THE LIBRARY

OF

THE UNIVERSITY

OF CALIFORNIA

PRESENTED BY

PROF. CHARLES A. KOFOID AND

MRS. PRUDENCE W. KOFOID

"V

A MONOGRAPH ON JOHNE'S DISEASE

{ENTERITIS CHRONICA PSEUDOTUBERCULOSA BOVIS)

A MONOGRAPH

ON

JOHNE'S DISEASE

(ENTERITIS CHRONICA
PSEUDOTUBERCULOSA BOVIS)

BY

F. W. TWORT, M.R.C.S. Eng., L.R.C.P. Lond.

SUPERINTENDENT OF THE BROWN INSTITUTION, UNIVERSITY OF LONDON

G. L. Y. INGRAM, M.R.C.V.S.

LATE VETERINARY SURGEON TO THE BROWN INSTITL TION

ILLUSTRATED WITH NINE PLATES

LONDON

BAILLIERE, TINDALL AND COX

8, HENRIETTA STREET, COVENT GARDEN

1913

[All rights rese-med]

KJ

PREFACE

Johne's disease, the subject of this small monograph,
is a condition that has recently attracted considerable
attention in this country. In 1895 Johne and Frothing-
ham discovered the causative bacillus in the lesions,
and since that time the disease has been investigated
by many other workers. Although at the present day
Johne's disease is frequently mistaken for tuberculosis,
strongylosis, and other conditions, it is rapidly be-
coming more generally recognized. It is known that
besides cattle, sheep, deer, and goats may become
infected, and that the disease is widely distributed over
the globe, in some areas the loss to stockowners from
this condition being greater than that from tuberculosis.
An important article by Professor Penberthy dealing
with the disease and its economic importance, and the
need for State legislation, has recently appeared in the
Journal of the Bath and West of England Agricultural
Society.

In the following pages we have attempted to sum-
marize existing knowledge on the subject, and have
brought before English readers the investigations of
Continental workers — notably those of Professor Bang
of Copenhagen and of Professor Miessner of Hanover
— which have not been generally available to veterinary
surgeons and stockowners in this country. We have
also included our own investigations, which have been

R ;• '^1' j!r <r> O ''^ r-

vi PREFACE

carried out at the Brown Institution during 1910, 191 1,
1912, and the first three months of the present year.

These investigations have thrown a considerable
amount of work on the staff of the laboratories, and
here we should like to record the industry and care
with which the chief attendant, Mr. Frankham, has
performed the various duties allotted to him.

The book consists of nine chapters, and throughout
the descriptions and details refer to cattle unless
otherwise specified. The first five chapters are allo-
cated to the history of the disease, its importance to
stockowners and breeders, its clinical features, methods
of diagnosis and treatment, and the pathological lesions
found post mortem.

In Chapter VI. we deal with the cultivation of
Johne's bacillus, and describe in some detail the ex-
periments that have been carried out in this direction
by different workers from the time of Johne and
Frothingham to the present day; we also include a
description of the bacillus, and the appearance of the
cultures on fluid and solid media.

In Chapter VII. we describe the method of preparing
a specific diagnostic vaccine, and the earliest experi-
ments carried out with avian and other tuberculins are
also given. Chapter VIII. contains some results with
agglutination and complement- fixation tests, which
were performed by Dr. C. C. Twort. Chapter IX,
contains most of the recorded experiments with infec-
tive material and pure cultures of the bacillus. Here,
too, we include a large number of experiments by
Dr. C. C. Twort and Mr. T. Craig on the small labora-
tory animals.

Research work on a disease which affects any of the
larger domesticated animals is necessarily very costly,
not only from the expense of the experimental subjects

PREFACE vii

but also from the cost of feeding and keeping them.
On this account our experiments, though covering
a fairly wide field, have not been so numerous in some
cases as we should have wished. In view of the impor-
tance of this disease to agriculturists, the question is
one which should be investigated with public money.
In our work on this disease, however, we have re-
ceived no assistance from the Board of Agriculture or
from the Development Fund Commissioners, even
though applications for a grant have been made after
the essential part of our work — the cultivation of the
bacillus — had been verified by the Danish Government
veterinary bacteriologists. Under these circum-
stances, we wish to give especial notice to the
generosity and kindness of many owners and breeders
of cattle who have assisted our work with gifts of
affected animals for experimental purposes, to the
help aff'orded by a large number of veterinary surgeons
who have sent specimens and answered questions on
diff'erent points of interest, to the Royal Society for
the monetary grants which made the earlier experi-
ments possible, and to the University of London for
the grants from the ** Henry Dixon" Fund for research.
We wish to thank Professor Dr. Miessner of Hanover,
Dr. Halfdan Holth of Copenhagen, and Professor
Penberthy for the loan of papers and for references to
other authors. Monsieur Vukovic of Livno (Bosnia) for
sending a paraffin block and details of his own work
on the disease as it aff'ects sheep, and Messrs. De Vine,
Hamilton, Angwin Le Sueur, and other veterinary
surgeons for kind assistance in many directions.

F. W. T.

G. L. Y. I.

April, 1913.

CONTENTS

Chapter I. :

PAGE

NOMENCLATURE ...... i

DEFINITION ... - . - - 3

HISTORY AND DISTRIBUTION ----- 3

Chapter II.:

ECONOMIC IMPORTANCE - - - - "14

ETIOLOGY ....--. i6

Chapter III. :

CLINICAL symptoms - ----- 21

DIFFERENTIAL DIAGNOSIS - - - - '30

Chapter IV. :

PROGNOSIS - - - - - - - 41

TREATMENT .------ 42

PROPHYLAXIS .-.-..- 44

Chapter V. :

POST-MORTEM EXAMINATION AND PATHOLOGICAL ANATOMY 46
PATHOLOGICAL HISTOLOGY - - - - "55

Chapter VI. ;

DESCRIPTION OF JOHNE'S BACILLUS - - - - 62

CULTIVATION OF THE BACILLUS - - - - 65

NATURE OF THE " ESSENTIAL SUBSTANCE" - - - 92

DESCRIPTION OF CULTURES - - - - - IO3

Chapter VII. :

DIAGNOSTIC AND OTHER VACCINES - - - - 106

ix

X CONTENTS

Chapter VIII.:

PAGR

AGGLUTINATION REACTIONS - - - - "135

THE COMPLEMENT FIXATION TEST - - - - 137

Chapter IX. :

THE PATHOGENICITY OF JOHNE'S BACILLUS :

INOCULATION EXPERIMENTS WITH INFECTED MATERIAL I40
INOCULATION EXPERIMENTS WITH PURE CULTURES OF

JOHNE'S BACILLUS - - - _ . 144

THE PATHOGENICITY OF THE BACILLUS FOR SMALL

ANIMALS - - - - - - 153

Bibliography - - - • - - - 174

LIST OF PLATES

I. A SHORTHORN COW SUFFERING FROM JOHNE'S DISEASE Frontispiece

FACING PAGE

II. THE LOWER PART OF THE ILEUM FROM A COW AFFECTED

WITH JOHNE's disease - - - - - 48

III. SECTION OF ILEUM ( X 250 DIAMETERS) - - * 5^

IV. SECTION OF ILEUM (x I,000 DIAMETERS) - - " 5^

V. FILM OF JOHNE'S BACILLUS ( X I,000 DIAMETERS) - - 62

VI. CULTURES OF JOHNE's BACILLUS ON MEDIA CONTAINING

EXTRACTS OF B. PHLEI - - - - - 82

VII. CULTURES OF JOHNE'S BACILLUS, THE HUMAN TUBERCLE

BACILLUS, AND THE AVIAN TUBERCLE BACILLUS - I04

VIII. CULTURE OF ACCLIMATIZED JOHNE'S BACILLUS GROWING

ON GLYCERINE-BEEF BROTH _ . - - 106

IX. CULTURES OF JOHNE's BACILLUS ISOLATED FROM AN EX-
PERIMENTAL CALF AND TWO EXPERIMENTAL GOATS - I50

XI

JOHNE'S DISEASE

CHAPTER I

NOMENCLATURE, DEFINITION, HISTORY, AND
DISTRIBUTION

Nomenclature — English. — Johne's disease. Chronic
pseudo-tuberculous enteritis. For the condition in
sheep, Scrapie or Scrapy (doubtful). Chronic bacterial
dysentery (America).

French. — Entente spezifique chronique des boeufs.

German. — Die spezifische chronische Enteritis des
Rindes. Die chronische pseudotuberkulose Darment-
ztindung des Rindes.

Danish. — Kvaegets kroniske smitsomme Tarmbetaen-
delse. Lollandske Syge.

Swiss. — Kaltbrandigkeit.

In Great Britain the disease to be described in this
book is usually spoken of as ''Johne's disease" — from the
name of the discoverer of the acid-fast bacillus present
in the lesions of cattle — as being less cumbrous than
the more descriptive term, "chronic pseudo-tubercu-
lous enteritis," which is commonly used in other
countries. The latter name was suggested by Pro-
fessor B. Bang in 1906, in which year he showed that
the condition was a distinct disease in no way con-
nected with tuberculosis; so that it has really more

I

2 JOHNE'S DISEASE

claim to be called " Bang's disease," and its causal micro-
organism "Johne's bacillus."

An objection to the term " pseudo-tuberculous
enteritis " exists in the fact that there is never any
resemblance between the macroscopic lesions of
Johne's disease and those of tuberculosis, though
under the microscope it is rarely possible to differen-
tiate the causal micro-organisms. However, though
for more than ten years after the discovery of acid-
fast bacilli in the lesions the disease was regarded as
a form of tuberculosis, and although the thickening of
the bowel had been noticed by other observers previous
to Johne and Frothingham, yet the name "Johne's
disease" has been adopted in England, and nothing
would be gained by attempting to alter it. In America
it is known as " chronic bacterial dysentery," to which
it may be objected that the passage of blood with
the faeces is not a very common feature of the
disease.

The popular terms for this disease are somewhat
varied, and differ in different localities — " skinters,"
"scanters," "piners," and "wasters," are all terms
applied to animals showing emaciation and diarrhoea.
Possibly the term "waster " is more commonly applied
to tubercular animals, in which the diarrhoea is less
marked ; but no reliance can be placed on the use
of these terms by farmers.

"Scrapy" seems to be a term somewhat loosely
applied to the disease when it affects sheep. Stockman
mentions this term in his various articles on the sub-
ject, but states that it may be the irritable skin condi-
tion to which reference is made under this name, and
which in sheep seems to be associated in some cases,
with bacterial enteritis.

" LoUandske Syge," or Laaland disease, is the old

DEFINITION— HISTORY AND DISTRIBUTION 3

name for the disease in Denmark, arising from the
prevalence of the condition in cattle on that island.

" Kaltbrandigkeit," according to Meyer, is the com-
mon term for Johne's disease among Swiss farmers, in
whose stock it occurs. The term merely describes the
symptoms of " thirst without feverishness."

Definition. — A chronic specific enteritis, affecting
cattle, more rarely sheep and deer, and probably goats
and allied animals, caused by the multiplication, in the
intestinal mucous membrane and mesenteric glands, of
a specific micro-organism known as "Johne's bacillus,"
which produces a diffuse thickening of the bowel and
an interference with food absorption, leading to
diarrhoea and wasting.

History and Distribution.— The older writers on
veterinary subjects devoted most of their attention
to horses and horsemanship. From the time of
Solleysel in France and Blundeville in England, both
of whom wrote about the middle of the seventeenth
century, the authors of numerous works on farriery
that we have consulted merely give, as an appendix,
a few prescriptions for common ailments of cattle.
Skellet, in 1806, wrote a book on " Parturition in Cow^s
and Diseases of Neat Cattle," in which he makes no
reference to any condition comparable with Johne's
disease. Some of these old writers, however, do
mention that certain forms of diarrhoea in cattle are
very chronic and incurable, and are " due to rotten-
ness."

Hurtrel d'Arboval, who, in 1826, compiled, in French,
a dictionary of the veterinary art, mentions, under the
head of "Enteritis in Cattle," a thickening of the
mucous membrane of the large and small intestines
associated with chronic diarrhoea ; but some of his

4 JOHNE'S DISEASE

cases appear to have shown distinct ulceration, and
were probably tubercular.

In the Vetermarian for 183 1, Farrow and Cartwright
both mention diarrhoea and wasting in cattle, and
describe lesions which leave little doubt that the
disease existed in England over eighty years ago.

B. Bang, of Copenhagen, states that, in 1881, Hansen,
a veterinary surgeon of Nysted, in the island of
Laaland, brought to his notice certain cattle in which
chronic diarrhoea was accompanied by a thickening of
the intestine. Later, Nielsen, on the same island,
observed a similar condition on a large estate in his
district. Bang made a post-mortem examination of
a cow from this estate, but in this particular case the
thickening was not very marked, and he was led to
ascribe the diarrhoea to the irritation caused by small
intestinal strongyles which were present.

In 1895, Johne, professor in the veterinary school at
Dresden, and Frothingham, an American doctor work-
ing with him, first drew attention to the presence
of acid-fast bacilli in the thickened intestine. Harms,
a veterinary surgeon of Oldenburg, applied the tuber-
culin test to a six-year-old Oldenburg cow suffering
from diarrhoea which he suggested was tubercular
in origin. The injection of an ordinary dose of diag-
nostic tuberculin caused a rise of i '6° C. in the tempera-
ture of the animal, the maximum reached being 39*6° C.
As a result of this test the animal was slaughtered.
Neither in the lungs nor in the lymphatic glands was
any tubercular lesion demonstrable. The small intes-
tine and caecum were sent to Johne at Dresden. He
found the ileum to be thickened in the manner now
regarded as characteristic of Johne's disease, and on
staining portions of the bowel with carbol-fuchsin,
decolorizing with sulphuric acid, and counterstaining

HISTORY AND DISTRIBUTION 5

— the method of staining now known as Ziehl-
Neelsen's — he discovered the acid-fast micro-organ-
isms which were then believed to be tubercle bacilli.
The condition was therefore described as a hitherto
unrecorded form of tubercular infection of the intes-
tine. Cultures from parts of the intestine rich in
bacilli were made on to glycerine-agar, but no growth
was obtained. The inoculation of guinea-pigs with
infective material also gave negative results. How-
ever, from the appearance of the bacilli in the lesions,
the authors were led to attribute the condition to an
infection with avian tubercle bacilli. Specimens were
submitted to Robert Koch, who gave it as his opinion
that the disease was either a true tuberculosis or
was due to some modified or degenerate form of the
tubercle bacillus.

The authors (Johne and Frothingham) concluded
from their researches that the condition was a pre-
viously unrecognized form of tubercular enteritis due
to an infection with the tubercle bacillus, or the avian
tubercle bacillus, or a variation of one of these micro-
organisms brought about by some change in nutrition,
etc. Such a change, they thought, might be re-
sponsible for the alteration in virulence and for the
negative or very slight reactions produced by the
inoculation of ordinary diagnostic tuberculin into
affected animals.

After Johne and Frothingham's original paper, the
next important publication with regard to this disease
came from Markus of Utrecht, in 1904. He stated that
in Holland the disease had been recognized for many
years by Koorevaar, who had repeatedly drawn atten-
tion to the thickened intestines of emaciated cattle
showing no gross lesions in any other organ. He
also stated that in some districts the condition in cattle

6 JOHNE'S DISEASE

was known as " Scheisser," a name which is probably
equivalent to the English term "scourer."

Markus himself found the causal bacilli in cases of
Johne's disease, and introduced the intra vitam method
of diagnosis that consists in taking scrapings from the
rectal mucous membrane and staining films made from
the material with carbol-fuchsin. He stated that at the
abattoir in Amsterdam, van der Sluys had recognized
eleven cases in one year. Markus made numerous
cultural experiments which were entirely negative,
and he inoculated rabbits, guinea-pigs, goats, and hens,
with infective material without obtaining any positive
result.

In 1905, Lienaux and van den Eeckhout, in the
*' Belgian Annals of Veterinary Medicine," gave an
account of a study of the disease occurring as an
enzootic in a herd of Jersey cattle, and also in native
breeds in which they had encountered cases. After
numerous experiments they concluded that they were
dealing with a form of tuberculosis, being misled, in
all probability, by the coexistence in some cases of
tuberculosis and Johne's disease in the same animal.
With experimental material from such animals tuber-
cular lesions were produced in other animals by inocu-
lation.

In the same year Borgeaud of Lausanne reported
two cases — one in a five-year-old cow which was not
markedly emaciated, and another in an animal which
had suffered from periodic attacks of diarrhoea for
about six months. In the latter case there was also
tuberculosis of the bronchial glands and pleura. Inocu-
lation of guinea-pigs with material from the infected
gut produced abscesses in which there were acid-fast
bacilli.
In 1906, Matthis of Lyons published an account of

HISTORY AND DISTRIBUTION 7

the cases which he had met with in his neighbour-
hood ; and in Germany, at the Berlin abattoir, Bongert
investigated many cases. Both of these writers came
to the conclusions arrived at by Johne and Frothing-
ham, and considered the disease to be tubercular in
nature.

In the same year B. Bang published an account of
his inquiries into the disease in Denmark, and this
authority must be credited with making the first real
advance in our knowledge of this condition since the dis-
covery of the acid-fast bacilli by Johne and his colleague.
Bang first established the fact that the disease is a
specific infection distinct from tuberculosis, and he
suggested the name chronic pseudo-tuberculous (or
paratuberculous) enteritis. He showed the existence
of the disease among red Danish cattle and other
native breeds, as well as in imported tubercle-free
J erseys. *

By feeding two calves with the mucous membrane
of the intestine of an infected animal, he was able to
reproduce the disease. His attempts to cultivate the
bacillus and produce lesions in guinea-pigs and rabbits
were negative, and he decided that the condition was
not a true tuberculosis. He also demonstrated its
chronic nature and the long period of incubation. In
the same year Bang gave an account of the disease
before the National Veterinary Association at Liver-
pool, and showed specimens in illustration. He pre-
dicted that it would be recognized in this country,
and mentioned that he had found it in tubercle-free
Jersey cows imported from the Channel Islands. In
the discussion that followed this paper, many of the
practitioners present stated that they had been aware
of the condition in their practices, but had not ascribed
it to its true cause, intestinal strongyles usually being

8 JOHNKS DISEASE

considered as giving rise to the diarrhoea when prob-
ably Johne's disease was the cause.

In 1907 an account of six cases was given by
M'Fadyean in the Journal of Comparative Patholog)>,
These cases occurred in Shorthorn, Sussex, and Jersey
cattle. An attempt to cultivate the bacillus was negative,
and inoculation experiments also failed. Since this
date very many cases have been recognized, and it is
now generally admitted that the disease is prevalent
all over England. Chase has seen it in imported Jerseys
in South Africa ; and Beebe, Pearson, and Melvin, have
reported cases in North America. Throughout Ger-
many the disease has been reported, and also in
Schleswig-Holstein.

Meyer of Berne, in Switzerland, in 1908, gave a good
description of Johne's disease — calling it " enteritis
hypertrophica bovis specifica " — as it occurs in several
of the Swiss cantons, and showed that it was in reality
responsible for conditions commonly supposed to arise
from strongylosis.

D. Meadows, of the Indian Civil Veterinary Depart-
ment, has informed us that a colleague of his has
observed a case in India, in Lahore, typical bacilli
being present in smears made from rectal scrapings.

In England, Johne's disease is certainly very preva-
lent, much more so than is usually supposed. From
inquiries made by owners of diseased cattle, and
from veterinary surgeons, and also from replies to
a circular letter sent to a large number of private
practitioners and to those superintendents of municipal
abattoirs who are veterinary surgeons, "we have evi-
dence of the existence of the disease in almost every
county.

The condition is becoming much more frequently
recognized and differentiated from other causes of

HISTORY AND DISTRIBUTION 9

diarrhoea in cattle, but some considerable differences
occur in the answers we have received. Generall}^
speaking, the Midland Counties seem to be the worst
affected. We have evidence of cases occurring in
Devon and Cornwall, in Westmorland, and in Kent.
Somerset appears to be badly affected, especially in
the low-lying district round Bridgewater.

Edwards of Mold (Flintshire) states that on an
average he meets with twenty cases a year. He
writes : '* I have not seen more than two cases on the
same premises at the same time, but have had seven
in the same place in one year. 1 think that a large
number of cases are never attended by a veterinar}^
surgeon ; the farmers regard them as * wasters,' and
either attempt no treatment or use home remedies. . . ."
In the North of Scotland cases are probably less
common, though we have received specimens from
Ross-shire and from the county of Lanark.

Sampson of Sheffield considers that the disease is
on the increase in his district. Leicestershire in some
parts is badly affected; so, too, is Northants, in which
county one breeder has lost sixty beasts from this
disease in the last seven 3^ears.

Perhaps the most accurate information can be
gathered from a private communication from De Vine,
who has taken a great interest in this condition. In
the municipal abattoir at Birmingham about 25,000
head of cattle are slaughtered each year, or roughly
500 a week. Out of these 500 animals, about three
cases of Johne's disease are noticed by De Vine in the
ordinary course of meat inspection — that is to say,
the animals are sufficiently affected to present macro-
scopic lesions. On making post-mortem examinations
of animals killed from six to twelve months after
inoculation with pure cultures, the present authors

10 JOHNE^S DISEASE

have been able to recover the bacilli from the deeper
layers of the mucous membrane of the intestine ; but
the thickening of the bowel in some of these cases
was scarcely noticeable, and, indeed, infection could be
proved only after a careful microscopic search, or by
the cultivation of the bacillus. Yet, of those animals
which on post-mortem examination show only slight
lesions of Johne's disease, many are thin and in poor
condition, being obviously affected by the bacilli or
their products. It is reasonable, therefore, to sup-
pose that in addition to the cases reported in the
Birmingham abattoir, a good many occur which it is
not possible to detect in the ordinary course of meat
inspection. Besides these well-marked and slight
cases, there must be added those animals which die
of the disease, or are slaughtered at home as being
useless to send to a properly inspected meat market,
where, on account of extreme emaciation, they would
be condemned. On the other hand, Parker of
Newcastle, in answer to an inquiry, states that the
condition is very rarely met with in the abattoirs in
that city; and he accounts for this by the fact that
only very good-class bullocks are slaughtered there.

In 1910, Riddoch, in inspecting about 2,800 dairy
cows kept in the city of Edinburgh, diagnosed three
cases by the examination of rectal scrapings ; but he
states that slaughterhouse statistics in the city would
be of little value, as all the extreme cases would be
totally condemned on account of emaciation, the cause
of which would not be stated.

At Belfast the disease seems almost unknown, and
at the Dublin abattoir distinctly uncommon. From
County Armagh, Thompson writes that he is unaware
of the existence of the disease in his district, and
though the disease is met with in Limerick, Wallis

HISTORY AND DISTRIBUTION 11

Hoare, of Cork, has not seen any cases in that
district. From answers to other inquiries in Ireland,
we suspect that many more cases occur than are
recognized.

The Channel Islands are known to suffer heavy
losses from Johne's disease, and are also remarkable
for the scarcity of cases of tuberculosis in cattle.
We have received most interesting information from
Le Sueur of St. Heliers, who with Olaf Bang has in-
vestigated many cases of the disease.

He writes that he sees in his district, on the island
of Jersey, about twenty-five cases a year, but rarely
more than four on one farm. Only one breed of cattle
is kept — Jersey — and the disease occurs most commonly
in cows about two and a half years old, frequently after
their first calf. He writes : " Johne's disease on the
island chiefly affects young stock, and heifers after
their first calving, and especially those reared on
boggy land. Likewise among farmers who are milk-
sellers, which means that they feed their calves, not
on milk, but on some condiment, the animals being
rather underfed. I also believe there is some tendency
to hereditary transmission, having noticed many cases
in the same family. I have also found that mature
healthy animals (cows of five to six years old), when
put on an infected farm, do not develop the disease."

The occurrence of the disease upon marshy or boggy
land is generally recognized. Bang has found it
prevalent in low-lying districts. Townsend finds it
very common in the Fen districts of Lincolnshire, and
many other observers (Le Sueur, Edwards, Scott) are
agreed upon this point. Le Sueur writes : " The
disease is unknown on some parts of the island — on
high lands and under good farmers."

With regard to the prevalence of the disease in sheep

12 JOHNE'S DISEASE

it is difficult to obtain reliable information. In 1907,
Vukovic of Bosnia found cases of the disease in cattle,
and in the following year he observed the same con-
dition in sheep. We have received a communication
from him with regard to the sheep, and he states that
he has met with several badly contaminated flocks,
although the majority of the flocks appear to be free
from the disease. The cases mentioned by him were
entirely in mountain sheep, and he makes the interest-
ing observation that the disease seems to be most
prevalent in leprous districts. Sections of the bowel
of affected animals (made in 1909), sent to us by this
authority, show acid-fast bacilli in enormous numbers.

Craik of Alnwick has brought cases of scrapie to the
notice of the English Board of Agriculture, and Stock-
man has found acid-fast bacilli in the intestinal lesions
similar to those of Johne's disease in cattle. From
inquiries which we made by a circular letter to a large
number of veterinary surgeons we have evidence of
the existence of a similar condition in sheep with the
clinical symptoms of Johne's disease, but usually the
disease was not confirmed on post-mortem examina-
tion by the demonstration of the bacillus.

M'Fadyean, Sheather, and Edwards, have described
a case in a Welsh ewe in which acid-fast bacilli were
found in large numbers in the thickened wall of the
intestine.

In the early part of this year the present writers
received from Northampton a well-marked specimen
of the disease in a sheep's gut, and it was from this
specimen that we cultivated the sheep strain of Johne's
bacillus mentioned later.

M'Fadyean, in his annual report to the Royal
Agricultural Society, stated that he had met with a
case in a herd of deer kept in a park, but, as far as we

I

HISTORY AND DISTRIBUTION 13

have been able to ascertain, this is the only case
recorded in this species of animal.

Quite recently a case has been reported in a horse by
Lienaux; but this animal, which came from a farm, also
showed definite evidence of tuberculosis in two of the
abdominal glands, and as the lesions in the gut were
evidently not typical, the case must be considered
doubtful.

CHAPTER II

ECONOMIC IMPORTANCE AND ETIOLOGY

Economic Importance.— Until Johne's disease has for
some time been scheduled under the Contagious
Diseases of Animals Act, it will be difficult to estimate,
with any degree of accuracy, the number of cattle
affected with this disease. It may be granted that any
animal which shows clinical symptoms is a source of
loss to the owner; but an animal may become poor
in condition and generally unthrifty from Johne's
disease, though it may be killed before the onset of
acute diarrhoea. The condition is not as yet generally
recognized by stockowners and farmers. Among the
smaller men especially, the "scanter" or "waster" is
sold at a low price to a butcher or dealer, and in the
latter case passed on to spread infection at the next
market.

Harvey, of St. Columb, who has had many years'
experience both of small farmers and of cattle breeding,
points out the danger of the custom of selling a
"scanter" with three or four sound bullocks, and
suggests that the sale of an animal known to be
affected should be made a punishable offence. This
cannot be, however, until a test with a specific diag-
nostic vaccine has been recognized, and State legis-
lation adopted.

In the milking breeds, Johne's disease is certainly

14

ECONOMIC IMPORTANCE AND ETIOLOGY 15

of the greatest importance, as it usuall}' attacks cows,
and causes death just at the time when they should be
at their highest value — i.e., after the second calf — when
giving the best yield of milk. We have seen valuable
three-year-old Jersey cows dying of this disease within
a few weeks of calving, and a pedigree bull on the
same farm reduced in value in the course of twelve
months from over ;^ioo to thirty shillings.

The loss sustained by an owner who attempts to
treat the disease wnth extra food and drugs is fourfold ;
there is no return for the extra expense in feeding;
there is less milk; a poor, weakly, or dead calf; and
when in the end the animal is sent to the butcher a
very low price is obtained. In the worst cases the
animals are sent to the nearest kennels, or are buried
on the farm. Besides these losses, there is the con-
stant danger that healthy animals on the same land or
in the same byre will become infected, and the loss is
likely to become an annual one. The same may be
said to hold good for tuberculosis in cattle ; but while
it is possible for a cow extensively affected with tuber-
culosis to be in fairly good condition, it is rare to find
an animal badly affected with Johne's disease which is
not so emaciated as to be wholly condemned in a
properly inspected meat market.

A farmer who buys a good and recently calved dairy
cow at the average price of ;^2o, and only obtains
250 gallons of milk instead of 500 to 600, loses on the
milk alone about £6. At the end of a year, in the case
of a sound animal, he would expect to have a cow
worth about £ig, and a calf worth at least ^1 ; but if
the animal be badly affected with Johne's disease, he
would be fortunate in obtaining ;^5, so that a loss of
;^2o is not an excessive estimate, as the milk has only
been calculated at 6d. a gallon.

16 JOHNKS DISEASE

According to the returns of the Board of Agriculture
for the year 191 1, there are in Great Britain about
4,200,000 head of cattle over two years of age ; of these
2,825,000 are cows in milk or in calf. If we take the
low estimate of i per cent, of the milch cows alone as
being affected with, and dying from, Johne's disease
(or sold to a butcher when they should be at their best
for milk purposes), and if we assume the loss on each
to be that estimated above — i.e.^ ;^20 — then the annual
loss in Great Britain would be over ^500,000. But
this takes no account of 1,250,000 beef animals, or of
the extra value of pedigree animals, or of the possi-
bility of many infected animals among the 26,000,000
sheep in Great Britain.

Bang quotes the figures in a circular issued by the
Kustos Insurance Company, which insures a great
number of cattle in Denmark (October, 1909). Accord-
ing to Bang, it is estimated that of 40 herds of Jersey
cattle insured for about 1,000,000 kroner, 150 animals
died in 29 herds. As compensation for deaths from
this disease alone, the company paid out 28,000 kroner,
which represents 3-5 per cent, of the total value for
which the animals were insured. Presuming that the
disease is equally prevalent among the 2,825,000 milch
cows in Great Britain, and valuing them at ;^20 per
head, the loss per annum involves over ;^i,ooo,ooo,
and since it is unlikely that the disease is twice as
prevalent in Denmark as in this country, our estimate
of ;^5oo,ooo is probably too low.

Etiolog-y. — As was mentioned when dealing with the
history of the disease, the association of a thickened
intestine with symptoms of chronic diarrhoea and
wasting had often been observed prior to Johne and
Frothingham's discovery of acid-fast bacilli in the
lesions. When B. Bang described the condition at

ECONOMIC IMPORTANCE AND ETIOLOGY 17

Liverpool in 1906, English veterinary surgeons at once
recognized it as occurring in their experience.

Although Johne and Frothingham first observed the
bacilli in 1895, they regarded them as tubercle bacilli in
a modified form, and their opinion was upheld until
1906, v^^hen B. Bang proved the condition to be distinct
from tuberculosis. He was able to reproduce the
disease in calves by feeding them with large quantities
of infected intestine; and Miessner and Trapp and
Malm have confirmed his results.

In 1910 the present writers prepared a medium on
vv^hich the bacillus was grown, and in the following
year showed that the cultures fulfilled Koch's postu-
lates; thus the undoubted relation of the bacillus to
the disease was proved. For fuller details of this
work, and a description of the morphology of the
bacillus, the reader is referred to Chapter VI.

In the later stages of the disease the bacilli are
discharged in large numbers in the faeces of the
animal; and it is with these bacilli that the food
supply and water become infected and the disease
conveyed to other animals. There is now no doubt
that the disease is not confined to cattle, but may
attack sheep, deer, goats, and possibly allied animals.
It occurs in animals of both sexes and all ages ; though,
from its chronicity, it is never observed in the very
young. As has already been pointed out, a damp,
marshy district seems to predispose to infection,
possibly by allowing the specific bacillus to retain its
vitality for a longer period outside the animal body.

The length of time the bacillus can live and remain
infective outside the body has not been accurately
determined, but it is probably very considerable.
Although it is highly improbable that the bacillus can
multiply in soil, it is quite conceivable that dung,

18 JOHNE^S DISEASE

hauled in large quantities on to land that is used for
growing swedes or other roots, is capable of infecting
the roots, and so by this means the disease may be
spread, as housed cattle often receive thirty to forty
pounds of such roots daily.

Under natural conditions it is improbable that the
majority of animals which ingest the specific bacillus
would die of Johne's disease, even if kept alive for a
sufficient time for the disease to develop. If this were
so, then in a byre containing one badly infected animal
among twenty healthy beasts, a much larger proportion
would contract the disease than usually happens.
According to statistics which we have been able to
gather from veterinary surgeons, as a rule, not more
than one or two cases occur on a farm in a year. The
number of abortive infections must play an important
part in determining the true infection of an animal, and
possibly some strains of bacilli are more virulent than
others, although inoculation experiments seem to show
that the disease is not so readily contracted as tuber-
culosis.

Many authors are inclined to consider certain breeds
of cattle, notably the Jersey, more susceptible than
others to Johne's disease, while O. Bang states that the
future of the Jersey breed in Denmark depends upon
the possibility of eradicating pseudo-tuberculous
enteritis from the herds.

M'Fadyean has recorded cases in Shorthorn, Sussex,
and Jersey cattle ; Angwin in Jerseys and Guernseys ;
Male in Jerseys and Devons. Chase has seen cases in
South Africa which occurred in imported Jerseys ; and
one of the first cases to be reported in North America
by L. Pearson occurred in a Jersey cow bred in that
country. Townsend has seen cases in pedigree red-
polled cattle, while M'Fadyean, Sheather, and Edwards

ECONOMIC IMPORTANCE AND ETIOLOGY 19

have recorded cases in Jerseys, Welsh, Shorthorn, and
Devon cattle. The present writers have investigated
cases in Shorthorns, Devons, Jerseys, and Herefords.

There seems to be but little doubt that in Denmark
a greater percentage of Jersey cattle is affected than of
the native breeds. This is shown by the Kustos
Insurance Company in the figures relating to the
compensation for losses due to Johne's disease in
Jersey and Danish cattle. In 40 herds of Jersey cattle
insured, the compensation paid was 28,000 kroner, and
in 20 herds of native cattle, 6,000 kroner ; from which
it would appear that the disease is roughly twice as
prevalent in Jersey cattle as in the native races.

Miessner and Trapp have made a very careful study
of the condition, and consider it to be a stall disease
(Stallseuche), or a disease occurring in animals kept
housed for a great part of the year.

In a herd of Hereford cattle kept specially for beef
purposes, the proportion of old cattle is very small
when compared with the proportion of aged cows in
a herd of Jerseys or milking Shorthorns. Where the
sole or chief object is to produce milk, the animal is
kept housed for a great part of the year, and especially
is this the case in Denmark, where the system of dairy
farming is more intensive than in England. It is also
well known that farmers prefer to house their cows,
with the idea that the greater warmth avoids the loss
from the utilization of food for maintaining the animals'
temperatures, and that by this means a better yield of
milk is obtained. Then again, in a milk breed it is
necessary for each cow to calve regularly, and the
strain on the animal economy is very great. The
well-known tendency of Johne's disease to appear
suddenly soon after calving may be ascribed to the
drain on the cow (she being already slightly affected

20 JOHNFS DISEASE

with Johne's disease) in providing for the needs of the
growing foetus, from the act of parturition, and from
the sudden onset of the full activity of the mammary
gland.

The possibility that one cow will infect a number of
others may be greater when the animals are closely
housed and are lying in pairs in stalls than when they
are at pasture; for, as is well known, cattle usually
avoid feeding on or near the masses of rank grass
which grow where dung has been deposited. On the
other hand, the infective material must be fairly gener-
ally distributed by rain, etc., so that the greater
frequency of the disease in stall-fed animals is probably
due more to a lowered resistance than to greater
opportunities of infection.

On the whole, it is probable that the prevalence of
the disease in a particular herd or race is determined
rather by the condition of life under which the animals
are kept and their feeding and general hygiene than
by any inherited tendency towards infection. The
cases seen by Le Sueur, occurring in succeeding
generations of Jersey cows, are probably the result
of the calves becoming infected from the faeces of the
cows, and not of an infection m utero which is exceed-
ingly unlikely to take place.

The frequency with which pedigree and "milk
record " animals are sent about the country to shows,
etc., and bought and sold or hired for breeding purposes
makes it probable that there are very few districts free
from the disease.

The etiological factors which determine the spread
of the disease in other susceptible animals have not
been accurately determined, but probably they are
much the same as for cattle.

CHAPTER III

SYMPTOMS AND DIAGNOSIS

Clinical Symptoms. — In the early stages of Johne's
disease there are no symptoms from which a positive
diagnosis can be made. In inspecting a herd of cows
in a byre at milking time, one or two may be picked
out which are apparently unthrifty. Their coats are
erect or staring, and their general condition is poorer
than that of their neighbours, although they have
received the same care and attention. Usually the
history of these animals shows that the extra food
given to improve their condition has produced no good
effect. In course of time they have slight attacks of
diarrhoea, lose more flesh, and give less milk. In the
case of a pregnant cow^ the additional strain of supply-
ing the needs of the growing foetus hastens the
progress of the disease, the diarrhoea becomes more
frequent and severe, and emaciation more marked.
Parturition, and the consequent activity of the mam-
mary gland, often produces a fatal result within a few
months of calving. Sometimes the symptom of diar-
rhoea is not shown until after calving, and yet the
cow may die within a few months of its onset. The
course of the disease depends somewhat on the diet
and general hygiene. Although Miessner considers it
a disease of stall cattle (Stallseuche), as a rule the
symptoms are increased in severity when the cattle

21

22 JOHNKS DISEASE

are turned out to pasture. In some cases it is possible
to prolong the life of an affected animal for many
months by placing it in a warm house and supplying
an easily digestible nitrogenous diet. The diet seems
to play an important part in the progress of the con-
dition. We have produced the disease in several
calves in inoculation experiments : six months after
inoculation the animals v^ere killed, and found to have
lesions of the disease, and to shov;^ acid-fast bacilli in
these lesions. When killed, the calves v^ere in poor
condition, thin and hide-bound. For some days one
was unable to rise without assistance; but on post-
mortem examination this animal showed tuberculosis
of the mediastinal glands as well as Johne's disease.
None of these calves, however, had shown signs of
diarrhoea. From the time of inoculation onwards the
faeces, examined frequently for the presence of acid-
fast bacilli, gave negative results, although for about a
fortnight the faeces of one calf contained a large number
of semi-acid-fast bacilli. These calves were fed for a
time on milk (previously boiled to avoid tubercular
infection) with the addition of a little cod-liver oil;
later they were given good meadow hay and occasion-
ally a little lucerne or other green food.

Bang noticed diarrhoea in calves at the eighth month
after feeding with large quantities of infected intestine,
and probably, at grass, our own animals would have
shown symptoms of diarrhoea ; but in naturally infected
animals, the period from the time the bacilli begin to
multiply in the intestinal mucosa to the onset of
diarrhoea is rarely less than six months, and in well-
fed animals it may be much longer. Probably it will
be shortened in the case of animals in exposed situa-
tions on poor pasture, in pregnant cows, and in those
which are suffering from tuberculosis, strongylosis, or

SYMPTOMS AND DIAGNOSIS 23

actinomycosis of the tongue or maxillae. The initial
quantity of infective material taken in, and the fre-
quency with which the infection is repeated, will, of
course, play a considerable part in determining the
severity of the symptoms and the time of their appear-
ance.

In no case have we been able to ascertain that the
act of rumination is suspended, or that there is a rise
of temperature for any length of time. Most authors
agree that the temperature rarely rises above normal,
and that it is often subnormal, though in some cases
Male has noticed a temperature of from 103° to 105° F.
Angwin has rarely found it above 101° F. The possi-
bility that fever, in cases of Johne's disease, may be
due to some coexisting disease, such as tuberculosis,
or to secondary infection with intestinal bacteria, must
not be forgotten. In a naturally affected animal at the
Brown Institution, kept under close observation for
over twelve months, the temperature was almost
always subnormal in the morning — about 99° F. — and
rarely, if ever, rose above 103° F. Nor, in inoculated
calves, has any important rise of temperature been
noted that could not be attributed to some temporary
digestive derangement or coexisting tuberculosis, etc.

Consequently, there are none of the classical symp-
toms of fever, and, unless the animal is also suffering
from tuberculosis or pulmonary strongylosis (Hoosej,
there is no cough. The muzzle is moist, and the eyes
are bright, though they may be sunken from the
absence of intra-orbital fat. In advanced cases, the
mucous membranes are pale and anaemic. The pulse
is sometimes weak, and may be faster than normal.

As a rule the appetite is unimpaired, and the animal
ruminates to the end. Even when very badly affected
a cow will usually eat cake or corn, though hay may be

24 JOHNFS DISEASE

refused. Coarse, innutritious food, frozen roots, en-
silage, etc., tend to aggravate the diarrhoea.

In spite of the absence of fever, there is usually great
thirst, as a result of the watery condition of the faeces.
This thirst, with no sign of fever, has given rise to the
popular name ''Kaltbrandigkeit," applied to the disease
by Swiss farmers.

Angwin states that it is often difficult to get affected
cows in calf; but the point is not of very great impor-
tance, as such animals should be slaughtered rather
than bred from. He states also that he has never
observed any oedema of the sternum or dewlap, or in
the intermaxillary space, but admits that he has seen
this point mentioned by lay writers. In this connection
Le Sueur has given some useful information. With
regard to the disease in the island of Jersey, he writes :
"... the guiding symptoms which decide the farmer
to destroy animals are oedematous swellings in the
intermaxillary space and under the sternum, with, of
course, diarrhoea, etc. . . ." We, too, have recently
seen an oedema of the intermaxillary space in several
instances.

The general symptoms are such that tuberculosis is
usually suspected, and a negative result with ordinary
tuberculin has almost come to be regarded as a positive
proof of the existence of Johne's disease— other obvious
conditions being eliminated.

Some observers, including Miessner, have stated
that the faeces are frothy and full of air-bubbles. This
is true is some cases, but cannot be considered as
peculiar to the diarrhoea that arises from Johne's
disease. The colour of the faeces is not, as a rule,
markedly different from that of healthy animals on
the same diet. Undigested particles of food can
be detected, and when the diarrhoea is very acute,

SYMPTOMS AND DIAGNOSIS 25

shreds ot blood-stained mucus may be seen in the
dejecta.

The diarrhoea is often intermittent in character, and
in some cases appears to recur at more or less regular
intervals of about three or four weeks. Between the
attacks the animal improves somewhat in condition.
We have noticed that the bacilli may be very numerous
in the faeces at the height of an attack of diarrhoea, and
that as the dung becomes more normal in consistence,
they are much less numerous. This point, however,
has not been confirmed by the examination of a large
number of cases.

In the more chronic cases, the attacks of diarrhoea
seem to vary in intensity. Miessner and Trapp, who
have made most careful records of cases of pseudo-
tuberculous enteritis, tested the urine in several
instances, but found no marked abnormalities. These
authors also made blood counts in some cases, but
found the normal cells in their usual numbers. They
found the milk reduced in quantity; but after pro-
longed centrifuging at a high speed they were unable
to find any bacilli in the sediment. Their inoculation
experiments with centrifuged milk, urine, etc., are
described in Chapter IX.

Symptoms in Sheep. — As has already been mentioned,
Johne's disease in sheep has been described in Bosnia
by Vukovic and in Great Britain by Stewart Stockman.
More recently M'Fadyean, Sheather, and Edwards have
reported a case in a Welsh ewe. In Scotland, where
the first case of the disease in Great Britain was dis-
covered in the investigation of an outbreak of a disease
known locally as " Scrapy," the condition is said to be
spreading. The following interesting note b}^ Stock-
man appeared in the Report of the Board of Agriculture
for 1909 :

m JOHNE^S DISEASE

" Scrapy. — This is a disease of sheep which is
only known in a limited area of Scotland ; but it
has been reported that the area is extending. On
the latter point, however, the evidence available is
not convincing, and it is difficult to get full infor-
mation on account of the reticence observed by
farmers. Apparently the disease only affects
ewes after they have had two or three crops of
lambs. The symptoms are rather peculiar, and
may at first be confounded with scab. One of
the first signs is an itchy condition of the skin,
which gradually becomes worse, and compels the
animals to constantly rub themselves against fixed
objects. The skin, however, shows no eruption as
in the case of scab, and no acari can be found. The
affected animals become very much emaciated.
Small abscesses which are probably only the in-
direct result of the disease may appear at various
parts of the body, particularly about the head.
Eventually the animals die from emaciation if
they are not previously slaughtered ; but affected
animals may live on in a miserable condition for
several months. Dipping has no curative effect;
on the contrary, it seems to intensify the symptoms.
If we accept the local statement that the disease is
spreading, one is justified in thinking that this may
be a disease which is transmissible either by direct
or indirect infection. It has not been possible to
obtain a great deal of material, and from local
inquiry by one of the Board's Veterinary Inspectors
it would appear that shepherds and farmers are
inclined to class more than one disease with totally
different symptoms under the head of Scrapy,
although some of the ailing animals do not show
the most prominent symptom of scratching, from

SYMPTOMS AND DIAGNOSIS 27

which the disease has received its local name. In
the case of one animal which had been affected for
several months a condition of the bowel somewhat
resembling that present in Johne's disease was
discovered at the post-mortem examination. Acid-
fast bacilli, indistinguishable from those found in
Johne's disease, were also present in large numbers
under the microscope in a small area of the intes-
tine. This important lesion, however, which
seemed at first to throw an unexpected light upon
the cause of the disease, has not again been dis-
covered in a very restricted number of autopsies
which it has been possible to make on other affected
animals."

In another report on the subject this extract is
quoted, and further details are given by the same
author. He wrote :

"Since the above report was written further
material has been received for which the writer
has to thank Mr. Craik, M.R.C.V.S. The material
in question was sent from a farm upon which the
disease known as 'Scrapie' had not previously
appeared ; but the owner had, a year before,
bought sixty-five sheep from another farm on
which the disease had existed for several years. . . .
Twelve animals out of sixty-five had died with
similar symptoms to the one sent up for examina-
tion."

Mr. Craik's report stated that the animals had started
to purge very severely during the last six weeks after
they had been put on roots, and that the disease seemed
to affect sheep in the same manner as Johne's disease
affects cattle. As the result of further inquiry it was

28 JOHNE'S DISEASE

stated that the sheep in question were not known to
have suffered from itching of the skin, but the emacia-
tion had been *' continuous and prolonged."

The author concludes that the disease known as
" Scrapie" is not necessarily a form of Johne's disease,
as it is possible, of course, that two different diseases
may affect the same animals. It is to be noted, more-
over, that the three sheep examined, and mentioned in
the extract from the annual report given above, showed
the characteristic skin irritation ; but in only one could
the lesions of Johne's disease be found ; while in the
later outbreak classical symptoms of Johne's disease
were observed, but no itching of the skin was noticed.

In the case examined by M'Fadyean, Sheather, and
Edwards, the subject, a Welsh ewe, was suspected of
suffering from parasitic gastro-enteritis, and on post-
mortem examination many worms (Stroitgyhts cervi-
cornis) were found in the abomasum. The frequency
with which sheep and lambs are attacked with parasitic
gastro-enteritis, and the high mortality often observed,
suggest, in such cases, the need for careful post-mortem
examinations ; as it is easy to overlook slight lesions
of Johne's disease, especially in the presence of an
apparently obvious cause, such as Strongyhis contortus.

M'Gowan and Rettie have recently published an
account of an examinatipn of four sheep suffering from
scrapie. They give as the usually recognized symp-
toms : " great emaciation, pruritus, wool rubbed off,
ulcers on nose, top of head, and legs ; bare callosities at
root of tail and on either side of it ; . . . rubbing against
walls and fences, etc., bleaching of wool over back, but
persistence of appetite. A secondary anaemia was
present, and the temperature varied within normal
limits." These authors found sarcosporidia in all four
cases, and no acid-fast bacilli. A description of the

SYMPTOMS AND DIAGNOSIS 29

post-mortem examinations and a discussion of this in-
fection is given on p. 37.

From the descriptions given by different authors,
it is probable that many of the symptoms described
under the heading of "Scrapy" and "Scrapie" are
not caused by Johne's bacillus, but are due to entirely
different causes. Indeed, it is unlikely that in an un-
complicated case of Johne's disease in a sheep the
symptoms are very different from those observed in
cattle. The skin lesions probably belong to another
condition, and the name " Scrapy " should be reserved
for this disease; while "Johne's Disease" should be
the name given to the disease of sheep produced by an
infection of Johne's bacillus in the gut and mesenteric
glands. This view is now held by most workers.

The present writers observed no symptoms, except
some wasting, in five sheep experimentally inoculated
with pure cultures of Johne's bacillus isolated from
a cow ; but the animals were killed when the disease
was in an early stage, and we know that in cattle
the disease must be advanced before definite clinical
symptoms become manifest.

In 191 1 we inoculated two young goats with pure
cultures of Johne's bacillus. Seven months later the
animals were apparent!}^ in good health, except that
one of them occasionally showed some slight diarrhoea.
No rise of temperature had been noted, their appetites
remained normal ; yet on post-mortem examination
both were found to be affected with Johne's disease,
and showed the typical lesions (pp. 53 and 151).

In the case described by Lienaux in a horse, diar-
rhoea, emaciation, and fever were present ; but as this
animal was also suffering from tuberculosis, it is prob-
able that the fever, at least, was caused by the tuber-
cular lesions.

30 JOHNE'S DISEASE

Differential Diagnosis (Clinical). — The causes that
may give rise to diarrhoea in cattle are very varied,
and include errors in feeding, frosted roots, mouldy
corn or hay, irritants such as ricin or sand in feeding
cakes, and various mineral poisons ; also intestinal
strongylosis, often associated in young stock with
bronchial strongylosis ; and, lastly, coccidiosis and
tubercular enteritis.

The acute febrile conditions, such as anthrax, rinder-
pest, and mahgnant catarrh, may also give rise to diar-
rhoea, but need not here be considered.

In some parts of the country certain land seems
to predispose to diarrhoea in cattle pastured on it.
In Somerset such land is known as " teart " land or
" scouring land." The exact reason for the effect that
this land has on the animals is not known ; but with
such land the diarrhoea occurs when the animals are
turned out, and ceases when they are removed. The
disease also attacks a number of animals at the same
time.

The irritants, which arise from unsound food,
poisoning, etc., can be considered together. They are
naturally the first causes to be sought for by a prac-
titioner in conducting an inquiry on a farm. There
will probably be several animals attacked at once.
The symptoms as a rule are acute, and the diarrhoea is
well marked before the animal has suffered sufficiently
from toxic effects to become emaciated. It may be
accompanied by fever, loss of appetite, and cessation
of rumination ; and an examination into the dietetic
and hygienic management of the farm will soon reveal
the cause.

There remain to be considered strongylosis, cocci-
diosis (psorospermosis), and tubercular enteritis.
Miessner and Trapp have mentioned a case of sarcoma-

SYMPTOMS AND DIAGNOSIS 31

tosis of the bowel ; the animal showed all the symp-
toms of pseudo-tuberculous enteritis, and the true
cause of the symptoms was only discovered on post-
mortem examination. This condition is probably
sufficiently rare to be ignored from a practical point
of view, though, from a pathological standpoint, the
case is of interest.

Strongylosis. — Cattle and sheep, especially young
stock, are frequently the hosts of various species of
strongyles. These small parasites — worms, as they
are commonly called — vary in length from 3 to 4 milli-
metres up to 20 to 30 millimetres, and they inhabit the
abomasum, or fourth stomach, and the small intestine.
There are numerous varieties, of which the com-
monest are :

In cattle ... Strongy his convohthts^ S. gracilis.
In sheep ... Stro7igylus cei^vicornis, S. contortus.

The distinguishing features of these worms are their
size, and the markings on their caudal appendages or
bursae. They affect sheep up to a year old, and some-
times adult animals ; they also affect cattle up to two
and a half years old. In adult animals their effects are
very much less marked. The chief symptoms of their
presence are diarrhoea, wasting, loss of appetite, fever,
and thirst. As a rule, a number of animals are at-
tacked at the same time, and in lambs the course of
the disease is very rapid, and the mortality very high.
In cattle the affection takes a more chronic course.
The affected beasts shows signs of anaemia ; their
coats are rough and their skin inelastic (hidebound).
There is a foetid watery diarrhoea, and cases may
be mistaken for tuberculosis or Johne's disease.
M'Fadyean, Sheather, and Edwards have recorded
a case of Johne's disease, in a Welsh ewe, in which

32 JOHNE'S DISEASE

a considerable number of worms — Strongylus cervi-
cofnis — were found in the abomasum. Husk, or hoose
(parasitic bronchitis), is frequently associated with
intestinal strongylosis. Treatment is very often un-
successful, and animals left untreated rarely recover.
On post-mortem examination the mucous membrane
of the stomach and intestines may be found to be
ulcerated, and may show catarrhal inflammation. It
is often slightly thickened, and of a dark livid colour ;
while a certain amount of gelatinous exudate is
present. Very frequently the large intestine has the
same appearance, and there may be some fluid in the
peritoneal cavity.

The chief characteristics that distinguish this con-
dition from Johne's disease are a high temperature —
105° to 106° F. — and a more rapid course. In some cases
animals, if severely affected, die in three to four days,
and they rarely live more than a month. The post-
mortem lesions are those of a catarrhal enteritis, and
in the faeces or stomach contents the worms, embryos,
or ova, can be found on microscopic examination. The
best way to discover the parasites is to take a small
quantity of the contents of the stomach, dilute well
with water in a flat glass vessel, stir, and allow to
settle. On decanting the water, and examining the
sediment with a hand-glass, one can see the worms,
or, if their appearance is familiar to the observer, the}^
can be detected with the naked eye. To determine
the species, the worms should be picked out with a
needle for examination under a low power of the micro-
scope. Seen with the naked eye, the parasites have
the appearance of shreds of cotton, and are greyish
white in colour, though in some instances they are
brown or reddish from contained blood.

The discovery of these parasites in large numbers,

SYMPTOMS AND DIAGNOSIS 33

and the history of the case and of the farm on which
it occurs, are, as a rule, sufficient to differentiate the
two conditions. In the event of the discovery of
only a few of these parasites, it is well to make
film preparations from scrapings of the. mucosa of the
intestine, and to examine for acid-fast bacilli.

Treatment of such cases is usually best carried out by
applications of lime or salt to the land and by proper
drainage. It is also recommended that young sheep
should not be allowed on land where old sheep have
recently been kept.

CocciDiosis, Psorospermosis, Dysentere Rouge. —
These terms are all applied to the disease of cattle that
is caused by the Coccidium {Eimeria) bovis^ discovered
by Zurn in 1878. Cases have been reported in Great
Britain by Gair (1898); also in Switzerland, France,
Italy, Germany, East Africa, the Soudan, and the
United States of America. When it attacks adult
animals and assumes a chronic form, it may be mis-
taken for Johne's disease. We have seen this con-
dition described under the latter name in an American
agricultural journal. The chronic cases are, as a
rule, sporadic, but in young animals it occurs as an
enzootic.

The coccidium oocyst, the form in which it is
most commonly met with in the faeces of cattle, is a
round or oval body measuring from 14 to 16 /x, or
in some cases from 20 to 30 ft (two to gw inch).
This is smaller than the Coccidium oviforme of the
rabbit, which is met with in many parts of Great
Britain.

The cysts have " a highly resistant envelope, show-
ing a doubly contoured outline when viewed through
the microscope. In freshly voided faeces the contents
of the oocyst in many instances entirely fill the shell,

3

34 JOHNKS DISEASE

through a rounding-up and collection of the cytoplasm
into a granular ball-shaped mass lying within the
doubly contoured cell of the oocyst. In such cases a
clear space appears to intervene between the envelope
of the cyst and its contents. A nucleus of fair size
can usually be distinguished in fresh preparations."
From this account of the coccidium, given by Jowett,
who investigated cases at Capetown, it is obvious that
an examination of the faeces of affected animals should
allow a diagnosis to be made easily. Small pieces
of mucus from the bowel may contain innumerable
oocysts.

In Switzerland, where the condition is not rare, and
where it has been studied by Hess and Guillebeau, it
most frequently attacks animals at grass ; on a pasture
or in a cowshed it may spread in rapid succession
from one animal to another until the majority or all
of the cattle become affected. It is most commonly
met with in summer or autumn. The coccidia are
supposed to be taken into the alimentary tract with
the food and water, and by migrating into the epi-
thelium they set up a severe inflammation (Friedberger
and Frohner). The lesions are those of acute enter-
itis ; the bowel contents may contain blood and the
mucous membrane may be swollen, and in some places
may show haemorrhages.

Ostertag states that Maske commonly found coc-
cidia, which caused tubercles about the size of a pin's
head, in the fourth stomach and intestines of sheep.
No mention is made of their causing symptoms, but
more recently Martin has found coccidia in the intes-
tines of young kids similar to those already described
by Marotel under the name of Coccidium Arloingi.
"The internal aspect of the intestine was strewn with
a large number of small nodules or tumours . . . re-

SYMPTOMS AND DIAGNOSIS 35

sembling coccidian tumours in the intestine of a
sheep." Some were deposited in the centres of the
regions of Lieberkuhn's glands, which were hyper-
trophied and dilated. The symptoms reported were
"sudden onset of the disease, refusal of food, rapid
emaciation and decubitus, and death in convulsions in
three days." Hertzog states that the symptoms in sheep
are similar to those in cattle, but that the coccidia are
not found in the faeces. In the acute stages in cattle
the presence of the coccidia in the faeces, and in
sheep the discovery of the coccidia in the intestinal
mucosa on post-mortem examination, should serve to
differentiate this condition and Johne's disease.

Tubercular Enteritis. — According to Friedberger
and Frohner, from statistics of researches made
throughout the German Empire, tuberculosis of the
intestines is found in i per cent, of cattle suffering
from tubercular disease.

Nocard, writing of tuberculosis in cattle, says :

It is not rare to find miliary tubercles de-
veloped within the thickness of the mucosa, or
in the submucous cellular tissue of the intestines.
These tubercles may be isolated or agglomerated ;
in either case they quickly undergo softening, and
empty their contents into the intestinal canal, thus
causing a small wound of the mucosa, which has
no tendency to heal ; these ulcers, at first small
and isolated, generally tend to spread. Such
lesions are especially numerous in the last portions
of the small intestine and in the caecum. Perfora-
tion of the intestine is quite exceptional. . . .
Intestinal and mesenteric tuberculosis causes fre-
quent colic with profuse diarrhoea, alternating with
obstinate constipation.

36 JOHNE'S DISEASE

From this description — a translation of Nocard's
article — it is evident that tubercular enteritis is not
common. Examination of the faeces would, of course,
be useless, as, if acid- fast bacilli were found, it would
be impossible to decide whether they were Koch's or
Johne's micro-organisms. An application of the tuber-
culin test should give a positive result, and as the
disease is very unlikely to be cured or to show
any improvement, immediate slaughter is advisable.
The symptom of obstinate constipation, noted by
Nocard, is important, as we have been unable to
discover any record of such a complication in Johne's
disease.

An interesting point has been raised by the dis-
covery of M'Gowan and Rettie that four sheep suffer-
ing from scrapie and examined by them were heavily
infected with Sarcocystis tenella. To a greater or less
extent all the domesticated animals are from time to
time found to be affected with sarcosporidiosis, and
occasionally the parasites are sufficiently numerous
to give rise to symptoms. Moule has found 98 per
cent, of cachectic sheep and 34 per cent, of healthy
sheep infected. He has also found the parasite in
46 per cent, of goats examined, and in 37 per cent,
of oxen condemned for extreme emaciation. Besnoit
and Robin, quoted by Gray, found a sarcocyst present
in great numbers in a ten-year-old cow which showed
marked skin lesions, thickening, depilations, etc.
Though they were unable to inoculate the disease,
they succeeded in causing death in a rabbit by injecting
subcutaneously a glycerine extract of some of the
larger lesions. Gray states that sarcocystin, consisting
of a glycerine extract of the parasites, was found by
Laveran and Mesnil, and also by Pfeiffer, to be toxic to
rabbits, producing, when injected under the skin.

SYMPTOMS AND DIAGNOSIS 37

diarrhoea and paralysis, and causing death in seven to
eight hours.

M'Gowan and Rettie, in the four cases of scrapie
mentioned, found, post mortem, no naked-eye lesions
except strongylosis of the lung and alimentary canal.
The extent of this invasion of strongyles is not men-
tioned. The lesion common to all the animals was
a marked sarcosporidiosis, which was only seen on
microscopic examination. The parasites were found
in the panniculus carnosus, in the muscles of the jaws,
pharynx, larynx, neck, pelvis, and hind hmbs ; also in
the intercostals, heart, oesophagus, and diaphragm.
Though carefully sought for, no acid-fast bacilli were
found in the intestinal canal or in the mesenteric
glands.

In swine the sarcocyst infection is said to cause
paralysis of the hindquarters, stiffness, and cutaneous
eruption ; but none of these symptoms, mentioned by
various writers, can be said to be characteristic, as
they may be met with in so many other diseases of
swine.

However, it may be shown by future investigations
that the skin lesions and pruritus are due to a sarcocyst
infection, and that diarrhoea and emaciation are the
only symptoms caused by the acid-fast bacilli in the in-
testines of sheep, as in cattle. The bacterial infection
of the bowel may assist the sarcocysts in becoming
dangerously numerous, and these parasites may lower
the power of resistance in the sheep so that the bacilli
can establish themselves. M'Gowan and Rettie's
suggestion as to the pruritus, etc., being caused by
the sarcocysts bears out Stockman's theory that the
enteritis and the skin lesions in these cases are distinct.

Diagnosis (Bacteriological).— Since the recognition
of Johne's disease is becoming more general in Great

38 JOHNE'S DISEASE

Britain, the question of a certain means of diagnosis is
now of great importance. Recently the case of a
warranted cow developing Johne's disease within a
month of sale has been the subject of legal comment
in an agricultural newspaper.

As a rule the attention of a practitioner is called to
a cow in poor condition which is suffering from diar-
rhoea, and there may be a history of other animals
having been similarly affected during the past year.
If he applies the ordinary tuberculin test with a nega-
tive result, the suspicion of Johne's disease is certainly
increased, and the faeces should then be examined for
acid-fast bacilli.

Meyers procedure is probably the best A small
quantity of liquid faeces should be placed on a flat
dish, and in this thin layer it may be possible to see
small shreds of mucus. The blade of a small knife
may then be passed through the flame of a spirit-lamp
and a piece of mucus picked out and rubbed over the
centre of an ordinary glass microscope slide. The
film is dried and stained by Ziehl-Neelsen's method
(p. 64). The presence in such a film of acid -fast
bacilli in every way resembling Johne's bacillus is
strong evidence that the animal in question is suffer-
ing from Johne's disease; but there are acid-fast bacilli,
such as dung bacilli (mist bacillus of Moeller) and the
timothy-grass bacillus, which may easily be mistaken
for the bacillus of pseudo-tuberculous enteritis.

A better method is to take scrapings from the rectal
mucous membrane or to pinch off a small portion of
this with the finger-nails by inserting the arm into the
rectum, since the bacilli so found are more likely to be
the specific infecting micro-organism. The latter pro-
cedure, however, is open to the objection that although
films made from the walls of the ileum and caecum and

SYMPTOMS AND DIAGNOSIS 39

part of the large intestine may show enormous numbers
of bacilH, yet the disease may not extend to such parts
of the bowel as are within reach of an arm or of a
curette. This is well illustrated in the graphic records
of the comparative numbers of bacilli in scrapings
taken from the mucosa of each lo feet of the in-
testines of cattle killed while suffering from Johne's
disease, drawn up by M'Fadyean, Sheather, and
Edwards. These authors' results show that by far
the largest number of acid-fast bacilli are found in the
neighbourhood of the ileo-caecal valve, and that the
number falls off almost to nil after the rectum is
reached, though in some cases films from rectal
scrapings are very rich in acid-fast bacilli. This
agrees with the experience of the present writers.

For these reasons the examination of scrapings from
the rectum cannot be relied upon, and a negative result
is valueless. Reichel and Deubler, as the result of an
examination for tubercle bacilli in the faeces of bovines
suffering from tuberculosis, arrived at the following
conclusion : Microscopical examination of faeces and
rectal scrapings is of no value, on account of the
frequent presence of bacilli with the morphological
and staining characters of the tubercle bacillus, but
which are not tubercle bacilli.

The problem of obtaining a reliable method of diag-
nosis was nearer solution when O. Bang, in 1909,
found that a certain proportion of cattle suffering
from Johne's disease reacted to avian tuberculin, in
the same way that tubercular cattle react to ordinary
tuberculin. In the absence of an autogenous vaccine,
which it was impossible to obtain until the specific
bacillus had been cultivated outside the animal body,
O. Bang's reagent was the best so far obtained ; but in
the hands of English users, and in our own experience.

40 JOHNE'S DISEASE

it is not sufficiently reliable to be used with reasonable
safety, as the large dose which has to be given (8 to
10 c.c. in adult bovines) is likely to give a reaction
with other conditions (see discussion under Vaccines,
Chapter VII.).

The present writers have obtained marked rises in
the temperatures of naturally affected animals and of
inoculated calves, goats, and sheep, by using a diag-
nostic vaccine prepared from pure cultures of Johne's
bacillus. The cultures w^ere grown on special media
and also on ordinary broth after acclimatization of the
bacillus. Holth also has obtained a typical reaction,
with an autogenous vaccine, in a calf which had been
inoculated three months previously with a pure culture
of Johne's bacillus, although in this instance an extract
ot the tubercle bacillus was also present in the fluid.
In the opinion of the writers, a vaccine prepared from
cultures of the specific bacillus is the only reliable
diagnostic reagent for Johne's disease, and in all
doubtful cases tests with such a vaccine should be
carried out. For full details the reader must refer to
Chapter VII. It only remains to be noted here that
very advanced cases — which, however, can be diag-
nosed quite easily from the clinical symptoms, etc. —
may fail to react, a result which is also frequently
obtained when advanced cases of tuberculosis are
tested with tuberculin. Our experiments indicate
that the autogenous vaccine for Johne's disease is
quite as reliable as tuberculin is for tuberculosis.

CHAPTER IV

PROGNOSIS, TREATMENT, AND PROPHYLAXIS

Prognosis. — It has been stated that in some cases of
Johne's disease recovery takes place. Le Sueur has
informed us that in Jersey he has noted that adult cows
put on to affected land do not contract the disease. If
these animals have not been previously in contact with
affected cattle or exposed to risk of infection, it must
be supposed that the natural resistance to the disease
is increased with age ; but it may be that the animals,
having already contracted the disease in a mild form,
and having subsequently recovered before the develop-
ment of clinical symptoms, have acquired thereby an
active immunity.

M'Fadyean mentions a case that he believed to be
one of recovery. He says : ** The exceptional case was
a yearling which, along with a cow on the same farm,
developed symptoms of the disease. . . . Soon after-
wards the cow was killed and the disease verified at
the post-mortem examination. The yearhng, although
not medicinally treated, gradually ceased to scour, and
gained a little in condition. It was killed six months
after admission, and the post-mortem examination
showed that the intestine was normal in appearance,
while none of the characteristic bacilli could be de-
tected with the microscope." In the case thus de-
scribed the animal had certainly been exposed to great

41

42 JOHNE^S DISEASE

risk of infection; but as, apparently, no acid-fast
bacilli were at any time found in the faeces, and none
in the bowel wall at the post-mortem examination, it is
possible that the original diarrhoea was due to some
other cause, especially as the animal was a young one.

B. Bang mentions that he has noticed that cows, after
showing symptoms of the disease (such as diarrhoea,
slight wasting, etc.), and then apparently recovering
for a while, die in the end of Johne's disease. We
have been unable to find any record of a case, where
the bacilli have been detected in the faeces, in which
the animal has been known to recover and to remain in
normal health for any considerable time afterwards.
It is highly probable that once the bacilli multiply to
any extent in the intestinal mucosa the animal will die
of the disease after a period of from three months to
two years, depending on the sex of the animal, its food
supply and hygienic management, and on the presence
or absence of other diseases, such as tuberculosis, etc.

It may be assumed, therefore, that a prognosis of
recovery is not possible in any clinically affected
case. The possibility of treatment with a curative
vaccine, when the disease is discovered in a very early
stage by means of a diagnostic reagent, can only be
determined after a considerable number of experiments.
Nevertheless, our knowledge of allied diseases such as
tuberculosis leads us to believe that such treatment
might lead to recovery.

Treatment. — Unfortunately, there is no specific treat-
ment for Johne's disease. We know of no drug that
will kill the bacillus in the pathological lesions or
neutralize the toxins which it produces. Moreover,
the risk that one diseased animal will infect others on
the farm is so great that by far the safest, and in the
end the cheapest, course is to kill all the infected

TREATMENT 43

animals — not only those which shov^^ clinical symptoms,
but also any that may react to a dose of diagnostic
vaccine prepared from cultures of Johne's bacillus. As
to whether an attempt should be made to improve the
condition of an affected animal before it is sent to the
butcher, the circumstances of the case must decide.
Should, however, an attempt be made to cure an
affected animal, complete isolation should be insisted
on ; the dung, litter, etc., should be burnt, and a
separate pail kept for milking, if necessary.

All the drugs that are usually employed as intestinal
astringents, as tonics, and as stimulants seem to have
been tried in Johne's disease, but in no case with
success. Miessner and Trapp record that tannoform,
creolin, starch gruels, iron, and various tonics, have all
given negative results in the hands of practitioners
who have brought cases of the disease to their notice
in the north-west of Germany.

In England, dilute sulphuric acid, tobacco in the
form of balls, nitro-hydrochloric acid, cyllin, solutions
of copper sulphate, perchloride of iron, and other similar
reagents, combined with such foods and gruels as are
usually employed in cases of diarrhoea, have been given
without success. Astringents and tonics (combined
with good, easily digestible nitrogenous food, with
small quantities of roots or green food, and a restricted
allowance of water) may possibly be used in some
cases with temporary advantage, but there is no
certainty in the results.

Curative vaccines, analogous to the curative tuber-
culins used in the human subject, can, of course, be
prepared. Indeed, in testing a badly affected animal
with various weak diagnostic vaccines, we have
noticed some considerable improvement in condition
after the administration of these reagents, and it is

44 JOHNE^S DISEASE

only lack of opportunity from financial reasons which
has prevented our experimenting on these lines. If
the disease were detected in an early stage with the
diagnostic vaccine, such treatment might be both
useful and of practical value in the case of pedigree
bulls.

Prophylaxis. — In the present state of our knowledge
the question of prophylaxis is of much greater impor-
tance than that of treatment. As soon as the diseased
animals are- removed from the farm, all buildings, etc.,
in which they have been housed should be effectively
disinfected. Where possible, the floor of the cowshed
should be scraped, and if the floor is an earthen one,
the scrapings should be burnt. Cement floors should
be washed with hot solutions of some reliable disinfec-
tant, and probably roughening the old floor and the
application of a fresh layer of cement would be a good
procedure, if combined with several white-washings of
the rest of the building and its fixtures with lime mixed
with carbolic acid, etc. All dung, litter, stale fodder,
etc., from the neighbourhood of affected animals should
be burnt or ploughed in on land used for root crops,
though the latter is not free from all risk. Scrapers,
milking-stools, milkers' coats and aprons, spans, neck
chains (or ropes), buckets, and any other utensils in
the byre or milking shed should be treated with dis-
infectants or thrown away, and new articles substituted.

When affected animals have been isolated in a
particular field, or where a field is known to be badly
contaminated, it might be advisable to plough it up
and bare fallow it during the summer, and then use
it for growing some crop that will not be given to cattle
or sheep. It is improbable that the disease can affect
pigs, and as only one doubtful case has been recorded
in the horse, these animals are not likely to contract

PROPHYLAXIS 45

infection ; cattle or sheep, however, should be kept off
land known to be infected.

Sheep must be considered as carriers of the infection ;
and in carrying out prophylactic measures on a farm
these animals should be inspected. Weakly and
emaciated members of the flock should be carefully
isolated, and if showing signs of diarrhoea, the faeces
should be examined microscopically. Such animals
should also be tested with a diagnostic vaccine pre-
pared from Johne's bacillus.

In buying new stock, whether sheep or cattle, their
history should, if possible, be known, and cattle should
certainly be tested before being allowed to run with
other animals on the farm. In the case of cows known
to have come from an affected farm, it might be ar-
ranged in a warranty that the purchases have passed
a test with a specific vaccine shortly before the sale.
To be really effectual, all these prophylactic measures
should be carried out under the personal supervision
of a veterinary surgeon, and the best means of eradi-
cating the disease is to test the whole herd every three
months in order to get rid of reacting animals before
they commence to excrete the causal bacilli in their
faeces in large numbers, and so expose their neighbours
to the risk of infection.

CHAPTER V

POST-MORTEM EXAMINATION, PATHOLOGICAL

ANATOMY, AND PATHOLOGICAL

HISTOLOGY

Post-mortem Examination and Patholog-ical Anatomy.

— In making a post-mortem examination of a case of
Johne's disease one should follow the ordinary proce-
dure. It is well in the case of a cow to examine
the supramammary lymphatic glands for evidence of
tuberculosis. The abdominal cavity should be opened,
and the stomachs and intestines, together with their
lymphatic glands, must be removed for a closer
scrutiny. The remaining abdominal organs, the thorax
with its contents and glands, and the pharyngeal lym-
phatic glands, should also be inspected for evidence of
tuberculosis and other pathological conditions. The
stomachs having been opened, they should be examined
for any abnormalities ; this applies particularly to the
abomasum, in which strongyles may be found.

The intestines must be slit up their entire length
with bowel scissors, or with a sharp knife with a cork
on its point, and lightly washed to rid them of ingesta,
a large sink being convenient for this purpose.

Perhaps the most striking feature of this disease is
the insignificant character of the lesions met with in
comparison with the state of emaciation to which
affected animals are, as a rule, reduced, and the fact

46

POST-MORTEM EXAMINATION 47

that, beyond the intestinal mucosa and the colic and
mesenteric lymphatic glands, no other lesions con-
taining bacilli in a naturally contracted case have been
recorded. As a rule the ileo-caecal valve and the last
part of the ileum ma}^ be expected to show the most
marked lesions.

Even when experimental animals are fed with in-
fective material, or pure cultures of Johne's bacillus,
or are inoculated intravenously, intraperitoneally, or
subcutaneously, the disease is developed in the small
intestine ; and except in the case of subcutaneous
injections, which in some cases have caused a small
localized abscess, no other lesions containing bacilli
are demonstrable post mortem.

Before cutting into the intestine one can often
perceive the thickening of the small bowel by taking
it between the finger and thumb and comparing it in
various places. In a bad case some parts are appre-
ciably thicker than others, but there is never any
evidence of the disease on the peritoneal membranes.
A variable length of the intestinal tract seems to be
affected. Measured from the ileo-caecal valve, the
small intestine for about 30 feet often shows evidence
of disease ; sometimes the length infected is as much
as 70 feet. The initial part of the duodenum in cattle
seems never to be affected ; but the disease may
extend as high up as Vater's ampulla, and in one of
our naturally infected cases this was extensively
involved.

A badly -affected piece of bowel shows marked
thickening, and the mucous membrane is thrown into
characteristic corrugations. These corrugations are
often broken up into small islands which give a warty
appearance to the surface of the gut. In a piece of
normal small intestine from which the fat, etc., in the

48 JOHNE^S DISEASE

mesentery has been cleared away, and which has been
cut open longitudinally, a number of small folds are
seen ; but if the bowel is stretched as widely as pos-
sible these folds disappear. In a case of Johne's
disease it is not possible to make these corrugations
assume the level of the normal intestine. The same
remarks apply to the caecum, which is very frequently
affected, though in this part of the bowel the lesions
are inclined to be more patchy.

Occasionally the thickening is hmited to small
portions of the bowel; a few feet may be markedly
affected, then an interval of sound or apparently sound
intestine may intervene, and then another portion of
diseased intestine may be found. The small intestine
may escape entirely, and lesions be present only in
parts of the large bowel. Apparently the naked-eye
evidence of the disease is no guide to the number of
bacilli that may be found beneath the surface. This
has been well shown in a case examined by M'Fadyean,
Sheather, and Edwards, in which, post mortem, the
small intestine showed no macroscopic evidence of
disease ; yet smears taken from the mucosa of the
last 90 feet were found to be quite rich in acid-fast
bacilli.

If a portion of intestine is badly affected acid-fast
bacilli are, as a rule, to be found in the neighbouring
lymphatic glands; but this is subject to considerable
variation, as in some cases the bacilli are unexpectedly
numerous, and in others none can be found. The
colour of the corrugated mucous membrane in a well-
marked case is rather characteristic, being of a pinkish-
yellow tint, with occasional patches of congestion ; but
there is never any erosion of the surface of the mucosa,
or any tendency to ulceration, unless the animal has
been killed shortly after a dose of diagnostic vaccine

PLATE II.

[Photo by F. Holmes, Bristol

LOWER PART OF ILEUM FROM A COW AFFECTED
WITH JOHNE'S DISEASE.

[ 2b face page 48.

POST-MORTEM EXAMINATION 49

has been given, when we have found that haemorrhages,
and even superficial ulceration, may occur. In some
cases, however, the mucosa is quite pale and anaemic.
The rectum frequently shows transverse markings, and
is often congested. The lymphatic glands are usually
enlarged ; but are rarely so to any extent, as may be
the case in tuberculosis. They are more or less pig-
mented, and when cut into the pigmentation is found
to be especially marked in the medulla. An oedematous
condition of the glands is present, and on section a
watery fluid exudes.

From the above it will be clear that, when well
marked, the lesions are easily appreciable to the naked
eye ; but on post-mortem examination of a suspected
case frequent scrapings should be taken throughout the
length of the ileum and large intestine. In several
cases the authors have been able to isolate the bacillus
in pure culture when apparently there were no lesions,
and when half an hour's search was necessary to find
an area showing any bacilli ; so that in a doubtful case
cultures should be made on the special media. The
ileo-caecal valve and adjacent lymphatic glands should
be examined most carefully, as these are the situations
in which the disease is most frequently found in very
early cases.

In some naturally infected animals we have observed
a fatty condition of the liver, a condition that may also
be found in cases of tuberculosis.

In the post-mortem appearances of sheep there
appears to be nothing to distinguish the lesions from
those occurring in cattle. The condition is of sufficient
interest to warrant the quotation, m extenso^ of the
descriptions of the cases investigated by Stockman,
and by MTadyean, Sheather, and Edwards.

The sheep forwarded to the Board of Agricul-

4

50 JOHNE^S DISEASE

ture for post-mortem examination are described as

follows :

**The lungs and heart were normal; the liver
was fatty; the kidneys were normal, the rumen^
reticulum, and the omasum were also normal ; but
the abomasum showed a few small areas of con-
gestion. The small intestine was thickened, and
showed numerous small haemorrhages under the
mucous membrane. The surface of the mucous
membrane was coated with a white creamy mucus,,
but it showed no corrugations similar to those
found in Johne's disease in cattle. The large
intestine showed zebra markings and a few small
haemorrhages under the mucous coat.

^^Microscopical Examination. — Scrapings from
the mucous surface of the small intestine showed
enormous numbers of acid-fast bacilli, mostly
arranged in dense clumps, and indistinguishable
from those found in Johne's disease of cattle.
Scrapings from the large intestine also showed
a considerable number of the same bacilli, but the
microbes were much fewer than in preparations
made from the small intestine. The same bacillf
were also found in considerable numbers in smears
made from the mesenteric lymphatic glands. Sec-
tions of the small intestines, made after embedding
in paraffin, showed a colossal number of acid-fast
bacilli. These were present as single elements
and as dense clumps. They were most numerous^
towards the surface of the mucous membrane, but
were also present in considerable numbers in
the deepest part of the glandular layer. Micro-
scopical sections of the mesenteric glands also
showed numbers of bacilli, and nothing resembling
a tuberculous formation could be seen.

POST-MORTEM EXAMINATION 51

*' Inoculation experiments performed on the
small animals of the laboratory showed that the
acid-fast bacilli were not those of tuberculosis."

M'Fadyean, Sheather, and Edwards, give the follow-
ing details of a post-mortem examination of a Welsh
ewe, examined in April, 191 2 :

"A considerable number of worms {Strongyhts
cervicornis) were found in the abomasum, which
was nearly empty, but no abnormal appearance
of the mucous membrane was observed. The
large and small intestines were slit up along their
entire length and searched for worm parasites.
One tapeworm was found.

'* There was distinct thickening of the wall in
the case of the large intestine, this being most
marked in the caecum ; there was also some
irregular wrinkling in this position. The thicken-
ing, although not so pronounced, was continued
throughout the large intestine. The posterior half
of the small intestine was appreciably thicker than
normal, and at some places there was abnormal
permanent wrinkling, though not so marked as is
usually the case in cattle.

" Microscopic Examination of Smears. — This
showed a rich invasion of the intestinal mucous
membrane with small acid-fast bacilli quite indis-
tinguishable from those usually found in Johne's
disease of cattle. . . . The smear taken from the
caecum proved to be extraordinarily rich."

Bacilli were found in parts of the small intestine,
but onl}^ a few in the colon.

In two sheep, which the present writers inoculated
with pure cultures of Johne's bacillus isolated from
a cow, and which were killed ten weeks later — after

52 JOHNE^S DISEASE

obtaining positive reactions with a specific diagnostic
vaccine — the following condition was observed post
mortem : The animals were somewhat wasted, and
showed enlarged glands throughout the abdominal
cavity and thorax. When opened, the glands showed
an oedematous condition, and some pigmentation most
marked in the medulla.

In the animal inoculated intravenously the intestines
were distinctly thickened, especially in the vicinity of
the ileo-caecal valve; but in the animal inoculated
intraperitoneally there was no evidence of thickening.

Both cases showed only a few bacilli in the lesions,
and none were found in the thoracic glands.

The condition of these sheep was identical with that
observed in the experimentally inoculated calves which
w^ere killed before the disease had become advanced.

The liver in the sheep appeared to be fatty, and
presented the same appearance as that sometimes met
with in cattle.

M'Fadyean, in his Annual Report of the Royal
Veterinary College, published in the Journal of the
Royal Agricultural Society^ 1907, wrote :

**.... In ascertaining the cause of chronic
diarrhoea among deer kept in a park, and when
one of the diseased animals was killed the post-
mortem examination unexpectedly disclosed the
fact that it was the subject of Johne's disease.
The fact that deer may suffer from the disease
is not one of much direct interest, but it is of
importance as suggesting that other ruminants,
such as the sheep, may not be immune."

In July, 191 1, the present authors inoculated two
goats with pure cultures of Johne's bacillus. Goat i
received intravenously 3 c.c. of an emulsion of bacilli

PATHOLOGICAL ANATOMY 53

in sterile 0-85 per cent, sodium chloride. Twelve
months later the animal was killed, and showed typical
and well-marked lesions of Johne's disease throughout
the intestines. The mesenteric glands were enlarged
and oedematous. The bacilli were present in the
lesions in fair number. Goat 2, which had received
I c.c. intraperitoneally, showed the disease in a very
early stage, and only very few bacilli were found.
The lesions present in these animals showed the same
characters as those already described in the case of
bovines, and, in spite of the methods of inoculation,
were confined to the intestine and neighbouring glands.
The bacillus was afterwards recovered from these
cases in pure culture (p. 152).

Lienaux gives the following account of the lesions
found in the horse which he recently described. The
account is taken from the translation which appeared
in the Veterinary Record of April 19, 191 3 :

" Post mortem, two lymphatic glands — one in
the great mesentery and one in the meso-colon —
were found absolutely caseous, though little en-
larged. These were the only lesions of tubercu-
losis present. But extensive and serious lesions of
the intestine existed, which were especially well
marked upon the caecum and double colon. The
walls of these two viscera were thicker and more
consistent than normal, and the small intestine
was altered in the same direction, though to a
much less degree. The lymphatic glands dis-
posed along the caecal and colic arteries were
slightly tumefied and turgescent, and along the
same vessels large distended lymphatic trunks,
filled wnth yellowish lymph, could be distinguished.

" The mucous membranes of the caecum and
large colon were very thick. Their free surface

54 JOHNKS DISEASE

was irregular, and was strewn with projections
reaching up to 2 millimetres (yV inch) in height,
and of very variable surface dimensions. Some-
times these projections took the form of well-
defined nodules of hemp-seed size, in other cases
— and these were more frequent — they took the
form of more or less extensive raised plates,
between which the mucous membrane appeared
smooth and comparatively normal in aspect.
Nodules and plates were in close proximity to
each other, so that the depressed areas between
them generally formed narrow folds. The sur-
faces of these nodules and plates showed losses
of substance, which were punctiform upon the
nodules but larger upon the plates, where it was
impossible not to recognize their ulcerous nature.
These ulcers were always shallow, and were
nearly always covered with vegetable debris,
which adhered to them rather closely.

" Microscopical examination of scrapings from
the lymphatic glands involved, stained by Ziehl's
method, showed bacilli isolated or in clusters, as
they are found in the mesenteric glands of cattle
affected with Johne's disease."

Lienaux also remarks —

" From the anatomo-pathological point of view,
it is interesting to note the abundant ulcera-
tion in this horse. Ulceration is rare in Johne's
disease in cattle."

From the description given by Lienaux, we should
nesitate before accepting this as an undoubted case ot
Johne's disease in the horse. Not only had the animal
definite tubercular lesions in the mesenteric glands,
but the lesions in the intestine appear, from the

PATHOLOGICAL HISTOLOGY 55

description, to be more typical of tuberculosis than
of Johne's disease.

Patholog-ical Histolog-y. — Organs for histological
examination should be removed from the animal as
soon as possible after death, as post-mortem changes
rapidly take place. Small pieces of gland and bowel
should be placed to harden in methylated spirit, or
in formalin -alcohol, or in Mailer's fluid containing
ID per cent, of formalin. When one stains for the
bacillus, it is preferable to harden in 5 per cent,
formalin without the addition of Mailer's solution. If
Mailer's solution is used, the bottles should be placed
in a cupboard away from the light, to avoid the pre-
cipitation of the bichromate. If alcohol is used, it is
advisable to start with a weak solution, and transfer the
tissue to solutions of increasing strength, since by this
means contraction of the tissue is very largely avoided.
After twenty-four hours in the hardening fluid, the
pieces of tissue are washed in running water for about
the same period of time or longer, and to insure
thorough washing, it is best to cut the tissue into thin
slices before the commencement of this process. The
pieces of tissue are then placed in 90 per cent, alcohol
for twelve to twenty-four hours, and, if they are not
too thick, the paraffin blocks can be completed on the
next day by the following procedure : Place in abso-
lute alcohol for one hour, and then change into fresh
alcohol and leave for the same time ; then transfer to
chloroform, xylol, toluol, or carbon bisulphide, and
leave for two hours, the solution being changed at the
end of the first hour. Before transferring from the
alcohol to the xylol or chloroform, the pieces of tissue
may be placed in a mixture of these substances. Now
remove from the chloroform, and place into the
paraffin bath in a mixture of equal parts of paraffin

56 JOHNE^S DISEASE

and chloroform or xylol, and at the end of an hour
transfer to pure paraffin for two hours, the paraffin
being changed once. The tissue should then be set in
blocks of paraffin, in the ordinary way, by means of
two L blocks.

The sections should be cut as thin as possible —
about 4 fjb — a good microtome, such as a Cambridge
rocker, being necessary for this purpose. It is best
to cut the bowel in a transverse direction at right
angles to the surface, in order to obtain a clear view
of the villi and their relation to the deeper tissues.
Sections, after being treated with xylol and absolute
alcohol, are stained by Gram's method, or, better, with
carbol-fuchsin and a contrast stain such as methylene
blue (Ziehl-Neelsen's method). If stained by the
latter method the bacilli are coloured a bright red,
in contrast to the tissues, which are coloured blue.
Histological changes can be observed better if the
sections are stained with haematoxylin and eosin.

We have seen that on post-mortem examination the
macroscopical appearances of the gut which are
especially characteristic are the general thickening
of the organ and the great prominence of the corruga-
tions. There is no caseation, and no sign of any
ulceration or fibrosis of the tissues, and although in
some cases vascular congestion may denote a certain
amount of inflammation, this is by no means always
present. Thus the post-mortem appearances of the
gut lead one to assume that Johne's bacillus does not
give rise under natural conditions to an acute in-
flammation, and microscopical examinations of both
early and advanced cases confirm this view.

As we have seen, the alimentary tract may be
affected from the duodenum to the margins of the
anus, and Johne's bacillus may be found within these

PLATE III.

Phoio bv F. E. Baniani, London.

SECTION OF ILEUM OF COW AFFECTED WITH

JOHNE'S DISEASE.

(jNIag-nification about 250 diam.)

The masses of bacilli in the mucous and submucous membranes
are black in the plate.

[To face page 56.

PATHOLOGICAL HISTOLOGY 57

limits. Sections of badly-affected bowel, stained with
carbol-fuchsin, decolorized with 25 per cent, sulphuric
acid, and counter-stained with methylene blue, may
contain so large a number of bacilli that the section
presents a magenta colour to the naked eye. As a rule
the number of bacilli present varies directly with the
amount of pathological change seen microscopically,
and thus they are usually most numerous in and
around the ileo-caecal valve, and it is in this situation
that the most marked histological changes are found.

In the small intestines the villi are seen to be swollen
and to have a club-shaped appearance, while many of
them are flattened on the surface and are stunted.
They often contain a large number of clumps of bacilli,
mostly at the apex and base, while there is a cellular
reaction consisting of epithelioid and round cells and a
few giant cells. The bacilli are usually most numerous
between the glands and in the lymphoid tissue forming
the solitary follicles, but in advanced cases the sub-
mucous tissue may be extensively invaded ; the bacilli
are, for the greater part, in dense clumps, and are both
intracellular and extracellular, while they are some-
times found in the characteristic wreath formations.
We have not often met with this wreath-like forma-
tion of the bacilli, and consider that it is less frequent
than is generally supposed. The bacilli seem to spare
the gland cells for a long time, as even in advanced
cases many of the glands appear to be fairly normal,
containing, perhaps, an isolated cell here and there full
of bacilli ; however, the glands may be atrophied to a
certain extent, presumably by the mechanical effect
of the hyperplasia of the adjacent connective tissue.
Between the glands, and deep down in the solitar}^
follicles, there are usually a large number of epithelioid
and lymphatic cells and a few giant cells. The last-

58 JOHNKS DISEASE

mentioned are less frequently found in the intestines
than in the lymphatic glands.

Johne and Frothingham, in their original paper,
remarked on the scanty number of typical giant cells
compared with the quantity usually found in bovine
tuberculosis. The increase in thickness of the gut is
thus seen to be due to the infiltration of new cells,
which are chiefly of an epithelioid type. In advanced
cases, in which very many bacilli are present, the
normal tissue may be extremely atrophied, while the
fibrous tissue stroma remains with an infiltration of
epithelioid cells. In cases in which the submucosa is
affected, it is found to be thickened generally, while
the muscularis mucosae is always easily distinguish-
able. The muscular and serous layers of the organ
are normal.

Various authors, including B. Bang, have stated
that there is some necrosis with desquamation of the
epithelium. Bang has since pointed out that this is a
post-mortem change, and is not seen in sections of
fresh gut that have been hardened in a solution of
formalin immediately after the animal is killed.
Nodules similar to those found in tuberculosis are
rare, and it is probable that true caseation is never
found in this situation. In at least loo carefully
examined sections, Johne and Frothingham were able
to find only three sites in which there was any simi-
larity to nodules of tubercular disease. These nodules
consisted of epithelioid cells and lymphocytes, with
one or two giant cells containing bacilli. In this con-
nection the possibility of coincident tuberculosis must
be remembered. In the large intestine the condition
found is more or less similar to that in the small
intestine, but the lesions are usually not so far advanced
and the number of bacilli present is less.

PLATE IV.

Photo by F. E. BaniaicL London.

SECTION OF ILEUM — SAME AS PLATE III.— SHOWING
THE LARGE MASSES OF BACILLI IN THE MUCOUS
MEMBRANE.

(Magnitication about looo diam.)

\ To /ace page 5^-

PATHOLOGICAL HISTOLOGY 59

The extent of the alteration in the bowel seems to
bear little, if any, relation to the intensity of the clinical
symptoms that are shown by the animal. B. Bang
states that in cases in which the clinical symptoms are
very marked there is often only a slight thickening of
the mucous membrane, and sections show but a
trifling infiltration with epithelioid cells. The extent
of the pathological changes found in the mesenteric
and colic lymphatic glands is usually proportional to
the extent of the lesions in the gut; in some cases,
however, this is not so, and the glands may be but
little affected in a fairly advanced case. The extent of
the glandular lesions varies considerably in different
cases, and in an advanced stage of the disease the
condition of the glands may be oedematous, or may
show a considerable destruction of the normal lym-
phatic tissue.

The usual change found microscopically is an
oedematous condition of the organ, the intracellular
connective tissue of which is prominent ; while the
lymphatic cells, instead of being closely packed, as in
a normal gland, are somewhat loosely arranged. This
is especially marked in the centre of the gland. In
many cases no other lesions can be found, and the
presence of bacilli, even after a careful search, cannot
be demonstrated. In these simple cases, however, it
is usual for a small number of bacilli to be present.
Sometimes the glands are markedly changed, and
numerous epithelioid and giant cells, accompanied by
masses of acid-fast bacilli, are found.

It is well to remember that, if an animal dies or is
killed shortly after the inoculation of a diagnostic
vaccine, microscopical examination of the organs shows
an intense congestion of the tissues, especially in the
lymphatic glands. This may give rise to an entirely

60 JOHNE'S DISEASE

erroneous conception of the original pathological
condition.

The congested vessels of the intestines may take up
quite a large proportion of the microscopic field ; not
only is this so of the submucosa, but also, to a less
extent, of the mucous layer. The rest of the organs,
with the exception of the liver, appear to be free from
bacilli and histo-pathological lesions. In some cases
the liver has presented a fatty appearance; but in
naturally affected cases we have never been able to
find bacilli in this situation, and the condition does
not appear to have been recorded by other workers,
except by M'Fadyean, Sheather, and Edwards in one
cow, and by Stockman, who noted it in an affected
sheep. In this connection, the effect of Johne's
bacillus on the liver of rabbits, which is described in
Chapter IX., is interesting.

The histo-pathological changes found in the in-
testines of sheep are more or less identical with those
found in bovines. In 1908 Vukovic found cases of
Johne's disease in sheep in Bosnia, and he was kind
enough to send us a paraffin block of the intestine of
one of these animals. Very large numbers of acid-fast
bacilli were found, the majority being situated in the
villi. The general condition of the intestine was
similar to that described above in the cases reported
by Stockman and by M'Fadyean.

In our experimentally inoculated sheep the liver
showed a small round-celled infiltration, and the gland
cells of the organ stained badly.

From what has been said it is clear that the patho-
logical condition caused by Johne's bacillus is strilcingly
different from that found in tubercular disease that has
existed for any length of time, although, as M'Fadyean
points out, the disease in the early stages is really very

PATHOLOGICAL HISTOLOGY 61

similar to tuberculosis. The absence of caseation (and
consequently of ulceration) is one of the most im-
portant differences, and, indeed, cellular degeneration
can rarely be recognized, except that the nuclei of the
cells may lose to a certain extent their power of stain-
ing. The general diffusion of the lesions and the
absence of any fibrous -tissue capsule are also dis-
tinctive features ; but perhaps the most prominent
difference between this disease and tuberculosis is to
be found in the number of causal micro-organisms
present. Owing to the comparatively slight effect of
a large number of Johne's bacilli on the surrounding
cells, many authors have contended that in all proba-
bility Johne's bacillus does not secrete toxins in the
same way as the tubercle bacillus does. For this
and several other reasons, which are discussed in
Chapter IX., we agree with this view; but at the
same time it may be mentioned that in animals that
do not suffer naturally from the disease, and in which
the bacilli do not appear to be able to multiply, the
caseous peritoneal glands are indistinguishable from
tubercular glands macroscopically, and only differ
microscopically as regards the number of bacilli and
a slighter degree of caseation. This question is con-
sidered more fully in Chapter IX.

CHAPTER VI

DESCRIPTION OF JOHNE'S BACILLUS— CULTIVATION

OF THE BACILLUS— NATURE OF THE ** ESSENTIAL

SUBSTANCE "—DESCRIPTION OF CULTURES

Description of Johne's Bacillus. — The specific bacillus
of pseudo-tuberculous enteritis, commonly known as
"Johne's bacillus," belongs to the acid-fast group of
bacteria, and is allied to the various tubercle bacilli.
According to the classification of micro-organisms
adopted by Lehmann and Neumann, it would be more
correct to describe it as a mycobacterium, and the
scientific name of the micro-organism would then be
Mycobacterium enteritidis chronicce pseudoiuberculosce
bovis Johne, the name by which we have suggested
it should be known. At the same time, in English
literature it is usually referred to as "Johne's bacillus,"
and in Continental countries as the " paratuberkel-
bazillus," or bacillus of pseudo-tuberculous enteritis.
In the lesions of the disease the bacilli often appear
in extremely large numbers. They are present as
slender rods, sometimes slightly bent, and are usually
between i and 2 a* in length. Sometimes they show a
beaded appearance, but this is not so marked as in
the tubercle bacilli. Their size and appearance are
such that it is not possible to detect any pecuHarity by
which they can with certainty be differentiated from
tubercle bacilli (see Plate V.).

62

PLATE V.

Plioto by F. E. Bciniard, London.

FILM FROM PURE CULTURE OF JOHNE'S BACILLUS
ON GLYCERINE - SALINE - TIMOTHY - GRASS
BACILLUS-EGG MEDIUM.

(Magnification about looo diam.)

[ To face page 62.

DESCRIPTION OF JOHNE'S BACILLUS 63

When first cultivated from the animal body on any
of the special media (media containing the dead bodies
of other acid-fast bacilli), the bacilli may grow longer
and thicker, and lie side by side in a manner very
similar to the tubercle bacillus. This is especially so
if the medium is a little dry or old, or for some reason
is not particularly good. On such media they ma}^
attain a length of 4 /x or more, and may show
definite dichotomous branching, with club formation,
and very distinct beading. When subcultured on to
moist and good media, the bacilli soon regain their
slender and short form, and in vigorous growing
cultures, especially if fluid media is used, they may
show little or no beading, and may become very short,
appearing almost like cocci.

M'Fadyean, Sheather, and Edwards have noticed
"numerous bacilli of quite exceptional length" in
smears made from a piece of rectal mucous membrane
taken from an animal intra vitam. The dimensions ot
these bacilli are not given, but they are stated to have
been much above the average length of Johne's bacillus,
and arranged in bundles, in which all the bacilli were
approximately parallel. Cultures were made from the
tissue on to ^g^ medium containing dead tubercle
bacilli, when the bacilli grew ; but on ordinary Dorset's
^gg medium no growth took place. These authors have
not observed any definite branching of the bacilli.

Spore formation has not been present in any of our
strains, and in none of our cultures have we been able
to detect any evidence of motility. In these respects
Johne's bacillus agrees with the other members of the
acid-fast group.

Holth states that in morphology and staining
characters his strain of Johne's bacillus agrees with
the description given in our original paper.

64 JOHNFS DISEASE

As has already been mentioned, Johne's bacillus
belongs to the acid-fast group, and is best stained by
the method known as Ziehl-Neelsen's. For this pro-
cess the following solutions are required :

Carbol-Fuchsin. — Fuchsin, i gramme ; "Absolute
alcohol, 10 c.c. Dissolved and added to 90 c.c.
of a 5 per cent, aqueous solution of carbolic
acid.

Sulphuric Acid. — A 25 percent, aqueous solution.

Loeffler's Methylene Blue. — Saturated alcoholic
solution of methylene blue, 30 c.c, mixed with
caustic potash solution (i to 10,000 of water), 100 c.c.

The simplest way of staining by this method is to
spread a thin film of the suspected material (scraping
of the faeces or intestine) on a slide, and dry by gently
heating. Then boil a little of the carbol-fuchsin solu-
tion in a test-tube, and pour sufficient on the slide to
completely cover the film (in place of carbol-fuchsin
carbol-gentian violet may be used). To insure that
the film remains covered, the slide must be left on a
flat bench, and should be stained for three or four
minutes. The stain must then be washed off with
water, and the film treated with sulphuric acid solu-
tion, the washing and acid treatment being repeated
until no more of the magenta colour can be extracted.
After washing with water, the film should be counter-
stained with the methylene blue solution, and this
washed off after a few seconds. The film should now
be dried, and examined under a yV oil-immersion lens ;
it is not necessary to use a cover-glass.

In the case of films made from faeces, it is not easy
to extract all the fuchsin from certain portions of the
undigested food, but this in no way interferes with
the recognition of the bacilli, if present. By this

DESCRIPTION OF JOHNE'S BACILLUS 65

method of staining, Johne's bacillus is coloured a
bright magenta and the rest of the material blue.

When staining films made from pure cultures,
counter-staining with methylene blue is not necessary.

Johne's bacillus may also be stained by Gram's
method, and is, therefore, Gram- positive. In this
method films of bacilli are treated with hot aniline-
gentian violet for three or four minutes ; the stain is
then poured off, and Lugol's iodine solution floated
over the surface of the film, and allowed to remain for
about a minute ; the films are then treated with abso-
lute alcohol until no more colour can be extracted. If
examined under the microscope, the bacilli are found
to be stained a blackish-violet colour.

From what has been said it is clear that in morph-
ology and staining reactions Johne's bacillus agrees
with the various varieties of tubercle bacilli. It shows
no character that cannot be met with under certain
conditions in the tubercle bacilli.

Cultivation of the Bacillus. — Johne and Frothingham,
after making their original discovery of the presence
of acid-fast bacilli in the intestinal mucous membrane
obtained from cases of pseudo-tuberculous enteritis,
attempted to cultivate the bacilli outside the animal
body. As a medium they used glycerine-agar, expect-
ing to obtain cultures of some variety of the tubercle
bacillus. Their cultures, however, remained sterile,
and on microscopic examination they found no evi-
dence of multiplication of the bacilli. In 1904, Stuur-
man obtained, from a case of Johne's disease at Leyden,
a pure culture of an acid-fast bacillus, and again, in
1908, from the inguinal gland of a guinea-pig inoculated
with a small piece of the intestine of an animal affected
with Johne's disease he obtained another culture,
which showed characters similar to the first. These

66 JOHNE^S DISEASE

cultures, which were isolated on glycerine-serum^
glycerine-agar, and other media, some of which con-
tained decoctions of the grass Phlemn p7^ate7ise, were
submitted to Koch and Rabinowitsch, who declared
them to be avian tubercle bacilli. Inoculation experi-
ments with these cultures failed to produce Johne's
disease, but caused lesions in the small laboratory
animals which proved them to be the avian type of
tubercle bacillus. Mettam, in a private communication,
states that he has also isolated a culture of the avian
tubercle bacillus from a case of Johne's disease. We
consider these results to be secondary infections with
the avian tubercle bacillus. This view is supported
by a large number of authorities, including Bang,
Miessner, MTadyean, Markus, and Meyer, who, pre-
vious to the year 191 1, experimented with infected
material from cows, and failed to obtain cultures of the
specific bacillus. It will be shown, also, that when the
bacillus has been isolated on the special medium, it
can be subcultured on ordinary laboratory media only
after prolonged cultivation and acclimatization outside
the animal body, which proves definitely that the
bacillus is not a variety of the avian tubercle bacillus.
In 1908, Bugge and Albien published a short note to
the effect that they had succeeded in obtaining a pure
culture of Johne's bacillus {Paratuberkelbazillen) from
the mesenteric gland of an affected animal. No par-
ticulars as to the medium were given in this paper;
but in 1910, Albien published a further note giving the
constitution of the medium used. This was as follows :

Nahrstoff Heyden 5-10

Agar I0-20

Glycerin 20-30

Kochsalz ^

NormallQsung von Kristallsoda (28 : 100) ... 5

Aqua dest 1,000

CULTIVATION OF THE BACILLUS 67

Hesse, in 1899, first tested Ndhrstoff Heyden, which
is a preparation of Qgg albumen, for the cultivation of
the tubercle bacillus. On the medium given, Albien
states that he obtained a growth on eight tubes out
of several hundred inoculated ; but the cultures were
not tested on calves, and no confirmation of the results
has been published. Moreover, our own attempts to
isolate and grow Johne's bacillus on such a medium
have given uniformly negative results.

In 1909, Melvin of America stated that he had
obtained a luxuriant growth of an acid-fast micro-
organism from a case of chronic bacterial dysentery
(Johne's disease) occurring in Oregon. The culture
was made on an egg medium, but the bacillus was
overgrown by a saprophytic micro-organism, and no
subculture could be made. As it is stated in the
Report of the Bureau of Animal Industry for 1910 that
attempts to grow Johne's bacillus had failed, it seems
clear that the acid-fast bacillus grown was not Johne's
bacillus, and we know from more recent experiments
that the bacillus gives no growth on a simple egg
medium.

Early in 1910 we started some experiments with the
object of cultivating Johne's bacillus, and of preparing
a diagnostic vaccine from the culture obtained. A
prehminary note on the results of this work was
included in a paper by one of us on the cultivation of
the lepra bacillus of man, published in 1910. In June
of that year we obtained from Mr. De Vine, of Birming-
ham, a specimen of the bowel and mesenteric glands
of a cow suffering from Johne's disease. The ileum
showed well-marked lesions of pseudo-tuberculous
enteritis, and films made from the gut revealed the
presence of a large number of acid-fast bacilli. The
specimen was fresh, and cultures were made

68 JOHNE^S DISEASE

immediately on to all the ordinary laboratory media.

In our experiments the diseased tissue was placed

either directly on to the medium to be tested or after

previous treatment with a i per cent, watery solution

of ericolin to kill contaminating micro-organisms. The

method of making direct cultures was as follows :

The gut and glands were thoroughly washed with

water, and the surface of an infected area seared with

a hot spatula ; microscopic films were made from the

tissues beneath the part seared to prove the presence

of the specific bacillus. Small pieces of tissue were

then removed with sterile scissors and rubbed over the

surface of the culture medium to be tested. If the

tissues were contaminated, the indirect method of

cultivation was adopted — i.e.^ the tissue was previously

placed in a sterile i per cent, watery solution of

ericolin and heated for an hour at 37° C, after which

the particles of tissue were removed and rubbed over

the media to be tested. The ericolin was used to kill

off most of the contaminating micro-organisms, for, as

has already been pointed out by one of us (F. W. T.),

the acid-fast group of bacilli are but little affected by

the action of this substance.

In the first instance we used as media unheated
extracts and tissues of normal cattle. The fresh
extracts of glands and organs, including the intestines,
were made and sterilized by passing through a
porcelain filter. These extracts were placed in sterile
tubes, and each was tested as a medium per se, and
also added in various proportions to the ordinary
laboratory media. Small sterile portions of bovine
organs, especially lymphatic glands, were also obtained
and placed in sterile tubes. All these media were
tested in various combinations, with and without
glycerine, cholesterine, various sugars, fresh blood,

CULTIVATION OF THE BACILLUS 69

and other substances. The cultures were placed in
an incubator at 39** to 40° C. On none of these media
could any definite growth of the bacillus be detected.

With the object of testing for the possible presence
of an ultramicroscopic virus working in symbiosis
with Johne's bacillus, we prepared an extract of the
infected intestine of a cow suflfering from Johne's
disease and passed it through a Doulton white filter.
The sterile filtrate so obtained was added to various
media, and the whole series inoculated with small
portions of intestine affected with Johne's disease.
These media all gave negative results.

From the experiments conducted on this case we
came to the conclusion arrived at by most other
workers, namely, that the specific bacillus would not
grow on any artificial medium known to bacteriologists,
and that if successful cultivation were to be achieved,
some new medium would have to be prepared. We
considered also that the failure of growth of the specific
bacillus must be due, either to the poisonous action of
some substance in the medium, or to the absence of
some foodstuff or stimulant necessary for its vitality
and growth.

At the same time we were struck by the apparent
close relationship existing between this micro-organism
and the tubercle bacillus ; and as the bacillus of pseudo-
tuberculous enteritis and the tubercle bacillus both
grow in the same species of animal, we thought it
highly improbable that there could be any substance
in the ordinary laboratory media which would act as a
poison to the one bacillus and not to the other. This
possibility was accordingly excluded, and we were
forced to conclude that the failure to grow the bacillus
must be due to the absence of some necessar}'- food-
stuff.

70 JOHNE'S DISEASE

Considering again the apparent close relationship
between the tubercle bacillus and the bacillus of
pseudo-tuberculous enteritis, and the fact that both
these bacilli live in the bodies of bovines, we judged it
probable that they would require the same chemical
substances for building up their protoplasm, certain of
which substances could be elaborated from artificial
media by the tubercle bacillus but not by the bacillus
of pseudo-tuberculous enteritis — in other words, that
the latter bacillus has lived a pathogenic existence from
such remote ages that it has lost the original power of
its wild ancestor — whatever bacillus that may have
been — and can no longer build up all its necessary
foodstuffs outside the animal body.

It was thought probable that if these substances
could be obtained ready formed, and were added to
some good artificial medium (Dorset's egg medium) the
bacillus would grow, and, further, that these substances
might be elaborated by allied micro-organisms such as
the tubercle bacillus, and even stored up as reserves in
their envelopes. On this reasoning, which led to a
partially successful cultivation of the lepra bacillus of
man, we decided to prepare media containing these
allied bacilli, which had been killed by heat.

We had, at the time, in our possession about three
hundred strains of tubercle bacilli, mostly isolated from
human tuberculous material on Dorset's egg medium.
Some of these cultures were taken, and after the
necessary subcultures had been made, they were killed
by steam. The growth was then scraped off, care being
taken to avoid any admixture of the medium which
might contain the waste products of bacillary growth
and be toxic to the bacillus of pseudo-tuberculous
enteritis ; more recently we have found this precaution
to be unnecessary. The growth of tubercle bacilli thus

CULTIVATION OF THE BACILLUS 71

obtained was ground in a mortar with glycerine and
saline, steamed for half an hour, and added to the white
and yolk of new-laid eggs in the following proportions :
^SSt 75 parts; oS per cent, sodium chloride in re-
distilled water, 25 parts. These were thoroughly
mixed, and to the mixture were added tubercle bacilli
I per cent, and glycerine, 5 per cent. The medium was
placed in sterile test-tubes; these were plugged with
cotton-wool, and heated in a hot-water bath at 60° C.
for one hour on three successive days, the tubes being
incubated at 37° C. in the intervals between steaming.
Finally the tubes were inspissated in slopes at 85°
to 90° C.

A second case of pseudo-tuberculous enteritis was
now obtained from Mr. De Vine. Specimens of in-
testine and glands were received on July 28, 1910.
Both the intestine and glands showed the typical
characters of the condition, and large numbers of
Johne's bacilli were present in various parts of the
tissues. Unfortunately, owing to the hot weather pre-
vailing at the time, the specimens on delivery had
commenced to decompose; but, in spite of this, we
prepared some cultures in the manner previously
described, both directly, and indirectly after treating
with ericolin solution. The cultures were made on
several of the media tested with the first cases, as well
as on a number of tubes of the special tubercle bacillus
medium. The tubes were capped with gutta-percha
tissue, and incubated at 39° to 40° C. After two days'
incubation all the direct tubes were badly contami-
nated, yet those inoculated with ericolinised material
showed only a few contaminating colonies. Sub-
cultures were made from uncontaminated areas of
most of the latter tubes on to fresh tubes of the same
medium ; but, owing to the small amount of the

72 JOHxNE'S DISEASE

tubercle bacillus medium then prepared, only one of
these tubes was subcultured. This one was made from
a gland. Films from these subcultures were examined
microscopically at intervals of about four or five days.
After nineteen days the subculture on the special
medium showed quite definite evidence of multiplica-
tion ; the bacilli had grown larger and thicker ; they
were well stained, and were present in large, close
masses. Subcultures were made from this tube on
to fresh tubes of various media, including one tube of
the special tubercle bacillus medium. These were ex-
amined at intervals as before, and the subculture on
the special medium showed microscopic evidence of
growth in ten days. Both the first and second sub-
cultures showed growth visible to the naked eye after
four weeks, and this gradually increased, reaching a
maximum in about eight weeks.

These tubes were easily subcultured on to fresh
tubes of the same medium, but on none of the ordinary
laboratory media were we able to get any evidence of
growth.

The third case of pseudo-tuberculous enteritis was
obtained from Mr. Hamilton. Specimens of intestine,
but no glands, were received on September 23, 1910.
They showed the typical lesions of the disease, and
a very large number of Johne's bacilli were present
in the tissues. When delivered, the specimens had
already commenced to decompose; but from them
cultures were made as previously described, both
directly, and indirectly after treatment with ericolin
solution on various media, including tubes of the
special tubercle bacillus medium. The tubes were
capped with gutta-percha tissue, and placed in an
incubator at 39° to 40° C. The results were the same
as in Case 2 ; all the direct tubes were badly contami-

CULTIVATION OF THE BACILLUS 73

nated, and those tubes which had been inoculated
with material previously treated with ericolin solution
grew only a few contaminating colonies. Of the
latter the cultures on the tubes of special medium
were subcultured from uncontaminated areas on to a
number of fresh tubes of various media, including the
special medium. The subcultures on the ordinary
media remained sterile, but those on the tubercle
bacillus medium grew Johne's bacillus in pure growth,
and were without difficulty subcultured on to fresh
tubes of the special medium. Naked-eye evidence of
growth was present in the first subcultures after about
six weeks.

The fourth case was obtained from Mr. De Vine, a
specimen of the intestine being received at the In-
stitution on January 26, 191 1. It showed the typical
lesions of pseudo-tuberculous enteritis, and a large
number of the specific bacilli were present in the
lesions. Since the specimen was quite fresh, cultures
were made, as previously described, from the ileum,
caecum, and ileo-caecal valve, directly on to nine tubes
of the special tubercle bacillus medium ; these were
capped, and placed at 39° to 40° C. After three weeks'
incubation two tubes were found to be contaminated,
whilst the remainder were covered with extremely
minute colonies of Johne's bacillus, and showed no
contaminations. The cultures grew well, and were
subcultured without any diflficulty on to the special
medium. Subcultures made on to Dorset's egg
medium, glycerine-agar, and various other media gave
no growth.

Case 5 was obtained from Mr. Hamilton, and was
received at the Institution on February 8, 191 1. The
specimen, consisting of ileum and ileo-caecal valve,
showed the typical lesions of pseudo-tuberculous

74 JOHNKS DISEASE

enteritis, and a considerable number of acid-fast bacilli
were present in the lesions. Cultures were made
from several parts of the specimen directly on to
twelve tubes of Dorset's egg medium. They were
taken in the manner already described, but as the
specimen was fresh on arrival, previous treatment with
ericolin solution was unnecessary. The tubes were
capped with gutta-percha tissue, and placed in the in-
cubator at 39° to 40° C. On the following day they
were examined, and found to be free from contami-
nating colonies, so the tiny pieces of tissue were
removed from three of these tubes and placed on to
three tubes of the special tubercle bacillus medium.
These were capped, and placed in the incubator with
the other tubes at 39° to 40° C. Six weeks later the
three tubes of special medium showed a few tiny
colonies of Johne's bacillus. Compared with the pre-
vious cases the rapidity of growth was very slow, and
was slight in amount, due, as was proved later, to the
unsuitability of the particular strain of tubercle bacillus
incorporated in the medium. Subcultures from these
tubes on to tubes of a fresh batch of tubercle medium
grew well. All the original cultures on Dorset's egg
medium remained sterile, as also did subcultures from
the special medium on to Dorset's egg medium.

The experiments with these cases (2 to 5) prove
definitely that it is possible to isolate from the intes-
tines of cattle suffering from Johne's disease an acid-
fast bacillus which will grow on a medium contain-
ing the dead bodies of human tubercle bacilli, but
will not grow on any of the ordinary laboratory
media.

Slight modifications in the constitution of the medium
were now made, and it was found that media containing
about 4 per cent, of glycerine yielded the best results.

CULTIVATION OF THE BACILLUS 75

Growth, however, took place with lo per cent, of
glycerine, and in the presence of a much higher per-
centage of salt; but in the complete absence of glycerine
growth was not nearly so good. It was found better
to dry the tubercle bacillus after killing it, and before
making it up into medium. This was done in vacuo
over caustic potash. The improvement in this case
may have been due to the formation of breaks in the
continuity of the covering of the bacillus, thus allowing
the " essential substance " to diffuse more easily into
the medium. Of the dried bacilli, J to i per cent,
was found to be sufficient to add to the ^gg medium.
Alterations in the normal alkalinity of the egg medium
produced no good results. The bacillus seems to
prefer a slightly alkaline medium.

In a further series of experiments the dead tubercle
bacilli were added to ordinary glycerine - agar and
glycerine-broth, but these media did not, as a rule,
give such good results, though glycerine-peptone-beef
broth, containing J to i per cent, of dried tubercle
bacilli, gave a fairly satisfactory growth. In the case
of the subcultures on the agar medium, growth occurred
along the needle-track as small, white, elevated colonies,
which at first were smooth and discrete, but after six or
eight weeks they coalesced and presented an irregular
surface. When older, the growth turned a dull yellowish-
white, and in tubes with a single colony this spread
and became more heaped up in the centre, while the
margins were thinner and irregular. The growth,
although slow, was very similar to that of the tubercle
bacillus. The growth in the broth subcultures occurred
as tiny granules at the bottom of the flask, and these
little masses of bacilli increased in size and number,
though very slowly. At the end of six months one
flask showed a very thin film on the surface of the

76 JOHNE'S DISEASE

medium. This slowly increased, spreading over the
surface of the medium and becoming more heaped
up. The colour was a dull white, slightly tinged with
yellow. These broth cultures were used for preparing
our first series of diagnostic vaccines.

We now proceeded to test our strains of Johne's
bacillus on media in which the dead tubercle bacillus
was replaced by various other micro-organisms. We
soon found that some strains of human tubercle bacilli
were more suitable than others; and, further, that if
the human tubercle bacillus was replaced by the bovine
type, no growth of Johne's bacillus took place, and that
this was so even when subcultured from strains that
had been growing outside the animal body for a year.
Several strains of tubercle bacilli isolated from cats
were also tested, but gave negative results. The cat
strains were distinctly bovine in character, and, as was
the case with the other tubercle bacilli, they were
grown on Dorset's egg medium. (A discussion of
these results and some details of further experiments
with the bovine strains are given later.)

In view of Johne andjFrothingham's original sugges-
tion that the bacillus of pseudo-tuberculous enteritis
was either an avian tubercle bacillus or some variety
of it, and the support this view received from the re-
actions to avian tuberculin obtained by Bang in animals
suffering from Johne's disease (vtde Chapter VII.), the
avian tubercle bacillus naturally suggested itself for
incorporation in media. Two strains, therefore, were
tested ; one was obtained from Krai, and the other
isolated by ourselves from a natural case of tubercu-
losis in a hen. The bacilli were grown on large tubes
of Dorset's egg medium, and when the growth had
reached a maximum it was killed, scraped off, dried,
and made up into medium as already described for the

CULTIVATION OF THE BACILLUS 77

human type. On this medium our strains of Johne's
bacilli usually grew, but only to a slight extent, and
the medium proved to be quite unsuitable for practical
purposes.

From the fact that since the remotest times the
timothy-grass bacillus {Bacillus phlei Moeller) found
on Phleum pratense must have been continually in-
gested by ruminants in their food, the possibility of
this bacillus being the wild ancestor from which Johne's
bacillus has arisen is at once obvious, and if such be
the case one might expect, from what has been already
said, that B. phlei would be a very suitable variety to
use in preparing the medium. To the Qgg medium,
therefore, this bacillus, grown on ordinary broth, was
added to replace the human tubercle bacillus, J to i per
cent, of the dried bacillus being used. Johne's bacillus
on this medium grew quickly and well, the growth
being better than on any of the media so far tried.
A slight growth was visible along the needle-track
in one week, and in six weeks the growth closely
resembled that of a bovine tubercle bacillus freshly
isolated from the animal bod}^

In exactly the same way other acid-fast bacilli were
tested — i,e.^ incorporated, when dead and dried, in
Dorset's egg medium. The following were experi-
mented with :

1. The smegma bacillus of Moeller.

2. The nasenschleim bacillus of Karlinski,

3. The fish tubercle bacillus of Dubard.

4. The butter bacillus of Rabinowitsch.

5. Bacillus from urine, Marpmann.

6. Pseudoperlsucht bacillus of Moeller.

7. Bacillus from butter, Grassberger.

8. Three of the Tobler group of acid-fast bacilli;
Tobler, I., IV., and VI., Krai.

78 JOHNE^S DISEASE

Johne's bacillus, when inoculated on to media con-
taining Nos. I, 2, 5, 6, and 7, gave a good growth ; but
on the rest growth was usually absent, though a very
slight film was occasionally present on the media con-
taining some of the Tobler varieties.

A number of blastomyces, streptothrices, cocci, and
bacilli that are not acid-fast, were also tested, but no
growth was obtained on media containing any of these
micro-organisms. In all such experiments we used
strains of Johne's bacillus that had been isolated on
the tubercle bacillus-egg medium, and that had been
growing outside the animal body for a considerable
time, and it was thought that the good growth ob-
tained with B, phlei medium might be due to the fact
that our strains were becoming acclimatized to arti-
ficial culture media. To test this point we made
primary cultures on to a B. phlei medium. A further
specimen of diseased gut was obtained through the
kindness of Mr. De Vine. In this case the disease was
in a very early stage, and the thickening of the intes-
tines was only quite moderate. Films were made from
the ileum and ileo-caecal valve, but only a very few
acid-fast bacilli could be found after a prolonged search.
Cultures, however, were made from the regions show-
ing most bacilli, on to tubes of Dorset's egg medium,
and also on to tubes of B, phlei medium. After five
weeks' incubation there was no evidence of growth
on Dorset's egg medium, but on the B. phlei medium
definite evidence of multiplication was observed.
Subcultures from the B. phlei medium on to fresh
tubes of the same medium grew well, as also did those
on a medium containing the human tubercle bacillus,
but on ordinary laboratory media there was no growth.

The bacillus isolated resembled in every way the
bacilli isolated from the four previous cases, and the

CULTIVATION OF THE BACILLUS 79

cultural characters were also the same. More recently
we have isolated Johne's bacillus on media containing
some of the other acid-fast bacilli mentioned above
— i.e.^ Nos. I, 2, 5, 6, and 7.

From these experiments it is clear that Johne's
bacillus will grow on media containing the dead
timothy-grass bacillus, or bacilli Nos. i, 2, 5, 6, or 7,
not only after it has been cultivated in the laboratory
for a considerable period, but also when taken direct
from the diseased gut of cattle.

Having determined the various acid-fast bacilli most
suitable for the growth of Johne's bacillus, we at-
tempted to extract the " essential substance " (the
substance necessary for the growth of Johne's bacillus)
from certain of these bacilli. The timothy - grass
bacillus was chosen, chiefly because it gave the best
results in the experiments with the whole bacilli, also
because it is harmless to man, and grows quickly on
simple media, thus enabling a large quantity to be
obtained in a short time.

For this purpose Dr. W. Bulloch kindly gave us a
quantity of this bacillus, besides various dead and
dried tubercle bacilli, which had been given to him
by Professor Bang about eight years previously.
Many of these had already been extracted by Bulloch
and Macleod when investigating the acid-fast proper-
ties of the tubercle bacillus. In a preliminary series
of experiments the different bacillary powders were
made up into media, the tubercle bacillus of our
original medium being replaced by one or other in
quantities of J per cent. Tubes of each were inocu-
lated with a fresh culture of Johne's bacillus, and the
results may be summarized as follows :

Dried timothy-grass bacillus {B. phlei) gave very
good results.

80 JOHNE^S DISEASE

Dried human tubercle bacillus gave good results,

but inferior to B. phlei.
Dried bovine tubercle bacillus gave negative

results.
Dried swine tubercle bacillus gave negative

results.
Dried tubercle of uncertain source, freed from

wax and fat, gave negative results.
Dried tubercle of uncertain source, freed from

wax, fat, and proteid, gave negative results.

The timothy-grass bacillus and the human tubercle
bacillus were found to be equally good when previously
autoclaved in normal saline for thirty minutes at
120° C. The results proved conclusively that the
"essential substance" contained in these bacilli is
comparatively stable, remaining undiminished in the
timothy-grass bacillus and the human tubercle bacillus
that had been killed and dried eight years previously,
and also after they had been autoclaved. It is inter-
esting to note that the bovine type, when added to
media, again gave negative results, and that the
bacillus of uncertain source, which had been extracted
with acid-alcohol, etc., also failed to give any growth
with Johne's bacillus.

We now took i gramme of the dried B. phlei
powder, and extracted with 20 c.c. of 0*8 per cent,
sodium chloride in redistilled water containing 4 c.c.
of glycerine. The mixture was autoclaved for half an
hour at 120° C, and then filtered. The filtrate was
added to the yolk and white of hens' eggs in the pro-
portion of I part of filtrate to 3 parts of egg. The
residue was washed several times with normal saline,
dried, and made up into medium with egg^ so that
J per cent, of the residue was present. The same
process was followed with another gramme of the

CULTIVATION OF THE BACILLUS 81

B, phlei^ except that redistilled water was used in
place of the glycerine and saline for extraction. We
found that Johne's bacillus grew on the glycerine-
saline extract medium, and on that containing the
residue. It also grew on that containing the residue
after extraction with redistilled water, but it failed to
grow on the medium containing the distilled-water
extract. From this we concluded that the ** essential
substance " is only very slightly, if at all, extracted
by redistilled water, but that it is soluble in a
glycerine - saline solution, although in the above
case it is clear that some of it remained in the
residue.

A further series of experiments was made, using
ethyl alcohol as the solvent. Two grammes of dry,
powdered B. phlei were placed in a Soxhlet
apparatus with loo c.c. of absolute alcohol and ex-
tracted for three hours. The residue was dried in
an incubator, and the alcoholic extract evaporated to
dryness, leaving a dark yellowish, sticky mass. The
extract and residue were then weighed separately,
and it was found that the bacilli were reduced in
weight from 2 grammes to i'25 grammes, the difference
being represented by the extract. (In later experiments
we have extracted very much larger quantities of
various acid-fast bacilli, sometimes using as much as
100 grammes of dry bacilli, and we have found that
the proportion of extract to residue varies consider-
ably, the proportion of extract usually being lower
than that given above.) Media were prepared with
the residue and with the extract thus obtained : some
contained only i or J per cent., and some i per cent.,
of the extract or residue. Tubes of each were inocu-
lated with a young growth of Johne's bacillus and
incubated at 39° C. Good growths were obtained on

6

82 JOHNE'S DISEASE

all the media containing the extracts, but as a rule
there were none on the residues.

These experiments proved that the substance in the
timothy-grass bacillus and in allied bacilli that is
essential for the growth of primary cultures of Johne's
bacillus is extracted by hot ethyl alcohol. As is well
known, if this hot extract is allowed to cool, a yellowish
sticky sediment and a white flocculent precipitate of
wax, etc., form, and can be removed by filtration. The
clear-coloured filtrate, when evaporated to dryness,
leaves a thick oily residue, which becomes firmer on
cooling. Part of this residue is soluble in hot and in
cold chloroform, leaving an insoluble portion, part of
which floats on the surface of the chloroform. This can
be removed by filtration, and, unlike the chloroform
soluble portion, it is found to be readily soluble in water.

Each of these portions was now tested in media,
and all gave positive results with Johne's bacillus, the
best being that which is insoluble in chloroform (see
Plate VI.).

Our researches with regard to media for the cultiva-
tion of Johne's bacillus had reached this stage when
the results were communicated to the Royal Society
(November 5, 191 1), and, with some animal inoculation
experiments and the results of certain vaccine tests
to be discussed later, formed the subject of the Brown
Lectures given at the Royal College of Surgeons early
in January, 191 2.

In May, 191 2, Halfdan Holth, working in the labora-
tories of Professor Jensen in Copenhagen — to whom,
early in 191 1, we had sent cultures of Johne's bacillus
— published an account of experiments with this
bacillus. (Results communicated March 21, 191 2.)

In March, 191 1, from a natural case of the disease in
a Jersey cow which had reacted to avian tuberculin,

Fig. I.

PLATE VI

Fig.

Pholc

I'.rislc!.

CULTURES OF JOHNE'S BACILLUS.

Ym I —Streak culture on an ea:g medium made up with saline and
containing 4 per cent, glycerine and i per cent, of the chloroform
insoluble portion of the cooled alcoholic extract of B. pliki.

Fig. 2.— Isolated colonies on a glycerine-agar medium containing dog's
filtered ascitic fluid and the same extract of B. plila as Fig. i.

Fig. 3.— Streak culture on same medium as Fig. 2.

[To face page 82.

CULTIVATION OF THE BACILLUS 8S

Holth inoculated the following media, on which he
obtained good growths :

(a) Blood-serum (horse) with 4 per cent, glycerine
and 2 per cent, dead tubercle bacilli added. The
bacilli were obtained from a two months' old broth
culture, and, before being added to the medium, were
mixed with a small quantity of salt solution and heated
to 100° C. for one and a half hours. The bacilli were of
the human type.

{b) Blood -serum with a quarter volume of liver
broth, 2 per cent, dead tubercle bacilli, and 4 per cent,
glycerine.

{c) Serum agar with 2 per cent, dead tubercle bacilli
and I per cent, glycerine.

{d) Egg medium as used by ourselves.

The tubes were sealed with paraffin and placed at
37° C, and in six weeks multiplication of the bacilli
was found to have taken place.

Holth found that the best growth occurred on {b).
At the end of six weeks the whole surface of the
medium was covered with colonies visible to the naked
eye and varying in size from J to i millimetre. Sub-
cultures on to other similar media showed good growth
after four weeks. To avoid the presence of tubercle
bacilli in the medium, a glycerine extract was prepared.
The bacilli from the growth on about 200 c.c. of a two
months' broth culture were mixed with about 40 c.c.
of glycerine, and, after shaking, the mixture was heated
for an hour at 100° C. After standing at room tem-
perature for several days, the pale yellowish-brown
liquor was decanted. The addition of 5 per cent, of
this liquid to the media in place of the tubercle bacilli
was found to give good results.

Holth states in his paper that the bacilli cultivated
agreed in their morphology and staining reactions with

84 JOHNKS DISEASE

the cultures obtained from us. In liver broth to which
blood-serum and the glycerine extract mentioned
above had been added, Holth observed, after an inter-
val of about four weeks, a slight growth which
appeared as a sediment at the bottom of the tubes.
This growth gradually increased in amount, and later
formed a film on the surface of the medium. The
surface growth was often observed to be thick and
knobby. Attempts to subculture the bacillus on to
media not containing extracts of tubercle bacilli or
their dead -bodies were unsuccessful, with the possible
exception of serum-agar containing i per cent, of
somatose and i per cent, of Heyden's "Nahrstoff," on
both of which a slight growth might have been
detected.

Regarding the addition of serum to the medium as
advocated by Holth, we found that it did not improve
our original egg medium, so performed no further
experiments on these lines until after the publication
of Holth's paper, when we tested dog's ascitic fluid
with liver broth-agar containing an extract of B. phlei.
The unheated ascitic fluid was added just before
setting the agar, and for one batch the fluid was
first passed through a Doulton porcelain filter. This
series of experiments showed the agar to be improved
by the addition of the fluid, especially when it was
filtered before being added to the agar. With the
addition of blood-serum, however, we did not obtain
such good results, but this may have been due to the
fact that we used rabbit serum, while Holth used
horse serum.

Since Holth's paper, M'Fadyean, Sheather, and Ed-
wards have published similar results. These workers
isolated the bacillus from twenty-three naturally in-
fected cases, and from four experimentally inoculated

CULTIVATION OF THE BACILLUS 85

animals, and this they did on media containing the dead
bodies of acid-fast bacilli, glycerine extracts of such
bacilli, or the products of their growth. These authors
prefer an agar or a serum-agar foundation for their
media as being less troublesome to prepare than
egg media. They were unable to obtain growth on
any of the ordinary media, but they obtained good
growths on liquid media made up of two parts of
ordinary broth containing 5 per cent, glycerine, and
one part of a 5 per cent, glycerine-broth culture
of the B. phlei that had been steamed for two hours,
and then centrifuged to remove the bacilli. Their
cultures on various media are illustrated by excellent
photographs in the Journal of Comparative Pathology^
September, 1912.

Regarding the use of a glycerine extract of the
tubercle bacillus for making media, the authors state :

" As previously stated, at the outset we used
the egg medium first recommended by Twort and
Ingram, and nearly always with success. It is,
however, a medium with some notable defects.
In the first place it is a little troublesome to pre-
pare, especially when perfectly fresh eggs are not
available. Secondly, it is an opaque medium, and
therefore does not allow one to observe the appear-
ance of any growth on it by transmitted light. On
this account peculiarities of growth are apt to be
overlooked, and accidental impurities are not so
easily detected as on a transparent or translucent
medium. Finally, the abundant presence of tuber-
cle bacilli in the substance of the medium is very
objectionable when in doubtful cases one wishes
to determine by microscopic examination whether
any multiplication of the implanted Johne's bacilli
has taken place.

86 JOHNE^S DISEASE

" For these reasons, and quite independently of
Holth's attempts in the same direction, we en-
deavoured to obtain some other medium for the
cultivation of the bacilli.

" As it appeared to be probable that the essential
substance present in the mixture of eggy tubercle
bacilli, and glycerine was something extracted
from these bacilli by the glycerine, it was resolved
to endeavour to obtain a strong glycerine extract
of tubercle bacilli, which when added to agar or
other transparent medium might render that suit-
able for the growth of Johne's bacilli.

" The dead bodies of tubercle bacilli cultivated
on the surface of 5 per cent, glycerine broth were
therefore extracted with hot glycerine, and after
removal of the bacilli the glycerine extract was
added to various media, which were then inoculated
with Johne's bacilli. This immediately yielded
positive results, and enabled us to obtain good
growths on media that were at once transparent
and free from tubercle bacilli.

" For purposes of extraction various strengths
of glycerine were employed, but a better extract
appeared to be obtained with diluted glycerine
than with the pure substance."

In our paper (Royal Society, B. 84, 1912, communi-
cated November 7, 191 1), to which the authors in their
paper have frequently referred, we do not suggest that
media must be made of egg and tubercle bacilli ; various
modifications are discussed, including those mentioned
by M'Fadyean, Sheather, and Edwards. On p. 525 we
stated :

" Experiments showed that ^ to i per cent, of
the dried tubercle bacillus was the most suitable

CULTIVATION OF THE BACILLUS 87

quantity to add. To obtain the best results, the
dried bacilH should be ground up with the glycer-
ine which has been mixed with an equal quantity
of o*8 per cent, saline, and the remainder of the
saline added later. The emulsion so obtained
should then be steamed for fifteen minutes, and,
when cool, added to the Ggg. The probable ex-
planation for this is that the glycerine acts as a
solvent for the essential substance, and some ex-
periments to be described later tend to confirm
this suggestion."

On p. 529 we discussed these later experiments thus :

** Some further experiments were now made :
I gramme of dried timothy-grass bacilli was
taken and extracted with 20 c.c. of o'8 per
cent, sodium chloride and 4 c.c. of glycerine.
The mixture was autoclaved for half an hour at
120° C. and passed through filter-paper. The
filtrate was then added to the white and yolk
of hens' eggs in the proportion of one part of the
filtrate to three parts of egg. Another batch of
medium was prepared by taking the residue of
the timothy-grass bacillus, washing it repeatedly
with normal saline, filtering it, and drying the
residue. This residue was made up into medium,
the tubercle bacillus of the original tubercle
egg medium being replaced by ^ per cent, of the
residue of the timothy-grass bacillus. . . . We
found that Johne's bacillus grew well on the
medium containing the glycerine saline extract
and on that containing the residue."

On p. 525 we also state :

** In another series of experiments the egg was
replaced by various other substances, such as

88 JOHNE'S DISEASE

broth or agar. These, as a rule, did not give such
good results, although ordinary glycerine-peptone-
bouillon, made distinctly alkaline and containing
i to I per cent, of dried tubercle bacilli, gave a
fairly satisfactory growth. This, with other ex-
periments to be described later, proved that
Johne's bacillus can grow quite well in the absence
of albumen."

In the same paper, p. 537, we wrote :

" As has been stated, no growth occurs on any
of the artificial media in general bacteriological
use, such as peptone-bouillon, agar, gelatine, serum,
potato, or eggf even when such substances as
glycerine, sugars, amino-acids, fresh blood, etc.,
are added. It is absolutely essential that certain
previously detailed bacteria or extracts from them
be added to one or other of the media, before any
growth of Johne's bacillus takes place, and this is
equally true for strains of Johne's bacillus which
have been freshly isolated from the animal body,
and for stains which have been cultivated on
artificial media for fifteen months or more."

From the quotations given it will be seen that the
question of obtaining a clear medium and one free
from the bodies of tubercle bacilli, or allied bacilli, was
fully considered in the paper to which we have referred,
and, as we also pointed out, the " essential substance "
in these bacilli can be extracted more efficiently by hot
alcohol than by glycerine if a Soxhlet's apparatus is
used when extracting with the former. In another
part of their paper M'Fadyean, Sheather, and Edwards,
mention some of these experiments.

In their paper {Journal of Comparative Pathology^
vol. XXV., part iii.) M'Fadyean, Sheather, and Edwards

CULTIVATION OF THE BACILLUS 89

also give an account of experiments carried out to
test the relative value of the human, bovine, and
avian types of tubercle bacilli. On pp. 267-268 they
state :

'* Twort and Ingram found that the best growth
v^as obtained on Dorset's egg medium to which
timothy bacilli (bacillus phlei) had been added, but
apparently tubercle bacilli of the human type were
in this respect only slightly inferior. On the other
hand, they obtained little or no growth on media
with which the avian type of tubercle bacillus had
been incorporated, and the results were entirely
negative when bovine tubercle bacilli were em-
plo3^ed.

" Our results are not at all concordant with these.
In comparative experiments we have generally
found that tubercle bacilli of the avian type gave
the best results, and that human bacilli were
slightly superior to the bacillus phlei. The word
'generally' has been used in the preceding sen-
tence because absolutely uniform results are not
obtainable with different lots of media prepared
in the same way, and it has therefore occasionally
happened that no difference could be observed
between parallel cultures according as the medium
contained the one or the other kind of acid-fast
bacillus. The most striking difference of opinion,
however, has arisen in connection with the use ot
tubercle bacilli of the bovine type. As stated above,
Twort and Ingram found this type of organism
useless for the purpose in view, and they appear to
have thought that in this fact they had discovered
evidence contrary to the view that the human and
bovine types are only slightly different varieties of
one and the same organism. We have found that

90 JOHNE^S DISEASE

the bacillus of Johne's disease can be cultivated,
either as primary or as subcultures, on media to
which bovine tubercle bacilli or extracts prepared
from them have been added, and our results would
not justify us in stating that in this respect the
bovine type is inferior to the human type or to the
bacillus phlei. And it ought to be stated particu-
larly that our successes have been obtained with
quite typical bovine baciUi, isolated directly from
bovine lesions by ourselves, markedly dysgonic,
and proved by experiment to be virulent for bovine
animals and rabbits."

The results obtained with the bovine type by these
authors are certainly different from those obtained by
ourselves, and given in our paper in the Proceedings
of the Royal Society and later in the Centralhlatt fur
Bakteriologie. The different results are probably
accounted for in part, at least, by the difference in
the media on which the bovine tubercle bacilli were
grown. Experiments bearing on this point are described
later in this chapter.

The authors (M'Fadyean, Sheather, and Edwards),
however, appear to have misunderstood our remarks
on the relationship of the human and bovine types of
tubercle bacilli. We made an observation incidentally
on this point, and suggested that the differences we
had found were worthy of further investigation. At
the same time we pointed out that we did not consider
that the different results we had obtained with the two
types represented an important biological difference, but
that the difference in the bacilli was probably physio-
logical in nature. Our remarks were :

** Whatever this difference between the two
types of bacilli may be due to, it does not in our

CULTIVATION OF THE BACILLUS 91

opinion necessarily represent an important bio-
logical difference : it is probably physiological in
nature, and may be due to the presence or absence
of some reserve food material existing, or other-
wise, outside the strictly vital portion of the
bacillus, or it may be due to some fat, wax, or
other covering material preventing this substance
from being utilized by Johne's bacillus. In the
light of some recent experiments the latter possi-
bility seems improbable, as we have been unable
to extract any substance suitable for the growth
of Johne's bacillus. These experiments are being
continued.

"While in this paper we cannot enter into the
controversy concerning the relationship between
the human and bovine types of tubercle bacilli,
yet, incidentally, we venture to remark that,
in spite of all that has been written in this
country, we are not yet convinced that the human
and bovine types are only slightly different varieties
of one and the same micro-organism. In this con-
nection the difference between the two bacilli
described above may be worthy of note and
further investigation ;"

In the same paper we wrote :

" We have not tested man}^ strains of the bovine
bacillus, and it is possible that Johne's bacillus will
grow on some bovine strains, or on those strains
which have been described as occupying an inter-
mediate position between the typical human and
typical bovine bacilli."

In describing the results obtained by M'Fadyean,
Sheather, and Edwards, and the results obtained by

92 JOHNE^S DISEASE

ourselves, we have thought it advisable to quote at
some length in order that the reader may fully
appreciate our respective opinions.

Nature of the ** Essential Substance."— Since the

publication of our experiments with media containing
acid-fast bacilli, or extracts obtained from the bacilli,
we have attempted to isolate the substance — the
"essential substance" — contained in these bacilli and
extracts, in the hope that we might be able to throw
some light on its chemical nature. To carry out our
experiments we obtained large quantities of B. phlei
by growing the bacillus on glycerine-peptone-beef
broth for fourteen days at 2>7'^ C. ; the cultures were
then filtered, and the growth washed and dried. We
prepared the alcoholic extract as before, and from
this we attempted by means of various solvents to
separate the " essential substance." In the first place,
the wax was obtained in a fairly pure state by
repeatedly dissolving in hot alcohol, precipitating by
cooling, and filtering. The wax, when added to media,
gave negative results, and need not be considered
further.

The two portions of extract soluble in cold alcohol,
when evaporated to dryness, were extracted with such
solvents as ether, petroleum ether, acetone, and methyl
alcohol, but with very unsatisfactory results, the
essential substance being mixed up with most of the
portions obtained. The experiments took a consider-
able time to carry out, as the only way in which we
could test for the substance was by preparing media
containing the separate portions, and by testing the
growth of Johne's bacillus on the various batches
of media. Usually it was necessary to incubate the
cultures lor at least two months before we could
be certain that no growth of Johne's bacillus had

NATURE OF THE " ESSENTIAL SUBSTANCE " 93

occurred, and, as we have already remarked, in
point of fact some growth took place on most of the
media.

The portion that was insoluble in chloroform, but
soluble in water, always gave a good growth, and
we were able to reduce the amount of essential sub-
stance in the chloroform soluble portion by extract-
ing with water, especially by dissolving it in a little
chloroform and shaking with water in a separating
funnel.

In the case of the bovine tubercle bacillus grown on
Dorset's egg medium, we obtained apparently the
same extracts as with the B, phlei, yet Johne's
bacillus failed to grow on media containing the ex-
tracts of the bovine tubercle bacillus.

To investigate the nature of the "essential sub-
stance," we now carried out experiments to ascertain,
if possible, the general nature of the substance or
substances from which it was formed, and from these
experiments we soon obtained some interesting results.
We experimented with B. phlei grown on various
media, and found that when grown on glycerine-agar,
or glycerine-broth, the bacilli formed a good medium ;
but, on the other hand, when grown on Dorset's egg
medium, the results were not nearly so good, and
when grown on glycerine-liver broth for four weeks
at 37° C, the bacillus was useless for making media
for Johne's bacillus. From B. phlei grown on liver-
broth we obtained apparently the same extracts as
before, but the extracts were unsuitable for making
into media. The same results were obtained, as a rule,
with the other acid-fast bacilli. The human and avian
tubercle bacilli made the best media for Johne's bacillus
when they had been grown on glycerine-beef broth, or
glycerine-beef broth-agar, although the avian type,

94 JOHNE^S DISEASE

even in this case, was not so good as the human
type. We also obtained some positive results with a
bovine strain grown on glycerine-beef broth, but here
again the results were not nearly so good as with
B. phlei.

A similar experiment was carried out with a freshly
isolated strain of Johne's bacillus grown on the
timothy-grass bacillus-egg medium, and with a strain
of Johne's bacillus acclimatized to grow without the
essential substance and grown on ordinary glycerine-
peptone-beef broth. In this experiment a freshly
isolated strain of Johne's bacillus grew on an ^gg
medium in which was incorporated i per cent, of the
dead acclimatized Johne bacilli which had been grown
on the glycerine-beef broth ; but, on the other hand,
it failed to grow on an Qgg medium in which were
incorporated the dead bodies of a freshly isolated
Johne bacillus which had been grown on an Qgg
medium containing i per cent, of dead B. phlei. Thus
we obtained the same result with Johne's bacillus
as with the bovine tubercle bacillus. This experiment
shows also that Johne's bacillus has not become
acclimatized to grow without the essential substance,
but that it has acquired the power of forming it from
the medium ; and it is interesting to note that the
strain of Johne's bacillus that has become acclimatized
to grow on glycerine-beef broth still gives no growth
on ordinary Dorset's egg medium. So presumably it
cannot form the essential substance from that medium,
although, of course, like an unacclimatized strain of
Johne's bacillus, it will grow well on the tgg medium
if it contains some essential substance obtained from
some other acid-fast bacillus, including the same
acclimatized strain of Johne's bacillus grown on
ordinar}^ glycerine-beef broth.

NATURE OF THE "ESSENTIAL SUBSTANCE^' 95

The fact that our strain of the bovine tubercle
bacillus, when grown on Dorset's egg medium, appears
to contain no essential substance does not prove that
no essential substance is formed, but that no reserve is
formed that can be extracted; in other words, that only
just sufficient is formed for the immediate require-
ments of the bacillus; and the same is probably true
in the case of B. phlei grown on glycerine-liver broth.
Therefore in most of these experiments that we have
classed as negative a small quantity of the essential
substance may have been formed for the immediate
use of the bacillus.

Our experiments also showed that certain strains of
the human tubercle bacillus seemed to form but little
reserve essential substance when first isolated from
the body on Dorset's Qgg medium ; and we thought
that the difficulty experienced in isolating this bacillus
from certain body fluids, such as pleural fluids, even
though their presence can be demonstrated by the
inoculation of a guinea-pig, may be due to a temporary
inability of the bacillus to form the essential substance.
To test this we tried to isolate the bacillus from these
fluids on media containing the essential substance, and
we tested tubercular pus from cows in the same way.
Our experiments were limited in number, but they
certainly indicated that a small quantity of the essential
substance (or of bacilli containing the essential sub-
stance) improves a medium used for isolating these
bacilli.

On reviewing the experiments so far carried out, it
is obvious not only that the essential substance is
soluble in water and present in the alcoholic extract
of B. phleiy only in a very small quantity, but also
that the medium on which the bacillus is grown plays
an important part in determining the production or

96 JOHNE^S DISEASE

otherwise of the essential substance, and the same
rule holds good for other acid-fast bacilli, including
the bovine type of tubercle bacillus.

Reasoning now on the same lines that first induced
us to try the addition of other acid-fast bacilli to a
medium for growing Johne's bacillus, we thought
that as the essential substance was necessary for
the growth of Johne's bacillus, so, too, the material
from which such organisms as the B. phlei formed
the essential substance, would be more or less neces-
sary for the growth of B. phlei, for the simple reason
that the essential substance, or an allied substance,
or one of a number of allied substances, must be
formed by B. phlei before that micro-organism can
grow.

Now, it is well known that fatty substances play an
important part in the metabolism of the acid-fast group
of bacilli, and also that certain alcohols, such as glycer-
ine and mannite, greatly improve the media on which
the bacilli can be grown. These and allied substances,
then, were the first to receive attention. They were
added to ordinary peptone-beef broth, a number of
other fluid media being prepared as controls. On
each of the media B, phlei was tested, not only as
regards growth, but also as regards the formation of
the essential substance. The following are the most
important of the media that were tested :

Naegeli's fluid + 4 per cent, glycerine.

Naegeli's fluid + 4 per cent, glycerine + i per cent, peptone.

Watery extract of hay.

Watery extract of carrot.

Gordon's lemco medium.

Gordon's lemco medium -|- 4 per cent, glycerine.

Watery extract of fish + i per cent, peptone.

Watery extract of brain -f- 1 per cent, peptone.

Watery extract of mammary gland of cow -f i per cent, peptone.

Watery extract of beef + i per cent, peptone.

NATURE OF THE "ESSENTIAL SUBSTANCE'' 97

Besides these we tested an extract of beef, to portions
of which were added one of the following substances
in the following quantities :

Glycerine

Glycerine

Glycerine

Glycerine

Mannite

Mannite

Erythrite

Erythrite

Dulcite

Methyl alcohol

Methyl alcohol

Absolute alcohol ...

Absolute alcohol ...

Propyl alcohol

Isopropyl alcohol ...

Per
Cent.

I
2

4

lO

I

2
I

2

I
2

5

2

5

I
I

Per

Cent.

Tertiary butyl alcohol

Glucose

Dextrose

Laevulose

Lactose

Maltose

Saccharose

Rhamnose

Galactose

Raffinose

Potassium sodium tar-
trate ,

Amygdahn ,

Asparagin

Glycol

To all, except the Naegeli media, ^ per cent, of sodium chloride
was also added.

B. phlei was grown on flasks of all the media, many
of which were also used for growing the "nasen-
schleim" bacillus and Grassberger's bacillus. The
quantity of growth obtained with B. phlei varied
considerably, the best being on the broth containing
glycerine; but growth was also fairly good on the
media containing mannite, erythrite, glucose, dextrose,
laevulose, 2 per cent, absolute alcohol, or 2 per cent,
propyl alcohol, and also on Gordon's fluid + glycerine
and on the hay extract. In the case of the " nasen-
schleim " bacillus and Grassberger's bacillus much the
same results were obtained, and in every case the
bacilli had formed the essential substance — that is,
when killed and dried and added to egg^ they made
good media for Johne's bacillus. The media, however,
varied, and although space will not allow us to give
details of the many hundreds of experiments carried

7

98 JOHNE^S DISEASE

out, we may say that from the results we were inclined
to believe that the essential substance was formed
from such substances as glycerine, mannite, and
absolute alcohol.

If such be the case, the next problem was to find out
how these substances might be changed by B. phlei
in the process of forming the essential substance for
its metabolism. We know that in the process of fer-
mentation hydration and oxidation take place. Even
in the case of alcoholic fermentation, in the late stages
of the process, when the yeast is growing as a film on
the surface of the fluid, the alcohol may become oxi-
dized and almost entirely disappear, and the proba-
bility is that either outside the cell or inside the cell
some organic acid is one of the final products formed
for the benefit of the bacterial or yeast metabolism, and
it may be taken as certain that this acid varies in the
case of different bacilli. It may be, in fact, that the
carboxyl group (COOH) has to be formed in certain
compounds before such micro-organisms can obtain
their carbon from sugars and alcohols.

In the case of B. coli^ the large quantity of acid
produced in media containing sugars may be the same
process, the conditions being such that more acid is
produced than is necessary or beneficial, a phenomenon
of over-production so often met with in lowly organized
life.

If, as we believe, some acid is necessary, then it
is quite clear that B. phlei and allied bacilli must con-
tain a special oxidizing enzyme, in order that they
may produce from such substances as glycerine the
particular acid which is necessary for each bacillus.

Now, from our experiments we were led to believe
that the essential substance might be an organic acid,
and that the reason why it is necessary to add this

NATURE OF THE " ESSENTIAL SUBSTANCE " 99

substance to egg before any growth of Johne's bacillus
can be obtained is that Johne's bacillus has lost its
power of producing the necessary oxidizing enzyme,
such an enzyme being unnecessary in the animal body,
where the special acid (or acids) is produced by the
ordinary cell metabolism of the host. It is not to be
presumed that the acid is formed for the benefit of the
bacterium, but that the acid being formed by the host,
the bacterium, when in remote ages it commenced to
become pathogenic, either found it suitable for its own
metabolism, or it mutated, so that it could utilize the
special acid present in the host. In either case the
bacillus would no longer require an enzyme to pro-
duce the acid, and as it became more strictly patho-
genic, it would no doubt cease to be capable of forming
both the enzyme and the acid. We know that bac-
teria do vary, or mutate, in this manner ; for instance,
in the typhoid-coli group the most pathogenic mem-
bers, compared with the non- pathogenic varieties,
have very largely, though not completely, lost their
power of producing acids. On the other hand, one
of us (F. W. T.) has induced the typhoid bacillus to
acquire the power of producing acid from lactose ;
this was done by subculturing the bacilli for a long
period of time in a poor medium, containing lactose
and no other sugar, thus forcing the bacilli either to
utilize the lactose, or hunger strike and perish. Now,
the special acid in the animal body may not be a waste
product of the cell, but may be essential to the host,
although it may only be present in the tissues in an
exceedingly small quantity. This, if true, might
account for the long incubation period in such con-
ditions as Johne's disease and leprosy; for the small
supply of acid would soon be used up by the bacilli,
and there would be no more available until the host

100 JOHNE'S DISEASE

reacted, so as to produce some more acid for its own
use. But we know that in the process of repair there
is usually over-production, a rule that is well illus-
trated in the formation of antitoxins ; so that one
would expect an over-production of the acid, and this,
again, would be more favourable to the bacilli, or, if
these had been killed, to a second infection of bacilli.

Although a great deal of what has been said is
theoretical, it is not entirely so, as our experiments
alone suggested that the essential substance might be
an organic acid ; so we again started to investigate the
alcoholic extract of B. phlei. The extract obtained
from loo grammes of dried bacilli as a whole showed
but slight acidity; but when separated into different
portions with the solvents already mentioned, we
found that some were markedly acid, and, further, that
those portions that were most acid made the best
media for Johne's bacillus. Using that portion of the
extract which is insoluble in chloroform and soluble in
water, we attempted to precipitate the acid with baryta
water. Only a small precipitate formed, so the fluid
was evaporated to dryness, and the sticky mass so
obtained heated with methyl alcohol. Most of the
mass dissolved, but a very small insoluble portion
(about 0*007 gramme) remained. This was incor-
porated in 30 c.c. of egg medium, which was dis-
tributed in tubes, and sterilized as usual. A good
result was obtained — that is, Johne's bacillus grew
well on the medium, and no growth took place on
a control medium made up at the same time with the
same mixture of eggs. The test was repeated on
several tubes of both media with another strain of
Johne's bacillus, and the same result was obtained.
Control media, containing small quantities of barium
hydrate, gave negative results. This experiment is

NATURE OF THE "ESSENTIAL SUBSTANCE" 101

interesting, not only because it supports our theory,
but also because it shows what a small quantity of the
essential substance is sufficient to stimulate the growth
of Johne's bacillus.

More recently we have attempted to obtain a pure
acid from the barium and sodium salts, but without
success, the chief difficulty being to obtain a sufficient
quantity of the barium salt. In view, however, of the
positive results obtained with the barium compound,
we now directed our attention to known organic acids.

In the first place it will be remembered that from
our experiments with B. phlei we came to the con-
clusion that the essential substance might be formed
by this bacillus from certain alcohols and sugars, so as
substitutes for the essential substance we now tested
acids that can be formed from these substances. The
following is a list of acids investigated : Glycerine,
tartronic, gluconic, saccharic, mucic, fumaric, malic,
aconitic, glycolic, glyoxylic, succinic. Glyceric alde-
hyde and glycerose were also tested.

Each of these substances was made up into media,
and as a basis the same glycerine-saline-egg mixture
was used as in the previous experiments. Each acid
was tested in percentages varying from o'oi to I'o,
and in several series of media the acid was previously
neutralized with sodium hydrate or baryta water.
Tubes of each batch of medium were inoculated with
Johne's bacillus, and after incubation for some weeks
at 37° C, a slight growth was obtained on certain of
the tubes containing glycerinic acid, and to a less
extent on those containing tartronic acid, and on those
containing gluconic acid.

In all these cases about i per cent, of acid was
present, but it had been almost completely neutralized
with NaOH before making: into medium. We also

102 JOHNE^S DISEASE

obtained slight growth on a medium that contained
0*2 per cent, of glycerose. The strain of Johne's
bacillus had been grown on timothy-grass bacillus-
egg medium for about twelve months, but still gave
no indication of growth on ordinary Dorset's egg
medium.

Although from the experiments it appears that not
one of the acids enumerated above is identical with
the essential substance, yet the results obtained with
glycerinic, tartronic, and gluconic acids are interesting,
and it is possible that the acid constituting the essen-
tial substance is allied to the acids mentioned. More
recently we have tested other organic acids, including
various tartaric, oxalic, and phosphoric acids. We
have also tested acids that contain other elements —
chlorine, nitrogen, or sulphur — in combination ; but
all the results were negative.

Although we have been unable to discover an
efficient substitute for the essential substance, our
experiments, considered as a whole, certainly suggest
that it is some organic acid. However, whatever its
nature may be, it will probably be difficult to obtain
sufficient material to determine its constitutional for-
mula, although in the future it may be found possible
to discover the nature of the constituent elements, and
the structure of the essential organic radicle in the
molecule.

The relationship of Johne's bacillus to the tubercle
bacilli and other acid-fast bacilli, and the possibility
that the essential substance may influence the patho-
genicity of Johne's bacillus, are discussed more fully at
the end of Chapter IX.

In the light of future research, some of the theories
we have put forward may prove to be wrong ; but we
have thought it advisable to give them, not only that

DESCRIPTION OF CULTURES 103

the reader may be able to follow the reasoning which
led us from one series of experiments to the next, but
also because we believe that the same fundamental
laws will be found to apply to Johne's bacillus and to
the lepra and tubercle bacilli. We hope, too, that
the experiments that we have carried out may help to
throw some light on the vital chemical changes that
occur in the life-history of these bacilli, both when
outside and when inside the animal body.

Description of Cultures.— In giving the experiments
on the cultivation of Johne's bacillus, we have fre-
quently described the cultures obtained, but in most
cases the description refers only to young primary
growths, so that a more complete description of
vigorous growing and older cultures is not out of
place. Details of the preparation of the media have
already been given.

Egg Media containing the Essential Substance. — These
media, as we have seen, may be made with tubercle
or other acid-fast bacilli that contain the essential
substance, or with the essential substance extracted
from these bacilli ; they must contain about 4 per cent,
by volume of glycerine. On such media Johne's
bacillus in primary cultures grows as tiny, dull white
colonies, which are rarely visible to the naked eye in
less than four weeks. The colonies are irregularly
round, and either remain quite small and discrete, or
they may coalesce. If only a few are present, they
increase in size, and when older become more elevated,
especially in the centre, and turn dull yellowish-white
in colour. The edges of the colonies remain thin,
and from the margins numerous irregular elevations
gradually rise towards the centre, and end in a peak,
which is sometimes very markedly elevated. The
elevations are most marked on dry media and on those

104 JOHNKS DISEASE

in which the essential substance is not mixed well with
the egg. These elevated colonies are well shown in
Plate IX., Figs, i and 2. When streak subcultures
are made on to the same media with a platinum loop,
growth occurs along the needle track. The growth in
this case does not greatly differ from that obtained in
primary cultures, except that it is more rapid, and the
colonies usually coalesce to form a continuous irregu-
larly heaped-up growth. This is shown on a tube of
medium that contains the chloroform insoluble portion
of a cooled and filtered alcoholic extract of B. phlei,
and is illustrated in Plate VI., Fig. i. If, in place of the
saline in the medium one uses peptone-beef broth, the
growth is certainly more vigorous ; this is illustrated
in Plate IX., Fig. 3. If the broth is made from brain,
such as sheep's brain, the improvement is still more
marked (see Plate VII., Fig. i). In this case the growth
cannot be distinguished from that of a vigorous-growing
culture of the human tubercle bacillus on ordinary
Dorset's egg medium (see Plate VII., Fig. 2), and is more
copious than that of either the bovine or the avian
tubercle bacillus grown on Dorset's egg medium. A
culture of the avian type of bacillus grown on egg
medium is shown in Plate VII., Fig. 3. If, in these
media, the egg is replaced by blood-serum or ascitic
fluid, Johne's bacillus does not grow so well.

Agar Media containing the Essential Substance. —
When the essential substance or a bacillus containing
the essential substance is added to glycerine-peptone-
beef broth-agar, the growth of Johne's bacillus is slower
and not so vigorous. The growth, however, does not
greatly differ from that which occurs on the egg media,
except that it is usually more coloured. The agar
medium, however, is improved by the addition of
blood-serum, or ascitic or pleuritic fluid, and growths

PLATE VII,

Fig. I.

Fig. 2.

Fig. ^,

Photo by F. Holmes, Bristol.
CULTURES OF JOHNE'S BACILLUS.

Fig. I.— Streak culture of Johne's bacillus on an egg medium made up

with sheep's brain broth containing peptone and glycerine, and

containing i per cent, of dead B. phki.
Yic, 2.— Streak culture of the human tubercle bacillus on Dorset's egg

medium.
Fig. 3.— Streak culture of the avian tubercle bacillus on Dorset's egg

medium.

[Tofacc pa$c 104.

DESCRIPl^ION OF CULTURES 105

on two tubes of one such medium, which contains dog's
unheated ascitic fluid which had been passed through
a sterile Doulton white filter, are shown in Plate VI.,
Figs. 2 and 3.

Fluid Nutrient 7l/^^/<7.— Ordinary peptone-beef broth
containing glycerine and the essential substance, or a
bacillus containing the essential substance, does not
form a good medium for a freshly isolated strain of
Johne's bacillus. There is great difficulty in inducing
the bacillus to grow on the surface of the medium.
After three or four weeks, growth occurs at the bottom
of the flask as tiny yellowish-white grains, and these
gradually increase in size and number. It is usually
some months before any growth takes place on the
surface of the medium, and when it first appears it
occurs as a very delicate film. In subcultures the
growth becomes thicker and more vigorous, although
in this case it rarely covers the whole surface of the
medium. After subculturing several times, surface
growth may occur as very thick, knobby, and irregular
masses. We found this knobby condition particularly
well marked with a strain of the bacillus which, after
two years, had become acclimatized to grow on
glycerine-beef broth without the essential substance ;
such a growth is shown in Plate VIII. The growth
extends Irom these knobby masses by forming fresh
rings of thin growth, which in turn also become heaped
up and knobby, or in the same way entirely fresh areas
may form. Several of the thin patches may be seen
very faintly in the plate. As the bacillus becomes
better acclimatized to the glycerine-peptone-beef broth
medium, the growth ceases to be characteristically
knobby, and becomes more like that of the tubercle
bacilli.

CHAPTER VII •

VACCINES : DIAGNOSTIC AND CURATIVE

The premature announcement in Germany, in 1891, of
the discovery by Koch of tuberculin and its curative
properties was followed by the administration of ex-
cessive doses. This caused a temporary discredit to
fall on the use of the reagent as a curative vaccine for
phthisis in man, and to the present day opinion is still
divided on this subject. But as a diagnostic reagent
for cattle, tuberculin, in competent hands, has proved
of the greatest value in eradicating tuberculosis from
affected herds, and in preventing its reintroduction by
newly-bought animals. Probably it was the success
claimed for tuberculin which led the Russians, Kalning
and Helman, to prepare mallein on the same lines.
Independently of the reduction of the equine popula-
tion owing to mechanical traction, the proportion of
horses affected with glanders in Great Britain has
been enormously reduced, and Hunting's work on this
disease in London has probably been the means of
preventing the infection of many men whose work
brings them in contact with large numbers of horses.

The tuberculin test as applied to cattle is now familiar
to all intelligent stock-owners. The preparation of
the reagent varies in small details in different labora-
tories, but, broadly speaking, a glycerine-broth culture
of the tubercle bacillus is killed by heat, filtered to

106

PLATE VIII.

PJioto by F. E. Barnard, Loinioii.

CULTURE OF ACCLIMATIZED JOHNE'S BACILLUS ON
ORDINARY GLYCERINE-PEPTONE-BEEF BROTH.

Note — Dense growth on surface.
,, Small granules of growth at bottom of fluid.
,, Very delicate surface growth in several places.

[To face page io6.

VACCINES : DIAGNOSTIC AND CURATIVE 107

remove the bacilli, and the filtrate evaporated to one-
tenth its original volume. Before it is sold to be used
by veterinary surgeons it is diluted with nine times
its volume of a weak solution of carbolic acid in lo per
cent, glycerine. The dose of the diluted vaccine for
an adult bovine is usually about 3 to 4 c.c. when
given as a subcutaneous injection under the skin of
the neck or behind the shoulder. If the vaccine is
prepared so that its strength differs from that given
above, then the dose will vary accordingly. The
temperature of the animal to be tested is taken for
one or two days, and if found to be within normal
limits, the injection is then given. The temperature is
noted at the time of injection, and also at the ninth,
twelfth, fifteenth, and eighteenth hours following the
inoculation. In an animal in which the temperature at
the time of inoculation was not above 103° F., a rise of
temperature to 104° F. or over is considered as a posi-
tive reaction — i.e., the animal is tubercular. If the
initial temperature is above 103° F., the test should be
postponed for a few days. With this test the tempera-
ture frequently rises to 105° or 106° F., and in such
cases the animal shows signs of fever, loss of appetite,
etc. In experienced hands the proportion of errors is
exceedingly small, although in very advanced cases a
negative result is not uncommonly obtained.

There are several modifications of the tuberculin
test.

The ophthalmic test is performed either with Cal-
mette's tuberculin or with a i per cent, solution of
Koch's Old Tuberculin. Calmette's preparation consists
of an alcoholic precipipate of tuberculin to which no
preservative has been added. Wolff-Eisner gives the
following description of the method of preparation : A
glycerine-broth culture of the bovine tubercle bacillus,

108 JOHNKS DISEASE

six weeks old, is autoclaved at iio° C. for twenty
minutes, and then evaporated at 80° to 90° C. to a tenth
of its volume. The fluid is now filtered and precipi-
tated with 95 per cent, alcohol ; the precipitation is
hastened by the addition of a small quantity of sodium
chloride. The fluid is now passed through filter-paper
and the precipitate scraped off and dried in vacuo.
The precipitate can be purified by repeating the pro-
cess. For use, the white precipitate is made up into a
I per cent, solution with sterile water.

To carry out the test, a few drops are placed into
one of the eyes of the animal, and the other eye is
used as a control. The presence or absence of a reac-
tion is judged by the amount of conjunctival inflam-
mation produced in the treated eye. In a tubercular
animal this reaction may be well marked, and a muco-
purulent secretion may result for a few days. The
ophthalmic or eye test has been used for the detection
of tuberculosis in animals that have been inoculated
by fraudulent dealers with large doses of tuberculin
just before sale in order to prevent any reaction if tested
shortly afterwards in the ordinary way. The conjunc-
tival reaction is not prevented by a previous subcu-
taneous inoculation of tuberculin.

Intradermal injections of tuberculin, which cause an
inflammation or thickening of the skin in tubercular
animals, have also been used, and a tuberculin prepared
with a non-irritant lanoline base may be given as an
inunction into sound or slightly scarified skin. These
methods, however, are not of practical value on a large
scale, having no advantages as primary tests over the
simple subcutaneous injection.

It is often not necessary to use an autogenous vaccine
to produce a reaction. Man, suffering from certain
forms of leprosy, reacts to a human tuberculin if suffi-

VACCINES: DIAGNOSTIC AND CURATIVE 109

cient is given. On the other hand, it must be remem-
bered that vaccines prepared from almost any acid-fast
bacillus will produce a reaction in such conditions as
tuberculosis, leprosy, and Johne's disease, provided
that a sufficiently large dose is given ; but with such a
dose animals suffering from other diseases, and normal
animals are very liable to react, and from this it follows
that any reaction obtained in the test animal is worth-
less or even misleading.

To obtain a specific and reliable diagnostic vaccine,
it should be prepared from the same species of bacillus
that causes the particular disease, in which case it is
necessary to give only a relatively small dose to pro-
duce a reaction, in fact, a dose sufficiently small to
avoid a definite reaction in animals suffering from
allied diseases or by normal animals. Other factors
remaining constant, it may be said in general that the
closer the relationship between the specific variety of
bacillus causing the disease and the bacillus from
which the vaccine is prepared, the more reliable and
specific the test becomes.

In the case of Johne's disease the absence of a pure
culture of Johne's bacillus led several workers to
investigate vaccines prepared from allied acid-fast
bacilli. O. Bang, in 1907, tested cattle suffering from
pseudo-tuberculous enteritis with a vaccine made from
B. phlei, and the present writers have since tested a
strain of B. phlei in the same way, both on naturally
infected and on artificially inoculated animals.

In 1907-08 O. Bang tested cattle suffering from
Johne's disease with a tuberculin prepared from
tubercle bacilli isolated from birds. The vaccine used
was prepared from a four months' old glycerine-broth
culture. The filtrate obtamed from this culture was
evaporated to one-tenth the original volume, and one

110

JOHNKS DISEASE

part of this concentrated liquor was added to four
parts of a J per cent, aqueous solution of pure carbolic
acid. Of this reagent Bang gave the following doses :

Animals over two years old
Animals one to two years old
Animals six months to one year old

lo c.c. subcutaneously.
7-8 c.c.
5-6 c.c.

It should be noted that the dose given (lo c.c. of a
I : 4 dilution) is about six times the dose of tuberculin
used (3-4 c.c. of a 1:9 dilution) when testing an adult
animal for tuberculosis.

O. Bang found that in very advanced cases of the
disease a good reaction was not obtained with avian
tuberculin. In his report he gives the results of about
fifty tests, from which we have selected seven of the
most marked reactions. The temperatures are given
in degrees Centigrade in the following table :

Number

At Injec-

8th to Qth

nth

13th

15th

1 7th

19th to 20th

of Case.

tion.

Hour.

Hour.

Hour.

Hour.

Hour.

Hour.

4

38-2

40-4

40-9

40-6

407

41*0

41 'O

5

387

38-5

39-1

39*6

40*2

40-6

39'4

8

387

40*2

40-4

40-4

41-1

40* I

39-8

9

38-6

40*6

41-2

40*0

40-3

40-4

39'6

17

38-8

40*0

41-1

407

40-9

39'5

391

26

38-0

397

407

40-8

40-4

39'9

28

38-0

38-8

38-6

39*9

40- 1

40-8

40*2

If the temperatures detailed above were the result of
tests with ordinary diagnostic tuberculin in tubercular
cattle, slaughter of all animals would be justified, as in
every case the maximum exceeds 105° F. (40*6° C), and
in some cases it reaches 106° F. (4ri° C).

With the same vaccine O. Bang tested about 1,700
animals, mostly herds of tuberculosis-free Jersey cattle,
and he found that from 6 to 35 per cent, of adult cattle
reacted. Of those that reacted 34 animals (tuber-

VACCINES : DIAGNOSTIC AND CURATIVE 111

culosis-free) were tested again with avian tuberculin
eight months after the first test, and only 88 per cent,
gave a positive result. This may have been due to
spontaneous recovery in the interval between the tests,
or to a curative effect of the vaccine ; or, again, it may
have been due to failure on the part of the vaccine.
Other observers have found that an animal may give
quite different results within a month with the same
dose of avian tuberculin (vide infra).

O. Bang recommends mixing avian tuberculin with
ordinary diagnostic tuberculin with the object of
testing for Johne's disease and tuberculosis at the
same time. He concludes that if an animal reacts to
avian tuberculin it may safely be assumed to be suffer-
ing from pseudo-tuberculous enteritis, but a negative
result would seem to be of doubtful value.

In 1909, Le Sueur of Jersey, in conjunction with
O. Bang, tested 194 head of cattle with avian tuber-
culin, and in a private communication Le Sueur
writes: "With regard to the avian tuberculin test I
do not regard it as practical, and feel convinced that
the reaction which takes place after injection is simply
due to the large amount of tuberculin injected." In
the same year Malm tested with avian tuberculin two
calves in which the disease had been produced by
feeding with infected mtestine. The first animal was
tested subcutaneously on five occasions, with two
positive and three negative results. In the tests
that proved positive large doses were given. The
ophthalmic test was applied with avian tuberculin
three times, two reactions being positive and one
uncertain. Of intradermal tests one was positive and
two were negative. The animal was slaughtered, and
the diagnosis of Johne's disease confirmed by B. Bang.
The second calf, with avian tuberculin, gave three

112 JOHNE'S DISEASE

positive and two negative reactions. When killed it
showed the characteristic lesions of the disease.

In his paper {Norsk Veterincer-Tidsskrift, No. 8,
August, 191 1), Malm gives the following notes on the
two calves :

CALF A.

3.4.08. Calf fourteen days old, fed with infected intestine in milk.

14.5.08. Again fed from another animal. No fever, no diarrhoea,

no bacilli in faeces till 23.6.09.

10.3.09. 2 c.c. avian tuberculin ; well-marked reaction — 38*8 to 40*9.

No diarrhoea, no fever next day.

2.4.09. ^ gramme Old Tuberculin ; negative reaction.
5.4.09. Uncertain reaction (ophthalmic) with avian tuberculin.

13.4.09. Good ophthalmic reaction in other eye.

23.4.09. I c.c. fish tuberculin + i c.c. blindworm tuberculin sub-
cutaneously ; negative reaction.

17.6.09. Intradermal reaction, avian tuberculin, positive.

23.6.09. Bacilli in fasces for first time.

25.6.09. Simultaneous, intradermal, and ophthalmic tests with avian
tuberculin. Ophthalmic test, positive.

28.6.09. Intradermal test, negative.
7.7.09. Diarrhoea noticed first time. 0*35 gramme avian tuber-
culin ; negative reaction.

28.9.09. Avian tuberculin ; negative reaction.
19. 1 1.09. Diarrhoea again noticed.

13.12.09. I gramme avian tuberculin ; negative reaction.

20. 1. 10. Large number of acid-fast bacilli in faeces.

29. 1. 10. 2 grammes avian tubercuhn ; good reaction (38*5 to 40* 2).
31. 1. 10. Killed. All the characteristics of a typical case of Johne*s

disease. Diagnosis confirmed by B. Bang, then in

Christiania.

Scrapings of bowel were inoculated into three
rabbits and eight guinea-pigs with negative results.

CALF B.

16.4. 10, Fourteen days old. Fed with 150 grammes mucosa of

bowel of Case 13 in warm milk.
2 1.6. 10. Positive reaction to \ gramme of avian tuberculin

(38-9 to 40-1). Marked diarrhoea after reaction, and

many acid-fast bacilli in faeces.
8.8.10. Old tuberculin ; negative reaction.

VACCINES : DIAGNOSTIC AND CURATIVE 118

27.9.10. ^ gramme avian tuberculin ; positive reaction (39*1 to 40' i).
6. 1. 1 1. Same dose of Old Tuberculin ; negative.

20.1. 1 1. Reacted with avian tuberculin (40'i).
1.4. 1 1. No acid-fast bacilli in faeces.

4.4. 1 1. I gramme of avian tuberculin gave a negative reaction.

10.4.11. I gramme Old Tuberculin ; negative.

18.4. 1 1. I gramme Old Tuberculin ; negative.

10.5.11. Killed. Proved to be a typical case of Johne's disease.

Miessner and Trapp, using avian tuberculin received
from Bang, tested a series of experimentally inoculated
animals. Their results are given in the table on
p. 114.

Here the first five calves certainly "reacted," and
sufTered an attack of diarrhoea as a result of the
disturbance.

G. P. Male has used avian tuberculin as a test for
this condition, the reagent being supplied to him by
Stockman. The following are his results : Herd i —
Jersey cattle. One bull, ten cows, and eight heifers
and calves were tested, and two cows and two heifers
gave the following reactions :

Animal.

Temperatures (Fahrenheit Scale).

Post Mortem.

Time of
Injection.

9th Hour.

1 2th Hour.

15th Hour.

(b)
(c)

id)

100-4

loi-o
loi-o

IOI"4

I04'0
I02'0
I02'0
10 1 "4

103*2
104-0
104-0
103-2

lOI-O
101-4
105-0

99-2

+ Johne's disease
+ Johne's disease
+ Johne's disease
+ Johne's disease

Case (d) was examined by Stockman post mortem,
who gave it as his opinion that the animal was affected
with Johne's disease, though no acid-fast bacilli were
found. None of the animals showed any evidence
of tuberculosis post mortem. Herd 2 : Thirty-two

8

114

JOHNE'S DISEASE

Animal.

Date of
Infection.

Infected with.

Method of
Injection.

Tempera-
ture
before

Injection,
Centi-
grade
Scale.

Highest
Tempera-
ture
reached.
Centi-
grade
Scale.

Hour
Maxi-
mum
reached.

Other

Effects.

Calf 62

16.2.09

Intestinal
mucosa
(Cow 6)

Per OS

38-9

41-5

36

Diarrhoea

Calf 63

16.2.09

Mesenteric

gland

(Cow 6)

Per OS

39'o

41-1

48

Diarrhoea

Calf 72

28.4.09

Mesenteric

gland
(Calf 182)

Subcu-
taneous

39'2

40-5

36

Slight
diarrhoea

Calf 73

28.4.09

Mesenteric

gland
(Calf 182)

Subcu-
taneous

39-0

40*3

8

Slight
diarrhoea

Calf 74

28.4.09

Intestinal

mucosa

(Calf 182)

Per OS

39' I

40-1

8

Severe
diarrhoea

Calf 122

3.9.09

Intestinal
mucosa
(Cow 9)

Per OS

39' I

39-3

36

None

Calf 104

(?)

From

mother

—

387

38-8

12

—

Calf 200

—

Control

—

39*1

39'4

10

—

Goat 105

37.09

Intestinal
mucosa
(Cow 8)

Per OS

40-0

40-8

16

—

Goat 106

3-7.09

Intestinal
mucosa
(Cow 8)

Per OS

39'i

39-6

10

—

Dog 76

28.4.09

Intestinal

mucosa

(Calf 182)

Per OS

39-0

39'5

10

—

Dog 107

3-7-09

Intestinal
mucosa

(Cow 8)

Per OS

39'5

397

8

—"

Dog 108

3.7-09

Intestinal
mucosa
(Cow 8)

Per OS

39'5

39-6

8

—

Dog 250

—

Control

—

39-8

39'9

14

—

VACCINES : DIAGNOSTIC AND CURATIVE 115

Jerseys of various ages gave the following re-
actions :

Animal.

Temperatures (Fahrenheit Scale).

Number.

Time of Injection.

9th Hour.

i2th Hour.

15th Hour.

5

IOI-2

lOI'O

103-8

103-8

8

lOI'O

103-2

101*4

lOI-O

20

lOI'O

104*0

102*4

102*2

24

lorS

lorS

103-2

104-0

29

10 1 'O

ioi"o

105-0

I02'0

Classed as

doubtful :

26

I02-0

I02'0

103-2

103-6

These cases were not confirmed by post-mortem
examination, but were sent away at once from the
farm. In the twelve months following the injections
no other cases of Johne's disease occurred.

Two suspected cases on farms on which there was
no history of Johne's disease were classed as negative
from the following results :

Case

Number.

Temperatures (Fahrenheit Scale).

Time of
Injection.

9th Hour.

1 2th Hour.

15th Hour.

1 8th Hour.

I
2

10 ro

IOO-2

IOI-4
102*0

I02*0
IOI'2

IOI*2
lOI'O

loi-o
loi-o

Both these cases had been affected for some months
with profuse diarrhoea, but on post-mortem examina-
tion they showed no characteristic corrugations as
seen in Johne's disease, though the small intestines
in both cases were inflamed and somewhat thickened :
no acid-fast bacilli could be found in either case. Both

116 JOHNKS DISEASE

animals had given a negative result when tested with
ordinary diagnostic tuberculin.

In only two of the nine cases detailed above, which
Male classed as reactions, did the temperature rise
above 104*0° F. Such reactions cannot be considered
as very definite, and it is doubtful whether State
legislation could be enforced and compulsory slaughter
ordered, unless a more marked reaction can be pro-
duced. In the case of animals housed specially for the
test and unused to being handled, the taking of the
rectal temperature and the use of necessary restraint
might lead to serious errors.

Since the publication of Male's results, the present
writers have performed some tests with avian tuber-
culin. The vaccine in our hands gave negative results,
and we concluded that, although some cases of Johne's
disease undoubtedly react to large doses of avian
tuberculin, the test is very uncertain, and is not of
much practical value. Details of these experiments
are given at the end of this chapter, where we discuss
vaccines prepared from cultures of Johne's bacillus.

More recently MTadyean, Sheather, and Edwards
have published results obtained with avian tuberculin.
The test was applied to fourteen bovines, all of which
were proved, on post-mortem examination, to be
affected with pseudo-tuberculous enteritis. The dose
of avian tuberculin given was 8 c.c. ; it was given
subcutaneously, and the results are shown in the
table on p. 117, which we have compiled from the text
of their paper.

In this table Cases XL and XVI. may be con-
sidered positive on the first test, although the reac-
tion in Case XI. is not very good. If one were testing
with ordinary tuberculin for tuberculosis. Cases VI.
and VIII. would be classed as doubtful.

VACCINES : DIAGNOSTIC AND CURATIVE 117

Case No.

Temperatures in Degrees Fahrenheit.

Result.

1

Time

of In-

jection.

3rd

Hour.

6th
Hour.

Hour.

i2th
Hour.

13th
Hour.

15th

Hour.

2ISt

Hour or
Later.

I.

IOI-8

102*8

103-4

102*0

I02-8

_

III.

Id -6

...

101*6

102*0

102*2

102-8

...

—

IV.

IOI'2

...

101*6

102*4

1 02 '7

...

I02*4

27th
Hour.

V.

loo-o

101*2

102*4

100-4

101*0

...

102*4

IOI-4

~

VI.

I02-0

103*0

1036

103*0

103*2

...

103*0

loi-o

-(?)

VII.

101*0

102*0

102*0

100*8

102*2

...

101*4

loi-o

—

VIII.

IOI'2

103*0

103*8

IOI-2

103-4

103-0

ioi*4

-(?)

XI.

IOI*2

T02-2

103*6

102*2

103*0

...

104*0

loi-o

A-

XII.

IOI*2

I02*0

101*8

103*0

102-4

...

I02-0

...

-

XIII.

IOI*6

101*8

103*0

103-0

102-2

...

1020

...

—

XIV.

101*6

101*8

103*2

102*8

102-2

_

102*0

24 th
Hour.

—

XVI.

IOI-6

102*2

101*2

104*6

106-4

103-6

103-2

+

XVII.

lorS

101*8

102*0

I02*2

102*2

...

102-0

...

-

i

27th

Hour.

XVIII.

102*5

101*4

101*4

ior6

I02-I

102*0

I02-I

—

Re-

tested :

III.

ioi*4

103*4

104*4

102 2

103*0

...

103-4

IOI-6

+

VII.

ioi*o

ioi*4

I02-8

102*2

101*4

I02-2

...

—

VIII.

101-4

101-4

103*0

105-8

103-4

...

102*6

...

+

XI.

102-6

102-0

104*0

105-4

105*2

...

105*0

+

XVI.

101*8

101*4

102*6

ioi*6

103*0

103*0

...

Retested, special dose :

Case IV. Time of injection, io2-6 ;

eighth

hour, 105-0

; next

morning, 104-2 ; evening, 103*6 ; f

ollowin

g days variec

i from

ioo"o to 104*0.

Case I. Left pleura and bronchial glands tubercular.

Case VII. Tubercular right pharyngeal gland on post-mortem examination.

Case III. was retested twelve weeks after the first
test, and the figures given above " represent a fairly
good general reaction produced rather early after the
injection of the tuberculin."

118 JOHNE'S DISEASE

Case IV. was retested after six weeks. A special
vaccine was given : Fifty milligrammes of avian
tubercle bacilli grown on the surface of glycerine-
broth, and made into an emulsion with 2 c.c. of physio-
logical salt solution, were injected into the jugular
vein. *' The temperature at the time of injection was
io2-6° F., and by midnight it had risen to 105° F. On
the following day the morning and afternoon tempera-
tures were 104-2° and 103-6° F. respectively." This
may be taken as a positive result, and the animal was
afterwards found to be suffering from Johne's disease.

Case VIII. was retested after eight weeks. "A
very good reaction was provoked on this occasion."

Case XL was retested after the same lapse of time,
and ** a good general reaction was again produced."

Case XVI., which gave a marked reaction on the
first test, on being retested after about nine weeks,
gave what the authors considered was a " slight " reac-
tion. Certainly the second set of figures in this case
would not be sufficiently definite for a practitioner
to risk the slaughter of a valuable animal. On post-
mortem examination this case showed no decided
macroscopic lesions, but gave *' a decidedly positive
result with regard to the presence of acid-fast bacilli."

Case XVII. had been previously tested by a
veterinary surgeon with avian tuberculin, with a
negative result.

We consider that the results obtained by these
workers afford further proof that the avian tuberculin
test is not a reliable means of diagnosing cases of
Johne's disease, for in spite of the large doses of
vaccine that were given, the majority of the cases
failed to react, a result which is in agreement with
that obtained by other investigators.

M'Fadyean, Sheather, and Edwards have also tried

VACCINES : DIAGNOSTIC AND CURATIVE 119

ophthalmic, intradermic, and endermic tests on certain
of the cases enumerated above.

The ophthalmic test with concentrated human tuber-
culin was applied with the object of discovering if any
of the animals were tubercular. The reactions are
described as follows :

Case III.: First test, "slight"; second test, "slight"; third test,

negative.
Case V. : One test, " fairly distinct."
Case VI. : One test, " absolutely negative."

Case VII. : First test, " pretty good " ; second test, " very well marked."
Case VIII. : First test, "extremely slight " ; second test, " slight."
Case XL : One test, " negative except for a trace of muco-pus."
Case XII. : One test, "fair."
Case XIII. : One test, " failed to provoke any."
Case XIV. : First test, "very feeble" ; second test, "distinct."
Case XVI. : First test, negative ; second test, negative ; third test,

negative.
Case XVII. : One test, " no indication."
Case XVIII. : One test, " failed to produce any."

The ophthalmic test with concentrated avian tuber-
culin gave the following reactions (the authors do not
state whether the tuberculin used for Case VIII. was
concentrated or not) :

Case V, : One test, " very distinct."
Case VI. : One test, " no reaction whatever."
Case VII. : One test, " discharge of muco-pus."
Case VIII. : One test, "good reaction."
Case XI. : First test,i"no indication whatever" ; second test,
negative.

Two intradermic tests were performed with con-
centrated avian tuberculin (Cases VII. and VIII.), and
two tests with avian tuberculin the concentration of
which is not definitely stated (Cases XI. and XVI.).
The reactions were as follows :

Case VII. : One test, "only just an appreciable."
Case VIII. : One test, "quite negative."
Case XI. : One test, negative.
Case XVI. : One test, negative.

120 JOHNE'S DISEASE

One intradermic test on Case III. with concentrated
human tuberculin gave a "pronounced" reaction.

Two endermic tests with concentrated human tuber-
culin were negative (Cases XVI. and XVIII.).

The authors do not state the conclusions to be
drawn from their experiments, and insufficient avian
ophthalmic, intradermic, and endermic tests have been
made to condemn these methods of diagnosis; but it
seems improbable that these tests will be of more than
scientific interest.

With regard to the avian tuberculin used as a sub-
cutaneous test, the number of negative results may be
due partly to the fact that the cases tested were some-
what advanced. O. Bang has found that very advanced
cases do not react, and this, of course, is true with
tubercular cattle inoculated with bovine tubercuhn.

In many cases the temperature reactions obtained
were not high, in spite of the large doses given — a
result which is in agreement with what we have
already stated in the beginning of this chapter with
regard to heterogeneous vaccines. The results ob-
tained by O. Bang were, however, the best that could
be got in the absence of pure cultures of the bacillus.

In 1910 the present writers started experimenting
on Johne's disease with the object of cultivating the
causative bacillus and of preparing a reliable and
specific diagnostic vaccine from the cultures obtained.
After cultivating several strains of the bacillus on
solid media, these strains were subcultured on to fluid
media {vide Chapter VI.), and from these cultures
vaccines were prepared.

Vaccine No, i. — In the first experiments we used an
alkaline peptone-bouillon containing 4 per cent, of
glycerine and i per cent, of dried human tubercle
bacilli. This was placed in Duclaux flasks, and steri-

VACCINES : DIAGNOSTIC AND CURATIVE 121

lized by steaming. These flasks were inoculated with
pure cultures of Johne's bacillus, and the main opening
of each was capped with gutta-percha tissue. The
flasks were incubated at 39° to 40° C. After the lapse
of a month small, yellowish-white grains of growth
became visible. These grew just above the sediment
at the bottom of the flasks, and gradually increased in
size and number. No film formation was observed.
After two months the flasks were steamed, their con-
tents passed through a Doulton white filter, and the
filtrate so obtained placed in small sterile phials in
quantities of 2 J and 5 c.c. The vaccine was not evapo-
rated to obtain a more concentrated solution, as we con-
sidered this unnecessary for preliminary experiments.

Vaccine No, 2. — A second batch of vaccine was pre-
pared in a manner exactly similar to the above, except
that the dried human tubercle bacillus was replaced
by the dried timothy-grass bacillus.

Vaccine No. 3. — A third batch was prepared by
growing Johne's bacillus in a broth medium containing
a glycerine-saline extract of the timothy-grass bacillus,
the extract representing i per cent, of dried bacilli and
4 per cent-, of glycerine. The medium used for pre-
paring this vaccine was filtered and autoclaved before
being inoculated, and in it the specific bacillus grew
fairly well in tiny masses on the bottom of each flask.

Vaccine No. 4. — A fourth batch was prepared from the
cultures of Johne's bacillus on the special timothy-grass
bacillus-egg medium, the growth being scraped off and
an emulsion made with Vaccine No. 3, described above.

Vaccine No. 5. — This was made in a similar manner
to No. 4, except that the cultures of Johne's bacillus
were suspended in o*8 per cent, sodium chloride in
place of Vaccine No. 3.

As controls to the above vaccines we used diagnostic

122 JOHNE'S DISEASE

tuberculin prepared at the Pasteur Institute, diagnostic
avian tuberculin obtained from the Royal Veterinary
College, and a special timothy-grass bacillus vaccine
prepared by ourselves. This last was made by grow-
ing the bacillus for about three weeks in a glycerine-
broth medium, which was then steamed and filtered
through a Doulton white porcelain filter. The sterile
filtrate was placed in small sterile phials without
previous concentration.

The results of tests conducted with these vaccines are
summarized in tabular form on p. 123. Bovines Nos. i
2, 4, and 5 are mentioned also in Chapter IX., where a
full account of the pathological lesions present in each
animal is. given. Bovines 2, 4, and 5 are calves.

A + sign is used to indicate a positive reaction, and
a — sign a negative one. On referring to the table,
one will notice that Vaccine No. i gave a positive
result with bovines Nos. i, 2, and 5, a doubtful re-
action with No. 4, and no reaction with the control
bull. It is also seen from the tuberculin tests and
from post-mortem examination that animals i, 2, and
5 had contracted tuberculosis.

In considering the results, it must be remembered
that Vaccine No. i was prepared by growing the
specific bacillus on a medium containing the tubercle
bacillus, and it might be expected that tubercular
animals would react to the vaccine on account of the
substances dissolved from the tubercle bacilli in the
medium, in which case the positive results do not
prove the presence of pseudo-tuberculous enteritis.
That the rises of temperature with Vaccine No. i may
have been caused by the presence of these substances
is also indicated by the absence of any reaction in the
control bull, and also by the negative results obtained
with Vaccines Nos. 4 and 5, which contained no

VACCINES : DIAGNOSTIC AND CURATIVE 123

e's

Hi

red
ure.

John

Bacc

recove

in Cult

1

1

1

+

•

•

•

+

V.

. ^.-^6

Post
rtem
ohne
iseas

+

1

1

+

1

1

1

+

i-Q

^ii

^e3

m

+

+

1

+

+

+

+

+

4)t5

t-(

(I

M

M

tS

M

w

°i

o

o

o

r^

t^

t^

i>i

O

^

-4"

i-i

ci

On

M

ON

M

t>.

A .

J) n

ci

O

.So

rtcd

d I

CO

=

!

•

•

'■

;

*. s

o

o

o

O

.S d

J

o 1

6 t

•

•

•

d 1-

S'

rt "

00

00

m

> "

c

§1d

o

o

o

o

o

o

"t d

6 1

GO

o 1

o 1

•

o 1
to

d 1

lO

d 1

HH O 1

00

H

«••: •

.E"^ S

o

o

o

o

o

o

d 1

6 1

d I

:

o 1

d 1

d 1

:

GO

vn

u^

in

in

li-)

•S

"3 S

o

o

o

d

d

d

u '^

i+

d +

6 1

•

d +

d 1

d 1

•

p

c<

c<

w

n

N

.

IJ

^ +

c3 1

o
^ 1

:

11'

uo

C4

<*- c

O O

O

^ 5

05 .i cJ

1 ^

c :

:

:

c^

u O

u u <u

u O

o ^

1

21

•—1 >

-

-5

o :

t! O

w

CO

li

Date

Inoculated.

o

o

2

s .

o'Z

h-t

w

^

U-5

o

o

3 _h

124 JOHNE^S DISEASE

tubercle bacilli. It may, however, be noted that the
bull was a very advanced case, and that Vaccines
Nos. I and 5 were weak.

From the results obtained with Vaccine No. i, it is
clear, as might have been expected, that to obtain
a specific reagent for pseudo-tuberculous enteritis the
tubercle bacillus must not be incorporated in the
medium. It will be seen from the table that a timothy-
grass bacillus vaccine caused no rise of temperature
in normal animals or in animals suffering from tuber-
culosis or pseudo-tuberculous enteritis, although, being
filtered through a porcelain filter, the vaccine was
probably weakened. It will be seen also that Vaccines
Nos. 4 and 5, which were prepared by growing Johne's
bacillus in media containing the timothy-grass bacillus,
caused no reaction in experimental or in control
animals.

It may seem surprising that no reaction was obtained
with Vaccines Nos. 4 and 5 in the animals affected with
Johne's disease; but, in point of fact, the negative
results might have been anticipated, as 'the greater
part of the bacillary emulsion used was obtained from
growths on solid media, the bacilli being made into an
emulsion without any previous grinding. It must also
be remembered that infected animals rarely, if ever,
show a rise of temperature during the course of the
disease, so that a more concentrated vaccine might be
required for Johne's disease than for tuberculosis. If,
as is held by many authors, the tuberculin reaction is
an anaphylactic phenomenon, and is in no way related
to the ordinary temperature changes found in a tuber-
culous animal, then the absence of a temperature in
Johne's disease should be no guide to the quantity of
specific vaccine likely to be necessary to produce
a thermal reaction.

VACCINES : DIAGNOSTIC AND CURATIVE 125

In the light of the results obtained with the vaccines
described above, we now prepared a sixth batch.

Vaccine No. 6. — This was made by growing Johne's
bacillus on ordinary glycerine-peptone-beef broth con-
taining a glycerine -saline extract of B. phlei (the
timothy-grass bacillus). This culture was grown for
nine months at 39° C. ; the whole was well shaken to
form an emulsion of the bacilli, placed unfiltered into
small phials, and heated for one hour at 62° C. The
vaccine was first tested on three fully-grown bovines,
and on five calves about seven months old, and was
inoculated intravenously in doses varying, according to
the size of the animal, from 5 to 10 c.c.

No. I, a Jersey bull, No. 2, a shorthorn cow, and
No. 3, a Jersey cow, all showed clinically the typical
manifestations of advanced Johne's disease, which they
had contracted naturally. Before we obtained these
animals, they had been tested several times with
diagnostic tuberculin with negative results, though the
bull had been in our possession for over eighteen
months, and had not been tested for tuberculosis
during this period. Each animal received 10 c.c. of
the special Vaccine No. 6. No. i gave a maximum
temperature of 105° F., which was reached an hour
after the inoculation. No. 2 gave a maximum tempera-
ture of io6-i° F.five hours after inoculation, and on the
following day developed a violent diarrhoea, which
persisted after the temperature had fallen. The faeces
contained blood and mucus. No. 3 gave a maximum
temperature of 104*8° F. This was reached in four
hours, and the temperature was not taken again.

Bovine No. i developed symptoms of tuberculosis,
so was killed (October 17, 1912). Post-mortem examina-
tion revealed typical Johne's disease in the intestine
and mesenteric glands. The disease was in a very

126

JOHNE'S DISEASE

Definite
Clinical

Specific
Vaccine of

Specific
Vaccine of

Number.

Animal.

Evidence of
Johne's Dis-

Diagnostic
Tuberculin.

Johne's

Bacillus.

No. 6

Intra-

Johne's
Bacillus.

ease with

No. 6 Sub-

Diarrhoea.

cutaneous.

venous.

I

Adult Jersey bull

+

_

+

2

Shorthorn cow

+

-

4-

3

Jersey cow

+

-

+

"experimental calf No. la

-

+

...

Experimental calf No. 2a

-

-

...

Experimental calf No. 3a

-

..

-

...

Experimental calf No. 4a

-

..

+

...

Experimental calf No. 5a

-

..

+

...

Experimental goat No. i

-

..

...

+

Experimental goat No. 2

-

..

+

4

Jersey cow

+

-

5

Shorthorn cow

4-

-

6

Shorthorn cow ...

4-

-

7

Shorthorn cow

+

-

8

Shorthorn cow

-

-

9

Shorthorn cow ...

+

—

lO

Shorthorn cow ...

+

-

II

Shorthorn cow

-

...

12

Shorthorn cow

—

13

Shorthorn cow

-

14

Shorthorn cow ...

-

...

15

Lincoln cow

-

16

Shorthorn cow

—

17

Shorthorn cow

—

18

Shorthorn cow ...

—

19

Shorthorn cow ...

-

20

Shorthorn cow

—

21

Shorthorn cow

22

Shorthorn cow ...

—

23

Shorthorn cow ...

+

24

Dutch cow

—

25

Shorthorn cow

+

VACCINES : DIAGNOSTIC AND CURATIVE 127

Specific
Vaccine of
Johne's
Bacillus.
No 7 Sub-
cutaneous.

Maximum
Tempera-
ture after
Vaccine of
Johne's
Bacillus.

Hour
Maximum
Tempera-
ture
reached.

Diarrhoea
started or
increased

after
Vaccine.

Post-
Mortem
Evidence

T. B.

Post-
Mortem
Evidence
Johne's
Disease.

Cultures of
Johne's
Bacillus
obtained

Post
Mortem.

Number

of
Animal.

105-0

I

+

+

+

I

..

io6'i

5

+

-

+

+

2

..

104*8

4

+

-

+

4-

3

104-6

3

~

-

+

4-

..

I02-6

5

-

4-

4-

102-6

4

-

+

-

-

105-0

6

-

+

+

+

..

105-4 ?

24?

-

+

+

+

I06-6

9

+

-

+

+

..

105-4

10

—

+

4-

+

104-6

6?

+

-

+

...

4

-

102-2

3

+

-

+

+

5

+

106-2

7

+

+

+

6

+

104-6

9

...

...

7

—

—

8

+

106-0

*6

+

-

4-

9

+

104-0

10

+

...

10

-

...

...

-

II

+

104-2

4

-

...

12

+

104-4

10

—

...

13

-

101-4

6

-

...

...

14

+

103-6

6

+

...

15

-

I0I-6

10

-

...

...

16

-

101-4

4

-

...

17

-

loi-o

10

-

...

...

i8

-

101-2

10

-

...

19

-

I02-0

10

-

20

-

IOI-8

6

-

...

21

-

101-4

6

-

22

+

104-2

4

+

...

23

-

IOI-8

6

—

...

24

+

104-0

6

"

...

...

25

128 JOHNE'S DISEASE

advanced stage, the bacilli being extremely numerous
in the films and sections from the diseased gut. Tuber-
culosis, which must have been contracted while the
animal was in our stables, was present in the lungs
and bronchial glands.

Bovine No. 2 died three weeks after the inoculation
(September 17, 1912), and on post-mortem examination
showed the typical lesions of Johne's disease. The
gut was very congested, and some haemorrhages were
present. There was no evidence of tuberculosis.

Bovine No. 3 died (August 26, 191 2). Post-mortem
examination showed advanced Johne's disease in the
intestine and mesenteric glands, the bacilli being fairly
numerous in films and sections from the diseased gut.

The five calves tested were those that are also
mentioned in Chapter IX., and numbered la, 2a, 3a,
4«, and 5a, respectively. They had been inoculated
with the strain of Johne's bacillus recovered from calf
No. 5 (pp. 123 and 148). The animals did not thrive well,
but they showed no clinical manifestations of Johne's
disease. All were tested with Vaccine No. 6 about six
months after inoculation with the living cultures;
calves Nos. 2a and 3a each received 3 c.c, and calves
Nos. la, 4a, and 5a each received 5 c.c. The following
results were obtained : calves Nos. 2a and 3a showed
no rise of temperature in six hours, and through an
error were not tested again until twenty-four hours
after the inoculation, when the temperatures were
normal. The temperature of calf No. la rose to
104*6° F. in three hours, that of calf No. 4a to 105° F.
in six hours, while calf No. 5a showed no rise in ten
hours, but when taken the following morning — twenty-
four hours after the inoculation — the temperature
registered 105*4° F., and was dropping.

A few days after the vaccine tests the calves were

VACCINES : DIAGNOSTIC AND CURATIVE 129

killed and post-mortem examinations performed. The
results are summarized in the table, and a detailed
description of the pathological lesions present is
given in Chapter IX., pp. 150 and 151.

We now tested the vaccine by means of subcutaneous
inoculations, and as at the time we had no more
bovines at our disposal, we used goats. The goats
tested were two which we had inoculated in July, 191 1,
with living cultures of Johne's bacillus. These animals
are also mentioned (p. 151).

At the time of testing the vaccine, eleven months
after the intravenous inoculation of the living culture,
goat No. I was thin, but otherwise there was no mani-
festation of the disease. Goat No. 2, which had been
inoculated into the peritoneal cavity, was apparently
quite healthy. Each animal received 3 c.c. of the
vaccine subcutaneously. The temperature of goat No. i
rose to io6*4° F. in five hours, reached 106*6° F., and re-
mained at 106° F. or over for fully eight hours ; the rise
of temperature was accompanied by some diarrhoea.
The temperature of goat No. 2 reached a maximum of
105*4° F. at the tenth hour, but no diarrhoea ensued.
Both animals were killed and post-mortem examina-
tions made. Goat No. i showed Johne's disease
throughout the intestines and mesenteric glands, and
goat No. 2 showed the disease in a very early stage.
In both cases the bacilli were found in the diseased
tissues, and were isolated on special media (vide
table, p. 126).

Satisfactory results with Vaccine No. 6 having been
obtained, a further batch — Vaccine No. 7 — was prepared
as follows : We used our strain of Johne's bacillus
that had become acclimatized to media containing no
other acid-fast bacilli or extracts of such bacilli. It
was grown for about four months on broth containing

9

130 JOHNKS DISEASE

I per cent, peptone, J per cent, sodium chloride, and
4 per cent, by volume of glycerine. The cultures were
shaken and the large masses of growth allowed to
settle. The fluid containing the smaller particles was
heated to 65° C. for one hour in a water-bath, and
was used without previous filtration. This vaccine
was tested on a number of adult bovines. Some were
tested at the Brown Institution and others on farms by
veterinary surgeons to whom we sent the vaccine.
The results are summarized in the table, and we
regret that we have been unable in most cases to
obtain a post-mortem examination.

In the table the animals are numbered 4 to 25
inclusive, and it is unnecessary to describe each in
detail, although the following points may be noted :
No. 5, which failed to react to the vaccine, was a very
advanced case of Johne's disease. It showed extensive
lesions in the ileo-caecal valve, in the lower part of the
ileum, and in the caecum. The upper part of the small
intestine and the large intestine were less affected. An
enormous number of bacilli were present in the mucous
and submucous coats of the ileum (see Plates III.
and IV.). The abdominal lymphatic glands were also
extensively involved, and showed a large number
of bacilli. In the opinion of the writers there is no
doubt that this case failed to react owing to the very
advanced stage of the disease. Cases Nos. 4, 6, and 9,
on post-mortem examination, also showed extensive
lesions of Johne's disease, but not so marked as in case
No. 5. In each case the condition was most evident
in the lower part of the ileum and in the ileo-caecal
valve. The abdominal lymphatic glands were only
moderately involved. In all three cases a considerable
number of bacilli were present in the ileum and a fair
number in other parts of the intestine and in the

VACCINES : DIAGNOSTIC AND CURATIVE 131

abdominal glands. Case No. 6 also showed tubercu-
losis of the bronchial glands. Cases Nos. 12 to 25
inclusive were all on the same farm, and were tested
by ourselves. The animals were in a bleak, open
position, and all were tested on a cold day. It is
well known that when tuberculin is used under such
conditions the temperature of a tubercular animal may
not rise to the same extent that it would under more
normal circumstances, and one would expect the same
rule to hold good in animals affected with Johne's
disease. If this is so, then the temperatures recorded
in animals Nos. 12, 13, 23, and 25 all indicate good
reactions, and in case No. 15 a fair reaction. It may
be noted, too, that the temperature of each animal was
subnormal before the vaccine was inoculated, and we
were only able to take it at the fourth, sixth, and tenth
hours following the inoculation of the vaccine. Case
No. 22 showed definite clinical signs of tuberculosis,
and gave no reaction with the vaccine.

More recently we have tested Vaccine No. 7 on five
sheep experimentally inoculated. No. i was inoculated
by the mouth, Nos. 2 and 3 intravenously, and Nos. 4
and 5 intraperitoneally. Ten weeks after inoculation
the animals were tested with the vaccine, this being
injected subcutaneously. The temperatures were taken
hourly after the third hour, up till the eleventh hour
following the inoculations, and they were taken again
at the twentieth and twenty-fourth hours.

No. I gave a maximum of io5'o° F. in 8 hours.
No. 2 „ „ 105*2° F. in 6 hours.

No. 3 „ „ 107*1° F. in 6 hours.

No. 4 „ „ 106*8° F. in 7 hours.

No. 5 „ „ 107*8° F. in 6 hours.

It will be observed that in every case the maximum
temperature was registered between the sixth and

132 JOHNKS DISEASE

eighth hours following the inoculation. None of the
animals showed any evidence of diarrhoea.

On reviewing the tests carried out on cattle, sheep,
and goats, we may at once exclude those with Vaccines
Nos. I to 5. These vaccines were prepared from early
cultures of Johne's bacillus, and were too weak to
cause any definite reaction in the animals. The
results obtained with Vaccines Nos. 6 and 7 show that
a vaccine made from a good glycerine-broth culture of
Johne's bacillus is as reliable a reagent for Johne's
disease as tuberculin is for tuberculosis, and, indeed,
it would have been surprising had it been other-
wise.

In the case of Johne's disease, it is interesting to
note that the maximum temperature was usually
recorded before the tenth hour — that is, much earlier
than when tubercular animals are tested with tuber-
culin. The situation of the disease in the intestine
and lymphatic glands might possibly account for this
early rise, but a more feasible explanation would
appear to be found in the nature of the lesions.
Nearly all tubercular lesions are surrounded by a
fibrous capsule, or are encompassed by a large quan-
tity of lymphocytes, while in Johne's disease the
lesions are never encapsulated, and gradually blend
with the normal tissue.

As regards the specificity, we may note that all our
tubercular animals that were free from Johne's disease,
including some that are not given in the chart, failed
to react in the slightest degree to a vaccine prepared
from cultures of Johne's bacillus ; while in animals
suffering from Johne's disease, whether tubercular or
not, a marked reaction was obtained. Exceptions,
however, are found, as in very advanced cases, which
may fail to react, and we know that this is often the

VACCINES : DIAGNOSTIC AND CURATIVE 133

case when testing advanced cases of tuberculosis with
tuberculin. As some of our cases of advanced Johne's
disease were suffering from concomitant tuberculosis,
the question arises as to whether the presence of the
latter disease did not possibly mask the reaction in
a few cases.

As in most biological tests, specificity can only be
obtained by an exact system of dosage, and it is for
this reason that the use of avian tuberculin in Johne's
disease has been proved to be practically worthless.
We have seen that in Johne's disease undoubted
reactions were obtained by Bang, Miessner, etc., by
the use of an avian tuberculin, but the reactions were
more often negative than positive; and there is no
doubt that to attain a larger percentage of positive
results the dose of this vaccine would have to be such
that it would be beyond the limits of reliability, and
tubercular or even sound animals might give a re-
action.

It is probable that in the future modified vaccines
will be prepared from cultures of Johne's bacillus, and
that other methods of inoculation will be resorted to.
In the case of tuberculins many different methods of
preparation and administration have been published,
and we have seen that Bang, Malm, Male, and, more
recently, M'Fadyean, Sheather, and Edwards, have
tested some of these modifications on animals suffering
from pseudo-tuberculous enteritis. It appears to us,
however, unlikely that there will be much improve-
ment on the subcutaneous inoculation of a vaccine
prepared from an ordinary glycerine-broth culture of
Johne's bacillus, although the ophthalmic test with
the same vaccine may be useful under certain
circumstances.

At the present day nearly all cattle have to undergo

134 JOHNKS DISEASE

the tuberculin test before being transported from one
country to another, and it is to be hoped that the
agricultural authorities of this and other countries will
soon enforce the same regulations as regards Johne's
disease, since a reliable diagnostic vaccine for this
disease can now be prepared.

CHAPTER VIII

THE AGGLUTINATION REACTION AND THE
COMPLEMENT FIXATION TEST

The Agg-Iutination Test. — The presence of agglu-
tinins in the serum of bovines suffering from Johne's
disease can be demonstrated in the ordinary way
(C. C. Twort); but, as in the case of tuberculosis,
a positive reaction cannot be obtained with a highly
diluted serum. Animals experimentally inoculated
also produce agglutinins ; not only bovines which
subsequently contract the disease, but also such
animals as rabbits and fowls, to which, up to the
present time, the disease has never been transmitted.
Needless to say, the sera of experimental animals
should always be tested before the first injection is
made, as the normal serum may possibly agglutinate.
Although we have not found any normal rabbits give
positive reactions, we have had to discard one or two
fowls on this account. In considering the dilutions of
the sera, they refer in all cases to a complete reaction,
a partial agglutination often being obtained with a
much higher dilution.

Technique. — This is extremely simple, and the same
as that used for testing a suspected tubercular serum,
with the following difference :

To obtain the best results in the last-mentioned
cases it is usual to employ a homogeneous broth-

135

136 JOHNE'S DISEASE

culture of the tubercle bacillus as recommended by
Arloing. Owing to the slow growth of Johne's
bacillus in the substance of a fluid medium, even
though it contains a certain amount of an alcoholic
extract of B, phleij one is obliged to have recourse
to an emulsion of the bacilli grown on solid media.
As will be remembered, Koch and Romberg used an
emulsion made from a growth on solid medium in their
tubercle agglutination reactions. To obtain an emul-
sion of Johne's bacillus, the growth is mixed with
saline, placed in a stout bottle containing glass beads»
and shaken in an ordinary electric shaker. To obtain
a good homogeneous emulsion, the usual precautions
must be taken. The culture should be young, and
after shaking should be filtered or centrifuged, to free
the emulsion from large clumps of bacilli. Fresh
unheated serum should be used, and it is best to per-
form the test as soon as possible after the withdrawal
of the blood. This precaution does not refer to fowl
serum, which agglutinates as well after heating at 56° C.
as before. In this case, too, the serum may be kept in
an ice-chest for months without any apparent diminu-
tion in activity. As in testing a tubercular serum, the
mixture must be incubated for some time — two to
four hours. If the microscopical method of examina-
tion is used, two hours is usually sufficient, and this
method in our opinion is far more reliable than the
macroscopic test. A control with the fresh unheated
serum of a normal animal of the same species as that
from which the suspected serum was obtained must,
of course, be made. It is well also to have a second
control, containing no serum, as spontaneous agglu-
tination of the emulsion is liable to take place, especi-
ally if living bacilli are used.

The sera of seven naturally infected cattle, six of

THE AGGLUTINATION TEST 137

which are numbered i, 2, 3, 4, 5, 6 in the table on
p. 126, were tested at the Brown Institution, and all
gave positive reactions in a dilution of from i in 10 to
I in 15 ; but it must be remembered that the serum of
normal cows will usually agglutinate an emulsion of
tubercule bacilli in a dilution of i in 5, or even i in 10
(Descos).

After testing the sera of five normal calves, two
normal sheep, and two normal goats, which were all
negative, the animals were inoculated with a pure
culture of Johne's bacillus (see pp. 150 and 151). Six
months later samples of blood were again taken, when
it was found that the five calves gave positive results
in a dilution of i in 5, whilst the remaining animals
showed no trace of an agglutinin.

Amongst the ordinary laboratory animals the fowl
appears to be by far the most suitable for the pro-
duction of these antibodies ; but even in this class of
animal there is considerable variation as regards the
amount of agglutinin obtained. In some cases the
serum may agglutinate after a single subcutaneous
inoculation in dilutions of i in 20 to i in 40, whilst in
others repeated inoculations fail to produce anything
like such a strong serum.

As might be expected, the experiments demon-
strated that the agglutination reaction is not specific
within the acid-fast group of bacilli. The serum of a
tubercular animal will agglutinate an emulsion of
Johne's bacillus, and vice versa; the dilution of the
serum is, however, usually higher with its homologous
bacillus.

The Complement Fixation Test. — This reaction can
also be obtained in the same category of animals as
mentioned under the agglutination test ; but it is a
more difficult and elaborate test than the latter, and

]38 JOHNE^S DISEASE

can hardly be performed to give any degree of
accuracy except by those habitually carrying it out.

The technique to be followed is similar to that used
in performing the reaction with a suspected tuber-
cular serum. Special care should be taken to titrate
accurately the complement before each series of bloods
is tested. At the Brown Institution a simple emulsion
of bacilli was used as antigen, but it is possible that
a better antigen would be obtained if an extract made
from diseased bowel were added to the bacillary emul-
sion, as recommended by Hammer for tuberculosis.
On the other hand, it is worthy of note that Miessner
and Kohlstock were unable to obtain any satisfactory
results when they used as antigen an antiformin
extract, etc., of diseased gut which they called
" enteritidin."

The tests which are detailed below were carried
out on seven naturally infected bovines at the Brown
Institution, the animals being the same as those used
for the agglutination tests (Nos. i to 6; see table,
p. 126). Of these animals, Nos. i, 4, 5, 6, and 7 gave
positive reactions. No. 3 a doubtful reaction, and No. 2
a negative. A few of the animals were tested several
times, and on every occasion gave the reaction with
more or less the same intensity. It may be mentioned
here that in most of the animals the disease was in
an advanced stage. In five experimentally inoculated
calves (Nos. la, 2a, 3^, 4^, 5«, Chapters VII. and IX.),
mentioned also under the agglutination tests, which
had received an emulsion of Johne's bacillus six
months previously, the reactions were negative, except
with No. s^t which gave a slight positive result.
At the time the blood was taken, these animals were
really in the incubation period of the disease, as they
showed no diarrhoea or any other symptoms ; but, on

THE COMPLEMENT FIXATION TEST 139

post-mortem examination, in most cases the gut was
thickened, and pure cultures of acid-fast bacilli were
recovered from the intestines of four of the cases.

In rabbits experimentally inoculated no difficulty
was found in demonstrating the presence of a com-
plement fixing antibody, but in the case of fowls all
attempts proved unsuccessful. Unfortunately the
complement fixation tests, like the agglutination re-
actions, proved to be specific within the acid-fast
group of bacilli only in a very limited degree.

Since avian tuberculin has been used as a diagnostic
reagent in this disease by O. Bang, Male, Miessner
and Trapp, Malm, and others, it was thought advisable
to carry out a number of comparative tests with the
immunity reactions described above. These experi-
ments, however, gave no indication that Johne's
bacillus is more closely allied to the avian tubercle
bacillus than to the human and bovine types. From
the results obtained, it appears highly probable that
neither the agglutination nor the complement fixation
reaction will prove of much value as a practical diag-
nostic method in Johne's disease. As in tuberculosis,
so, too, in Johne's disease ; the inoculation of a diag-
nostic vaccine has been shown to be a far more delicate
test, and, what is perhaps of still greater importance,
it is more specific, and at the same time easier to
perform.

CHAPTER IX

THE PATHOGENICITY OF JOHNE'S BACILLUS : INOCU-
LATION EXPERIMENTS WITH INFECTED MATERIAL
—INOCULATION EXPERIMENTS WITH PURE CUL-
TURES OF JOHNE'S BACILLUS— THE PATHOGEN-
ICITY OF THE BACILLUS FOR SMALL ANIMALS

Inoculation Expepiments with Infected Material. — As

no cultures of Johne's bacillus were available at the
time, all the early experiments regarding the patho-
genicity of the bacillus were performed with infective
material that was obtained at post-mortem examina-
tion.

The first to carry out systematic experiments in this
direction were Johne and Frothingham, who sought to
prove the tubercular nature of the condition by inocu-
lating subcutaneously two guinea-pigs with small
portions of the submucous tissue of the caecum of
a cow affected with the disease. In neither case was
there any reaction at the site of inoculation ; but the
animals became considerably thinner, so that in spite
of the absence of any local affection, they considered
that the animals were suffering from a generalized
tuberculosis. About five weeks after the inoculation,
however, they were astonished to find that the con-
dition of the animals had improved, and at the end of
eight weeks both guinea-pigs were again in a perfectly
sound condition. As a similar state of affairs may

140

EXPERIMENTS WITH INFECTED MATERIAL 141

sometimes occur with the avian tubercle bacillus, these
authors concluded that they were dealing here with
this micro-organism. It is now known that this was
not so, and in the light of further experiments it is
possible that the transitory illness of these animals
was due to contaminating intestinal micro-organisms
in the inoculated mucous membrane, and not to any
toxic effect of Johne's bacillus.

In 1904, Markus inoculated subcutaneously six
guinea-pigs and two rabbits, and fed two rabbits and
three hens with infective material. The animals, all
of which had increased in weight, were killed within
periods of from 1 50 to 300 days, and were found post
mortem to be quite normal. Stuurman, by feeding
a rabbit with material from the intestine of an affected
cow, produced abscesses in the intestines, and from
these cultivated in pure growth an acid-fast bacillus
which was afterwards proved to be the avian type of
tubercle bacillus. B. Bang experimented with the
diseased intestines and glands that he obtained from
fourteen cows by inoculating a number of small
animals. The material obtained from two of these
cows, which came from tuberculous herds, produced
typical tuberculosis in guinea-pigs, rabbits, and calves.
Bang, however, produced Johne's disease in three
calves by feeding them with large quantities (from i to
3 pounds) of the mucous membrane of a cow which was
killed while suffering from the disease.

Miessner and Trapp, at the Agricultural Institute at
Bromberg, made a number of animal inoculations with
material from eight cases of naturally acquired Johne's
disease. One of the experimental calves on post-
mortem examination showed well-marked lesions of
Johne's disease, but no signs of tuberculosis. The
following experiments were made with material from

142 JOHNE'S DISEASE

a cow which was suffering from Johne's disease, but
was free from tuberculosis : The milk was centrifuged,
and the sediment inoculated into six guinea-pigs and
one calf, with negative results in six months. The
urine was centrifuged, and the sediment inoculated
into six guinea-pigs, which also gave negative results.
The mucous membrane of the intestine was also inocu-
lated into a number of animals. These are tabulated
below, and show negative results with all the animals.

Six guinea-pigs subcutaneously, negative in four to five months.
Six guinea-pigs intra-abdominally, negative in four to five months.
Three rabbits subcutaneously, negative in four to five months.
Three rabbits intra-abdominally, one died in nine days (peri-
tonitis), and the remainder were negative in five months.
Six pigeons subcutaneously, negative after five months.
One calf intravenously, negative after six months.
One calf per os, negative after six months.
Three hens per os, negative after four months.

A large series of inoculations was also carried out
with the lymphatic glands attached to the mesentery.
These are tabulated belov/, and in all cases show
negative results.

Six guinea-pigs subcutaneously, negative after five months.

Six guinea-pigs intra-abdominally, negative after two to six

months.
Three rabbits subcutaneously, negative after four months.
Three rabbits intra-abdominally, negative after three and a half

months.
Two hens per os, negative after three and a half months.
One goat intravenously, negative after eight months.
One dog intravenously, negative after three and a half months.
One calf intravenously, negative in three months.
One calf intravenously, died in two days.

Negative results were also obtained with the spleen,
udder, and lymph glands of the pleura, as shown below.

Spleen inoculated into four guinea-pigs subcutaneously, negative
in four months.

EXPERIMENTS WITH INFECTED MATERIAL 143

Udder inoculated into two guinea-pigs intravenously, one nega-
tive in two months, one died in two days.

Lymph glands of pleura inoculated subcutaneously into two
guinea-pigs, negative in two months.

Although in the naturally infected cow the bacilli
were particularly numerous in the caecum, it is
interesting to note that all the inoculations, even in
the calves and in the goat, gave negative results.

These authors, however, made similar experiments
with seven other cows which showed lesions of Johne's
disease ; five of these were also tubercular. In some
cases the small animals inoculated with infective
material from those cows, which were tubercular,
contracted tuberculosis ; but, although a very large
number of animals (including a sheep) were inoculated
by various channels with infected intestine, Miessner
and Trapp were unable to reproduce the disease in
any species with the exception of three calves. Of
these calves one was inoculated intravenously with
mesenteric gland, and the remaining two with the
intestinal mucous membrane of a cow suffering from
Johne's disease. In these cases the disease was demon-
strated on post-mortem examination. In each case the
intestine was thickened, and films from the thickened
portions showed numerous acid-fast bacilli. With the
intestinal mucosa of one of these calves two more calves
were fed and another inoculated subcutaneously. All
three animals reacted to avian tuberculin five or six
months later (see p. 114).

Working in 1907, M'Fadyean failed to produce any
lesions in rabbits and guinea-pigs by inoculating the
animals with infected intestine, and many other authors
(Lienaux, Matthis, Freger) have made similar un-
successful attempts to infect the small laboratory
animals. In other cases tuberculosis has resulted.

144 JOHNKS DISEASE

due, no doubt, to coincident tuberculosis in the cow
from which the infected material was obtained.

Inoculation Experiments with Pure Cultures of the
Bacillus. — When in 1910 the present writers obtained
pure cultures of Johne's bacillus on a special medium
(see Chapter VL), a series of animal experiments was
started. Care was taken to subculture the growths
for several generations, in order that cultures might
be obtained free from the diseased tissue that was
placed on the original tubes. The strain used was the
first that we isolated, and was obtained from the gland
of the naturally infected case No. 2 (see p. 71). In the
first instance we inoculated a cow and five calves, all
of which were obtained from a herd of cattle that had
been under the observation of a veterinary surgeon
for three years, during which period no case of Johne's
disease or any similar condition had occurred. One
calf died seventeen days after the inoculation, and need
not be considered further. Of the remaining cases,
each is described below individually and in detail.
Bovines Nos. i, 2, 4, and 5 are also referred to on p. 122.

Bovine No. i. — A shorthorn cow about eight years
old and six weeks pregnant was tested in October,
1910, with ordinary tuberculin, and was found to be
free from tuberculosis. On November 19, 1910, the
animal was inoculated with a pure culture of Johne's
bacillus. The growth was eight weeks old, and the
third subculture distant from the original culture from
the mesenteric gland. The growth was made into an
emulsion with sterile o'8 per cent, sodium chloride, and
the whole given by the mouth to the cow, which had
had no food for twelve hours. No immediate symptoms
followed. From the time of feeding with the bacilli the
temperature of the animal was taken twice daily, and
it remained within normal limits, the averages for the

EXPERIMENTS WITH PURE CULTURES 145

morning and evening over a long period being ioi"2° F.
and io2*2° F. respectively. Repeated examination of
the faeces failed to reveal any acid-fast micro-organism
resembling Johne's bacillus. On June 5, 191 1, the cow-
calved. Parturition was normal and the calf quite
healthy.

On October 23, 191 1, she was killed, and a post-
mortem examination made. The carcass showed
marked emaciation, with practically complete absence
of subcutaneous and intra-abdominal fat. The bronchial
glands showed advanced tubercular lesions, while the
pleura and the thoracic surface of the diaphragm
showed a few recent tubercular patches. A few
tubercular nodules were also present in the lungs.
Some of the intestinal lymphatic glands also showed
tubercular lesions of recent origin. All the remaining
organs and tissues of the body, with the exception of
the intestines, appeared normal. When the intestines
were opened, a considerable thickening of the mucous
membrane was observed. This was most marked in
the vicinity of the ileo-caecal valve, the terminal portion
of the ileum, and the first part of the large intestine.
No ulceration or caseation of the mucosa was present.
The general thickening and corrugation of the mucosa
presented the characteristic appearance of Johne's
disease, and films made from beneath the mucous
membrane showed a few small acid-fast bacilli. From
the tubercular bronchial gland we obtained a pure
culture of a bovine strain of tubercle bacillus ; but all
the cultures taken from the intestinal mucosa on to
the special media remained sterile. It is unfortunate
that the specific bacillus was not isolated from this
case. Nevertheless, from the naked-eye appearance,
and still more from the histological examination of the
intestine, there is no doubt that this animal had con-

10

146 JOHNKS DISEASE

tracted Johne's disease as a result of the experiment,
and it is to be presumed that the coincident tubercu-
losis was an accidental infection contracted by the
animal while in the stables.

Bovine No. 2. — Heifer calf. Previous to inoculation,
the temperature of this calf was taken twice daily for
twelve days, and was found to be normal for an animal
of its age. The faeces were also examined several
times for acid-fast bacilli, with negative results. On
November 10, 1910, when sixteen days old, the calf
was inoculated with a culture of Johne's bacillus. The
growth was three weeks old and the third subculture
distant from the primary culture from the diseased
gland. The growth was washed off with sterile
o*8 per cent, sodium chloride, and an opalescent
emulsion obtained. This was inoculated into a vein
of the left ear, but no immediate symptoms followed.
The faeces were frequently examined for the presence
of acid-fast bacilli, and from December 15, 1910, to
January 17, 191 1, films occasionally showed some
thick acid-fast bacilli, which, however, did not resemble
Johne's bacillus. These were probably some sapro-
phytic " dung bacilli," and from the latter date no
acid-fast micro-organisms were found.

On October 24, 191 1, the animal was killed. On
post-mortem examination, the carcass showed no
emaciation, and no pathological lesions were found in
any of the organs or tissues of the body with the
exception of the bronchial lymphatic glands. These
were slightly enlarged, and showed several caseating
centres in which tubercle bacilli were demonstrated.
On microscopical examination, the intestinal lymphatic
glands failed to reveal any acid-fast bacilli. Cultures
were made in the usual manner from one of the tuber-
cular bronchial glands and from beneath the intestinal

EXPERIMENTS WITH PURE CULTURES 147

mucosa. Those from the gland grew tubercle bacilli ;
the rest remained sterile. The fact that this animal
accidentally contracted tuberculosis, while it resisted
the inoculation of the culture of Johne's bacillus,
demonstrates, with other experiments, a comparatively
low virulence for Johne's bacillus.

Bovine No. 3. — This was a red and white male calf
which was received at the Institution when three days
old. Taken for about three weeks, the temperature
remained normal for a young animal, and repeated
examination of the faeces failed to reveal any acid-
fast bacilli. On December i, 1910, the calf was in-
oculated intraperitoneally with ^ c.c. of a thick
emulsion of Johne's bacillus, the growth being the
second subculture distant from the primary culture
from the gland. No immediate symptoms followed,
and the temperature remained normal. Daily exami-
nation of the faeces, however, from December 29 to
January 11, showed an increasing number of short,
thick, acid-fast bacilli with rounded ends. Though
these did not resemble Johne's bacilli as found in pure
culture or in lesions, we decided to kill the calf and
examine the intestine. This was done on January 11,
191 1, and the post-mortem showed no pathological
lesions. A few acid-fast bacilli were found in the
mucous membrane and in sections of the bowel, but
they appeared to be in the crypts of the membrane,
and were probably not Johne's bacilli. We consider
the result of this experiment negative, the calf being
killed before the disease had had time to develop.

Bovine No. 4. — This was a brown male calf, and
when received at the Institution (January 7, 191 1) was
two days old. The faeces showed no acid-fast bacilli,
and the temperature remained normal up to Feb-
ruary 21, when the calf was inoculated into the peri-

148 JOHxNE^S DISEASE

toneal cavity with about 5 milligrammes of a moist
growth of Johne's bacillus. This growth was the
fourth subculture distant from the primary culture.
From the time of inoculation onwards the animal
showed no definite symptoms, no acid-fast bacilli
could be detected in the faeces, and, with the exception
of the periods when various vaccine tests were made,
the temperature remained normal. On October 16
the calf was killed. The animal was in good condi-
tion, and no lesions either of tuberculosis or of Johne's
disease could be found. Films made from beneath the
intestinal mucosa showed no acid-fast bacilli, and cul-
tures from this situation remained sterile, so that the
result was entirely negative.

Bovine No. 5 {Calf No. 5). — This was a red and white
male calf, and was received at the Institution January 7,
191 1. Repeated examination of the faeces failed to
reveal any acid-fast bacilli, and the temperature re-
mained normal. On February 21 the calf was inocu-
lated intravenously with about 7 milligrammes of a
moist culture of Johne's bacillus. The culture used
was the fourth subculture distant from the primary
growth. No immediate S3^mptoms followed, and there
was no rise of temperature. The condition of the calf
remained good during March and the beginning of
April, but towards the end of the month it became
thin and weak. These symptoms increased up to
May II, when the calf was not able to rise without
assistance, though the appetite was good and the
temperature normal. In the early part of June the
calf could again walk without assistance, but it re-
mained very thin. There was no diarrhoea, nor could
any acid-fast bacilli be found in the faeces at any time.
On July 12, 191 1, the animal was killed. The carcass
showed extreme emaciation, and a small tuberculous

EXPERIMENTS WITH PURE CULTURES 149

nodule was found in one of the bronchial glands ; but
there was no other evidence of tubercular disease in
the body. The remaining organs and tissues, with the
exception of the intestines, showed no pathological
lesions, though there was very little fat present in any
of the usual situations. The intestine, on examination,
showed a moderate degree of thickening, especially in
the ileum and caecum near the ileo-caecal valve. Films
were made in the usual manner from beneath the
mucous membrane, and after a prolonged examination
several typical Johne's bacilli were found in those from
the region of the ileo-caecal valve. Cultures were
made from a number of the glands and from the sub-
mucous tissue of the gut on to tubes of Dorset's egg-
medium. The tubes were capped with gutta-percha
tissue and incubated at 39° C. After five days the
small pieces of tissue were removed from the Dorset's
egg medium and placed on to tubes of tubercle bacillus-
egg medium and timothy-grass bacillus egg-medium.
One tube of the latter medium, on which was placed
material from the ileo-caecal valve, showed definite
microscopic evidence of growth in twenty-five days.
This culture, the only one obtained, was easily sub-
cultured on to fresh tubes of the special medium, but
failed to grow on ordinary Dorset's egg medium. The
acid-fast bacillus in the bronchial glands was isolated
on Dorset's egg medium, and it possessed all the
characteristics of a typical bovine tubercle bacillus.
It is interesting to note that although this animal had
contracted typical Johne's disease, yet, in spite of the
tuberculosis, which might be expected to lower the
resistance of the animal, the disease was in a very early
stage. This experiment is further evidence of the
slow progress of the disease.
The strain of Johne's bacillus isolated from this calf,

150 JOHNE^S DISEASE

as has been mentioned, was obtained originally from
the naturally infected case No. 2 described on p. 71.
The culture obtained from the calf was again grown
for about six months outside the animal body, and
occasionally subcultured. A growth obtained in the
third generation, which was about two and a half
months old, was then inoculated into five calves.
Calves Nos. la and 2a were inoculated intravenously,
Nos. 3« and 4a intraperitoneally, and No. s^ subcu-
taneously.

After being tested with various vaccines, these
animals were killed a little over six months from the
date of inoculation, and, post mortem, all but one
proved to be more or less affected (see table, p. 126).

Calf No. la showed typical lesions of Johne's disease
in the intestines and mesenteric glands. The bacilli
were most numerous in the tissues of the ileo-caecal
valve, but were also present beneath the mucous
membrane of other parts of the gut, and in the glands.
The animal showed no evidence of tuberculosis.

Calf No. 2a showed slight lesions of Johne's disease
in the intestine and in the mesenteric glands, which
were somewhat larger than normal. Several acid-fast
bacilli were found in the glands, and a few beneath
the mucous membrane near the ileo-caecal valve. No
tubercular lesions were found.

Calf No, 3a showed typical tubercular bronchial
glands, but there was no evidence of Johne's disease
in the intestine or mesenteric glands.

Calf No. 4a showed tubercular bronchial glands,
which were caseous and in places calcareous. Johne's
disease was present in a moderately advanced stage.
The bacilli in the intestinal wall were most numerous
near the ileo-caecal valve, a situation in which we have
usually found the lesions to be most pronounced. The

PLATE IX.

Fig

Fig. 3.

Photo by F. Holmes, Brislol.
CULTURES OF JOHNE'S BACILLUS.

Fig. I. — Primary culture from experimental goat No. i on an egg medium
made up with saline and containing i per cent, of dead B. phlei and
4 per cent, glycerine.

Fig. 2. — Primary culture from experimental goat No. 2 on same medium
as Fig. I.

Fig. 3.— Streak culture from experimental calf No. 5 on an egg medium
made up with peptone -beef broth and containing i per cent, of dead
B. phlei and 4 per cent, glycerine.

[To face page 150.

EXPERIMENTS WITH PURE CULTURES 151

disease in the mesenteric glands was particularly well
marked, due, no doubt, to the method of inoculation.
Films from these glands showed a fair number of
Johne's bacilli in each field.

Calf No. 5a was small and emaciated. On post-
mortem examination it showed typical tubercular
bronchial glands and early tuberculosis of the apex
of the right lung. There was no definite thickening
of the mucous membrane of the intestine, and the
mesenteric glands were not much enlarged. Very
few acid-fast bacilli were found beneath the intestinal
mucosa, but several were present in films from one
of the mesenteric glands.

All five calves showed some congestion of the
mucous membrane of the intestines, with occasional
haemorrhages. Some of the glands also showed
haemorrhages, probably caused by the dose of vaccine
which each animal had recently received (p. 128). These
conditions were present only in a slight degree in
calf No. la.

From all five cases cultures were made in the usual
manner from the intestinal mucosa and abdominal
lymphatic glands, and in all cases except No. la the
bacillus was recovered in pure culture on a timothy-
grass bacillus medium. Tubes of ordinary media
inoculated at the same time from the intestine and
glands all remained sterile. Thus, out of five calves,
four had contracted the disease.

In July, 191 1, we inoculated two young goats with
living cultures of Johne's bacillus. The growth from
one tube of medium was made intp an emulsion with
10 c.c. of sterile 0-85 per cent, sodium chloride. Goat
No. I received 3 c.c. of this emulsion intravenously ;
goat No. 2 was given i c.c. in the peritoneal cavity.

Eleven months later, after being tested with a vaccine

152 JOHNE^S DISEASE

(p. 129), these two goats were killed. At this time
one of the animals (goat No. i) was thin, and the
inoculation of the vaccine had caused severe diarrhoea ;
but in goat No. 2 there were no clinical manifestations
of the disease. On post-mortem examination, goat
No. I showed typical lesions of Johne's disease
throughout the intestine and in the abdominal lym-
phatic glands. In films from both places the bacilli
were present in fair numbers. Goat No. 2 showed the
disease in a very early stage, and several bacilli only
were found. From both these cases, however, follow-
ing the usual procedure, we were able to isolate the
bacilli on the special media (see Plate IX., Figs, i and 2).

From the positive results obtained in the two goats
it is highly probable that these animals, like bovines,
can contract the disease naturally, although, as has
already been pointed out, Miessner and Trapp
obtained negative results with goats that were fed
with diseased gut from a cow.

Quite recently we have reproduced Johne's disease
in sheep (Vetermary Record^ Aprils 1913)- Five animals
were inoculated with cultures of Johne's bacillus iso-
lated from a cow. Sheep No. i was inoculated by the
mouth, Nos. 2 and 3 intravenously, and Nos. 4 and 5
intraperitoneally. Ten weeks after the inoculations
the animals were tested with a diagnostic vaccine.
Nos. I and 2 gave doubtful reactions, and Nos. 3, 4,
and 5 good reactions {vide Chapter VII., p. 131).

Shortly after the vaccine tests, animals Nos. 3 and 5
were killed and post-mortem examinations made.
Both showed definite lesions of Johne's disease, and
no evidence of tuberculosis.

In the case of sheep No. 3, the intestines showed
a moderate degree of thickening, most marked in the
caecum and ileo-caecal valve, but only a few bacilli

EXPERIMENTS WITH PURE CULTURES 153

were found. The mesenteric glands were consider-
ably enlarged, but in these we were unable to demon-
strate the presence of Johne's bacillus.

Sheep No. 5 showed no definite thickening of the
intestine, but several acid-fast bacilli were found in
the walls of the ileo-caecal valve. The mesenteric
glands were much enlarged and presented the typical
appearance of Johne's disease, although only a few
bacilli were present.

These experiments are interesting, since they prove
that Johne's bacillus, isolated from a naturally infected
bovine, is pathogenic for sheep, and is capable of
producing a condition in these animals which is
identical with that found in cattle, and we feel justified
in assuming that the disease in naturally infected cattle
and sheep is caused by one and the same micro-
organism.

The Pathog-enicity of the Bacillus for Small Animals.

— Numerous attempts have been made to infect the
smaller laborator}^ animals, such as rabbits, guinea-
pigs, etc. As we have already mentioned, the earlier
investigators, Johne and Frothingham, B. Bang, etc.,
used portions of the diseased bowel obtained from
cattle suffering from this condition. The results,
except when the inoculated material also contained
tubercle bacilli, appear to have been entirely negative,
indicating that these animals are endowed with a
natural immunity against the disease in the same way
as they are immune to the human lepra bacillus.

At the Brown Institution, working with pure cultures,
the authors have inoculated pigeons, hens, rabbits,
guinea-pigs, mice, and rats, subcutaneously and intra-
peritoneally, while several pigeons, hens, and rabbits
were inoculated intravenously. These animals, to-
gether with some mice, rats, and rabbits, which were

154 JOHNE^S DISEASE

fed with pure cultures of the bacillus, gave entirely
negative results. In all cases single inoculations were
made, and the animals were examined in periods vary-
ing from two months to two years after the inoculation.
In one rabbit, inoculated intraperitoneally, a caseous
nodule about the size of a large pea was found em-
bedded in the intestinal wall ; the nodule involved the
peritoneal and muscular layers, and although a few
acid-fast bacilli were found microscopically, they could
not be recovered in culture, and were probably dead.
The result must be considered negative.

More recently C. C. Twort and T. Craig have
investigated the effect on rabbits and hens of massive
doses of Johne's bacillus, not so much with the idea of
reproducing the typical disease as with the intention
of studying the toxic effects of the bacillus in com-
parison with other members of the acid-fast group, and
the mechanism of the immunity of these small animals.
The results detailed below refer to the experiments of
these workers, and if one takes into account the size
of the doses, they demonstrate the relatively low
toxicity of Johne's bacillus.

The Intravenous Inoculation of Rabbits. — The in-
oculation of a single dose of 30 to 120 milligrammes of
Johne's bacillus produces apparently no ill-effect on
the animals, which eat well and show no loss of weight,
while in a young animal the normal growth is unim-
paired. There is no immediate or subsequent rise of
temperature, or, at the most, a rise of 0*5° F. on the day
following the injection. If a second injection is made
three to five days later, there is again apparently no
ill-effect, except, perhaps, some slight loss of appetite
for a day or two, and the temperature remains prac-
tically constant. In a large number of animals inocu-
lated with Johne's bacillus the rise of temperature

THE BACILLUS FOR SMALL ANIMALS 155

produced by the bacilli never exceeded i° F., and in no
case has a maximum of 104° F. been recorded. The
normal temperature of a rabbit lies between 100°
and 104° F., and, as is well known, slight causes pro-
duce considerable variation. Thus a rise of tempera-
ture that fails to reach 104*5° F. is not of much
significance, except in those animals in which the
temperature has been consistently low — i.e., 100° to
102° F. before the inoculation.

Animals receiving a second inoculation usually
remain quite healthy, but if a further dose is given five
days later, some, after four, five, or six weeks gradually
become emaciated, and ultimately die. If a second or
third dose is given fifteen to thirty days after the first
inoculation, a large proportion of the animals die
within four weeks. Here, undoubtedly, one is dealing
with animals that have already developed specific
anticorps in their blood, etc., and the violent reactions
sometimes observed shortly after the second inocula-
tion are not dissimilar to ordinary anaphylactic shock,
and the ultimate death of the animals is probably due
to this cause.

PosT-MoRTEM Examinations. — A single intravenous
injection produces no macroscopic change in any of the
organs, with the exception of the kidneys and lungs.
Twenty-four hours after the inoculation the kidneys,
on histological examination, are found to be congested ;
and this condition is more evident in animals killed on
the third day. If the animals are killed after three
or four weeks, no congestion or other pathological
change can be detected. From the first day the lungs
are usually somewhat congested, and may show
haemorrhagic points, although after a few weeks the
normal condition is again found. Animals inoculated
intravenously with two or more doses within five days,

156 JOHNE^S DISEASE

and killed shortly afterwards, show much the same
condition as is met with after a single injection. In
those killed at a later period the organs usually show
additional changes. The kidneys may, or may not, be
congested, and sometimes they present slight evidence
of cirrhosis. The lungs are usually normal in appear-
ance, but they may be a little cedematous. The spleen
is often somewhat enlarged, while the liver is more or
less fatty throughout.

Animals that die after receiving a second intravenous
dose fifteen to thirty days after the first injection, and
those that succumb to repeated inoculations given at
short intervals, can be considered together. In these
cases post-mortem examination reveals an acute
nephritis, and sometimes the presence of fluid in the
peritoneal cavity. More rarely the pleural cavities
also contain fluid. There is usually fluid in the peri-
cardial sac, which is often distended to such a degree
that one has no hesitation in attributing death to the
presence of the fluid in this situation. The visceral
layer of the pericardium is often somewhat rough, but,
except for the presence of the fluid, there is not much
evidence of pericarditis. The lungs, in most cases,
are pneumonic. The liver may be congested, or pale
and fatty, according to the length of time that the
animal has survived. The spleen is generally normal,
though it may be slightly enlarged. The bladder is
usually distended with urine, whilst the lymphatic
glands, such as those of the axilla, are often congested,
sometimes intensely so. In none of these cases has
any pathological change been found in the intes-
tines, and nodular formations are absent from all the
organs.

Microscopical Examination of the Organs. — The
kidneys are congested from the first day, and already

THE BACILLUS FOR SMALL ANIMALS 157

show evidence of tubal desquamation, which appears
to be most marked in the tubuli contorti. In the
animals that receive a single injection, this inflamma-
tory condition does not increase much in severity, and
in a very short time the organ regains, more or less,
its healthy state. When multiple doses are given, the
changes described above are intensified, the kidneys
show marked haemorrhagic tubal nephritis, and in
cases that have survived for any length of time an
early interstitial nephritis may also be present. The
nodular formations which follow the intravenous
injection of most of the other acid-fast bacilli have not
been observed with Johne's bacillus, and we have been
unable to trace the passage of the bacilli through the
kidney by means of stained sections. However, the
bacilli are undoubtedly excreted by this organ, since
they can occasionally be demonstrated in smears made
from the stringy albuminous material usually present
in the pelvis of the kidney. All attempts to obtain
cultures from the urine have failed, although numerous
specimens were taken from twenty-four hours to two
months after the inoculation of the animal.

Congestion of the lungs is evident after twenty-four
hours, and masses of acid-fast bacilli are found sur-
rounded by a few epithelioid cells. The cells rapidly
increase in number, and small foci appear in the inter-
stices of the alveoli ; these foci resemble on casual
observation the early stage of a miliary tubercle. The
majority of the baciUi are quickly phagocytosed, but
those in clumps remain extracellular, and are sur-
rounded en masse by epithelioid cells, lymphocytes, etc.
The intracellular bacilli are often found in characteristic
wreath-like formations, but they disappear completely
on the tenth to the fifteenth day after the last inocula-
tion. In animals receiving a single injection, the lung

158 JOHNKS DISEASE

gradually resume their normal healthy state, although
in some cases the walls may remain somewhat
thickened. In those that die as a result of multiple
injections, the lungs are more or less completely
solidified, and present a state of static pneumonia.

The spleen is not much affected except for a certain
amount of congestion, although acid-fast bacilli are
present from the first day, and persist for at least
thirty days. From the beginning practically all are
intracellular, but they invariably resist the action of
decolorizing agents, and remain well formed or
become somewhat granular. The Malpighian bodies
are usually quite free from bacilli.

When present in the spleen, the bacilli are found
also in the liver, and may be present from the first
to the twentieth or thirtieth day after the last injec-
tion. They are phagocytosed by the interstitial cells
(Kupffer's cells, sessile macrophages of Metchnikoff),
but the true gland cells remain free from bacilli. Some
investigators have maintained that the liver gland cells
may, under certain circumstances, show phagocytic
properties. C. C. Twort and Craig have obtained
a remarkably clear picture of the phagocytic power of
the interstitial cells, with total inactivity of the gland
cells ; the interstitial cells in many cases are crammed
with bacilli. The liver soon becomes congested, and
from the second to the third day, or even earlier, shows
evidence of degeneration; the protoplasm becomes
granular, whilst the nuclei remain well formed and
stained. The condition is more marked in the hepatic
than in the portal zone, as might be expected from
the accompanying congestion of the organ. About the
third day a lymphatic invasion commences around the
portal vessels and bile-ducts ; but in those animals that
receive only a single injection it is not extensive, and

THE BACILLUS FOR SMALL ANIMALS 159

rapidly disappears, leaving a loose fibrous tissue. On
the other hand, where the injections have been re-
peated, a large proportion of the parenchyma may be
replaced by this loose fibrous tissue, and a general
fatty condition of the remaining liver substance may
supervene.

The intestines, when examined at different levels,
show a complete absence of pathological changes, and,
so far, it has been found impossible to demonstrate the
presence of acid-fast bacilli in this situation.

In animals that receive several injections at long
intervals, the axillary glands may be very congested,
and may show small haemorrhages, while acid-fast
bacilli are often present.

As only a comparatively small number of bacilli
seem to be excreted by the kidney, but, on the other
hand, are rapidly absorbed by the hver, it was thought
that many might pass through the bile-ducts into the
intestines.

In order to prove this the following experiments
were performed :

Two rabbits were inoculated intravenously with
30 milligrammes of the bacilli, one being killed twenty-
four hours, and the other forty-eight hours, after the
inoculation. A few drops of the urine and bile were
placed on to separate tubes of the special medium
necessary for the growth of Johne's bacillus, and incu-
bated at 39° C, while the remainder of each of the
fluids was centrifuged, and the deposit examined
microscopically for bacilli. A careful examination of
the deposits failed to reveal the presence of any micro-
organisms, and the cultures appeared to be sterile at
the end of three weeks. However, a week later
several minute colonies were visible in the tube con-
taining the bile from the rabbit killed forty-eight hours

160 JOHNE^ DISEASE

after inoculation, and, on staining, bacilli with all the
characters of Johne's bacillus were found.

A similar series of experiments, consisting in feeding
rabbits with pure cultures of Johne's bacillus, were
then carried out, but no bacilli could be found in the
bile or urine, and cultures made from these fluids
remained sterile.

The Intraperitoneal Inoculation of Rabbits. — The intra-
peritoneal inoculation of loo to 200 milligrammes
of Johne's bacillus is easily tolerated by rabbits, and
seems to produce no ill-effect upon the general health ;
there is no rise of temperature or loss of appetite
or weight, and no deaths directly due to the bacilli
have been noted. If small quantities of the fluid con-
tents of the peritoneal cavity are pipetted off a few
hours after making the injection, and a microscopical
examination made, there is evidence of leucocytosis,
and the bacilli are found to be phagocytosed, only
a few remaining free after twenty-four hours. Johne's
bacillus, however, has a very great resisting power,
and may be found well stained and well formed inside
the phagocytes for several weeks.

In animals killed four weeks after inoculation all
that can be seen on post-mortem examination is a very
small amount of thick stringy pus in the peritoneal
cavity. Rabbits inoculated into the peritoneal cavity
and kept for two or three months are of special interest,
as they are the first animals in which any evidence of
nodular formation has been found. Usually these
nodules are not numerous, and are limited to the
abdominal cavity. They vary in size from a match-
head to a bean, and are indistinguishable from an
ordinary caseous tubercular mass. The nodules occur
on the under surface of the diaphragm, on the peri-
toneal covering of the liver and spleen, and in the

THE BACILLUS FOR SMALL ANIMALS 161

large omentum ; often there is a fair-sized nodule on
the caecum. The last mentioned is frequently lobu-
lated (like a gland), and may be simply attached to the
intestine by a broad pedicle ; it may, however, be
embedded in the organ, involving the peritoneal coat.

The liver is often somewhat fatty, and on micro-
scopical examination the condition of this organ and of
the spleen is more or less identical with that found
when the intravenous method of inoculation is used.
The distribution of the bacilli in these organs is very
similar ; they are present twenty-four hours after the
inoculation, and, as in the intravenous cases, may
persist for a month. The caseous nodules referred
to often contain enormous numbers of small acid-fast
bacilli, presumably Johne's bacilli ; but it has been
found impossible to recover them in pure culture, due,
no doubt, to their being dead. Sometimes the number
of bacilli is small, as in the intestinal nodule of the
rabbit inoculated intraperitoneally by the authors.

Acid-fast bacilli have not been demonstrated in any
of the remaining organs, all of which appear to be
normal.

The Subcutaneous Inoculation of Rabbits. — The sub-
cutaneous inoculation of Johne's bacillus into rabbits
produces a caseous abscess at the site of inoculation.
These abscesses persist for a great length of time, but
the most interesting feature is the resistance of the
bacilli to destruction in this situation ; they can be
found in large numbers many months after the inocu-
lation, and remain well stained and formed.

If dead bacilli are inoculated in place of living they
show an equally marked resistance to destruction.
Up to the present time not a single acid-fast bacillus
has been found in any of the internal organs of these
animals after subcutaneous inoculation.

II

162

JOHNES DISEASE

Fowls inoculated intravenously with Johne's bacillus
develop lesions more or less similar to those found in
rabbits after intravenous inoculation, but only a very
limited number of these animals have been tested.

A Comparison with Other Acid-fast Bacilli. — C. C.
Twort and T. Craig have performed a considerable
number of experiments on rabbits with other members
of the acid-fast group, their object being to compare
the toxicity of these bacilli with that of Johne's
bacillus. The following bacilli were tested :

1. Bacillus phlei (Moeller).

2. Smegma bacillus (Moeller).

3. Marpmann's bacillus from

urine.

4. Paratubercle bacillus (Binot).

5. Mist bacillus (Moeller).

6. Pseudoperlsucht (Moeller).

7. Nasenschleim bacillus (Kar-

linski).

8. Duval's ''so-called" leprosy

bacillus.

9. Tobler I.

10. Tobler II.

11. Grassberger's bacillus.

12. Fish tubercle bacillus (Du-

bard).

13. Grass bacillus (Moeller).

Nearly a hundred animals were inoculated with
these bacilli, and a subsequent examination of the
organs was made. In the majority of rabbits B. phlei
was used ; only two or three animals were inoculated
with each of the remaining types of bacilli.

It is upon the following points that the chief differ-
ences appear to rest :

1. The general toxicity of the bacilli and the length
of time the animals survive after the inoculation.

2. The power of the bacilli to cause a definite rise of
temperature.

3. The excretion of the bacilli by the kidney, and the
production of nodules in this organ.

4. The production of caseous nodules.

5. The cultivation of the bacilli from the urine of the
inoculated animals.

The bacilli detailed above may be divided into two

THE BACILLUS FOR SMALL ANIMALS 163

groups, according to their toxicity. Nos. i to 7 may
be considered as more or less toxic, and the remainder
as comparatively non-toxic. The description given
belov^ applies entirely to the intravenous method of
inoculation. With Nos. i to 7 there is always a
definite rise of temperature to 105° or 106° F. ; it
reaches 105° F. twenty-four hours after inoculation, and
rises another 0*5° or 1° F. on the following day. On
the fourth or fifth day there is a fairly sudden fall, and
at the end of the week the normal is again reached.
Loss of appetite and consequent wasting are also
present, the latter being in most cases very marked.
The animals usually die within five to ten days,
although they may temporarily recover, and not
succumb until two or three months later, while in
some cases recovery appears to be permanent.

From the second to the third day onwards the
bacilli are excreted in large numbers by the kidneys,
and may be recovered in pure culture from the urine
twenty-four hours after inoculation. The kidneys are
riddled with nodules after the fifth day, these nodules
being formed entirely of lymphocytes, bacilli, and cells
of the involved kidney substance. If the animal sur-
vives, the bacilli rapidly disappear, and the nodules
become absorbed without any caseation. It is worthy
of note that in this situation the bacilli attain a great
length, and are easily decolorized after staining.
Caseous nodules may be found in the lungs, liver,
spleen, and peritoneum, and in some cases nodules
formed of invading lymphocytes are present in the
heart muscle and suprarenal glands. Of the six re-
maining bacilli, Duval's so-called leprosy bacillus is
the only one that produced a temperature of 105° F.,
and this result has been obtained in two rabbits.
Animals inoculated with these six varieties of bacilli

164 JOHNKS DISEASE

do not die, the bacilli being less toxic than Nos. i to 7.
The only varieties that produce nodules in the kidney
are Tobler I. and Grassberger's bacillus, but the
nodules are very few in number. Cultures have been
obtained from the urine with Nos. 9 to 11, whilst those
made from the urine of animals injected with Nos. 8,
12, and 13 have all proved negative. In animals inocu-
lated with the fish tubercle bacillus and with Duval's
bacillus, caseous nodules have been found in the peri-
toneum, but attempts to trace the passage of the
bacilli through the kidney by microscopical examina-
tion of sections of this organ have all given negative
results.

All the bacilli under consideration are more or less
non-toxic if inoculated into the peritoneal cavity or
subcutaneously, and the same is true if they are killed
by heating and injected intravenously. By the term
"non-toxic" is meant that there is no rise of temperature,
or loss of appetite or weight, and the animal does not
die after the inoculation of 100 to 200 milligrammes of
the bacilli. That some of these bacilli are, to a certain
degree, pathogenic for animals if inoculated into the
peritoneal cavity or subcutaneously, has frequently
been demonstrated by many investigators. The lungs
of some of the rabbits inoculated subcutaneously with
B, phlei have consisted, to a large extent, of necrosed
tissue, yet the animals, while alive, appeared to be
quite healthy. In one case hardly one-third of the
normal lung tissue remained.

On reviewing the comparative experiments, one is
forced to assume that the difference between these
acid-fast bacilli is one of degree only ; but in dividing
them into groups, toxic and non-toxic, it is clear, from
what has already been said, that Johne's bacillus is
one of the least toxic of the non-toxic group. When

THE BACILLUS FOR SMALL ANIMALS 165

inoculated intravenously, it is highly probable that all
the varieties are excreted, to some extent at least, by
the kidney. It is easy to understand the negative
cultural results obtained v^^ith the fish tubercle bacillus
and with Moeller's grass bacillus, both of which are
probably rapidly destroyed in the animal body owing
to the existing temperature ; it seems, however, rather
curious that it is impossible to obtain cultures of
Johne's bacillus, since the temperature here does not
come into play. It must be remembered that only a
limited number of animals have been used in these
experiments, so that a certain amount of reserve must
be exercised in drawing conclusions from the results.

The Resisting Pozver of Johne's Bacillus to the De-
structive Agents in the Animal Body. — While showing
a high degree of resistance to decolorizing reagents,
such as mineral acids and alcohol, Johne's bacillus is,
at the same time, very difficult to destroy in the animal
body. As we have seen, when inoculated into the
peritoneal cavity of rabbits, the bacilli are rapidly
phagocytosed ; but they may be found a month later
within the leucocytes, resisting well, after staining,
decolorizing reagents, and being either normal in ap-
pearance, or, at the most, somewhat granular. B. phlei
and the human tubercle bacillus are also rapidly
phagocytosed, but they soon disappear almost entirely
from the peritoneal fluid, and the same has been found
to occur in the peritoneal cavity of mice. Caseous
nodules produced by tubercle bacilli in the peritoneal
cavity contain but few bacilli, while those caused by
Johne's bacillus may be crowded with bacilli. In
rabbits immunized by repeated subcutaneous inocula-
tions of dead human tubercle bacilli, and subsequently
inoculated into the peritoneal cavity with living bacilli
of the same species, or with Johne's bacillus the

166 JOHNE'S DISEASE

tubercle bacilli are found to disappear the more
rapidly. This might be expected, but, on the other
hand, in animals immunized with dead Johne's bacillus,
and then inoculated with one of the two living bacilli
as before, it is again the human tubercle bacillus that
first disappears.

If, as is thought by some authors (Wolff- Eisner, etc.),
the tuberculin reaction be due to the action of the
specific lysin on the tubercle bacilli, or particles of
them, then one would expect the rise of temperature
in the tuberculin test to take place at an earlier hour
than in animals suffering from Johne's disease, and
treated with a diagnostic vaccine prepared from Johne's
bacillus. The contrary, however, seems to take place,
since the tuberculin reaction appears about the ninth
hour to the eighteenth hour, whilst, as we have seen,
the reaction in the case of Johne's disease usually
takes place before the ninth hour. It is, however,
possible that the comparatively early disappearance of
tubercle bacilli is not due to lysis, but to the fact that
they are more toxic than Johne's bacillus to the cells,
which break down more quickly, and liberate the
bacilli, these subsequently becoming disseminated
throughout the animal body. It is also well known
that in an encapsulated caseous nodule tubercle bacilli
are usually not numerous, whilst we have seen that
Johne's bacillus is often present in enormous numbers.
The same appears, in a general way, to be true as
regards the destruction of the bacilli in subcutaneous
abscesses.

That (lysed ?) Johne bacilli are as toxic as the
(lysed?) tubercle bacilli is proved by the general dis-
turbance caused in animals by the inoculation of a
diagnostic vaccine. The same is shown by experi-
ments performed on rabbits that have been immunized

THE BACILLUS FOR SMALL ANIMALS 167

by intravenous inoculation of Johne's bacillus, B. phlei^
or the human tubercle bacillus. Five to ten milli-
grammes of any of these bacilli often produce rapid
death of the animal if inoculated intravenously, while
in many cases a previous high rise of temperature is
noted. This rise of temperature is usually preceded
by a well-marked fall, which occurs during the first
hour or two following the injection. When the inocu-
lation is made subcutaneously, the temperature often
rises but httle, and the experiment never terminates
fatall3^ It requires no larger dose of dead Johne's
bacillus to kill an animal immune to the human tubercle
bacillus than it does of the latter to kill an animal
immune to Johne's bacillus. The bacillary emulsions
have not been accurately titrated to find the minimal
fatal dose, and it is, of course, assumed that the dose
would be smallest in those cases in which the homolo-
gous bacillus is used. Control animals, immunized
with emulsified Dorset's ^gg medium to eliminate any
effect of the tgg albumin in these reactions, were
negative, both as regards the production of a rise in
temperature and the death of the animal.

If mice are inoculated into the peritoneal cavity with
dead Johne's bacillus, B. phlei, or the human, avian, or
bovine types of tubercle bacilli, the most prominent
feature is the more rapid disappearance of the four last-
mentioned varieties as compared with Johne's bacillus.
It is about a week or more after the injection that the
difference is most noticeable. At the same time animals
inoculated with Johne's bacillus do not appear to die
so frequently as when inoculated with the other bacilli.

Experiments m vitro on the toxicity of the bacilli to
guinea-pig's leucocytes are also interesting. The leu-
cocytes can be obtained in the usual way by the in-
traperitoneal injection of Mellin's Food,| or similar

168 JOHNKS DISEASE

substance. They are collected, centrifuged, and
washed, and six drops are added to one drop of a
homogeneous emulsion of the bacillus to be tested,
together with two drops of a normal guinea-pig's serum,
and the mixtures are incubated at 37° C. In every
case, if the emulsion be not too thick, phagocytosis is
complete in about twenty-four hours. The comparative
non-toxicity of Johne's bacillus for the leucocytes is
shown by the fact that the remaining tubes containing
human, bovine, or avian tubercle bacilli, or B. phlei^
soon become contaminated, showing death of the leuco-
cytes ; while the tube containing Johne's bacillus re-
mains sterile for a longer time, and the leucocytes
appear normal. On the other hand, these are partially
degenerated in the tube containing B. phlei, and are
completely so in those containing tubercle bacilli.

From what has been said above it is clear that
Johne's bacillus has a low degree of toxicity, especially
for such animals as rabbits. As we have seen, in
cattle that suffer naturally from Johne's disease toxic
symptoms are very little in evidence, and the tempera-
ture remains constant throughout, showing a lack of
any general disturbance of the animal economy, whilst
the absence of local necrosis seems to indicate that the
bacillus has no very harmful influence on the neigh-
bouring cells. The rise of temperature sometimes
met with in very advanced cases is probably due to a
secondary infection with intestinal micro-organisms
the general resistance of the animal being low owing
to malnutrition. In some cases the number of bacilli
found on post-mortem examination is small, and from
this certain authors have assumed that the symptoms
accompanying the later stages of the disease are
directly caused by highly toxic substances secreted by
the bacilli. The view, however, generally held is

THE BACILLUS FOR SMALL ANLMALS 169

that the toxicity of Johne's bacillus is not great,
and that a diseased animal is but little affected until
the large masses of bacilli, and the pressing of new-
tissue on the glands, lead to derangement of food
absorption, malnutrition, and diarrhoea ; all the obser-
vations and experiments of the authors tend to confirm
this view. In an intestinal disease the number of
factors influencing the general health must be con-
siderable, and, as we have already seen, an exactly
opposite condition of affairs may exist — />., the pre-
sence of a large number of bacilli with a comparatively
robust state of health of the animal.

We have alread}^ seen that certain investigators
reproduced the disease in calves, but the doses of
infected material used were often very large, and some-
times repeated. The experiments of the authors with
pure cultures of the bacillus gave the same results.
To reproduce the disease in a susceptible animal, it
was necessary to inoculate much larger doses of
Johne's bacillus than would have been necessary to
produce tuberculosis with the tubercle bacillus.

But, even when the typical disease is present, it
must be remembered that the bacilli and lesions are
confined to the intestine and glands, and the question
arises as to whether there are not, in the case of
Johne's bacillus, special factors, besides the production
of toxins by the bacillus and the active resistance of
the host, which help to determine its pathogenicity,
and in the case of susceptible animals, its distribution.
It may be that in the body, as in artificial culture media,
special foodstuffs are necessary for its vitality, and
that these exist in sufficient quantity only in the walls
of the intestine and in the mesenteric glands of certain
ruminants. It is to be presumed that such foodstuffs
cannot exist in large quantities, even in the intestine,

170 JOHNE^S DISEASE

otherwise Johne's bacillus would grow on a broth pre-
pared from such tissues, or on pieces of fresh gland
removed aseptically, and this we know is not the case.
What is far more probable is that the necessary food
exists in very small quantity, but is constantly re-
plenished as it is used by the bacillus. As we have
pointed out (Chapter VI.), we have evidence that the
substance contained in the body of B, phlei, which
stimulates the growth of Johne's bacillus in artificial
cultures, is an organic acid ; and it is probable that it
is some such acid — possibly in combination — existing
in the intestinal wall, which stimulates the growth of
the bacillus in this situation. Such an acid might be a
product of metabolism of the neighbouring glands, or
a product of digestion passing through the intestinal
walls, or, indeed, it might be produced from such sub-
stances as grass by the action of certain intestinal
bacteria, and be absorbed by the intestine, thus reach-
ing the vicinity of the baciUi. However this may be,
the facts remain that the disease is confined to the
intestine and mesenteric glands of certain ruminants,
and that a large dose of infective material is necessary
to reproduce the disease experimentally. From this it
seems probable that under good hygienic conditions a
healthy bovine has but little chance of infection if a
single dose of infective material is taken with the food.
Repeated ingestion of infected food, especially by
animals poor in condition, is probably essential for
infection in the majority of cases.

Considering now the case of rodents and birds, we
have already seen that these do not naturally contract
the disease, and that very large doses, and in some
cases repeated doses, are necessary to produce patho-
logical changes ; but even in this case the bacilli
rapidly die, and cannot be recovered in pure cultures.

THE BACILLUS FOR SMALL ANIMALS 171

In rabbits the temperature is never high, and the
absence of other signs of intoxication is a marked
feature. The natural immunity of these animals to
Johne's bacillus is not sufficient to safeguard them
against a fatal termination of the experiment, and we
have seen that the saprophytic B. phlei may cause
rapid death of the animal, although the latter is en-
dowed with an immunity against this bacillus. This
may be appreciated more easily by a brief considera-
tion of what takes place in animals inoculated with
B. phlei. Although B. phlei is a saprophytic micro-
organism, when inoculated intravenously into rabbits
it causes a rise of temperature within the first twenty-
four hours. This is presumably due to a toxin secreted
by the bacillus, the animal possibly producing anti-
bodies to the toxin, which may neutralize it completely
by the fourth or fifth day after the injection, at which
time, as we have seen, the temperature falls more
or less to the normal.

An intraperitoneal or subcutaneous inoculation pro-
duces no rise of temperature. In these cases it is
probable that the toxin is absorbed by the liver or
subcutaneous tissue, and thus does not reach the heat-
regulating centres of the brain. That the crisis is not
caused by the death of the bacilli inside the animal
body, or by the excretion of the bacilli, is proved by
following closely the condition of the kidneys. It is
true that cultures may be obtained from the urine
twenty-four hours after inoculation, but on micro-
scopical examination of the organs the number of
bacilli present is very small, and in some cases they
cannot be found even after a prolonged examina-
tion.

On the other hand, it is not until the third or fourth
day that the kidney becomes invaded by a large number

172 JOHNE'S DISEASE

of bacilli, and the bacilli increase in number up to the
eighth or ninth day, long after the temperature has
fallen. However, it must be remembered that at this
stage, although the bacilli are numerous, they are
surrounded by a dense wall of small round cells, and
it is significant that the appearance of these cells
coincides with the fall of the temperature. A detailed
discussion of these facts is hardly within the scope
of this book ; it only remains to be borne in mind
that such a condition of affairs does not exist when
Johne's bacillus is inoculated. The ultimate death of
the animal appears to be due to the pathological condi-
tion of the kidney.

If now we consider Johne's bacillus, we find a condi-
tion similar to that obtained when using killed B. phlei.
The absence of a rise of temperature is probably due
either to the rapid death of the micro-organism, or to
the lack of formation of any free toxin ; we are inclined
to favour the latter hypothesis in view of the similar
condition found in naturally infected bovines. The
survival of rabbits is undoubtedly due to the main-
tenance of the normal condition of the kidneys, through
which the bacilli are slowly filtered, and owing to the
death of the bacilli, or to their inability to grow in this
situation, the kidneys remain more or less in an
uninjured state.

Thus it appears that although Johne's bacillus is
pathogenic for certain ruminants, it is, nevertheless,
one of the least toxic of the acid-fast group of micro-
organisms, and we have seen that the majority of
animals possess a natural resistance against the
bacillus. We believe, too, from the distribution of
the bacillus in naturally affected animals, and from the
experiments on the cultivation of the bacillus, that the
question of a special food-supply may play an impor-

THE BACILLUS FOR SMALL ANIMALS 173

tant part in determining the pathogenicity of the
bacillus and its distribution in the body of a naturally
affected animal.

Since this book was written C. C. Twort has suc-
ceeded in reproducing in a rabbit a pathological condi-
tion similar to that found in cattle and sheep in the
early stages of Johne's disease. This he has done by
the intravenous inoculation of a culture of Johne's
bacillus which has become acclimatized to grow ^n
ordinary glycerine-beef broth. On post-mortem ex-
amination the mucous membrane in the vicinity of the
ileo-caecal valve was thickened, and the abdominal
lymphatic glands were large and oedematous ; bacilli
were found in both situations, and there was no
evidence of caseation. The disease in the intestine
did not extend more than a foot from the ileo-caecal
valve. It may be noted that the animal was inoculated
with two doses of Johne's bacillus.

BIBLIOGRAPHY

As far as the authors have been able to ascertain, the following
is a complete list of all the publications bearing on the subject
of Johne's disease. It has not been found possible to consult
all the references here given in the original, some being out of
print or unobtainable for other reasons. The references are
given in the alphabetical order of the names of the authors.

Albiex. — " tjbcr die Ziichtung des Erregers der ' Enteritis chronica
infectiosa bovis,' " Berliner Tierarztliche Wochenschrift, p. 793,
No. 41, October, 1910.

Angwin, J. T. — " Johne's Disease in Cattle," Proceedings Southern
Counties Veterinary Association, June 27, 1907.

" Further Notes on Johne's Disease in Cattle," ibid., May 14,

1912.

Arloing. — Compts. rend. Acad, de Science, Mai 9, Mai 13, 1898.
Bang, B.— "Chronische pseudotuberkulose Darmentziindung beim
Rinde," Berliner Tierarztliche Wochenschrift, No. 42, S. 759, 1906.
Proceedings National Veterinary Association, Liverpool, 1906.

" Die spezifische chronische Enteritis des Rindes," Ninth

International Veterinary Congress, Hague, September, 1909.

" Kvaegets smitsomme kroniske Tarmbetaendelse," 66^^ Beret-

ning fra den Kgl. Veterinaer-og Landbohoejskoles Laboratorium,
January, 1910.

Bang, O. — " Om Anvendelse af Tuberkulin af Fjerkraetuberkel-
baciller som diagnostisk Middel ved Kvaegets kron. smits.
Tarmbetaendelse," 66^^ Beretning fra den Kgl. Veterinaer-og
Landbohoejskoles Laboratorium, January, 1910.

Beebe, W. L. — American Veterinary Review, September, 1908.

BONGERT. — I. " Beitrage zur Lehre von der Entstehung der Tuber-
kulose." n. " Die Tuberkulose Infiltration der Darmmukosa
beim Rinde, eine Form von Darmtuberkulose, welche durch
abgeschwachte Saugetiertuberkelbazillen hervorgerufen wird,''
Deutsche Tierarztliche Wochenschrift, S. 229 and 241, Bd. 14^
1906.

175

176 JOHNE^S DISEASE

BoxGERT, J. — Die spezifische chronische Enteritis des Rindes. Ninth

International Veterinary Congress, Hague, 1909.
BORGEAUD. — "Contribution a I'Etude de I'Enterite Chronique des

Bovides," Schweitzer Archiv fiir Tierheilkunde, S. 221, Bd. 47,

1905.

Progres Veterinaire, p. 205, Mars 25, 1905.

BUGGE AND Albien. — " Vorlaufige Mitteilung iiber die Enteritis

chronica bovis pseudotuberculosa," Berliner Tierarztliche

Wochenschrift, No. 10, S. 175, 1908.
BUGGE AND CoRDSEN. — " Einige Beobactungen iiber die Enteritis

chr. bovis pseudotub.," Zeitschrift. fiir Infektionskrankh. parasit.

Krankh. und Hyg. der Haustiere, S. 133, Bd. 5, 1908.
Bulloch and Macleod. — " The Chemical Constitution of the

Tubercle Bacillus," Journal of Hygiene, vol. iv., 1904.
De Jong. — " Intraveneuze injectie van vogeltuberkelbazillen bij

geiten," Veterinaire Pathologic en Hygiene, S. 221, 3^ Reeks,

1905.
Descos. — " La Sero-reaction chez I'Enfant," Jour, de Physiolog. et

de Path. Generale, January, 1903.
De Vine. — " Johne's Disease," Midland Counties Veterinary Associa-
tion Proceedings, May 14, 191 2.
Freger. — " Enterite Chronique Hypertrophiante des Bovides,"

Jour, de Med. Vet. et de Zootechnie, p. 287, tome 57, 1906.
Gray. — " Sarcosporidiosis in Sheep," Wallis Hoare's " System of

Veterinary Medicine," vol. i., p. 1139, 1913.
Hammer, Carl. — " Die Komplementbingungsreaktion bei Tuber-

kulose," Miinch. Med. Wochenschr., No. 32, 1912.
Hertzog. — "Disease-Producing Micro-Organisms," 191 1.
HoLTH. — " Reinziichtung des Bazillus der spezifischen chronischen

Darmentziindung des Rindes (Paratuberkelbazillus)," Zeitschr.

fur. Infekskr. parasitkr und Hyg. der Haustiere, Bd. i, Heft 5.
HORNE. — " Enteritis chronica pseudotuberculosa bovis oder die

Johne'sche Seuche Konstatiert in Norwegen," Norsk Veterinar-

tidsskrift, S. 72, 1908.

Autoreferat in Berliner Tierarztliche Wochenschrift, S. 235

1908.

Tidsskrift for det Norsk Landbrug, S. 139, 1908 ; S. 88, 1909.

Berliner Tierarztliche Wochenschrift, Ss. 192, 329, 1909 ;

S. 109, 1910.
HuRTREL d'Arboval. — Dict. de Med. et de Chir. Vet., vol. i., p. 516,

1826.
JOHNE AND Frothingham. — " Ein cigentiimlicher Fall von Tuber-

kulose beim Rinde," Deutsche Zeitschr. fiir Tiermedizin und

vergleich. Path., S, 438, Bd. 21, 1895.

BIBLIOGRAPHY 177

JowETT. — " Coccidiosis of the Fowl and Calf," Journal of Compara-
tive Pathology, vol. xxiv., part 3, 191 1.

Koch, M., and Rabinowitsch.— " Die Tuberkulose der Vogel und
ihre Beziehungen zur Saugetiertuberbulose," Virchow's Archiv,
Bd. 190, S. 246, 1907.

Koch and Romberg. — Deutsche Med. Wochenschr., Nos. 18, 19, 1901.

Leclainche. — " L'EnteriteChronique Hypertrophiante des Bovides,"
Revue Generale de Med. Vet., p. 305, tome 9, 1907.

LiENAUX. — "Considerations en faveur du Saprophytisme Initial
probable du Bacille Tuberculeux Bovin," Annales de Med. Vet,,
p. 598, 1905.

" Sur la Nature Tuberculeuse de I'Enteritc Hypertrophiante

diffuse du Boeuf : Nouvelles Experiences," Annales de Mdd.
Vet., p. 433, 1907.

" Johne's Disease in the Horse," Veterinary Record, April 19,

1913 (abstract from Annales de M6d. Vdt.).
LiENAUX AND VAN DEN Eeckhout. — " Contribution a I'Etude d'une

Enterite Tuberculeuse speciale et de la Diarrhee chronique du

Boeuf," Annales de Med. Vdt., 1905, pp. 65 et 125.
LuDWiG. — " Tuberkulose Infiltration des Darmes." Bericht iiber das

Veterinarwesen in Konigr. Sachsen fiir das Jahr., 1904, S. 82.
M'GowAN AND Rettie. — " Sarcocystis tenella in Sheep," Proceedings

Pathological Society of Great Britain and Ireland, January, 191 3.
M'Fadyean. — " Johne's Disease : a Chronic Bacterial Enteritis of

Cattle," Journal of Comparative Pathology, p. 48, 1907.

Annual Report for 1907 of the Principal of the Royal

Veterinary College, Journal of the Royal Agricultural Society of
England, vol. 68, 1907.

M'Fadyean, Sheather, AND Edwards. — "Johne's Disease: (i) Re-
cords of Examinations of Natural Cases ; (2) Cultural Characters
of the Bacilli," Journal of Comparative Pathology, vol. xxv.,
part iii., September, 191 2.

Male, G. P. — "The Clinical Aspect of Johne's Disease and the Avian
Tuberculin Test," Royal Counties Veterinary Association,
April 28, 191 1. (Published in Vet. News, May, 191 1.)

Malm, O. — " Om den specifike kroniske tarmbetaendelse hos
kvaeget," Norsk Veterinaer-Tidsskrift, No. 8, August, 191 1.

Medicinsk Revue (Bergen), 191 1.

Referat. Deutsche Tieriirzthche Wochenschrift, No. 37, 191 2.

Mathis. — " Lesions de la Diarrhee chronique des Bovides," Bulletin

de la Soc. des Scien. Vet. de Lyon, No. 2, 1906.
Markus, H. — "Een Specifieke Darmontsteking bij het Rund, Waar-
schijnlijk van Tuberkuleusen aard," Tijdschriftvoor Veeartsenij-
kunde, 1903, Deel 30, Bldz, 195.

12

178 JOHNE'S DISEASE

Markus, H. — " Eine Spezifische Darmentziindung des Rindes
wahrscheinlich Tuberkul5ser Natur./' Zeitschr. fiir Tiermediziii,
1904, Bd. 8, S. 68.

" Die Spezifische Chronische Enteritis des Rindes," Ninth

International Veterinary Congress, Hague, 1909.
Mellon and M'Ginnis. — "Johne's Disease," Journal of the Depart-
ment of Agriculture for Ireland, No. 2, vol. xiii., January, 1913.
Melvin. — "Chronic Bacterial Dysentery," Annual Report, Bureau of

Animal Industry, U.S.A., 1909-10.
Meyer. — " Uber die durch Saurefeste Bakterien Hervorgerufene
diffuse Hypertrophie der Darmschleimhaut des Rindes," Arbeiten
aus dem Inst, zur Erforschung. der Infekskr. in Bern aus den
Laborat. des Schweizer. Serum u. Impfinst., Heft 2, 1908.
" Bacillus Para-Tuberculosis Johne," Proceedings Phila-
delphia Pathological Society, October, 191 2.
MiESSNER. — " Der Infektiose Darmkatarrh des Rindes," Ninth

International Veterinary Congress, Hague, 1909.
MiESSNER AND KoHLSTOCK. — " Immunisicrungsversuche beim Chroni-
schen Infektiosen Darmkatarrh," Berliner Tierarztliche Wochen-
schrift, November 25, 1912.
MiESSNER AND Trapp. — " Der Chronische Infektiose Darmkatarrh des
Rindes," Mitt. d. Kaiser Wilh. Institut fiir Landwirtsch. in Brom-
berg., 1910, Bd. 2, Heft 3, pp. 219-286.
Nocard. — "Tuberculosis," Diet, de Med. Vet., etc., Bouley et Reynal

vol. xxi., p. 422.
Pearson. — " The Presence of Chronic Bacterial Dysentery in Cattle
in America," American Veterinary Review, vol. xxxii,, No. 5,
February, 1908.
Penberthy. — "Johne's Disease," Journal of Bath and West of

England Agricultural Society, January, 1913.
Reichel and Deubler. — " Examination of the Fasces of Cattle for
Tubercle Bacilli," Journal of Medical Research, vol. xxiv.,
January, 191 1.
RiECK. — " EigentiimHche Darmtuberkulose," Bericht iiber das

Veterinarwesen im Kgr. Sachs, 1896, S. 118.
Schmaltz. — Berliner Tierarztliche Wochenschrift, No. i, 1909.
Sluys, van der. — Generalversammlung der Niederlandischen Tier-

arzte in Utrecht, September, 1902.
Stockman. — " Johne's Disease," " Scrapie in Sheep," Reports of
Chief Veterinary Officer to the Board of Agriculture, 1909, 191 1.
"Johne's Disease in Sheep," Journal of Comparative Path-
ology, vol. xxiv., March, 191 1.
Stuurman. — " Die spezifische hypertrophische Darmentziindung des
Rindes," Ninth International Veterinary Congress, Hague, 1909.

BIBLIOGRAPHY 179

TowNSEND, C. W. — "Johne's Disease in Cattle," Proceedings
Lincolnshire Veterinary Association, June, 1909.

TwoRT, C. C. — " The Agglutination and Complement-Fixation Re-
actions in Animals Experimentally inoculated with Johne's
Bacillus, etc.," Centralblatt fiir Bakt. Originale, Bd. 66, 1912,
Heft 2-4.

TwoRT, C. C. AND Craig, T.— "The Pathogenicity of Johne's
Bacillus compared with that of Other Acid-Fast BacilH for Some
of the Laboratory Animals," Centralblatt fiir Bakt. Originale,
Bd. 68, 1913, Heft 5-6.

TwoRT, F. W.— " The Fermentation of Glucosides by Bacteria of the
Typhoid Coli Group and the Acquisition of New Fermenting
Powers by Bacillus dysenteric^ and Other Micro-organisms," Pro-
ceedings Royal Society, B., vol. 79, 1907.

" The Influence of the Glucosides on the Growth of Acid-
Fast Bacilli and a New Method of isolating Human Tubercle
Bacilli directly from Tuberculous Material contaminated with
Other Micro-Organisms," Proceedings Royal Society, B.,
vol.81, 1909.

A Method for isolating and growing the Lepra Bacillus of

Man," Proceedings Royal Society, B., vol. 83, September,
1910.

TwoRT, F. W. AND Ingram, G. L. Y. — '' A Method for isolating and
cultivating the Mycobacterium enteritidis chroniccB pseudotuber-
culoscB bovis Johne, and some Experiments on the Preparation
of a Diagnostic Vaccine for Pseudotuberculosus Enteritis of
Bovines," Proceedings Royal Society, B., vol. 84, 1912.

" Further Experiments with the Mycobacterium

enteritidis chronias pseudotuberculoses bovis Johne, and with
Vaccines prepared from this Micro-Organism," Centralblatt fiir
Bakt., Bd. 67, Hefts, 1912.

" Further Researches on Johne's Disease," Veterinary

Record, September 14, 191 2.

" Johne's Disease in Sheep," Veterinary Record,

April 12, 1913.
VUKOVic. — Quoted by Bang, 66*^^ Beretning fra den Kgl. Veterin-

aer-og Landbohoejskoles Laboratorium, p. 39.
Wolff-Eisner, A.—" Friihdiagnose und Tubcrkulose-Immunitat,"

Wiirzburg, 1909.

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CALKIN'S Protozoology.

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FLEMING'S Text-Book of Veterinary Obstetrics.

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FLEMING'S Text-Book of Operative Veterinary Surgery.

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GALVAYNE'S XXth Century Book on the Horse.

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An Abridged List of IVorks Published by

GRESSWELL S Veterinary Pharmacopoeia and Manual of

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HOARE'S Manual of Veterinary Therapeutics and Phar-

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KITT S Text-Book of Comparative General Pathology.

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LANDER'S Veterinary Toxicology.

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LAVERAN & MESNIL'S Trypanosomes and Trypanosom-
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LAW'S Text-Book of Veterinary Medicine.

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LITTELJOHN'S Meat and Its Inspection.

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MAYALL'S Cows, Cow-houses, and Milk.

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MAYALL'S Sheep and Goats.

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MERILLAT'S Animal Dentistry and Diseases of the

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MERILLAT'S Principles of Veterinary Surgery.

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MULLER'S Diseases of the Dog and their Treatment.

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NEUMANN'S Treatise on the Parasites and Parasitic

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NUNN'S Veterinary Toxicology.

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OSTERTAG'S Handbook of Meat Inspection.

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REEKS' Common Colics of the Horse.

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REEKS' Diseases of the Horse's Foot.

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ROBERTSON'S Meat and Food Inspection.

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SESSIONS' Cattle Tuberculosis.

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SMITHS Manual of Veterinary Physiology.

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SMITH'S Manual of Veterinary Hygiene.

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Aft Abridged List of Works.

SMYTHE'S Veterinary Parasitologfy.

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THOMPSON'S Elementary Lectures on Veterinary

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WILLIAMS' Principles and Practice of Veterinary

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WINSLOW'S Veterinary Materia Medica and Therapeutics.

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WOODRUFF'S Economics of Feeding- Horses.

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TROPICAL VETERINARY BULLETIN.

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