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PATHOGENICITY OF LEPTOSPIRA POMONA FOR HAMSTERS

By
Joyce Elaine Trier

AN ABSTRACT

submitted to the College of Science and.Arte.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
Year 1958

Approved:::§?fiéizgz:4odb¢V\~

0

 

Joyce E. Trier

The purpose of this study was to determine the effects
of 4 different strains of Leptospira pomona upon young ham-
sters, especially the bacteriological, serological, and
hematological aspects of the infection.

Four groups of 30 to 50 hamsters each, approximately
4 weeks of age, were infected with.;, omona, each.group
with a different strain of this serotype. One cc of a 1:5
dilution of guinea pig blood obtained at the febrile period
of the infection was inoculated intraperitoneally into each
animal. Control groups of animals were inoculated with.a
1:5 dilution of normal guinea pig'blood. At 2 or 3 days
after infection and at suitable intervals thereafter groups
of 5 animals were bled aseptically from the heart for dup-
licate cultures in:modified Chang's media. Throughout the
eXperiment, at intervals of several days, control hamsters
were also sacrificed and hematological and serological stud-
ies performed on the bloods. The hamsters were then sac-
rificed and the Jugular vein severed. One to 2 cc of this
blood was introduced into approximately 10 mg of powdered
heparin to be used for serological and hematological stud-
ies. The urine was observed for turbidity and in same cases
the benzidine test was carried out. Normal hamster urine
is turbid. It has been observed that urine of recently

infected rodents becomes clear.

Joyce E. Trier

2

Hemoglobin, erythrocyte, and leucocyte values were de-
termined from.the blood and in some cases hematocrit levels
were determined. Agglutination-lysis tests were performed
on the plasma samples obtained from the remaining heparin-
ized blood. After 2 and 4 weeks of incubation, the cultures
'were examined.microsc0pically for the presence of leptospirae.

Strain‘Wickard produced a subclinical infection in
hamsters with minor erythrocyte destruction and a mild leuc-
ocytosis. A high antibody titer was evidenced by day 13,
which helped remove the organisms before extensive harm
had occurred.

Strain'Wickard (hamster lethal variant) caused wide-
spread lysis of erythrocytes and a high.mortality rate as
early as day 6. Considerable leucocytosis occurred, but
the antibody titer remained quite low in the early stages
of the disease. The extreme lysis of erythrocytes with lit-
tle antibody protection probably caused death.

Strain Brooks produced a subclinical infection in hams
sters, and only minor hematological changes occurred. The
high antibody titer by day 6 probably resulted in destruc-
tion of the leptospirae.

Strain Ohio was lethal, causing severe central nervous
system disturbances and coma in all animals on day 7. There
was severe lysis of erythrocytes and only minor leucocytosis.
The lack of antibody develogment and the overWhelming effect
of the organisms on the animals probably were the main fac-

tors causing death.

PATHOGENICITY 0F LEPTOSPIRA PGMONA FOR HAMSTERS

By
Joyce Elaine Trier

A THESIS

Submitted to the College of Science and.Arts 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

AC KN O‘flEDGI-EENT S

The author wishes to express her sincere thanks to
Dr. Erskine V. Morse, whose guidance and interest made
this investigation possible. She is also grateful to Dr.
Lloyd C. Ferguson and Dr. Robert F. Langham for their help-
ful assistance, and to her husband, whose moral support

was mlmtede

TABLE OF CONTENTS

LIST OF TABLES....................................
INTRODUCTION......................................
LITERATURE REVIEW.................................
ZMATERIALS AND METHODS.............................
RESULTS...........................................
Part I. Strain‘Wickard.......................
Part II. Strain Wickard (Hamster Lethal).....
Part III. Strain Brooks......................
Part IV. Strain Ohio.........................
DISCUSSION AND CONCLUSIONS........................

SMIARYOOOOOOOIOO0.0...0......OIOOOOOOOOOOIOOOOOOO

T-ABIIESOOCICCOOOOOOOO0000......OOCOOOOOOOOOOOOOOOOO

WNCESOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO
umm.’I.0......00.0.0.0...OOOOOOOOOOOIOOOOOOOO

Page

12
12
14
15
16
18
23
25
29
32

TABLE
TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

II

III

VI

VII

VIII

LIST OF TABLES

NORMAL BLOOD VALUES 0F HAMSTERS.........

AVERAGE HBII‘IAT OLOGICAL AND AGGLUTINATION
VALUES FOR HAMSTERS INFECTED WITH STRAIN
WICMRDANDCOIITROIJSOOCOC.0...‘.........

AVERAGE HEMAT OLOGICAL AND AGGLUTINATION
VALUES FOR HAMSTERS INFECTED WITH STRAIN
WICKARD (HAMSTER LETHAL) AND CONTROLS...

AVERAGE HENATOLOGICAL AND AGGLUTINATION
VALUES FOR HAMSTERS INFECTED WITH STRAIN
BROOKS AND CONTROLS.....................

AVERAGE HEMATOLOGICAL AND AGGLUTINATION
VALUES FOR HAMSTERS INFECTED) WITH STRAIN
OHIO AND CONTROLSOOOOOOOOO0.00.00.00.00.

HEMATOLOGICAL AND SEROLOGICAL VALUES--
STRAIN WICMDOOOOIOOOOOOOOOOOOOOOOCOCOO

HEMATOLOGICAL AND SEROLOGICAL VALUES--
STRAIN WICKARD (HAITSTER LETHAL) . . . . . . . . .

HEMATOLOGICAL AND SEROLOGICAL VALUES--
STRAIN BRO0KS...........................

HEMATOLOGICAL AND SEROLOGICAL VALUES--
STRAIN mloeeeooeeooeeeeeeeoeeeeoeeeeeso

Page

25

26

2'7

28

53

S4

35

56

INTROD TCTION

The leptospiroses, in the last 15 years, have received
renewed and increased attention and interest throughout
the world. In the United States, due to the prevalence
of bovine and porcine Leptospira Ramona infections. research
efforts have been directed toward the elucidation of the
host-parasite relationship, epizootiology, and.public health
aspects of this group of contagions.

Some reasons for increased interest in the leptospiroses
are as follows: (1) rodents and domestic animals serve as
resevoirs of infection, (2) there are many newly discovered
species of Leptospira that infect both.man and animals,

(5) some diseases formerly thought to be viral in origin
actually have been found to be due to leptospirae, (4) the
leptospiroses may vary greatly in severity and assume biz-
arre clinical manifestations, and (5) the infections may
be mistaken for brucellosis, typhoid fever, poliomyelitis,

meningitis, and influenza.

LITERATURE REVIEW

Leptospirosis was first reported in farm.animals in
the United States less than 15 years ago (27). Today bov-
ine and porcine leptospirosis is present in all states.
However, long before this, other forms of leptospirosis
had been found in various parts of the world. In 1905
Stimpson observed'Weil's disease, or Lepto§pira icterohemr
ogghagiae infection of man (17). In 1923 canine leptospir-
osis was recognized in North.America by Kirkwood and Horning
(17). Sohofield (30) described hemoglobinuria in dairy
cows in Ontario, which.he concluded was due to Clostridium
‘wglchii. .Also in 1933 Rose and.Edgar (29) described ictero-
hemoglobinuria in calves in Australia and again concluded
that 9;. welchii was the etiological agent. Further work
failed to confirm these assumptions. These reports may
have been dealing with bovine leptospirosis.

ZMichin and.Azinov (21) in Russia reported spirochetal
jaundice in cattle in North.Caucasus during 1935. The or-
ganism which was isolated proved pathogenic for guinea pigs
and mice. The authors indicated that infection was spread
by the contamination of pastures. water, and salt licks
by urine and feces. and that insects and rodents played a

role in transmission. The agent was termsd.§, icterohaemr

oglobinuriae.

Brits, in 1942, reported in Kansas that a bovine hem-
oglobinuria of undetermined etiology affected cattle of
all ages.(4) . The next year Smith (31) described an "id-
iopathic hemoglobinuria" of cattle in Oklahoma with a mor-
bidity rate of 5 to 35 percent and a mortality rate which
approached 100 percent. It, too, affected animals of all
ages. The same year 3 I'mysterious" deaths occurred in a-
dult cattle in Connecticuttr(l'7). The symptoms were anor-
exia, subnormal temperature, and convulsions on approximate-
ly the sixth day of illness. The livers had a "nutmeg"
appearance. Multiple areas of sinusoidal congestion with
hemorrhages and necrosis of the liver pyrenchym were ob-
served. Levaditi stained sections showed leptospiral-like
bodies. This was the first report of bovine leptospirosis
in the United States. . .

L. pomona, the organism under study in this paper, was

first isolated in 1937 from a dairy farmer in Queensland,
Australia by Clayton _e_t_ 21. (5). A mild type leptospirosis
prevailed and in man was called the "seven day fever“.
The same organism was isolated from swine in Australia in
1942 by Johnson (16) , and was incriminated as the etiolog-
ical agent of Swineherd's disease by Gsell (12) in Switz-
erland.

Serological surveys have shown that L. pomona infect-
ions are widespread among cattle ('7, 1'7, 22, 23, 28) and
swine (3, 11, 23, 28). Isolations were made from a kidney

of an apparently healthy pig (3). Most porcine infections
are subclinicalg however, serious losses result in droves

of pregnant sows which abort. Leptospirosis is communic-
able among swine and transmission readily occurs to other
domestic animal species as has been shown experimentally.

In addition to £0 pomona, L. canioola and L. icterohemorr-
hagiae may be found as etiological agents in bovine (8,

15, 36) and porcine (9, 25) leptospirosis. It is, therefore,
imperative that the infecting serotype be identified in all
outbreaks of leptospirosis.

The course of leptospirosis is rather variable, but
in general it follows a basic pattern in susceptible hosts.
The incubation period is from 4 to 12 days , after which a
demonstrable leptospiremia occurs. At this time there is
a rise in body temperature to 104 to 106 1?, depending on
the species. Acute symptoms of diarrhea, anorexia, depress-
ion, and polypnea occur at this time. Hanoglobimria and
icterus occur at the end of the acute symptomatic phase.
These clinical manifestations occur at about '7 to 12 days
after infection. Antibodies also appear at this time and
remain at high levels for about 3 weeks.

At day 12 to 14 a chronic leptospiruria begins and
lasts about 3 months or longer, depending upon the host
species , the infecting serotype, and the pH of the urine--
pH 7.0 is optimal for the organisms. Kidney damage follows
and grayish white foci up to 1 cm in diameter are found. in

the cortices. Edema of the renal lymph nodes may also be
observed, as reported by Langham _e_§ EL. (18). MicrosoOp-
ically these foci contain lymphocytes, macrophages, and plas-
ma cells. Some fibroblasts and collagenous fibers are also
present. Degeneration and necrosis of the proximal and
convoluted renal tubules is a comes finding.

In pregnant animals abortion often occurs and does so
at 20 to 30 days after exposure.

Horton (24), in 1942, first reported on the suscept-
ibility of hamsters to leptospiree. in his work with L.
ganicola. These animals are most useful in isolating lep-
tospirae from nun and lower animals; however, Larson (19)
indicated that L. canicola killed hamsters. In 1944 Ran-
dell and Cooper (26) reported that the hamster was the an-
imal of choice for isolating leptospirae; guinea pigs and
mice are relatively resistant, while laboratory white rats
are totally refractory. Both L. canicola and L. icterohem-
oghagiae killed hamsters in 5 to 10 days.

Randy and Ferguson (13) reported that hamsters surviv-
ed infections with L. mona, Hardacre strain, following A
15 serial hamster passages. 0n the sixteenth passage the
animals died on the fourth day after inoculation. A vir-
ulence decrease for cattle was observed on the twenty-third
hamster passage.

According to Bauer (2), strain Wichard, which causes
a mild infection in hamsters, becomes lethal after 4 pass-

ages in media. .After 12 passages a steady decrease in vir-
ulence was noted. ‘He also found that 4, 7, and l5'week
old.hamsters were very susceptible to L. pomona, and that
the susceptibility of hamster erythrocytes to leptospira
hemolysin was not associated*with.the hemolytic effect of
the organisms upon the hamsters.

Very little work has been reported in the literature
concerning'hematological values of normal hamsters. Mat-
erial for Table I was taken from.a review of the literature
by Gardner (10), as contributed by Stein (33, 34), Stewart
(35), and.Hu (14).

This thesis deals with.the relationships between the
following‘bacteriological, serological and hematological
aspects of leptospirosis (L. moss) in hamsters during the
course of infection:

1. Hemoglobin, leucocyte and erythrocyte values of

hamsters infected with the various strains.

2. Cultures of the heart blood.which demonstrate pre-

sence or absence of leptospira in the blood.

3. Agglutination-lysis tests which.indicate antibody

formation.

4. Appearance of the urine, whether turbid or non-

turbid, and examination for hemoglobin and for

leptospirae.

TABLE I
NORMAL BLOOD VALUES OF I-LAMSTERS

 

 

 

 

meters mid Values
Age No , Erythrocytes Leuc 05 Hem0g5 Reported
sex EQanMMWM
20 days 2Ma1e 5.06 5.03-5.08 ' Stein
33-34 da 514 ' 5.96 5.36-6.69 Stein
36-37 da 4M 17.04 Stein
1-2 mo 12H 7.0 '7 ,470 16 .4 Stewart
l-2 mo 11F 7 .01 8 ,210 16 .9 Stewart
40-43 da 3M 6 .98 6 .4-7 .55 Stein
47-50 da 3M . 18 .3 Stein
57-62 (18. 6M 8.60 8.16-9.29 Stein
66-70 da 511 18.98 Stein
85-100 da 511 9 .14 9 .06-9 .22 Stein
2-4 no 2514 7 .50 8 ,150 17 .1 Stewart
2-4 mo 24F 7 .10 ‘7 ,870 16.4 Stewart
108-131 da 514 15 .65 Stein
140-226 da M 8.8 Stein
9 mo plus 2414-1? 7.5 8,560 17.6 Stewart
Adult 10 9 .57 10 ,070 Hu
Adult M 20.6 Stein

 

1 Multiply by 106 for erythrocytes per can blood
Leucocytes per cm blood
3 Grams hemoglobin per 100 cc blood

MATERIALS AND METHODS

In the first experiment 50 hamsters approximately 4
weeks of age were inoculated intraperitoneally with ‘._L_. 22-
m, strain Wickard. The inocula consisted of 1 cc of
a 1:5 dilution in sterile saline of infected guinea pig
blood obtained at the height of the febrile response. Strain
Wickard was isolated from the urine of an infected dairy
cow in Wisconsin during 1953 by Morse gt 2.1.:- (22). The
microorganism had been maintained in continuous guinea pig
passage to assure retention of virulence. This strain was
not lethal for hamsters. Thirty hamaters, which were sim-
ilarly inoculated with 1 cc of a 1:5 dilution of normal
guinea pig blood, served as controls for hematological and
serological data.

In the second experiment 35 hamsters, approximately
4 weeks of age, were inoculated intraperitoneally with 1
cc of a 1:5 dilution of guinea pig blood infected with the
Wickard strain (above) which had been found to be lethal
for hamsters, as described by Bauer (2) . Twenty additional
hamsters were inoculated with 1 cc of a 1:5 dilution of
normal guinea pig blood to serve as controls.

In the third experiment 30 hamsters, approximately
4 weeks of age, were inoculated intraperitoneally with 1
cc of a 1:5 dilution of guinea pig blood infected with L.

pomona, strain Brooks. This was the sixth guinea pig pas-
sage of this microorganism following isolation from bovine
urine in Michigan. Similarly, 20 hamsters were inoculated
with a 1:5 dilution of normal guinea pig blood to serve as
controls.

In the fourth experiment 30 hamsters, approximately
4 weeks of age, were inoculated intraperitoneally with E-
pomona, strain Ohio. The inocula consisted of 1 cc of a
1:5 dilution of infected guinea pig blood. Twenty hamsters,
to serve as controls, were similarly inoculated with 1 cc
of a 1:5 dilution of normal guinea pig blood.

Determinations as to the exact number of leptospirae.
present in guinea pig blood inocula were not made. However,
previous work in this laboratory has shown that 104 to 105
leptospirae per cc are present in the blood of guinea pigs
at the height of the febrile response (105-106 F).

In each of the four experiments, groups of five animals
were sacrificed 3 days after inoculation and at appropriate
intervals thereafter. The intervals depended upon the course
of infection. A11 sick or moribund animals were killed as
soon as detected. The hamsters were anesthetized with et-
her and then bled aseptically from the heart on the days
when leptospiremia was expected. Blood was inoculated in
modified Chang‘s media (23) which contained 10 percent ster-
ile bovine serum and .01 percent hemoglobin (Difco). Two

screw cap tubes, each containing 10 cc of media were inoc-

10

ulated with 0.02 to 0.1 cc of blood. The cultures were
incubated at 30 C and examined for the presence of lepto-
spirae at 2 and 4 weeks employing darkfield microscopy
(590x).

Hamsters were exsanguinated and the blood collected
in tubes containing approximately 10 mg of powdered hepa-
rin. Approximately 1-2 so were obtained in this way for
hematological and serological examinations.

The urine was collected where possible and examined
for turbidity and the presence of leptospirae by darkfield
microscopy (590K).

Hemoglobin values were determined by placing 0.02 cc
of the heparinized blood in a tube containing 5 cc distil-
led water. A drOp (0.025-0.04 cc) of ammonium hydroxide
was added per tube and the tubes were shaken vigorously for
10 seconds. The tubes were then allowed to stand 10 min-
utes and were read on a Bausch and Lomb Spectronic 20 at
465 mu. This is an oxyhemoglobin method.

Erythrocyte counts were made using a Spencer Briteline
counting chamber. Heparinized blood, 0.005 cc, was diluted
to 1.01 cc with 0.85 percent saline or Hayem's solution in
a red cell pipette. Five-hundredths cc of heparinized blood
was diluted to 1.1 cc with a 1 percent acetic acid solution
for counting leucocytes.

The agglutination-lysis test was conducted on all plas-

ma samples. Living cultures of L. pomona, E. canicola,

11

and L. icterohemorrhagiae AB were used as antigens in the
tests. The plasma was diluted from 10'1 through 10's, the
antigen added, and the tubes were incubated in a 3'7 0 wat-
erbath for 2 hours. The mixture was then examined for ag-
glutination-lysis using a microscope with an Abbe conden-

sor fitted with a star diaphragn (100X) . This provided
a modified darkfield arrangement.

RESULTS

Part I. Strain Wickard

The Wicleard strain produced only minor hematological
changes following intraperitoneal inoculation in healthy
young hamsters. During the first 13 days after inoculation,
the hemoglobin and red cell values were quite constant,
varying from 15.1 to 17.1 gn hemoglobin, and from 6,650,000
to 7,352,000 red blood cells. These were minimal and max-
imal averages of the groups of 5 sacrificed animals for the
different days.

At the same time the controls ranged from 16.1 to 16.5
gm hemoglobin and 7,042,000 to 7,142,000 erythrocytes.

0n the fifteenth day a decrease in the hemoglobin and
erythrocyte values occurred for infected animals; 11.3 to
15.7 gm hemoglobin as the range and 13.5 as the average.
The erythrocytes ranged from 4,230,000 to 6,430,000 with
an average of 5,464,000 cells. On the twenty-first and
twenty-fifth days the counts were slightly elevated (Table
II).

The leucocyte counts showed considerable variation.
The second day the values were very low for some unexplain-
ed reason, perhaps stress. The levels at this time aver-
aged 3,825. Aside from this, they remained at about 6,000
to 7,000 until day 10 when an increase to 10,300 was obser-

13

ved. A gradual decrease occurred until day 25, when again
an increase to 11,420 was noted. The controls remained
quite constant at approximately 5,000 to 7,000 per cm.

Antibody titers, as determined by agglutination-lysis
(A-L) tests, rose slowly from day 7 until day 13, when a
maximum of 10'”6 was recorded. At this time there was some
drop in red blood cells and hemoglobin. The titer decreased
slightly in the following days (Table II). Plasma from the
non-infected animls was used as a negative control and it
remained serologically negative.

The cultures were examined at the indicated time in-
tervals. Three of 5 animals were positive as early as day
2 following inoculation. On day 7 only one of five was
positive, and thereafter no positive cultures were obtained.
Therefore, 5 days before any drop in hemoglobin and eryth-
rocytes, the leptospirae were not present in the blood and
had presumeably become localized in the kidneys. However,
no leptospirae were found in the urine by darkfield examin-
ationat any time. Hemoglobin was not found in the urine
as determined by the benzidine test. The animals did not
show any clinical symptoms or signs. The titers started
to decline and the blood values approached normal. This
would indicate that a subclinical disease occurred in ham-

sters infected with L. pomona, Wickard.

14

Part II. Strain Wickard (Hamster Lethal)

A variant of the'Wickard strain, lethal for hamsters,
was isolated by Bauer (2). This variant, when inoculated
intraperitoneally into hamsters, gave rise to more pronoun-
ced hematological changes than the nonlethal parent strain.
Hematological values until day 6 were fairly constant and
compared closely with the controls. On day 7 the animals
‘were'moribund and one died. Hemoglobin values averaged 11.9
gm with an average of 5,587,000 erythrocytes which.repres-
ented a decrease of 3.4 gm hemoglobin and 1,517,000 red cells
from the averages for the animals sacrificed on the prev-
ious day. That evening 4 more animals were found sick and
sacrificed. The hemoglobin and erythrocyte values decreased
to 10.5 gm.and 5,362,000 respectively, which.was a signif-
icant depression in a few'hours. One animal was ill on
day 8: the red cell count was depressed to 3,980,000. For
the next 2 days the hemoglobin and red blood cell counts
rose to 21.1 and 9,250,000 respectively, due to‘hemoconcen-
tration. These animals were moribund and very little blood
could be obtained at sacrifice.

The leucocyte counts rose from.an average of 8,090 on
day 6 to 11,430 on day 7, and then to 28,300 on day 8.

Then they decreased to averages of 7,120 and 6,300 on days

9 and 10 respectively, but increased slightly to over 9,000
on days 11 and 12. The 4 remaining animals appeared normal
on the-2 last days of the experiment, days 11 and 12 (Table
III).

15

On day 6 the urine of 4 of 5 animals appeared non-tur-
bid and 2 samples were positive for hemoglobin. The turbid
or normal urine of 1 hamster was negative for hemoglobin.
It was not possible to obtain urine from 2 animals.

On days 7 and 8 urine samples were red in color: on
days 9 and 10 urines were non-turbid again, but the benzi-
dine tests were positive. On days 11 and 12 the urines
'were still partially cloudy or approaching the normal.

The antibody titers rose gradually to 10"7 on day 7
and 10"8 on day 9. They decreased to 10"5 and 10"4 on days
11 and 12 respectively. There were positive blood cultures
on 4 of 5 animals on days 3, 5, and 6. It was not possi-
ble to obtain sufficient blood on succeeding days to make
suitable cultures. The agglutination-lysis tests for the

controls were negative throughout the experiment.

Part III. Strain Brooks

The Brooks strain of L, pomona produced slight hema-
tological change in hamsters. The'hemoglobin and red cell
values and leucocyte counts compared closely with.the con-
trols through day 20.- The average values of the infected
as well as the control animals were somewhat lower than
those of the hamsters used in Part I and Part II.

The antibody titers increased from.10'1 on day 3 to
10'6 on day 6. These receded to 10‘5 on days 10, 15, and
20.

16

Media were inoculated with the blood from all animals
but none proved to be bacteriologically positive. No clin-
ical symptoms were evidenced and the hematocrit values amm-

pared quite well with those of the controls (Table IV).

Part IV. Strain Ohio

The Ohio strain, although lethal for hamsters, pro-
duced little observable change in the blood constituent
values. However, on some days considerable variations among
different animals did occur. Through day 6 the values were
found to be quite normal: but on day 7 the range varied from
9.3 gm hemoglobin to 19.9 an and from 4,910,000 to 9,320,000
red cells. These animals were moribund, and the hematocrit
value for the animal having 19.9 gm hemoglobin was 63 per-
cent. This was considerably higher than the expected nor-
mal of 46 percent, and indicated considerable hemoconcen-
.trati0n (Table V).

Other hamaters killed at 1:00 P.M. and 4:00 13.11. of
day 7 had hemoglobin levels of 18.1and 16.9 gm, while the
red cell counts were 7,063,000 and 7,440,000 respectively.
Controls on day '7 averaged 16.0 gm hemoglobin and 6,743,000
red blood cells. Again hemoconcentration was in evidence
for the infected group.

The white cell count rose from 7,350 on day 6 to 16,040
on day 7 at 8:00 A.M., while the maximum was 26,000. The
averages remained high for the remaining hamsters killed
on that day.

17

The antibody titer rose to 10‘5 on day 7 (4:00 P;M.).
Cultures were positive in most cases, for 28 of 30 animals,
from.day 5 through day 7. The animals became moribund and
died before the agent left the blood.

As early as the fifth day, the animals showed extreme
central nervous system disturbances. The legs stiffened
in extensor rigidity, the head was held back, and paralysis
occurred. In some cases the animals trembled violently.
The urine was non-turbid but did not contain visible hemo-

globin.

DISCUSSION AND C ONCLUSIONS

The hemoglobin values obtained using the Wickard strain
as the infectious agent varied only slightly from the con-
trols. The values remained constant until day 15, and then
decreased to a minimum of 11.3 gm. Shortly thereafter a
rise occurred, indicating that hemoglobin loss was slight,
transient and probably minimal. The erythrocyte values
dropped prOportionately; 4,230,000 being the lowest noted
on day 15 when again the normal level was gradually attain-
ed. This suggests that smme degree of lysis of cells occur-
red around the fifteenth day, but this, too, was transient.
This reaction was probably caused by the presence of lepto-
spiral components in the blood which were released.when the
organisms were lysed by the antibody. The antibody titer
rose significantly on day 13, which would tend to support
this concept. It is assumed here that decreases in hemoglo-
bin and erythrocytes is due to lysis of the cells; however
it is possible that some of this may be caused by a depres-
sed state of the blood forming organs due to the infection.

A leucocytosis was evidenced on day 10. This may have
been stimulated by the migration and localization of
leptospirae in the kidneys. The diffuse nephritis produced
during renal residence would tend to lead to a leucocytosis.
[Approximately 3 days would allow for multiplication and

develoPment of renal inflammatory reactions.

19

Strain Wickard caused a subclinical infection in ham-

sters as shown by the following observations:
1. There was no apparent illness, and.no apparent
loss in body weight.
2. Leptospiremia was of low degree and insufficient
organisms were present at day 15 to cause a
significant drOp in hemoglobin and red cell values.
Hematuria or hemoglobinuria was absent.
3, A relatively mild leucocytosis occurred, which
suggests that renal inflammatory reactions due to
leptospiral colonization had occurred on day 15.
4. The high titer which.developed by day 13 was
responsible for removing leptospirae from.the
blood. I
The infected hamsters reacted quite differently to Wickard
strain which is lethal to hamsters. The hemoglobin levels
remained quite constant until day 7, at which.time the values
fell rapidly to 11.9 gm. ‘Within 9 hours the average levels
were 10.5 gm; the lowest was 7.3 gm. The red cell counts
decreased accordingly; the lowest was 4,100,000.

On day 6, 4/5 of the animals were positive and on day
7 in the morning, 2/3 were positive. Hereafter it was imp
possible to obtain enough blood for cultures; therefore, it
was difficult to conclude whether the organisms were begin-
ning to leave the blood on day 7. The enormous numbers of

organisms in the animals probably led to the lysis of

20

erythrocytes, and the leucocytosis which reached 28,300
cells probably resulted from.the severe nephritis.

Hemoconcentration was observed When the animals became
moribund. This may have been a result of dehydration due
to the toxic effects of the disease. Accurate hematological
determinations were difficult.

The antibody titers increased, but not fast enough to
counteract leptospiremia whidh eventually overwhelmed the
animals.

It is suggested that death occurs with this strain due
to:

1. Lack of development of antibody titer during the

earlier phases of infection.

2. The considerable number of organisms which caused
widespread lysis of erythrocytes.

3. Erythrocyte destruction was further evidenced by
hemoglobinuria as indicated.by positive benzidine
teets and the red color of the urine.

The Brooks strain also produced a subclinical infection
since the hamsters did not become visibly ill or moribund,
Also significant decreases in hemoglobin or red cells were
not noted, which suggests that there were insufficient
organisms present to cause lysis of erythrocytes. On day
20 two samples were lower than normal. ‘At this time lep-
tospirae would not normally be present in the blood. The
titer increased quickly to 10's. This may'also have influ-

21

enced the course of the infection. Since cultures of blood

were negative, it might be conjectured.that the leptospirae

‘were removed from the blood before day 3, and were localized
in the kidneys.

Leucocyte counts remained low in this experiment which

indicates that the kidney infection was probably minimal.

The Brooks strain was determined to be relatively non-

pathogenic for hamsters as evidenced by:

1. Lack of lysis of erythrocytes.

2. Only minimal concentration of organisms in the
kidney and probably a low grade renal damage as
evidenced by a normal leucocyte count.

3. Rapid increase in antibody titer to 10"6 on day 6.

Strain Ohio was shown to be very pathogenic for hams-

ters, since death often occurred by day 7. The hemoglobin
and erythrocyte values decreased to a low of 9.3 gm.hemo-
globin and 4,910,000 red cells on day 7, after normal val-
use were recorded on day 6. Shortly thereafter increases
to 20 gm.hemoglobin and 9,300,000 erythrocytes were observ-
ed which.indicated hemoconcentration. The hematocrit values
substantiated this conclusion. The antibody titer increas-
ed to 10‘; on day 7. This lack of antibody may have hast-
ened death.by allowing the leptospirae to overWhelm the
hamsters. Blood cultures were all positive through day 7,
which indicated that the agent was still present in the

blood at the time of death. In contradiction the average

22

‘white cell count rose, which.mey indicate kidney localiza-
tion as well as severe generalized infection. Upon closer
examination, however, it can be seen that the high White
cell counts occurred only where hemoconcentration was evi-
denced. '

Hon-turbid urine, which.is abnormal, was Observed for
hamsters Which died. The urine would.have undoubtedly'be-
came red due to the presence of large amounts of heInOglo-
bin, had the animals survived. Death.oocurred before org»
anisms left the blood and migrated with.definite renal lo-
calization.

It is suggested that hamsters die of infection with
Strain Ohio due to:

1. Lack of antibody develoyment to prevent the over-

whelming leptospiremia.

2. Lysis of erythrocytes.

3. Damage to the central nervous system, as evidenced

by coma and paralysis. This:may be due to toxic
principles elaborated.in,13!g,‘but not demonstra-

ble in vitro.

SIM/1353'!

The purpose of the investigation was to ascertain the
hmtological and serological effects caused by infection
of hamsters with L. pomona, and to examine the differences
in pathogenicity of various strains of this serotype.

Four experiments were conducted. In each 50 to 50
young hamsters were inoculated with L. pomona, strain Wick-
ard, strain Wichard (hamster lethal), strain Brooks, or
strain Ohio. At indicated intervals, hamsters were sacri-
ficed and bacterological, serological, and hematological
studies perfomed.

Strain Wickard caused only minor hematolOgical changes,
but marked antibody titers developed by day 15. Beginning
on day 10 and continuing to the conclusion of the experiment,
small decreases in erythrocyte and hemoglobin values occur-
red; and small increases in white cell counts were noted.
This strain was shown to produce a subclinical infection
in hamsters. _

Strain Wickard (hamster lethal) produced much more se-
vere hematological damage. The organisms produced death
within '7 to 10 days. The blood constituents were as low
as 7.3 gs hemoglobin and 3,980,000 red cells. The leuco-
cyte counts increased moderately. Antibody titers of 10'7

were observed on day '7.

24

Strain Brooks produced very little erythrocyte lysis
or observable illness in hamsters. Antibody levels increas-
ed to 10‘5 by day 7, which may have had a limiting effect
upon the infection. It is suggested that the very mildest
of subclinical infections occurred.

Strain Ohio infections proved to be the most pathogen-
ic and resulted in considerable hematolOgical change. Min-
imal levels of 9.3 an hemoglobin and 4,910,000 erythrocytes
were observed. A leucocytosis occurred but this may have
been the result of hemoconcentration. This strain was prov-
ed to be lethal for all animals by day '7. Severe central
nervous systan disturbances were noted. Failure of the host
to produce agglutinin-lysins may have been responsible for,
or at least contributory to, the lethal effect. The high-

est titers observed were 10's.

TABIE II

AVERAGE HEMATOLOGICAL AND AGGLUT INATION VALUES FOR I-LAI'ISTERS

INFECTED‘WITH STRAIN'WICKARD AND CONTR0L3*

 

Day

2

20'

8C
10
13
15
150
21
25
25C

 

Hemg- 1 Eryth- 2 Leucoz Aggl'n gult Remarks
16.5 6,650,000 5,825 Neg. 5/5
16.5 7,110,000 5,200 Neg. ---
15.6 7,046,000 6,650 Neg. 0/5
16.5 6,870,000 5,840 Neg. 5/5
15.8 6,980,000 6,700 10'5* 1/5
17.1 7,552,000 6,450 10'5 0/5
16.1 7,042,000 5,940 Neg. ---
15.1 7,250,000 10,500 10‘4 0/5
15.5 6,856,000 9,840 10‘7 ---
15.5 5,464,000 7,020 10"6 ---
16.2 7,142,000 6,950 Neg. ---
15.0 6,910,000 7,970 10-6 ---
14.5 6,107,000 11,420 10".6 --- 6 Hamsters
16.2 7,584,000 6,590 Neg. ---

 

GNP *

Values based on the average for 5 animals sacrificed

on the various days except where noted.

Grams hemoglobin per 100 cc blood.

Number of cells per cm blood.

Nmnerator--mmber of animals with positive cultures.
Denominator-number of animals tested.

0 represents values for control animals.

1' Majority of reactions in all tubs dilutions 25% agglut-

ination.

TABLE III

26

AVERAGE HEMATCLOGICAL AND AGGLU'I‘INATION VALUES FOR HAMSTERS

INFECTED WITH STRAIN WICKARD (HAMSTER IETHAL) AND CONTROLS“

 

 

Hemo-

Eryth-

Leuco-

Aggl ' n

Cult-

__

 

 

Day globin:L rocytes cartes‘2 Titer ures:5 Remarks

5 14.9 7,126,000 6,410 10"1 4/5

50' 15.8 6,940,000 6,640 Neg. ---

5 16.0 6,898,000 6,940 10"?" 4/5

6 15.5 7,104,000 8,090 10-48 4/5

60 15.7 7,290,000 7,050 Neg. ---

7 AM 11.9 5,587,000 11,450 10"5 --- 5 Hamsters, M

7 PM 10.5 5,562,000 10,900 10'“7 --- 4 Ham.. M

8 15.5 5,980,000 28,500 10'"7 --- 1 Hash, M

8c 16.7 7,552,000 7,170 Neg. ---

9 20.0 9,055,000 7,120 10‘8 --- 2 Han” M
10 21.1 9,250,000 6,500 10'8 --- 1 Ham., M
11 18.8 8,155,000 9,575 10"5 --- 2 Ham.
12 18.0 7,790,000 9,125 10'4. --- 2 Ham.
120 15.2 6,560,000 7,110 Neg. ---

1_ * Values based on the average for 5 animals sacrificed
on the various days except where noted.
1 Grams hemoglobin per 100 cc blood.
g Number of cells per mm blood.

Denominator-number of animals tested.
' C represents values for control animals.
f Reactions in all dilutions no greater than 25% aggl'n.
fl Complete or 100% agglutination no higher than 10" .

M Moribund.

numerator-number of animals with positive cultures.

2'7

TABLE IV
AVERAGE HEMATQLOGICAL AND AGGLUTINATION'VALUES FOR.H4MSTERS
INFECTED WITH STRAIN BROOKS AND CONTROLS”

 

 

 

 

 

my 25311 25252 3:25:35 4453:? 23:25
5 14.6 5,626,000 8,510 neg. 0/5
50' 14.4 6,105,000 7,570 Neg. ---
6 14.5 5,894,000 7,070 10"6 0/5
60 13 . 7 6 .460 .000 6 .600 Neg. ' «-

10 15.6 5,552,000 6,920 10"6 0/5

100 15.5 6,515,000 7,600 N53. ---

15 14.9 6,446,000 6,940 10"6 0/5

150 15.6 6,707,000 7,150 Neg. ---

20 14.9 6,541,000 7,185 10"6 0/5

200 16.0 7,555,000 6,470 Neg. ---

 

* Values based on the average for 5 infected animals
and 3 control animals sacrificed on the various days.

1 Grams hemoglobin per 100 cc blood.

2 Number of cells per cm blood.

3 Ntmeratorummber of animals with positive cultures.
Denominator-number of animals tested.

' 0 represents values for control animals.

28

TABLE‘V
AVERAGE HEMATOLOGICALAAND.AGGLUTINATION'VALUES FOR HAMSTERS
INFECTED WITH STRAIN OHIO AND 001118018"

 

 

 

Day Hsmo- Eryth- Leuco- Aggl'n Cult- Runarks
globinl ‘ rocytes‘3 -1 gfiesz 1731333“ 33991“ b
5 15.0 6,508,000 6,660 Neg. 1/5
50' 14.7 6,270,000 7,820 Neg. ---
5 14.9 6,046,000 6,950 Neg. 5/5
50 ' 14.9 6,575,000 6,850 Neg. ---
f
6 15.8 6,656,000 7,550 10-1 4/5

7 All 15.5 7,287,000 16,040 107-?f 5/5 Moribund

fl
7 1PM 18.1 7,065,000 18,558 10“? 7/7 7 Hamsters. M

7 4H: 16.9 7,440,000 16,610 10'2“ 6/7 7 Ham" M
70 16.0 6,745,000 7,280 Neg. ---

* Values based on the average for 5 infected animals
and 3 control animals sacrificed on the various days.
1 Grams hemoglobin per 100 cc blood.
2 Number of cells per cum blood.
3 Numerator--number of animals with positive cultures;
' Denominator-number of animals tested.
0 represents values for control animals.
M Moribund. ,
‘t Reactions in all dilutions no greater than 25% aggl'n.
it Only one serum had 50% at 10'1; other reactions all
25% agglutination.-

1.

2.

3.

4.

5.

6.

'7.

8.

9.

10.

11.

12.

29

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' tospira pomona Infection. J.A.V.M.A., 124. (19 :
4 ' 0

Bauer, D. C. Studies on the Virulence of Le tos ira
omona. M.S. thesis. Michigan State UniversI't'y.
East Iansing, 1957.

Bohl. E. H. and Ferguson, L. C. Leptospirosis in Dom-
eStic Animals. J.A.V.MOAO’ 121, (1952):421'4280

Brite. Allen. A Bafflin Disease of Midwest Cattle.
Vet. made, a, (1942 3386-388.

Clayton. Ge Es Be, Derrick. Es He, and CilantO, R. W}
The Presence of Leptospirae in a Mild Type (Seven
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Collier. W. A. and Mochter, A. Die Komplementbindung
bie der Diagnose der Leptospirosis in Niederland-
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572.

Erdheim. M. Leptospirosis in Cattle. Some Selected
Herd Histories. No As Vet., ii, (1953)322‘240

Field. H. I. and Sellers. K. C. Leptospira icterohaem- )
orrha iae Infection in Calves. Ve . Rec. __62 (1950 :
5—‘65—11-51 . ' '

Field, H. I. and Sellers. K. C. Legtosgira icterohasm-

orr§giae gnfection in Piglets. e . Rec.. Q3.
3 " 1.

Gardner. M. V. The Blood Picture of Normal Laboratory
Animals--Review of Literature. 1936-1946. Biochem-
ical Research Foundation. Newark, Delaware.

Cochenour. w. 8.. Jr.. Johnson, H. V.. and Ya er, . H.
Porcine Leptospirosis. A..T.V. Res.. 15, 1952):
158-160.

Gsell. 0. Le tos ira omona die Schweinehuterkranhheit.
Schweiz. Me . Wschr.. ___'76 . (1946):25'7.

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14.

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17.

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22.

23.

24.

25.

26.

30

Hamdy, A. H. and‘Ferguson. L. C. Virulence of Lepto-
8'011‘9. mom in Hamaterss AstVs R350 , E; 1 7)
33:15. '

Hu. C. H. and Pai. H. C. Chinese Med. J. Suppl.. (1938):
131.

Ingram. P. L.. Jack, E. J.. and Smith J. E. An Outbreak
of Le tos ira icterohaemorrha iae Infection in Calves.
Vet. Eec.. 5;, (I952):335.

Johnson. D.‘W. The Discovery of a Fifth Type of Lep-
tOBpira. MCds Jo 1111811., 2:, (1942)3431'4339

Jungherr. E. Bovine Leptospirosis. J.A.V}MQA.. 105,
(1944):276-281. “'

Langham. R. F..‘Morse. E. V.. and Mortar. R. L. A.J.V.Res.,
lg. (1958):395-400.

Larson. C. L. Experimental Leptospirosis. Pub. Hea. Rep..
52. (l944):522-527.

Harsh. H. Leptospirosis in Bovine Icterohemoglobinuria.
J.A.V4M.A.. 107. (1945):119-121.

Michin. N. A. and.Azinov. S. A. Spirochetal Jaundice
of Cattle in North Caucasus (translated title).
Sovyet Vet., 19. (1935):25-27. Original not seen.
Abstract in Vet. Bull.. 13, (1942):551.

Mbrse. E. V...Allen, V.. Krohn, A. F.. and Hall, R.
Leptospirosis in‘Wisconsin. I. Epizootiolo and
Clinical Features. J.A.V}M.A., 127. (1955 :417-
421.

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

IMorton. H. E. Susceptibility of Syrian Hamster to Lep-
tospirosis. Proc. Soc. Expt'l Biol. Med.. 43, (1942):
566-568.

Nisbet. D. I. Leptospira icterohaemorrha iae Infection
in Pigs. J. Comp. Path. TE r.. 5;. (1951):155-160.
Randall, R. and C00per. H. The Golden Hamster as a

Test Animal for the Diagnosis of Leptospirosis.
Science, 100. (l944):522-527.

2'7.

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51

Reinhard. K. R. Bovine Leptospirosis. Symposium on
the Leptospiroses. Med. Sci. Pub. No.1..ArmyiMed.
Serv. Grad. School. (l952):190-192

Reinhard. K. R. Present Knowledge and Concepts of Lep-
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487-493.

Rose. A. L. and Edgar. G. Entero-toxemic Jaundice of
Sheep and Cattle. Aust. Vet. J.. 12, (1936):212-
220.

Schofield. F.‘w. Bovine Hemoglobinuria Associated with
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Stewart. M; O. Florio. L.. and Mugrage. E. R. J. Expt'l
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van der Hoeden. J. LeptosEira canicola in Cattle.
J. Comp. Path There, _____, 1955;:§:§-2839

APPENDIX
Detailed Data Pertaining to the Investigations

TABLE VI
fI-IEI’IATOLOGICAI. AND SEROLOGICAL VALUES--STRAIN WICKARD*

fiem o- Erythr - Leuco Aggan
Day globin1 cyteg “cytess Titer Cultures4 Remarks

 

 

 

 

H 17.1 7.03 4.900 Neg. 3/5 l++x
2 M 16.2 6.65 4.400 11680 2 "'
L 15.3 6.11 2.900 Neg. 2 --
. H 1 g o 0 ,1 0 63. ""
20 M 16.6 7.08 5,150 Neg.
L 15 '7 6 75 4 350
H 16.4 7.58 .700 Neg. 5(-.7
H 15. 7 7.02 6. 600 Has.

 

3 '7 6 68 5 500 Ne
M 16.2 7.01 5,500 Neg. 2 ("T
L 15 5 6 06 4 450 A
H 16.6 10- +1
7 M 16.27.20 7,550 10-3 --

 

 

 

L 15 7 5 76 4 100 10-2
o o s "1
8 M 16.6 7.45 5,650 10:5
L 15 5 6 78 4 400 0-3
""""H" 15.1" ”8'45" """'“"7,“_"Looo Neg . «-
8c M 16.2 7.02 6,250 Neg.

 

L 15 .3 5 .83 4 .500 Neg.
H 6. e s - (")

10 M 15. 7 7.65 10, 050 10-4

 

 

 

 

 

 

15 0 6 41 9 60010'4
*‘T “-16.2 2.5—7—1620-000 ---
M 15.56.68 9, 550 10-6
4 5 6 25 7 650 0"5
W3 1 . 6. 00 ---
15 M 15.7 5.75 6,150 10-5
L 11.5 4.25 4.0%0 10"5
H 17.6 .83 ' 0 N68. nun-s
150 M 15.7 7.55 7,000 Neg.
E 15.5 5.91 5.700 Negs
s s g ""
21 M 15.0 6.70 7,950 10"6
a 15.7 6.17 6.250 10-5 A
1 e e g I 0 """
25 M 14.5 6.05 15,550 10'6 6 Hamsters
E 12.0 4.85 6.750 .0 5 e
e e g 08. "'
250 n 16.6 7.68 6,700 Neg.
L 14.1 6.10 4.000 Neg.

 

*Values represent the high. median and low values.

1 Grams hanoglo in per 100 cc blood.

2 Multiply x 10 for red cells per cm blood.
Number of white cells per cm blood.

4 Number ositive animals/number animals tested.

‘3‘ Duplicae tubes; number animals per tube combination.
C represents values for control animals.

TABLE VII

HEMATOLOGICAL AND SEROLOGICAL VALUES--

STRAIN WICKAR'D (HAMSTER LETHAL)*

34

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Day Hemo- 1 Erythr - Leucoa A881'“ Cultures4. Ranarks
globin _cytes Mes Titer
H 15 .5 7.55 8,550 10-1 4/5 4 + «334
5 N; 15.0 7.08 6,550 10-1 1 ~-
4 5 6 46 4 600 Ne
e O 9 38- "'
50‘ M 15.7 7.08 6,200 Neg.
15 o 6 45 5 600 Ne; A
7 ;. , z 0,. s o-
5 N: 16.2 6.95 6,600 10-1 5-+-)
L 15 5 6 40 5-800 1.-1 --
1. .3 "800 10. ' 4'-)
6 n: 15.5 7.20 8,500 10-4 1 --)
5 0 6 45 6 900 10-2
s e 9 N08. ""'"'
60 1M 15.7 7.40 6,600 Neg.
L 5 5 6 92 6 150 Ne
e e g - "'" THEMSLBI‘E
7 AM MI 12.0 5.78 11,500 10”6 1 dead. 5 111
L 10 1 4 50 10 150 10-5 Bloody urine
4. Q , - -‘- I Hams era
7 EMIM 10.1 4.55 10,850 10-8 1 dead. 4 111
L 7.5 4.10 8.200 10-5 Bloo§y urine
8 15 .3 3.98 28.300 10-7 "’ 1 i1
Bloody urine
0 s 9 68. ""
8C MI 16.6 7.25 7,150 Neg.
6 2 7 02 5 800 Ne
H . . , 0 - --- ‘2’111"“'
L 19 8 50 6 550 10-7 Urine clear
15 21.1 9.25 6.350 10'7 --- hematocr -62%
1 111
II”'TH"I9:9“’ 8.45 ‘97755"‘10=5:: --- No --
L 17 6 7 82 9 000 10-5 Urine turbid
1 1 19. . 0 ,000 0-0 --- Not {11732
L 16.5 7.18 8.250 10'4 Urine turbid
H 160 O . Neg. “'-
120 M. 15.5 6.62 7,800 Neg.
L 15.7 5.55 4.850 Neg.

 

* values represent the high. median and low values.
1 Grams hemoglobin per 100 cc blood.

2 multiply X.105 for red cells per cmm blood.

5 Number of white cells per cmm'blood.
Number positive animals/number animals tested.

f Tubes in duplicate; number animals per tube combination.
C represents values for control animals.

TABLE VIII
Im’LATOLOGICAL AND SEROLOGICAL VALUES-~STRAIN BROOKS“.

 

 

Hano— Eryth- Leuco- Aggl'n 4 :q 1:
Day globinl rocytes2 cytess Titer Cultures -mar s

 

 

 

 

 

 

 

 

 

 

 

H 15.7 6.27 9,250 Neg. 0/5 5(--)x
5 M 15.0 6.01 8,650 Neg.
L 12.5 4.51 6.850 Neg. g
' H 1 .0 6.71 ' .800 lies. ---
3C M 15.0 6.42 7.000 Neg. 3 Hamsters
L 15.5 5.18 6.500 Neg. fifi
H 1 s s 4 s 10- 0 'q
6 M 14.5 6.05 6,750 10-5
1. 15 7 5 51 5 600 10"5
14.1 6.7 ' 00 Neg. ""
60 M 13.7 6.36 6.100 Neg. 3 Hamsters
L 15 5 6 2g_ gmoo Ne T fi
10 M 14.1 5.55 6,750 10-5 Hematocrito-zléfé
L 15.0 5.29 5.850 10-4 fl
H 16.6 6.86 9,900 Neg. ---
100 M 15.5 6.56 6,700 Neg. 5 Hamsters
L 4 1 6 12 6 200 Ne
1 Q 6. ’ 00 1 - 0 (")~ v-
15 M 15. 6.50 6,750 10-5
5 15.5 5.8% 5.55% Neg. HT 5'2“
1 . . . eg. --- F .-cmaT.-- /0
15C M 15. 6.63 8.150 Neg. Hanat.--51%
' L 15 0 6 34 5 050 Ne 3 Hamsters
e s s - "" m 0"
20 11 14.5 6.55 7,600 10"5 Hemat.--50%
L 12 0 4 58 4 500 1 -1 6 Hamsters
H 16.2 7.75 7.000 Neg. ‘ ‘ Heznat.--Z§7§
200 M 16.2 7.65 6.200 Neg. 5 Hamsters

 

* Values represent the high. median and low values.
Grams hemoglo in per 100 cc blood.

2 Multiply X 10 for red cells per cm blood.

3 Number of white cells per cm blood.

4 Number positive animals/number animals tested.

3" Duplicate tubes; number animals per tube combination.
C represents values for control animals.

TABLE IX
HEMATOLOGICAL AND SEROLOGICAL VALUES-~STRAIN 0H10*

 

 

Hemo- Erythro- Leuco- Aggl'n ult f R ‘3:
Day globinl cytos2 _qytes:5 Titer 0 use“ emar s

 

 

 

 

 

H 15.7 6.99 7.450 10?? 1/5 1 ff Hemato-
3 :M 15.3 6.75 6,850 N38. 4 -' OIit--45%
I. 13.7 5.23 5.800 Neg.
' H 1 . 6. . N98. ““ Hemat.'-50%
3C M 15.0 6.20 7.400 Neg. 3 Hamsters
L 13 3 5 83 6 000 Ne .
. . . 0 eg. {77+} Urine ~cIear
5 M 15.0 5.83 9.000 Neg. 3 +- Nervous
L 14 l 5 38 3 600 Ne smtoms
H 15.7 7.02 7.255 Neg. --- Hemat.--45%
5C M. 14.5 6.65 ' 6.650 Neg. 3 Hamsters
L 14 5 6 05 6600 Ne
H 13.6 7.42 15.850 10"! 575 5(++) ‘UrIne chr
6 M 16.2 6.49 7,950 Neg. 5 111
L 15 0 5 9'7 4 050 Ne
l .9 9. .0 10' 5 + ems s.--
7 AM M 16.2 7.08 16,800 10"1 65, 55, 54%
9 3 4 9 6 600 Ne Moribund
6 -

. . . 6(1)...) '7 MorIEund

1 m M 18.1 6.95 18.700 10'1 1 +-
L 15 7 6 64 14 200 10' ‘

T—‘f—W—‘E‘T’M 1 .1 8. 2 ,2 0 1‘0T- 677 6??) 3““‘1‘6‘7 Mor und

4 PM M 17.1 7.62 18,250 10"1 1 --

1g.5 6.18 6,900 10-1

18.1 ,6.98 8.350 Negg‘fi‘-- Hemat.--49%

16.2 6.80 7,200 Neg. 5 Hamsters
15.7 6.45 6,500 Neg.

Values represent the high. median and low values.
Grams hemoglobin per 100 cc blood.

Multiply X 105 for red cells per cm blood.

Number of white cells per cm blood.

Number positive animals/number animals tested.
Duplicate tubes: number animals per tube combination.
C represents values for control animls.

 

 

 

s!
O

 

dunner-HI: Haunt-i