•iv'!'tv».'^T<.V

bn fl^b

THE

ETIOLOGY AND PATHOLOGY

OF

GROUSE DISEASE & FOWL ENTERITIS

THE

ETIOLOGY AND PATHOLOGY

OF ,

GROUSE DISEASE

FOWL ENTERITIS

And some other Diseases afiecting Birds

E. KLEIN, M.D., F.R.S.

\\

LECTURER OF GENERAL ANATOMY AND PHYSIOLOGY IN THE MEDICAL
SCHOOL OF ST. BARTHOLOMEW'S HOSPITAL, LONDON

WITH FIFTY-THREE ILLUSTRATIONS

iLontion

MACMILLAN AND CO.

AND NEW YORK
I 892

All rights rcsoi'cd

LIBRARY
G •

Printed by R. &. R. Clark, Edinburgh.

TO

SIR JOSEPH LISTER, BART., M.D., LL.D., F.R.S.

THIS BOOK

IS RESPECTFULLY DEDICATED

BY

THE AUTHOR

703403

PREFACE

Having been engaged for the last five years in
collecting data as to the etiology and pathology of
grouse disease, I find that the time has arrived
when I am enabled to give a coherent account of
the subject ; the number of grouse, dead or dying
naturally from the disease, which I have dissected,
and which were derived from different parts of
England and Scotland, sent to me during several
years through .the kindness of the Editor of the
Field, is very considerable, as will be fully referred
to later ; the number of cultivations from the grouse,
the number of experiments with cultivations on birds
and mammals has been sufficiently large to enable
me to draw definite conclusions as to the nature ofj
those cultivations. To the Editor of the Field, and
through him to many gentlemen, both in England
and Scotland, interested in grouse, my best thanks
are due for the valuable assistance which I received
from them. The second disease treated here is a

viii PREFACE

highly infectious and fatal disease of fowls — fowl
enteritis, which was at first believed to be fowl
cholera, to which it bears a certain resemblance,
but from which it materially differs, not so much
as to its mortality and high degree of infectiousness,
but in respect to its pathology and causation. In
order to show these differences I have prefaced the
chapters on fowl enteritis with a short chapter on
fowl cholera. The first material of fowl enteritis
on which I worked was placed at my disposal by
Mr. W. Cook of Orpington, the well-known author
on, and successful breeder of poultry, through the
intervention of Mr. C. Wellington ; both these
gentlemen, notably Mr. Cook, have rendered me
invaluable service, for which I beg to return them
my best thanks.

The third disease, about which I intend to make
a few remarks, is the infectious disease of young
pheasants known to pheasant - breeders as "the
cramps." While engaged in Ayrshire for the Field,
in the summer of 1887, in inquiring into the
pathology and etiology of the grouse disease, I
had the opportunity of studying the cramps at
Blairquhan, and to the keeper there, Mr, Douglas,
my thanks are due for supplying me then and after-
wards with materials both as livino- and dead
specimens.

PREFACE . ix

In conclusion I wish to thank my friend Mr.
Andrew Pringle for having kindly prepared many
of the micro-photograms, illustrating the microscopic
appearances, both of the grouse disease and of the
fowl cholera and fowl enteritis, and to my friend
and pupil, Mr. E. C. Bousfield, for some of the
micro-photograms and of the photograms of the
cultivations.

St. Bartholomew's Hospital
January 1892.

CONTENTS

PART I

THE GROUSE DISEASE

CHAPTER I

PAGE

Nature of the Disease ...... i

CHAPTER n
Symptoms and Pathology . . . . . . i^

CHAPTER HI

Character of the Bacillus of the Grouse Disease . 24

CHAPTER IV
Experiments with Culture by Subcutaneous Inocula-
tion 33

CHAPTER V
The Autumnal Disease ...... 44

CHAPTER VI
Further Characters of the Bacillus . . . .51

CHAPTER VII
General Considerations . , . , . -58

CONTENTS

CHAPTER VIII

PAGE

A Pathogenic Bacillus resembling the Bacillus of

THE Grouse Disease ...... 63

PART II

FOWL CHOLERA, FOWL ENTERITIS, AND "CRAMPS"
IN YOUNG PHEASANTS

CHAPTER IX
Fowl Cholera ........ 79

CHAPTER X

Fowl Enteritis — Nature of the Disease ... 86

CHAPTER XI

Cultivation of the Bacillus of Fowl Enteritis . 92

CHAPTER XII

Experiments on Fowls . . . . . .100

CHAPTER XIII
Attenuation of the Virus of Fowl Enteritis . . 107

CHAPTER XIV
" Cramps " in young Pheasants . . . . .121

Figures i to 53 . . . . . • 132-140

PART I
THE GROUSE DISEASE

CHAPTER I

NATURE OF THE DISEASE

The epidemic disease which affects Red Grouse
(Lagoptcs scoticMs) in Scotland, the North of England,
Wales, and Ireland during the spring and the summer, ^
and which, particularly during April, May, and June
of some years, makes such sad havoc amongst them,
is the one which goes under the name of the Grouse
Disease. Not that grouse and allied species are
exempt from other affections, but the disease which
for years past, in one or another part of the north
of the British Isles, has carried away annually large
numbers of these birds, is the one known as the
"Grouse Disease." Nor do we mean that this
disease occurs amongst the red grouse in spring and
summer-time only, for there is good evidence to show
that though these are the seasons in which the plague,
when occurring, is most conspicuous owing to its
extensive mortality, the autumn and winter are not free

THE GROUSE DISEASE chap.

from the presence of the malady, which owing to
various circumstances to be mentioned hereafter is
then less conspicuous, and may even be altogether
overlooked.

The fact that year after year in one or another
tv<<(ii^-'j part of the country the disease is or becomes
epidemic, is in itself strong prima facie evidence
that it is an infectious illness ; further, the now well-
recognised occurrence of isolated fatal cases of the
same illness amongst the grouse in one or another
part of a given district during autumn and winter,
makes it certain that the disease has become endemic,
and is ready, when favouring opportunities offer them-
selves, to assume the epidemic character. This is
the case with some other acute infectious endemic
maladies affecting men and animals which, at certain
times of the year and under certain other favouring
conditions, assume the epidemic character. Such a
change is, however, a slow and gradual development.
Take, for instance, Asiatic cholera, endemic in Bengal.
It is present in isolated cases all the year round in
some part or another of Bengal. During the cold
season the number of cases gradually increases ; by
the end of the cold season, during the spring and
beginning of summer, the cases become so numerous,
and the spread of the disease so conspicuous, even
outside the endemic area, that the disease possesses

NATURE OF THE DISEASE

the epidemic character ; then, with the commencement
of the rainy season, it gradually diminishes again, and
reaches its lowest level towards the end of this and
the commencement of the cold weather. This same
character is observed in countries in which cholera is
not endemic, and which remain free from it for years,
e.g. Syria, Egypt, and European States. Cholera im-
ported into these areas gradually and steadily works
itself up into an epidemic, and equally gradually
again declines, the summer and spring being the most
favourable seasons in which it thus becomes epidemic.
During autumn, and towards the winter, the cases
become less numerous and milder, and as such may
occur as isolated cases all through the winter.
Towards spring they become again more numerous
and more severe, and in spring may cause a
recrudescence of the epidemic. Scarlet fever, diph-
theria, and typhoid fever are other well-known
instances in which acute endemic infectious disorders
during certain seasons show marked exacerbations.
In grouse disease the month of April, and the com-
mencement of May, are the times in which the
disease, as a rule, gradually by numbers and inten-
sity (fatal cases) becomes conspicuous. During the
end of May and the commencement of June it is at
its height, and then again declines during July ; but
fatal cases occur also during August, September,

THE GROUSE DISEASE

October, and later, but their numbers are, for obvious
reasons, smaller during this time. That a moor, or
even a whole district, is and remains free from the
1 epidemic during one or more years may be, and as
a rule is, due to a variety of conditions which also as
regards epidemics of other infectious diseases are
known to occur ; some of these we do know and
call secondary or accessory conditions. Assuming
the disease to be an infectious disease, there is
nothing extraordinary in the fact, occasionally ob-
served, and used by some as an argument against
the infectious nature of the malady, to wit, that in
a given country a series of moors are seriously im-
plicated, while here and there one or another moor
is only slightly affected, or apparently remains free
from the disease. I say there is nothing extraordinary
in this because there may be, for all we know, a
variety of circumstances at work which favour, or
disfavour, the spread of infection from one moor to
the neighbouring moor. In human epidemics this
fact is observed repeatedly. One locality becomes a
hot-bed for the disease ; the neighbouring locality
remains fairly free from disease, or has only few cases.
In a third locality at a greater distance the disease
is again rife, and so on. Whether, in the case of the
grouse disease, birds are in better condition to resist the
disease in one locality than in the neighbouring one ;

NATURE OF THE DISEASE

whether birds affected with the disease carry it from
one locality, not to the one lying next, but to a moor
some distance off; whether in one moor there are
present conditions as to situation, heather, water, etc.,
which are favourable or unfavourable to the develop-
ment and spread of the disease, — all these are points
which may, and probably (see below) do, play a part
in the dissemination of the plague.

A disease which year after year assumes the char-
acter of an epidemic at particular seasons, which com-
mences as isolated cases, and gradually works itself
up into a serious epidemic, i.e. spreads from a few to
many individuals, and again in the same way gradually
declines ; further, a disease which has such uniform
and typical characters, both as to symptoms and
pathology ; bears on the face of it the character of an
acute infectious disease.

The late Dr. Cobbold, in his well-known brochure
on the grouse disease, enunciated that the cause of
the grouse disease is a parasite, in fact a nematoid
worm belonging to the group of strongyles i^Stron-
gyhis pergracilis), for in the intestines of grouse
dead of the disease this parasite was constantly /
present. Numerous grouse which he examined, and
which succumbed either in the epidemic of 1872 or
1873, and others examined in later years, contained
these parasites in abundance, though " without doubt "

6 THE GROUSE DISEASE chap.

(Cobbold, Parasites, p. 439) "the occasional presence
of numerous tape-worms {Tcenia calva) hastened the
consequent fataHty." This view has always been
strenuously opposed by various writers and observers,
both lay and veterinary or medical, inasmuch as it
was considered quite insufficient to account for the
disease ; for it was pointed out by Dr. Farquharson
and Mr. Wilson (see below), and can be easily veri-
fied, that the same parasites occur also in birds not
subject to the disease. Now, taking into consideration
that the disease is well characterised both in its symp-
toms and its pathology ; that the same parasites un-
doubtedly occur in grouse not afflicted with the grouse
disease ; that grouse dead of the disease are found
sometimes plump, sometimes emaciated ; and, above
all, that grouse dead of the disease, in several instances
when examined, showed no presence of the stron-
gylus : it must be evident that the parasites some-
times present in the intestine cannot be the real cause,
the causa cattsans, of the disease. That cause must be
of a different nature. It must be of a nature which
works rapidly, or fairly rapidly, which affects the
system as a whole, and which easily spreads amongst
the birds. We shall presently see that one of the
most prominent pathological changes in the diseased
grouse is an acute congestion of one or both lungs,
and that this change, whether very severe or less

NATURE OF THE DISEASE

severe, is independent of the presence of strongylus.
I dissected numerous birds dying or dead of the
disease in 1887, and showing the characteristic lung
change, and must confess that I was extremely sur-
prised to find the strongylus in some cases entirely ivH|p6\h.
absent, in others present in very small numbers,
though taenia calva was present in aH in large num-
bers, just as large as in birds killed but not affected
with the grouse disease.

Amongst various writers on the grouse disease, none
deserve a higher place than Dr. H. Colquhoun and
Dr. D. G. F. Macdonald. By selecting the opinions
of these two writers amongst a host of writers who,
by letters to the Times and the Field or other publica-
tions, have contributed to this question, I am practi-
cally quoting in a condensed form the most note-
worthy summary of the various opinions offered. All
these opinions as to the cause of grouse disease do
not in reality touch the cmisa caitsans at all, but
the conditions or secondary causes, which favour, and
play an important part in, the appearance of the
grouse disease in the epidemic form. Amongst these
conditions may be mentioned : — {a) bad season ;
{b) bad or insufficient food ; and {c) overstocking.
It must be obvious that as in many other acute in-
fectious disorders amongst men or animals these
conditions play a very important part, inasmuch as

\

8 THE GROUSE DISEASE chap.

they create a less resisting, more susceptible material
for the carrying out of -infection. The more adverse
the season — inclemency of the weather, frost, etc. —
the more sparse or inferior the natural food, the less
hardy and strong are the birds when the time arrives
which is most favourable for the development of the
disease, and therefore the more easily do they take
infection. This is well understood as applicable to
other infectious disorders. A strong individual, not
weakened by bad and insufficient food or by bad or
inclement weather, will resist the infection much more
easily than one previously brought down in general
health and strength. Suppose there are, on a given
moor, a large number of birds in this weakened state,
and suppose there exists on this moor the active
cause of the disease, it stands to reason that, there
being present a good many birds of no great resisting
power, one or the other of them will take the infec-
tion, from these others will take it, and so on, so
that, after a short time, a large number of foci will
thus be started acting not only on the remainder of
the weaker but also on the stronger birds. But sup-
posing, on the other hand, the birds in early spring
are well and strong ; though the active cause of the
disease be present on this moor, yet it will not easily
find, as it were, a susceptible individual, and conse-
quently the disease will show itself either only in

NATURE OF THE DISEASE

Sporadic cases, or will be barely appreciable, or will
be absent altogether.

And so also with overstocking and over-preserva-
tion. This is admitted by most to have an injurious
effect, inasmuch as it is liable to produce too great
a struggle for existence and a consequent weakening
of the birds on the one hand, and, on the other, a
possibility, owing to the great number of birds left,
of cases of disease lingering on through the winter
as mild cases, but ready in the following spring to
cause an epidemic of the severe fatal disease (see
below). Dr. R. Farquharson, in a letter to the Lancet,
Sept. 1874, was the first to state the opinion that
the grouse disease was of the nature of a con-
tagious fever, since " the idea of an epidemic and
infectious fever fits the facts already obtained better
than anything else" {Grouse Disease, Macdonald,
p. 129).

He argued that, as great differences prevail in the
degree of loss of flesh observed — some dead or dying
birds being found as plump as in their healthy con-
dition, whilst others are found reduced to mere skele-
tons,— this is in favour of its specific or constitutional
nature ... "so that whilst those dying from a very
acute attack, or whose constitutions are unable to
withstand its debilitating influence for more than a
brief period, retain the outward appearance of health,

THE GROUSE DISEASE

Others present all the conditions of excessive exhaus-
tion " (/.f. pp. 130 and 131).

I may here at once state that this explanation
offered by Dr. Farquharson, as to the difference
of condition observed in birds dead of the grouse
disease, is one which I think perfectly correct and
one for which I shall be able to offer direct experi-
mental evidence. Moreover, it is one which from
analogy, i.e. such as is observed in other infectious
fevers, would, a priori, suggest itself. The next
noteworthy contribution made to our subject is by
Mr. Andrew Wilson, Lecturer on Zoology and Com-
parative Anatomy, Edinburgh, who in the Edinburgh
Medical Jottrnal gave the first accurate description
of the pathology of the disease. '* Mr. Wilson has
had many opportunities (Macdonald, pp. 145 and 146)
afforded him for the examination of grouse dying from
the disease, and he states that, in most of the birds
he examined, he observed a markedly congested
appearance of the mucous surface of the digestive
and respiratory tracts. Repeated dissections and
careful observations satisfied him beyond doubt of
the almost invariable presence of this lesion in birds."
Mr. Wilson "unhesitatingly" prefers the theory of
Dr. Farquharson, i.e. that the grouse disease is an
infectious fever ; " and the reasons for enlistment
under the banner of the epidemic hypothesis, rather

NATURE OF THE DISEASE

than under that of parasitism, are chiefly those de-
rived from the consideration that the former theory
fully comprehends all the conditions which the
phenomena of the grouse disease may present ; and
further, that the theory may be applied to the ex-
planation of points and facts, of which the theory
of helminthiasis can take no heed.

** We thus find that these birds are almost invariably
affected by both kinds of entozoa — most specimens
to a moderate extent, some to a very great degree.
But it is exceedingly rare to meet with a bird in
which not a single parasite of either kind is present.
Out of many cases examined not a single bird was
found actually free from the characteristic tape-worm,
and in one or two doubtful instances only could it be
asserted or suggested that none of the round worms
were present. '

" That the presence of these parasites was uncon-
nected with the death of the great majority of the
birds examined is, I think, proved by the want of
any causal or obvious relationship between the infesta-
tion and the fatal issue ; and also by the absence, in
most cases, of the signs of fatal parasitism — such as
inanition, producing the * pining ' condition, actual per-
foration, morbid appearance of the muscular tissues,
etc. No one can deny that the presence of para-
sites in large numbers undoubtedly causes death in a

cU

12 THE GROUSE DISEASE chap, i

specific and readily understood manner ; but this fact
will not explain the nature of the cause or disease
which operates so fatally, without any exceptional
development of parasites, and certainly without the
slightest appearance of inanition or other concomitant
symptoms of helminthiasis. Outside the parasitic
hypothesis, applicable as that theory is to a certain
class of cases, there lies, I am convinced, the great
bulk of fatal instances, the exact cause of which fatal-
ity must be sought for in some lesion analogous to that
involved in the idea of the epidemic theory. We
must thus account for the death in numbers of grouse
which, with what one may consider a normal and
actual degree of parasitism, yet die and succumb to
disease, and which on examination present well-
nourished bodies, firm, healthy, muscular tissue, and
other signs of presumed health " (Macdonald, pp.
147 and 148).

CHAPTER II

SYMPTOMS AND PATHOLOGY

It must be obvious that in a disease affecting birds
that cannot be kept in captivity for any sufficient
time, the accurate and systematic observation of the
symptoms of the disease is fraught with great diffi-
culties, and it is therefore impossible to give a con-
nected account of the course of the disease. From
what I can gather of shrewd and observant keepers,
and what I myself observed on the grouse in 1887
while staying on the moors in Ayrshire and in Cum-
berland, thanks to the editor of the Field, and the
kind and generous hospitality of Mr. G. Bailey
Worthington, then holding Blairquhan, and of the
late Sir Frederick Graham of Netherby Hall, on
the borders of Cumberland, and also from what I
observed of ammers and other birds experimentally
infected with the disease in the laboratory, I fully
agree with Dr. Farquharson that the grouse disease
is an acute infectious fever — being, in fact, an acute

'4

THE GROUSE DISEASE

infectious pneumonia. In the severe forms, such as
are observed during epidemics in May and June,
the birds, once affected, rapidly, in a day or two,
show great loss of muscular power and seek the
water. The loss of muscular power is apparent by
the impeded flight, this being sluggish, being per-
formed with unwillingness, and not for any normal
time, that is to say, if on the wing the birds soon
drop again. Wherever there is a trace of water the
affected birds will seek this, and in many instances
in severe epidemics their bodies are picked up in
numbers on the shores of such water. As the post-
mortem examination shows, the congestion of the
lungs forms a conspicuous pathological symptom ;
and it is therefore clear that, as in acute congestion
of the lung in man and animals, also in the grouse,
under these conditions, fever must be a prominent
symptom, and the above appearances during life
easily account for this.

Also the peculiar alteration in the call of the
diseased birds, this being somewhat feeble and
hoarse, would be a natural result of the lung con-
gestion, since this condition would prevent the birds
from uttering their call with normal force, and the
congested state of the mucous membrane of the
larynx would cause the sound to be less clear and
sharp, and more hoarse. The more or less ruffled

II SYMPTOMS AND PATHOLOGY 15

condition of the feathers of the back and throat,
or rather the absence of the smooth condition and
metallic lustre of the feathers of the back observed
in diseased birds, could also be accounted for by
their feverish condition.

The colour of the eyelids, bright in the normal
state, is not of the normal bright orange colour in
birds dead of the disease. In every severe congestion
of the lungs, such as obtains in the severer forms of
the grouse disease, there is a general venous conges-
tion of all parts consequent on the engorgement of the
venous part of the heart supplying the pulmonary
artery ; and therefore also the skin all over the body,
including the eyelids, would participate in this venous
congestion. The natural aspect of the bright pigment
of the eyelids would therefore become altered by the
presence of this venous congestion, and the eyelid
would look duller.

Pathological appearances. — The birds that die dur-
ing a severe epidemic, such as obtains in the spring
and early summer, show the following post - mortem
appearances : the crop is generally more or less full
of undigested heather ; as a rule this is the more
conspicuous the better nourished and the plumper
the bird, and the better preserved the pectoral
muscles are. I have dissected birds which were
emaciated, the legs bare of feathers, and in which

1 6 THE GROUSE DISEASE riiAi'.

the crop contained only little or no food ; and in
such cases the disease of the lungs is as a rule,
but not without exception, not very extensive, only
a portion of one or both lungs being congested.
Such is the case in some birds in the spring and
summer epidemic ; such is the case in birds that die
in the autumn. In the majority of instances in which
the dead birds are found plump — as for instance
during the height of the spring epidemic — the lungs
show a great deal of congestion, the crop is full, and
the legs are not bare. I conclude from this that, in
these cases the disease being severe, it rapidly kills
the animal, or more correctly, if the animal rapidly
succumbs to the disease it will show less loss of
flesh, and the crop more full of heather than when
the animal lingers on for some time — for then the
fever would inevitably cause great loss of flesh, and
the animal, as is to be expected, would present appear-
ances of emaciation. This view, enunciated by Dr.
Farquharson and Mr. Wilson, seems to me the best
to harmonise with the facts.

The chief changes are undoubtedly those found
in the lungs. The mucous membrane of the larynx
and trachea is of a dark colour, hypera^mic ; this
is the more pronounced the more marked the
lung change. The latter shows deep coloration
in the greater part of either one or both organs,

1 1 S YMP TOMS AND PA THOL OGY 17

the hind portions being, in these cases, the ones
chiefly affected ; or both lungs are uniformly con-
gested, being in some cases of a dark purple-red
colour. On microscopic examination it is found that
in the congested parts the large and small blood-
vessels are uniformly distended and filled with blood,
and that the air-spaces of the more deeply affected
parts are uniformly filled either with a homogeneous
or granular exudation, or with blood ; so that in these
parts we have a solidification of the lung which com-
pares with the condition known as the red hepatisa-
tion in Pneumonia, There is, however, no fibrine in
the form of threads noticeable in the air - spaces ;
the smaller air-spaces contain blood eiz masse, while
the large ones are filled with a homogeneous albu-
minous exudation. From this we conclude that rup-
ture of small vessels had taken place during life

(Fig- I)-

The spleen is not enlarged, and appears of a dark

colour. The liver is uniformly congested and soft ;

it is either of dark red colour or appears almost black.

On microscopic examination the large blood-vessels

as well as the capillaries of the lobules are distended

and filled with blood corpuscles. In some cases the

liver, on post - mortem examination, is blackish, or

rather is of a dark olive-green colour. In these

instances the liver cells appear granular and more or

c

1 8 THE GROUSE DISEASE chap.

less disintegrating, and contain dark brown pigment
granules ; besides, numerous clumps of dark greenish
pigment masses are found in the spaces of the intra-
lobular capillary vessels, while the large vascular
branches are full of blood. Evidently in these cases
the disease is of longer standing than a few days, for
the capillary blood-vessels are not found filled with
blood corpuscles, as would be the case in recent cases,
but contain the remnants of disintegrated blood cor-
puscles, viz. masses of amorphous pigment. The
dark greenish-black condition of the liver is always
associated with great emaciation of the animals, and,
as is proved by microscopic examination, is associated
with a large amount of blood pigment, so that from
this we conclude that there must have existed, some
time before death, stasis in the blood-vessels, disin-
tegration of the blood corpuscles, and change of the
blood pigment. This is in agreement with what we
mentioned on a previous page as to the emaciation
of birds indicating that the disease must have lasted
for some time. In all instances, however, the liver
cells themselves contain brown pigment granules.
The kidneys are congested, in some instances leading
to haemorrhage into the tissue of the kidney. The
intestinal mucous membrane shows patchy congestion,
and the same is the case with its serous covering,
which in most instances is congested in many places,

II SYMPTOMS AND PATHOLOGY 19

sometimes to a considerable extent. In some cases
the peritoneum appears very moist in these locaHties ;
that is to say, there is a small amount of exudation. I
have seen a few cases in which haemorrhage has taken
place, showing itself in the form of petechise in the
peritoneum.

From this we see that the grouse dead of the
disease show a definite series of pathological appear-
ances, in which the congestion of the lungs and liver
is very conspicuous.

The above considerations led me to conclude that
the disease was an acute infectious disease, and owing
to the constantly congested and diseased condition of
the lungs and liver, I thought it possible that the
disease belonged to the group of diseases in which
the microbe caused the disorder by its multiplication
in the blood. The first birds dead of the disease
which I dissected in June 1887, ^^ Ayrshire, w^ere
examined with the view of discovering the microbe
in the blood. Cover-glass specimens were made of
the heart's blood and of the blood of the lung, i.e.
a thin film of blood was dried on cover-glasses and
then stained in the usual manner with aniline dyes
— methyl blue or gentian violet ; but on micro-
scopic examination no bacteria could be discovered. ;
Numerous tubes, some containing nutrient gelatine, '\
others nutrient Agar, were inoculated in the usual

THE GROUSE DISEASE

manner with the heart's blood ; they all remained
sterile on incubation and free of any growth. The
heart's blood of at least half-a-dozen grouse dead
of the disease, or killed while diseased, was thus
examined, but no bacteria could be discovered in it.
Lungs, liver, and kidneys were taken out from the
animals after death, and were cut into small bits and
preserved in spirit or Muller's fluid. Sections were
then cut and stained with aniline dyes and mounted
in the usual way, but here also no bacteria could
then be found. This was the position of things
which I described in my first report in the Field in
July 1887. I have in 1888, 1889, and 1890 exam-
ined many birds that died of the disease in April,
May, June, or July ; and also in them I could not
discover in the blood of the general circulation, either
in cover-glass specimens or by cultivation, any bac-
teria. From this I am enabled to say that in a large
number of the grouse dead of the grouse disease in
the spring and summer, — that is, dead with the char-
acteristic pathological appearances of the lungs and
liver, — the blood of the general circulation does not
contain any microbes discoverable by the cover-glass
or culture test. In 1888 therefore I directed my
attention to examining in the same way, in the fresh
condition, the tissue of the lung and liver ; and,
with the exception of one or two doubtful cases, in

SYMPTOMS AND PATHOLOGY

all Others, whose number is considerable, I obtained,
by the culture test in nutrient gelatine, evidence of the
presence in the lungs and the liver of numbers of
bacteria belonging to one and the same species ; for qr(: ^
when a particle of the lung or liver tissue is rubbed over
the slanting surface of nutrient gelatine, or when it is
shaken up in a test-tube with melted sterile nutrient
gelatine, and the latter is then poured into a sterile
glass dish, allowed to set and then incubated — that is
if a plate cultivation in gelatine is made — colonies of
one single bacterial species are obtained. By making
sections through the inflamed lung or liver, after har-
dening, and staining these sections for several hours
in methyl blue, or better still in rubin and methyl
blue, it is found that in some parts some of the
capillary blood-vessels, both in the lung and in the
liver, contain continuous masses, plugs or emboli, of
the same bacterial species (Figs. 2, 3, 4, 15 and 16).^
But there are seen under the microscope extensive
parts in which the blood-vessels do not contain them.
It is from this easy to see why I failed in 1887 to
obtain the microbe from the lung or liver in cover-
glass specimens or in culture, viz. I used in both
cases a droplet of the blood of the lung or liver,

1 In sections through the hardened liver stained with rubin, then
with blue, the clumps of microbes are brought out with great dis-
tinctness as blue masses on a red ground.

THE GROUSE DISEASE

and from what has been stated just now it is clear
that it would amount to an extremely distant chance
to get some of those capillary plugs in the droplet of
blood. In 1888 I made a good incision with sterile
scissors or scalpel into the substance of the lung or the
liver — the lung or liver as a whole having previously
been well dipped in saturated solution of mercuric
bi-chloride to disinfect the surface — and from the
cut surface a particle of the tissue was cut out with
sterile scissors and used for cultivation ; here, of
course, the chances of hitting one of the capillaries
containing the bacterial masses are very much greater.
And working on this plan I have obtained sufficient
evidence to enable me to state confidently that the
lung and liver of the grouse that are affected with
- the disease in the spring and early summer con-
tain a definite species of bacteria forming continuous
masses or plugs in some of the capillary blood-vessels,
but that in the blood of the general circulation they
cannot be demonstrated. From this then it follows
that the bacteria find their way into the lung and
liver, in whose capillaries they settle and by their
multiplication produce those embolic plugs. This
is nothing exceptional, since we know of infectious
diseases, both acute and chronic, in which the specific
microbe does not thrive in the circulating blood, i.e.
does not find in this a suitable nidus, and is there-

SYMPTOMS AND PATHOLOGY

fore absent from this, though it uses the blood circula-
tion for getting to its proper nidus — in our case the
lung and liver. In human croupous pneumonia it
is the lung ; in tuberculosis the lymphatic tissues,
lung, liver, and spleen.

CHAPTER III

CHARACTER OF THE BACILLUS OF THE GROUSE
DISEASE

In the grouse disease the cultivations obtained, as
I said above, in almost all cases are of exactly the
same character, and it is these which I now proceed
to describe.

(i.) Cultural characters : —

The characters of the microbe in the gelatine plate,
in gelatine stab- and streak-cultivation are quite dis-
tinct. After 24-36 hours' incubation at 20° C. of
a gelatine plate, the first indications of the super-
ficial colonies are noticed as grey, translucent, fiat,
angular dots, visible under a magnifying-glass. They
spread rapidly in breadth, so that after 3-4 days
they reach a diameter of 3, 5, and more millimetres ;
they remain fiat and thin and are slightly thicker and
folded at the margin, which is much crenated. In
reflected light their aspect is whitish ; they look dry,
and when viewed obliquely have a fatty satiny lustre.
In transmitted light they look pale brown and trans-

CHAP. Ill CHARACTER OF BACILLUS 25

lucent. About the end of the week their maximum
growth is reached (Fig. 5). As with other species, so
also here, when the colonies of the surface are placed
at great intervals they grow much larger than when
they are near together. Those that develop in the
depth of the gelatine plate are very small as com-
pared with those growing on the surface, and remain
minute round dots, whitish in reflected, brownish in
transmitted light. The gelatine is never liquefied by
the growth.

Sub-cultures made of a colony on the surface of
gelatine as streak-cultures, — i.e. when with the end of
a platinum wire a colony is touched and is then
drawn in a line over the surface of the gelatine, — show
after one or two days along the line of inoculation
a narrow, grey, translucent band. This band rapidly
broadens, and remains flat, and possesses a crenated
margin. After a week its breadth is 5-10 millimetres
and more, it is flat, thin, translucent, dry, smooth, and
possessed of a satiny or fatty lustre ; the margin is
slightly thicker than the middle, translucent, and
plicated. In stab-culture the line of inoculation —
the stab — becomes visible after a day or two as a
grey line. Under a glass it is composed of minute
spherical dots. On the upper surface of the stab
a thin, translucent, whitish-grey film gradually spreads
over the gelatine with irregular margin, which in

26 THE GROUSE DISEASE chap.

about a week has covered nearly the whole free
surface of the ofelatine in the test-tube. But even
at the maximum growth the clots constituting the
stab remain small (Figs. 6, 7), and are brownish in
transmitted light.

After a few days' incubation of the stab culture there
are generally in the deeper parts one or two large
flat gas-bubbles to be noticed, fixed on to the growth
with one end, and placed, therefore, eccentrically to
the line of the growth. The gas -bubbles slant
generally in an upward direction. The development
of gas-bubbles in the deeper parts of the growth is
well and easily shown by making a "shake culture"
in gelatine — that is to say, by distributing (by shak-
ing) a limited number of the microbes in melted
nutrient gelatine contained in a test-tube and then
allowing this gelatine to set, by keeping it in cold
water for a short time before incubation. After
two days numerous colonies are visible as minute
dots distributed through all the layers of the gelatine,
and attached to each dot or colony in the deeper
parts is a small flat gas-bubble. Such a culture
looks extremely characteristic (Fig. 8). The gas-
bubbles increase in size, and, if the colonies are
numerous, i.e. if a considerable number of the
microbes had been originally introduced into the
gelatine, the lower half or two-thirds of the culture

in CHARACTER OF BACILLUS 27

are crowded with gas-bubbles, the upper layers of the
gelatine remaining free from them. Gradually, how-
ever, the gas-bubbles disappear, i.e. escape on to
the surface, and by the end of eight or ten days
all but the deeper situated bubbles have disappeared.
The escape of the gas -bubbles between the gela-
tine and the glass wall of the test-tube can be
easily ascertained.

On nutrient Agar at 36-37° C. the microbes grow
with great rapidity ; in two or three days the sur-
face of the Agar becomes covered with a greyish-
white thin film.

In alkaline beef broth (with i per cent peptone),
incubated at 36-37° C, there is, after 24 hours' growth,
strong uniform turbidity, which reaches its maxi-
mum in about 3 days ; at the same time there is
a copious greyish floccular or granular precipitate.
After about 5-7 days there is noticed, just at the
point where the surface of the fluid is in contact
with the glass of the test-tube, a ring of whitish
growth ; but there is at no time a distinct pellicle
formed on the surface. On potato, previously steril-
ised in the steam steriliser, the microbe grows well
^t Z^-ZT C., forming in a few days a light yellow-
brown, limited film, which looks like paint, and
does not spread much over the surface of the
potato.

28 THE GROUSE DISEASE chap.

(2.) Character of the microbe in the fresh state : —
The microscopic examination of the microbe in
the Hving or fresh condition, i.e. in a drop of saHne
solution, or serum, or broth, shows it to be a spheri-
cal, or more generally an oval corpuscle, occasion-
ally more distinctly rod-shaped or cylindrical, with
rounded ends. The microbes occur either single or,
more commonly, double, i.e. as dumb-bells (Fig.
9). The great majority are without any movement
of their own, showing only the oscillating move-
ment that all granules suspended in a fluid show,
namely, the Brownian molecular movement. But
occasionally one or the other shows active locomo-
tion, either spinning round and round, or darting,
under oscillation of its body, through the field of
the microscope. Such actively motile individuals
are to be seen fairly frequently in a fresh prepara-
tion made of a recent colony from a gelatine plate,
or from a recent gelatine tube-culture, or from Agar
culture, from the fresh lung -tissue or from the liver.
By means of the platinum wire a particle of the
culture or of the tissue is suspended or distributed in
sterile salt-solution — or better, in broth — and examined
either in the " suspended drop," i.e. in the hollow
glass slide, or simply mounted on an ordinary glass
slide. The number of non-motile bacteria is always,
under the most favourable conditions, greatly in

Ill CHARACTER OF BACILLUS 29

excess of those that are possessed of motility ; the
number of these latter being limited. In prepara-
tions made of recent culture, except potato or broth
cultures, the number of motile forms is appreciably
greater than from a culture of some days' standing ;
in older cultures they are missed. If of any gela-
tine cultivation, say a streak culture or a plate cul-
ture, fresh preparations are examined from day to
day, the same tube or the same colony being used,
it is noticed that while in the first few days the
number of motile bacteria is sufficiently great to
see in every field several or even many examples,
about the end of the week, and later, few motile
forms are to be found in the whole slide. And if
now fresh sub-cultures are started, it is again noticed
that, while recent, they show the motile forms fairly
abundant ; if of some standing, only non-motile ones
are present. I conclude from this that there is
produced in the cultures, by the growth, a chemical
substance which inhibits and is adverse to the
motility. It cannot, I think, be explained by saying
that the later offsprings are devoid of the organs
of motility, i.e. the flagella, while the earlier off-
springs possess them, because when they are planted
on fresh nutritive medium they again show motility,
and on potato or broth, even after 24 hours' growth
no motile forms are found. It can hardly be said

30 THE GROUSE DISEASE chap.

that in one case they produce offsprings with flagella
and in the other without. I have not examined them
with regard to demonstrating the presence or absence
of flagella, and therefore cannot say positively that it
is not so, but it seems to me more probable that
the loss of motility is due to some chemical poison
produced by the bacteria. In the fresh blood of
grouse (dead in the late summer and autumn) the
bacteria are always present, as I shall point out
below, and motile forms, though not very abundant,
can nevertheless be easily detected. So also in the
fresh blood of animals and birds infected by inocula-
tion with the cultures of the microbe (mice, guinea-
pigs, yellow-ammers, and buntings), motile forms can
be found. In the grouse and in the sub-cultures from
the grouse the oval forms abound. Some are so
short that they appear like cocci ; but it must be
added that, though at first sight many appear like
spherical cocci, on more careful focussing with high
powers it can be shown that in reality most of
them are oval, but appear spherical when viewed
foreshortened or in optical transverse section. In
sections through the liver or lung of the grouse
dead of the disease, or of other animals infected
with the cultures, the blood capillaries, as men-
tioned above, are found plugged with the microbes.
Now it is precisely in such masses that the individuals,

Ill CHARACTER OF BACILLUS 31

even under a moderately high power, e.g. under a
power of 300 to 400, appear as spherical cocci ; but
on examining carefully with oil immersions the
marginal parts of the masses, where the individuals
are loosely arranged, it is clear that most of them
are distinctly oval or even rod-shaped or cylindrical.

When the microbe is examined in mice or guinea-
pigs that have died after inoculation with the culture,
it is found that the number of rods and cylindrical
forms is very much greater than in the grouse or in
the sub-cultures of the grouse. This is well shown
in Figs. 10 and 11.

In the ammer, bunting, greenback, and finch,
which are highly susceptible to the disease, many
individuals are rod-like or cylindrical ; and also, in
the blood of the grouse dead during late summer
and autumn, rod-shaped individuals abound.

Whatever the source of the organism when grown
in broth, — or better still, when grown in or on nutri-
tive gelatine to which 5 per cent, solid sea salt (Sted-
man's sea salt) has been added, — the bacteria grow
out into long cylindrical threads, most of them smooth
and uniform, but some made up of individual cylin-
drical bacilli (see Fig. 12). There can then be no
doubt that we have to deal with a form which corre-
sponds to those groups of bacterial species known as
bacilli.

THE GROUSE DISEASE

\SX^

Measurements made of the organisms in cover-
glass preparations, dried and stained, give the follow-
ing figures : —

The spherical forms are 0.4 /^^ in diameter ;

The oval forms are 0.6 yu, long, 0.4 yu- thick ;

The rods and cylindrical forms 0.8-1.6 /x long,
0.4 /A thick.

1 I /x = o.ooi millimetre, or about 2TW0 P^^^ of an inch.

,:jt .-a .Lift. . ic<l

' IC0V4 •tCl

CHAPTER IV

EXPERIMENTS WITH CULTURE BY SUBCUTANEOUS
INOCULATION

Experiments were made on tame mice with cultiva-
tions,— either with a particle of a gelatine- or potato-
or Agar culture distributed in, or mixed with, sterile
salt solution, the mixture being slightly turbid ; or
with a broth culture 24-48 hours to 3 days old. Of
the salt mixture or of the broth culture a few drops
(3-5 minims) are injected under the skin of the back.
The following day the animals are found quiet, and
they do not feed well; the "eyes are almost closed, the
back is curved, the coat rough, they look " lumpy " ;
the breathing is rapid, and, while motility becomes
weaker and weaker, they die either before 36 hours
are over or during the following night, i.e. before
48 hours. If the inoculation is made with a broth
culture 24-48 hours old, most of the mice inoculated
are dead before the end of the second day ; some
live till the end of the third day ; a few remain quiet

and ill and gradually recover. In the latter cases a

D

i uJru«.K

34 THE GROUSE DISEASE chap.

more or less extensive necrosis of the skin and sub-
cutaneous tissue at and about the seat of inoculation is
noticed.

As stated just now, the majority of the mice
inoculated with a recent broth culture (3-5 minims)
die. Gelatine cultures less than 10 days old, and
potato- or Agar cultures less than 4 or 5 days old,
used as a slightly milky salt mixture, prove viru-
lent : old cultures of gelatine and old cultures of
Agar are apt to show less virulence, this may
become almost lost with age. In the former case a
similar proportion of the mice succumb, but later than
after inoculation with recent cultures ; in the latter
no death follows on inoculation of even large doses.
In order to ensure virulence I use recent broth
cultures. These do not fail, no matter whether they
are sub-cultures of old or recent cultures on gelatine,
Agar, or broth, I have tested in this respect cultures
that have been carried on in sub-cultures for many
generations — in gelatine for more than three years
and a half; and I find that when a broth sub-culture
is made of an old gelatine culture, say one or several
months to half a year old, and is inoculated into
mice, it proves virulent. The highest degree of
virulence is obtained by making a sub-culture in
alkaline broth to which a piece of boiled white of
egg is added (the whole being sterilised before use).

IV CULTURE BY SUBCUTANEOUS INOCULATION 35

The vitality of the microbe is a very great one,
since gelatine cultures, kept at the temperature of
the room for more than a year and a half, proved
still alive, i.e. good and virulent sub-cultures could
be easily obtained from them.

On making a post - mortem examination of the
mice that die, the following appearances are con-
stantly found : — Right heart contains fluid blood, left
heart contracted. The lungs show oedema and
congestion ; this latter extends generally over both
organs, and is marked by their being redder
than normal — sometimes deep red or dark purple-
red. The liver is dark red, and congested ; the
spleen is of a dark colour, and slightly enlarged ;
both kidneys are congested. At the seat of inocula-
tion the subcutaneous tissue shows sometimes slight,
sometimes distinct, congestion, or even sanguineous
infiltration. In the heart's blood the microbes can
be easily detected by cover-glass specimens, and ^
particularly by cultivation ; a droplet of blood yield-
ing, in plate cultivations, or in tube cultivations
(rubbed over the slanting surface of solid nutrient
gelatine by means of a platinum wire), sometimes
many, sometimes innumerable colonies. A section
having been made with sterile scissors into the
tissue of the congested lung, a cover-glass impres-
sion is taken of the cut surface ; this is then dried

36 THE GROUSE DISEASE chap.

and stained in methyl blue or gentian violet,
washed in water, dried, and mounted. Examined
under the microscope, it shows the outlines of the
air-cells marked by innumerable microbes: round or
oval forms, singly and particularly in dumb-bells,
rods singly and in dumb-bells, and a few cylindrical
forms (Figs. 13 and 14). In some cases their
number is not so great, but in acute fatal cases they
are always easily discovered in cover-glass specimens
made of the lung juice. Cultivations made of the lung
tissue, the liver, and the spleen, always yield a great
abundance of colonies. As the result of inoculations
made with the culture of the grouse into the sub-
cutaneous tissues of guinea-pigs, about 50 per cent of
the animals die during the third or fourth day ; the
others, though they survive, develop a firm tumour
at the seat of inoculation. But all the guinea-pigs
are distinctly ill 24 hours after inoculation ; they show
a slight soft swelling at the point of inoculation,
and are quiet and off their food ; some grow weaker
during the second and third day, cannot move, are
shaky when they attempt to move, and gradually
sink and die. At the post-mortem examination the
subcutaneous tissue at and about the seat of inocula-
tion, on the abdomen, and even on the chest, is
oedematous and shows haemorrhage. The blood of the
right heart is fluid, both lungs are much congested ;

IV CUL TURK BY S UBC UTA NEO US JNOCULA TION 37

the liver is also congested ; the spleen is not enlarged,
but dark ; the peritoneum, the kidneys, and the supra-
renals are much congested. The peritoneum is
moist, and there is sometimes a small amount of
exudation ; petechiae are often noticed in the
omentum. The heart's blood, and particularly the
lungs, contain the microbes in large numbers (Fig.
17). Cover-glass specimens and cultivations of the
blood, and particularly of the lung juice, show the
microbes in very large numbers. So also the peri-
toneum, especially the serous covering of the liver.
In the heart's blood the microbes occur as single or
dumb-bell oval bodies, and as rods; they are either
in the plasma or they are contained within the
white blood -cells — in the latter case the cells are
very much swollen up, and some are disintegrating.
In Fig. 17 this condition is well shown.

[The intracellular occurrence of the bacilli in the
guinea-pig, and, as will be mentioned presently, also
in the ammer, does not indicate that there is going
on a process of combat on the part of the cells as
against bacilli, as is only too readily assumed by
those who with Metschnikoff assume this to be
the case wherever cells are met with that enclose
bacilli ; for in our cases we are dealing with a dis-
ease which is rapidly fatal, and in which the bacilli
abound in the blood.]

38 THE GROUSE DISEASE chap.

As regards the shape and motiHty of the microbe
in the guinea-pig, the same holds good that has
been said of the mouse.

The inoculated guinea-pigs which survive show
at the seat of inoculation a firm flat tumour which
extends on to the chest. About the end of ten days or
so, necrosis and sloughing of the indurated part of the
groin sets in, which gradually leads to complete healing.

In several instances guinea-pigs which had been
inoculated with virulent culture, but had survived,
were, at the end of twelve to fourteen days, re -inocu-
lated with a virulent culture, but no result followed
this second inoculation.

Rabbits inoculated subcutaneously with culture
that proved virulent on mice and on guinea-pigs show
only a local result. A thickening about the seat of
inoculation, due to caseous purulent infiltration, sets in,
but remains circumscribed, and in some cases opens
as an abscess, which rapidly heals ; or the tumour
becomes firmer and smaller and gradually disappears.
Only once have I seen a rabbit die during the second
day, but neither cover-glass specimens nor cultivations
revealed any microbes either in the blood or in the
lung.

In pigeons and fowls the subcutaneous inoculation
is not followed by any, not even a local, positive result,
the animals remaining lively and well.

IV CULTURE BY SUBCUTANEOUS INOCULATION 39

The most striking results were obtained on the
common bunting and yellow-ammer (greenback, cock- sfe*Ucc&t
bit, and finch), for the injection of a small drop of a
broth culture into the thigh is followed by fatal results.
The injected leg is swollen before the end of twelve
hours, the birds do not move that leg, and sit perched
on the other leg. Towards the end of the first day
they are quiet, " lumpy," their feathers are ruftled,
their breathing is very rapid, their eyes are closed ;
the animals are drowsy and have diarrhoea. Most of
them die towards the end of the first day, about the
20th-2 2nd hour; some die at about the 24th hour;
a few survive longer — even to the fourth day. On
post - morte^n examination haemorrhagic infiltration is
found in the muscles of the thigh at the seat
of the inoculation, the lungs are deeply con-
gested, the liver is congested, and so is the mucous
membrane and the serous covering of the intestine.
The spleen is dark and small. There is then a com-
plete analogy in these respects with the appearances
found in the grouse. The blood of the heart and the
sanguineous juice of the congested lungs teem with the
microbes (Figs. 18 and 19), either as single or dumb-
bell spherical and oval forms, or as single or dumb-bell
rods with rounded ends. Many of the white blood
cells in the lung juice contain in their substance the
bacilli : in some the bacilli are few in numbers, in

40 THE GROUSE DISEASE chap.

Others the cell substance is crowded with them (Fig.
20). Examined in the fresh state, some of the bacilli
show mobility. In dried and stained cover-glass speci-
mens and in cultivations from the heart's blood or
lung juice ; in plates, gelatine streak and stab, in
Agar, on potato, and in broth, the microbe shows
the same characters as that of the mouse (Fig. 21).

The buntings and ammers possess even greater
susceptibility to the disease than the mice ; this is
shown by the fact that gelatine and Agar cultures,
which through age have become attenuated for mice
and guinea-pigs, still produce fatal results in these
birds.

Sections made through the lung show many
capillaries and small vascular branches almost com-
pletely blocked by continuous masses of the microbe.
Staining the sections in rubin (2 per cent) for two
or three hours, then washing in water, then staining
in methyl blue aniline water for seven or eight
minutes, washing, dehydrating, passing through xylol,
and mounting in Canada balsam solution, shows the
cell nuclei and the bacilli blue, the rest of the
tissue red.

Sparrows were also inoculated subcutaneously.
The great majority (70 per cent) die under the same
symptoms and with the same post-mortem appearances
as the ammers ; the microbes are present in the lungs

IV CULTURE BY SUBCUTANEOUS INOCULATION 41

and the heart's blood ; in the latter, however, they are
not numerous ; in some cases they could not be found
in the blood, though they were present in the inflamed
lungs.

I have further tested, as regards virulence, the
sub-cultures derived from the blood and lung of the
mouse, and from the blood and lung of the ammer,
on mice, guinea-pigs, and ammers, and have found
that there is no difference between them and the sub-
cultures obtained from the lung of the grouse. While,
then, by these experiments it follows that the cultures
of the microbe act virulently when subcutaneously
injected into mice, guinea-pigs, buntings, ammers,
and sparrows, they do not produce fatal infection in
rabbits, and do not produce any result in pigeons or
fowls ; and for these reasons alone the microbe as
well as the disease it produces is well defined from
chicken cholera, which, as we shall show later on,
is very characteristic as regards the virulence of its
specific microbe on rabbits, fowls, and pigeons. As
we shall also show, there exists a certain similarity
in shape, in certain cultural characters, and in the
non- liquefaction of the nutrient gelatine, between the
microbe of grouse disease that we have been hitherto
considering, and the microbe of chicken cholera ; but
the differences in the growth of the two microbes in
the various culture media are sufficiently striking

42 THE GROUSE DISEASE chap.

to enable us to say that they are two well-defined
species.

Experiments of feeding and air infection : —

Numerous experiments were made on mice, guinea-
pigs, and ammers to see whether, by introducing the
bacilli into the digestive organs, infection could be
produced. For this purpose broth cultures, the viru-
lence of which was proved by subcutaneous inocula-
tion, were mixed with the food given to mice and
guinea-pigs, but in no single instance was any result
produced. In the case of the ammers, several drops
of the broth culture were directly dropped into the
mouth, the beak being held open, and readily swal-
lowed. No result followed.

From this it follows, then, that by ingestion the
microbe does not act, and therefore must be said to be
destroyed by the gastric fluid, and does not reach in a
living state the small intestine.

It is otherwise with the respiratory organs, for
infection by these has been ascertained in the case of
the ammers. Several experiments were made in
this respect ; in one set a bird was inoculated with
virulent culture, and then placed in a cage with two
normal birds. The inoculated ammer was found
very ill the next day, with the one leg drawn up,
feathers ruffled, breathing very rapid, eyes closed.
The bird died during the second day ; the other two

IV CULTURE BY SUBCUTANEOUS INOCULATION 43

were found ill the day following the death of bird j^^^^^ ^.j^
No. I, and died in the course of the third day with "1 '^"^'^
the symptoms and appearances of the disease ; in
their blood and lung the microbe was present in great
numbers. In a second set two wire cages were placed
close side by side ; one contained an ammer that was
affected with the disease after inoculation ; this bird
had been inoculated with attenuated culture (see
below), and remained in the diseased condition for
several days ; in the adjoining cage were six normal
ammers. The two cages were covered with one
cloth. All the six neighbours became ill with the
disease after two days, and died with the typical
symptoms and appearances during the following day.

CHAPTER V

THE AUTUMNAL DISEASE

In 1888, 1889, and 1890, I received, thanks to the
editor of the Field, during August, September, Octo-
ber, November, and even December, many grouse
from various parts of Scotland and the north of
England which had died of the grouse disease, that
is to say in which, on post-mortem examination, the
congestion of one or both lungs, the congestion of
the liver, the small dark spleen, and the patchy redness
of the intestine and the peritoneum, were present.
In many of these birds the heart's blood was examined
for the presence of the specific microbe, both in cover-
glass preparations and by culture, and, unlike the birds
dead during the spring and early summer in which the
microbes are absent from the blood of the general
circulation, the heart's blood of those birds always
contained them in large numbers. In fresh speci-
mens of the heart's blood they can be identified as
oval or rod -shaped single or dumb-bell bacteria,
some actively motile. As a matter of fact it was

CHAP. V THE A UTUMNAL DISEASE 45

this observation which led me to re - investigate
the microbes obtained from the spring grouse, for of
them I had in my earlier communications on the
subject stated that they are non-motile. On re-
investigation, I ascertained that this is not so, that in
every specimen made of the cultures there are some
individuals which show mobility, but the number of
which, as stated above, decreases in older cultures, and
which are absent in recent potato and broth cultures.

The bacteria in the blood are, as a rule, more
rod-shaped than those taken from the lung, though
also in the blood some are spherical or slightly oval ;
the rods are also here commonly arranged as dumb-
bells (Fig. 22).

Cultivations made with a drop of the blood in
gelatine plates, or by rubbing a droplet of blood over
the slanting surface of solidified nutrient gelatine,
show, after 24-48 hours, innumerable colonies ; the
slanting surface of the gelatine is so covered with
them that, having become in many places confluent
by proximity, they form an almost continuous film.
As regards the character of the microbes in cover-
glass specimens, fresh or dried and stained, made of
the cultures, these microbes are identical with those
derived from the lung, both of the spring grouse
and of the autumn birds, and there is nothing to
Specially add to what has been stated in a former

46 THE GROUSE DISEASE chap.

chapter. The microbe from the heart's blood culti-
vated in gelatine plates, in streak and stab in gelatine,
in Agar, in broth, and on potato, is in every essential
the same as already described ; so that there can
be no doubt that the microbe of the lung and liver
of the spring grouse, and of the heart's blood of the
autumn grouse, is the same in morphological and cul-
tural respects ; and since the pathological appearances
of the grouse dead in the autumn are identical with
those dead during the spring and early summer, we
are justified in saying that the autumnal disease is
also the real grouse disease. Now, on inquiry, it is
found that the disease in the late summer and autumn
is of less frequent occurrence ; this is, of course, to be
expected, seeing that the total number of the grouse
rapidly diminishes during the shooting season. But
when saying that the disease is less frequent during
the summer and autumn, I am not referring to such
a cause of diminution. I am referring to moors in
which no epidemic disease has been prevalent during
the spring, or in which no great amount of shooting
has been going on in August. I have received birds
during August, September, October, and the be-
ginning of November, from moors in which very
little or no disease had been present in the spring
and early summer, and yet birds died now and then
during the autumn. We may from this conclude that

V THE AUTUMNAL DISEASE 47

in these cases the disease, though present, was of the
sporadic character. If this be the correct view, it
would point to the danger existing on such moors, as
far as they themselves as well as their neighbours
are concerned, and it would explain at the same time
how, by lingering on during the winter, an epidemic
outburst of the disease may occur in the following
spring. Though I do not think that this is the only
way in which the spring epidemic may arise, it is at
any rate one very probable way. It has been pointed
out already, and will be again referred to, that the
vitality of the microbe, as proved by experiments with
cultures,, is of a very high degree, and it is quite pos-
sible that the microbes left in a moor from a previous BatXl*' u**^
epidemic, and remaining sheltered in the soil under .;,

the heather, grass, or otherwise, may on a future
occasion give rise to infection, and thus start a new
epidemic. But the theory that cases of a previous
spring and summer epidemic linger on through the
autumn and winter as sporadic cases in any one part
of a district, would furnish a more direct cause for
an epidem.ic in the ensuing spring than the other
condition, which supposes that the microbes derived
from birds that died in the spring or summer may
retain their vitality, and remain alive on the moors
through the autumn and winter till the next spring,
when they might give rise to fresh outbreaks.

48 THE GROUSE DISEASE chap.

In this disease, as in others, certain accessory or
secondary conditions are required in order to enable
the disease to assume the epidemic character. This
has been already mentioned ; and now we wish to
add that the microbe of the autumn disease seems
distinctly less virulent than that of the spring
disease. Inoculations of mice were made with recent
broth cultures derived from the heart's blood microbe
of autumnal cases, but in the majority of cases no
fatal result was produced, although all the animals be-
came ill. In a series of such experiments comprising
ten mice, all were quiet and off their food the day
after inoculation ; one died after three, one after seven
days, the other eight survived ; while of six mice
inoculated for control with recent broth culture of
the microbe of the lung of the spring disease, four
died in 48 hours, one during the third day, and only
one survived.

Buntings and ammers so highly susceptible to
the spring microbe are also found less affected by
the autumn microbe ; most of them die, it is true,
but conspicuously at a later period than after inocu-
lation with the spring microbe ; they are ill for several
days, some die after the fourth, others after the fifth
day, their heart's blood containing the microbe in
large numbers. To prove that the autumn microbe
is of an attenuated virulence, I re-inoculated, after

V THE A UTUMNAL DISEASE 49

two to three weeks, those mice (80 percent) which
had survived the first inoculation with the autumnal
microbe, with virulent culture of the spring microbe
— tested on control mice — and found that they proved
refractory, neither disease nor death ensuing.

These experiments seem to me at any rate
indicative of a means of discovering a protective
vaccine for the grouse, i.e. a material which would
only produce a transitory illness without fatal result.
For if cultures of the autumnal microbe became still
more attenuated, say by certain age of the sub-culture,
it might be found just of the sufficient degree of
activity to cause slight disease, transitory in character,
but protective against infection with the fatal dis-
ease. To ascertain this a long series of experiments
would be required, which I have not had leisure to
make. I have myself made some experiments with
the gelatine sub-cultures of the autumnal microbe ;
these sub-cultures were one week old, and were
inoculated into mice, but though the animals showed
slight illness, no fatal result followed ; and I can quite
imagine that if such or similar cultures were tried
by inoculation on young grouse on the moors, — a not
at all impossible proceeding considering that most
keepers know of most of the grouse nests, — it might
be possible to produce a slight transitory illness,
protective of the fatal disease. But this is a

50 THE GROUSE DISEASE chap, v

suggestion, the carrying out of which, from what I
know of moor-owners, would meet with great diffi-
culties ; difficulties not on the part of scientific men,
who, I am sure, would be willing to undertake such
work, but on the part of the grouse-owners them-
selves, who, with some notable exceptions, would
probably be unwilling to consent to such experiments
unless immediate success could be guaranteed.

CHAPTER VI

FURTHER CHARACTERS OF THE BACILLUS

One of the important points in the life-history of a
bacterium in general, and of pathogenic microbes in
particular, is the question of spore formation. A
bacterium that is possessed of the power of forming
permanent seeds or spores possesses at the same time
the power of preserving vitality and of insuring new
generations to a far greater extent than one that is
not possessed of this power, because spores are, as a
rule, capable of retaining their vitality and the power
of germinating under conditions and circumstances
such as easily affect the vitality of non-spore-bear-
ing forms, e.g. heat, cold, drying, absence of suf-
ficient nutriment, chemically obnoxious substances.
While it is well known that continued cold, even
if above freezing-point, or heat far below the point
of boiling water (even 55° C), acting for many hours,
or deficiency of nutriment, drying, acids and alka-
lies, affect considerably the vitality of non-spore-
bearing forms — many in fact perishing under those

52 THE GROUSE DISEASE chap.

conditions — those that form spores, and as spores,
resist all the above influences. In the case of
pathogenic bacteria those that form spores, e.g. the
bacillus antkracis, the bacillus of the classical malig-
nant oedema, the tetanus bacillus, — having formed
spores, as spores are, for the above reasons, of
the utmost importance in etiological respects ; for
the spores, even when finding themselves under very
unfavourable conditions, retain their vitality, and,
having access to a suitable animal body, are capable
of setting up the disease. In the case of non-spore-
bearing forms this can only happen if they are for-
tunate enough to be shielded in one way or another
from these obnoxious influences.

Now the bacillus of grouse disease belongs to the
group of pathogenic organisms which do not form
spores. On microscopical examination of fresh and
of stained specimens there is no indication of the
presence of anything that will compare with the
spores in those species of bacilli in which spores do
occur, and the experiments on the influence of heat
and of drying fully bear this out. A culture of the
grouse bacillus exposed to a temperature of 60° C. for
five minutes becomes hereby completely sterilised ;
no new sub-culture can be produced from it. Or, if
a culture medium in a test-tube or flask is inoculated
with the living grouse bacilli, then exposed to 60° C. for

VI FURTHER CHARACTERS OF THE BACILLUS 53

five minutes, and then incubated, it is found to remain
free of any growth — that is, to remain sterile ; while
a culture tube not exposed to this temperature, on
incubation produces good and normal growth.

Whatever the age of a culture, and whatever the
medium in which the bacilli have been growing, a
temperature of 60° C, acting for five minutes, kills
all life of the bacilli of the grouse disease. This fact
is, so far as our present knowledge goes, sufficient to
justify us in concluding that in those cultures no spores
have been formed.

I have made experiments with regard to the pos-
sibility of attenuating the virulence of the bacilli by
temperatures below the killing point, viz. below 60° C,
in order to obtain easily procurable definite materials
by which, on inoculation, owing to the decreased
virulence, a slight transitory ailment could be pro-
duced which would protect against a second fatal
infection ; for I had already ascertained, as mentioned
on a former page, that mice, after inoculation with
attenuated virus (the cultures derived from the blood
bacilli of the grouse dead from the autumn disease),
and after recovery from the transitory illness, remain
refractory to a second inoculation with virulent cul-
tures.

I exposed, therefore, virulent broth cultures to a
temperature of 50° Fahr., five, ten, fifteen, and twenty

54 THE GROUSE DISEASE chap.

minutes respectively, and then tested their virulence
by inoculation on mice and buntings, but no difference
in the virulence of the so-heated cultures could be dis-
covered, for the animals inoculated with them died in
the same way and time as the control animals inocu-
lated with the culture previous to the heating. I then
tried heating the virulent broth cultures to 55° C. for
ten minutes and for twenty minutes respectively. The
result was unsatisfactory, since the cultures so heated
proved still virulent. I need hardly add that cultures
heated to 55° C. for twenty minutes contain the bacilli
in a living condition. Sub-cultures made from such
heated cultures all yield normal good growths on
inoculation.

In connection with the action of heat on broth
cultures, I wish to mention here the following : —

Broth cultures incubated from two to three days
at 35-37° C. contain chemical substances which them-
selves are capable of producing the death of mice
on inoculation in sufficient quantities, for when such
a broth culture is sterilised, i.e. is exposed for five
minutes to 60° C, when hereby all the bacilli are
killed, and when from such a sterilised broth culture
five minims are injected into mice under the skin
of the back, the animals die under the same symp-
toms and in the same time as the control mice
inoculated with the same amounts of the same broth

VI FURTHER CHARACTERS OF THE BACILLUS 55

culture previous to sterilisation. Of course the mice
that die after being inoculated with the sterilised
broth culture do not contain any bacilli either in
the heart's blood, or lung, or spleen. In a later
chapter, after having described and discussed a
species of pathogenic bacilli (aerobic bacillus of
oedema) occurring in the soil, which is in many-
respects similar to the bacillus of grouse disease, I
shall have occasion to return to an interesting physio-
logical action of the grouse bacilli when inoculated
simultaneously with the aerobic oedema bacillus. At
present I wish to conclude this chapter by the
observations I made with resfard to the influence
of drying on the bacilli of grouse disease.

The experiments of drying a particle of the growth
prove that hereby the vitality of the bacilli is com- I^ax^v^ •
pletely annihilated. Of a living broth culture a thin
film is spread over a sterile cover-glass (sterilised
previously by heat), then kept covered up, while the
film is dried either over a dish of sulphuric acid or in
the air-pump : if with such a dried film a tube con-
taining nutrient broth is inoculated and incubated,
the broth remains sterile, proving that the drying
has killed the bacilli. But the experimental result is
different — if, for instance, a particle of the growth is
removed from a culture on gelatine, Agar, or potato,
and dried on a cover-glass. In a percentage of cases

56 THE GROUSE DISEASE chap.

it will be found that the particle, though apparently
dry, nevertheless still includes living bacilli ; for
on inoculation of broth with it, and after incubation,
the broth becomes turbid with the grouse bacilli, a
new growth having taken place. This is evidently
due to the fact that the particle becomes dry on
the surface only ; the central portion not becoming
quite dry, being protected by the outer dry part,
still harbours living bacilli. The larger the particle,
the greater the chance of the bacilli surviving in the
centre. Now this may be applied also to the con-
ditions obtaining on a moor. A bird dies of the
disease on the moors. Its body becomes lacerated
or opened in some way, by animals of prey or
otherwise, portions of the lungs and liver dry up,
and may retain living bacilli which may remain
sheltered for some time, and, when finding access to
a suitable bird, say next spring, may set up the
disease, and thus produce a focus for the develop-
ment of the epidemic. I do not mean to say that
such an occurrence can be or has been proved, but it
must be admitted that such an occurrence is possible,
and that it is justified by the experimental facts
with regard to the influence of incomplete drying.

From all that has preceded it will be evident
that the only measures of dealing with the disease in
order to check its spread and epidemic appearance,

FURTHER CHARACTERS OF THE BACILLUS 57

lie in the direction of what is popularly known as
the stamping-out process. Any grouse on the moors
at all suspicious at any time should be destroyed,
and every grouse thus destroyed or that dies of the
disease should be carefully removed and burned. I
am not sure that those who burn the heather on
moors on which grouse - disease prevails are not
following a right plan, for the reason that hereby
they remove that which most likely harbours the
adhering infective material ; nor am I sure that, on
moors on which disease has been present, an exten-
sive battue of grouse would not also remove many
of the birds that have the disease in a mild form, and
that might carry it on, as it were, through the autumn
and winter. It is therefore especially important that
during autumn, and possibly the winter, a careful
look-out should be kept for suspicious grouse, for
it is these which require removal particularly.

CHAPTER VII

GENERAL CONSIDERATIONS

We have shown in the preceding account that in
the grouse dead of the disease there occurs con-
stantly in the diseased lungs and liver a species of
bacilli in a definite distribution ; that this species
grown on and in various media shows definite char-
acters ; and that with the cultures of the bacilli a
definite acute fatal disease is produced in mice and
guinea - pigs, in buntings, yellow-ammers, and spar-
rows ; that the disease thus produced in the bunt-
ings and other birds, in its pathological appearances,
compares in a remarkable degree with the disease
found in the grouse ; and that, further, the disease in
the bunting can be set up, not only by subcutaneous
inoculation, but also can be communicated directly
from a bird ill with the disease to a healthy bird ;
and that, lastly, in the animals infected with cultures
of the bacilli, these latter are found again in definite
distribution. All these facts justify us in saying
that this species of microbe occurring in the diseased

CHAP. VII GENERAL CONSIDERA TIONS 59

lung and liver of grouse dead of the grouse disease
is the causa causans of the disease. It will no doubt
be argued that the chain of evidence as to this par-
ticular microbe being the cause of the grouse disease
is in so far incomplete as no infection of normal
grouse has been carried out with it. The force of
this argument I fully admit, and I have endeavoured
to meet it ; but owing to the difficulty which I en-
countered in getting permission to inoculate with
the microbe young grouse on the moors on which
no grouse disease prevails, all my endeavours have
failed. It is clear that such experiments must be
carried out on moors on which no grouse disease
exists, for if the experiments were made in a
locality where grouse disease occurs, the objection
could with justice be raised that accidental or
natural infection of the experimented grouse was
not excluded. I do not think that it would be
very difficult to separate a few young grouse
— although grouse do not easily bear seclusion
in a cage — before they go for cover, and to in-
oculate them. But this would require my going to
and staying at the moors, and getting permission to
do it, an expensive experiment which I am not in
a position to carry out. But I tried to meet the
difficulty in a different way, viz. by getting eggs of
grouse and having them hatched by hens. With

6o THE GROUSE DISEASE chap.

very great difficulty I succeeded, with the kind help
of the Editor of the Field, in getting together about
two dozen eggs that could be tried. I received more
than that from many gentlemen who responded to
my appeal ; but owing to the insufficient care with
which the eggs had been packed, and the great
injury they received on the long journey south, they
were quite unfit for experiment.

Out of the two dozen eggs thus obtained and
subjected to hatching ^ only one yielded a bird, but
unfortunately, also, this expired a few hours after
appearing. I was therefore reluctantly forced to
abandon the attempt of carrying my experiments fur-
ther. But, notwithstanding the absence of this last
link in the chain of evidence, I think I may say that
there can be little doubt that the bacillus which I de-
scribed is the real cause of the disease, — its constant
and definite presence and distribution in the diseased
lung and liver of the grouse dead of the disease, its
virulence on buntings and other birds, the character of
the disease produced in the birds and the transmission
by the air from bunting to bunting, with the same
distribution of the microbe in the latter, prove to my
mind conclusively that we are dealing with a bacillus

^ Messrs. Woodin and Aldous deserve my grateful acknowledgment
for having placed at my disposal their farm, near East Grinstead,
Surrey, for purposes of this experiment.

VII GENERAL CONSIDERATIONS 6i

which is not only accidentally present in the grouse
disease, and which most certainly is not of the nature
of a harmless saprophyte, but which possesses a
definite causative relation to it.

This bacillus belongs to a group of pathogenic
bacilli which have certain important characters in
common, but which, owing to certain other import-
ant distinguishing characters, both in culture and
in physiological action, represent several definite
species. This group of bacilli comprises :

The bacillus of fowl cholera, the bacillus of fowl
enteritis, the bacillus of duck cholera, the bacillus
of the Middlesbrough pneumonia, the aerobic bacillus
of malignant cedema described by me, the bacillus
of Frettchenseuche, the bacillus of Wildseuche, the
bacillus of swine -plague of the Germans, the bacillus
of English swine- fever and American hog cholera.
All these bacilli are morphologically similar ; they
are oval in shape, single, but chiefly in dumb-bells ;
amongst them are rods and even cylinders, single or
in dumb-bells, some motile, others non- motile; they
are aerobic, they do not liquefy the gelatine, they
grow in a similar but not identical way in gelatine,
in plate, streak, and stab culture, but they show con-
siderable and definite differences as regards rapidity
of growth ; they produce in rodents an acute fatal
disease which, generally speaking, partakes of the

62 THE GROUSE DISEASE chap, vii

character of a hsemorrhagic septicaemia, but there are
well -pronounced differences as regards the duration
of the disease and as regards the species of rodents
and birds that are susceptible or insusceptible to the
different species of these bacilli.

CHAPTER VIII

A PATHOGENIC BACILLUS RESEMBLING THE BACILLUS
OF THE GROUSE DISEASE

In the course of the spring and summer of 189 1 I
found in garden earth, in very large numbers, a species
of bacilli which have a powerful virulent action on
rodents, and which, when introduced into them sub-
cutaneously, produce fatal infection ; their action on
the mouse, and to a certain lesser extent on the guinea-
pig, is not dissimilar to that of the grouse bacilli. In
cultivations in various media the bacillus presents a
great many points of resemblance with the bacillus
of grouse disease, for which it might be easily mis-
taken ; but the two microbes are two distinct species,
and therefore the occurrence in numbers of the patho-
genic bacillus in earth — whether it also occurs in earth
on the moors I am unable to say, I got it from garden
earth that had been manured within recent times —
cannot be brought in any relationship to the bacillus
of grouse disease. In order to obviate such a rela-
tionship between the two species of bacilli being

64 THE GROUSE DISEASE chap.

even suspected, I will fully describe this bacillus and
its action.

It is well known that recently manured garden
earth contains the anaerobic motile spore - bearing
bacillus of Koch, which, inoculated into guinea-pigs
subcutaneously, produces acute malignant oedema,
malodorous, rapidly spreading, and leading to gan-
grene of the muscular and cutaneous tissues, and to
the death of the animal in from 20 to 30 hours. This
anaerobic bacillus of malignant oedema grows in a
characteristic and typical way in the depth of grape-
sugar gelatine and Agar, and produces, as it grows,
numerous gas bubbles ; it liquefies the gelatine, and
forms chains or threads of various lengths.

Desirous of obtaining this classical bacillus of
Koch's malignant oedema, I introduced in the custom-
ary manner subcutaneously into the groin of the guinea-
pig a small quantity of recently manured garden earth.
Next day the animal showed extensive oedema in the
groin, on the abdomen, and thorax, was very shaky on
its legs, and died in less than thirty- six hours. The
sanguineous oedematous fluid was crowded with
motile short bacilli, a few longish chains and threads
amongst them, also a few that contained the oval spores
characteristic of the anaerobic malignant-oedema bacillus
(Fig. 24). Cultivations were made in ordinary nutrient
gelatine and in grape-sugar gelatine, and it was found

VIII A PATHOGENIC BACILLUS 65

that the very great majority^ of the bacilH present in
the oedema fluid belonged to a species of short motile
bacilli which grow best aerobically, i.e. on the surface
of the gelatine, which do not at any time liquefy the
gelatine, and which do not form spores. By putting
a drop of the above oedema fluid into about 10 cc. of
sterile salt solution, and, after shaking, making from
this mixture, with one platinum loop, a gelatine -plate
cultivation and cultivations in the depth and on the
slanting surface of ordinary gelatine or of sugar
gelatine, innumerable colonies of the same species of
aerobic, non- liquefying, non- spore -bearing bacilli were
obtained. Out of six tubes containing grape-sugar
gelatine, inoculated from the salt mixture, only in one
tube in the depth did a colony of the classical liquefy-
ing oedema bacillus come up, so that the number of
these, as compared with those of the aerobic non-
liquefying bacilli, was insignificant.

A drop of the oedema fluid inoculated into guinea-
pigs, rabbits, and mice produced, in the first two
species of animals, the same malignant oedema, the
oedema fluid again malodorous and crowded with the
aerobic non-liquefying short bacilli (Figs. 23 and 24).
In mice no oedema occurred at the seat of the inocula-
tion, just as in the classical malignant oedema. The
heart's blood of the guinea-pigs, rabbits, or mice,
dead from the classical malignant oedema or from our

F

66 THE GROUSE DISEASE chap.

oedema, contains only a few bacilli ; the spleen in the
guinea-pig and rabbit is not enlarged, in the mouse
only slightly so, and contains few of the bacilli. In
all these respects there is, therefore, a complete
analogy between the classical malignant oedema and
the one under consideration.

Cultivations injected in sufficient quantities sub-
cutaneously in guinea-pigs and mice and rabbits
produce the same fatal disease as the garden earth,
and there exists also in this respect an analogy
between the classical malignant oedema and our
disease, since it is well known through Gaffky and
others that in order to produce a fatal result with
cultivations of the classical oedema bacillus, large
doses must be injected. There is, however, a slight
difference between the two diseases consisting in this,
that in the classical malignant oedema the oedema
is generally more extensive, more distinctly mal-
odorous, and more clearly associated with gangrene
of the adjacent muscle than in our disease, though
also in this latter the changes are sometimes very
severe and not inferior in degree to those in the
classical malignant oedema.

Owing to the bacillus in our malignant oedema
growing well on the surface of ordinary nutrient
gelatine, cultures of the bacillus can very easily be
obtained from the oedema fluid. These cultures

viii A PATHOGENIC BACILLUS 67

in gelatine plate cultivations, in gelatine streak,
stab, and shaken cultures, on and in Agar, on potato
and in broth, so much resemble the cultures of the
grouse bacillus that unless the two cultures are simul-
taneously inspected and compared one with the other
it is extremely difficult to distinguish them. The
character of the cultures of the grouse bacillus having
been already described in previous chapters, it is
unnecessary to state them again.

Making cultivations of the two species at the
same time and keeping them under exactly the
same conditions, the differences noticed may be
stated thus: (i) In plate cultivations of both these
bacilli, it is found that the superficially situated
colonies of the grouse bacillus are more rapid in
appearing and more rapid in spreading, they are
flatter, their edges thinner than in those made of
our aerobic oedema bacillus ; (2) in gelatine streak
culture the band formed in the line of inoculation is
broader, flatter, and thinner, and grows quicker in
the case of the grouse bacillus ; (3) in stab culture
the plate -like growth on the upper free end of the
stab is broader, thinner, and spreads out more ; (4)
in broth the turbidity of the fluid and the granular
precipitate is greater and more copious in the grouse
bacillus.

In shaken gelatine cultures in both cases numerous

68 THE GROUSE DISEASE chap.

flat gas bubbles appear all through the gelatine, one
in connection with each individual colony, except
in the superficial layers ; the gas bubbles situated
in the depth remaining longest ; in respect of these
gas bubbles no difference is noticeable between the
two species. On potato the difference noticeable
between the two microbes is this, that the oedema
bacillus grows faster and spreads out quicker over the
surface of the potato than the grouse bacillus, the former
producing a more brownish growth than the latter.

As regards the morphological appearances, the
gelatine and Agar cultures of our malignant oedema
bacillus, examined fresh, show more motile forms
than the cultures of the grouse bacillus, and the
individual bacilli are distinctly longer and more
cylindrical — both in the animal and in the culture —
in the case of our malignant oedema than in the
case of the grouse bacillus ; this is a definite and
constant difference easily detected.

Measurements of the bacilli of our malignant
oedema in dried and stained cover-glass specimens,
show : —

In the oedematous fluid of the guinea-pig :
Thickness 0.7 /^
The majority in length 0.8-1.2 yu
Some ,, ,, 1.6-2,4 /^
Few up to . . 2.4 yu long.

viii A PATHOGENIC BACILLUS 69

In specimens made of broth and Agar cultures :
The majority in length 1.6-2.4 A*"

In specimens from gelatine cultures :
Length of the majority 0.8 /x.

By comparing these figures with those given of
the bacilli in grouse disease it will be seen that
our malignant oedema bacilli are distinctly longer
than those of the grouse disease.

Another difference is the distribution of the bacilli
in the animals (mice and guinea-pigs) infected : the
grouse bacillus is present in the blood and spleen
in greater abundance than our malignant oedema
bacillus ; the cedematous swelling in the guinea-pig
after inoculation with oedematous fluid or cultures
of our oedema bacillus is always more pronounced
than after inoculation with the cultures of the grouse
bacillus. Rabbits are susceptible to our malignant
oedema bacillus, but insusceptible to the grouse
bacillus.

If guinea-pigs or rabbits are inoculated with a
drop of the oedema fluid, or with a good dose
of our malignant oedema bacillus, the animals are
found ill and quiet as early as 12 hours after inocula-
tion ; they are found dying or dead in from 20 to 36
hours with considerable oedema and haemorrhage in
the groin, on the abdomen, and chest. Sections made
through the skin, the subcutaneous and muscular

70 THE GROUSE DISEASE chap.

tissues, show the tissue of the cutis and the sub-
cutaneous tissue crowded with the bacilH — the bacilh
either uniformly distributed or aggregated in clumps ;
owing to the oedematous condition, the interfascicular
spaces are distended and contain numerous leu-
cocytes ; the adjacent muscular fibres are oedema-
tous, disintegrating, and in the lymph-spaces of the
connective tissue between the muscular fibres are
crowds of the bacilli. Now after inoculation with
the grouse bacillus such a state is not noticed. The
leucocytes of the oedematous subcutaneous tissue
just mentioned, in very acute cases, include the bacilli,
some only one or two, others are crowded with them
(see Fig. 27).

If for inoculation of a guinea-pig a small dose of
a cultivation of the malignant oedema bacillus is
used, say one or two minims of a recent broth cul-
ture, the animal does not die ; it is ill after 24 hours,
and shows an oedematous swelling, but this is not
by any means so large as after a large dose. The
animal, as just stated, does not die, and the soft
oedematous swelling in a few days decreases, be-
comes firm, and forms a solid cord or band in the
subcutaneous tissue ; this firm swelling in some cases
leads to necrosis and sloughing of the skin with
complete healing, or it gradually, during a week
or fortnight, becomes smaller and smaller till it

A PA THO GENIC BA CILL US 7 r

entirely disappears ; rarely, the animal dies between
the end of the first and the end of the second week.
If the tumour of such a "chronic" animal is hard-
ened and examined in sections, stained first with
rubin and then with methyl blue, one finds numerous
bacilli in the tissue of the tumour. That they are
living can be shown by the successful cultures made
of the fresh tumour tissue. What is, however, re-
markable is that while the substance of the leuco-
cytes in the subcutaneous tissue at and about the
seat of inoculation in the acute fatal cases en-
closes the bacilli abundantly, there is nothing of the
sort to be seen in the leucocytes of the "chronic"
tumour. This is rather contrary to what we ought to
expect if the theory of phagocytosis (Metschnikoff),
i.e. the swallowing and destruction of the bacilli by
the leucocytes, were applicable here ; for it would be
serving no object if the leucocytes of the oedematous
tissue in the acute cases were destroying them,
seeing that the tissue in general is flooded with
the bacilli, and the animals die rapidly ; whereas in
the "chronic" cases, if the resistance of the animal
were due to a salutary part played by the leucocytes,
to wit, acting as phagocytes : taking up and destroy-
ing the bacilli and thus preserving the life of the
animal, we should expect to find at least some of
the leucocytes enclosing the bacilli ; but evidence

72 THE GROUSE DISEASE chap.

of this kind is altogether absent in the chronic
cases.

Just as in the case of the grouse bacillus, so also
with our malignant oedema bacillus ; animals that
have passed through a mild attack and recover with-
stand a second inoculation with a fatal dose.

As has been mentioned in a previous chapter,
the bacillus of grouse disease does not form spores ;
the same holds good for the bacillus of our malignant
oedema. Complete drying kills the latter ; heating cul-
tures to 60° C. for five minutes kills all life ; no further
sub-cultures can be made. It is on these grounds
easily possible to separate in garden earth the
classical (Koch) anaerobic spore-bearing bacillus from
our aerobic non-spore-bearing bacillus. By exposing
garden earth mixed with a little sterile salt solution
in a test-tube to a temperature of 70-75° C. for five
minutes, the spores of the classical anaerobic bacillus
are preserved, while our bacillus is killed ; with such
garden earth I inoculated guinea-pigs subcutaneously,
and now produced the classical malignant oedema,
the oedema fluid crowded with the classical anaerobic
spore-bearing bacilli ; by cultivation this cedema fluid
can be shown to be free of our aerobic bacillus.

In order to ascertain further differences between
the bacillus of grouse disease and our aerobic 'bacillus
of malignant cedema, I inoculated yellow - ammers

VIII A PATHOGENIC BACILLUS 73

with recent broth cultures (one to two minims) of
the latter. The birds were found dead or dying be-
fore 24 hours were over, the heart's blood and the
blood juice of the hypersemic lungs containing but
comparatively few of the bacilli, in strong contrast to
the condition described of the behaviour of the
bacillus of grouse disease.

An important difference, however, exists between
the action on pigeons of our aerobic oedema bacillus
and that of the grouse bacillus. As has been stated
in a previous chapter, pigeons are refractory to even
large doses of virulent broth culture of the grouse
bacillus, but on injection with broth culture of our
malignant oedema bacillus they are anything but
refractory. On the day following the subcutaneous
inoculation with 5 minims of a recent broth culture
of our malignant oedema bacillus, the pigeons were
found distinctly ill, quiet, off their food, when made to
move they were seen to be very shaky on their legs,
and they were very unwilling to move ; this condition
lasted for several days, but eventually they recovered.

A very remarkable antagonistic action exists be-
tween the two bacilli if they are injected at the same
time into the same mice. Recent (24-48 hours old)
broth cultures of either of them, injected separately
under the skin of the back of white mice, in doses
varying between 2 and 5 minims, produce illness and

74 THE GROUSE DISEASE chap.

death In one to three days in the great majority
of the animals ; they die with the same symptoms
and with very similar post-mortem appearances : no
oedema at the seat of the inoculation, congestion of
both lungs, the spleen slightly enlarged ; the heart's
blood and the lungs and the spleen contain the bacilli
abundantly. Now I inoculated mice with broth
culture (24 hours old) of our oedema bacillus, each
animal receiving 2-5 minims ; other mice were in-
oculated at the same time and in the same doses with
broth culture (24 hours old) of the grouse bacillus,
and a third lot were inoculated at the same time with
a mixture in equal parts of the very same broth
cultures of our bacillus of maligfnant oedema and of
the grouse disease. The result was very remarkable.
Three such series were made : in each series two mice
being used for each experiment. In series one, both
mice inoculated with the malignant oedema bacillus
were ill and weakly, one died before the second day
was half over, the other survived ; the two mice done
with the grouse culture both died before the second
day was over ; and of the two mice done with the
mixture, one died on the sixth day, the other survived.
In series two, both the mice done with the malignant
oedema bacillus died ; one of the two mice done with
the grouse culture died on the second day, the other,
though very ill, recovered ; of the two mice done with

VIII A PATHOGENIC BACILLUS 75

the mixture, one died on the fifth day, the other
survived. In series three, both mice done with the
culture of maHgnant oedema were found dead on the
second day with abundant bacilH of maHgnant oedema
in the blood ; both mice done with the culture of the
grouse bacillus were found dead on the second day
with abundant bacilli of the grouse disease in the
blood ; and both mice done with the mixture were
alive and well on the seventh day. Now in this last
series the amount of the mixture injected in the last
two mice was double the amount of the culture of
malignant oedema and of grouse separately, and al-
though the two mice that were inoculated with the
same amount of malignant oedema alone, as also the
two mice inoculated with the same amount of grouse
culture alone, succumbed, yet the two mice inoculated
with the mixture, that is, with double the amount,
survived. Clearly there is then a decided antagonism
within the body of the mouse between the two species,
by which the action of the one is neutralised by that
of the other. If the two species were the same, then
the two mice done with the mixture, having received
a double dose, ought to have succumbed so much
more readily, whereas on the contrary they remained
alive ; it follows from this that the two microbes are
two totally different species. This conclusion must,
therefore, remove all doubt as to any close relation

76 THE GROUSE DISEASE chap, viii

existing between our bacillus of malignant oedema
occurring in earth and the bacillus of grouse disease ;
and it is obvious that notwithstanding certain striking
cultural similarities between the two species, no valid
reason exists to accuse our bacillus of earth, i.e. that
of our malignant oedema, as causally connected with
the grouse disease.

PART II

FOWL CHOLERA, FOWL ENTERITIS, AND
"CRAMPS" IN YOUNG PHEASANTS

CHAPTER IX

FOWL CHOLERA

During the early part of 1 889 I became informed of
the existence of an acute and fatal epidemic disease
amongst fowls at a poultry-farm in Kent, which was
represented to me as being chicken cholera, and as I
had not till then come across or heard of the exist-
ence of chicken cholera in England, I was anxious to
obtain material from that farm. The fowls which I
received w^ere carefully examined and used for micro-
scopic examination, for cultivation, and for animal
experiment, and it very soon became evident that the
disease with which these fowls had been affected was
clearly distinct from chicken cholera ; it was a disease
which, though similar to, was in many important
points different from chicken cholera ; I have called
\x. foivl enteritis. In order that the reader may be in
a position to correctly understand these points of
similarity and difference, I will first describe the
nature of chicken cholera, known so well and so com-

8o THE GROUSE DISEASE chap.

pletely by the researches of Perroncito, Toussaint,
Pasteur, Kitt, and others.

The disease fowl cholera or chicken cholera, rife
in France and Germany, is an acute infectious dis-
ease, rapidly fatal, and producing great devastations
amongst fowls. After an incubation of from i6 or
1 8 hours to a day or a day and a half, the fowls
show the first symptoms of the disease — diarrhoea of
thin, greenish, mucoid evacuations, unwillingness to
move, and quietness ; they refuse food ; they soon
become drowsy and somnolent, and this rapidly in-
creasing, leads to death in from 24 to 36 or 48
hours. On post-mortem examination the heart is
distended and filled with blood that rapidly clots,
the lungs, liver, spleen, intestines, and peritoneum
are found much congested ; in the lungs, liver, and
intestine, particularly the duodenum and in the peri-
toneum, haemorrhages occur in the form of petechias
and patch-like blood effusions. The blood of the
heart and the general circulation is crowded with
minute oval or short rod-like bacteria, known as the
bacilli of fowl cholera ; their number is generally as
great, often greater, than that of the blood-corpuscles
(Figs. 32, 33, and 34). In stained preparations the
rods show a characteristic deeply stained granule at
each end, the middle part of the rod being clear ;
hence at first sight it appears like a dumb-bell. Some

FOWL CHOLERA

of the rods form dumb-bells or even short chains.
The size of the single rods in stained preparations is
o.2/i thickness, the spherical ones 0.2 yu- in diameter,
the oval and rod-shaped 0.4-0.5 ^i in length, some are
longer, 0.8 /a; there are some which measure 1-1.5 /i,
but on careful examination it will be found that these
are really dumb-bells of rods. ^

The bacilli are present in large numbers in the
blood of all organs, and almost in pure cultivation in
the mucoid contents of the intestine.

Inoculation into fowls, pigeons, and rabbits, of
a drop of blood or of a drop of a broth culture of
the microbe, produces Invariably the disease and
death in from 20-36 hours under the same symptoms,
and the same distribution of the bacilli. In the
rabbit, death as a rule occurs within 24 hours, some-
times in as short a time as 18-20 hours, the blood
teeming with the bacilli ; haemorrhages in the liver,
the intestine, and peritoneum being generally found.
In the rabbit dead of the inoculated disease, the
bacilli are oval or rod-like and of the same character
as those in the fowl and pigeon, but there are a great
many that are almost spherical, far more than in the

^ The size of the bacilli generally given in text - books (Fliigge,
Baumgarten) is too large. The above figures given by me were
ascertained with exact measures. In the illustrations given both by
Fliigge and by Baumgarten, the microbe is also represented of too
large a size, as is proved by photographs.

G

THE GROUSE DISEASE

fowl and pigeon. Specimens made of cultivations in
broth, on gelatine, or on Agar show, besides the char-
acteristic oval rods, also numerous dumb - bells or
chains of four.

If a drop of blood of an animal dead of the
disease, or of a recent culture, be examined in the
fresh condition, the bacilli are not possessed of active
motility, all appearing non-motile or only showing
Brownian molecular movement. A trace of a droplet
of the heart's blood of a fowl, pigeon, or rabbit dead
of the disease, used for a gelatine plate cultivation or
rubbed over the slanting surface of nutrient gelatine
and kept at 20° C, yields innumerable colonies. The
first indications of the colonies visible to the unaided
eye do not appear before 48 hours are over, though
with a magnifying-glass they can be just discerned
after 36 hours. When easily visible to the un-
aided eye, that is, during the third and fourth days,
and even later, they appear as minute dots, greyish-
yellow in reflected light. Under a lens they are
round, sharply outlined, and somewhat prominent,
brownish and granular in transmitted light. The
colonies growing on the surface are a little larger
and have a slightly uneven outline, recognisable as
such under a glass, those in the depth remain very
small spherical dots (Fig. 35). In gelatine streak
culture, after an incubation at 20" C, there is visible,

FOWL CHOLERA 83

but not before 48 hours, a thin greyish-yellow line of
growth which gradually broadens during the next days,
its outline rounded and knobbed, but even after a
week to a fortnight does not reach in breadth more
than a few millimetres. In stab culture the stab
becomes marked as a line composed of minute dots,
brownish in transmitted light, whitish-yellow in re-
flected light ; at the upper free end of the gelatine
is a small rounded plate of growth slightly raised,
with irregular outline, and, after a week or ten days.
measurinof a few millimetres. The orelatine is never
liquefied. Alkaline beef-broth peptone incubated at
35-37° C. becomes slightly turbid by the growth of the
microbe in 24 hours ; the turbidity increases during
the next days, but, even when the maximum develop-
ment has been reached, is never very pronounced.
There is then present a small amount of a greyish-
white powdery precipitate. On the surface of Agar
the growth forms a thin, translucent, grey film ; on
potato sterilised in the steamer, and then kept at
35-37° C, the growth is slow and remains fairly
limited to the region of inoculation, forming a whitish
or light brown film spreading slowly in breadth.

As has been mentioned above, a drop of a broth
culture 24-48 hours old, or a drop or two of a salt
mixture of the growth taken from a fairly recent
gelatine or Agar culture, invariably produces fatal

84 THE GROUSE DISEASE chap.

infection in fowls, pigeons, and rabbits. And the
virulence of the cultures, be they derived from fowl,
pigeon, or rabbit, is maintained unimpaired for an
unlimited number of sub-cultures in broth, gelatine,
or Agar, provided these be used when recent. I
have had sub-cultures which had been carried on
on gelatine for from several months to over two
years ; fresh broth sub - cultures started from them
proved always virulent. Fowls can be infected not
only by subcutaneous injection but also by feeding,
but the result is not by any means so certain ; since
some animals escape infection. As has been men-
tioned above, the intestinal contents teem with the
bacilli, and it is therefore justifiable to assume that
under natural conditions the food picked up from
the ground by the fowls becomes contaminated with
the microbes of the dejecta. Before concluding this
description, which is introduced here merely for the
sake of comparing this disease with the disease to
be described presently, viz. with the fowl enteritis, I
wish to mention two further points : —

(i.) The enormous rapidity with which the microbe
multiplies in the rabbit. A rabbit of known weight
was inoculated with a drop of broth culture containing
about 20,000 bacilli ; the animal was dead with the
typical disease in 20 hours. The number of the bacilli
present in the heart's blood was then ascertained, and

FOJVL CHOLERA 85

assuming the bacilli to be distributed throughout the
blood in a uniform manner, it was calculated that,
provided the relation of blood to body weight be
about I to 14 or 15, there were present in the total
blood over 1 200 millions of bacilli, that is to say, that
the 20,000 bacilli introduced into the animal had
multiplied in 20 hours so as to yield 1200 millions,
or in other words, that each one of the introduced
bacilli had produced a crop of 60,000 bacilli.

(2.) On examining sections of the hardened liver of
animals — fowl, pigeon, or rabbit, — dead of the disease,
there are always found in each section patches of the
liver acini in which the capillary vessels are distended
and filled with the bacilli ; the liver cells around being
dark, granular, and disintegrating ; in the fowl they
are filled with fat droplets, but in all parts of the
section all the blood-vessels contain crowds of the
bacilli, and many of them are present outside the
vessels between and around the liver cells (Figs.
38 and 39).

The illustrations which are given in Figs. 32-40
give an accurate representation of the nature of the
gelatine cultivations, of the shape and size of the
microbe, and of their distribution in the blood and
the organs.

CHAPTER X

FOWL ENTERITIS NATURE OF THE DISEASE

In 1888 Mr. W. Cook, well known through his works
and writings on fowls and fowl-breeding, had on his
poultry-farm, then at Orpington, in Kent, in about
two acres of land, a fatal epidemic of fowls by which
he lost, between March 1888 and March 1889, over
400 birds. The greatest number of birds, nearly
200, died during March and April 1888, occasionally
6 per diem ; during the beginning of 1889 the number
of fatal cases had decreased considerably — to about 7
per week. Mr. C. Wellington, then at Mr. Cook's
farm, was kind enough to supply me, with Mr. Cook's
permission, with birds affected with the disease and
either dying or recently dead. From these birds I
obtained materials with which I commenced experi-
ments on normal fowls and other animals. On
making post-mortem examinations the difference be-
tween this fowl disease and fowl cholera — for such
was the disease considered — was distinct. All the
fowls that I received from Mr. Cook's farm, and

CHAP. X FOWL ENTERITIS— NATURE OF DISEASE 87

those experimental fowls which I afterwards had
occasion to examine, presented the same kind of
post - 7nortein appearances : the heart was full of
coagulated blood ; this condition of the blood was
noticed very soon (^-i hour) after death. The lungs
in all animals were normal, no congestion, no haemor-
rhage ; the intestines showed redness in the mucous
membrane and in the serous covering, but there was
no haemorrhage anywhere ; in the caecal appendages
there was a good deal of mucus, either in the form
of flocculi or as continuous yellowish, greyish, or
brownish masses ; the rectum contained greenish-
yellow fluid faecal matter. The spleen was enlarged
to twice or thrice its normal size, but its substance
was not excessively bloody, since from a cut surface
there was not that oozing out of sanguineous fluid
commonly observed of a hyperaemic spleen. The
liver was somewhat enlarged, soft, and flabby, there
were no haemorrhages ; the surface of the liver and
of the peritoneum was moist, occasionally there was
a small amount of exudation, but no haemorrhages.
Examining the fresh blood, it was at once noticed
that though bacteria could be found here and there
in each field of the microscope, they were very few
and far between, one or two small rods only being
met with in one field. Making cover-glass specimens,
and, after drying and staining, examining them under

88 THE GROUSE DISEASE chap.

the microscope, this condition, viz. the relative scarcity
of the bacteria in the blood, was confirmed (Fig. 41).
It was at the same time noticed that the bacteria were
distinctly thicker and longer than in fowl cholera, they
occurred also as single rods or as dumb-bells with
rounded ends. This condition, as regfards the bacilli
in the blood, was uniform in all the fowls that I got
from Orpington, as well as in those which were in-
fected in the laboratory from the Orpington fowls.

Thus far it was quite clear from these observa-
tions that the Orpington disease differed from fowl
cholera: (i) in the absence of hsemorrhagic changes
so common in fowls dead of fowl cholera; (2) in
the scarcity of the bacteria in the blood ; (3) in the
difference in size between the bacteria present in
the blood of the Orpington fowls and those of the
typical fowl cholera. Further differences established
were these : the spleen of the Orpington fowls,
examined in cover-glass specimens, contained the
bacteria somewhat more numerously than the blood
(Fig. 42), but very far less numerously than in fowl
cholera, and also the bacteria of the spleen of the
Orpington fowls were distinctly thicker and longer
than those of the fowls dead of fowl cholera.

Measurements of the bacilli of the blood or spleen
tissue, or of cultures in dried and stained cover-glass
specimens, showed : —

X FOWL ENTERITIS— NATURE OF DISEASE 89

The thickness of the bacilli . 0.3-0.4 /ti.

The length of the single bacilli . 0.8-1.6 /x.
Some as long as 2 /a.

The mucus of the intestine of the Orpington
fowls contained the bacteria, just as the mucus of
the fowl -cholera fowls, in almost pure cultivation,
either as spherical or oval coccus -like organisms,
singly or as dumb-bells, or as distinctly cylindrical
bacteria (Figs. 43 and 44). Examining in salt
solution or broth the fresh bacteria of the blood or
spleen of a fowl dead of fowl cholera, or of gelatine
cultures of the same, there are no motile bacilli to
be met with. The bacteria of the Orpington fowls
are also devoid of motility, for in no preparation,
either of the blood or spleen, or of gelatine or other
cultivations, did the bacilli reveal anything like
motility. As has been already mentioned, inoculat-
ing subcutaneously with the blood or spleen-juice
of a fowl dead of fowl cholera, fowls, pigeons,
or rabbits, invariably a fatal result is produced
in one or two days, or even less, with the
characteristic symptoms and post - mortefn appear-
ances of fowl cholera. Experiments of the same
character were therefore instituted with the blood
and spleen tissue of the Orpington fowls, but the
result was totally distinct from that obtained in fowl
cholera, for the pigeons proved quite insusceptible ;

90 THE GROUSE DISEASE chap.

SO were also the rabbits that had been inoculated
with the blood or spleen of the Orpington fowls.
All pigeons and rabbits remained alive and well.
The fowls that were so inoculated remained well for
the first four or five days, then they left ofT feeding,
became quiet, and showed diarrhoea of greenish-yellow
evacuations ; a day or two later they were found
dead, but in no case did they show the somnolence
characteristic of fowl cholera. I experimented on a
large number of fowls with blood and spleen tissue
of the Orpington fowls, or of fowls that succumbed
after inoculation from the Orpington fowls, also on
a very large number of fowls that I inoculated with
cultures of the blood or spleen tissue, dead from the
natural disease or after inoculation, and in no single
instance have I found any difference in the results, in
no single instance have I seen any such rapid course
as characterises fowl cholera. The earliest date at
which the animals showed illness was the fourth
day, and this, amongst a series of experiments com-
prising several dozens of fowls, occurred only once or
twice. Most animals remained well for the first four
days at any rate. Death in no single instance occurred
before the sixth day, in the majority of instances
during the seventh day, occasionally the eighth or
ninth day. On account of the enteric disturbance
being a prominent symptom, and to distinguish the

X FOWL ENTERITIS— NATURE OF DISEASE 91

disease from fowl cholera, I have called it fowl
enteritis. I have described the nature and character
of the disease in the Centralblatt filr Bacteriologie
und Parasitenkunde, 1889, v. 21 and following; and
although the different size and scarcity of the bacteria
in the blood in the Orpington fowls was clearly-
pointed out, as also the difference in the appearances
at the post-inorte7n examination in fowl enteritis and
fowl cholera ; the remarkable and striking fact that
pigeons and rabbits were found insusceptible to the
disease, and other differences, were clearly stated,
yet some critics ventured to offer the opinion that
the Orpington disease, i.e. the fowl enteritis, is not
sufficiently well differentiated from fowl cholera to
be declared not to be such. To this I can only
say, — and in this 1 am sure every pathologist and
bacteriologist who has any personal knowledge of
fowl cholera will agree with me, — that those critics
either are not sufficiently, certainly not directly, ac-
quainted with fowl cholera, or they have not read my
statements as regards the Orpington disease, or both.

Other equally striking differences are noticed be-
tween the two diseases if the cultures obtained from
the Orpington fowls are studied.

CHAPTER XI

CULTIVATION OF THE BACILLUS OF FOWL ENTERITIS

A DROPLET of blood taken from the heart and used
for a gelatine plate cultivation, or rubbed over the
slanting surface of gelatine, yields always a pure culti-
vation of colonies of the bacilli of fowl enteritis. The
number of colonies of a droplet of blood of a fowl dead
of fowl enteritis compared with the number of colonies
of a droplet of blood of a fowl dead of fowl cholera, as
may be expected from what has been said above, is
strikingly different. This difference can be made very
conspicuous by diluting the blood first and then mak-
ing cultivations. A measured drop of blood — one
minim or one division of a Pravaz syringe — of a fowl
dead of fowl cholera is added to lo cc. of sterilised
(boiled) salt solution, and well mixed ; from this a
droplet is taken by means of the platinum loop, and
with it a gelatine tube is inoculated, and from it a
plate cultivation is made ; or it is rubbed over the
surface of the slanting gelatine contained in a tube.
The number of colonies making their appearance in

CHAP. XI CULTIVATION OF THE BACILLUS 93

such cultivations on incubation is still very consider-
able. But if on exactly the same lines plate cultiva-
tions or slanting gelatine tube cultivations be made of
the blood of a fowl dead of fowl enteritis, the number
of colonies is extremely limited, — some tubes remain
free of colonies, others contain only a very limited
number.

The number of colonies that make their appear-
ance when using a small particle of the spleen tissue
in fowl enteritis is, as might be expected from what
has been said in the preceding chapter, considerably
greater than when using the heart's blood. The
character of the colonies and the rate of growth are
different in the two diseases. In the case of the fowl
enteritis, the first signs of the colonies are noticed
with a glass very distinctly after 24 hours' growth as
greyish translucent dots. During the second and
third days the colonies situated on the surface rapidly
enlarge into greyish -white translucent discs with
thinner angular margin ; they remain small spherical
dots when situated in the depth. In transmitted
light the superficial as well as the deep colonies are
brownish, the latter of course more so than the former.
The superficial colonies grow steadily in breadth, be-
come more angular, are large and thin as compared
with those of fowl cholera (Fig. 45), and do not at any
time look yellowish-white like the latter. The chief

94 THE GROUSE DISEASE chap.

difference, however, is shown in the rate of growth :
the colonies of the fowl -cholera bacillus grow con-
spicuously slower and remain smaller than those of
the fowl - enteritis bacillus. I have repeatedly had
occasion to make comparative experiments ; in this I
was ably assisted by Dr. Wood, of Melbourne, while
working in my laboratory : cultivations of the fowl-
cholera bacillus and of the fowl-enteritis bacillus were
made at the same time under the same conditions in
the same stock of nutrient gelatine, kept then under
precisely the same conditions, and always, without ex-
ception, the differences described above in the aspect,
and particularly the rate of growth, of the two microbes
were conspicuous and constant. The time at which the
colonies of the fowl - enteritis bacillus appeared, and
the rate with which they grew afterwards, the first
much shorter, the latter very much greater, were
appreciably different from those of the colonies of the
fowl-cholera bacillus.

In sub-cultures in gelatine, in streak and stab, the
growth is distinct even during the second day : in
the streak culture a greyish-white flat band appears,
which gradually broadens during the following days ;
its margin is irregularly knobbed and sinuous. After
about two weeks the band is 3-5 millimetres broad,
but it remains flat ; in reflected light it looks whitish, in
transmitted light, light brown. In stab culture, during

XI CULTIVATION OF THE BACILLUS 95

the second and third days, the line of inoculation
becomes visible as a greyish-white line, which under a
glass is seen to be made up of spherical dots ; on
further incubation these enlarge, but remain always
separated ; on the upper free end of the stab the
growth is a thin translucent greyish-white film with
crenate margin.

The differences in the rapidity of the growth in
streak, stab, and shake culture shown by the bacillus
of fowl cholera and of fowl enteritis are very marked,
that of the latter being considerably more rapid than
that of the former.

The gelatine is never liquefied by the growth.

In faintly alkaline broth kept at 35-37° C. the
bacillus of fowl enteritis grows rapidly, forming
uniform turbidity in twenty-four hours ; the turbidity
gradually increases, and, compared with that produced
by the fowl -cholera bacillus, is always at the same
distance of time markedly greater ; there is also a
greater amount of whitish precipitate in the broth
cultures of the bacillus of fowl enteritis than in those
of the bacillus of fowl cholera ; there is never any
formation of anything like a pellicle. Broth cultures
of the bacillus of fowl enteritis kept at 35-37° C.
begin to get clearer in the superficial layers by the end
of the week. On nutrient Agar incubated at 35-37°
C. the bacillus of fowl enteritis forms a whitish-grey

96 THE GROUSE DISEASE chap.

translucent film with sinuous margin ; after about a
week it has spread almost over the -whole surface of
the Agar, but it remains a thin film.

Equally marked differences between the bacillus
of fowl cholera and fowl enteritis are shown by their
behaviour on potato ; while the bacillus of fowl cholera
shows distinct growth on boiled or steamed potato, as
has been described in a former chapter, the bacillus of
fowl enteritis practically does not grow on potato —
even after a week at 35-37° C. incubation the point of
inoculation is only just perceptible as a whitish patch ;
of anything like an appreciable amount of growth
nothing is to be made out.

As regards aspect and size of the bacilli of fowl
enteritis there is not much to be added to what has
been said already ; in all media the bacilli are either
oval or rod- or cylindrical-shaped organisms, either
single or in dumb-bells, their ends rounded, and when
examined fresh in salt solution or in broth, never show-
ing motility. Their length, however, varies somewhat
in gelatine and Agar cultures. Cover-glass impression-
preparations of gelatine plate cultivations, dried and
stained, show that in some colonies the bacilli are
conspicuously more cylindrical than in others. Fig. 48
is a photograph taken from an impression specimen of
a colony on the surface of a gelatine plate, and here
the bacilli are all conspicuously cylindrical, so much so

XI CULTIVATION OF THE BACILLUS 97

that on account of their uniformity I was at first
inclined to doubt their genuineness, but I have, by-
repeated examination and by sub-cultures, convinced
myself of their being genuine fowl enteritis bacilli.
Some colonies, however, show many of the bacilli
shorter and not of such uniformity in length (Fig. 49).
A comparison of photographs made of impression
specimens of the colonies of the bacillus of fowl
cholera and of the bacillus of fowl enteritis shows
the difference in thickness and length of the two
species very conspicuously (Figs. 36 and 48). In
broth cultures of the bacillus of fowl enteritis there
are many bacilli which are cylindrical in shape, some
in chains, and some even forming filaments.

It has been mentioned in Chapter IX. that the
mucus in the intestine in fowl enteritis is crowded
with the bacilli ; cover-glass specimens show this suffi-
ciently well (see Figs. 43 and 44). This fact is con-
firmed by cultivation. The intestine — best the caecal
appendages — having been opened with sterile scissors,
a particle of the mucus is removed with a platinum
loop and placed in a test-tube containing a few cubic
centimetres of sterilised salt solution or beef broth ; it
is then well shaken. From the mixture, by means of
the platinum loop, inoculations are made of gelatine,
used either for plate cultivations or the droplet is
rubbed over the surface of the slanting gelatine and

H

98 THE GROUSE DISEASE chap.

then incubated. Large numbers of colonies of the
bacillus of fowl enteritis make their appearance with
scarcely any other contaminating bacteria : I have in
this way obtained, directly from the intestinal mucus,
pure cultivations with numerous colonies on the slant-
ing surface of gelatine.

Dr. F. H. Andrewes has in my laboratory ascer-
tained that various species of microbes, which, when
grown on the ordinary nutritive media, occur chiefly
as oval or short rod-like organisms, — of swine fever,
Middlesbrough pneumonia, grouse disease, — when
grown on similar media, but to which previously had
been added Stedman's sea salt, to the amount of five per
cent, always form filamentous and thread-like bacilli,
either straight or wavy, singly or apparently branched ;
and when cultivated back on the ordinary media
again produce growths of the oval or short rod-like
forms. Dr. Wood repeated for me these experiments
with reofard to the fowl cholera and fowl enteritis
bacilli, and he ascertained as a uniform result that
while the bacillus of fowl cholera does not grow in
the salted media (gelatine, Agar, beef broth), the
bacillus of fowl enteritis grows well therein, form-
ing peculiar curved filaments of various lengths,
sometimes very considerable, the threads being
single or apparently branched : but this latter may
be only apparently so, because a matting together

XI CULTIVATION OF THE BACILLUS 99

and a consequent appearance of branching cannot
be easily excluded.

I have also compared the two bacilli in their
behaviour in gelatine to which previously a trace of
litmus had been added — sufficient to render the gelatine
of a slight bluish tint. While the bacillus of fowl
enteritis decolorises the litmus gelatine markedly at
and around the growth, the bacillus of fowl cholera
does this only very slightly indeed. (As is the case
with other organisms, tested by various observers,
this method of growing microbes in litmus-gelatine,
since its introduction .by Buchner, is a common method
of cultivation for the sake of differentiation.) The
decoloration produced also by the bacillus of fowl
enteritis is more rapid and more pronounced near the
surface than in the depth.

From all these observations, then, we are in a
position to affirm that also in respect of the character
of the cultivations in various media the bacillus of
fowl enteritis is a distinct species, well differentiated
from the bacillus of fowl cholera.

CHAPTER XII

EXPERIMENTS ON FOWLS

I. I HAVE already mentioned in Chapter IX. that
several experiments of subcutaneous inoculation of
fowls were carried out with blood or spleen tissue of
the Orpington fowls, dead naturally from fowl
enteritis. Altogether eight fowls were thus in-
oculated ; four of these were ill on the 5th-6th, four
on the 6th-7th day, being quiet and off their food and
having diarrhcea ; five died on the 7th, two on the
8th, and one on the 9th day. The post-mortem
appearances were in all these animals the same, and
such as have been already stated. The bacilli were
demonstrated in every one of these in cover-glass
specimens, and in cultivations of the heart's blood, the
spleen tissue, and the intestinal mucus.

2. Two fowls were inoculated with the blood or
spleen tissue of one of the above experimental fowls
that had died of the disease after inoculation ; the
result was exactly the same as above.

3. Inoculations of fowls were also made with

EXPERIMENTS ON FOWLS •,.' .'',;' •' lOi

cultivations derived from the blood of the 0?f)ingtcfr|i>':'; ;\,' j ,V
fowls. From a broth culture (first sub-culture), after
twenty-four hours' incubation, about lo minims were
inoculated subcutaneously into each of the two fowls.
On the 6th day both animals were found quiet and dull,
off their food, and suffering from diarrhoea. On the
7th day both were found dead with the characteristic
post-mortem appearances ; cover-glass specimens and
cultivations of the heart's blood, spleen tissue, and
intestinal mucus yielded abundant evidence of the
presence of the bacilli.

4. From a second broth sub-culture twenty -four
hours old, which had been established from a previous
gelatine sub-culture, two fowls were inoculated. Both
showed the first signs of illness on the 6th day, being
quiet, off their food, and affected with diarrhoea.
One of these fowls was found dead on the 7th day,
with the typical appearances, and the bacilli in the
blood, spleen, and intestinal mucus. The second
one was still quiet and had diarrhoea on the 8th
day. On the 9th day it still had diarrhoea, but
appeared a little more lively and was seen feeding.
On the loth day the diarrhoea was less, and the fowl
was distinctly better. The diarrhoea gradually sub-
sided, and at about the 1 8th -20th day the fowl seemed
to have perfectly recovered.

Two other fowls were inoculated subcutaneously

THE GROUSE DISEASE

with several minims of a broth sub-culture, twenty-four
hours old. Both birds showed the first signs of illness
on the 5th day ; they were quiet, off their food, and
had diarrhoea. The condition was the same on the
6th and 7th days ; on the 8th day the diarrhoea was
less, the birds were livelier, moved about, and
fed also a little ; they gradually recovered from the
diarrhoea, and by the end of the third week had
seemingly quite recovered.

[I shall have occasion to refer further below to a
large number of inoculation experiments on fowls made
with broth cultures, but as they were made with broth
cultures previously exposed to certain higher degrees
of temperature, with the object of attenuation, they will
be described in connection with the protective inocula-
tions.]

In order to see whether the infection of fowls can
be produced by feeding, the following experiments
were instituted :

5. Some of the intestinal contents of a fowl that
had died of the disease after inoculation with blood
and spleen tissue of an Orpington fowl, were poured
down the throat of two fowls. One of these birds
showed the first signs of illness — viz. slight diarrhoea
on the 6th day, on the 7th day the diarrhoea was pro-
fuse, the bird was quiet, dull, and off its food. This
animal was found dead on the 8th day. On post-

xii EXPERIMENTS ON FO WLS 103

mortem examination the typical appearances were
found. The blood, spleen tissue, and intestinal mucus
contained the bacilli ; cover-glass specimens and culti-
vations proved that the intestinal mucus was almost
a pure culture of the bacilli of fowl enteritis. The
second fowl remained lively and showed no diarrhoea.
After the lapse of eighteen days since the feeding,
this fowl was inoculated subcutaneously with a particle
of a first sub-culture on gelatine. On the 5th day the
bird was quiet and had diarrhoea, on the 6th the
diarrhoea was profuse, and on the 7th the fowl
was found dead with the characteristic appearances
and the typical distribution in its body of the bacilli
of fowl enteritis.

6. Two fowls were fed with a mixture, prepared
by mixing broth, Agar, and gelatine sub-cultures, the
two former about one month old, the last about six
weeks old. By the 7th day no sign of illness was
noticed in these fowls. They were fed again with a
gelatine sub-culture, eleven days old, but no illness
was noticed by the 8th day.

From this series it follows that by feeding with
the intestinal contents the disease is producible, but
with cultures the experiment has not yielded positive
results ; it ought to be noted, however, that the
cultures used in Experiment 6 were of considerable
age, and it must be added that the second feeding

I04 THE GROUSE DISEASE chap.

of the same fowls with gelatine sub-culture, eleven
days old, proved without success, although the same
gelatine sub-culture when inoculated subcutaneously
(Sub-experiment 5) proved virulent. We conclude,
therefore, that although some bacilli — e.g. of the in-
testinal mucus — when introduced by ingestion, escape
the action of the stomach and prove capable of infec-
tion (Experiment 5), this is the case only to a limited
degree.

Experiments on other Animals

7. Pigeons, notoriously susceptible to fowl cholera,
are quite refractory to fowl enteritis ; in no single
instance have I been able to produce illness after
inoculation with large doses of virulent material ; that
is, material which proved virulent for fowls, even in
small doses.

8. As is well known, rabbits are extremely sus-
ceptible to fowl cholera ; but to fowl enteritis their
behaviour is altogether different. A drop of blood of
a fowl dead of fowl cholera, or a drop of a recent
broth sub-culture of the bacillus of fowl cholera, injected
subcutaneously into a rabbit, produces infection and
death in from 20 to 30 hours without fail. Rabbits were
injected subcutaneously with 5-10 minims of blood
and spleen tissue of the Orpington fowls, as also of

EXPERIMENTS ON FOWLS 105

fowls dead after inoculation from these fowls, but no
result followed — the rabbits remained alive.

Rabbits were then injected subcutaneously with
broth sub-culture of the bacillus of fowl enteritis.
Altogether six rabbits were inoculated, each receiving
\ to I Pravaz syringeful of a culture twenty - four
hours old ; the same culture was tested on fowls and
proved virulent. Of the six rabbits five remained
well, the sixth was found dead on the fifth day. On
post-mortem examination both lungs were found much
congested, the spleen was dark and very slightly (?)
enlarged, the liver was slightly congested, no apparent
change in the intestine. Cultivations on gelatine were
made of the heart's blood and incubated. A very
limited number of colonies made their appearance.
Three drops of blood yielded altogether thirty -four
colonies, all of them typical colonies of the bacillus of
fowl enteritis.

From this it is quite clear that fowl enteritis and
fowl cholera are two different diseases ; further proof
will be furnished later on by showing the behaviour
towards fowl cholera of rabbits that survived a first
inoculation with fowl enteritis.

9. Guinea-pigs were also inoculated by injecting
them subcutaneously, some with blood and spleen
tissue of a fowl dead of fowl enteritis, others with
active broth culture of the bacillus of fowl enteritis.

io6 THE GROUSE DISEASE chap, xii

but no result was obtained ; the animals remained
alive and well.

lo. Mice (two in number) were inoculated under
the skin of the back with a large dose of a gelatine
culture of the heart's blood of an Orpington fowl.
Both animals were found ill after twenty-four hours ;
coat rough, "lumpy," breathing rapid. After forty-
eight hours the animals were worse, very quiet, eyes
closed. After three days they were so bad that they
looked dying. One died during the day. On post-
mortem examination the liver was found congested,
the spleen dark and distinctly enlarged ; no other
change. Cultivations were made of the heart's blood
and the spleen juice, but on incubation only a few
colonies of a liquefying coccus were found, no colonies
of the bacillus of fowl enteritis. The second mouse
was still ill, but alive on the fifth day : it was killed,
and cultivations were made of the heart's blood and
the spleen tissue, but no colonies of any bacteria made
their appearance. I take it, then, that the two mice
were made ill on account of having injected into them
a large dose of the chemical products of the bacilli,
the bacilli themselves not surviving or multiplying in
the body of the mouse, and therefore the mouse must
be also considered as refractory to the bacillus of fowl
enteritis, altogether different from fowl cholera, to
which illness mice are very susceptible.

CHAPTER XIII

ATTENUATION OF THE VIRUS OF FOWL ENTERITIS

The next question that I wished to answer was,
whether it is possible to find a means of attenuating
the virulence of cultures of the bacillus of fowl enter-
itis, in order to discover, if possible, a means of pro-
tective inoculation ? For this purpose two preliminary
questions had to be answered : ( i ) Do the bacilli
form spores ? (2) Does a mild attack of the disease
protect the animal against a second fatal attack ?

I. As regards the formation of spores. In all
experiments of attenuating the virulence of a microbe,
the presence or absence of spores is of first import-
ance. The spores being possessed of a greater power
of resistance would, therefore, evade the attenuating
influence of adverse conditions, such as certain degrees
of higher temperatures, chemical agencies, etc. The
bacilli of a culture not having the power of forming
spores might become attenuated in virulence, though
of course not killed, by degrees of temperature such as
55° C. and more, or by the addition of small doses of

io8 THE GROUSE DISEASE chap.

certain antiseptic media or other substances ; whereas
if spores are formed by the bacilli, the application of
such conditions would be altogether productive of a
different result.

Now the experiments of feeding which have been
recorded in the preceding chapter point strongly to
the absence of spores, because if such were present in
the intestinal contents, or in the cultures used for
the feeding, the positive results would have been more
numerous than they have actually been. But these
results do not absolutely demonstrate the incapability
of the bacilli of forming spores, and for these reasons :
under natural conditions the fowls, just as in fowl
cholera, contract infection most probably by picking
up food from a soil tainted with the evacuations of
birds affected with the disease ; and it might be pos-
sible that the bacilli on and in the soil produce
spores, but they do not do so within the living body
of the animal, or in the artificial cultures that had
been used.

A careful microscopic examination of old cultures
shows that there are present numerous bacilli which,
from their swollen-up condition and their abnormal
shape (spindle-shaped and abnormally thick), are no
doubt comparable to involution forms. Besides there
are present numerous bacilli which, from their not
taking the stain, compare to dead bacilli, of which

xni ATTENUATION OF VIRUS OF FOWL ENTERITIS 109

only the sheath is left. (Fig. 50 shows well such
forms.) In old broth cultures, three and more weeks
old, filamentous and cylindrical bacilli are met with
numerously, which clearly show the granular de-
generation and gradual disappearance of the proto-
plasm, so that then only the empty sheath is left. Such
forms contain spherical or oval or irregular granules,
which still take the dye ; but the older the culture,
and the more numerous these forms, the less likely
is it found that the culture has retained vitality ; that
is to say, the less likely is it possible to start a new
living sub-culture from them. I have made a large
number of experiments, and have had broth cultures
from several weeks to several months old, in which
such forms {i.e. cylindrical or filamentous bacilli, con-
taining from place to place, in an almost empty sheath,
only here and there a larger or smaller stained par-
ticle) abounded, yet no living sub-culture could be
started from them. These stained particles do not
present any uniformity either in shape, size, or posi-
tion, and there would be, therefore, no justification
for regarding them as spores ; besides, the death of
old broth cultures containing those forms would clearly
negative their being of the nature of spores. But
also, by direct experiment, it is possible to show
that neither in the bacilli in the fowl, nor in the
cultures, is there anything present that is capable

no THE GROUSE DISEASE chap.

of resisting those tests generally considered indica-
tive of the presence of spores. Simple but thorough
drying in a thin film kills all bacilli ; exposure to a
temperature of 60° C. for ten minutes sterilises the
bacilli completely. Numerous experiments were
made in this direction ; the result was in all cases
uniform. While heating to 60° C. for five minutes
was not reliable, heating to 60° C. for ten minutes
in all instances killed the bacilli.

From recent and old broth, gelatine, and Agar
cultures numerous inoculations were made into gela-
tine and into alkaline broth in tubes ; these were then
exposed to 60° C. for ten minutes, placed in the incuba-
tor, the first at 20° C, the latter at 35-37° C, but they
all remained sterile. Broth cultures from twenty-four
hours to three weeks old, and gelatine cultures from
one to six weeks old, were tested for the vitality of the
bacilli by establishing sub-cultures from them, then ex-
posing the original cultures to 60° C. for ten minutes.
After this sub-cultures were again made. While the
sub-cultures made from the original culture before heat-
ing proved successful, the sub-cultures made from the
heated original culture remained sterile. It follows
then from these experiments that there Is no evidence
that the bacillus of fowl enteritis is capable of forming
spores.

2. Does one mild attack of fowl enteritis protect a

XIII ATTENUATION OF VIRUS OF FOWL ENTERITIS in

fowl against a second attack ? It must be evident
that unless this is the case, that is, unless an animal
becomes refractory to a second attack by having passed
through a first mild attack, a primary inoculation with
attenuated virus would be useless ; it is for this reason
imperative to ascertain whether, in a particular in-
fectious disease, an animal having had the disease in a
mild form becomes thereby possessed of immunity
against a second attack. We have mentioned in a
previous chapter experiments (Experiment 4) in which
fowls had been the subject of inoculation with cultiva-
tions of the bacillus of fowl enteritis, and had become
ill but had recovered. In addition to these fowls
(three in number) five more fowls had recovered from
the disease after inoculation with culture — these five
animals will be mentioned below in connection with
other series of experiments — so that there were eight
fowls that had passed through an attack of the disease
but had recovered. When they had seemingly quite
recovered — they were lively, fed well, and appeared
in all other respects well — they were subjected to a
second inoculation, some with considerable doses of a
recent first broth sub-culture, that is, a broth sub-culture
made from a recent gelatine culture of the heart's
blood of a fowl dead of the disease ; others with blood
or spleen tissue of a fowl dead of the disease. The
same broth culture was used at the same time for in-

THE GROUSE DISEASE

oculation of two control fowls ; both these latter be-
came ill on the fifth day with the typical diarrhoea, they
were quiet and off their food. No control experiments
were made for testing the virulence of the blood and
spleen tissue, because I considered this an unnecessary
repetition ; the experiments described in the previous
chapter were so clear and decisive as to the virulent
character of the blood and spleen tissue of a fowl that
had died of fowl enteritis, that it did not require any
further confirmation. The above eight fowls that
were thus subjected to a second inoculation with viru-
lent material showed no signs of any illness, they
remained lively, fed well, and at no time showed any
diarrhoea, and they remained well for weeks after, till
they were discarded. It follows from these experiments
that fowls that have passed through a first mild attack
of the disease are possessed of immunity against a
second attack. It was now most important to discover
the material that could be relied upon as producing a
distinct but transitory attack of fowl enteritis.

The first experiments were made with broth sub-
cultures from a gelatine culture of the heart's blood of
the rabbit mentioned in the previous chapter (sub. 8),
as having been the only rabbit out of six that had
died after inoculation with broth culture. Seeing that
rabbits are so little susceptible to this disease, I thought
it not improbable that the bacilli taken from the body

XIII ATTENUATION OF VIRUS OF FOWL ENTERITIS 113

of the rabbit might have lost some of their virulence,
since the body of this animal is evidently not a favour-
able medium for their growth and multiplication. On
testing by experiment this supposition, it did not prove
correct. Twelve fowls were inoculated with broth
sub-cultures of the bacillus derived from the rabbit.
The result was this — that all twelve birds had
diarrhoea on the 5th or 6th day, and that nine died
(between the 7th and 9th day) and only three ^ sur-
vived. A broth sub-culture derived from the rabbit
proved, therefore, of a considerable degree of virulence,
worse than useless for protective inoculation, since
75 per cent of the animals inoculated died.

Another set of experiments was this : a fowl had
died of fowl enteritis, after inoculation with culture, on
the loth day, that is later than usual, and I surmised
that possibly the bacilli in this fowl might be of an
attenuated virulence. From gelatine cultures of the
heart's blood of this fowl broth sub-cultures were then
made and after 24 hours' incubation at 'i^^* C. were
used for the inoculation of twelve fowls. Seven of
these died of typical fowl enteritis, the other five,-
though ill between the 5th and 9th day, survived.

1 These three fowls were mentioned above as having been after-
wards used for a second inoculation with virulent material and proved
refractory.

2 These five fowls were mentioned above as having been used for
a second inoculation with virulent material and proved refractory.

I

114 THE GROUSE DISEASE chap.

This broth culture proved, therefore, also useless for
protective inoculation.

The next attempts were directed towards attenu-
ating virulent broth cultures by heat : ( i ) by exposing
broth cultures incubated at 37° C. from 24 to 48 hours
to a temperature of 50 C. for 10 minutes. Of such a
culture about ^ of a cubic centimetre was injected
subcutaneously into each of ten fowls. All these
fowls became ill with fowl enteritis, eight died, two
survived. These two were then re-inoculated with
virulent broth culture, and proved refractory.

(2) Of broth culture exposed to 50" C. for 20
minutes, about \ oi 2. cc. was injected into each of
eleven fowls. All became ill, four died, seven survived.
These seven were also re-inoculated and proved
refractory.

(3) Of broth culture exposed to 55^ C. for 15
minutes about l of a cc. was injected into each of
eight fowls. All these fowls were quiet on the 6th, 7th,
and 8th days, were off their food, but had no diarrhoea ;
seven survived and became well again, ^ but the eighth
was found dead on the 8th day with enlarged spleen ;
cultivations made of the heart's blood proved that the
animal had died of fowl enteritis. This series seemed,
therefore, a considerable step in advance of the former.

1 Also these seven fowls were re-inoculated with \irulent broth
culture, but proved refractory.

XIII ATTENUATION OF VIRUS OF FOWL ENTERITIS 115

(4) In this series the broth culture was heated to
55° C. for 20 minutes and then inoculated in the same
quantity as before into eight fowls. They were all quiet
and off their food on the 6th and 7th days, but there
was no diarrhoea ; on the loth day they seemed to all
appearances right again. This was then the result
aimed at, and it now only remained to be proved that
these same fowls were capable of withstanding a
second inoculation with virulent material. For this
purpose a broth culture, 24-48 hours incubated at
'^']'' C, was used for inoculation, two control fowls
being at the same time inoculated with the same
broth ; each of these ten fowls (that is the eight that
had been subjects of the first inoculation and the two
control fowls) received one whole Pravaz syringeful,
i.e. a considerable dose. The two control fowls be-
came ill and were dead on the 7th day from the typical
fowl enteritis, the eight other fowls remained perfectly
well. From this, I think it follows that a recent broth
culture heated to 55° C. for 20 minutes can be taken
to represent a fluid suited for protective inoculation.
Such a broth culture when tested by sub-culture
yields good and virulent growths, so that while the
heating to 55° C. for 20 minutes impairs the virulence,
it does not impair the vitality of the bacilli.

(5) A series of experiments which deserves mention
was instituted to see, whether under natural conditions

ii6 THE GROUSE DISEASE chap.

infection is carried from animal to animal by air, or
whether, as we mentioned in a former chapter as very
probable, infection is produced by ingestion — the
animals picking up food from soil tainted with the
evacuations of a diseased fowl. Mr. W. Cook, of
Orpington, kindly placed at my disposal a plot of
ground, not situated at or near his poultry-farm. This
plot was divided by wire netting into two adjacent
sections ; the wire netting separating these was made
double, and a space or passage of about i8 inches
was left free between them. Into each section were
placed ten healthy fowls, which we will call lot A and
lot B respectively. All fowls of lot A were then
inoculated with broth sub-culture of the bacillus of
fowl enteritis, the fowls of lot B remaining untouched.
All of lot A were distinctly ill on the fifth day ; they
were quiet and had diarrhoea between the fifth and
eighth day ; seven died, three survived.^ While the
disease was rife amongst lot A, one fowl escaped from
lot B and found its entrance amongst lot A.- After a
few days sojourning amongst them it became also ill
and died of the fowl enteritis ; also a strange fowl not
belonging to either of the two lots got access to lot A :

^ After a fortnight, when they seemed quite well again, they were
re-inoculated, but proved refractory.

2 The two plots of ground had a covering of wire netting, but this
was not perfect.

XIII ATTENUATION OF VIRUS OF FOWL ENTERITIS 117

this too became ill with the fowl enteritis and died.
But the nine fowls of lot B remained well many days
after the disease had come to an end in lot A. So
that though a wire netting separation with only 18
inches interval divided the fowls of lot A from those
of lot B, the disease from lot A was not communicated
to the latter fowls ; but on the other hand, the two
accessory fowls that had lived with the diseased fowls
of lot A in the same enclosure became infected.

The nine control fowls of lot B were afterwards
inoculated with broth cultures, they all became ill on
the fifth day, and suffered from diarrhoea ; eight died,
one survived.

From what has been stated in the preceding two
chapters, it is firmly established, I think, that both in
the nature and course of the disease, and in the
character and distribution of the bacilli, and in
the susceptibility of some and non-susceptibility of
certain other animals, fowl enteritis and fowl cholera
are two well - differentiated diseases. As has been
shown, particularly in the last chapter, the uniformity
in the symptoms and the duration of the incubation
period of the disease observed on a very large number
of fowls — nearly five dozen fowls were experimented
upon in the last five series of experiments — prove
conclusively the difference between the two diseases.
But I have some further experiments to record that

THE GROUSE DISEASE

confirm this in a striking manner, though they were
undertaken with a different object. While investi-
gating the action on animals of different pathogenic
microbes, when introduced simultaneously or sepa-
rately into the same animal, I tested also in this
direction the action of the bacillus of fowl cholera and
fowl enteritis. Pigeons, as we mentioned on a former
page, are very susceptible to fowl cholera, but quite
refractory to fowl enteritis. I inoculated [a) two
pigeons with fowl enteritis culture (broth culture 48
hours old), {p) two pigeons with fowl cholera culti-
vation (broth culture 48 hours old), and {c) two
pigeons with a mixture in about equal parts of the
same two broth cultures.

The pigeons a remained well and alive, the two
pigeons b and the two pigeons c were found dead after
48 hours, from typical fowl cholera. The two pigeons
a were then after about ten days inoculated with fowl
cholera cultivation (broth culture 48 hours old) ; both
were found dead before 30 hours were over from
typical fowl cholera.

One fowl and one pigeon were inoculated with a
broth culture of the bacillus of fowl enteritis. The
fowl became ill with the typical fowl enteritis, but the
pigeon remained, as a matter of course, unaffected.
The fowl, though at first very ill, nevertheless did not
die, but recovered gradually. After the lapse of 14

XIII ATTENUATION OF VIRUS OF FOWL ENTERITIS 119

days the fowl seemed perfectly well again ; it and the
pigeon were then inoculated with broth culture of the
bacillus of fowl cholera. At the same time one con-
trol fowl and one control pigeon were inoculated with
the same culture. Both the pigeons were dead in
1 8 hours of typical fowl cholera ; both fowls were
very ill during the second, third, and fourth days, very
drowsy, had copious diarrhoea, but both gradually
recovered.

From these experiments it follows, (i) that while
both the pigeon and fowl are susceptible to fowl
cholera, the pigeon in a higher degree than the fowl,
this is totally different as regards fowl enteritis, since
the pigeon is refractory and the fowl very susceptible ;
(2) that a first attack from fowl enteritis does not
protect the fowl from an attack of fowl cholera.

Although the two diseases are different, the mode
of their spread and the general behaviour of the two
species of bacilli are similar, and therefore the rules
that should guide us in the prevention of the spread
of either should be the same. These may be sum-
marised in saying — (i) every fowl that shows any
suspicion of the disease should be at once removed,
killed, and burned; (2) the remaining fowls should be
at once transferred to new ground, and, if practicable.

I20 THE GROUSE DISEASE chap, xiii

should be subdivided in separate small lots ; (3) the
ground from which the affected fowls have been
removed should be turned, disinfected with quicklime,
and not used for fowls for a considerable time. These
seem to me the best and easiest ways to prevent the
healthy fowls contracting the infection by picking up
food tainted with the evacuations (full of the specific
bacilli) of the diseased fowls.

CHAPTER XIV

"cramps" in young pheasants

During my stay (June 1887) at Blairquhan, Mr.
Douglas, the head gamekeeper, then in the employ of
G. Bailey Worthington, Esq., called my attention to a
great mortality from "Cramps" existing amongst the
young pheasants then reared by him. He was in
great distress on account of the helplessness in which
he found himself in the matter. The utmost care, the
most scrupulous attention to all details necessary in
the rearing of pheasants exercised by him and those
serving under him, availed nothing to neutralise or
stem the progress of the disease ; daily new birds
showed signs of the disease, and daily, when he in the
morning went round the grounds and inspected the
coops, several new deaths had occurred amongst the
young birds. From what I have observed myself, the
first signs of illness manifest themselves in lameness
of one leg and in the unwillingness of the birds to
move ; next day both legs are lame, the birds are
sitting quietly, and when made to move are seen to

THE GROUSE DISEASE

drag, more or less, their feet along the ground ; gener-
ally next day, or the day following, this condition
becomes more intensified, the birds do not move at
all, their eyes are closed, they do not of course feed
any more, and soon after the animals are found
dead. This disease is known to keepers and pheasant-
breeders as the "cramps," but I think it would be
more appropriate to call it paralysis, which would be
more in harmony with the inability to move, though
in reality it is neither the one nor the other. During
the two days that I stayed at Blairquhan I saw a con-
siderable number of animals thus affected or dead
from the disease, and I had the opportunity of making
a post-mortem examination on a good number. After-
wards Mr. Douglas forwarded to me in London a con-
siderable number of young pheasants that had died of
the disease, and these also were carefully examined.
All the birds were a few days to a few weeks old, the
disease occurring most commonly during the second
to third week ; and all without exception showed, as
the only constant and conspicuous feature, a disease of
the bones of the extremities — always of the legs, in
some few instances also of the bones at the anterior
extremities. The disease may be compared to an
acute periostitis and osteomyelitis. The femur of
both legs, and the tibia very often, showed either
at the upper or the lower end of the shaft, or at

XIV ''CRAMPS" IN YOUNG PHEASANTS 123

both, a localised congested state of the periosteum,
often haemorrhage into this and into the surrounding
muscle ; the bone itself is thinned at these places or
completely dissociated either in a circular line com-
prising the whole circumference, or obliquely, so much
so that between the actual condition and what one
would consider a fracture — simple fracture in the
former, oblique or compound fracture in the latter —
there seemed a complete analogy. The first few birds
dead of the disease that I dissected at Blairquhan
showed the fracture at the upper or lower end of the
shaft of the femur and tibia, particularly the former, so
pronounced that Mr. Douglas himself in the next few
birds could easily discover this condition. Haemor-
rhage into the periosteum and the surrounding muscle
occurs oftener in the upper and lower ends of the
shaft of the femur than in the tibia, and chiefly in
connection with oblique fracture. I thought at first
that the condition of the bones might have been
produced accidentally, either by the foster-mother or
by other animals attacking the little pheasants. But
Mr. Douglas assured me that this is quite out of
the question, and, in fact, impossible that such a thing
should happen unknown to him and his assistants, the
rearing being carefully watched night and day. The
large number of birds that died from the same disease
during June of that year, day after day, and that had

124 THE GROUSE DISEASE chap.

died in previous years, notwithstanding the utmost
care and attention, made it, therefore, clear that we
had to deal with an epidemic disease. The principal
symptom of the disease, viz. the impaired movement
observed by the keepers in many parts of the country,
south and north, and the cause of its appellation, viz,
"cramps," made it also clear that the disease is con-
nected with disease of the limbs, and it is hardly
credible that this should everywhere be the same, and
the result of an accident that had everywhere escaped
the watchfulness of the attendants. But what did
surprise me is the fact that both in the epidemic in
Scotland, which I then observed, as well as in young
pheasants sent to me through the Field from the
south of England that had also died of the "cramps,"
the remarkable condition of the bones — femur and
tibia — should have entirely escaped notice. And,
moreover, a correspondent of the Field thought it
"absurd" my saying that the young pheasants dying
of the " cramps " have this condition of the bones,
viz. partial and complete dissociation of the ends of
the shaft. I can only repeat that in no single instance
of young pheasants dying of the " cramps " have I
missed this condition, but I must add that I know of
an epidemic disease among young pheasants leading
to death in which this condition is not found, and
which animals do not die of the "cramps," but are

XIV ''CRAMPS'' IN YOUNG PHEASANTS 125

affected with a localised necrotic disease of the mouth,
throat, eyelids, and skin of the lips, contracted from
their foster-mothers, the hens, affected with a well-
known chronic localised necrotic thickening of the
mucous membrane of the tongue and throat. The
two diseases, "cramps" and necrotic stomatitis and
dermatitis in young pheasants, are in symptoms and
anatomical changes utterly distinct diseases.

As mentioned above, the principal anatomical
change in " cramps " of young pheasants is found in
the periosteum and the bone near the ends of the
shaft of the femur and tibia, occasionally but rarely
also of the humerus.

On making sections through the affected parts of
the bone and its periosteum, the changes are those
which constitute periostitis and osteomyelitis. The
vessels of the periosteum at the ends of the shaft
are engorged with blood, extravasated blood being
noticed in the tissue of the deep layer of the
periosteum ; this, at the same time, is thickened
and crowded with round - cells or inflammatory
corpuscles. The inflammation or infiltration with
round - cells or leucocytes extends into the bone,
all Haversian spaces and Haversian canals being
densely infiltrated with them, the bone matrix either
totally or partially destroyed, absorbed ; the infiltra-
tion with round -cells of the bone extends into, and

126 THE GROUSE DISEASE chap.

forms a continuity with, the inflamed central marrow,
this showing its blood-vessels much distended by
blood and its tissue a continuous mass of round-cells.
From the places of greatest inflammation, i.e. at the
ends of the shaft, the engorgement of the vessels and
the infiltration with leucocytes of the bone and the
central marrow extend a little way beyond. This
condition of periostitis and osteomyelitis is present in
all the cases which I examined, and the rarefaction
and absorption of the bone at the end of the shaft and
the subsequent dissociation is hereby easily explained.
In cover-glass specimens made of the inflamed
tissues, and in sections through the inflamed parts, a
few minute oval or cylindrical bacilli are constantly
met with (they require for demonstration prolonged
staining of the sections — a couple of hours and more
in methyl blue aniline water ; or staining for half an
hour to an hour in gentian violet aniline water shows
the bacilli well). Of course it is necessary after the
staining to well decolorise the section in methylated
spirit in order to remove as much as possible the dye
from the tissue cells, the bacilli retaining the dye more
persistently. From several cases cultivations were
made ; with sterile scissors the bone is laid bare, with
another pair of sterile scissors an incision is made into
the inflamed part, then with a sterilised platinum loop
a small particle of the marrow is removed and trans-

XIV "CRAMPS" IN YOUNG PHEASANTS 127

ferred to test-tubes containing nutrient gelatine and
nutrient Agar set with slanting surface, and is well
rubbed over the slanting surface. The gelatine tubes
are incubated at 20° C, the Agar tubes at 36-37° C.
All the tubes thus inoculated developed several
colonies of one and the same species of bacilli. In
the gelatine tube I noticed after 2-3 days the first
indications of the colonies in the shape of minute
translucent greyish droplets ; in Agar the colonies
are noticeable after one day's incubation. The
colonies, even when well developed, are from the size
of a pin's point to that of a millet seed. Streak sub-
cultures on gelatine are very characteristic, this being
due to two facts — {a) the slowness of the growth, the
line of inoculation becoming marked as a line of small
greyish transparent droplets, not before two to three
days ; (b) as the droplets enlarge they have a tendency
to become more or less confluent, but they always
remain recognisable as individuals — there is not a con-
tinuous band-like growth produced, as in most streak
cultures of bacilli. The same applies to Agar streak
cultures, only of course the growth is faster, but also
here the individuality of the colonies remains more or
less preserved even where the colonies are situated
closely and uninterruptedly side by side. In old
gelatine- and in old Agar cultures the colonies be-
come whitish, raised, knob-like, their outline slightly

128 THE GROUSE DISEASE chap.

crenate. In stab cultures the line of inoculation
becomes marked as a grey line made up of separate
droplets. The gelatine is never liquefied by the
growth.

In fresh specimens made of the colonies, i.e. ex-
amining a particle of the growth in sterilised salt
solution or in broth, the bacilli are without motility ;
in such specimens, as also in cover-glass specimens
dried and stained, the microbes are short bacilli
rounded at their ends, many are short ovals, some
cylindrical ; there are always some cylindrical and
rod-like bacilli present which are thicker in the middle
portion than at the ends, they look, therefore, spindle-
shaped ; they occur singly or often as dumb-bells. In
specimens made of gelatine cultures the bacilli differ
somewhat from those grown on Agar, inasmuch as in
the former most of the microbes are short ovals, and
in stained specimens show at each end in the trans-
parent sheath a stained granule. These look very much
like the typical fowl cholera bacilli ; but also in speci-
mens of gelatine culture there are some which are
rod-shaped or even distinctly cylindrical ; but not so
numerous by any means as in Agar cultures. In
these latter the majority are rod-shaped or cylindrical,
and amongst them occur such as are characteristically
spindle-shaped ; but also in Agar cultures there occur
a good many that are short ovals. Figs. 52 and 53

XIV ''CRAMPS" IN YOUNG PHEASANTS 129

show well the differences between the bacilli in gela-
tine and Agar cultures respectively.

Measurements made in dried and stained cover-
glass specimens give the following numbers : thick-
ness about 0.3 micro-millimetres ; there is no differ-
ence in this respect between the bacilli grown on
gelatine and those on Agar ; the cylindrical spindle-
shaped bacilli are thickest and measure as much as
0.4 to 0.5 /A in the thickest portion ; the length of the
majority of the bacilli from gelatine cultures is 0.4 /i to
0.6, a few 0.8 /i, very few more than this — 1.6 ix. In
Agar cultures the majority are 0.6 to 0.8 to i.o /x long,
some up to 1.6 /i. The cultures and sub-cultures which
I prepared from the young pheasants in June and
July 1887, and which I described in the Field, July
30, 1887, I put away^ during the following vacation,
August and September, in order to resume the inquiry
on my return towards the latter half of September,
but unfortunately, to my great discomfiture, they were
all found dead ; every tube put aside, though it re-
mained free of all accidental growth and looked in
all respects preserved, was tested by sub-cultures, but
with no result ; none could be started from them, and
therefore all further experimentation with them came
to an end. I have not been able hitherto to procure

1 A few experiments on guinea-pigs and mice by subcutaneous
inoculation of culture did not yield any definite result.

K

I30 THE GROUSE DISEASE chap, xiv

new material, i.e. young pheasants affected with the
" cram])s," and am, therefore, unable to say anything
further on this subject. But of this I am clear, that
the disease is in pathological respects a well-defined,
acute periostitis and osteomyelitis, and that it is
associated with a definite and well -characterised
species of bacilli, present in the inflamed bone, and
obtainable in all cases by cultivation.

EXPLANATION OF ILLUSTRATIONS

All Figures are reproductions from Photograms, except Figures
15 and 16, which are from drawings by Mr. M. H. Lapidge.

Figures 10, 11, 14, 17, 18, 19, 22, 32, 33, 34, 36, 41, 42, 43, 44,
48, and 50, are by Mr. Andrew Pringle, the remainder by Mr. E. C.
Bousfield.

l-'lG. 1.

Section through the Lung of (Irouse dead of the Grouse
Disease. Magnifying power about lo.

The air-cells appear solid, being filled with blood and exuda-
tion. Two large air-spaces are shown, filled with homogeneous
exudation.

Fig. 2.

Section through the Liver of Grouse dead of the Grouse
Disease. Magnifying power looo.

a. Part of a large blood-vessel filled with blood-corpuscles.
I?. Capillary blood-vessel plugged with the bacilli.
c. Liver cells.

Fig. 3.

A similar preparation as in Fig. 2. Magnifying power 1000.
Several capillary vessels are shown, plugged wdth the bacilli.

Fig. 4.

Section through the Lung, showing a mass of capillary blood-
vessels plugged with the Bacilli. Magnifying power 650.

Fig. 5.

Plate cultivation of the Bacillus of Grouse Disease. Natural
size.

Each of the small dots is a colony growing in the depth. Each
of the larger white patches is a colony on the surface of the gelatine.

Fig. 6.

Streak-culture on gelatine of the Bacillus of Grouse Disease.
Natural size,

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig, 11

Fig. 12

Fig. 7.

Stab - culture in gelatine of the Bacillus of Grouse Disease.
Natural size.

Fig. 8.

Shake-culture in gelatine, showing gas bubbles in the depth of
the gelatine. Natural size.

Fig. 9.

Cover-glass specimen of the Lung Juice of Grouse dead of the
Grouse Disease. Magnifying power 1000.

One dumb-bell of the bacilli is shown and several blood discs.

Fig. 10.

Cover-glass specimen from a Culture in gelatine of the Bacillus
of the Grouse Disease. Magnifying power 1000.

Fig. II.

Cover-glass specimen from a Culture in gelatine of the Bacillus
derived from the blood of an inoculated Mouse. Magnifying
power 1000.

Fig. 12.

Cover-glass specimen from a "Salted" Culture of the Bacillus
of Grouse Disease (see p. 31). Magnifying power 1000.

Fig. 13.

Cover-glass specimen of the Lung Juice of an infected Mouse.
Magnifying power 1000.

The baciUi are present in large numbers, singly and in masses.

Fig. 14.

A similar preparation as in Fig. 13. Magnifying power 1000.
A swollen leucocyte filled with the bacilli.

Fig. 15.

From a section through the Liver of Grouse dead of the Grouse
Disease, showing capillary blood-vessels filled with the Bacilli. Low
power.

Fig. 16.

From a similar preparation as Fig. 15, but more highly mag-
nified.

Fig. 17.

Cover-glass specimen of the blood of an infected Guinea-pig.
Magnifying power 1000.

Numerous bacilli are seen, free and enclosed in leucocytes, the
latter much swollen and disintegrating.

Fig. 18.

Cover-glass specimen of the blood of an infected Ammer.
Magnifying power 1000.

Amongst the blood-corpuscles are very numerously the bacilli.

Fig. 13

• ^

Fig. 16

Pig. 17

Fig. 18

ar .aiH

-i .jii"^

Fig. 19

Fig. 20

Pig. 21

Fig. 22

Fig. 23

Fig. 19.

Cover-glass specimen of the Lung Juice of an infected Ammer.
Magnifying power 1000.

Numerous bacilli are seen amongst the blood-corpuscles.

Fig. 20.

Cover-glass specimen of the Lung Juice of an infected Ammer.
Magnifying power 1000.

Amongst the blood discs (of which only the nucleus is shown)
there are isolated bacilli. Two leucocytes include in their substance
the bacilli.

Fig. 21.

Cover-glass specimen from a cultivation of the Bacilli derived
from the blood of an infected Ammer. Magnifying power 1000.

Fig. 22.

Cover-glass specimen of the Heart's Blood of a Grouse dead of
the Autumnal Grouse Disease. Magnifying power 1000.

Amongst the blood discs (of which only the nucleus is shown)
are seen the bacilli.

Fig. 23.

Cover-glass specimen of the Subcutaneous CEdema Fluid of a
Guinea-pig infected with the .^■Erobic CEdema Bacilli. Magnifying
power 1000.

Fig. 24.

Similar preparation as in Fig. 23. Magnifying power 1000.

Fig. 25.

Cover -glass specimen from an Agar culture of the Orotic
GEdema Bacillus. Magnifying power 650.

Fig. 26.

Similar specimen as in Fig. 25, but from a gelatine culture.
Magnifying power 1000.

Fig. 27.

Leucocytes of the Subcutaneous CEdema Fluid of an infected
Guinea-pig ; the cells contain the Bacilli in their interior. Mag-
nifying power 1000.

Fig. 28.

Plate culture of the .^robic CEdema Bacillus. The small points
are colonies in the depth ; the large white patches are colonies on
the surface of the gelatine. Natural size.

Fig. 29.

Streak culture on gelatine of the /Erobic GEdema Bacilli.
Natural size.

Fig. 30.

Stab-culture of same. Natural size.

Fig. 25

Fig. 26

Fig. 27

Fig. 28

1^" .- "it-

Fig. 29

Fig. 3C

Fig. 32

Fig. 31

Fig. 33

Fig. 34

yig 86

Fig. 36

Fig. 31.

Shake-culture of same, showing a few gas bubbles in the deeper
parts of the gelatine. Natural size.

Fig. 32.

Blood of Fowl dead of Fowl Cholera. Numerous Bacilli of
Fowl Cholera amongst the blood-corpuscles. Magnifying power
1000.

Fig. 33.

Blood of Pigeon dead of Fowl Cholera. Magnifying power 1000.

Fig. 34.
Blood of Rabbit dead of Fowl Cholera. Magnifying power 1000.

Fig. 35.

Plate culture in gelatine. Natural size.
The colonies are all extremely minute dots.

Fig. 36.

Cover-glass specimen (impression specimen) of a colony of the
Fowl Cholera Bacillus. Magnifying power 1000.

Fig. 37.

Section through the Liver of a Fowl dead of Fowl Cholera.
Magnifying power 1000.

Many blood-vessels contain the bacilli very numerously.

Fig. 38.

Section through the Liver of a Rabbit dead of Fowl Cholera.
Magnifying power 1000.

Fig. 39.

Streak culture on gelatine of the Fowl Cholera Bacilli. Natural
size.

Fig. 40.
Stab-culture of same. Natural size.

Fig. 41.

Blood of Fowl dead of Fowl Enteritis. Magnifying power 1000.
Very few baciUi amongst the blood-corpuscles.

Fig. 42.

Cover-glass specimen of Spleen Juice of a Fowl dead of Fowl
Enteritis. Magnifying power 1000.

Several baciUi amongst the tissue cells.

Fig. 37

Fig. 38

Fig. 39

Fig. 40

Fig. 41

Fig. 42

Fig. 43

Fig. 44

Fig. 46

I'm

Fig. 46

Fig. 47

Fig. 48

Fig. 43.

Cover-glass specimen of the Intestinal Mucus of a Fowl dead of
Fowl Enteritis. Magnifying power 1000.

The bacilli are present in great numbers and in pure culture.

Fig. 44.

Cover-glass specimen of the Intestinal Mucus of a similar Fowl
as in Fig. 43. Magnifying power 1000.

Fig. 45.

Plate cultivation in gelatine of the Bacillus of Fowl Enteritis.
Natural size.

The small dots are colonies in the depth ; the large patches are
colonies on the surface of the gelatine. The difference between the
plate cultivation of the bacillus of Enteritis and that of the bacillus of
Fowl Cholera (Fig. 35) is obvious. Both plates were made at the
same time, and were kept under precisely the same conditions.

Fig. 46.

Streak culture on the surface of gelatine of the Bacillus of Fowl
Enteritis. Natural size.

Fig. 47.
Stab-culture of same. Natural size.

Fig. 48.

Cover-glass specimen, — impression of a Superficial Colony from
a plate culture, the margin of a Colony being shown here. Magni-
fying power 1000.

Fig. 49.

Cover-glass specimen from a culture on gelatine of the Bacillus
of Fowl Enteritis. Magnifying power 1000.

Fig. 50.

Cover-glass specimen from an old broth culture of the Bacillus
of Fowl Enteritis, showing involution forms and degenerating
filaments of the Bacilli. ■ Magnifying power 1000.

Fig. 51.

Section through the Liver of a Fowl dead of the Fowl Enteritis.
Magnifying power 1000, showing numerous bacilli in the capillary
blood-vessels, these latter being seen cut across.

Fig. 52.

Cover-glass specimen of an Agar culture of the Bacillus of " the
Cramps " of Young Pheasants. Magnifying power 2000.

Fig. 53.

Cover-glass specimen of a gelatine culture of the same Bacillus.
Magnifying power 2000. The difference in the shape of the bacilli
between those from an agar- and those from a gelatine culture is
striking.

Fig. 49

Fig. 30

Pig. 51

Fig. 52

Fig. 53

MESSRS. MACMILLAN & CO.'S PUBLICATIONS.

WORKS BY THE SAME AUTHOR.

MICRO-ORGANISMS AND DISEASE. An Introduction into
the Study of Specific Micro-Organisms. Illustrated. Third Edition,
Revised. Crown 8vo. 6s.

ACADEMY : — "No present line of medical research is so likely to prove
fraught with benefit of diminishing disease and spreading health as that to which
Dr. Klein's little book otfers an introduction, lucid and concise. . . . Dr. Klein's
exposition is specially opportune as offering to the general public, in the few clear
pages of an authority and expert, a short and safe way out of the tangle of diverse
opinion. When, as here, tlie authority of experience is modest, and skill is
tentative and cautious, they have most weight. This book in a marked degree
shows these to be the graces of its author as an observer and teacher, and the
discovery increases one's previous sense of his trustworthiness."

S A TURD A V REVIEW:—" By far the most important book among those
relating to matters of health and disease which are now before us is that of Dr.
Klein on Micro-Organisms. . . . Dr. Klein is well known as a first-rate observer
and careful reasoner, and has, moreover, for many years given untiring study to
the questions which he now handles. lie is therefore fully qualified to give, as
he here does, a succinct account of what is known regarding this difficult matter. . . .
Dr. Klein's book, while mainly intended for the expert, is written with sufficient
clearness to be of considerable interest to all who may wish to know what is being
done regarding the important question of the germ-origin of disease."

THE BACTERIA IN ASIATIC CHOLERA. Crown 8vo. 5s.

LANCET : — " No single observer has contributed more to the knowledge on
this subject than Dr. Klein, and the work before us is a thoroughly scientific
exposition of it in all its bearings ... a perusal of his book cannot fail to leave
the impression that there is still much to be learnt concerning cholera and its
relationship to bacterial life."

MANCHESTER GUARDIAN :—"Dx. Klein is one of our best English
bacteriologists, and in this book any one interested in the subject will find the
clearest description of the delicate experiments made to prove the nature of the
comma-bacilli. There is throughout the volume the tone that should pervade all
scientific works, of candid acknowledgment of the work and opinions of opponents,
and at the same time of determination to subject their conclusions to the most
rigorous and searching tests."

VIENNA JVEER'L V NEIVS:—" Fiokssoi- Klein's criticism, which is
absolutely free from personalities, is based on the results of careful microscopical
and bacteriological examinations carried on, for many years, in England and India.
The book contains a full synopsis of the present state of the etiology of cholera,
and being written in a clear style, will initiate the student into the study of bac-
teriolog)'. It will, moreover, be a valuable guide to beginners by showing them
how cautious one must be in drawing conclusions especially in bacteriological
matters. By a number of excellent illustrations the publishers have added to
the special features of the book, which is sure to obtain a large circulation in the
medical world."

MACMILLAN AND CO., LONDON.

MESSRS. MACMILLAN & CO.'S BOOKS
ON BIOLOGY, ANATOMY, AND PHYSIOLOGY.

LESSONS IN ELEMENTARY PHYSIOLOGY. By Thomas H.
Huxley, F.R.S. Illustrated. New Edition. Revised. i8mo. 4s. 6d.

ELEMENTARY BIOLOGY. By T. H. Huxley, P'.R.S., and H. N.
Martin, F.R.S. New Edition. Revised and extended by G. B. Howes
and D. H. Scott. Crown 8vo. los. 6d.

AN ATLAS OF PRACTICAL ELEMENTARY BIOLOGY. By
G. B. Howes. With a Preface by T. II. Huxley, F.R.S. 4to. 14s.

COMPARATIVE EMBRYOLOGY (A Treatise on). By F. M.
Balfour, M.A., F.R.S. Illustrated. In 2 Vols. Vol. I., i8s. ;
Vol. II., 2IS.

THE ELEMENTS OF EMBRYOLOGY. By Professor M. Foster,
M.D., F.R.S., and the late Francis M. Balfour, F.R.S. Third Edition,
Revised. Edited by Adam Sedgwick, M. A., and Walter Heape. ids. 6d.

A TEXT-BOOK OF COMPARATIVE ANATOMY. By Dr.

Arnold Lang, Professor of Zoology in the University of Zurich, formerly
Ritter Professor of Phylogeny in the University of Jena, ^^'ith Preface to
the English Translation by Prof. Dr. Ernst PIaeckel, Director of the
Zoological Institute in Jena. Translated into English by Henry M.
Bernard, M.A. Cantab., and Matilda Bernard. Part I. 8vo.
17s, net.

TEXT-BOOK OF PHYSIOLOGY. By Professor Michael Foster,
M.D., F.R.S. With Illustrations. Sixth Edition, largely revised. In
Three Parts. 8vo. Parts I. and II., los. 6d. each. Part III., 7s. 6d.
Part IV., los. 6d.

PRACTICAL PHYSIOLOGY AND HISTOLOGY: An Elementary
Course. By Professor M. Foster, F.R.S., and J. N. Langley, F.R.S.
Crown 8vo. 7s. 6d.

ZOOTOMY (Vertebrata) : A Course of Instruction in. By Professor
T. J. Parker. Illustrated. Crown 8vo. 8s. 6d.

ELEMENTARY LESSONS IN ANATOMY. By St. George
Mivart, F.R. S. With 400 Illustrations. l8mo. 6s. 6d.

ELEMENTS OF COMPARATIVE ANATOMY OF ^VERTE-
BRATES. Adapted from the German of Robert Wieders'heim. By
Professor W. N. Parker. With 270 Illustrations. 8vo. 12s. 6d.

THE OSTEOLOGY OF THE MAMMALIA (An Introduction to).
By William Henry Flower, LL.D., F.R.S. Third Edition. Revised
with the assistance of Hans Gadow, Ph.D., M.A. los. 6d.

THE MYOLOGY OF THE RAVEN {Cofvus corax sinuatus). A
Guide to the Study of the Muscular System in Birds. By R. W. Shueeldt.
Illustrated. 8vo. 13s. net.

ELEMENTARY PRACTICAL HISTOLOGY. By Willl\m Fearn-
LEY. With Illustrations. Crown Svo. 7s. 6d.

LESSONS IN ELEMENTARY BIOLOGY. By Professor T.
Jeffrey Parker, B.Sc, F.R.S. Illustrated. Crown Svo. los. 6d.

DISEASES OF FIELD AND GARDEN CROPS, chiefly such as are
caused by Fungi. By Worthington G. Smith, F.L.S. Fcap. Svo. 4s. 6d.

TIMBER AND SOME OF ITS DISEASES. By H. Marshall
Ward, F.R.S., Professor of Botany Royal Indian Engineering College,
Cooper's Hill. Cr. Svo. 6s.

MACMILLAN AND CO., LONDON.

MESSRS. MACMILLAN & CO.'S BOOKS.

BIOLOGY.

HANDBOOK OF FIELD AND GENERAL ORNITHOLOGY.

By Prof. Elliott Couks, M.A. Illustrated. 8vo. los. net.
ORGANIC EVOLUTION as the Result of the Inheritance of Acquired

Characters, according to the Laws of Organic Growth. By Dr. G. II. T.

Elmer. Translated by J. T. Cunningham, F.R.S.E. 8vo. 12s. 6d.

Works by ALFRED RUSSEL WALLACE, LL.D.

DARWINISM: An Exposition of the Theory of Natural Selection.

3d Edition. Crown 8vo. 9s.
NATURAL SELECTION : AND TROPICAL NATURE. New

Edition. Crown 8vo. 6s.
ISLAND LIFE. New Ed. Crown 8vo. 6s.

Works by the Right Hon. Sir JOHN LUBBOCK, F.R.S., D.C.L.

THE ORIGIN AND METAMORPHOSES OF INSECTS. Illus.

Crown 8vo. 3s. 6d.
ON BRITISH WILD FLOWERS CONSIDERED IN RELATION

TO INSECTS. Illustrated. Crown 8vo. 4s. 6d.
FLOWERS, FRUITS, AND LEAVES. Illustrated. 2d Edition.

Crown 8vo. 4s. 6d.

BOTANY.

PHYSIOLOGICAL BOTANY. I. Outlines of the Histology^ of
PhDsnogamous Plants. II. Vegetable Physiology. By George Lincoln
GoODALE, M.A., M.D., Professor of Botany in Harvard University. 8vo.
los. 6d.

STRUCTURAL BOTANY, OR ORGANOGRAPHY ON THE
BASIS OF MORPHOLOGY. To which are added the Principles of
Taxonomy and Phytography, and a Glossary of Botanical Terms. By Prof.
Asa Gray, LL.D. 8vo. los. 6d.

FIRST LESSONS IN PRACTICAL BOTANY. By G. T. Bettany.
i8mo. IS.

A COURSE OF PRACTICAL INSTRUCTION IN BOTANY.
By F. O. Bower, D.Sc, F.R.S., Regius Professor of Botany in the Uni-
versity of Glasgow. Crown 8vo. 10s. 6d. Abridged Edition.

[/« preparation.

THE FERTILISATION OF FLOWERS. By Hermann Muller.
Translated by D'Arcy W. Thompson, B.A., Professor of Biology in Uni-
versity College, Dundee. Preface by C. Darwin, F.R.S. Illustrated.
8vo. 2 IS.

Works by Daniel Oliver, F.R.S.

Late Professor of Botany in University College, London.

LESSONS IN ELEMENTARY BOTANY. Illustrated. Fcap.

8vo. 4s. 6d.
FIRST BOOK OF INDIAN BOTANY. Illustrated. Extra fcap.

8vo. 6s. 6d.

Works by Sir JOSEPH HOOKER, F.R.S., &c.

PRIMER OF BOTANY. Illustrated. i8mo. is.
THE STUDENT'S FLORA OF THE BRITISH ISLANDS. 3d
Edition, revised. Globe 8vo. los. 6d.

MACMILLAN AND CO., LONDON.

MESSRS. MACMILLAN & CO.'S PUBLICATIONS.

BOOKS ON MEDICINE.

A MANUAL OF PUBLIC IIEALTIL By A. Wynter Blyth, M.R.C.S.

8vo. 17s. net.

LESSONS ON BRESCRirTIONS AND THE ART OF PRESCRIBING.

By W. H. Griffiths. Adapted to the Pharmacopoeia, 1885. i8mo. 3s. 6d.

A TEXT-BOOK OF PATHOLOGY, SYSTEMATIC AND PRACTICAL.

By D. J. Hamilton, F.R.S.E., Professor of Pathological Anatomy, University of Aberdeen.
Illustrated. Vol. I. 8vo. 25s.

A TEXT-BOOK OF GENER.\L THERAPEUTICS. By \Y. Hale White,

M.D., Senior Assistant Physician to and Lecturer in Materia Medica at Guy's Hospital.
Illustrated. Cr. 8vo. 8s. 6d.

TEXT-BOOK OF PATHOLOGICAL ANATOMY AND PATHOGENESIS.

By Prof. E. Ziegler. Translated and Edited by Donald Macalister, M.A., M.D.
Fellow and Medical Lecturer of St. John's College, Cambridge. Illustrated. 8vo.

Part I.— GENERAL PATHOLOGICAL ANATOMY. 2d Ed. 12s. 6d.
Part II.— SPECIAL PATHOLOGICAL ANATOMY. Sections I.-VIII.
2d Edition. 12s. 6d. Sections IX. -XII. 12s. 6d.

\Yorks by T. LAUDER BRUNTON, M.D., F.R.S.,
Examiner in Materia Medica in the University of London, in the Victoria
University, and in the Royal College of Physicians, London,

A TEXT-BOOK OF PHARMACOLOGY, THERAPEUTICS, AND
MATERIA MEDICA. Adapted to the United States Pharmacopeia, by F. H. Williams,
M.D., Boston, Mass. 3d Edition. Adapted to the New British Pharmacopoeia, 1885, and
additions, 1891. 8vo. 21s. Or in 2 Vols. 22s. 6d.

TABLES OF MATERIA MEDICA ; A Companion to the Materia Medica
Museum. Illustrated. Cheaper Issue. 8vo. 5s.

THE CROONIAN LECTURES FOR 1889.
THE INTRODUCTION TO MODERN THERAPEUTICS. Being the

Croonian Lectures in the relationship between Chemical Structure and Physiological Action
delivered before the Royal College of Physicians in London, June 1889. By T. Lauder
Brunton, M.D., D.Sc. Edin., LL.D. Aberd., F.R.C.P., F.R.S. 8vo, cloth, 3s. 6d. net.

DISORDERS OF DIGESTION: THEIR CONSEQUENCES AND

TRE.AT.MENT. By T. Lauder Brcxton, M.D. This work contains, in addition to the

Lettsomian Lectures, a number of other Papers by the Author on similar subjects. 8vo, los. 6d.

EDINBURGH MEDICAL JOUR \'AL. — "Distinguished by accurate observation and

original thinking, these papers have been largely instrumental in moulding current medical

opinion in regard to many of the subjects with which they are concerned."

LONDON MEDICAL RECORD.— '^\Vs can cordially recommend the volume, as one
which combines much practical and technical information and sound common sense."

MACMILLAN AND CO., LONDON.

\\

^
^

nN

THIS BOOK IS DUE ON THE LAST DATE
STAMPED BELOW

AN INITIAL FINE OF 25 CENTS

WILL BE ASSESSED FOR FAILURE TO RETURN
THIS BOOK ON THE DATE DUE. THE PENALTY
WILL INCREASE TO 50 CENTS ON THE FOURTH
DAY AND TO $I.OO ON THE SEVENTH DAY
OVERDUE.

BIOLOGY

LIBRARY

-. - r

LD 2l-5)n 7,'37

703403

lOLOGY
JGRARy

G

UNIVERSITY OF CALIFORNIA LIBRARY