THE PATHOGENESIS OP LEPTO SPIRAL INFECTIONS
(Leptospira pornona) DURING PREGNANCY
IN CATTLE AND SWINE

By
Raymond Lione Morter

A THESIS
Submitted to the School for Advanced Graduate Studies
of Michigan State University of Agriculture and
Applied Science in partial fulfillment of the
requirements for the degree of
DOCTOR OP PHILOSOPHY
Department of Microbiology and Public Health

I960

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Raymond Lion© Morter

ABSTRACT
All of tan pregnant heifers exposed to Lentosnira pomona devel­
oped either clinical or serological manifestations of leptospirosis*
The heifers were killed 10 to 60 days after esqc>08ure* None of the
fetuses developed leptospirosis* Leptospira© were not demonstrable
in the fetal fluids oT tissues and with one exception the placental
tissues*
The histopathological lesions of the heifers closely resembled
those observed in other reported experimental L* pomona infections of
cattle* The presence of a cytopathogenic agent produced b y the lepto—
spirae is suggested* This proposed agent is probably responsible for
alterations in the maternal—fetal placental relationship that interfere
with fetal development and may cause fetal death*
Two fetuses were infected with L* pomona b y direct inoculation
in utero* The fetal leptospirosis was similar to the postnatal infec­
tions* Leptospiremia was observed in one fetus on postexposure day 7
and by day lh the leptospirae were demonstrable in the kidney but
no other tissues* The histopathological lesions of the placenta
resembled those associated with maternal infections*
Five sows had been experimentally infected with L. pomona 10 to
Ih months previously* The sows were re-exposed during the last onehalf of the gestation period. The immunity produced b y the active
Infection was sufficient to protect the pregnant sows against chal­
lenge with a virulent strain of L. pomona*

Approved

ACKNOWLEDGEMENTS
The interest and guidance of Drs. L. C® Ferguson and E. V.
Morse whose direction and support made this work possible are
highly appreciated.

The suggestions and sincere interest of the

entire committee were most helpful*

The development of those parts

of the thesis that relate to pathology benefited greatly from the
counseling of Dr. R. F. Langham.
The technical assistance of Mrs. A. Lundberg and Mr* L. Eames,
whose aid made phases of this work possible, is acknowledged.
The National Science Foundation, whose support permitted the
author to devote two years to research and postdoctoral study is
acknowledged.

TABLE OF CONTENTS
Page
Introduction

1

Literature Review

3

Materials and Methods

7

Results

13

Discussion

22

Conclusions

33

Tables

*....................

35

Figures

39

References

57

INTRODUCTION
The ability of the female to maintain pregnancy and give birth
to normal young is dependent on a complex of interrelated factors.
Obviously the genital tract must be anatomically normal.

The dam

should be free of disease, in particular those diseases which are
known to have specific effects on the female genital tract.
are a manifestation of such diseases.

Abortions

It is frequently possible to

recover the etiological agent from the aborted fetus and often from
the female genital tract.

Infectious diseases can alter the physio-

logical blance or placental relationships and cause abortion without
actual infection of the fetus or the genital tract.

The endocrine

system of the dam must maintain a delicate hormonal balance to meet
requirements through all stages of gestation.

The nutritional status

of the dam must be adequate to meet the added metabolic requirements.
Leptospirosis (Leptospira pomona) frequently has been asso­
ciated with abortions in cattle and swine.

Leptospirae have been

regularly isolated from aborted swine fetuses.

Attempts to recover

leptospirae from aborted bovine fetuses have failed rather consis­
tently.

Invasion of the fetus by leptospirae is an important cause

of porcine abortions.

Bovine abortions

causes, but the conditions incompatible

probably result from other
with fetal life have notbeen

well defined.
It was the purpose of this work to ascertain the effects on
the bovine fetus ofj l) experimental L.

pomona infections of thedam,

and 2) experimental L. pomona Infection of the fetus in utero.

2

Purther, pregnant sows were re-exposed to L. pomona to determine if
the maternal leptospiral antibodies were capable of protecting the
fetuses against infection.*

* Parts of this phase of the research appeared in the Americas
Journal of Veterinary Research (i9 6 0 ) 21g 95-98.

3

LITERATURE REVIEW

Jungherr (18) demonstrated leptospirae in kidney sections of
diseased cattle, however leptospirae were not isolated from these
cases and the serotype remained -unknown.

Earlier reports of ictero-

hemoglobinuria and idiopathic hemoglobinuria may he considered bovine
leptospirosis (2 2 ,*4-0).

The symptoms of icterus, hemoglobinuria and

abortions are similar to those associated with L. pomona infections
of cattle.
In 19*4-6 Mathews (2 3 ) demonstrated leptospirae in tissue sections
and was able to transmit the disease from cattle to calves and guinea
pigs.

Blood taken during the febrile stage of the disease was used

for passage.
The first cultural demonstration of L . pomona from cattle in
the United States was accomplished by Baker and Little (l). Abortion,
usually occurring during the last trimester of pregnancy, has been
reported as a common manifestation of bovine leptospirosis (6 ,9 *1 1 »1 3 »
1^+,1 5 ,17,28,31,3^,^2 ,^3) •

Only two reports of isolation of L . pomona from the aborted
bovine fetus have been found (9 ,3 *4-). The report of Podgwaite

al.

(3 h) has been reviewed by Eerguson et a l . (1 5 ) and possible incon­
sistencies in the report were discussed.

Bacres and Kiesel (9)

recovered leptospirae from an aborted bovine fetus by inoculating
chinchillas with thoracic fluid and subsequent blood culture in
Chang* s semisolid medium.

The inability to isolate leptospirae from

the aborted bovine fetus, fetal fluids or placentae (1 3 ,14-,1 5 ,2 8 ,3 1 ,
35)

suggests that other mechanisms may play a more important role than

I*

invasion of the fetus in leptospiral abortions.

Te Punga and Bishop

(^3 ) suggest three causes for 'bovine abortionss 1.) pyrexia and sys­
temic reaction resulting in abortion; 2 ) interrupted transfer of
metabolites, due to localized lesions in the maternal-fetal cotyledonary Junction, with subsequent fetal death; and 3 ) actual invasion
of the fetus by the leptospirae, with death and expulsion following
active fetal infection.
Ferguson et^ al. (15) found an apparent lack of erythrocytes in
the circulatory system of aborted bovine fetuses.

A suggested mech­

anism for this phenomenon was the release of a toxic material from
the leptospirae which was able to cross the placental barrier and
lyse the fetal erythrocytes.
Leptospirae were not demonstrated in fetal or placental
material from experimental or field cases by Borg-Fetersen and
Fennestad (1 3 ,1 ^) but the authors considered the findings compatible
with the hypothesis that leptospiral abortion is most often due to the
fetal leptospirosis of the fetus.

The failure to isolate leptospirae

was thought due to the inability of the organisms to survive in the
environment of the autolyzing fetus.

Fetal infection probably results

from invasion of the fetus at the time of the maternal leptospiremia
and acute infection of the fetus follows an incubation period of a
week or more (1 3 )*
Leptospira—like bodies have been demonstrated in fetal and
placental tissues by silver impregnation techniques (6 ,35#*+3) . Hone
j

of these observations was substantiated by bacteriological findings.
Determining the presence of leptospirae in the placenta and fetal
tissues by silver impregnation cannot be considered a definitive

5

diagnosis unless such tissues are found to be bacteriologically
positive (3 0 ).
Examination of aborted fetuses in experimental studies failed to
reveal leptospirae by cultural or animal inoculation techniques (2 8 ,
35)*

Experimentally infected heifers were slaughtered 1 0 -lh and 25=33

days after exposure.

All fetuses were alive but leptospirae were not

demonstrated (2 8 ,3 1 ).

Leptospiral antibodies were not found in the

fetal sera by the agglutination-lysis test, indicating that maternal
antibodies had not been transferred to the fetus and fetal antibodies
had not been produced in response to invasion by leptospirae.
Morter (29) reported a series of histopathological changes in
the cotyledons which might interfere with the development of the fetus
and result in fetal death and abortion.
Turner e_t al. (^4) isolated Leptospira canicola from the urine
of a newborn calf.

It is presumed that the infection was contracted In

utero as the calf was only two days old at the time the urine sample
was collected.

Morter at al. (3 0 ) suggested that serotypes other than

L. pomona might be involved in bovine abortions.
Fennestad and Borg=Petersen (ll) have reported experimental in
utero infection of the fetuses of six immune dams by performing a
laparotomy and intrap lace ntal inoculation of viable cultures of lepto—
spirae.

They concluded that leptospiral abortion in cattle is usually

the result of fetal death following invasion of the fetus by lepto­
spirae during leptospiremia of the dam.
fetal death and exp'ud.sio11 of

Further, the interval between

fetus exceeds the survival time of

the leptospirae in the dead fetus.

6

Occasionally the bovine fetus may be capable of producing
antibodies in utero (1 2 ).

Two fetuses infected with Leptospira

saxkoebing produced specific antibodies _in utero and survived intraplacental inoculation.
L. pomona infections have been demonstrated by experimental
and clinical studies to be an important cause of porcine abortions
(5»7»8,10).

Leptospirae have been recovered from the aborted swine

fetus (5 .7 .8 ).
It has been inferred that swine previously infected would be
refractive to subsequent exposure and able to complete a normal ges­
tation period (7 ) •
Morter (29a) reported an apparent incidence of leptospirosis
of 10 percent in Iowa swine and that abortions were the most marked
clinical manifestation of the disease.
Leptospiral abortions occxir in other species.

Leptospiral

abortions in sheep in Illinois have been reported, but the etiological
agent was not identified (3.^)-

Experimental L . pomona infections in

pregnant ewes failed to produce any abortions but leptospirae were
recovered from the cotyledon of one ewe (2 0 ,2 1 ).
Smith at al. (4l) recovered leptospirae from the fetal tissues
or fluids of 3 of 17 ewes experimentally infected.

The isolations

were accomplished by hamster inoculation with fetal tissues collected
at necropsy on postinoculation day 3 .
Van der Hoeden (45) described an outbreak of caprine lepto­
spirosis in Israel cause by Leptospira grippotyphosa. A majority of
the pregnant goats aborted.
A mare aborted during an outbreak of L. pomona infection in horses (37).

7

MATERIAL AND METHODS
Fifteen apparently normal purebred Holstein-Friesian heifers 2 h
to 3 0 months old were used as experimental animals for the study of
experimental leptospirosis in pregnant cattle.

The animals were pro­

cured at 8 to 12 months of age from the State Prison of Southern
Michigan,

They were maintained free of contact with other cattle dur­

ing the period preceding the experiments-

No leptospiral antibodies

were observed in the serum of any heifer-

The modified agglutination-

lysis test (27) was employed using L. pomona (strain Johnson), Li.
canicola and L- icterohemorrhagjae(AB) living antigens.

End point

titers were interpreted as the greatest tenfold dilution in which at
least 50 percent agglutination or lysis occurred.

This serological

method was used throughout all phases of the work.
official Brucella abortus strain 19 vaccinates.

All heifers were

The heifers were bred

artificially with semen furnished by the Michigan Artificial Breeders
Cooperative.

All heifers were in the ^th to 8 th month of pregnancy

(table 2 ) when experimentally infected.
The heifers used in experiment one were divided into three
groups.

The distribution is set forth in table 1.

lated subcutaneously.

They were inocu­

Heifers 5, 1» 8 and 9 were assigned to group 1

(table l) . Heifer 9 served as a control.

The heifers were exposed to

L. pomona strain Wickard (2^) which had been maintained in continuous
guinea pig passage since its original isolation.

The inoculum con­

sisted of 5 nil of heparinized guinea pig blood harvested at the peak
of the febrile response (I0h*5“106.0 F) . Heifer 8 received 5 ml of
normal c&vian blood.

8

Group 2 was composed of heifers 07. 08. 88 and heifer 3 serving
as control (table l) . The inoculum was 5 ml of calves® blood per
animal taken during pyrexia,

Three serial blood passages in guinea

pigs had preceded the inoculation of the calves.

The control received

5 ml of normal calf blood.
Group 3 composed of heifers

7, 10, 11 and 6 as control, was

exposed to a hamster-lethal variant of L. pomona (strain Wickard).
This variant has been designated as wWickard Lw by Bauer (2).
serial blood passages in hamsters were made.

Four

The inoculum was pre­

pared from 10 ml of heparinized blood and 30 ml of a 10 percent
emulsion in sterile 0.85 percent saline of kidney, liver and spleen
harvested from moribund hamsters (table l).
Animals 13 and 23 were used for inoculation directly into the
fetus.

The heifers were given 8 ml of Diquel^ (Jen-Sal) intravenously.

A T~block of the left paralumbar fossa was accomplished by local in­
jection of xylocaine*

The laparotomy site was clipped and scrubbed.

The animals were restrained in lateral recumbency on the right side
with legs mechanically extended.

The site was draped with sterile

towels and an incision was made in the posterior part of the paralumbar
fossa extending from the level of the tuber coxae to the fold of the
flank.

The uterus was exteriorized and manually fixed to present the

muscle mass of the fetal thigh in suitable position for inoculation.
A sterile 16 gauge needle 2j- inches long served as a cannula and was
passed through the wall of the uterus.

Sterile 2 by 2 gauze sponges

saturated with bovine antileptospiral (L.pomona) serum were packed
about the cannula.

One and one-half ml of a 7-day culture of L.

pomona (strain GLD) was inoculated into the fetus by passing a 6-inch

9

needle through-the cannula into the fetal thigh*

This strain of L.

pomona was originally isolated from dogs hy Morter ejt al. (32) and
was in the fifth passage in culture.

As the syringe and its attached

needle i/ere withdrawn sponges saturated with antileptospiral serum
were used to prevent contamination of the surface of the uterus with
the inoculum.

The exposed area of the uterus was flooded with anti­

leptospiral serum.

The cannulating needle was withdrawn and the

uterus returned to its normal position.

The incision was closed in

three layersj the peritoneum, the muscle layers, and the skin.

A

sterile dressing was applied.
Tor the re-exposure of pregnant sows, a susceptible boar, a
susceptible barrow and five pregnant sows served as experimental
animals.

The sows had been experimentally infected with L. pomona

10 and 1*4- months previously.

Urinary shedding had not been demon­

strated for 8 to 10 months (26).

Breeding was by natural service 75

to 95 days prior to re-exposure.

Serum antibodies for L. pomona were

demonstrated in the sows at the end point titers of 1 0~ 3 and 1 0 “^.
The challenge strain of L. pomona. of porcine origin, had been
maintained in continuous guinea pig passage in this laboratory.
single passage in hamsters was made.

A

The inoculum consisted of hepar­

inized hamster blood and 10 percent suspension of hamster kidney and
liver tissue in 0.85 percent sterile sodium chloride solution.

Four

of the sows, the boar and the barrow each received 7 - 5 ml of the mix­
ture of blood and tissue suspension.

Sow ^928 served as a control and

received 7 - 5 ^ 1 of normal hamster blood and tissues.

10

The inoculum for each of the experimental groups was titrated in
hamsters.

The highest dilution which caused death of the hamsters or

stimulated antibody response was considered the end point.
10

Less than

organisms are known to he infective for hamsters (2 ) . The number

of organisms per ml of inoculum varied from 1 0 ^ to more than 1 0 ^ when
prepared from blood or tissues (table l) . The culture used to inocu­
late the two fetuses _in utero was found to contain > 1 0 ^ organisms
per ml.
All animals were observed for clinical manifestations of
disease.

Rectal temperatures were taken twice daily (table 2) until

agglutination-lysis end point titers of 10 -=2 or greater were observed
or the animals were necropsied.
Animal inoculation techniques were employed to determine the
presence of leptospirae in tissues or fluids.

Tissues were processed

in tissue grinders with sterile 0 . 8 5 percent saline to give a final
tissue concentration of approximately 10 percent.

The material was

injected intraperitoneally into young guinea pigs weighing approxi­
mately 250 grams or
2

to 5-week-old hamsters.

ml of the inoculum and hamsters 1 ml.

G-uinea pigs received

Undiluted amnio=allantoic

fluids, fetal stomach contents, urine and blood were also injected
intraperitoneally in the same amounts into hamsters or guinea pigs.
Approximately three weeks postinoculation the animals were killed and
sera checked for agglutination=lysis reactions.

The presence of

leptospiral antibodies was considered indicative of leptospirae in
the original materials.
Hemocultures were prepared with bovine blood in fluid media
on postexposure days 7 and 8 or later at the time of a temperature

11

rise and with swine blood on days 5 to 8 postexposure.

Blood was col­

lected aseptically from the jugular vein of the cattle or the precaval
venous well of swine.

Each of five tubes of Chang® s fluid medium (2 b-)

or Stuart® s fluid medium (Difco) was inoculated with five drops of
blood and incubated at 29 C.

Each tube was examined microscopically,

utilizing darkfield illumination (6 0 0 x)» at ten-day intervals for the
presence of leptospirae.

Each of two guinea pigs or hamsters was

inoculated with 0 . 5 ml of blood from the heifers at the same time
media were inoculated.

Positive agglutination-lysis titers in the

laboratory animals 18 to

days postinoculation was considered indic­

ative of leptospirae in the inoculum, i.e. heifers were in the
leptospiremic phase of the disease.
To procure arterial blood samples from the heifers for compar­
ative evaluation of fetal and maternal hematological determinations,
the carotid artery was surgically fixed to the skin of the midcervical
region.

Surgery was performed under local anesthesia accomplished by

injection of 2 percent xylocaine into the area.
and prepared for surgery.
the jugular furrow.

The site was clipped

The skin was incised dorsal and parallel to

The skin was reflected dorsally, the cervical

muscles separated exposing the carotid which was dissected free of the
surrounding connective tissue.
beneath the skin.

A loop of the carotid was positioned

The muscles were sutured together belox* the carotid

and the loop of the vessel was fixed in the subcutaneous area by
nonabsorbable sutures.
mattress sutures.

The skin incision was closed by interrupted

A minimal recovery period of two weeks intervened

between the surgical fixation of the carotid and experimental exposure
to L. pomona.

12
So obtain fetal blood samples a hysterotomy was performed under
local anesthetic and tranquilizer as previously described.

The sur­

gical procedures to expose the uterus were the same as those for the
in utero inoculation of the fetus.

Arterial and venous blood sanples

were obtained from the umbilical vessels.

The heifers were then

exsanguinated.
Heifers were killed from postinoculation days 10 to 60 in an
attempt to delineate the pathogenesis of bovine leptospirosis during
pregnancy.

The maternal tissues collected at necropsy were? kidney,

adrenal, liver, spleen, lung, uterus, cotyledon, mammary gland and
brain.

Fetal tissues included? thymus, lung, heart, liver, spleen,

kidney, adrenal and brain.

The swine were anesthetized by intravenous

administration of sodium pentobarbital, exsanguinated and postmortem
examinations conducted.

Fetal and maternal kidney, liver, brain,

adrenal, heart and placenta were collected.

Tissues were collected

for both animal inoculation procedures and histopathological evaluation.
One or more fixatives were used: Zenker*s, 10 percent buffered formalin,
or CarnoyBs.
to 8 u.

Tissues were embedded in paraffin and sectioned at 7

Hematoxylin and eosin, analine blue and Sudan XV were the

staining procedures employed.

13

RESULTS
1. Exposure of Pregnant Heifers.

The clinical manifestations of leptospirosis observed from
post inoculation days 1 through 10 included polypnea, anorexia and
increased body temperature.

Maximal rectal temperatures varied from

102.5 to 106.3

with seven heifers having temperatures greater than

104 IP (table 2).

Other symptoms were variable and not distinctive.

The temperature rise was accompanied by a transient anorexia, a slight
increase in respiratory rate, some constipation and a dulling of the
hair coat.
Leptospirae were isolated from five heifers by direct culture
or animal inoculation or by both procedures during leptospiremia.

No

isolations were made from five of the exposed heifers or from the
three controls.

The agent was recovered from the kidneys or urine of

four heifers and from the cotyledons of one.

The agent was not iso­

lated from any of the fetal tissues or fluids nor the kidneys or urine
of the control heifers (table 3 )•
The initial agglutination-1ysis reactions were observed from
days 5 to 14.

All 10 of the infected heifers developed serum titers

in a dilution of 10“^
negative.

or greater.

The controls remained serologically

No agglutination-lysis reactions were demonstrable in any

of the fetal sera, body fluids or amnio-allantoic fluids.

The sero­

logical results are summarized in table 4.
Each animal in group 1 except the control was given 5 ^1 of
cavian blood that contained 10** organisms per ml.

Heifer 5 had a

rectal temperature of 104.4 E on postexposure day 8; the agent was
isolated culturally from blood on day 8; and from the kidney by

14

animal inoculation on day 10.

The highest rectal temperature recorded

for heifer 1 was 102.5^ on postejposure day 8 . Leptospirae were re­
covered by hemoculture and animal inoculation on day 10 and from the
kidneys on day 23*

The agent was not recovered from the H o o d or

kidneys of heifer 8 although a rectal temperature of 104.5 3T was
recorded on post inoculation day 3-

Hemocultures were not made until

day f ive.
The first demonstrable titers for the heifers in group 1 were:
heifer 5» 1 0 "^ on day 9; heifer 1 , 1 0 “ 3 0n day 13; and 1 0 "*^ for heifer
8

on day 14.

The titers persisted until the animals were killed

(table 4) .
Heifers 07, 08 and 8 8 of group 2 each were inoculated with 5 ml
of calves* blood which contained more than 10 ** organisms per ml.
Hemocultures prepared on postexposure days 8 or 9 were positive for
all three of the infected animals.

Heifer 07 died on day 33 as the

result of an embolus that detached from the intima of the carotid.

A

severe chronic necrotic endarteritis was found at the point of sub­
cutaneous fixation of the carotid.

Death was attributed to the embolus

lodging in blood vessels of the brain.

Isolations of leptospirae were

not attenpted from fetal or maternal tissues of this animal.
Guinea pigs inoculated with kidney tissue from heifer 8 8 on day
40 developed a positive serum titer for L>. pomona. Leptospirae were
not recovered from the kidneys of heifer 08.

The three heifers

developed significant agglutination-lysis reactions (table 4).
The heifers in group 3 inoculated with the hamster blood and
tissue (table l) were observed to have the maximal rectal temperatures
on days 1 through 5 with temperatures from 103-0 to IO6 . 3 F recorded.

15

Attempted isolations from the blood stream all gave negative results.
Ho hemocultures were made before the appearance of agglutinins to L.
pomona in the sera of all four exposed heifers (table h) . The agent
was demonstrated in the kidneys of heifer h on day JO but not in the
kidneys of heifers 7 , 10 or 11 (table 3 ).
The significant gross lesions observed at necropsy were limited
to the maternal kidneys and were extremely variable according to size
and number.

Some kidneys had only two or three grayish-white foci one

to two mm in diameter.

In other kidneys, multiple lesions up to 8 mm

in diameter were observed.

On cross section the less marked lesions

were superficial, i.e. involving one to two mm of the cortex.

The

more pronounced lesions invaded the medullary portion of the kidney.
No gross lesions were found in any of the fetuses.
Microscopically the foci in the kidneys consisted primarily of
lymphocytes with a few plasma cells and macrophages.

Within the areas

of dense lymphocytic infiltration much of the parenchyma had undergone
degeneration.

Many of the tubules had atrophied.

The epithelium of

some tubules was granular, vesicular or desquamated, and pyknotic nuclei
were observed in many of the tubular epithelial cells.

In heifer 08

the central portions of the lymphocytic foci were less dense than the
perimeter of the lesions.

The nuclei of the lynphocytes in the central

portion were less dense than normal.

Repair by regeneration of renal

parenchymatous cells of fibroblast proliferation was not apparent
(figure l) . The cortex was contracted at the sites of the foci of
lymphocytic infiltration in heifer 7-

Connective tissue proliferation

was marked at the cortico-medullary junction but decreased in amount

16

toward the capsule.

In the subcapsular portion of the cortex the

concentration of lymphocytes remained practically unchanged from an
early lesion.

There was a

marked depression in the cortical surface

at the site of the foci (figure 2 ) .
Small foci of lymphocytes had infiltrated the areas adjacent to
interlobular bile ducts (figure 3 ).

In heifers 5 and 7 the hepatic

perivascular spaces were infiltrated with eosinophils and an occasional
polymorphonuclear granulocyte (figure h) . No microscopic lesions were
found in the kidney or liver of any of the control animals.
The microscopic lesions in the cotyledons were most pronounced
in those heifers killed during the first thirty days postexposure.

On

day 10 in heifer 5 > an acute focal placentitis was characterized by
necrotic chorionic villi and maternal crypts filled with tissue debris
and blood clots.

Alterations in the relationship between the fetal

and maternal placentae consisted of changes in the respective epi­
thelial surfaces.

Some maternal crypts were devoid of epithelium.

The chorionic epithelium was hydropic and contained pyknotic nuclei.
Pyknosis was most apparent in the diplokaryocytes (figure 5) - Necrotic
fetal placental tissue had lost its normal structure by day 23.

Some

of the fetal cells were in close proximity to the maternal epithelium.
The maternal epithelium no longer formed a well defined demarcation
between itself and the necrotic fetal villi (figure 6 ).

In figure 7

the pyknosis of the maternal epithelium was marked. One crypt was devoid
of epithelium.

Increased amounts of fibrous connective tissue sur­

rounded the maternal crypts (figure 7) . By postexposure day

the

presence of large cells, some with hyperchromatic nuclei and a loss
of the normal appearance of the maternal epithelium, suggested

17
regenerative hyperplasia.

The fetal placenta showed no apparent

damage (figure 8 ) . The other heifers in this group showed similar
changes in cotyledons with the most marked changes in those killed
within thirty days after inoculation.

Comparable changes were not

observed on the control sections studied.
IX. Direct Inoculation of the Fetus an utero.
Heifer 23 had an uneventful postsurgical recovery period.
clinical signs of leptospirosis were observed.

No

It proved to be inpos-

sible to determine fetal viability following inoculation.

The four-

month fetus was not palpable, being located well below the pelvic brim.
An enlarged edematous cervix was found by rectal palpation on post­
inoculation day 8 and thereafter.

On day 8 the heifer had a positive

serum titer of 10“^ which rose to 10“^ on day lh.

Leptospirae were

not isolated in hemocultures prepared on post inoculation days 6 , 7 or 8 .
At necropsy, on day 1^, the uterus was cyanotic, the cotyledons
were pale and the maternal and fetal placentae separated easily.
fetus was dead and edematous.

All body cavities and tissue spaces were

filled with a serosanguineous fluid.
empty of blood.

The

The heart and large vessels were

Microscopically, nucleated erythrocytes were found in

the blood vessels of all fetal tissues examined (figure 9) - No mature
erythrocytes were observed.

In tissue sections of a noninfected

control fetus of comparable age no immature erythrocytes were observed.
The maternal crypts were devoid of fetal villi and we re char­
acterized by increased amounts of connective tissue and disappearance
of the maternal epithelium (figure 10) . Foci of lymphocytic infiltra­
tion were not observed in any fetal tissues.

A few scattered foci of

lymphocytes were observed in the maternal liver.

18

No fetal blood sample was obtained.

Thoracic fluid had a

detectable agglutination-lysis reaction, probably nonspecific.

The

amniotic fluids were negative.
Inoculation of guinea pigs with amniotic fluid, fetal liver,
spleen, muscle, lung and kidney demonstrated the presence of lepto­
spirae only in the fetal kidney (three of five guinea pigs).

Of

the maternal tissue samples only the kidney was found to contain lepto­
spirae by the guinea pig inoculation technique.

Routine cultural

procedures did not yield any evidence of bacterial contamination of
the fetus.
Heifer 13 was killed on postinoculation day 7•
were found in any of the maternal tissues.

No gross lesions

The fetal placentae were

not firmly attached to the maternal placentae, separating without dif­
ficulty.

The uterus was normal in appearance.

and closed by a mucous plug.

The cervix was normal

The 7 ”Dionth fetus was viable but appeared

weak and the heart ceased to beat shortly after opening the uterus.
The fetus made no respiratory movements.
served.

No gross lesions were ob­

Microscopically small foci of immature lymphocytes and plasma

cells were observed in the cortex of the fetal kidney (figure 1 1 ) and
adjacent to the large vessels of the liver.

A diffuse placentitis

involved the epithelium of the chorion and maternal placentae (figures
12

and 1 3 ) . The remnants of the chorionic epithelium were hydropic and

the nuclei of many diplokaryocytes were pkynotic.

Necrosis of the

endometrium was characterized by numerous pyknotic nuclei.

Lympho­

cytes had infiltrated the minor calyces but were not present in the
parenchyma of the maternal kidney (figure lh) .

19

Antibodies were not demonstrated by the agglutination-lysis
techniques in the amniotic fluid, fetal blood or fetal stomach con­
tents.

The heifer was serologically negative on both post inoculation

days 6 and 7«

—1

The dilutions used were 10

, 10

»2

=1

and 10= .

Heifer 13 was in the leptospiremic phase of the disease at the
time of necropsy.

Leptospirae were isolated in hemocultures inoculated

on day 7 and were demonstrated by the guinea pig inoculation technique
in the maternal liver, kidney and cotyledons.

All guinea pigs inocu­

lated with fetal stomach contents, amniotic fluid, liver, spleen, lung,
skeletal muscle and kidney were serologically positive to L. pomona.
III. Re-exposure of Pregnant Sows.
Hone of the pregnant sows, control or challenged, demonstrated
any clinical signs of leptospirosis.

The boar and the barrow had tem­

perature rises to 10h.6 P on postinfection days 5 and 6 . Concurrent
constipation and anorexia were noted.
Leptospirae were isolated from the blood of the boar and the
barrow by cultural methods on post inf ect ion days 5 * 6 and 7 - Isola­
tions were not accomplished from the blood of pregnant sows.

Lepto­

spirae. were demonstrated by guinea pig inoculation in the kidney and
brain of the barrow on postinfection day
spleen.

Leptospirae were present

7

* but

not from liver or

in the kidney

ofthe boar on post­

infection day 1 3 ; all other tissues including the testes were negative.
The control sow, h9 2 8 , was
liver and apleen, amnio-allantoic

killed on day 3-

Maternal kidney,

fluid, and fetalblood, kidney and

liver were inoculated into guinea pigs, but leptospirae were not
demonstrated.

Seven 90-day fetuses appeared normal.

serum gave an agglutination-lysis titer of 10“^.

The maternal

Urine antibody was

20

demonstrated at a dilution 10“"3. The fetal sera and amnio-allantoic
fluids were serologically negative.
Sow *+929 was killed on postchallenge day 6.

Nine apparently

normal fetuses at approximately the 95th day of gestation, were present.
3?etal and maternal tissues did not contain leptospirae.

The terminal

agglutination-lysis titer of the maternal serum was

and maternal

urine 10““3 . No antibody reaction was observed in the fetal sera.
At necropsy, 11 days after challenge, 10 apparently normal, 86day fetuses were present in sow *+958.
from the fetuses or the sow.

Leptospirae were not isolated

The end point titer of the maternal

serum was 10*“®; the urine titer was 10“*^.

Leptospiral antibodies were

not observed in the fetal sera.
Seventeen days after challenge, sows *+923 and ^ 9 6 9 farrowed
litters of 9 and 10 respectively, which developed

normally. Agglutin­

ation-lysis reactions occurred in a 10=^ dilution of sera of newborn
pigs three hours after birth.
were negative.

Samples procured from the pigs at birth

Two pigs from each litter were killed when *+8 hours

old, and tissues were inoculated into guinea pigs.

The results were

negative.
Sow *+969 was killed on post challenge day ^1 and

sow h923 on day

62. Leptospirae were not isolated from the kidneys or urine.
At necropsy the gross changes (greyish-white foci, measuring
1

to *+ mm in diameter) were confined to the kidneys of all mature pigs,

including the control sow.
the cortex into the medulla.
to be normal.

On section, a few lesions extended through
The placenta and fetal kidneys appeared

21

Microscopically, many of the foci in the kidneys of the chal­
lenged sows consisted of an infiltration of lymphocytes, a few plasma
cells and some macrophages (figure 15) .

Some areas were characterized

"by active proliferation of fibroblasts, while others were primarily
cicatrical tissues (figures 1 6 , 1 7 ) •
occasionally atrophied.

In these foci, a tubule was

The renal corpuscles had some thickening of

Bowman* s capsules and the glomerular tufts due to connective tissue
proliferation.

The epithelium of the proximal convoluted tubules had

and increase of fat (figure 18) .

The kidney lesions of the boar and

the barrow were composed of small areas of predominantly lymphocytic
infiltration similar to the type described by Langham e^b al. (1 9 ) An active inflammatory process, typified by foci of lymphocytic
infiltration, was an unexpected finding in the kidneys of the control
sow.

Some of the foci extended only 1 to 2 mm below the cortical

surface, while others penetrated to depths of 8 to 9 mm, reaching the
medulla.
dulla.

Connective tissue proliferation was most marked in the me­
No evidence of leptospirae was observed in the silver-impreg­

nated kidney sections.

22

DISCUSSION
Abortions due to leptospirosis (L. pomona) are a major cause of
economic losses in cattle and swine-

In beef cattle and swine, abor­

tions automatically limit the potential income of the producer.

In

dairy cattle the loss of a calf has less importance but production
throughout the ensuing lactation period may be seriously curtailed.
The apparent difference in the ability of leptospirae to invade
the porcine fetus and not the bovine fetus may be attributed to the
species differences in placentation.

The sow has a diffuse placenta

with the entire surface of the amnio-chorion forming a placenta with
the endometrial surface in apposition to it.

The cow has a cotyle-

donary type of placenta with 100 to 1 2 0 specific points of attachment.
By classical histological definition the placenta of the sow is of
the epitheliochorial type with six cell layers intervening between the
maternal and fetal circulation.

The maternal intraepithelial capil­

laries of the porcine placenta are frequently in direct contact with
the chorion.

Fetal capillaries extend between the cells of the chorion

and are found in direct apposition to maternal

capillaries.

From the

middle of the gestation period to term the porcine placenta can be
classified as endotheliochorial or endothelioendothelial with as few as
2 cell layers intervening between the fetal and maternal systems.

The

bovine placenta is considered to be syndesmochorial lacking the mater­
nal epithelium and five cell layers separating the fetal and maternal
circulatory systems.

This is not actually the cases the bovine pla­

centa normally maintains three maternal and three fetal cell layers.
Only in the intercotyledonary spaces does the surface of the endome­
trium become denuded and even then the relationship between the fetal

23
and maternal circulation is less intimate than in the sow.

The more

complex placental harrier in the cow may prevent invasion of the fetus
by leptospirae.

Other characteristics of the placenta, trophoblastic

enzymes or membrane potential may differ enough to permit invasion of
the swine fetus but not the bovine fetus.
The bovine fetus furnishes a suitable environment for parasit­
ism by L. pomona as demonstrated by the establishment of the experi­
mental in utero fetal infections which appeared to follow a course
similar to postnatal infections.

Fetal leptospiremia occurred on

postinoculation day 7 (fetus 1 3 ) with leptospirae present in all tis­
sues and fluids examined.

Leptospirae were demonstrated in the kidney

of fetus 23 on post inoculation day 1^.
other tissues.

No leptospirae were present in

The appearance of circulating antibodies is positively

correlated with the cessation of the leptospiremia and establishment of
the renal carrier state in typical cases of leptospirosis.
spiral antibodies were not present in the fetal serum.

Lepto-

Survival of

leptospirae in the kidney and no other tissue cannot be explained by
the protection afforded the organisms against antibodies in the kidney
tubules.

Possibly the physiological requirements of the organism can

be satisfied only in the kidney.

In any event, the typical course of

the disease, except for antibody production, was mimicked by the course
observed in the fetus in utero. Fetus 23 was dead and resulting autolytic changes may not have been compatible with the survival of the
leptospirae in other tissues.

This would suggest some special type of

protective environment furnished by the kidney permitting their
survival -

2b

Leptospirae crossed the placental harrier from the fetal to the
maternal tissues following the leptospiral infection of the “bovine
fetus.

Leptospirae were isolated from kidney of one heifer and from

the blood, liver, kidney and cotyledons of the second dam.

Positive

hemo cultures were obtained on post inoculation day 7 and positive serum
titers on day 8 .

The organisms appeared to have traversed the pla­

cental harrier, movement from fetus to dam, rapidly and produced con­
current infections of the dam and the fetus.

The maternal infections

could have resulted from contamination of the peritoneal surface of
the uterus: however, the precautionary measures employed minimized this
possibility.
Conditions may exist that permit the leptospirae to traverse the
placental barrier from the fetus to the dam that are different than
those conditions that appear to prevent invasion of the fetus as a resuit of maternal infection.

A difference in membrane potential could

permit movement in one direction but prevent retrograde movement.
charge at the maternal surface might repel the organisms.

The

The direct

infection of the fetus could produce alterations rendering the placen­
tal barrier more permeable to the passage of leptospirae.

The validity

of the latter hypothesis is questionable as the serological and cul­
tural findings indicate that the infection of the dam occurred at
approximately the same time as that of the fetus, and before marked
alterations in the placenta could occur.
Several hypotheses have been suggested to explain the inability
of several workers to isolate leptospirae from the bovine fetus (1 3 *
1^,15i31»35):

inability of the organisms to survive in the dead

fetus during the interval prior to expulsion, Z) invasion of the fetus

25

occurs during the leptospiremia of the dam and a secondary incubation
period must follow before significant numbers of leptospirae are found
in the fetus, 3 ) ability of the fetus to produce antibodies against the
leptospirae with their elimination from the fetal system, h) production
of toxic materials in the maternal system by the leptospirae which tra­
verse the placental barrier and cause fetal death, 5 ) cytotoxic effects
upon the fetal-maternal placental junction which might interfere with
the development of the fetus resulting in fetal death and abortion.
Three of these hypotheses assume an actual infection of the fetus; two
suggest that leptospirae do not invade the fetus.
If leptospirae invade the bovine fetus, proper application of
cultural methods should result in recovery of the causal agent.

Insuf­

ficient numbers of organisms in the material examined, interference by
antibodies or lack of sensitivity of the techniques employed could con­
tribute to the failure to recover leptospirae from the bovine fetus if
fetal infection occurred.

The ability of the animal inoculation tech­

nique to determine the presence of as few as five organisms (L. pomona)
has been reported (2) , and one virulent leptospira (L. pomona)may
establish infection in guinea pigs or hamsters (h6) . Leptospirae were
recovered from the experimentally infected fetuses by animal inocula­
tion.

Fetal tissues did not interfere with the results.

Even if very

few organisms were contained in the inoculum the animal inoculation
technique would signify their presence.
Rudge (3 8 ) reported interference with demonstration of lepto­
spirae in kidney tissue from a calf with leptospiruria by specific
antibodies.

The calf had a titer of 10

and the organisms were inac­

tivated during the preparation of the tissue suspension for inoculation

26

into guinea pigs.

No demonstrable agglutination-lysis reactions were

observed with the fetal serums in these experiments.

Interference by

antibody could not have influenced the results of this work since the
fetal sera in these experiments have uniformly been free of leptospiral antibodies.
The gross and microscopic lesions in the dead fetus (heifer 25)
support the theory of fetal death due to the effects of hemolytic toxin
(15) * An overwhelming hemolytic anemia is suggested by the absence of
blood in the heart and large vessels, pleural and peritoneal cavities
filled with a serosanguineous fluid, and the predominance of immature,
nucleated erythrocytes observed in tissue sections.

It could be pos­

tulated that a lepto spiral toxin produced severe hemolytic anemia.

A

compensatory release of immature erythrocytes from the hemopoietic
centers occurred.

Increased permeability of the vessel walls and a

reduction in colloidal osmotic pressure resulted in the paucity of
blood in the vascular system and the extravascular sero sanguineous
fluid.
The systemic reactions of the heifers did not affect fetal life
although temperatures as high as 106.3 I* were recorded.

The hypoth­

esis that maternal pyrexia and systemic reactions can cause fetal death
lack proof.

The reactions to experimental infection of the dam are

usually mild and transient and seldom severe enough to produce an
abortion.

Further, the interval between the maternal leptospiremia

and abortion suggests the two are not directly related.
Alterations in the chorionic-maternal placental relations reported by Morter (29) were progressive in nature.

The early lesions

were not extensive but increased in severity with time following

27

infection*

To ascertain the effect of the experimental L. pomona in-

fections of the pregnant heifers on the placental relationships
selected tissues from the uterus and cotyledons of each heifer were
examined microscopically.

Placentitis was present in either the chor­

ion or the maternal epithelium in all cotyledons examined from
infected heifers.

Similar changes were not observed in the 3 controls.

The placentitis was similar to that previously reported C3 0 ) hut
in no case as extensive as the changes observed following abortion*
The most marked lesions were observed in those heifers killed approxi­
mately 3 0 days after inoculation.

According to observations recorded

in various published papers, most leptospiral abortions occur 3 to 4
weeks after infection in both naturally occurring and experimental
infections (ll,13*15* 28,31) • This is about the same time the most
severe lesions were found.

By day 45 the areas of placental damage

were being repaired by regenerative hyperplasia.

The placental lesions

appeared to increase in severity until day 30 and then regress and in
no case were severe enough to cause fetal death.
Leptospirae were isolated from the cotyledons of one heifer.
Previous reports (20,28,41) indicated that sporadic invasion of the
cotyledons of cattle and sheep occurs.
be recovered from the placenta.

In most cases the agent cannot

The occurrence of a ple.centitis with

no organisms demonstrable in these tissues suggests that a cytopathogenic factor may produce the changes.

In bovine leptospirosis, infec­

tion of the placental tissue cannot be demonstrated routinely and is
probably not important in the pathogenesis of placental lesions.

If

the tissue changes result from a cytopathogenic agent, the amount or
activity of such a substance could result in great variation in damage

28

to the placenta.

Death of the fetus would depend on the degree of

interference with placental exchanges and, as in "brucellosis, might
not always occur.

The placentitis produced "by the experimental infec­

tion of the heifers was not severe enough to cause fetal death.
Serological or cultural evidence of leptospirosis was observed
in all 10 heifers exposed experi men tally to L. pomona but not in any of
the three controls.

No bacteriological, serological or histopatho-

logical evidence of infection was found in any of the fetuses from
these 10 heifers.

All of the fetuses were viable at the time of

necropsy and consequently leptospirae could not have been destroyed
by the autolytic products of a dead fetus.

Fifty guinea pigs or ham­

sters were inoculated with fetal tissue emulsions or fluids from each
fetus.

The animal inoculation technique is sensitive, to small

numbers

of leptospirae and the presence of organisms would have been detected
had they been present in the fetuses.

Under the conditions of this

experiment organisms were unable to traverse the placental barrier and
establish fetal infection.
60 days postexposure.

The heifers were necropsied from 10 to

If a secondary incubation period were required

for the establishment of fetal infection such an event should have
been determined by this procedure.
The ability of an organism to parasitize a host depends upon
invasion of the host and establishment of an infection.

This is

usually followed by the production of lesions as a result of the in­
fection.

Bauer (2) reported a difference in virulence between strains

of L. pomona. Two strains of L . pomona. Wickard (2*0 and Wickard L (2),
known to differ in virulence, were employed experimentally to infect
the heifers.

Neither strain proved capable of invading the fetuses.

29

Virulence of an organism alters with, continuous passage in media
or adaptation to a particular host.

Leptospirae maintained in fluid

media “become less virulent - nearly, if not completely, apathogenic.
Strains of L. pomona maintained in serial passages of kidney tissue in
guinea pigs retain a high degree of virulence for other animals (21,26,
27.28,31)*

Each group of heifers received an inoculum prepared in a

different manner.

The inocula were prepared in guinea pigs, hamsters

and calves (table l) . The calf passage was used in an attempt to de­
velop a bo vine-adapted population of leptospirae, but the inoculum from
calves elicited a response similar to that produced by the inoculum
of guinea pig blood.

The hamster lethal variant and the large number

of organisms employed in inoculating group 3 were designed to create
a more severe manifestation than produced in the other groups.

The in­

cubation period was shortened, antibodies were detectable sooner,
maximal temperatures higher, but the other manifestations were no more
marked than in other animals.
Those heifers receiving the material lethal to hamsters were
given 6 0 0 times as many organisms as the groups receiving guinea pig
or calf blood.

Demonstrable agglutination-1ysis reactions were ob­

served in the serum of all four of these heifers by postinoculation
day 6 . The average incubation period in experimental L>. pomona infec­
tions of cattle is 6 to 8 days, and antibodies are usually not detected
before day 7 or 8 . Apparently the large inoculum had shortened the
incubation period.

A similar phenomenon, i.e. leptospiremia on post­

inoculation days 1 to h, has been reported in sheep and calves when
large numbers of leptospirae were inoculated subcutaneously or intra­
venously (20,21,36,^1).

30

Blood taken during pyrexia provides an inoculum of rapidly multiplying organisms.

Such a population may differ widely in virulence

from the population that localizes in the kidneys.

Organisms shed in

the urine provide the usual means for natural transmission of lepto­
spirosis.

The use of porcine or hovine urine from renal shedders as

an inoculum might produce leptospirosis of a different character than
that observed in these experiments.
The heifers were of similar genetic background, having been se­
lected from a single herd of purebred Holstein-Friesian cattle.

A

degree of inherent resistance to leptospirosis could have minimized the
effects of the experimental disease.

A subsequent naturally occurring

outbreak in the parent herd was very mild.
serological diagnosis.

Recognition was limited to

The heifers were in excellent health, well

housed and were receiving an adequate ration.
of the heifers was lactating.

Being primiparous, none

Many of the factors thought to enhance

susceptibility of animals naturally infected were minimized by the con­
ditions of the experiment.

Under different conditions of management,

environment and nutrition, leptospirosis might be more severe and
sporadic infection of the fetus occur.
The inability to recover leptospirae from the fetuses of eleven
heifers experimentally infected with L. pomona has been previously
reported (28,31).

Alterations in the placental relationships that

could interfere with fetal development were also found (3 0 ).

In this

series of experiments 10 pregnant heifers were infected with L . Pomona
and in no case was fetal infection produced.

Direct infection of the

fetus must be sporadic and may depend upon fortuitous conditions of
exposure, nutrition, parasitism, lactation or nonspecific stress factors.

31
Leptospirae must be recovered from the aborted bovine fetus by accep­
ted techniques to establish fetal leptospirosis as the cause of fetal
death.
Maternal antibodies are unable to cross the placental barrier of
the sow.

It was postulated that leptospirae might escape the effect

of the maternal antibodies, cross the placental barrier, and survive in

the antibody-free fetal environment.

The pathogenicity of the chal­

lenge strain used in these experiments was demonstrated by the course
of the disease in susceptible swine.

The demonstration of L. pomona

in the brain of the barrow indicates an invasiveness of the organism
for this tissue.

Encephalitis or meningitis, a reported clinical mani­

festation of porcine leptospirosis (2 ^,3 9 )» could result from direct
infection of tne central nervous system by leptospirae.
Since leptospirae were not demonstrable in fetal tissues, and
fetal deaths or abortions did not occur, the maternal antibodies fur­
nished adequate protection against challenge and uterine invasion
apparently did not occur.

Gestation and parturition were normal.

Colostral antibodies were detected in the sera of newborn pigs three
hours after birth.

Protection against possible postnatal infections

was afforded.
Marked increase in serum antibody levels was observed following
challenge, with endpoint titers increasing by a factor of 10 ** in the
immune sows* The response was transient; the serum titer of one sow
decreased from 10 ^ to 10 ^ by postchallenge day ^4-1.
The barrow had leptospiremia with concurrent serum titer of
10“** on postinfection day 7-

Leptospirae were isolated from the blood

in the presence of homologous antibody.

The early immune response may

32

produce an incomplete antibody, which is unable to react with all
the leptospirae.

Leptospirae can be successfully maintained through

several passages in media containing homologous antibody (3 3 ).

This

phenomenon suggests a possible mechanism for in vivo selection or vari­
ance.

Not all leptospirae are agglutinated by homologous antiserum

in vitro. Morse at a l . (27) suggested that agglutination is rarely
complete in the agglutination-lysis test.

Neutralization of the cir­

culating antibody permits survival of some organisms.
The histopathological changes in the susceptible swine were
comparable to the renal changes previously described (8,19).

Langham,

Morse and Morter (19) described microscopic lesions of repair and
active inflammatory reaction 18^ days after exposure.
were observed in the kidneys of the re-exposed sows.

Similar lesions
Active renal

inflammation could have been the result of challenge, i.e. the exposure
of sensitized renal tissue to the leptospiral antigen.

However, this

would not explain the active inflammatory processes in the kidneys of
the control sow.
10

Since active inflammatory processes persisted for

to 1 ^ months after initial infection, some antigenic principles

of the leptospirae, capable of stimulating a cellular reaction, may
be retained in localized areas after renal shedding is not detectable.

33

SUMMARY AND CONCLUSIONS
All of ten pregnant heifers exposed to Leptospira. pomona devel­
oped either clinical or serological manifestations of leptospirosis.
The heifers were killed 10 to 60 days after exposure.
fetuses developed leptospirosis.

None of the

Leptospirae were not demonstrable

in the fetal fluids or tissues and with one exception the placental
tissues.

Direct invasion of the fetus by I. pomona does not appear

to play an important role in bovine leptospiral abortions.
The histopathological lesions of the heifers closely resembled
those observed in other reported experimental L. pomona infections of
cattle.

The presence of a cytopathogenic agent produced by the lepto­

spirae is suggested.

This proposed agent is probably responsible for

alterations in the maternal-fetal placental relationship that interfere
with fetal development and may cause fetal death.

The placental

lesions were the most marked at about thirty days after exposure.
T ifteen days later regenerative hyperplasia of the maternal placental
epithelium indicated that the lesions were undergoing repair.
Sporadic infections of the bovine fetus by L. pomona occur but
probably are influenced by special conditions of virulence and sueceptibility.

The preponderance of the experimental evidence indicates

that fetal leptospirosis is not the major cause of abortions.
Two fetuses v/ere infected with Ij. pomona by direct inoculation
in utero. The fetal leptospirosis was similar to the postnatal infec­
tions.

Leptospiremia was observed in one fetus on post expo sure day 7

and by day 1 ^ the leptospirae were demonstrable in the kidney but
no other tissues.

The histopathological lesions of the placenta

resembled those associated with maternal infections.

3h

Five sows liad "been experimentally infected with L. pomona 10 to
lh months previously.

The sows were re-exposed during the last one-

half of the gestation period.

The immunity produced by the active

infection was sufficient to protect the pregnant sows against chal­
lenge with a virulent strain of L. pomona.
Active inflammatory processes persisted in the porcine kidney
for 10 to lh months after initial infection and 8 to 10 months after
leptospirae were demonstrated in the urine.
Although direct invasion of the porcine fetus has been con­
sidered the cause of porcine leptospiral abortions the exact mechanisms
of action on the fetus have not been determined.

L. pomona infections

in swine are very mild and frequently almost asymptomatic except for
abortion by the pregnant sow.

Further elucidation of the toxic

principles and the pathogenesis of the porcine abortions is indicated.
The chemical entities which are proposed as active in bovine abortions
may have similar effects on the porcine fetus.

Infection of the por­

cine fetus could be a manifestation of species susceptibility to the
invasiveness of the organism and the toxic materials produced either
in the maternal or fetal system.

35

TAB LB 1
Source and Type of Leptospira pomona Used in Exposure of Heifers
Heifer
Number

Source

Material

Passage*

Amount
ml

Number^
per ml

GROUP 1 - Wickard strain
Infected
5*1,8
Control 9

guinea pig

blood

10‘

guinea pig

blood

0

GROUP 2 - Wickard strain
Infected

07,08*88

calves

blood

Control 3

calf

blood

3 GP &
1 calf

10

GROUP 3 - Wickard L strain
Infected
^,7,10,11

hamster

blood &
tissue

Control 6

hamster

blood

h hamster

3^

10

-

3

* Serial passages of blood at pyrexia.
t Titration in hamsters, log10; less than 10 organisms infective dose.
^ 2 ml hamster blood subcutaneously 2h hours after original inoculum.

36

TABLE 2
Observations on Eebrile Response and Stage of Gestation

Group
number

Heifer
number

Maximum body temperatures
Degrees
Postinoculation
F.
day

Month of
testation

5

8

10b A

iii

1

8

102.5

8

8

3

10*+.5

*+

Control

9

5

100.9

H

2 Infected

07

8

1 0 3 .1

5

08

8

10*+.*+

7

88

9

10*+.0

6

Control

3

6

1 0 0 .6

8

3. Infected

*+

3

1 0 3 .0

7

7

3

1 0 6 .3

b

10

5

1 0 5 .1

5

11

1

1 0 5 .5

74

6

2

101.2

2+

1 Infected

Control

37

TABLE 3
Isolation of Leptospirae from Heifers and Fetuses

Heifer
number

Postinocu­
lation
£ s x ________

....... Isolations
Blood*
:
Animal
Cultural inoculat ion
%

of Leptospirae..
:________Tissues
Day of
necropsy Maternal

Fetal

GROUP 1
Infected 5
1

8

+

10

8
Control

9

10

23
34

-

5^

10

33^

08

8

37

88

9

40

3

“

17

Infected 4

7-10

30

7

7-10

45

10

7-10

60

11

7-10

42 calved normally

6

-

10

GROUP 2
Infected 07

Control

not attempted

+

GROUP 1

Control

* Culturally in media; guinea pig or hamster inoculation.
cultures on postinoculation day 5«
Hysterotomy to obtain fetal and placental specimens.
^ Died of unrelated causes.

+

First hemo-

38

TABLE L
Antibody Titers for Leptospira pomona

Heifer
number

Initial

Final
Titer*

Day

„ Fetal sera
Titer* and fluids

GROUP 1
Infected

Control

5

9

2

10

2

0

1

13

3

23

7

0

8

14

2

3^

5

0

0

5

0

0

9

GROUP 2
Infected

Control

07

13

1

35

3

0

08

10

3

37

5

0

88

12

5

40

5

0

0

17

o

0

3

GROUP 1
Infected

Control

4

6

1

30

5

0

7

5

1

45

5

0

10

6

2

60

4

0

11

5

2

42

6

0

0

10

0

0

6

* Reciprocal of the logarithm of the greatest serum dilution in
which at least 50 percent of the organisms were agglutinated or
lysed*

39

-■
v ti

FIGURE 1

Vsr-kv^\
Kr -•;•V

Bovine kidney from heifer experimentally infected with
li* Pomona at day 37 with marked lymphocytic infiltration
of periphery of focal lesion and less dense infiltration
in center. xlOO.

*+0

-V'* c .v'wr-7.
.

FIGURE 2

'•V.v/T''.:-, 'Mi

Bovine kidney from heifer experimentally infected with
•L.* Pomona, at day ^5 * Contraction of the cortex at
site of foci of lymphocytic infiltration. xlOO.

FIGURE 3* Bovine liver from heifer experimentally infected with
li- Pomona at day 3?» Small focus of lymphocytes
adjacent to the interlobular bile duct, xlhO.

'+2

v.y<

FlG-URE b. Eosinophilic infiltration of hepatic perivascular spaces
of bovine liver. xlOO.

43

FIGURE 5* Cotyledon at day 10. Separation of maternal and fetal
tissues? a) hemorrhage, b) tissue debris, and c) pyknotic
nuclei of diplokaryocytes. xlQO.

44

»
* ■'«.
!>

-■

<•

* * & > ••

i

/_

*V.j\,
*v
* ■ £ & * & • ’■*

-, ... * n
*,. *

*.

Si
.■

> * R
'

/ 'y
•1f* | w*,m

v\

v .#*•* ff
«v•I■•*

0*
*'

FlG-URH 6. Cotyledon at day 23. Necrotic fetal tissue with some
fetal cells adhering to the maternal epithelium. xlhO.

^5

v

FlG-UBE 7. Cotyledon at 23 days. Marked pyknosis of the maternal
epithelium and proliferation of fibrous connective
tissue. xlOO.

h6

0 <Q
- r.

* *

. »®
Vf

, V, • t , , ”

v
;

>

•
V

\ *,

' >

r

'*-*«

•’ : { ■ * / * , • . < V «'
* ' ® . i . ;.. •*'"
I „ ,«*, "V#u

r# ;>*

*

<

•.*

V-

^4
tr*Uw*. .# S
/i
V ~ v *.• -vii *V« •* f?
V

-j 4

$

.«V
v
iii " • >
/ *
- * - v t - y . - , y . t v
'* # '
-'■
“
, «4-Tk.
T * . 4*
.
‘ '.* \ . « v
,
‘. * , ft V
V f .. * ^
^
^ 1
V v
<» t* ' * ,
•

__

* *•#*.
w .

v

•
*

1

^.
k.

A
*,

FIGURE 8. Regenerative hyperplasia of maternal epithelium.
Bovine cotyledon day ^5* x280.

FIGURE 9. Nucleated erythrocytes in vessel of bovine fetus. xlb-O.

1(8

-v

*. *
v

:

S * 5 ^

‘I V -

•

.
\ #. -A W
*' Cvv *
, - •*
: W-. -.■
v* >
. ’
* .jr,i,,-7r"*• ^■ <*.
°\• v „
'!!* vc
* >if
*£
.
‘
T.
*
J V.
<£
> f • tf^*a
BTS.W > ’ o ’vV if",'**j ‘ *'1
'

,

2f*i

♦ aTc%4 •■
'■ »* ■ v . JWVh>*v.i

i f l - O v 4'
:'-%vl

tV •>

"5 ^

V

*■

FIGURE 10. Jja utero infection, cotyledon at day Ih, Absence of
fetal villi and maternal epithelium with increased
amount of connective tissue. x70.

49

J-W*-

I I * ’ f c •*:>;i ^ w

»n*

^

FIGURE 11. Kidney of "bovine fetus at day 7* Small foci of
immature lymphocytes and plasma cells. xlAO.

50

FIGURE 12* Cotyledon at day 7 postinoculation of bovine fetus
in utero» Diffuse placentitis. x?0.

51

'kV
:

w
.u » X :\w
. v # - v :€
a V j 2w
3 v •*' *

FIGURE 13.

Another area of diffuse placentitis.
day 7. xl^O.

Cotyledon at

52

FIGURE l^K Maternal bovine kidney at day 7 of jjn utero infection.
Lyir^hocytic infiltration of the minor calyces. x70.

53

FIGURE 15. Marked infiltration of lymphocytes in cortex of porcine
kidney, a) Note atr.ophy and disappearance of tubules,
b) Pyknosis of nuclei of tubular epithelium. XI3O .

5^

-rjfZ--J MrT-'

FIGURE 16. Extensive increase in fibrous connective tissue and
some lymphocytes "between tubules of medulla of
porcine kidney. xl30*

55

i, V‘|

* *

.

•**L

6

■#«

.®r

#

*

W

\
!

“

f

1

•w^s
*

f -

FIGURE 17. Higher magnification of figure 16. x360.

56

v

& %
h>

FIGURE 18. Medulla of porcine kidney to show fat vacuoles in
terminal portions of the proximal convoluted tubules
and some intertubular infiltration with lymphocytes.
xl30.

57

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