HESIGPATHOLOGY 0?" FEB BOVINE
PLACENTA IN ALEFTOSPIRA: POMONA ENFECTION

Thesis for film Degree of M. S
MICHEGAN STATE UNIVERSITY

Ray L. Morter
1958

“ID.
1148318

The research reported herein is an extension of investigations
conducted by the candidate and the majoradvisor at the University
of'Wisconsin. Materials collected in connection with this work
have been processed and evaluated by the candidate at Michigan State
University. The appendix contains two reprints of published articles
dealing with the experimental design, bacteriological and serological
studies pertaining to the histopathological results delineated in

this thesis.

 

 

 

 

 

HISTOPATHOLOGY OF THE BOVINE PLACENTA IN

LEPTOSPIRA POMONA INFECTION

 

by

Ray L. Morter

A THESIS

Submitted to the College of Veterinary Medicine
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1958

ACKNOWLEDGMENT

The author wishes to acknowledge with
appreciation and gratitude the helpful
assistance of Dr. E. V. Morse and Dr. R. F.

Langham.

INTRODUCTION.

TABLE

METHODS AND MATERIALS.

RESULTS

DISCUSSION
CONCLUSIONS

REFERENCES

APPENDIX

OF CONTENTS

12
13
15

LIST OF FIGURES

Figure Page

1. X175 Apparently normal bovine cotyledon.
(A) Fetal villus, (B) Maternal epithelium
with little connective tissue stroma, (C)
Diplokaryocyte. (Separation of fetal and
material tissues in an artifact.). . . . 16

2. X325 Apparently normal bovine cotyledon.
EA; Fetal villus, (B) Maternal epithelium,
C Diplokaryocyte. . . . . . . . . 16

3. X300 Maternal—chorionic junction; area of
hemorrhage and hemolysis the tenth day after
infection. . . . . . . . . . . . 17

4. X300 Cotyledon of heifer 2, 25 days after
infection showing vacuolization of fetal
villus. . . . . . . . . . . . . 17

5. X135 Twenty-eight days after infection
showing increased fibrous connective tissue
and vacuolization of the fetal villi. (A)
Vacuolar fetal villus, (B) Pyknotic nuclei
in the maternal epithelium, (CT) Maternal
connective tissue . . . . . . . . 18

6. X300 Higher power of Figure A. . . . . . 18

7. X135 Heifer 7, forty-six days after infection
showing marked proliferation of connective
tissue stroma of the cotyledon (CT), and
empty crypts (A) . . . . . . . . . l9

8. X300 Cotyledon of heifer empty crypts with
pyknotic nuclei and lack of continuity in
the maternal epithelium . . . . . . . 19

9. X135 Heifer 3 which aborted on the twenty-
ninth day after infection demonstrating
the loss of the normal cotyledonary archi-
tecture. (A) Necrotic remnants of fetal
villi, (B) Absence of material epithelium,
(CT) Maternal interstitial fibrous con-
nective tissue . . . . . . . . . . 20

10. X300 Higher power of Figure 9. . . . . . 20

INTRODUCTION

Abortions, usually occurring during the last trimester
of pregnancy, have been reported as a manifestation of
bovine leptospirosis.[2,3,4,5,8,9,10,13,1A] In the United
States bovine leptospiral abortions are due almost exclu-

sively to Leptospira pomona infections and abortions may be

 

the only manifestation of bovine leptospirosis. The patho-
genesis of the abortions has not been defined. Te Punga and
Bishop [15] have postulated three possible causes for
leptospiral abortions: (a) pyrexia and systemic reaction
resulting in abortion; (b) interrupted transfer of metabolites
due to localized lesions in the maternal-fetal cotyledonary
Junction, with subsequent fetal death; (0) actual invasion
of the fetus by the leptospiras with death and expulsion
following active fetal infection. Ferguson gt a1.[4]
propose the release of a toxic material from the leptospiras
which is able to cross the placental barrier where it is
presumed to destroy the red blood cells of the fetus. Alter—
ation of the epithelium of the maternal crypts, the site of
production of the hormones of pregnancy in some species,
could result in hormonal imbalance and abortion.

Abortions occur two to three weeks after the acute
systemic phase of leptospirosis and after the expelled fetus
probably had been dead 24 to 48 hours. [2}59] Attempts to

isolate leptospiras from fetal tissues, fetal fluids or the

2

placentas have generally been unsuccessful, indicating that
L; pomona does not cross the placental barrier in pregnant
cattle.[2,3,4,10,1l] The report of Podgwaite gt al.[l2] of
isolation of L; pomona from three aborted bovine fetuses has
been reviewed by Ferguson.[4] The demonstration of lepto-
spira-like bodies in fetal material by silver staining
techniques has been reported but not substantiated by bacter-
iological evidence,[2,l5] Leptospiras may be unable to
survive in the environment of the autolyzing fetus. However,
attempts to isolate the agent from living bovine fetuses,
removed at necropsy, at various times following maternal
infection, have been unsuccessfu1.[10,ll]

Histopathological examination of the cotyledons of
pregnant heifers, killed at selected intervals following
infection, was undertaken to determine if alterations in the

maternal-fetal cotyledonary relationships had occurred.

RESULTS

The clinical manifestations of the infection were
mild. During the acute phase of the disease, the experi-
mental heifers showed rectal temperatures of 103.2 to 104.4
F. Six of the animals evidenced anorexia, tachycardia and
depression. Hemoglobinuria, icterus or diarrhea were not
observed. The animals were killed 10 to 46 days following
exposure.

Heifer 5 was sacrificed on the tenth post-infection
day during leptospiremia. The fetus was approximately seven
months old, viable and appeared normal at necropsy. Signif-
icant microscopic lesions were not observed in the fetal
tissues. Grossly, the uterus and cotyledons appeared normal.
Microscopically, the cotyledons showed large areas of
hemorrhage with erythrocytic hemolysis and hemosiderosis at
the junction of the allantochorion and the maternal tissue
(Figure 3). The relationship of the chorionic villi to the
maternal crypts was quite normal. Some vacuolation of the
chorionic cells was observed. Similar changes occurred in
the cotyledons of one heifer sacrificed twelve days after
infection.

Heifer 2 was killed 25 days after infection. The fetus
was viable and apparently normal, as were the uterus and

cotyledons. Areas of hemorrhage at the Junction of the

5

chorion and maternal tissue were observed microscopically.
In the deeper portions of the cotyledons there were some
hemorrhagic areas and degeneration of the fetal villi charac-
terized by vacuolization (Figure A). Mitotic figures were
occasionally seen in the epithelium. The maternal crypts
exhibited increased fibrous connective tissue.

A viable four-month fetus was obtained from heifer 6
at necropsy on post—exposure day 28, and the only gross
lesion observed was one small grey-white area in the dam's
renal cortex. The most pronounced histopathological
findings involved the chorionic villi (Figure 5), some of
which showed a vacuolar degeneration. Cellular detail was
lacking in these villi and pyknosis was pronounced. The
epithelial lining of the maternal crypts was disrupted in
some areas and evidenced pyknosis (Figure 6). This would
indicate that a degenerative process had resulted in a loss
(iffetalqnaternal relationship in these areas. Increased
amounts of connective tissue were present in the stroma of
the maternal crypts.

Forty-six days after infection, heifer 7 was sacrificed.
The fetus was viable and no gross lesions were observed in
the fetus, placenta or uterus. Numerous grayish-white foci
were evident in the maternal kidneys and extended through
the cortex into the medulla. Microscopically the cotyledons
were found to have many empty crypts, some nearly devoid of

an epithelial lining. Adjacent crypts contained cellular

debris or degenerating villi (Figures 7 and 8). There was
an increase of fibrous connective tissue in the maternal
crypts.

Forty-seven days after infection the fetus of heifer
A was alive. Gross lesions were limited to the maternal
kidneys. The microscopic lesions of the cotyledons resembled
those of heifer 7. These changes included increased amounts
of connective tissue in the maternal cotyledon, degenerate
chorionic villi and separation of the villi from the crypts.

Heifer 3 aborted 29 days after experimental infection
or 22 days after showing a systemic reaction to the infection.
The fetus appeared to have been dead 12 to 24 hours at the
time of abortion and mild autolysis was evident. The fetal
organs appeared edematous. The pleural and peritoneal
cavities of the fetus contained increased amounts of a
serosanguineous fluid as did the pericardial sac. The
placenta was retained. The animal was sacrificed 24 hours
following abortion. Microscopically, the uterus was edematous
and the bases of the maternal crypts were congested. The
normal architecture of the cotyledon was missing, the con-
nective tissue proliferation obliterating most of the maternal
crypts (Figure 9). The crypts were devoid of the cuboidal
epithelial lining. The few remainingfetal villi were markedly
necrotic (Figure 10). Leptospiras were isolated from the
maternal kidneys but from none of the fetal nor placental

tissues.

The gross and microscopic lesions in the maternal
kidneys were similar to those reported previously.[5]
Microscopically the capsules of the kidneys stripped with
very little difficulty. The cortex had numerous grayish-
white foci which measured up to five millimeters in diameter
and in some instances the lesions involved the medulla and
hilus. These lesions when observed microscopically revealed
a marked infiltration of lymphocytes, a few plasma cells and
macrophages. In the same areas some tubules showed atrophy
and necrosis. About 47 days postinfection, fibrous connec-
tive tissue proliferation caused a thickening of some of
the Bowman‘s capsules of the renal corpuscles. Increased
fibrous connective tissue in the intertubular areas led to
some tubular atrophy and necrosis.

Terminal serums from the heifers gave agglutination-
lysis reactions at dilutions of 1:1,000 to l:1,000,000.
Leptospiras were not isolated from any of the fetuses nor
were they demonstrated by the silver staining technique

applied to fetal and placental tissues.

DISCUSSION

One of seven heifers experimentally infected with L.
pomona aborted 22 days after the acute phase of the disease.
The aborted fetus appeared to have been dead 12 to 2A hours
and only slight autolysis was observed. The other six
animals were killed 10 to 47 days following infection.

Their fetuses were viable at the time of necropsy and
appeared normal macroscopically as did the uteruses and
placentas.

Microscopic changes, progressing from the tenth to
the forty-seventh day, were found in the cotyledons, and
were most marked in the cotyledons of the heifer which aborted.
Ten days after infection areas of hemmorrhage and hemolysis
appeared at the chorionic-maternal junction (Figure 3).

By the twenty-eighth day the chorionic villi had a vacuolar
appearance, pyknosis and karyorrhexis of the epithelium
lining the maternal crypts was evident, and proliferation
of the interstitial connective tissue between the crypts
was apparent (Figures 4, 5, and 6). Forty-six and forty-
seven days after infection there was an increase in fibrous
connective tissue in the maternal caruncle, loss of epithe-
lium lining the crypts and an absence of fetal villi
(Figures 7 and 8). The normal architecture of the placen-

tomes of the heifer which aborted was almost completely

masked by the increased amounts of connective tissue

(Figures 9 and 10). Very few necrotic villi were present

and a morphological relationship between the fetal and
maternal tissues which would permit normal function appeared
to be almost completely lacking. The pathogenesis of bovine
leptospiral abortions apparently can be explained, therefore,
by the alterations in the intimate relationships between the
fetal and maternal systems that are necessary to support
fetal development. The microscopic lesions demonstrate a
series of changes of increasing severity from the time of

the activeinfection in the dam until abortion occurs. These
alterations in the placentome can interfere with the transfer
of essential materials across the placental barrier resulting
in fetal inanition and death. The dead fetus becomes, in
essence, a foreign body and is expelled.

Direct infection of the fetus by E; pomona lacks
unequivocal bacteriological proof at this writing. The
inability to demonstrate leptospiras in any of the seven
fetuses is in agreement with most reports.[2,3,4,11] Six
of the fetuses were viable at the time materials were taken
for bacteriological examination. Therefore, destruction of
leptospiras in the environment of an autolyzing fetus could
not account for the negative bacteriological results. Iso-
latiOns were accomplished from the cotyledons of one heifer
during the leptospiremic phase of the disease but not from

its fetus. This indicates that the organisms

10
approached but failed to cross the placental barrier during
the leptospiremia of the dam. Similar results were obtained
by Lindquist, Morse and Lundberg[6] with experimentally
infected pregnant ewes. Leptospiras were isolated from the
cotyledons of one ewe during leptospiremia. The fetal
organs, blood and fluids from this ewe and all ewes sacrificed
during the experiment were bacteriologically negative.
Positive results must be obtained by accepted bacteriological
methods to confirm the presence of L; pomona in fetal tissues.
An inoculum composed of a 10 per cent emulsion of 200 mgs.
of tissue in 0.85 per cent sterile saline, containing ten
or less leptospiras will infect guinea pigs or hamsters.[l]

If only a few leptospiras are present in tissues, they may

not be recognized by darkfield examination or staining tech-
nique but can be recovered by guinea pig inoculation.[l6]
Silver impregnation techniques can be valuable in determining
the presence of leptospiras in tissues but can not be con-
sidered a definitive diagnostic means for L. pomona infections,
unless the tissues are found to be bacteriologically positive

for leptospiras. L. sejroe, L. icterohemorrhagiae and L.

 

canicola infections may result in the invasion of the bovine
fetus. The leptospiras observed by some infetal tissues
stained by silver impregnation techniques may actually be
one of the above three serotypes and not L. pomona. Clari-
fication of the entire matter awaits experimental proof

and should not be based upon conclusions drawn from naturally

ll
occurring infections. Various argyrophilic tissue compon-
ents can interfere with proper interpretation of silver
stained histological preparations by untrained personnel.

Bovine leptospiral abortions occur three to four weeks
following infection of the dam. The fetuses apparently die
shortly prior to abortion. If fetal death were associated
with the systemic reaction of the dam it is questionable if
abortion would be delayed up to four weeks.

Fetal hematological determinations were not undertaken
but intact erythrocytes were present in the histological

preparations of the fetal tissues.

CONCLUSIONS

One of seven pregnant heifers infected with L. pomona
aborted 29 days following infection.

Leptospiras were not isolated from the aborted fetus
or any of the six fetuses which were viable at the time the
dams were killed. Direct infection of the bovine fetus did
not appear to be responsible for the abortions under these
conditions. A series of histopathological changes was found
in the cotyledons which could interfere with the development

of the fetus and result in fetal death and abortion.

IO.

REFERENCES

Bauer, D. C. and Morse, E. V.: Variation and Hemolysis
Production in Relation to Virulence of Leptospira
pomona. Proc. Soc. Expth. Biol. and Med. (In
press.)

 

Fennestad, K. L. and Borg-Petersen, C.: Studies on
Bovine Leptospirosis and Abortion. II. Experi-
mental Leptospirosis in Pregnant Heifers. Nord.

Vet. Med. 8, (1956): 815-833.

Fennestad, K. and Borg-Petersen, C.: Studies on Bovine
Leptospirosis and Abortion. III. Attempts to
Demonstrate Leptospria in Cotyledons from Aborting
Cattle. Nord. Vet. Med., 8, (1956):882-886.

Ferguson, L. C., Ramge, J. C., and Sanger, V. L.:
Experimental Bovine Leptospirosis. Am. J. Vet.
Res.. 18. (1957): 43-49.

Hadlow, W. J., and Stoenner, H. G.: Histopathological
Findings in Cows Naturally Infected with Leptospira
pomona. Am. J. Vet. Res., L6 (1955): 45-56.

 

Lindquist, K. J., Morse, E. V., and Lundberg, A. M.:
Experimental Leptospira pomona Infection in
Pregnant Ewes. (In print.)

 

Manual of Histologic and Special Staining Technics.
Armed Forces Institute of Pathology. (1957): 184.

Morse, E. V., Allen, V., Krohn, A. F., and Hall, H.:
Leptospirosis in Wisconsin. I. Epizootiology and
filinical Features. J.A.V.M.A., 127, (1955): 417-

21.

Morse, E. V., Allen, V., Pope, E. P., and Krohn, A.:
Leptospirosis in Wisconsin. II. Serological
Studies. J.A.V.M.A., 127, (1955): 422-426.

Morse, E. V., and McNutt, S. H.: Experimental Lepto-
spirosis. I. The Course of Leptospira pomona
Infection in Pregnant Heifers. J.A.V.M.A., 128,
(1956): 225-229. _“

 

l4

Morter, R. L., and Morse, E. V.: Experimental Lepto-
spirosis. II. The Role of Calves in the Trans-
mission of Leptospira pomona Among Cattle, Swine,
iheep, and Goats. J.A.V.M.A., L28, (1956): 408-

13.

 

Podgwaite, G. G., Tourtellotte, M. E., Jacobs, R. E.,
Helmbodt, C. F.,Easterbrooks, H. L., Williams,
L. F., Jungherr, E. L., and Plastridge, W. N.:
Isolation of Leptospira pomona from Three Aborted
Bgvine Fetuses. Ved. Med., g. No. 4, (1955): 164-
l 5.

 

\'

Ringen, L. M., Bracken, F. K., Kenzy, S. G., and
- Gillespie, R. W. H.: Studies on Bovine Lepto-
spirosis. I. Some Effects of Dihydrostreptomycin
and Terramycin on the Carrier Condition in Bovine
Leptospirosis. J.A.V.M.A., L26, (1955): 272-276.

Stoenner, H. G., Crews, F. W., Crouse, A. E., Taschner,
L. F., Johnson, C. E., and Wohlef, J.: The
Epizootiology of Bovine Leptospirosis in Washington.
J.A.V.M.A., 122, (1956): 251-259.

Te Punga, W. A., and Bishop, W. A.: Bovine Abortion
Caused by Infection with Leptospira pomona, N. Z.‘
Vet. J., 1, (1953): 143-149.

 

Walch-Sorgdager, B.: Leptospirosis. League of Nations
Bull., Health 0rg., 8, (1939): 1-2, 143-386.

APPENDIX

l6

.opzoOSstoamHm Mow
assesses Amy .asaaa> Hesse e
oQH>on asshoc maucoamam< mmm.x

.ESHHonpado
.conoampoo

3

A.poeeanns

as QH modmmfip Hmcaopws was Hapmm mo

soapmhsmomv .opmoohamxoamfim on .mEoAum

manna» o>apoocsoo mapufia Spas Esfiaocpamo

Hangouts Amv .mSHHH> Hench A<v .coomampoo
oQH>on asshos haucohmmm¢ mud x qflldwwm.

 

l7

.mSHHH> Hmuom mo soap
IwNfiHOSom> mcfizonm coapoomsa ampmm mmmm mm
.m woman: mo cocoahuoo oom x .: .mam

 

.GoapooMGH ampmw mac swamp can mammHoEon
can owmchaosos mo some “coauocsn
oasoaaononamnhopmz oom x . Hm

 

18

 

. .oSmme
o>apooccoo asshopmz ABOV .Esfiamzpfiqm
anchoume can Ga Hoaosc capocth Amy
.msaaa> assoc neaosoe> Aev .HHHH> amuse
on» mo coapmnaaosom> can osmmfip o>apomc
Icoo mSOanm commohoca wCHzonm commommsfi
noses mane newao-sucoze mma x .m .mam

 

l9

.Edfiaonuamo
Hangouts on» CH mufiscapsoo mo xoma
was Hoaosc capocxmm spas mumhmo madam
.e sedans no soooasooo oom x .m .wam

 

 

.A<v mpahno

madam one .Aeov couoampoo on» mo mEoppm

osmmap m>apomcc00 mo soaumaoMHHoam

Umxpme wcHBonm coapoomca momma mhmo
Kamuzumom .N pomaom mma N .w . Hm

1.6.! «5.-
.u WOQ‘flW
w .3-

’
.nd.’

       

 
 

20

 

.mdmmfiu m>fiuooqcoo adompam Hmfipfipm

lacuna anchopwz Aeov .ESHHoQuHQo Hmahouws

mo mommmnd Amv .HHHH> Hmuom mo mpcwssop

capoaOmz A<v .oHSQoopHnomw mammoomahuoo

asshoc on» mo mmoa on» wcaumhpmcoEoU

coapoomca moumm amp sumacnzpcoZm 6mm :0
coupons sodas m somaom mma x .m . Hm

 

Reprint from tho JOURNAL of the American Vetorirury Modicai Association, vol. 128, No. 5, March I, 1956, pp. 225-229.

Experimental Leptospirosis. I.

The Course of

Leptospira Pomona Infection in Pregnant Heifers

ERSKINE V. MORSE, D.V.M.. Ph.D.. and S. H. McNUTT. D.V.M.
Madison, Wisconsin

LEPTOSPIRA POMONA infection is a zoonosis
and as such warrants the attention of both
veterinarians and physicians. The disease
is currently widespread in both cattle and
swine in the United States. Leptospirosis is
estimated as the third most important
malady of cattle in the United States.5
Leptospiras are known to be present in
the urine, milk, blood, and other tissues of
infected cattle during various phases of the
disease. Recent experimental work deals
with the course of the infection in calves‘1
and the efficacy of antibiotic therapy in
cattle.“ The disease in cattle, as indicated
by surveys and field investigations, appears
to be most acute and severe in the pregnant
and lactating animal.8 The study to be re-
ported was directed toward elucidation of
the course of L. pomona. infection in the
pregnant, nonlactating, dairy heifer.

MATERIALS AND METHODS

Seven, apparently normal, grade Holstein-Frie-
sian heifers approximately 22 months of age served
as the experimental animals. The serums of the
animals at the outset were negative to the agglu-
tination-lysis test for L. Pomona or Leptospira ic-
terobaemorrbagiae at the 1:10 dilution] and were
negative, i.e., reacted no higher than the 1:25 dilu-
tion, when examined by the rapid plate and tube
agglutination tests for bovine brucellosis as recom-
mended by the U. S. Department of Agriculture.

 

From the Department of Veterinary Science, University
of Wisconsin. Madison. Dr. Morse is now at Michigan
State University, Department of Microbiology and Public
Health, Easr Lansing. Mich.

Published with the approval of the director of the Wis-
consin Agricultural Experiment Station. Supported in part
by a grant from the Research Committee of the Graduate
School from funds provided by the Wisconsin Alumni Re-
search Foundation.

The cooperation and assistance of W. D. Stovall. M.D.,
director, and Miss Virginia Allen, bacteriologist, State
Laboratory of Hygiene, are gratefully acknowledged.

Conception resulted from artificial insemination.
During the course of the investigation, each heifer
was maintained in an individual unit of an isola-
tion building.

Heifers 1 through 4 were exposed to L. pomona,
strain Wickard, during the sixth or seventh month
of pregnancy, while heifers 5 through 7 were in-
fected during the third to fourth month of gesta-
tion. The leptospiral strain was isolated by the
senior author in 1953 from the urine of a cow in
southern Wisconsin. The microorganism had been
maintained by serial passage in guinea pigs for a
number of months and was known to be virulent.
The exposure inoculum consisted of heparinized
blood (1 cc.. of heparin to 8 or 9 cc. of blood)
obtained from 3 to 5 guinea pigs during the pyretic
period of the infection. Each heifer was given 5
cc. of the blood by the subcutaneous route.

Heifers 1 through 4 were examined daily, f.e.,
rectal temperature, heartbeat, and respiratory rates
were determined for three days previous to ex-
posure and for 15 days following exposure. There-
after, examinations were made at five— to nine-day
intervals until the experiment was terminated.
Blood and urine samples were obtained for bac- ‘
teriological or serological examination on alternate
days during the 15-day postexposure period and
subsequently at the time of each physical examina-
tion.

Heifers 5 through 7 were examined daily for
two days previous to infection and for 14 days
following infection; subsequent examinations were
made on alternate days until the twenty-fourth day
and at weekly intervals thereafter. Blood and urine
were obtained for bacteriological or serological
examination daily on days three through 14 follow-
ing exposure and each day the heifers were ex-
amined thereafter. ‘

At the time of necropsy, liver, spleen, kidney,
lung, cotyledons and udder were obtained from
the heifers and fetuses. Blood, 'urine, fetal stomach
contents, and fluids from the uterine and fetal
cavities were inoculated into five tubes of modified
Chang's fluid medium." '

The tissues were homogenized in sterile 0.85 per

(225)

 

 

 

226

ERSKINE V. MORSE AND S. H. MCNUTT

J.A.V.M.A.
MARCH 1. 1936

 

cent sodium chloride solution, and approximately
2 to 3 ml. of the 10 per cent tissue emulsion was
inoculated by the intraperitoneal route into each of
2 to 4 guinea pigs weighing 200 to 250 Gm.; the
guinea pigs were exsanguinated three to four
weeks after inoculation and the serums examined’
for the presence of L. pomona agglutinins. Urine
and udder secretions of all heifers were inoculated
intraperitoneally while the blood of heifers 1
through 4 was injected by the subcutaneous route
into each of the 2 to 4 guinea pigs. The latter pro-
cedure was not followed for heifers 5 through 7
since it was found that L. pomona usually could
be isolated directly, using modified Chang's me-
dium." '

Agglutination-lysis tests,7 employing L. pomona
strain Johnson as antigen, were conducted on each
blood sample from the cattle and their fetuses.
Udder secretion as well as fetal fluids, i.e.,
amniotic, chorionic, or allantoic, when suitalble,
were examined by the agglutination-lysis test using
L. pomona as the antigen.

RESULTS

During the acute or early phase of
the infection, i.e., postexposure days one
through 12, leptospiremia, fever, anorexia,
depression, anemia, icterus, diarrhea, or
hemoglobinuria sometimes occurred.“-13
Heifers 3, 4, and 5 were mildly to moder-

ately pyretic during this period. The ob-
served highest rectal temperatures of these
3 animals were 103.6 F., 103.8 F., and
104.2 F., respectively. Malkmus and Opper-
mann6 indicated that the normal bovine
body temperature range is 100.4 to 103.1
F. The marked temperature rise of 105.0
F. to 106.0 F., commonly reported for bo-
vine leptospirosis, was not observed.

During the period of leptospiremia, an-
orexia and depression occurred in 4 of 7
animals. Evidences of icterus were not de-
tectable upon physical examination. How-
ever, the buccal mucous membranes of
heifer 5 appeared to be paler than normal.
None of the cattle were observed to void
bloody urine during the course of the ex—
periment. The urine of heifer 2 appeared
to be darker than normal on the seventh
day following L. pomona exposure.

During the febrile period, six to nine
days following exposure, either the respir-
atory or heartbeat rate was slightly ele-
vated6 for 6 of the animals. Heifer 5 ap-
peared to have an acute pneumonia, and a
copious, clear nasal discharge was evident.

N ecropsies were performed ten to 46 days
following exposure. Multiple small white

TABLE i—Summery of Symptometological. Pathological, end Bacteriological Observations of 7
Pregnant Heifers with Experimental Leptospira Pomona Infection

 

Heifer

Clinical features, normal
unless indicated’

 

Necropsy
findings

Isolation of
L. pomona from:

 

1

Temperature 103 F. and heartbeat
88 on day 6; anoretic, urine
slightly turbid.

Highest temperature 102.4 F.,
heartbeat 100 on day 7; urine
darker than normal on day 7.

Temperature 102.6 F. on day 8,
103.6 F. on day 9; heartbeat 84
and 80, respectively; anorctic
day 9; urine dilute on day 8;
aborted at 29 days.
Temperature 103.8 F.; heartbeat
96; anoretic on day 7.

Temperature 103 F., respirations
38 on day 7; temperature 104.2 F.,
rcspirations 30; anoreric. clear
mucous nasal discharge. loose fetid
feces on day 8; temperature 103.1
F., respirations 45. decreased nasal
discharge, pale buccal mucous
membranes. and some appetite on
day 9, heartbeat—slightly elevated
Temperature 103 F., respirations
38 on day 7, heartbeat slightly
elevated

Highest temperature. 102 F. on

day 6, heartbeat slightly elevated

Performed on day 12-——
focal, interstitial nephritis.

Performed on day 25—focal,
interstitial nephritis.

Performed on day 30—-
diffuse peritonitis. septic metriris,
retained fetal membrane; focal.
interstitial nephritis.

Performed on day 47—focal,
interstitial nephritis.

Performed on day lo—large
areas of subcapsular cortical
renal hemorrhage; abnormal
amount synovial fluid in major
ioinrs; liver enlarged.

Performed on day 28——one small
area of focal, interstitial nephritis.

Performed on day 46—numerous
areas of focal. interstitial
nephritis in borh cortex and
medulla.

Blood on days 6 and 8+;
urine on day 123.

Blood on day 8+; kidney”.

Urine on days 22 and 268;
kidneysx.

Blood on days 6+ and 8+#;
urine on days 13 and 25:
kidneys#.

Blood on days 4 through 10+:
kidneys, lungs, udder. liver-v
spleen and cotyledon“.

Urine on day 19#.

Blood on day 5+; urine on
days 13. 19, 21 and 29,3.

 

 

*All days are following exposure. + Chang’s medium isolation. +3 Leptospiras observed but nor in pure
culture. 1,! inoculated guinea pigs' serums positive on agglutination-lysis resr.

 

MMMA.
acrt 1, 1956

LEPTOSPIROSIS IN PREGNANT HEIFERS

227

 

foci of interstitial nephritis11 were present
in the cortex and extended several centi-
meters into the medullary portions of the
kidneys of all the animals except heifer 6
which had only one such area in one kidney.
The other organs of heifers 1, 2, 4, 6, and 7
as well as those of their fetuses appeared
to be normal.

Heifer 3 aborted 29 days following ex-
posure. The fetus, it was concluded, had
been dead 12 to 24 hours at the time of
abortion. The fetal membrane was edema-
tous and friable; manual removal was not
possible. The uterus contained a flocculent,
mucoid, nonodorous fluid. The fetal organs
and muscles of their abdominal and thoracic
walls were edematous and moderate autol-
ysis was evident. Approximately 200 cc. of
bloody transudate was present in both the
thoracic and abdominal cavities. The peri-
cardial sac contained approximately 75 cc.
of a similar fluid.

The day following abortion, the body
temperature of heifer 3 was 101.8 F. and
the heart rate was 140. The animal was
killed and necropsy revealed an extensive
edema of the posterior portion of the pelvic
cavity, uterus, and bladder and a mild
peritonitis and concurrent septic metritis.
The omentum contained small areas of
petechiation, and hemorrhagic areas, 0.5
to 1 cm. in diameter, were present on the
cortical surfaces of the kidneys. At the
time of abortion, the serum agglutination-
lysis titer was 1 :1 million, and the titer of
the milk whey was the same.

Heifer 5 was killed ten days following
exposure. With the exception of the kid-
neys, all organs appeared to be normal.
The kidneys contained numerous areas of
subcapsular and cortical hemorrhage. The
animal had been reluctant to move during
the three days prior to necropsy, and it
was concluded that an acute arthritis or

synovitis was present. Approximately 20
cc. of synovial fluid was contained in each
of the scapulohumeral and hip joints. Lepto-
spiras were not isolated from the synovia.

Leptospira pomona was obtained from
the blood of 5 of the animals by direct
culture in Chang’s fluid medium. Lepto-
spiras were present in the renal tissue of
4 cows as proved by the guinea pig-inocu-
lation technique. Leptospiruria or renal
residence of leptospiras or both were de-
monstrable by guinea pig inoculation pro-
cedures for all 7 heifers.

The presence of leptospiras in lungs, ud-
der, liver, spleen, and cotyledons of heifer 5
was indicated by the inoculation of guinea
pigs and subsequent development of agglu-
tination-lysis titers.

Leptospiras were not isolated from any
fetal materials obtained. Except as speci-
fied, leptospiral isolations were not made
from the genital tract, liver, spleen, udder,
or lungs of the heifers.

The clinical features, gross pathological
and bacteriological observations made of
these animals are recorded (table 1).

Leptospira pomona agglutinin-lysin titers
of 1 :10 appeared in the serums of the cattle
seven to nine days following exposure. By
the thirteenth postexposure day the titers
were 1:100 to 1 :10,000. The highest agglu-
tinin titer observed was 1:1 million; it
occurred 30 days following exposure. A
milk whey titer of the same magnitude as
the serum titer was observed. Agglutinin-
lysins were not present in the urine, fetal
fluids, or extrafetal fluids from any of the
cattle examined. The results are summarized
(table 2).

DISCUSSION

The major problem in the control of
leptospirosis appears to be the detection of
the animal which sheds leptospiras in the

TABLE 2—Serological Results Obtained with the 7 Heifers Infected with Leptospira Pomona

 

\

Positive serum dilution.

 

 

 

 

Heifer Day“ 2-7 9 11 13 20 25 30 47
1 o 10'1 10" ..
2 0 10‘1 10“ 10" 10" 10" ..
3 o to-1 10" 10-4 104 10-6 'o-H-
4 0 10'1 10'2 10" 10“ 10" 10'” 10"
Day” 3-6 7-9 11 13 15 17 22 24 26 28-34 35-46
5 0 10'1 10-1
6 0 10" 10'1 10'2 10“ 10" 10" 10'3 104 10:3
7 0 10-1 10-2 10—3 104 10-4 10.: 104 10-8 10.: 10-: to 104

 

‘Agglutination-lysis teSt using L. pomona. strain Johnson, as antigen. Titer =

highest dilution showing

evidence of agglutination or lysis or both. I""‘Day following exposure to L. pomona, strain Wickard. tMilk

whey agglutinins present at 10" dilution level.

 

 

 

 

228

urine. Therapeutic efforts are generally
directed toward alleviation of the acute and
severe manifestations of the disease. The
obviously ill animal is naturally considered
as a prime source of contagium, but the
occult case, as a urinary shedder of lepto-
spiras, is equally as dangerous. The control
of this disease in cattle must, therefore, be
based not upon individual animal consider-
ations but upon detection of the disease in
all carrier animals within the herd. The
asymptomatic carrier may shed L. pomona
in the urine for at least 45 days and prob-
ably longer. Slaughtering of acutely ill ani-
mals will not eradicate the infection from
the herd. Probably the majority of cattle
which have agglutinin—lysin titers have
been carriers and shedders at some period.
In addition, a control program for lepto-
spirosis must be based upon the broadest
epizootiological concepts because of the Wide
host range of L. pomona, e.g., swine,‘
horses,15 sheep,1 goats,9 and mans“,16

Leptospira pomona serum agglutinin-lysin
titers may appear as early as seven to
nine days following exposure. By the thir-
teenth day, the titer may be 1:100 to 1:
10,000. After 25 days, a peak of 1:10,000
to 1:1 million may be anticipated.

The Leptospira antibody titers ascend
rapidly and remain at 1:1,000 to 1:10,000
for 29 to 52 months.8 Whey titers in colos-
tral milk are comparable to those obtained
for the serum of an individual animal.

There is little doubt that L. pomona is
responsible for bovine abortions. The agent
was isolated by the authors from the coty-
ledons of 1 experimental heifer. Leptospiras
were also found, by Podgwaite et al.,1° to
be present in 3 aborted bovine fetuses. The
exact mechanism of abortion is unknown.
It appears that interference with the pla-
cental and fetal circulation, as evidenced by
the extensive edema, may have been the
cause or at least a contributing factor. The
fetus examined in this investigation had
been dead 12 to 24 hours prior to the abor-
tion, which occurred 29 days following ex-
posure. Evidence obtained from investiga-
tions’»8 of a number of natural occurrences
of bovine leptospirosis indicates that abor-
tions take place three to four Weeks follow-
ing exposure to L. pomona. The abortion is
not a manifestation of the acute phase of
the disease. The serum and milk whey
agglutinin levels at the time of abortion
were 121,000 to 1:1 million".8

Gross renal lesions are usually observed

ERSKINE V. MORSE AND S. H. MCNUTT

J.A.V.M.A.
MARCH 1, 1956

even in the milder and asymptomatic cases
of bovine leptospirosis. Considerable scru-
tiny at necropsy may be required to locate
the characteristic yellow or white foci on
the renal cortex11 which descend 2 to 3 cm.
into the medullary portions of the kidneys.
Attempts to discover these lesions are well
warranted, especially in the recently in-
fected animal. _

Icterus, anemia, and hemoglobinuria or
hematuria are not constant findings in
experimental or naturally occurring lepto-
spirosis in pregnant heifers. Respiratory
distress in the severely ill bovine animal is
a striking feature of the malady. Gross
pulmonary lesions are apparently absent.

Stiffness and reluctance to move have
been reported in naturally infected cattle.”
One of the experimental heifers experienced
a severe, acute synovitis. This clinical fea-
ture of leptospirosis, though not constant,
may be observed during the early phases
of the disease.

SUMMARY

Seven pregnant heifers were infected
with Leptospira pomona by subcutaneous
inoculation. One became severely ill, 1
aborted, and 5 remained essentailly asymp-
tomatic. Leptospiremia endured for one to
five days in 5 of the animals while all 7
developed at least a transitory leptospiruria
as evidenced by isolation of the agent from
urine or kidneys. Leptospira pomona was
recovered from the udder, cotyledon, liver,
spleen, lungs, and kidneys of 1 acutely
and severely ill animal killed ten days after
the onset of infection. Leptospiras were
not isolated from the tissues of any of the
fetuses from these heifers. Agglutinin-
lysin serum antibodies were demonstrable
within ten to 14 days following exposure
to L. pomona. The experimental evidence
indicates that infected cattle, even in the
absence of visible manifestations of the
disease, constitute a potent reservoir of
animal infection and human exposure.

References

1Beamer, P. D., Hardenbrook, E., Jr., and Mor-
rill C. C.: Studies on Leptospirosis in Domestic
Animals. 1. Leptospirosis in Sheep. Vet Med., 48,
(1953): 365-366.

’Chang, S. L.: Studies on Leptospira Icterobae—
morrbagiae. J. Infect. Dis., 81, (1947): 28-34.

aClayton, G. E. B., Derrick, E. H., and Clento,
R. W.: The Presence of Leptospirosis of a Mild
Type (Seven Day Fever) in Queensland. Med. J.
Australia, I, (1937): 647-654.

‘Gochenour, W. 8., Jr., Johnston, R. V., Yager,

 

J.A.V.M.A. anrosrmosns IN PREGNANT HEII-‘ERS

MARCH 1, 1956

229

 

R. H., and Gochenour, W. 8.: Porcine Leptospiro-
sis. Am. J. Vet. Res., 13, (1952): 158-160.

‘Agricultural Research Service, U.S.D.A: Losses
in Agriculture. A Preliminary Appraisal for Re-
view, (June, 1954): 129-137.

‘ kmus, B., and Oppermann, T.: Clinical
Diagnostics of the Internal Diseases of Domestic
Animals. 11th ed. Alexander Eger, Chicago, 1941.

'Morse, E. V., Allen, V., Krohn, A. F., and Hall,
R.: Leptospirosis in Wisconsin. I. Epizootiology
and Clinical Features. J.A.V.M.A., 127, (1955):
417-421.

'Morse, E. V., Allen, V., Pope, E. P., and Krohn
A. 17.: Leptospirosis in Wisconsin. 11. Serological
Studies. J.A.V.M.A., 127, (1955): 422-426.

’Morter, R. L., and Morse, E. V.: Unpublished
data, 1955.

”Podgwaite, G. D., Tourtellotte, M. E., Jacobs,
R. E., Hemboldtz, C. F., Esterbrooks, H. L., Wil-
liams, L. F., Jungherr, E. L., and Plastridge, W.
N.: Isolation of Leptospira Pomona from Three
Aborted Bovine Fetuses. Vet. Med., 40, (1955):
164-165. '

”Reinhard, K. R.: A Clinical Pathological Study
of Experimental Leptospirosis in Calves. Am. J.
Vet. Res., 12, (1951): 282-291.

”Reinhard, K. R.: Bovine Leptospirosis. Sympos-
ium on the Leptospiroses. My Med. Ser. Grad.
School, Med. Sci. Pub. No. 1 (1952): 126-139.

”Reinhard, K. R.: Newer Knowledge of Leptos-
pirosis in the United States. Exptl. Parasitol., 2,
(1953): 87-115.

“Ringen, L. M., Bracken, F. K., Kenzy, S. G.,
and Gillespie, R. W. H.: Studies on Bovine Lep-
tos irosis. I. Some Efiects of Dihydrostreptomycin
andP Terramycin on the Carrier Condition in
Bovine Leptospirosis. J.A.V.M.A., 126, (1955):
272-276.

”Roberts, S. J., York, C. J., and Robinson, J.
W.: An Outbreak of Leptospirosis in Horses on a
Small Farm. J.A.V.M.A., 121, (1952): 237-242.

1‘Shaefler, M.: Leptospiral Meningitis. An In-
vestigation of a Waterborne Epidemic Due to
L. pomona. J. Clin. Invest, 30, (1951): 670-671.

 

 

 

 

Reprint from the JOURNAL of the Amerlcan Veterlntl'y Medical Association, vol. 128, No. 8, Apr" 15, 1956, pp. 408-413.

Experimental Leptospirosis.

II. The Role of Calves in the

Transmission of Leptospira Pomona Among Cattle,
Swine, Sheep, and Goats

RAYMOND L MORTER. 8.5.. and ERSKINE V. MORSE, D.V.M.. Ph.D.

Madison,

LEPTOSPIROSIS (Leptospira pomona infec-
tion) has become recognized during the
past ten years as a threat to the livestock
population of this country. The epizooti-
ology has been defined but requires further
elucidation. Cattle may be renal carriers of
leptospiras for periods of three months.10
Leptospira pomona. infections can be trans-
mitted experimentally from infected calves
to susceptible calves.1 Burnstein and
Baker3 showed that infection may be
transmitted from diseased pigs to other
pigs and calves but they were unable to
show the reverse, that leptospirosis can be
transmitted to pigs by contact with L.
pomona-infected calves. The role of the in-
fected calf in transmission of leptospirosis
to other cattle, pigs, sheep, and goats has
not been thoroughly delineated. This work
was undertaken to further the epizootio-
logical knowledge and to make possible a
study of the course of L. pomona infection
in several domestic animal species.

MATERIALS AND METHODS

The strain of L. pomona selected for exposure
was originally isolated from bovine urine obtained
during active infection in a Wisconsin dairy herd.1
It had been maintained since isolation by continual
passage through guinea pigs and had been desig-
nated as strain Wickard.’

Six calves, 4 pregnant heifers, 4 weanling pigs,
4 sheep, and 2 goats which were negative to the
serum agglutination-lysis test' at the initiation of
the experiment served as experimental animals.
The calves, which were from 1 week to 4 months
of age, were infected with L. pomona by subcu-
taneous injection. They then served as carriers to

 

From the Department of Veterinary Science, University
of Wisconsin. Veterinary science publication N.S.194. pub-
lished with the approval of the director of the Wisconsin
Agricultural Experiment Station.

This study was supported in part by a grant from the
Research Committee of the Graduate School from funds
provided by the Wisconsin Alumni Research Foundation.

The cooperation and assistance of W. D. Stovall, M.D.,
director, and Miss Virginia Allen, bacteriologist, State
Laboratory of Hygiene, and the counsel of Dr. C. A.
Brandly, Department of Veterinary Science, are acknowl-
edged by the authors.

The present address of Mr. Morter is Division of Vet-
erinary Medicine, Iowa State College, Ames, and of Dr.
Morse, Department of Microbiology and Public Health,
Michigan State University, East Lansing.

Wisconsin

which the other animals were exposed. The preg-
nant, grade Holstein-Friesian heifers were in the
fifth to seventh month of gestation at the time of
contact exposure. The pigs, which were approx-
imately 8 weeks old, were held in isolation for
five days before being placed in isolation units with
the infected calves. The sheep, which were 3 and
4 years old, were procured from a flock which had
no clinical history to indicate the presence of lep-
tospirosis. Two, 4-month-old goats were obtained;
they had been confined in a small pen since birth.

The animals were kept in four pens of a mod-
ern isolation unit in which principles of strict iso-
lation were exercised and in a cattle shed where
contact with other livestock was impossible and
where human and vehicular traffic to the premises
was restricted to authorized personnel. This re-
duced the potential of accidental infection to a
minimum. The isolation pens provided 70 sq. ft.
of floor space and were cleaned daily with warm
(180.0 F.) water under pressure. The shed unit
was 16 ft. by 20 ft. with approximately one half
of the floor area paved with concrete and the re-
mainder being a dirt floor. No drainage was pro-
vided. This unit was not cleaned during the course
of the experiment.

The animals in the isolation pens were pro-
vided with a mixed ground grain ration with
dried beet pulp incorporated as bulk for the ru-
minants. No litter was provided. The grain ration
was fed in metal baskets or metal troughs, and
the water was provided in suitable garbage cans.
The uneaten hay provided litter for the animals in
the shed unit.

The animals were initially dispersed (table 1)
in the various units as follows: Each of three iso-
lation pens was stocked with 1 infected calf, 1
heifer, and 1 pig; the fourth isolation unit con-
tained l infected calf and 1 heifer; and the shed
unit contained 2 infected calves, 1 pig, 4 sheep,
and 2 goats. At the termination of the experiments
involving 14 of the animals, the 6 remaining ani-
mals were placed in two pens of the isolation unit.
The first pen contained 2 infected calves and 2
goats, while the second contained 1 infected calf
and 1 ewe. The two groups were maintained for
an additional 14 days and the experiment was then
concluded.

The 6 calves were inoculated subcutaneously
with 5.0 cc. of heparinized blood obtained from in-
fected guinea pigs. This blood was obtained when
the maximal pyretic response (105.0 to 106.0 F.)
was manifested.” The normal animals were placed
in contact with the infected calves seven days fol-
lowing inoculation. Rectal temperatures were taken

(408)

 

 

 

 

 

 

 

 

 

 

 

J-A-V-M-A- EXPERIMENTAL LEPTOSPIROSIS 409
APRIL 15, 1956
TABLE l—Distribution of Experimental Animals for Transmission of Leptospirosis
Initial distribfititTn'i' Secondsryfdistr—ihu-t—iont
Isolation unit Ali-pen No.) —Tsolation unit (pen N0.)
Animals 1 __2.__. 3 4 Shed Animals 1 2
Calves“ l 1 1 1 2 Calves" 2 l
Heifers l l l 1 .. ............
Swine 1 I 1 .. 1
Sheep .. .. .. .. 4 Sheep .. 1
Goats . .. 2 Goats 2
* All calves experimentally infected and used as sources of infection for other animals.
'l' Exposure for 30 hours to 34 days.
:1: Exposure for an additional 14 days.
daily and the animals were observed for clinical ford calves but not by the 3 infected

signs of leptospirosis. Whenever a temperature
rise, anorexia, polypnea, depression, or lethargy
were manifested by any of the animals, blood
samples were secured on that day and on two
successive days. A minimum of five tubes of modi-
fied Chang's fluid medium" ' were inoculated with
approximately 0.05 cc. of the blood. Cultures were
placed in an incubator at 28 C. and held for a maxi-
mum of 30 days. All tubes were examined at ap-
proximately weekly intervals by dark field micro-
scopy for evidence of leptospiras. Tubes of the
medium which proved to be contaminated were
discarded. Serum was collected from the animals
at three- to fiveday intervals following the observa-
tion of symptoms and at weekly intervals thereafter
until destroyed. The agglutination-[ysis test was
employed throughout for serological examinations.”

Approximately 3.0 cc. of urine, milk, and 10.0
per cent tissue emulsion of kidney in 0.85 per cent
sodium chloride solution were inoculated intraper-
itoneally into each of 3 to S guinea pigs and each
of 5 hamsters. Lactation resulted from 1 of the
heifers from the nursing efforts of the infected
calf. Chorionic or amniotic fluids, stomach con-
tents, and saline emulsions of fetal bovine kidney,
liver, and spleen were also inoculated into guinea
pigs. Guinea pigs and hamsters were held for 18
to 30 days prior to exsanguination. Their serums
were examined by the agglutination-lysis test.’
Titers of 1:100 or higher were considered to be
indicative of infection and proof of the presence
of L. pomona in the original inoculum.

Animals were destroyed at various intervals dur-
ing the course of the experiment to obtain gross
pathological, serological, and bacteriological data.

EXPERIMENTAL RESULTS

1) Experimentally Infected Carrier
Calves—Rectal temperatures of the experi-
mentally infected calves, taken daily,
showed a febrile reaction with maximal
temperatures of 103.8 to 106.5 F. at three
to seven days postinoculation. Other symp-
toms observed for periods of two to three
days were lethargy, constipation, mild ca-
tarrhal to purulent rhinitis, excessive lac-
rimation, polypnea, a slight nonproductive
cough, and excessive micturition. Hemo-
globinuria was manifested by all 3 Here-

Holstein-Friesian calves. Hereford calf 67
died of acute leptospirosis eight days after
inoculation; gross lesions were not found
on necropsy. Hereford calf 69 had extreme
hemoglobinuria on postinoculation days 12
to 15 and died on the twenty-fifth day; the
course of the infection was complicated by
a persistent diarrhea. Leptospiras were
isolated from blood which was collected
during pyrexia. Leptospiremia was proved
for all 6 calves and 4 of them were found,
by means of the guinea pig inoculation
technique, to be shedding leptospiras in
their urine. Calf 67, which died during
acute leptospirosis, and calf 68 were never
proved to be renal shedders.

Serum agglutination-lysis titers were
first detected on six to 15 days after inocu-
lation. When the 4 surviving calves Were
destroyed for necropsy, at 44 days post-
inoculation, their titers were l:10,000,000
and leptospiras were not isolated from
their kidneys or urine. Their kidneys con-
tained numerous gray-white foci over the
entire cortical surface. These foci extended
several centimeters into the cortex and, in
some instances, into the medulla. There
was considerable resistance to the knife
blade, indicating extensive fibrosis, when
longitudinal slices were made of the kid-
neys (fig. 1). The livers of 2 calves were
firm and mottled. Accumulation of peri-
toneal and"pericardial fluids was observed
in some of the calves. Calf 71 was ex-
tremely emaciated and unthrifty.

The clinical features and bacteriological
findings for the 6 calves are shown in
table 2.

2) Animals Infected by Contact Trans-
mission—Heifer 46 had a temperature of
104.0 F. six days after 30 hours of contact
with calf 67 before the calf died. Pyrexia
was followed by slight constipation. Other
clinical signs were not observed. The heifer
was slaughtered 33 days postexposure. On
necropsy, the chief lesions were pronounced

 

 

 

410

gray-white foci of both kidneys; the other
organs appeared to be normal. A living
41/2-month-old fetus was present in the
uterus. The chorionic, allantoic, and amni-
otic fluids were straw colored, and the cap-
sules of both kidneys were extremely ede-
matous. Attempts to isolate leptospiras
from the maternal blood, urine, or kidneys,
as well as from the fetal organs, were not
successful. The terminal serum agglutina-
tion-lysis titer of heifer 46 was 1:1,000,000.

Heifer 73 was in contact with infected
calf 70 for 19 days. A primary temperature
rise to 104.0 F. occurred seven days follow-
ing exposure and a serum titer of 1:10
appeared on the fifteenth day postexposure
and, on the nineteenth day, she had a tem-
perature of 104.4 F. and leptospiras were
isolated from her blood and milk. On the
twenty-fifth day following exposure, when
she was destroyed, her temperature was
105.0 F., her respiratory rate 103 per min-
ute, and she had a cloudy vaginal dis-
charge. Agglutinins were demonstrable in
a serum dilution of 1:10,000,000. Her 5-
month—old fetus was alive, had strong heart
action, but made no respiratory attempts.
Amniotic fluids and fetal stomach contents
were viscid, turbid, and yellow. The fetal
viscera and mesenteries were slightly ede-
matous. The cortices of the maternal kid-
neys contained discrete, grayish foci. All
guinea pigs which were inoculated with

RAYMOND L. MORTER AND ERSKINE V. MORSE ’

J.A.V.M.A.
APRIL 15. 1956

fetal kidney and spleen material died 24 to
72 hours later. Leptospiras were not iso-
lated from any of the fetal organs or fluids
but were isolated from the maternal kid-
neys.

Heifer 76 showed clinical signs of lepto-
spirosis during the twenty-fourth day of
exposure to infected calf 72. Her tempera-
ture was 103.2 F. and her titer was 1:100,-
000; a titer of 1:10 had first appeared on
the fifteenth day postexposure. At 27 days
postexposure, when her temperature was
103.4 F. and her respirations 104 per min-
ute, she was destroyed for necropsy. Her
kidneys were friable and mottled with
grayish white foci which extended deep
into the cortex and medulla and there were
numerous petechial hemorrhages in the
renal cortex. The liver was firm in consist—
ency and distinctly mottled. The gallblad-
der was slightly edematous. The fetus was
alive but, on necropsy, showed edema of
the kidney capsule, pericardium, and mes-
entery. Leptospiras were not demonstrable
in the maternal kidneys or liver nor in the
fetal kidneys, spleen, amniotic fluids, or
stomach contents.

Heifer 84, which had been in contact
with infected calf 68 which was not proved
to be shedding leptospiras in the urine, de-
veloped no signs of leptospirosis except a
temperature of 103.6 F. on the third day
postexposure. Significant serum agglutina-

 

Fig. l—Kidneys and renal lymph nodes of calf 7|. Extensive cortical and medullary lesions can he
observed. indicating chronic type of Leptospira pomona infection.

 

 

J.A.V.M.A.
APRIL 15. 1956

EXPERIMENTAL LEPTOSPIROSIS

411

TABLE 2—Evidence of Leptospirosis Observed Among Experimentally Infected Caves“

 

 

 

 

POStexposurc Clinical signs Bacteijglogicaires—ultst

Animal Breed day Temp. (F.) Hemoglobinutia Blood: Urine: Kidney
67 Hereford 6 10 .8 6-7 — ‘-
68 Holstein-Friesian 3 103.2 : :iS) ) r‘ '—
69 . . 6 105.0 + +(4-7) +(10-20) —
70 Friesian 5 106.5 -- + (6,7) + (16-26) — ‘
71 Friesian 7 105.8 -- + (5-7) +(10-28) “
72 5 104.8 + +(5-10) +(12-28) -

 

" Subcutaneous exposure to Leptospira pomona, strain Wickard. These calves were used as infecred animals

for transmission experiments.

TBIQOd cultureddirectly in Chang's medium. Urine or kidney homogenates inoculated via intraperitoneal
1’0““! In guinea P189; develoPment of serum-agglutininlysins indicated presence of Leptospira organisms In

inoculum.

1: Numbers indicate the days or period postexposure on which leptospiras were demonstrable.

tion—lysis titers failed to develop. She was
destroyed for necropsy ‘at 33 days post-
exposure. Her kidneys contained a few
scattered, grayish foci which extended only
a few millimeters into the cortex. Serums
of guinea pigs and hamsters, which were
inoculated with maternal kidney or fetal
kidney, liver, spleen, amniotic fluid, or
stomach contents, failed to give positive
agglutination-lysis titers.

All 4 pigs developed leptospirosis follow-
ing contact with the experimentally in-
fected calves. Thermal responses were evi-
denced from the eighth to the eighteenth
days postexposure, with peak temperature
rises of 104.6 to 106.5 F. One pig appeared
lethargic and developed a diarrhea concur-
rent with the pyrexia. The other 3 pigs
remained asymptomatic except for the fe-
brile responses. Leptospiras were isolated
from the blood of 2 pigs. Serum agglutinin
titers of 1:108 to 1:108 were present seven
to twelve days following pyrexia in 3 of the
pigs. The fourth, pig 99 which was in con-
tact with calf 68, had a questionable ag-
glutination-lysis serum reaction on the
eighth day postpyrexia when it was de-
stroyed. By guinea pig and hamster inocu-
lations, leptospiras were proved to be in
the kidneys or urine of all 4 pigs. Pro-
nounced kidney lesions were not observed
in any of the 4 pigs.

None of the 4 sheep manifested clinical
signs of leptospirosis, none developed
agglutination-lysis serum titers, and lepto-
spiras Were not isolated from their organs,
blood, or urine.

The 2 goats were in contact with calves
69 or 71 for 26 days without developing
clinical leptospirosis and their serums did
not contain leptospiral agglutinins. Both
goats were then placed with infected calves
70 and 72. Nine days later, goat 87 had
a temperature of 104.0 F., was lethargic,
anorexic, evidenced general malaise, and

leptospiras were isolated from the blood
in Chang’s medium.“-7 Goat 87 was slaugh-
tered five days after the pyrexial response.
The terminal agglutination-lysis test titer
was 1:100,000. Three of 5 guinea pigs
which were inoculated with urine and 3
of 5 guinea pigs which received kidney
emulsion from goat 87 developed significant
temperatures of over 104.0 F. and lepto-
spiras were isolated from their blood. Coat
88 remained asymptomatic as well as sero-
logically and bacteriologically negative.
Symptomatological and bacteriological find-
ings for the animals infected by contact are
summarized in table 3.

Agglutination-lysis tests of serums of
all species were performed using L.
pomona, strain Johnson, and Leptospira
icterohemorrhagiae AB, as antigens. The
results indicated a cross serological reac-
tivity. Some of the serums which had a
titer of 1:100,000,000 for L. ponwna evi-
denced an agglutination-lysis reaction in
dilutions of 1 :100,000 for L. icterohaemor—
rhagiae, AB. Most serums with a 1:1,000
L. pomona reaction produced agglutination
or lysis of L. icterohaemorrhagiae AB, an-
tigen at a dilution of at least 1:100. The
serological data are summarized in table 4.

DISCUSSION

A possible inference that breed or type
may affect susceptibility to leptospirosis is
indicated by the death of 2 Hereford calves
as well as by the acute hemoglobinuria
which was observed only in the Herefords.
Only mild transitory signs of leptospirosis
were observed in the other calves. Marsh6
reported leptospirosis in Hereford cattle
that was characterized by a severe hemo-
globinuria and a 90 per cent mortality
among the calves. In Wisconsin, leptospiral
infections have involved calves in only
three of 41 dairy herds studied, with hemo-
globinuria being uncommon or inconstant.’

 

 

 

412

RAYMOND L. MORTER AND ERSKINE V. MORSE

J.A.V.M.A.
APRIL 15. 1956

 

However, a rapid, acute course, a rather
constant finding of hemoglobinuria, and
some deaths have been reported for beef
cattle and dairy calves.5

Small numbers of leptospiras may be
shed in the urine of calves.3 Ringen et at.“
suggested that approximately 750 lepto-
spiras (L. pomona) per inoculum are re-
quired to produce 100 per cent infection
in guinea pigs. Sixty-six guinea pigs were
inoculated, in the present experiment, with
2.0 cc. of urine from known infected calves.
The serums of 18 of these guinea pigs
were negative for leptospiral agglutinins
and 13 of the 18 negative serums were
from guinea pigs which received the urine
of calf 68, which apparently contained in-
sufficient organisms to be infective for
guinea pigs. Infection was not transmitted
from calf 68 to the heifer but it was to
a pig in the same pen, which suggests the
possibility of a greater degree of suscepti-
bility of swine than of cattle. Forty-four
days after subcutaneous inoculation, lepto-
spiras were not detected in the urine or
kidneys of the 4 surviving calves by guinea
pig or hamster inoculation. The renal-car-
rier and shedder state in these calves was
of short duration but of a serious nature.

Evidence of transmission, by pen con-
tact, from known infected calves to 3, of
4 pregnant heifers, all 4 pigs, and 1 of 2
goats indicates that calves may be im-

portant sources of infection. This seems to
be the first authenticated occurrence of
leptospirosis (L. pomona) in a caprine host
in North America but Leptospira grippoty—
phosa. infections in goats have been re-
ported from Israel.12

Leptospirosis occurred in 1 heifer follow-
ing 30 hours Of contact with an infected
calf. The ease of transmission from calves
to other susceptible hosts is evidenced by
the fact that these animals, with the ex-
ception of the group kept in the shed, were
maintained in sanitary surroundings much
better than those which would be main-
tained under ordinary farm conditions.

The fact that the sheep did not become
infected, either in the shed or the isolation
unit, may have been due to their age (3 to
4 years) or to a species characteristic of
low susceptibility. Leptospira 2201er in-
fections in sheep have been described.'~’
Further studies on the susceptibility of
sheep to L. pomona and on the transmis-
sion among sheep and from sheep to other
species are indicated.

Abortions did not occur and leptospiras
were not isolated from the bovine fetuses,
all of which were alive when the heifers
were subjected to necropsy. Marked edema
of the fetal kidney capsule was the most
prominent gross pathological lesion. Serum
agglutination-lysis titers were not evident
in the fetuses of infected cattle.

TABLE 3—Bacteriological, Clinical, and Necropsy Observations of Animals Which Evidenced Lepto-
spirosis" Following Contact Transmission

 

Clinical

Bacteriologicalzl:

Necropsy

 

Blood Urine Kidneys Dayt

Observations

 

 

 

 

Animal Daytllemp. (F.) Respirations Other
Heifer 46 6 104.0 60/min. Consripation. —
Heifer 73 7 104.0 ............ ....... _ +(19)
25 105.0 103/min. Lochia. _
Heifer 76 24 103.2 ...... . ................. ~—
27 103.4 104/min. ............
Fig 77 s 105.4 ........................ —
18 104.8 ........................
Pig 78 14 106.5 ........................ + (16)
Pig 99 13 104.6 ... ......... —
Pig 600 15 105.4 ............ Diarrhea. +(13)
depression.
Goat 87 9 104.0 ............ Depression, +(10)

anorexia.

Dangray-white, renal foci.
Fetus: alive; fluids in body cavities,
edematous kidney capsules.

- - 33

Dam: same as for heifer 46. .
Fetus: alive; edema of. mesenteries
and viscera; yellow, VISCId allanto-

amniOtic fluids.

+(25) + 25

Dam: petechiae, gray-white foci on
kidneys; liver firm and mottled.
Fetus: alive; edema of renal capsule

and mesenteries.
Kidneys: ecchymotic hemorrhages,
few gray-white foci.

+0” + 21

17 Kidneys: few scattered gray foci.

+
+(21) + 21 Kidneys: few hemorrhagic foci.
+ 21 Kidneys: cortical petechiae and dis-
crete gray-white foci.

+(14) + 14 No significant lesions.

 

‘ All experimental animals with demonsrrable serum'agglutinin titers of 1:100.000 (or higher) except pig 99.

1- Days are postexposure. Goat 87 data represented for second exposure period.

3 Blood cultured directly in Chang’s medium. Urine and kidneys proved by guinea pig or hamster inoculation tech
nique. Numerals in parenthesis indicate days postexposure when isolations were made.

 

l.A.V.M.A.
APRIL 15. 1956

EXPERIMENTAL LEPTOSPIROSIS

413

 

TABLE 4—Agglutination-Lysis Titers of Serums of Animals Infected by Contact Transmission“

 

Dayipostexposure

 

 

 

 

Animal 0 to 5 6 to 10 11 to 15 16 to 20 21 to 25 26 to 30 31 to 40
Heifer 46 - - - 1:10 1:103 1.10" 1210‘
Heifer 73 —- - 1:10 1:10 1:107 ........
Heifer 76 -‘ — 1:10 1:10 1:105 1:105 ........
Heifer 84 - — — -— 1:10 1:10 ........
Pig 77 - - '" 1:103 1:10“ ........

Pig 78 — - 1:10’ 1:10I 1:10' ................
Pig 99 — - - — 1.10 ........
Pix 600 — — 1:10' 1:10a 1:10a ................
Coat 87 — — 1:105? ,,,,,,,, ,,,,,,,,
l’Titer expressed as dilution end point reading. Considered positive if 50 per cent agglutination or lysis

or bath occurred.
tDays after start of second exposure period.

A public health problem is suggested by
the isolation of leptospiras from the milk
of heifer 73. The concentration of lepto-
spiras in 4.0 ml. of milk was sufficient to
infect guinea pigs and might well be a
threat to human health.

The serological results here recorded in-
dicate a cross reaction between L. pomona,
strain Wickard, and L. icterohaemorrha—
giae AB.

SUMMARY

Six young calves were experimentally
infected with Leptospira pomona. Only the
3 Herefords developed hemoglobinuria; 1
died on the eighth day postinoculation
and another on the twenty-fifth day. All
6 developed leptospiremia and 4 shed lep-
tospiras in their urine.

This infection was spread, by contact, to
3 of 4 pregnant heifers, to all of 4 pigs,
to 1 of 2 goats, but to none of 4 sheep.
Clinical signs of infection appeared six to
24 days after exposure. The course of lep-
tospirosis in calves, heifers, pigs, and a
goat, as well as the serological results ob-
tained following infection, are discussed.

References
1Baker, J. A., and Little, R. B.: Leptospirosis in
Cattle. J. Exptl. Med., 88, (1948): 295-308.

’Beamer, P. D., Hardenbrook, 11., Jr., and Mor-

rill, C. C.: Studies on Leptospirosis in Domesti-
cated Animals. 1. Leptospirosis in Sheep. Vet.
Med., 48, (1953): 365-366.
urnstein, T., and Baker, J. A.: Leptospirosis in
Swine Caused by Leptospira Pomona. J. Infect.
Dis., 94, (1954): 53-64.
‘Chang, S. L.: Studies on Leptospira Ictero-
bacmorrbagiae. J. Infect. Dis., 81, (1947): 28-34.

“Ferguson, L. C., and Bohl, E. H.: The Lepto-
spiral Diseases of Animals. Proc. Book, AVMA
(1953): 87-93.

‘Marsh, H.: Leptospirosis in Bovine Icterohemo-
globinuria. J.A.V.M.A., 107, (1945): 119-121.

1Morse, E. V., Allen, V., Krohn, A. F., and Hall,
R.: Leptospirosis in Wisconsin. 1. Epizootiology
and 4Clinical Features. J.A.V.M.A., 127, (1955):
417- 21. '

'Morse, E. V., Allen, V., Pope, E. P., and Krohn,
A.: Leptospirosis in Wisconsin. 11. Serological
Studies. J.A.V.M.A., 127, (1955): 422-426.

’Morse, E. V., and McNutt, S. H.: Experimental

tospirosis. 1. The Course of Leptospira Pomona

I ection in Pregnant Heifers. J.A.V.M.A., 128,
(1956): 225—229.

"Reinhard, K. R.: Present Knowledge and Con-
cepts of Leptospirosis in Farm Animals. J.A.V.-
M.A., 123, (1953): 487-493.

uRingen, L. M., Bracken, K., Kenzy, S. G., and
Gillespie, R. W. H.: Studies on Bovine Leptospiro-
sis. 1. Some Effects of Dihydrostreptomycin and
Terramycin on the Carrier Condition in Bovine
Leptospirosis. J.A.V.M.A., 126, (1955): 272-276.

1,van der Hoeden, J.: Leptospirosis Among Goats
in Israel. J. Comp. Path. and Therap., 63, (1953):
101-111.

"Wolfe,
spirosis.
1953.

. V.: Laboratory Diagnosis of Lepto-
arlcs Thomas Co., Springfield, 111.,

 

-....

. w “Mk—mgs.
)

 

 

717113162
Mortar, Raymond L
MS 1958 MPH
Histoyathology of the bovine

placenta in.Leptg§gir§ pgmong
infection.
/ r /"\ I . -1 ./:'u A

116 rter’ 'Raymond 11
VS 1958 MPH
Histopathology of the bovine

placenta in W mane
infection.

 

Illl
5
III
I
l
l
i
l
in
II
II
III
l
ll
l7
‘1'
l
I
II
I'-

3 03142 9313