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HELENA MONOCYTOGENES:
A FIELD SURVEY AND
LABORATORY OBSERVATlONS

Thesis for flu Dogru of M. 5.
MlCHIGAN STATE COLLEGE
Mifchell L. Gray
1949

TH ESlS

This is to certih] that the

thesis entitled

Listeria Monogztogenes: A Field Survey and
Laboratory Observations

presented bl]

Mitchell L. Gray ‘

has been acceplvd towards fulfillment

ul lllt‘ requirements {or

Bacterlology and Public
Health

5&5: degree in ,

 

Date, Marv}?! DR,

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A :‘_‘L ._-' " ~33”, .

 

LISTERIA MONOCYTOGENES: A FIELD SURVEY
AND LABORITERY UBSERVATIONS

 

by
Mitchell L. Gray

A Thesi-

Snbmitted to the School of Graduate Studies of
Michigan State College of Agriculture and
Applied Science in partial fulfillment
of the requirements for the degree

of

MASTER OF SCIENCI

Deyartncnt of Bacteriology and Public Health
1949

l.
l
I
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ll

{Ill-l.
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I

(3,!

Affectionately dedicated
to
Nell

216988

ACKNOWLEDGEMENT

The author wishes to exPress his sincere
appreciation to Dr. H. J. Stafseth for his con-
structive criticism and benign influence. To
Dr. Frank Thorp, Jr. for the keen inspiration
and compelling influence here reflected. To
Drs. L. B. Shell, R. A. Runnells and R. F.
Langham for their part in the necropsy examina-
tions. Also remembered are the veterinarians
in the field for their cooPeration in making
the material available for study in the labor-
atory. Thanks also are given to the technicians

for their assistance.

The author is grateful to Merck & 00.,
Rahway, N. J., for the streptomycin; Commercial
Solvents Corp., ferre Haute, Indiana, for the
bacitracin and Park Davis & 00., Detroit, Mich.,
for the chloromycetin.

TABLE OF CONTENTS

Page
Introduction 1
Part I. A Field Survey
Methods of Isolation for Diagnosis 4
Report of Cases 7
Discussion ' 17
Summary 25
Illustrations, 27
Part II. Laboratory Observations
Isolation from the Bovine Brain 42
Ocular Instillation 46
Viability 53
Inhibition 57
Sodium Azide 58
Potassium Tellurite 58
Acetic Acid - Deep Brain Medium 60
The Effect of Antibiotics 61
Streptomycin 61
Bacitracin 89
Penicillin 91
Chloromycetin 92
Pathogenicity 93
Immunological Reactions 9?
Biochemical Reactions 99
Summary 101
Illustrations 104

Bibliography 115

INTRODUCTION

In the nineteen years that have passed since
Gill (1931) showed Listeria monocytogenes to be the
etiological agent of circling disease in sheep, lis-
teriosis has become a disease of considerable economic
importance. It has been established that its presence
is world wide; that its hosts are not only sheep, but
also cattle (Graham 33 31, 1943; Hatch, 1945; Boucher,
1946; Cole, 1946; Pounden‘gt'gl, 1947A; Gray 33'31,
1948), swine (De Blieck and Jansen, 1942, 1943; Gra-
ham gt'gl, 1943; Kerlin and Graham, 1945; Rhoades and
Sutherland, 1948), goats (Graham.gt.§l, 1943; Kaplan
and Lager, 1945; Gifford and Jungherr, 1947), dogs
(Cox, 1945 and Chapman, 1947), chickens (Graham gt 21,
1943; Jansen and Pepperkamp, 1947), some forms of wild
.life (Lilleengen, 1942; Graham gt 21, 1943; Gifford and
Jungherr, 1947; Jansen and Pepperkamp, 1947), man
(Kaplan, 1945; Handelman £3 21, 1946; Wheeler, 1948).
Subsequently it has been reported in sheep by Graham 3t
21, 1943; Pounden and Edington, 1947; Gray 23 El, 1947;
Ryff and Lee, 1948. There is, as yet, no method of con-
trol nor adequate treatment of its victims. The next

two decades may find it one of the leading killers of

livestock.

The presence in Michigan of this bacterium and

listeriosis, the disease caused by it, was first sus-
pected by Chang (1945). He reported a two-month old
lamb suffering from a disorder of the central nervous
system, but did not culture the brain at necrOpsy.
However, histological sections of the medulla revealed
the characteristic "perivascular cuffing". Gray 33's;
(1947) confirmed its presence in sheep by isolation and
identification of‘L. monocytogencs. This report includ-
ed three ewes and one lamb. The ewes exhibited typical
encephalitis while the lamb displayed a septicemia.

One other ewe showed similar symptoms but Listeria was

not isolated.

The following year Listeria was shown by Gray
23’s; (1948) to be present not only in sheep but also
in cattle.fren Michigan. That year gram positive bac-
teria were isolated from five cows and one ewe. These
bacteria were subsequently identified as L. monocytogenes.
During the winter and spring of 1948 Listeria was iso-
lated from nine sheep and nine cows. In a one month
period from December 15, 1948 to January 15, 1949‘Lig-
123}; was isolated from seven head of cattle. From
that date until April 28, 1949 Listeria was isolated
from three more cows and 17 sheep. The records compiled
during this study indicate that this disease has killed
at least 104 head of livestock. A killer of food pro-

ducing animals such as this, which is also a menace to

l‘ II [III II. I lll| I I

the health of their owners, warrants a complete study
to determine, if possible, effective methods of diag-
nosis, mode of transmission, methods of treatment and

a system of control.

For convenience sake this thesis is divided into
two parts. The first consists of a report of the prob-
lem of listeriosis as it affects livestock in the state
of Michigan. The second part presents the experimental
procedures undertaken in an effort to contribute to a
better understanding of the behavior of the etiological

agent.

Part I

A FIELD SURVEY

Part I. A FIELD SURVEY
methods 2; Isolation for Diagnosis

The first attempts to isolate L. monocytogenes
from suspected material resulted in failure. The tech-
nic employed consisted of scaring the incised portion
of the medulla oblongata at its junction with the spinal
cord. A sterile inoculating 100p was inserted and the
material adhering to the loop after removal was plated
on tryptose agar and sheep blood agar plates. Incubation
at 37° C for periods up to 96 hours failed to produce
any significant bacterial growth.

With the adoption of a modification of a technic
described by Biester and Schwarte (1939) isolation of

Listeri§_was accomplished. This procedure consisted

 

of scaring the exposed surfaces of the medulla after

its removal from the skull. A large portion of this
material was placed in a sterile mertar tagether with
10-15 ml nutrient broth and ground to a fine pulp. This
material was transferred to a sterile bottle containing
glass beads and put in a shaking machine for from 10

to 20 minutes. Approximately 0.5 ml of the resulting
material was plated on tryptose and sheep blood agar
plates. Incubation at 37’ C for 18 to 24 hours produced

a heavy growth of Listeria on both media.

Both the above described technics were carried
out on the three cases of listeriosis in ewes reported
by Gray 33 a; (1947). In no instance was Listeria iso-
lated when the brain material was not macerated as describ-

ed above.

When, in the spring of 1947, several bovine brains
were submitted to culture for Listeria, the inoculated

 

plates remained sterile. Some of the animals concerned
had shown what seemed to be a clinical picture of the
disease, yet the causative agent could not be isolated
(figure 15). However, it was found that after the pre-
pared brain suspensions had been refrigerated at 4. C
for varying lengths of time, Listeria could be demon-
strated by colonies in quantity comparable to those found
in sheep brain isolations. These original observations
were reported by Grayugt.§1 (1948A) and are further con-
firmed in this thesis.

Recently a further modification was made. This
consisted in the employment of a Waring blendor with a
micro head attachment to macerate the brain material.

A portion of the scared medulla was placed in the blender
together with approximately 15 m1 of nutrient broth.

This was agitated for three or four minutes. A 0.2-

ml portion was plated on tryptose agar plates. The in-

cubation time and temperature were the same as above.

Except for the economy of time and convenience the blendor
has no particular advantage in sheep brain isolations.
However, it would seem that the number of colonies in
bovine isolations was increased. To prevent excessive
heating the running time of the blendor should not ex-

ceed four minutes.

In examining the incubated plates for the pres-
ence of Listeria a dissecting microscope was employed
as described by Huddleson (1946). White light was direct-
ed obliquely (approximately 45°) through the stage of
the microscope. The colonies of Listeria were a bright
green with a finely textured surface. This was found
to be so characteristic that Listeria colonies can be
identified even in cultures with extreme contamination
(figure 1). This may safely be used as a diagnostic
technic and was used in all but the first four isolation

attempts reported.

By employing these technics the isolation of
Listeria did not fail in any clinically described case.

 

The diagnoses were substantiated by demonstration of

the histo-pathology in hematoxylin eosin stained sec-
tions of the medulla. In listeriosis a marked so-called
'perivascular cuffing" and areas of focal necrosis

were noted. ‘These areas were characterized by both

mononuclear and polynuclear cells (figure 19).

ll‘l!lllll

-7-

Report 2; Cases

Outbreaks, 1946.

Case.l. march 23, 1946. St. Johns.

yearling ewe was admitted to the pathology labora-
tory for diagnosis, March 22. The animal was lethargic
and showed rather indefinite symptoms of central
nervous system origin. Kctosis was suspected but
clinical pathological tests of the blood and urine
revealed no significant change in glucose value and
no kctcne bodies in the urine. After three days'
observation the animal was sacrificed. At necropsy
the brain showed some congestion. There were no
other significant findings. Cultures prepared from
the medulla were positive for Listeria.

Case‘g. April 4, 1946. East Lansing.
In eight-day old lamb was presented for routine
necropsy. The lamb had been weak since birth. The
cmentum and diaphragm adhered to the liver. White
necrotic foci were uniformly spread throughout the
liver substance. There were numerous whitish grey
foci throughout the lungs. Cultures were prepared
from the liver, lung, and heart blood. Listeria
was isolated from the liver and heart blood.

0380 so m 7, 1946o hat main‘e
I'Iour-year old ewe was found staggering on the morn-
ing of May 6. After 250 ml glucose was administered
intervenously, she seemed no better. The next morn-
ing 200 ml saline with 5’ glucose were given intra-
peritoneally. She died in the evening of that day.
At necropsy there was an acute bronchopneumonia.
The kidneys showed parenchymatous degeneration. There
were no other lesions. Cultures prepared from the
medulla were positive for Listeria.

Case's. lay 14, 1946. East Lansing.

six-year old ewe was noticed staggering on the
morning of lay 13. She was given 200 ml saline with
5’ glucose intraperitoneally. Sulfathiazole, 210
grains, was administered orally. Sulfathiazolc ad-
ministration was repeated in the evening. The owe
was found dead the following morning. Necropsy
findings were confined to a marked hemorrhagic duo-
denitis. Cultures prepared from the medulla were
positive for Listeria.

-8...

Outbreaks, 1947.

Case 5. February 27,1947. Clare.
I nine-month old bull was showing symptoms of lister-
iosis. It was killed February 26, and the head sub-
mitted for culture. Cultures prepared from the medulla
were negative after 24 hours' incubation at 37' C, but
when the brain suspension, which had been stored
in the refrigerator for three months, was again
plated, there was a heavy growth of Listeria.

Case 6. march 24, 1947. Kalamazoo.
ThIs animal was the third in a group of steers. The
first two died after showing symptoms of cerebral
disturbance. The first died about six weeks before;
the second about two weeks before. This animal be-
gan walking in circles and seemed to be blind in
the right eye. It crowded into the corner of the
stall and showed symptoms of agony. It would go
down and had to be helped up each day. At necropsy
the mucous membrane of the fourth stomach showed
general congestion. The small intestine showed dif-
fuse congestion and blood stained contents throughout
its entire length. A few petechial hemorrhages
were found on the heart. Upon removal of the brain
an excess of cerebral spinal fluid was noted. Cul-
tures prepared from the medulla showed the presence
of Listeria. Examination for rabies was negative.

Case 7.. April 5,1947. Rose Lake.

six-month old heifer was admitted to veterinary
clinic on March 31. She had been circling to the
left since March 30. She was killed for necropsy.
Except for some congestion of the brain, findings
were negative. Cultures prepared from the medulla
were badly contaminated and Listeria was not observed.
After refrigeration for two months it was possible
to observe colonies of Listeria among the contaminants.

Case 8. April 15, 1947. Climax.

yearling heifer entered veterinary clinic on April
11,1947. The right ear drooped, the right eye was
somewhat inflamed and the animal circled to the right.
The tongue was hanging out, and there was excessive
salivation. There was some twitching of the peripheral
muscles. (The owner had lost one heifer about a week
before which had shown similar symptoms.) The animal
was killed for necropsy. The abomasum showed consid-
erable congestion. There was marked patchy congestion
throughout the small intestine. The lungs and kidneys

were mildly congested. There was an excess of core—
bral fluid, and the brain was markedly congested.
Cultures prepared from the medulla were negative.
Three weeks later it was possible to demonstrate
numerous colonies of Listeria from the refrigerated
brain suspension.

Case 2. April 24, 1947. Rose Lake.

yearling ewe had shown a tendency to turn her head
to the right, and to circle. These symptoms persist-
ed for three days. She had been kept in the same
pen as the heifer in Case 7. She was killed for no-
cropsy. The abomasum showed several small erosions
of the mucosa. The lungs and kidneys showed some
congestion. There was marked congestion of the brain.
No other lesions were noted. Cultures prepared from
the medulla were positive for Listeria.

Case 10. May 8, 1947. Springport.
nTIEQ'3 a two-year old cow developed anorexia and
difficulty in swallowing. Temperature was 104.2.
May 5 the temperature was 105. The tongue was hanging
out and there was hay in the mouth. The animal showed
an inability to swallow water. The right eye was
inflamed, the right ear drooped and she circled to
the right. The brain was submitted for culture.
Listeria was isolated from the medulla.

Outbreaks, 1948.

Case.ll. December 31, 1947. Manchester, Indiana.
he head of a five-year old cow was submitted for
culture. She had been raised on this farm and no
new stock had been brought in. She had shown symp-
toms of a central nervous disorder and walked in
circles. One lip, ear, and eyelid were paralyzed.
Temperature was slightly elevated. She lived for
six days after these symptoms appeared and became
violent before paralysis developed. Cultures
prepared from the medulla were positive for Listeria.

Case pg. January 9, 1948. Jackson.

The head of a cow was sent for examination. This was
the fourth cow showing symptoms of a central nervous
system disorder. The first died about three weeks
before. The second apparently recovered. The third
was killed and the brain sent to the state health
laboratory to be checked for rabies. This was re-
ported as being negative.

| [I I 1“

 

- 9 -

were mildly congested. There was an excess of cere-
bral fluid, and the brain was markedly congested.
Cultures prepared from the medulla were negative.
Three weeks later it was possible to demonstrate
numerous colonies of Listeria from the refrigerated
brain suspension.

Case 9. April 24,1947. Rose Lake.

yearling owe had shown a tendency to turn her head
to the right, and to circle. These symptoms persist-
ed for three days. She had been kept in the same
pen as the heifer in Case 7. She was killed for no-
cropsy. The abomasum showed several small erosions
of the mucosa. The lungs and kidneys showed some
congestion. There was marked congestion of the brain.
No other lesions were noted. Cultures prepared from
the medulla were positive for Listeria.

Case n10. May 8,1947. Springport.

3 a two-year old cow developed anorexia and
difficulty in swallowing. Temperature was 104. 2.
May 5 the temperature was 105. The tongue was hanging
out and there was hay in the mouth. The animal showed
an inability to swallow water. The right eye was
inflamed, the right ear drooped and she circled to
the right. The brain was submitted for culture.
Listeria was isolated from the medulla.

Outbreaks, 1948.

Case 11. December 31,1947. Manchester, Indiana.
The head of a five-year old cow was submitted for
culture. She had been raised on this farm and no
new stock had been brought in. She had shown symp-
toms of a central nervous disorder and walked in
circles. One lip, ear, and eyelid were paralyzed.
Temperature was slightly elevated. She lived for
six days after these symptoms appeared and became
violent before paralysis developed. Cultures
prepared from the medulla were positive for Listeria.

Case 12. January 9,1948. Jackson.

ho head of a cow was sent for examination. This was
the fourth cow showing symptoms of a central nervous
system disorder. The first died about three weeks
before. The second apparently recovered. The third
was killed and the brain sent to the state health
laboratory to be checked for rabies. This was re-
ported as being negative.

-10..

All the affected animals showed the same symptoms:
elevated temperature, eyes protruded, staggered

gait, walked in circles, leaned against any station-
ary object as if unable to stand and after about
four days went down. They all continued eating until
they went down.

There were 25 animals of all ages in this lot. All
but these four were suffering from winter dysentery.
There had been no new animals brought on this farm
in the past two years. One cow on a farm across the
road had died after showing similar symptoms.

Cultures prepared from the medulla were positive
for Listeria.

Case 13. February 2, 1948. Manchester.

I'ten-month old ewe was presented for necropsy. It
was the sixth animal in this flock to die. Three
others were sick. Symptoms developed rapidly. Some
died suddenly. Others lived for three or four days.
it necropsy the abemasum of this ten-month old ewe
showed mild congestion. The brain was markedly con-
gested. No other lesions were noted. Cultures pre-
pared from the medulla were positive for Listeria.

Case 14. February 3, 1948. Manchester.
History the same as that of the ewe in Case 13. In
this ten-month old wether no lesions were noted at
necropsy other than congestion of the brain and an
excess of cerebral fluid. Cultures prepared from the
medulla were positive for Listeria.

Case 12. February 11, 1948. Benton.

aged ewe was presented for necrOpsy. Three other
animals had died during the past month. They all
staggered, showed a discharge from the nose, and an
excessive salivation. They died about 24 hours
after the onset of symptoms. The abomasum was con-
gested. The small intestine showed congestion through-
out its entire length. The lungs, kidneys and brain
showed a mild cengestion. Cultures prepared from
the medulla were positive for Listeria.

Case 16. February 11, 1948. Fenton.
hf? sheep was from the same flock as reported in Case
15. Only the head was submitted for examination.
The brain was badly decomposed, but Listeria was
isolated from the medulla.

Casoilz. larch 11, 1948. Bronson.
The head of a cow which had been shot after showing

-11..

symptoms of a central nervous system disorder was
presented for culture. The bullet was found lodged
under the pituitary fossa. There was marked hemorrhage
throughout the brain and the meninges. Listeria

was isolated from the medulla.

08.80 .1__8_o April 9, 1948e Homer.
The head of a cow which showed circling was present-
ed for culture. The brain was markedly congested.
Listeria was isolated from the medulla.

Case 12. April 14, 1948. Grand Ledge.
yearling ewe was presented for necropsy. The day

before she was noticed to circle around the fence,
and press into corners. She went down and showed
kicking and running motions. She died during the
night. There were a few hemorrhages in the heart,
the lungs were congested and there was a marked eon-
gestion of the brain. Cultures prepared from the me-
dulla were positive for Listeria.

Blood picture on April 13 was: WBC - 8,450; BBC -
10,000,000; EB - 8.9 gm ’3 P - 82; L - 16; I - 2.

Case 29. April 21, 1948. Pewame.
The head of a ewe was presented for culture. All
together the owner had lost seven ewes. They tended
to circle to the right. The temperature of this ewe
Just before death was 107.6. The brain showed con-
siderable congestion. Cultures prepared from the
medulla were positive for Ldsteria.

Caso'gl. April 22, 1948. Williamsten.
he owner had lost one ewe April 20. Another ewe,
five years old, began to circle the night of April
21. The head was thrown backward and to one side.
She was killed for necrOpsy. There was some conges-
tion of the abomasum; the brain was slightly congest-
ed. No other significant lesions were noted. [Lig-
teria was isolated from the medulla.

Blood picture: NBC - 14,100; RBC - 9,520,000;
HB - 10.1 gm i; L - 3; P - 79; M’- 15; B - 3.

case 33o May 4, 1948o Clark8'1110e
The owner had lost two animals previous to this one.
The head of this two and a half-month old ewe lamb
was presented for culture. They all showed symp-
toms of circling to the right. There was hemorrhage
around the spinal cord at the base of the brain.
no other lesion was noted. Listeria was isolated
from the medulla.

-12...

Case 23. may 9, 1948. Laingsburg.
The—head of a cow was presented for culture. She
seemed to be blind in one eye. She showed anorexia
and drooling. The face was paralyzed prior to death.
The brain was congested and there was an excess of
cerebral fluid. Listeria was isolated from the
medulla. A rabies examination was negative.

Case.2g. May 24, 1948. Albion.
The head of a yearling bull was presented for culture.
The animal had shown twitching of the nose, slobbering
and an inability to swallow. The right eye appeared
to be turned inward. These symptoms persisted for
six days before death. Listeria was isolated from
the medulla.

Case-29. June 21, 1948. Dansville.
The head of a yearling bull was presented for culture.
The animal was off feed and showed a pharyngeal paraly-
sis. The head was held down, there was marked saliva-
tion and the animal was weak and staggering. The
temperature was 107.0 at the onset of symptoms. It
had been treated with sulfonamide and penicillin.
Cultures prepared from the medulla were negative for
Listeria. When the refrigerated brain suspension
was cultured, August 5, there was a heavy growth of
Listeria.

Case'gg. July 16, 1948. Brown City.
The head of a cow was presented for culture. A
clinical diagnosis of rabies had been made. The
medulla was cultured for Listeria and the remain-
der of the brain was sent to the State Health De-
partment for rabies examination. Cultures were
positive for Listeria; rabies examination, negative.

Outbreaks, 1949.

Case 21. December 17, 1948. Rochester.
The head of a steer showing symptoms of an encephalitis
was presented for culture. Several other animals
in this same herd were showing similar symptoms. The
affected animals circled, had a paralysis of the eye-
lid and a profuse salivation. These symptoms persist-
ed thrce or four days. Listeria was isolated from
the medulla of the head presented.

Case fig. December 21, 1948. mason.
The head of a bull was presented for culture. The

- 13 -

animal had difficulty in swallowing, excess salivation.
rigid Jaw, temperature of 106, and black diarrhea.

The owner reported that the animal had been injured

by a rusty nail. Tetanus was suspected. It had

been treated with procaine penicillin and tetanus
antitoxin. The brain appeared normal. Cultures

from the medulla were positive for Listeria.

 

Case 22. December 21, 1948. Grass Lake.
I cow head was submitted for culture. There was no
history given. Listeria was isolated from the medulla.

Case 30. December 23, 1948. Stockbridge.
A'TIve year old cow was presented for necrOpsy. For
two days she had shown an unsteady gait, and occasion-
ally she had gone down. The small intestine was
slightly congested throughout. There was one small
area of mottling on the liver. The meninges were
congested, and there appeared to be a fluid with
gas bubbles in the fissures of the brain. Listeria
was isolated from the medulla.

Blood count: WBC - 10,900; HB - 14.3 gm p; L - 39;
P - 41; M - 4; B - 1; E - l5.

Case.§l. January 12, 1949. Bewamo.

I yearling bull was presented for necropsy. It was
the second one with like symptoms to die. Two days
previous the bull was seen to fall against a fence.
The animal seemed to be in pain. There was slight

pulmonary edema, and the right cerebral hemisphere

was congested and edematous. Listeria was isolated
from the medulla.

Caselgg. January 14, 1949. Jackson.
cow head was presented for culture. There was
no history given. Listeria was isolated from the
medulla.

Caso.§§. January 14, 1949. Grass Lake.
The head of a three-year old heifer was presented
for culture. There was no history given. Listeria
was isolated from the medulla.

Case134. February 5, 1949. Charlotte.
A ewe from this flock had died after showing symptoms
similar to this ewe which was admitted to veterinary
clinic. She had been circling to the right for three
days and seemed to be blind. There was twitching of
the peripheral muscles. At necropsy there seemed to
be a general congestion of the visceral organs. ‘Lig-

-14...

teria was isolated from the medulla.

Blood picture: '
Feb. 4: WBC - 6,400; BBC - 12,790,000; L - 20;
P - 69; M - 8; B - 3.
Feb. 5: NBC - 2,200; BBC - 14,830,000; L - 25;
P-683H-7e

Case 35. February 11, 1949. Charlotte.
tie-year old ewe showed symptoms similar to those
in Case 34. At necropsy the small intestine, stomach
and meninges showed congestion. The lungs were
edematous and congested. Cultures from the medulla
were positive for Listeria.

Case 36. February 12, 1949. Charlotte.
Th1? ewe was noticed to be circling in the morning,
died in the afternoon of same day. Other than areas
of congestion in the gastric mucosa, necrOpsy find-
ings were negative. Listeria was isolated from the
ne‘ullae

Case 37. February 15, 1949. Eaton Rapids.
I'TFe-year old ewe which had been circling was pre-
sented for necropsy. The small intestine was con-
gested throughout its entire length. The lungs showed
an extensive pulmonary edema and congestion. The
heart showed hemorrhages in the epicardium. The
brain and all visceral organs were mildly congested.
Listeria was isolated from the medulla.

 

Case‘gg. February 22, 1949. Ionia.
The head of a ewe which had shown symptoms of a comp
tral nervous disorder was presented for culture.
One other ewe had died after showing similar symptoms.
Ldsteria was isolated from the medulla of the head
presented.

Case.§2. February 22, 1949. Lansing.
I yearling heifer developed symptoms of a central
nervous disturbance, February 17. There was extensive
salivation. She walked in a straight line with the
head pulled to the left side and extended. Tempera-
ture ranged from 104 to 101. She was treated with
penicillin and sulfapyridine. At necropsy there
was slight congestion of the ileum, a few hemorrhages
in the gastric mucosa, lungs were edematous, kidneys
were pulpy and hemorrhagic and there were numerous
hemorrhages on the heart. Listeria was isolated
from the medulla. ‘—""‘""

Blood count, Feb. 18: WBC - 9,600; L - 37; P - 56;
H-l;B-l;E-'5o

-15..

Case 40. February 22, 1949. Romeo.
1'33» head was presented for culture. The animal
had shown circling, salivation and difficulty in
walking. Cultures prepared from the medulla were
positive for Listeria.

Case 41. February 24, 1949. East Lansing.
cur; ram was found prostrate on February 22. There
was a serous discharge from the nose, pupils were
dilated, the head was held to the right and extended
with cars drooped. There were muscular tremors
throughout the body, hypersensitivity, and anorexia.
Temperature was 100.3. It was treated with 175
grains sulfapyridine and 200,000 units of penicillin.
At necropsy the small intestine was congested, the
lungs showed edema, and there was some gastritis.
Cultures prepared from the medulla were positive
for Listeria.

Blood picture: WBC - 13,900; EB - 10.8 gm p; L - 33;
F - 64; H - 3.

Case‘gg. March 1, 1949. Ionia.
The head of a ewe was submitted for culture. This
ewe was from the same flock as reported in Case 38.
Listeria was isolated from the medulla.

Case‘gg. March 4, 1949. East Lansing.

yearling ewe from the same flock as Case 41 had
shown similar symptoms for two days. At necropsy
pulmonary edema was found in the lungs, numerous
adhesions were present between the liver and dia-
phragm and intestines, the stomach showed some gas-
tritis, the small intestines were very reddened with
some mucoid degeneration, the large intestine had
some nodular worm lesions, the kidneys and mesenteric
lymph nodes were congested. Cultures prepared from
the medulla were positive for Listeria.

Bleed picture:
March 2: NBC - 14,050; KB - 13.1 gm p; L - 55; P - 45.
larch 3: NBC - 14,000; HB - 14.2 3! ’3 L - 24;
P-75;H-l.

Case 44. march 4, 1949. East Lansing.
ee-year old ewe was found lying on its back.

When placed on its feet it circled to the left. The
animal breathed with a marked rattling sound. The
upper air passages contained a very heavy mucopurulent
exudate. At necrOpsy there were no significant lesions.
Cultures prepared from the medulla were positive for
L s eria.

Blood picture: WBC - 18,300; RB - 9.8; L - 21; P - 79.

-16..

Case 45. March 9, 1949. Okemos.
The-head of a cow was presented for culture. The
animal had shown an inability to swallow. After
five days it showed loss of the use of the hind quart-
ers and was destroyed. Cultures prepared from the
medulla were positive for Listeria.

Case 46. March 14, 1949. Ypsilanti.
I'FEarling ewe which had shown symptoms of a central
nervous system disorder was presented for necrOpsy.
Three other lambs had died recently. They all died
within one day after symptoms were noted. They
circled and pressed their heads against partitions.
No gross lesions were found in the yearling ewe
presented for necropsy. Cultures prepared from the
medulla were positive for Listeria.

Case 47. March 16, 1949. Williamston.

'EIx-year old ewe which had shown signs of a central
nervous system disorder was presented for necropsy.
Two other ewes had died showing similar symptoms.
This six-year old ewe showed no gross lesions at
necropsy other than a dull nodular appearance of the
meninges. These tiny nodules were a yellowish gray
in color and were uniformly distributed. They had
the appearance of a suppurative meningitis. Cultures
prepared from the medulla were positive for Listeria.

Case‘gg. march 24, 1949. Charlotte.
I yearling ram was presented for necropsy. It had
been circling to the right, the right ear drooped,
and there was very marked salivation. It had been
treated with sulfathiazole and 400,000 units of pen-
icillin. The small intestine showed numerous scat-
tered areas of congestion and petechial hemorrhages
to about the middle of the ileum; here the mucosa
became thicker for about three feet and did not show
any congestion nor hemorrhages. The remainder of
the ileum was hemorrhagic and congested. The lungs
were edematous. There were hemorrhages scattered
throughout the heart. Lymph nodes were congested.
The large intestine was congested and showed a few
petechial hemorrhages. Cultures prepared from the
medulla were positive for Listeria.

Blood picture: march 28: WBC - 17,150; HB - 10.8 gm T;
L - 34; F - 64; M - 2.

Case 49. March 31, 1949. Big Rapids.
One of two ewes which had died after showing symp-
toms of circling was presented for necropsy. Ad-
vanced post mortem changes prevented detection of
lesions. Cultures prepared from the medulla were
positive for Listeria.

- 17 -

Case 59. April 7, 1949. Albion.
ewe was admitted to the veterinary clinic showing

symptoms of central nervous system disorder. Four-
teen other ewes died showing similar symptoms. They
lived for only one or two days after the onset.
Pregnancy disease was suspected. This ewe which had
been admitted to the veterinary clinic was killed
and necropsied. There were no gross lesions ex-
cept that the meninges appeared to be thickened and
stipled with minute yellowish foci resembling a
suppurative meningitis. Cultures prepared from the
medulla were positive for Listeria.

Case‘gl. April 18, 1949. Romeo.
The head of one of two rams that had died after
showing symptoms of circling for 24 hours was sub-
mitted for culture. This flock of 160 sheep was
crowded into an area sufficiently large for only
about 60 sheep. No further outbreaks were reported.
Listeria was isolated from the medulla of the brain
submitted.

 

Case.§§. April 20, 1949. Manchester.

o one-month old lambs were admitted to the vet-
erinary clinic. They both showed symptoms of circling.
They would lean against any stationary object.

There was marked salivation, drooping of the ears,
strabimus, and terminally, prostration. Four ewes
in this flock had died since March 15, and two other
ewes were showing similar symptoms. One of the lambs
died April 20. The other was comatose on April 21
and killed. Necropsy findings were negative in each.
Listeria was isolated from the medulla of both lambs.

 

The case reports revealed that there were no
pathognomonic symptoms nor lesions in listeriosis such
as those encountered in hog cholera, for example. Symp-
toms ranged from the so-called "textbook description"
to a mere pharyngial paralysis. The duration of the
disease was short in some cases; in others it extended
as long as ten days. This was true in both species

studied but generally the course was much shorter in

-18..

sheep than in cattle.

Listeriosis in sheep could easily be confused
with ketosis or with overeating disease. In flocks
where these diseases were concurrent they could be dis-
tinguished only at necrOpsy. The fatty liver found
in ketosis and the hemorrhages characteristic of over-

eating disease were not found in cases of listeriosis.

Case 52 was of significance in that one month
old lambs that were down with head pulled back were
often considered to be suffering from entrotoxemia.
It was quite likely that if more of these lambs had been
available for culture, Listeria could have been isolated
from them. In entrotoxemia death is so sudden that

symptoms are seldom noticed.

In cattle listeriosis was most easily confused
with rabies or with poisoning. Some cows became quite
violent in the terminal stages. All rabies suspects
sent to the laboratory proved to be listeriosis. In
the milder cases symptoms closely resembled poisoning
but where gastric analyses were performed no poisons

could be demonstrated.

The similarity of the majority of the cases, how-
ever, did render certain symptoms characteristic of

listeriosis. At the onset of the disease the infected

-19..

animal would usually separate itself from the rest of
the herd. It would crowd into a corner or lean against
a fence or manger as if it were unable to stand unsup-
ported. If the animal did walk it would usually go in
a circle. The animal became very depressed and refused
feed. There usually was some elevation of temperature.
A conjunctivitis in one or both eyes developed in some
cases. The head was held back to one side with the car
on that side drooped. There was usually stringy saliva-

tion and a marked discharge from the nose.

As far as could be determined the mortality was
near 100%. All affected animals admitted to the veterin-
ary clinic terminated fatally. However, a few veterin-
arians reported that some animals suffering with that
which they had diagnosed as listeriosis, did recover.

No ante mortem isolations were made so these diagnoses

could not be confirmed.

Lesions at necrOpsy ranged from none to rather
extensive damage. As far as could be determined all
but one of the cases (case 3) were uncomplicated. A
marked enteritis was shown in many cases (figure 2).

It was felt that this lesion may have some significance
but it was not clearly understood. This was also shown
in some rabbits artificially eXposed. A number of

cases were characterized by a marked pulmonary edema,_

- 20 -

but this too was not consistent. (figure 20)

A number of animals, particularly sheep, were
presented for necrOpsy showing symptoms suggestive of
listeriosis. NecrOpsy findings failed to establish a
diagnosis. Repeated cultures of the medulla, however,
failed to reveal the presence of Listeria. Histo-patho-
logical examination of the medulla failed to show peri-
vascular cuffing or areas of necrosis. The only excep-
tion to this was a horse submitted for necropsy. .Lig-
335$; was not isolated but tissue sections of the medulla
showed lesions resembling those of listeriosis. This

case has not been positively diagnosed.

From the foregoing it was evident that listeriosis
could be positively diagnosed only by isolation and

identification of‘L. monocytogenes.

In seven of the cases (35, 36, 37, 39, 41, 44, 51)
the lungs, spleen, liver and kidney were cultured. An
area from each organ was seared and macerated in the
Waring blendor. About 15 m1 nutrient broth was added
to make a suspension. A 0.2-ml portion was plated on
tryptose agar and incubated at 37’ C for 24 hours. In
no instance was Listeria isolated. Tissue sections pre-
pared from these organs failed to show lesions. Fetuses

removed at necropsy from cases 36 and 37 were negative

-21..

for Listeria following the usual culture procedure.

 

In four cases (43, 44, 46, 50) cultures were
prepared from the cerebrum. Listeria was not isolated.
The cerebellum was cultured in two cases (46, 50).

Listeria was isolated in small numbers from both cases.

 

Four cases (17, 23, 25, 26) all bovine, showed

Listeria in larger numbers in the cervical cord immedi-

 

ately posterior to the medulla than in the medulla itself.

Frequently conjunctivitis and keratitis accompanied
the natural occurrence of the disease. Cultures prepared
from conjunctival swabs failed to reveal the presence
of Listeria. Saliva and nasal mucus were negative for
Listeria. With one exception, Case 2, Listeria was not

isolated from any source other than the brain.

It may be of some significance that in young
animals listeriosis invariably occurs as a septicemia.
Burn (1936) reported its presence in the liver of infants.
De Blieck and Jansen (1942, 1943), Kirlin and Graham
(1945) and Rhoades and Sutherland (1948), all reported
isolation of Listeria from the liver of young pigs.

Grini (1943) recovered Listeria from the liver of a six-
day old foal. None of these reports indicate whether
Listeria was also present in the brain of the affected

 

animals. This evidence suggested that the disease may

-22..

have been septicemic at some time prior to the appearance
of nervous symptoms or that there may have been an al-
tered host-parasite relationship in the young. If the
former was true, a monocytosis could be eXpected in the
peripheral blood at the time of exposure. Intravenous
inoculation of experimental animals invariably produced

a monocytosis. However, during the course of this study
several flocks of sheep having active cases of listeriosis,
were observed. The exposed animals were bled but no
significant deviation from normal hemocytological values
was evident. Subsequently, in one flock, deaths due

to listeriosis occurred at about three and six-week
intervals. The postulation of an altered host-parasite
relationship could not be made until the specific nutrient
requirements of the bacterium, or the physiological
differences in the young and adult were more clearly
understood. Hunter,(1942) reported some growth require-
ments of Listeria. Graham‘gt'al (1943) reported that

the sera of a relatively high percentage of cattle with
no history of exposure to Listeria contained agglutinins
for the organism. This could indicate that either there
were no specific agglutinins for Listeria, that many
animals were exposed to the disease but never showed
symptoms, or that antibodies were present which cross
agglutinated with Listeria antigen. When a number of
sheep brains from a flock which had had an outbreak of

_ 23 -

listeriosis were cultured, Listeria was not isolated

 

from any of them. The sheep had died of supposedly
natural causes. Tissue sections of the medulla revealed
no evidence of pathology. There was no evidence that
these animals were carriers or that they had been exposed

to the disease.

The evidence presented by two outbreaks in the
spring of 1947 suggested that listeriosis may have a
long period of incubation. Two cases, 6 and 8, occurred
22 days apart. There was no known direct contact between
the two affected herds. However, feeder cattle that
had been imported from the same source in the state of
Texas were involved in each outbreak. Numerous other
owners had reported that the affected animals, especial-
ly in cases involving cattle, had been imported from
the Southwestern or Western feeding ranges. Coupled
with this was the fact that during the four years of
this study the disease had recurred in only one flock
of sheep. In only one instance were both sheep and cattle
affected on the same premises (cases 7 and 9). In this
outbreak a sheep died from listeriosis 19 days after a
cow had died. Another outbreak (cases 34, 35, 36, 48)
occurred where cows were kept in the same barn with the
infected sheep. In fact, the infected sheep had access
to a box stall shared by cattle. The exposed cattle

- 24 -

failed to show symptoms of listeriosis.

Case 50 also was interesting for its epizootical
implications. Animals in direct contact became infected
while animals sharing a common feed rack, but not in
direct contact, did not show evidence of the disease.
This would substantiate the conclusion that listeriosis
was not transmitted in this instance by contaminated
food or water. One experiment (Thesis, Part II) sup-
ported this. Similar findings are reported by Gill (1937),
Paterson (1940A), Schwarte and Biester (1942), Graham
.22.2i (1943). However, Julianelle (1941) was able to
infect white mice fatally when broth cultures were given
as the sole source of fluid intake. -

It had not been possible to reproduce listeriosis
experimentally except by extremely drastic means such
as intracerebral inoculation. Numerous investigations
had established this; i.c., Biester and Schwarte (1939),
Schwarte and Biester (1942), Olofson (1940), Muth and
Morrill (1942), Graham‘gt.§l (1943) and Part II of this
thesis.

Numerous investigators had attempted transmission
by intranasal sprays. These reports were in such com-

plete disagreement that they will not be discussed here.

It was shown by Graham gt_gl (1943) and Gray 33

- 25 -

‘al_(l948A) (Thesis, Part II, Rabbit No. 9) that the ner-
vous form of listeriosis could follow conjunctival in-
stillation. Graham 33 El (1943) reported that one of
two month old pigs died 11 days after instillation of
culture. There was no conjunctivitis or keratitis.

Listeria was recovered from the medulla. Gray 33 El

 

(1948A) reported the death of a rabbit, 23 days following
ocular instillation. The symptoms were suggestive of

listeriosis. There were no significant lesions at necrop-
sy. Listeria was isolated only from the medulla. Tissue

sections of the medulla showed perivascular cuffing.

However, transmission under field conditions
could not be established. Cultures of drinking water,
straw, feed, litter, etc. from infected premises failed
to establish the presence of Listeria in these materials.
The nostrils and blood of exposed animals were cultured
with negative results. Until the natural reservoir of
this bacterium could be determined it was unlikely that
its epizoology could be established.

Summary

Fifty two cases of listeriosis, occurring in a
four-year period, were reported. In 29 of these sheep
were involved, while cattle were involved in 23. The

known losses totaled.more than 104 animals of both species.

-26..

Certain aspects of the epizoology of the disease are

disoussed.

Several technics were presented for the success-
ful isolation of the bacterium from both species of an-
imals. A method of readily identifying colonies of

Listeria was described.

Fig. 1.

Map indicating areas where listeriosis has
occurred. Red denotes outbreaks in sheep,
blue denotes outbreaks in cattle.

 

Fig. 2.
Case 7e

Figo so
0330 43c

Green appearnace of Listeria colonies
when viewed through a dissecting micro-
scOpe using oblique light. This is at
the time that Listeria was first visible
among the contaminating bacteria.
Kodachrome print.

Intestine showing the marked congestion
of the small intestine seen in many of
the necropsied animals. Kodachrome
print.

m — ~—__— —— —.— _ _ _ _— ‘—

—____——_—

 

Figs 2

 

Fig. 4. Primary isolation from bovine medulla

Case 29. using the grinding and shaking technic
for the preparation of the brain sus-
pension. Only two of the colonies
visible are Listeria.

Fig. 5. Primary isolation from bovine medulla
Case 29. using the Waring blendor for the pro-
paration of the brain suspension.

Twenty two of the colonies visible
are Listeria.

-29-

 

Fig. 4

 

Fig. 6. Primary isolation from bovine medulla

Case 33. using the grinding and shaking technic
for the preparation of the brain sus-
pension. Only two of the colonies
visible are Listeria.

Fig. 7. Primary isolation from bovine medulla
Case 33. using the Waring blendor for the pre-
paration of the brain suspension.

Fifty six of the colonies visible are
Listeria.

-50..

 

Fig. 6

 

Fig. 7

Fig. 8. Primary isolation from the bovine medulla.
Case 17.

Fig. 9. Primary isolation from the spinal cord
Case 17. immediately posterior to the medulla.

-31-

 

Fi‘e 8

 

Figs 9

Fig. 10. Primary isolation from the bovine medulla.
Case 25.

Fig. 11. Primary isolation from the spinal cord
Case 25. immediately posterior to the medulla.

~52-

 

Fig. 11

 

Fig. 12. Liver showing the large white necrotic
Case 2. foci.

 

Fig. 12

Fig. 13. Photomicrograph of liver showing
Case 2. focal areas of necrosis (HdE).

r A.
amt.
.

 

Fig. 14. High power photomicrograph of a minute
Case 2. focal area showing bacteria as dark
stained bodies (Gram-Weigert).

 

Fig. 15.
Case 8.

Yearling heifer. Note the position of
the head, drooping right ear, protrud-
ing tongue, and excessive salivation.
There is also marked conjunctivitis

of the right eye. Primary culture of
the medulla was negative for Listeria.
Print from a Kodachrome transparency
from the collection of Dr. C. S. Bryan.

 

Fig. 15

Fig. 16. Ewe. Note position of head, droOped
Case 34. right ear and protruding tongue.

 

Fig. 16

Fige 17o
Case 34.

Ewe showing tendency to lean against
stationary objects. Note mucOpurulent
exudate from nostrils, protruding
tongue, and snow on the wool indicat-

ing that the ewe could not maintain
her equilibrium.

.4..-—

     

 
 
  

. .. w w .- '
,._',"'..'-7’_'.',‘? . ,_ "1; 9:956 35;" '
1 1":nf ,..AI..' ‘

Fig. 17

Fig. 18. Lamb showing the head drawn back and

Case 52. tongue protruding. The neck and fore-
legs are tense and rigid while the
hind quarters are relaxed.

 

Fig. 18

Fig. 19. Photomicrograph of bovine medulla show-
ing perivascular cuffing and areas of
necrosis. Note also necrosis of
neurons (HdE).

 

Fig. 19

Fig. 20. Edematous lungs seen in many of the
Case 41. sheep presented for necrOpsy.

-41..

 

Fig. 20

Part II

LABORATORY OBSERVATIONS

- 42 -

Part II. LABORATORY OBSERVATIONS

Isolation from the Bovine Brain

 

Using the technics of isolation described in
Part I, it was relatively easy to isolate L. monocy-
togenes from the brain in cases of ovine listeriosis.
However, the direct isolation of the microrganism from
the bovine brain met with only partial success. Several
bovine brains (case 5, 7, 8, 25, 39) failed to yield

Listeria upon primary culture. After refrigeration of

 

the prepared brain suspensions it was possible to de-
monstrate Listeria in all cases where listeriosis was
suspected and where tissue sections revealed perivas-
cular ouffing and necrosis in the medulla. In all cases
the number of colonies obtained in primary isolations
from bovines was small compared to that obtained by
primary isolations from ovines. This is shown in

table I and figures 24 to 39.

The evidence presented here strongly suggested
that the bovine brain possessed a factor bacteriostatic
for Listeria. The presence of such substances in animal
tissues might account for the specific immunity to in-
fection inherent in certain tissues. Listeriosis in
the ovine was generally extremely acute with a short

fatal termination. In the bovine the disease was less

-43...

acute and recoveries were reported (Biester, 1941;
Graham, 1943; and others). Pounden (1947A) reported
one outbreak of an extremely acute nature, but that
could be considered as atypical. Primary isolation of

Listeria from the ovine seldom resulted in failure,

 

but in the bovine primary isolations were made in less
than a third of the suspects cultured even though lis-
teriosis was confirmed by histopathologic examinations.
These observations were similar to those reported by

Gifford and Jungherr (1947).

The mechanism of inhibition exhibited in cul-
tures from the bovine brain was not understood. There
seemed to be a correlation between the number of colonies
obtained at primary isolation and time that had elapsed
between death and preparation of culture. Animals
that had died of the disease in the veterinary hospital
and were necrOpsied immediately presented the greatest
difficulties with respect to primary isolation. Bovine
heads received through the mail seldom gave negative
results on primary isolation. No direct relationship
between time of death and preparation of culture could
be established because the time of death was not given
in all cases. This evidence strongly suggested the
presence of an unstable substance bacteriostatic for

I Listeria in the bovine brain. However, to date this

-44..

T

was not established because experimental results were

not conclusive.

Whole bovine brains that had yielded a small
number of colonies at primary isolation were kept at
4' C for varying lengths of time. Weighed portions of
the medulla suspended in a constant volume of broth
were cultured at intervals of a few days. It was found
that within five days it was no longer possible to
isolate Listeria from these brains. Similarly, sheep

 

brains bedame sterile after about seven days. This in-
dicated that Listeria could not survive for many days
in the brains of its victims, although the microorgan-

ism remained viable in prepared brain suspensions.

Apparently normal bovine brains removed immediate-
ly after slaughter were prepared, employing the usual
technic. A 0.2 ml portion was cultured to check for
sterility. The suspension was immediately inoculated
with a known number of Listeria. A 0.2-ml portion was
plated and incubated at 37' C for 24 hours. There was
no evidence that the growth of Listeria was inhibited
by the brain material.

Attempts were made to establish the number of
microorganisms in the ovine and bovine brains by means
of histological sections of the medulla. The results
of this work were inconclusive due to the difficulty in

-45..

properly staining the bacteria. Added to this was
the difficulty of distinguishing between the bacteria

and the numerous fibers of the supporting cells.

Table _1_

Increase in number of colonies after refrigeration of
brain suspensions

 

 

Days

Case 0 2 4 6 8 10 15
10 90 250 1,500 - 18th day
11 33 27 21 22 340 I - 20th day
12 56 161 1360 3300 I
18 15 35 55 200 312 890 1,000 - 12th day
23 55 61 82 189 3,200 - 18th day
24 2,300 - 3 days
25 0 O O I - 45th day
27 C C I (still many contaminant:
29 1 3 3 9 3 82 I - 28th day
29b 24 70 68 62 87 85 717 - 20th day
30 C 4 4 4 I - 25th day
30b 4 9 14 . 300 I - 20th day
31 125 240 800 1500 - 5th day
32 9 15 26 500 900 I
33 2 14 4 22 47 1,000 - 14th day
33b 56 108 220 I
39 O 1 2 3
40 S F
45 39 .1500

I - too numerous to count

C - contamination

S - spreader

p -

few colonies, obscure

A 0.2 ml portion of each suspension was used for
each count. All counts were made in duplicate. The
figure shown was the average of the two. A streak plate
was used for primary isolations. For the counts a sterile
'bent glass rod was employed to distribute the suspension

more evenly. Difcc tryptose agar was used throughout.

Ocular instillation
Morris and Julianelle (1935) and Julianelle and

Pons (1939) found Listeria to produce a characteristic
inflammatory reaction when instilled into the eye of
rabbits. Suitable bacterial suspensions were prepared
by adding approximately eight ml tryptose broth to an
18-24 hour tryptose agar slant culture. The growth was
loosened with a sterile leap. The tube was then agitat-
ed to break the clumps of organisms, after which it was
allowed to stand for 20-30 minutes. Two to four draps
from a capillary pipette were dropped directly into the
conjunctival sac.‘ All cultures isolated in the first
year of this study were tentatively identified by their
ability to produce a conjunctivitis in susceptible animals.
However, the following year it was found that recently
isolated bovine strains did not consistently produce
this reaction. In some instances there was merely a
slight congestion of the lid. In cases where there was
a more marked reaction it was delayed for as long as
six days. Whereas with strains isolated from sheep the
conjunctivitis appeared within 24-36 hours. It was
postulated that this phenomenon was responsible for

the failure, in many cases, to isolate EEEIEEEE from

cows that had succumbed to this disease.

- 47 -

Rabbit EEpll

Two to four drops of culture no. 11 (bovine - 4 days after
isolation) instilled into the left eye of rabbit.

 

 

Days Condition WBC L P M B E
0 Normal. 16,600 24 6O 4 7 5
1 Normal. 22,900 45 4O 3 7 5
2 Normal.

3 Normal. Eye swabbed. 13,450 41 54 1 1 3
4 Slight congestion of 18,300 30 63 O 4 0
lids(?) Culture shows
Listeria.
5 'SIIEEt‘Injection of up- 24,800 37 62 l 0 0
per lid.
6 Normal. 22,350 45 52 O 3 O
12 Normal. 24,000 56 42 0 2 O

 

Rabbit _N_o_. 3;

Two to four drops of culture no. 11 (bovine - 76 days after
isolation) instilled into the left sip of rabbit.

 

Days Condition Temp. WBC L P M B E
0 Normal. 102.6 8,200 46 54 O O 0
1 Normal. 102.2
2 Moderate congestion
in both lids. Eye
swabbed. 102.7 9,650 69 29 1 l 0
3 Increased vascular-

isation. Purulcnt
exudate. Culture
negative. 102e4

4 No change.$wabbed 103.2 11,900 51 47 0 l 1
6 Nermal(?) Watery

exudate. Culture

negative. 101.8 12,450 64 3O 2 3 l
7 Normal.
8 Normal.

 

From these data it was observed that this culture four days
after isolation failed to produce a conjunctivitis in a suscep-
tible animal, whereas 76 days after isolation a marked con-
junctivitis was produced in 48 hours.

- 43 -

Rabbit Ego E

Two to four drOps of culture no. 12 (bovine - 24 hours after
isolation) instilled into left eye of rabbit.

 

Days Condition WBC L P M’ B E
0 Normal 9,150 59 39 O 2 O
1 Normal 10,600 61 36 0 3 O
2 Normal 17,350 67 31 0 O l
3 Lacrimation(?) 15,400 42 57 0 O l
4 Normal 11,750 44 54 O l O
5 Normal 11,350 70 28 0 2 0
6 Normal
9 Slight reddening(7)

10 Reddening more marked

11 No change(?)

12 Slight injection of 11,800 55 39 3 3 0
upper lid(?)

13 Normal

 

Rabbit pg. g

Two to four drops of culture no. 12 (bovine - 69 days after
isolation) instilled into left eye of rabbit.

Days Condition Temp. WBC L P M B E

 

0 Normal 103.0 8,400 81 18 1 0 0
l Nictating membrane 102.0
and conjunctiva
slightly injected. . .
2 Increased vascular- 102.7 7,550 60 40 O O O
ization but no con-
gestion nor exudate.
Swabbed.
3 Increased vascular- 103.2
ization of cornea.
Culture negative.
4 No change. Swabbed. 103.0 12,750 84 16 O 0 0
6 Slight improvement. 102.6 9,300 64 32 2 l 1
Culture negative.
Swabbed.
7 Approaching normal.
Culture negative.
Swabb ede
8 Normal. Culture
negative.

 

From these data it was observed that this culture 24 hours
after isolation failed to produce a conjunctivitis in a suscep-
tible animal, whereas 69 days after isolation a marked con-
junctivitis was produced in 24 hours.

- 49 -

Rabbit Ego 3

Two to four drops of culture no. 14 (ovine - ten days after
isolation) instilled into left eye of rabbit.

 

Days Condition
0 Normal.
1 Normal.
2 Considerable white purulent exudate. Eye closed.
marked congestion.
3 Eye partially cpen. Exudate somewhat dry.
4 No change.
5 Less exudate. Cornea slightly injected. Swabbed.
6 No change. A few colonies of Listeria.
8 No change.
10 Eye open. Pinkish in color. Swabbed.
11 No change. Numerous colonies of Listeria.
12 Improved.
14 Normal.
16 Normal. Swabbed.
17 Normal. Culture negative.

 

Rabbit E2. 2

Two to four drops of culture no. 15 (ovine — two days after
isolation)_instilled into left egg of rabbit.

 

Days Condition
0 Normal.
1 Normal e
2 Eye completely closed. Considerable white purulent
exudate. Marked congestion.
3 Eye slightly cpen. Less exudate. No other change.
4 No change.
5 Increased amount of exudate. Marked vascularization
of cornea. Swabbed.
6 No change. Heavy growth of pure Listeria.
7 No change.
8 Eye cpen. No other change.
10 Small ulcer lmmm dia. on cornea. Eye nearly opaque
and has a pale pink appearance. Swabbed.
11 No change. Few colonies Listeria.
l2 Opacity less marked. Right eye Has considerable
watery exudate.
13 Improved.
14 No change.
16 Normal(?) Swabbed.
l7 Slight amount of purulent exudate. Culture negative.
18 Normal.

 

-50..

Rabbit N2. 1

Two to four drops of culture no. 16 (ovine - two days after
isolation) instilled into left eye of rabbit.

Days Condition

 

O Normale

1 Normal.

2 Eye nearly closed. Considerable white purulent exudate.

Marked congestion.

3 No change.

4 N0 change.

5 Less exudate. *Eye open, still congested. Swabbed.

6 No change. Heavy growth of pure Listeria.

7 No change.

10 Mere exudate. No other change. Swabbed.

11 No change. Few colonies of Listeria.

12 Small ulcer on cornea. Cornea shows marked injection.
Cornea opaque. Exudate gone.

13 No change.

14 Considerable exudate. Cornea is clear.

15 No change.

17 Ulcer gone. Less congestion. Swabbed.

18 No change. Culture negative.

19 Improved. Still considerable exudate.

20 No change.

21 Slight amount of exudate. Eye appears normal.

22 Normal. Swabbed.

23 Normal. Culture negative.

 

Rabbit No. _8_

Two to four drape of culture no. 19 (ovine - three days after
isolation) instilled into left eye of rabbit.

Days Condition

 

0

Normal e

Considerable purulent exudate. Marked congestion
and vascularization of lids and cornea.

Less exudate. No other change.

no change e

No change.

Exudate is dry. No other change.

No change.

Cornea red and opaque. Considerable watery exudate.
No change. Swabbed.

No change. Heavy growth of Listeria.

commons-mm H

l-'

 

No further secord was kept of this reaction.

 

-51..

Rabbit .132. 2

Two to four drops of culture no. 8 (bovine - two days after

isolation)* instilled into left eye of rabbit.
Days Condition WBC L

P

In

B

E

 

10,500 57
Normal
Normal 11,650 74
Normal
Right eye negative. Nic-
tating membrane of left
eye congested. Swabbed.
No change. Culture shows
few colonies of Listeria.
No change. 8,250 69
No change.
Eye less congested.
No change. Swabbed. 9,600 68
No change. Culture
negative.
12 Slightly more congested. 14,900 58
White purulent exudate
in anterior corner. Eyes
awabb ed.
13 No change. Not eating.
Cultures negative.
14 Marked conjunctivitis.
Exudate increased. De-
pressed. Has not eaten.
Eye swabbed.
l5 Numerous colonies Listeria
Eye partially closed. Not
eating. Drinking water
gone e
16 No change.
17 Eye improved. 15,800 40
18 Culture negative. Eye
shows marked improvement
with only slight conges-
tion throughout. Increas-
ed appetite.
19 Eye only very slight con-
gestion. Swabbed. Gen-
eral condition appears
normal.
20 Culture negative. Head
down on floor. Does not
seem to be able to raise
it. Seems to circle to
right. Puts head in cor-
ner and pushes. Rubs nose
with forepaws. Breathing
difficult with slight
wheezing sound. Ears hot,

IFGNHO

 

HOQQO‘ cm

38

24

29

56

3

- 52 -

 

Days Condition was L P M B E

 

temperature not taken.
not eating, but seems

to want water. Cannot
lift head to drink ex-
cept with great diffi-
culty. Hind legs twitch-
ed when held. Feces were
mucoid and adhered to
anus c

21 No change.

22 No change.

23 Comatose. Exudate from 3,900 31 66 l 1 1
right eye. Heavy mucus
from nose. Killed by
other inhalation. No
significant lesions at
necropsy. Cultured
both eyes, nose, liver,
blood and brain.

24 Cultures negative for
Listeria. Medulla
ground and cultured.

25 Culture shows moderate
growth of Listeria.
Approximater 260 col-
onies.

 

,*Culture originally considered negative.

Rabbit No. 9 represented a true case of encephalitis due
to Listeria following ocular instillation.

From Rabbit No. 5, 6, 7 and 8 it was observed that recently
isolated ovine cultures produced a marked conjunctivitis
within 48 hours. Listeria could be isolated from the ex-
posed eye for at least II days folowing instillation.
Following ocular instillation, it was found that in
spite of the severity of reaction the infection was
confined to the eye. The infected eye usually acquired

a resistance to reinfection. However, no generalized

—53-

immunity was established. This was evidenced by the
fact that the unexposed eye could be infected or that
intravenous injections of cultures readily caused death.
Conversely, rabbits having high agglutination titers

in the circulating blood showed no immunity to ocular
instillation. These data were not at variance with

those reported by Julianelle (1941).

Viability

In the study of the etiology of this disease it
was necessary to have an understanding of the micro-
organism's resistance to its environment. Straw, hay,
wood shavings, calf pellets (feed) and rabbit pellets
(feed) were placed in individual culture tubes. These
were autoclaved at 15 pounds pressure for one hour on
three consecutive days. Sterility was checked by adding
3-5 ml sterile nutrient broth to several tubes and in-
cubating them at 37° C for 48 hours. After incubation
a standard loopful of the material was plated on tryp-
tose agar and incubated an additional 48 hours. A broth
culture, case no. 6, which had been incubated at 37° C
for 48 hours was added to each tube in amounts of 2-3
ml. The tubes were allowed to remain at room tempera-
ture for 24 hours. The broth was removed aseptically

.by aspiration. The tubes were placed in a pasteboard

- 54 -

box covered with Kraft paper and stored in an appropriate

place in the animal pathology barn on June 19, 1947.

The viability of the organisms on each substance
was checked at weekly intervals. A tube of each of the
materials was removed and 2-3 ml nutrient broth was

added. The tubes were then incubated at 37° C for 24
hours. A lOOpful of this material was then plated on

tryptose agar plates and incubated at 37' c for 24 hours.
Growth was checked for purity by the use of the oblique
lighting previously described. Under the above experi-
mental conditions the viability of L. monocytogenes was

found to be as follows:

Straw - 6 weeks
Hay -16 weeks
Wood shavings - 6 weeks

Calf pellets (feed) -16 weeks
Rabbit pellets (feed)-26 weeks

An observation of incidental interest was that
in the late stages (above 20 weeks) many of the tubes
were found to be contaminated with fungi and large gram
positive bacilli. Apparently the cotton plugs in the
tubes covered with Kraft paper did not afford sufficient

protection in a dust laden atmosphere.

Cultures recovered from rabbits which had been

inoculated intracerebrally were transferred to sheep

- 55 -

blood agar slants on October 16, 1946. These were in-
cubated at 370 C for 24 hours and placed in the refriger-
ator at 4° C. At approximately one-month intervals
transfers to tryptose agar slants were made to check

for viability. Incubation of the subculture at37° c
for 18-20 hours produced a heavy growth of Listeria.

This continued until November, 1948, a period of 25
months. The transfers of December, 1948, showed good
growth after 48 hours' incubation at 37' C. The last
transfers made April 20, 1949 still showed a heavy growth
of Listeria after 48 hours' incubation at 37' c. The

 

agar in the original slants was completely dehydrated
and very hard and brittle. None of this material was
transferred. The transfer 100p was merely scraped along

the hard surface of the agar.

‘

A similar experiment was begun January 3, 1947.
The slants in this instance were maintained at room
temperature. Good growth was evident after 18-20 hours'
incubation at 37' 0 through April 22, 1947. The trans-
fer of .Nmy’16 was negative. At that time the slants
were completely dehydrated. A few ml of nutrient broth
was added to the original slants June 5, 1947. Incuba-
tion at 37“ c for 18-20 hours showed Listeria still to

be viable.

From the foregoing it is evident that Listeria

-56..

might remain viable for long periods of time under vary-
ing environmental conditions. Witts and Webb (1927)

and Schwarte (1942) reported that cultures maintained
on artificial media for a considerable period of time
lost their pathogenieity. One of the cultures that had
been kept at 4° 0 without transfer for 17 months was
inoculated intravenously into a rabbit. The case

history follows.

Rabbit‘lg. July 29, 1943.

0.5 m1 culture no. 3c standardized to McFarland Nepholomet-
or Tube no. 1 inoculated 1.7.

Days Condition WBC L P M B E
0 Normal 14,350 70 27 1 l l
l Profuse exudate 12,350 23 71 l 3 2

from both eyes.
Listless. Cultured

2 Cultures negative 10,450 26 66 6 3 O
Trembling

3 Trembling. Loss 17,200 11 70 15 3 l
of coordination.
Feces wet and s

sticky
4 No change
5 Dead

Necropsy findings were limited due to advanced post mor-
tem changes. 'Listeria was isolated from the heart blood,
liver and medulla.

On this same date this culture when instilled in-
to the eye of a susceptible rabbit produced a mild con-

junctivitis in 48 hours.

Thirty months after the beginning of the experi-
ment this same culture (3c - 0.5 ml standardized to Me

-57..

Farland Tube no. 1 I.V.) when inoculated intravenously
into a susceptible rabbit caused death in less than 72
hours. Listeria was isolated from the heart blood,
liver and kidneys. This same culture also produced a
mild conjunctivitis in a susceptible rabbit in 72 hours.

These data indicate that Listeria may remain
viable for long periods with no loss of pathogenieity.

Inhibition

Early in the course of this study it became ob-
vious that if Listeria was to be successfully isolated
from material containing other aerobic microorganisms
a satisfactory differential or enrichment medium would

have to be devised.

Snyder and Lechstein (1940) demonstrated the
value of sodium azide as an inhibitory substance for
gram negative bacteria. Packer (1943) described a
method for the isolation of streptococci and Erysipolo-
BEES! rhusiopathiac in which sodium azide and crystal

 

violet were employed to inhibit the growth of gram nega-
tive bacteria. It was shown by Pike (1945) and by Mueller
and Miller (1946) that potassium tellurite was of value
in isolation of gram positive microorganisms from throat
swabs. Owen (1946) reported good results in inhibiting
gram negative bacteria when acetic acid was added to

meat infusion tubes. Potassium tellurite, sodium azide,

-58..

and acetic acid in deep brain medium were chosen for

Studye

Sodium azide.

A 1.0% stock solution was prepared and autoclaved
at 15 pounds pressure for 20 minutes. ApprOpriate por-
tions of this stock were added to sterile tryptose agar,
pH 7.0, to make the following concentrations: 0.2%, 0.1%,
0.05% and 0.03%. Plates were then poured in the usual

manner.

Potassium tellurite.

Two different methods were used in the prepara-
tion of the potassium tellurite plates. The first
method consisted in adding the appropriate concentra-
tion of potassium tellurite to the tryptose agar before
autoclaving. This procedure rendered the agar black
in color and made it difficult to distinguish differences

in colonial morphology.

The second method was the same as that used for
the sodium azide plates, i.c., addition of a sterile
stock solution to sterile tryptose agar. The concentra-

tions employed were 0.1%, 0.05%, 0.03% and 0.01%.

Pure cultures of L. monocytogenes either alone
or in mixture with both gram positive and gram negative
bacteria were streaked on the various concentrations

of media using tryptose agar as a control. It was ob-

-59-

served that sodium azide would have little or no appli-
cation in the isolation of Listeria, as it was complete-
ly suppressed in concentrations as low as 0.03%. Many

_of the other test organisms showed growth in concentra-

tions of both 0.03% and 0.05%.

Potassium tellurite, on the other hand, showed
considerable selectivity. Growth of Listeria was not
greatly suppressed in 0.1% concentrations and growth
was uninhibited at concentrations of 0.05% and less.
These concentrations were sufficiently high to suppress
most gram negative bacteria. However, micrococci and
streptococci also grew freely at the higher concentra-
tions. The use of the dissecting microscoPe, as previous-
ly described, was employed at this point. The Listeria
colonies appeared black as do all colonies on this medium,
but the characteristic green color was evident at the
periphery of the colony. The micrococci were pinkish
yellow at the periphery, intensely black at the center,
and extremely glossy in appearance. The streptococci

were smaller, pinkish grey in color with a dull surface.

A less artificial method of determining the ef-
fectiveness of this medium for the isolation of Listeria
consisted of swabbing the nasal passages of supposedly
normal sheep. The swabs were placed in nutrient broth,
three dmpps of a 24 hour broth culture of Listeria was

added. The tubes were incubated from 4 to 8 hours at

- so -

37° c and plated on potassium tellurite plates and tryp-
tose agar. Identification of colonies of Listeria was
invariably impossible on the tryptose agar but they
were found without difficulty on the potassium tellurite

plates.

Excellent results were obtained when 0.05% potas-
sium tellurite was added to plain nutrient broth and
used as an enrichment medium. The material to be cul-
tured, nasal swabs, straw, feces, etc. were added direct-
ly to the broth and incubated 6 to 24 hours at 37° C.

A leapful of this material was then plated on plain
tryptose agar and incubated for 20-24 hours at 37° 0.
Examination of the plates with the aid of the dissect-
ing microscOpe revealed the absence or presence of Lig-
teria. With this method nearly pure cultures of’légy
teria can be obtained providing the number of micrococci

in the material is not too high.

Deep brain medium.

Deep brain medium was prepared and acetic acid
added to make concentrations of .Ol%, .15%, 0.1%, 0.5%,
1.0%. This medium was inoculated as described above
and incubated at 37° c for 20-24 hours. A loopful of
the material was plated on tryptose agar plates. These
were incubated for 20-24 hours at 57° C. Listeria grew
freely in all concentrations. The gram negative bacteria
were markedly suppressed especially in the higher con-

centrations. The results were comparable to those of

-61..

potassium tellurite. Potassium tellurite was adopted,

however, because of the convenience of its preparation.

The Effect of Antibiotics

The effects of various therapeutic agents in
treating infections with members of the genus Listeria
had been reported. Porter and Hale (1939) showed that
sulfanilamide and sulfapyridine were of value in treat-
ing Swiss mice eXperimentally exposed to Listeria.
Graham 22 El (1943) reported the use of sulfanilamide
in field cases of listeriosis. Their work indicated
that this drug was of little or no value in naturally
occurring cases of the disease. Foley, Epstein and Lee
(1944) reported that Listeria cultures will grow freely
in 40 times the concentration of penicillin necessary
to inhibit other gram positive organisms. Handelman
£§_§l (1946) reported apparent recovery from Listeria
meningitis in a six week old infant treated with sodium
sulfadiazine after penicillin therapy had failed. Gib-
bons (1948) claimed success in treating some cases of
bovine listeriosis with penicillin and sodium sulfamethazine.
Discouraging results with treatment were cited Thesis,
Part I. This review would Justify further search for

a satisfactory treatment of this disease.

Streptomycin
Tryptose agar, pH 7.0, plates containing 0.5,

- 52 -

1.0, 3.0, 6.0 and 10.0 units of streptomycin (hydrochlor-
ide) were prepared. Seven cultures of ovine origin

and eight cultures of bovine origin were used. These
stock cultures were maintained on tryptose agar slants.
Their age ranged from two years to those recently isolat-
ed. They were transferred through nutrient broth daily
for three days prior to exposure to the antibiotic. A
1.0 mm platinum 100p was used to inoculate the plates.
They were incubated for 24 hours at 37° C. It was ob-
served that most of the cultures grew well on plates
containing 0.5 unit of streptomycin but were almost com-
pletely inhibited by one unit. Incubation for an addi—
tional 24 hours showed a marked growth of Listeria at

one unit and slight grewth at 3.0 units. The cultures
containing higher concentrations were negative (table I).
These results were similar to those reported by Coles
(1948). Because of the similarity of results, four cul-
tures of ovine and four cultures of bovine origin were
chosen for further study. Colonies were picked from

the plates containing one unit and transferred to plates
containing three and six units. Colonies were then picked
from the three and six unit plates and plated on 10

unit plates. By this adaptive procedure it was possible
to produce good growth in 24 hours on the 10 unit plates.
Tables 11, III, and I? show the results up to 6 units
after 24 hour incubation periods at 37° 0. An additional

-63...

24 hours' incubation invariably produced sufficient
growth for transfer to a higher concentration of strep-

tomycin.

During the course of this study it was observed
that there was an apparent individual resistance to
streptomycin. Where discrete colonies were observed
there was a marked difference in colony size. This had
never been observed in growth on plain tryptose agar

(figure 40).

One of the large and one of the small colonies
.were picked from a plate containing one unit and from
a plate containing 10 units and transferred to broth and
plain agar slants, incubated for 24 hours and plated on

streptomycin plates. The results are shown in Table 7.

These exposed cultures were then transferred
daily for seven days through broth and on tryptose slants
and re-exposed to the same concentrations of streptomycin.
The results were essentially the same. These cultures
were then left without transfer at 4° 0 and at 21° C for
a period of six weeks. Re-eXposure to streptomycin

yielded approximately the same results.

In lefl tests were carried out using rabbits.
The streptomycin used was the calcium chloride complex.
In each experiment one animal was treated at exposure,
one 12 hours following exposure and one left as an un-

treated control. Temperatures were recorded at eight

-64..

hour intervals the first day and every 24 hours there-
after. Leukocyte and differential counts were taken

at 24 hour intervals. Dosage ranged from 2,000 units
per day per rabbit to 150,000 units per day per rabbit.
Because the number of rabbits used, 21 in all, was

small no definite conclusions could be made but in each
case the animal treated initially showed the least temp-
erature rise, but two of these animals were the first

to die. The monocytosis following infection was incon-
clusive but tended to be somewhat lower in the treated
animals. The histories of the individual rabbits are
given on pages 68 to 88. Cultures isolated from the
treated animals were used to inoculate tryptose agar plates
containing streptomycin. There was some evidence that
these organisms had acquired a resistance to the anti-

biotic but the results were not consistent and are not

given here.

-65...

TABLE I
Cultures initially exposed to streptomycin.

 

Units streptomycin /”I1 agar

 

24 hr. incubation 48 hr. incubation
Culture 0 0.5 1.0 3.0 0.5 1.0 5.0__g
l ++++ +++ ; - ++++ + t
2 ++++ +++ - - ++** 3 1 colony
3 ++++ ++t $ - ++t+ 3 1 001°n¥
4 ++++ ttt i - ++++ + l colony
5 ++++ +++ s - ++++
6 ++t+ +++ ¥ - t+++ + ;
7 ++++ +++ 3 - ++~+ + z
8 ++++ +++ 3 - *t*+ + i
9 ++++ t++ i - ++t+ + i
10 ++++ +++ 5 - ++++ 1 3 colonies
11 ++++ +e+ - - ++++ 3 2 colonies
12 ++++ ttt ++ - ++++ +-- +
++tt +++ 3 - ++++ t 1
B ++++ +t+ 3 - ++t+ 1 i
++++ +++ + - +++¢ + *

 

slight growth t - marked growth ; - trace

I contaminated
strain obtained from Dept. Bact. and Pub. Health, M.8.C.

+0
II

strain obtained from Mich. Dept. Pub. Health Lab.
strain obtained from Va. Poly. Inst., Blacksburg, 7a.

0 U b 0
II

-66...

' TABLE II
Initial exposure
Units streptomycin / m1 agar

Culture 0 0.5 1.0 3.0
2 +t++ +++ - -
4 ++++ +++ T -
B +++o +++ T -
9 ++++ +++ 4 -
7 ++++ +++ - -
10 ++++ +++ + -
12 ++++ +++ T -
C ++++ +++ - -
T 8 Trace

Cultures 2, 4, B and 9 are of ovine origin; 7, 10, 12
and B of bovine origin.

TABLI III
Colonies picked from 0.5 u plates
units streptomycin / n1 agar

Culture
2 ++++ ++++ +++ -
4 t+++ ++++ ++ -
B ++++ ++++ ++ -
9 ++++ ++++ ++ -
7 ++++ ++++ + -
10 ++++ ++++ + -
12 ++++ ++++ ++ -
C ++4+ ++++ + -

-67..

TABLE IV
Colonies picked from 1.0 u plates
Units streptomycin / n1 agar

Culture 0 1.0 3.0 6.0 10.0
2 +++# ++++ ++ T -
4 ++++ +c++ ++ + -
B 444+ ++++ e T -
9 ++o+ ++++ +++ +++ -
7 ++++ - T - ,
10 ++++ ++++ - - -
12 ++o+ ++++ + - _
C ++++ ++++ + + -
TABLE V

Units streptomycin / ll agar

12 LY ++++ ++++ +++ + 4
12 87 ++++ ++++ T - T
10 LII +++o ++++ - - -
10 SII ++++ ++++ - - -
10 control +t++ T - - -

L7 8 1argo colony from 10 u LII
87 = small colony from 10 u SII

large colony from 1 u

small colony from 1 u

- 58 -

Rabbit pg

0.5 m1 of a saline suspension of culture no. 9 stand-
ardized to a HacFarland tube No. 1 inoculated I.V.
This animal was an untreated control.

Days Temp. Hb WBC L P M B E
0 102.8 11.4 9,800 35 61 0 0 4
1 106.0 8,200 26 70 3 1 0

(not eating) (blood culture - 3 colonies)
2 105.0 12.0 5,000 26 59 14 O 0

(snuffles - exudate in left eye - breathing hard
running nose)

3 100.0 8,700 18 51 31 O 0
(semi comatose)

31222.91

Considerable exudate from both eyes and thick mucus
from the nose.

Liver: Marked fatty changes and a few white foci.

Heart: Fiabby, yellow, slightly congested.

Spleen: Swollen with numerous white foci 1 mm. in diameter.
Stomach and intoetino were nearly empty.

111 other organs appeared normal.

Cultures: both eyes, nose, saliva, lung, liver, spleen,
kidney and heart blood.

Positive cultures: blood, 1 colony; spleen and lungs,
a few colonies; and liver, heavy
growth.

- 69 -

Rabbit 63

 

0.5 m1 of a saline suspension of culture no. 9 stand-
ardized to a MaeFarland tube No. 1 inoculated 1.7.
1,000 u streptomycin given at time of inoculation and
every 12 hours thereafter until death.

Days Temp. Hb WBC L P M B E
0 102.5 14.1 6,950 41 54 1 3 1
1 103.6 7,550 18 88 O 0 0
2 105.0 12.9 5,400 41 56 3 O 0

Culture of 1 shows 7 colonies Listeria. Trembling,
drinking lots of water, not eating.

Necropsy

Liver: severe fatty changes and numerous white foci
throughout all lobes.

Spleen: black and swollen.

Lungs: congested.

Heart: flabby and yellow and somewhat congested.

All other tissues appeared normal.

Cultures: lungs, liver, kidneys, spleen and heart blood.

Positive cultures: liver, heavy growth; one kidney
and spleen, a few colonies.

The other cultures were covered by a spreading contaminant
and no Listeria could be observed.

-70-

Rabbit 15

0.5 m1 of a saline suspension of culture no. 9 stand-
ardized to a HacFarland tube No. 1 inoculated 1.7.
1,000 u streptomycin was given every 12 hours starting
12 hours after inoculation.

Days Temp. Hb WBC L P M B E
0 102.3 10,900 55 44 0 1 O
1 104.6 9,100 18 75 2 3 2
2 106.2 11.1 10,950 45 43 12 0 0

(Diarrhea - cnufflec - blood culture negative)
5 103.0 9,950 so 22 as o 0
(Breathing hard)
4 99.8

This animal was down and seemed to be near death.
Suddenly it lunged out of its cage striking the cement
flocw with its head. He was dead when picked up.

Necropsy

There was considerable mucus from the nose.

Lungs: 01d abscesses throughout all lobes.
Some areas of both cold and active pneumonia.

Heart: Ilabby and slightly ingested and congested.

Liver: Patty degeneration with a few white necrotic
foci about 1 mm. in diameter.

Kidneys: Appeared normal but each contained blood tinged
“anc

Spleen: Swollen and numerous areas of fecal necrosis
approximately 1 mm. in diameter.

Uterus: Red and swollen.

All other viscera appeared normal.

Cultures: liver, spleen, heart blood and lung.

Positive cultures: liver, heavy growth; spleen, a
few colonies.

-71..

Rabbit pg

 

0.5 m1 of a saline suspension of culture no. 9 stand-
ardized to a MaeFarland tube No. 1 inoculated I.V.
1,000 u streptomycin given at time of inoculation and
every 12 hours thereafter until death.

Days Temp. Hb WBC L P H. B E
0 102.0 11.4 14,900 23 73 3 1 O
1 106.0 15,850 14 78 8 0 O
2 105.5 8.9 17,000 10 77 12 O l
3 104.4 14,350 21 59 19 1 0
4 103.6 9.2 16,250 11 75 12 2 0
5 103.8 40,000 6 64 28 2 0
6 104.6 17,250 17 63 20 O O

20 38 48 12 0 2
(monocytes old and degenerate - estimated
count - 10-15,000)

32 4,800 51 43 3 0 3

This animal never completely recovered from the effects
of the disease. It was thin and emaciated, took very
little food or water.

Necropsy

Liver: Swollen and leukemic in appearance.

Lungs: Some cold pneumonia.

Heart: Pals and with the veins injected.

Spleen: Black and swollen to twice normal size with
numerous white foci throughout.

Kidney: Swollen - ri ht has a large abscess at anterior
end (section .

Cultures: both .y... kidney, lung, liver and blood.

Positive cultures: kidney, 2 colonies; lung, liver and
blood, each 5 colonies.

_ 72 -

Rabbit 25

 

0.5 ml of a saline suspension of culture no. 5 stand-
ardized to a lacFarland tube No. 1 inoculated 1.7.
1,000 n streptomycin given at time of inoculation and
every 12 hours thereafter until death.

Days Temp. Hb WBC L P M B E
0 103.4 12.5 7,000 23 62 O 7 0
1 104.8 10,800 8 89 2 1 O
2 105.5 11.4 8,750 21 68 8 3 0
(blood culture - 2 colonies)

3 105.0 10,700 9 63 26 2 0

4 104.7 10.8 16,550 6 so 25 1 o
(crippled)

5 100.4 26,300 3 74 22 1 o
(crying as if in pain)

6 96.2 21,000 8 73 18 1 0

(still crying - emaciated)
Killed with other.

10.2.0121

There was excessive purulent exudatefrom left eye.

Liver: A few necrotic areas 1 mm in diameter. 01d
ccccidia lesions along margin and hepatic artery.

Spleen: Swollen, a few small foci.

Kidney: A few old abscesses.

Brain: There seemed to be an excess of fluid and the
medulla appeared congested.

Cultures: Kidney, lung, blood, liver, spleen, cerebral
fluid, brain, aqueous humor, both eyes, mouth
and nose.

Positive cultures: kidney and liver, heavy growth;
lung, spleen, brain, cerebral fluid,
and blood, a few colonies.

-73-

Rabbit 10a

0.5 ml of a saline suspension of culture no. 5 stand-
ardized to a MacFarland tube No. l inoculated 1.7.
1,000 n streptomycin given every 12 hours, starting
12 hours after exposure.

Days Temp. Hb BBC L P Hf B E
0 103.5 10.1 12,000 47 52' 1 O 0

1 106.5 7,900 5 82 13 0 0

2 105.3 12.9 15,450 17 50 32 0 l

3 104.8 26,200 10 58 32 0 0

4 103.2 10.1 22,650 16 56 28 0 0

5 102.4 28,000 10 44 46 O 0

6 103.4 19,200 13 54 32 l 0
20 17 67 16 0 0

This animal never completely recovered. It was thin,
refusing feed and water most of the time. Has not
eaten for the past 3 days.

£22m

Liver: Humorous white necrotic areas about 1 mm in
diameter throughout.
Kidneys: Left swollen to twice normal size.
Right slightly swollen, soft and degenerated.
Stomach and intestine: Empty.

Cultures: liver, blood and kidney.

All cultures were covered with spreadcrs and no Listeria
could be observed.

-74..

Rabbit pp

0.4 ml culture no. 5. This animal was an untreated
control.

D838 Temp c WBC L P

B

102.5 10,400 so 19 l

107.01.u.12,350 40 53 1

106.9P.M.

107.4 14,500 26 62 9 o
o
2
6

N03

0

1

2

(drinking - anorexia)

3 106.4 33,100 9 65 22
(trembling, nervous, no activity)

4 106.0 29,600 20 67 11
(bad fits)

5 105.0 26,400 43 40 11
(snuffles - difficulty standing)

6 Dea‘o

OOOFCRQOH

Necropsy

Liver: Swollen, a few small white foci and 01d coccidia
lesions.

Spleen: Black and swollen.

No other gross lesions.

Cultures: liver, heart blood.

Positive culture: liver, few colonies.

-75-

Rabbit‘gp

0.8 ml culture no. 5 1.7. 10,000 u streptomycin given
at time of inoculation and 5,000 u given every 8 hours
thereafter. 0.8 ml was given in error.

Days Temp. WBC L P M B E

0 102.3 11,600 62 34 2 2 O
(sore on left eye)

1 106.6 15,100 62 23 9 6 0

(left eye closed - some exudate - many platelets
and baskets)

2 106. 0 41, 800 53 31 14 2 0
(many platelets and baskets - snufflcs - eating)

3 105.3 33,100 73 10 16 1 0
(anorexia - snuffles - nervous - crippled)

4 . 94.8 50,150 51 4 42 3
(comatose)

Killed with other.

Necropsy
Liver: Large areas that appeared necrotic - old
coccidia lesions along the margin.
Spleen: Pitted - a few necrotic foci approximately

1 mm. diameter.

Intestine: Empty - injected throughout the entire
lon‘tho

Spinal cord: Pinched in thoracic region and congested
throughout. ,

No other gross lesions were observed.

Cultures: blood, liver, urine, medulla.

Positive cultures: liver, few colonies.

-76..

denied

3 drops of culture no. 9 instilled into the left eye.
10,000 u streptomycin at time of exposure and 5,000
u given every 8 hours thereafter.

(clearing - when in center-eultur )
17 8,600 41 44 7

Days 4 Temp. NBC L P M B E
O 102. 2 9 ,350 64 3O 2 2 2
1 103.0 10,950 66 23 9 2 0
2 105.2 8,650 69 27 1 3 O

( eye closed tight - streaming exudate )
3 103.0 8,450 64 23 4 7 2
(no change)
4 103.3 12,400 69 20 7 4 0
(eye closed - exudate dry)
5 103.7 11,300 52 38 10 0 0
(no change)
C 102.9 10,400 54 38 6 2 0
(open, but opaque - no exudate)
11 103.6 10,200 48 36 8 4 4
(opaque (Kodachrome ) culture)
14 103.1 9,700 55 35 4 6 O
o
6 2

—77..

Rabbit 42

0.4 ml culture no. 5 1.7. 10,000 u streptomycin given
12 hours after exposure and 5,000 u given every 8 hours
thereafter.

Days Temp. WBC L P M B E
0 103.3 6,350 58 41 1 0 O
1 103.31.M. 11,550 34 59 4 0 0

106.3P.M. -
2 105.4 8,750 19 54 22 0 5
3 106.3 17,700 15 48 30 O 7
(trembling - shuffles - heavy breathing)
4 106.0 16,250 14 69 13 4 0
(worse - relaxed - exudate in left eye)
5 103.3 11,850 34 42 17 7 0
(no change)
8 103.6 29,100 31 62 5 2 0
(thin, soft feces - improved)
11 105.4 18,100 25 38 53 4 0
(normal)
14 103.3 13,900. 53 37 1 9 0
(normal)
17 8,600 48 28 11 3 0

(normal)

- 73 -

2.911151

0.4 m1 culture no. 8L 1.7. This animal was an untreated
control.

Days Temp. WBC L P M B E
0 103.4 5,750 91 8 1 0 0
1 A. N2104.0 8,250 87 13 0 0 0

P. M.104.7
2 106.6 8,850 56 34 8 1 1
5 105.2 10,150 75 15 6 5 1
(difficulty‘walking)
4 104.9 12,400 47 20 28 3 2
(slight trembling)
5 104.1 14,350 59 35 3 3 0
8 103.4 11,100 56 34 7 1 2
11 102.9 8,550 50 27 21 2 0
14 102.9 12,850 46 33 12 8 1
(normal) *
17 8,800 24 51 15 9 1

68 Dead

Necropsy

Both eyes: considerable exudate
left eye somewhat injected especially along
the lower margin of the cornea.

Kidneys: slightly pale and swollen.

There were no other gross lesions.

Cultures: heart blood, liver, spleen, kidney, brain,
lung, and both eyes.

Positive cultures: None.

-79..

321131121

‘0.4 ml culture 8L 1.7. 10,000 u streptomycin given
12 hours after exposure and 5,000 u given every 8 hours
thereafter.

Days Temp. NBC L P N’ B E
0 103.6 7,800 78 22 0 0 0
1 do]. 104.8 9,150 29 64 l 0 l

P.N. 106.0
2 106.5 6,050 37 35 21 3 4
3 105.2 8,650 40 24 31 5 0
(breathing hard)
4 104.0 16,550 74 15 7 4 0
(slight trembling)
5 104.6 9,850 71 14 13 2 0
8 103.6 10,800 64 20 12 4 0
(thin)
11 103.0 8,950 42 30 16 9 3
14 103.2 7,950 52 35 12 l 0
(normal)
17 7,550 49 40 7 4 0

-80..

3221111

Three drops of culture no. 9 instilled into the left
eye. This animal was an untreated control.

Days Temp. WBC L P M’ B E
0 104.2 6,900 76 23 1 0 0
1 103.5 10,500 61 36 3 0 0
2 103.5 9,500 60 32 5 3 0

(slight exudate)
3 105.5 10,100 68 24 2 1 0
(very slight injection and exudate)
4 103.5 12,550 69 23 6 2 0
(no change)
5 103.4 12,850 81 12 7 0 0
normal)
8 03.5 10,150 67 24 2 2 0
(normal - eye culture negative)
11 105.2 10,500 59 28 9 4 0
(normal - 4 colonies Listeria)
14 103.0 9,000 77 I7 3 3 0
(normal - eye culture negative) -
17 7,250- 46 40 7 6 1

- 81 -

Rabbit 82

0.4 ml culture no. 9 1.7. This animal was an untreated
control.

Days Temp. WBC L P M B E
0 105.0 10,100 72 26 o 1 1
1 1.n.105.4 7,650 45 55 o 2 0

P.H.106.1
2 107.0 7,850 55 64 2 1 0
(left eye closed)
3 106.6 9,800 53 43 2 0
(head swaying)
4 105.8 15,500 33 53 12 2 0
(trembling - twitching)
5 104.5 14,650 56 4e 7 9 o
(no change)
8 104.6 17,450 61 27 6 6 0
(left eye closed - othdrwioc normal)
11 104.1 17,100 33 54 8 5 0
(no change)
14 105.5 9,700 54 29 6 1 0
(eye open)
17 6,250 44 37 10 9 0

(normal)

-82..

Rabbit 22

0.4 ml culture n0. 9 1.7. 10,000 units streptomycin
given 12 hours later and 5,000 units given every eight
hours thereafter.

Days Temp. WBC L P M B E
0 102.8 7,600
1 104.6A.M.9,950 25 75 0 0 0
106.6P.M.
2 106.4 9,050 48 51 8 3 0
3 106.5 15,650 53 39 7 1 0
4 106.1 15,450 33 60 7 0 0(anorexia)
5 104.5 15,900 51 54 12 2 1
8 104.6 26,550 47 45 5 3 0
(not drinking)
11 104.0 no blood (right eye bad)
14 104.9 22,700 41 55 2 1 1
(right-bad - very thin)
17 11,450 64 29 4 3 0
19 PoMc Dead.

Right eye:
Heart:

Intestine:
Liver:

Necropsy

marked conjunctivitis.

firm, yellow mass at apex measuring about
1.5 cm. in die. This same material ex-
tended into the walls and septa of both venp
tricles. The entire organ appeared pale

and flabby.

empty and somewhat congested.

a few old coccidia lesions.

No other gross lesions.

Cultures:

eyes, heart, heart blood, liver brain and
urine.

Positive cultures: none.

Rabbit 10b

0.4 m1 culture no. 9 1.7. 10,000 u streptomycin given
at exposure and 5,000 u every 8 hours thereafter.

Days Temp. WBC L P M’ B E
0 102.2 9,000 84 16 0 0 0
1 1.N. 102.7 -10,250 41 53 0 2 4

P.N. 105.1

2 106.3 8,500 62 34 O 4 0

3 105.0 19,050 60 31 d 5 0

4 104.9 18,150 68 25 3 4 0

(off food)
5 104.0 15,100 56 40 0 4 0
(eating again)

8 103.3 15,400 64 33 0 3 0
11 17,600 55 32 5 6 2
14 102.7 15,400 58 37 2 3 0
17 11,550 31 56 7 5 1

241414.11

0c4 I1 Onlture 817 Ich
time of exposure and 5,000 u given every 8 hours there-

afte

r.

1.1.
P.H.

Temp.

103.0
103.5
106.3
106.2
105.3
104.3
104.0
103.0
102.6
103.3

WBC

5,650
8,400

9,550
15,100
11,000
15,400
12,250

9,850

7,050
12,250

- 84 -

p...
Nwhomomm 00 K

mentioned-700.74 l-‘O w

10,000 u streptomycin given at

GIN FI-‘H

0(not eating)
0(eating)
1(normal)
1(norma1)
0(norma1)
1(normal)

- 35 -

Rabbit‘lp

0.75 m1 of culture no. 9 1.7. This animal was an un-
treated control.

Days

0 7:00
3:20
9:45

2 P.M.

Liver:

Spleen:
No other

Cultures:

Temp. WBC L P M B E

103.04.M. 8,650 67 25 2 6 0
105.8P.M. (breathing hard and twitching)
106.8P.M.

106.0A.M. 5,200 53 45 0 2 0
105.8P.M. (many nucleated and other im-
mature reds - unsteady)

102.7 10,200 60 36 4 0 0
(limp, unable to stand)

" DCaic

A number as small pinpoint areas of necrosis,
severe fatty degeneration, and a few old coccidia
lesions along the margin. Slightly swollen.
Black but otherwise normal.

gross lesions.

liver, heart blood, kidney.

-86...

Rabbit _2_._

0.75 ml culture no. 9 1.7. 50,000 u streptomycin 1.M.
every 3 hours.

Days Temp. WBC L P M’ B E
0 7:00 103.3A.M. 10,700 74 24 1 0

5:20 105.5r.n. (breathing hard)
9:45 105.52.n.

1 106.01.!3 4,800 31 66 1 2 0
105.6P.M.
2 105.5 10,550 41 42 14 2 1
(some immature red cells)
5 105.1 15,900 55 29 16 2 0
(not many immature red - eating?)
5 9,400 66 1e 11 5 1
7 10,600 74 24 1 1 0
10 12,200 63 28 6 1 2
14 9,900 64 24 ‘ 6 5 1

-87...

Rabbit 33

0.75 ml culture no. 9 1.7. 50,000 u streptomycin at
exposure and 25,000 u every 3 hours thereafter.

Days Temp. WBC L P M’ B E
0 7:00 102.7A.M. 10,850 66 28 2 4

3:20 105.6P.H. breathing hard.)
9:45 106.9P.M.

1 106.01.u. 6,850 45 49 4 2 0
105.7r.u.
2 105.9 5,900 59 41 16 2 o
5 101.2 15,550 45 5e 17 0 o
5 20,400 65 19 15 1 1
7 16,550 67 6 5 2 o
10 17,400 69 27 4 o o
14 14,450 79 14 6 1 o

-88..

Rabbit 2

0.75 ml culture no. 9 1.7. 50,000 u streptomycin
every 3 hours.

Days Temp. WBC L P M B E

5:20 106.4PJI.
9:45 106.5PJI.

1 106.01.u. 6,450 10 88 0 2 o
106.2P.M.
2 105.0 9,650 22 49 26 1 0
5 105.5 15,600 38 41 21 o o
5 9,600 60 26 12 1 1
7 11,850 56 29 11 4 0
10 10,050 55 26 12 5 o
14 6,600 65 15 21 5 o

Bacitracin

- 89 -

Considerable interest had been shown in baci-

tracin since its isolation by Johnson 23.21 (1940).

.12 vitro tests were carried out using four ovine (1, 9,
13, 48) and four bovine (8, ll, 27, 45) strains of Lie-

teriao

Difcc tryptose agar, pH 7.0, was used as a base

medium and bacitracin added to make the following con-

ccntrations:

Oo125, Oc25, 0.5, 1.0 and 2.0 unitSo

plates were inoculated with a 1 mm loOpful of 24 hour

broth culture of each strain.

incubation at 37. C for 24 and 48 hours.

Plates were read after

The results

are shown in tables 71, VII and 7111.

TABLE 71
Initial exposure

These

 

Units bacitracin 7 ml agar
24 hr. incubation

48 hr. incubation

 

Culture 0 0.125 0.25 0.5 1.0 2.0 .5 1.0 2.0
1 4444 4444 4444 i - L- +4 - -
9 4444 4444 4444 4 - - 444 - -
13 4444 4444 4444 i - - 4+4 - -
48 4444 4444 4444 - - - 4 - -
8 4444 444+ +444 444 - - 444 f -
11 4444 4444 4744 444 - - 444 E -
27 4444 4444 4444 - - - 3 ~ -
45 +444 4444 4444 - - - - - -

 

- 90 -

TABLE VII
Colonies picked directly from 0.5 u plates

 

Units bacitracin 7 m1 agar
24 hr. incubation

 

 

Culture 1.0 2.0
l - -
9 t -
is g ..
8 t -
11 E -
TABLE VIII

Cultures picked from 0.5 u plates and maintained in
nutrient broth for 16 days.

 

Units—bacitracin 7—mI agar
24 hr. incubation

 

Culture 0.5 1.0
1 - -
9 - -
13 - -
8 E -
11 f -

27 ' - -

 

These data suggest that certain strains may be
slightly more resistant to bacitracin than are others.
However, all strains failed to grow in the presence of

1.0 unit of the antibiotic. The resistant bovine strains

- 91 -

are more resistant than are the resistant ovine strains.
These cultures were not able to adapt themselves to baci-

tracin as they did to streptomycin.
Penicillin

Similar tests were carried out using penicillin.
The results obtained were not at variance with those
reported by Foley'gt.gl (1944) and Handelman 21.31 (1946).

The results are shown in Table 11.

TABLE IX

Initial exyosure

 

Units penicillin lfiml agar
24 hr. incubation 48 hr. incubation

 

Culture 0 0.125 0.25 0.5 1.0 0.5 1.0
1 4444 4444 4444 444 444 444 444

9 4444 4444 4444 444 4 44 44

18 . 4444 4444 4444 444 4 444 44
48 4444 4444 4444 444 44 444 44

8 4444 4444 4444 44 - - 4

11 4444 4444 4444 444 1 444 4
27 4444 4444 4444 444 - 444 4
45 4444 4444 4444 44 f 44 1

 

These data corroborate other investigations show-
ing that penicillin alone had little or no value in
treating Listeria infections. This was further substantiated

by data on clinical cases cited in Part I.

-92..

Chloromycetin

Recently considerable attnntion had been focused
on chloromycetin, an antibiotic isolated by Ehrlich 23
.21 (1947). Although this substance had shown its great-
est effectiveness against certain viruses, rickettsiae
and gram negative bacteria, Smadel and Jackson (1947),
its effect on Listeria was determined. From the results
shown in table.I it appeared unlikely that it will have
any value in treating infections involving Listeria.
Susceptible microorganisms were inhibited in concentra-

tions as low as O.l-0.05 micrograms per ml of medium.

TABLEJI
Initial exposure

 

iierograms I_ml agar
24 hr. incubation

 

Culture 0 0.5 1.0 1.5 2.0
1 4444 4444 4444 4 -
9 4444 4444 4444 444 E
15 4444 4444 4444 44 -
48 V4444 4444 4444 44 4
3 4444 4444 4444 1 -
11 4444 4444 4444 4444 4
27 4444 4444 4444 4 i
45 4444 4444 4444 444 5

 

PathOgenicitx

the case histories of the rabbits in the strep-
tomycin experiments were included to show the typical
reactions following intravenous inoculation of Listeria.
The most striking characteristic was the high percentage
of nonoeytes in the peripheral blood. This reaction
was reported by Murry‘gt'gl (1926) in the first descrip-
tion of L! monocytogenes. It was this characteristic
which led them to give 'monccytegenes' as a species
name to the organism. This reaction had been used
by Witts and Webb (1927), Bloom (1928). Conway (1938,
1939) and numerous other hematologists in investigations

on the origin and nature of the monocyte.

Grahan'gt‘al (1943) reported that there was no
evidence showing that a circulating monocytosis was
present in affected ruminants. This.was borne out in
part by cases presented in Part I. However, case 21
showed a monocytosis of 15$ and case 34 a monocytosis
of 7 and 8 $ on consecutive days. These values were
sufficiently high to suggest that an increased number
of monocytes may be present in ruminants suffering from
listeriosis. Chapman (1947) reported increased numbers

of monocytes in the dogs from which 'he isolated Listeria.

After ocular instillation an increase in circulat-
ing nonocytes was not consistently shown. This was in

accord with the findings of Julianelle (1941) and Graham

-94-

‘21 a; (1945). Julianelle and Pens (1939) state that
large numbers of monocytes were found in the exudate
of eyes following ocular instillation. A few smears
made from this exudate and stained with Wright's stain
showed many polemorphonuclear leucocytes but few mono-
cytes. However, so few smears were made that this was
not intended to be contrary to the findings reported

above.

It will be of interest to note that rabbits ex-
posed to Listeria by intravenous inoculation showed "sludge
blood" as reported by Knisely'gt’gl (1947). Animals
that survive for longer than three days became so
badly affected that it was difficult to obtain suitable
samples for hemocytological examination. this condition
persisted for several days after which time, if the

outcome of the disease was favorable, the blood regained

its normal fluidity.

The presence of basophilic staining granules in
the polymorphonuclear leucocytes was observed in almost
all animals exposed to Listeria. These granules were
present in naturally occurring cases as well as in those
artificially exposed. This was true by any route of
infection; ocular instillation, intravenous inoculation,
or ingestion. It was felt that they were similar to the

so-called "toxic granules'l found in infections (Downey 1938).

-95-

To determine the effect of oral administration
of Listeria a rabbit was given 250-300 ml of 24 hour
broth culture as the sole source of fluid intake for
a period of 20 days. Two days following initiation of
this regime the rabbit was irritable and displayed slight
difficulty in walking. The leukocyte count was increased
by 6,000 cells per mm3, and the differential blood count
showed 6* monocytes. The lymphocyte-polymorphonuclear
leukocyte ratio was not altered. This condition per-
sisted for two days after which time the animal appeared
normal. By the sixth day the blood values had returned
to normal limits. There was no further reaction noted.

The temperature did not vary throughout the 20 day period.

The significant finding of this experiment was
that Listeria could pass through the entire length of
the digestive tube. Listeria could be isolated from
the feces in large numbers throughout the 20 day period
and for at least five days after the termination of the
experiment. Nine days later Listeria could not be iso-
lated from the feces. In no instance was Listeria iso-

lated from the urine.

In attempting to isolate Listeria from the feces,
a few fecal pellets were macerated in the Waring blendor
together with approximately 50 ml nutrient broth. One

ml portion of this suspension was transferred to broth

_ 95 _

tubes containing 0.05% potassium tellurite. These were
incubated at 37° C for 24 hours and plated on tryptose
agar. The plates were incubated for 24 hours at 37° C
and examined with a dissecting microscope using oblique
light. Listeria was often isolated in nearly pure cul-

ture.

Other modifications of this procedure were

tried, but the one described proved to be most satis-

factory.

Also of significance was the fact that an ocular
reaction could not be produced in this rabbit with a
culture that readily produced a conjunctivitis in sus-

ceptible animals.

Six rabbits were inoculated intracerebrally under
other anesthesia. in inoculum of 0.2 ml of a saline
suspensions of organisms produced death in from 20 hours
to 4 days in all but one. Listeria was isolated from
the liver and brain of these animals. One animal sur-
vived for a week but it was nervous and irritable and

killed by other inhalation. Listeria could not be isolated.

Six rabbits inoculated subcutaneously with 0.5
ml of a saline suspension of organisms showed no ill
effects after one week period of observation. These

same animals were inoculated intravenously with 0.5 ml

-97...

of a saline suspension of Listeria. Death followed in
periods from 24 hours to three days. At necr0psy all
but one animal showed a large abscess with a necrotic
center at the point of the subcutaneous inoculation.
Listeria could be isolated from the liver and heart

blood but not from the abscess.

Immunological Reactions

The serological reactions of Listeria had received
considerable attention from Seastone (1935), Schultz
gt a; (1938), Paterson (1939, 1940), Julianelle (1941),
Graham 33 El (1943), and Drew (1946).

Schultz gt 2% (1938) and Julianelle (1941) sug-
gested the presence of two serological groups. Julianelle
(1941) designated these as Type I, or rodent strain;
and Type II, or ruminant strain. He postulated that the
ultimate origin of the strains isolated from infective
cases could be determined by this method. This was in
disagreement, however, with Paterson (1940) who found,
within the genus Listeria, four serological groups based
on somatic and flagellar antigens. He concluded that
there was no host specificity for the four suggested.
Drew (1946) found two groups based on precipitation
reactions which corresponded to the two groups of

Schultz 21.2; (1938), Julianelle (1941) and to groups

-98..

I and IV of Paterson (1940).

It was not the purpose of this study to venture
into the serological characteristics of this group.
However, antigens prepared from cultures one through
22 and cultures A, B, C, and D* were tested for agglut-
inability with antisera prepared from cultures 9 (ovine)
and 5 (bovine). Antisera were prepared by repeated
intravenous injections of living organisms into a group
of rabbits that previously had been exposed to the specif-
ic culture and had survived. It was of interest that
these animals displayed no alterations in hemocytological
values during the 24 day immunization period. A second
group of rabbits was immunized using a heat killed cul-
ture. These, too, showed no variation in hemocytological
values. At the termination of immunization there was
no significant difference in antibody titer between the
groups receiving living and those receiving killed anti-
gen. This was also observed by Graham‘gt‘al (1943).

The agglutination titer of the ovine strain anti-
sera ranged from 1:2560 to 1:5120 while that of the
bovine strain antisera ranged from 1:640 to 1:1280 with

their homologue antigens.

Antigens were prepared from the various cultures

 

'*Hnman strain, Larkin 6288. Received from Dr.
W. Wheeler, Childrens' Hospital, Columbus, Ohio.

- 99 -

by suspending in physiological saline the washed off
growth from a 24 hour agar slant culture. Cultures
were killed by heating at 90° c for 1 hour and stand-
ardized to the density of a McFarland Nepholometer tube
no. 2. Tests were performed as described by Julianelle

(1941).

It was found that of the 26 strains tested, all
but three (8, 12, 20) showed agglutinability with the
two types of antisera. Cultures 3 and 26 showed only
slight agglutinability. The remaining cultures showed
high agglutinability in dilutions of 1:2560 and 1:5120
with the antisera prepared from the ovine culture, and
in dilutions of 1:640 to 1:1280 with the antisera pre-

pared from the bovine culture.

These tests revealed that of the strains isolated
during this study at least two distinct immunological

groups were represented.

Biochemical Reactions

The biochemical and fermentation reactions of

Listeria had been reported by numerous investigators;

 

Seastone (1935), Julianelle (1941), Harvey and Faber
(1941), Graham gt El (1943), and many others. As point-
ed out by Julianelle (1941) the saccharolytic reactions

- lOO -

of the various strains were so similar that they do not
form a sufficient basis for classification of the various
strains. Harvey and Faber (1941), however, suggested
that the fermentation of melizitose might form a basis

of division. The division of groups by the fermentation
of melizitose closely resembles the serological division
of Paterson (1940). Fermentation reactions were carried
out on the following fermentable substances: glucose,
galactose, levulose, arabinose, xylose, lactose, maltose,
sucrose, trehalose, raffinose, dextrin, inulin, inositol,
duloitol, mannitol, sorbitol, glycerol, salicin. These
substances were added to a basal medium containing bacto-
tryptose, beef extract, sodium chloride and distilled
water. Brom cresol purple was used as an indicator.

All media were sterilized by autoclaving at 15 pounds
pressure for 15 minutes with the exception of dextrin'
and maltose which were filtered through a fritted glass
filter. All reactions were observed for 14 days. Doubt-

ful reactions were checked with a Beckman pH meter.

All strains produced acid but no gas after 24
hours in glucose, levulose, trehalose and salacin. Acid
production was slow and variable in arabinose, galactose,
lactose, maltose, sucrose, dextrin, sorbitol and glycerol.
There was no fermentation of xylose, raffinose, inositol,

inulin, duloitol, nor mannitol.

- lOl -

Biochemical reactions were carried out using media
and methods indicated in "The Manual of methods for the
(Pure Culture Study of Bacteria". Nitrates were not re-
duced. Indole was not produced. Gelatin was not liquefied.
All strains showed a slight beta hemolysin on thin sheep
blood agar plates. Starch was not hydrolysed.

All strains produced H23 in 24 hours using a
medium consisting of liver infusion, Witte's peptone,
sodium chloride and distilled water. Lead acetate strips
were used as an indicator. As there were no reports
of the production of H25 by Listeria, several other ac-
cepted media for the production of H23 were inoculated
with cultures taken at random. H23 was not produced in
any of these media. Thus it is evident that H23 can
be produced with the proper medium.

The above fermentation and biochemical reactions
were carried out on cultures 1 through 47 and A, B, C

and. De

Summary

The difficulty of successful isolation of Listeria
from the bovine brain was discussed. It was demonstrated
that refrigeration of brain suspensions yielded an in-
creased number of colonies when a 0.2 ml portion was

plated. Evidence was presented that recently isolated

- 102 -

bovine strains might not produce a conjunctivitis in a
susceptible rabbit. It was postulated that these pheno-
menon are related to an unstable substance in the bovine

brain inhibitory to Listeria.

It was shown that Listeria could survive for
six weeks on straw and wood shavings; 16 weeks on hay
and calf feed; and for 26 weeks on rabbit feed. It
remained viable without transfer at 4° C for at least

30 months with no loss in pathogenicity.

The effects of various selective media incorpor-
ating sodium azide, potassium tellurite, and acetic acid
were reported. Potassium tellurite, 0.05fi, in nutrient

broth was found to be most effective.

The effect of streptomycin, bacitracin, penicillin,

and chloromycetin on Listeria cultures was presented.

There was little evidence that these antibiotics could

be of therapeutic value.

Listeria was shown to be pathogenic for laboratory

 

rabbits following intravenous and intracerebral inocula-
tion. Oral administration produced only a slight reac-
tion. Subcutaneous inoculation was followed by abscess

formation at the site.

Immunological, fermentation, and biochemical re-

actions were presented. The cultures studied could be

divided into at least two serological groups. Fermenta-

- 103 -

tion and biochemical reactions were similar for all

strains.

Fig. 21. Liver at necr0psy. The white necrotic
Rabbit 6a. foci are a characteristic lesion at
necrOpsy of rabbits that have died
following intravenous inoculation.
This lesion is the same as seen in
the lamb reported in case 2. See
also figure 12. Kodachrome print.

Fig. 22. Typical conjunctivitis following ocular
instillation of Listeria culture.
Kodachrome print.

- 104 —

 

Fig. 21

 

 

Fig. 23. Heavy confluent growth of Listeria

Case 5. after the brain suspension Had Been
refrigerated for three months. This
had originally been considered negative.

Fig. 24. Primary isolation from bovine medulla.
Case 10. Ninety colonies Listeria.

- 105 -

 

Fig. 23

 

Fig. 24

Fig. 25. Primary isolation from bovine medulla.
Case 12. Forty four colonies Listeria.

Fig. 26. Plating of brain suspension after re-
Case 12. frigeration at 4° C for eight days.

A 0.2 ml portion was plated in each
instance.

- 106 -

 

Fig. 25

 

Fig. 26

Fig. 27. Daily increase in colony numbers during
Case 12. an eight day period of refrigeration
of the brain susPension. The first
plate is also shown in figure 25; the
last is shown in figure 26.

-lO7-

5 .34

 

Figs 280

Primary isolation from bovine medulla.
Case 18s

Eighteen colonies Listeria.

Fig. 29.

Plating of brain suspension after re-
CESO 180

frigeration at 4' C for 11 days. A

0. 2 ml portion was plated in each in-
stance.

-108-

 

 

Fig. 29

 

Fig. 30. Primary isolation from bovine medulla.
Case 23. Fifty four colonies Listeria.

 

Fig. 31. Plating of brain suspension after re-
Case 23. frigeration at 4° C for 17 days. A

0.2 m1 portion was plated in each in-
stance.

— 109 -

 

Fig. 30

 

Fig. 51

Fig. 32. Primary isolation from bovine medulla.
Case 32. Nine colonies Listeria.

 

Fig. 33. Primary isolation from bovine medulla.
Case 31. One hundred twenty five colonies Listeria.

 

- 110 -

 

Fig. 32

 

Fig. 33

 

 

Fig. 34. Primary isolation from bovine medulla.
Case 28. See also figure 38.

Fig. 35. Primary isolation from bovine medulla.
Case 24. These two cases presented the highest
number of colonies at primary isolation
from the bovine. At least 36 hours
had elapsed between death and culture
in each case.

-111-

 

Fig. 34

 

Fig. 35

Fig. 36. Primary isolation from ovine medulla.
0386 go

 

Fig. 37. Primary isolation from ovine medulla.
08.89 14o

- 112 -

 

Fig. 56

 

Fig. 37

Fig. 38. Primary isolation from ovine medulla.
Case 15o

 

Fig. 39. Primary isolation from ovine medulla.
Case 16.

-113-

 

 

 

Fig. 40. Difference in colony size of Listeria
on tryptose agar containing streptomycin.

 

- 115 -

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ROOM USE OWLY,

 

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