominis. A case regarded as authentic by Darling
(1910a) was reported by Kartulis (1893). There appears to be no doubt
about one described by Baraban and St. Remy (1894). In this case the
infection occurred in the larynx, the muscle fibres of which were distended
to about four times their normal thickness (Fig. 330). The first case
described by Darling was in a negro. The parasites were discovered in
portions of the biceps muscle, which had been removed owing to suspicious
signs of trichinosis. The patient was actually suffering from typhoid
fever, and it is concluded that the pains complained of in the muscles
were actually due to the typhoid infection, and not to the Sarcosporidia.
The largest parasites found had a length of 84 microns and a breadth of
27 microns, while the spores measured 4-25 by 1-75 microns. The second
case described by Darling (1919) was in an East Indian who had died of
malaria. Sections of the tongue revealed the parasites in the muscle
fibres. Manifold (1924) described an infection of the muscle fibres of a
human heart. The spores in this case were over 10 microns in length.
Sarcosporidia in Animals.
Though Miescher (1843) discovered the Sarcosporidia in the muscle
fibres of the mouse, they were first named by Kiihn (1865) Synchytrium
miescher ianum. As they evidently did not belong to this genus, Ray
Lankester (1882) established the genus Sarcocystis, by which name they are
now known. As already remarked, many forms have received distinctive
names because of variations in size and their occurrence in different hosts.
Alexeieff (1913a) justly remarks that there is no means of distinguishing
the supposed species. He concludes that they all belong to the one
species, S. miescheriana (Kiihn, 1865). The following forms have been
recorded :
Recorded Species of Sarcocystis.
Mammals :
8. lindemanni
Rivolta, 1878
Man
8. muris
Blanchard, 1885
Rat, mouse
S. hirsiita
Moule, 1887
Ox
S. cruzi
Hasselmann, 1923
Ox
8. blanehardi
Doflein, 1901
Buffalo
8. fusiformis
Railliet, 1897
Buffalo
8. siamensis
V. Listow, 1903
Buffalo
8. tenella buhali
Willey, Chalmers, and
Philip, 1904
Buffalo
8. tenella
Railliet, 1886
Sheep
8. miescheriana
Kiihn. 1865
Pig "^
8. bertrami
Doflein, 1901
Horse
8. hueti
Blanchard. 1885
Seal (Ofaria californica)
8. leporum
Crawley, 1914
Rabbit
GLOBIDIUM
769
Mammals — Continued .
S. pitymysi
S. sp.
S. cameli
8. richardi
8. sp.
8. S]).
8. aucheniw
8. gazeUce
8. Icortei
8. (jyaciJi.^
8. monh'i
8. CHiiicali
8. darlingi
8. hubalis
8. woodJioHsei
Birds:
8. rilei/i
8. hoyrdthi
S. falcatula
8. sp.
8. turdi
8. coin
8. setophagce
8. aramidis
8. ammodromi
Lizards:
8. platydaetyli
8. gongyli
Splendore, 1918
Krause, 1863
Mason, 1910
Hadweii, 1922
Hadweii, 1922
Hadweu, 1922
Brumpt, 1913
Balfour, 1913
Castellani and Chalmers,
19(19
Von Ratz, 191)8
Xeveu-Lcniaire. 1912
Brumpt, 1913
Brumpt, 1913
Dooiel, 1916
Dogiel, 1916
utiles, 1893
Von Eatz, 1908
.Stiles, 1893
Barrows, 1883
Brumpt, 1913
Fantham, 1913
Crawley, 1914
Splendore, 1907
Splendore, 1907
Field vole {Pitymys savii)
Do,2;, cat
Camel
Seal {Phoca richardi)
Reindeer
Caribou
Llama
Gasella rufifrons
Macacus rhesus
Deer
Goat
Rabbit
Opossum
Bubalis cokei
Gazella grant i
Duck
Chicken
Uabia ludoviciana
Parula piUayiim i
Merula merula
Colixs ciiithinitidon
8etoph,ni„ lulirilh,
Aram ides sardrnra
Ammodromus manimhe
(Bertram, 1892 Gecko {Platydactylus facetanus)
\Cliatton and Avel, 1923 Gecko {Tarentola mauritanica)
Trinci. 1911 Govgylns ocellatus
Globidium Flesch, 1884.
These parasites, which are probably related to the Sarcosporidia, have
the form of spherical cysts up to 5 millimetres in diameter embedded in the
mucosa of the alimentary canal or skin of mammals (Fig. 331, i). Each
is enclosed by a membranous capsule, and when fully grown consists of
groups of spores which resemble those of the Sarcosporidia.
Flesch (1883) was the first to discover one of these parasites in the small
intestine of the horse. He (1884, 1884a) gave it the name Globidium
leuckarti. This species was rediscovered by Hobmaier (1922), and has
been studied by Kupke (1923). Blanchard (1885) saw a similar parasite
in a kangaroo and, believing it to be related to the Sarcosporidia, named it
Sarcocystis tniicosce. Moussu and Marotel (1902) observed a form in the
sheep, and regarded it as a developmental stage of the coccidium, Eimeria
faurei. It was studied by Gilruth (1910), and in the same year by Chatton
(1910), who named it Gastrocystis gilruthi. A similar parasite was discovered
in the subcutaneous tissue and muscles of a cow by Besnoit and Kobin
(1912), according to wdiom it was named *S'. hesnoiti by Marotel in 1912.
Franco and Borges (1916), who studied this organism, came to the conclusion
I. ' 49
770
GLOBIDIUM
that it differed sufficiently from other members of the genera Sarcocystis and
Gastrocystis (Globidium) to justify the creation of a new genus, Besnoitia.
It is evidently very similar to the members of the genus Glohidium, in
which it seems better to retain it at present as G. hesnoiti.
,, «'.'f'-"';:'.'--'.""''r^"':V*<2VsT^.iOT»»
^ ^^^^^*'^''-^--
Fig. 331.— Globidium gilruthi from the Mucosa of the Stomach of Sheep
AND Goats. (After Chatton, 1910.)
1. Section (if mucosa showing cyst ( Xca. 100).
'2. Section of portion of immature cyst more highly magnified ( Xca. 250).
3. Section of portion of mature cyst filled with spores ( Xca. 500).
4-fi. Method of development of spores from multinucleated cytoplasmic bodies ( Xca. 500).
7. Individual spores ( Xca. 2,000).
Gilruth and Bull (1912) described a series of parasites which they found
in the intestinal mucosa of the kangaroo, wallaby, and wombat of Australia.
In the kangaroo {Macropus sp.) there were large and small cysts, which
they supposed belonged to different organisms. The larger was named
GENEEAL ORGANIZATION 771
lleocystis tnacropodis, and the smaller one Lymphocystis macropodis. A large
one in the wombat {Phascolomys latifrons) was called /. wombati. Believing
the form in the wallaby {Petrogale sp.) to be a sarcosporidian, they gave it
the name S. macropodis. The large cysts, which were named I. wombati,
had thick walls, and reached a diameter of about 93 to 113 microns. In
structure they resembled G. gilruthi. The smaller cysts, named L. macro-
podis, occurred in the connective tissues of the mucosa in large numbers.
When mature, they had a diameter of about 8-4 microns and were filled
with spores. The cyst wall was merely a membrane, which appeared to
consist of the remains of a mononuclear cell, the nucleus of which could be
detected as a flattened structure at one side. It is possible that L. macro-
podis is really a small form of I. macropodis, in which, however, free spores
were not seen, though stages showing many nuclei and what appeared to
be commencing spore formation occurred. The parasite called S. macro-
podis was also in the mucosa, measured 150 to 700 microns in diameter,
and was filled with spores. It appears to be a species of Globidiuyn, though
Chatton (1912c) has suggested placing it in a new genus, Haplogastrocystis.
Recently the writer and Scott (1925) and Triffifct (1926) have seen in the
wallaby {M. bennetti) parasites like /. macropodis and L. macropodis. It
was not possible to determine whether the smaller form was actually of the
same species as the larger one, though this would seem not improbable.
The whole of the connective tissue of the mucosa was filled with the
smaller parasite, while numerous free spores were scattered between the
cells. In addition, the muscle fibres of the intestine contained elongate
vacuolic spaces filled with spores, which appeared very similar to those of
the parasite of the Lymphocystis type. Whether this parasite, again, is
a species of Sarcocystis or is another stage of Lymphocystis could not be
decided. If three parasites are represented, then in this portion of the
intestine there occurred four distinct species, as an Eimeria was present in
the epithelium. It is possible that the parasite described by Blanchard
(1885) and named S. mucosce is identical with /. macropodis. Cunha and
Torres (1924) record a species {G. tatusi) found by them in the armadillo.
The structure of the cysts of G. gilruthi of the sheep and goat was
described in detail by Chatton (1910). The cysts have a diameter of
200 to 500 microns, and are situated within little opalescent elevations of
the mucosa (Fig. 331, i). Each is enclosed by a definite wall, which has
concentric striations. At one place in the wall there is a large flattened
nucleus, which may be 80 microns in length by 10 in breadth (Fig. 331, 2).
According to Chatton, the cyst wall represents the remains of a very much
hypertrophied and altered cell which may be connected by a kind of neck
with the connective tissue. Within the mature cyst is a mass of spores,
each of which measures 10 by 1-5 microns (Fig. 331, 7). One end is blunt
772
GLOBIDIUM
and the other pointed, and, as in the spores of the Sarcosporidia, there is a
nucleus near the blunt end, while the other end is clear and hyaline. The
cytoplasm contains granules, one of which lies between the nucleus and
clear pointed end and is distinctly larger than the others. In cysts which
have not completed their development there occur spherical bodies with
numerous nuclei arranged over the surface (Fig. 331, 4). Portions of
cytoplasm, each with one of the nuclei, then grow out from the surface as
pointed buds, which gradually assume the character of spores. The latter
remain attached to the residue of cytoplasm till they break loose and are
scattered within the cyst (Fig. 331, 5, 6).
The infection is very common in the
abomasum. In the majority of animals the
infection is small, but it is sometimes heavy.
As the cysts, when mature, rupture into the
stomach, in heavy infections haemorrhages
may be caused and serious symptoms follow.
Triffitt (1925) has found G. gilruthi in as
many as 92 per cent, of British sheep.
According to Franco and Borges (1916),
infections of the skin with G. besnoiti may
occur, as also of the connective tissue and
fasciae of the muscles. In the latter case,
the muscles may appear studded over with
white nodules due to the presence of the
parasite, so that the flesh has to be con-
demned as unfit for food.
Kupke (1923) has studied G. leuckarti of
the intestine of the horse. He finds that the
parasite is embedded in a very much hyper-
trophied cell, the nucleus of which can often
be detected lying at one side (Fig. 332). The
parasite itself is an ovoid body consisting of a thick capsule which, in serial
sections, can be seen to possess a definite pore at one end. The mass of
cytoplasm within the capsule becomes multinucleate and divides into
a number of separate bodies, each of which develops a number of nuclei.
Presumably, each body gives rise to a cluster of spores.
The various species of Globidium agree fairly closely with that of the
sheep described above. Some of the forms have been described as siDecies
of Sarcocystis, to which they undoubtedly bear some resemblance.
Hobmaier (1922) has expressed it as his belief that the parasites are
really fungi, and not Protozoa. It seems probable that the organisms
are related to Rhinosporidium, described below (Fig. 336).
Fig. 332. — Globidium leu-
ckarti FROM Intestinal
Mucosa of the Horse
( X 600). (After Kupke,
1923.)
Section showing opening at the
pole and still undivided con-
tents.
HAPLOSPORIDIA
773
The following species have been described:
G. leuckarti
G. gilruthi
G. besnoiti
G. mucosa'
G. {Ileocjjstis) rnacropodis
G. sp. {Lympliocystis macro-
podis)
G. sp. {Sarcocystis macro-
podis)
G. ivombati
G. tat us i
Flescli, 1883
Chatton, 1910
Marotel, 1912
Blanchard, 1885
Horse
Sheep and goat
Cattle
Kangaroo {Macropus peni-
cillatus)
CTilrutli and Bull, 1912 Kangaroo [Macropus sp.)
Gilruth and Bull, 1912 Wallaby [Petrogale sp.)
Gilruth and Bull, 1912 Wombat {Phascolomys lati-
frons)
( 'unlia and Torres, 1 923 Armadillo
Under the name of Fihrocystis tarandi, Hadwen (1922) describes certain
cysts which occur in the fibrous tissue, especially that covering the tendons
and the periosteum, of the reindeer and caribou. The cysts have a
diameter of 100 to 450 microns, and consist of three layers enclosing
numerous spores. In the reindeer it gives rise to a condition known as
" corn-meal disease," on account of the gritty feel of the affected parts.
When in the periosteum, the cysts cause the bone to atrophy, so that it
becomes pitted. Both the reindeer and the caribou suffer from sarco-
sporidiosis of the muscles, and though the cysts of F. tarandi differ
structurally from the Sarcosporiida, which resemble those of sheep, this
difference may be due to their development in the fibrous tissue.
Haplosporidia Liihe, 1900.
Under this heading are included a number of parasites which in many
respects resemble the Microsporidiida. They are found in aquatic inverte-
brates and fish, and occur as small uninucleated amoeboid bodies or as
multinucleate plasmodia. They float freely in the body-cavity fluid of
the invertebrates, or infest the cells such as those of the intestine. In
fish they attack the gills or other tissues, giving rise to white nodules.
After growth and multiplication have taken place spores are produced,
but these are not provided with polar capsules. The spore is spherical or
ovoid, and the surface may be variously marked with ridges or tubercles.
In some cases it is provided with a tail-like process. The genus Bertramia,
established by Caullery and Mesnil (1899), includes forms which are
parasitic in the body-cavity fluids of aquatic worms and rotifers. The
minute uninucleate body develops into a cylindrical or sausage-shaped
Plasmodium containing many nuclei. It finally divides into a number of
uninucleate forms. Roughly spherical and irregularly marked spores are
produced. The genus Ichthijosporidium Caullery and Mesnil (1905) includes
several species which infect fish. As in the case of Microsporidiida, white
nodules are produced in the tissues or on the gills, and these are seen to
774
HAPLOSPORIDIA
consist of an encapsuled multinucleate j)lasmodium in which occur ovoid
spores. They may be confused with Microsporidiida, a mistake which was
made by Thelohan (1895), who placed /. giganteiim in the genus Glugea.
The members of the genus Hajjlosjwridimn Caullery and Mesnil (1899) are
parasitic in marine annelids. They give rise to spherical cysts, in which the
Plasmodium breaks up into a number of uninucleate bodies, each of which
divides into four to form four ovoid spores. Each spore has one end
flattened. Granata (1914) described in detail the development of H. lim-
nodrili parasitic in the intestinal epithelium of Limnodrilus ndeJiemianus
T^iG. .333. — Section of Intestine of Limnodrilus ndel-emiaims infected with
Haplosporidium limnodrili Granata, 1914 ( x 750). (After Granata, 1914.)
y. Young form with single nucleus; p, older forms with two or more nuclei; s, form with four
nuclei (schizont); m, young forms resulting from division of schizont; g, forms which give
rise to spores, gametes, or zygotes; sp, spores.
(Fig. 333). The genus Urosjwridium Caullery and Mesnil (1905) is closely
related to H aplosporidium. The spore is provided with a long caudal
process. The genus Coelosporidiuin Mesnil and Marchoux, 1897, was estab-
lished for certain parasites of the kidneys of Crustacea. Crawley (1905)
placed in this genus a parasite of the Malpighian tubes of the cockroach
which had been taken for a microsporidian [Nosema periplanetcB) by Lutz
and Splendore (1903). It occurs as amoeboid bodies and ovoid spores in
the cytoplasm of the cells. Another genus is Serumsporidium Pfeiffer,
1895, which includes parasites of the coelomic fluid of Crustacea. They
have been studied by Noller (19206) and Stempell (1921). Noller
GENERAL ORGANIZATION 775
described S. fnelusince from the body cavity of Simulium reftans. The
smallest uninucleated forms measured 5 to 7 by 3 to 4 microns. This
develops into a multinucleate plasmodium, which becomes enclosed in
a cyst from 25 to 70 microns in diameter. Within the cyst the plas-
modium divides into a large number of the uninucleate forms. Stempell,
describing the parasites from the crustacean Herpetocypris strigata, has
noted the formation of spores which differ as regards their shape, external
markings, and contents. He recognizes several genera.
A parasite in the form of small uninucleate bodies and multinucleate
spheres was discovered by Calkins (1900) in the lymphatic system of trout
which were dying in an epidemic. Calkins gave the name Lymphospori-
dium truttce to the parasite. The parasite which Woodcock (1904) dis-
covered in plaice and flounders in the form of small white nodules on the
./■
Fig. 334. — Helieosporidimn parasiticmn (x about 3,000). (After Keilin, 1921.)
1-6. Stages in schizogony.
7. Mature spores with coiled filament and three amoeboid bodies.
8. Ruptured spore showing escape of filament.
surface of the internal organs was named by him Ly mpJiocysfis joJinsfonei.
The nature of the organism is not known, some thinking it to belong to the
Microsporidiida.
A curious parasite, Helicosporidiumparasiticum, has been described by
Keilin (1921) from thelarvse of Dasyhelea obscura, a ceratopogon (Fig. 334).
The body spaces of the larvae are invaded by small round cells, which
multiply by schizogony. The schizonts are 4 microns in diameter, and give
rise to four to eight merozoites. The remarkable feature of the parasite
is its spore, which is a spherical body 5 to 6 microns in diameter. The
capsule encloses four cells, three of which are amoeboid, while one develops
into a long coiled and resistant filament which appears to be free within
the cyst. When the host dies, the spores rupture apparently by aid of
76
RHINOSPOKIDIUM
the spirally coiled filament which is discharged and remains free in the
medium. It is 60 to 65 microns in length, and the nucleus of the cell
from which it was derived is still present 15 microns from one extremity.
The spores do not resemble those of any known microsporidian, for the
filament is not developed in a polar capsule, and is a much stouter structure
than those discharged from the spores of Microsporidiida.
It will be evident that the parasites which have been considered under
the heading Haplosporidia form a very heterogeneous group. It seems
highly probable that some of them, at least, are really fungi.
Rhinosporidium Minchin and Fantham, 1905.
Under this heading are included certain organisms which give rise to
polypi, especially in the nose, of human beings and horses. They are
/■■
:!|
A 1^
Fig. 335. — Section of Nasal Polyp from a Case of Infection with Bhino-
sporidium seeberi (A, x 60; B, x 260).
(Microjjhotographs of sections of tissues given to the writer by Professor J. H. Asliworth.)
undoubtedly vegetable parasites, as conclusively demonstrated by Ash-
worth (1923), but they are considered here, as for a long time they were
regarded as Protozoa. Their structure and development appear to throw
light on the forms described above, the Protozoon affinities of which are
extremely doubtful.
GENERAL ORGANIZATION
777
Rhinosporidium seeberi (Wernicke, 1903). — This organism was first
seen by Seeber (1900) in a nasal polyp in South America (Figs. 335, 336).
As pointed out by Ashworth (1923), it was referred to by Belou (1903) as
%, "^
'/3
Fig. 336. — Stages in the Development of Bhmosporidium seeberi from Xasal
Polyp of Man. (After Ashworth, from Trans. Boy. Soc, Eclin., liii., 1923.)
1. Very early stage 6 /< in diameter lying between connective tissue cells ( x 1,600).
2. Later stage 65 /t in dianu-ter witli single nucleus ( x 400).
.3. Section of later stage with si.\ty-four nuclei ( x 400).
4. Section of stage with aliovit ."ioo nuclei. The envelope is composed of a thin chitinous external
layer and a thick inner celluluse layer. The position of the future pore is indicated by a
depression in the celhilDsc layer ( ■ 400).
5. Section of stage in which the contents of sporangium has subdivided into about 4,000 nucleated
cells ( x 800). 6. Discharge of mature spores through pore of sporangium ( x 200).
7. Section of a spore (10 x 7 /()> showing nucleus with karyosome and cytoplasm containing
vacuoles, three of which include refrin<j;ent spherules.
Coccidium seeberia Wernicke, 1900. Minchin and Fantham (1905) named
it R. kinealyi, but undoubtedly Wernicke's name, R. seeberi, has priority,
as pointed out by Hartmann (1921). Minchin and Fantham regarded the
778 EHINOSPORIDIUM
organism as a Protozoon belonging to the Haplosporidia, but Asbwortb
has shown conclusively that it is a vegetable parasite allied to the fungi.
The organism produces nasal polypi in man (Fig. 335), and has also been
seen in polypi of the conjunctiva, lacrimal sac, and ear, in a papilloma of
the penis, and in the uvula. According to Ashworth (1923), who has given
a complete account of the organism, the younger forms are spherical
bodies about 6 microns in diameter embedded in the cytoplasm of con-
nective tissue cells. Each has a capsule enclosing a mass of cytoplasm with
a single nucleus and a number of deeply-staining granules of reserve food
material (Fig. 336, i). Growth takes place, nuclear multiplication by
mitosis occurs, and the cytoplasm becomes charged with numerous food
granules. Eventually, the central cytoplasm segments into uninucleate
masses, and this process spreads towards the periphery of the cyst till the
contents are completely divided. Multiplication by fission of the separate
masses may occur. Eventually, each separate mass becomes enclosed in a
membrane. Meanwhile, the parent cyst, which now has a diameter of
250 to 300 microns, develops a thick lining composed of cellulose and a
definite pore forms at one point. Through this pore the daughter cysts
are discharged to spread the infection to neighbouring tissues Each
daughter cyst is taken u'p by a mononuclear cell and commences to grow.
The infection, which is of a very chronic type, has been recorded from
India, Cochin-China, Ceylon, Argentina, and North America.
Zschokke (1913) described R. equi from the nasal septum of a horse
in South Africa. According to Hartmann (1921), Frey and Hartmann
arrived at conclusions regarding the nature and development of the
organism similar to those put forward by Ashworth for R. seeberi. That
there is any specific difference between the human and equine form seems
doubtful.
END OF VOL. T.
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