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THE AMOEBAE LIVING IN MAN

A ZOOLOGICAL MONOGRAPH

BY

CLIFFORD DOBELL, M.<.A., F.R.S.

Assistant Professor of Protistology and Cytology in the Department of Biology,
Imperial College of Science and Technology, London ;
Late Fellow of Trinity College, Cambridge.

‘* Veritas Temporis filia, non Authoritatis.”’
—Bacon, Wov. Org.

PuBLISHED FOR THE MepicaAL RESEARCH COMMITTEE
BY
JOHN BALE, SONS & DANIELSSON, LTD.
83-91, GREAT TITCHFIELD STREET, OXFORD STREET, LONDON, W.1.
I9Ig.

PREBACE,

] HAVE attempted in the following monograph to give a concise and
accurate account of all the amoebae which live in human beings.
Although our knowledge of these protozoa has grown apace in the last
few years, there is at present—so far as | am aware—no published work
which gives a correct description of them from the standpoint of
modern protozoology. The works already published—the various
treatises on the Protozoa, and innumerable papers scattered through
zoological and medical periodicals—reveal, if taken together, a sad
state of confusion in the minds of men : and much that is now certainly
known appears uncertain, merely because of the conflicting and con-
tradictory statements with which the literature of this subject abounds.
That correct solutions of the chief problems of the past, and a true
account of the state of knowledge at present, would be welcomed by
many zoologists and medical men—especially by those whose work lies
in the tropics—I have good reason to believe: and it is in the hope of
contributing something towards the attainment of both these ends—the
clarification of ideas, and the codification of facts—that the following
pages have been written.

This little treatise is addressed to all who are interested in the
amoebae of man from a zoological standpoint, and is intended for those
who already possess some acquaintance with the science of protozoology.
To these it is offered with the hope that the work which the author has
expended on it may lighten their labours in the same field. It is, more-
over, the work of a biologist and not of a medical man—of one whose
chief interest is, and has ever been, in the Protozoa themselves and not
in the diseases which they produce—of one whose point of view has
been determined not by practice as a physician but by the study of other
amoebae and their kindred. Few zoologists have hitherto had an oppor-
tunity of studying the amoebae of man, and few medical men have
possessed the zoological knowledge necessary for a proper investigation
of the material in their hands. What has already been published demon-
strates conclusively the need for further work on this subject by proto-
zoologists skilled in all the intricacies of their science; and if I have
presumed to take so great a task upon myself, it is because no other
worker has yet volunteered for this difficult but urgent service.

I have devoted a great part of my working life to the study of

Vi. PREFACE

amoebae of all sorts, for they have always been with me favourite objects
of investigation ; and I probably realize more clearly than most men
how very little is really known and understood concerning these remark-
able organisms. I confess, indeed, that I still regard them as in many
ways the most difficult to investigate of all living animals. No one man
will ever succeed in solving all the myriad problems which they present :
and it is merely as a small contribution to what must ultimately be the
work of many, that these pages are submitted to the judgement of all
fellow-workers seeking after truth.

April, 19109. C.D

VI.

WIT.

VIETE

CONTENTS.

INTRODUCTION

A Note on MATERIAL AND METHODS ne ae ee

THE PRESENT STATE OF KNOWLEDGE OF THE AMOEBAE LIVING

IN Man

THE GENERA OF AMOEBAE LIVING

Genus Entamoeba.
(1) Entamoeba histolytica
(2) Entamoeba colt iu
(3) Entamoeba gingivalis

Genus Endolimax.
Endolimax nana

GENuS Jodamoeba.
Todamoeba biitschlit ...

Genus Dientamoeba.
Dientamoeba fragilis...

Tue AMOEBAE FOUND IN
MoNKEyYS

IN MAN

oe eee see wee

URINE, IN Docs, AND IN

NOTES ON CERTAIN OTHER AMOEBOID ORGANISMS DESCRIBED

FROM Man

BIBLIOGRAPHY ...

INDEX...

Piates—at end of Volume.

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125

134
142

152

THE AMOEBAE LIVING IN MAN.

I
INTRODUCTION.

FIRST became interested in the amoebae which live in man some
twelve years ago. I was then investigating the amoebae of frogs,
and the observations which I made led me to study much of the

published work dealing with the related rhizopods occurring in man and
other animals. At that date Schaudinn’s views were generally accepted ;
and so great was his prestige, that to question any of his pronounce-
ments in protozoology was almost to stamp oneself—in the estimation of
most zoologists and medical men—as an inexperienced or incompetent
worker. Nevertheless, my own observations soon constrained me to
believe that Schaudinn’s conclusions regarding the intestinal amoebae
of man were mostly incorrect—as time has since shown convincingly:
but although | have always thought that my conclusions concerning
the development of the amoebae of frogs would ultimately prove to be
equally applicable to the species living in man, I was long unable to
verify this owing to my inability to obtain adequate material for a study
of the latter organisms.

In the meantime, the wrong road of inquiry opened up by Schaudinn
was eagerly explored by many other workers: with the result that our
“knowledge” of the intestinal amoebae was soon a muddle of facts,
misinterpretations, malobservations, and fanciful speculations, from
which—only a few years ago—it seemed almost impossible to extricate
the truth.

For some years I followed, with the greatest interest, all the work
published on this subject—noting the various “new species” from time
to time discovered, reading the writings of the older observers as oppor-
tunity occurred, and forming my own opinions of them to the best of
my ability. But I very soon realized that my opinions were almost
worthless without a very extensive first-hand acquaintance with all the
organisms under discussion: and consequently, whilst I criticized and
controverted many of the accepted views in conversation, in correspon-
dence, and in my lectures at the Imperial College, I did not permit
_ myself to express any opinions in print. Of this Iam now glad, because
experience has taught me that it is impossible to draw correct con-
clusions from merely studying what has been written on this subject.
I thought too, and still think, that it is a mistake to confine oneself, in
dealing with such a subject, to the parasitic forms ; and my own work
has, accordingly, been carried out as a part of a more comprehensive

study of the amoebae generally—both parasitic and free-living.
T

2 THE AMOEBAE LIVING IN MAN

The War unexpectedly gave me an opportunity of studying, on
a large scale, all the intestinal protozoa of man. In 1915 large bodies
of troops from the Eastern War Areas began to arrive in this country.
It was at first believed that amoebic dysentery had been responsible for
a very large proportion of the invalidism in our troops from Gallipoli
and Egypt: and although we now know that this was a mistake, it
called for protozoological examination of the stools of a very large
number of military patients invalided home with a diagnosis of ‘‘ dysen-
tery.” No machinery for coping with such diagnostic work on a large
scale existed at the time, and it thus became necessary to train and
Organize a number of workers specially for the purpose. This work was
begun by Dr. C. M. Wenyon, who was at that time most qualified to
undertake such a difficult task; but after he had made a beginning, his
services were demanded elsewhere, and at the request of the Medical
Research Committee I took charge of the work at the end of 1915.”
Since that time I have, with the permission and assistance of the
Imperial College, devoted myself uninterruptedly to the practical study
of the intestinal protozoa of man. A large part of my time has, of
necessity, been occupied with routine work of diagnosis, with teaching,
and with the investigation of methods of treating amoebic dysentery.
But nevertheless I have had, during the whole of this period, great
opportunities for studying the human intestinal protozoa from the
zoological standpoint. I believe, indeed, that no zoologist has ever
before had such an immense amount of material at his disposal for
a study of the intestinal amoebae of man.

During the War I have naturally followed, with the keenest interest,
all the work which has been done upon the present subject—so far as
the results of other workers, both at home and abroad, have been
ascertainable. I have also recently read, or re-read, most of the pre-
viously published work, in order to correct my earlier impressions and
clear up my former difficulties in the interpretation of the results of
many other workers. In the light of present knowledge, and with a
large personal experience to fall back upon, I now find but little diffi-
culty in understanding many observations which formerly puzzled me:
and so far as the intestinal amoebae of man are concerned, I believe
that it is now possible to claim that all the chief problems of the past
have been solved. For my own part, at all events, I can now say that
almost all my own doubts and difficulties have disappeared.

Having thus reached definite conclusions on many points, and
having corrected or verified many of the observations of others, so that
their findings are reconcilable with my own, I have thought it a not
altogether thankless task to record the main results. I have endeavoured,
in the following pages, to deal fairly with the work of all my prede-
cessors; and I have done my best to cope with the immense and
scattered literature on amoebae and amoebiasis. I hope I have not
omitted to study any previous work of material importance from the
zoological standpoint. If so, it is through ignorance of its existence, or
ihability to gain access to it. On the other hand, I have knowingly
ignored much purely medical work; and for the reason that it is not

* A full account of the circumstances here briefly alluded to will be found in the
Introduction to Special Report No. 4 (1917), published by the Medical Research
Committee.

INTRODUCTION 3

germane to my present purpose, which is to collect and coérdinate all
our present zoological knowledge of the amoebae which live in man.
My original intention was to confine myself to the intestinal amoebae ;
but this soon became almost impossible, as the others had so frequently
to be discussed or compared. I decided, therefore, to attempt to deal
with all the amoeboid organisms which have been described from
human beings. My work is obviously incomplete and faulty in many
ways, but even these deficiencies may have their uses: for the mere
exhibition of a glaring defect, or the clear definition of a difficulty,
frequently hastens the advance of science by indicating a pitfall or
a wrong road of inquiry.

Although I have always formed my opinions independently, and
attach—as every honest worker must—particular importance to my own
personal observations, I have undoubtedly been infiuenced, in the
present work, by the advice and assistance which I have received from
my fellow-workers. Such help is impossible to assess, or even to define.
My obligations in this respect are none the less immense, and I can at
least attempt to acknowledge them. In the first place, my indebtedness
to Dr. C. M. Wenyon—now Colonel, A.M.S.—is gratefully recorded.
When I began to study the amoebae of man in earnest, his help—always
most generously given—was invaluable. I learnt more in conversation
with him than I had ever previously learnt from books. My ignorance
of medicine has to some extent been compensated by working in
collaboration with other medical friends, who have always given me
help and instruction in the kindest possible manner. In this connexion
I am glad of an opportunity to acknowledge my obligations to Dr. H. H.
Dale, F.R.S., with whom I had the good fortune to work in the early
days; to Dr. A. C. Stevenson, of the Wellcome Bureau, who has shared
much arduous work with me throughout, and with whom I have dis-
cussed—with much profit to myself—most of the questions here
considered ; and to Dr. G. C. Low, whose extensive clinical experience
has always been freely placed at my disposal. For much good advice
and frequent assistance in bacteriological matters I am further indebted
to Capt. S. R. Douglas, I.M.S. (ret.) and to the late Dr. H. S. Gettings.

The friends, pupils, and fellow-workers who have helped by giving
me material, by supplying me with information, or by directing my
attention to various points of interest, are innumerable. But in this
connexion I would again thank the following for their assistance on
divers occasions: Captain F. W. O’Connor, R.A.M.C., Dr. P. P.
Laidlaw of Guy’s Hospital, Dr. E. H. Kettle of St. Mary’s Hospital,
Dr. G. T. Western of the London Hospital; and Messrs. H. A. Baylis,
the late W. O. Redman King, A. Malins Smith, J. R. Matthews,
Geoffrey Paget, R. E. Savage, A. G. Thacker, and Hugh Watson. Miss
M. W. Jepps, who for a time acted as my assistant, has also given me
much help. I am further indebted to Captain F. L. Armitage, N.Z.M.C.,
for specimens from a case of amoebic abscess of the brain; to Dr.
J. W. Scott Mache for material from a case of “urinary amoebiasis” ;
and to Mr. T. Goodey for some preparations of Entamoeba gingivalis.
Several patients with interesting amoebic infections have also given me
the greatest assistance by submitting themselves to repeated and irksome
examination. Though nameless here, they are neither forgotten nor
unthanked.

Much of the work whose results are here recorded has been carried

4 THE AMOEBAE LIVING IN MAN

out in London at the Wellcome Bureau of Scientific Research. My best
thanks are again offered to the Director, Lieut.-Col. Andrew Balfour,
C.B., C.M.G., and all his staff, for their hospitality and unfailing help
at all times and on all occasions. The work itself has been done with
the aid of grants from the Medical Research Committee, and without
this assistance would have been impossible. In addition to this general
acknowledgement of my indebtedness to the Committee, I should like
to place on record my deep obligation to their Secretary, Sir Walter
Fletcher, K.B.E., who not only enabled me in the first place to begin
the work, but who also, by constant help and encouragement, insured
its accomplishment.

To all those mentioned and to many others my thanks are due and
gratefully offered. Nevertheless, I take full responsibility for all the
views expressed in the following pages; and if I have fallen into
errors, the fault is mine, and not attributable to those who have helped
me. In compiling the present memoir I have endeavoured on all
occasions to give credit to every worker for his own discoveries, and
any omissions in this respect—for such oversights are, I fear, inevitable
—are unintentional. I am well aware, moreover, that in a subject of
such magnitude and complexity there is little to be said that is really
new. My aim has been to set down what is true rather than what is
novel. For myself, therefore, I would only say, in the words of that
wise old philosopher, John Locke: ‘Truth needs no recommendation
and error is not mended by it; in our inquiry after knowledge, it
little concerns us what other men have thought.”

If.
A NOTE ON MATERIAL AND METHODS.

I DO not propose to describe in detail all the methods which I have
used in studying the amoebae of man. It seems to me unnecessary to
do so, because no special or peculiar methods are required for such a
study. Any good cytological or protozoological methods of obser-
vation, fixation, staining, etc., may be used successfully: and since I
have tried most of them, it would be merely wearisome to enumerate
them all individually. I wish to say a few words here, however, upon
certain points which are of particular importance.

First, I would note that it is impossible to obtain a correct know-
ledge of the amoebae living in man without studying an abundance
of material. The study of only a few infections, or of insufficient
material from many, is very apt to lead to error. Secondly, the material
must be as fresh as possible, as all the amoebae which live in human
beings degenerate and die rapidly outside the body. Most of the
mistakes which have been made with regard to the morphology and
life-histories of these organisms have been due to failure to take
these two elementary precautions : and it is tragic to note, in reading
the literature, the vast time and trouble that have been wasted in the
study of wholly inadequate quantities of material of the poorest quality.
Whatever mistakes I may have made myself have not been due, as a
rule, to any lack of material. For a study of the intestinal amoebae I
have had more than enough. During the last few years I have per-
sonally examined over 10,000 human stools of all sorts: I have also
had at my disposal nearly 150 kittens experimentally infected with
E. histolytica: and 1 have been able, through the kindness of many
friends, to examine a considerable amount of pathological material
from cases of amoebic dysentery and other diseases. The obtaining of
living amoebae in a really fresh and healthy condition suitable for
careful microscopic study—both alive and after treatment by good
cytological methods—has not been easy. But the difficulties encoun-
tered have mostly been overcome, and I now feel that I have had
sufficient experience to enable me to speak with some confidence about
the intestinal amoebae at least. My confidence concerning most of the
facts recorded in the following pages has been greatly strengthened
by the confirmatory observations of a large number of fellow-workers
and pupils. The majority of my own observations have, indeed, become
the common everyday knowledge—confirmed time after time in the
daily routine—of dozens of workers who have been engaged with me in
this field of work during the War.

I would note here, however, as a word of warning to many who have
studied this subject only from the practical standpoint of diagnosis, that

6 THE AMOEBAE LIVING IN MAN

the methods which suffice for this purpose are not adequate for obtaining
a complete knowledge of the amoebae themselves or of their develop-
ment. For example, if an amoeba found in the stools is so degenerate
as to be unidentifiable, its species may, in practice, often be deter-
mined by the finding of its cysts. But this does not mean that these
amoebae are really undeterminable as to their genus or species; nor does
this empirical method permit us to dispense with a study of the normal
organisms when considering them from a protozoological standpoint.
As a matter of fact, I believe all the species of amoebae living in the
human intestine are easily determinable if examined in a fresh and
normal state, though often undeterminable under the conditions imposed
by laboratory practice. Makeshift methods of examination and deter-
mination are only of value for purposes of ordinary laboratory diagnosis,
which has often to be made from material which 1s quite unsuitable for
zoological study; and it is hardly necessary to point out that a mere
examination of amoebae or cysts in saline or iodine solution—which
is usually sufficient for diagnostic purposes—cannot supply that detailed
information requisite for a true understanding of the organisms
investigated. This information, as I would again emphasize, can be
obtained only by studying an abundance of the best material with the
aid of all the cytological resources now at the disposal of protozoologists.

The following special points concerning technique may be noted
briefly here, as they have some interest or importance.

It is absolutely essential to study the living organisms as well as fixed
and stained specimens. Every competent protozoologist now recognizes
this as a general principle; but we have already had, nevertheless,
numerous descriptions of “amoebae” from man which have never even
been seen alive. After studying the living amoebae or cysts, an examina-
tion of them in iodine solution* is often very helpful—especially for
cysts, in which the iodine-reaction of the contained glycogen often
supplies important information. In iodine solution, also, owing to the
fixation which takes place, the nuclei become easily visible; and their
structure can thus be studied roughly, and their number counted accu-
rately. Any good cytological method of fixation will usually fix amoebae
well, but the fixation of cysts is often much more difficult. The best
fixative for cysts is, in my experience, Schaudinn’s sublimate-alcohol
solution to which 4 to 5 per cent. of glacial acetic acid has been added.
Cysts—especially those of E. coli—are sometimes difficult to stain: and
it is useful to remember that they are, in general, more readily permeable
to watery than to alcoholic solutions. Grenacher’s borax-carmine, used
warm, and acidified with a small quantity of glacial acetic or hydro-
chloric acid, will often stain the contents of cysts when all other methods
have failed. For ordinary routine purposes I have found Mayer’s
haemalum by far the most reliable and rapid stain for cysts of all sorts ;
though it cannot, of course, replace the finer cytological stains—such as
the various iron-haematoxylin methods. My alcoholic iron-haematein

* I do not know who first introduced this method for the study of intestinal amoebae
and cysts. The method is, of course, one of the earliest ever used for studying pro-
tozoa. McCarrison (1909) Kuenen and Swellengrebel (1913) and others, have used it for
studying the cysts of Etamoebae ; and since its recommendation by Wenyon (1915) as
a routine method, it has come into general use. Iodine should be used as a strong
aqueous solution in potassium iodide—the stronger the better.

A NOTE ON MATERIAL AND METHODS 7

method* is very good for all amoebae, but usually not reliable—owing
to unequal penetration—for staining cysts.

I have obtained beautiful preparations of amoebae and their cysts,
and of sections of intestinal ulcers and other tissues containing E. histo-
lytica, by employing a modified form of Mann’s stain. As I have used
this method for some years, and taught it to many people, I may mention
it here. I use Mann’s well-known methylblue-eosin mixture+ made up
in the usual way, but differentiate the preparations with a very dilute
solution of Orange G in 70 per cent. alcohol—instead of using the
alcoholic solution of caustic soda which he employs. Very fine results
can be obtained by this method—not only with tissues and amoebae, but
often with cysts as well. For sections I have also found Borrel’s magenta
and picro-indigo-carmine method very useful: but a modification of
this, in which I use acid fuchsin instead of basic fuchsin, gives even
better results with sections containing E. histolytica, as the amoebae can
be differentially stained by this method. Safranin may also be used
instead of magenta, and gives excellent results. For demonstrating
glycogen in the cysts of amoebae, Best’s specific carmine staint is very
useful—both as a control for the reactions obtained with iodine, and
as a method by which permanent preparations showing glycogen can
be obtained. Cysts may be fixed previously in Schaudinn’s sublimate-
alcohol solution, or with Carnoy’s chloroform-alcohol-acetic-acid mix-
ture: and if it is desired to show the nuclei as well as the glycogen,
they can be coloured by previously staining the cysts in Weigert’s
alcoholic iron-chloride haematoxylin—without removing the glycogen
or affecting its staining powers.

All the specimens figured in the Plates have been drawn, with the
aid of the camera lucida, at a uniform and exact magnification of
2500 diameters. All the drawings were made from preparations examined
under a 2 mm. apochromatic objective (Leitz), with N.A. = 1.40, using
compensating oculars and an achromatic aplanatic condenser. The
methods by which the specimens figured were fixed and stained are
all noted in the descriptions of the Plates. I will only add here that
for studying and drawing organisms stained with red stains—such as
fuchsin or carmine—I have found it an advantage to use a green light
for illumination, as details can then be resolved with greater ease and
precision. I have founda Wratten colour screen (“B. Filter,” No. 58),
placed below the condenser of the microscope, very useful for this
purpose. This is, of course, a general method, and not one peculiarly
suited to the study of amoebae. I mention it here merely because all
specimens actually stained red, but depicted in black in Plates III-V,
have been drawn from preparations examined in this manner.

* Vide Dobell (1914).
+ Vide G. Mann: “ Physiological Histology” (1902), p. 216.
+ Vide ¥. Best (1906): Zeztschr. f. wiss. Mikrosk., xxiii, 319.

IIT.

THE PRESENT STATE OF KNOWLEDGE OF THE
AMOEBAE LIVING IN MAN.

BEFORE I attempt to give detailed descriptions of the amoebae which
live in man, I will devote a short space to a general account of our
knowledge of the whole subject ; my object being to indicate, by means
of a brief historic survey, the present state of knowledge concerning all
the amoebae which live in human beings, and the more important of the
steps by which this knowledge has been acquired.

By far the greater part of the literature on this subject deals with the
amoebae which live in the intestine. These have always been of par-
ticular interest to the physician, on account of their relation—real or
supposed—to dysentery and other pathological conditions. But it is
only within the last half-dozen years that the medical problems con-
nected with these organisms have been fully elucidated: and it is
probable that many more years will yet elapse before the knowledge
which we now possess becomes generally current in zoology and
medicine. It is a familiar observation that it is often more difficult to
establish a truth than to perpetuate an error : and of this the history of
the present subject already supplies abundant illustrative instances.

Amoebae appear to have been first found in man by Gros (1849) in
Russia, who discovered and briefly described the amoeba now known as
Entamoeba gingivalis, which lives in the mouth. Steinberg (1862),
Grassi (1879a), Kartulis (1893), and Prowazek (1904), all rediscovered the
same organism later, and in recent years it has acquired some notoriety.
The earlier workers appear to have regarded it as a harmless organism :
but Bass and Johns (1914, 1915), Smith and Barrett (1915), and other
more recent investigators—especially in America—have upheld the
hypothesis that it is the cause of pyorrhoea alveolaris, and consequently
a pathogenic parasite of considerable medical importance. Much work
has since been done on this subject, and it now appears more than
probable that the earlier workers—as is so often the case—had the
clearer vision. At the present moment all the evidence points to the
conclusion that E. gingivalis is an inoffensive commensal, which man
very commonly, but unsuspectingly, lodges in his mouth. It seems
unlikely that the future will reveal any further facts of fundamental
importance concerning this amoeba.

It is generally said that the intestinal amoebae of man were dis-
covered by Lambl, in Prague. This is, I think, an error. It is true
that Lambl (1860) reported the finding of “amoebae” in the intestine
of a child dead of enteritis; but as he also found ‘‘ Diffiugia” and
“‘Arcella”’ in the intestinal contents, his observations are open to grave
suspicion—as Leuckart (1863) long ago pointed out. His ‘‘ amoebae,”
moreover, can hardly have belonged to any of the species now known

THE PRESENT STATE OF KNOWLEDGE OF THE AMOEBAE 9

to live in the intestine. They measured only 4 to 6p in diameter, and
were almost certainly small degenerating individuals of the flagellate
Trichomonas hominis. Although this has been pointed out by Leuckart
(1863)* and Grassi (1888), modern workerst still continue to cite Lambl
as the discoverer of the intestinal amoebae of man.

So far as I have been able to ascertain, the intestinal amoebae of
man were really discovered by Lewis (1870) and Cunningham (1871),
in the course of their investigations on cholera in India. The amoebae
which they studied probably belonged, for the most part, to the species
now known as Entamoeba coli—as will be shown later. It is therefore
not surprising that these workers concluded that the intestinal amoebae
of man are not pathogenic. Soon after the publication of their observa-
tions, however, another amoeba was discovered by Lésch (1875), in
the stools of a patient suffering from dysentery, in Russia: and the
discovery of this organism initiated a discussion which subsequently
engaged the attention of a large number of workers and lasted for some
forty years. The chief point at issue was whether the intestinal amoebae
of man do or do not cause dysentery. Opposite views were held by
different workers and at different periods—the consensus of opinion
swinging first to one side and then to the other.

There appear to me to be two chief reasons why the comparatively
simple problems connected with amoebic dysentery remained so long
unsolved. First, there was a failure to realize that the amoebae constitute
a large group of organisms, containing many species belonging to many
different genera. Of these, man harbours not one but several different
kinds; and all are forms which have nothing to do with the free-living
amoebae. The second obstacle in the way to knowledge was the failure
to realize that “ dysentery ” is not one disease, but a symptom of several
different pathological conditions. There is no one specific “cause”
of all dysenteric symptoms; and it is now even difficult to conceive
how anybody could ever have thought that he had ruled out amoebae, —
as a cause of dysentery, by simply demonstrating that certain bacteria
can also cause it.

We now know that the amoeba which Lésch discovered was
Entamoeba histolytica—one of several different species living in the
human bowel, and the only one, so far as is known, capable of causing
dysentery or any other disease in man. We now know also, however,
that this organism does not usually cause dysentery, which is most often
the result of infection with certain bacteria. At the time of Lésch’s
discovery bacillary dysentery was not a clearly recognized condition,
and all amoebae were regarded—at least by physicians—as suspiciously
alike. Lésch himself was not convinced that the amoebae which he
found were the cause of his patient’s illness, and he seems to have ~
regarded them rather as a secondary or accessory factor in the causation
of dysentery. Much of the earlier work on the intestinal amoebae was

* Leuckart (1863, p. 140) drew attention to the similarity of Lambl’s figures—
which he reproduced—to an organism described from fowls by Eberth. This organism
(** Trypanosoma” ebertht Kent) is now known, since the work of Martin and Robertson,
to be a Zrichomonas.

+ Many of them cite a paper published in the Vrerteljahrschr. 7. prakt. Heilkde.,
Prag, 1859. In this, however, amoebae are not even mentioned. I fancy few people
have really seen Lambl’s work of 1860, which is very difficult to obtain.

i0 THE AMOEBAE LIVING IN MAN

done by the Italians—Grassi (1879—1888), Calandruccio (1890), Celli
and Fiocca (1894, 1895), Casagrandi and Barbagallo (1895, 1897)—whose
observations were in many cases perfectly correct, but unfortunately
marred by their failure to recognize the existence of more than one
species of amoeba in man. They identified all their amoebae with
those of Lésch, whereas they were really those studied by Lewis and
Cunningham; and they wrongly concluded, from their investigation
of a harmless species, that all intestinal amoebae are equally harmless.

Lésch’s amoebae were studied, during the same period, by many
other workers. Among these Kartulis (1885—1893), Councilman and
Lafleur (1891), Kovacs (1892), and Kruse and Pasquale (1894) must be
specially mentioned. To Councilman and Lafleur especially belongs
the credit of having first stated clearly that there is a particular kind of
dysentery caused by amoebae—“ amoebic dysentery,” as they first called
it. Before their time there were workers—such as Kartulis—who
apparently believed that “dysentery” in general is invariably and
exclusively the result of amoebic infection. Councilman and Lafleur
also confirmed the observation of Kartulis (1887) that amoebic dysen-
tery may be followed or accompanied by the formation of hepatic
abscesses ; and that in such cases amoebae, apparently identical with
those found in the stools, may be present in the liver—as Koch” first
showed. That “tropical” hepatic abscess is definitely associated with
“tropical ”’ dysentery had, however, been recognized long before by the
Anglo-Indian clinicians, whose observations thus found their true
explanation.

I do not know who first suggested that more than one species of
amoeba may inhabit the human bowel. Schuberg (1893) attributes the
idea to Kartulis (1891), but it was expressed also by Councilman and
Lafleur (1891) and Lutz (1891) at about the same time. Schuberg
himself, and Lutz, and most other workers at this period, concluded that
there was insufficient evidence to prove the existence of more than one
species. Councilman and Lafleur, it is true, believed in the existence
of more than one species, but they adduced no evidence in support of
their belief. This evidence, however, was promptly supplied by Quincke
and Roos (1893); but by one of those curious blunders which so often
arrest the progress of science, their observations were almost ignored
by their contemporaries, and never received the attention which they
merited.

By the year 1897 all the main facts necessary for understanding the
relation of amoebae to dysentery had been discovered. It was clear
from the work of the bacteriologists+ that epidemic dysentery is usually
caused by bacteria and not by amoebae. It was equally clear, however,
that there is a particular kind of dysentery caused by amoebae—as the
work of Lésch, Kartulis, Councilman and Lafleur, Kovacs, Kruse and
Pasquale, and Quincke and Roos had demonstrated. It was, moreover,
evident, from the work of Grassi, Calandruccio, Celli and Fiocca, and
Casagrandi and Barbagallo, that all intestinal amoebae do not cause
dysentery. Furthermore, it had been shown by Quincke and Roos how
the different species of intestinal amoebae can be distinguished from

* Vide Koch and Gafftky (1887).
t Consult especially Janowski (1897) in this connexion.

THE PRESENT STATE OF KNOWLEDGE OF THE AMOEBAE II

one another zoologically: and it had been proved, by Casagrandi and
Barbagallo, that the intestinal amoebae have nothing to do with the
free-living species, from which they differ both morphologically and in
the character of being uncultivable in artificial media. It is truly
astonishing, in reading the works on the intestinal amoebae of man,
such as Behla’s (1898), and the larger medical and zoological treatises
published at the end of the last and the beginning of the present century,
to observe the blindness which appears to have descended upon every-
body who studied this subject at this period. Instead of illumination,
darkness followed ; and the twentieth century began with a period of
nearly a dozen years of chaos.

For this period of confusion Schaudinn was, in my judgement,
chiefly responsible. Notwithstanding his great services to the science
of protozoology in other respects, his influence upon the present subject
was almost wholly bad. His work, published in 1903, produced a pro-
found effect, though it was merely a brief preliminary statement of his
views—dogmatic, full of errors, unillustrated ; and his conclusions, had
they been presented by any other worker, would probably not have
been accepted without further evidence. There was, indeed, but one
fundamental point in which he was not mistaken—his assertion that
there are two different amoebae, one pathogenic and the other harmless,
inhabiting the human bowel: and nobody who has read the works of
earlier observers can give Schaudinn much credit for this “ discovery,”
in which he had been forestalled repeatedly. Nevertheless, the fact
remains that when Schaudinn said it, everybody realized its truth ;
whereas the words of earlier observers fell upon deaf ears. Schaudinn’s
‘‘life-histories ”’ of the two forms were almost entirely wrong. Some
of his observations and experiments are, indeed, so incredible* that it is
difficult to believe that they were not sheer inventions. Certain it is, at
all events, that no competent worker will ever repeat them. In his
revision of the nomenclature of the intestinal amoebae he was equally
unfortunate, and for his errors of judgement we still suffer.

Another cause of the arrest of progress in this subject at the
beginning of the century was undoubtedly the work of Musgrave and
Clegg (1904, 1906), carried out in the Philippines. These workers
upheld the thesis that “all amebas are or may become pathogenic.”
Their chief reason for believing this, apparently, was their inability to
distinguish one species of amoeba from another. For them all amoebae
were alike. They appear to have been almost uninfluenced by the
earlier work of others, and to have thought it unnecessary to study
protozoology or cytological methods. Mixing up all species of amoebae
indiscriminately, and studying none of them properly, they soon reached
the conclusion that “the whole of the surface flora of the Philippine
Islands carries a large number of these parasites [i.e., ‘ amoebae’
generally]. Some of which, at least, belong to the class [= species]
which produces disease in human beings.” The importance they
attributed to morphology can be gauged by their statement that “at
the present time no value can be attached to it.” Upon zoologists
generally the expression of such views could naturally produce but

* See, for example, his amazing experiment by which he proved that £. histolytica
forms minute spores capable of surviving, in a condition infective to cats after complete
desiccation. This experiment is, to me, still quite inexplicable.

12 THE AMOEBAE LIVING IN MAN

little etfect. Upon medicine, however, Musgrave and Clegg’s work has
still left its impression ; and from time to time others still fall into the
same errors. The chief merits of their work consisted in their improve-
ments in the methods of cultivating free-living amoebae, and _ their
introduction of the now current term “ amoebiasis,” to denote a state of
“infection with amebas.”

The publications of the first decade of the present century make,
for the most part, unpleasant reading. The German workers—Prowazek,
Hartmann, and others—continued along the path of error indicated
by Schaudinn. Craig, in America, and many others, followed in their
train, and their “confirmations” of Schaudinn’s work, coupled with the
discovery at intervals of new “species” of amoebae in man, served
only to make matters worse. A present-day seeker after the truth,
completely ignorant of the subject, would obtain more reliable infor-
mation by consulting the works published prior to 1900 than those
which appeared during the next ten years. In the latter period almost
the only observation of any real value was made by Huber (1903), but
it was stifled by the authority of Schaudinn and the other German
workers. Huber rediscovered and first investigated the cysts of the
dysentery amocba—formerly found by Quincke and Roos—and really
supplied the chief deficiency in our knowledge of this organism. The
rediscovery of these cysts by Viereck (1907), Hartmann (1908), and
Elmassian (1909),—who all described them as belonging to new species
—did not mend matters. No real advance was effected until the com-
pletion of the epoch-making work of E. L. Walker in the Philippine
Islands. This work must now be considered.

Walker made an unpromising beginning. His first paper (1908)
repeated many of the errors of his predecessors. He was unable to
distinguish the free-living from the parasitic species of amoebae, and
confused forms which he was able to cultivate, with those found in the
intestinal contents of man and other animals. A second paper, pub-
lished three years later, marked an immense stride forward. Walker
(1911) was then able to draw the following conclusions* from his
researches :

(1) The amoebae found in the Manila water supply, and those
cultivable from the intestinal tract of man, all belong to the genus
Amoeba Ehrenberg. These species are not parasitic in the intestine
of man. When amoebae are obtained in cultures from the intestinal
contents, they are derived from ingested cysts which have passed
unchanged through the intestine.

(2) The amoebae parasitic in the intestinal tract of man belong to
a distinct genus, Entamoeba Casagrandi and Barbagallo. They are
obligatory parasites, and cannot be cultivated. Two species are recog-
nizable — one non-pathogenic (Entamoeba coli) and one pathogenic
(E. histolytica). The first forms cysts containing 8 nuclei; the second
cysts containing 4 nuclei. The organisms are transmitted from man
to man by means of these cysts.

In his final paper, published in collaboration with Sellards (1913),
Walker was able to supply proofs of all these statements, as a result of

* I have modified and condensed these conclusions somewhat, so that they are not
given here in Walker’s own words exactly.

THE PRESENT STATE OF KNOWLEDGE OF THE AMOEBAE 13

a series of carefully planned and executed experiments on human beings.
This work finally solved all the chief problems connected with the
relation of amoebae to dysentery. It confirmed and vastly extended
the earlier observations of the Italian workers, of Quincke and Roos,
and of others, and placed our knowledge of the subject on a firm
foundation of fact which is still unshaken and probably unshakable.

There are, however, certain minor details in which Walker’s
conclusions must now be qualified. His statement that the free-living
amoebae belong to the genus Amoeba is incorrect. The forms which he
studied—the so-called *‘limax amoebae ”—certainly do not belong to
the genus Entamoeba ; but it is equally certain that they do not belong
to the genus Amoeba Ehrenberg, but to other genera. Further, it is now
certain that there are not merely two intestinal Entamoebae in man.
There are, as later work has shown conclusively, at least five different
species belong to four distinct genera, Of these, however, only one—
E. histolytica—is pathogenic, as Walker maintained.

Apart trom these later additions to our knowledge, there is little to
be changed in Walker’s conclusions. His work is, and will probably
remain, one of the most brilliant contributions ever made to medical
zoology. For my own part, I regard the chief problems connected with
the relation of intestinal amoebae to dysentery and other diseases as
solved finally by Walker’s work. The researches of later workers are,
if read in the light of his results, all easily comprehensible and confirma-
tory. Among these workers Wenyon, Darling, and James, must be
specially mentioned, on account of the important additions which they
have made to our knowledge: but their work will be considered in
greater detail later.

From time to time writers have since relapsed into the old mistakes ;
but it is clear that this has always been due to ignorance of the facts or
failure to understand the knowledge already acquired. In recent years,
for example, Gauducheau (1915) has questioned the correctness of
Walker’s conclusions—but without understanding them, and from the
standpoint of the early days of confusion. Again, Knowles and Cole
(1917) have counselled us to go back to the same period, by attempting
to show that all the intestinal amoebae of man belong to one species.
Their proposal to call this hypothetical “species” by the inadmissible
name of ‘ Entamoeba coli communis” shows how little qualified they are
to express any opinion on questions of protozoology, biological
systematics, and nomenclature. Many other recent workers have not
only been unable—on account of their imperfect zoological knowledge
—to distinguish different genera and species from one another, but they
still continue to confuse these organisms with cells belonging to the
human body. The recent works of Bartlett (1917) and Thomson and
Thomson (1916 a) supply instances of this: but the just criticisms of
Bahr and Willmore (1918) fortunately make it superfluous to discuss
these observations further. Thomson and Thomson (1916) have even
published work undertaken to ascertain whether the dysentery amoeba
lives in the sand in Egypt. Their views, apparently, are closely similar
to those of some of the earliest workers, and their standpoint is that of
Musgrave and Clegg,—which has really been untenable since the days
of Casagrandi and Barbagallo, and inexcusable since the work of Walker
and Sellards, Many other recent mistakes could easily be cited : but as
they all rest upon a similar ignorance of facts which are no longer even

14 THE AMOEBAE LIVING IN MAN

debatable, it is unnecessary to argue about them. Indeed, one can but
express surprise when a worker so experienced in other fields as
Marchoux (1918) now asks us to reconsider whether there is, after all,
such a thing as amoebic dysentery. Opinions such as this can only be
regarded as anachronisms, which time will eventually set right.

Although it seems probable that our knowledge of the oral and
intestinal amoebae of man is now in a state approaching finality,* this
cannot, unfortunately, be said of some of the other amoeboid organisms
which have been described from other situations in human _ beings.
There is little doubt, however, that many of these are not amoebae at
all, but tissue cells or other bodies mistaken for amoebae. I shall con-
sider some of the more important of these later. It will suffice here
merely to notice the possible existence of such organisms.

After this brief introductory survey I shall now pass on to consider
in detail the individual species of amoebae which live in man. But
before doing so it is necessary to say a few words about the genera to
which these species belong—a subject which is by no means free from
difficulties owing to the present limited state of knowledge of the
amoebae in general.

* It is, perhaps, not superfluous to point out that although we now possess much
exact and probably definitive knowledge of the intestinal amoebae of man, the literature
of the subject is still in the greatest confusion. There is not a single text-book, either
zoological or medical, which contains an even approximately correct account of these
amoebae. Medical works such as those of Brown (1910), Craig (1911), Rogers (1913),
and Phillips (1915), on amoebae and amoebiasis, and the zoological treatises of Doflein
(1911), Minchin (1912), Brumpt (1913), and others, are compact with errors of all sorts.
Inexperienced workers should be specially warned against the work of Rogers (1913),
which—though it contains many excellent clinical and other observations—does not con-
tain reliable information concerning the amoebae of man. The figures of “amoebae”
in this work are quite unrecognizable, and most misleading.

15

IV.
THE GENERA OF AMOEBAE LIVING IN MAN.

ALL the older writers who dealt with the amoebae of man placed them
in the genus Amoeba, which originally included a very heterogeneous
collection of naked rhizopods. As knowledge accumulated, however, it
became clear that this genus would have to be dismembered ; and one
of the earliest attempts in this direction was made by Leidy (1879), who
proposed to separate the amoeba parasitic in the cockroach (Amoeba
blattae Biutschli) from the free-living forms. For this organism he
proposed the new genus Endamoecba.* Consequently, the type species
of Endamoeba Leidy is E. blattae Biitschli.

Casagrandi and Barbagallo (1895 a), apparently in ignorance of Leidy’s
work, proposed a new genus Entamoeba for the amoebae which they
studied from man. At first (1895 a) they called their organism Eztamoeba
coli, believing it to be the same as that described by Lésch (1875) and
called by him ‘Amoeba coli”; but they renamed it later Entamoeba
hominis (Casagrandi and Barbagallo, 1897).

It is clear from their papers that the actual organism to which these
names were applied was the large harmless amoeba of the human colon
—now generally known, since the time of Schaudinn (1903), as Enl-
amoeba coli. It follows that the type species of the genus Entamoeba
Casagrandi et Barbagallo is E. colt.

The genus Entamoeba of Casagrandi and Barbagallo was accepted by
Schaudinn (1903) for E. coli and also for E. histolytica, the dysentery
amoeba: and since the appearance of his work it has been customary to
refer almost all the parasitic amoebae to this genus. A curious belief
seems, indeed, to have grown up that there are but two genera of
amoebae—free-living species all belonging to the genus Amoeba, and
parasitic forms all belonging to Entamoeba. Walker (1911, 1913), for
example, in his admirable works on the amoebae of man, speaks as
though no other genera exist; and most medical writers who have
studied amoebae apparently share this belief. No zoologist, however,
can now hold such a view for a moment; for it is certain that, from the
zoological standpoint, both the free-living and the parasitic species of
amoebae belong to many different genera.

There can be little doubt that the two intestinal amoebae of man
commonly known as Entamoeba coli and E. histolytica are co-generic.
The characters supplied by their nuclear structure, their cysts, and their
development and morphology generally, warrant their inclusion in the

* Leidy’s paper was long overlooked, and his genus forgotten until attention was
called to it by Chatton (1910). It may be noted here that Leidy repeated his definition
of Endamoeba in his large work on the freshwater rhizopods (Leidy, 1879 a, footnote
p. 300), which also seems to have been generally overlooked,

10 THE AMOEBAE LIVING IN MAN

same genus—whatever that may be. Moreover, the amoeba living in
the mouth of man (E. gingivalis) cannot at present be distinguished
generically from these two forms. But should these three species be
placed in the same genus as the amoeba of the cockroach ? Should
they, in other words, be put in the genus Endamoeba Leidy ?

At the present moment this question cannot be answered con-
clusively. Chatton and Lalung-Bonnaire (1912) and Alexeieff (1912)
have answered it in the negative, basing their views on the differences
in development described by Mercier (1910) in the parasite of the
cockroach. It therefore appeared to them necessary to introduce a new
generic name for the amoebae of the type of E. coli, because these
workers—and all others, apparently—consider that “‘ Eidamoeba” and
“Entamoeba” are mere variant spellings of the same name. Chatton
and Lalung-Bonnaire (1912) accordingly proposed the new generic
name Léschia, whilst Alexeieff (1912) proposed the new name Proci-
amoeba, Alexeieff’s paper was published about a month after that of
Chatton and Lalung-Bonnaire; and consequently, when this was
pointed out by Chatton (1912), he withdrew his name later (Alexeieff,
1912 a), as a synonym of Ldschia. It would thus appear, at first sight,
that the amoeba of the cockroach should be placed in the genus
Endamoeba Leidy, whilst the three best-known amoebae of man (E. colli,
E. histolytica, E. gingivalis) should be placed in the genus Léschia.

There are, however, two real difficulties in the way of accepting this
solution. First, it is by no means certain, at the present moment, that
the amoeba of the cockroach and the amoebae of the FE. coli type are
generically distinct. The distinction was drawn at a time when these
amoebae were believed to possess quite dissimilar life-histories. Accord-
ing to Mercier (1910), FE. blattae has a sexual phase at the beginning of
its life-cycle. The cysts liberate broods of large or small amoeboid
gametes which conjugate in pairs heterogamously. The previous
development within the cysts is simply a process of straightforward
nuclear division. On the other hand, according to Schaudinn (1903)
and Hartmann (1908) respectively, £. coli and E. histolytica (then called
E. tetragena) display a process of autogamy within their cysts; so that
a later gamete-formation and conjugation, like those of E. blattae,
appeared to be excluded from their development. At the present
moment, however, the position is very different. We now know, since
the work of Walker (1911) and others—which agreed with my original
observations (1908, 1909) on the closely related amoeba of the frog—
that there is no autogamy in the cysts of E£. coli, E. histolytica, or any
of their congeners. The development within the cyst is a straight-
forward process of nuclear division, like that described by Mercier and
others in E£. blattae. Furthermore, Mercier’s account of the sexual
process in this species has not yet been confirmed, and the corres-
ponding stages in E. coli and E. histolytica have never been studied.
Whether they are alike or different in this respect has therefore still
to be determined; and upon this determination the decision as to
whether they should be placed in the same or in different genera will
largely depend.

A second difficulty is this. Ltthe (1908), believing Schaudinn’s (1903)
incorrect account of the development of E. histolytica to be true, pointed
out that this parasite could not be placed in the same genus with E. coli
and related forms. He therefore proposed the new genus Poneramoeba

Fes FT eee ee
,

THE GENERA OF AMOEBAE LIVING IN MAN 17

for E. histolytica. Although the grounds for the proposal are now
known to have been fallacious, the name was introduced for an easily
recognizable organism ; and there seems to be nothing in the rules of
nomenclature which can.render it invalid. Moreover, Lihe’s name
seems to be the first generic name available for the dysentery amoeba,
if it is decided to remove it from the genus Endamoeba (= Entamoeba) :
and since E. coli and the other harmless forms related to it are co-generic
with E. histolytica, it follows that all these organisms might have to be
placed in the genus Poneramoeba. This would be a most unfortunate
interpretation of the laws of nomenclature; for it would place all the
harmless species, which constitute the greater part of the group, in a
genus designed for, and designatory of,* the one exceptional species
which is known to be pathogenic.

Chatton and Lalung-Bonnaire (1912), believing £. histolytica to be
subgenerically distinct from the amoebae of the E. coli type, proposed
a new subgenus. Viereckia to contain it. According to their nomen-
clature the organism should be called Léschia (Viereckia) tetragena.
From Chatton’s later publications it may be gathered that he has
abandoned this view, for he now calls the dysentery amoeba Entamoeba
dyseiteriae. The name Viereckia appears, in any case, to be a synonym
of Poneramoeba—if the dysentery amoeba is to be placed in a genus
apart. At the moment, however, there seem no adequate grounds for
separating it from other amoebae such as £. coli, E. muris, or E. ranarum.

In my opinion it is most undesirable to change the names of the
amoebae living in man unless this course becomes absolutely necessary.
I] propose, therefore, provisionally to retain the name Entamoeba Casa-
grandi et Barbagallo, 1895, for the organism to which it was given—
namely, E. coli—and for all those species which are clearly co-generic
(E. histolytica, E. gingivalis, E. muris, etc.) ; and to retain also the genus
Endamoeba Leidy, 1879, for the one organism—E. blattae—for which it
was proposed. It may be argued that Endamoeba and Entamoeba are
different spellings of the same name, or that they differ too slightly from
one another to be kept separate. This has already been urged by many
writers, and is doubtless justifiable. Nevertheless, nobody can say at
present whether the organisms originally called Endamoeba and
Entamoeba respectively are generically the same or different: and if the
difference is at present so slight and uncertain, then a slight difference
between their generic designations might not inappropriately express it.
Whether ultimately shown to be right or wrong, this course is, I think,
the one which will give rise to the least confusion at present. I cannot,
for my own part, accept with equanimity any drastic change in nomen-
clature which will certainly lead to confusion—however plausible a case
may be made for it by those who care more for the “correctness” of
names than for the codification of knowledge. What, for example,
should we gain by calling the dysentery amoeba, which every worker in
England has known for years as Entamoeba histolytica, by the new name
Poneramoeba coli? And yet a very plausible case indeed can be made
out for this combination.

I shall therefore continue to refer three of the common amoebae
of man—namely, E. coli, E. histolytica, E. gingivalis—to the genus

* Poneramoeba, from movnpés, causing pain, harmful.

18 THE AMOEBAE LIVING IN MAN

Entamoeba Casagrandi et Barbagallo, 1895 ; whilst provisionally I reserve
the separate genus Endamoeba Leidy, 1879, for the amoeba of the
cockroach. On this system, the type species of Entamoeba is E. coli,
and the type of Endamoeba is E. blattae. They have not one common
type.

oa may perhaps remark here that although it is clearly incorrect to
attribute to any authority a name which he did not employ, this has
frequently been done in the case of the names under discussion.* For
example, I note that Craig (1912, 1912 a) writes ‘‘ Entameba Casagrandi
and Barbagallo” ; later (Craig, 1913 6, 1914) ‘‘ Entamocba Leidy” ; still
later (Craig, 1917) “ Endameba Leidy.” Not one of these is the name
employed by the authority cited. Moreover, I would protest against the
suppression of the diphthongs in Amoeba, Entamoeba, and Endamoeba,
which has now become habitual with most American writers. They
may be justified in translating the English word “amoeba”’ into the
American “ameba’”’ (plural, “amebas’’): but it is difficult to see what
grounds they can have for altering the Latin language—for generic
names like “Entamoeba” are, theoretically at least, Latin and not
American. For my part, I find “Endameba” almost as unpleasant as
the quite unjustifiable and offensive ‘‘Entaméba”’ which German writers
frequently employ. Obviously, ‘ Amoeba” (and all derivatives such as
Entamoeba or Endamoeba) is orthographically correct as a generic name
only when written thus in its original form.

In addition to the three amoebae belonging to the genus Entamoeba
there are three other species which have to be noted here. First, there
is the organism named Entamoeba nana by Wenyon and O’Connor
(1917). ‘This organism clearly belongs to a different genus from that
typified by £. coli. From examination of the evidence I consider that
it should be placed in the genus Endolimax Kuenen et Swellengrebel
(1917). This question will be considered in greater detail, however, in
the discussion of the nomenclature of this species. (Vide infra, p. 101.)
Secondly, there is the peculiar binucleate amoeba for which—in a joint
papert—I have proposed the name Dientamoeba. The nomenclature of
this organism has already been discussed in detail in the earlier paper.
Thirdly, there is another intestinal organism which is described in the
present work, but which has previously been known in a disconnected
and incomplete manner. This is the organism called Entamoeba biitschlit
by Prowazek (1912.4), but whose cysts were called ‘Iodine cysts” by
Wenyon (1916). As this amoeba cannot be placed in any of the existent
genera, I shall propose the new genus Jodamoeba for it. The nomen-
clature of this organism will be discussed later, in the description of
the species.

In the next section I shall attempt to give a systematic account of all
the species of amoebae from man. Before doing so, however, I may
summarize the conclusions reached in this section. They are set forth
in the following synopsis, which will show the genera, species, and
types at a glance, and will also serve as a key to the species described in

* In addition to the instances cited I may also note that Hartmann (1913) writes
“ Entamoeba Leidy emend. Schaudinn”—a remarkable combination when it is
remembered that Leidy’s real name (Zudamoeba) was unknown to Schaudinn, who
used Hatamoeba Casagrandi et Barbagallo.

+ Vide Jepps and Dobell (1918).

THE GENERA OF AMOEBAE LIVING IN MAN 19

the ensuing sections. It should be added that the doubtful organisms
discussed in a later section (p. 134) are purposely excluded from this
synopsis, as our knowledge of them is still too uncertain to allow of
their classification—supposing them to be amoebae. It is probable,
however, that many of them are not even protozoa.

SYNOPSIS OF GENERA AND SPECIES OF AMOEBAE LIVING IN MAN.

Genus 1. ENTAMOEBA Casagrandi & Barbagallo, 1895.
(nec Endamoeba Leidy, 1879.)

Synonyms :
Poneramoeba Lihe, 1908.
Léschia
Viereckia
Proctamoeba Alexeieff, 1912.

[Amoeba (pro parte), Endamoeba, Entameba, Endameba,
Entamoba, Auctt.]
Type: E. coli (Grassi) Casagrandi & Barbagallo.

Species in Man: E. coli (Grassi) Casagrandi & Barbagallo.
E. histolytica Schaudinn (emend. Walker).
E. gingivalis (Gros) Brumpt.

Chatton & Lalung-Bonnaire, rgr12.

Genus II]. EwDoLivax Kuenen & Swellengrebel, 1917.

Only species, hence type: E. nana (Wenyon & O’Connor)
Brug.

Genus III. /ODAMOEBA nov. gen.
Only species, hence type : I. bi#tschlii (Prowazek) Dobell.

Genus IV. D/ENTAMOEBA Jepps & Dobell, 1918.
Only species, hence type: D. fragilis Jepps & Dobell.

20

Vie
GENUS ENTAMOEBA CASAGRANDI & BARBAGALLO, 1895.

THREE species belonging to this genus are found in man. I shall
begin with the most important—

(1) ENTAMOEBA HISTOLYTICA SCHAUDINN, 1903 (EMEND.
WALKER, 1911).

“ Amoeba coli’? Lésch, 1875.

? “Amoeba urogenitalis”’ Baelz, 1883.

? Amoeba vaginalis Blanchard, 1885.

? Amoeba intestinalis Blanchard, 1885.

“ Amoeba dysenteriae’? Councilman & Lafleur, 1891.
Amoeba coli (Lésch)

Amoeba dysenteriae (Councilman & Lafleur)
“ Amoeba coli felis’? Quincke & Roos, 1893.
Amoeba lobosa var. coli Celli & Fiocca, 1894.
Entamoeba histolytica Schaudinn, 1903.
Entamoeba dysenteriae (Councilman & Lafleur) Craig, 1905.
[Entamoeba] “ tetragona” (Schaudinn) Huber, 1906.

Entamoeba coli var. tetragena Viereck, 1907.

Entamoeba africana Hartmann, 1907.

Entamoeba tetragena (Viereck) Hartmann, 1908.

“Entamoeba Schaudinni’”’ Lesage, 1908.

Poneramoeba histolytica Lithe, 1908.

Entamoeba minuta Elmassian, 1909.

Entamoeba nipponica Koidzumi, 1909 (pro parte).

Entamoeba hartmanni Prowazek, 1912.

Entamoeba coli Werner, 1912 (pro parte).

Léschia (Viereckia) tetragena Chatton & Lalung-Bonnaire, 1912.
Entamoeba brasiliensis Aragao, 1912 (pro parte).

Léschia histolytica (Schaudinn) Mathis, 1913.

Entamoeba venaticum Darling, 1915.

Entamoeba minuta Woodcock & Penfold, 1916 (mec Elmassian, 1909).
‘‘ Non-pathogenic £. fetragena” Shimura, 1916 (pro parte).
Endamoeba coli (Losch) Aragao, 1917.

Endamoeba dysenteriae (Councilman & Lafleur) Pestana, 1917.
Entamoeba tenuis Kuenen & Swellengrebel, 1917.

Entamoeba minutissima Brug, 1917.

Endamoeba histolytica (Schaudinn) Craig, 1917.

Entamoeba coli conmunis Knowles & Cole, 1917 (pro parte).

Kovacs, 1892.

HISTORIC.
Entamoeba histolytica, the dysentery amoeba of man, was discovered
by Lésch in 1873 (vide Lésch, 1875), in the stools of a young Russian
peasant named Markoff, who had come, from his village in the Govern-

trite hah ti Bf ad

ENTAMOEBA HISTOLYTICA 21

ment of Archangel, to look for work in Petrograd. Here he contracted
dysentery, and after spending about 5 months in hospital, died from
an intercurrent attack of pneumonia. Lésch has left descriptions of the
patient, the parasites, and the post mortem findings, which leave no room
for any doubts concerning the interpretation of his case.

Lésch’s patient suffered from a persistently relapsing dysentery, with
bloody mucous stools in which large numbers of very active amoebae
were often present. They measured, when rounded, 20-30 w as a rule,
though sometimes more ; and “not seldom”’ they contained red blood
corpuscles, and occasionally leucocytes and fragments of epithelial cells,
Their ectoplasm was clearly differentiated from their endoplasm, and
each amoeba possessed a vesicular nucleus with a well-defined membrane
and a minute central nucleolus—points which are all illustrated by good
figures. Attempts were made to infect 4 dogs, by mouth and rectum,
with fresh stools containing the parasites. One dog contracted dysentery,
with numerous amoebae in its evacuations, and was ultimately killed.
A post mortem examination showed that its large intestine was ulcerated,
amoebae being present in the ulcers and in the intestinal contents.
When the patient died, the autopsy revealed a similar condition of
ulceration in his large intestine. Lésch considered that his amoebae
were different from any previously described, and proposed to call them
“ Amoeba coli.” Though he remained in doubt as to the precise relation
of the parasites to the patient’s disorder, he appears to have believed that
they were not the primary cause of the dysentery, but acted rather as
mechanical irritants which prevented the healing of the dysenteric
ulcers originally caused by some other agency.

Losch studied another case of amoebic dysentery later at Kieff. It
was recorded by Massiutin (1889), who described four other cases of
amoebic infection—all probably (?) infections with E. colimand who
also considered that amoebae do not directly cause dysentery. The
“ Amoeba coli” found by Grassi (1879 a), and studied by him and other
Italian workers subsequently, was—in all probability—wrongly identified
with Lésch’s amoeba. It was, for the most part, Entamoeba coli—not
E. histolytica.

The next discovery of importance, after Lésch’s observations, was
made by Koch in 1883, though not published until a few years later
(Koch and Gaffky, 1887). In conducting inquiries for the Cholera
Commission sent to Egypt and India in 1883, Koch had occasion to make
post mortem examinations of 5 cases of dysentery—2 of them compli-
cated with liver abscess, Sections of the intestinal ulcers of 4 of these
cases revealed “peculiar amoeboid structures,’ of variable shape and
“about 14-2 times as large as white corpuscles.” They were present in
sections only, or in material from the bases of the ulcers—never in the
dejecta or gut contents: and in one case they were also present in the
capillaries round the wall of the liver abscess. Koch appears to have
regarded these bodies as “amoebae,” but his observations, at the time,
were by no means unequivocal. There can be little doubt now, how-
ever, that he actually observed EL. histolytica, apparently for the first time,
in the primary lesions in the bowel and also in its secondary site of
infection in the liver.

Further observations upon the occurrence of amoebae in dysentery
were soon recorded in Egypt by Kartulis (1885, ef seq.). In his first
paper (1885) he described, from 6 cases, “ giant amoebae ?” which do

22 THE AMOEBAE LIVING IN MAN

not appear to have been amoebae at all. What they were I cannot
determine from his account.* In a second paper, however, published
in the following year (Kartulis, 1886), he gives a very different descrip-
tion of “amoebae.” He says he studied 150 cases of undoubted
dysentery in Egypt, and found amoebae in all. In sections of the
intestinal ulcers of 12 of these cases he also succeeded in finding the
parasites—thus confirming the observations of Koch. Control cases,
not suffering from dysentery, were never found infected. The amoebae
are said to have measured 12-30 w in diameter, and other characters are
also noted ; but his description of them is very inferior to Lésch’s. At
this date Kartulis could record only negative results from his attempts
to infect laboratory animals, and to cultivate the parasites. He con-
sidered, however, that the amoebae were the cause of “ tropical dysen-
tery,’ and appears to have identified them with Lésch’s “ Amoeba
coli.” Although it is now certain that many of Kartulis’s amoebae
really were E. histolytica, it is inconceivable that he could really have
found this parasite in every one of 150 cases of clinical dysentery
examined in Egypt, and in no non-dysenteric cases. Some at least
of his dysenteric patients must have been cases of bacillary dysentery,
and it would be impossible now for any competent worker to examine
many non-dysenteric persons in Egypt without finding E. histolytica.+

A year later Kartulis (1887) published another important paper,
announcing that he had found his amoebae in the pus of liver
abscesses—thus confirming the much earlier suspicions of the Anglo-
Indian doctors, and the observation of Koch, that “tropical”’ liver
abscess is a sequel to a certain form of dysentery and due to the same
cause. A fuller account of his observations was published two years
later (Kartulis, 1889) ; and still later he recorded that he had been able
to infect cats with the amoebae, and thereby to produce dysentery in
them experimentally (Kartulis, 1891). In this he appears to have been
anticipated by Hlava (1887), working at Prague. Unfortunately,
Kartulis (1891) also claimed to have cultivated the dysentery amoebae
(in straw infusions, exposed to the air), and to have produced dysentery
in cats by injecting the cultures. These obviously fallacious experi-
ments were soon seized upon and discredited by other workers, and
served rather to weaken than to strengthen his contention that intestinal
amoebae are the cause of “tropical”? dysentery and liver abscess. The
last important contribution made by Kartulis to our knowledge of the
dysentery amoeba was his discovery of the parasite in abscesses of the
brain (Kartulis, 1904). That the brain, like the liver, may be secondarily
infected is now a well established fact—foreshadowed long ago in the
work of Morehead and the Anglo-Indian clinicians, and fully confirmed
by Legrand (1912) and others.

The discovery of amoebae in post-dysenteric liver abscesses was soon
confirmed by Osler (1890) in America, whose observations led Council-
man and Lafleur (1891) to undertake an extensive investigation into the
pathology of “‘amoebic dysentery” and “amoebic abscess of the liver”

* Although called “giant” forms, their sizeis given as ‘‘ 0’°00015—0'000222 mm.”’ but
the figures—stated to be magnified about 100 diameters—show much larger bodies.
No structure can be made out in them, and none is described. It is not stated that
the “amoebae” were motile when alive.

+ Compare, for example, the findings of Wenyon and O’Connor (1917).

ENTAMOEBA HISTOLYTICA 23

—terms which they introduced. Although their work is still a classic
‘from the standpoint of pathology, it added almost nothing to our know-
ledge of the amoebae concerned. From a zoological point of view their
account of £. histolytica is greatly inferior to Lésch’s.

Kovacs (1892) studied 2 cases of amoebic dysentery, and experi-
mentally infected 5 kittens with the amoebae, of which he gave a clear
description. There can be no doubt that he studied E. histolytica, and
that his work was an important confirmation of the earlier observations.
He observed typical intestinal lesions in his cats, but failed to cultivate
the amoebae. Fuller confirmation of the facts discovered by Lésch,
Koch, Kartulis, and Councilman and Lafleur, was published soon after-
wards by Kruse and Pasquale (1894), as a result of investigations carried
out in Egypt. An important new point which they* brought to ligh-
was the fact that the amoebae in liver abscess pus—which is bacterio
logically sterile, as Kartulis (1887) first showed—are able, if injected per
rectum, to infect a cat and give it dysentery. They successfully per-
formed this experiment three times out of seven attempts.

The experiment clearly indicated two important conclusions: namely,
that the amoebae associated with dysentery are identical with those found
in the pus of liver abscesses, and that the parasites are causally connected
with these diseases. A counterpart to this experiment was furnished
later by Harris (1901), who succeeded in infecting puppies with the
amoebae from a human case of dysentery. Not only did they acquire
amoebic dysentery—as in Lésch’s (1875) experiment—but two of them
also developed amoebic liver abscesses subsequently. At the same time
it was shown that the bacteria cultivated from the stools of the patient
did not cause dysentery when introduced into the dog’s intestine.
Several workers have since produced amoebic liver abscesses experi-
mentally in cats by a similar procedure.t

The most important zoological work at this early period, however, is
that of Quincke and Roos (1893) and Roos (1894), carried out in Kiel;
for it not only showed that more than one species of amoeba inhabits
the human bowel, but it also showed with equal clearness how these
species may be differentiated, and how man becomes infected with them.
It is astonishing that this fundamentally important work has hitherto
received so little attention.

Although Quincke and Roos studied only a single case of amoebic
dysentery, they studied it very carefully; and they controlled their
observations by a study of the amoebae occurring in non-dysenteric cases.
The amoebae found in the patient suffering from dysentery are well
described, and recognizably figured.t The sharp demarcation between
the ectoplasm and the endoplasm, the appearance of the nucleus, the

* Schuberg (1893) states that this experiment was first performed by Kartulis (1891).
This author says, however, that he cultivated the amoebae from a hepatic abscess—z7
pure culture, free from all bacteria : and with this culture he claimed to have infected
a kitten, which acquired typical amoebic dysentery and showed typical ulceration of its
gut post mortem. In the light of our present knowledge the interpretation of this result

by no means free from difficulties.

+ Craig (1905), Huber (1909), Wenyon (1912), Baetjer and Sellards (19142), Dale
and Dobell (1917).

t By Roos (1894). The woodcuts in the first communication by Quincke and Roos
(1893), are very crude.

24 THE AMOEBAE LIVING IN MAN

frequent presence of red corpuscles in the cytoplasm, and the striking
activity of the organism, are all noted. In their account of all these
characters Quincke and Roos confirmed the observations of Lésch,
Kovacs, and other early workers. But in addition, they discovered
the cysts of the parasite in the stools of their patient. They are
described as rounded, refractile structures with a thin but definite
wall, smaller than the active amoebae, and measuring 10-12 in
diameter. By means of careful experiments they proved that a cat
can be infected, and acquire amoebic dysentery with characteristic
intestinal lesions, by causing it to swallow the cysts, or by injection of
the active amoebae per anum. All these characters were emphasized as
distinctive of the amoebae associated with dysentery in man: for they
found that the other amoebae which they studied (in reality E. colt)
differed in all the characters noted. They were sluggish, contained
ingested foreign bodies, but never red corpuscles, formed larger cysts
(16-17 w) with a thicker wall, and were non-infective and non-pathogenic
_ for cats. From their observations they drew the conclusion that man
acquires his infection with the dysentery amoeba by swallowing its cysts
—as they had shown to be possible in the case of the cat. The only
thing of any importance that Quincke and Roos failed to do was to
investigate the cytological details of the amoebae and their cysts. They
merely noted that the latter contain “ nucleus-like structures,” but they
did not study these properly nor count them. Roos’s figures show cysts
with one or two nuclei (indistinct in some), and indications of chromatoid
bodies. In the matter of nomenclature they were unfortunate; for
though they rightly identified their pathogenic amoeba with Lésch’s
‘Amoeba coli,” they wrongly proposed to change its name to “ Amoeba
coli felis,” on account of its pathogenicity for the cat.

The dysentery amoeba was restudied and redescribed by many workers
in the following decade, but nothing material was added to our knowledge
of it. Jiirgens (1902) confirmed the earlier observations on the amoebae,
and Schaudinn (1903) renamed them. But neither of them studied the
cysts again, or understood the life-history of the organism. Schaudinn,
indeed, added a wholly incorrect account of its development, far behind
that of Quincke and Roos. He failed to find the cysts, and substituted a
highly imaginative account of sporulation” in place of encystation.*

In the very same year that Schaudinn’s erroneous statements made
their appearance, a real discovery was made by Huber (1903) : but so great
was the authority of Schaudinn, that Huber’s work was—and is—almost
completely ignored. Huber (1903) confirmed the observations of
Quincke and Roos. He studied a typical case of amoebic dysentery, he
saw the amoebae and their cysts, and he infected cats with the former per
rectum and with the latter per os. | He added the important observation |
that the cysts contain 1, 2, or 4 nuclei, but never more, and also chromatin
masses and blocks, and can thus be distinguished from the cysts of the

* According to Schaudinn, Z. histolytica does not encyst but forms resistant spores,
3-7 # in diameter. These are described as being formed by a kind of “ budding,’
which is now generally supposed to have been a degenerative fragmentation. Craig
(1908) “ confirmed” this account, and published figures of the stages: but notwith-
standing the circumstantial evidence brought forward by both these workers, I am
unable to decide what these “spores” really were. Craig has since recanted, but he
has not wholly explained his previous findings.

.

ENTAMOEBA HISTOLYTICA 25

“ordinary” amoeba (i.e., £. coli, as described by Schaudinn). Huber
told Schaudinn* of his observations—which were perfectly correct—but
neither the latter nor anybody else who knew of them seems to have
attached any importance to them at the time. After Schaudinn’s death
the cysts were once more “discovered” by Viereck (1907) and by
Hartmann (1907),t who regarded them as belonging to new species
of Entamoeba—named by them respectively E. tetragena and E. africana.
Elmassian (1909) again “ discovered”’ them two years later, and regarded
them—together with the precystic amoebae which form them—as another
new species, which he named LF. minuta. These various “ discoveries,”
and others made during this period, only served to add to the existing
confusion. Quincke and Roos’s observations were forgotten, Huber’s
were ignored, everybody looked for—and some found—the non-existent
development of E. histolytica described by Schaudinn. No real advance
in our knowledge of the dysentery amoeba took place until the work of
Walker (1911) made its appearance, followed soon after by his later
memoir, in collaboration with Sellards (1913), which solved most of the
roblems connected with E. histolytica.

Walker (1911) first showed that E. histolytica and E. coli are quite
distinct and easily separable species, though possessing a_ similar
development. The first forms cysts containing, when mature, 4 nuclei ;
the second cysts containing 8 nuclei. In the cysts of both, development
occurs in a straight-forward manner by the repeated division of an
originally single nucleus—without any “autogamy” or other mysterious
processes such as were described by Schaudinn. Then he showed that
E. histolytica, E. tetragena,and E. minuta are all different namesfor one and
the same species. Finally, with Sellards (1913), he proved conclusively
by experiments on human beings that man becomes infected by ingesting
the cysts of these amoebae; and that infection with E. histolytica may give
rise to dysentery, while E£. coli is harmless to its host. To Walker (1911,
1913) we also owe the conception no less than the discovery of the
“carrier” condition in E. histolytica infections—a conception which
cleared up all the difficulties which previously prevented the life-history
and activities of this organism from being properly understood. Certain
details of Walker’s work will be considered later. Here the historic
importance of the work as a whole is all that immediately concerns us.

All the correct observations made since the appearance of the results
of Walker and Sellards merely confirm or elucidate the facts which they
established. It will therefore be unnecessary to consider, at this point, all
the minor details contributed by numerous subsequent workers; but I
would especially mention here the names of Darling (1912, et seq.),
Wenyon (1912 ef seq.), and James (1914), who have all made valuable
later contributions to our knowledge of E£. histolytica.

NOMENCLATURE.

The nomenclature of the dysentery amoeba has been for some time a
very vexed question. It has already been discussed ad nauseam by
numerous writers : and my only excuse for reopening the question is my
desire to reach finality in the matter. I have already discussed the

* Vide Huber (1906, 1909).
+ Vide Hartmann and Prowazek (1907).

26 THE AMOEBAE LIVING IN MAN

nomenclature of this organism briefly elsewhere (Dobell, 1918), and will
begin by recapitulating what I there said.

The dysentery amoeba was first described by Lésch (1875), who named
it ‘Amoeba coli.” Consequently, if his name is accepted, and the
parasite is placed in the genus Entamoeba, its correct name—according
to the rule of priority—is Entamoeba coli Lésch. This name, however,
was most unfortunately assigned to the large harmless amoeba of the
human colon by Schaudinn (1903), in his revision of these forms : and
since then it has been used with no other signification. To transfer
this name now can only lead to the direst confusion. So far as I am
aware only one writer has hitherto had the temerity to advocate such a
course—Aragao (1917, 1917 a), who considers that we should henceforth
call the dysentery amoeba E£. coli, notwithstanding the confusion it will
create, in order to conform to the law of priority. Many other workers*
now call the dysentery amoeba E. dysenteriaet, on the grounds that this
specific name was given to it by Councilman and Lafleur in 1891, and
therefore has priority over histolytica Schaudinn (1903). This course
was first recommended by Craig (1905), who abandoned it later when
Stiles (1905) showed that it was not justifiable. Stiles’s revision of
“Amoeba coli’”’ is, however, no longer acceptable, because he did not
know all the facts of the case.t He showed, nevertheless, that dysen-
teriae Councilman and Lafleur, 1891, is not available as a name for the
dysentery amoeba—as I have also pointed out (1918) in ignorance
that Stiles had already done so. Councilman and Lafleur proposed to
call Lésch’s “ Amoeba coli”? by the new name “amoeba dysenteriae ”’
solely because they considered the former inappropriate.§ Their name
is therefore a synonym of “ Amoeba coli” Lésch, if these names are con-
sidered to have any standing. For my own part I consider “amoeba
dysenteriae’ to be unquestionably synonymous with “Amoeba coli”
Lésch ; but I also regard it as having no systematic status whatever.
It was written in ordinary type, without a capital letter for the generic
name; and, moreover, as the context shows, it was proposed as a
descriptive term and not asa binominal Linnaean name. On no grounds,
apparently, can E. dysenteriae be justified as the name of the dysentery
amoeba.

The singular point in the nomenclature of those who call the
dysentery amoeba E, dysenteriae, is that they all, with few exceptions,
give the name E. coli to the species to which Schaudinn gave it.|| This
curious inconsistency I have already pointed out (1918). It appears

* For example Brumpt (1913), Mathis and Mercier (1916), and many other workers
in France and America.

t Kartulis (1893) and numerous medical writers have used the term “ Amoeba
dysenterica ’’—presumably in mistake for “dysenteriae.”

_ £ Stiles (1905) concluded that if there is only one amoeba in man, its proper name
is #. coli Losch ; but if there are two—a pathogenic anda non-pathogenic—then their
names are respectively Z. Azstolytica and E. coli, as determined by Schaudinn.

§ “We have called the organism, which was first described by Loésch under the
name of amoeba coli, the ‘amoeba dysenteriae.’ The name given to it by Lésch is not
distinctive . . etc.” Councilman and Lafleur (1891), p. 405.

|| Pestana (1917) is the only exception I can recall. He names the dysentery
amoeba £. dysenteriae and the non-pathogenic species &. hominis—which abolishes
the name co/z altogether, though with no apparent justification.

ENTAMOEBA HISTOLYTICA 27

to be quite unjustifiable. If Lésch’s name “coli” is accepted at all,
it must, according to the rules, be given to the dysentery amoeba, and
to no other: and it is not permissible to use its synonym “ dysenteriae”
to replace it, and then to bestow “coli” upon another species. One
way out of this difficulty has recently been suggested by Mesnil (1918),
who thinks that Lésch originally gave the name Amoeba coli to a
mixture of species — one of which was later called dysenteriae by
Councilman and Lafleur. Unfortunately there is no evidence that this
was the case. There is absolutely nothing to indicate that Losch
studied more than one species, and to my mind there can be no
doubt as to which this was—namely, the dysentery amoeba. Schaudinn
(1903), it is true, was “unable to decide”’ whether Lésch studied the
pathogenic or the harmless species, and consequently gave his name
to the latter : but no experienced modern worker can share his doubts.

Schaudinn (1903) displayed a singular lack of judgement in his
revision of the name “ Amoeba coli.” Beyond a doubt he should have
called the dysentery amoeba Entamoeba coli Lésch and the non-
pathogenic species E. hominis Casagrandi et Barbagallo. This would
have created no confusion at the time, as people were then accus-
tomed to call the dysentery amoeba Amoeba coli.* Since Schaudinn
made his mistake, however, his names have been almost universally
adopted ; and they should now, in my opinion, be preserved at all costs.
I believe this can be done, moreover, without violation of the Rules
of Zoological Nomenclature, though it appeared to me impossible
when I last discussed the problem (1918). I now offer the following
solution :

“ Amoeba coli” Lésch (1875) may be regarded as a descriptive term,
and nota binominal Linnaean name within the meaning of the code.
It was introduced in the following words : “ Da die von mir beschrieb-
ene Amdbe, so viel mir bewusst, tberhaupt mit keiner der bisher
bekannten Formen vollkommen iibereinstimmt, so scheint es mir
gerechtfertigt, dieselbe bis auf Weiteres mit einem besonderen Namen
zu bezeichnen und nach ihrem Fundorte etwa Amoeba coli zu nennen ”
(Lésch, 1875, p. 208). The generic name is written with a capital—as
it would be, in any case, in German—but in ordinary type. There is
nothing to indicate that Lésch did not employ it as a mere descriptive
term{+—in the customary medical manner (i.e., like the ordinary names
of bacteriology, or Councilman and Lafleur’s “amoeba dysenteriae”’).
Undoubtedly many of the earlier workers regarded it in this light
(e.g., Councilman and Lafleur), The name Amoeba coli written in italic
type, and as a proper zoological name, seems to have been first used by
Grassi: and the organism to which he gave it was probably the non-
pathogenic form—not the dysentery amoeba—owing to a misidentifica-
tion. Consequently, I regard “Amoeba coli” Loésch, 1875, as a
synonym of E. histolytica Schaudinn, 1903, but not a valid zoological
name; and Amoeba coli Grassi, 1879, as the first valid name given to
the organism which Schaudinn (1903) later called Entamoeba coli—as

* Musgrave and Clegg (1904) say that the amoebae in human stools were “usually”
called Amoeba dysenteriae when large and containing red corpuscles, and Amoeba coli
when smaller, devoid of blood corpuscles, and believed to be non-pathogenic. The
earlier literature does not bear out this statement.

+ And with some hesitation, as the word “etwa”’ before it seems to imply.

28 THE AMOEBAE LIVING IN MAN

Casagrandi and Barbagallo (1895) had done before him. I regard
“amoeba dysenteriae’? Councilman and Lafleur, 1891, as ruled out
because it is a synonym of ‘“ Amoeba coli” Lésch, and in addition not
a valid name, but a descriptive term. Similarly the name ‘“ Amoeba coli
felis,’ employed for the same organism by Quincke and Roos (1893),
is not a valid Linnaean name, but a trinominal descriptive phrase.*
This name has no status in zoological nomenclature. There are three
other possibile early names for the dysentery amoeba—‘‘ Amoeba
urogenitalis”” Baelz, 1883, Amoeba vaginalis Blanchard, 1885, and
Amoeba intestinalis Blanchard, 1885. The first two of these depend
upon the identification of the amoebae found in human urine, which
is such a large subject that I shall devote a separate section to it (vide
uifra, p. +25). I conclude there, however, that neither of these names
should be employed. The other name—A. intestinalis Blanchard—
cannot properly be used for any organism, I think. Blanchard (1885)
introduced it for some amoebae which are stated by Leuckart (1879)
to have been seen by Sonsino in the stools of a child suffering from
dysentery. They are merely stated to have been 8-10 times the size
of a red blood corpuscle and therefore apparently larger than Lésch’s
amoebae. They may have been the dysentery amoeba, but they are not
identifiable.

Whilst it is true that the terms A. coli and A. dysenteriae were some-
times used correctly as zoological names, yet they were never used
with clear specific conceptions before the time of Schaudinn. We
constantly fnd the name ‘A. coli’’ used indiscriminately for two
different species. It ought not to have been—after Quincke and Roos,
Kovacs, and Casagrandi and Barbagallo—but nevertheless it was. Thus
we find even so competent a zoologist as Doflein (1901) describing
“A. coli Lésch”’ as the one and only amoeba from the human intestine
—illustrated by Lésch’s figures of the free forms of E. histolytica and
Grassi’s figure of the cyst of E. coli. 1 therefore regard Schaudinn’s
name for the dysentery amoeba as the first proper zoological desig-
nationt of the species; and I shall, accordingly, continue to call this
organism Entamoeba histolytica Schaudinn, 1903. Whether right or
wrong, this name is the only one which can now be used without
creating chaos in the nomenclature of the amoebae of man.

The chief synonyms of £. histolytica are given in the list which heads
this section. A few explanations may be given here, however, in justifi-
cation of the inclusion of certain names.

Amoeba lobosa var. coli is the name proposed by Celli and Fiocca
(1894a), in accordance with their system of nomenclature, for Lésch’s
amoeba. It is therefore a synonym of E. histolytica. “ Amoeba lobosa”’
is not a proper name for any amoeba, and Losch’s organism cannot
therefore be classified as a variety of a non-existent species. “ Entamoeba
Schaudinni”’ Lesage (1908) is presumably a name intended, by this

* Like “ Bacillus coli communis” and many other bacteriological names which are
clearly not formed in accordance with the rules cf nomenclature.

+ Though his description is not, of course, by any means the first. Schaudinn
himself considered that the best account was that of Jiirgens (1902)—who did not
name the amoebae, and later (Jiirgens, 1903) adopted Schaudinn’s nomenclature.
ein ignored or underrated the work of many of his predecessors—e.g., Kovacs,

00S, etc.

ENTAMOEBA HISTOLYTICA 29

author, to denote F. histolytica. Consequently it becomes a synonym
of the latter. Entamoeba nipponica Koidzumi (1909) almost certainly
included* E£. histolytica and possibly also E. coli and other amoebae (?)
and tissue cells. Darling (1913), by misquotation, renamed it E. nippon-
ensis. E. hartmannt Prowazek (1912 a) was certainly for the most part
E. histolytica—a strain producing small cysts. E. tenuis Kuenen and
Swellengrebel (1917) and FE. minutissima Brug (1917) are similar strains
of E. histolytica. Swellengrebel (1917) has described these small cysts as
belonging to the flagellate Chilomastix mesnili: Aragao (1912) has included
them with cysts of F£. coli in his “F. brasiliensis” ; Woodcock and
Penfold (1916) call them £. minuta, though this is not the E. minuta of
Elmassian (1909), which was the common strain with cysts about 12 p
in diameter. Everybody will admit now that E£. tetragena and E. africana
Hartmann (1907) are synonyms of FE. histolytica. Nevertheless, a word
may be said here about the former, as it has a curious and little known
history.

From the papers of Huber (1903, 1906, 1909) it is clear that he re-
discovered the cysts of F. histolytica at about the time when Schaudinn
(1903) named this parasite and described its ‘‘ spore-formation.’”? Huber
showed the cysts to Schaudinn, who would not admit that they belonged
to E. histolytica: but he told Huber that they belonged to a different
species, which he had studied in one case himself, and which he was
going to call E, tetragena. Bya misprint in the original version (Huber,
1906), this name is written “‘ fetragona’’—a mistake+ which Huber (1909)
corrected later. When Viereck (1907) rediscovered the cysts, he tacitly
adopted Schaudinn’s name, and followed him in regarding the species
as distinct from E. histolytica. However, he appears to have considered
that it was really a variety of E. coli, and proposed tetragena as a varietal
_ and not as a specific name. Hartmann (1908), after rediscovering
_ “E. africana,” decided that it was identical with Viereck’s species, and
named it E. tetragena Viereck—still supposing it to be a new species.
The muddle thus created by the German workers, as a result of ignoring
Huber’s work and supporting Schaudinn’s wrong observations and
interpretations, has survived in certain quarters to the present day.
E. tetragena and E. histolytica are names of the same organism, and there
is no justification whatever for employing the former. Nevertheless,
Kuenen and Swellengrebel (1913) and some other modern workers refuse
to give itup. A variant on the name (E. “ tetragina’’) was introduced
by Walker (1911), through a mistake of some sort. The sooner all these
names—tetragena, tetragona, tetragina—are forgotten, the better will it
be for both zoology and medicine.

It remains to add that the “amoeba” described by Noc (1909) from
cases of dysentery was partly E. histolytica. His forms from the stools,
containing red blood corpuscles, undoubtedly belonged to this species :
but the forms which he cultivated were just as certainly free-living
amoebae, and not E. histolytica. The “E. histolytica” which Lesage (1905)
cultivated from dysenteric stools, and with which he performed some

* Some of the forms of “4. xipponica” are stated to have contained red blood
corpuscles. If these really were amoebae—and not endothelial cells—then they must
have been &. Azstolytica.

+ At first sight this looks like a pun on Viereck’s own name—but it seems to have
been unintentional.

¢

30 THE AMOEBAE LIVING IN MAN

incredible experiments on cats (Lesage, 1907 a), were similar free-living
forms. The ‘Amoeba Il” of McCarrison (1909), regarded by him as
possibly E. histolytica, was clearly in reality a cell and not an amoeba.
The ‘‘ non-pathogenic Entamoeba tetragena” of Shimura (1916, 1918) is,
apparently, chiefly E. histolytica from healthy carriers. The “ E. coli”
of Werner (1912) apparently included E. histolytica also—to judge from
his figs. 32-38, Pl. I]. And it is not improbable that the smallest
amoebae of “FE. williamsi’”’ described by Prowazek (1911, fig. 19) were
the precystic amoebae belonging to a strain of EF. histolytica which forms
small cysts. But it would take too long to note all the names given to
E. histolytica by numerous authors who have casually included a few
individuals or cysts of this species in their descriptions or figures of
others. Specially noteworthy instances will be mentioned later.

There are still two groups of amoebae which have to be taken into
account in discussing the nomenclature of E. histolytica; namely, the
amoebae found in human urine, and those found in the intestines of
various animals—especially dogs and monkeys. These will be considered
later (see p. 125 et seq.), as they cannot be conveniently discussed here :
but I will forestall the conclusions there reached by noting at this point
that I find sufficient justification for the inclusion of the names “Amoeba
urogenitalis’’ Baelz (1883), Amoeba vaginalis Blanchard (1885), and
Entamoeba venaticum Darling (1915), in ‘the list of synonyms of E. his-
tolytica. On the other hand, I find as yet insufficient evidence for the
inclusion of any of the names given to the amoebae of monkeys.

Entamoebae in many ways Closely similar to E. histolytica have also.
been found in several other animals. These, too, have been named, and
the synonymy of the dysentery amoeba cannot be completed without
taking them into account.

Entamoeba ranarum (Grassi) Dobell occurs in frogs and toads (cf.
my papers 1908, 1909, 19094). It resembles E. histolytica so closely in
certain ways as to suggest that it may be the same species. The precystic
amoebae and the cysts of the two forms are sometimes indistinguishable.
Their identity was suggested by Alexeieff (1914), and appeared to merit
further inquiry. Experiments which I made in 1916 seem, however, to
show clearly that E. histolytica and E. ranarum are distinct species. At
all events, it was found impossible to infect tadpoles with the cysts
of E. histolytica. (Vide Dobell, 1918.) Another amoeba whose cysts
closely resemble those of E. histolytica is E. aulastomi Noller (1912),
which lives in the hind-gut of a leech,* Aulastomum gulo Moq.-Tandon
(= Haemopis sanguisuga L.). At present there is no proof that this
amoeba is not identical with E. histolytica or E. ranarum, though it will
probably, I think, turn out to be a distinct species.

Conclusions regarding Nomenclature of the Dysentery Amoeba.

The conclusions which I draw from the facts noted in the foregoing
paragraphs may be summed up briefly as follows :
The name used for the dysentery amoeba of man should be
Entamoeba histolytica Schaudinn, 1903, as this is the first zoo-
logical name correctly given to this species, and the only one

* Not of “the eagle,” as Craig (19134, 1914, 1917) states—apparently as a result
of mistranslation of the German word “ Egel.”

ENTAMOEBA HISTOLYTICA 31

which can now be used without creating confusion. The names
Entamoeba coli or E, dysenteriae should in no case be employed.
The former can be justified, from the standpoint of nomenclature,
but not from that of common sense. The latter name has no
status in zoological nomenclature.
A full summary of the nomenclature and synonyms of this species
is provided in the list of names which heads this section (p. 20).

DESCRIPTION.

Entamoeba histolytica has been so frequently described that I shall
confine my description to its chief characters, and shall then discuss
several points of importance on which there is not yet a general agree-
ment among the various workers who have already described the
parasite.

The active forms of E. histolytica show great variation in size, ranging
from about 18 in diameter up to about gow. Asa rule, however, they
measure between 20 w and 30y when rounded. The living animals,
when fresh and healthy, are extremely active. They flow along in a
slug-like manner with great rapidity, and show no conspicuous differen-
tiation between ectoplasm and endoplasm. When they have been out-
side the body of their host for some time, however, at a temperature
lower than that of the body, they present quite different though equally
characteristic movements. They then remain in one place, throwing
out large, hyaline, blade-like pseudopodia composed of ectoplasm
sharply separated from the endoplasm. This movement is, in my
opinion, seen only in animals which are already in some degree
degenerate. But it is very characteristic of this species, and serves to
distinguish it from E. colt.

The endoplasm of the parasite contains numerous small granules
(microsomes), which may be easily stained intra vitam with neutral
red,* flavine, and several other dyes. Apart from these granular con-
stituents it is homogeneous and colourless, resembling ground glass in
optical texture. The cytoplasm as a whole is characterized by its clear-
ness and freedom from inclusions. Degenerate amoebae often contain
bubble-like vacuoles, but these are never present in healthy individuals.
They begin to appear soon after the amoebae have left the body. Food
vacuoles may or may not be present. When present they contain red
blood corpuscles, and occasionally leucocytes and fragments of other
cells. Normally they do not contain, in my experience, any other
inclusions.t Red corpuscles may be present in very large numbers. I
have seen an amoeba in which I could count over 4o. As a rule, how-
ever, they are not so numerous—r to to being most commonly seen.
These ingested red corpuscles are, of course, a very characteristic feature
of this species: and it is probably safe to assume that any amoeba

* It is not true, as Cutler and Williamson (1917) appear to believe, that Z. histolytica
is the only intestinal amoeba of man which stains with neutral red zzfra vitam. A few
simple experiments with other species will suffice to show the fallacy of this view.

+ Wenyon and O’Connor (1917) describe and figure (Text fig. 4) specimens of £. hzs-

tolytica containing spores of a bacillus (&2. megatherium ?) which they had apparently
ingested.

32 THE AMOEBAE LIVING IN MAN

found in a human stool is E. histolytica if red corpuscles are present in
its protoplasm. (Cf. Wenyon and O’Connor, 1917.) As the corpuscles
are digested in the food vacuoles, they usually appear more or less
eroded or fragmented when inside the amoebae, and distinctly smaller
than those seen in the blood or in bloody stools. Contractile vacuoles
are, of course, invariably absent—as in all Entamoebae.

The nucleus-of E. histolytica is very characteristic. It has the general
structure typical of the genus Emtamoeba, but contains less chromatin
than that of most other species. In the living animal it is inconspicuous
or invisible, but becomes prominent and distinct as the organism dies.
It is vesicular and usually spherical, measuring about 4 p to about 7 p in
fixed and stained specimens. Its structure can be satisfactorily studied
only in amoebae which have just left the body; for example, those
which have just been passed per anum, or which have just been scraped
out of an intestinal ulcer or other amoebic lesion. The nuclei of nearly
al] the amoebae seen in a dysenteric stool, examined in the usual way,
are generally degenerate,—to a greater or less extent; and most of the
descriptions and figures of the nuclei of E. histolytica hitherto published
appear to be based largely upon such degenerate individuals.

The normal nucleus, when properly fixed and stained, always shows
the same structure. (See figs. 1, Pl. I and 16, Pl. II.) It has a very
delicate achromatic membrane externally,* which is lined usually by a
single layer of small chromatin granules—thus giving the nucleus an
annular appearance in optical section. As a rule the granules are of
very uniform size, and are either in contact or only very slightly
separated from one another. The centre of the nucleus is occupied
by a small spherical karyosome, about 0°5 w in diameter, composed, in:
all probability, entirely of chromatin. I find no evidence that it contains
a centriole—as is so frequently alleged. The most carefully fixed and
stained preparations, examined with the best optical apparatus, invari-
ably show the karyosomes in all healthy normal individuals to be quite
homogeneous. A most important point about the karyosome is its
position in the nucleus. In typical healthy individuals it lies at the
centre. An eccentric karyosome 1s exceptional. (Cf. figs. 1, 16.)

In addition to the chromatic karyosome just described there is
frequently a more or less definite achromatic capsule-like structure
which surrounds it, giving to the whole the appearance of a deeply
stained central granule surrounded bya paler halo. This structure is
perhaps best seen in specimens stained by Mann’s method (cf. fig. 1,
Pl. 1), in which the karyosome itself is stained red, and the “halo” blue.
In some individuals which otherwise appear perfectly normal this
appearance cannot be clearly seen, and | am still undecided as to its
correct interpretation. I think the halo should be regarded as a part
of the karyosome; but it is possibly merely a coagulum formed by
fixation and deposited on the chromatic part, which represents the true
karyosome. In my view the karyosome is probably a composite
structure, consisting of a central core of chromatin surrounded by a
more delicate cortex—the “halo”—of achromatic substance. This

——

* As stated by Walker (1911), though previously denied by Schaudinn (1903). The
achromatic membrane can often be easily seen in badly fixed specimens in which the
chromatin granules have been artificially separated from it.

Poe Te

ENTAMOEBA HISTOLYTICA a3

interpretation is supported by comparison with E. coli, 1n which the
cortical layer is more conspicuous.

The area between the karyosome and the peripheral layer of
chromatin granules is normally free from chromatin. In fixed and
stained individuals it appears to be filled with a network of linin, often
with definite threads radiating from the karyosome (see figs. 1, 16). This
“network’”’ I regard as the optical appearance of an alveolar structure
probably formed by fixation. In degenerate or badly fixed individuals,
and in deeply-stained specimens, “ chromatin” granules may sometimes
be seen in the linin network. But these are always, I believe, artifacts
or chromatin fragments or granules detached from the periphery or from
the karyosome.

The nuclear characters just enumerated—the uniform layer of small
chromatin granules at the periphery, the central karyosome with its halo,
and the absence of chromatin in the intervening space—serve to dis-
tinguish E£. histolytica with certainty from other intestinal species of the
genus Entamoeba. Unfortunately, one has often, in practice, to make
a diagnosis from organisms which are largely degenerate, and in which
the nuclei are abnormal ; and as it is frequently impossible in such cases
to determine the species of a given amoeba from the structure of its
nucleus, the red corpuscles in the cytoplasm often afford a more service-
able specific criterion of E, histolytica.

Some writers have laid considerable emphasis on the position which
the nucleus occupies in the organism. It is frequently stated* that in the
genus Entamoeba the nucleus is eccentric,+ whereas in the genus Amoeba
and other free-living forms it is central. It is true that the nucleus in
most entamoebae is not centrally placed: but it is not true that the
nucleus is usually central in species of the genus Amoeba, The belief
appears to me to have originated from the fact that the small free-living
amoebae—commonly but incorrectly referred to the genus Amoeba, and
wrongly called collectively “ Amoeba limax’’—usually have more or less
centrally placed nuclei when fixed and stained. In these amoebae,
however, as in all others, the nucleus constantly changes its position
with the movements of the organism : and it is, consequently, no more
typically central in them than in other forms. The central position of
the nucleus in a fixed and rounded small organism is, I think, an artifact,
which can be explained in a purely mechanical manner. It depends
merely upon the size of the organism and the physical state of its proto-
plasm whether its nucleus is or is not centralized in the process of fixa-
tion. At all events, in large amoebae—of any genus—this phenomenon
is not seen.

Many other descriptions have already been given of the nucleus of
E, histolytica. There are also plenty of figures already published show-
ing blocks and irregular masses of chromatin on the nuclear membrane,
chromatin in the achromatic zone, and broken up karyosomes. Even
parasites without karyosomes have been described and figured. That

* For example, see Phillips (1915): ‘‘ The Entamoebae have an eccentric nucleus
in the resting organism instead of a centrally placed one” (p. 8): whereas in the
“genus Amoeba (Ehrenberg) . . . the nucleus in the resting organisms .
is in a more or less central position ” (p. 6).

+ This was, I believe, first emphasized—in the case of the intestinal amoebae of man
—by Schaudinn (1903).

3

34 THE AMOEBAE LIVING IN MAN

these descriptions are all based upon defective and insufficient material
anybody can easily convince himself, if he will take the trouble to obtain
really fresh and healthy amoebae, and study them with sufficient care
and with proper cytological technique. All the abnormal forms of
nuclei so often described as normal can be made at will by simply
keeping normal individuals until they degenerate and die.

Hartmann (1908, ef alibi) lays great stress upon the occurrence of
“ cyclical changes around the karyosome” in E. histolytica. In normal
individuals, however, these are never seen. His “cycle” represents
an arbitrary series of degenerate forms with various arrangements of
chromatin between the karyosome and the nuclear membrane. All these
forms are common in stale stools or liver-abscess pus. But they are not
encountered in really fresh and well-fixed material, in which the nuclear
structure is constantly as I have just described it.

The differences so often described between the nuclear structure of
E. histolytica and ‘‘ E. tetragena” are clearly due to misunderstandings of
various sorts, since these names are synonymous. I do not understand
the views of those who still—whilst admitting the synonymy—distinguish
between “histolytica” and “ tetragena’’ forms of this amoeba. The
distinction is, in any case, quite unjustifiable.

General Outline of the Life-history.—The life-history of E. histolytica in
man is very simple. The active amoebae just described live in the tissues
of the gut wall, where they multiply by division. In a typical “ normal”
infection, a certain proportion of the amoebae constantly leave the ulcers.
and pass into the lumen of the large bowel, where they encyst and later
pass out, in the encysted form, with the faeces. The precystic amoebae,
which are thus free in the lumen of the bowel, are smaller than the ordinary
forms which continue to multiply in its tissues. They are entirely free
from cytoplasmic inclusions, and are in all probability formed simply by
division of the larger tissue-inhabiting individuals. The cysts, which are
found only in the faeces, are slightly smaller than the precystic amoebae,
and when fully developed are quadrinucleate. They vary greatly in size ;
and it has been shown that there are at least several distinct races of the
parasite distinguishable by the size of the cysts which they form. When
the cysts are swallowed by a human being, they probably hatch in the
small intestine and liberate small amoebae, which pass down with the
intestinal contents into the large bowel. Here they attack and invade the
tissues, and thus begin the cycle of development anew. No other stages
are known to occur normally in the life-history. In exceptional circum-
stances, however, a complication in development may result from the
migration of the amoebae from their primary site of infection into the
liver, brain, or other organs. If they succeed in establishing themselves in
these secondary sites they cause abscesses, which may give rise to further
pathological complications. The amoebae in secondary infections of the
organs are always of the typical tissue-inhabiting form—precystic
amoebae and cysts being found in the intestinal contents only. Such
secondary infections are clearly accidents in the life-history of the
amoeba. They do not form a part of its normal cycle of development,
but must be regarded as side-issues resulting from the straying of certain
individuals from their normal or primary habitat, which is undoubtedly
the wall of the large intestine. However important such migrations
may be for man from a medical point of view, they are of no use to the

ENTAMOEBA HISTOLYTICA 35

amoebae; since the erring individuals are unable to complete their
development, and are thus inevitably lost to the species as a whole.

Other life-histories of E. histolytica have been described, but they
appear to rest upon misinterpretations of various sorts. The more
important of these will be considered later. I would note particularly
here, however, that there is no ‘‘spore-formation” such as Schaudinn
(1903) described in this species, and that ‘‘schizogony,” or multiple
fission, does not occur ; and further, that no conjugation or other sexual
process has yet been shown to take place at any stage in the life-history.

The chief points in the habits and life-history, as outlined above, will
now be considered in further detail.

Habitat.—The normal habitat of E. histolytica, as already noted, is
the tissue of the large intestine* of man. The parasites are here found
in the mucous, submucous, and occasionally the muscular layers, where
they cause a typical ulceration. I need not here discuss the morbid
anatomy of the various forms of intestinal amoebiasis. Good descrip-
tions are to be found in the publications of Councilman and Lafleur
(1891), Dopter (1905, 1907), Kuenen (1909), Christoffersen (1917), and
others, to whose works the reader may be referred.

The commonest secondary site of infection is the liver, where the
parasites give rise to the formation of abscesses. There can be little
doubt that they reach this organ by way of the portal vein. From the
liver they may pass in the blood stream to the lungs, brain, and possibly
other organs, where they may also settle down and cause the formation
of abscesses. Amoebic abscess of the brain is a very uncommon disease,
only about 50 cases having been recorded.t| Amoebic abscesses in the
spleen have been described by J. P. Maxwell (1909) and Rogers (1913),
and infections of the urinary system may occur.{ I have studied several
cases of liver abscess and specimens from one cerebral abscess—thanks
to my friend Capt. L. Armitage, N.Z.M.C.—and I can confirm the
observations of many other workers that the amoebae in these situations
are indistinguishable from the forms in the gut wall. The pathological
anatomy of these various lesions does not concern us here.

E. histolytica has been stated by Lesage (1907) to occur at times in
the blood ; in the skin, by various workers (cf. p. 140) ; and in other
situations. The evidence for such statements is, however, still far from
convincing. Job and Hirtzmann (1919) have recently alleged that
E. histolytica (“ Amoeba dysenteriae’) is intracellular during its youngest
stages of development in the liver. But their account is too brief and
unconvincing to overthrow all the observations of other workers—none
of whom have ever found truly intracellular stages of this parasite.

Mode of Nutrition.—A study of sections of intestinal ulcers and liver
abscesses, and of the amoebae themselves as they occur in fresh
dysenteric stools, makes it abundantly clear that E£. histolytica nourishes
itself at the expense of the tissues. ‘The amoebae penetrate the tissues
by destroying the cells. In all probability they do not force their way
mechanically into the healthy tissues, but secrete a powerful cytolytic

* Infection of the small intestine by Z. Azstolytica is extremely rare. It has been
described by Harris (1898) and Kuenen (1909) in man, and I have seen two instances
in the cat (cf. Dale and Dobell, 1917).

t See especially Legrand (1912), Sitsen (1913), and Armitage (1919).

t Cf. p. 125 ef seg.

36 THE AMOEBAE LIVING IN MAN

ferment which first dissolves them. Schaudinn’s name “ histolytica”’ is
most appropriate for this parasite; but his description of the amoebae
forcing their way through the tissues and dislocating the cells by means of
their tough and powerful pseudopodia appears to be unfounded.* Good
sections show clearly that the amoebae apply themselves to the tissues,
which then break down; and the organisms thus come to lie in pools of
histolysed tissue which they evidently absorb as nutriment. As already
noted, they may also ingest red blood corpuscles or fragments of cells,
but this is probably the exception rather than the rule. Their chief food
is, in all probability, derived from the destroyed tissues, but it is absorbed
and not bodily ingested in the manner typical of amoebae. In acute
amoebic dysentery, when much blood is present in the intestinal
contents, a large proportion of the amoebae passed in the stools may
contain red corpuscles : but in sections of intestinal ulcers the proportion
is, in my experience, considerably less.

A belief that EF. histolytica depends, in some mysterious way, upon
certain bacteria with which it lives in “symbiosis,” is continually
encountered in the literature. It appears to have arisen at a time when
it was believed—from the work of Kartulis, Celli and Fiocca, and others
—that the amoebae cultivated from stools were identical with the
dysentery amoeba: and it has been maintained by Musgrave and Clegg
(1904) and more recent workers apparently for a similar reason. It is
true that the small free-living amoebae can only be cultivated together
with bacteria; but this, of course, is because these micro-organisms
form the food upon which such amoebae live. There is here no question
of a ‘‘ symbiosis,” in the proper sense of the word. Moreover, there is
no evidence whatsoever that E. histolytica depends for its existence
upon bacteria of any sort. It does not eat bacteria, like the free-living
amoebae, and no concrete evidence has ever been brought forward to
show that it lives in symbiosis—properly so called—with any other
organism.t The hypothesis at present adds an unnecessary complica-
tion to the life-history of the parasite, and 1s not worth discussion until
some facts can be produced in its favour.

Pathogenesis.—Only since the publication of the admirable work of
Walker (1911, 1913), has the relation ot EF. histolytica to man been
correctly understood. All the more recent work—including the vast
experience gained during the War—has abundantly confirmed his con-
clusions.{ It is thus possible, I believe, to deal now with these, and
their consequences, as facts; and to give them here with that brevity
which certain knowledge alone will permit.

E. histolytica, although a tissue parasite, does not usually cause
dysentery or any other clinical symptoms in its host. As it destroys the
tissues, these regenerate, and the parasite and its host live in a state

* Schaudinn appears to have taken this notion from Jiirgens (1902). It has found
favour also with Craig and others.

+ The suggestion that attacks of amoebic dysentery are due to some unexplained
co-ordinated action between £. Azstoly¢ica and certain unknown bacteria, appears to
be equally unfounded. The idea has been frequently expressed, however, since the
time of Janowski (1897), though on what grounds I am unable todiscover. It originated,
I believe, through a confusion of amoebic with bacillary dysentery.

t The important work of Wenyon and O’Connor (1917) requires special mention in
this connexion.

ENTAMOEBA HISTOLYTICA 27

of equilibrium.* This is the “normal” or most usual condition, and
an infected individual in such a state of equilibrium is called—following
Walker (1911, 1913)—a “carrier”? of the parasite. He can only be
distinguished from uninfected individuals by the presence of the cysts
of E. histolytica in his stools. His large bowel is ulcerated, more or less,
but this is not visible externally and gives rise to no clinical symptoms.

When the parasites and their host do not live in harmony with

one another—as happens in a certain proportion of cases—pathological
conditions result. These affect both the host and the parasite. In the
case of the former, they are manifested as various diseases, which are
of three main types: (1) Irritation of the intestine, producing most
commonly diarrhoea and intestinal irregularities of divers sorts, and
leading, in severe cases, to a typical form of dysentery (Amoebic
Dysentery). (2) Generalized effects resulting from the destruction of
the lining of the bowel, but not manifested as local intestinal diseases
(General Amoebiasis). (3) Secondary disorders consequent upon the
wandering of the parasites from the gut into other organs, such as the
liver, where they give rise to inflammatory and suppurative conditions
(Amoebic Hepatitis, Hepatic and Cerebral Abscess, etc.). All these
diseased conditions of man are harmful to the parasite also, for they
disturb its food supply, interrupt its normal life-history, and lead to
a great wastage and mortality among the amoebae concerned. In
amoebic dysentery, for example, the amoebae are cast out of the body
in large numbers before they can encyst ; and they consequently perish
and are unable to propagate their species. Similarly, in the case of
secondary infections of the organs, the parasites may enjoy a brief spell
of reproductive activity; but they do not encyst in any situation save
the gut, and are, in the organs, cut off from the outside world with no
means of continuing their race. The various amoebic diseases are thus
“‘ diseases” for the parasites as much as they are for their hosts. And
it is clear that infection with E. histolytica cannot invariably, or even
usually, be accompanied by acute dysentery; for if it were the parasite
would soon be exterminated.
_ Entamoeba histolytica is thus a pathogenic parasite in a restricted
sense. It is always a destroyer of tissue, but by no means always
productive of disease. The usual type of human infection is that
exemplified by the carrier of the parasite. The carrier state will there-
fore be considered here rather more fully, as it involves several matters
of importance.

Carriers—The carrier of E. histolytica was first defined by Walker
(1911, 1913). It is true that others had previously spoken of amoebic
carriers, but they used the term in a different sense, and without com-
prehending the facts upon which Walker’s conception rests. For
example Martini (1908) and Vincent (1909) both described and discussed
“carriers” of E. histolytica; but they were ignorant of the life-history
of the amoeba, and of the part played by its cysts. They apparently
regarded “carriers” merely from the clinical point of view, and on
analogy with the carriers of various bacillary infections. Walker’s
conception of the carrier of E. histolytica is, however, in many ways
different: his ‘‘ carrier” is, in fact, something sui generis—by no means

—

*I have already elaborated this conception somewhat in an earlier publication
Dobell, 1918 a).

38 THE AMOEBAE LIVING IN MAN

exactly comparable with the bacillary “carriers”’ of various sorts. It
is, perhaps, unfortunate that the same term should have been employed
for both; but since it is in current use, it seems inadvisable to try to
change it now.

We can now see that the carrier of E. histolytica is merely the
ordinary individual in the normal state of infection. He is the indi-
vidual who is naturally adapted to his parasites, and who suffers no
appreciable harm from their presence. From the diagnostic point of
view, he is the individual who passes the cysts of the amoeba in his
stools. If the amoebae do him no harm, and find his bowel a comfort-
able environment in consequence, they develop in their normal manner
—completing their life-cycle by encystation. The carrier of E. histolytica
can therefore be accurately defined as the individual who passes cysts
of the parasite in his stools.

The carrier obviously “carries” the active amoebae in his tissues.
He isacarrier of amoebae. He is not properly called a “ cyst-carrier”
—a term which has unfortunately been introduced into many languages,
and which is now used by many workers—because he does not “ carry”
cysts* in any ordinary sense. He deposits cysts as soon as they are
formed. Nobody would call a man infected with an intestinal worm an
“‘egg-carrier’”’: because if he “carries” anything, it is clearly the worm,
—not the eggs which it lays and which he discharges in his stools.
“‘Cyst-carrier ” is similarly a misnomer, and does not correctly represent
the facts.

Walker (1913) divides carriers into two classes—contact carriers and
convalescent carriers. The former are those people who have never
suffered from amoebic dysentery; the latter, those who have had an
attack of amoebic dysentery, but who have then recovered clinically
without losing their infections. On clinical grounds it is important to
recognize these two categories; for the contact carrier 1s typically a
healthy individual, whose infection does him no appreciable harm,
while the convalescent carrier is the individual who has shown himself
susceptible to the action of the parasite. He has already suffered, and
frequently continues to suffer, from their presence. Clinically, he is
often a case of relapsing dysentery, with intermissions of variable
duration when he passes into the carrier state. There is, of course,
no hard and fast line between the typically healthy carrier and the
patient suffering from acute amoebic dysentery. They are the extreme
manifestations of one common condition—intestinal amoebiasis—con-
nected by all intermediate states, any of which may be seen in different
individuals or in the same individual at different times.

Carriers are of importance from two different standpoints: the
practical, because they alone are the source of infection to others ; the
theoretical, because they explain all the apparent contradictions which
previously prevented people from understanding the part which E. histo-
lytica plays in the causation of amoebic dysentery. It is now easy to
understand—though unfortunately still not generally understood—how

* We even read, in recent papers, of carriers of £. histolytica being “infected with
cysts.”. This shows a most incomprehensible ignorance of the true conditions. It is
doubtless due to the same confusion of ideas that leads people to talk of “ cyst-carriers,”
and to ask for methods of treatment that will enable them to “kill the cysts” inside
an infected person.

Fa

ENTAMOEBA HISTOLYTICA 39

it is that E. histolytica is the cause of amoebic dysentery and other
diseases, and yet usually ‘‘non-pathogenic”: how amoebic diseases
are not contracted from persons actually suffering from them: and
consequently, how amoebic dysentery and liver abscess come to be
endemic and never epidemic in their incidence. For every abnormal
individual suffering from dysentery, but non-infective to others, there are
dozens of comparatively healthy infected individuals—carriers—who
show no clinical signs of infection, but whose amoebae undergo their
normal development, and whose cyst-containing faeces are infective
to others.

There can be little doubt that E. histolytica, even when it causes no
dysenteric or other recognizable symptoms, must always live at the
expense of its host’s tissues. Every healthy carrier has the lining of his
large bowel more or less ulcerated ; though the ulceration may be, and
probably often is, superficial and almost invisible post mortem to the
naked eye.* But even quite extensive ulceration may exist without any
dysenteric symptoms being evident. This is clearly shown by the post
mortem findings of Musgrave (1910), Bartlett (1917), and others; and it
is undoubtedly incorrect to suppose—as many still do—that because an
infected person does not suffer from dysentery his intestine is therefore
not ulcerated. A point of importance in this connexion is the fact that
a person may suffer from an amoebic abscess of the liver or other organ
without ever suffering from dysentery. Now E. histolytica reaches the
liver by way of the portal vein. To get into this the amoebae must
traverse the wall of the gut, and to do so they must damage the tissues.
Consequently, the parasites must have caused at least some ulceration of
the intestine before they gained access to the liver. The case described
by Armitage (1919) is particularly interestingt in this respect: for the
patient was always, so far as his intestinal infection was concerned, a
contact carrier of E, histolytica, who had never suffered from dysentery
or other intestinal trouble. But he acquired a typical amoebic abscess
of the liver, and when this was almost cured, an amoebic abscess of the
brain, from which he died. In his stools the cysts of the parasite were
present, and in the abscesses the typical tissue-inhabiting amoebae. Such
cases as this show clearly that the contact carrier is not infected with
a non-pathogenic strain of amoebae—as some would argue; and also
that ulceration of the intestine may be present in the absence of all
dysenteric symptoms.

Walker (1913), Wenyon and O’Connor (1917), and most other com-
petent observers appear to share these views as to the pathology of the
carrier condition; and no other interpretation which has been put
forward is, I think, compatible with all the facts. But I shall have
occasion to refer to the discrepancies and difficulties in these other
interpretations later in another connexion.

At present there is little to show what percentage of the persons who
acquire infection with £. histolytica will become healthy carriers, and
what percentage will suffer from amoebic dysentery or other diseases.
The only figures are those furnished by the experiments of Walker

* I have seen—in the cat’s intestine—ulceration which is only recognizable with
certainty in sections examined under the microscope.

+ I cite this case because it is one in which I was able, through the kindness of
Capt. Armitage, to take a personal interest.

40 THE AMOEBAE LIVING IN MAN

(vide Walker and Sellards, 1913). Of 18 men who were experimentally
infected with E. histolytica, only 4 (222 per cent.) developed symptoms of
amoebic dysentery—the rest (14, or 77°8 per cent.) becoming contact
carriers. Some of them were under observation for over two years, and
never showed any signs of dysentery or other amoebic disorders.

It should be noted that 2 of the 4 subjects who developed dysentery
had very mild attacks, which might have been overlooked if they had
not been under close observation. Moreover, Wenyon and O’Connor
(1917) found 106 carriers among 1979 healthy men examined in Egypt ;
and of these 106 infected individuals they say “only 16 gave any history
of dysentery, and it is certain that the latter figure is too high, for in no
case can we be certain of the type of dysentery from which the case
suffered.” Taking these points into consideration, and allowing for the
error due to the small number of cases studied, I think too much
importance should not be attached to the exact percentages recorded by
Walker. It is extremely difficult to obtain reliable information on this
subject; but from my own experience I am persuaded that Walker’s
percentage is too high. I do not believe that more than ro per cent. of
persons who become infected with E. histolytica ever suffer to any
appreciable extent from their infections; and I think it very probable
that even this is much too high an estimate.

Infections with E. histolytica appear to be remarkably persistent ;
and there is good reason to believe that, when an individual once
acquires an infection, it will usually—unless he is subjected to specific
treatment—persist for the rest of his life.* Cases are known in which
infection has lasted for at least 16 years, and probably for much longer
periods (Dobell and Stevenson, 1918). Infected individuals may remain
healthy, or may show continuous or intermittent symptoms of intestinal
derangement, such as diarrhoea or dysentery. A man may be a
comparatively healthy carrier for months or even years, and then suffer
from an acute attack of dysentery: or he may at any time contract a
liver abscess or secondary infection of some other organ from the
primary focus in his intestine. But the factors which determine
exacerbations, or the spontaneous abatement of symptoms, or the
origination of secondary infections of the various organs, are still too
obscure for discussion of them to be profitable at present.

Multiplication.—The only process of reproduction which I have ever
observed in E. histolytica is equal binary fission. Dividing organisms
are excessively rare in the stools of human beings, even when suffering
from dysentery and passing large numbers of amoebae. In my ex-
perience they are so rare, indeed, that they may practically be said to be
absent. This is not surprising, when 1t is remembered that the organisms
live —and therefore probably multiply—in the tissues. Those which are
washed out from the intestine invariably die, and as arule rapidly. It
thus seems clear that the reproductive stages must be sought in the
ulcers in the wall of the bowel. As this is impossible in the case of
human infections, I had recourse to the cat: for in this animal the
amoebae multiply with great rapidity, and it is therefore comparatively
easy to obtain ulcers in any desired stage of development and amoebae

* Cf. especially Walker and Sellards (1913), Wenyon and O’Connor (1917), Dobell

and Stevenson (1918).

ENTAMOEBA HISTOLYTICA 4t

in unlimited numbers.* After various trials, I found that dividing
organisms could only be studied satisfactorily in sections. The infected
kitten must be killed, and not allowed to die; and the ulcers in its
intestine must be fixed immediately after it has been killed. The least
delay in removing the tissues causes the disappearance of dividing
organisms or the appearance of abnormal division forms. Scraping the
amoebae out of the freshly excised ulcers also gave me unsatisfactory
results as a rule. The following account is based, therefore, upon a
study of the dividing amoebae found in the ulcers of kittens experi-
mentally infected with E. histolytica, and studied in serial sections of
material fixed and stained by various reliable methods. I have now
studied a large amount of material from kittens—obtained during the
investigations undertaken with Dr. H. H. Dale in 1916+—and I have
been able to study the process of division in considerable detail. It may
be noted, however, that it is by no means so easy to obtain all the stages
of division as one might suppose. One may section many ulcers without
being rewarded by finding a single stage.

The process of division in £. histolytica is closely similar to that
of E. ranarum, and the division of the nucleus is almost identical with
that which I have previously described in the cysts of the latter species
(Dobell, 1909). I have illustrated the chief stages in E. histolytica in
figs. 43-54, Pl. III, which almost describe themselves.

The first recognizable stage in division (fig. 43) shows an increase in
the volume of the nucleus, a fragmentation of the karyosome, and
apparently a migration of chromatin from the nuclear membrane to-
wards the centre. Such stages are easily recognized, and are connected
by numerous transitional stages with organisms showing ordinary resting
nuclei (eg., ig. 1, Pl. 1). The chromatin granules gradually become
more numerous, the outline of the nucleus becomes oval, and achromatic
threads appear within it—usually more or less longitudinally disposed
(fig. 44). I have been unable to make out any definite arrangement of the
chromatin granules at these stages : and although the nuclear membrane
stains readily now, and at all subsequent stages, it seems to be no longer
studded with chromatin granules on its inner surface—as in the resting
nucleus. The appearances suggest that these granules have passed, for
the most part, towards the centre, where they form part of the mass.
of granules now seen (fig. 44).

The nucleus next becomes fusiform, at first having the shape of a
small stumpy spindle (fig. 45), and later of a long slender one (fig. 46)
which ultimately stretches right across the organism (fig. 47). I have
studied all these stages with great care, but they are very puzzling. At
first sight the spindles suggest mitotic figures, with chromosomes and
achromatic spindle-fibres. More careful investigation has always con-
vinced me, however, that the irregular masses and threads within the
nuclei cannot be resolved into the definite chromosomes and other
structures of a true mitotic figure. Fig. 45, which is drawn from a
rather small individual at an early stage, will illustrate my meaning.
The granules and threads within this nucleus strongly suggest a mitosis.

* Contrary to Swellengrebel and Schiess (1917), and some other observers, I find
that the amoebae in the cat are morphologically identical with those in man. Cf. Dale
and Dobell (1917).

+ Vide Dale and Dobell (1917).

42 THE AMOEBAE LIVING IN MAN

I cannot count the “ chromosomes,” however, and I cannot resolve the
structures here seen into a typical mitotic figure. Similar stages have
similar appearances, but the numbers of “chromosomes ’”’ and fibres
appear to be variable. In later stages (fig. 46) the figures are even worse
to study, and show a most confusing arrangement—or apparent lack
of arrangement—of stainable threads, masses, and granules. There are,
however, usually several definite achromatic fibres which pass from one
end of the spindle to the other. They can sometimes be seen with great
clearness, and even counted (fig. 47), but their number seems to be
inconstant and their arrangement often varies. Not infrequently—as in
fig. 47—they are crossed or twisted towards the middle of the spindle.
(This figure is drawn from an iron-haematoxylin specimen, very strongly
differentiated.)

The greatly elongated spindle now constricts in the middle, its
internal structure undergoing no obvious change (fig. 48). The con-
striction becomes more marked (fig. 49), and the two ends now pull
apart (fig. 50), but still remain connected for some time by a thread.
This then snaps, and the resulting daughter nuclei become rounded and
vesicular (fig. 51). At this stage they begin to show a definite arrange-
ment of the chromatin once more—some of the granules passing to the
periphery, and one of them, slightly larger or more conspicuous than
the others, often being recognizable as the karyosome of the new nucleus.
During the earlier stages of division the spindle is typically sharply
pointed ; and the points frequently persist until quite a late stage (fig. 50).
Sometimes the ends become rounded earlier, however, before the
daughter nuclei are fully formed (fig. 49). The specimens figured have
been selected to show these slight variations.

The end stages in fission are very simple. The two daughter nuclei
undergo reconstruction, into the form of the resting nucleus, by gradual
rearrangement of the chromatin granules on the nuclear membrane and
differentiation of the karyosome in the centre. The whole organism
becomes elongated, and the nuclei pass to its ends (fig. 52). A constric-
tion then appears in the middle of the animal (fig. 53) and gradually
deepens until complete constriction inte two is effected (fig. 54). Rem-
nants of the thread which originally connected the two daughter nuclei
often persist for a considerable time, as little knobs or outgrowths on the
daughter nuclei (cf. figs. 52,53). Late stages in fission, it may be added,
are extremely difficult to obtain. This is because the dividing amoebae
are usually tightly packed together in the bases of the ulcers: and when
division of the cytoplasm takes place, they still remain closely crowded.
It is thus very difficult, in sections, to be certain whether two small
amoebae in close apposition are dividing or divided forms, or merely
two small unrelated organisms accidentally in contact. Figs. 53 and 54
were drawn from specimens lying in the mucus on the surface of an
ulcer, where they were somewhat isolated. They were found with
difficulty, as dividing organisms are rare in such situations.

The foregoing account of the nuclear division is based, as already
noted, on a study of a large number of dividing organisms. It is some-
what unsatisfactory because the nuclear divisions are so peculiar that they
are difficult to describe in ordinary terms. Whether chromosomes are
present I am still unable to decide, as the nuclear figures are very difficult
to interpret. The illustrations are drawn as carefully as possible, so that
the reader will, I hope, be able to put his own construction on the actual

ENTAMOEBA HISTOLYTICA 43

events depicted. For my own part, I do not consider the nuclear
division of E. histolytica to be a regular mitosis. On the other hand,
I cannot call it an amitosis. It seems rather to belong to an intermediate
category, like the nuclear division of E. ranarum.

A word may be added regarding the possible presence of a centriole.
It will be noted that I have not so far described a centriole—for the
reason that I have not found one. In the early stages—e.g., that of
fig. 43—I have never succeeded, by any method of staining, in detecting
a centriole. In later stages, such as figs. 44 and 45, “centrioles” can,
no doubt, be discovered by the ingenious. Their selection is not
difficult, to the willing, from the number of granules and threads at
one’s disposal. Of the existence of a real centriole, however, I am
unable to convince myself. I have made a special study of all the
spindle figures such as those shown in figs. 45-47: because it seemed
to me that if centrioles were present they should here be discoverable
at the poles. But I have not found them. Occasionally—as in
fig. 47—a granule may be visible at one pole, or more rarely at both.
Sometimes, also, threads appear to join these granules, forming a
“‘centrodesmose.” But | attach little importance to these, as they may
be found in different positions in different nuclei; and if they represent
centrioles or their derivatives, then these structures must display a degree
of mutability and variation in behaviour which makes their relation to
nuclear division open to the gravest doubts. In short, I have found no
structures which I can regard, with any confidence, as centrioles or
centrosomes in E. histolytica; and in this respect my findings agree
completely with those previously recorded for E. ranarum (Dobell,
1909, 1914).

The division of E. histolytica has been partially described by several
workers, but nobody appears to have studied all the stages previously.
Schaudinn (1903) stated that the organism divides into two, and that
the nuclear division is an amitosis; but he did not describe the process.
Most of the published figures depict organisms in various stages of
degeneration—not in division. For example, the “dividing” forms of
Werner (1908), and Hartmann’s (1908, 1912 a) “prophases” with
“dividing centrioles,’ are probably not division stages at all. It is
significant that Hartmann never found the later stages of division.
Brumpt (1913, p. 24, fig. 9) has figured some division stages in amoebae
from the cat, but these also are incomplete and partly abnormal. The
nuclear division is not completed in the way suggested by his figures.
Job and Hirtzmann (1916) say that the nucleus divides by “amitosis,”
but give no description or figures. It seems probable that they never
observed real dividing forms. Mathis and Mercier (1916 b) have given
a brief description of division in E. histolytica, but it is incomplete and
faulty, as they apparently saw only a very few stages in human stools.
I cannot confirm their statement that a centriole is present; but until
they publish a fuller account, with figures, of the stages which they
actually saw—apart from their interpretations—it is impossible to discuss
satisfactorily the discrepancies in their description. It should be noted,
however, that they deny that the “ histolytica forms” of E. histolytica—
the large tissue-inhabiting forms, which contain red corpuscles —undergo
division at all. These, of course, are the very forms which do divide,
and which constitute the bulk of the species. They are the forms whose
divisions I have just described in detail.

44 THE AMOEBAE LIVING IN MAN

The earliest observations* on the division of E. histolytica appear to
have been made by Harris (1894), who saw division occur in the living
organisms. He saw several specimens divide into two, but gave very
imperfect figures. A division into three which he once saw was
doubtless a pathological process of fragmentation. According to
Harris’s account, the daughter individuals remain attached for a time
by a slender connecting thread of cytoplasm. I have never been able
—in spite of many attempts—to observe division in living specimens of
E. histolytica. Harris says he sought for it in vain for three years. He
finally saw it in the stools of only one patient suffering from acute
amoebic dysentery. At his first observation he saw one amoeba divide
into three; at a second he saw “several amoebae” divide into two ;
and at a third, “division was again observed” (into two?). No
cytological details were made out.

It should be added here that the binucleate specimens of E. histo-
lytica, which sometimes occur in the stools, and which many observers
have seen and noted, are not normal division stages. They are, I
believe, forms which were undergoing division at the time when they
were passed out of the body, and in which nuclear division has then
been completed without fission of the cytoplasm following. As is well
known, the sudden cooling of cells or organisms during division usually
causes an arrest or regression of the process; and the appearance of
binucleate individuals of E£. histolytica in stools is probably to be
similarly explained as a result of the sudden change of temperature
experienced on leaving the host. These binucleate forms I have some-
times watched for a considerable time, but they have never completed
their division.

Cutler (1919) has recently published an account of the division of
E. histolytica which is largely incorrect, and is based upon a study
of degenerate individuals in human stools—so far as I can judge. He
does not appear to have seen most of the stages which I have here
described. He believes that there is a peculiar ‘‘ chromatin extrusion ”
during the division of the nucleus ; but from his account it seems to me
probable that this is a degenerative phenomenon. Certainly no such
process occurs in the organisms which I have studied—nor in the
nuclear division of any other protozoon with which I am acquainted.

A process of multiple fission or schizogony has been described in
* Amoeba dysenteriae”’ (=E. histolytica) by Job and Hirtzmann (1916).
What their ‘‘ morulae” supposed to be so formed really were I am
unable to decide from their description and figures. They certainly
were not stages in the normal development of FE. histolytica, in any case,
as Mathis and Mercier (1916 b) have already pointed out. The whole
account is highly suspicious and unconvincing. More recently the
same authors (Job and Hirtzmann, 1919) have reaffirmed their belief in
the existence of a schizogony, but without adducing any evidence.

Encystation.—Before the amoeboid forms of E. histolytica encyst they
undergo a reduction in size, with the formation of precystic individuals

* Kruse and Pasquale (1894) figured what they thought might be dividing amoebae,
but they were unable to observe division. Their figure appears to depict either two
separate amoebae in contact, or else a couple of large cells from the stool. Doflein
(1901) suggested that these forms might be stages in “ conjugation” —a suggestion
which seems quite unjustified at the present day.

ie ete ss

ENTAMOEBA HISTOLYTICA 45

which differ in several respects from the tissue-inhabiting forms. The
reduction of size is doubtless effected by division, though the process
has not yet been observed. The small precystic amoebae were originally
described by Elmassian (1909) as a distinct species—E. minuta—and are
sometimes still called ‘‘ minuta”’ forms in consequence. Their correct
interpretation* we owe to Walker (1911, 1913), though I had previously
observed and described an exactly comparable phenomenon in the
development of E. ranarum (Dobell, 1908, 1909).

The precystic forms of E. histolytica are intermediate in size between
the large tissue-forms and the cysts—the smallest of them being, of
course, of the same size as the cysts which they form. There is thus a
considerable range of variation in their dimensions. Those races which
produce cysts of small size have correspondingly small precystic forms,

whilst the strains with large cysts have precystic stages of appropriately

larger sizes. In all strains, however, the precystic amoebae have the
same general structure. (See figs. 77-80, Pl. IV.) They are entirely
free from all food inclusions—which are eliminated by digestion or
excretion befere encystation—and are, when alive, sluggish or sessile.
Their nuclei have the same general structure as those of the large forms,
but the peripheral chromatin is often in a slightly thicker layer, and the
karyosome is often slightly larger in proportion, and sometimes—though
not often—slightly displaced from the centre. There is also, sometimes,
a small amount of chromatin in the zone between the karyosome and
the nuclear membrane (cf. fig. 2, Pl. 1). In all these respects the pre-
cystic amoebae approximate in structure to E. coli, from the precystic
forms of which those of EF. histolytica are often difficult and sometimes
impossible to distinguish. (Cf. figs. 2 and 13, Pl. I.)

The precystic amoeba comes to rest, becomes rounded, and secretes
a cyst wall, thus becoming completely encysted. Encystation occurs
only in the bowel—the precystic amoebae which are passed out with the
stools being apparently unable to complete their development outside
the body. In my experience they invariably die without encysting.
The “encystation’” of E. histolytica outside the body has recently been
described by Yoshida (1918) ; but his figures unmistakably depict a
variety of abnormal and degenerate amoebae undergoing fragmentation
and other pathological changes. His experiments appear to show,
however, that E. histolytica may, under certain conditions, survive—in
a more or less degenerate state—outside the body for a considerable
time (up to 72 hours).

Cysts—The normal development of the cysts of E. histolytica is as
follows. The encysted or encysting organism forms, in its cytoplasm,
blocks or masses of a highly refractile substance which gives all the
reactions of chromatin. 1 call these chromatoid bodies,t but they have
been given various other names (chromidia, crystalloids, inclusions, etc.).
Whether they are formed from the chromatin of the nucleus, or are
secreted in the cytoplasm, is still uncertain. Their staining reactions

* Confirmed by Darling, Wenyon, and many other workers soon afterwards,

{ I have used this term for some years, as it seems to me the most suitable. (Cf,
Dobell and Jepps, 1917.) It is unnecessary to enter into the prolonged argument which
has recently taken place between Chatton (1917, 1918 a) and Mathis and Mercier(1917e,
1917) on this subject. It merely shows the lacunae in our knowledge, and the different
interpretations which different observers can put upon the same appearances.

46 THE AMOEBAE LIVING IN MAN

prove nothing* in this respect, and all that can be said with certainty
is that they appear in the cytoplasm, where they increase in size. When
fully developed they are most commonly in the form of a few fairly
large blocks or bars with rounded ends (cf. figs. 3-5, Pl. I).

At the time when the chromatoid bodies make their appearance,
a vacuole also forms in the cytoplasm (fig. 3). It is of variable size, and
sometimes two or even three are formed instead of one. The vacuole
is stained brown in iodine solution, but not so deeply as the corres-
ponding structure in the cysts of the other intestinal amoebae. In
iodine solution its edge is not as a rule sharply defined. The brown-
staining substance in the vacuole is glycogen ; for it not only gives the
iodine reaction just noted, but also shows the characteristic solubilities
of this substance, and can be stained by Best’s specific carmine methodt

ea Pa ad Boa it

The cyst when first formed is uninucleate (fig. 3, Pl. 1). Its nucleus
typically measures about one third of the diameter of the whole cyst, or
slightly less. The nucleus later divides into two (fig. 4), and each of the
daughter nuclei then again divides, so that four nuclei are finally formed
(fig. 5). In this quadrinucleate stage the diameter of each nucleus is
approximately one sixth of that of the entire cyst, or about half that of
the nucleus in the uninucleate cyst. The nuclei of the binucleate cyst
are intermediate in size. (Cf. figs. 3,4, and 5, Pl. 1, and figs. 72-76, Pl. 1V.)}
The nuclear divisions within the cyst appear to be exactly like those in
the free amoebae, except for the progressive reduction in size. They are
also almost exactly like the nuclear divisions in the cysts of E. ranarum,
which I have elsewhere figured in detail (Dobell, 1909). At all stages in
development the resting nuclei in the cysts of £. histolytica have
a structure exactly like that seen in the free forms. It is therefore
unnecessary to describe it again. One noteworthy feature may be
mentioned, however. In 2-nucleate or 4-nucleate cysts, the nuclei often
show a characteristic condensation of chromatin at one pole, so that the
“ring” of chromatin appears slightly thickened at one side (see fig. 5).
This also I have described previously in £. ranarum.

When the cyst has reached the 4-nucleate condition, no further
nuclear divisions occur. The glycogen vacuole, however, generally
disappears, so that the mature cyst comes to stain uniformly pale brown
throughout in iodine solution. If the mature living cysts are kept under
observation, they can also be seen to lose their chromatoid bodiest

* James (1914) apparently believes that the chromatoid bodies must be cytoplasmic in
origin because they can be coloured blue by Mann’s method. But they can also be
coloured red by this stain (cf. figs. 3-5, Pl. 1), and it is even possible to stain some blue
and some red in the same cyst. I have excellent specimens showing them blue, red,
and all intermediate shades of purple in the same preparation.

t As first shown by Kuenen and Swellengrebel (1913).

t Malins Smith (1918) has recently stated that “the supposition of some writers.
(Hartmann, 1912, James, 1914) that chromatoid bodies tend to disappear as the cyst
becomes mature is not borne out by the facts.” That the mature cysts, with 4 nuclei,
lose their chromatoids when kept outside the body, is, however, a fact. I first showed
this to occur in £. ramarum, and have since confirmed it repeatedly in Z. histolytica.
It is also confirmed by Chatton (19174). If Smith means that the cysts do not lose their
chromatoids in developing from the uninucleate to the quadrinucleate stage, then he is
probably correct. Nobody has yet maintained that this occurs, however, so far as I am
aware. Smith appears to have misunderstood what Hartmann wrote—as reference to the:
passage which he quotes from him seems to indicate.

ENTAMOEBA HISTOLYTICA 47

gradually in the course of a few days. Cysts which, when freshly passed,
show chromatoid bodies in the majority, at the end of about a week
show no chromatoids in the majority. The chromatoids can also be
seen to grow smaller and disappear in individual cysts examined from
day to day. It thus appears probable that both the glycogen and the
chromatoid bodies represent reserve materials of some sort—the former
being used up while the cyst is developing, and the latter being absorbed
in the mature cyst whilst it is waiting to be ingested by a new host. As
a rule the cysts contain no other inclusions than those already noted.

In size the cysts of E. histolytica are subject to great variation. Their
diameters range from about 5 as a minimum in the races producing
small cysts, up to about 20” as a maximum in races forming large cysts.
Each race produces cysts of a constant average size, though showing the
usual degree of variation round the mean. The figures on Plates I and
IV will give a good idea of the difference in size observable in different
races. The cysts shown in figs. 72-74, Pl. IV belong to a race with
cysts having an average diameter of 6°6u: those shown in figs. 3-5,
Pl.1 are from a race with cysts of an average diameter of 13°5; and
those in figs. 75, 76, Pl. IV are from a race forming cysts with an average
diameter of 15 4.* Except for their size, there are no constant morpho-
logical differences between the cysts belonging to different strains.

In form, the cysts of E. histolytica are typically spherical or ovoid,
though they are not as a rule perfectly symmetrical ; but their asymmetry
is often extremely slight.

The cyst wall, as in other Entamoebae, is colourless and perfectly
smooth. It is formed of a single layer, so far as I can determine, and
measures about 0°5 w in thicknesst in cysts of medium size (ca. 12 p);
but it is slightly thicker in larger and correspondingly thinner in smaller
cysts.

The specific gravity of the cysts of E. histolytica is about 1065,
according to Ujihara (1914), who also found that the cyst wall is almost
insoluble in gastric juice, readily soluble in trypsin, slightly soluble in
bile, but resistant to lipoid solvents, such as sodium taurocholate and
saponin. Its exact chemical composition is still uncertain.

The cysts of E. histolytica will survive for several weeks outside the
body of man, if they are kept moist and cool. They will live in damp
faeces or in water without showing any conspicuous change save the
loss of their chromatoid bodies. As a rule, if the cysts are kept under
observation, it will be found that some of them remain alive much
longer than the others. In water or faeces some will usually be found
dead at the end of a week, many more after the lapse of a fortnight, and
after this period only isolated survivors will be discoverable. The
longest time of survival which I have observed is five weeks (cysts
kept in water), but as a rule they will not live so long. Desiccation
kills them immediately, and they degenerate much more rapidly at a high

* These figures are the average sizes of the living cysts. In stained specimens the
cysts show an apparent reduction in diameter of about Io per cent., as I have shown
elsewhere in a joint paper (Dobell and Jepps, 1918).

+ Some observers (Kuenen and Swellengrebel (1913), Woodcock and Penfold (1916),.
Brug (19174), etc.), state that the cyst wall has only a “single contour” when seen
under the microscope. It has only one layer, it is true ; but in optical section both the
inner and the outer surface can be seen and the distance between them measured. The
“single contour” appears to be due to an incorrect adjustment of the microscope.

48 THE AMOEBAE LIVING IN MAN

than at a low temperature. At body temperature they generally dic
within a few days at most. Degeneration of the cysts is readily recog-
nizable. The nuclei first become unnaturally distinct in the fresh cysts
—owing to the coagulation which occurs on the death of the proto-
plasm—and then break up. As the cysts die they also become permeable
to aqueous solutions of various stains (eosin, etc.). The cytoplasm
becomes vacuolated, and finally disintegrates.

Cysts are passed in the faeces in the uninucleate, binucleate, or
quadrinucleate stage. Those containing less than 4 nuclei never
develop to maturity outside the body, and usually die much sooner
than the mature cysts. Even cysts with dividing nuclei do not complete
their nuclear divisions. Spindle-fgures and other stages arrested in
division can be seen to remain unchanged within the cysts until
degeneration takes place.

Numerous observations on the vitality of the cysts have already
been recorded. (Cf. Kuenen and Swellengrebel (1913), Wenyon and
O’Connor (1917), etc.) My observations are in general agreement with
those of others, and with my earlier observations on the cysts of
E. ranarum.

Several common variations in the contents and form of the cysts
of E. histolytica must be noted. These concern chiefly the chromatoid
bodies and the shape of the cyst asa whole. As regards the former, it
may be noted first that the chromatoids are sometimes formed in the
precystic amoebae before the cyst wall is secreted (see fig. 82, Pl. V).
This is not very uncommon. Secondly, the chromatoid bodies show
considerable variation in form. Although typically few and in the form
of thick short rods, they may be very numerous (fig. 70, Pl. IV) and of
many different shapes—long thin rods, filaments, round or irregular
masses, granules, etc. (Cf. figs.3-5, 70, 72-76.) Sometimes they are
completely absent (fig. 71) in cysts at all stages of development.

Malins Smith (1918) has recently attempted to determine the
frequency with which chromatoid bodies occur in the cysts of E. histo-
lytica. He found that they were present in 27 per cent., absent in
65 per cent., and doubtful in 8 per cent. of 1162 cysts which he
examined. Unfortunately he did not take into account the fact that
the chromatoid bodies disappear gradually from the cysts after they
have left the body, and he does not state how long his cysts had been
kept before he examined them. Moreover, his figures are based upon
the study of living cysts in saline, or those examined in iodine solution*™
—not upon stained specimens. It is certain that he would have
found a much higher percentage containing chromatoid bodies had he
examined only freshly-passed cysts in carefully stained preparations.
His figures can thus hardly be accepted as a correct estimate of the
frequency with which chromatoid bodies occur normally in the
development of the cysts of E. histolytica.

Although the cysts of £. histolytica are typically fairly symmetrical,
they may have the most bizarre shapes. Slight irregularities in outline
are commonly seen, and occasionally an infected person will pass a
stool in which almost every cyst is irregular in shape. Abnormalities in

* It should be noted that small chromatoid bodies—and sometimes even large ones
—are difficult to distinguish in cysts mounted in iodine solution.

ENTAMOEBA HISTOLYTICA 49

shape may take almost any form—bulgings, constrictions, and various
distortions—so that they are difficult to describe. In general they are
similar to the irregularities so frequently encountered in the cysts of
I. bittschlii (cf. text-fig. 2, p. 115). 1 have seen pear-shaped, rod-shaped,
hourglass-shaped, L-shaped, and variously shaped cysts of other forms,
including many which are rhomboidal or triangular in outline. I have
twice seen ‘‘ twin” cysts, formed of two complete cysts united at their
point of contact, and with their contents completely continuous.

Nuclear abnormalities are not uncommon. Cysts containing three
nuclei—one large and two small—or nuclei of different sizes and
abnormal shape or structure may often be found.

Do the Cysts of E. histolytica ever contain more than Four Nuclei ?—As
a rule the cysts of FE. histolytica contain, when mature, four nuclei. At
this stage development ceases. Some workers, however, believe that
occasionally all the nuclei again divide, so that a cyst containing eight
nuclei is finally formed. No worker claims to have observed more than
eight nuclei in a cyst of this species: but as this number is that
characteristic of E. coli, which so frequently accompanies E. histolytica,
it is of some importance to ascertain whether the latter species ever
produces similar cysts.

Kuenen and Swellengrebel (1913) claimed to have found cysts of
E. histolytica containing 5, 6, and 8 nuclei, and this was reasserted later
by Swellengrebel and Schiess (1917). The evidence adduced is,
however, most unconvincing : and I think it more than probable that
these authors mistook small cysts of F. coli, occurring in a mixed
infection, for those of E. histolytica. Kuenen and Swellengrebel (1913)
state that E. coli occurs “only seldom” in Deli, where they worked.
This can hardly be correct, for E. coli is so common everywhere else ;
but it implies—to my mind—that they overlooked or misinterpreted
many £. coli infections. Again, they cite a case in which the 8-nucleate
cysts of E. histolytica were found in company with “ minuta” amoebae
of this species containing bacteria and starch grains. Now the pre-
cystic amoebae of E. histolytica do not eat either of these things, but
small EF. coli amoebae, which are easily mistaken for them, frequently
do. I have no doubt that their patient was really infected with E. coli, on
this evidence alone. Further, these authors give 16 was the minimum
diameter of the cysts of E.coli, and they evidently reckoned all cysts
with a smaller diameter as belonging to E£. histolytica—a very common
mistake: yet typical 8-nucleate cysts, undoubtedly belonging to E. coli,
may be found with considerably smaller diameters—at least down to
11 yw. Another point which requires notice is their statement that
the 8-nucleate cysts of EF. histolytica which they found “must” have
belonged to this species because the cyst walls showed only a “ single
contour.” This, as already noted,* is an incorrect statement about
either species, and rests upon an error in observation. It seems to me
certain, therefore, that Kuenen and Swellengrebel were mistaken, and
that their 8-nucleate “cysts of E. histclytica”’ were really in every case
cysts of E. coli.t

Brug (19176) also believes that he has seen at least one cyst of

* See p. 47, footnote.

+ The structure of the nuclei within these cysts is not described, though this is the
chief character by which a correct specific determination can be made.

4

50 THE AMOEBAE LIVING IN MAN

E. histolytica containing 8 nuclei. His evidence is fairly strong, but is
not completely convincing, as Smith (1918) has already pointed out ;
for he has not excluded the possibility of a mixed infection. He does
not say how often he examined the stools of his patient: he merely
states that they were “often” examined, and that E. coli cysts were
“ always absolutely absent ’’—a very rash statement to make about any
stool. He does not sufficiently appreciate, apparently, the fact that
the stools of a person infected with E. coli may be examined on scores
of occasions without the infection being detected.* The 8-nucleate cyst
which he found measured 12 w in diameter: but undoubted cysts of
E. coli also occur of this size. The cytological characters of the nuclei
are not given, and without knowing these it is impossible to be
absolutely certain of the species to which his cyst belonged.t

Mathis and Mercier (1917 b) deny that E. histolytica ever forms cysts
with more than four nuclei. Most other workers who have had a very
large experience are also extremely doubtful regarding their occurrence :
and there can be no doubt whatever that, if they do occur, they are
excessively rare.[ I have examined some hundreds of thousands of
cysts of E. histolytica, and I have seen but three which I believe to have
been 8-nucleate cysts of this species. That this interpretation is correct,
Iam unable to prove conclusively, on account of the great difficulty—
amounting in practice almost to an impossibility—of excluding a con-
comitant infection with E. coli. I have not yet succeeded in finding
an 8-nucleate cyst of E. histolytica in my stained preparations ;§ and
the cytological details, especially as regards the finer nuclear structure,
cannot be made out with complete certainty in iodine solution—the
medium in which my cysts were examined.

It seems almost certain, however, although the evidence so far is
inconclusive, that 8-nucleate cysts of £. histolytica must occur. All the
other cyst-forming amoebae of man occasionally form supernucleate
cysts, containing a double number of nuclei. Cysts of E. coli with
16 nuclei are not excessively rare; 8-nucleate cysts of E. nana also
occur : and binucleate cysts of I. biitschlii may, on very rare occasions,
be found. Of the related amoebae in other animals, E. ranarum—
whose 4-nucleate cysts are very closely similar to those of E. histolytica
—very rarely forms 8-nucleate cysts, according to Epstein and Ilovaiski
(1914). I have never seen cysts with more than 4 nuclei in this species
(cf. Dobell, 1909), although I have now studied a considerable number.
E. aulastomi, another entamoeba with 4-nucleate cysts very like those of
E. histolytica and E. ranarum, also very rarely forms 8-nucleate cysts:
for Ndller (1912), who discovered this species, once found a single cyst
with 8-nuclei. In all these organisms there is little chance of the cysts

* For some important data bearing on this subject see my earlier work (1917), and
also compare Smith (1918).

+ Brug does not use the “single contour” of the cyst wall as a specific character,
but for the singular reason that only a ‘“‘single contour” is observable in the cysts of.
both £. colz and £. histolytica /

{ Vide Wenyon and O’Connor (1917), Dobell and Jepps (1917).

§ I have, however, seen a stained cyst in a preparation made from one of my cases
by Miss Jepps: and from the cytological characters of this cyst, and the history of
the patient from whom it came—a patient whom I examined many times—I have
little doubt that it is an 8-nucleate cyst of Z. Azstoly¢ica, and not of £. colz. '

ENTAMOEBA HISTOLYTICA 51

having been incorrectly identified, or of their having been confused with
those of other species. The supernucleate cysts of E. coli, E. nana, and
I, biitschlii are not to be confused with the cysts of any other amoebae in
the same situation ; and neither the frog nor the leech is infected—so
far as is known—with a species ot Entamoeba normally forming
8-nucleate cysts.

I think it certain, therefore, that E. histolytica sometimes forms super-
nucleate cysts containing 8 nuclei: but this happens so very rarely that,
for all practical purposes, such as diagnosis, their occurrence can be
ignored.

Races distinguishable by the Size of their Cysts——As | have already
discussed this subject fully elsewhere in a joint paper (Dobell and Jepps,
1918), I shall here merely recapitulate the chief conclusions there drawn,
and make one or two additions.

The fact that E. histolytica is a species which is composed of different
races distinguishable by the size of their cysts, was first definitely stated
by Wenyon and O’Connor (1917). I reached the same conclusion
independently, and have given the evidence for it in two previous joint
papers (Dobell and Jepps, 1917, 1918). In the second of these we were
able, I think, to place the matter beyond all reasonable doubt. It was
there shown, by the analysis of careful measurements of large numbers of
cysts from different infections, that “ E. histolytica is a collective species.
It comprises a number of distinct races, strains, or pure lines,
distinguishable from one another by the size of the cysts which they
produce. How many such distinct races exist is still undetermined, but
we have demonstrated the existence of at least five. There is no
evidence that the different races differ in their geographical distribution,
or in any character save size. These races remain constant in character
within a given host; and the dimensions of the cysts are not determined
by the action ot the host upon the parasite, since two different races
may coexist side by side in the same host.”

The five races of E. histolytica which we specially studied had cysts
with mean diameters* of 6°6 », 8°3 w, 11°6 w, 13°34, and 15m. Text-fig. 1,
here reproduced (p. 52) from our paper, will show at a glance the striking
differences in the dimensions of the cysts belonging to four of these
races (Cases H.8, H.7, E.42, B.1). The curves were plotted from
measurements of 500 cysts from each race, and were superposed
subsequently. The ordinates show the number of cysts measured, the
abscissae their diameters in microns.f

Races of E. histolytica which produce cysts of the smaller sizes
(usually 74—9 yw in diameter) were long overlooked. They appear to
have been first recorded by Prowazek (1912a), who found them in,
Samoa, and regarded them as belonging to a distinct species which he
named “ Entamoeba hartmanni.” In the same year they were also found
by Aragao (1912) in Brazil, and combined by him with E. coli to form

* These figures were calculated for living cysts—the measurements having been
made upon fixed and stained specimens. The differences in size between living cysts
and those fixed, stained, and mounted in balsam, were investigated in detail, and the
factors determining these differences ascertained. Vide Dobell and Jepps (1918).

+ The diameters here shown are those of stained cysts mounted in balsam, in which
medium the apparent diameter of a cyst is about 1o per cent. less than its true
diameter when measured alive in saline solution.

§2 THE AMOEBAE LIVING IN MAN

another ‘‘ new species.” called ‘“ E. brasiliensis.” They were seen later
by Ujihara (1914) in Formosa, and correctly referred to E. histolytica.
Ujihara* apparently studied five different strains of E. histolytica, with
cysts of the following mean diameters approximately: 6°8 uw (1 case),

8°5 w (3 Cases), 10°24 w (3 Cases), 11°95 mw (7 cases), 13°6 w (4 Cases)
Unfortunately the author does not state how many cysts he measured
from each case, nor how he made his measurements. It is impossible
to know what exact value to attach to his figures in consequence.

210
200
190
180
170
160
150)

42\ [B.1

LK

—
a

Ay 5 (Gat 9.10 111.12 1914.15 16017 eee
TEXT-Fic. 1.—After Dobell and Jepps (1918).

Nevertheless, there is a striking similarity between four of his races and
four of those which I studied with Miss Jepps, as will be apparent from
the following table :—

Ujihara (1914). Dobell and Jepps (1918).
Race I aus ee 68 hu eee ee 66 uw
aoe els nae 85 bu Bas nae 83 u
538 10°24 # bas bie —-
ree! TI‘Q5 wu ues Il'6R
99/5 136 uw aa wie 133 ¢
” 6 Ps see aes Ison

* In my earlier papers (Dobell and Jepps, 1917, 1918) I unfortunately overlooked
the observations of this Japanese worker—partly, I think, because he himself did not
call attention to the significance of his findings, which were merely recorded in a table
with other data. I did not realize their importance until I recently 1e-read his paper.

5 lal eal
a Lela

ENTAMOEBA HISTOLYTICA 53

As regards the race called “3” in this table, it should be added that
Wenyon and O’Connor (1917) have studied one somewhat similar (their
case “ Kettlewell”’) : and I have also seen at least one similar infection,
in which the cysts ranged round a mean of about 10 w.* The race
called ‘‘6”’ in the table, is absent from Ujihara’s series. It undoubtedly
exists, however, as I have studied several cases; and Wenyon and
O’Connor (1917) have recorded a case of the same type (case “ Healy”),
with large-sized cysts reaching 18 w in diameter. The large cysts of
these strains of E. histolytica are sometimes attributed to E.coli. I know
of several actual instances where this mistake has been made in the
laboratory.

James (1914) found cysts of E£. histolytica measuring 7-10 mw in
diameter, and referred them to their proper species.t Woodcock and
Penfold (1916) again described small-sized cysts of E. histolytica, but
proposed to call them provisionally “ E. minuta.”’ This was an unfortu-
nate application of the name originally proposed by Elmassian (1909) ;
and although Woodcock (1917) still adheres to this nomenclature, there
is clearly no justification for it.

Swellengrebel (1917) has described small cysts of E. histolytica, but
attributed them to the flagellate Chilomastix mesnili. Kuenen and
Swellengrebel (1917) in the same year again described them, but regarded
them as the cysts of a new species of Entamoeba, which they proposed
to call FE. tenuis. It may be noted as a curiosity, however, that Kuenen
and Swellengrebel (1913) had previously referred Prowazek’s E. hart-
manni, which was the same thing—.e., a race of E. histolytica producing
small cysts—to “£. tetragena,” which is their name for E. histolytica.
They have given no reasons for these inconsistent statements. Brug
(1917) has also found the small cysts of E. histolytica, and has also
regarded them as belonging to a new species, for which he proposed
the name E. minutissima. Later, however, Brug (1918) withdrew this
name in favour of that of Kuenen and Swellengrebel. It is thus diffi-
cult to understand what the Dutch workers’ present views really are
about the strains of E. histolytica which produce small cysts; and it is
possible that they will undergo still further changes when they have
studied the publications of others. At present they appear to be
unaware of the large amount of work which has been done on this
subject by English investigators.

Mathis and Mercier (1917 g) are unwilling to believe in the existence
of the strains of E. histolytica with cysts of different sizes. Their
doubts were expressed, however, before the appearance of my full paper
on the subject (Dobell and Jepps, 1918). Various objections which
they raised have there been answered, though some of their criticisms
appear to me irrelevant. It may be noted that they regard all cysts
of E. histolytica with diameters of less than 10 mw as ‘‘ abnormalities ”"—
a view which is certainly untenable. It may be noted further, as

* Vide Dobell and Jepps (1918), p. 540.

t Called by him &. ¢e¢ragena. James appears to have regarded the small cysts as
abnormal, for he says “ probably some circumstance of environment was responsible
for their condition” (1914, p. 187). This hypothesis can hardly be maintained now
that it has been proved that two strains—a small and a large—may exist in the same
host simultaneously (Dobell and Jepps, 1918).

¢ Cf. Dobell and Jepps (1917, 1918).

54 THE AMOEBAE LIVING IN MAN

somewhat curious, that these authors—who deny that E. histolytica has
races producing cysts of small size—have nevertheless included ‘ E.
hartmauni Prowazek, 1912” in their list of synonyms of “ E. dysenteriae,”
which is the name they give to E. histolytica (Mathis and Mercier
(1916) p. 644); though Prowazek’s “E. hartmanni’ was actually a
strain of E. histolytica producing small cysts.

Malins Smith (1918) has recently cast doubts upon the existence of
more than two strains of E. histolytica. He admits that there is an
“ordinary strain” having cysts with an average diameter of 12°6 mw, and
a “small strain” with cysts of 7°7 uw. He seems, however, to have reached
this conclusion as a result of a fallacious method of investigation. He
measured small samples of cysts from 30 different infections (not more
than 50 from any individual case), and thus obtained measurements of
the diameters of 1000 cysts. Plotting these graphically, he obtained a
bimodal curve with modes at 7's wand 12°2 w. He then says: “ There
seems to be no doubt from the curve that the cysts of this species
divide themselves naturally into two strains, differing only in size, with
dimensions as indicated in the curve.” But this conclusion is surely
unwarranted. If there are strains of E. histolytica which differ in the
diameters of their cysts—as the figures which Miss Jepps and I have
published do, 1 think, prove conclusively—then Smith’s curve does not
show how many such strains there are, and what mean diameters they
individually possess, but merely the frequency with which strains of
different sizes occurred in the sample of 30 infections which he studied.
His curve merely shows—on the basis of small samples from each—that
strains having the larger-sized cysts were most plentiful in his material,
strains with small cysts less numerous, and those with cysts of inter-
mediate and very large size absent. His own figures (his Table I, p. 34),
so far as they go, seem to me quite consistent with our results : but his
conclusion that there is only evidence of the existence of two strains,
with average cyst-sizes of 7°7 w and 12°6 yw, can hardly be deduced from
his curve. He has not even demonstrated the existence of a single
strain with either of these diameters as its mean.* No adequate number
of individuals was studied from any one strain; and how any legitimate
conclusions can be drawn by treating the problem in such a manner
I do not understand. One might as well attempt to show that there are
no differences in stature among the various European races of man by
measuring the heights of a few individuals from each race and then
striking an average for the whole lot! It is clearly impossible to
show by Smith’s method whether racial differences do or do not exist.

It seems to me, therefore, that there is a fundamental fallacy in the
method which Smith has adopted; and consequently I do not think it
necessary to discuss the details of his work further. Nevertheless, I am
glad to note that he admits that the species E. histolytica can be divided
into at least two different races, distinguishable by the sizes of their
cysts; and | have but little doubt that, if he continues his investigations,
he will be able to convince himself of the existence of others.

* His Cases 24, 25, and 26 have an average diameter of 12°6 u, but only 50, 20, and
37 cysts respectively were measured from each of these. With such small samples
the conclusion hardly amounts to a demonstration. I can find no single case recorded
in which the cysts had an average diameter of 7°7 «—a figure which seems to result
from taking the mean of at least two distinct strains.

ENTAMOEBA HISTOLYTICA 55

It would be interesting to know whether cysts of different dimensions
belong to strains having tissue-inhabiting amoebae of correspondingly
different sizes. Up to the present I have not been able to determine
this point. An attempt to infect a kitten by means of small-sized cysts
—made in conjunction with Dr. H. H. Dale in 1916—was unsuccessful ;
but the large tissue-forms could doubtless be obtained in this manner.*
There is no doubt, of course, that the precystic amoebae of different
strains correspond in size to the dimensions of the cysts which they
form—strains with large cysts having large precystic amoebae, and
strains with small cysts small precystic forms. (Cf. figs. 77-80, Pl. V.)
But I have never yet been able to study a case, known to be infected
with a strain producing small cysts, in the acute dysenteric stage of the
infection : and with the strains producing larger cysts I have not been
able to estimate the size of the amoebae with sufficient accuracy to
render any comparison possible.

The only relevant figures which I have been able to collect are those
published by Ujihara (1914), who has recorded the dimensions of the
active amoebae observed in the stools of a number of patients suffering
from acute amoebic dysentery, and the dimensions of the cysts found in
the faeces of the same cases subsequently. Unfortunately he gives no
indication of the number of specimens measured or of his method of
measurement—both very important points. His table shows that there
is no correlation between the size of the active amoebae and the size
of cysts which they produce. Some of the largest amoebae, in fact,
were found in cases which subsequently showed the smallest cysts in
their faeces. For example, his Case 1 passed, during a period of acute
dysentery, amoebae whose diameter is stated to have been 44°63 p,
whilst the cysts from this patient measured 6°83 ». The corresponding
figures from his Case 5 are 30°7u and 8°45 pw.

I may add that my friend the late Mr. W. O. Redman King told me
that he had studied a mild case of amoebic dysentery which recovered
clinically without treatment. In the stools—of which he sent me a
specimen—cysts measuring 8-9 in diameter were present after the
recovery. He informed me that the amoebae passed during the
dysenteric attack were not noticeably smaller than those which he had
seen in cases infected with strains producing cysts of the larger sizes
(about 12); but unfortunately he made no permanent preparations of
the amoebae, and did not measure many of those which he saw alive.

At the present moment, therefore, the only certain conclusion which
can be drawn is that this question merits further investigation.

The Early Stages of Development. Excystation.—Although the vegetative
amoebae and cysts of E. histolytica are now fairly well known, the earlier
stages of development are still undiscovered. Since Quincke and Roos
(1893) first put forward the suggestion, it has been amply proved that
man acquires his infection with the parasite by ingesting its cysts ;+ but

* Cutler (1919) has stated recently that he infected a cat by means of cysts of a “small
type.” The animal “developed dysentery and died,” and in its intestine he found “a
small variety of tissue-invading amoebae.” It is stated that sufficient material from this
case has not yet been examined, but it is to be hoped that further and more precise
information will be forthcoming.

+ Numerous workers have, of course, experimentally infected cats and_ other
animals by means of the cysts of £. histolytica. See p. 67 infra. Quincke and Roos

50 THE AMOEBAE LIVING IN MAN

the stages intervening between ingestion and the establishment of the
amoebae in the tissues of the large intestine require further investigation.

Walker (vide Walker and Sellards, 1913) fed 20 men on E. histolytica,
sometimes encysted, and sometimes free—a special technique being
employed in the latter case to enable the amoebae to pass through the
stomach. He succeeded in infecting 18 out of the 20 men fed by these
methods. The infections were established — as determined by the
appearance of cysts in the stools—in times varying from 1 to 44 days,
the average period being g days. The time which elapsed between
the infective feed and the attack of dysentery—in four cases which
developed dysentery—was 20, 57, 87, and 95 days (average 64°8 days).
This last figure is clearly of doubtful value, since it ignores all the cases
which did not have dysentery—any of which might subsequently
develop amoebic dysentery at any time, and so change the “average
incubation period” within indeterminate limits. It may be noted that
the “incubation period” in the cat—which, if infected at all, invariably
suffers from dysentery, and does not become a carrier — is usually
about a fortnight, if the infection is conveyed to it by cysts per os.
With intrarectal injection of active amoebae it is much shorter as a
rule, infection being sometimes established within twenty-four hours.*

Since it is impossible to observe the earliest stages in the develop-
ment of E. histolytica in human beings—their normal host—it appears
probable that recourse must be had to animal experiments if further
information on this subject is to be obtained. The cat at once suggests
itself as a suitable subject for investigation ; but unfortunately, it is not
so suitable as one might suppose. In the course of work undertaken
with Dr. H. H. Dale in 1916, attempts were several times made to infect
kittens with cysts administered fer os ; and then, by killing them later,
and examining the contents of their intestines, to discover the earlier
stages in the development of the amoebae in this animal. Unfortunately
all these attempts failed. The only definite results which we obtained
showed clearly that cats are not easily infected by this method ; and
that as a rule the majority of the ingested cysts die in the cat’s intestine,
though a few will pass through unchanged. We made altogether 17
attempts to infect kittens by these means, using cysts from 7 different
human infections, but we succeeded in infecting a kitten only once.
It thus seemed to me probable that it would be necessary to make a
very large number of experiments, and a great sacrifice of kittens, to
obtain conclusions of any value in this way. The attempt was therefore
abandoned after a few preliminary failures.

Chatton (1917b) appears to have been more successful with this
method. He found that the cysts of E. histolytica, when swallowed by a
cat, passed through its stomach without undergoing any change save the
“digestion ’”’+ of their chromatoid bodies. In the small intestine,

made the suggestion on analogy with their results with cats. It should be remem-
bered, however, that Grassi and Calandruccio had previously shown that man acquires
infection with Z. co/z by swallowing the cysts of this species.

* With a strain of amoebae which was passed through 106 kittens by rectal injection,
the average incubation period was slightly over 2 days (Dale and Dobell, 1917).

t Chatton does not say whether the chromatoids are “ digested” in the stomach by the
host or the parasite. I take it that actually he merely observed no chromatoids ; and
that he interprets their absence as an indication that the amoeba has assimilated them

Tees? ee

ENTAMOEBA HISTOLYTICA 57

however, the cysts hatched, and each liberated a 4-nucleate amoeba.
These amoebae passed down the intestine with the gut-contents, and
were found in the large bowel and the evacuations. According to
Chatton the newly hatched amoebae actively ingest bacteria, become
vacuolated, and their four nuclei become clumped together. Further
development was not seen.

Chatton believes his observations to indicate that the cysts of
E. histolytica normally hatch in the small intestine, liberating undivided
4-nucleate amoebae, which first live upon bacteria and then pass on and
establish themselves in the tissues of the large bowel. He interprets the
ingestion of bacteria in a phylogenetic sense—as an indication that this
species was primitively a commensal, like EF. coli, before it took to preying.
upon the tissues. I do not share this view. I think it more probable
that his amoebae were degenerate, and invaded by bacteria.* The fact
that they were vacuolated supports this interpretation ; for vacuolation is
one of the first signs of degeneration in E. histolytica. The fact that the
nuclei were later found to be agglomerated still further supports it ; for
agglomeration of the nuclei is often seen in multinucleate protozoa during
degeneration.t It is, of course, impossible to know whether the cats in
which these amoebae were found would have become infected if they
had not been killed : but Chatton states that none of his controls became
infected.{ It seems probable, therefore, that the cats which were killed
would also have remained uninfected. I am thus inclined to think that
the cysts, in these experiments, hatched in an abnormal manner, and the
amoebae afterwards degenerated and gradually died. The intestine of
the cat was sufficiently like that of man—their normal environment for
development—to enable them to emerge ; but it was not sufficiently like
the human intestine to enable them to develop. The experiments
support the view, which is at present the only probable one, that the
cysts hatch in the small intestine. But they by no means prove that
the cyst normally liberates a 4-nucleate amoeba in man ; or, if it does,
that its later development is similar to that observed in the cat. Chatton
himself says that “the incomplete development in the cat is due to the cat
being not a normal host of the amoeba.”

Ujihara (1914) had previously stated that the cyst wall of E. histolytica
is soluble in trypsin, but almost insoluble in gastric juice. This seemed
to indicate the small intestine as the place where the cysts normally
hatch : and Penfold, Woodcock, and Drew (1916) have stated that they
were able to cauze cysts to hatch by placing them in liquor pancreaticus.
(Benger),—in which, however, “ only a small proportion excyst.” They
say: “ We have tried pepsin, in an acid medium, bile, and pancreatic
extract, either alone, consecutively, or together, as appeared indicated,§
but the only success we have had has been with pancreatic extract used
alone.” They observed the emergence of a single amoeba from the cyst,

* See p. 62.

+ For instance in 77richosphaertum and Actinosphaertum.

+t He says : “ None of the cats not sacrificed in my experiments has contracted
dysentery as a result of ingestion of the cysts.”

_§ It would be interesting to know the “indications” for acting upon cysts with a
mixture of trypsin, in acid solution, and alkaline pancreatic juice. I cannot help think-
ing that an amoeba—even if quite willing to emerge—would be as much perplexed by
being placed in such a mixture as I am in trying to conceive the indications for its use.

58 THE AMOEBAE LIVING IN MAN

through a small aperture. The nuclei of these amoebae were not inves-
tigated, however, and they state that “from our living (sic) observations,
we could not tell whether the amoebae which excysted had always four
nuclei.” Nevertheless, the authors “are strongly of the opinion that this
is the normal method of excystation.” The evidence for such strong
views is not indicated. The excysted amoebae are stated to have thrown
out pseudopodia, but they underwent no further development, and
finally died.

Shortly after the publication of these experiments, Dr. A. C. Stevenson
and I attempted to repeat them. Although we carefully followed the
methods described, we never succeeded in causing the cysts to undergo
any development. After a number of failures, we came to the con-
clusion that E£. histolytica will not usually excyst in liquor pancreaticus.
We thought it highly probable that among the cysts which Penfold,
Woodcock, and Drew employed, there were some which had only
recently been formed ; and that the walls of these might occasionally
be digested by the pancreatic fluid, and so liberate newly-encysted
organisms still possessing some powers of movement. We could find
no evidence to prove that the authors had ever caused a mature
4-nucleate cyst to hatch—in spite of their firm conviction. I still think
that this is a probable explanation of their observations.

More recently Cutler (1919) has recorded similar experiments. He
states that “all the excysted amoebae were uninucleate,” and agrees with
the explanation just advanced. But he claims to have obtained more
satisfactory results by treating the cysts with liquor pepticus “tor a short
time,” followed by liquor pancreaticus—a method which apparently failed
with Penfold, Woodcock, and Drew. By this method, the majority of
excysted amoebae are stated to be 4-nucleate, the author having been
“able to stain them with methyl green during their emergence.” No
further development of these excysted amoebae was observed. But
Cutler adds that though he has “not seen the intermediate stages there
is evidence that these quadrinucleate amoebae ultimately divide to form
four small amoebulae.” Except, however, for a figure of a small amoeba
—whose origin is not indicated—the evidence is withheld.

It will thus be clear, I think, that the early stages in the development
of E. histolytica are still in doubt. Beyond a definite indication that the
cysts hatch in the small intestine, and some inconclusive evidence that
each liberates one 4-nucleate amoeba rather than four small uninucleate
organisms, there is little to go upon. It is probable that the 4-nucleate
amoebae divide into uninucleate individuals—if they really emerge in
this condition : but whether the new individuals, however formed, are
gametes which conjugate in pairs—as I suggested in E. ranarum (Dobell,
1909), and as Mercier (1910) claims to have shown in E. blattae—is still
a matter for speculation.

Are there Races of E. histolytica differing in Pathogenicity ?—It has
frequently been suggested that there are races or strains of E. histolytica
which differ in virulence or pathogenicity. This view is generally put
forward owing to a misconception of the carrier condition, or through
false analogies drawn between E. histolytica and the Bacteria. It merits
some consideration here, however, as it involves several important points
in the life-history of the parasite.

In the first place, it may be pointed out that the existence of patho-
genic strains—causing amoebic dysentery, liver abscess, etc.—and non-

ENTAMOEBA HISTOLYTICA 59

pathogenic strains—such as are seen in contact carriers—is, on a priori
grounds, almost inconceivable. A “highly virulent” strain of the
parasite, which always causes acute amoebic dysentery to its host, could
not come into existence unless it simultaneously underwent a radical
change in its life-cycle. Amoebic dysentery, as already noted, is disad-
vantageous to the parasite as much as to its host ; for during a dysenteric
attack the amoebae are cast out of the body and are unable to encyst.
The “virulent strain,” if it ever arose, would therefore be unable, in
nature, to transmit itself beyond its first host. As I have elsewhere
pointed out: “If the dysentery amoeba were always to cause acute
dysentery in every human being it infected, it would become extinct
within a period of time immeasurably less than that necessary for its
extermination by any conceivable human agency” (Dobell, 1918). It
thus seems clear that “virulent” and “non-virulent” races of the parasite
do not occur in nature.

The clinical differences between a healthy contact carrier of E. his-
tolytica and a person suffering from acute amoebic dysentery are easily
and simply explained if they are referred to differences in the suscepti-
bility of the hosts. Some individuals are unable to tolerate the parasites,
and react to their presence by developing acute dysentery; but such
individuals are the rare exceptions, and the disharmony which results
when they accidentally become infected is as disadvantageous for their
parasites as it is for themselves. The ordinary individual, when he
acquires an infection, becomes a carrier; and an equilibrium is at
once established between his parasites and himself—a balance is struck
between the regenerative powers of the host, and the destructive powers
of the parasite, resulting in a condition which is not distinctly harmful
to either.

That this explanation of the apparent differences in pathogenicity of
E. histolytica in different hosts is the correct one, there is abundant
evidence to show. We constantly see amoebic dysentery patients who
make a complete clinical recovery without losing their infections. They
become convalescent carriers indistinguishable clinically from contact
carriers who have never had dysentery, though remaining still infected
with the same strain of amoebae. But the cysts from the stools of such
convalescent carriers, who are not suffering from dysentery, will, if
swallowed by a susceptible host, again produce amoebae which cause
dysentery. This experiment has been made on man by Walker, and has
been frequently made with cats, which are so susceptible to the action
of the parasite that they invariably acquire dysentery if they become
infected. The cat never becomes a carrier, no matter what strain of
amoebae is used to infect it; and consequently the infection in these
animals can only be maintained by artificial means. Such experiments
have been performed with the cat by many different workers, and there
can be no doubt as to the facts. 1 may cite as a particularly instruc-
tive instance, however, an experiment made by Wenyon and O’Connor
(1917), who gave a kitten a most acute and fatal dysentery by feeding
it with cysts from the stools of a perfectly healthy man who had never
himself had dysentery. Experiments such as this show clearly that it
is the susceptibility or resistance of the host, and not a difference in the
virulence of the parasite, which determines ‘whether any given infected
individual does or does not suffer from amoebic dysentery.

Equally conclusive experiments have been made upon man himself.

60 THE AMOEBAE LIVING IN MAN

Walker (vide Walker and Sellards, 1913) infected a man by feeding him
with cysts from the stools of another man—a convalescent carrier, who
had previously suffered from amoebic dysentery. The second man,
however, became a contact carrier. He acquired an infection with the
same strain of amoebae, but did not develop dysentery. From the cysts
in his stools a third man was then similarly infected. He also became
a contact carrier. But from the cysts in his faeces a fourth man was
infected, and he developed a typical attack of acute amoebic dysentery
twenty days after ingesting the cysts. It is difficult to conceive of any
more conclusive proof that it is the host, and not the parasite, which
determines whether an infection is “ pathogenic” or “non-pathogenic.”

Baetjer and Sellards have recently attempted to show that there are
strains of E. histolytica with different degrees of virulence.* Although
this appears highly improbable, their experiments may be briefly noted
here, as their conclusions have been accepted by some workers. The
fullest account is given in the paper by Sellards and Baetjer (1915), in
which three different “strains of E. histolytica” are described. The first
was obtained from a patient (Case A) with intestinal symptoms but no
dysentery. The amoebae in the stools were scanty, sluggish, and con-
tained no red blood corpuscles. Their nuclear characters were not
those typical of any of the intestinal amoebae of man.+ “ Cyst-like
bodies” with 1, 2, or 3 nuclei were also found in the stools. A
specimen containing chiefly the “ cyst-like bodies” was injected into
the caecum, ileum, and stomach of a kitten. Some weeks later the
animal passed a few ‘‘ amoebae” and “ cysts” containing 4, 5, or 6 nuclei.
These are all figured, and are strikingly like cells from the kitten’s
intestine, though unlike any developmental stages of E. histolytica. The
kitten was killed and its ‘‘amoebae”’ inoculated into two others. One
of these was also killed later, and ‘‘amoebae”’ were found in its large
intestine post mortem. They were ‘‘not very well preserved, but
apparently approached the histolytica type.”

In the second case (Case B) amoebae ‘‘somewhat intermediate
between the coli and the histolytica types”’ were found. No figures are
given. A kitten inoculated with these ‘amoebae ”’ developed dysentery,
but recovered ‘‘ before any entirely satisfactory specimens were obtained
for morphological study.” In the third case (Case C), no amoebae were
ever found in the patient—only ‘‘ cyst-like bodies containing from one
to three nuclei.”’ These were inoculated into the caecum and stomach
of a kitten, which developed a watery diarrhoea a month later, with
‘““amoebae”’ like those of Case A in its stools. Its symptoms abated,
and it was finally killed. Post mortem neither amoebae nor evidences of
amoebic infection were discoverable.

It will be obvious from this brief summary that there is not much
evidence that Cases A, B, or C, or any of the experimentally “ infected ”
kittens, ever harboured amoebae at all. Nothing greatly resembling
Entamoeba histolytica is described or figured from any human case
or kitten.

* The authors consider that they were dealing with “ atypical strains” of Z. histo-
/ytica modified by environment.

+ The authors only refer to &. coli and E. histolytica, and are apparently unaware
that any other species occur in man.

ENTAMOEBA HISTOLYTICA 61

Sellards and Baetjer believed that the atypical strains of amoebae
which they studied produced atypical symptoms in their patients ; and
that similar atypical infections with corresponding symptoms were
produced in the experimentally infected kittens. Careful consideration
of the recorded details of these experiments and the figures of their
findings leaves me, however, in no doubt that they mistook cells of
various sorts for amoebae and cysts. I can find no good evidence that
amoebae of any sort were present in their patients or transmitted to
their kittens, and it therefore seems superfluous to discuss whether their
“atypical strains” were varieties of any particular species of Entamoeba.
As they stand, their recorded observations supply no evidence whatever
in support of the hypothesis that there are strains of E. histolytica which
differ in virulence ; and their hypothesis that strains producing atypical
symptoms in man produce similar symptoms in experimentally inoculated
kittens, is flatly contradicted by the well established fact that strains
producing no symptoms in man produce the most acute dysentery in
kittens. Indeed, every kitten which has ever been infected per os by
means of the cysts of E. histolytica furnishes evidence against their view.

Can E. histolytica live as a Commensal ?—There are still a few
workers who find it difficult to believe that E. histolytica is a tissue-
parasite always. They consider—if I understand them aright—that the
ordinary healthy carrier of the parasite cannot have an ulcerated gut,
because he manifests no clinical symptoms of disease: and they believe,
apparently, that in healthy persons infected with E. histolytica the para-
sites must be living as harmless commensals—like F. coli. This view
appears to me to be inconsistent with nearly every fact that is definitely
known about E. histolytica. It is,in my opinion, unsupported by any
concrete evidence, and seems to rest largely upon a misunderstanding
of the carrier condition.

On purely a priori grounds the hypothesis is highly improbable. It
is unlikely that an amoeba which is generally dependent upon living
tissues for its nourishment should at times completely change its habits
and become a feeder on bacteria. I know of no analogous instance
in favour of such a supposition. Moreover, if it were proved that
E. histolytica undergoes such radical changes in its habits from time to
time, then the whole question of the pathogenicity of intestinal protozoa
would require reconsideration. For example, there is no reason why
E. coli should not also be able to undergo similar drastic changes in
habit, and thus acquire pathogenic powers. But at present there is
absolutely no evidence that either species is able to perform the remark-
able transformations which the hypothesis demands of them.*

The hypothesis has been advanced especially by Kuenen and Swellen-
grebel (1913, 1914), and adopted by Woodcock (1917), Brug (1917 b),
and a few other workers. Kuenen and Swellengrebel (1913) state that
E. histolytica lives, during what they call its “ minuta phase,” as a
saprozoic or commensal organism. It feeds on the gut contents, and
not upon the tissues. Brug (19176) even asserts that the “ minuta”’
forms are as “omnivorous” as E£. coli. The only concrete evidence

* The hypothesis appears to me to rest partly on a false analogy with bacteria, as is
shown by the fact that some writers speak of Z. A7s¢olytica living “‘ saprophytically ’—
a term improperly applied to anything which is not a plant.

62 THE AMOEBAE LIVING IN MAN

which I can find for these and similar statements is, however, that given
by Kuenen and Swellengrebel : and it appears to be contained in their
statement that they have seen amoeboid forms of E. histolytica con-
taining bacteria and other foreign bodies.

The explanation of this is very simple. In my experience E. histo-
lytica, in a freshly passed stool, does not as a rule contain bacteria in
its cytoplasm. This is true of all amoebae, whether large tissue-forms
containing red corpuscles, or precystic individuals. The latter are,
indeed, especially characterized by the absence of all ingested bodies
from their cytoplasm. They are the forms which, far from ingesting
solid bodies of any sort, have got rid of any inclusions which they may
previously have contained (red corpuscles, fragments of tissue cells,
etc.) in preparation for their encystation. They digest or egest food
particles, and never ingest them. In this respect they resemble the
encysting forms of E. ranarum and every other amoeba—both parasitic
and free-living—with which I am acquainted. That bacteria are
generally absent from these amoebae in a freshly passed stool is, I think,
an indisputable fact. Itis, however, equally true that bacteria can usually
be found in E. histolytica amoebae, of every sort, in stools which are not
fresh. In stale stools, or liver abscess pus, the majority of the amoebae
often contain bacteria; and as a rule the staler the material, and the
more degenerate the amoebae in it, the more plentiful are the bacteria
contained in them. A similar observation can be made on the amoebae
in the tissues. In sections of ulcerated human intestines, which are very
rarely obtainable immediately after death, most or all of the amoebae
are usually degenerate: and many of them, as a rule, contain bacteria
in greater or less numbers. If a cat with acute amoebic dysentery is
killed, and its intestine fixed immediately by a good cytological method,
and then sectioned, as a rule not a single amoeba will be found to
contain bacteria. Although these are abundant in the gut contents
and the older necrotic tissues, the areas occupied by the amoebae, no
less than the amoebae themselves, are remarkably free from bacteria of
every sort. If, however, the infected cat is allowed to lie dead for some
time before its tissues are fixed, then the amoebae in the ulcers will
often be found subsequently to contain bacteria. In such circumstances
the amoebae are always more or less degenerate, and all more or less
full of bacteria—exactly as they are in human tissues obtained ost
mortem.

Examination of really fresh material will convince anybody, I think,
that E. histolytica, when in the amoeboid state, normally never contains
bacteria. Since these only appear in the amoebae when they are
degenerating or dead, the most reasonable way to account for this is,
obviously, to infer that dead and dying amoebae are subject to bacterial
invasion. Cells and dead protoplasm of all sorts, when present in the
gut contents, are readily invaded by bacteria ; and there is no obvious
reason why amoebae should not share the same fate. I have not the
slightest doubt that this is usually the correct explanation of the fact
that E. histolytica amoebae sometimes, and in certain circumstances,
contain bacteria.

When individuals of E. histolytica are found to contain bacteria, then
careful examination always shows that the amoebae are degenerate.
Precystic amoebae, for example, containing numerous bacteria, are very
common in stools which are not perfectly fresh ; but as a rule every such

ENTAMOEBA HISTOLYTICA 63

individual will be found to have an abnormal nucleus, and to show other
typical signs of degeneration. Fig. 81 (Pl. V) depicts a common type of
precystic amoeba from a stale stool. The structure of its nucleus at once
stamps it as a dying or dead organism.

E. histolytica may also—though this is very rare—be parasitized by
bacteria. I have made a careful study of three such infections, and have
satished myself of the correctness of the observations. I do not know
whether the parasitic bacteria occur in the tissue-invading forms within
the intestinal ulcers: but they are certainly present in the precystic
amoebae. Fig. 83 (Pl. V) shows one of these from the case which I -
studied in the greatest detail. The patient in whom this infection
occurred was a convalescent carrier of E. histolytica, and the amoebae
were obtained by the administration of a saline purgative. The prepara-
tions were fixed almost immediately after the amoebae left their host, so
that a post mortem invasion of their protoplasm by bacteria can, I think,
be excluded. A large percentage of the amoebae, in this instance,
contained the micro-organisms shown. They were also present in the
cysts (fig. 84, Pl. V); but almost all such infected cysts were uninucleate.
There were uninfected amoebae also, and normal uninfected cysts con-
taining 1, 2,or4 nuclei. It thus seems highly probable that the parasitic
bacteria in the precystic amoebae did not prevent encystation, but
arrested the development of the cysts at the uninucleate stage. The
bacteria were easily visible inside the living uninucleate cysts ; and when
these were kept they rapidly degenerated—the bacteria apparently
increasing in numbers within the cysts, which finally contained merely
disintegrated protoplasm and many bacteria in active Brownian move-
ment. All the parasitized amoebae and cysts showed more or less
degenerate nuclei (cf. figs. 83 and 84).

Another case of the same sort I studied with Dr. A. C. Stevenson,
who kindly permits me to record our observations. This patient also
was a convalescent carrier, and was under observation for about six
months, during which time his stools were examined repeatedly. They
almost always contained numerous cysts of E. histolytica ; and these
were almost invariably uninucleate, and parasitized by bacteria (fig. 85,
Pl. V). They frequently contained chromatoid bodies also, but these
were generally large and easily distinguishable from the bacterial in-
clusions. Binucleate cysts were very rare, and 4-nucleate cysts were
never found. As in the preceding case, the cysts died very rapidly out-
side the body on every occasion when they were kept under observation.
We came to the conclusion that the cysts were infected with a parasitic
micro-organism which inhibited their development, but we did not
obtain amoebae from this case. Infected cysts were passed by this
patient for about five months, during which time he was unsuccessfully
treated several times. At each relapse after treatment he passed infected
uninucleate cysts once more. As only degenerate uninucleate cysts.
occurred, we were often in doubt as to whether many of them were
really cysts of E. coli. After a final thorough course of treatment, how-
ever, with emetine bismuth iodide, all the cysts vanished from the stools
and never reappeared subsequently, although the examinations were
continued over a considerable period.

The first patient was also treated with emetine bismuth iodide, and
likewise cured of his infection. Both cases were, I think, undoubtedly
infected with a strain of E. histolytica which was itself infected with a

64 THE AMOEBAE LIVING IN MAN

bacterial parasite. In both these cases, and at least one other which I
have seen, the parasitic bacteria appeared to be identical. They were
cocco-bacilli of the form shown in figs. 83-85, Pl. V.

I would also note here that the chromatoid bodies of E£. histolytica
may be mistaken for bacteria when, as sometimes happens, they are in
the form of slender rods, filaments, or small granules. As noted else-
where, these bodies may occur not only in the cysts but also in the pre-
cystic amoebae (fig. 82, Pl. V) ; and such amoebae may easily be misin-
terpreted as precystic individuals containing ingested bacteria.

When precystic or other amoeboid forms of E. histolytica contain
‘‘ ingested bacteria” it is therefore necessary to prove (1) that these are
not really chromatoid bodies, or, if really bacteria, (2) that they are
not parasites or (3) organisms which have invaded the dead or dying
amoebae. I cannot find, however, that Kuenen and Swellengrebel, or
any other workers who share their view, have attempted to exclude any
of these sources of fallacy. Until they do, their statement that E. his-
tolytica can feed upon bacteria is not supported by any evidence. The
mere finding of “bacteria”’ in amoebae by no means proves that E. /is-
tolytica is capable of living as a commensal.

Kuenen and Swellengrebel (1913) also describe a case in which they
found “minuta” forms of E. histolytica containing starch grains, in
addition to bacteria. At first sight this appears puzzling. But the
explanation is at once found when, a little later, we read that this case
also passed 8-nucleate cysts of E. histolytica. 1 have not the slightest
doubt that this case was really infected with £. coli, and that the small
amoebae containing starch grains and bacteria, and the 8-nucleate cysts,
all really belonged to this species. To the three postulates made in the
previous paragraph it thus seems necessary to add a fourth—that when,
in a stool, “amoebae” are found containing bacteria, some proof that
they belong to E. histolytica shall be required.

From the foregoing considerations it therefore appears to me highly
improbable that E. histolytica can alter its mode of life so fundamentally
as to become a mere commensal like E. coli: and in support of such
a view—which is entirely opposed to my own experience, and to me |
almost inconceivable—I can find no evidence whatsoever.

Sexual Phenomena. Conjugation.—No conjugation of any sort has
been shown to occur at any stage in the life-history of E. histolytica. It
is sometimes stated that Schaudinn (1903) described an autogamy in the
cysts of this species: and although this is incorrect, such a process
was actually described by Hartmann (1908) in “EF. tetragena”—really
the same species. As I pointed out at the time (Dobell, 1909), his inter-
pretation was not justified, and the alleged “‘autogamy” was disproved
later by Walker* (1911).

Sexual stages in the life-history have been suggested by several
workers, but are still unsupported by evidence. Job and Hirtzmann

* Walker gives me the credit of having pointed out that the development in the
cysts is by “straightforward nuclear division”: but he is not quite correct in stating
that I came to this conclusion in the case of £. co/z. 1 proved it in &. ranarum, and
pointed out that the development of £. Aistolytica (then called “ Z. ¢etragena”) was
probably ‘‘almost identical.” At that time I was still in doubt about the development
of £. coli, owing to the very definite statements of Schaudinn (1903), and Wenyon’s
(1907) “ confirmation” in Z. muris.

ENTAMOEBA HISTOLYTICA 65

(1916) describe a stage which they believe to be “a female gamete”; but
they observed no conjugation or other sexual process, and no reasons
are given for their peculiar interpretation. Mathis and Mercier (1916 a,
1917 0) have asserted that there are two different kinds of cysts in this
species—‘‘ microcysts,” measuring 12°5 w in diameter, and “ macrocysts”
of 14m: and they suppose that these, when ingested, liberate micro-
gametes and macrogametes respectively, which conjugate with one
another. It has been shown (Dobell and Jepps (1918) ; Malins Smith
(1918)) that this dimorphism of the cysts is not really observable ; and
consequently the rest of their hypothesis is deprived of its chief evidence.

It is not impossible, however, that conjugation does occur at the
stage in the life-cycle suggested by Mathis and Mercier — namely
between the amoebae liberated from the cysts in the small intestine at
the beginning of their life-history. Nevertheless, this has yet to be
observed. 1 originally suggested (1909) that such a development might
occur in the very closely related species E. ranarum, and Mercier (1910)
has since described it in E. blatiae. But his observations have not yet
been confirmed, and at present there is no proof that conjugation occurs
in any other parasitic amoeba.

Other Interpretations of the Life-history.—It is now generally recognized
that the life-history of EF. histolytica follows the course here described.
Since the appearance of the works of Walker (1911, 1913), any other
interpretation of the development of this parasite has, I think, become
untenable. My own experience has, at all events, convinced me over
and over again that his conception of the life-cycle as a whole is the only
correct one. It agrees entirely, moreover, with that of E. ranarum, which
I had worked out previously. In both species there is a large amoeboid
form which represents the ordinary vegetative and reproductive stage.
In certain circumstances this form produces—probably by simple
division—a smaller precystic form which does not feed in the usual
way, but gets rid of any food it may happen to contain, and then
encysts. Within the cyst a nuclear multiplication occurs, by successive
divisions, until four nuclei are formed. The cyst is then mature, and
ready to infect a fresh host. The whole life-history is extremely simple,
and is only complicated, in E. histolytica, by the occasional wandering of
the parasite from its normal habitat in the gut wall into other tissues.
The chief difference between £. ranarum and E, histolytica is in habit—
the one being a simple commensal, the other a true parasite, incapable of
nourishing itself upon anything but living tissue.

Several other views have been advanced concerning the life-history of
E. histolytica. Schaudinn’s (1903) description is now universally admitted

to have been incorrect, and merits no further discussion, Kuenen and

Swellengrebel (1913, 1914), among more recent workers, hold peculiar
views concerning the life-history of E. histolytica. They believe (vide
Kuenen and Swellengrebel, 1913) that the parasite, which they call—
without any justification—E. tetragena, occurs in three “ phases’: a
“ histolytica phase,” in the tissues in dysentery, liver abscess, etc.; a
“minuta phase,” living as a harmless commensal like £. coli in the lumen
of the gut; and a “‘ tetragena phase” in which the parasite appears in the
encysted form. They consider that the “minuta phase,’ which is
saprozoic, can “reproduce” the “histolytica phase,” which is parasitic
in the tissues, and vice versa. They do not regard the “ minuta” forms as
precystic amoebae ; though—if I understand them correctly—they believe

5

66 THE AMOEBAE LIVING IN MAN

that the “ minuta” amoebae give rise, in some way, to the cystic “ tetragena
phase.” Swellengrebel and Schiess (1917) even say that this peculiar
development has been “demonstrated ’”’; but it appears to me to rest upon
a misconception of the part played by the precystic amoebae in the life-
cycle of the parasite.

As we have seen,* Kuenen and Swellengrebel (1913) have never
proved that the ‘‘ minuta’’ forms are capable of living saprozoically, and
it is more than probable that they are not. So far as I understand their
views, they rest upon a misinterpretation of the following facts, which are
well known to everybody familiar with E. histolytica. (1) A patient
suffering from acute amoebic dysentery passes the large tissue-invading
forms of the parasite only (their “ histolytica phase”), and not precystic
amoebae or cysts. (2) When the symptoms abate, the patient suffers from
diarrhoea, and passes chiefly precystic amoebae (their “ minuta phase”’).
(3) When the symptoms disappear, and the patient’s stools become solid,
he passes cysts only (their “tetragena phase’’). All these events are very
simply explained—as I have already shown—without having recourse to
their hypothesis. In the ordinary carrier of E. histolytica all the “ phases”
actually exist simultaneously. The cysts are in his stools ; the precystic
amoebae are in the lumen of his gut—from which they can easily be
obtained at any time by the administration of a purgative ; and the tissue-
invading forms are in the ulcers in his gut wall—from which they could be
obtained by scraping the ulcers, though not, as a rule, by the administra-
tion of purgatives. I do not know how Kuenen and Swellengrebel
account for the appearance of large numbers of cysts in the faeces of a
carrier every day for long periods, unless they suppose that the cysts
multiply in the large intestine : nor how they would account for the fact
that the carrier has an ulcerated intestine containing parasites of
“‘ histolytica”’ form ; nor yet how they would explain the case of a carrier
with ‘ tetvagena phase”’ cysts in his faeces, an abscess full of ‘‘ histolytica
phase” amoebae in his liver, and ‘“‘ minuta phase” parasites discoverable
in his stools as often as a purgative is administered to him. But their
hypothesis will require much further elaboration and far more evidence
than they have hitherto adduced if it is to explain the facts and gain any
adherents. At present I cannot find any evidence in favour of it in the
publications of Kuenen and Swellengrebel (1913, 1914), Swellengrebel
and Schiess (1917), or any other workers.

Another peculiar interpretation of the life-cycle of E. histolytica is that
put forward by Mathis and Mercier (1916). These authors, who call the
parasite E. dysenteriae, believe that it occurs in three chief “forms” or
“types,’—a “tetragena form,” free in the gut; an encysted form, in
normal stools; and a “histolytica form” in dysenteric stools. Of the
last they say: “‘ The fact that the histolytica type is seen exclusively in the
bloody mucous stools of acute attacks of amoebiasis, allows us to admit
that this type does not belong to the developmental cycle of the parasite.”
And they conclude—if I understand them correctly—that the ordinary
forms of E. histolytica, which occur in all the tissues capable of invasion,
and which constitute the major part of the species, are a kind of
developmental abnormality. Their reasons for taking this curious view
are not clear to me: and consequently, beyond noting that their use of
the terms “ histolytica”’ and “‘ tetragena” seem to have but little historic

* See p. 61 ef seq.

ENTAMOEBA HISTOLYTICA 67

justification, and are therefore confusing, I am unable to criticize their
conceptions. I find nothing to support their view, and do not understand
why they 4o not adopt the simple, obvious, straightforward, and consistent
interpretation of Walker and most other workers.

Animal Infections—Entamoeba histolytica is the only amoeba which
has been proved to be able to live in more than one host. Man 1s its
normal host, in all probability, but other animals can be experimentally
infected. Lésch (1875), as already noted, infected a dog; and Hlava
(1887), Kruse and Pasquale (1894), Harris (1901), and Dale and Dobell
(1917) also succeeded in infecting this animal. The animal most readily
infected is, however, the cat, to which the amoeba has been successfully
transmitted by a large number of workers.* The earlier workers infected
cats by injecting amoebae from human dysenteric stools into the large
intestine per anum, and this method is frequently successful. They
found, as was to be expected, that no infection took place if the amoebae
were administered fer os—a fact often since confirmed. Quincke and
Roos (1893) found, however, that cats can be infected by causing them
to swallow the cysts of E. histolytica, and this too has been frequently
confirmed by later workers.t There is no good evidence that the cat
can be infected in other ways.t

The cat, when infected, typically aquires acute and fatal amoebic
dysentery, closely resembling the comparable condition seen in man.
Spontaneous recovery takes place very exceptionally, and the cat, in all
probability, never becomes a carrier. The parasite appears to be in-
capable of forming cysts in the cat’s intestine (cf. Dale and Dobell, 1917).
The ‘“cysts’”’ described in cats by several authors were almost certainly
not cysts of EF. histolytica, for no competent workers who have studied
the infections in cats have ever seen cyst-formation in this animal.
There is already a large amount of evidence on this head. For example,
Dale and I (1917) studied about 150 kittens infected with E. histolytica,
but in spite of the most careful search never discovered any cysts
in them. This is in complete agreement with the observations of
Wenyon (1912) and many other careful workers.

Darling (1913 c) found “ uninucleate cysts” in an infected kitten, but
it is highly probable that they were merely rounded amoebae. The
“cysts” of E. histolytica observed in cats by Sellards and Baetjer (1915)
were obviously cells. Their description and figures make this clear—
their “‘ cysts” being quite unlike those of £. histolytica, and containing
sometimes 5 or 6 nuclei. In an earlier paper (Baetjer and Sellards, 1914)
they stated that “encystment frequently occurred” in their infected
cats, but advanced no evidence. They have also stated (Baetjer and
Sellards, 1914@) that they produced “a carrier state” in cats; but they

* See, for example, Hlava (1887), Kartulis (1891), Kovacs (1892), Quincke and Roos
(1893), Kruse and Pasquale (1894), Jiirgens (1902), Wenyon (1912), Darling (19132),
Baetjer and Sellards (1914), Dale and Dobell (1917), etc.

+ Cf. Huber (1903, 1909), Kuenen and Swellengrebel (1913), Wenyon and O’Connor
(1917), Dale and Dobell (1917), etc.

{ The fantastic experiments of Lesage (1907 a) are hardly worthy of mention. He
claimed to have infected cats by placing stale dysenteric stools or liver abscess pus up
their noses, by injecting similar material hypodermically, by putting the dried blood of
an eae cat into the nose of another, and by other extravagantly impossible
methods.

68 THE AMOEBAE LIVING IN MAN

appear to mean by this that they simply observed “periods of apparent
health alternating with acute relapses ’’—a very different thing from the
true carrier condition seen in man.

Cutler (1918) somewhat casually mentions that he found “a few
cysts’ in two experimental cats (Nos. 1 and 2), although his account of
the second animal makes it probable that it was never really infected with
E. histolytica. In a more recent paper (Cutler, 1919) he says: “in the
cat’s gut cyst formation probably never occurs” (p. 131); again, “‘in
cat infections, ... cyst formation ... is rare” (p. 140); and again,
“Tf Dale and Dobell mean ... that they found no evidence of cysts
(sic) formation in cats, I am in entire agreement.’ Cutler has thus twice
asserted and twice denied that cysts are formed in the cat. This seems
to me sufficiently contradictory to render further criticism superfluous.

Amoebic abscess of the liver has been produced both in dogs and in
cats, as a sequel to intestinal infection. It was first produced experi-
mentally in the dog by Harris (1901), and Kartulis (1913) has recorded
a spontaneous case. Craig (1905), Werner (1908), Huber (1909), Wenyon
(1912), Dale and Dobell (1917), and others, have observed amoebic liver
abscesses in cats. The first case was probably Huber’s, though not the
first to be described. Kartulis (1913) states that he has seen a case of
spontaneous amoebic dysentery and liver abscess in a badger, and this
may have been due to accidental infection with E. histolytica. In the
cat, the amoebic liver abscesses closely resemble the early abscesses seen
in man.

Although carnivores seem most easily infected with E. histolytica,
there is now evidence that rodents can also harbour the parasites. The
most interesting case is that of the guinea-pig. Kartulis (1886), Hlava
(1887), Kruse and Pasquale (1894), and Werner (1908), were unable to
infect this animal; but Baetjer and Sellards (1914a) and Chatton (1917a,
1918, 1918), have since succeeded. From the observations of these
workers it appears that E. histolytica infection in the guinea-pig is not
accompanied by dysentery. It is sharply localized in the caecum, where
it gives rise to remarkable lesions resembling neoplasms, and described
by Chatton as a “ lympho-sarcomatoid hyperplasia.” The infection can
be brought about by cysts per os or by amoebae per anum (Chatton), as
in the cat, and it appears to be usually fatal to the guinea-pig. A fuller
account will be found in the papers cited. It only remains to add that
“amoebiasis” in the guinea-pig appears to be a‘disease of a type different
from the dysenteric infections of man and carnivores. Whether it is
ever paralleled in human beings remains to be seen. Possibly some
cases of “latent amoebiasis” in man are of a similar character. No
cysts of E. histolytica have yet been described, however, from the faeces
of infected guinea-pigs. And it is worthy of note that this animal has
an amoeba * of its own, resembling £. coli and E. muris. Leger (1918)
has recently given an account of an epizootic, believed to be due to
intestinal amoebae, among guinea-pigs in Cayenne (Guiana). The
reasons for connecting the amoebae with the disease are, however, far
from obvious, and certainly inconclusive.

Kartulis (1886), Hlava (1887), Dale and Dobell (1917), and others

* Cf. Chatton (1917a@), etc. This organism was probably first noted by Walker
(1908). Its correct name appears to be Entamoeba cobayae Walker emend. Chatton
(syn. £. caviae Chatton, 1918 4)—if really a distinct species.

ENTAMOEBA HISTOLYTICA 69

have unsuccessfully attempted to infect the rabbit. But Huber (1909)
claims to have succeeded, and his observations, though little noticed, are
of considerable interest. He says he succeeded in infecting 4 out of 8
rabbits fed on human stools containing cysts of E. histolytica. They
did not acquire dysentery or diarrhoea, nor did they pass amoebae with
their faeces. Three died after 3—5 weeks, and the fourth was killed.
Post mortem examination showed that the amoebae were localized in
ulcers in the caecum. The ulceration was characterized by great inflam-
matory thickening of the submucosa, and was different from that usually
seen in man and the cat. It would be interesting to know whether the
rabbit can become a carrier of E. histolytica—like a human being ; and
Huber’s results seem to indicate the importance of fuller inquiry.

Unsuccessful attempts have been recorded to infect rats (Werner
(1908), Dale and Dobell (1917), Chatton (19180), etc.) ; mice (Chatton,
1917), 1918b) ; and a gerbil (Kruse and Pasquale, 1894). Lynch (19150),
however, claims to have succeeded in the case of the rat, and also to
have observed spontaneous amoebic dysentery in this animal. Although
he gives a circumstantial account of the lesions, and appears to be in no
doubt about the identity of his amoebae with E. histolytica, his descrip-
tion of the parasites is very unconvincing. He seems, moreover, to
be unaware that rats are sometimes infected with another species of
amoeba (E. muris ?), which may possibly account for his findings—as
Chatton (1917a) has pointed out. Lynch’s conclusions can hardly be
accepted without further evidence. Kartulis (1891), it may be added,
had previously stated that rats may suffer from spontaneous amoebic
dysentery.

Experiments on monkeys, and the amoebae naturally occurring in
these animals, will be considered in another place. (Vide p. 131 infra.)

Dissemination.—It is clear that E. histolytica infection is normally
conveyed from man to man by the contamination of food or drink
with faecal matter containing the cysts of the parasite. The spread of
infection must always depend, therefore, upon defective sanitary condi-
tions—as is verified by the evidence which has accumulated showing
that infections with this organism are commonest in the tropics and
other places where hygienic conditions are worst. Infection is probably
acquired in many cases by drinking water polluted with faeces containing
cysts of E. histolytica. It has also been shown by Wenyon and
O’Connor (1916, 1917), that flies may act as spreaders of infection : for
flies will feed readily upon human faeces containing cysts, and can pass
these intact through their bodies and out in their own faeces. It thus
seems probable that the fly is often an important agent in spreading
E. histolytica in nature. It should be noted, however, that some other
workers have regarded the activities of the fly in this connexion from a
different standpoint. Roubaud (1918) suggests, indeed, that the fly is
beneficial rather than harmful : for its faeces rapidly undergo desiccation
when deposited ; and any cysts which it may pass are thus, in ordinary
circumstances, rapidly destroyed.

Cultivation.—Many workers have, in the past, attempted to cultivate
E, histolytica. The earlier of them often claimed to have succeeded, but
it is now generally recognized that they merely cultivated free-living
amoebae from the stools. The original claim of Kartulis (1891) was
disposed of by Celli and Fiocca (1894, 1894a, 1895) and Casagrandi and
Barbagallo (18952, 1897, 18974), who arrived at the true explanation of

79 THE AMOEBAE LIVING IN MAN

his observations ; but since then history has repeated itself many times.*
The conclusive experiments of Walker and Sellards (1913) finally placed
the matter on a thoroughly scientific basis, and during the last decade
claims to have cultivated any parasitic amoeba have become fewer and
fewer. There are probably very few workers now who believe that
E. histolytica is cultivable by any known method.

Recently Cutler (1918) has published a description of experiments
which lead him to believe that he has discovered methods for the
cultivation of this parasite. His work therefore requires notice here.
Although his account appears at first sight to prove that his claims are
justified, they have not yet been confirmed by other workers. In con-
junction with Capt. S. R. Douglas, I.M.S. (ret.), I attempted to cultivate
E. histolytica by his method, but all our attempts were complete failures.
This work, in conjunction with further information concerning his
methods kindly given to me subsequently by Mr. Cutler himself, has
shown me that his observations may be capable of a different interpre-
tation from that which he has put upon them. His experiments were not
quite so simple as they seem from his brief account ; and consequently
I cannot yet regard his claim as fully justified until further evidence and
explanations, with independent confirmation by others, are forthcoming.

Treatment.—It is not my intention to discuss the treatment of
E, histolytica infections here : but one aspect of this subject is so remark-
able that it is impossible to omit all reference to it even in a purely
zoological memoir. I refer, of course, to the specific action of emetine
on human infections with E. histolytica. Ipecacuanha has been used for
centuries as a cure for dysentery, but it is only comparatively recently
that its alkaloids—especially emetine—have been proved to be of the
highest therapeutic value in the treatment of infections with E£. histolytica.
Upon human infections with this amoeba emetine has a remarkably
specific action. It was originally thought, as a result of Vedder’s (1912)
observations, that emetine is a peculiarly ‘amoebicidal” substance,
and that it acts by killing the parasite directly when administered to an
infected human being. It is almost certain now, however, that the
alkaloid is not particularly poisonous to amoebae, and that its action is
primarily upon the host and not upon the parasite (Dale and Dobell,
1917). Whatever the mechanism of this action may be, there can be no
doubt that emetine has a truly remarkable therapeutic efficacy when
administered in a suitable manner ;}+ and this specific reaction of the
parasite to the drug has done much to clear up the problem of the
species of amoebae inhabiting the human bowel.

Geographical DistributionIt is now certain that the present
geographical distribution of E. histolytica is world-wide. It is not
restricted to the tropics—as is frequently assumed—but occurs in every
country in which it has been sought by competent observers. Indigenous
infections are now known to occur, for example, in Russia (Lésch, 1875 ;
Yakimoff, 1917); in France (Gailliard and Brumpt (1912), Paviot and
Garin (1913), Landouzy and Debré (1914), Ravaut and Krolunitzki
(1916), etc.); in Germany (Jiirgens, 1906); in the Northern United

* Cf. Musgrave and Clegg (1904, 1906), Lesage (1905, 1907), Walker (1908), Noc
(1909), Gauducheau (1912), etc.

{ For further information on this subject see especially Wenyon and O’Connor
(1917), Dobell (1916, 1917), Dobell, Gettings, Jepps, and Stephens (1918).

ENTAMOEBA COLI 7t

States of America (Giffin, 1913) ; and in the British Isles (Yorke, Carter,
Mackinnon, Matthews, and Smith (1917), Laidlaw (1918), Matthews
and Smith (1919), Baylis (1919), etc.)—in addition to all the tropical
and subtropical countries where its occurrence, as known from the
diseases which it produces, has long been famous. It is hardly possible
to doubt that EF. histolytica occurs wherever man occurs—though the
frequency of infection is probably not equal in all places and among
different populations.*

(2) EVTAMOEBA COLI (GRASSI, 1879) CASAGRANDI & BARBAGALLO,
1895 (VEC LOSCH, 1875).

“ Amoebae”’ Lewis, 1870.

“ Amoebae” Cunningham, 1871.

‘‘ Psorospermi”’ Grassi, 1879 (pro parte).

Amoeba coli Grassi, 1879 (nec Lésch, 1875).
Protomyxomyces coprinarius Cunningham, 1881 (pre parte).
? Amoeba intestinalis Blanchard, 1889.

Amoeba coli mitis

Amoeba Ones Quincke & Roos, 1893.
Entamoeba coli Casagrandi & Barbagallo, 1895.
Entamoeba hominis Casagrandi & Barbagallo, 1897.
Entamoeba coli Schaudinn, 1903 (mec Losch, 1875).
“Entamoeba Loeschi”’ Lesage, 1908.

Amoeba coli Brumpt, tgto0 (nec Lésch, 1875).
Entamoeba williamsi Prowazek, 1911.

Entamoeba hartmanni Prowazek, 1912 (fro parte).
Entamoeba brasiliensis Aragao, 1912 (pro parte).
Léschia coli Chatton & Lalung-Bonnaire, 1912.
Entamoeba coli communis Knowles & Cole, 1917 (pro parte).
Endameba intestinivulgaris Aragao, 1917.
Endamoeba intestino-vulgaris Aragao, 1917.
Endameba coli Craig, 1917.

Endameba hominis Pestana, 1917.

HISTORY AND NOMENCLATURE.

The species here named Entamoeba coli was probably discovered by
Lewis (1870) in India. He saw ‘‘amoebae” in the stools of patients
suffering from cholera, but from his account it is impossible to identify
their species with certainty. At about the same time, however, his col-
laborator Cunningham (1871) made similar observations ; and from the
latter’s publications it is possible to identify their findings with some
certainty. Cunningham (1871) says that he found “amoebae” in 18 per
cent. of the choleraic stools which he examined in Calcutta; and as he
has carefully noted some of their characters, there can be no doubt that
what he called “amoebae” really were these organisms—not cells or

* It seems equally certain that there is only one species of amoeba responsible for
all the amoebic diseases—dysentery, liver abscess, etc.—all the world over. At all
events, no amoeba other than Z. histolytica has ever been proved to be pathogenic to man

72 THE AMOEBAE LIVING 1N MAN

other protozoa. Moreover, it 1s clear that the amoebae which he
observed were intestinal amoebae, and not free-living species. These
are probably the first recorded observations on the intestinal amoebae of
man; and as Cunningham’s early work has hitherto received but scant
attention,* and is not easily accessible, I shall consider it in some detail.

Cunningham was fully alive to the possible errors involved in examin-
ing stools for amoebae. ‘‘ There is,” he writes, ‘a considerable amount
of difficulty and numerous sources of fallacy to be encountered in pro-
ceeding to the consideration of amoebae” (1871, p. 44)—a remark which
his successors might have taken to heart with much profit to science and
themselves. He notes further that he distinguished his amoebae from
cells in the stools by their ‘ power of free progression,” and he observes
that they rapidly die outside the human body. He speaks of both free
and ‘‘encysted”” amoebae, but most of the latter—though probably not
all—were merely rounded and motionless individuals. For the first
time he observed and recorded that the intestinal amoebae differ from
free-living species in possessing no “contractile vesicles.” It is clear
from his later work—though not from the earlier—that Cunningham
(1881) saw both the free forms and the cysts of Entamoeba coli. ‘‘ They
occur in the excreta during health, as well as in cases of cholera and
other morbid conditions affecting the intestinal canal” (Cunningham
(1881), p. 248). His text-fig. 4 depicts the cysts, and text-figs. 5 and 6
show the free amoebae—the last being an unmistakable FE. coli con-
taining ingested Blastocystis (“sporoid bodies”). « “In. the eneysied
condition, when their form is more or less spherical or elliptical, they
frequently attain a diameter of 25 w or even more, and they may range
downward from this until their diameter only amounts to 8 w” (p. 249).
From the last remark it seems probable that he observed the cysts of
more than one species. He notes the variable size and shape of the
amoebae, the ‘changeable vacuoles, often of considerable size,” and
again the absence of a contractile vacuole, Of the nucleus he says that
it was not always visible, that it ““may or may not include a visible
nucleolus,” that it may attain a diameter of 7-9 w; and that “it is
circular and apparently discoid, but in some cases may appear annular
from the presence of a thickened margin” (p. 249). I believe nobody
who reads Cunningham’s account and studies his pictures can fail, if he
knows this organism, to recognize in them the commonest of the
intestinal amoebae of man—Entamoeba coli.

Unfortunately, in his later work Cunningham was led astray by the
flagellates and other organisms which developed in his material : and he
combined together all the organisms which he found in faeces—includ-
ing the intestinal amoebae and flagellates, which he had accurately
observed—to form the life-history of a single organism which he named
“ Protomyxomyces coprinarius,’ and regarded as a sort of Mycetozoon.

After Cunningham, Grassi (1879@) described amoebae from human
faeces, and identified them with the organisms then recently discovered
by Lésch (1875). Accordingly, he named them ‘‘ Amoeba coli Lésch.”
Now there can be little doubt that Grassi was mistaken in this. The

* Craig (1908) has stated that Grassi (1888) “was probably the first investigator to .
demonstrate the occurrence’ of amoebae in the faeces of healthy individuals” (p. 332) ;
and he adds that Cunningham “was probably observing developmental stages of
flagellates.” Few readers of Cunningham’s works are likely to share these views.

ENTAMOEBA COLI 73

organisms which he found were, for the most part, Entamoeba coli—the
same as the amoebae observed by Cunningham: and they were not
Lésch’s “ Amoeba coli,” which was Entamoeba histolytica. This wrong
identification has been responsible for much of the confusion which
arose later. Grassi, moreover, not only saw E. coli in the amoeboid
stage: he also saw its cysts. But he mistook these for coccidia,* and
described them as such (Grassi, 1879). His figures (1879, figs. ro and
II) are conclusive in this respect. Later, he himself confessed that his
“coccidia””’ were “resting amoebae,” or cysts (Grassi, 1882, 1883, 1888).
Grassi’s second account (1882, 1883) of his ‘‘ Amoeba coli”’ is brief, and is
accompanied by an unrecognizable figure of the organism. He says it
occurs in healthy people, as well as in those suffering from diarrhoea or
other intestinal ailments : but as it lives in the more fluid part of the
contents of the colon, it is most abundant in the stools when these are
soft or diarrhoeic. With Cunningham he regarded the organism as non-
pathogenic. It was stated to measure 8-22 w in diameter, its nucleus
being 2.2—5.5 w and round in shape, “ with 2 (? always) nucleoli.””, The
ectoplasm was said to be very thin, the endoplasm granular and con-
taining numerous vacuoles filled with food of all sorts—starch grains,
bacteria, etc.

In a later paper Grassi (1888) gives some further details concerning
his “ Amoeba coli.” He says the organism encysts in the same way as
Endamoeba blattae.+ The cysts are smaller than the amoebae; they
contain “ more or less numerous (3, 6, 9) nuclei,” and are diagnostic of
the species. He adds the important statement that he and Calandruccio
—with whose collaboration the above observations were made—have
shown “by repeated experiments” that human beings, when they
swallow the cysts, acquire infection with the amoebae, which multiply
by fission in their new host. Calandruccio (1890) has confirmed these
statements. Grassi (1888a@) reaffirms his belief that the amoebae are
harmless to man—‘ they are simple commensals, altogether innocuous.”
He says he has found them present in enormous numbers in the stools
not only of persons suffering from dysentery, but also of those with
many other disorders—typhoid, cholera, pellagra, simple diarrhoea ab
ingestis, etc.—and in the stools of perfectly healthy people, who often
continue for months to pass the cysts in large numbers. The infection
experiments noted above were not followed by dysentery or other intes-
tinal derangement.

From the foregoing it is abundantly clear that Grassi studied chiefly
the amoeba here described as Entamoeba coli, though he did not count
the nuclei in the cysts correctly—the numbers he givest being very
unusual, and the typical) number (8) in the mature cyst not being
mentioned. It should be noted that Grassi mentions (1883, 1888) that
his “ Amoeba coli” will ingest red blood corpuscles, when these are
present in the intestinal contents. It thus seems probable that he also
saw FE. histolytica, but did not distinguish it from the commoner form.

* For further details concerning Grassi’s “coccidia” see my paper on the coccidia
of man (Dobell, 1919).

+ Grassi (1888) incorrectly called this organism “Amoeba blattarum.”

t Calandruccio’s (1890) statements concerning the number of nuclei in the cysts
of £. coli are identical with Grassi’s.

74 THE AMOEBAE LIVING IN MAN

I therefore conclude that Grassi’s “ Amoeba coli” was, for the most part,
Entamoeba coli; but that it included also, in all probability, some
individuals at least of E. histolytica.

The next important work to be considered is that of Quincke and
Roos (1893) and Roos (1894), whose admirable work on E., histolytica
has already been noted. These observers also studied Entamoeba coli,
of which they gave an easily recognizable description and good figures.
They found the organism in the stools of a patient with colitis, and in
9 healthy human beings out of 24 whose stools they investigated.
Unfortunately, the clinical condition of the first case led them to believe
that his amoebae were a distinct species from the others, though they
found no morphological differences in either the amoebae or their
cysts to justify this conclusion. They named this “pathogenic” form
“Amoeba coli mitis,” and the form from healthy people “ Amoeba
intestini vulgaris.” From the descriptions and figures these amoebae
were identical, except as regards their provenance ; and both these names
were therefore given to the same species—namely, Entamoeba coli.
I would point out here again that the names which Quincke and Roos
gave to their amoebae should not be considered in discussing priority
in the nomenclature of these organisms. The names are trinominal
descriptive terms, and not binominal names bestowed in accordance
with the Rules of Nomenclature. They have no status and are not valid
zoological names. It is therefore not justifiable to attempt to revive
them, as Aragao has recently done.. He proposes (1917) to replace the
name £. coli by “ Endameba intestinivulgaris” or (1917 a) “ Endamoeba
intestino-vulgaris Quincke and Roos, 1893”; though these are not their
names at all, but Aragao’s emendations which attempt to make one of
their designations of this organism conform to the Linnaean system—
a system which they did not follow. Aragao’s names are thus synonyms
without validity.

Quincke and Roos gave a good description of FE. coli, and of the
characters which distinguish this species from E. histolytica. They
describe the amoeba as sluggish, with no sharp demarcation between
ectoplasm and endoplasm, with food-vacuoles containing many ingested
foreign bodies but never red corpuscles. The nucleus is also noted
and recognizably figured. The cysts are distinguished from those of
E. histolytica by their larger size (16—17 «) and thicker walls, and are
said to contain one or more nuclei. On this point their description is
defective. They found further that this species is non-pathogenic to the
cat, and they did not succeed in infecting this animal either with cysts
per os or with amoebae fer anum ; but Roos (1894) points out that man
probably acquires his infection by swallowing the cysts, in the same
way that the cat may become infected with E. histolytica—as their
experiments had proved. It is difficult to understand why these really
excellent observations should have had so little influence on later
workers, Their only real blemishes were errors in nomenclature and
insufficient investigation of cytological details.

Soon after Quincke and Roos, Casagrandi and Barbagallo (1895,
1897) made important contributions to our knowledge of Entamoeba
coli. Unfortunately they contradicted the conclusions of the former
workers—without adequate evidence—and maintained that there is only
one species of intestinal amoeba in man. They maintained— with
Grassi—that this amoeba is harmless, and occurs in both dysenteric

ENTAMOEBA COLI 75

and non-dysenteric persons. Further, they identified the organism
which they studied with Lésch’s ‘‘ Amoeba coli,” though it was
really Grassi’s “Amoeba coli,’ and not Lésch’s organism, At first
(1895a) they named their amoebae Entamoeba coli—the new generic
name being proposed in ignorance of the similar name (Endamoeba)
previously introduced by Leidy (1879). In their second publication
(1897), however, they renamed the species completely, calling it Eit-
amoeba hominis, Pestana (1917) has recently attempted to revive this
name.

It may be noted that Casagrandi and Barbagallo (1897) introduced
the name E. hominis ina most irregular manner. It occurs only twice
in their publication—first as an implied synonym of “Amoeba coli
(Lésch)” in the title of their paper, and a second time in the summary
of their conclusions (p. 163), where it is again implied that it is syn-
onymous with “ A. coli.” There can be little doubt that these authors
actually studied the species here considered; but it seems probable
that they also saw, and confused with it, other species. For example,
they found individual amoebae as small as 5 in diameter, and these
cannot have been E. coli. They also saw amoebae containing red blood
corpuscles, and these must have been E. histolytica. They found cysts
with diameters down to 8 p, and these were, in all probability, those of
E. histolytica or E. nana. They did not describe the nuclei in the
cysts correctly—even as regards their number; for they stated that the
cysts may contain “from 1 to 11 and more,’ although they figured some
typical 8-nucleate specimens. Certain of their other observations will
be considered later: for the present it will suffice to note that their
“* Entamoeba coli” or “ E. hominis’ was, for the most part, the organism
here described under the first of these names.

Schaudinn (1903) restudied and redescribed this organism, adding
numerous details—mostly wrong—to the earlier accounts. He con-
sidered that the amoebae studied by Quincke and Roos (1893) were all
uncertain species, because “infections of cats” . . . ‘‘cannot be
used as a specific criterion ’’*—which shows how superficial was his
knowledge of the amoebae of man and of the work of these authors.
Casagrandi and Barbagallo (1897), however, he allows to have studied
his own “harmless form” from man with accuracy. Since they
identified their amoeba with the ‘‘ Amoeba coli” of Lésch, Schaudinn
considered that this specific name (coli) should be used for the species
which they described. He accepted, also, their generic name Entamoeba
in place of Amoeba: and he concluded that the correct name of
this organism is “ Entamoeba coli (Lésch) emend. Schaudinn.” This
determination was not justified by the facts, as can easily be shown.
Nevertheless, the organism has borne this name ever since.

Schaudinn’s contributions to our knowledge of Entamoeba coli con-
sisted chiefly in the discovery of a ‘‘schizogony”’ in the free amoebae
and an ‘‘autogamy”’ in the cysts—both, in all probability, non-existent.
He also confirmed the observations of earlier workers in regard to the
structure and general appearance of the organism and its cysts, and as
to its non-pathogenicity. The more important of his statements will

* This is, of course, true as a general statement. But in the present case it so
happens that this criterion is a good one: and moreover it was by no means the only
one used by Quincke and Roos.

76 THE AMOEBAE LIVING IN MAN

be considered later. His only real discovery of any importance was
that the mature cysts of this species are typically 8-nucleate: they do
not contain the indefinite numbers of nuclei attributed to them by the
earlier Italian workers.

For nearly a decade Schaudinn’s work was generally accepted and
“confirmed.” The work of E. L. Walker (1911), followed by his later
publication with Sellards (1913), then for the first time placed our
knowledge of this organism on a sound scientific foundation. He
proved conclusively that it is a distinct species, and by experimentally
infecting human beings showed that it is non-pathogenic. From this
date onwards little of importance has been discovered. Walker’s work
will be considered in greater detail later, but it is necessary to notice
it here on account of its capital importance in the history of our
knowledge of this species.

A few further words must now be said about the nomenclature of
this amoeba. I have already discussed the subject very briefly elsewhere
(1918), and have had to refer to it previously in the present work: but
I cannot evade the question here. The present position is briefly this :
“Amoeba coli” Lésch (1875), was not Entamoeba coli but Entamoeba
histolytica—using these names to denote the species here described under
them. ‘‘ Amoeba coli’ Grassi (1879 a) was, however, chiefly—if not
entirely—Entamoeba coli: and so were Entamoeba coli Casagrandi et
Barbagallo (1895 a), Entamoeba hominis Casagrandi et Barbagallo (1897),
and Entamoeba coli Schaudinn (1903). The organism has been called
E. coli by almost every worker since, and it is generally recognized by
no other name. Now Schaudinn was unable to decide whether Lésch
studied the form which he himself called E. coli or that which he called
E. histolytica. His inability to do so can be explained by supposing
that he had not studied Lésch’s paper properly, or that he had not
studied the amoebae of man properly—either or both of which may
account for his singularly unfortunate pronouncements on the question :
for there can be no doubt that Lésch’s ‘‘ Amoeba coli”? was Schaudinn’s
Entamoeba histolytica, and not his Entamoeba coli. There is not a
vestige of evidence that Losch ever saw the latter species.* Con-
sequently, Schaudinn was in error when he founded his species £. colz
as a part of Lésch’s species “Amoeba coli.” As already noted in
discussing EF. histolytica, Schaudinn ought to have called this form
Entamoeba coli—using the specific name first given to it. The first
specific name available for the non-pathogenic species would then have
been hominis—proposed by Casagrandi and Barbagallo (1897). His
own “Entamoeba coli” would then naturally have been called Entamoeba
hominis, and there would have been no further trouble. However,
he made his mistake and nobody corrected him at the time. But it
will create endless confusion if the name Entamoeba coli is now trans-
ferred to the dysentery amoeba; and, as I have already stated, I should
consider such a change—on the grounds of priority, or for any other

* As already noted on an earlier page, Mesnil (1918) believes that Lésch studied
a mixture of species, and that the name co/z can be given to one of these—the harmless
one, as proposed by Schaudinn. M. Mesnil’s judgement—for which I have the greatest
respect—is so rarely at fault that I have, since reading his remarks, re-read the whole
of Lésch’s paper with the greatest care: but I am still unable to find the slightest
evidence in support of his reading.

ENTAMOEBA COLI Th

reason whatsoever—as contrary to the spirit, though probably conform-
able to the letter, of the Laws of Zoological Nomenclature. Accordingly,
I accept Schaudinn’s mistake and its consequences. I would point out,
however, that the specific name coli was first given to the organism in
question by Grassi in 1879—not by Loésch: and that the generic name
Entamocba was introduced, and combined with this specific name, by
Casagrandi and Barbagallo in 1895. Consequently, if this amoeba con-
tinues to be known in future as Entamoeba coli, then its full title should
be Entamoeba coli (Grassi, 1879) Casagrandi et Barbagallo, 1895 ; and not
* Entamoeba coli Losch emend. Schaudinn ”’—as Schaudinn would have
it. It was not permissible to Schaudinn to amend Lésch’s name so as
to make it designate a different organism altogether: and if the name
Entamoeba coli is retained in Schaudinn’s sense, then the authorities
cited for it should be the workers who first used it in this sense—
namely, Casagrandi and Barbagallo, and not Lésch and Schaudinn.

The chief other synonyms of this organism are given in the list
which heads this section. It only remains to add that Entamoeba coli is
probably the amoeba that Schuberg (1893) found in non-dysenteric
people; the ‘‘ dimoeba coli” of many earlier authors, and some later
workers—e.g., Massiutin (1889, Cases 2—5), Brumpt (1910); the
“Amoeba 1” of McCarrison (1909)—as he himself surmised; and pre-
sumably the organism referred to as ‘‘ Entamoeba Loeschi” by Lesage
(1908). To this species also is referable the greater part of the “ Eut-
amoeba coli communis” of Knowles and Cole (1917), as Brug (1917 a)
and Malins Smith (1918) have already pointed out. ‘The 8-nucleate cyst
of “ E. hartmanni” described and figured by Prowazek (1912 a), the same
author’s ‘“‘£. williamsi”’ (Prowazek, 1911), and the larger cysts of
“ E. brasiliensis” described by Aragao (1912, 1914), also all belong, in all
probability, to this species. On the other hand, the “Entamoeba coli”
of Werner (1912) probably comprises not only this species but also
E. histolytica, E. nana, and J, biitschlii. Hartmann (1913, et alibi) con-
siders that Entamoeba minuta Elmassian (1909) is a synonym of E. coli
—which is obviously incorrect, as everybody who reads Elmassian’s
paper with any care must admit. Whether any of the organisms (and
cells ?) called “FE. nipponica’’ by Koidzumi (1909) were E. coli I am
unable to determine with certainty. The E. coli of Akashi (1913) was
chiefly this species, but apparently included E. nana as well. Brumpt
(1913)—without stating his reasons—gives as synonyms of E. coli not only
E. hartmanni, E, williamsi, E. minuta, E. nipponica, and E. brasiliensis,
but also E. biitschlit Prowazek (1912 a), E. polecki Prowazek (1912), and
the numerous “species” of amoebae cultivated from human stools by
Celli and Fiocca (1894a). There is clearly no justification for the inclu-
sion of these free-living forms. Fantham (1911) would include the
free-living forms “ Entamoeba tropicalis’ Lesage (1908) and “ E. hominis”
Walker (1908) in Entamoeba coli—with which they have no connexion :
and Calkins (1912) would include ‘‘ Amoeba lobospinosa”’ Craig (1912),
another free-living form. Craig (1917) even goes so far as to say that
“E. tropicalis Le Sage” is certainly E. coli, though there is nothing in
the original work of Lesage (1908) to substantiate this statement. From
his description it was probably a mixture of free-living amoebae.

Whether any of the Entamoebae of monkeys are really identical with
E. coli it is still impossible to determine (see p. 131), but the close similarity
of E. legeri Mathis (1913) and the probably identical forms observed by

78 THE AMOEBAE LIVING IN MAN

Wenyon (1908), Brumpt (1909), and others, should not be overlooked.
Rodents—e.g., mice—also harbour Emtamoebae which are not at present
distinguishable with certainty by any character, save their habitat, from
Entamoeba coli. Brumpt (1910), indeed, formerly included the amoebae
from mice, rats, guinea-pigs, and monkeys under E. coli—or, as he
wrongly called it, “ Amoeba coli Lésch.” At present, however, this
comprehensive conception of the species seems premature.

DESCRIPTION.

Entamoeba coli is one of the largest of the amoebae of man, though
subject to great variation in size. Rounded individuals, belonging to the
ordinary active stage of the organism, may have any diameter from about
18 » to 40m. As a rule, however, in my experience, their diameter lies
between 20 w and 30. As regards size, therefore, this species closely
resembles E. histolytica. The living organisms, examined immediately
after leaving the human body, are typically distinguishable from the
latter species by the following characters :

They are far less active, as a rule merely showing changes of shape
unaccompanied by active locomotion. Sluggish movements are charac-
teristic of this species ;* and the sudden extrusion of clear, blade-like
pseudopodia—so often seen in E. histolytica—is never observable. Their
ectoplasm is also far less differentiated, and the line of demarcation
between it and the endoplasm usually not conspicuous. Their endo-
plasm is very bulky and granular, and usually heavily charged with food
vacuoles containing various inclusions, but not red blood corpuscles or
tissue elements. The nucleus is usually very conspicuous, appearing as
a round or oval beaded ring, lying as a rule in an eccentric position.
There is, of course, no contractile vacuole; but £. coli often contains
vacuoles of a peculiar form,t+ resembling clefts or dilated cracks with
pointed ends, and containing a liquid—possibly water. These vacuoles
are quite different in appearance from the bubble-like vacuoles so com-
monly seen in degenerate E. histolytica. Whether, like these, they are
formed as a result of degeneration I have not determined.

The food vacuoles may contain all manner of food derived from the
contents of the bowel, and they give this species a very characteristic
appearance in life. The structure of the cytoplasm is difficult to make
out in consequence, but as a rule it appears coarser and more granular
than that of E. histolytica. lt often appears somewhat greenish in
colour—probably owing to the contained granules. The colour, though
slight, is quite distinct when individuals of the two species are seen
lying side by side—E. histolytica appearing whitish and E. coli greenish. {

* Wenyon and O’Connor (1917) state that Z&. colz may sometimes show movements
quite as active as those of Z. Azstolytica. Though I do not question their observation,
I may say that I have never been fortunate enough to see such lively specimens, and
they must be very rare.

+ These are shown in the amoeba depicted in fig. 55, Pl. IV.

{ This description refers to the appearance of the amoebae under an ordinary
achromatic lens. Under an apochromat, with good illumination, there is no appre-
ciable difference in colour. The difference usually seen depends, therefore, in all
probability, upon the difference in the granulation of their cytoplasms. It is purely an
optical effect, and not due to the presence of any colouring matter.

ENTAMOEBA COLI 79

Apart from the cytoplasm and its inclusions—which will be referred to
again later—E. coli can be recognized most readily by its nucleus.

The nucleus of £. coli, like that of all other Eutamoebae, contains
most of its chromatin in a thin peripheral layer and a comparatively
small karyosome. Its structure is shown in figs. 12 (PI. [), 17 (PI. II),
and 55 (PI. IV). It is typically round or slightly oval, and its diameter
is usually from about 4, in the smallest individuals, up to about 8 pw in
the largest. In structure it is closely similar to the nucleus of E. his-
tolytica, already described ; but it differs in the following features. The
external achromatic membrane is slightly thicker : the layer of chromatin
within it consists of rather larger granules, more closely set together,
though equally evenly disposed: the chromatic part of the karyosome
is relatively somewhat larger—measuring about I pw in good-sized indi-
viduals : the cortex—or “ halo ”—of the karyosome is more definite and
solid in appearance: the karyosome is nearly always eccentric—not
central: and definite chromatin granules are generally present in the
area between the karyosome and the peripheral chromatin layer. These
distinguishing characters will be readily seen on comparing figs. 1 and 12
(Pl. I) and 16 and 17 (Pl. II). They are quite constant in perfectly fresh
specimens which have been properly hxed and stained: but is is fre-
quently impossible to distinguish £. coli from E. histolytica by its nuclear
characters in poor preparations, or when the organisms are in the least
degenerate.

Most of the published descriptions and figures of E. coli have been
drawn, apparently, from a study of more or less degenerate amoebae.
The large, discrete masses of chromatin so often shown at the periphery
of the nucleus are only seen in degenerate organisms. The “ring”
is really, in a normal animal, very uniform. The karyosome, to which
too little attention has generally been paid, is rarely described in its
normal form. It breaks up and disappears altogether in dying or dead
organisms : and this accounts for the fact that in many figures it is
depicted as an irregular mass of granules, or is conspicuous by its
absence. Too little attention has also been paid to its position. In
the nuclei of the vast majority of individuals it is not central, though
often depicted in this position.* The presence in E£. coli of chromatin
granules in the zone between the karyosome and the peripheral layer is,
in my experience, constant; and the importance of this character—as
distinguishing the species from E. histolytica—has not been sufficiently
emphasized, I think, because chromatin granules are so commonly
seen in this position in the degenerate nuclei of the latter species.
In £. coli, moreover, the peripheral chromatin layer is usually very
resistant,t though the other constituents of the nucleus are not; and it
may often be seen as a distinct ring of beads in an unfixed organism
which has been dead even for days. Dead individuals of EF. histolytica
break up much more rapidly, and their nuclei disintegrate usually
within an hour or two at most.

* In a certain number of individuals the karyosome will appear to be central, of
course, even if really eccentric—if the nucleus is viewed from the pole towards which
it is displaced.

+ And consequently, preparations of #. co/z, showing fairly respectable nuclei, can
often be made from stale material in which all the amoebae have long since died.
It is unnecessary to emphasize the errors which may result from examining such
preparations.

80 THE AMOEBAE LIVING IN MAN

Food.—E. coli, as already noted, is a voracious and omnivorous
feeder. In its food vacuoles may be found bacteria of all sorts, and
every kind of vegetable débris from the contents of its host’s colon
(cf. fig. 17, Pl. II). Individuals may often be found containing starch
grains and other plant remains. They also sometimes ingest the cysts
of other protozoa such as lamblia (Giardia intestinalis),* coccidia
(Isospora hominis),t and E. histolytica:} and they may even eat unen-
cysted protozoa, according to some observers. Grassi, and Casagrandi
and Barbagallo, found free trichomonads and Giardia within them,
and O’Connor (1919) has confirmed the latter observation. In my
cases I have seen only the cysts of this flagellate inside E. coli. §

Red blood corpuscles and tissue cells appear to be about the only
things which £. coli will not eat. It is true that various observers—for
example, Craig (1911)—note the occasional presence of such elements
in the vacuoles of this species; but nobody has yet adduced sufficient
evidence to place this observation beyond doubt. It is highly probable,
indeed, that the individuals with ingested red corpuscles were really
E, histolytica and not E. coli, the cases studied having been infected with
both species. Mixed infections are extremely common; but E. coli
containing red corpuscles—if they really do occur—are so uncommon
that I have never encountered them. My observations agree in this
respect entirely with those of Wenyon and O’Connor (1917). These
workers also attempted to cause E. coli to ingest red corpuscles in vitro,
but with negative results. There can be no doubt that it is sufficiently
correct for all practical purposes to state that E. coli does not ingest red
corpuscles; and that if an intestinal amoeba| is found containing these,
then it belongs almost certainly not to this species, but, with the greatest
probability, to E. histolytica. This fact is of the greatest service in
diagnosis. It is also of interest biologically, for it marks the profound
difference in food-habits between the two species—a difference clearly
correlated with the great difference in their mode of life and patho-
genicity.

It is now unnecessary to insist upon the fact that £. coli is a harmless
commensal, possessed of no pathogenic powers whatever—so far as
evidence is available: for only the inexperienced or prejudiced hold
a contrary opinion. The early views of Cunningham, Grassi and Calan-
druccio, Casagrandi and Barbagallo, and many others, have, in this
respect, been completely vindicated. It may be added that some of the
Italian workers—especially Casagrandi and Barbagallo (1897)—have even
gone so far as to maintain that E. coli is beneficial to its host; for it
removes and destroys bacteria and waste matters of all sorts, and so
acts as a useful scavenger of the large intestine.

Habitat.—All the evidence available goes to show that E. coli lives in
the large intestine. The active forms live and multiply in the soft or

* Vide Grassi (1888 a), Casagrandi and Barbagallo (1897), O’Connor (1919).

| Vide O’Connor (1919).

{ Vide Wenyon and O’Connor (1917).

§ Capt. O’Connor has, however, shown me stained preparations of £. codz containing
unencysted lamblia.

|| The body in question must, of course, be really an amoeba. Endothelial cells in
human stools—especially in cases of bacillary dysentery—are often mistaken by the
inexperienced for £. Azstoly/ica when they happen to contain red corpuscles.

ENTAMOEBA COLI sI

liquid contents of the upper part of the colon; and in the more solid
and formed faeces in its lower parts, encystation occurs—the fully-
developed cysts being normally discharged in the stools. This was first
pointed out by Grassi (1882, 1883), and all subsequent work has shown
that his conclusions were correct. At present there is no evidence that
E. coli can live in any other part of the human body, or in any other
manner—that it can invade the tissues, or give rise to secondary infec-
tions of the organs.

Multiplication.— There can be little doubt that £. coli, like other
Entamoebae, reproduces by fission into two. This has been stated to
occur by Grassi (1888) and many later workers; but nobody has yet
described the process, and I doubt whether anybody has ever seen it.
Schaudinn (1903) alleged that E. coli reproduces “ by simple division,”’
with an “‘amitotic” division of its nucleus: but he never described the
process properly, and the rest of his observations on this organism are
so full of errors that it is impossible to place much confidence in his
mere statement. The earlier observations on “dividing” organisms,
made by Casagrandi and Barbagallo (1897), are also very questionable.
I think they merely saw binucleate amoebae—as many others, including
myself, have done since—but not real division stages. Craig’s (1911)
figures of ‘‘ multiplication by simple division in E. coli” (his fig. vi) I
take to be diagrams drawn from imagination. Hartmann and Whitmore
(1912) saw binucleate amoebae, and described “beginning stages” in
nuclear division, with “heteropolar” and “tripolar karyosome-spindles.”
Hartmann (1912) has even asserted that 20-30 per cent. of all individuals
of E. coli are found to be in early stages of division : but this is certainly
incorrect, and a very fantastic interpretation of the appearances which
he saw. In the “apparent division” stages of EF. coli figured by James
(1914), degeneration is, to me, far more apparent than division. Many
other workers—for example Mathis and Mercier—refer to the division
of E. coli as though it were well known to occur. Doubtless it does
occur, but I can find no satisfactory evidence that anybody has yet
observed it.

In stools containing large numbers of £. coli one can generally find
individuals containing two nuclei, and sometimes nuclei which resemble
the degenerate division stages occasionally seen in E, histolytica in human
stools. Save for these questionable forms, I have never been able to
find division stages of E. coli, in spite of much search: and consequently
—although I have no doubt that it must occur, and often—I cannot
give any description of the process of multiplication in this species.

Several workers have described a process of multiple fission or
schizogony in E.coli. It was first suggested, I believe, by Casagrandi and
Barbagallo (1897), who figured what they believed to be an 8-nucleate
amoeba (their Pl. II, fig. 13) : but they were unable to prove that multi-
plication occurs in this way. Schaudinn (1903) later alleged that E. coli
undergoes multiple fission into 8 small amoebae—the process being
preceded by a multiple division of the nucleus. No competent observer
has ever confirmed this observation, although it has become a favourite
with writers of text-books. It has been accepted by Craig (1911) and
others, though the nearest approach to confirmation appears to be that
of Akashi (1913): but judging from his figures, the “schizonts”’ are
ordinary cysts of E£. coli—showing various appearances commonly met
path tn preparations—and the brood of young amoebae formed by their

82 AMOEBAE LIVING IN MAN

’

“schizogony ’
E. nana.

More recently, Mathis and Mercier (1917 d) have attempted to show
that £. coli forms two kinds of cysts—“schizogonic”’ and “‘ gamogonic.”’
The latter will be considered later. In the former, they believe that the
nucleus divides repeatedly until 8 to 16 are present in the cyst. The
nuclear multiplication may take place partly in the amoeboid condition ;
and nuclei of variable size are often found, which are believed to
reproduce by budding. The fuliy-formed cysts vary from 14 mu to 26 p
in diameter, but measure usually 17 w. Their protoplasm may contain,
even within the fully formed cysts, the remains of ingested food; and
it is stated to be more alveolar, vacuolate, and eosinophile than that of
the ordinary (‘‘ gamogonic’’) cysts. At the end of the period of nuclear
multiplication, the protoplasm segments, the cyst bursts, and a brood of
small amoebae is liberated. All this is said to occur in the same host,
as a normal process of auto-infection.

I have never seen anything resembling this process, although I have
studied a very large number of E. coli infections. I have, however, seen
most of the forms which Mathis and Mercier interpret in this manner.
Measurements of the cysts give no support whatever to their belief that
there is a dimorphism of the type which they describe. Smith (1918)
and Matthews (1919) have shown conclusively that it does not exist, and
my own observations are entirely in agreement. The cysts with more
than 8 nuclei, and with nuclei of unequal size, are, in my opinion,
abnormalties. They do not occur in every infection, and similar nuclear
abnormalities may be found in E. histolytica—a species in which they
admit that no schizogony occurs. The ‘foreign bodies” in the
‘“‘schizogony cysts’’ are merely small chromatoid bodies, such as so
frequently occur in £. coli cysts—notwithstanding that Mathis and
Mercier deny that they are ever found in this species. And _ their
“amoebae” with more than one nucleus are merely, I believe, irregularly
shaped cysts such as one often meets with in E. coli infections.*

One naturally attaches much importance to the statement of Mathis
and Mercier that they have seen the emergence of the broods of small
amoebae from their “schizogony cysts.” On this point their words are
unequivocal. Not only do they say that it is “‘not rare” to find this
process taking place in fresh stools, but they also state that they have
watched the living amoebae emerge. Their only figuref of the process,
however, is highly unconvincing. The formation of the 8 young
amoebae is not shown—merely a body, supposed to be a cyst of E. coli,
with 4 small amoebae. Two of these are depicted within the cyst, and
two without; and their nuclei are strikingly different from the nuclei
which the authors figure in the earlier stages. Mathis (1y13) had
previously figured} a similar stage, which it is interesting to compare.
One would be tempted to think that both figures were drawn from the

is apparently a suitably-posed group of 8 individuals of

* Casagrandi and Barbagallo were misled, I believe, in the same way by these
cysts—as several other workers have also been since. Irregular cysts may easily be
mistaken for amoebae in stained preparations, because—as shown elsewhere (Dobell
and Jepps, 1918) for Z. Azstolytica—the cyst wall becomes invisible when the cyst is
mounted in balsam.

1 Vide Mathis and Mercier (1917 @), fig. 15.

$ Vide Mathis (1913), Pl. II, fig. 16.

ENTAMOEBA COLI 83

same specimen, were it not for the fact that the position occupied by
a nucleus in the earlier is occupied by an amoeba in the later figure.
Except for this, and a slight difference in the position of the amoebae
outside the cyst, the two cysts are strikingly alike.* They both contain
a vacuole of a peculiar shape. So far as Iam aware the presence of
a large nucleus in the first cyst has not been explained ; but it seems
inconsistent with the later account of the process of schizogony. On
such evidence, I am unable to believe in the “schizogony” of E. coli:
and I believe that any worker who has studied this species with care,
will, if he compares these figures with one another, and with the forms
usually encountered in the stools, find little to convince him of the
correctness of the interpretations of Mathis and Mercier. In spite of
the most careful search—with many other workers—I have never suc-
ceeded in finding cysts of E. coli liberating broods of small amoebae ;
and the statement that this phenomenon is “not rare” is indubitably
incorrect. It is so rare in fact that one may examine many hundreds of
stools containing E. coli, and hundreds of thousands of cysts, without
seeing any indication of it whatsoever. I regard the ‘‘ schizogony” of
Mathis and Mercier as an incorrect and arbitrary series of stages, and
lam wholly unconvinced of the existence of this phenomenon in the
life-history of E. coli.+

Encystation.—Before encystation FE. coli undergoes a considerable
reduction in size, forming precystic amoebae very closely similar to
those of E. histolytica. These precystic amoebae (fig. 13, Pl. I) are very
sluggish, or even motionless, entirely devoid of all food inclusions,
and consequently colourless and hyaline in appearance. They usually
measure from 15 w to 18 w in diameter. When alive their nuclei are
rather more conspicuous than those of E. histolytica at the corresponding
stage. The karyosome is slightly larger, as a rule, and more frequently
eccentric ; and the chromatin between it and the peripheral “ring” is
rather more abundant. But all these differences are very slight, and
not always observable. Since E. histolytica, as ] have already noted, tends
to assume similar characters at this stage, it is, in my opinion, fre-
quently impossible to distinguish the precystic forms of the two species
with certainty—either when alive, or in the best stained preparations.
That these forms are often mistaken for one another I know only too
well from experience. I have repeatedly been perplexed and mistaken
in my attempts to determine them. If only these forms are present in
a stool, it is, in practice, unwise to pronounce a definite diagnosis. The
only safe course is to continue the examination of the stools until the
ordinary vegetative forms—with their characteristic structure and cyto-
plasmic inclusions—or the cysts, make their appearance.

The precystic amoeba is doubtless formed—as in E. histolytica—by
division of a large organism: but the dividing forms I have never been
able to find. Its size also, as in this species, is proportionate to the size
of cyst which it will produce.

* The second figure of ‘‘ schizogony’’ differs also in being inverted.

t Mercier and Mathis (1918) state that 2. anarum, a species apparently more
closely related to £. histolytica than to £. colz, also forms cysts of two sorts—schizo-
gonic and gametogonic. The latter are the cysts which I previously described in this
species, whereas the former appear to be the amoeboid forms undergoing multiple
fission originaliy discovered and described by Collin.

84 AMOEBAE LIVING IN MAN

Encystation occurs in the same manner as in E. histolytica. In
typical cases, the precystic amoeba becomes rounded and secretes its
cyst wall, which also consists of a single layer, but is slightly thicker
than that of E. histolytica. Inside the cyst the nucleus increases in size,
and then divides into two. Each daughter nucleus again divides,
forming four nuclei; and bya further division of each of these, 8 nuclei
are finally formed. A glycogen vacuole appears in the cytoplasm at an
early stage, and reaches its maximum development in the binucleate
cyst. It is then absorbed, and the mature 8-nucleate cyst is typically
without one. Chromatoid bodies may or may not be present at any
stage in development. All these stages are shown in PI. IV, figs. 56-62.
After this brief summary of the development of the cyst, I may now
consider several points in more detail, with special reference to the
differences between E. coli and E. histolytica—since these are of great
diagnostic importance.

Size of Cysts —Cysts of E. coli may be found with all diameters from 10 w
up to 30m or even more. Cysts smaller than 10 w have been described,
but I have never seen them, and it is not unlikely that these very
small cysts really belonged to a different species. They have all been
described, at all events, by observers unacquainted with the smaller
species livingin man. Very largecysts—over 30 ~—are very uncommon,
and are generally supernucleate abnormal forms. The largest I have
ever found measured 33°5 w, but Wenyon and O’Connor (1917) have
recorded a still larger specimen, measuring 38 wp X 34 pu

There can now be little doubt that EF. coli, like E. histolytica, is a
composite species consisting of a number of different races distinguish-
able by the sizes of the cysts which they produce. This was suggested
by Wenyon and O’Connor (1917), and the measurements which I have
made leave no doubts in my mind as to the correctness of their view.
The question has recently been carefully studied by Matthews (1919),
who has reached the conclusion that there are at least three, and pro-
bably four, distinct races of £. coli, with living cysts of the following
mean sizes: 15 , 18°7 w, 21°7 w, and probably 16°5 w. In all these races
there is, of course, the usual variation in size around the modal value.

Malins Smith (1918) measured 1,000 cysts of E. coli from many
different infections, and found their average size was 17°3 pw. This
method, however, as I have already pointed out in considering E. histo-
lytica, throws no light upon the existence of races. From Smith’s
figures, as Matthews (1919) points out, it seems probable that he studied
at least several distinct races. That these races actually exist in E. coli,
I think Matthews’s figures show conclusively. He adopted similar
methods to those which I used for E£. histolytica, and his results are
closely comparable. I believe, moreover, that the races of E. coli which
he has demonstrated by no means exhaust the number actually occurring
in this species.

The measurements of E.coli cysts published by Kuenen and Swellen-
grebel (1913) and Mathis and Mercier (1917 b) are too few to have any
value in this connexion. Their curves—based in each case on measure-
ments of only 100 cysts—are peculiar, but need not be further con-
sidered here. Their shortcomings have already been emphasized by
Smith (1918) and Matthews (1919).

It has been proved beyond question that size is no certain criterion for
distinguishing between the cysts of E£. coli and E. histolytica. The sizes

ENTAMOEBA COLI 85
of the cysts in these two species overlap to such an extent that only the
extreme limits of size are of any diagnostic value. Cysts of both species
may measure anything between 10 w and 20; but a cyst measuring
less than to is probably not a cyst of E. coli, and one measuring more
than 20 probably does not belong to E. histolytica. Most English
workers with a large experience are now in complete agreement on this
point. *

Cyst Nuclei.—The resting nuclei within the cysts of EF. coli have, at
all stages of development, the same structure as the resting nuclei of the
free amoebae. The eccentric position of the karyosome, the presence
of chromatin granules on the linin network between it and the peripheral
layer, and the other slight differences noted as observable between the
nuclei of E. coli and E. histolytica, are constant at all stages. Far too
little attention has previously been paid to this fact, most observers
having emphasized the differences or resemblances in the number of the
nuclei rather than in their structure. The structure is, however, of far
greater diagnostic value than the number, in most cases ; for uninucleate,
binucleate, and quadrinucleate stages in development are, of course,
common to both species. The structural differences between the nuclei
in the cysts of E. coli and E. histolytica will be readily appreciated by
comparing figs. 3, 4, and 5 with fig. 14 (Pl. I), and figs. 62, 63, 66—69,
with figs. 7o—76 (Pl. IV). The position of the karyosome should be
specially remarked. It is typically central in every nucleus in a cyst of
E, histolytica, and eccentric in the nuclei of E.coli. The greatest careless-
ness has been shown by many workers in depicting the nuclei in the cysts
of both species, but especially in those of E.coli; of which there are many
published figures showing central karyosomes in every nucleus in the
cyst—though such appearances are practically never met with in healthy
cysts with normal nuclei. In properly fixed and stained specimens it is
usually possible to determine with certainty whether a given cyst belongs
to one species or the other from the nuclear structure alone.

The size of the nuclei within the cysts of E. coli is also an important
character. With successive nuclear divisions, the nuclei diminish in
size, as in the case of E. histolytica. In typical, mature, 8-nucleate cysts,
the nuclei are uniform in size; and they generally have a diameter which
is between 3 and 3 of that of the entire cyst. (Cf. figs. 14 (PI. I), 62, 68
(Pl. IV), etc.) The uninucleate cyst at the beginning of development
possesses typically a nucleus which has approximately twice the diameter
of each of the nuclei in the mature 8-nucleate cyst. The size rela-
tions are thus similar to those seen in E. histolytica. Consequently, in
binucleate and quadrinucleate cysts of E.coli the nuclei are larger—rela-
tively to the size of the cyst—than they are at the corresponding stages in
E. histolytica. This will be clearly seen by comparing figs. 4 and 5 (Pl. I)
or 71, 73, 74, 76 (Pl. IV) with figs. 59, 61, and 69 (PI. IV). It is often
possible to say with certainty whether a given 4-nucleate cyst belongs to
E. coli or E. histolytica merely from the relative size of the nuclei (cf.
figs. 69 and 71)—apart from the characteristic differences in their structure.
Of the early stages in development of the cysts of E. coli, the 4-nucleate
is that least often found in the stools. Binucleate and 8-nucleate stages

* Cf. Wenyon and O’Connor (1917), Dobell and Jepps (1917), Malins Smith (1918), etc.
The measurements here given refer to living cysts, or those examined in iodine solution.

86 AMOEBAE LIVING IN MAN

often occur in abundance, whilst the intermediate 4-nucleate stage may
be rare or even absent. This is probably due to the fact that the cyst
rests for a long time in the binucleate stage, and then rapidly completes
the two further nuclear divisions. There is probably a very brief resting
period at the 4-nucleate stage. This is also indicated by the fact that
the majority of 4-nucleate cysts of E. coli, if carefully examined, show
one or more of their nuclei preparing to divide or actually in the form of
a spindle (cf. fig.61, Pl. 1V). This is often another very useful character
for discriminating between the 4-nucleate stages of the two species.

It should be noted that the size of the nucleus in the uninucleate
cyst of E. coli is very variable. At the beginning of development
(fig. 56, Pl. 1V) it is comparatively small, resembling the nucleus in the
precystic amoeba (fig. 13, Pl. 1). For the first nuclear division it then
increases in size enormously (fig. 57, Pl. IV), finally forming a typical
spindle (fig. 58, Pl. IV). The subsequent nuclear divisions are all
similar, and resemble those of E. histolytica. Sometimes definite
chromosomes appear to be present, however, as in the nuclear spindle
shown in fig. 58 (Pl. IV): but I have not yet been able to satisfy myself
of their invariable occurrence at all stages. I hope to return to this
question later, when I have been able to examine more preparations.
I would merely note here that, as in E. histolytica, the dividing nuclei in
cysts discharged in the stools very soon become abnormal; and unless
the cysts are fixed immediately, they show most misleading and abnormal
nuclear figures. All the divisions appear to be accomplished in the
same manner—the nuclear spindles, etc., in succeeding stages differing
only in their size. Division of the nuclei is not always synchronous,
and this occasionally leads to the production of cysts containing—
probably only temporarily, in ordinary circumstances—odd numbers of
nuclei, such as 3, 5, or 7.. Cysts with 6 nuclei may also be found some-
times. Supernucleate cysts also occur from time to time. Instead of
the nuclei entering into a permanent resting stage in the 8-nucleate cyst,
one or more may again divide. In this manner cysts containing any
number of nuclei from g to 16 may be formed. Fig. 67 (Pl. IV) shows
a 16-nucleate cyst which is, as is generally the case, of unusually large
size. Such cysts are not common, and do not represent normal stages
in development, in my opinion.* They are, I think, supernucleate
“freaks,” like the 8-nucleate cysts of E. nana or the binucleate cysts of
I. biitschlii, which are both equally rare. I have very rarely found cysts
of E. coli containing more than 16 nuclei. I have seen cysts—examined
in iodine solution—containing 18 nuclei, on several occasions; and
I have once found one containing more—at least 20. The nuclei, which
lie at different levels, are very difficult to count with certainty in such
cysts, and the only safe way is to draw them very carefully with the
camera lucida.t

Nuclei of different sizes are sometimes seen in the cysts of E. coli.
I do not attach any importance to these, but regard them as abnormali-
ties. They are, on the whole, uncommon: but occasionally an infected

* Cf. Mathis and Mercier (1917 @), who regard them as “ schizogonic ” cysts.

+ Asan instance of the ease with which mistakes can be made, I may mention that
I began to draw the cyst shown in fig. 67, Pl. IV in the belief that it contained 12 nuclei.
It was only after every nucleus had been outlined with great care with the camera lucida
that I realized my error.

ENTAMOEBA COLI 87

person may pass a stool in which almost every cyst shows these atypical
nuclear forms. They are probably the result of some change in environ-
ment caused by the diet of the host, or some similar factor.

The supernucleate and other abnormally nucleate cysts have already
been noted by many workers. Casagrandi and Barbagallo (1897) were
probably the first to notice these forms, and they appear to have believed
that the number of nuclei in the mature cysts is inconstant. They seem
also to have supposed that the nuclear multiplication within the cyst
takes place by a peculiar process—a kind of endogenous budding.
Schaudinn (1903) was really the first observer to emphasize the fact that
the mature cyst is typically 8-nucleate. According to Smith (1918) some
87 per cent. of all E. coli cysts, encountered in random examinations of
stools, contain this number of nuclei. It is thus an excellent specific
character for purposes of diagnosis.

Schaudinn (1903), as is well known, described a remarkable process
of “autogamy” during the development of the cyst of E. coli. There
is not the slightest doubt that he was entirely mistaken in his interpreta-
tion—notwithstanding the “confirmations”’ which have been published
by others. As Walker (1911) and Whitmore (1911) first pointed out,
the development of the cysts of E£. coli is perfectly straightforward and
simple—just as I had previously described it in E. ranarum, and as is
probably the case in all Entamoebae. The observations of Walker and
Whitmore have. been abundantly confirmed by every competent
observer since, and all my own observations are in entire agreement
with their interpretation.*

Glycogen.—E. coli typically contains more abundant glycogen in its
cysts than E. histolytica, but it is present only in the earlier stages of
development. The glycogen is often formed in the precystic amoebae,
before the formation of the cyst wall. This can easily be demonstrated
by treating them with iodine solution, when the glycogen shows itself in
the form of one or more brown patches of variable size and stained
with different degrees of intensity. In the uninucleate cyst the glycogen
occupies an area of variable size. Sometimes it appears to be within
the protoplasm, and sometimes between the encysted organism and its
cyst wall. (Cf. fig. 56, Pl. 1V.) As already noted, the glycogen is most
abundant in binucleate cysts, in which it often occupies the greater part
of the available room. In such cysts it appears as a solid mass, with a
well-defined edge. It stains deeply in iodine solution, and gives the
characteristic reaction with Best’s specific carmine stain (fig. 15, Pl. I).
In stained preparations which have been passed through water, the
glycogen, is, of course, removed; and the area which it occupied is
therefore seen as a vacuole (figs. 57-60, Pl. IV), usually with an irregular
outline. A small amount of glycogen can often be demonstrated in
4-nucleate cysts of E£. coli; but its presence in 8-nucleate cysts is
extremely rare. It appears to be used up during development from the
binucleate to the 8-nucleate stage. Cysts may occasionally be found
devoid of glycogen in the early stages of development, but in my expe-
rience this is very rare.

* Although most other workers are very familiar with this fact, I emphasize the
point here because Schaudinn’s erroneous statements still unfortunately find acceptance
with most writers of text-books—both zoological and medical.

88 AMOEBAE LIVING IN MAN

The glycogen in the cysts of E. coli appears to have been first noted
by McCarrison (1909); though he did not recognize it as such and
merely recorded the presence of a “port-wine staining area” in the
2-nucleate and 4-nucleate, but not in the 8-nucleate, cysts of his
“Amoeba I,” when they were examined in iodine. His figures and
account leave no doubt that he was dealing with the cysts of E. coli.
Kuenen and Swellengrebel (1913, 1914) investigated the contents of the
cysts more carefully, and showed for the first time that the iodine-staining
substance is glycogen. Their observations have been repeatedly confirmed
since. In my opinion the glycogen in the cysts of Eutamoebae is a most
important and useful diagnostic character (cf. Dobell and Jepps, 1917).

Several authors—including Wenyon and O’Connor (1917) and Mathis
and Mercier (1917d)—regard the cysts of E. coli which contain glycogen
as abnormal forms, incapable of further development. This view seems
to have originated with Schaudinn (1903), but I think if is a mistake, and
one due largely to the fact that too much importance has been attached
to the appearances of the glycogen-containing cysts in stained prepara-
tions. In these, the area originally occupied by the glycogen appears as
a clear space (cf. figs. 57-60, Pl. IV), or vacuole : and such “ vacuolate”
cysts may well appear to be degenerate if the fact that they once con-
tained glycogen is overlooked. These workers, at all events, never even
mention that the “vacuole” really contains glycogen—though this can
be readily demonstrated. The “vacuole” is not an empty space, nor
the result of degeneration. And when it is remembered that the majority
of binucleate cysts of FE. coli contain this glycogen “ vacuole,” and
that similar glycogen inclusions occur in the cysts of most other para-
sitic amoebae at some stage in their development, it is difficult to believe
that it isan abnormality. It is difficult to believe that most individuals of
E. coli should store up glycogen—for no purpose—and then die without
completing their development. That cysts containing a large glycogen
“ vacuole” are, moreover, capable of further development has been
shown by Wenyon himself (1907) in the case of FE. muris.*

Chromatoid Bodies—As already noted, chromatoid bodies are not
always present in the cysts of E.coli. They are often in the form of
small granular or rod-like bodies in the earliest stages of development
(fg. 56, Pl. IV), and are frequently larger and more abundant in
binucleate cysts (fig. 60). In mature 8-nucleate cysts they are as a rule
apparently absent : but well-stained preparations of freshly-passed cysts,
if examined with care, generally show one or more very small chromatoid
bodies lying among the nuclei (cf. figs. 14 (Pl. 1), and 62 (PI. IV)).
Sometimes, however, the cysts of E. coli contain, at all stages of develop-
ment, chromatoid bodies which are quite as abundant as those usually
seen in the cysts of E. histolytica. They differ, as a rule, in form ; being
typically acicular or filamentar, and often aggregated into sheaves (figs.
63—66, P]. IV). In general, they may be compared with fragments of
splintered glass.

Sometimes the cysts of E. coli contain chromatoid bodies of a remark-
able filamentar type (fig. 65, Pl. 1V). The filaments are long and slender,
and wound in asort of skein inside the cyst, around and among the

* He has observed and figured the “vacuole” at all stages in development of a
living cyst of this species, though without noting the presence of glycogen in it.

ENTAMOEBA COLI 89

nuclei. That these filaments are really chromatoid bodies, and not
inclusions of some other sort, I think there can be no doubt: for stages
intermediate (fig. 64) between the typical filamentar form (fig. 65) and
the more usual acicular sheaves (fig. 63) are not difficult to find. These
cysts with filamentar chromatoid bodies appear to be those which
Prowazek (1911) described in his new species “ Entamoeba williamsi,”*
They were also, apparently, included in “ £. brasiliensis” by Aragao
(:912, 1914), and have been seen and figured by other workers.

According to Malins Smith (1918) chromatoid bodies occur in 5°5 per
cent of the cysts of £. coli. This figure, however, is certainly too low ;
for it was arrived at by considering only those cysts which contain
conspicuous chromatoids. All those with small chromatoid bodies, only
demonstrable with certainty in carefully stained specimens, were left out
of account.

Mathis and Mercier (1917 a, b, et alibi), apparently alone among recent
workers, deny that chromatoid bodies ever occur in the cysts of E. colt.
So far as I understand their views, they regard the deeply stained bodies
in the cysts of this species as artifacts, formed by a deposition of stain
(iron-haematoxylin) in folds of the cyst wa!l or in the protoplasm. It
is easy to convince oneself that this interpretation is incorrect. The
chromatoid bodies are easily visible in living cysts, and readily stained
by carmine, haemalum, and other progressive methods. I can only
suppose that these authors have devoted too little attention to the study
of living cysts, and too much to iron-haematoxylin preparations. From
their more recent papers (Mathis and Mercier, 1917 ¢, f) I gather that
they now admit that chromatoid bodies are sometimes present in £. coli
cysts, though they call them by a different name from that which they
give to the chromatoids of E. histolytica.t This seems to me an attempt
to fix an arbitrary verbal distinction where none exists in nature.

The chromatoid bodies of F£, coli are probably formed in the same
manner, and subserve the same functions, as those of E. histolytica.
Their origin in the cysts is equally difficult to determine, and at present
doubtful. I have never seen them in the precystic amoebae of this
species, though they are possibly formed before encystation occasionally,
asin &, histolytica. This is a very difficult point to determine, owing to
the great similarity of the precystic amoebae of both species, and the
frequency of mixed infections.

Chromatoid bodies were probably first noted in the cysts of E. coli by
Grassi (1879). They were seen later by Casagrandi and Barbagallo
(1897), who regarded them as a sort of reserve material (‘alimento

* Prowazek (1911, 1912) gives as a further character of this “ species” the occurrence
of cysts containing 10 or 14—15 nuclei. These, as already noted, sometimes occur in
ordinary £. co/z infections. It should be noted that Prowazek also stated that his
“ E. williamsi” occurred in company with £. colz.

+ They now call the chromatoids of £. histolytica “‘batonnets sidérophiles,” and
those of £. coli “ plages sidérophiles” ; which clearly shows the undue importance
which they give to the iron-haematoxylin method. Cf. Mathis and Mercier (1917 e).
Chatton (1917, 1918 a) has already criticized their statements, and I have elsewhere
pointed out the incorrectness of their observations in this respect (Dobell and Jepps,
1917).

{ Their figure 18 shows, in my opinion, a cyst of £. co/z containing a chromatoid
body. Viereck (1907), however, regarded it as a cyst of £. Azstolytica—falling, I
believe, into the very common error of attributing every cyst which contains chroma-
toid bodies to this species.

go AMOEBAE LIVING IN MAN

immagazzinato”’). Schaudinn (1903) called them “chromidia,” and
since then they have generally been so named. With the “chromi-
dium ” of the shelled Rhizopods, however, they probably have nothing
in common.

Mode of Infection—What has been said already about the vitality
and powers of resistance of E. histolytica cysts is equally applicable to
E. coli, and need not be repeated. That the cysts are the forms which
convey infection from man to man there can be no doubt. Grassi
(1888) and Calandruccio (1890) state that they were able to infect
human beings by causing them to swallow the cysts of this species.
Schaudinn (1903) later asserted that he had twice* successfully performed
this experiment upon himself. Carefully conducted experiments made
later by Walker and Sellards (1913) have proved conclusively that infee-
tion is brought about in this manner. They fed 20 men on cysts of
E. coli, belonging to 5 different strains (i.e., from the stools of 5 different
persons), and successfully infected 17 of them. Infection, as judged by
the appearance of cysts in the stools, was established in from 1 to 11
days after feeding—the average time being 4°7 days.

The early stages in the development of the cyst in its new host are
still unknown. On analogy with other species, it is probable that the
cysts hatch in the small intestine, and liberate broods of 8 amoebae—or
possibly a single 8-nucleate amoeba, which later divides into 8 young
organisms—which pass into the large bowel, and there establish
themselves.

Casagrandi and Barbagallo (1897) believed that they had been able
to observe these stages in cats. They claimed to have seen the cysts
hatch in the large intestine, and small amoebae emerge; but they
believed that this development could only be observed in cats whose
intestines had been previously irritated and inflamed by a special method
of treatment. Their account is, to me, very unconvincing—as also is
their figure, which resembles an artificially ruptured cyst,+ but is
supposed to show the emergence of the small amoebae. Schaudinn
{1903) stated that he had confirmed these observations, and that ‘“ the
results of my infection experiments on myself, and on experimental
animals, agree completely with the findings of the two Italian inves-
tigators.” No other workers, apparently, have ever been able to infect
cats or any other animals with £. coli, or to observe any stages in their
development in animals fed upon the cysts. Quincke and Roos, Darling,
Wenyon, Craig, and many other workers always obtained negative

* This statement may well be questioned. Schaudinn stated that the infection
lasted on each occasion for only 2 months, and then disappeared spontaneously. It is
a remarkable observation, if true: for there can be no doubt that infections persist
usually for many months at least, and even for years,and I have never seen a single
case which has lost an infection with certainty whilst under observation. Apparent loss
is very common—the stools of infected persons often remaining “negative” for long
periods. Cf. Dobell (1917). Schaudinn’s observations appear to me valueless, in the
absence of any adequate control experiments. It is not improbable that he was infected
with £. coli before. during, and after his experiments, but merely failed to find the cysts
at certain examinations.

t Werner (1912) has figured a similar burst cyst—stained—which is supposed to
illustrate the same stage ; but, as he naively remarks, the bursting in this case was “the
result of a trauma.” It is by no means difficult to obtain such burst cysts—especially
with certain fixatives ; but it is difficult to see what connexion they can have with the
development of the amoeba.

ENTAMOEBA COLI gI

results. I do not believe it is possible to infect the cat with £. coli, or to
obtain any stages of development in this host. In the experiments
carried out with Dr. Dale in 1916 (vide Dale and Dobell, 1917) I studied
a number of kittens which had ingested the cysts of this species, but I
was unable to observe any development, and none of the kittens ever
acquired an infection. I found, on several occasions, that some of the
ingested cysts passed unchanged through the kitten’s gut, and were
discharged in its faeces ; but a larger proportion degenerated and died
in transit, and I generally failed to find any forms—either cysts or
amoebae—1in the faeces of kittens which had ingested cysts of E. coli.

Conjugation.—At present there is no evidence of the existence of sexual
phenomena of any sort in the life history of E.coli. The ‘‘autogamy”
described in the cysts by Schaudinn (1903), and “confirmed” by several
later workers, was certainly a mistaken interpretation—as already noted.
“Conjugation”’ phenomena observed by other workers appear to be
equally speculative, and are hardly worth even mentioning.

Mathis and Mercier (19170, d) maintain that the cysts (“ gamogonic
cysts”) of E. coli are of two sorts,—‘‘ macrocysts’’ and ‘‘ microcysts,”
distinguishable by their dimensions. They are supposed to liberate
macrogametes and microgametes respectively. The only concrete evidence
advanced in support of their view is a remarkable curve—based upon
measurements of only 100 cysts—incidentally introduced in one of their
papers (1917 6). The much more extensive series of measurements of
E. coli cysts made by Smith (1918) and Matthews (1919) have shown
conclusively that Mathis and Mercier’s conclusions are not justified.
The dimorphism which they postulate in the cysts of EF. coli does not
exist. My own measurements also show clearly that there is no evidence
of the existence of ‘‘ microcysts”’ and “ macrocysts.”’

Whilst there is thus no direct evidence of the existence of asexual cycle
in E. coli, it is by no means certain that conjugation does not occur. If
it does, then the probability is that it takes place between the young
amoebae recently issued from their cysts. To this extent I agree with
Mathis and Mercier. I originally suggested a similar possibility in the
development of £. ranarum, and Mercier (1910) soon afterwards stated
that he had observed the whole sequence of events in Endamoeba blattae.
His observations supply the only real grounds for supposing that a
sexual cycle occurs in the development of any of the intestinal amoebae
of man or other animals.

Cultivation.—E. coli has not been cultivated by any worker. It is
certain, and will be conceded by everybody acquainted with the subject,
that the “£. coli” cultivated by many of the earlier and some later
investigators (e.g., Fantham, 1911), was in reality some free-living species
and not Entamoeba coli.

Occurrence.—There is now so much information regarding the
occurrence of E. coli, and the facts are so generally recognized, that our
knowledge of the distribution of this organism can be dismissed in a
sentence. £. coli has been found living as a harmless commensal in the
colon of man wherever and whenever it has been sought : no race, nor any
country, has yet been discovered in which infections with this species
are not common.

Treatment.—An E. coli infection cannot be eradicated by any known
method of treatment. Emetine, various intestinal disinfectants, and other
substances which have been tried are all inefficacious. There is a large

Q2 AMOEBAE LIVING IN MAN

body of evidence on this question which it will be unnecessary to discuss
here. See especially Wenyon and O’Connor (1917), Dobell (1916, 1917),
Dobell, Gettings, Jepps, and Stephens (1918), etc.

(3) ENTAMOEBA GINGIVALIS (GROS, 1849) BRUMPT, 1913.

“ Amoebea gengivalis” Gros, 1849.

Amiba buccalis Steinberg, 1862.

Amoeba dentalis Grassi, 1879.

Amoeba kartulisi Doflein, 1901.

Entamoeba buccalis Prowazek, 1904.

Entamoeba maxillaris Kartulis, 1906.

? Amoeba pyogenes Verdun & Bruyant, 1907.

Amoeba gingivalis (Gros) Brumpt, 1910.

Entamoeba kartulisi Doflein, 1911.

? Entamoeba pulmonalis Brumpt, 1913 (uec Artault, 1898).
Endameba buccalis Bass & Johns, 1915.

Endameba gingivalis (Gros) Smith & Barrett, 1915.
Endamoeba gingivalis (Gros) Smith & Barrett, 1915.
Endameba gengivalis (Gros) Lynch, 1915.

Endamoeba gingivalis (buccalis) Craig, 1916.

“ Endamoeba gingivalis (Gros emend. Prowazek) ” Craig, 1916.
? Endamoeba confusa Craig, 1916.

“Endamoeba Gros” Hecker, 1916.

HISTORY AND NOMENCLATURE.

The amoeba of the human mouth is of historic interest because
it is probably the first parasitic amoeba discovered—not only in man, but
in any animal. Like the dysentery amoeba, it was discovered in Russia.
Its discoverer, Gros (1849), found it in the tartar on the internal surface
ofthe teeth. His description is very brief, but his figures are recognizable.
He named the organism ‘“Amoebea gengivalis’’—apparently intended
for Amoeba gingivalis—and suggested that it might be spontaneously
generated in the human mouth.

Some years later Steinberg (1862), also in Russia, found apparently the
same organism, and named it Amiba buccalis.* Grassi (1879 a)subsequently
observed amoebae in the human mouth, and called them Amoeba dentalis,
noting that they were possibly identical with Steinberg’s forms. A few
years afterwards, however, he expressed doubts as to whether the things
which he studied really were amoebae or simply cells (Grassi, 1882,
1883).

noe (1892) and Kartulis (1893) found amoebae in the pus from
maxillary abscesses. Kartulis found them in an abscess of the lower
jaw of an Arab in Egypt. They were described and figured by him, but
not named ; though he threw out the suggestion that they might be the

* The original paper by Steinberg—sometimes cited as Sternberg—I have not been
able toconsult. A translation of the part dealing with the amoeba of the mouth is given
by Smith and Barrett (1915), to whom I am indebted for my information concerning
Steinberg’s observations.

ENTAMOEBA GINGIVALIS 93

same as the ‘‘ Amoba buccalis”’ of Steinberg and the amoebae of Grassi:
and he also hinted that they might possibly be identical with ‘‘ Améba
dysenterica,” which they were said greatly to resemble. The amoebae of
Kartulis were named “ ? Amoeba kartulist” by Doflein (1901), Entamoeba
maxillaris by Kartulis himself (1906), and later Entamoeba kartulisi by
Doflein (1911). I agree with Smith and Barrett* (1915) that these
names are probably synonyms of F£. gingivalis.

Prowazek (1904) redescribed this amoeba, and renamed it “ Entamoeba
buccalis n. sp.”—apparently in ignorance that it had already been
observed and previously named buccalis. He was impressed by its
resemblance to £. coli, and apparently considered it equally harmless.

Brumpt (1910) amended the name of the organism to Amoeba
gingivalis, and later (1913) to Entamoeba gingivalis Gros, which appears
to be the correct name of this species,—as Smith and Barrett (1915) have
already pointed out + in their detailed analysis of the nomenclature. It
is, moreover, the name now generally in use, though some recent workers
still call the organism E. buccalis. Craig (1916), indeed, has used the
curious combination “ Endamoeba gingivalis (buccalis),” though in the
same work he also names the amoeba “ Endamoeba gingivalis Gros 18409,
em. v. Prowazek 1904.” Both these names are clearly incorrect: for the
first is not in accordance with the rules of nomenclature, and the
second attributes to Prowazek a name which he never used.

The amoebae described from an abscess in the malar region by Verdun
and Bruyant (1907, 1907 a), and named by them Amoeba pyogenes, appears
to me to have been E. gingivalis, though Smith and Barrett (1915) regard
it as probably a distinctspecies. I also consider that Endamoeba confusa

Craig (1916) is probably synonymous with E. gingivalis. It is a name
proposed for an oral amoeba not yet properly described. From Craig’s
statements it appears to be closely similar to E. gingivalis, though said to
be smaller. Its chief distinctive character seems to be “the liability
of confusing this species with the smaller examples of E£. gingivalis.”
At present there is no evidence to prove that such a confusion would be
unwarranted. The name “Endamoeba Gros” employed by Hecker
(1916) is evidently applied to E. gingivalis. I take it to be a term
formed on analogy with the peculiar names used in bacteriological
nomenclature. ¢

The earlier workers made very few observations of any value upon
E. gingivalis : and among the more recent workers there are but few who
have supplied really accurate data from the zoological standpoint.
Originally the organism excited but little interest. Indeed, it received
no real notice until Smith and Barrett (1915) and Bass and Johns (1915)
first § advanced the hypothesis that E. gingivalis is the cause of pyorrhoea
alveolaris (Riggs’ Disease); Bass and Johns (1915) maintaining the

* These authors unfortunately give both the names incorrectly, writing “ Exdameba
kartulist Doflein” and ‘‘ maxilaris.”

7 They use the generic names Exdamoeba or Endameba, however, on grounds
of priority ; though they presumably regard them both as interchangeable with
Entamoeba. 1 do not follow Lynch (1915) in using Gros’s (1849) original spelling
of the specific name, which is clearly a misprint (“ gexgivalis” for gingivalis).

~ Such as “ Bacillus Flexner,” “ B. Shiga,” etc.

§ Both these authors published preliminary papers in the preceding year (1914).
Bass and Johns (1915) give the priority in the “ discovery ” to Smith and Barrett.

Q4. AMOEBAE LIVING IN MAN

thesis that ‘“‘the specific cause of pyorrhea dentalis and alveolaris is
endamebas.”’ All these workers went a step further and claimed that
emetine has a specific therapeutic action in ‘oral entamoebiasis ’—
comparable with its action on E. histolytica in amoebic dysentery. These
statements have been the subject of much controversy, and have been
productive of a considerable amount of literature on the medical side.
As they have now been to a great extent disproved, and as the controversy
has little zoological interest, it will be unnecessary to consider it here in
detail. The special points arising from it—in so far as they are concerned
with the amoebae themselves—will be considered later.

On account of the supposed “specific relation” of the organism
to disease, and the ‘‘ specific action” of emetine upon it, some workers
have even been led into discussing whether EF. gingivalis is not really
identical with E. histolytica. Smith and Barrett (1915), for instance,
suggested this possibility; but later (1915a) they abandoned the hypothesis,
and concluded that the two organisms are specifically distinct.* Although
the characters which distinguish £. gingivalis from the intestinal amoebae
of man appear at first sight somewhat indefinite, there can be no doubt
now, I think, that it is an entirely independent species. Its resemblance
to E. coli, noted by Prowazek (1904), or to E. histolytica, as stated by
Smith and Barrett (1915), is not, I think, very striking to anybody
familiar with all these species.

It may be added that the organism called “ Amoeba pulmonalis”’ by
Artault (1898) may possibly have been £. gingivalis: but I consider that
the bodies which he saw were probably not amoebae but cells. Brumpt
(1910, 1913), however, says he has also seen the organism, and gives
some diagrams of it. He calls it Entamoeba pulmonalis (Brumpt, 1913),
and says it may be identical with EL. gingivalis. It seems to me not
improbable that Brumpt’s “ FE. pulmonalis” really was E. gingivalis, and
was wrongly referred to Artault’s species. Consequently, I have taken
this view in enumerating the synonyms of E. gingivalis at the head of
this section. It also seems not unlikely that the ‘“ameba” found by
Lynch (tg15 a) in the lower jaw of an American negress suffering from
‘suppurative and hyperplastic osteoperiostitis,” was really E. gingivalis.
The author considers that it belonged to a new species, but as he has
not studied or described the organisms properly—having accidentally
destroyed his preparations—it is unnecessary to discuss this possibility.
Since Kartulis and Flexner described “amoebae” from maxillary
abscesses, many similar cases have been recorded, and at present there
seems to be no sufficient reason to suppose that the organisms observed
were not EF. gingivalis in every case. Most of the descriptions are too
meagre, however, to prove this conclusively : and I have little doubt
that some at least of the “amoebae” found in suppurative conditions in
and about the mouth have been endothelial or other cells, and not
amoebae of any sort.

Craig (1916) records it as his opinion that “ in all probability, further

* These authors (1915 @) attempted to infect kittens with £. gingivalis by feeding
them on pyorrhoeal pus rich in amoebae, and by injecting the amoebae into the rectum,
or into the colon afterlaparotomy. They also injected amoebae fer rectum into two
puppies. All the experiments had negative results. They concluded that £. gingtvalis
is specifically distinct from Z. Azstolytzca, and that “ intestinal infestment” is “ probably
impossible.”

ENTAMOEBA GINGIVALIS 95

research will demonstrate that still other species of amoebae occur in
the human mouth”; and he adds “it will be strange indeed if both
Endamoeba histolytica and Endamoeba coli, the common intestinal end-
amoebae of man, are not sometimes encountered in this locality.” This
last statement appears to me to be a wild and wholly unwarranted pre-
diction, which, if fulfilled, will indeed be strange. It will, indeed,
be more—it will revolutionize our present conceptions not only of the
amoebae but also of parasites generally. For the moment, however,
there is no evidence whatever to support Craig’s view, and there is very
little in favour of the hypothesis that more than one species of amoeba
inhabits the mouth of man.

DESCRIPTION.

Entamoeba gingivalis is a small amoeba which appears to show great
variation in size. Prowazek (1904) gives its diameter as 6-32 wp;
Chiavaro (1914) gives 5-20 w; Mendel (1916) gives 5-40 «4; Goodey and
Wellings (1916) give 7°5-27 «; Goodrich and Mosely (1916) 10-30 p;
and Nowlin (1917 a) 12-40 w; whilst Smith and Barrett (1915) state that
the usual diameter is 30-35 w, and that organisms may measure even
more—up to 60 w. In my experience this amoeba is usually much
smaller, and its diameter is usually between 10 wand 20 w in fixed and
stained specimens. I have not seen living organisms with a greater
diameter than 25 w, and as a rule they have been considerably less.
My impression is that this species is typically smaller than E£. coli, but
I have studied only a limited amount of material.

The organism when alive resembles £. coli in general appearance,
but is usually more active. Ectoplasm and endoplasm are fairly sharply
differentiated, and the pseudopodia are well-developed, lobose, and
typically rounded—not pointed. Individuals showing their pseudopodia
extended are much more frequently seen in stained preparations of this
species than in those containing £. coli or any of the other intestinal
amoebae of man—all of which tend to become spherical when fixed.
The most striking feature of the living amoeba is its cytoplasm, which
is usually filled with numerous food vacuoles containing peculiar in-
clusions. In this respect it often resembles a well-fed small individual
of E. coli, The nature of these inclusions will be considered later, but
it should be noted here that they have a greenish, refractile appearance,
and are roundish in shape, so that they are not unlike ingested red
corpuscles seen with ill-adjusted illumination. Red corpuscles are,
however, in my experience, invariably absent from the endoplasm.

The nucleus of £. gingivalis is typically spherical and vesicular,
inconspicuous during life, and closely resembles those of E. coli and
E, histolytica in structure. (See Pl. V, figs. 93, 94.) Its diameter in my
stained specimens is generally from 2°5 w to 3 w, and it appears to be
slightly smaller, relatively to the diameter of the organism, than the
nucleus of E. histolytica or E. coli. The size is variously given by
different observers as 2'5-4 w (Goodey and Wellings, 1916), 2-5 w (Smith
and Barrett, 1915), or 3-6 w (Goodrich and Moseley, 1916). There is
probably a very thin achromatic membrane surrounding the whole
nucleus, as in other Entamoebae. As a rule this is invisible, however,
and the chromatin granules which are massed against its inner surface
completely hide it. These granules are, in healthy and well-fixed

96 AMOEBAE LIVING IN MAN

specimens, of uniform size and closely packed together, so that the
nucleus appears as a very definite and even ring in optical section
(figs. 93, 94). The ring is of approximately the same thickness as in
E. coli, but is usually more uniform. The spherical karyosome (figs.
93, 94), which measures about 0°5-0'75 yw in diameter, is sometimes—but
not always—surrounded by a paler area, like the “halo” in £. coli and
E. histolytica. The karyosome itself appears to consist entirely of
chromatin. According to Prowazek (1904) it consists of several separate
granules, but I have not seen such a structure in normal individuals, and
1 believe his statement is incorrect. In position the karyosome may be
either eccentric (fig. 93) or central (fig. 94). In this character the
organism appears to occupy an intermediate position between E. coli
and E. histolytica ; its karyosome being not so constantly eccentric as in
the former, nor so constantly central as in the latter. Between the
karyosome and the peripheral “ring” of chromatin there is a clear
space, which appears to me to be entirely free of chromatin or other
granules in normal well-stained specimens. In this respect E. gingivalis
thus resembles E£. histolytica, and differs from E. coli.

Smith and Barrett (1915) state that a “centriole” is sometimes
present in the karyosome. Prowazek also believed that there is a
centriole—to judge from the statements of Hartmann (1913)—though
according to his earlier interpretation he regarded the whole karyosome
as a “nucleus” within the real nucleus (cf. Hartmann and Prowazek,
1907, p. 316). Smith and Barrett (1915, 1915 a) state that the nucleus
itself is more often central in position than in E, histolytica. The exact
position occupied by the nucleus of an amoeba is, however, difficult to
define : and the “ eccentric’ nucleus of the Entamoebae is a character
upon which too much reliance should not be placed.

The endoplasm of £. gingivalis, as already noted, usually contains
numerous food-vacuoles which enclose peculiar ingested bodies. These
are generally round or oval, and stain very intensely with nuclear stains ;
but if the stain is suitably extracted they are seen to be composed of
granules of variable size. In specimens deeply stained with iron-
haematoxylin they appear black and homogenous, and resemble the
ingested red corpuscles in £. histolytica. That they are not red
corpuscles, however, can easily be demonstrated by suitable staining
and by examination of the living animal. From their staining reactions
the bodies consist mainly of chromatin. According to Goodey and
Wellings (1916) they are the “nuclei of degenerated and disrupted
salivary corpuscles” occurring in the mouth. Goodrich and Moseley
(1916) say that they are the “nuclei of lymphocytes or other mononu-
clear leucocytes.” But Smith and Barrett (1915) appear to believe that
the inclusions are partly leucocyte nuclei and partly red corpuscles.
They believe that red corpuscles are frequently ingested by E. gingivalis ;
but they say that these are soon haemolysed after ingestion, so that they
rapidly disappear. Goodey and Wellings (1916) deny that E£. gingivalis
ever ingests red corpuscles or polymorphonuclear leucocytes, but
Goodrich and Moseley (1916) state that both of these “ have been seen”
within the organisms. Prowazek (1904) merely stated that the food
consists partly of ‘leucocytes,’ but he did not specify the kind.
Nowlin (1917 a) refers to the “‘solid masses” in the cytoplasm, but
never observed E. gingivalis ingesting red corpuscles or leucocytes.
This worker expresses the peculiar opinion that the organism “absorbs

ENTAMOEBA GINGIVALIS G7

its food mainly, taking in by osmosis the fluids of leucocytes or other
media on which it rests.’ It is dithicult to reconcile this view with the
fact that the amoeba is frequently filled with what are obviously the
remains of nuclei of some sort, and also with the same worker’s earlier
statement that the amoeba “can load up with a great cargo of cells and
tartar.” The most astonishing view, however, is that expressed by
Craig (1916), who conjectures—for no apparent reason—that the inclu-
sions in £, gingivalis are “some species of protozoan organism.”

As I have already noted, | have never seen red corpuscles in
E. gingivalis; but | am not prepared to deny that they are ever ingested
by this species, in view of the stateinents of Smith and Barrett, Goodrich
and Moseley, and others. That they are not ingested as frequently as
the former authors believe, however, there seems good reason to suppose.
Most of the inclusions are undoubtedly the remains of nuclei, either of
salivary corpuscles or of other leucocytes or cells. I believe polymor-
phonuclear leucocytes are occasionally ingested. The organism figured
in Pl, V, fig. 93, contains, I believe, the remains of one. Apart from
these nuclear residues, E. gingivalis also ingests bacteria, which are
usually present in large numbers in its vacuoles. These have been seen
and described by Prowazek (1904), Smith and Barrett (1915), Goodey
and Wellings {1916), and most other observers. Nowlin (1917 a) states
that she has observed the living amoebae ingesting bacteria. I have not
been able to watch this process myself, though it must occur frequently
when the animal ts in its normal environment.

E. gingivalis probably reproduces by division into two, but I have
observed no stages in the process. Prowazek (1904) mentions simple
fission as its chief mode of multiplication, and a figure of his, showing
an amoeba with a ‘‘dividing nucleus,” has been several times repro-
duced (cf. Hartmann and Prowazek (1907) p. 316, Hartmann (1913)
p. 641, etc.) and interpreted in various ways. Originally it was said to
show one nucleus dividing inside another ; later, to be a single nucleus
undergoing “‘promitosis.” Stages in a process of mitosis are referred to
by Chiavaro (1914), but not clearly described. A late stage in division is
figured by Goodey and Wellings (1916), but they found no earlier stages.
Goodrich and Moseley (1916) state that reproduction is effected by
“binary fission,” but the process does not appear to have been observed
in detail. Nowlin (1917) records a ‘‘mitosis” in this organism, but
gives highly unconvincing figures. She also believes that “ budding ”
or “multiple fission” takes place, ‘‘ merozoites” being formed to the
number of “8 or 9 to more than a dozen.” From the description and
figures it appears probable that these were cellular elements of some
sort from the mouth, and not amoebae. Other authors mention division,
but no one has yet described it properly.

Cysts of E. gingivalis have been described by several workers. Smith
and Barrett (1915) state that they “ have found ‘dauer’ cysts, but thus
far no reproductive cysts”—whatever that may mean. No descriptions
are given. Craig (1916) describes both “ cysts ” and “ precystic amoebae.”
The former are said to measure 8-10 in diameter, and to be uninucleate.
“Larger cysts are sometimes observed,” but all sizes are said to
be very rare. According to Goodrich and Moseley (1916) Craig’s
cysts probably belonged to “limax” amoebae, which they believe to
occur occasionally in the mouth. Craig truly says of the “cysts”
previously found by Chiavaro (1914) that they are “far from convincing.”

7

98 THE AMOEBAE LIVING IN MAN

Nowlin (1917) also found “cysts” of E. gingivalis. Their size is not
given, but they are said to be smaller than the active amoebae. ‘“ They
usually show some faint, rounded inclusions, probably the remains of
food vacuoles.” (If this is correct, then they differ from all other amoebic
cysts.) The nucleus is not mentioned, and the illustration of the “ cyst”
is very unconvincing. Mendel (1916) maintains a judicial reserve regard-
ing the occurrence of cysts. I have never been able to find any cysts in
the mouths of persons infected with EF, gingivalis. Careful search for
them has also been made by Smith and Barrett (1915), Goodey and
Wellings (1916), and Goodrich and Moseley (1914). None of them
succeeded in finding any, nor did Prowazek (1904). It seems to me very
probable that this organism does not form cysts at all, but is disseminated
in an unencysted condition by simple contact between mouth and
mouth—as Goodrich and Moseley suggest. At all events, all the “cysts ”’
hitherto found have been so imperfectly described—almost every im-
portant cystic character being left out of the descriptions—that it is
impossible to accept any of them, on the evidence so far presented, as
cysts of FE. gingivalis.

Craig (1916) says he has observed in E. gingivalis a process “ which
1 regard as conjugation.” It is not described, however, and the observa-
tion is not confirmed by any other worker ; and at present there is no
evidence of the existence of any sexual development in this species.
Prowazek (1904) had previously thrown out the equally unfounded sugges-
tion that E. gingivalis “ sporulates” in the same way as E. histolytica—as
erroneously described by Schaudinn (1903).

Occurrence and Habitat.—E, gingivalis commonly occurs in the tartar
of the teeth—where it was originally found by Gros (1849)—and also in
the materia alba between and around them. It seems to be specially
common in the Leptothrix masses on the inner surface. The organism is
often abundant in the pus found in pyorrhoeal pockets, and in other
oral suppurations. According to Smith, Middleton, and Barrett (1914)
it also occurs in the crypts of the tonsils, and on the tongue according
to Lynch (1915). I have once found it among the spirochaetes and
fusiform bacteria in the throat of a patient with Vincent’s angina.
Probably the organism may occur in any part of the mouth, though it
seems to be particularly abundant in suppurative conditions. Lynch
(1915) found E. gingivalis on the healthy gums and false teeth of two
old women who had lost all their own teeth, and who showed no signs
of pyorrhoea or other dental disease.

Goodrich and Moseley (1916) have made the interesting observation
that an amoeba “‘indistinguishable” from E. gingivalis occurs in pyo-
rrhoeal pus from the mouths of dogs and cats. It thus appears probable
that the organism is not confined to man.

Pathogenicity—Bass and Johns (1914) and Smith and Barrett (1915)
originally claimed that E. gingivalis is the cause of pyorrhoea. They
found it almost invariably present in this condition, and constantly
absent from healthy mouths. Their observations, however, have not
been confirmed; and almost all the recent investigators who have
devoted careful attention to the matter have concluded that E. gingivalis
is probably a harmless organism.* Observations which have been pub-

* This is the opinion of Goodey and Wellings (1916), Goodrich and Moseley (1916),
and many other workers. It was also the view of Prowazek (1904).

ENTAMOEBA GINGIVALIS 99

lished by Lynch (1915), Mendel (1916), Goodey and Wellings (1916),
Goodrich and Moseley (1916), Williams, Sholly, Rosenberg, and Mann
(1915), Mitchell, Culpepper, and Ayer (1916), and others, show conclu-
sively that E. gingivalis occurs in normal healthy mouths. For example,
Mitchell, Culpepper, and Ayer say: “it is evident that a very large per
cent of normal mouths harbour” this organism. They found amoebae
in the mouths of 21°6 per cent. of children with normal gums; and
Mendel found them in 8 out of 36 children, and in 24 out of 42 adults,
all of whom showed no evidence of pyorrhoea. It seems equally certain,
on the other hand, that E. gingivalis occurs more abundantly in the
mouths of persons with pyorrhoea and unhealthy gums. Thus, Mendel
(r916) found the organism in 38 out of 40 persons with pyorrhoea;
Williams, Sholly, Rosenberg, and Mann (1915) found them in 70 per
cent. of children with ‘spongy, bleeding gums’’; and Mitchell,
Culpepper, and Ayer (1916) found them in 74°4 per cent. of children with
“receding, spongy, bleeding gums.” As E. gingivalis feeds largely upon
bacteria and the nuclei of disintegrated cells in the saliva, it seems not
improbable that a condition such as pyorrhoea—with abundance of
bacteria and broken-down pus cells—is particularly favourable to their
growth. This would easily account for the greater frequency of the
amoebae in suppurative conditions.

E. gingivalis is by no means always present in pyorrhoeal pus, or in
dental abscesses. I have examined at least one case of pyorrhoea, and
the pus from two dental abscesses, with completely negative results after
a very exhaustive search. Similar results have been recorded by others.
The heaviest infection which I have seen was in a man with only
slight pyorrhoea, but with a very dirty and ill-kept mouth. Scrapings
from all parts of the teeth and gums showed large numbers of amoebae,
often accompanied by Trichomonas and immense numbers of spiro-
chaetes. I have also seen E. gingivalis on one occasion in a syphilitic
lesion of the mouth—a mucous patch on the lower lip.

Hecker (1916) attempted to cause pyorrhoea by injecting E. gingivalis
—washed bya special technique, to free the amoebae from bacteria—
into the gums of a guinea-pig and a man. Repeated inoculations did
not succeed in establishing an infection or in causing pyorrhoea.
Prowazek (1904) found E. gingivalis in carious teeth, but the observa-
tions of Chiavaro (1914) and Mendel (1916 a) seem to show that dental
caries is not caused by its presence, as some writers have suggested.

At present, therefore, there seems to be no good evidence to support
the hypotheses that E. gingivalis attacks the tissues, that it is the cause
of pyorrhoea, or that it is in any way pathogenic. The suggestion that
the amoeba acts as a pathogenic agent by means of a “symbiotic rela-
tion” with certain bacteria, as suggested by Smith, Middleton, and
Barrett (1914), seems equally unfounded. The organism appears rather to
be a harmless commensal, like E. coli. Chiavaro (1914) considers that it
is ‘most probably an adjuvant in the autodisinfection of the mouth ate:
whilst Goodey and Wellings (1916) suggest that, since E. gingivalis is a
“scavenger ’”’ of bacteria, it ‘‘ may therefore be considered as a useful
rather than a harmful organism.” The available evidence, however,
appears to me to afford but little ground for regarding it as either harm-
ful or beneficial.

Treatment.—As already noted, Bass and Johns (1914, 1915), Smith,
Middleton, and Barrett (1914), Lynch (1915), and others, claimed to

roo THE AMOEBAE LIVING IN MAN

have found that emetine has a specific action upon E. gingivalis, and
they therefore advocated the administration of this drug in pyorrhoea.
It will be sufficient to note here that these claims have never been sub-
stantiated, and that many workers have now found that emetine is not a
specific cure for pyorrhoea or amoebic infections of the mouth.
Goodrich and Moseley (1916), Mendel (1916), and others, have failed to
observe any effects produced upon E. gingivalis by giving emetine to its
host. Lynch (1915), who apparently believes in emetine as a specific for
“ oral endamebiasis,” cites a number of cases in which it appears to have
been useless. It thus seems very probable that emetine has no specific
action upon E. gingivalis, and that the original claims were based upon
insufficient evidence. No other substance has, up to the present, been
regarded as a specific for infections with this amoeba.

Io!

VI.

GENUS ENDOLIMAX KUENEN & SWELLENGREBEL, 1917.

THERE is only one species belonging to this genus, namely :
ENDOLIMAX NANA (WENYON & O’CONNOR, 1917) BRUG, 1918.

? Entamoeba phagocytoides Gauducheau, 1908 (pro parte).

Chlamydophrys stercorea Elmassian, 1909 (nec Cienkowski, 1876).

? Entamoeba nipponica Koidzumi, 1909 (pro parte).

“Small amoeba” Wenyon, 1912.

? Vahlkampfia punctata (Dangeard) Chatton & Lalung-Bonnaire,
1912 (pro parte).

Entamoeba coli Werner, 1912 (pro parte).

? Vahlkampfia Whitmore, 1913.

Vahlkampfia Craig, 1913.

Entamoeba coli Akashi, 1913 (pro parte).

‘‘ Free-living amoebae ”’ James, 1914 (pro parte).

Tetramitus mesnili Wenyon, 1915 (pro parte).

Amoeba limax Wenyon, 1916 (nec Dujardin, 1841).

Vahlkampfa Flu, 1916 (pro parte).

“ Non-pathogenic E. tetragena” Shimura, 1916 (pro parte).

Entamoeba nana Wenyon & O’Connor, 1917.

“ Limax”’ Swellengrebel & Mangkoe Winoto, 1917.

Entamoeba nana (Wenyon & O’Connor) Dobell & Jepps, 1917.

Endolimax intestinalis Kuenen & Swellengrebel, 1917.

Vahlkampfia nana (Wenyon & O’Connor) Brug, 1917.

‘* Limax amoeben” Flu, 1918.

HISTORY AND NOMENCLATURE.

Endolimax nana is so common an inhabitant of the human bowel
that it is remarkable that it escaped recognition for so long. This was
partly due, I believe, to its confusion with other amoebae—especially the
small coprozoic and easily cultivable organisms commonly, but incor-
rectly, called ‘‘ Amoeba limax.”

Gauducheau (1908) described a small amoeba which he named
Entamoeba phagocytoides and which he said lived ‘‘in the intestine of
man ”—a statement since reiterated many times. He believed he had
been able to cultivate this species, but the forms in his cultures were
clearly free-living amoebae. It seems possible, therefore, that he culti-
vated these from stools containing E. nana, and wrongly assumed the
two organisms to be identical. His ‘‘ £. phagocytoides” was probably, at
all events, a mixture of different organisms: for it was found in fresh
faeces, and isolated in cultures from stools, from liver abscess pus, and
from water. It has been regarded by Gauducheau as having a genetic

102 THE AMOEBAE LIVING IN MAN

connexion with E. histolytica and Trichomonas, and has had other
~remarkable and incredible characters attributed to it at different times.

Elmassian (1909) found some small amoebae in human stools and
regarded them as naked forms of Chlamydophrys stercorea, which
Schaudinn (1903) had stated to occur in human faeces. The figures
(Elmassian, 1909, figs. 39, 40) apparently depict E. nana. They pro-
bably do not represent the rhizopod which Cienkowski (1876) called
Chlamydophrys, and which J have never seen in human stools.

It seems to me probable that several of the Japanese workers have
also seen E. nana, but misinterpreted it. The “merozoites” of ‘ Ent-
amoeba nipponica” described by Koidzumi (1909), the “‘ young amoebae ”’
formed by the ‘‘schizogony” of £. coli, described and figured by
Akashi (1913), and similar forms of a “non-pathogenic tetragenous
amoeba” figured by Shimura (1916, 1918), probably or possibly all
depict E. nana. Wenyon (1912, 1913) undoubtedly saw E. nana and
recognized it as a distinct species (cf. Dobell and Jepps, 1917). He
figured a cyst later (Wenyon, 1915), but considered that it might belong
to the flagellate Chilomastix (‘ Tetramitus”) mesnili. He found the
amoebae again in patients from Gallipoli (Wenyon, 1916), and called
them “Amoeba limax”; but later, in a joint work (Wenyon and
O’Connor, 1917), named the species Entamoeba nana.

As already pointed out elsewhere (Dobell and Jepps, 1917), the “‘free-
living amoebae from the human intestine” described by James (1914),
in Panama, were probably for the most part E. nana. This author states
that he saw preparations of Wenyon’s amoebae, and that they were the
same as hisown. This is probably correct; for I have also seen Wenyon’s
original preparations, and they certainly contain E. nana. But James
says further that his amoebae were the same as those called “ Vahl-
kampfia punctata Dangeard”’ by Chatton and Lalung-Bonnaire (1912).
Now these authors believed that they had found a “limax’’ amoeba
living in the intestine, and had succeeded in cultivating it. From their
account it seems certain that they really did cultivate “ Amoeba punctata”’
—which is Dimastigamoeba gruberi (Schardinger, 1899) Alexeieff, 1912},
a common free-living form which I have also cultivated from human
faeces, soil, and water. It has been described under many other names,
This organism, however, does not live in the intestine; and moreover it
will not usually grow in cultures kept at the temperature of the human
body. It appears highly probable, therefore, that Chatton and Lalung-
Bonnaire were mistaken in supposing that the amoebae present in the
intestine of their patient were the same as those in their cultures. What
the intestinal forms really were it is impossible to tell from their account.
They may have been E. nana, but they may also have been J. biitschlii.
If James saw preparations of their cultivated form (“ A. punctata’”’ =D.
gruberi), then he was also mistaken in supposing them to be identical
with the forms which he himself had found (E. nana).

E. nana also appears to be the form that Craig (1913 6) saw in James’s
preparations, and which he says was “a typical Vahlkampfia” : anda
similar form may have been seen by Whitmore (1913), who mentions and
depicts a “vegetative form of Vahlkampfa” from a human stool. Flu
(1916) also found a “ Vahlkampfia” which was said to live partly free and
partly in the intestine. Later (Flu, 1918) he appears to have come to
the conclusion that this was E. nana ; but he considers that this species is
merely a harmless ‘‘limax amoeba adapted to a parasitic mode of life.”

ENDOLIMAX NANA 103

Werner (1912) apparently observed the cysts of E. nana but con-
sidered them to belong to E.coli. At all events, his crude figures (PI. I,
figs. 19, 20, 23, 29, 30) are far more like cysts of E. nana than those of
E. coli, to which they are attributed.

E. nana was described as a separate species by Swellengrebel and
Mangkoe Winoto (1917) under the peculiar name “limax” : and later in
the same year Kuenen and Swellengrebel (1917) described apparently the
same form again, naming it Endolimax intestinalis. Brug (1917) pointed
out that the organism described as Entamoeba nana by Wenyon and
O’Connor (1917) could hardly be placed in the genus Entamoeba—as
was, indeed, obvious: but his proposal to place it in the genus
Vahlkampfia instead—as Vahlkampfia nana—was even more open to
objection. No matter what interpretation is put upon this genus—and in
my opinion the name Vahlkampfia should not be used for any organism
—it is clear that E. nana has nothing whatever to do with any of the
free-living amoebae for which it was intended. On the appearance of
Kuenen and Swellengrebel’s (1917) work, Brug (1918) corrected his
earlier opinion; and as the work of Wenyon and O’Connor appeared
before that of Kuenen and Swellengrebel, he pointed out that the specific
name (ana) proposed by the former must stand, though the generic name
(Endolimaxz) of the latter should be used instead of Entamoeba for the
organism in question. Its correct name would thus be Endolimax nana.

This organism can hardly be placed in the genus Entamoeba, on
account of the peculiarities of its nuclear structure and its cysts. On the
other hand, there was—prior to the introduction of the name Endolimax
—apparently no genus to receive it. Although I regard this generic
name as inappropriate, in that it implies * a resemblance of this form to
“« Amoeba limax,” | believe Brug’s amendment must be accepted, as it is
in accordance with the rules of nomenclature.

I would here call attention, however, to the apparent similarity of
E. nana to a curious organism discovered by Minchin (1910) in the
malpighian tubules of fleas (Ceratophyllus fasciatus), and named by him
Malpighiella refringeus. The systematic position of this parasite is still
uncertain, though it is described as “amoeboid”: and it forms 4-nucleate
cysts with a structure apparently very closely similar to those of E. nana.
Similar organisms have since been recorded by Alexeieff (1913) from the
vagina of a leech (Hirudo medicinalis), and by Ndéller (1914) from dog-
fleas and rat-fleas. Neither of these authors, however, has ascertained
definitely whether Malpighiella is an amoeba or not. The former thinks
it is, the latter that itis not. If it really is an amoeba, then it seems not
improbable that E. nana may have to be placed eventually in the genus
Malpighiella, from which it seems at present to differ in no characters of
generic magnitude. Unfortunately I have not yet been able to investigate
Malpighiella with a view to deciding this question.

Attention may also be directed to an amoeba described from the
faeces of frogs by Epstein and Ilovaiski (1914), and named by them
Naegleria ranarum. The organism is said to be “ semi-parasitic,” and it
certainly cannot be placed with propriety in the genus “ Naegleria.” The
cysts of this species appear to be very like those of E. nana in certain

* This, at all events, appears to have been the authors’ intention : though it may be
remarked that Zimax is the name proper to a slug, and therefore Endolimax would be
a more suitable name for a parasitic mollusc than for a protozoon.

104 THE AMOEBAE LIVING IN MAN

respects, and it is possible that the organism may eventually be found to:
belong to the same genus.

DESCRIPTION.

Endolimax nana has already been described more or less completely
by Wenyon and O’Connor (1917), Swellengrebel and Mangkoe Winoto
(1917), and Kuenen and Swellengrebel (1917). I have also given a
brief account of it in an earlier joint paper (Dobell and Jepps, 1917), and
shall therefore now merely note the points of chief importance.

E. nana 1s a small amoeba which usually measures, when rounded,
from 6 4 to 12 w in diameter. Living amoebae average about 8 pz, but
fixed and stained specimens are generally about 1 yw less in diameter.
The living forms somewhat resemble small free-living amoebae (so-called
“imax” amoebae) at first sight, but this resemblance vanishes on closer
study. They possess no contractile vacuoles, their nuclei are not clearly
visible when alive, and their movements rapidly cease outside the body.
Such movements as are usually observable under the microscope are like
those of a small individual of E. coli—sluggish creeping, with few blunt
pseudopodia showing a variable degree of distinctness between endoplasm
and ectoplasm, soon followed by mere change of shape without locomo-
tion, and ending in cessation of all movements. The cytoplasm usually
contains many food-vacuoles filled with micro-organisms.

The most characteristic feature of the free amoeba is its nucleus,
which can only be studied satisfactorily in well fixed and stained
specimens from very freshly passed stools, and under high powers of
the microscope. The nucleus is vesicular, and measures from about
1 w to 3 w in diameter, according to the size of the individual. As a
rule its diameter lies between 2 yw and 2°5 w. (See PI. I, fig. 7, and
Pl. II, figs. 18-23.) There is a peculiar karyosome in the nucleus of this
organism, distinguished by the great diversity of form which it displays.
It contains most of the nuclear chromatin and consists usually of one
fairly distinct mass connected by threads or processes with one or more
smaller masses. The main mass of the karyosome is usually eccentric
in position, and consequently the organism often appears, when deeply
stained or when seen under a comparatively low magnification, to con-
tain a nucleus with a single rather small and eccentric karyosome-
Figures of some of the commoner forms of nucleus are shown in
Pl. I, fig. 7, and Pl. II, figs. 18-23 ; and fig. 24 (Pl. II) shows 8 other
nuclei from other individuals. These drawings will convey a clearer
idea of the nuclear peculiarities of this species than pages of description.
It should be added that very careful study of the nuclei of these
organisms—in really good preparations, and with the best lenses and
critical illumination—shows that hardly any two individuals present
precisely the same nuclear appearance. Several distinct types of nucleus
can be distinguished—such as those represented by figs. 18, 19, 22, etc.—
but there are many variants and intermediates, and the appearances
differ, of course, according to the orientation of the nuclei in relation to
the observer.

Apart from its karyosome the nucleus of E. nana presents no remark-
able features. It possesses a well-marked nuclear membrane in which
minute granules—possibly of chromatin—can sometimes be seen (figs.
18 21, etc.); and between the membrane and the karyosome there is

ENDOLIMAX NANA 105

the usual “clear zone,” sometimes traversed by radiating strands of
‘“linin” (fig. 21). I have not been able to convince myself of the
existence of any “ peripheral chromatin” in the clear zone.

The nuclear appearances just described are those characteristic of
this organism immediately after its discharge from the human body.
If the amoebae are kept for some time subsequently, so that many of
them degenerate and die, they show a considerable difference in nuclear
structure. The karyosome segments run together into a more or less
homogeneous mass, which then generally comes in contact with the
nuclear membrane at one pole of the nucleus. The latter then has the
appearance of a signet ring—the karyosome representing the signet. Such
amoebae are certainly abnormal, as anybody can convince himself by
examining a good series of stained preparations made from a stool con-
taining large numbers of amoebae and fixed at different intervals of time
after it was passed. It will then be found that it is only in the freshest
samples that the true nuclear structure of E. nana can be seen.

The typically irregular forms of karyosome in this species were noted
by Wenyon and O’Connor (1917). Swellengrebel and Mangkoe Winoto
(1917) and Kuenen and Swellengrebel (1917), however, appear to regard
a spherical karyosome as the normal form—apparently because they did
not study sufficiently fresh material. Had they done so, they could
hardly have considered—as they do, apparently—that E. nana is a kind
of “limax amoeba.” From all such organisms it is readily distinguish-
able by its karyosome alone. James (1914) shows an organism, which I
believe to be E. nana, containing a nucleus with an irregular karyosome
(see his fig. 117, Pl. xv). He probably saw the usual forms of the karyo-
some in this species, but mistook them for stages in division: for he says
(James, 1914, p. 198) he saw ‘many organisms in various stages of
division” in his preparations. But even in the freshest preparations
division stages are excessively rare; and unfortunately he does not
describe or figure his dividing forms.

Endolimax nana doubtless multiplies, like other amoebae, by division
into two. I have not yet succeeded in finding more than a very few
organisms which may show stages in nuclear division, and I am
therefore unable to describe the process of fission at present. I would
add that the various forms of karyosome encountered in individuals of
this species appear to bear no relation to nuclear division : and although
the different forms can be easily arranged in series with one another,
there is at present no possible means of ascertaining whether such a
series corresponds with a serial or cyclical change which takes place
in the nucleus of one and the same organism during its life.

I have not succeeded in cultivating E. nana in any medium,—nor
has any other worker, so far as I can ascertain. I may note, however,
that I have cultivated free-living amoebae (‘ limax amoebae’’) from
stools which originally contained FE. nana, and | believe that other
observers (such as Gauducheau, Noc, and Lesage) may have done the
same : which may partly account for their belief that they had culti-
vated amoebae which normally live in man. ;

The habitat of E. nana is the human intestine, but the exact site of
infection in the bowel is still in doubt. There are some reasons for
believing that it may live in the small intestine (cf. Dobell and Jepps,
1917), but this has still to be verified. That it lives in the contents
of the intestine, and is not a tissue parasite, is, however, certain.

106 THE AMOEBAE LIVING IN MAN

I would note here that Endolimax nana is sometimes parasitized by
a micro-organism belonging to the genus Sphaerita Dangeard. The
infected amoebae, when alive, are very conspicuous objects, containing
one or more spherical morulae of brightly refringent spores of the
parasite. The spores are very symmetrically disposed, and at first
sight resemble a mass of ingested micrococci enclosed in a food
vacuole. Further study of such organisms, however, will show earlier
stages in the development of the Sphaerita (cf. fig. 88, Pl. V), and dis-
closes their true character. The spores stain deeply with iron-haema-
toxylin or haemalum (cf. figs. 87, 88), and on being carefully decolorized
show very little internal structure (fig. 89). They measure about 0.75
in diameter, and are consequently very difficult to study. Other stages
are correspondingly minute, and I shall not describe them here. The
parasite lives in the cytoplasm, and does not attack the nucleus (like the
closely related form Nucleophaga), though the nuclei of many parasitized
amoebae appear more or less degenerate. I have not seen Sphaerita
within the cysts of E. wana—only in the free amoebae.

A closely similar form has been described in ‘‘ Aimoeba limax” by
Chatton and Brodsky (1909), to whose work the reader is referred for
further details (and literature) concerning these curious parasites of
Protozoa. They have been described in several different free-living
rhizopods and flagellates, but so far as I am aware have never pre-
viously been recorded in any parasitic amoebae. A related parasite—a
Nucleophaga—has, however, been described in the nucleus of Endamoeba
blattae by Mercier (1910).

I have now seen several E. nana infections in which a considerable
proportion of the amoebae were infected with Sfhaerita. The one
which I was able to follow for the longest time was under observation
for about three months, and I always found individuals parasitized by
Sphaerita when free forms of F. mana were present in the stools. It
thus appears probable that the infection is persistent. It seems, never-
theless, to have little effect upon the amoebae as a whole, for most of
the Sphaerita infections which | have studied were in persons heavily
infected with E. nana, who usually passed large numbers of normal
cysts in their faeces.

I mention this parasite of E. mana here because of its interest and
because the infected amoebae have—to my knowledge—already puzzled
several people who have seen them. One worker who encountered
them mistook the parasitized individuals for a new species of amoeba—
the spore morula of Sphaerita being mistaken for a nucleus, whilst the
nucleus of the host was overlooked. Other workers have mistaken
E. nana individuals, with deeply-stained sporangia, for Dientamoeba
and for cells containing masses of micrococci. Sphaerila has such a
characteristic appearance in living amoebae that it cannot easily be
overlooked, nor can its spores be taken for “nuclei” by a careful
observer.

Cysts—The cysts of E. nana are very characteristic structures, and
contain, when mature, four nuclei. Precystic amoebae contain no food
vacuoles, and their cytoplasm is consequently very clear. They are not
distinctly smaller than ordinary active forms. Such individuals assume
a rounded or oval form, and then secrete a thin cyst wall, which is colour-
less and perfectly smooth—as in most other intestinal amoebae. When
newly formed, the cyst contains a single nucleus (fig. 25, Pl. II), anda

ENDOLIMAX NANA 107

variable number of very small refractile granules. These are more
clearly seen in the living cyst than in stained specimens. They give
some of the metachromatic staining reactions of volutin, and are
probably composed of this substance. They probably do not consist
of chromatin, and are therefore not comparable with the chromatoid
bodies in the cysts of Entamoebae and many free-living species. In
uninucleate cysts the nucleus is usually relatively large (up to about 3 yp),
and its karyosome relatively small, often having the form of a larger
eccentric mass of chromatin united by a fine thread with a much
smaller granule (cf. fig. 25, Pl. I1)—a type of nucleus specially noted by
Wenyon and O’Connor (1917). The nucleus later divides into two (fig.
26), and each of these again divides into two (fig. 27). Except for their
progressive reduction in size, the resting nuclei in the uninucleate,
binucleate, and quadrinucleate cysts show no change of structure. The
arrangement of the chromatin in them is very hard to study accurately,
on account of their very small size, but they show no conspicuous
differences from the nuclear types observable in the active organisms.

Mature living cysts of E. nana are typically oval, and measure 8-10 «
in length and 7-8 win width. In fixed and stained specimens they appear
slightly smaller. The nuclei in the mature cysts (stained) have a diameter
of about 1 wz to 1:3. (Cf. fig. 8, Pl. I, and figs. 27-29, Pl. II.) Except
for the volutin granules already noted, the mature cysts typically con-
tain no visible contents besides their four characteristic nuclei. The
latter often lie near together towards one end of the cyst, but they may
occupy any positions in relation to one another. In iodine solution the
cysts stain a uniform yellow colour, and their nuclei are, as a rule,
inconspicuous; though their karyosomes can generally be made out,
with a little care, in this medium, and sometimes their nuclear membranes
as well.

If a stool containing a large number of encysting amoebaeand newly-
formed cysts is examined in iodine solution, it will generally be found that
many of the former show diffusely stained brown patches in their
- cytoplasm : and among the encysted forms there is usually but little
difficulty in finding some which show definite contained masses giving a
typical glycogen reaction. These glycogen masses are usually particularly
prominent in binucleate cysts—as in E. coli; but they are also found
sometimes in quadrinucleate and uninucleate cysts. They give other
characteristic reactions of this substance, and show typical staining with
Best’s specific carmine method (see fig. 9, PI. 1). Glycogen cannotalways
be demonstrated in the cysts of E. nana, and it is usually commoner in
the cysts contained in soft and diarrhoeic stools—in which free and
encysting forms and young cysts are numerous—than in those found in
formed and solid stools—which contain a large proportion of mature
cysts and no free amoebae. Mature cysts of E. nana remain unchanged
in human faeces for several weeks, if they are not desiccated. But the
glycogen, if present originally in them, disappears completely— as noted
already by Swellengrebel and Mangkoe Winoto (1917).

Several variations in the cysts of E. nana require further notice.
Their form may range from the typical oval to that of a sphere. Very
rarely they are of irregular shape, showing slight constrictions or bulgings
which give rise to a variety of different forms. Their size, too, varies.
Very small cysts, down to 6 w in mean diameter, and very large ones, up

‘II w or slightly more, may occasionally be met with (cf. figs. 30 and

108 THE AMOEBAE LIVING IN MAN

31, Pl. II). And it is possible—and perhaps probable—that there are
difierent strains of this species which can, as in E. histolytica and E. coli,
be distinguished by the dimensions of their cysts.

As a rule the mature cysts of £. xana contain only four nuclei. But
cysts containing eight nuclei are to be found (fig. 86, Pl. V), though these
are very rare. Up to the present I have not seen more than a dozen
such cysts among very many thousands examined. Swellengrebel and
Mangkoe Winoto (1917) say that they have seen cysts containing five and
six nuclei, but I cannot confirm their observation, and from their figures
I doubt its accuracy. Cysts showing at first sight more than four
nuclei are often met with: but careful study of these shows generally
that the supernumerary “nuclei” are really deeply-stained “ volutin”
granules, or that a single nucleus with a bipartite karyosome has been
counted as two nuclei. The same authors hint ata possible “ autogamy ”
within the cysts of E. nana, and at other nuclear phenomena. It is
certain, however, that such suggestions are unjustifiable, and that
development in the straightforward manner described above is the rule.
In all the thousands upon thousands of cysts of this organism that I
have examined from hundreds of infections, there is not the slightest
indication of any nuclear phenomena beyond those which I have
described.

_ Occasionally the cysts of FE. nana contain peculiar inclusions
resembling rods or granules. They are sometimes long and filamentar,
and sometimes in the form of definite bundles of short rods or heaps of
coccus-like granules. Two cysts with such inclusions are shown in figs.
28 and 29, Pl. Il. Those with filaments in them can be mistaken for
small cysts of lamblia (Giardia intestinalis). The inclusions are visible
in the living cysts, and are well seen in those stained with iron-
haematoxylin ; but they are not easily distinguishable in cysts stained by
most other methods. Such cysts appear to be characteristic of the
infections in certain individuals. At all events, Ihave found that when
they are present in the stool of a given individual on one occasion, they
can be found again in his stools subsequently for a period of at least
several months. What these inclusions are I am unable to state. From
their forms one might suppose that they are parasitic or symbiotic
bacteria; but they may possibly be structures comparable with the
chromatoid bodies of £. histolytica and E. coli.

Occurrence.—E. nana is one of the commonest inhabitants of the
human bowel. Since the appearance of the work of Wenyon and
O'Connor it has been found by every competent worker who has made
a protozoological study of human stools. It occurs in the faeces of
persons who have never left the British Isles, and is plentiful among
British troops invalided to England from the tropics and from France.
There are now numerous records available showing its frequency (see,
for example, Wenyon and O’Connor (1917), Dobell and Jepps (1917),
Mackinnon (1918), etc.). The cysts of this organism are small, and
easily overlooked. Consequently, the incidence of infection is higher
than would appear from most published records. A series of British
soldiers—156 cases, consisting entirely of dysenterics from abroad, and
all infected with E. histolytica—which I examined (in conjunction with
Miss Jepps) with great care, in order to determine the exact incidence of
infection with this organism, showed that it was present in no less than
33°3 per cent. Every case was examined at least seven times, and many

ENDOLIMAX NANA 109

of them oftener. (Vide Dobeil, Gettings, Jepps, and Stephens, 1918.)
Other series have shown comparable figures,—allowance being made for
the degree of thoroughness with which the examinations have been
carried out. There can thus be no doubt that E. nana is of frequent
occurrence in man.

Pathogenicity.—Although most of the infections with £. nana hitherto
recorded have been found in persons who have had intestinal ailments,
there is no reason to suppose that the organism is pathogenic. Wenyon
and O’Connor (1917) found it in the stools of healthy men, with no
history of dysentery or bowel trouble, and I can confirm their observations
in this respect. From such figures as are available | can find no appreci-
able difference in the frequency of infection with this amoeba in healthy
people and in those with intestinal disorders. Moreover, E. nana appears,
in its habits of life, to resemble £. coli. It feeds chiefly upon bacteria in
the contents of the gut, and there is no evidence that it can injure the
tissues. It seems to me certain, therefore, that E. wana is a harmless
commensal, and nota pathogenic parasite.

Treatment.—I may ada here that no treatment has yet been found
which will remove an infection with E. nana. Emetine administered to
an infected individual—either hypodermically or per os—never removes
the organism permanently, though it may disappear temporarily from the
stools during treatment. I have now studied a large number of cases who
have received emetine treatment, and have seen no exception to the
generalrule. No other substances—such as various intestinal antiseptics,
etc.—which have hitherto been tried, have any action upon the organism
in the human body.

TIO

VII.
GENUS IODAMOEBA NOV. GEN.

Up to the present only a single species belonging to this new genus
is known. This is the form which I shall call:

JODAMOEBA BUTSCHLII PROWAZEK, 1912 (EMEND.).

Entamoeba biitschlii Prowazek, 1912.

? Entamoeba tetragena Hartmann, 1912 (pro parte).

? Entamoeba coli Werner, 1912 (pro parte).

? Vahlkampfia sp. Chatton & Lalung-Bonnaire, 1912 (pro parte).
? “ Free-living amoebae” James, 1914 (pro parte).

‘‘ Spherical bodies,’’ Wenyon, 1915.

‘* Todine cysts ”’ or ‘‘ I. cysts,” Wenyon, 1916.

«1. cysts,’ Wenyon & O’Connor, 1917.

“ Joodcysten,” Brug, 1917.

‘“* Pseudolimax,” Kuenen & Swellengrebel, 1917.
Entamoeba tetragena Flu, 1918 (pro parte).

Endolimax Williamsi Brug, 1919 (nec Prowazek, 1911).

HISTORY AND NOMENCLATURE.

The first recognizable account of this amoeba seems to me to be
that of Prowazek (1912 a), who named it Entamoeba biitschlii—at the
same time noting that “the designation Entamoeba is provisional,
since we do not know the life-cycle.” His description is very imperfect.
He saw but a single infection, in a child from the Caroline Islands in
Saipan (Ladrones). The case was also infected with £. coli and other
organisms.

Prowazek states that his amoebae measure 10-24 w (presumably in
diameter, when rounded), and that their nuclei are vesicular, with a
round central karyosome containing acentriole. Between the karyosome
and the membrane there is a network with chromatin granules dis-
tributed on it. ‘Cyclical processes” are observable around the karyo-
some, and stages in ‘‘nuclear division” are described. ‘ Division”
stages with two and three nuclei are figured. The cyst is also figured,
but not described. It is merely said to be round, with a distinct
membrane, and entirely different from that of EF. coli. The specimen
figured is said to have measured 14°8 yw. It is uninucleate, with its
protoplasm apparently shrivelled.

Hartmann (1912) has figured an organism which he calls a “ young
amoeba” of Entamoeba tetragena (his fig. 2 a, b), and two other
organisms—described as “degenerate forms” of the same species (his
figs. 15, 16)—which appear to me to be probably the form under dis-
cussion. They certainly do not look like E. histolytica, at all events.

IODAMOEBA BUTSCHLII Ilt

In the same publication Werner (1912) figures three structures which he
calls “‘uninucleate cysts of E. coli with vacuoles” (see his figs. 16, 17,
22): and these also appear to me to have been drawn from the present
species. But the figures are extremely bad, and at all events quite unlike
the cysts of E. coli.

It seems to me possible that the ‘free-living amoebae’? which
Chatton and Lalung-Bonnaire (1912) found in the human intestine, and
identified as Vahlkampfia punctata Dangeard, were really I. biitschlii.
As noted already—in considering E, nana—it is highly probable that
they were not ‘ V. punctata,” which these workers cultivated from the
same stool. Their description of the intestinal forms is too meagre,
however, for me to identify them with certainty ; but to judge from the
figures they were certainly not unlike the form under consideration. The
same remarks are applicable to some of the “ free-living amoebae from
the human intestinal tract” noticed by James (1914) in Panama. His
figures 114-116 are certainly suggestive of I. biifschlii rather than E.nana,
to which species the rest of his “ limax” amoebae should probably be
referred.

Wenyon (1915 a) briefly described and figured some “spherical
bodies,” containing an “iodophilic” inclusion, found in human faeces.
Somewhat later (Wenyon, 1916) he redescribed them under the name of
“Todine cysts” or ‘‘ I. cysts.” A fuller account, with some further facts.
concerning them, was published by Wenyon and O’Connor (1917), who
gave them the same name. Wenyon (1915 a) regarded these bodies as
“‘ probably of a vegetable* nature,” and stated later (1916) that this “is
proved by the fact that they germinate when kept in faeces.” Dr.
Wenyon called my attention to these bodies in 1915, and since then
I have studied them in stools from many different persons. At first I
agreed with him that they were probably vegetable organisms, but I now
know that they are really the cysts of the amoeba described in this.
section. They have become familiar—since Wenyon’s account—to
most workers in England under the name of “I. cysts,” or “1. bodies.”
Brug (1917), in Java, redescribed them as “ Joodcysten,” and regarded
them as probably “a new sort of parasite’? altogether—what sort, he
did not suggest.

Later, Kuenen and Swellengrebel (1917) found both the “I. cysts”
and the amoebae which form them in a single case, and provisionally
named the organism ‘ Pseudolimax’”—a name which they fortunately
state to be not subject to the laws of nomenclature. They made no
reference to the work of others on the same form, and apparently regarded
it as a kind of “ limax amoeba ”’—which it certainly is not. Brug (1918)
subsequently pointed out that their ‘ pseudolimax” was the organism of
the ‘I. cyst.” Another Dutch worker, Flu (1918), has since found the
“T,. cysts’? once more, and has concluded that they are degenerate
cysts of E. histolytica—or, as he terms it, “E. tetragena.” This is
undoubtedly incorrect.

I first saw the living amoebaet which form the “I. cysts,” I believe,

* Matthews (1918) also states that “ they probably represent some stage in the life-

history of a vegetable organism.’

_ + 1 may say that all my own work on this organism has been done in entire
ignorance of the simultaneous investigations of the Dutch workers, whose papers I was
not acquainted with until my own work was completed.

112 THE AMOEBAE LIVING IN MAN

in a preparation which Col. Wenyon showed me in the summer of 1917.
At that time, however, their connexion with the “1. cysts” was uncertain,
though it was one of several possibilities which we discussed. So far as
I am aware, Col. Wenyon did not pursue the investigation of these forms
further. I continued to look for the amoebae again, however, in other
cases; and I saw them once more in another case which I studied at
the end of 1917, and in yet another at the beginning of 1918: but |
was unable to prove conclusively that the “I. cysts”” and amoebae were
genetically connected, and on both occasions the amoebae were mosily
dead and degenerate. During 1918 I studied several further cases of
infection, but with little better success until the autumn. When my
assistant, Miss M. W. Jepps, who had learnt of the ‘‘I. cysts” and
amoebae from me, left me in the spring, and went to Southampton, |
suggested that she should continue to study cases of “I. cyst infection ”
with a view to clearing up the matter. This she did, and in the late
autumn I received from her a preparation containing numerous
“T. cysts’? and amoebae, which she had observed alive, from a case
which she was studying. A few days later I was fortunately able to
study the living amoebae, the cysts, and all intermediate stages, in great
detail, in another case which Major G. C. Low kindly allowed me to
examine. After a very careful study of Miss Jepps’s preparation and
_ those which I had myself made from Major Low’s case, I felt convinced
that the ‘I. cysts”’ and the peculiar amoebae are stages in the develop-
ment of the same organism. Since then I have encountered the
amoebae and cysts again in other cases, and Capt. F. W. O’Connor has
very kindly allowed me to study further specimens containing both
amoebae and cysts of this species from cases which he investigated in
Egypt in 1917. I have now no doubts as to the general correctness of
the description given below. There is, I think, no reason to doubt that
the “I. cyst” —in spite of its remarkable structure—is really the cyst of
an amoeba; and that this amoeba is, moreover, the organism which
Prowazek (1912 a) imperfectly described, and named Entamoeba biitschlit.

Brug (1919), in a paper just published, has returned to the study of
this organism and identified it with Prowazek’s “ Entamoeba Williamsi”’
—which was really, as has already been pointed out, £. coli, Brug
considers that the organism should be referred to the genus Endolima-.
The type of this genus is, however, E. nama—an organism whose cysts
and nuclear structure are entirely different. His conclusion that the “I.
cysts are the cysts of Entamoeba Williamsi, Prowasek (sic). The latter
should be called: Endolimax Williamsi”, is one in which no proto-
zoologist with a systematic knowledge of the amoebae can possibly,
I think, concur.

It is clear that this organism cannot be placed in the genus Entamoeba,
on account of its nuclear structure and the characters of its cysts. Nor
is there any other genus of amoebae in which it can be correctly placed ;
and it thus seems necessary to create a new one to receive it. The name
“‘ Pseudolimax,”’ given by Kuenen and Swellengrebel (1917), was not
proposed in accordance with the rules of nomenclature as a generic
name, and is also inappropriate. As I think it desirable to preserve the
historic connexion between the amoeba and Wenyon’s “ Iodine cysts,”
which are already so well known to many workers, I therefore propose
the generic name given above—Jodamoeba—for this organism. Its
name, accordingly, becomes Jodamoeba biitschlii Prowazek, 1912,

IODAMOEBA BUTSCHLII 113

It may be added that Brumpt (1913), James (1914), and Pestana
(1917), have considered Prowazek’s “ Entamoeba” biitschlii to be E. coli.
James, indeed, says that its “ cysts can in no way be differentiated ” from
those of the latter. This I conceive to be a quite unjustifiable interpreta-
tion. Kuenen and Swellengrebel (1913) suggested that “ E£.”’ biitschlii is
a degenerate “ minuta” (i.e., precystic) form of E. histolytica, and Flu
(1918) seems to consider it also as a degenerate form of this species.
This also I regard as very wide of the mark. I believe that none of these
authors had sufficient material at their disposal to enable them to form
a correct opinion regarding this organism. Wenyon and O’Connor
(1917) have called attention to the resemblance of Prowazek’s “ E.”
biitschlii to E. nana, but note that “this author’s description is too
meagre to allow of any comparison being made”; and they add that
‘““he does not describe any encysted forms”—which is not quite correct,
though it might perhaps be asked how far Prowazek’s rough account
can properly be termed a “description.”

DESCRIPTION.

Iodamoeba biitschlii 1s, as a general rule, a small amoeba intermediate
in size between £. coli and E. nana. 1 believe I have, in the past,
sometimes mistaken the living organisms for small individuals of the
former species or large ones of the latter. The diameter of the living
amoebae, when rounded, is usually about 9—13 » ; but larger individuals
up to 17—20 yp, and very tiny ones down to 5 n, are also found. I have
never seen any as large as 24—the maximum size mentioned by
Prowazek (19124). Since he studied a case infected with E. coli also, it
is possible that he mistook some individuals of this species for J. biitschlii.
According to Kuenen and Swellengrebel (1917) the amoebae measure
10—12 yw in diameter: according to Brug (1919) 12—20 p.

In general form and habit this organism is closely similar, when
alive, to a small specimen of E.coli. Ectoplasm and endoplasm, pseudo-
podia, and the sluggish movements when outside the body, are closely
alike in the two species. The cytoplasm of J. biitschlii is also frequently
filled with food-vacuoles containing numerous bacteria and other foreign
particles ; and a contractile vacuole is, of course, likewise absent. Apart
from its usually smaller size, the only obvious character which distin-
guishes J. biitschlii in the living state is its nucleus, which is almost
invisible—in fact, often quite invisible, in organisms containing much
food. In this respect it differs greatly from E£. coli, whose nucleus—
appearing like a beaded ring—is so conspicuous in the living amoeba.
As a rule, I. biitschlii becomes rounded and begins to degenerate very
soon after leaving the human intestine.

In good stained preparations this organism is easily distinguished by
its nucleus from any of the other amoebae living in man. (See PI. I,
fig. 10, and PI. II, figs. 32—34.) The resting nucleus is similar to that
of many of the small free-living amoebae. It is vesicular, with a
moderate-sized central karyosome, and measures from about 2‘op in
small to about 3°5 « in large individuals. Its diameter is usually between
one fourth and one fifth of that of the whole organism—when fixed and
stained ; and the diameter of the karyosome varies from about one half
to one third of that of the entire nucleus. The karyosome stains intensely
with chromatin stains. With iron-haematoxylin it may appear homo-
geneous (fig. 34), or may show a paler centre (fig. 32). Occasionally a

8

114 : THE AMOEBAE LIVING IN MAN

granule can be seen in the centre—as figured by Prowazek (1912 a,
figs. 13, 16)—but there seems no justification for calling this a centriole.
I have discussed similar appearances in other amoebae elsewhere (1914),
and shall not discuss the interpretation of them further here.

The nuclear membrane is fairly well developed, and stains readily.
Occasionally it shows very fine granules (? chromatin) imbedded in it,
but these cannot usually be seen clearly. Between the karyosome and
the membrane there is the usual “ clear zone,” which is occupied in the
present species by a layer of fairly large granules—so-called “ peripheral
chromatin.” These granules usually lie in a single layer. They stain
somewhat deeply with iron-haematoxylin, but on extraction give up the
stain more readily than the karyosome. They can thus be completely
decolorized, and counterstained with eosin or other plasma stains,
whilst the karyosome remains deeply coloured (cf. fig. 32). In ordinary
iron-haematoxylin preparations they are often overstained, so that they are
confounded with the karyosome ; or they may be completely decolorized,
so that they disappear. Very often, in such preparations, only their
outlines are visible ; and this gives rise to an optical effect suggesting the
presence of a network or series of septa connecting the karyosome with
the nuclear membrane. This is the structure described in the nucleus
of “ E.” biitschlii by Prowazek. By using suitable counterstains and good
lenses with proper illumination it 1s not difficult to convince oneself that
his interpretation was incorrect, and that the clear zone is really occu-
pied by a layer of small granules and not bya network. (Cf. figs. ro,
32, 34.) I note that Kuenen and Swellengrebel (1917) have already
observed the presence of “ peripheral chromatin” in the nucleus of this
form. It may be noted further that these granules are generally disposed
in a single layer in the active amoeba; and that they are often distinctly
separated from the nuclear membrane but apparently imbedded in the
karyosome, whose outline often appears slightly stellate in consequence

fig. 32).

Bie some stress on these nuclear characters, as they supply the only
means of distinguishing the amoebae of this species with certainty.* All
active amoebae, of whatever size, appear to possess the same nuclear struc-
ture. In very small forms, however, it is impossible to make out all the
details with precision. Such organisms (fig. 33), and also many degenerate
or badly fixed and stained individuals, cannot always be distinguished
with certainty from similar small or degenerate specimens of FE. nana. It
may also be very difficult, or impossible, to distinguish very small speci-
mens of I. biitschlii from small uninucleate individuals of Dientamoeba
fragilis.

or biitschlii appears to feed chiefly upon small micro-organisms in the
intestinal contents. To judge from the inclusions in its food-vacuoles,
its food habits are closely similar to those of E. nana.

The habitat of the organism has not been determined with certainty :
but from the close parallel between the appearance of the free and
encysted forms and those of E. coli, in the stools of persons infected
with both species, I judge it to be probably—like the latter—an

* That is to say, of distinguishing them from other amoebae found in the same
situation. Many of the small free-living amoebae possess, of course, a nuclear structure
which is very closely similar.

IODAMOEBA BUTSCHLII II5

inhabitant of the large bowel. I. biitschlit always dies very quickly
outside the intestine, and has not yet been cultivated.

Nuclear “divisions” have been described by Prowazek (1912 a) in
I. biitschlii, but his figures are unconvincing. I have not been able to
study the division of this amoeba in detail, and the few apparently
dividing nuclei which I have seen are different from those described
by him. The usual method of multiplication is, no doubt, by simple
bipartition—as in most other amoebae ; but stages in the process are
extremely difficult to obtain. Binucleate amoebae, usually of large
size, are sometimes found; and they probably represent forms which
have been arrested in division by discharge from the intestine—as in the
other species. A binucleate organism has been figured by Prowazek
(1912 4, Pl. xvi, fig. 14), but I am not certain whether this figure was
really drawn from a free amoeba. The forms which I have seen usually
have two resting nuclei exactly like those of the active forms, with
central karyosomes : whereas his figure shows a form with nuclei like
those in the cysts. He states further that multiple division occurs, but
his figures are capable of a different interpretation (vide infra). I believe
there is no evidence of schizogony in this species.

TEXT-FIG. 2.—Outline drawings of cysts of J. dii/sch/zz, in iodine solution. (Magnification
1,500 diameters.)

Cysts.—The cysts of I. biitschlii are very remarkable structures,
differing considerably from those of the other intestinal amoebae of
man. They have become familiar to many workers since Wenyon
first described them—as “I. cysts”—from the stools of dysenteric
patients from Gallipoli in 1915. The living cysts may be nearly
spherical or oval, but they are very frequently irregular in outline.
They may be kidney-shaped, pear-shaped, fusiform, rhomboidal, or
almost triangular or square in outline, and their forms are often such
as almost to defy description. Text-fig. 2, A to O will give some idea

116 THE AMOEBAE LIVING IN MAN

of the shapes commonly met with. Oval or spherical cysts usually
measure about gy to 12m in diameter: but it is extremely difficult to
state the dimensions of the more irregular forms even approximately.
Attempts to measure the more regular cysts have shown that their
commonest size is about 9-10 in width by Io-12y in length. I have
generally found that the simplest way to gauge the size of these cysts, in
practice, is to measure their greatest length and their greatest width, and
then take the mean as the “size” of the cyst. If this is done for a
number of specimens it wiil usually be found that the average “ size” lies
between 10 andi1yp. Cysts of all sizes from about 6 w to 16 w occur,
however, but those with such extreme dimensions are uncommon.

The living cysts have a fairly thick wall. They are of a clear white
colour, and usually contain two clearly visible inclusions—a number of
very brightly refractile granules, and a dull area of variable size, and
usually of a more or less spherical shape. The granules may be few or
many, collected together or dispersed through the cyst. They resemble
micrococci, and range in diameter from o0'25 wu or even less up to about
Iu. After fixation they take up nuclear stains, but give them up on extrac-
tion more readily than the chromatin in the nuclei, and then readily
stain with plasma stains. They may show metachromatic staining (red)
with methylene blue or haematoxylin, but their reactions are difficult to
study on account of the impermeability of the living cysts to most stain-
ing reagents. Neutral red, when it can be got to enter the cyst, colours
them bright red; but as a rule the contents of fully-formed cysts remain
quite colourless in watery solutions of this stain. The bright granules
do not stain with Best’s carmine, and as a rule stain feebly with borax
carmine or paracarmine. They are insoluble in water, alcohol, chloro-
form, acetic acid, and most ordinary reagents. It appears to me pro-
bable, therefore, that they are not chromatin granules, and that they are
chemically different from the chromatoid bodies or granules in the cysts
of the Entamoebae. They seem, on the other hand, to consist of a sub-
stance similar to volutin, and I shall therefore speak of them as volutin
granules for the present. They are shown in the cysts figured in PI. I,
fig. 11, and PI. I], figs. 38, 40-42. They have been omitted, however,
from the cysts outlined in Text-fig. 2.

The dull inclusion in the living cysts of J. biitschlii is very strikingly
stained when they are placed in an aqueous solution of iodine. It then
assumes a dark mahogany colour, and appears as a solid body with a
well-defined outline. (See text-fig. 2, A to O.) This reaction at once
suggests that the inclusion is a mass of glycogen; and this is confirmed
by its equally well-marked reaction when treated with Best’s specific
carmine stain for this substance (fig. 39, Pl. 11). Itis, moreover, insoluble
in alcohol and chloroform, but readily soluble in water ; so that in cysts
stained in watery solutions—such as iron-haematoxylin or haemalum—it
is completely extracted, and its place is represented by a vacuole (cf. figs.
40-42, Pl. II). This glycogen mass is, of course, the structure which
Wenyon has termed the ‘‘iodophilic body,” and from which he named
the cysts “Iodine cysts.” It is doubtless homologous with the similar
glycogen masses or vacuoles found in the cysts of £. coli, E. histolytica,
E. nana, and other protozoa.

The glycogen masses in the cysts of J. biitschlii are most readily
studied in cysts suspended in iodine solution. (See text-fig. 2, A to O.)
They may be very small (fig. 2 F) or even absent (fig. 2 B), very large

IODAMOEBA BUTSCHLII [17

(figs. 2 J, M), and of variable shape. Usually they are more or less
rounded. At times two (fig. 2 K) or even three separate masses of
glycogen may be present inacyst. They gradually disappear from the
cysts if they are kept for some days, being apparently absorbed. It
thus seems probable that they represent a reserve of food material.

In addition to the volutin granules and the glycogen mass, the
cyst of J. biitschlii contains a single nucleus. This differs in structure
from that of the active amoeba, and to understand the difference it is
necessary to study the changes which occur during encystation. I
shall therefore revert to the active form at this stage, in order to
describe this process.

Unlike E. histolytica and E. coli, I. biitschlii does not undergo any
diminution in size prior to encystation. The precystic amoebae are, in
fact, some of the largest forms of the organism met with ; so that they
are not, in this respect, comparable with the “minuta” forms of E.
histolytica. Amoebae which are preparing to encyst get rid of all the
food contained in their vacuoles, and their cytoplasm becomes beautifully
clear and transparent. At the same time the nucleus increases in size
(see figs. 35, 36, Pl. II). This increase is chiefly noticeable in the
zone between the karyosome and the nuclear membrane. This zone,
which contains the layer of granules of “ peripheral chromatin” in the
active amoeba (figs. 32, 34, Pl. II) now becomes filled with much more
numerous granules, often forming several distinct layers (figs. 35, 36).
At this stage the nucleus has increased from about 2—2°5 w in the active
form to 3 w or even 4 in diameter. The amoebae are now strikingly
different from the vegetative forms. Their protoplasm is clear and
white in the living organism, and in well fixed and stained specimens
appears finely alveolate and remarkably uniform (figs. 35, 36). These
amoebae are very sluggish, soon become non-motile, and then more or
less rounded, when they secrete their cyst walls (fig. 37).

With the formation of the cyst wall further changes take place within
the organism. The encysting, or partly encysted, amoeba shows at first
a small and diffusely stained brown patch in its protoplasm when
examined in iodine solution. This is the forerunner of the glycogen
mass, and it can be seen to become larger, more deeply stainable, and
with a definite contour, in individuals at later stages in development.
Simultaneously the volutin granules appear in the cytoplasm. At first
they are extremely small, and indistinct, but later they are seen to be
larger and highly refractile. At the time of their formation they do not
seem to have any definite relation to the nucleus or the glycogen mass,
but make their appearance in any part of the cytoplasm. The most
striking change occurs, however, in the nucleus. The karyosome, which
is central in the active and precystic amoebae (figs. 32-36), gradually
passes towards the periphery (fig. 37), until it lies as a large and compact
mass in contact with the nuclear membrane in the fully-formed cyst
(figs. 40-42). Fig. 37 shows an organism which has just formed its cyst
wall. The volutin granules are already fairly numerous: the glycogen
mass was small, and is represented by a small vacuole in the stained
specimen. The karyosome is passing to the periphery of the nucleus,
and the abundant “peripheral chromatin” has a characteristic appear-
ance. Comparison of this figure with fig. 36—an earlier stage—and
fig. 4o—a mature cyst—will show at a glance the chief changes which
occur during encystation.

118 THE AMOEBAE LIVING IN MAN

In the karyosome of encysting amoebae a differentiation into a paler
cortical part and a more deeply staining central body can often be seen
in iron-haematoxylin specimens (cf. figs. 36, 37). This is not usually
visible in the mature cyst. In the latter, the nucleus has the appearance
of a signet-ring—especially in cysts examined in iodine (text-figs. 2A
to O), in which medium the peripheral granules are invisible, though the
karyosome and the nuclear membrane are usually to be made out with
ease. The granules in the nucleus of the cyst often stain very intensely,
so that it is necessary to stain the cysts very carefully in order to obtain
a correct picture of the structure of the nucleus. Sometimes one or
more of the granules will retain the stain more strongly than the
remainder, so that occasional dark granules may be seen lying among
the others (cf. fig. 42).

The cysts of this amoeba are very apt to undergo shrinkage during
fixation, staining, and mounting; and there is thus often aspace between
the contents and the wall in mounted specimens. Very generally, also,
there is some shrinkage of the cytoplasm surrounding the nucleus, so
that the latter often appears—in stained preparations—to lie in a vacuole
(cf. figs. 38-40, 42). These appearances are, of course, artifacts ; but the
remarkable forms of the cysts encountered in good preparations—such
as figs. 41 and 42, for example—are not the result of fixation or other
manipulation. They are equally visible in living cysts. Fig. 38 shows a
badly fixed cyst of large size, in which there has been much cytoplasmic
shrinkage. I have drawn it because it shows a common appearance of
these cysts in stained preparations. It bears a striking likeness to the
cyst of “ Entamoeba” biitschlii depicted by Prowazek and said to measure
148m (19124, Pl. xviii, fig. 21); and consequently, it supports my
identification of this form with that here described.

Cysts of I. biitschlii containing more than one nucleus are very
uncommon. Brug (1917) states that most unusually large cysts are
binucleate, but this is incorrect. I have seen several containing two
nuclei, but none with more. I regard them as abnormal supernucleate
forms, like the 8-nucleate cysts of E. nana or 16-nucleate cysts of £. coli.
I think Prowazek’s binucleate and trinucleate ‘‘ amoebae” (his figures 14
and 15) are really similar cysts, of irregular form, and not stages in
division or schizogony. It may be noted that James (1914) has inter-
preted Prowazek’s fig. 15 as an enucleate amoeba of E. coli containing
three ingested yeasts—not three nuclei. This seems to me a very far-
fetched explanation.

The remarkable forms so often assumed by the cysts of J. biitschlii
would seem to indicate that they are formed under some peculiar
conditions of stress or pressure. An amoeba naturally tends to assume
a spherical form when at rest, and about to encyst: and it has occurred
to me that the strange shapes of the cysts of this organism may possibly
be due to the fact that the amoebae are crowded together in the crypts
in the large intestine at the time of their encystation, so that their cysts ©
become distorted by mutual pressure. Similar strangely shaped speci-
mens of other protozoa, such as coccidia, which are normally oval or
spherical, may often be seen in cases of heavy infection where they are
closely packed and pressed together in the tissues.

The mature uninucleate cysts of J. biitschlii undergo no further
development outside the human body. They will remain unchanged—
except for the disappearance of the glycogen, as already noted—in faeces

IODAMOEBA BUTSCHLII 119

or water for two or three weeks; but they are unable to withstand
drying, like those of the other intestinal amoebae. Their development
in a new host has still to be determined. Doubtless they hatch in the
small intestine, and each liberates a single uninucleate amoeba.

Descriptions and figures of the cysts of I. biitschlii have already been
given by Wenyon (1915 a), Wenyon and O’Connor (1917), Kuenen and
Swellengrebel (1917), Brug (1917, 1919), and Flu (1918). All have noted
their striking appearance in iodine solution, but only Kuenen and
Swellengrebel have stated that the “iodophilic body” is composed of
glycogen, Wenyon and O’Connor note that they vary greatly in size and
shape, and give their size as 72 to 15 w “ormore.” They do not mention
the volutin granules in the cysts; but Kuenen and Swellengrebel observed
them, and apparently considered them to represent the ‘peripheral
chromatin” of the nucleus extruded into the cytoplasm. Brug (1917) also
noted these granules, but was unable to interpret them. He calls them
“‘coccoid corpuscles,” and says they may be parasites, ‘‘saprophytes ”
(sic), or metabolic products. He describes the “ peripheral chromatin ”
of the nucleus as being disposed in the form of a crescent—an appear-
ance resulting, apparently, from defective fixation. The size of the
cysts is given by Kuenen and Swellengrebel (1917) as 10 w to 12 p—
the same size as their amoebae. Flu (1918) describes the cysts as
“spherical bodies with a large vacuole, which . . . assumes a more
or less brown colour” in iodine solution. His figures are very poor,
and he considers the “ bodies”—as already noted—to be degenerate
cysts of E. histolytica.

I believe that there are different races of J. biitschlii which are
distinguishable by the sizes of their cysts—as in E. histolytica and E. coli.
Certainly a distinct difference is sometimes observable in the size of
the cysts passed by different persons. But the cysts are so difficult to
measure that I cannot place much confidence in such measurements
as I have made, and I am therefore unable to offer any proof of the
existence of such races at present.

Occurrence.—There can be no doubt that I. biitschlii has a wide
geographical distribution. It occurs in Egypt (Wenyon and O’Connor),
the South Seas (Prowazek), the Dutch East Indies (Kuenen and Swellen-
grebel, Brug, Flu), and I have found the cysts in the stools of patients
who have been invalided to England from all the chief areas of the
present war. The infected cases examined have included persons from
all the five continents ; but it is, of course, impossible to discover where
or when their infections were acquired. The organism occurs also in
persons who have never left the British Isles.*

Wenyon (1916) found the cysts of J. biitschlii in the stools of 29 out
of 556 cases of dysentery and other intestinal ailments invalided to
England from Gallipoli in 1915. They have since been found in a
similar proportion of cases of dysentery, etc., from all the theatres of
war, by many other workers in England. My own records show that
they must occur in at least 5 per cent. of all the cases examined,
but the exact incidence I cannot determine: for different series of
patients have been examined with different degrees of thoroughness, and
I have examined many stools selected because they contained the cysts,

* Cf. Matthews and Malins Smith (1919).

120 THE AMOEBAE LIVING IN MAN

which have often been sent to me for identification. The largest series
of cases from which I can form an approximate estimate of the preva-
lence of this organism is that which I examined at Epsom (vide Dobell,
Gettings, Jepps, and Stephens, 1918). Here I examined 1,300 men and
found 29 infections. As the cases were examined on the average only
twice each, it is certain that more were really infected. If the resuits
can be interpreted—as seems highly probable from the figures at my
disposal—in the same manner as the findings of E. histolytica (vide
Dobell, 1917), then the whole series was probably infected to the
extent of some 4 or 5 per cent. My figures also appear to indicate
that infections with J. biitschlii are commoner in persons who have
been in the tropics and the Near East than in those who have been
in France and the British Isles only.

Wenyon and O’Connor (1917) have published statistics showing the
incidence of infection in various groups of people examined by them in
Egypt. The figures range from 2 per cent. up to 14'8 per cent.—the
latter being the figure for natives in Hadra Prison. The percentages are
derived from series examined only once per case on the average, and are
therefore all too low. They were—with one exception—always lower
than the percentage infections with E. histolytica. In my experience the
frequency of J. biitschlii as compared with E. histolytica infection for the
same group of cases lies as a rule between 1:3 and1:5. Itis certainly
the least common of the intestinal amoebae of man (except Dientamoeba),
but probably occurs—so far as I can judge—in at least some 3 to 5 per
cent. of all human beings.

It is a curious fact that J. biitschlii very frequently occurs in company
with £. histolytica—far too frequently for it to be due to chance. When
not accompanied by this species, it is generally in company with E£. coli
or E. nana, and all the four species occur together fairly often. I have
not yet found with certainty a single pure infection with J. biitschlit.
There can be no doubt that it is an entirely independent species, how-
ever ; and I can offer no plausible explanation of its almost invariable
association with other amoebae.

Pathogenicity—Although I. biitschlii has usually been found in
the stools of persons who have previously suffered from dysentery or
other intestinal disorders, this is probably merely because the stools of
healthy people are not usually investigated. I have found infections in
healthy people with no history of dysentery or persistent diarrhoea.
Wenyon and O’Connor (1917) have found the cysts in the faeces of
healthy white troops and natives in Egypt, and Matthews and Malins
Smith have found them in residents in the British Isles. There is
at present no evidence that the organism is pathogenic, and it seems
almost certain that it is not.

Treatment.—One of the most remarkable characters of J. biitschlii is
its prompt disappearance when emetine is administered to its host.
Wenyon and O’Connor (1917) noted that emetine hydrochloride given
either hypodermically or by the mouth to infected persons causes the
cysts to vanish from their stools. In none of their cases did they sub-
sequently reappear during the period of observation. I have found that
the administration of emetine bismuth iodide has the same effect,
and have already recorded the apparent cure of 5 cases by this means
(vide Dobell, Gettings, Jepps, and Stephens, 1918). I have studied other
cases, both before and since, and some who were treated with emetine

IODAMOEBA BUTSCHLII 121

hydrochloride hypodermically. In no case have I found any evidence
of the persistence of this amoeba after the administration of a thorough
course of emetine in any form. To judge from my own records,
I. biitschlii may even respond more readily to emetine than E. histolytica,
for I have never yet seen a case “relapse” after treatment. This may
be a coincidence, however, as I have studied only about a dozen fora
sufficient period : and, moreover, all these cases were infected with
E. histolytica—for which the emetine was given—and were all apparently
cured of infection with this parasite at the same time.

This behaviour of J. biitschlii towards emetine administered to its
host is to me extremely puzzling. If emetine were an “ amoebicidal”
substance, having a specific lethal action upon amoebae generally—
as was once believed—such a reaction might even have been predicted :
but since it is now highly probable that emetine acts primarily upon
the host, and not upon the parasite (cf. Dale and Dobell, 1917), it is
difficult to understand how it eradicates infections with J. biitschlii.
The fact that emetine will not cure an infection with E. coli or E. nana
appeared to be clearly correlated with the difference in habit of these
species as compared with £. histolytica—neither of them preying directly
on its host. But in J. biitschlii we appear to have an organism which
lives upon the intestinal contents, like FE. coli and E. nana, but which
responds to emetine treatment like the tissue-parasite E. histolytica.
This may conceivably indicate that there is some peculiarity in the
habits of J. biitschlii which has not yet been discovered, or that emetine
is directly toxic to it. At present the facts seem inexplicable.

122

VIII.
GENUS DIENTAMOEBA JEPPS & DOBELL, 1918.

THERE is only one species of this genus known—Dientamoeba fragilis,
which I have already described in detail in a joint paper (Jepps and
Dobell, 1918). I shall, therefore, merely summarize here as briefly as
possible what has there been said.

HISTORY AND NOMENCLATURE,

D. fragilis was probably discovered, though it was not described, by
Wenyon in rgog. It was later rediscovered independently by Miss M. W.
Jepps and by myself in 1917. We described seven cases of infection,
which, with Wenyon’s original case, make 8 known cases in all. I have
found no others since, and, so far as J] am aware, none have been.
recorded.* The organism is probably rare, but not so rare as these
figures appear to indicate; for itis extremely delicate, and perishes soon
after leaving the body. Consequently, infections must often be over-
looked. The nomenclature has been fully discussed in my earlier paper
with Miss Jepps, and need not be reconsidered here. There can be no
doubt that this organism is generically distinct from all other amoebae
of the human gut.

DESCRIPTION.

D. fragilis is a very small amoeba. When rounded it measures from
3°5 w to 12 w in diameter, its usual size being about 8-9 pw. It is
thus similar in size to E. nana and the smallest precystic forms of
E. histolytica.

The living amoebae are actively motile. They show a well-marked
differentiation between their ectoplasm and endoplasm. The pseudo-
podia are typically composed almost entirely of ectoplasm, and are few,
flattened, leaf-like, and often lobed or indented. The endoplasm is
granular, and usually contains numerous food-vacuoles; but a con-
tractile vacuole, as in other parasitic amoebae, isabsent. During locomo-
tion the animal has asomewhat snail-like appearance—the hyaline pseudo-
podia being in advance, the granular endoplasm concentrated in a fairly
definite rounded mass posteriorly. Like other intestinal amoebae, the
organism is colourless.

The most characteristic feature of D. fragilis is its nuclear system.

* Since the publication of the description of this organism five cases of infection have
been reported to me privately by others. On investigation none of these proved to be
infections with Dzextamoeda in reality. I record them to show the errors which can occur
in diagnosis. Two of them were small free-living “/iax” amoebae, in stale stools.
Two others were leucocytes, in stools containing pus; and the remaining one was an
infection with Z. zama, in which many of the amoebae were parasitized by Sphaerita—
the spore-morulae cf which were mistaken for nuclei.

DIENTAMOEBA FRAGILIS 123

Each individual is typically binucleate (figs. go, gt, Pl. V), the two nuclei
being identical in size and structure. Their size is proportional to that
of the whole organism, and their diameters range from about 0°8 u in very
small amoebae up to about 2°3 w in very large. On the average the
nuclei measure about 2 w, in stained specimens. They are invisible or
inconspicuous in the living organism ; but in well fixed and stained indi-
viduals, obtained immediately after they have left the human body, they
have a very characteristic structure—unlike that of the nuclei of any of
the other intestinal amoebae of man. Each nucleus is spherical and
vesicular. The chromatin is all situated in a fairly large central
karyosome, which, in well-differentiated iron-haematoxylin preparations,
can be seen to consist of a number of granules apparently embedded
in a plastin matrix (see figs. 90, 91). The granules are not always
uniform in size, and one of them is often larger and more conspicuous
than the others (cf. the lower of the two nuclei in the amoeba depicted
in fig. gt, Pl. V). No centriole is demonstrable in the karyosome. The
whole nucleus is bounded externally by a very delicate and feebly
staining nuclear membrane, separated by a clear zone from the karyo-
some. Linin threads of extreme tenuity can be seen, in well stained
individuals, radiating from the karyosome to the nuclear membrane. As
a rule, excessively minute granules can be resolved on or in the nuclear
membrane—usually at the points where the linin threads are attached
to it. Apart from these granules, of doubtful composition, there is no
“peripheral chromatin” either in the clear zone outside the karyosome
or on the nuclear membrane. The structure of the nuclei will be evident
from inspection of figs. go-g2 (PI. V).

The two nuclei of a Dientamoeba may occupy any position, relatively
to one another, in the body of the organism. They are sometimes in
pe (fig. 91), or may be separated by an interval of variable extent
(fig. 90).

Most individuals are binucleate, but in every infection a certain
number of uninucleate specimens can always be found (fig. 92). Careful
examination of over 1,000 individuals from three different cases showed
that about 80 per cent. of them were binucleate and 20 per cent.
uninucleate. Uninucleate individuals may be found of all sizes, from
the smallest to very large; but they are, on the whole, somewhat smaller
than the binucleate forms. These, however, are sometimes of extremely
small size (3°5 ).

Division stages are extremely rare in the stools, and all that have so far
been found represent organisms witha single dividing nucleus. From this
fact—and others just noted—I have been led to suppose that D. fragilis
differs from the other known binucleate species of amoebae (such as
“Amoeba” binucleata Gruber, and ‘‘ Amoeba” diploidea Hartmann et
Nagler) in its mode of reproduction. It seems probable that the organism
is, when full grown, binucleate; and that when division occurs, it con-
sists in a simple fission of the cytoplasm, resulting in the formation of
two uninucleate daughter individuals. The nucleus in each of these
divides into two during the growth period, thus giving rise to the adult
binucleate form once more.

D. fragilis undergoes degeneration in a very characteristic manner.
Soon after leaving the body the amoebae usually become rounded and
motionless, and filled with vacuoles. These then coalesce into a single
large central vacuole, surrounded bya thin layer of protoplasm. The

124 THE AMOEBAE LIVING IN MAN

organism then appears under the microscope as a delicate ring of proto-
plasm, containing the two nuclei and any food inclusions which may
have been present, with a large space in the middle. At this stage it has
a striking resemblance to a Blastocystis. Degenerating organisms may
remain in this condition for a long time before they finally disintegrate.

Although a prolonged and very careful search has been made for the
cysts of this organism, none have ever been found. In this respect
D. fragilis resembles E. gingivalis. How the former is transmitted from
host to host is still a matter for speculation. Only when the stools of
an infected individual are liquid or soft can the amoeba be found in
them. When they are hard and formed, no trace of the amoeba, and
no resistant stages, can be detected. The free amoeba usually perishes
so rapidly outside the body that it is highly improbable that it can be
transmitted in this form.

The habitat of D. fragilis is probably the colon, but up to the present
it has been found only in the stools. It is not known to occur in any
animal but man, and has not yet been cultivated.

To judge from the food contained in its vacuoles, D. fragilis lives
exclusively on bacteria and other small vegetable micro-organisms in
the gut contents. It is therefore, in all probability, a harmless com-
mensal like £. coli and E. nana. No method of treatment is known to
have any effect upon it.

The geographical distribution of this remarkable and interesting
organism is at present uncertain, but it seems probable that it is wide.
Of the seven cases of infection which I have studied, one was in a
healthy resident in Great Britain, who had never been abroad ; two were
in British soldiers invalided home to England from Salonika, suffering
from dysentery; one was in a British soldier invalided from Salonika to
England with malaria; and the remaining three cases were New Zealand
soldiers who had served in France and England only. Although most
of these cases had suffered from dysentery, there is no evidence that it
was in any way due to infection with D. fragilis: and none of them
were suffering from dysentery at the time when their infections were
discovered.

IX,

THE AMOEBAE FOUND IN HUMAN URINE, IN DOGS,
AND IN MONKEYS.

] HAVE already mentioned that there are several problems connected
with the amoebae which have been described from human urine, and
also with those which have been found in certain animals other than
man—especially dogs and monkeys. It is not possible to deal thoroughly
with several problems in the nomenclature of the amoebae of man with-
out taking these organisms into account; and I shall therefore say
something about all of them in this section, and endeavour to clear up
some of the difficulties which they offer at present.

I shall deal with the amoebae from urine, from dogs, and from
monkeys, separately and in this order.

THE AMOEBAE FOUND IN HUMAN URINE,

There are now over a dozen records of amoebae discovered in the
urine or urinogenital organs of human beings, and a condition of “urinary
amoebiasis” is recognized by some clinicians. Since the amoebae con-
cerned are supposed by some workers to be E. histolytica, and since they
have received various names, it has already been necessary to notice
them when dealing with the nomenclature of this species.

Amoebae appear to have been first recorded in the urine by Baelz
(1883), who found them in a young Japanese woman with “ tuberculosis
of the urinogenital apparatus and lungs.’’ She entered hospital the day
before her death. Her urine, removed by catheter, was bloody and
contained much pus and necrotic tissue; and, in addition, Baelz found
enormous numbers of actively motile amoebae measuring 50 w in
diameter, and, except for their rather larger size, “in every way” like
Lésch’s ‘“‘Amoeba coli.” According to Baelz, “they consist of a
granular body-substance with a large vesicular nucleus”: and he says
they were present in the bladder and vagina, and he believed that they had
wandered from the latter into the former. He does not record that they
contained ingested red corpuscles, and apparently he did not examine the
stools. No post mortem examination was made. He remarks that “if
the parasite, as is probable, represents a new species, then it may be
given the name Amoeba urogenitalis.” Blanchard (1885), in recording
this case, names the organism “ Amoeba vaginalis Baelz, 1883 ”—which
is incorrect, and presumably a lapsus calamt.

Jiirgens (1892) has described mucous cysts “ filled with amoebae”
which he found in the bladder of an old woman—a case of chronic
cystitis and endometritis. The “amoebae” are not properly described :
but it is stated that they put out pseudopodia when warmed, and that
some of them had no nuclei. Their resemblance to cells was noted, and

126 THE AMOEBAE LIVING IN MAN

they are said to have been present in the vagina as well as the bladder.
I am at a loss to know why these structures were supposed to be
amoebae. It seems highly probable that they were cells, though of what
sort precisely one cannot determine from the inadequate description.

Kartulis (1893, footnote p. 2) says he found ‘‘ amoebae ”’ in the urine
of a Cretan, but he gives no proper description of them—merely noting
that they measured 12-20 pw, had “finely granular” protoplasm, and
showed a nucleus in stained preparations. His reasons for regarding
these bodies as amoebae are not given.

Posner (1893), in Berlin, describes and figures ‘‘amoebae” found in
the urine of a man with haematuria. They changed shape slowly, were
not motile, and usually measured 25-30 in diameter. Although
the author speaks with confidence, and says that they “ immediately
struck everyone who saw them as amoebae,” his figures and descriptions
are not likely to impress anybody familiar with these protozoa. There
can be no doubt, I think, that the objects he saw were really cells. At
all events, there is no evidence that they were amoebae. Wyjnhoff
(1895) studied four similar cases of ‘urinary amoebiasis” at Utrecht :
but although he describes ‘‘ amoebae,” “cysts,” and “ multiplying ” forms,
it is clear from his account that the structures which he encountered
were not amoebae but cells of various sorts occurring in the urine.

Craig (1911, p. 233) mentions that he has studied a case of urinary
amoebiasis. The bladder is said to have been infected with E£. histolytica,
and a minute fistula between it and an intestinal amoebic ulcer is said
to have been detected post mortem. No details are recorded.

Fischer (1914) found amoebae in the urine of a Chinaman at Shanghai.
After one visit to the hospital—when the amoebae were found—the
patient unfortunately left. No proper history was obtained, and the case
was not followed up. A diagnosis of “? Cystitis” was made. The
stools were not examined, and it is uncertain whether the patient had
ever had dysentery. The amoebae are stated to have been actively motile
and much vacuolated, and measured about 20-25 w in diameter. They
were not properly described, but are said to have been indistinguishable
from “ Entamoeba tetragena,” as seen in local cases of amoebic dysentery.
It is not stated whether they contained red corpuscles or not, and their
mode of entry into the urine was, of course, undetermined.

Lynn (1914) has recorded the case of “an intelligent coloured man”
who washed out his rectum and then his bladder with the same syringe—
without sterilizing it—with the result that blood and pus appeared in his
urine, and “urinalysis’’ revealed ‘“‘ motile amoebae (Entamoeba tetra-
gena),” according to a laboratory report. No amoebae were found in
the stools. This case is said to have “responded” to emetine treat-
ment—though the evidence for this is, to say the least, meagre. In
the absence of any further evidence concerning the “amoebae,” it is
impossible to accept them as E. histolytica.

Walton (1915) has published an important paper dealing with a case
of urinary amoebiasis. His patient was an Indian native with chronic
Bright’s disease, whose urine contained very numerous highly active
entamoebae. Nearly all of these contained red blood corpuscles.
Rounded specimens measured usually 25-35 mw in diameter in stained
preparations. No cysts were found. The amoebae are carefully
described, and the author concludes that they were “E&. tetragena.”’
Amoebae, apparently of the same species, were found in the stools.

AMOEBAE IN URINE 127

The case was treated with emetine hydrochloride hypodermically (1 grain
daily), and on the fifth day the amoebae disappeared completely and were
not found again, though the case was carefully controlled for over a
month. An attempt to infect a kitten per anum with amoebae from the
urine gave negative results, as also did a similar experiment with the
amoebae from the stools.

Macfie (1916) describes one case in a European, and records four
others in natives, of urinary amoebiasis seen on the Gold Coast. The
parasites were found in the urine, but never in the stools. They were
generally “ quiescent,” but sometimes showed sluggish movements; and
they were usually degenerate, owing, itis suggested, to the harmful action
of urine on them. Sometimes they contained red blood corpuscles.
They measured 7-33 in diameter, but 50 individuals measured on
one occasion gave an average of only tom. The nucleus possessed
“a well-defined nuclear membrane, refractile, and clearly differentiated
from the surrounding endoplasm. On the inner side of this membrane
there was usually a large amount of chromatin arranged in irregular
nodular masses, and throughout the nuclear substance similar chromatic
matter was distributed. The karyosome was large and showed a centriole.”
It is difficult to see much resemblance to Entamoeba histolytica in this
account, but the author states it as his belief “ that these amoebae cannot
be differentiated from Entamoeba histolytica (tetragena).” He also states
that he found cysts in the urine, ‘but none was seen containing more
than four nuclei.” These “cysts” are not figured—nor, indeed, are the
amoebae. (It may be recalled that the “cysts ” of amoebae which Macfie
(1915) described previously from a monkey are clearly shown by his
figures to have been Blastocystis.) Dr. Macfie very kindly sent me some
preserved sediment from the urine of one of his cases. I have examined
it very carefully, but have been unable to find anything but pus and
various tissue-cells in it. No recognizable amoebae, and nothing even
resembling cysts of E. histolytica, have rewarded my search. I am
therefore still unconvinced that the structures which he observed were
really amoebae.

Ward, Coles, and Friel (1916) discovered “amoebae”’ in the urine
of a case of jaundice, and proposed to call them ‘ Amoeba urinae
granulata.” From their account, and the later revelations of Friel
(1917), there can be no doubt that the bodies in question were not
amoebae at all. It will suffice to note that “the bodies were totally
unlike Endamoeba coli or Endamoeba histolytica’? (Ward, Coles, and
Friel, 1916) ; and that ‘“‘it is probably not an amoeba in the proper
sense of the term,” but “resembles a vegetable cell, recalling the ‘ proto-
coccus’ met with in stagnant water” (Friel, 1917).

Chalmers and O'Farrell (1917) describe a case of “ urinary amoeb-
iasis” in a Greek woman in the Sudan, and mention several others in
which they found amoebae “always more or less encysted, and more
or less degenerate.’ In the case described they were non-motile,
“roundish,” and measured about 18 w in diameter, with a nucleus of 4 p.
They were believed to be a “pre-cystic stage”’ of E. histolylica. On one
occasion “numerous degenerating cysts appeared in the urine.” The
authors find “ no reason to doubt”’ that their identification was correct:
but their description and figure (a photomicrograph) convince me that
they were mistaken. 1 cannot recognize E. histolytica in either; and
their belief in the patient’s “‘response”’ to emetine treatment seems by

128 THE AMOEBAE LIVING IN MAN

no means justified by the facts given. It is important to note also that
the faeces were twice examined carefully (after a purgative), and no
amoebae were found. Equally unconvincing to me is the account of
“amoebae” found in the urine of a woman at Sierra Leone by Wright
(1917). They were usually “quiescent,” and measured usually 20-25 wu
in diameter. Cysts ‘‘ were numerous,” and many had “ 2 and 3 nuclei.”
The “cysts”? had a diameter of about 20 to 25 w. Although the author
states that he “is persuaded to regard the amoebae as belonging to the
‘histolytica’ type,” I am unable to find any grounds for such per-
suasion: nor is the statement that the patient “responded readily to
specific treatment with emetine hydrochloride” likely to command
assent from anybody who reads the recorded details in a critical spirit.

Finally, Aravantinos and Michailides (1918) record the finding of
amoebae in the urine of a Greek boy with cystitis. They are said to
have been active, and uninucleate ; but they are not described or figured,
and were not investigated cytologically. The authors consider that the
amoeba was not E. histolytica. The case is supposed to have been cured
by lavage of the bladder with an infusion of ipecacuanha. During
treatment “cysts” appeared in the urine, but they are not described.
This is another very unsatisfactory case.

It appears to me probable that most of the cases of “urinary
amoebiasis’ hitherto described should be rejected, since there is not
sufficient evidence that the ‘“ amoebae”’ which were found really were
amoebae. There is, in fact, little to indicate that the bodies were even
protozoa in most cases—still less that they were amoebae or that they
belonged to any particular species. For my own part, I have little doubt
that Jurgens (1892), Kartulis (1893), Posner (1893), Wijnhoff (1895),
Macfie (1916), Chalmers and O’Farrell (1917), and Wright (1917) really
found cells of various sorts and mistook them for amoebae. The
‘‘amoebae” of Ward, Coles and Friel (1916) and Friel (1917) are self-
condemned; whilst the “amoebae” of Craig (1911) and Lynn (1914)
cannot be discussed in the absence of all descriptions of them. The
“amoebae” of Aravantinos and Michailides (1918) were possibly Tricho-
monas vaginalis. The degenerating amoeboid forms of this flagellate
can, as 1 know from experience, be mistaken for “amoebae,” and they
occur in the bladder and urethra of males as well as of females. It is
possible that some of the other “amoebae” described were also this
organism in reality.

By far the most satisfactory case of urinary amoebiasis yet recorded
seems to me to be that of Walton (1915). I see no reason to doubt that
he really found amoebae, and that these were—as he believed—E.
histolytica. There is no reason why this parasite should not occasionally
occur in the urine, though infection of the urinary system must always
be secondary to a primary infection of the large intestine. If E. histolytica
is found in the urine, therefore, a concomitant infection of the bowel
should also be present. This was so in Walton’s case; but the absence
of amoebae or cysts from the stools of some of the other cases speaks
against the supposition that the ‘‘amoebae” in the urine really were
E. histolytica. It is against all analogy to suppose* that the organisms

* This I take to be the explanation which Macfie (1916) offers to account for the
absence of an intestinal infection in his case. He says he is “inclined to believe. .
that this patient may at some time have unconsciously harboured amoebae in his large
intestine,” and that they somehow “found their way .... to the neighbourhood of
the seminal vesicles,” and so emerged by way of the urethra.

AMOEBAE IN DOGS 129

would migrate ew masse from the gut to the secondary site of infection.
Moreover, if the urinary system were secondarily infected, one would
not expect to find cysts of the parasite in this situation. It is a general
rule that E£. histolytica does not encyst in the secondary sites of infection,
but only in the primary site in the bowel. The occurrence of cysts
in the urine, therefore, as recorded by Macfie (1916), and Chalmers and
O'Farrell (1917), would—if a correct observation—be surprising. On
@ priori grounds it seems to me to weaken rather than strengthen their
case. The “cysts” of Wright (1917) were clearly, from their size and
structure, not cysts of E. histolytica,

Walton’s case * I regard as a true case of secondary infection of
some part of the urinary system with E£. histolytica. The negative experi-
ments which he made with kittens do not invalidate this conclusion, as
similar negative results can often be obtained with undoubted E. histoly-
tica from stools. The case of Fischer (1914) was, I believe, in spite of
its obviously deficient investigation, a similar case of E. histolytica infec-
tion, and I see no reason why Baelz’s (1883) original case should not be
included in the same category. If it be included, then “Amoeba
urogenitalis ” Baelz, 1883, becomes a synonym of £. histolytica—which
is, I think, the most justifiable interpretation at present. Amoeba vagi-
nalis Blanchard, 1885, being another name for the same organism, is
then likewise a synonym of E. histolytica.

I regard Baelz’s name ‘‘ Amoeba urogenitalis” as a provisional
descriptive term and not as a Linnaean name subject to the law of
priority. He did not determine whether his organism was a new species
or not, and his name was proposed in case it should turn out to be new.
Chalmers and O'Farrell (1917) refer to “‘the unpleasant point” that “if
the rules of zoological nomenclature are pressed, we ought to call the
amoeba of dysentery by Baelz’s name.’ Unfortunately they do not state
how they arrive at this conclusion, which seems to me wholly without
foundation. If Baelz’s name is regarded as valid, then Liésch’s name
(“Amoeba coli”) has at least an equal right to recognition. Now the
latter was undoubtedly given to the dysentery amoeba. Baelz’s amoeba
either was or was not the same organism. If it was, and “the rules are
pressed,” then Amoeba wurogenitalis Baelz, 1883, is a synonym of
Amoeba coli Lésch, 1875, and therefore cannot be used. If Baelz’s
amoeba was not the same species as Lésch’s, then it was not the
dysentery amoeba, and Amoeba urogenitalis is the name of a different
organism. It therefore appears to be impossible to call the dysentery
amoeba by Baelz’s name-—whatever interpretation is adopted. I hold
that both these names should be cancelled, on the grounds already stated.

THE AMOEBAE FOUND IN DOGS.

Amoebic dysentery is said to occur spontaneously in dogs. The
parasite which causes it apparently resembles £. histolytica closely, and
has been named. It is therefore necessary to consider this organism.

Kartulis (1891) says that he has seen spontaneous amoebic dysentery
in a dog in Egypt; and that the amoebae were indistinguishable from
those in human amoebic dysentery, and produced a similar ulceration

* I may say that I attach particular importance to this case because of my personal
knowledge of the.author and of his skill and competence as a protozoologist.

9

T30 THE AMOEBAE LIVING IN MAN

of the gut. Later (Kartulis, 1913) he states that he has observed two
dogs with amoebic dysentery, and that one of these also developed a
liver abscess. Darling (1915),* in Panama, studied a fatal case of
amoebic dysentery in “a large hound used for hunting deer.” The
dog’s colon was ulcerated, and many amoebae were present in the
ulcers and in the bloody mucous stools. They resembled “E. tetra-
gena’'—to judge from his description, they were indistinguishable from
this species (E. histolytica). Darling considers the possibility of his
dog having acquired its infection from man, but says (incorrectly) that
“dogs are not susceptible to infection by E. tetragena,’ and accordingly
proposes provisionally to name the Entamoeba of the dog E. venaticum.t
Ware (1916) has since described an outbreak of amoebic dysentery
among foxhounds in India. Altogether nine dogs were affected, of
which one died and eight were apparently cured by the hypodermic
administration of emetine. The amoebae were active organisms, some
of them containing ingested red blood corpuscles. ‘Mr. Shunker
pronounced them to be extremely like entamoeba histolytica” (sic).
They were therefore believed to belong to this species.

Now it is known that dogs can be experimentally infected with
Entamoeba histolytica from man. Lésch (1875) injected 4 dogs “per os
et anum” with amoebae from his celebrated original case of dysentery.
One of them contracted amoebic dysentery, with typical ulceration of
the bowel. Hlava (1887) also is said to have obtained positive results
with 2 out of 17 dogs similarly treated, though Kartulis (1891) failed to
confirm these experiments. Kruse and Pasquale (1894) record that they
infected a dog with dysentery amoebae from man. Harris (1901)
succeeded in infecting 3 puppies—though 4 older dogs were negative—
by means of rectal injections of bloody mucous stools containing active
amoebae from a human case of amoebic dysentery. All the infected
animals had dysentery, with typical amoebic lesions of the bowel post
mortem, and two of them developed amoebic abscesses of the liver in
addition. Dale and I (1917) have also infected 2 puppies by rectal
injection of E£. histolytica belonging to a strain propagated through a long
line of kittens.

It is thus clear that the dog, like the cat, is susceptible to infection
with E. histolytica. In both animals infection results in acute dysentery,
and may be followed by liver abscess—as in man. The dog—again like
the cat—apparently does not become a carrier of the parasite: at all
events, nobody has yet discovered the cysts of E. histolytica in the stools
of infected animals, and Darling (1915) expressly notes their absence in
his case. The two infected puppies which I have seen both recovered
spontaneously, and neither subsequently passed cysts. They were
specially examined to determine this point. One of them was finally
sacrificed, and showed no signs of ulceration of its intestine.t It
seems to me doubtful whether emetine has any curative action on
amoebic dysentery in the dog—in spite of the findings of Ware (1916) :
for it is possible that his dogs might have recovered without treatment,
and it has been found (Dale and Dobell, 1917) that emetine will not cure
amoebic dysentery in the cat—which behaves in other ways similarly to

* First recorded, but not fully described, in Darling (1912).
+ This is presumably a mistake—venatica (or possibly venaticorum) being intended ?
{ Vide Dale and Dobell (1917).

AMOEBAE IN MONKEYS 131

the dog when infected with E. histolytica. If Ware’s conclusion is
correct, however, it emphasizes the resemblance between the Entamoeba
found in the dog and E£. histolytica.

It seems to me highly probable that the spontaneous amoebic
dysentery and liver abscess observed in dogs by Kartulis, Darling, and
Ware were all due toinfection with E. histolytica, The cases all occurred
in countries where human faeces containing cysts of this parasite cannot
be uncommon: and the habits of the average native and the average dog
are quite compatible with the hypothesis that dogs, if susceptible, may
occasionally acquire an accidental infection with E. histolytica as a
consequence of ingesting cysts deposited by human carriers. It therefore
seems justifiable to conclude—in the absence of evidence to the contrary
—that Entamoeba ‘ venaticum” Darling is a synonym of E. histolytica.

THE AMOEBAE FOUND IN MONKEYs.,

Many monkeys harbour species of Entamoeba which are not with
certainty distinguishable from those of man. As some of them have
received names, it is necessary to consider them.

Kruse and Pasquale (1894) unsuccessfully attempted to infect a
monkey with amoebae from a case of dysentery. Musgrave and Clegg
(1904) believed that they were able to infect monkeys (Macacus cynomolgus
and M. philippinensis) with amoebae from their cultures, thereby pro-
ducing dysentery in them. Walker and Sellards (1913), however, were
unable to infect the same species with E. histolytica. Musgrave and
Clegg state that they “have occasionally observed cases of naturally
contracted amebiasis in monkeys” in the Philippines: which may
account for their “‘ positive” results (obtained with harmless free-living
amoebae), and the ulceration of the colon described and figured in their
experimental animals.

Wenyon (1908) records that he found “ cysts of an amoeba indistin-
guishable from those of Entamoeba coli”’ in ‘‘ the intestine of a monkey ”
at Khartoum. Brumpt (1909) found a similar organism, with 8-nucleate
cysts, in Macacus sinicus. Attempts to infect 4.cats were negative. Noc
(1909) tells us further that the “ macaques” of Saigon commonly pass
amoebic cysts 10-12 w in diameter in their faeces, and that they suffer
from spontaneous dysentery.

Castellani (1908) described a spontaneous case of amoebic liver abscess
ina Ceylon monkey (Macacus pileatus),and named the parasite Entamoeba
nuttalli. He found no lesions in the bowel, and from his account and
figures (from dried films) the organisms cannot be identified, as all
cytological details are lacking. According to Kartulis (1913) Strong
observed a case of amoebic appendicitis and liver abscess in an orang-
utan at Manila. Chatton (1912 a) found small amoebae—many containing
chromatoid bodies—in the faeces of a dead Macacus sinicus. They closely
resembled the precystic forms of E. histolytica, but he found no mature
cysts, and no lesions in the large intestine, and therefore did not name
the organism—beyond referring it to his genus Léschia (= Entamoeba).
Franchini (1912) claims to have infected “a monkey” with “ Amoeba
tetragena” froma human case of dysentery, but his figures and description
are far from convincing. * Ujihara (1914) also claims to have infected a
monkey with this species, but his experiment is very questionable.

* Some of the “ amoebae” figured appear to be Lamd/ia.

132 THE AMOEBAE LIVING IN MAN

Mathis (1913 a) has shown that the Tonkin monkeys (Macacus rhesus
and M. tcheliensis) are commonly infected with two species of Entamoeba
which appear to be indistinguishable from £. coli and E. histolytica.
The one, with 8-nucleate cysts, he named Léschia legeri ; the other, with
4-nucleate cysts containing chromatoid bodies, L. duboscqi. Mathis
thus regarded these species as distinct from the closely similar forms in
man, and Mathis and Mercier (1917 c) have since attempted to show that
morphological differences exist between FE. coli and E. legeri. They fail
to prove their case, however, and advance no character of the latter which
may not also be discoverable in the former.

Prowazek (1912 a) had previously found an Entamoeba in an orang-
utan (Simia satyrus), and named it E. pitheci. This “species” appears to
be a mixture of Mathis’s two species. Later, Behrend (1914) found
amoebic cysts in Mf. rhesus—said to resemble those of £. coli, but
measuring 8-25 mw in diameter, and apparently also belonging to both
the species of Mathis.

Swellengrebel (1914) has also described an Entamoeba, which he
named E. chattoni, from M, rhesus. It appears to resemble a strain of
E. histolytica which forms small cysts—the diameter of the cysts of
E. chattoni being given as 8-9. It is said further, however, that no cysts
with more than two nuclei were ever found. Swellengrebel identifies his
amoeba with that of Chatton (1912 a), but not with those of other authors.
Macfie (1915) has recorded an amoeba from a monkey (Cercopithecus
petaurista) on the Gold Coast. It was believed to have caused a fatal
dysentery to its host, though the evidence appears inconclusive. It is
said to measure 12-30 w in diameter (stained), and to contain both bacteria
and red blood corpuscles. No cytological characters of any systematic
value are recorded. The organism is said to form cysts, with a diameter
of 12» to 33m: but the figures of them show unmistakable specimens of
Blastocystis. Theamoeba was named Entamoeba cercopithect.

Finally, Eichhorn and Gallagher (1916) in America record an out-
break of spontaneous amoebic dysentery among captive spider monkeys
(Ateles ater). Eight showed typical amoebic ulceration of the intestine,
and two developed amoebic liver abscesses. The amoebae are quite
unrecognizable from the figures. The authors state that “no special
attempt was made to determine the species of the amebas concerned” ;
and they also state that attempts to infect cats by feeding them on
dysenteric stools containing the amoebae were unsuccessful. They
conclude that ‘these negative transmission experiments suggest that the
parasite found is of a different species from that in man.” It is obvious,
however, that such an experiment is meaningless, and would have been
equally negative if they had made it with active forms of E. histolytica.

From the foregoing * it seems fairly clear that several different species
of monkey are infected with at least two different Entamoebae, which are
not yet distinguishable with certainty from EF. coli and E. histolytica.
Monkeys appear, moreover, to be subject to amoebic dysentery and

* I have not included some observations recorded by Greig and Wells (1911), who
state that they were able to cultivate amoebae from the faeces of 53 monkeys (Aacacus
sp. ?); because in their tables I can find no evidence,that they found parasitic amoebae in
any of their animals. Craig’s (1912 a) statement that these authors found amoebae
indistinguishable from those of man in all the 53 monkeys they examined therefore
appears to me to be incorrect.

AMOEBAE IN MONKEYS 133

amoebic abscess of the liver. It seems probable, therefore, that they
harbour a non-pathogenic Entamoeba like E. coli, with an 8-nucleate
cyst, and a pathogenic parasite like E. histolytica, with a 4-nucleate cyst.
These two species, if really distinct and valid, should probably be named
as follows :—

(1) Entamoeba pitheci Prowazek, 1912, emend.

Syn. :
Entamoeba pitheci Prowazek, 1912 (pro parte).
Léschia legeri Mathis, 1913.
? Entamoeba cercopitheci Mache, 1915 (pro parte).
Entamoeba legeri Mathis & Mercier, 1917.

Non-pathogenic. As yet indistinguishable from E. coli. Observed
also by Wenyon (1908), Brumpt (1909), and Behrend (1914), but not
named by them.

(2) Entamoeba nuttalli Castellani, 1908.

Syn. :
Entamoeba pitheci Prowazek, 1912 (pro parte).
Léschia sp. Chatton, 1912.
Léschia duboscqi Mathis, 1913.
Entamoeba chattoni Swellengrebel, 1914.
Entamoeba cercopitheci Macfie, 1915 (pro parte).

Probably a facultatively pathogenic tissue-parasite—causing dysentery
and liver abscess. At present indistinguishable from E. histolytica.
Observed probably by Musgrave and Clegg (1904), Strong (vide Kar-
tulis, 1913), Noc (1909), ? Franchini (1912), Behrend (1914), and Eich-
horn and Gallagher (1916), but not named by any of these authors.
Swellengrebel’s form is, perhaps, a distinct species, but this is still
unproved. )

I have observed the cysts of both these amoebae in the faeces of
Macacus rhesus* which I examined in London in the course of my work
with Dr. H. H. Dale (vide Dale and Dobell, 1917). At present I am
unable to distinguish them from those of E£. coli and E. histolytica
respectively, and I think it by no means impossible that the amoebae are
really identical with these species. If this is so, then E. pitheci Prowazek
(with its synonyms) becomes a synonym of £. coli (Grassi) Casagrandi et
Barbagallo, and E. nuttalli Castellani (with its synonyms) a synonym of
E, histolytica Schaudinn. At present there are not sufficient data to
determine this point, and the question can only be decided by further
observation and experiment. There is, at all events, as yet no proof that
monkeys harbour Entamoebae in any way different from those of man.
LEE SUES BETES a UES SR BS aR peheh ced aes By RE Seg SONNE DOW MER SEND rd NS NeoPa

* Attempts which were made to infect two of these monkeys with Z. histolytica
were negative.

134

X.

NOTES ON CERTAIN OTHER AMOEBOID ORGANISMS
DESCRIBED FROM MAN.

I HAVE already had occasion, in the preceding pages, to notice a
number of questionable amoebae which have been described from human
beings. Some at least of these are probably not really amoebae, but
other organisms or even cells belonging to the human body. Many of
the “amoebae” found in urine, for example, undoubtedly belong to
this last category. There are, however, several other “amoebae” which
have not yet been discussed, but which must be mentioned in any work
which aims at dealing with all the amoebae of man.

It has already been noted that the “amoebae” described originally
by Lambl (1860) were, in all probability, not amoebae but Trichomonas.
It has also been noted that the original ‘‘ giant amoebae” of Kartulis
(1885), from cases of dysentery, were also probably not amoebae, though
what they really were I have been unable to determine. In addition,
we have seen that the amoebae described by Noc (1909), Gauducheau’s
(1908) “Entamoeba phagocytoides,” and several other amoebae supposed
to be more or less parasitic in man, were all either free-living organisms
or mixtures of these with one or other of the amoebae which really
live in the human intestine. It is not necessary to say more about
these “species” here, and I shall therefore now confine my attention to
certain other ‘“‘amoebae”’ which have not yet been discussed.

The Amoebae cultivated from Human Stools and Liver-abscess Pus.—
It is now generally recognized that none of the amoebae of man can be
cultivated in the media ordinarily used for the cultivation of free-living
forms: and it will now be generally admitted that all the species obtained
in cultures made from stools or from liver-abscess pus belong to the
latter category. The following “species,’’ which have been named, have
been cultivated from human faeces:

Amoeba lobosa guttula, A. lobosa oblonga, A. spinosa, A. diaphana,
A. vermicularis, A, reticularis—all described and named by Celli and
Fiocca (1894 a).

Amoeba gruberi Schardinger (1899).

Entamoeba tropicalis Lesage (1908).

Amoeba hominis Walker (1908).

Amoeba lobospinosa Craig (1912).

There are others, but these are the species most often cited, or con-
fused with the amoebae living in man. In my opinion not one of these
species, with the exception of Schardinger’s,* is identifiable from the

* This is the organism renamed “ Amoeba punctata” by Dangeard (1910)—an easily
recognizable and common form which has been frequently studied and almost as
frequently named.

DOUBTFUL AMOEBOID ORGANISMS 135

description given. Consequently, I think all the names will have to be
abolished. There are also many other imperfectly described amoebae
recorded from stools, but fortunately most of them have not been named.

Several different workers have cultivated amoebae from the pus of
liver abscesses. In every case these were free-living species, and not
E. histolytica—so far as can be determined from the descriptions.
Gauducheau (1912) obtained his “ Entamoeba phagocytoides” from this
source, and Musgrave and Clegg and others had previously obtained
other amoebae in a similar manner. Liston and Martin (rg11) and
Martin (1911) described two species of “amoebae from liver abscesses ”
—a “large” and a “small.” Their cultures were obtained originally
from Wells, in India, whose paper on “ Aerial contamination as a fallacy
in the study of amoebic infections by cultural methods” (Wells, 1911)
probably contains the true explanation of the origin of their organisms.
Liston and Martin’s “small amoeba” I am unable to identify, from the
incomplete description. Their “large amoeba” is—as I have elsewhere
(1914) pointed out—a species closely related to the forms placed in the
genus Hartmannella by Alexeieff (1912 a), of which the type is H.
hyalina Dangeard (= Amoeba hyalina Dangeard, 1910).

“Leydenia gemmipara Schaudinn, 1896.”—In ascitic fluid from two
patients with abdominal malignant growths, Leyden and Schaudinn
(1896) discovered some peculiar bodies which they interpreted as
amoebae. To these Schaudinn gave the new name Leydenia gemmi-
para; but he stated subsequently (Schaudinn, 1903) that they were
really the naked amoeboid forms of the shelled rhizopod Chlamydophrys
stercorea Cienkowski (1876). This organism, according to Schaudinn,
lives coprozoically in human faeces, but the amoeboid forms of it may
be found occasionally in the intestine. “Leydenia” has been regarded
as a very questionable rhizopod by many workers already, and | fully
share their doubts.

I have never encountered Chlamydophrys in human faeces ; and the
amoeboid forms, if they ever do occur in the intestine of man, must be
excessively uncommon. It should be remembered that Schaudinn
recognized only two species of amoebae in man—E. coli and E.
histolytica: and I suspect that his ‘‘ Chlamydophrys” amoebae were really
E. nana, which occurs so commonly but which he did not know. I have
already pointed out that this is probably the correct interpretation of
the “Chlamydophrys” found by Elmassian (1909), and so far as I am
aware no other worker has “confirmed” Schaudinn’s observation. I do
not doubt that Chlamydophrys may occasionally occur in stale human
faeces, though I have never encountered it. It certainly occurs in the
dung of various animals, and in sewage beds.* But that there is any
connexion between this organism and “ Leydenia,’ or any of the
amoebae of the human bowel, I regard as highly improbable. We do
not know how Schaudinn convinced himself of the identity of these
different forms, as he adduced no evidence for his statements ; and it is
not to be forgotten that most of his other statements about the amoebae
of man were incorrect.

I have little doubt that “Leydenia” itself was not an amoeboid

* I have studied the organism in the faeces of frogs and toads (Dobell, 1909): and
I may record that my friend the late Mr. C. H. Martin showed me, some years ago,
a fine culture of Ch/amydophrys which he obtained in material from a sewage farm.

130 THE AMOEBAE LIVING IN MAN

organism at all. The “amoebae” were really cells from the body-cavity.
Their movements, which consisted chiefly in change of shape and the
emission of “pseudopodia,” and their ‘‘ contractile vacuoles ’—which
contracted only once in a quarter of an hour—appear to me to supply
insufficient data for assuming that they must have been amoebae; whilst
the ‘‘plasmogamy” and the “reproductive” phenomena (budding,
division, etc.) observed and figured are far too much like aggregations
of degenerate body-cells to be accepted as evidence of rhizopod affinities.
Without making a careful study of the cellular elements in ascitic fluid
I am not prepared to identify Schaudinn’s “ organisms” more precisely.
But I find it impossible to regard “ Leydenia” as an amoeba without
very much stronger evidence, and its identification with Chlamydophrys
I regard as a mere speculation.

“Amoeba miurai Ijima, 1898.’—Shortly after the discovery of
“ Leydenia,’ Miura discovered a similar “organism” in a Japanese
woman. Itwas described by Ijima (1898), who named it Amoeba miurai.
According to him, it was present in the serous exudate from the pleural
and peritoneal cavities of the patient, who was diagnosed by Miura as a
case of “pleuritis and peritonitis endotheliomatosa.” Two days before her
death the same “organisms” were also found in the bloody mucous
stools. Ijima noted the resemblance of “A. miurai”’ to ‘ Leydenia,”’
and also that it was ‘ discovered under almost identical circumstances ”’ ;
but he regarded it as belonging to a different species. No movements,
save change of shape, were observed, and no contractile vacuoles; but
nuclei varying in number from one to three were present. Inspection
of the figures leaves no doubt in my mind that “ Amoeba miurai”’ is
closely similar to “ Leydenia,’ and is to be interpreted in the same way :
that is to say, it is not an amoeba, or protozoon of any kind, but a
misinterpretation of cells from the pleural and peritoneal cavities. This
“organism,” therefore, should be deleted from the list of amoebae living
in man.

“Amoeba pulmonalis Artault, 1898.’—I have already mentioned—
when discussing E£. gingivalis—that Artault (1898) found an “ amoeba”
in a lung cavity. He proposed to call it Amoeba pulmonalis, It was
present in small numbers in the sputum, among leucocytes, etc., and
is said to have undergone changes in shape, though apparently it was not
motile. Beyond the statement that the ‘‘amoeba” showed “a nucleus
and a vacuole” very clearly, no account of the morphology was given.
It was not figured ; and the author states that it was “ perhaps the same
as the Amoeba vulgaris’’—an organism with which I am unacquainted.
There is nothing in Artault’s description to indicate that he was dealing
with an amoeba; and even if he was, there is no character given which
can enable its species to be determined. It seems highly probable,
indeed, that his “amoebae” were really cells.

Brumpt (1910) states that he has also observed “A. pulimonalis.”
He gives figures of the “organism” which are not very convincing. In
his account I find no evidence that the things which he depicts are
really amoebae. They might equally well have been cells.

It may be added that two different amoebae may really occur
occasionally in the sputum—E. gingivalis, from the mouth, and E. his-
tolytica, from a lung abscess ora liver abscess which has ruptured into
the lung.

“Entamoeba undulans Castellani, 1905.” — Castellani (1905), has

DOUBTFUL AMOEBOID ORGANISMS 137
described an ‘‘amoeba” from the intestine of man and named it
“FE, undulans.” From his description and figures there can be no
doubt that these “ amoebae ’’—which possessed an undulating membrane
—were really degenerate forms of Trichomonas hominis, with which his
patient was said to be also infected. This has already been pointed
out by a number of workers (Wenyon (1913), Hartmann (1913), etc.).
The non-flagellate, amoeboid, degenerating forms of various species
of Trichomonas are now familiar to all workers who have studied these
organisms. It may be noted that they were probably first mistaken for
amoebae in man by Lambl (1860) ; and that they were seen and first
correctly identified as degenerating flagellates* by Cunningham (1871).
Since then they have many times been mistaken for amoebae.

“ Craigia.””—Craig (1906) has described a remarkable organism to
which he originally gave the name Paramoeba hominis—believing it to
be similar to the marine Paramoeba eilhardi Schaudinn.t Subsequently
it was placed in the new genus Craigia by Calkins (1912). The organism
is said to live in the intestine, to be pathogenic, and to possess both
amoeboid and flagellate stages in its life-history. The amoeboid forms
apparently resemble E. coli and measure 10-25 w in diameter. They
form multinucleate cysts which liberate broods of flagellates ‘‘ 10-20 p
in diameter.” Although Craig has published two accounts of this
organism (1906, 1910), and made frequent reference to it in other works,
he has never advanced adequate evidence for its existence. To prove
that an organism such as he postulates really exists, requires further
evidence,— evidence, moreover, of quite a different order from anything
which he has yet been able to adduce. There is, nevertheless, abundant
evidence in Craig’s works that he was not, when he wrote, sufficiently
familiar with the common intestinal amoebae and flagellates of man to
be able to distinguish a new organism of the type described—-supposing
it to exist. Obvious sources of error and confusion were not excluded ;
and in the absence of all essential information concerning the cytology
of “ Craigia,” I am unable to accept his conclusions.

So far as I am aware “Craigia” has been found subsequently by only
one other worker—Barlow (1915). This writer not only “ confirmed ”
Craig’s observations but extended them by discovering a new species.
According to him, there are really two species of “ Craigia,’”—C. hominis
and C. migrans. His descriptions of both, however, serve only to
confirm my suspicions that all these organisms are in reality a mixture
of other and more familiar species of amoebae and flagellates. 1 have
never found any organism which resembles Craigia: and until it has
been vouched for by some independent and competent protozoologist—
familiar with all the intestinal protozoa of man, and in possession of first-
rate preparations of all the stages described—I am unable to believe in
its existence. The name Craigia should therefore, in my opinion, be
regarded as a nomen nudum, or as a partial synonym of at least two other

* Cunningham, however, called the organism ‘“‘ Cercomonad A,” and was not aware
that it was a 7y¢chomonas.

+ Iam inclined to believe that P. ec/hardi is itself probably a fictitious organism,
formed by combination of a rhizopod with the swarmers of an alga. I find Schaudinn’s
account of it far from convincing.

138 THE AMOEBAE LIVING IN MAN

generic names (a flagellate and an amoeba), until adequate evidence can
be produced in favour of its retention.

“ Amoeba pyogenes Verdun & Bruyant, 1907.”—This name has been
given to an amoeba found in the pus from an abscess in the malar
region by Verdun and Bruyant (1907, 19074). It is said to be actively
motile and to measure 20-35 w in diameter, with a nucleus of 8-15 w;
and to form cysts measuring 6-15 and containing from one to four
nuclei. The endoplasm is described as granular, and filled with digestive
vacuoles containing red blood corpuscles and leucocytes. According to
its describers the species resembles “ Amoeba coli Losch” (i.e., E. histo-
lytica), and the organisms found in similar situations by Kartulis, Flexner,
and others (i.e., probably E. gingivalis).

In my opinion this species was probably a mixture of E. gingivalis
and cells of various sorts. At present there is insufficient evidence for
regarding it as new. Smith and Barrett (1915, 1915 a), however, regard
it as probably an independent species—related to, or possibly identical
with, some very questionable “amoebae” found by Ribbert in Stensen’s
duct. They also suggest that it has affinities with “ EF. mortinatalium,”
mentioned below.

The Amoebae described by McCarrison (1909).—1 have already identified
two of the intestinal amoebae studied by McCarrison (1909). His
“‘ Amoeba I” was certainly, as he supposed, £. coli, His “Amoeba II”
was equally certainly a name given to cells from the intestine—not to
E. histolytica as he surmised. It only remains to add that the “ third
amoeboid body” which he briefly described and figured was undoubtedly
the flagellate Giardia (=Lamblia) intestinalis, and therefore not an
intestinal amoeba at all.

“ Endamoeba mortinatalium Smith & Weidman, t1g1o.”’—Smith
and Weidman described, in 1910,* some “amoebae’”’ which they found
in the kidneys, liver, and lungs of a still-born foetus. They subse-
quently described another similar “infection” in a 2-months old
syphilitic child (Smith and Weidman, 1914). The amoebae in both
cases were believed to be of the same species, for which the name
E. mortinatalium was proposed. Smith and Weidman (1914) state that
similar “ protozoa” had been found in the kidneys of a syphilitic new-
born infant by Ribbert, and later by the same worker in the parotid
glands of non-syphilitic children ; and that Jesionek and Kiolemenoglou
found similar bodies in the kidneys, liver, and lungs of a syphilitic
foetus. Smith and Weidman consider their “amoebae” to be identical
“with those in at least Ribbert’s first case and in that of Jesionek and
Kiolemenoglou.”

“E, mortinatalium” is described as an amoeba measuring usually
22-30 w in diameter. Its nucleus is from one third to one half the
diameter of the whole organism, and contains a large central
karyosome. It should be noted that the “organism” was never seen
alive, so that although “ pseudopodia”’ are mentioned there is no
evidence of its motility. From the figures and description I am entirely at
a loss to know why the bodies in question are regarded as amoebae at all.

* According to Smith and Weidman (1914) their first paper was published in the
University of Pennsylvania Medical Bulletin, 1910, but 1 have not been able to
consult it.

DOUBTFUL AMOEBOID ORGANISMS 139

To my mind it seems probable that they are merely large cells in the
organs studied. It seems premature to discuss the systematic position
of this amoeba, therefore, before we know that it really is one.

It is difficult to understand how infection takes place if “ E. mortina-
talium” really is an amoeba. Smithand Weidman (1914) consider that in
both their cases the mother was primarily infected, and the child acquired
its infection “from some as yet unknown focus in her.” ‘ Doubtless,”’
they say, “‘ the parasites are harmless for the adult mother ; while for the
fetus, especially when impaired by luetic taint, they may well prove
pathogenic and capable of destroying life.” Doubtless: but one would
like some evidence before accepting these speculations as facts. For the
present it will suffice to notice that “ E. mortinatalium” differs radically
from all species of amoebae proved to occur in man.

“Entamoeba polecki Prowazek, 1912.”—Most of the species of
Entamoeba described at various times by Prowazek have already been
identified : but there remains one of his species which has not yet been
mentioned. This is an organism—or organisms—which he named
“ Entamoeba polecki” (Prowazek, 1912). It has been justly remarked by
James (1914) that “this writer worked with very insufficient material” :
and the present species forms no exception. James, indeed, even doubts
whether the name “EF. polecki’’? was really given to “entamoebae or
something else.”

In a very brief note Prowazek (1912) states that he found the
organism in a pig, and also in the stools of a child, in Saipan (Ladrone
Islands). It is said to be “ 10-12 p large” ; and it is stated further that
in “ older faeces’? amoebae measuring only 5 « were also observed, which
copulate in pairs and encyst. A few figures are given, but they are
difficult to interpret. There is nothing to show that this “species” is
not a mixture of amoebae* from pigs and human beings confused with
free-living species from stale human stools.

In view of the astonishing habitat and development ascribed to this
organism, the absence of evidence in support of Prowazek’s statements,
and the fact that no recognizable description has yet been published, I
consider that “ E. polecki’”” is a species which should be rejected.

“Entamoeba dysenteriae europeae Popper, 1917.”—Under this name
another “new amoeba” has recently been described by Popper (1917).
It was found in the stools of patients suffering from dysentery in Galicia
and Hungary. Although it is described at length, it is impossible to
recognize, in the description, anything which greatly resembles an
amoeba. Most of the cytological characters necessary for its determina-
tion are not recorded ; and the account shows such obvious ignorance
of amoebae generally, and of the intestinal amoebae of man in particular,
that it can hardly be doubted that Popper’s “new amoeba” is really
a mixture of cells—and possibly other bodies—from dysenteric stools.
I shall not discuss this “ organism” further. But I may note the following
as samples of the author’s observations, and in justification of my
rejection of this species. “ E. dysenteriae europeae” is a “ colourless cell”

* I note that Brumpt (1913) and Pestana (1917) regard “ Z. folecki”’ as a synonym
of E. coli, and that Hartmann (1913) retains it as an independent species. Neither of
these views appears to me justifiable.

140 THE AMOEBAE LIVING IN MAN

filled with “numerous granules” showing great activity caused by
“ protoplasmic streaming.” Its size is very variable, but is “‘ estimated ”’
at “30 and more.” The nucleus is large and vesicular or kidney-shaped.
The “amoeba” forms bud-like “‘ pseudopodia” very rapidly, but does
not undergo locomotion. The “cysts” are “large,” “without distinctly
recognizable membrane.” Their contents were not investigated, but
they are said to have been tightly packed with highly refractile masses.
It is said, finally, to resemble ‘‘ Amoeba coli Loesch” and to differ from
“Amoeba histolytica Schaudinn ”—which are, of course, two different
names for the same organism.

There is thus every justification for removing this improperly named
and probably non-existent “amoeba” from among the amoebae of man
and the causes of dysentery.

The Amoebae described from Human Skin Lesions.—It has already been
noted that amoebae have been described from the skin. Carini (1912,
1912a@) has recorded two cases in which a phagedaenic ulceration
developed in the tissues surrounding the wound made in operating
upon a liver abscess. He found numerous amoebae in the subcutaneous
tissues and in the exudate from the ulcers; and he regarded them as
belonging to E. histolytica—apparently believing that they invaded the
skin secondarily from the pus draining from the liver abscess. Dagorn
and Heymann (1912) have described a similar case, and a report upon
the amoebae found has been published by Gauducheau (19124). Several
other similar cases are also on record. The first appears to be that of
Nasse (1892), who did not see the amoebae alive, and whose material
was admittedly badly fixed and prepared.

It is, I think, still very doubtful, from the descriptions published,
whether the ‘‘amoebae”’ found in such phagedaenic lesions really were
E. histolytica. No proper account of them has yet been published,
and no protozoologist with an adequate systematic knowledge of the
amoebae appears to have studied any of these cases. Although Carini
merely says* that his amoebae had affinities with “ Amoeba tetragena,” he
appears to assume their identity with this species (i.e., Entamoeba histo-
lytica). He has given no proper description of them. Gauducheau
(1912) gives an account of his “amoebae” which leads one to doubt
whether they were amoebae at all. He did not attempt to identify them,
because he holds—if 1 understand him correctly—that it is not possible
to distinguish different species of amoebae from one another. It thus
seems clear that further investigations by a competent protozoologist
are necessary; and until more information is available, it seems to me
unprofitable to discuss the nature of these “ amoebae.”

In the same category may be placed the “amoebae” found by J. L.
Maxwell (1912) is cases of ‘‘ fistulous disease of the buttocks ” observed
in Formosa. Although the author states it as his opinion that the pus
from the fistulous tracks contained ‘‘ amoebae, conforming I believe to
the type of entamoeba histolytica,” and would regard these as the cause
of the condition, there seems at present to be no evidence of any weight
in support of such an interpretation. The exact systematic position of

* “ Les amibes présentent des caractéres les rapprochant de l’Amoeba tetragena”’
(Carini, 1912).

DOUBTFUL AMOEBOID ORGANISMS 141

these ‘‘ amoebae” can hardly be discussed before it has been shown that
they really are amoebae.

Many other amoeboid organisms have been described from man. In
fact there is hardly an organ in the body from which somebody, at some
time, has not obtained ‘‘amoebae.” In this respect the amoebae have
been maltreated in much the same manner as the coccidia, and it would
not be difficult to compile a long list of ‘‘pseudo-amoebae”’ from
medical literature. But further discussion of these appears to be
unnecessary ; and it seems best to leave them in that oblivion into
which they have, for the most part, justly fallen.

142

BIBLIOGRAPHY.

[THE following Bibliography has been compiled with great care. It contains all those works
to which reference is made in the text, but is not ‘‘ complete” in the sense that it contains
all works in which the Amoebae of Man are mentioned, or described at second hand. It is
hoped, however, that but few works of material importance from a zoological standpoint
have been omitted. With the exception of three works which I have not been able to obtain
anywhere, and which are duly noted, I have read or consulted in the original all the publica-
tions cited : and I have made every effort to eliminate and correct, by direct transcription
from the originals, the numerous and oft-repeated errors in most of the previously published
bibliographies dealing with the subject. ]

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10°

148 THE AMOEBAE LIVING IN MAN

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15

to

INDEX.

Acknowledgements, 3, 4.
Ameba, 18.
Amoba buccalis, 93.
—- dysenterica, 93.
Amoeba (genus), 12, 13, 15, 18, 19, 333 ortho-
graphy, 18.
— binucleata, 123.
— blattae, 15.
— colt, 20, 21, 27, 28, 71-77, 129.
— dentalis, 92.
— diaphana, 134.
— diploidea, 123.
— dysenteriae, 20, 27 n, 28, 35, 44.
— gingtualis, 92, 93.
— grubert, 134.
— honiints, 134.
— hyalina, 135.
— intestinalis, 20, 28, 71.
— kartulist, 92, 93.
— limax, 13, 33, 97, 101, 102, 103, 105, 114n,
122n.
— lobosa coli, 20, 28.
— — guttula, 134.
— — oblonga, 134.
— lobospinosa, 77, 134.
— miurai, 136.
— pulmonalis, 94, 136.
— punctata, 102, 134n.
— pyogenes, 92, 93, 138.
— reticularts, 134.
— spinosa, 134.
— tetragena, 140.
— urinae granulata, 127.
— urogenitalts, 129.
— vaginalis, 20, 28, 30, 125, 129.
— vermicularis, 134.
— vuloaris, 136.
Amoeba coli, 15, 20, 21, 26, 27, 28, 73, 75, 76,
125, 129, 140.
— — felis, 20, 24, 28.
— — mitis, 71, 74.
— dysenteriae, 20, 26, 27, 28.
— dysenterica, 26n.
— histolytica, 140.
— intestini vulgaris, 71, 74.
— urogenitalis, 20, 30, 125, 129.
“« Amoeba I,” 77, 88, 138.
‘*__ JJ,” 30, 138.
Amoebae cultivated from liver-abscess pus, 134,
135; from stools, 134.
—, cultivation of, 12, 22, 23, 69, 70, 9I, IOI,
102, 105, III, 115, 124, 132M, 134, 135.
—, Cunningham’s, 9, 71, 72.
—, genera of, in man, 15-19; synopsis, 19.
—, Lambl’s, 8, 9, 134, 137.
—, Lewis’s, 9, 71.

Amoebae, Noc’s, 29, 134.

—, species of, in man, 19.

Amoebea gengivalis, 92.

Amoebiasis (term), 12.

Amoebic abscess of liver (term), 22; patho-
genesis, 35, 37-

— dysentery (term), 10, 22; pathogenesis, 36

sq.

Amiba buccalis, 92.

Ateles ater, amoebae in, 132.

** Atypical strains of 2. hzstolytica,” 60, 61.

Bacteria in Z. histolytica, 62, 63, 64.

Badger, amoebae in, 68.

‘* Batonnets sidérophiles,”’ 89 n.

Best’s carmine, 7.

Bibliography, 142-151.

Binucleate forms of Z. col, 81; of &. histo-
lytica, 44.

Bladder, amoebae in, 125, 126.

Blastocystis, 72, 124, 127, 132.

Blood, amoebae in, 35.

Brain abscesses, amoebae in, 22, 35, 37; 39-

Budding, in Z. gingivalis, 97; in £. histo-
lytica, 240.

Caries (dental), amoebae in, 99.

Carriers of Z. histolytica, 25, 37S8q; contact,
38; convalescent, 38.

Cats, Z. coli in, 90, 91; £. histolytica in, 22,
23, 24, 56, 57, 60, 61, 67sq; liver abscess
in, 23, 68.

Cells mistaken for amoebae or cysts, 13, 14, 29,
60, 61, 80n, 97, 126, 127, 128, 134, 136, 138,
139, 140.

Centrioles, in Z. gingivalis, 96; in Z. histo-
lytica, 32, 433 in 1. btitschiét, 110, 114.

“© Cercomonad A,”’ 137 n.

Cercopithecus petaurista, amoebae in, 132.

Cerebral abscess, 22, 35, 37, 39-

Chilomastix mesnili, 29, 53, 102.

Chlamydophrys stercorea, 101, 102, 135, 136.

Chromatoid bodies, of £. coli, 88-90;
E. histolytica, 45-48, 64.

Chromidia, 45, 90.

“« Coccidia’’ of Grassi, 73.

Cockroach, amoebae of. See Exdamoeba blaitae.

Contact carriers, 38.

Convalescent carriers, 38.

Craigia (genus), 137, 138.

— homints, 137.

— migrans, 137.

Crystalloid bedies, 45.

Cunningham’s ‘‘amoebae,”’ 9, 71, 72.

Cyclical changes around karyosome (Z. Aésto-

lytica), 34.

of

INDEX

** Cyst-carriers,” 38.
Cystitis, amoebae in, 125, 126, 128.

Dientamoeba (genus), 18, 19, 122-124; type
species, 19.

— fragilis, 19, 122-124; cysts, 124; degenera-
tion, 123 ; description, 122 ; division, 123 ;
food, 124; habitat, 124; history, 122;
morphology, 122, 123; movements, [22 ;
nomenclature, 122; nuclei, 122, 1233
occurrence, 122, 124; pathogenicity, 124 ;
transmission, 124 ; uninucleate forms, 123.

Dimastigamoeba grubert, 102.

Discovery of intestinal amoebae of man, 9, 71;
of oral amoebae, 8, 92.

Dogs, amoebae in, 30, 67, 129-131; amoebae
transmitted to, 21, 23, 130; amoebic dysen-
tery in, 129, 130, 131; liver abscess in, 130,
ie tie

Doubtful amoeboid organisms, notes on, 134-
14!.

Dysentery, amoebic, 9 sq, 36 sq.

— bacillary, 9 sq, 36n, 80n.

** Eccentric nuclei” of amoebae, 33, 96.

Emetine, action of, on Z. co, 91; on £. gin-
givalis, 94, 100; on £&. histolytica, 70; on
E. histolytica in cats and dogs, 130, 131;
on £. nana, 109; on Z. biitschliz, 120, 121.

Endameba (genus), 18, 19.

— buccalis, 92.

— colt, 71.

— gengivalis, 92, 93 n.

— gingivalis, 92.

— hominis, 71.

— intestinivulgarts, 71, 74.

-- maxilaris, 93 0.

Endamoeba (genus), 15-19 ; type species, 15, 18.

— blattae, 15, 16, 17, 58, 65, 73.

— coli, 20.

— confusa, 92, 93-

— dysenteriae, 20.

— gingivalis, 92.

— gingivalis (buccalis), 92, 93-

— histolytica, 20.

— intestino-vulgaris, 71, 74.

— mortinatalium, 138, 139.

‘« Endamoeba Gros,”’ 92, 93.

Endolimax (genus), 18, 19,

species, 19.

— intestinalis, 101, 103.

— nana, 18, 19, 82, 101-109, III-114, 121, 122,
135; autogamy, 108; cultivation, 105;
cysts, 106 sq; description, 104-109;
division, 105; food, 104, 105; glycogen
in cysts, 107 ; habitat, 105 ; history, 1o1 sq;
inclusions in cysts, 108; morphology,
104 sq; movements, 104; multiplication,
105 ; nomenclature, IOI sq; nucleus, 104,
105; occurrence, 108; parasitized by
Sphaerita, 106; pathogenicity, 109; pre-
cystic forms, 106; strains, 108; super-
nucleate cysts, 108; synonymy, I01;
treatment, 109.

— Williamsz, 110, 112.

Entameba, 18, 19.

Entamiba, 18, 19.

Entamoeba (genus), 15-19, 20-100;

species, 15, 18, 19.

— africana, 20, 25, 29.

IOI-109; type

type

153

Entamoeba aulastomi, 30, 50.

— brasiliensis, 20, 29, 52, 71, 77, 89.

— buccalis, 92, 93.

— biitschiiz, 18, 77, 110, 112, 113, 118.

— caviae, 68 n.

— cercopithect, 132, 133.

— chatton2, 132, 133.

— cobayae, 68.

— coli, 19, 20, 26, 71-92, IOI, 103, 110, I12,
113, 131, 133; autogamy, 16, 25, 87, 91;
chromatoid bodies, 88-90 ; conjugation, QI ;
cultivation, 91; cysts, 84 sq; description,
78 sq ; division, 81 ; early stages of develop-
ment, 90 ; encystation, 83; food, 80; gly-
cogen in cysts, 87, 88; habitat, 80; history,
71 sq; infection, mcde of, 90 ; morphology,
78 sq ; movements, 78 ; multiplication, 81 ;
nomenclature, 26, 71 sq; non-pathogenicity,
80; nucleus, 78-79; occurrence, 9I ; pre-
cystic forms, 83; races distinguishable by
size of cysts, $4; sexual cycle, 91; super-
nucleate cysts, 86, 87: synonymy, 71 ; trans-
mission, 12, 90; treatment, QI.

— coli communis, 13, 20, 71, 77.

— colt var. telragena, 20, 29.

— dysenteriae, 17, 20, 26, 31.

— dysenteriae europede, 139, 140.

— gingivalis, 8, 16, 17, 19, 92-100, 136, 138 ;
animals infected with, 98 ; conjugation, 98 ;
cysts, 97, 98; description, 95 sq ; dissemina-
tion, 98 ; division, 97 ; food, 95-97 ; habitat,
98 sq; history, 92 sq; inclusions, 95-97 ;
inoculation into animals, 94 n, 99; mor-
phology, 95; movements, 95; nomencla-
ture, 92 sq; nucleus, 95, 96; occurrence,
98 sq; pathogenicity, 8, 93, 98 sq; pre-
cystic forms, 97; synonymy, 92; treat-
ment, 99.

— hartmannt, 20, 29, 51, 53) 54) 71, 77:

— histolytica, 9, 12, 13, 15-17, 19, 20-71, 73-76,
80, 94, IIO, 113, 120, 121, 125-131, 133,
138, 140; acquisition of infection, 55 ;
animal infections, 67sq; autogamy, 16,
25, 643 bacteria in, 61-64; carriers of,
378q, 66; chromatoid bodies, 45-48, 64;
commensalism, 61 sq; conjugation, 16, 35,
440, 64, 65; cultivation, 22, 23n, 69, 70;
cysts, 45sq ; description, 31 sq; dissemi-
nation, 69; division, 40sq}; encystation,
448q; excystation, 55sq; experimental
infection of man, 56, 60; food-vacuoles,
313 geographical distribution, 70, 71;
glycogen in cysts, 46; habitat, 35, 125
sq; history, 20-25; incubation period, 56 ;
life-history, 34, 65; morphology, 31 sq;
movements, 31; multiplication, 40 sq ;
nomenclature, 25-31; nucleus, 32; nu-
trition, 35; pathogenesis, 36sq; patho-
genicity, variation in, 58 sq; persistence of
infection, 40; precystic form, 45; races
distinguishable by size of cysts, 51 sq;
sexual phenomena, 64; supernucleate
cysts, 49 sq; synonymy, 20; treatment,
70 ; virulence, variation in, 58 sq.

— hominis, 27, 71, 75, 76, 77-

— kartulist, 92, 93.

— legeri, 77, 132, 133.

— maxillaris, 92, 93-

—_ minuta, 20, 25, 29; 45) 53, 65,77.

— minulissima, 20, 29, 53.

154 THE AMOEBAE

Entamoeba muris, 17, 64, 68, 69, 88.

— nana, 18, 101, 102, 103.

— nipponensis, 29.

— nipponica, 20, 29, 77; IOI, 102.

— nuttallt, 131, 133.

— phagocytoides, 101, 134, 135-

— pithect, 132, 133.

— polecki, 77, 139.

— pulmonalis, 92, 94.

— vanarum, 17, 30, 45, 46, 48, 50, 58, 64n, 65,
83 n, 87, 91.

— fenuts, 20, 29, 53+

— letragena, 20, 25, 29, 34, 53, 65, 110, 126.

— tetragina, 29.

— letragona, 20, 29.

— tropicalis, 77, 134.

— undulans, 136, 137.

— venalicum, 20, 30, 130, 131.

— williamst, 30, 71, 77, 89, 112.

Entamoeba Loeschi, 71, 77.

— Schaudinni, 20, 28.

Fistulous disease of buttocks, amoebae in, 140.

Fixation, methods of, 6.

Flies as disseminators of Z. histolytica, 69.

Foetus, amoebae in human, 138, 139.

‘* Free-living amoebae” (James), IOI, 102, IIO,
III.

Frogs, amoebae in. See Extamoeba ranarum.

““Gamogonic cysts,” of Z. colz, 82, 91; of
EL. ranarum, 83.

Genera of amoebae in man, 15-19; synopsis of,
19.

General amoebiasis, 37.

‘* Giant amoebae” (Kartulis), 21, 134.

Giardia ( = Lamblia) intestinalzs, 108, 131 0,
138; in £. coli 80.

Glycogen in cysts of £. co/z, 87, 88; of Z. his-
tolytica, 46; of E. nana, 107; of L. bzitschliz,
116, 117 ; staining methods for, 6, 7.

Guinea-pigs, amoebae in, 68, 78; inoculated
with £. gingivalis, 99; parasitized by £.
histolytica, 68.

Haematuria, amoebae in, 126.

Harimannella, 135.

— hyalina, 135.

Hepatic abscess, amoebae in, Io, 21, 22, 23, 68,
130, 131, 132; pathogenesis, 37,

Hepatitis, amoebic, 37.

‘* histolytica form,’’ 66.

“* histolytica phase,’’ 65, 66.

History, briefly summarized, 8 sq.

Intestinal amoebae of man, discovery of, 8, 9;
plurality of species of, 10.
Intracellular stage of Z. hzstolytica, 35.
Introduction, 1-4.
Todamoeba (genus), 18, 19, type
species, 19.
— btitschlii, 19, 102, 110-121; cysts (‘‘I.
cysts”), III, 112, 115-119; description,
113 sq; division, 115; encystation,
117 sq; food, 114; glycogen in cysts,
116, 117; habitat, 114; history, I10 sq;
morphology, 113 sq; movements, 113;
nomenclature, I10 sq; nucleus, 113, 114;
occurrence, 119, 120 ; pathogenicity, 120;
precystic forms, I17; races, 119}; super-

110-121;

LIVING IN MAN

nucleate cysts,
treatment, 120.
“‘ Jodine cysts” .(‘*I. cysts’); 18, 110j;maus
12.
Iodine, in diagnosis, 6; solution, 6 n.
Ipecacuanha, action on &, histolytica, 70.
Lsospora heminis, in E. colt, 80.

118; synonymy, I10;

**Joodcysten,’’ I10, III.

Lambl’s ‘* amoebae,’’ 8, 9, 134, 137-

‘* Large amoeba from liver abscesses ” (Liston
and Martin), 135.

Leech, amoeba of, 30, 50.

Lewis’s ‘‘ amoebae,”’ 71.

Leydenia gemmipara, 1355 136.

*¢ Limax,” IOI, 103.

““iimax”’ amoebae, 13, 33, 97, IOI, 102, 103,
105, I14n, 122n; in human mouth, 97.

‘* Limax amoeben”’ (Flu), 101, 102.

Liver abscess, amoebae in, I0, 21, 22, 233; in
badger, 68 ; in cats, 23, 68; in dogs, 23, 68,
130, 131; in man, 10, 37; in monkeys, 131,
132, 133; pathogenesis, 37.

Liver-abscess pus, amoebae cultivated from,
23n, 134, 135.

Loschia (genus), 16, 19.

— colt, 7l.

— duboscgt, 132, 133.

— histolytica, 20.

— legert, 132, 133-

— (Viereckia) tetragena, 17, 20.

Lung abscess, amoebae in, 35, 136.

Macacus spp., amoebae in, 131, 132, 133.
‘* Macrocysts” of Z. colt, 91; of Z. histolytica,

65.

Malpighiella refringens, 103.

Material and methods, 5-7.

Maxillary abscess, amoebae in, 92, 93.

Methylblue-eosin stain (Mann), 7.

Mice, amoebae in, 78. See also Autamocba
muris.

‘* Microcysts” of &. coli, 913; of Z. histolytica,
6

ss minuta” form of Z. histolytica, 45.
‘* minuta phase,” 61, 65, 66.
Monkeys, amoebae in, 30, 77, 78, 131-133.

Naegleria ranarum, 103. ;

Neutral red, staining of Z. hzstolytica with, 31.

Noc’s ‘‘ amoeba,” 29, 134.

‘‘Non-pathogenic Z. /e¢ragena,” 20, 30, 101,
102.

Nucleophaga, 106; in £. blattae, 106.

Paramoeba eilhardi, 137.

— hominis, 137.

Parasites of ‘A. dimax,’ 106; of Z. dlattae,
106; of £. xana, 106, 122n; of Z&. hasto-
lytica, 63, 64.

Parotid duct and gland, amoebae in, 138.

Peritoneal cavity, amoebae in, 135, 136.

Phagedaena, amoebae in, 140.

Pig, amoebae in, 139.

‘* Plages sidérophiles,” $9 n.

FPoneramoeba (genus), 16, 17, 19+

— coli, 17.

— histolytica, 20.

Present state of knowledge, 8-14.

INDEX

Proctamoeba (genus), 16, 19.

Protomyxomyces coprinartus, 71, 72.

** Pseudolimax,” 110, III, 112.

** Psorospermi”’ (Grassi), 71, 73.

Pyorrhoea alveolaris, amoebae in, 8, 93, 94, 98,
99.

Rabbit, infection of, with Z. Azstolptica, 69.

Rats, amoebae in, 78; amoebic dysentery in,
69; infection with Z. histolytica, 69.

Riggs’ disease, amoebae in, 93. See Pyorrhoea.

** Schizogonic cysts,’’ of Z. col’, 82, 83, 86n;
of Z. ranarum, 83 n.

Schizogony in &. colz, 81-83, 102; in Z. géng?-
valis, 97; in £&. htstolytica, 35, 443; in
L, btitschliz, 115, 117.

Simia satyrus, amoebae in, 132.

** Single contour” of entamoebic cysts, 47 n,
49, 50n.

Skin, amoebae in, 35, 140.

** Small amoeba ” (Wenyon), ror.

‘*Small amoeba from liver abscesses ” (Liston
and Martin), 135.

Small intestine, infection of, with Z. Azstolyttca,
35.

Species of amoebae in man, synopsis of, 19.

Sphaeritain ** Amocba limax,” 106; in &. nana,
106, I22n.

** Spherical bodies’’ (Wenyon), 110, III.

Spleen abscess, amoebae in, 35.

“*Spores’”’ of Z. hzstolytica, 24n.

155

‘¢Sporulation ”’ of Z. ezngivalts, 98; of Z. his-
tolyttca, 24, 35.

Sputum, amoebae in, 136.

Staining methods, 6, 7.

Supernucleate cysts of #. aulastomi, 50; of
E. colt, 50, 51, 86, 87; of &. histolytica, 49
sq; of Z. mana, 50, 51, 108; of &. ranarum,
50; of 7, d2itschliz, 50, 118.

Survival of Z. hzstolytica outside body, 45; of
cysts of Z. histolytica, 47, 48; of cysts of
E. coli, 90; of cysts of 2. nana, 107.

‘* Symbiosis ’’ of Z. szzgzvalis, 99; of ZB. htsto-
lytica, 36.

Synopsis of genera and species, 19.

“* tetragena form,” 66.

‘* tetragena phase,” 65, 66.

Tetramitus mesnilt, IOI, 102.

‘‘ Third amoeboid body,” 138.
Trichomonas, 9, 80, 102, 128, 134, 137.
— hominis, 9, 1373; in &. colt, 80.

— vaginalis, 128.

Urinary amoebiasis, 35, 125 sq.
Urine, amoebae in, 30, 125-129.

Vagina, amoebae in, 125, 126.

Vahlkanipfia, 101, 102, 103, TIO.

— nana, 101, 103.

— punctata, IOI, 102, III.

Viereckia (subgenus), 17, 19.

Volutin in cysts of 2. nana,
biitschliz, 116.

1077 OGMLs

Joun Bae, Sons AND Danietsson, Lrp., Printers, 83-91, Great Titchfield St., London, W. r.

PLATE I.

"All the figures in this plate are drawn from preparations fixed with sublimate-alcohol (with or
without acetic acid), and stained by Mann’s methylblue-eosin method (modified), unless a different
technique is indicated. Magnification 2,500 diameters.

Figs. 1—6, Entamoeba histolytica.

Fig. 1. Z. histolytica, active amoeba scraped from an intestinal ulcer (cat), showing typical
structure of nucleus, and partly digested remains of three ingested red corpuscles. (Fixed
in Bouin’s fluid.)

Fig. 2. Precystic amoeba, from human stool (subacute amoebic dysentery).

Fiys. 3, 4, 5. Uninucleate, binucleate, and quadrinucleate cysts respectively : from same case as
preceding. Strain forming cysts (living) with mean diameter of ca. 13°5. Note nuclei,
chromatoid bodies (red), and clear spaces in 3 and 4 representing glycogen vacuoles.

Fig. 6. Binucleate cyst showing glycogen (red patch). Fixed with Carnoy’s fluid, stained
with Best’s carmine.
Figs. 7, 8,9. “ndolimax nana.
Vig. 7. Free 2. nana, in human stool. Note nucleus, ingested bacteria, pseudopodium, etc.
Fig. 8. Mature 4-nucleate cyst of 2. zana, from same case.
Fig. 9. Binucleate cyst, containing glycogen mass (red). Fixed in sublimate-alcohol, stained
with Best's carmine.
Figs. 10, 11. /odamoela biitschlii.
Fig. 10. Free /. dytschiz?, in human stool. Note nucleus, ingested bacteria, etc.

Fig. 11. Mature cyst. Note nucleus, volutin granules (pink), and clear space occupied by °

glycogen mass in living cyst (‘*I, body”),

Figs. 12—15. Hntamoeba coli.

Fig. 12. Active amoeba, in human stool. Note structure of nucleus, ingested bacteria, etc. (and
compare with fig. 1).

Fig. 13- Precystic amoeba, from same case. Note nuclear structure, absence of ingested food,
etc. (and compare with fig. 2). .

Fig. 14, Mature 8-nucleate cyst, from same case. Note nuclear structure (compare with figs. 12
and 13, and figs. 1—5), and small chromatoid body (red).

Fig. 15. Binucleate cyst, containing large glycogen mass (red). Fixed i ’s fluid i
with Best’s carmine. (Cf. figs, 6 aK) : oe ee ea eee aegened

PLATE

Il:

PLATE Il.

All figures—unless otherwise stated—from specimens fixed in sublimate-alcohol and stained
with Heidenhain’s iron-haematoxylin and eosin. Magnification 2,500 diameters.

16. Lntamoeba histolytica. Large active amoeba containing three red blood-corpuscles,
nes from stool of a case of amoebic dysentery (human). Stained with Weigert’s iron-haema-
toxylin and eosin.

Fig. 17. Zntamocba coli. Large active amoeba, from human stool. Observe the nucleus,
ingested bacteria and other food-inclusions in endoplasm, etc. (Compare with fig. 16.)

Figs. 18—31. Zndolimax nana.

Figs, 18—23. Active amoeboid forms of Z. zana, from human stools: showing various forms of
nuclear structure, etc.

Fig. 24. Eight nuclei from other individuals of Z. naa, to show some of the commoner types of
karyosome seen in this species.

Figs. 25, 26, 27. Unioucleate, binucleate, and quadrinucleate (mature) cysts respectively.
Figs. 28, 29. Cysts (mature) of 2. nana with filamentar and granular inclusions.

Fig. 30. Very small mature cyst.

Fig. 31. Unusually large cyst.

Figs. 32—42. Jodamoeba biitschiii.

Figs. 32—34. Active /. d7itschlit amoebae from human faeces, Note nuclear structure, ingested
micto-organisms, etc. (Compare with figs. 16—23.)

Figs. 35, 36. Precystic amoebae. Observe the changes taking place in nuclear structure, freedom
from cytoplasmic inclusions, etc.

Fig. 37- Organism just encysted. Note structure of nucleus (compare with preceding figures),

volutin granules (pink) in cytoplasm, and small clear space representing glycogen mass in
living organism,

Fig. 38. A large cyst, badly fixed, and showing much protoplasmic shrinkage, etc. [For
comparison with the figure of the cyst of ‘* Entamoeba” biitschiri published by Prowazek
(19124, Pl. XVIII, fig. 21).]

Fig. 39. Mature cyst, containing large glycogen mass (red). Fixed in Carnoy’s fluid, stained
with Weigert’s iron-haematoxylin and Best’s carmine.

Figs. 40, 41, 42. Mature cysts, showing typical structure of nucleus, volutin granules, and
glycogen “ vacuole ” (represented as a clear space after removal of the glycogen). 41 and
42 are cysts of irregular shape, such as are often formed by this species. (The irregular

outlines are not artificially produced by the cytological reagents used.) Stained with
haemalum and eosin.

PICAGE Eas

PLATE III.

All drawings made from amoebae in sections of intestinal ulcers of kittens experimentally
infected with Entamoeba histoltica. All material fixed with Bouin's fluid, and sections stained
as follows:

Figs. 43, 44, 48. Heidenhain’s iron-haematoxylin, fuchsin S, and picro-indigo-carmine.
Figs. 45, 46, 49. Fuchsin S, picro-indigo-carmine.

Fig. 47, Heidenhain’s iron-haematoxylin, orange G.

Figs. 50, 51. Mann’s methylblue-eosin (modified).

Figs. 52, 53. Safranin and light green.

Fig. 54. Borrel’s stain (magenta and picro-indigo-carmine).

The drawings show successive stages in the normal process of division in 2. histolytica. For
fuller description see text, pp. 41-43. (The dark bodies in the cytoplasm are remains of
ingested red corpuscles.) Magnification 2,500 diameters.

PPAR ante

PLATE IV.

All figures drawn—unless otherwise stated—from specimens fixed in sublimate-alcohol and
stained as indicated. Magnification 2,500 diameters.

Figs. 55—69. Zntamoeba coli.
Fig. 55. Active Z. coli from human stool, showing ‘‘clefts” in cytoplasm, etc. (Weigert’s
iron-haematoxylin. )
Figs. 56—69. Cysts of £. co/i at various stages in development.

Fig. 56. Newly-formed uninucleate cyst, showing minute chromatoid bodies, glycogen space, etc.
(Stained haemalum.)

Fig. 57. Later cyst, with very large nucleus preparing for first division. Note chromatoid
bodies and glycogen *‘ vacuole."’ (Mann’s stain, modified.)

Fig. 58. First nuclear spindle, (Heidenhain’s iron-haematoxylin and eosin.)

Figs. 59, 60. Binucleate cysts. Note glycogen ‘‘ vacuoles,’’ chromatoid bodies, etc. (Compare
with fig. 15, Pl. I.) Fig. 59 from cyst stained by Mann’s method (modified): Fig. 60,
haemalum.

Fig. 61. Quadrinucleate cyst. Note that all nuclei are in early stages of division—as they
usually are in 4-nucleate cysts of this species. Fixed Bouin’s fluid, stained alcoholic ferric-
chloride iron-haematein.

Fig. 62. Typical 8-nucleate cyst (mature). Note structure of resting nuclei. (Heidenhain’s
iron-haematoxylin.)

Figs. 63—66. 2. coli cysts, at various stages of development, containing chromatoid bodies of
different forms. Fig. 65 shows a mature cyst with filamentar chromatoids—probably a cyst
of the type attributed by Prowazek to ‘* Entamoeba williamsz.”” (All these cysts stained
with haemalum, progressively.)

Fig. 67. Abnormal 16-nucleate cyst of Z. co/i. (Stained with haemalum.)
Fig. 65. Very small S-nucleate cyst of Z. col’. (Haemalum and eosin.)

Fig. 69. Very small 4-nucleate cyst of Z. co/i, containing 4 resting nuclei and chromatoid bodies.
Compare with fig. 71, ete. (Haemalum and eosin.)

Figs. 70—76. Entamoeba histolytica.

Fig. 70. Cyst of 2. Aistolytica (uninucleate), containing very abundant chromatoid bodies.
(Fixed alcoholic picro-acetic-acid, stained paracarmine.)

Fig. an Mature 4-nucleate cyst, without chromatoid bodies. (Haemalum.) Compare with
ig 69.

Figs. 72, 73, 74. Three very small cysts of Z. Aistolytica—uninucleate, binucleate, and quadri-

nucleate respectively—belonging to a strain producing cysts (living) with an average
diameter of 6°6u. (Haemalum.)

Figs. 75,76. Two very large cysts of Z. histolytica—respectively uninucleate and quadrinucleate
—belonging to a strain forming (living) cysts with an average diameter of 15. (Haemalum,)
Compare with preceding figs. 72-74, and with figs. of Z. coli on this plate.

PEATE

IV.

PLATE V.

All figures drawn from preparations fixed with sublimate-alcohol and stained with Heidenhain’s
jron-haematoxylin unless otherwise indicated. Magnification 2,500 diameters.

Figs. 77—85. LZntamoeba histolytica.

Figs. 77,78. Precystic forms of Z. histolytica belonging to strains producing small cysts—
such as those shown in figs. 72—74, PI. 1V. (Stained with haemalum.)

Figs. 79, 80. Precystic amoebae belonging to strains forming cysts of larger size—such as those
shown in figs. 3—5, Pl. I.

Fig. 81. Precystic amoeba from a stale stool. Degenerate and invaded by bacteria. Compare
nucleus with those in figs. 77—80.

Fig. 82. Precystic amoeba with rod-like chromatoid bodies—resembling ingested bacteria.
(Haemalum.)

Fig 83. Precystic amoeba parasitized by bacteria. (Alcoholic iron-haematein.)

Fig. 84. Degenerate uninucleate cyst, belonging to same strain—also parasitized by bacteria.
(Alcoholic iron-haematein.)

Fig. 85. Degenerate uninucleate cyst from another infection. (Weigert’s iron-haematoxylin.)
The cyst contains 3 chromatoid bodies, and a number of parasitic bacteria, like those in
figs. 83 and 84. [For fuller description of these forms see text, p. 63 e/ Seq.)

Figs. 86—89. 2ndolimax nana.

Fig. 86. Abnormal 8-nucleate cyst of Z. nana. (Haemalum.) Compare with figs. 27, 30, 31,
PI. IL.

Figs. 87, 88, 89. Individuals of #. nana parasitized by Sphaertta sp. Figs. 87, 88, from
specimens stained with haemalum: showing respectively an amoeba containing two
sporangia, and one with a single spore-morula and younger stages. Fig. 89, specimen
stained with iron-haematoxylin, containing one large and one small spore-morula.

Figs. 902-92. Dientamoeba fragilis.

Figs. 90, 91. Two ordinary binucleate individuals from human stools,

Fig. 92. A uninucleate specimen.

Figs. 93, 94. Zntamoeba gingivalis.

Fig. 93. An individual with various cytoplasmic inclusions, and a nucleus with an eccentric
karyosome. From human mouth, (Case of pyorrhoea alveolaris.)

Fig. 94. Similar organism. Nucleus with central karyosome. (Compare with figs. 77—80 on
this Plate, and figs. of Z. coli and Z. A7stolytica on Plate I.)

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