PATHOLOGY AND BACTERIOLOGY OF ABORTION AND PERINATAL

\

DEATH OF YOUNG IN RABBITS, SHEEP AND GOATS
INDUCED BY LISTERIA MONOCYTOGENES

By
Chintamani Singh

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Pathology

1956

VITA
Chintamani Singh
candidate for the degree of
Doctor of Philosophy

Final examination: 10 A.M., May 22, 1956, Room h57, Giltner
Hall

Dissertation: Pathology and Bacteriology of Abortion and
Perinatal Death of Young in Rabbits, Sheep
and Goats Induced by Listeria monocytogenes.

Outline of Studies:

Major subject: Animal Pathology
Minor subjects: Microbiology and Public Health, Animal
Husbandry

Biographical Items:

Born; November 30, 1922, Belawon, Muftiganj, Jaunpur,
U. P., India.

Undergraduate Studies: Intermediate Science (Biology),
Udai Pratap College, Banaras,
U.P., India, 19h}; Graduate of
Bihar Veterinary College, Patna,
India, 19h?

Graduate Studies: Master of Science, Michigan State
College, 1953

Experience: Veterinarian in charge civil Veterinary
Hospital, U.P., l9h7-l9h9; in charge Circle
Laboratory, Office of the Deputy Director,
Animal Husbandry, Allahabad, U.P., India,
l9h9-1950; Instructor, U.P. Veterinary
College, Mathura, U.P., India, 1950-1952;
Research Assistant, Livestock Research
Station, Mathura, U.P., India (about a
month); Graduate Assistant, Department of
Animal Pathology, Michigan State University,

Member of Indian Science Congress, Indian Veterinary
Association, Uttar Pradesh Veterinary Asso-
ciation, Sigma Xi, Phi Zeta, Michigan Branch
Society of American Bacteriologists.

ACKNOWLEDGMENTS

The author is deeply grateful to many persons and organi-
zations and for many kinds of assistance and encouragement.
They include particularly: Dr. C. C. Morrill, Head of the
Department of Animal Pathology for inspiration and guidance
in the histopathologic aspects of this investigation and in-
valuable help in the preparation of this manuscript; Dr. M.
L. Gray, Assistant Professor of Animal Pathology for his
unusual interest and understanding guidance in the experi;
mental phases and bacteriological aspects of this investi-
gation and considerable assistance in the preparation of
this manuscript including the figures; Dr. R. A. Runnells,
Professor and former Head of Department of Animal Pathology
for his understanding and encouragement; the late Dr. Frank
Thorp Jr. for his invaluable help and guidance during the
early part of the investigation; Dr. D. H. McWade, Depart-
ment of Animal Pathology for performing a Caesarian section
on one of the experimental goats; Dr. R. H. Nelson, Head,
Department of Animal Husbandry, and Dr. w. N. Mack, Professor,
Department of Microbiology and Public Health for their help-
ful suggestions; Dr. Hans Ruhland, Pathologist, Michigan
Department of Agriculture, for his invaluable help in the

many German translations; Mrs. Marie Kalivoda, librarian,

ii

I“

for her help in obtaining necessary reference material; Mrs.
Betty Beaton for typing the many necropsy reports; all the
other staff members of the College of Veterinary Medicine
and other colleges of this University who contributed so
unselfishly to the author's training and experience or gave
technical assistance during this investigation.

Thegauthor greatly appreciates the financial support
of scholarships and an assistantship provided by Michigan
State University and a grant from the Chas. Pfizer Company,
Inc., Terre Haute, Indiana, for partial support of the cost
of this investigation.

The author is highly grateful to Mr. and Mrs. E. D.
Harrison and other friends of the East Lansing community

under whose unfailing care he found a home away from home.

iii

I.
II.
III.
IV.

TABLE OF CONTENTS

PAGE
INTRODUCTION 1
HISTORICAL BACKGROUND 3
REVIEW OF LITERATURE 9
EXPERIMENTS 22
A. Rabbit Experiments 22
l. Conjunctival EXposure 22
a. Materials and Methods 22
b. Results: Bacteriology 25
c. Results: Pathology 30
2. Oral Exposure 52
a. Materials and Methods 52
b. Results: Bacteriology 55
c. Results: Pathology 56
3. Conjunctival Exposure of Rabbits Surviving
Previous Oral EXposure 78
a. Materials and Methods 78
b. Results: Bacteriology 78
c. Results: Pathology 80
h. Oral Exposure of Rabbits Surviving
Previous Conjunctival Exposure 80
a. Materials and Methods 80
b. Results: Bacteriology 81
c. Results: Pathology 82
S. Rebreeding of Infected Rabbits Without
Any Further Exposure 8h
a. Materials and Methods 8h
b. Results: Bacteriology 8h
c. Results: Pathology 85
B. Sheep Experiments 90
1. Oral Exposure 90
a. Materials and Methods 90
b. Results: Bacteriology 90
0. Results: Pathology 91

iv

 

 

.ll

 

 

TABLE OF CONTENTS (Cont.)

 

Page
C. Goat Experiments 97
l. Conjunctival Exposure 97
a. Materials and Methods 97
b. Results: Bacteriology 97
0. Results: Pathology 99
2. Oral EXposure ‘ 103
a. Materials and Methods 103
b. Results: Bacteriology 103
c. Results: Pathology 10h
V. DISCUSSION 112
VI. CONCLUSIONS 123
VII. SUMMARY 127
BIBLIOGRAPHY 132

 

ill

 

 

TABLE

I.

II.

III.

IV.

V.

VI.

VII.

VIII.

IX.

LI ST OF TABLES

Reports of Isolations of‘L. monogytogenes
from Abortions in Cattle . . . . . . .
Reports of Isolations of‘g. monogytggenes
from Abortions in Sheep and Goats . . .

Reports of Isolations of L. monogytogenes

from Perinatal Death of Young Animals (Calves,

Lambs, Foals, Pigs) . . . . . . . . .

Reports of Isolations of.§. monocytogenes from
Perinatal Death (Human Infants) . . . . . . .
Isolations of‘L. monocytogenes from Aborted

Fetuses Following Conjunctival Exposure of

Rabbits O O O O O O O I O O O O O O

Isolations of L, monocytogenes from Rabbits

(2-3 Weeks Pregnant) Following Conjunctival

Exposure 0 O O O O O I O I O O O O O

Isolations oflg. monocytogenes from Perinatal

Death of Young Following Conjunctival Exposure

of Rabbits . . . . . . . . . . . . . .

Isolations of L. monocytggenes from Non-Pregnant
Rabbits Following Conjunctival Exposure .
Isolations of L. monocytogengg from Aborted

Fetuses Following Oral Exposure of Rabbits

vi

PAGE

10

ll

12

13

26

27

28

29

57

 

LIST OF TABLES (Cont.)
TABLE Page
X. Isolations of L. monocytogenes from Rabbits
(2-3 Weeks Pregnant) Following Oral Exposure . 58
XI. Isolations of L. monocytggenes from Perinatal
Death of Young Following Oral EXposure of
Rabbits . . . . . . . . . . . . . . . . . . 59
XII. Isolations of L. monocytogenes from Young Rabbits
(Perinatal Death) Following Conjunctival Exposure

of the Dam Subsequent to Previous Exposure . . 79

vii

I. INTRODUCTION

The isolations of Listeria monocytogenes from various
clinical syndromes (meningo-encephalitis, septicemia, kerato-
conjunctivitis, abortions and perinatal death of young) in
30 species of animals including man have been reported from
31 countries all over the world. Attention has been drawn
particularly to recent epidemics of perinatal death (still-
birth and early death) of infants due t°.£° monocytogenes
in both sectors of Germany, where listeriosis has now been
designated a reportable disease.

In addition to infection in the reproductive system of
the human female, abortions and perinatal death of young
caused by L. monccytoggnes have been reported in cattle,
sheep, goats, swine, rabbits and horses.

This investigation was designed to study the role of
L. monocytogengg in abortions and perinatal death of young
iJI animals and with the hope that it might shed some light
cui the pathogenesis of the condition in man. The experiments
were conducted primarily on rabbits, sheep and goats and two
Immites of exposures (conjunctival and oral) were used. Con-
juuurtival exposure was chosen from academic interest to in-
vestigate the role of L. monocytogenes in the so-called

local eye reaction and the possibility of its dissemination

 

 

 

 

to other parts of the body following conjunctival instilla-
tion. Oral exposure was conducted because it is one of the
most common avenues of bacterial infection and thus might

be the route used by L. monogytogenes in causing the clinical
syndromes of abortion and perinatal death of young in both
animals and man.

While attempts were made to study infection by‘L. mggg;
cytogenes through these portals of entry, it was also felt
that an attempt should be made by histopathologic studies
to elucidate the pathogenesis of this condition.

Therefore bacteriology and pathology were employed

as companion studies in the eXperiments reported here.

 

 

31v: '1"

i:

II. HISTORICAL BACKGROUND

In May of l92u, Murray, Webb and Swan noticed six
cases of sudden death in young rabbits in the animal breed-
ing establishment of the Department of Pathology at Cambridge

University. Many more deaths followed in the subsequent 15

months. They isolated a small Gram-positive motile rod

which reproduced the disease characteristically in rabbits,
guinea pigs, and mice. The most striking character of this
disease was found to be the production of a marked monocytosis
and, as no reports of a similar microorganism could be found,
these authors called the microbe Bacterium monocytogenes in
1926.

Working independently, Pirie discovered the same bac-
terium in four wild gerbilles (Tatera lobengulae) in South
.Africa. Because of the marked necrosis of the liver pro-

<hiced in these animals, he called the bacterium Listerella

ihepatolytica in 1927 in honor of Lord Lister, the well-known

pioneer of modern aseptic surgery. As the generic name

Listerella had already been used for a mycetozoon (a group

 

cu? slime molds), and as the generic name Bacterium, used by
Murray 9_t_ 2.];- (1926) was not suitable because this micro-
organism did not possess the characteristics of the genus

Bacterium, the International Rules of Nomenclature invalidated

 

 

 

[II

 

the generic names given to this microbe. In 19h0, Pirie
proposed the generic name Listeria which was adopted in the
sixth edition of Bergey‘s Manug;.g§ Determinative Bacteriology
(19h8). This Manual describes L. monocytogenes as: small rods,
0.h to 0.5 by 0.5 to 2.0 microns with rounded ends, slightly
curved in some culture media; occur singly or in V-shaped

or parallel pairs and in short chains; motile, peritrichous,
with four flagella at ordinary temperature, with tendency
toward non-motility or single flagellum at 37° C; not acid-
fast; Gramppositive.

The bacterium also is urea-, indol-, V.P.- (Voges-
Proskauer test), M.R.- (Methyl red test), nitrite-, citrate-,
HZS" and geIatin-negative. It is catalase-positive; non-
sporing; non-capsulated; facultative anaerobic. It grows well
on tryptose agar with dew-drop-like colonies which appear
small, circular, slightly raised, finely textured, translu-
cent, and blue-green in color when examined by means of
<3blique1y transmitted light and a binocular scanning micro-
scope. (Fig. 1)

Murray (1953) reported that almost exactly twenty years
trfter the publication of his original description of this
rmicroorganism in 1926, he found a paper published in Sweden
by Hulpher in 1911. In this paper, Hulpher described a micro-

organism (which was undoubtedly L. monocytogenes) and, struck

 

s:

by the liver necrosis associated with it, he named it
Bacillus hepatig.

The first isolation of L. monocytogenes from a human
case of infectious mononucleosis was reported by Nyfeldt(l929).
Girard and Murray (1951) reviewed the literature and discussed
the relationship of L. monocytogenes to infectious mononu-
cleosis in humans. Burn (1936) was the first to report the
isolation of this bacterium from three cases of meningitis
in infants under one year of age. Since then many cases of
meningo-encephalitis associated with L. monocytogenes in
humans have been reported in the literature. Those reported
until 1952 were reviewed by Finegold gt 2;. (195h).

The first report of the isolation of L. monocytogenes
from sheep was published by Gill (1931) from an outbreak of
so-called "circling disease" in New Zealand. He isolated
the bacterium from the brain of the infected sheep and estab-
lished its relationship to the disease. Gray (195h) cited
that Pletnov and Stiksove (1950) claim the isolation of L.
monocytogenes from pigs in Russia as early as in 19211. ..

Graham 23 51. (1939) were the first to report the isola-
tion of _L_. monogtogenes‘ from an aborted bovine fetus. Since
then, abortions in other species of domestic animals, sheep,
goat, swine, horse and perinatal‘death of calves, lambs,

foals and pigs have been reported from all over the world

(Tables I, II and III).

 

 

HI

 

Graham, Levine and Morrill (l9h3) published a bulletin
"Listerellosis in Domestic Animals" reviewing previously
published cases in animals and presenting the results of
their field and laboratory investigations.

Gray (l95h) reviewed the literature pertaining to
various forms of listeric infections in animals and man.

He presented the isolations of L. monocytogengg from various
clinical syndromes in several species of animals and man in
tabular form as a readily available source of bibliographic

material. He also reported at least 20 different rough

colonial variants which develop in smooth cultures maintained

on artificial media. (Fig. 2)

Boise, Potel and Krebs (1951) described a disease in
human infants which they called "granulomatosis infant-
iseptica'. From.the visceral organs they isolated a small

Gramppositive rod which they called Corynebacterium infanti-

septica. (Potel, 1952). Seeliger (1952) suggested that this
bacterium was actually L. monocytogenes. This subsequently

 

has been confirmed by a number of investigators. It is now

generally conceded that this disease is identical to that
which was formerly known as “pseudo-tuberculosis" of the
newborn. This disease appears to be most prevalent in
Central Europe particularly in Eastern Germany and Czecho-
slovakia. Recently a similar condition has been reported

in Canada and in the United States. (Table IV).

 

Seeliger (1955), surveying the literature, reported
the following recorded cases of human listeric infections:

Probable cases since 1891 . . . . . 56

Cases reported between 1918-1939 . . 29

Cases reported between l9u0-19h9 . . NO

Cases reported since l950..more than 221

He also summarized the cases according to the reported
isolation of'L. monocytogenes from various clinical syndromes
as follows:

Heningo-encephalitis . . . . . . . . 85 cases

Infectious mononucleosis . . . . . . hl cases

Conjunctivitis . . . . . . . . 13 cases

Septic granulomatosis

Adults . . . . . . . . . . . . . h cases
Newborn . . . . . . . . . . . . 121 cases
Other manifestations . . . . . . . . 26 cases

(urethritis, endocarditis, pneumonitis)

The most recent and comprehensive review of literature
pertaining to the isolation of L, monocytogenes from various
'manifestations of disease in man and animals and other re-
lated.studies of the bacterium has been published in book
form under the title "Listeriose" by Seeliger (1955). This
book:a1so includes the reviews and discussions on diagnosis,

differential diagnosis, pathogenesis, transmission, public

health relationship, prophylaxis, treatment, and other phases
of the disease.

Murray (1955) stated that L. monocytogeneg has been
isolated and identified in a minimum of 27 species of animals
(rabbit, hare, guinea pig, gerbille, lemming, mouse, rat,
hamster, chinchfitut vole, sheep, goat, cow, pig, horse, fox,
dog, ferret, raccoon, chicken, canary, duck, goose, eagle,
capercailzie, unspecified birds, and man). To this list
should be added mink (Stenberg, 1955), Skunk (Bolin, gt 5;.,
1955), and owl (Reed, 1955). ‘

Murray (1955) also stated that the distribution of
listeriosis in various forms has been reported from.26
countries in five continents, ranging from the Arctic to
the tropics. To this list should be added Azerbaijan
(Khalimbekov, 1952), Belgium (Geurden and Devos, 195h).
Ceylon (Bandarnayake, 1953), Czechoslovakia (Pouska, 1950),
and Puerto Rico (Felsenfeld, 19MB). Thus the list comprises

at least the following 31 countries:

1. Argentina 12. England 22. Norway

2. Australia 13. Finland 23. Palestine

3. Austria 1h. France 2h. Poland

h. Azerbaijan 15. Germany 25. Puerto Rico
5. Belgium. 16. Holland 26. Russia

6. Brazil 17. Hungary 27. Scotland

7. Canada 18. India 28. South Africa
8. Ceylon 19. Italy 29. Sweden

9. Cuba 20. Japan 30. United States
10. Czechoslovakia 21. New Zealand 31. Uruguay

11. Denmark

III. REVIEW OF LITERATURE

The whole scope of infections due to _I_.. monocytogeneg

 

has been reviewed adequately by a number of workers as men-
tioned in the historical background. The reports reviewed
here are mainly concerning problems of this study.

In general, the encephalitic form of listeric infec-
tion is more commonly reported in mature large animals,
particularly ruminants, and man. The septicemia form.is
more frequently found in rodents, chickens and in aborted
fetuses, stillborn, premature, or newborn young animals and
man. However, there is a strong suspicion that atypical

or subclinical infections may occur both in animals and

man 0
Gray (l95h) in a review tabulated the known isolations

of _I_-_. monothogengg. This included all forms of the disease
in all species. He reported that symptoms of the encephalitic
:form.of listeric infection in sheep, goats and cattle are
similar but differ in severity. The following symptoms are
common: nasal discharge, inappetence, fever, leaning or
pushing against an object, "circling”, general depression

01- stupor, incoordination, torticollis, difficulty in
swallowing due to paralysis of facial and throat muscles,

uncontrolled running motions, complete prostration and death.

 

10

Gray (195k) reported that listeric abortions in cattle
usually occur between the hth and 7th months of gestation
and in sheep at or near term. Isolations of L; monocytogeneg
can be made from the liver, blood, fetal membranes and es-
pecially from the abomasal contents of the fetus. Positive
diagnosis depends upon such isolation. The following tables
are based upon the review of Gray (195h) with subsequent

additions of new reports as they appeared in the literature.

TABLE I

REPORTS OF ISOLATIONS OF‘L. MONOCYTOGENES
FROM ABORTIONS IN CATTLE

 

 

 

Author Year Country
Graham 33 51. 1939 U.S.A.
Evans and Sawyer l9h2 U.S.A.
'Uramby 19u2 Sweden
Olson 19h5 Sweden
Ferguson 23; 51. 1951 U.S.A.

Van Dorssen and Jansen 1951 Netherlands
Levi g}; 31. 1952 Israel
'Van Ulsen 1952 Netherlands
Eveleth gt; §_1_. 1953 U.S.A.
Annanzhulov 1953 Russia
Stockton 23.". 31. 1951+ Russia
Gray'gt £1. 19514. U.S.A.
De Lay and Shultz 1951i U.S.A.
Rowsell (unpublished) 1955 Canada
1r.d4 Schaaf (unpublished) 1955 Netherlands
Smith 21.; 2;. 1955 U.S.A.

 

——

TABLE II

11

REPORTS OF ISOLATIONS OF L. MONOCYTOGENES

FROM ABORTIONS IN SHEEP

 

 

 

 

Author Year Country
Paterson 19h0 England
Poppensiek l9hh U.S.A.
Olson 19h5 Sweden
Charles 1950 Australia
Eveleth'gg‘gl. 1953 U.S.A.
Amanzhulov 1953 Russia
Paulsen and Moule 1953 Australia
Hindmarsh and Blumer (1932)

(Cited by Paulsen and Moule, 1953) 1953 Australia
Stenberg I l95h Finland

Abortions in Goatg
Amanzhulov 1953 Russia

 

Gray‘s; 2;. (1956) reported a condition in cattle which

appears to be similar to the epidemic of perinatal death

of human infants reported by Potel (1953).

They observed

cases in which calves were born at or near term.but sur-

'vived.only for a few days. These calves were very weak

and showed marked bloody diarrhea. L. monocytogengg was

:isolated from.the livers of 6 calves.

12

Gray (195h) reviewed the incidence of perinatal death

of young in animals and man due to L. monocytogengg. The

following tables are based on this review with subsequent

additions of new reports which appeared in the literature.

TABLE III

REPORTS OF ISOLATIONS OF L. MONOCYTOGENES
FROM PERINATAL DEATH OF YOUNG ANIMALS

 

 

 

Author Year Country
. 9.9.1.122
Harbour l9h1 England
wramby l9hh Sweden
Rubarth and Hollarz 1951 Sweden
Gray 22,5}. 195h U.S.A.
Lambs
Jepsen 19h2 Sweden
Gray 22_§;. l9u9 U.S.A.
Stenberg 1953 Finland
Ms.
Krage l9hh. E. Prussia
Svenkerud l9h8 Norway
.De Isy'and Shultz l95h U.S.A.
Elsa
de Blieck and Jansen 19112 Netherlands
Kerlin and Graham 191.15 U.S.A.
Solomkin 1951; Russia
De Lay and Schultz 195)... U.S.A.

 

TABLE IV

13

REPORTS OF ISOLATIONS OF L. MONOCYTOGENES

FROM HUMAN INFANTS (PERINATAL DEATH)

 

 

—_’ —-—‘_—_‘:~

ti

 

Author Year Country
Tompkins 195 2 U . S .A .
Alex and Potel 1953 E. Germany

. Erdmann and Potel 1953 E. Germany
Hagemann and Simone 1953 E. Germany
Linzenmeier .e_t_ g}. 1953 W. Germany
Starck 1953 E. Germany
Potel 1953-5b, E. Germany
Patocka 23 El- 1953 Czechoslovakia
Stoot l95h Canada
De Lay and Shultz 1954 U.S.A.
Breunning and Fretzsche 19511 E. Germany
Portero and Despirito 19514 U.S.A.
Hahnefeld and Nisolk 19514 E. Germany
Martinek 19511 E. Germany
Detmold and Girgensohn 195Li E. Germany
Norman 1955 U .S .A .
Jormston gt 4. ' 1955 Canada

Reed _e__1; §_1_. 1955 Canada

Greey (Cited by Johnston 23 EL.) 1955 Canada

 

ll

 

v

 

Newborn human infants are either stillborn, or born
prematurely or die a short time after birth. Usually they
are weak and develop cyanotic attacks before death. It is
interesting to note that although perinatal infection is
almost 100 percent fatal, both in animals and humans, the
mothers show only transient or inapparent symptoms. Some
women complain of such nonspecific symptoms as headaches,
stomach-ache and fever 1-3 weeks before parturition and in
some, the movements of the fetus may decrease.

There are no reliable clinical tests for the detection
of listeriosis in its early stages or identification of pos-
sible carrier states. Hematological pictures, especially
in ruminants, do not give any indication of the specificity
of the disease. In some cases acetone is reported to be
present in the urine, which further confuses the disease
'with other symptomatically similar diseases, particularly
acetonemia, in cattle and sheep.

The significance of the triple-phosphate-producing
properties of this bacterium in edematous fluids as pointed
out by Hurray (1953) is still unknown. Further biochemical
studies on the peritoneal, pleural and pericardial effusions
which clot on exposure to air are needed.

Murray g£_gL. (1926) described greyish white foci of
necrosis in the liver of rabbits and guinea pigs. Jansen

(1915) (reported necrotic endometritis in a pregnant rabbit which

15

died after aborting a fetus. There are often no detectable
gross lesions found in animals which die of listeric en-
cephalitis. Congestion of the brain may be found in some
cases but is variable. Gray (l95h) pointed out that slight
clouding of the meninges or pin-point grayish white foci in
the brain may be seen rarely. He also reported that lesions
in other viscera are rare but occasionally fatty liver,
duodenitis or pulmonary edema are seen.

Pallaske (l9h0) reported marked focal necrosis of the
liver in an adult sheep. No such lesions have been reported
in adult cattle.

In listeric abortions in animals, pus in the uterine
cavity and vaginal discharge may be noticed in some cases.
There are usually no detectable gross lesions in aborted
fetuses. However, Smith at 31, (1955) reported a small
amount of yellow flocculent material in the pleural and
peritoneal fluid of aborted fetuses.

Harbour (l9h1) isolatedlL. monocytogengg from a calf
'which.showed focal hepatic necrosis and ulcerative enteritis.
This calf had diarrhea and died 9 days after birth.

Gray 33 Bl- (l9h9) isolated L. monocytogenes from the
liver of an 8-day-old lamb that showed focal necrosis.
Gray _e__t_ 9;. (1956) also reported a similar condition in
8 newborn calves which lived only a few days. They were

weak and showed marked gastritis and enteritis on necropsy.

16

Some calves showed a few pin-point grayish white foci in

the liver. These authors also noted the presence of thrombi

on the heart valves of a lamb and serosanguinous fluidinthe peri-
cardial sac. Similar cases have been reported in other

animals (Table III).

Isolations of L, monocytggengg from human infants in
perinatal death is reported by several workers. (Table IV).
Reiss, Potel and Krebs (1951) called this condition
"granulomatosis infantiseptica" on the basis of lesions ob-

served in the organs of the infants. The chief gross
lesions they described as miliary granulomata with necrosis,
found particularly in the liver, spleen, adrenals, lungs,
lymph nodes and brain.

Hagemann and Simon (1953) found gross lesions in the
liver, lungs, spleen and adrenals as well as in the lepto-
1meninges in perinatal death of infants associated with‘L.
:monocytogenes. They described the lesions in these organs
as numerous submiliary to miliary greyish-white nodule-like
foci. In some cases they found similar foci in the mucosa
of the mouth, tonsils, asephagus, stomach and small and
large intestine, including the rectum.

Detmold and Girgensohn (195A) found amnionic fluid
turbid in some cases in addition to the findings of other
*workers just described.

Johnston 2; 2;. (1955) reported gross lesions mainly in
both lobes of the liver of a newborn infant which died 72

17

hours after birth. The liver was uniformly enlarged and
firm, and its surface and substance were studded with sub-
mdliary greyish-white nodules which were neither raised nor
palpable. In another case the amnionic fluid was reported
as being "murky" but not foul-smelling.

The study of microscopic lesions of listeric encephalitis
in the brain of cattle, sheep and goats is reported in con-
siderable detail by many workers; among them are King (l9k0),
Olafson (l9h0), Biester and Schwarte (19hl), Graham.g£‘gL.
(l9h3), Ryff and Lee (l9h8), Jensen and Mackay (l9h9), Gray
and Moore (1953). and Tajima and Nishihara (1953). The
lesions are found primarily in the medulla and pens and are
characterized by marked perivascular infiltration or "cuffing"
and areas of focal necrosis which are variable in degree and
extent. The perivascular "cuff" is formed mainly by mono-
nuclear leukocytes (lymphocytes and monocytes) in all species
of ruminants.

Gray and Moore (1953) reported that polymorphonuclear
leukocytes (polymorphs) predominate more in the foci of
necrosis in sheep and goats than in cattle and may in some
cases even cause liquefaction and suppuration. These
authors also observed in contrast that focal lesions in
cattle usually are limited to edema and small focal collec-
tions of microglial cells and lymphocytes and are usually

not as extensive as in sheep.

18

Olafson (l9h0) and Pallaske (19u0) pointed out the
tendency toward suppuration of the necrotic lesions in the
brain of sheep.

Histopathology of the aborted fetuses in listeric
abortions is rarely reported. Smdth g; g;. (1955) reported
that kidney sections from aborted bovine fetuses showed ad-
vanced decomposition and masses of bacteria resembling L.
manggxtggengp. They found acute endometritis in the sections
of uterus of the dam but with no marked involvement or necro-
sis. The medulla and other tissues from the dam showed no
significant lesions.

The microscopic lesions in cases of perinatal death
of young in animals and man are variable in intensity and
extent.

Gray 93 2;.(1956) reported that the microscOpic hepatic
lesions in calves which died after birth were of three types,
including degenerative changes (fatty metamorphosis) in
hepatic cells, discrete focal necrosis without involvement
of adjacent hepatic cells and diffuse necrosis with almost
complete disappearance of hepatic cells. These authors
also reported extensive degeneration of endothelial lining
and thrombosis of hepatic arteries in varying degrees.

They also noted the presence of Gram-positive bacteria re-
sembling L. monocytogeneg in the necrotic areas. The same

authors observed hemorrhagic gastritis and enteritis with

l9

necrosis of intestinal epithelium and congestion and throm-
bosis of some blood vessels.

The histopathology in cases of listeric perinatal death
of human infants has been reported by several German workers -
Potel (1953-5h), Hagemann and Simon (1953), Hagemann gj‘gl.
(1953), Hahnefeld and Nisolk (195h), Detmold and Girgensohn
(195h) and others: Patocka‘gL 5;. (1953. Czechoslovakia),
and Johnston 2; EL. (1955, Canada). In general the micro-
scopic lesions described by these workers are in agreement.
The principal lesions in various organs (particularly in
the liver, spleen, adrenals, lungs, lymph nodes, and brain)
are characterized by areas of focal necrosis, with varying”
degree of leukocytic infiltration and fibroblastic prolifera-
tion. Hagemann‘gg filo (1953) described the lesions caused
by‘L. monocytogenes according to the tissue reactions in-
volved in these conditions. The most common lesion was
focal necrosis with the presence of varying numbers of
‘bacterium in ahmost all the foci. The organisms were usually
found at the periphery of the lesion rather than at the cen-
ter. In other cases they found similar lesions in the
imucosa.of the mouth, tonsils, esophagus, stmmach, small
and large intestine (including rectum) and placenta. Another
type of reaction observed by these workers was characterized
as a proliferating granuloma through an increase in mesen—

chymal cells, a proliferation in which both vascular and

20

connective tissues take part. The organisms are rarely
demonstrated in or isolated from this type of lesion.
This so—called "granuloma" was not seen in all cases of
infection.

Johnston gt g1. (1955) reported the microscopic lesions
of two newborn infants. One died h2 hours and the other 3
hours after birth. The first showed many small areas of
hepatic necrosis with little inflammatory reaction. These
lesions had no consistent relationship to any part of the
liver lobule. Many Gramppositive, short, slightly beaded
rods were observed in the lesions. They also found similar
foci in the spleen, adrenal medulla, thymus, bone marrow,
and the submucosa of the bladder. Sections of the brain re-
vealed an acute meningitis with polymorph infiltration. No
focal accumulations of mononuclear cells were found. The
myocardium was normal.

Sections of lung from the other infant who died 3 hours
after birth showed bronchitis and bronchiolitis with a radi-
ating bronchopneumonia. The pulmonary alveoli were filled
‘with.amnionic debris and there was early hyaline membrane
‘formatdon. Many small abscesses with a peripheral cell
response were seen in the adrenal gland. There was pus on
the suwfaoe of the gastric mucosa with a slight inflammatory
reaction in the mucous membrane. They also noticed Gram-

positive bacilli "in the adrenal abscesses, in the pus

21

covering the gastric mucosa, and diffusely throughout the

lungs. The liver, brain and remaining organs were normal."

22

IV. EXPERIMENTS

A. Rabbit Experiments

1. Conjunctival EXposure

a. Materials and Methods

Rabbits 2:3 week§_pregnant. Nine pregnant rabbits (2-3 weeks
gestation) ranging in age from 8 to 18 months were exposed
to‘L.'monocytogengg by instillation of 2 drops of culture into
the left conjunctival sac. The culture (12255) used in the
experiment was isolated from the medulla oblongata of a calf
which died of listeric encephalitis complicated by vitamin

E deficiency. Unless otherwise indicated, this culture was
used in all experiments. The culture was grown at 37° C on
tryptose agar (Difco) slants for lB-Zh hours. The suspension
was made with sterile distilled water to a density approxi-
mating the number 8 tube of the McFarland nephelometer when
compared in a Cenco-Sheard-Sanford photelometer. Two drops
of the culture were instilled into the conjunctival sac by
means of a capillary pipette. Swab cultures from the left
eye and vagina were taken each 3rd day after exposure for a
period of 2 weeks. All aborted fetuses were examined post

mortem and liver and stomach contents of each and cotyledon

23

and amnionic fluid and exudates when available were cultured.
Necropsies were done on all does which aborted and died or
were sacrificed. Liver, spleen, heartblood, kidney, uterus,
cotyledon, lung, brain, vagina, urine and eye were cultured.
Cultures were made on tryptose agar (Difco) plates, incubated
at 37° C for 18-2h hours after which they were examined by
means of a binocular scanning microscope and oblique light-
ing (Fig. l and 2) as described by Gray 22,3L. (l9u8).
Tissues were fixed in 10 percent formalin, dehydrated in
graded alcohols, cleared in xylene and embedded in paraffin
of melting point 56°-58° C. Sections were out 6-7 microns
thick and stained with hematoxylin and eosin by the Armed
Forces Institute of Pathology method (1953) with the modi-
fication of (1) using standard hemotoxylin solution diluted
50 percent with distilled water, and (2) introducing carbol
xylene (melted phenol and xylene, equal volumes) for 5
minutes for differentiation before the final two changes in
neutral xylene and mounting in Permount. Whenever necessary,
Mac Callumis Modification of Goodpasture's Method for bac-
terial stain in tissue section was used. (Mallory, 1938).

Rabbits near term. Seven rabbits 2-h days prepartum were

OXposed in the same manner.

NOld-pregnant rabbits. Six non-pregnant rabbits were exposed
Similarly.

 

Fig. 1. Ar anmement of binocular scanning microscope
and light source for examination of bect rial colonies.

 

Fig. 2. Brooth (A) and rough (B) colonies oi'my {‘ \
cvtoonnes as viewed through bi oculdr scannidr uic-o-

scope. (I c. l)

 

 

25

b. Results: Bacteriology

Rabbits 2-3 weeksgpregnant. 0f the 9 does, 3 aborted after
the 3rd day after exposure and died the following day or in
two days. One aborted 5th post-exposure day and was sacri-
ficed; two died before abortion; and two aborted 3 and 6

days after exposure, respectively, and survived. The results
of isolation of L. monocytogenes from aborted fetuses and

does are shown in Tables V and VI, respectively.

Rabbits near term. Seven does gave birth to 58 young of
which 16 were either stillborn or died shortly after birth;
the remaining young died within 8 days except for one which
survived. The does did not show significant manifestation
of illness and all except one survived. L. monocytogenes

was isolated from the young as shown in Table VII. Tissues

were not refrigerated.

INonepregnant rabbits. Six non-pregnant rabbits showed no
apparent signs of illness and were sacrificed. .L. monocyto-

genes was isolated as shown in Table VIII.

c. Results: Pathology

Lesions are described under the heading of the organ
or tissue involved. Organs showing no significant lesions

are not mentioned.

 

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Rabbits 2q3 weg§§:pregnant

Necropsy l9l6X - Rabbit 221 aborted 3 days after conjunctival

instillation of L.:monocytogene§ and died a day after abor-

tion.

Gross pathology:

Eye - marked conjunctivitis and profuse purulent exudate;
eyelids were adhered together and purulent exudate
oozed out on pressure. (Fig. 3)

Vagina - moderate sanguinous discharge.

Diffuse congestion of liver, lungs, spleen, intestine,
uterus.

Uterus - very severe inflammation characterized by necrotic
lesions with caseation and marked purulent exudate.

Cotyledons - extensively necrotic and disintegrating.

Histopathology:

Uterus - acute necrotic endometritis with very extensive
necrosis with caseation involving a large portion
of endometrium including uterine glands; some of
the remaining glands filled with purulent exudate;
cellular infiltration consisting predominantly of
polymorphs, but with some lymphocytes, plasma
cells and macrophages; edema of endometrium.
Myometrium shows thrombosis of blood vessels,

infiltration of leukocytes (mainly polymorphs),

and some necrosis.

31

 

Fig. 3. Left e'e of a rabbit showing marked conjunc—
tivitis and purulent exudate with adhesions of upper
and lower eyelids 3 days after conjunctival instil-
lation of L. monocytogenes.

 

Fig. h. Left eye of a rabbit showing karate-conjunc-
tivitis 9 days after conjunctival instillatiO“ of
L. monocytogenes.

 

 

 

.I. I _III {I I! I

 

Liver —

32

marked diffuse fatty metamorphosis, and congestion.

Spleen and lungs - some congestion and hemorrhage.

Kidney -

Necropsy l9l7X - Rabbit 21h aborted h days after conjunctival

instillation of.L. monocytogenes and died the day after abor-

tiOne

slight congestion.

Grossgpathology:

 

Eye - marked conjunctivitis with purulent exudate.

Vagina - slight sanguinous discharge.

Liver - congestion and a mild, diffuse, yellowish discolor-
ation with a few pale, greyish white foci up to
1 mm. in diameter.

Uterus - very severe inflammation with extensive necrosis
with caseation of endometrium and some purulent
exudate.

§§530pathology:

Uterus - acute necrotic endometritis with almost complete

necrosis of the uterine glandular epithelium and
the lumen of remaining glands filled with puru-
lent exudate. Cotyledonary portion of endome-
trium and myometrium show marked congestion and
necrotic foci with leukocytic infiltration (main-
1y polymorphs); clumps of bacteria in cotyledon,
placenta, and uterine glands. (Fig. 5 and 6)

Urinary bladder - slight congestion.

 

Fig. 5. (NecrOpsy 1917X) Endometrium from rabbit 21h
showing necrosis and purulent exudate. Hematoxylin-
eosin. 155x.

 

Fig. 6. (Necronsy 1917K) Uterine glands from rabbit
21h showing distention with purulent exudate. Hema-
toxylin-eosin. 325x.

k»)

k»)

Kidney -

Spleen -

Liver -

congestion.
mild hematogenous pigmentation (probably hemo-
siderosis).
mdld, diffuse fatty metamorphosis and moderate

focal coagulation necrosis.

Necropsy 1928X - Rabbit 216 aborted h days after conjunctival

instillation of.L. monocytogenes and died 2 days after abortion.

Gross pathology:

Eye -

Uterus -

marked conjunctivitis with purulent exudate.
congestion and extensive caseation necrosis of
endometrium, and filled with purulent exudate and

macerated remains of conceptus.

Histqpathology:

Uterus -

Liver -

Spleen -

acute necrotic endometritis and myometritis, with
marked necrosis involving almost all of the endo-
metrium including the uterine glands; some of the
remaining glands distended with caseo-purulent
exudate; marked congestion and edema, especially

in the endometrium; cotyledons and placenta showed
extensive marked necrosis and polymorph infiltra-
tion; several thrombi in vessels of the myometrium.

a few coccidial lesions unrelated to this experiment.
mild hematogenous pigmentation (probably hemo-

siderosis).

35

Necropsy 1929K - Rabbit 222 aborted 5 days after conjuncti-
val instillation of‘L. monocytogenqg and was sacrificed 2
days later.

Gross_pathology:

Eye - marked conjunctivitis and purulent exudate.

Vagina - slight sanguinous discharge.

Uterus - marked inflammation with caseation necrosis and
thick purulent exudate.

Histopatholqu:

Uterus - acute necrotic endometritis and myometritis with
marked but patchy necrosis of the endometrial
epithelium; a zone of necrosis and polymorph
infiltration noticed in the cotyledon above the
decidua basalis; clumps of bacteria more prevalent
on the maternal side of the zone of necrosis and
polymorph infiltration. (Fig. 7, 8, 9 and 10)

Liver - slight centrolobular fatty change; mild focal
infiltration of lymphocytes; mild infiltration

of eosinophiles in the sinusoids.

Necropsy 1935X - Rabbit 212 died 7 days after conjunctival
instillation of L. mpnocytogenes. Beginning 2 to 3 days be-
fore death the animal developed anorexia, torticollis, in-

coordination, and depression.

Gross pathology:

Eye - severe conjunctivitis with marked purulent exudate.

K»)
O\

 

Fig. 7. (Necropsy 1929X) Endometrium from rabbit 222
showing zone of endometrial necrosis and polymorph
infiltration. Hematoxylin-eosin. 1 x.

 

Fig. 8. (Necroosy 1929X) Endometrium from rabbit 222
showing extensive necrosis with caseation and leukocy-
tic infiltration, nrcdominantly nolymorphs. Hemato-
xvlin-eosin. 155x.

 

Fig. 9. (Necropsy 1929X) Endometrium from rabbit 222

showing Gram-positive bacteria resembling L. monocyto-

genes. thallum's Lodification of Goodpasture's Stain.
Ox.

 

Fig. 10. (Necropsy 1929X) Endometrium of rabbit 222
showing Gram-positive bacteria resembling L. monocyto-
genes in the cotyledonary portion. LcCallum's nodifi-
cation of Goodpasture's Stain. 820x.

37

‘0 ‘Mill-ia- . I ml .41...-

l . (RI B4517:

38

 

Vagina - slight sanguinous vaginal discharge.

Uterus - highly congested and inflamed; uterine cavity
filled with purulent exudate, macerated fetuses
and necrotic cotyledons.

Liver - slight yellowish discoloration.

All other visceral organs including brain - highly congested.

Histopathology:

Uterus - acute purulent metritis involving both endometrium
and myometrium; necrosis of almost all the endo-
metrium including uterine glands; a zone of necro-
sis with heavy infiltration of polymorphs at the
junction of the endometrium and retained cotyledon;
focal necrosis with polymorph infiltration and
thrombosis of several vessels in the myometrium.
(Fig. 11 and 12)

Cotyledon - limited necrosis with variable polymorph
infiltration.

Liver - moderate centrolobular fatty metamorphosis.

Spleen - mild hematogenous pigmentation; small amount of
hyaline material, probably amyloid, in the splenic
corpuscles.

Kidney - moderate subacute interstitial nephritis character-

ized by focal lymphocytic infiltration near the

blood vessels.

Medulla and cerebellum.- mild congestion and hemorrhage.

\O

‘

 

Fig. 11. Necropsy 1935X) Myometrium from rabbit 212
showing thrombosis and polymorph infiltration. Hema-
toxylin-eosin. 155x.

 

Fig. 12. (Necropsy 1935K) Endometrium from rabbit 212
showing caseous necrosis and polymorph infiltration.
Hematoxylin-eosin. 155x.

MO

Necropsy 19u7x - Rabbit 203 died 5 days after conjunctival

instillation of‘L. monocytogenes.

Gross_patholqu:

 

Eye - conjunctivitis and purulent exudate but not so
marked as in other cases.

Uterus - highly inflamed with necrotic to purulent lesions
and containing 9 fetuses.

Histopathology:

Uterus - acute necrotic endometritis with necrosis and poly-
morph infiltration in the upper portion of endo-
metrium including uterine glands; congestion and
edema.

Liver - marked diffuse fatty metamorphosis; coagulation
necrosis, and congestion.

Kidney - some subacute interstitial nephritis characterized

by mild focal lymphocytic infiltration.

Necropsy 1952X - Rabbit 209 was sacrificed 9 days after

conjunctival instillation of E. monocytogenes.

Gross pathology:

Eye -

Vagina -

Uterus -

severe karate-conjunctivitis with some purulent
exudate. (Fig. h)

slight sanguino-purulent discharge.

marked inflammation with necrosis and purulent
exudate, the left horn being relatively more

severely affected than the right one.

hl

Histopathology:

Uterus - subacute necrotic metritis with very extensive
necrosis, and polymorph infiltration of endometrium
involving most of the uterine glands; remaining
uterine glands highly distended and cystic; mild
lymphocytic infiltration of myometrium.with some
macrophages. (Fig. 13 and 1h)

Cotyledon - some necrosis and marked polymorph infiltration.

Placenta - purulent exudate, with some eosinophiles also.

Liver - mild subacute interstitial hepatitis; congestion;
some karyolysis of hepatic cells.

Kidney - subacute interstitial nephritis with lymphocyte,
macrophage and numerous eosinophilic infiltration.

Fetal livers - marked congestion and difiuse fatty meta-

morphosis.

gabbits near term

Necropsy 19S9X - Young of Hanoit 223. This doe gave birth

to 9 young 3 days after conjunctival eXposure. All the

9 young died within 6 days after birth.

Grossgpathology:

Liver - mild pale yellowish discoloration.

Histopathology:

Liver - congestion, patchy centrolobular fatty changes and
focal necrosis in various combinations and quanti-

tative variations. Necrosis usually appeared in

 

Fig. 13. (Necropsy 1952X) Endometrium from rabbit 209
showing cystic dilatation of uterine glands and some
polymorph infiltration. Hematoxylin-eosin. 155x.

 

Fig. lb. (Kocronsy 1952') Endomotrium from rabbit 209
Showing confluence of several cystic uterine glands,
Hematoxvlin—eosin. 15§X.

:-

[U

#3

small discrete foci in the vicinity of blood
vessels. In some cases extensive necrosis oc-
curred along the outer zone of a large blood
vessel; several thrombi were seen in the smaller
blood vessels in the necrotic areas. Necrotic
foci were usually infiltrated with polymorphs and
clumps of bacteria. In one case necrotic cell debris
was noticed in the bile ducts. (Fig. 16)

Gall bladder - limited focal necrosis and polymorph infil-

tration involving mucosa and submucosa. (Fig. 15)

NecrOpsy 19601 - Rabbit 22? gave birth to 9 young h days
after conjunctival exposure. Six died and 3 mutilated ones

.‘J

were sacrificed the same day. The doe was sacrificed 6 days

post partum.

Gross_pathology:

Uterus - some grayish white material in the uterine cavity.

Histopatholggy;

Uterus - subacute endometritis with infiltration of lympho-
cytes and plasma cells, early proliferation of
fibroblasts, slight purulent exudate in lumen of
the uterus.

Liver - mild subacute interstitial hepatitis with a few
foci of lymphocytic infiltration.

 

Fig. 15. (NecrOpsy 19S9X) Gall bladder from one of
the young of rabbit 223 showing a focal cell reaction
in which polymorphs predominate. Hematoxylin-eosin.
155x.

\
.‘ ‘
sag

    

Fig. 16. (Necropsy 1959X) Liver from one of the young
0f rabbit 223 showing necrotic cell debris in bile
ducts. Hematoxvlin-eosin. ISFX.

MS

Necropsy l96hX - Young of Rabbit 232. These 7 young were

born A days after conjunctival exposure of the doe. Six

died within 8 days and one survived.

Gross_pathglggy; .

Liver - diffuse yellowish discoloration; greyish white foci
observed in some cases with quantitative variation.

Histopathology:

Cotyledon - focal areas of necrosis with polymorph infil-
tration.

Liver - moderate centrolobular fatty changes and focal
coagulation necrosis with variable polymorph infil-
tration. In young which survived a week or more,
lymphocytic infiltrations and early proliferation
of fibroblasts and angioblasts were seen. (Fig.

17 and 18)

Heart - a single focus of necrosis with moderate polymorph
infiltration. (Fig. 19)

Stomach - focal superficial necrosis of mucosa; mild
purulent exudate on the surface. (Fig. 20)

Lungs - acute broncho-pneumonia with.marked congestion.(th 2D

Kidney - one focus of necrosis with moderate polymorph

infiltration. (Fig. 22)

Necropsy 1965x -'Young of Rabbit 23h. Ten young were born
h days after conjunctival instillation of the doe with'L.

monoc to ones, and died 3 days later. The doe herself died

3 weeks post partum. (Necropsy 1985X)

 

Fig. 17. (‘ecroosy l96hX) Liver from one of the yo ang
of re bbit 232 showing focal necrosis with
morrh infiltration.

. ild poly-
emitovylin—eosin. 152x.

 

Mg.l& Lecropsy l96hX)

;;\V.

Liver from
Ofrmm1f23’ snowing nroli‘er“‘ive ch°n~eo and “\
cvtic infiltza °

one of
ation.

the
(atoxrlin-eosio.

young
, ,urrxflio-
325x.

4

6

 

.J'

 

~“C"¢”)9Zl'

Fig. 19. Necropsy 196HX) Heart from one of the young
of rabbit 232 showing focal necrosis and polymorph in-
filtration. Rematoxylin-eosin. 155x.

    
  

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‘13:!
a n3. '3 I."

3
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e . ..
o ‘ v
‘-
I . e.
d...

My.2m flecropsy 196hX Stomach from one of the
ymmgofrabbit 232 showing focal necrosis with poly—
Immminfiltration and mildly purulent exudate on the
smimeofthe mucosa. Hematoxvlin-eosin. 155x.

 

 

Fig. 21. (Necropsy l96hX)

Lung from one of the young

of rabbit 232 showing focal infiltration of polymorphs.
Eematoxvlin-eosin.

155x.

 

Nita" ‘*

-uéaga1=.1

Mg.22.(Necropsy 196UX)

Kidney from one of the
ymmvofrabbit 232 showing focal necrosis and poly-
nmmhifliltration.

Yemetoxylin-eosin. 155x.

14.9

'088 Batholggl:

.ver 01' young - mild patchy yellowish discoloration and
several discrete necrotic foci.
.11 at. 02 atho lo 31 :

Liver - moderate centrolobular fatty changes to extensive

diffuse fatty metamorphosis, several small discrete
foci of coagulation necrosis with no polymorphs or
very little infiltration to a much larger focus of
necrosis with numerous polymorphs; fatty changes
and necrosis appear together in: some foci.

Lungs - mild congestion, very early pneumonia.

Cotyledon - several areas of focal necrosis with polymorph

infiltration.

Necropsy 1985K - Rabbit 23h. gave birth to 10 young LL days
after conjunctival exposure. All the young died within 3
days (Necropsy 196530; the rabbit died 3 weeks post partum.
Gross pathology:

Liver - mild, diffuse yellow discoloration.

Uterus - numerous fibrotic adhesions with intestine; very
pale in appearance; many firm abscesses in the
right horn ranging from pinpoint to 5 mm. in dia-
meter containing caseated pus. No such lesions

were seen in the left horn. (Fig. 23)

So

Histopatholqu:
Uterus - chronic suppurative metritis involving myometriumv
and endometrium with almost complete loss of uterine

glands. (Fig. 2h)

Necropsy 1983x - Young of Rabbit 2M9.were born 2 days after
conjunctival instillation of the doe, and all died within 3
days after birth.

Gross pathology:

Liver - a few submiliary greyish white foci.
Histopathology:

Cotyledon - limited focal necrosis with a few polymorphs.

Placenta - marked purulent exudate.

Liver - lesions varied from.mild centrolobular fatty changes to

several small focal areas of necrosis with polymorph
infiltration.

Nonppregnant rabbits

Necropsies 1931K - Rabbit 217
l937X - Rabbit 226
l9h8x - Rabbit 228
19531 - Rabbit 229
1955x
1961x

Gross pathology:

Necropsy revealed nothing of any significance in these non-

Rabbit 230

Rabbit 231

pregnant rabbits except in the case of Rabbit 231,

whose uterus appeared somewhat enlarged and congested.

 

 

It 'unc I .’ l 4 5 t 7 8

 

Fig. 23. (Lecropsy lQBSX) Uterus from rabbit 23h
opened to Show collections of caseo-purulent exudate.

.. t
;\ L"

n .I
‘3‘, \.

 

Fig. 2h. (NecrOpsy lCBSX) Uterus from rabbit 23h.

51

Light-stained area (lower left) is caseous cell debris
at the endometrial surface. All layers of uterine wall

show inflammatory infiltrations accompanied in some
areas by necrosis and even suppuration. T'emntoxylin-
eosin. 155x.

52

Histgpathology:

No microscOpic lesions of any significance could be noticed
in the tissues (liver, spleen, kidney, urinary
bladder and uterus) of these non-pregnant rabbits

except the following:

Necropsy 1931X - Rabbit 217
Liver - a few small foci of lymphocytic infiltration.
Uterus - marked infiltration of eosinOphiles with some

polymorphs, lymphocytes and plasma cells. (Fig. 25)

Necropsy 19h81 - Rabbit 228
Liver - a few small foci of lymphocytic infiltration.

(F180 26)
Uterus - a slight focal infiltration of lymphocytes in the

endometrium.

Necropsy 1961K - Rabbit 231
Liver - moderate centrolobular fatty changes, a few foci of

lymphocytic infiltration and some scattered eosinophiles.

2. Oral Exposure

a. Materials and Methods

Rabbits 2-3_weeksgpregnan . Six (2-3 weeks pregnant) rabbits
ranging in age from 8 to 18 months were exposed to‘E. mono-

cytogenes by adding dense suspensions of the bacterium to the

53

 

Fig. 25. (Necropsy l93lX) Endometrium from rabbit 217
showing infiltration of lymphocyte and eosinophile

and some cellular exudate on the surface. Hemato-
xylin-eosin. 155x.

 

22;

Fig. 26. (Necropsy l9h8 ) Liver from rabbit
showing focal lymphocytic irfiltration in the inter-
stitial tissue. hematoxylin-eosin. 325x.

5h

drinking water. Two of the pregnant does were exposed to
culture 1766X isolated by Potel (1953) from the liver of an
(aborted infant; the remaining four animals were exposed to
culture 12255. Thirty-two ounce "Sani-glas" prescription
bottles containing approximately 100 m1. of tryptose agar
(Difco) were inoculated with a distilled water suspension
of freshly grown culture and incubated at 37° c for 18-21;
hours. They were then filled with tap water, agitated to
'wash the culture from the agar surface, and the resulting
suspension poured into a ceramic watering dish. Thus each
rabbit daily had access to 32 ounces of a dense tap-water
suspension of‘L. monocytogeneg. This constituted the only
source of fluid intake for a period of 2 weeks. The average
density of these suspensions corresponded approximately to
the No. 2 tube of the McFarland nephelometer when compared
in a Cenco-Sheard-Sanford phctelometer. No attempt was made
to prevent contamination of the water by other organisms;
however, the water was cultured at frequent intervals and,
when badly contaminated, a clean dish was provided. All
aborted fetuses and animals which died or were sacrificed
were cultured as described by Gray gt‘gl. (19h8, 1951).
Tissues to be cultured were ground in a mortar together
with sterile water to form an80 percent suspension. A
large loopful (h.0 mm. die.) of this suspension was plated
on tryptose agar (Difco) plates. The remaining suspension

was stored in the refrigerator at h° C. The plates were

SS

incubated at 37° C for 18-2u hours after which they were
examined by means of a binocular scanning microscope and
obliquely transmitted light. Tissue suspensions which
failed to reveal the presence of L. monocytogenes were re-
plated at intervals of several weeks. If‘g. monocytogenes
was not isolated after 3 months of refrigeration, the sus-

pension was considered negative.

Rabbits near term. Two near-term.pregnant rabbits were ex- i

 

posed to culture 1766X and the remaining six were exposed to L
culture 12255.

Non-pregnant rabbits. Three non-pregnant females and one male
rabbit were exposed to culture 12255 and another male rabbit

was exposed to culture 1766x.

b. Results: Bacteriology

Rabbits 2-3 weeks pregnant. Of these 6 rabbits, 3 aborted on
the hth post-exposure day and died Z-h days after abortion

and 2 aborted 5-6 days after exposure and survived. Some
fetuses were macerated and others were mummified. There ap-
peared to be no difference in the reactions of the does
exposed to the 2 different cultures. Before death these does
showed anorexia and depression. Isolation ofIE. monocytogenes

from.the fetuses and from the does is shown in Tables IX and X.

56

Rabbits near term. There appeared to be no difference in

the reactions of the does exposed to the 2 different cul-
tures. Of the 55 young born to 8 does, 2h were stillborn

or died shortly after birth, none surviving more than u days.
None of the does died. They displayed no noticeable signs
of illness except that most of them did not take care of

the young. Isolation of Q. monocytogenes from.the young is

shown in Table XI.

Nongpregnant rabbits.» The non-pregnant and male controls
showed only a mild anorexia on the second and third days

of exposure, with one exception. On the 12th post-exposure
day one male showed some difficulty in breathing which per-
sisted for 3 days, at which time he was sacrificed. Necropsy
revealed a mild pneumonia and‘g. monocytogenes was isolated
from the lungs, nares, cecum, and small intestine. In spite
of the isolation from.the lung, the pneumonia appeared to be
of different origin as only one colony ofԤ.:monocytogenes
was observed among a profuse and otherwise pure growth of

an unidentified Gramrnegative rod. Ԥ.:monocytogenes was

not isolated on initial culture from the three sacrificed
female controls; however, it was isolated from the liver

of one after maceration and refrigeration for 2 months.

0. Results: Pathology

Rabbits 2-3 weeks pregnant

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60

Necropsy 1968X - Rabbit 238 aborted 2 fetuses the 5th day

after exposure and died the same day.

Gross pathology:

“Uterus -

Liver -

Lungs -

contained 10 additional fetuses and endometrium
appeared necrotic. In some cases amnionic fluid
was dark purple.

mild pale yellow discoloration and several greyish
white areas about 2 to 3 mm in diameter.

marked congestion.f

Fetal liver - mild yellow discoloration and several sub-

miliary to miliary greyish white foci.

Histopathology:

Uterus -

Cotyledon

Liver -

acute necrotic endometritis with necrosis involving
at places varying depths of endometrium.

- several areas of focal necrosis with polymorph
infiltration, clumps of bacteria, and several
thrombi with polymorph infiltration.

marked diffuse fatty changes and several foci of
coagulation necrosis with mild polymorph infil-
tration, some eosinophiles and a few lymphocytes,

some congestion.

Fetal tissues:

Liver -

moderate congestion, diffuse fatty change, several
foci of necrosis with polymorphs and bacteria, at
some places dense clumps of bacteria in the paren-

chyma and some in the vicinity of bile ducts. (Fig.
27 and 28)

 

61

 

Fig. 27. Necropsy 1968X) Liver from one of the young
of rabbit 238 showing Gram-positive bacteria resembling
L. monocytogenes. EcCallum's Lodification of Goodpas—

ture's Stain. 820x.

 

Fig. 28. Liver from one of the young
Of rabbit 238 showin? Gram-nositivo bacteria resembling
L. monocvtocones in the vicinity of the bile duct.
LcCallum's hodification of Goodpnsture‘s Stain. 820x.

(Necroosv 1968X)

.—

u. 1...:
“I,

. _1_i,

 

Kidney -

Heart -

Lungs -

62

foci of necrosis with polymorph infiltration and

bacterial clumps.

mild coagulation necrosis with clumps of bacteria.

marked congestion.

Necropsy 197OX - Rabbit Zhl aborted h days after exposure and

died 2 days after abortion.

Grossgpathology:

Uterus -

Kidneys -

Lungs -
Vagina -

congestion with pale streaks showing through serosa;
uterine cavity contained a large amount of caseated
necrotic thick gray material resembling pus, endo-
metrium.underneath the gray material appeared to be
gray and necrotic. There was one cotyledon in the
uterus which appeared to be entirely necrotic, and
almost black in color.

petechial hemorrhages and a few greyish white areas.
mild congestion with some edema.

slight sanguinous discharge.

Fetal liver - slight yellow discoloration.

Histopathology:

Uterus -

acute necrotic endometritis with an extensive
necrosis of endometrium and marked purulent exu-
date involving most of the uterine glands; edema
of the endometrium; areas of necrosis with poly-
mmrph infiltration and several thrombi in the
myometrium; some thrombi were relatively large and

infiltrated with polymorphs.

‘15

 

 

63

Cotyledon - several thrombi with polymorph infiltration;
numerous clumps of bacteria.

Fetal membrane - mild purulent exudate.

Kidney - subacute interstitial nephritis and small focal
areas of coagulation necrosis.

Liver - some centrolobular fatty change.

Lungs - mild congestion and edema.

Fetal tissues:

Liver - mild diffuse fatty change.

Necropsy 1976K - Rabbit 2&0 died 6th day after exposure

9!;

without aborting.

Gross_pathology:

Uterus - greatly distended and showed very marked congestion
and numerous gray streaks and plaques of what ap-
peared to be necrotic tissue. In the uterine
cavity nine partially mummified fetuses were found
tightly adhered to the endometrium. There was no
evidence of any amnionic fluid. In addition it
contained large amounts of caseous material adher-
ent to the endometrium; underneath it, the endo-

metrial surface appeared corrugated and necrotic.

 

The cotyledons were gray in color and completely

necrotic. (Fig. 29 and 30) The livers of the

 

fetuses were all pale yellow in color and no lesions

could be detected in the fetuses because of mummi-

fi catione

 

ig. 29. Necropsy 1976X) terus from rabbit 2&0 i3

‘LT‘
situ showing greyish streaks in the otherwise con-
C"

nut,

 

Fin. 30. (Necropsy 1976X) Uterus from rabbit 2&0
partly Opened to Show the caseous necrotic :wfnrial
on the fetal membranes and endometrium.

65

Liver - slightly pale in color and some submiliary greyish
white foci.
Histopathology:

 

'Uteunzs -

Cotyledon

acute necrotic endometritis and myometritis with
extensive necrosis of almost all the endometrium
including the uterine glands with marked purulent
exudate. The few remaining uterine glands were
filled with exudate. The myometrium showed edema
and contained numerous extensive thrombi with poly-
:morph infiltration. The serosal layer was exten-
sively necrotic and infiltrated with polymorphs.
(Fig. 31. 32. 33 and 3h)

- several extensive thrombi with polymorph infil-
tration, clumps of bacteria, extensive areas of

necrosis and purulent exudate, especially on the

outer surface. (Fig. 35)

Fetal membranes - enclosed an abundance of necrotic debris.

Liver -

Kidney -

(Fig. 36)

several foci of coagulation necrosis with moderate
polymorph infiltration. (Fig. 37)

nuld subacute focal interstitial nephritis with
focal lymphocytic infiltration. (Fig. 38)

Fetal tissues:

liver -

Lungs'-

moderate diffuse fatty changes.

nuld congestion.

 

 

U\

 

Fig. 31. (Necropsy 1976X) Endometrium from rabbit 2h0
showing necrosis and caseo-purulent exudate on the
surface. lie atoxylin-eosin. 155x.

    

. . ‘-. [if - ."
V '0 l I' I v s‘ , e
\k ’..-{ 5.. ‘\;\'. " 31".“ /' .. .

Fig. 32. (Necropsy 1976X) Ifyometriwn from rabbit 240
showing: necrosis and a predominantly polymorphic in-
filtration. IIcmatoxylin-eosin. 155x.

(‘1\

 

 

\II‘> “ I. . I
' '1" ' ..‘ :51"! .' .
V ‘t ‘h’w ’~",?S

Fig. 33. (Necropsy 1976X) Endometrium from rabbit 2M0
showing necrosis and polymorph infiltration. Hemato-
xylin-eosin. 155x.

   
 

" Max"
‘9', .I"
s. . '

  
 

 

» .. 5'-
‘ -~““ '. " 1"

s

  

  

Fig. flu (Necropsy 1976X) hyometrium of rabbit 2L0
shmdnr inflammatory infiltration of a venous thrombus
withrmlynornhs and localized disaopearance of endo-
flmlbnm Femntoxylin-oosin. 155x.

 

 

Fig. 35. (Necropsy 1976X) Cotyledon from rabbit 2&0
showing necrosis with purulent exudate on the surface.
Hematoxvlin—eosin. 155x.

 

Fig. 36. (Necropsy 1976 )
211.0 showing purulent exudat

V
A\
v

Fetal membranes from rabbit
c. Hematoxylin-eosin. 1551):.

 

 

e .7. ‘
lax ‘-
Fig. 37. (Iecropsy 1976X) Liver from rabbit 2L0

showing necrosis and moderate polymorph infiltration.
Hematoxrlin-eosin. 155x.

 

th 38.(Iecropsy 1976X) lilnev of rabbit 2&0 show-
insxumdoninsntlv lwnphocv‘ic infiltration. Nemato—

X‘lin-eoshi. lqfix.

 

7O

Necropsy 20031 - Rabbit 252 aborted macerated fetuses 1;. days
after exposure and died 5 days after abortion. (Fig. 39)
Gross pathology:

Uterus - greatly distended, dark purple in color and con-

taining large masses of necrotic debris and puru-
lent exudate. (Fig. 140)
Liver - slightly pale
Bistopathologz:

:
r: l.

Uterus - acute necrotic endometritis with very extensive

necrosis of most of the endometrium with infiltration

of cells, chiefly polymorphs. A zone of necrosis

with heavy infiltration of polymorphs was noted in
the cotyledonary attachment with the endometrium;
the myometrium showed extensive areas of necrosis,
polymorph infiltration, edema and numerous thrombi
with mild polymorph infiltration. (Fig. 11.1 and AZ)
Cotyledon - areas of extensive necrosis on the outer surface
with marked polymorph infiltration. Some bacteria

noted.

Fetal membranes - necrosis and caseo-purulent exudate.

Necropsy 2056K - Young of Rabbit 256 - Of 10 young born
after 3 days of oral exposure of the doe, 3 were found dead

and the rest died within 1;. days of post-natal life.
ngss pathology:

Liver - necrotic foci in the liver (studded with submiliary

 

 

Fin. 9. Necropsy 2000K) Aborted fetuses from rabbit
252 showing varying degrees of disintegrstion.

 

Fia.h0. (Necropsy 2003K) Uterus from rabbit 252
shmfinw abundant caSeo-purulent exudate covering a
diffimely reddened endometrium, and masses of
necrotic cotyledons.

 

            
 

72
9 . »‘ i
'27:”?! I .
a " r. l
H h
.0 ‘
- r
w, .
' Q
,‘V ' 4

uetrium from rabbit 252

Fig. hl. (Necropsy 2003K) En
showing very extensive necrosis and purulent exudate.
Hematoxylin-eosin. 155x

.‘ '
its

"a
R

a; P _
. --V§$.33

" i
V. I -‘
' “I g ('5‘? In "J ‘

‘

 

p
“l” 15)

U‘A‘.

Fig. A2. Iecropsy 2003K)
polymorph infiltration.

Lyonetrium of rabbit 252

Y

4..

showing throabosis (arrow) and necrosis with moderate
Yenatoxvlin-co

 

73

greyish white foci with an area of fatty change.
(Fig. hB)

Histgpathology:

Liver -

Kidney -

the lesions ranged from a few discrete foci of
necrosis with mild polymorph infiltration in some
livers to numerous foci of necrosis with moderate
to marked polymorph infiltration in others. In
one case several foci of necrosis with clumps of
bacteria were noted near a large blood vessel.

The smaller blood vessels showed localized dis—
appearance of endothelial lining and tendency to
thrombus formation. Some congestion and fibrinous
exudate was noted in sinusoids. Clumps of bacteria
were scattered through the parenchyma. (Fig. AM)
a discrete foci with mild polymorph infiltration

in one case.

Medulla and cerebellum - some congestion, quite marked in

meningeal blood vessels.

Necropsy 20581 - Young of Rabbit 268 - Three young born after

3 days oral exposure of the doe, died within h days of post-

natal life.

Gross pathology:

Liver -

numerous submiliary to miliary greyish white foci,

covered with a fibrinous exudate forming adhesions.

(F13. hS)

 

 

Fig. R3. (Necropsy 2056X) Liver from ne of the young
of rabbit 256 showing numerous submiliary to miliary
greyish-white foci and an irregularly shaped area
(left) showing fatty changes in addition.

 

Fig. uh. (RecrOpsy 2056K)
of rabbit 250 showinr Ioci
changes.

Liver from one 3 t
of necr sis and fsth
Hematoxvlin-eosin. 155x.

3!“ 77011.le
1L .,

 

. ‘ .4, , (Kali-.1.» 11

75

Histopathology:
Liver - the lesions ranged from.discrete foci with a mild
polymorph infiltration to numerous extensive foci
of necrosis and marked polymorph infiltration.
Most of the necrotic foci were in the vicinity of

blood vessels. In some instances the inflammation

involved extensive areas of the parenchyma and ex-

tended through the vessel walls apparently causing

thrombus formation and polymorph infiltration in
the thrombus itself. Bacteria could be seen in
several foci of necrosis, in some cases in clumps}
fibrinous exudate on the surface with polymorph
infiltration underneath. (Fig. no and h?)
Small intestine - a small focus of polymorph infiltration

in across with a fibrinous exudate overlying it.

(Fig. hB)

Non-pregnant rabbits.

Necropsy 1977K - Rabbit 2&2 was sacrificed after 8 days of

oral exposure to L, monocxtogeneg.

gross patholgglz No significant lesions.

EistOpathologl:

Liver - several small foci of lymphocytic infiltration
in the parenchyma.

 

Fin. MS. (SecrOpsy 2058X) Liver from one of the young
of rabbit 268 showing numerous submiliarv to miliary
greyish-white foci and covered with fibrinous exudate.

 

Fig. us. (Lecronsv 2078X) Liver from one of the venue
of rabbit 268 shovinn an extensive area of necrosi
and noleornh infiltration. Hematoxvlin-eosin. 1Ky3,

 

 

Fig. A7. (KecrOpsy 2OSEX) Liver from one of the young
of rabbit 268 showing fibrinous exudate on the surface

and an underlying zone of polymorph infiltration.
Hematoxylin-eosin. 155x.

 

Fig. QB.

(Necropsy 20§9X)
the young of rabbit 268 Showing fibrinous exudate on

the serosa accompanied by nolynerrh infiltration.
Hematoxylin—eosin. 155x.

Small intestine from one of

77

78

3. Conjunctival Exposure of Pregnant Rabbits

Surviving Previous Oral Exposure

a. Materials and Methods

Six pregnant rabbits near term.were exposed conjunc-
tivally to‘g. monocytogenes as described under initial con-
Junctival exposure. Of these, two (rabbits 258 and 259) were
previously exposed orally as non-pregnant animals and the re-
maining u were exposed near term and gave birth to infected
young from.which'§. monocytogenes was isolated.

All had been exposed orally Z-h months previously. In
the intervening period, all but one (rabbit 268) were bred as
soon as possible after the first exposure. However, at least
2 weeks elapsed between exposure and breeding, since the does
refused the buck. All delivered normal young. Circulating
antibody titers in the blood were not determined at the time

of second exposure.

‘b. Results: Bacteriology

The results of this exposure followed the pattern of
the results obtained in the case of rabbits which were ini-
‘tially'exposed.near term.by conjunctival instillation. 0f
37 young, 19 were stillborn or found dead, 1).; died within a
period of )4. days of postnatal life and I... survived. The does

did not show any apparent symptoms and all of them survived.

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1mm m Wm mm mm mm H .m. mm m wgow w a PMSMOQKH MCHUmohm
”M.RdSGm WWW. m. "Du. N W W“. w. W. ”W M MO 90952 SW...” .HO .HO 9035.32
pm. 9 a.” m u m. W. W.” J R0952 thOLUQ 036nm Duran OHGQ “finndm
9

 

 

mmDmomxm Q<mo mDOH>mmm OB Bzmzdummbm Eda flue mo Mmbwomxm M<>Heozbbzoo
JZHzoqqom Ame<ma A<B<ZHmva mBHmm<m 0230M 20mm mmzmooawoozoz .A mo mZOHB<AomH

HHX mqm<a

 

80

Isolation of.§. monocytogenes from the young is shown in
Table.XII. ‘E. monocytogenes could not be isolated from.the
does when they were sacrificed over a period ranging from 22

days to one year.

c. Results: Pathology

Grossly the liver of most of these young presented
the same lesions, as the young of previous exposures (pale
discoloration and submiliary to miliary greyish white foci of
necrosis. No other lesions were detected. No tissue sections
were taken from young.

Histopathology of liver, spleen, urinary bladder, small
intestines, large intestines, kidney, uterus of does, re-

vealed nothing significant.

h. Oral Exposure of Pregnant Rabbits Surviving

Previous Conjunctival Exposure

a. Materials and Methods

Two pregnant rabbits, one near term.and the other 2-3
weeks pregnant were exposed orally to Q. monocytogenes
(culture 12255) in the same manner as described under initial
oral exposure .

Rabbit 232 was exposed orally near term. She had been

exposed conjunctivally near term about 5 months before, and

 

81

had given birth to infected young. All but one of the young
had died within 7 days. ‘E. monocytogenes had been isolated
from the young which died.

Rabbit 220 was exposed orally in the third week of ges-
tation. She had been exposed conjunctivally in the 3rd week
of gestation about 6 1/2 months before. She had aborted and
‘L.‘monocytogene§ had been isolated from.the aborted fetuses.
Both rabbits had been bred during the intervening period and
had given birth to apparently normal young. Circulating
antibody titers in the blood were not determined at the time

of the second exposure.

b. Results: Bacteriology

Rabbit 232 gave birth to 5 young, h of which died within
the first 6 days of life. One survived but died due to an
infected leg injury at 3 weeks of age. ‘Q. monocytogenes was
isolated from the liver of 2 of 3 young which died on the 3rd
day after birth and from the liver, blood,kidney and brain of
the hth young which died on the 6th day after birth, showing
central nervous system.damage.

Rabbit 220 aborted 5 days after exposure and died 2
days laterv ‘E. monocytogeneg was isolated from the liver
and brain of two aborted fetuses and from several macerated
fetuses retained in the uterus. The bacterium was also iso-

lated from.the uterus, liver, spleen and urine of the doe.

82

0. Results: Pathology

Necropsy 2126K - Young of rabbit 232.

Gross_pathology:

Liver - lesions varied from pale yellow discoloration to
extensive focal necrosis. Necropsy of one young
(Fig. A9) showing symptoms suggestive of central
nervous system.damage revealed moderate hydroperi-
toneum and a few petechial hemorrhages in the
kidney and heart, in addition to numerous submiliary
to miliary greyish white foci in the liver.

Histopathology:

~Liver - lesions consisted of diffuse fatty changes and
multiple focal necrosis, with infiltration of the

latter with polymorphs in varying number. In one

case (Fig. 50) surface of the liver was covered with
fibrinous exudate accompanied by polymorph infil-

tration.

NecrOpsy 23SOX - Rabbit 232 sacrificed 3 months after giving

birth to infected young showed no gross or microscopic lesions.

Necropsy 2173K - The uterus of rabbit 220 was filled with
macerated fetuses and a large amount of caseous necrotic

debris. No tissue sections were taken.

83

 

Fig. he. Six day-old kindling of rabbit 232 showing
depression and incoordination suggestive of central
nervous system damage.

 

Fig. 50. (Necropsy 2126K) iver from kindling (Fig. h9)
showing surface fibr'nous exudate accompanied by poly-
morph infiltration and fatty changes in the parenchy-
ma. Nematoxylin-eosin. 153x.

81+

S. Rebreeding of Infected Rabbits Without Further Exposure
a. Materials and Methods

Two rabbits (2H9 and 250) were rebred 8 days after
giving birth to infected young following conjunctival ex-

posure.
b. Results: Bacteriology

Three weeks after rebreeding, rabbit 2&9 aborted 3
fetuses (Fig. 51) and rabbit 250 aborted one mummified fetus.
Rabbit 2M9 passed one cotyledon on the 6th day, 2 macerated
fetuses on the 7th day (Fig. 52), and one mummified fetus on
the 10th day following abortion. .20 monocytogenes was iso-
lated from the liver and stomach contents of all aborted
fetuses, from.macerated fetuses, cotyledons, and vaginal exu-
date. .

Six days after expulsion of the last fetus, rabbit 249
was again bred. Twenty-nine days later, eight masses of
thick yellow material which appeared to be purulent exudate
were found to be on the dropping pan of the cage (Fig. 53).
There were also several fragments of tissue which might have
been cotyledons. Later the same day this rabbit delivered
one very macerated fetus. 'L. monocytogenes was isolated from
the purulent exudate and macerated fetus. The bacterium

could not be isolated from blood, urine, liver, spleen,

n... .-‘-‘

 

85

kidney, either horn of uterus, nor vagina of rabbit 2h9 when
she was sacrificed 9 months after the third gestation.

c. Results: Pathology

Necropsy 1983X - Young of rabbit 2A9, from first gestation

(see under initial conjunctival exposure experiment near

term).

Necropsy 20lhx - Aborted fetuses following second gestation
(without further artificially induced exposure), showed no

significant gross or microscopic lesions.

Necropsy zoéux - Aborted fetuses following third gestation
(without further artificially induced exposure) showed marked
maceration and disintegration and were covered with purulent
exudate. Histopathology revealed necrosis of cotyledons

and caseo-purulent exudate on the skin of macerated fetus.

(Fig. 5h)

Necropsy 2h2OX - Rabbit 2&9 could not be bred any longer

in spite of numerous attempts, and was sacrificed 9 months

after the third gestation.

Gross pathology:

‘Uterus - left horn appeared quite large; right horn was
normal in size but very dark purple in color.
Both horns had a few white areas that appeared

to be old scars. There were several fibrotic

86

adhesions from the uterus to the parietal layer of

peritoneum.and intestine. (Fig. 55)

 

Ovaries - left ovary contained many abscesses about 1 mm.
in diameter, and a few small cysts. The right
ovary showed h small abscesses and several cysts.

Kidneys - several small focal pitted areas in the cortex
and many pale streaks in the medulla.

histopathology:

Liver - mild subacute interstitial hepatitis.

Uterus - extensive proliferative processes in the endo-
metrium (Fig. 56).

Kidney - mild subacute interstitial nephritis.

Necropsy 2h51x - Rabbit 250 was sacrificed due to complications

unrelated to the after-effects of this experimentation.

 

 

Fig. 51. (Necropsy 20th) Aborted fetuses from rabbit
2M9. This doe gave birth to infected young from in-
duced exposure, was rebred, and aborted the next time
without further known exposure.

human: 1 p x

 

Fig. 52. (Necropsy ZOth) hacerated fetuses of rabbit
2h9 expelled on the 7th day following abortion of
fetuSes shown above (fig. 51).

8?

(Fr-97f“ -M"I'_

 

 

 

 

 

 

88

 

Fig. S3. Grayish white vaginal discharge from rabbit
249 after abortion (the third gestation to be affect-
ed by a single exposure).

I I
’
s
‘ '.

   
   

My; --'

q

if}! .5 3"
.‘V-~-._,

  
     

‘- .~..-
a“: “

Fig. Sh. (Necropsy zoeax) C sec-purulent exudate on
the surface of the macerated skin 0- fttus from rabbit
2h9. Eematoxylin-eosin. 175x.

 

“fin-”mm

1

 

Fig. 55. (Necropsy 2h20X) Fibrotic adhesions on the
uterus from rabbit 2h9, extending to parietal peri-

toneum and intestinal serosa.

 

of rabbit 249

Fndometrium

Fig. 56. (Eecropsy 2h20X)
showing the nroliferstive nroccss.

 

90

B. Sheep Experimgnts
1. Oral Exposure

a. Materials and Methods

THO pregnant ewes near term were exposed to L. monocyto-
genes by adding suspensions of the bacterium to drinking
water. Thirty-two ounce ”Bani-glee” prescription bottles
containing approximately 100 ml. of tryptose agar (Difco)
were inoculated with a distilled water suspension of freshly
grown culture (12255) and incubated at 37° C fer 18-2h hours.
They were then filled with tap water and agitated to free the
culture from the agar surface. Each day 6h ounces of this
suspension were added to approximately 10 quarts of tap water
in a galvanized pail. This constituted the only source of
fluid intake for the ewes for a period of seven days. All
aborted fetuses, young which died or were sacrificed, and the
dams which died or were sacrificed, were examined post mortem
and the liver, spleen, kidney, lungs, heartblood, stomach
contents, urine, brain, spinal fluid, uterus, cotyledons

and exudates were cultured as described for the rabbit ex-

periments (oral exposure).

b. Results: Bacteriology

Sheep 1 gave birth to slightly premature stillborn

twins 6 days after oral exposure. L. monocytogeneg was

 

91

isolated from the uterus, liver, brain and udder of the

sheep and from liver, stomach content, blood, brain and

spinal fluid of the stillborn lambs. Sheep 2 gave birth

to a lamb 7 days after oral exposure which died 10 days later.
£9 monocytogeneg was isolated from cotyledons and from.the
liver, kidney, and brain of the lamb. This sheep was sacri-
ficed 37 days after the exposure. .L- monocytogenes could not

be isolated from any visceral organ including brain.

c. Results: Pathology

Necropsy 21h21 - Sheep 1 — During the next nine days after
parturition this ewe showed a thick blood-tinged, mucus-like
putrid discharge from the vagina. She was inappetent and
progressively became very weak, emaciated and dyspneic and
expired on the ninth post-partum day. There were no symptoms

suggestive of a central nervous disorder. (Fig. 57)

GrossApathology:

Uterus - very extensive necrosis of the endometrium and
myometrium: uterine cavity filled with a thick,

putrid, sanguine-purulent exudate containing
necrotic cotyledons. (Fig. 58)

Liver - moderately pale yellow and friable.

Kidney - mild pale yellow discoloration.

Intestine - moderate number of nodular worm lesions.

H1 stopathology:

Uterus - tissue section not taken.

'"27

\O
[U

 

Fig. 57. Sheen 1 showinm prostration and vaginal
exudate on the 8th post-par um day, one day hero

death.

 

Fin. :5. ( ncronsv Zl‘gx) Utnpus 'rcm sh en 1
Sheirinrf decro‘ric to nurulont 9:110 ct1i.tis'_-rith snu—
Ruino—p mrzlent exudnro contrininH shreTss of necrotic
tissue.

9.3

Liver - moderate centrolobular fatty metamorphosis.
Kidney - moderate fatty change.

Intestine - subacute necrotic enteritis with congestion.
Lungs - marked congestion and edema.

Spleen - marked congestion

Necropsy 2121K - Stillborn twin lambs of Sheep 1

Grossgpathology:

Kidney - hemorrhagic in both the lambs.

Mesenteric lymph nodes - hemorrhagic in both the lambs.

Liver - pale yellow in the female lamb and congested in
the male .

Brain - slight congestion in the male.

Lungs - petechial hemorrhages in female.

Heart - petechial hemorrhages in epicardium of each.

Histopathology:

Lymph node - extensive hemorrhage and congestion.

Lungs - acute bronoho-pneumonia with congestion, serous
and purulent exudate, and some necrosis.

Liver - moderate diffuse fatty changes.

Kidney - moderate subcapsular petechial hemorrhage, congestion.

Necropsy 2216K - Sheep 2 - Seven days after oral exposure to
‘L. monocytogenes, this ewe gave birth to what appeared to be
a full-term lamb. No clinical manifestation of any sickness

was noticed except slight inappetence. The ewe was sacrificed

one month postpartum.

 

9h

Grossgpathology:

Uterus - incomplete involution; some caseo-purulent material
in the uterine cavity.

Histopathology:

Uterus - subacute necrotic endometritis with marked conges-
tion; edema, cystic degeneration of uterine glands;
some proliferation of fibroblasts; infiltration
with eosinophiles and some polymorphs.

Lungs - mild patchy subacute broncho-pneumonia with con-
gestion, hemorrhage, infiltration with lymphocytes
and numerous eosinophiles.

Small intestine - mild subacute enteritis, with desquamation
and coagulation necrosis of mucosa and infiltration

with lymphocytes, plasma cells and eosinophiles.

Necropsy 211;.11 - Lamb of Sheep 2 was born after

the ewe was exposed orally to L. monocytogenes for seven
days. It was very weak and stood only with difficulty and
for short periods of time. Most of the time it lay down and
was never seen suckling the ewe. It became progressively
weaker and unable to stand. (Fig. 59) It showed symptoms
Suggestive of a central nervous system.disorder, including
twitching of the muscles of the face and forelegs, inco-
ordination, and torticollis. It expired on the eighth post-

natal day 0

 

95

Gross‘pathology:
Lungs - slight pneumonia of the apical lobes.

Histopathology:

Lungs - acute broncho-pneumonia with congestion, purulent
exudate, and some necrosis; atelectasis and comp
pensatory emphysema. (Fig. 60)

Liver - marked congestion.

 

96

   

Fig. 59. horibund lamb from Sheep 2 showing extreme
depression and weakness 7 days after birth.

 

Fig. 60. (Necropsy 2lth) Lung from the same lamb
(Fig. 61) showing purulent exudate, some necrosis,
atelectasis and compensatory emphysema.

 

97

C. Goat Experiments
1. Conjunctival Exposure

a. Materials and Methods

Three goats, one near termination of pregnancy, one
non-pregnant female, and one castrated male were exposed
by conjunctival instillation of _I_.. monocytogenes. The
culture (12255) was grown on tryptose agar (Difco) slants
at 37° C for lB-Zh hours. It was then suspended in sterile
distilled water to a density approximating the number 10
tube of the McFarland nephelometer when compared in a Cenco-
Sheard-Sanford photelometer.. Four draps of this suspension
were instilled into the conjunctival sac of the left eye by
means of a capillary pipette. All aborted fetuses, young
which died or were sacrificed and all dams which died or
were sacrificed, were examined post mortem.and the liver,
spleen, kidney, lungs, heartblood, stomach contents, urine,
brain and spinal fluid, uterus, cotyledons, and exudates

were cultured as described under the rabbit experiments (oral

exposure).

b. Results: Bacteriology

Two days following the instillation ofԤ.:monocytogenes
into the conjunctival sac of the pregnant goat, a slight

serous discharge from.the eye was noticed. This persisted

‘4 H. “a.“ 'fi
,-
.

 

98

for two days after which the eye appeared normal. The goat
did not show any apparent signs of sickness. On the 26th
day after exposure the goat appeared to be in labor. The
following day she was prostrate and unable to get up.
Caesarian section was performed under pentothal anesthesia.
Three apparently healthy full term kids were delivered. The
dam.was given 12 million units of penicillin and appeared

to be resting quietly. Three cultures from the cotyledons
and amnionic fluid remained sterile. The kids suckled colos-
trum in the evening and again late at night. The following
day the kids appeared normal and suckled a little milk from
the dam. However, the dam appeared quite ill and by mid-
morning showed symptoms of a central nervous system.disorder;
incoordination, convulsions, twitching of the facial muscles
and running motions suggestive of listeric encephalitis.
(Fig. 61) Rectal temperature was 106.5. In the evening she
was given 1000 m1 of cow's milk, but no medication. The
next day she was given 500 ml. of cow's milk. The rectal
temperature came down to 10h.0 and the animal was quiet and
calm. However, she made no attempt to get up and expired
in the evening, approximately 66 hours after Caesarian sec-
tion. ‘5, monocytogene§_was isolated on initial culture
from.the brain and liver. One of the three kids was sacri-
ficed on the seventh postpartum day. Necropsy and cultures

revealed nothing significant. Another kid was sacrificed at

 

99

the age of five months due to a scrotal injury. No other
lesions were detected and all cultures remained sterile.
The remaining kid is presently a year old and apparently
normal.

The non—pregnant female and castrated male goats ex-
posed in the same manner showed slightly increased lacrimation
in the exposed eye for 3 to h days following conjunctival
instillation of‘g. monocytogenes. After this the eye appeared
normal and there was no further indication of illness in
either animal. The castrated male goat was re-exposed in
both eyes 15 days after the first exposure and was sacri-
ficed 10 days later. ‘L. monocytogenes could not be isolated

from any tissue.

c. Results: Pathology

Necropsy 2102K - Goat 2, castrated male.

Gross pathology: revealed nothing significant.

Histopathology:t

Medulla - moderate lymphocytic perivascular "cuffing" and
perivascular hemorrhages. ‘

Cerebellar stalk - moderate lymphocytic perivascular cuffing
and some neuroglial proliferation.

Liver - moderate centrolobular fatty metamorphosis.

NecrOpsy 2286K - Goat 7 died about a month following conjunc-

tival exposure, after showing involvement of central nervous

 

 

100

system. (Fig. 61)
Q£g§§_pathology:
The goat had shown a serous discharge from.the eye
for 2 days but no appreciable inflammatory reaction could be
detected.
Uterus - a little caseo-purulent exudate and necrotic cotyle-
dons. -

Histgpathology:

Medulla - very marked perivascular infiltration with mono-
nuclear leucocytes (mainly lymphocytes) and occasion- 5
al polymorphs; areas of necrosis with very little :
infiltration to areas of necrosis with infiltration
of large number of polymorphs and tendency toward

liquefaction; some sections showed very extensive

 

early suppurative lesions; limited neuroglial pro-
liferation. (Fig. 62, 63 and 6h)

Cerebellar stalk - moderate lymphocytic perivascular infil-
tration.

Cerebrum - some edema and slight meningitis with leucocytic
infiltration, chiefly lymphocytes.

Liver - moderate centrolobular fatty metamorphosis.

No other tissue sections were taken.

 

lOl

   

Fig. 61. Goat 7 with listeric encephalitis 28 days
following conjunctival exposure to g. monocytogenes.

 

 

Fig. 62. (Lecropsy 2296K) hedulla oblongata of goat 7
showing perivascular "cuffs" formed by infiltration of
leukocytes, predominantly lymphocytes. Hematoxylin-
eosin. 155x.

102

 

Fig. 63. (Necropsy 2286K) Same tissue as Fig. 62.
325K.

 

 

Fig. 6h. (Necropsy 2286K) Ledulla oblongata of goat
7 showing necrosis and polymorph infiltration with
tendency toward liouefaction and early suppuration.
Hematoxylin-eosin. 155x.

 

103

2. Oral Exposure
a. Materials and Methods

Three pregnant goats and a castrated male goat were
exposed to‘L. monocytogenes by adding suspension of the
culture (12255) to the drinking water as described under

the sheep experiment (oral exposure).
b. Results: Bacteriology

Of the three pregnant goats exposed orally to Q. gong:
cytogenes, goat h on the eighth post-exposure day was list-
less and inappetent and reluctant to move. Although she
still could get up the ninth day, she died the tenth day after
exposure. 'Q. monocytogeneg was isolated on primary culture
only from.the liver and some cotyledons. Some cotyledons
contained the bacterium in large numbers while cultures pre-
pared from others remained sterile. Cultures from.the kid-
ney and brain became positive for‘E. monocytogenes after 36 days
of refrigeration. Three kids were in 23232. ‘9. monocytogenes
was isolated from the liver, stomach contents and brain of
both of the two female kids, from the lungs and kidney of
one of them, but there were no isolations from the one male
kid.

The other two pregnant goats showed no indications of

illness following oral exposure of'g. monocytogenes. However,

 

10h

goat 5 aborted twins on the 12th post-exposure day and goat

6 aborted triplets on the 13th post-exposure day. All five
aborted fetuses appeared to be in about the fourth month of
gestation. Only one appeared to have been dead for some time
before expuslion. A few small yellow necrotic foci were ob-
served in the livers of four. There were no other detectable
lesions. 1,, W was isolated from the uterus of
goat 6 (Necropsy 2217X) which was sacrificed 7 days after
abortion. The liver, stomach contents, kidney, brain,

lungs and heartblood of all 5 fetuses were positive for‘g.
monocytogenes._ The castrated male showed no apparent signs
of illness. 'L. monocytogenes was isolated from the kidney,
urine and nasal passages when it was sacrificed 10 days after

oral exposure.
c. Results: Pathology

NecrOpsy 2103K - Goat 3, castrated male.

Gross pathology:

Intestine - mucosa highly congested throughout the length.

Kidney - a few pale greyish-white focal areas in the cortex.

Histopathology:

Small intestine - marked congestion, desquamation of epi-
thelium at the tip of villi.

Large intestine - acute catarrhal enteritis with marked con-

gestion and a few areas of necrosis infiltrated

with some polymorphs; hyperplasia of lymphoid follicles.

AI...'-.l.r\ ’0 m—‘l'
. n
.e‘

‘s . ' .n‘u‘.\n

 

Liver -

105

moderate congestion, mild centrolobular fatty change.

Gall bladder - very marked congestion and desquamation of

epithelium.

Necropsy 21721 - Goat h died 10 days after oral exposure

without having aborted.

Gross_pathology:

 

Liver - moderate fatty metamorphosis.

Lungs - moderate edema.

Vagina - contained a small amount of mucopurulent exudate.

Uterus - contained 3 kids at about u months of gestation.
The liversof the kids showed some yellowish.dis-
coloration.

Histqpathology:

Liver - moderate centrolobular fatty metamorphosis.

Lungs - moderate edema and congestion.

Kidney - some hemorrhage in the cortex, marked degenerative '
changes (resembling fatty change and necrosis), in
the proximal convoluted tubules. It could not be as-
certained with certainty whether the karyolysis was
antemortem.

Spleen - diffuse marked congestion and hemorrhage.

Mesenteric lymph node - marked congestion and hemorrhage.

Heart -

marked congestion of coronary vessels.

Fetal liver - marked congestion and hemorrhage.

.s—.—- _-._._. .i-

 

106

Necropsy 2190K - Aborted fetuses of goat 5
groggupatholcgy:

Goat 5 aborted twin kids 12 days after oral expos-
ure. The male fetus showed numerous focal greyish white areas
of necrosis whereas the female showed only a few foci.

The female appeared to have been dead for a longer period
of time than the male.

Histopathology:

_ . .«i _au-.w—.— ~4:
I.

Liver - marked congestion, focal necrosis, containing
large clumps of bacteria near the margin of the

zone of coagulation necrosis. (Fig. 65 and 66)

Necropsy 2217K - Goat 6 aborted triplets (Fig. 67) on the
13th day of oral exposure and was sacrificed 7 days later.
Gross pathology: .

Uterus - incompletely involuted, edematous, filled with
purulent exudate and caseo-necrotic debris. (Fig.
68)

Histcpathology:

Uterus - subacute endometritis with marked edema and con-
gestion of endometrium; moderate infiltration of
lymphocytes; purulent exudate in the uterine

- cavity. (Fig. 69 and 70)
Liver - moderate centrolobular fatty changes.
Spleen - some congestion and hemorrhage.

Small intestine - marked congestion in the mucosa.

 

107

Kidney - marked congestion.

Necropsy 21911 - Aborted fetuses of goat 6

Gross pathology: '

Liver - several miliary greyish white foci in the livers of
all three fetuses. (Fig. 71)

gistopathology:

Liver - marked congestion, atrophy of hepatic cords,
moderate focal necrosis containing large clumps

of the bacteria. (Fig. 72)

 

 

 

Fig. 65. (NecrOpsy 219CX) Liver of one of the aborted
fetuses from goat 5 showing a large clump of bacteria.
Hematoxylin-eosin. 820x.

 

 

 

Fig. 66. (Necropsy 2190K) Liver of one of the aborted
fetuses from coat 5 showing clumps of bacteria near

the periphery of a necrotic focus. Homatoxylin-eosin.
820x.

109

 

Fig. 67. One of the aborted fetuses from goat 6
(about h months gestation).

   

 

Fig. 68. Necropsy 22171) Uterus from goat 6 showing
edema, incomplete involution and casoo-purulent exu-
date in the uterine cavity.

 

 

llO

   

i

Fig. 69. (Necropsy 22l7X) Endometrium from goat 6
showing edema, necrosis and polymorph infiltration.
Hematoxylin-eosin. 155x.

 

Fig.70. (Necronsy 2217K) Endoxetrium from goat 6
shwhm nurulont exudate on the surface. nemato-
mflhueosin. 155x.

lll

    

W m ,2
lwerhlc I
Fig. 71. (Necropsy 2191K) Portion of liver from one

of the aborted fetuses of goat 6 showing submilianf
greyish white foci.

    

' A
V!
-1. .

$4.364 “in '. 31“.:

Fhu'fl. Necropsy 2191

 

V ) Liver from one of the a-
bmtmifetuses 0‘ cont showing necrosis and large
Mumsof bacteria. E

X
. 6

- 0 “Fa
Ienfitoxylin-COSin. bdbx.

 

112

V . DISCUSSION

5

The results of the rabbit experiments revealed that

abortion or perinatal death of young can be consistently

induced by either conjunctival or oral exposure to L. mono-
gitogenes.

They also demonstrated that if the infected con-
ceptus were quickly and completely expelled, the doc usually
survived whereas if the infected conceptus and macerated

fetuses were retained in the uterus the doe died.

This
'may explain the death of several does in the course of the
experiment.

Anton (1931;) and Morris and Julianelle (1935) observed

that the instillation of the cultures of _I_.. monocytogenes

 

into the conjunctival sac of rabbit or guinea pig produced
a marked "local" purulent conjunctivitis and keratitis.

On
the basis of this specific reaction Julianelle and Pens

(1939) suggested it as a quick method of. identification of

 

L. monocytogeneg and since then this reaction has been accepted
as one of the standard technics along with other bacterio-
logical and biological tests for identifying this bacterium.
Julianelle and Moore (1914.2) further studied the ophthalmic
response in several species-of animals (especially in rab-

bits and guinea pigs) and reported that the ocular reaction

after instillation of _I_.. monocytogenes is characterized by

113

an inflamatory response in the conjunctiva and cornea

without appreciable involvement in the deeper structures

of the eye. They report that, "in general, the cellular

infiltration in the first few days consists principally of
lymphocytes but by the 5th day, the proportion of poly-

morphonuolear leukocytes increases until these cells pre-

dominate by the 10th day. Edema of the involved structures

is a prominent feature at the height of the infection. Vas-

cularization of cornea develops concomitantly with other

evidences of inflammatory response. The pathological

changes rapidly recede and the eye generally heals without
scarring 2 to 3 weeks after instillation of the organism."
This reaction led Julianelle and Moore (1914.2) and others to

believe that it is a "local” eye reaction.
However, Graham, 21; §_]_._. (1914.3) and Gray _e_t _e_l. (1914.8)
reported death due to listeric encephalitis of a pig and a

rabbit, respectively, following conjunctival instillation

of E. monocytogenes. Anton (1931+) observed death due to lie-

teric septicemia in a one-w‘eek-pregnant rabbit and abortion
in another rabbit late in gestation following conjunctival

instillation of Q. monocytogenes. Belin (19147) observed

abortion in a guinea pig following conjunctival instilla-

tion of the bacterium.
The results of this study confirm experimentally the

observation of these latter authors and clearly demonstrate

11k

that the bacterium is not confined in the so-called "local"

eye reaction (kerato-conjunctivitis) as previously supposed
by Julianelle and Moore (1911.2) and others. The results also
emphasize the fact that Q. monocytogenes is not only capable

of setting up an inflammatory process in a mucous membrane but
also may gain entrance to the body through the apparently in-

tact mucous membrane, and may cause abortion and perinatal

. .914...
1.

death of young in pregnant rabbits. This was further suggested

by the finding that conjunctival instillation of 1.3. monocytogenes

 

produced a listeric encephalitis in a pregnant goat 30 days
after exposure.

.-..A-_ _..i.. 44
A ' t

In a castrated male goat, histopathological
studies of the brain revealed a mild encephalitis, in spite

of the fact that the animal did not show noticeable symptoms

and the bacterium could not be isolated following conjunc-

tival exposure to _I_-. monocytogenes.

Asahi and Hosoda (1952 -- 1953) claimed the production of

encephalitis in goats following conjunctival exposure of _I;.

monocytogenes. These authors reported the bacterium reaches

the medulla by way of the trigeminal nerve and then spreads

to other brain tissues. While this claim needs further con-

firmation, it does appear that the pathogenesis of listeric

encephalitis. in ruminants has some unusual features. This

is suggested by the fact that encephalitis was not demonstrated
following conjunctival or oral exposure of rabbits or oral
exposure of sheep and goats, although. _L_. monocytogenes was

isolated from the brain in several cases representing all

I

 

115

three species. It is also interesting to note that.£.‘gggg;
cytogenes could not be isolated from the placenta, the young,
or the reproductive tract of the goat which died of listeric
encephalitis following conjunctival instillation (it was
isolated from brain and liver). The observation that no abor-
tion and perinatal death of young was noted in several natural
outbreaks of listeric encephalitis in pregnant sheep further Fm‘
suggests variation in pathogenesis. Though this study clear-
ly indicates that conjunctival exposure of‘g. monocytogenes

may incite listeric encephalitis, no isolation of the bac-

DM. .v .a—Hh-_il._
. . J A
q

terium has been reported from the eye of ruminantswwith
natural infection. Other portals of entry, especially intra-
nasal or via the respiratory tract, are suggested by Gill
(1933), and Olafson (l9h0) and hematogenous pathogenesis is
suggested by Graham g; a}. (l9h3) and Olson (1951). Osebold and
Inouye (195M) are inclined to favor a hematogenous patho-
genesis in spite of the fact that they could isolate £2.
monocytogenes in 5 out of 7 instances from.the brain of rab-
bits following intranasal exposure. Asahi and Hosoda (1953)
claim to have induced listeric encephalitis in h of 11 goats
following inoculation in the submucosa of the lip, and in 2
of 3 goats following exposure to rough feed contaminated with
‘E. monocytogeneg. These authors suggest entrance of the bac-
terium through small wounds caused by the rough feed. Fel-

senfeld (1951) reported an outbreak of listeric conjunctivitis

 

 

116

in workers in a poultry processing plant. He isolated L.
monocytogenes from the spleen of five apparently normal
birds which were processed at the plant. This incident
suggests the possibility of the conjunctival as one of the
portals of entry.

An oculoglandular form of listeric infection in Russia
is reported by Pletneva and Stiksove (1950) and Shmeleva f_?
(1953). They could not isolate‘L. monocytogenes from.the E
patients but reported the diagnosis on the basis of aggluti-
nation tests. Shmeleva (1953) reported direct contact with
animals in.h cases of conjunctivitis in human patients.

The fact that abortion and perinatal death of young in
rabbits, sheep and goats followed oral exposure to'g.‘gggg;
cytogenes re-emphasizes the contention that g..monocyt9genes
can pass through the intact mucous membrane.

Patocka (1953) has suggested the possibility of a fil-
terable form of Q. monocytogenes, but this has not been sub-
stantiated so far.

Olson (1956) reported the isolation of a listeriosis-
enhancing agent (LEA) which in combination with‘g; monocyto-
sgggg via intranasal exposure results in encephalitic form of
listenbsis in sheep. Lesionsyfime confined to the central
nervous system.

Listeriosis in the young of all species of animals so

far observed, including man, is usually septicemic and

117

terminates fatally. The experimentally induced disease as
observed in this study closely resembles that of the natural
epidemic of perinatal death of human infants in Germany and
elsewhere (Table IV) and that of perinatal death of calves,
lambs, foals and pigs reported elsewhere (Table III). The

does exposed near term.usually survived. This also is true

 

of the naturally infected mothers in all species. Isolation F*’
of g. monocytogenes from the viscera of young (especially 1
liver and stomach content) and from the placenta, vaginal 1
exudate and uterus of the dam supports the contention of g
Erdmann and Potel (1953) who suggest the sequence of events L

leading to fetal infection as follows: infection of the dam;
transfer of the bacterium across the placenta resulting in
septicemia with development of lesions in the fetus; shedding
of the bacteria through the urine, contaminating the amnionic
fluid; aspiration and swallowing of the contaminated amnionic
fluid resulting in a secondary infection of the fetus by

way of the respiratory and digestive tract. Similar views
are held by Linzenmekr 33 31. (19531, Hahnefeld and Nisolk
(l95h) and Flamm (1955).

Oral exposure of rabbits surviving previous conjunctival
exposure, and vice versa, clearly indicated that the previous
exposure (2-6 months later) did not provide protection to
further exposure. Circulating antibody titers were not deter-

mined immediately before the second exposure; however,

 

118

Julianelle (19hl) contends that agglutinating antibodies are
not a measure of protection against infection with L'.E222:
cytogenes.

.90 monocytogenes was found to survive at least h weeks
in the reproductive tract of a rabbit which died after con-
Junctival exposure. This conforms to the report of Levi gt
,El: (1952) who isolatedlg. monocytogenes from the ovary of
a heifer which was slaughtered two and a half months after
listeric abortion.

In the present study, conjunctival exposure to L. mggg:
W causedin one rabbit, in addition to infection of
fetuses of the current gestation, two subsequent abortions
without any further exposure. These abortions took place
about the 3rd week of gestation. It remains to be determined
as to how conception could occur and the fetuses develop up
to a certain stage of gestation in the presence of‘g.lmggg;
cytogenes. The question of some hormonal factor or fetal
tissue affinity is a subject for further research.

Other routes of infection, especially the urogenital
tract, must be considered in cases of abortion and perinatal
death of young. An intrauterine infection induced by vaginal
instillation of‘L. monocytogenes has been observed in an asso-
ciated study in this laboratory and fetal death and absorp-
tion of embryos in rabbits following intravaginal exposure
on the 7th day of gestation has been reported by Osebold and
Inouye (l9Sh).

 

 

119

The isolation of Q. monocytogenes from the urethral
exudate of 5 men with gonorrhea by Wenckebach (1953), from
vaginal mucus of a cow by Stenberg (1953), from the cervix
of a so-called repeat-breeder cow by Gray and McWade (l95h)
and from.vaginal exudate of a human patient by Stoot (195u),
suggests a venereal mode of infection.

It is quite clear from this study that uterine contents,
aborted fetuses and vaginal exudates contain L. monocytogengs
in large numbers. On expulsion they may constitute a source
of infection by contaminating food and water. Gray at 2;.
(1956) reported that one goat was infected by direct
contact with infected goats (which aborted following oral
exposure to Q. monocytogenqg and their contaminated environ-
ment. This is also supported by the observation of Poppen-
siek (l9hh) in which a cow died of listeric encephalitis
after grazing on the same pasture where an ovine abortion
thought to be due to E. monocytogenes had occurred. Van der
Schaaf (1955) reported 2 cases of listeric infection in a
farmer and in a veterinarian after handling a case of bovine
abortion. ‘9. monocytogenes was isolated from blood and spinal
fluid of the farmer who died and from blood of the veteri-
narian who survived following therapy. ‘

In the present study,‘£. monocytogenes was isolated in
one instance from.the milk of a rabbit and from.the udder of
a sheep which died following oral exposure. Potel (1953-5h)

 

120

isolated'L. monocytogenes from a case of bovine mastitis.
Gray and Thorp (1956) have shown on rabbit experiments that
'9. monocytogeneg passes into the milk following oral exposure
of the dos. Thus infection of the newborn could be induced
by suckling the orally exposed doe.

This study also demonstrates that Q. monocytogenes is
excreted through the urine and may be a source of contaminae
tion of feed, possibly by rodents. No cultures of fecal
material were made in this investigation; however, it may
serve as a source of infection. Potel (1953) reported that
meconium.of the infected human infant is a very good source
for the isolation of ;. monocytogenes. This should be fur-
ther investigated by experimentation on animals. Other
phases deserving investigation are the possible roles of
rodents, insects and parasites and the possibility of the
organismfls being a saprophytic and facultative pathogen.

The whole field of the transmission and intertransmissi-
bility of listeriosis is incompletely understood. No reliable
diagnostic or clinical tests are available for detection of
a carrier state or subclinical case. Therefore prophylactic
measures are lacking and need further research.

The presence of the bacteria can be demonstrated in the
tissue sections of most of the organs of aborted fetuses and
young which have died perinatally thus further confirming

the septicemic nature of the disease in these animals.

 

 

.121

Gray (1956) reported the isolation of Q. monocytogenes
from thrombi on the heart valves of a lamb. Hoeprich.g£,§;.
(1956) have reported a case of subacute bacterial endocarditis
due toIL. monocytogenes in a human patient. These reports in-
dicate that atypical forms of listeric infection should be
investigated.

The exact manner in which‘g. monocytogenes induces the

-..- .. Im‘rn Ila
-u
h

lesions is incompletely understood. Although the lesions

appear to be caused by some bacterial toxins, or toxins, the

.....9

production of endotoxin or exotoxin has not been demonstrated I
so far.

In this study, the tendency toward thrombus formation
in the liver of young and thrombosis with leukocytic infil-
tration and endothelial degeneration in the blood vessels of
the myometrium, indicate that L. monocytogenes may initiate
an inflammatory process in the blood vessels and cause thromp
bosis. This thrombosis would contribute in the causation of
necrosis by interfering with the circulation. This, in turn,
would interfere with elimination of toxic metabolic products
and thus aid in the furtherance of the necrotic processes.
This may also explain the edema of the endometrium.

The encephalitic lesions in the medulla in goats, es-
9361ally in the one which died following conjunctival exposure
t0_L_. monocytogenes, closely resemble the microscopic lesions

found in natural cases in sheep and goats. The lesions are

122

primarily found in the medulla. Asahi and Hosoda (1952 -
1953), on the basis of histopathologic examination of brain
and trigeminal nerve sections, claim that the bacterium fol-
lows the trigeminal nerve and produces lesions along the
pathway of the nerve and then in the medulla. They state
that infection may then spread outward even to the meninges.

The fact that encephalitic lesions were produced following

1‘ -> ’ .119 ' Ia"

conjunctival instillation in 2 goats in the present study,
tends to support this claim. However, further work is nec-
essary to establish definitely that.§. monocytogenes may g
follow branches of the trigeminal nerve. Hydroperitoneum 1
and fibrinous exudate on the visceral organs,especially on
the liver and intestine, were noted in two of the young
following oral exposures of pregnant rabbits near term. The
microsc0pic lesions in the gall bladder (necrosis and poly-
morph.infiltration), necrotic exudate and presence of the
bacterium.in the vicinity of the bile duct, found in this
study, apparently have not been previously reported in the
literature. The significance of these findings remains to
be further explored.

The number of positive isolations of'g. monocytogenes
from tissues cultured in the present study was considerably
enhanced by employment of the isolation technic described
by Gray _e_t _a_1_. (19118). This technic has been further em-
phasized in the reports of Gray e_t_ 5;. (1955. 1956).

123

VI. CONCLUSIONS

1. Conjunctival instillation of Q. monocytogenes in
pregnant rabbits results not only in conjunctivitis but
also in abortion or perinatal death of young.

2. Oral eXposure of pregnant rabbits, sheep and goats
to Q. monocytogenes causes abortion and perinatal death of

young 0

3. In case of retention of infected conceptus, the dam
may die from septicemia.

h. In perinatal infection of the young in rabbits,
sheep and goats, the disease is usually septicemic and
aLmost always fatal. The dam.usually survives with little
or no apparent symptoms of illness.

5. The bacterium may remain latent in the rabbit for at
least 3 weeks after kindling and may cause subsequent abor-
tion, if the animal is rebred within a week after abortion.

6. Evidence of immunity in rabbits (at least within
2-6 nmmths) following survival of any form.of eXposure under
conditions of these experiments is lacking.

7. Conjunctival and oral exposure of non-pregnant
rabbits and oral exposure of castrated male goats usually
fails to cause systemic disease. However, the organism may

be isolated from visceral organs in some cases as long as 10

days after exposure.

I‘MTI"

U. L7~ TIA.—

II???"

121+

8. '9. monocytogenes may be excreted in the urine of
rabbits and goats.

9. Conjunctival instillation of E. monocytogenes in
goats may cause listeric encephalitis.

lO. Listeric encephalitis may not accompany uterine

infection in goats and sheep following oral exposure to L.

1 —.—- i
1 ‘11
.

monocytogenes.

--K J‘.‘ .

11. The clinical manifestations of listeriosis apparently
vary with species, route of exposure and stage of pregnancy.

12. Gross lesions in aborted fetuses and cases of peri-

IP21-

natal death of young in rabbits, sheep and goats include,
with variations in degree and extent, the following: yellow
discoloration and submiliary to miliary greyish-white foci in
the liver and, less regularly, fibrinousadhesions on the
surface of the liver and hydroperitoneum.

13. Gross lesions of aborting and parturient does and
ewes include, with variations in degree and extent, the
.following: necrosis of endometrium, with caseous necrotic
:materdal, retained conceptus, and purulent uterine exudate.
Ibess consistently observed are: incomplete involution of the
uterus in sheep and goats; fibrotic adhesions on the uterus
of rabbits which abort several times; and yellow discolora-

titni and small greyish-white foci in the liver of dams dying

wi th septicemia .

 

12S

1h. Histopathologic examination of aborted fetuses and
cases of perinatal death of young in rabbits, sheep and
goats reveals, with variations in degree and extent, the
following: fatty changes and focal necrosis of the liver,
with polymorph infiltration and tendency of thrombus forma-
tion in the smaller blood vessels. Less frequent findings
include: foci of necrosis and polymorph infiltration in E
heart, lung, kidney, stomach and gall bladder, necrotic
exudate in the bile ducts of some young rabbits, and prolifer- 1
ation of fibroblasts and angioblasts in the liver. In addi- 1
tion to hepatic necrotic foci, acute bronchopneumonia may 1
be found prominently in the young of sheep and goats. Bac-
teria (sometimes in very large clumps) may be demonstrated
in several organs especially in liver, heart and kidney.
15. Histopathologic examination of uteri of aborting
and parturient does and ewes reveals, with variations in de-
gree and extent, the following: endometritis with necrosis
of endometrium.and purulent exudate on the surface of mucosa;
necrosis and distention of uterine glands with or without
cellular exudate; necrosis and thrombosis with polymorph
infiltration and endothelial degeneration in the blood ves-
sels of'the myometrium. Bacteria (sometimes in large clumps)
may be noticed in the endometrium, uterine glands, cotyledons
and myome trium.
16. Histopathologic examination of the liver of aborting

and parturient does and ewes shows, with variations in degree

126

and extent, the following: fatty changes, some coagulation
necrosis with or without polymorph infiltration; small foci

of lymphocytic infiltration and proliferative processes in
chronic cases. Subacute interstitial nephritis may be noted
in some cases. Edema of the lung is usually observed in sheep
and goats.

17. The most prominent microscopic lesions in the con-
junctivally exposed goats are seen in the medulla oblongata.
They consist of perivascular infiltration, chiefly with lym-
phocytes, and areas of necrosis with polymorph infiltration
and, sometimes, early suppuration.

18. This study supports the contention that intra-
uterine infection of the young in animals and man may take
place through the placenta.

19. The use of obliquely transmitted light and a binocu-
lar scanning microscope is helpful in identifying the colonies
of‘g. monocytogenes.

20. The maceration of tissues and storage under refriger-
ation for at least 3 months is helpful in the isolation of

,9. monocytogenes when primary cultures are negative.

"i I‘

4"!

 

 

127

VII. SUMMARY

1. Conjunctival exposure of 9 rabbits (2-3 weeks
pregnant) to Listeria monocytogenes caused abortion in 3-6
days and death of 5 does due to septicemia. Oral exposure
of 6 rabbits in the same stage of pregnancy caused abortion
in h-6 days and death of h does due to septicemia. L. mggg;
cytogengg was isolated from liver, spleen, blood, kidney,
uterus, cotyledon, brain and vaginal exudates of does, and
from the liver, stomach content, and amnionic fluid of
aborted fetuses.

2. Conjunctival exposure of 7 rabbits near term caused
perinatal death of 57 of 58 young due to septicemia within
7 days. Only one survived. All does except one survived
and showed no apparent signs of illness. Oral exposure of 8
rabbits in the same period of gestation caused the perinatal
death of all 55 young within u days due to septicemia. All
does survived and showed no apparent signs of illness. 1;.

monocytogenes was isolated consistently from the liver,

stomach content, and cotyledons and in most instances in which

brain, blood, spleen, intestine, lung, kidney and urine of

the young were cultured.

3. Conjunctival exposure of 6 and oral exposure of 3

non-pregnant rabbits failed to produce apparent infection.

 

128

E9 monocytogenes was isolated from the uterus of 2 rabbits

 

and liver of one rabbit conjunctivally exposed (tissues not
refrigerated) and from the liver of one rabbit orally exposed
(after refrigeration).

h. Retention of the infected conceptus usually was
followed by death from septicemia whereas the does usually
survived if the conceptus was expelled quickly and completely.

5. L. Wwas isolated from a uterine abscess
of one rabbit which died 3 weeks post partum following con-
junctival exposure.

6. Conjunctival exposure of 6 rabbits (near term) sur-
viving previous oral exposure caused perinatal death of 33
of 37 young; A survived. The results followed the pattern of
initial conjunctival exposure both in production of lesions
and isolation of E. monocytogenes from the young.

7. Oral exposure of 2 rabbits(one 2-3 weeks pregnant
and the other near term) surviving previous conjunctival
exposure was followed by abortion and perinatal death as in
the case of initial oral exposure.

8. One rabbit which was bred within a week after giving
birth to infected young following conjunctival exposure,
aborted about the third week of gestation. Rebreeding within
6 days after the abortion resulted in subsequent abortion in
the 3rd week of the 3rd pregnancy.

9. Oral exposure of 2 pregnant ewes near term resulted

in death of the young. One ewe died 9 days post partum due

.- ’ "n, when X11‘

44.. tv M'r,..g_xr~_~/;’asn —
d I

 

129

to septicemia. ‘L, monocytogenes was isolated from the uterus,

liver, brain, and udder of the ewe and from the liver, stomach
content, blood, brain, spinal fluid, and kidney of the young
and from.the cotyledons.

lO. Conjunctival exposure of 2 goats resulted in enceph-
alitis.

11. Oral exposure of 3 pregnant goats was followed by
abortion of all 8 fetuses and death of one of the dams.

‘2. monogytogenes was isolated from the liver, stomach con- 1
tent, kidney, brain, lungs and blood of most of the aborted
fetuses.

12. Aborted fetuses and young which were stillborn or
died soon after birth revealed liver lesions consisting
of varying degree and extent of yellow discoloration and
submiliary to miliary greyish white foci. HydrOperitoneum
and fibrinous exudate on the surface of the liver were
noticed in some young rabbits. Slight penumonia was noted
in one lamb.

13. Uteri of rabbits which aborted showed marked con-
gestion and caseous necrotic masses on the endometrial sur-
face with varying degree and extent of purulent exudate in
the uterine cavity. After removal ofthe caseous material
from.the uterine mucosa, the endometrium appeared reddened
and corrugated; in some cases it was necrotic throughout its

depth. The cotyledons were usually necrotic. Yellow

130

discoloration and small greyish-white foci were noticed in the
liveniof some does. Incomplete involution of the uterus with
some purulent exudate was noted in sheep and goats. The
uterus of one sheep which died was extensively necrotic and
filled with thick, putrid material containing necrotic cotyle-
dons.

1h. HistOpathologic examination of the livers of
aborted fetuses and young which died perinatally (rabbits,
sheep and goats) revealed (a) varying degree and extent of
fatty change in the hepatic cells, (b) varying sizes and
numbers of foci of necrosis, and (0) variable polymorph
infiltration of necrotic areas. Similar necrotic lesions
were noted in the heart, kidney, lungs, stomach, and gall
gladder in some cases. Fibrinous adhesions were seen on the
liver and intestine of some young rabbits. Bacteria could
be demonstrated in the liver, bile duct, kidney and heart
of rabbits and liver of goats. In one young rabbit, the
bile ducts were found to be filled with purulent exudate.

15. Histopathologic examination of the uteri of rabbits
which died or were sacrificed after abortion revealed acute
necrotic endometritis with varying extent of necrosis and
leukocytic infiltration (mainly polymorphs) of endometrium.
In some cases, edema of endometrium and dilatation of uterine
glands with or without cellular exudate were noted. Clumps

or bacteria were often seen in the endometrium, uterine glands

- «we 1 Mimi]

131

and cotyledons. The myometrium.often showed necrosis in
variable extent and thrombosis of blood vessels with leuko-
cytic infiltrations. In chronic cases following abortion,
proliferative processes and scarring of the uterus were ob-
served. The uteri of sheep and goats showed mild endome-
tritis with some necrosis and edema of endometrium and some
purulent exudate on the surface of endometrium. ;
16. The possible significance of the foregoing findings
and some of their implications with regard to further research

are discussed.

c .-n -r_a-m:—I
e t. e

132

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.10.. ~i .—

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1* '
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PATHOIDGY AND BACTERIOLOGY OF ABORTION AND PERINATAL
DEATH OF YOUNG IN RABBITS, SHEEP AND GOATS

INDUCED BY LISTERIA MONOCYTOGENES

By
Chintamani Singh

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of
'Michigan State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Pathology
1956

Approved éé‘.W
i r i

,1;th 21‘? "7’ . ‘7?! "

(In .019- I02...“
I

“U5 bA'dUD ULLLIAL’CAIUCUIJ. 01.11511

1

Studies on the pathology and bacteriology of abortion
and perinatal death (stillbirth, and early death) of young
in rabbits, sheep and goats induced by Listeria monocytogenes
were undertaken to help explain the role of the bacterium in
this syndrome.

Pregnant, non-pregnant and male rabbits and goats and
pregnant sheep were exposed either by conjunctival instilla-
tion of suspensions of the bacterium or by adding them to
the drinking water.

Conjunctival exposure of pregnant rabbits resulted not
only in a marked conjunctivitis but also in abortion when
the doe was exposed early in gestation; when the exposure
was late in gestation, the young were either stillborn or
died within a few days due to listeric septicemia. Con-
junctival exposure of non-pregnant rabbits resulted in
marked conjunctivitis and a low-grade and otherwise inappar-
ent infection. Conjunctival exposure of goats resulted in a
very mild eye reaction but caused death of a pregnant goat
due to listeric encephalitis and induced encephalitic lesions
in a castrated male goat.

These findings establish the fact that the infection
resulting from conjunctival instillation of L. monocytogenes
does not remain localized in the conjunctiva.

Oral exposure of pregnant rabbits, sheep and goats re-

sulted in abortion if the dams were exposed early in gestation;

Chintamani Singh
2

if the exposures were late in gestation, the young were
either stillborn or died within a few days due to listeric
septicemia. Non-pregnant animals exposed in the same manner

suffered a low-grade inapparent infection.

 

.E- monocytogenes was readily isolated from aborted
fetuses, young born at term, placenta and from the dams
which died due to septicemia.

The results suggest the uterus as the principal target
of infection in pregnant animals. If the entire conceptus
was expelled, the defense mechanism of the dam could success-
fully combat the relatively few remaining bacteria as in
the non-pregnant. However, retention of infected conceptus
(1) caused localized suppuration, (2) constituted a source
of infectibn which resulted in subsequent abortions if the
dam.was rebred within 6 days, or (3) caused death from
septicemia.

No evidence of immunity could be demonstrated in rabbits
(at least within 2-6 months) following survival of any form
of exposure under the conditions of these experiments.

The various clinical syndromes of listeric infections
apparently vary with species, route of exposure and state
and stage of pregnancy.

The most prominent pathological findings in aborted
fetuses and the young which died perinatally included necrotic
foci in the liver and less frequently in the heart, lung,

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Chintamani Singh
3

kidney, stomach and gall bladder, with necrotic debris, in

the hepatic bile ducts; fibrinous exudate on the surface of
liver and intestine; noderate hydroperitoneum. The bacterium
could be demonstrated in tissue sections of most of these
organs. Examination of dams which died or were sacrificed
revealed necrotic endometritis with variable amount of caseous
to purulent exudate. Numerous necrotic foci and thrombi were
noticed in the myometrium. The bacterium could be demonstrated

in tissue sections of the uterus including the cotyledons.

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