VIRULENCE FACTORS OF
LEPTOSPIRA POMONA

By
DIETRICH CHARLES BAUER

AN ABSTRACT
Submi t t ed t o t h e School f o r Advanced Graduat e St u d i e s
o f Mi chi gan S t a t e U n i v e r s i t y o f A g r i c u l t u r e and
Ap p l i e d Sci ence in p a r t i a l f u l f i l l m e n t of t he
r e q ui r e me n t s f o r t h e degree of
DOCTOR OF PHILOSOPHY

Department o f M i c r o b i o l o g y and P u b l i c H e a l t h
1959

Approved

Dietrich

C h a r l e s Bauer

ABSTRACT
The i n f o r m a t i o n a v a i l a b l e r e g a r d i n g t h e f a c t o r s which
c o n t r i b u t e t o t he v i r u l e n c e o f

leptospfrae

i s both meager and

confIicting.
To gai n some i n s i g h t

i n t o t h e mechanisms o f L. pomona

i n f e c t i o n s an i n v e s t i g a t i o n was under t aken t o demonst r at e a
f a c t o r or f a c t o r s r e s p o n s i b l e f o r t h e t o x i c e f f e c t s of t he
or gani sm.

Duri ng t he s t u d i e s t he o p p o r t u n i t y a l s o arose t o

examine l e p t o s p i r a l

e x t r a c t s f o r enzymes o f t h e c i t r i c

acid

eye I e .
L e p t o s p i r a e were separ at ed from c u l t u r e s by c e n t r i f u g a ­
t i o n and washed and resuspended t o
of c u l t u r e .

e x t r a c t t e s t e d f o r t o x i c and enzymati c

No t o x i c a c t i v i t y

i nt r a d e r m a l

inoculation

change i n h e ma t o l o g i c a l
following

could be demonstrated f o l l o w i n g

i n t o hamsters or gui nea p i g s .

No

v al ues coul d be observed i n hamsters

i n t r a c a r d i a l in o c ul a ti on of the e x t r a c t s .

which r e c e i v e d L.
and urea

volume

The c e l l s were d i s r u p t e d by soni c o s c i l l a t i o n

and t he c e l l - f r e e
activity.

l/lOO the o r i g i n a l

Hamsters

pomona c e l l s showed i ncr eased b i l i r u b i n

l e v e l s and reduced hemoglobin and h e m a t o c r i t

levels.

E x t r a c t s o f L. pomona were t e s t e d f o r s u c c i n i c dehydro­
genase, m a l i c dehydrogenase,
and a c o n i t a s e .
for a ll

dehydrogenase, fumarase

Va r y i ng degrees of a c t i v i t y

were demonstrabl e

o f t hese enzymes.
Further

o f L.

isocitric

i n v e s t i g a t i o n s on t he mechanism o f v i r u l e n c e

pomona were performed using t h e hemol ysi n found in

supernatant f l u i d s of

leptospiral

was found t o be p r e c i p i t a t e d

cultures.

The hemol ysi n

by 35$ s a t u r a t i o n wi t h ammonium

Dietrich
sulfate.

C h a r l e s Bauer

S e p a r a t i o n and c o n c e n t r a t i o n o f t he hemol ysi n
in y i e l d s o f about 30 per c e n t .

t h i s manner r e s u l t e d
The t o x i c i t y

o f t he c o n c e n t r a t e d hemol ysi n p r e p a r a ­

t i o n s was examined by i n t r a v e n o u s i n o c u l a t i o n o f young
Animal s r e c e i v i n g
cent

icteric

l oss o f hemogl obi n,

Inoculation.

The

and he mog l ob i nu r i a was e v i d e n t .

S m a l l e r doses o f hemol ysin r e s u l t e d

Lambs r e c e i v i n g

lambs.

l a r g e doses o f hemolysi n showed a 60 per

l oss o f hemoglobin 30 hours a f t e r

lambs were weak,

in

wi t h t he

i nt r a v e n o u s

i n a 30 t o 50 per cent

lambs appeari ng weak and p a l e .
i n j e c t i o n s o f washed L.

pomona

c e l l s devel oped an acut e h e mo l y t i c anemia, evi denced by a
65 per cent drop in hemoglobin and h e m a t o c r i t

levels.

J a un di c e ,

weakness and he mogl obi nur i a were e v i d e n t .
Gross and mi cr oscopi c exami nat i on o f t he t i s s u e s o f
t he

lambs showed a g e n e r a l i z e d

and p e t e c h i a l

copper c ol o r e d

hemorrhages on t h e k i d n e y .

t i o n and n e c r o s i s
distal

icterus,

in many areas o f t he

and proxi mal

livers

Areas of degenera­

l i v e r were not ed.

The

convol ut ed t u b u l e s o f t he ki dney showed

a r e a s o f d e g e n e r a t i o n and some t u b u l e s c on t a i n e d a homogenous,
dark red s t a i n i n g m a t e r i a l .

Animals r e c e i v i n g hemol ysi n or

whole c e l l s

l e s i o n s except

revealed s i m i l a r

group more e x t e n s i v e r e n a l
Anot her group o f
doses o f he mol y s i n.

lambs r e c e i v e d a s e r i e s of small
live

No evi dence o f h e mo l y t i c anemia

was observed in any o f t he a ni ma l s .
to i n t e r p r e t

nating antibody a f t e r

latter

damage was p r e s e n t .

They were t hen c h a l l e n g e d wi t h

organi sms or hemol ysi n.

were d i f f i c u l t

in t he

However, t he r e s u l t s

due t o the development o f a g g l u t i ­

r e c e i v i n g hemol ysi n p r e p a r a t i o n s .

Dietrich

C h a r l e s Bauer

Conc e nt r a t e d c u l t u r e super nat es o f a nonhemol yt i c
stfain a l s o s t i m u l a t e d t h e p r o d u c t i o n of a g g u l u t i n a t i n g
body.

anti­

Al though a n t i s e r u m i n h i b i t e d t h e hemol ysi n no evi dence

was o b t a i n e d t o

i n d i c a t e t h a t t h e hemol ysi n s t i m u l a t e d a n t i ­

body p r o d u c t i o n .
An assay pr ocedur e was devel oped f o r d e t e r mi n i n g
hemolytic a c t i v i t y

which was r a p i d and r e l a t i v e l y

accurate.

By means o f t h i s p r oc e d ur e , j j i v i t r o p r o p e r t i e s of
t h e hemol ysi n were i n v e s t i g a t e d .

The r e s u l t s

indicated that

t h e r a t e o f hemol ysi s was g r e a t l y

i ncr eased in i n c u b a t i o n o f

t h e t e s t s a t 37°C was f o l l o w e d by a 4°C i n c u b a t i o n .
it

Further,

was demonstrated t h a t a d s o r p t i o n o f t h e hemol ysi n to

eryt hr oc y te s occurs,
chemical
tact

reaction.

which i s pr obabl y a ph y s i c a l
Anti body

r a t h e r t han

i n h i b i t e d hemol ysi s i f

in con­

wi t h t he hemol ysi n b e f o r e t h e a d d i t i o n o f e r y t h r o c y t e s .

The a d d i t i o n o f a n t i s e r u m wi t h or a f t e r t h e e r y t h r o c y t e s
resulted

i n di mi ni s h e d i n h i b i t i o n .

On t h e b a s i s o f t he r e s u l t s o b t a i n e d , a mechanism
i s proposed f o r t h e development of h e mo l y t i c anemia a s s o c i a t e d
wi t h

leptospirosis.

It

i s a l s o concluded t h a t t h e hemol ysi n

i s not t he on l y t o x i n o f
icteric

l e p t o s p i r a e which can produce an

c o n d i t i o n and t h a t

virulence factors of

it

r e p r e s e n t s on l y one o f t h e

leptospirae.

The f a c t t h a t f i v e

enzymes o f t he c i t r i c

acid cycle

a r e demonst r abl e I n d i c a t e s t h a t t h i s m e t a b o l i c pathway i s
pr obabl y o p e r a t i n g

in

leptospirae.

VIRULENCE FACTORS OF
LEPTOSPIRA POMONA

By
DIETRICH CHARLES BAUER

A THESIS
Submi t t ed t o t h e School f o r Advanced Graduat e St u d i e s
o f Mi chi gan S t a t e U n i v e r s i t y o f A g r i c u l t u r e and
Ap p l i e d Sci ence in p a r t i a l f u l f i l l m e n t o f t he
r e q u i r e me n t s f o r t h e degree o f
DOCTOR OF PHILOSOPHY

Department o f M i c r o b i o l o g y and P u b l i c H e a l t h
1959

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2 (s o

%}

DEDICATION
To Loi s

ACKNOWLEDGMENTS
The a u t h o r wishes t o thank Dr s .
E. V. Morse who d i r e c t e d t h i s r e s e a r c h ,
and i n t e r e s t .
H. L.
this

C. Ferguson and

for th e ir

guidance

suggest i ons o f f e r e d by Dr .

S a d o f f and h i s encouragement and s i n c e r e i n t e r e s t
thesis
It

ance

The many h e l p f u l

L.

is g r e a t l y a p p r e c i a t e d .
i s a p l e a s u r e t o acknowledge t h e t e c h n i c a l

o f Dr .

S. S l e i g h t ,

assis

Mr. L« Eames and Mrs. A. Lundberg

w i t h o u t whose hel p much o f t h i s work would not have been
possi b l e .

in

TABLE OF CONTENTS
PAGE
INTRODUCTION...............................................................................................

I

LITERATURE REVIEW .................................................................................

3

MATERIALS AND METHODS .......................................................................

12

RESULTS.........................................................................................................

18

DISCUSSION....................................................................................................

36

SUMMARY AND CONCLUSIONS ...................................................................

49

TABLES.............................................................................................................

33

F I G U R E S ........................................................................................................

67

REFERENCES....................................................................................................

79

iv

INTRODUCTION
The p r o p e r t i e s o f b a c t e r i a which c o n t r i b u t e t o t h e i r
v i r u l e n c e may be sepa r a t e d

i n t o two broad c a t e g o r i e s .

First,

t h e r e are t h e c h a r a c t e r i s t i c s a s s o c i a t e d wi t h overcoming t he
host def enses and e n a b l i n g t h e p a r a s i t e t o e s t a b l i s h
Under t h i s gr oupi ng may be i ncl uded h y a I u r o n i d a s e ,
fib rin o Iysin,

leukocidin,

infection.

c oa gul a s e ,

and a n t i p h a g o c y t i c subst ances.

The

second group of v i r u l e n c e f a c t o r s a r e t hose which produce t he
lesions

in t h e h o s t ' s t i s s u e s ,

endotoxins.

The r e l a t i v e

t h e so c a l l e d

i mportance o f t he s e two groups in

t he d i s e a s e process i s e x t r e me l y v a r i a b l e .
examples are o f a n t h r a x and b o t u l i s m .
i nvades and m u l t i p l i e s
However,

an animal

e x o t o x i n s and

The c l a s s i c a l

Ba c i I I us ant hr ac i s

in g r e a t numbers in t he host t i s s u e s .

needs onl y t o

i ng e s t food

in which

C l o s t r i d i um b o t u l i num has grown and produced t o x i n t o d i e
o f b o t u l i s m and on l y r a r e l y
t he a f f e c t e d

i s t h e organism r ecovered from

individual.

I n f o r m a t i o n wi t h r egard t o t hese v i r u l e n c e f a c t o r s
among t h e

leptospirae

i s both meager and c o n f l i c t i n g .

b a s i s f o r t he t r e a t m e n t and c o n t r o l
all

phases of t he h o s t - p a r a s i t e

sidered.

Therefore,

As a

of an i n f e c t i o u s d i s e a s e ,

relationship

should be con­

f o r t he purpose o f g a i n i ng some i n s i g h t

i n t o t h e mechanisms o f L e p t o s p i r a pomona i n f e c t i o n s an i n ­
v e s t i g a t i o n was under t aken t o a t t e mpt t o demonstrate a
substance or substances r e s p o n s i b l e f o r t he t o x i c e f f e c t s
of t he organi sm.

Dur i ng t he course of t h e i n v e s t i g a t i o n s

t he o p p o r t u n i t y arose t o examine c e l l
I

f r e e e x t r a c t s from

2

L.

pomona f o r enzymes o f t h e c i t r i c

t h e b a s i s of t h e pa t ho ge n i c e f f e c t

acid cy c le .
of bacteria

Because
i s c h e mi c a l ,

t he more i n f o r m a t i o n a v a i l a b l e

r e g a r d i n g t h e chemical

activi

ties

i s t he o p p o r t u n i t y f o r

under­

o f a pathogen t h e g r e a t e r

st an di n g t h e b a s i s of i t s

virulence.

3
LITERATURE REVIEW
A.

Virulence Studies
Gsell

( 2 4 ) has de s c r i b e d t h e host response t o L e p t o s p i r a e

pomona i n f e c t i o n s as o c c u r r i n g

i n two phases.

The f i r s t

phase

i s c h a r a c t e r i z e d by t h e pr esence o f t h e s p i r o c h e t e s i n t he
circulatory

system.

The second phase i s a p e r i o d o f t i s s u e

damage i n v o l v i n g p r i m a r i l y t h e
erythrocytes.
of a n t i b o d y .

liver,

kidneys,

meninges and

T h i s stage t e r m i n a t e s u s u a l l y w i t h t h e appearance
I c t e r u s may or may not be p r e s e n t .

In man t h e

d i s e a s e i s most commonly a s s o c i a t e d w i t h muscul ar pai ns and
m e n i n g i t i s but i c t e r u s

i s r a r e l y observed ( 2 ) ,

as a r u l e show o v e r t si gns o f

Pi gs do not

i l l n e s s o t h e r t han a s l i g h t

t e m p e r a t u r e e l e v a t i o n and l oss of a p p e t i t e .

However,

pr egnant sows a b o r t i o n i s f r e q u e n t l y a s s o c i a t e d wi t h
spirosis

(7).

The di s e a s e i n c a t t l e

is v a ria b le .

in
lepto­

Young

ani mal s a r e more s e v e r e l y a f f e c t e d showing j a u n d i c e and
he mo g l o b i n u r i a
frequently,

(60).

leptospirosis

in 58 per c e n t ,

(53*55).

Bryan ( 8 ) s t u d i e d t he symptoms

in 125 herds o f c a t t l e .

A b o r t i o n occur r ed

reduced mi l k f l o w i n 47 per c e n t ,

i n 30 per cent and anaemia and j a u n d i c e in 15 t o
L.

pomona i n f e c t i o n s

hemogI obi nuf r a

19 per c e n t .

i n sheep a l s o seem t o vary

s e v e r i t y depending on t h e age o f t he a ni ma l s ( 3 7 ) .
out br ea k

Jess

but a b o r t i o n and reduced mi l k f l o w a r e a f r e q u e n t

result of infection
of

Ad u l t ani mal s appear i c t e r i c

in

A severe

in lambs in New Zeal and was r e p o r t e d by H a r t l e y

(25)

in which i c t e r u s and h e mog l ob i nu r i a were t h e promi nent c l i n i c a l

4
signs.
of

Webster and Reynol ds ( 6 9 ) d e s c r i b e d two ou t br e a k s

leptospirosis

in ewes and lambs.

A severe h e m o l y t i c

anemia wi t h h e m o g l o b i n u r i a and j a u n d i c e were a g a i n p r o mi ne n t .
A b o r t i o n and deat h o f ewes has been recorded by Beamer and
coworkers ( 5 ) .
Ex pe r i me nt a l
et a I .

(47).

p e r i me n t a l

o v i n e i n f e c t i o n s were s t u d i e d by Morse

A h e m o l y t i c anemia was observed

a ni ma l s w i t h accompanying h e m o g l o b i n u r i a .

j a u n d i c e was noted i n
Sever al

Gsell

t i o n and t he

I lamb.

(24)

l e s i o n s observed i n

leptospiral

stated t h a t the s e v e r i t y of the

di s e a s e p i c t u r e o f

picture.

intoxica­

He d e s c r i b e d t he

l e p t o s p i r o s i s as a gener al

i n which a l e p t o s p i r a l

intoxication

t o x i n causes t he t y p i c a l

was a d m i t t e d however,

lesions.

t h a t any i n t e r p r e t a t i o n o f t h e d i s ­

ease as being due t o a t o x i n was h y p o t h e t i c a l
such agent was y e t de mons t r a bl e .

r e l e a s e d from t h e

as long as no

Ferguson ,et a_l_. ( 1 8 )

suggested t h a t t h e cause o f bovi ne a b o r t i o n
was a t o x i n ,

infec­

i n t e n s i t y o f t h e s e p t i c e m i a de t e r mi ne t h e d i f f e r ­

ences observed in t h e c l i n i c a l

It

A mi l d

i n v e s t i g a t o r s have suggested t h e p o s s i b l e

causes o f t h e v a r i o u s
tions.

i n 5 of 8 ex­

in

leptospirosis

l e p t o s p i r a e by l y t i c

in t he dam, which e nt e r e d t h e f e t u s .

antibodies

Morse and coworkers ( 4 7 )

suggested a s i m i l a r mechanism as t h e cause o f t h e h e m o l y t i c
anemia i n sheep.

In t h i s case ant i body was t hought t o r e ­

l ease a h e m o l y t i c e ndot ox i n from t h e s p i r o c h e t e s or t h a t
a n t i b o d y would a c t wi t h a h e m o l y t i c a n t i g e n and lyse e r y t h r o ­
cytes

in t he presence o f complement.
As e a r l y as two years a f t e r

Inada and Ido ( 3 1 )

in

5
1915 i n c i r m i n a t e d L^. i cterohemmorhaq i ae as t he e t i o l o g i c a l
agent o f W e i l ' s d i s e a s e , Matsuzaka ( 3 9 )

r e p o r t e d t he t o x i c

p r o p e r t i e s o f heat k i l l e d

l e p t o s p i r a e f o r gui nea p i g s .

Fukushima and Hosoya ( I S )

and Hi guchi

of L.

( 2 7 ) found t h a t

Later,
cultures

i cterohemmorhag i ae which had been ma i n t a i n e d under

a n a e r o b i c c o n d i t i o n s or j_n vacuo a t 37°C f o r 2 t o 3 days,
produced i n c r e a s e d b i l i r u b i n

levels

in t h e blood o f gui nea

pi gs and hyperemi a o f t he bu l ba r c o n j u n c t i v a .

Stavitsky

(61)

made e x t e n s i v e a t t e mp t s t o c o n f i r m t he s e f i n d i n g s wi t h o u t
success.

Th i s same worker t e s t e d

hyaIuronidase, f ib r i o o l y s i n ,
wi t h n e g a t i v e f i n d i n g s
(67)

leptospiral

extracts for

l e u k o c i d i n and coagul ase a c t i v i t y

(62).

Su bs e q ue n t l y ,

s t a t in g t h a t hyaIuronidase a c t i v i t y

a r e p o r t appeared

was de mons t r a bl e .

F a i n e ( 15 ) approached t h e study o f

leptospiral

by comparing t h e f a t e o f v i r u l e n t and a v i r u l e n t L.
hemmorrhaq i ae c e l l s

injected

virulence

i ctero-

i n t o young gui nea p i g s .

Both

s t r a i n s were p h a go c y t i z e d by f i x e d phagocytes of t he r e 1 1 c u I o c e n d o t h e I i a I system.
obs e r v e d.

The onl y d i f f e r e n c e between t h e s t r a i n s was t h a t

virulent cells

s u r v i v e d whereas a v i r u l e n t di d n o t .

same i n v e s t i g a t o r
number o f

(16)

lesions.

per ml o f body f l u i d s

per ml.
of

s t u d i e d t he r e l a t i o n s h i p

l e p t o s p i r a e pr e s e nt

appearance o f

lesions.

L e u c o c y t i c phagocyt osi s was not

Thi s

between t h e

in gui nea pig t i s s u e s and t h e

A c o n c e n t r a t i o n of

10^ l e p t o s p i r a e

c o i n c i d e d wi t h demonstable hemmorhagic

Death occur r ed wi t h c e l l

concentrations of

I

Death could occur in t h e absence o f hi gh numbers

leptospirae.

With ani mal s i n f e c t e d wi t h doses near t h e

LDipO> death was observed 3 days a f t e r
serum a n t i b o d y .

t he appearance o f

6

Such o b s e r v a t i o n s a r e conf i r med by o t h e r
( 3 2 , 4 6 ) on t h e e f f e c t
st ages o f

of serum t h e r a p y duri ng d i f f e r e n t

leptospirosis.

Serum a d m i n i s t e r e d p r i o r t o or

s i m u l t a n e o u s l y wi t h exposure t o
tection,
after

but t h i s

studies

l e p t o s p i r a e a f f o r d e d pr o­

e f f e c t d i mi n i s h e d when serum was gi ven

exposur e.
In

1956 AI exander e t aj[.

( I ) and Russel I ( 5 7 )

re­

po r t e d t h e presence o f a s o l u b l e hemol ysi n in c u l t u r e s u p e r n a t e s o f some l e p t o s p i r a l

serotypes.

The hemol ysi n was
I t o 3 days a f t e r

f ound i n g r e a t e s t amounts i n t h e s uper nat es
maximum growth had o c c u r r e d .
activity

Only a small

was found i n d i s r u p t e d c e l l

was de s t r o y e d by h e a t i n g a t 56°C f o r

amount of h e m o l y t i c

preparations.
1C mi n u t e s .

Activity
Maximum

r a t e s o f hemol ysi s were a c hi e v e d a t 37 ° c » wi t h no hemol ysi s
o b s e r v a b l e a t 0 °C.

The hemol ysi n was oxygen s t a b l e ,

d i a l y s a b l e and i n h i b i t e d by l e p t o s p i r a l
c y t e s o f sheep,

antiserum.

nonErythro­

cows and goats were r e p o r t e d t o be most

s u s c e p t i b l e t o t h e hemolysi n

(I).

R a b b i t and hamster erythrocytes

were l ess s u s c e p t i b l e and bui nea pi g RBC were u n a f f e c t e d
A c t i v i t y was observed wi t h some s t r a i n s o f L.
ben jami n. L.
pomona, L.

( 57, 4 ) .

cani coI a . L.

c y n o p t e r i « JL. autumnal i s . L . a u s t r a I i s A, L.

bebdomadi s and L.

suggested t h a t

batavi ae.

t he h e mo l y t i c a c t i v i t y o f

The a ut ho r s ( I )
l e p t o s p i r a e was due

t o t h e presence o f a t o x i n .
Bauer and Morse ( 4 ) compared t he h e m o l y t i c a c t i v i t y
o f _L. pomona c u l t u r e s o f v a r y i n g degrees o f v i r u l e n c e wi t h
t h e i r LD50 f o r hamst er s.
c a t e d by t h e
activity

The lack o f c o r r e l a t i o n was i n d i ­

f a c t t h a t t h e c u l t u r e wi t h t h e h i g h e s t

had a l a r g e LD^q .

hemolytic

7
Imamura and coworkers ( 3 0 )
d e m o n s t r a t i o n o f a heat
o f L.

reported

in

1957 t he

s t a b l e substance i n sonic e x t r a c t s

i c t e r o h e m m o r r h a q i a e . which when i n j e c t e d

intracutaneousIy

i n t o gui nea p i g s or r a b b i t s produced an ar ea o f i nf l a mma t i o n
o f 20 t o 30 mm i n d i a m e t e r .

The n o n - p r o t e i n n a t u r e o f t he

substance was i n d i c a t e d by i t s n e g a t i v e b i u r e t
positive

results

in t h e Mol i sch and B i a l ' s o r c i n r e a c t i o n s .

In 1957 Kemenes (3^0 r e p o r t e d t h a t
intravenously with
ture

by i n j e c t i n g

150 t o 220 ml amounts o f

supe r na t e s o f hi gh h e m o l y t i c a c t i v i t y ,

and he mo g l o b i n u r i a were produced in 8 t o

dyspnea,

gr i ppot yphosa.

leptospirae.

o b s e r v a t i o n s had been made by e a r l i e r
A recent p u b l i c a t i o n

Daily
pr o­

pomona.

These workers observed

t h a t an i n h i b i t o r o f t h e hemol ysi n was pr e s e nt
serum used f o r c u l t i v a t i o n o f t h e

fever

Thi s jjn vi vo

was observed w i t h hemol ysi ns o f L.

can i coI a and L.

cul­

20 t o 50 ml,

duced a h e m o l y t i c anemia i n about 5 t o 7 days.

L.

lambs

leptospiral

16 hour s .

i n j e c t i o n s o f s m a l l e r amounts o f hemol y s i n,

hemolytic e f f e c t

r e a c t i o n and

in t h e r a b b i t
Similar

investigators

(I,

57).

( 5 6 ) has suggested t h a t t h e

hemol ysi n o f L • pomona may be a p h o s p h o I i p a s e .

Thi s con­

c l u s i o n was based on r e s u l t s a t t a i n e d by t he use o f phos­
p h o l i p i d s as i n h i b i t o r s o f t h e hemol ysi n.
B.

C i t r i c Aci d Cycl e Enzymes.
Pat hogeni c

and a l l

l e p t o s p i r a e r e q u i r e blood serum f o r growth

a t t e m p t s t o s u b s t i t u t e d e f i n e d n u t r i e n t s f o r t he

serum have been unsuccessf ul

(2).

F r a c t i o n a t i o n o f sera t o

a s c e r t a i n t h e components necessary f o r growth and r e s p i r a t i o n

8

has been a t t e mp t e d by s e v e r a l
(58)

reported th at

wor k e r s .

serum al bumi n c o n t a i n e d much o f t h e n u t r i e n t

v a l u e o f whole serum.

Albumin p r e c i p i t a t e d

ammonium s u l p h a t e was e f f e c t i v e
was n o t .

Schneiderman et a I .

but a l c o h o l

by 71 per cent
p r e c i p i t a t e d albumi n

The al bumi n c oul d not be r e p l a c e d by a m i x t u r e o f

amino acids which s i mu l a t e d t h e amino a c i d composi t i on o f t he
al bumi n as det e r mi ne d by m i c r o b i o I o g i c assay.

At t empt s by

o t h e r workers t o r e p e a t t he s e e x pe r i me nt s have been wi t h o u t
success ( I I ) .
F u l t o n and Spooner

( 2 0 ) t e s t e d v a r i o u s components of

serum f o r r e s p i r a t o r y a c t i v i t y
methods.

as measured by manometric

Serum p r o t e i n was found t o be as e f f e c t i v e as whole

serum i n s t i m u l a t i n g

respiratory a c tiv ity of

At t empt s t o show p r o t e i n breakdown however,
ful.

leptospirae.
were not success­

Serum p r o t e i n s were t hen e x t r a c t e d w i t h e t h e r t o d e t e r ­

mine whether a n o n - p r o t e i n f a c t o r was e s s e n t i a l
tion.
their

for respira­

The e t h e r e x t r a c t e d p r o t e i n s showed no r e d u c t i o n in
ability

to s t im u la t e r e s p i r a t i o n .

Nevertheless the

e t h e r s o l u b l e substances di d possess r e s p i r a t o r y a c t i v i t y .
Ph o s p h o l i p i d s were e x t r a c t e d from t h e e t h e r s o l u b l e f r a c t i o n
by acet one p r e c i p i t a t i o n .
the p r o t e i n f r a c t i o n

in s t im u l a t i n g

H e l p r i n and H i a t t
of

Th i s f r a c t i o n was as a c t i v e as

(26)

also inv estiga te d the e f f e c t

l i p i d s on t he r e s p i r a t i o n o f

f a c t i o n V (albumin)

leptospirae.

Human plasma

was found t o c o n t a i n e s s e n t i a l l y a l l

t h e serum r e s p i r a t o r y a c t i v i t y .
t r a c t i o n almost a l l

respiration.

However,

o f t he a c t i v i t y

t h e acet one p r e c i p i t a b l e

lipids

after

was l o s t .

of

acetone ex­
Addition of

back t o plasma f r a c t i o n V

9
restored the o r ig in a l

activity.

L i p i d s al one

inhibited

respiration.

A number of f a t t y

tory a c t i v i t y

wi t h and w i t h o u t t h e p r o t e i n f r a c t i o n .

absence o f p r o t e i n t h e f a t t y
t i o n but w i t h p r o t e i n a l l

a c i d s were t e s t e d f o r

possessed a c t i v i t y .

r e s p i r a t i o n was observed,

a c t i v e and b u t y r i c t h e
highly

stimulatory.

A correlation
t o st i mu­

a r a c h i d i c a c i d being t h e most
U n s a t u r a t e d a c i d s were a l s o

The a ut ho r s concl uded from t h e i r

t h a t the s ti m u la t o r y
respiration

least.

results

e f f e c t o f serum p r o t e i n s on l e p t o s p i r a l

i s due t o both t h e c o n t r i b u t i o n o f f a t t y

and a d e t o x i f y i n g

I n the

a c i d s di d not s t i m u l a t e r e s p i r a ­

between t h e number o f carbon atoms and t h e a b i l i t y
late

respira­

effect

acids

of t he a l bumi n.

R e c e n t l y G e r h a r d t and Ba l l

( 2 1 ) have r e p o r t e d t h a t

u t i l i z a t i o n o f amino a c i d s from r a b b i t

serum by l e p t o s p i r a e

can be demonstr ated by chr omat ogr aphi c and m i c r o b i o l o g i c a l
assay p r oc e d ur e s .

It

has been shown by s e ver al

workers t h a t

l e p t o s p i r a e do not m e t a b o l i z e c a r b o h y d r a t e s ( 2 0 ,
L e p t o s p i r a e r e q u i r e oxygen f o r growt h.
McLeod and Rodican
r i n g s of

oc c ur r e d 4 t o
was minimal

growth in t ubes of s e m i - s o l i d or s o l i d

10 per cent r a b b i t

serum.

The bands o f growth

10 mm below t h e s u r f a c e of t he medium.

or absent

dense ar e a s o f gr owt h.
that

C z e k a l ows k i ,

( 1 3 ) observed t h e f o r m a t i o n o f narrow

leptospiral

agar c o n t a i n i n g

9).

Growth

in t he c l e a r areas above and below t he
From t hese r e s u l t s

it

was concluded

l e p t o s p i r a e r e q u i r e amounts o f oxygen below t h a t o f t he

atmosphere and should be c onsi der ed m i c r o a e r o p h i I i c .
The g e n e r a t i o n t i m e o f
by s e v e r a l

l e p t o s p i r a e has been r e p o r t e d

i n v e s t i g a t o r s as between 24 and 68 hours ( 2 0 ,

9).

10
These r e p o r t s were based on r e s u l t s o b t a i n e d by obser vi ng
changes in t u r b i d i t y

in f l u i d

cultures.

Cox and Larson ( 1 2 )

devel oped a s o l i d agar medium f o r growing

l e p t o s p i r a e which

p e r m i t t e d growth o f t h e organisms t o be det ermi ned by t he
p l a t e count method.

They o b t a i n e d g e n e r a t i o n t i me val ues

o f f rom 6 t o 24 hours depending on t he c u l t u r e medium ( 3 6 ) .
The di s c r e pa n c y between t h e s e v al ues and t hose o f pr e v i ou s
r e p o r t s was suggested t o be due t o t he p o s s i b i l i t y t h a t
turbidimetric

e v a l u a t i o n s could onl y be performed duri ng

t h e d e c l i n i n g phase o f growt h, t h e number of
being t oo few duri ng t h e

leptospirae

l o g a r i t h m i c phase f o r t u r b i d i m e t r i c

evaluation.
The enzymes or enzyme systems o p e r a t i n g
have been s u b j e c t e d t o onl y
and coworkers

(13)

limited

in l e p t o s p i r a e

investigation.

Czekalowski

r e p o r t e d t h a t c a t a l a s e was not pr esent

and p e r o x i d e f o r m a t i o n coufd not be observed.

T e t r ame t h y l

p a r a - p h e n y I e n e d i a m i n e was added t o ar e a s o f dense l e p t o s p i r a l
growth i n a semi

s o l i d medium.

The f o r m a t i o n o f a dark bl ue

c o l o r over t h e a r e a s o f gr owt h,
indophenoIoxidase,
oxidase.

(23).

t he f o r m a t i o n of

demonstrated t h e presence o f b a c t e r i a l

T h i s same r e a c t i o n

leptospirae

indicating

i s obt ai ne d wi t h c o l o n i e s o f

L e p t o s p i r a e were found t o be s e n s i t i v e to

cya ni de and t h e a u t h o r s concluded t h a t a cytochrome system
was p r ob a bl y p r e s e n t .
F u l t o n and Spooner ( 2 0 )
effect

a l s o noted t he i n h i b i t o r y

o f cyani de and a l s o t h a t o f a z i d e .

e x a mi n a t i o n o f suspensions o f

S p e c t r os c o pi c

l e p t o s p i r a e showed a d s o r p t i o n

bands a t 550 mm and 525 mm cor respondi ng t o t he bands o f

11
cytochrome c.

On shaking

r eappear ed upon s t a n d i n g .

in a i r

t he bands di s a ppe a r ed but

M/ l O cyani de pr e v e nt e d t h e d i s ­

appearance o f t h e a d s o r p t i o n bands i n d i c a t i n g t h a t
o x i d a s e was o p e r a t i v e .
active

in i n h i b i t i n g

arsenite,

Ot he r enzyme i n h i b i t o r s which were

r e s p i r a t i o n of c e l l

ur e t ha ne and i o d o a c e t a t e .

mal onat e and f l u o r i d e were not
these studies
hy d r y l

it

suspensi ons were

Arsenate,

inhibitory.

pyr ophosphat e,

On t he b a s i s o f

was suggested t h a t enzymes c o n t a i n i n g

sulf-

groups were a c t i v e .
Recently the

been s t u d i e d
iron.

cytochrome

(17).

i r o n r e q ui r e me n t s o f

l e p t o s p i r a e have

Growth di d not occur

in t h e absence o f

When FeCI ^ or hematin was added growth o c c u r r e d ,

but

at a sl ower r a t e t han when hemoglobin or hemoglobin d e r i v a t i v e s
were added.
essential

It

was concluded t h a t

porphyrin-containg

hemoglobin t o

leptospiral

i r o n was needed t o form

compounds.

The a d d i t i o n o f

c u l t u r e media as a source o f

p r o p h y r i n compounds had been suggested by e a r l i e r
(14).

workers

12
MATERIALS AND METHODS
A.

V ir u le n c e Studies
Four s t r a i n s o f L . pomona were used in t h e s e s t u d i e s .

S t r a i n W was o r i g i n a l l y

i s o l a t e d from t h e u r i n e o f an i n ­

f e c t e d cow and had been ma i n t a i n e d in a v i r u l e n t
con t i n uo us passage i n young gui nea p i g s .
infective for
(45)

sheep ( 4 7 ) ,

and dogs ( 1 0 ) .

hamsters ( 4 ) .

It

swine ( 4 4 ) ,

was i n f e c t i v e

s t a t e by

Thi s s t r a i n was

cattle

(43),

but not

goats

lethal

S t r a i n IW was a v a r i a n t o f s t r a i n W.

v a r i a t i o n s occur r ed when s t r a i n W was i n o c c u l a t e d
and c u l t u r e d from t he s e a ni mal s duri ng

for
The

into

Ieptospiremia.

lambs
After

3 t o 4 t r a n s f e r s of t h e organisms in c u l t u r e media t hey
became l e t h a l
y e a r s ago,

f o r hamsters ( 4 ) .

this

Si nce i t s

s t r a i n has been ma i n t a i n e d in c u l t u r e wi t h

a passage t hr ough hamsters a f t e r
fers.

No change in i t s

been obser ved.

i s o l a t i o n two

lethal

every

10 to

p r o p e r t i e s f o r hamsters has

S t r a i n LB was a hamster

lethal

a s t r a i n o f J_. pomona i s o l a t e d from c a t t l e .
was produced in t he same manner as s t r a i n LW.
a non pat hogeni c

15 c u l t u r e t r a n s ­

va ri a n t of

Th i s v a r i a n t
S t r a i n J was

s t r a i n which has been ma i n t a i n e d f o r 5 y e ar s

In c u l t u r e .
Large volumes o f

leptospiral

in t h e f o l l o w i n g manner.

c u l t u r e s were o b t a i n e d

To one l i t e r

screw cap b o t t l e s

was added 450 ml o f s t e r i l e S t u a r t ‘ s medium ( 6 3 ) c o n t a i n i n g
10 per cent s t e r i l e

rabbit

serum.

The inoculum c o n s i s t e d

o f 2 ml of a 3 t o 5 day o l d c u l t u r e .

The c u l t u r e s were i n ­

cubated at 29°C and observed d a i l y f o r evi dence of t u r b i d i t y .

13
L e p t o s p i r a e were c o l l e c t e d from t he c u l t u r e s when
maximum t u r b i d i t y

had been a t t a i n e d .

The organisms were

removed from t h e c u l t u r e s by c e n t r i f u g a t i o n
model

AX c e n t r i f u g e a t

12, 000 RPM f o r

i n a Lourdes

30 mi n ut e s .

The c e l l s

were t hen washed t h r e e t i mes and resuspended t o t h e d e s i r e d
volume i n s a l i n e s o l u t i o n .
To o b t a i n

l a r g e volumes o f

leptospiral

hemol ysi n t he

c u l t u r e s were i ncubat ed f o r 2 t o 3 days a f t e r maximum t u r b i d i t y
had o c c u r r e d .
frozen u n til

They were t hen pl aced
ready f o r use.

in a S e i t z f i l t e r
filte r

disc.

At t h i s t i me t hey were f i l t e r e d

wi t h a He r c u l e s ST- I

The f i l t r a t e s

Following
sterility

fitted

in t h e r e f r i g e r a t o r or

sterilizing

were s t o r e d a t 4°C or f r o z e n .

incubation a l l

c u l t u r e s were t e s t e d f o r

by mi c r os c o pi c e x a mi na t i on and i n o c u l a t i o n o f

t h i o g l y c o l l a t e br ot h

(Difco).

T i t r a t i o n s of hemolysi n were made by two f o l d
dilu tio n s of culture
final

super nat es in s a l i n e s o l u t i o n ,

volume o f 0 . 5 ml.

An equal

i ncubat ed f o r

4 ° C.

The r e c i p r o c a l

wi t h a

volume o f a 2 per cent

suspension o f washed sheep red blood c e l l
tests

serial

was added and t h e

3 hours at 37°C and 12 t o

15 hours at

o f t h e h i g h e s t d i l u t i o n o f hemol ysi n

produci ng o b s e r v a b l e hemol ysi s was de s i gna t e d t h e number o f
u n i t s o f hemol ysi n

(HU) per ml.

Red blood c e l l s

(RBC) were o b t a i n e d from sheep, cows

and gui nea pi g s and pl aced
solution

(40).

frigerator

i n an equal

Such p r e p a r a t i o n s were st or ed

f o r p e r i o d s o f up t o

were d i s c a r d e d .

volume o f A l s e r v e r ' s

10 days,

after

in t he r e ­
which they

RBC were pr epar ed f o r h e m o l y t i c t e s t s by

14
washing t h r e e t i m e s and resuspendi ng

in s a l i n e s o l u t i o n or

phosphate b u f f e r e d s a l i n e s o l u t i o n pH 7 . 3 t o make a 2 per
cent suspensi on.

A f t e r 4 days t hese p r e p a r a t i o n s were d i s ­

carded.
work were 4 t o 6

The a ni ma l s used f o r e x p e r i me n t a l

week o l d hamst er s, 4 t o 7 week ol d guinea pi g s and lambs
f rom b i r t h
free of

t o 3 months o f
leptospiral

age.

All

ani mal s were known t o be

serum a n t i b o d i e s unl ess o t h e r w i s e s t a t e d .

The pr esence o f serum a n t i b o d i e s was det ermi ned by a m i c r o s c r o p i c a g g l u t i n a t i o n t e s t p r e v i o u s l y de s c r i be d

10 f o l d s e r i a l
tion

in

d i l u t i o n s o f serum were made

a total

volume o f 0.1

ml.

(47)

in which

in s a l i n e

solu­

A 0 . I ml volume o f a 3

t o 5 day c u l t u r e of s t r a i n J was added t o each t u b e .

After

i n c u b a t i o n f o r 2 hours a t 37°C» t h e c on t e n t s o f each tube
were d e p o s i t e d on a s l i d e and t he amount of a g g l u t i n a t i o n
de t e r mi ne d by mi c r os c opi c e x a mi n a t i o n .
Hemol ysi n i n h i b i t i o n t e s t s were performed by making

2 foId serial
final

d i l u t i o n s of serum in s a l i n e s o l u t i o n

in a

volume of 0 . 5 ml t o which was added 0 . 5 ml c f a d i l u ­

t i o n o f hemol ysi n c o n t a i n i n g approxi mat e Iy 50 u n i t s o f
hemol ysi n per ml .

The m i x t u r e s were incubat ed a t 37°C f o r

30 mi nut es a f t e r which 0 . 5 ml o f a 2 per cent suspension of
sheep RBC was added.
for

3 hours and 12 t o

of in h ib ito r

The t e s t s were then
15 hours at 4°C.

i ncubat ed at 37°C

The h i g h e s t d i l u t i o n

which pr event ed hemol ysi s was de s i g na t e d the

inhibition t i t e r .
Hemoglobin va l ue s were o b t a i n e d by t he CyanmethemogI obin
method ( 5 9 ) .

H e ma t o c r i t

d e t e r m i n a t i o n s were made in t he

15
st a n da r d manner

(59.

Blood c he mi s t r y d e t e r m i n a t i o n s o f

hamsters were made using micro-methods as o u t l i n e d by Na t e l s o n
(49).

Differential

l euc oc y t e counts were det ermi ned using

st a n d a r d h e m a t o l o g i c a l
leptospirae

pr ocedur es ( 5 9 ) .

The number o f

i n t h e plasma o f hamsters and lambs was e s t i ma t e d

by mi c r o s c o p i c e x a m i n a t i o n .

P r o t e i n d e t e r m i n a t i o n s were

perf ormed by t he method o f Lowry ( 3 8 ) .
B.

C itric

Acid Cycl e Enzymes

De t e r mi n i n g

if

citric

a c i d c y c l e enzymes were p r e s e n t

was suggested by t he f a c t t h a t
possess a cytochrome system,

leptospirae u t i l i z e

pl us the evi dence t h a t phospho­

l i p i d s a r e m e t a b o l i z e d and t he p o s s i b l e
hemol ysi n wi t h a p h os p ho I i pa s e .
nal

r e s p i r a t i o n of

oxygen and

identity

of the

Such a study o f t he t e r m i ­

l e p t o s p i r a e was made by t h e f o l l o w i n g

p r oc e d ur e .
The method used f o r o b t a i n i n g
in

l a r g e volumes has been de s c r i be d

and Methods,
actively

c u l t u r e s of

leptospirae

in Se c t i o n A of M a t e r i a l s

The medium was i n o c u l a t e d wi t h 2 ml o f an

growing c u l t u r e of L_. pomona s t r a i n LW.

After

in­

c u b a t i o n a t 3 CfC f o r 5 t o 8 days t h e c u l t u r e s were t e s t e d
for

c o n t a m i n a t i o n by i n o c u l a t i o n of t h i o g I y c o I I a t e broth

(Difco)

and by mi c r os c opi c e x a mi n a t i o n .

using c u l t u r e s ,
discarded.

Any r e s u l t s o b t a i n e d

subsequent l y shown t o be c ont a mi na t e d,

were

L e p t o s p i r a e were c o l l e c t e d from t he c u l t u r e s by

c e n t r i f u g a t i o n at

12, 000 RPM f o r

30 mi nut es.

Throughout

c e n t r i f u g a t i o n and subsequent procedures t he organisms were
ma i n t a i n e d at 0 t o 4° C.

The packed c e l l s

were washed t h r e e

16

t i m e s and resuspended in d i l u e n t t o
t u r e volume.

The d i l u e n t

1/100 t h e o r i g i n a l

cul­

used f o r d e t e r m i n a t i o n s o f a c t i v i t y

o f dehydrogenases was 0 . I 5 M N a C I .

For assayi ng a c o n i t a s e

and f umarase t h e d i l u e n t was a b u f f e r e d s o l u t i o n o f c i t r a t e
or f u m a r a t e ,

respectively.

were t r e a t e d f o r

The c o n c e n t r a t e d c e l l

15 mi nut es i n a 10 Kc Raytheon soni c o s c i l l a ­

t o r and kept a t 0 t o 4°C u n t i l
cell

suspensions

ready f o r use.

This disrupted

suspensi on c o n s t i t u t e d t h e enzyme p r e p a r a t i o n .
Isocitric

dehydrogenase a c t i v i t y

method o f Horecker and Kornberg

(29).

was assayed by t he
The r e a c t i o n mi x t u r e

c o n t a i n e d 0 . 2 ml o f 0. 5M Phosphate b u f f e r pH 7 . 0 ,
O. I M MgCI 2 , 0 . 0 2 ml o f 0.025M TPN, 0. 1
isocitrate,

0. 1

ml

0. 1

ml o f

ml of 0 . 0 0 5 M d - l

enzyme and H20 t o a volume o f 3 . 0 ml.

The r e d u c t i o n o f TPN was f o l l o w e d in a Beckman DU S p e c t r o ­
phot omet er a t 34-0 mu.
M a l i c dehydrogenase was assayed by t he method de s c r i be d
by Mehl er e t aj^.

(41).

The conver si on o f o x a l a c e t a t e t o

m a l a t e was f o l l o w e d by t he o x i d a t i o n o f DPNH t o DPN a t
mu.

340

The c u v e t t e s c o n t a i n e d 0 . 3 ml o f 0 . 2 5 M g I y c y I g I y c i n e

buffer,

pH 7 . 4 ,

oxalacetate,

0. 1

ml of 0 . 0 0 1 5 M DPNH, 0.1

pH 7 . 4 ,

0.1

ml of enzyme and 2 . 4 ml H20 .

S u c c i n i c dehydrogenase a c t i v i t y
r e d u c t i o n of t he dye,

ml of 0 . 0 0 7 6 M

was measured by t he

dichIorophenoIindophenoI.

was f o l l o w e d by obser vi ng t h e change in o p t i c a l
sured a t 600 mu.

c o n t a i n e d 0 . 2 ml of 0.2M sodium s u c c i n a t e ,
buffer,

d e n s i t y mea­

I n h i b i t i o n o f t he hydrogen t r a n s p o r t

was a c hi eved by t h e a d d i t i o n of c y a n i d e .

histidine

The r e a c t i o n

pH 6 . 5 ,

The r e a c t i o n

system
vessel

0 . 3 ml o f 0 . I M

0 . 2 5 ml of 0 . I M KCN, 0.1 ml of

17
0.05 M
phenoI

SO^, 0 . 2 ml

o f enzyme, 0 . 2

ml o f d i c h I o r o p h e n o I i n d o -

and HgO t o 5 m l .
Measurement o f

fumarase a c t i v i t y was accompl ished by

t he decreased o p t i c a l

d e n s i t y a t 300 mu o c c u r r i n g as mal at e

i s formed from f u ma r a t e ( 5 2 ) .
fumarate,

0. 1

One t e n t h ml o f . 0 I 7 M sodium

ml o f phosphate b u f f e r ,

pH 7. 3»

0 . 5 ml o f

enzyme and 0 . 5 ml o f H2O were added t o t h e c u v e t t e .
A s i m i l a r pr ocedur e de s c r i be d by Racker
employed f o r assayi ng a c o n i t a s e a c t i v i t y .
optical

(52)

was

The i n c r e a s e in

d e n s i t y a t 240 mu was measured as a c o n i t a t e was formed

f rom c i t r a t e .
sodium c i t r a t e ,

The r e a c t i o n m i x t u r e c ont a i ne d

1. 0 ml o f 0.3M

1. 0 ml o f .05M phosphate b u f f e r ,

pH 7 . 4 ,

0.5

ml o f enzyme and 0 . 5 ml o f H2O.
P r o t e i n was e s t i ma t e d by t he r a t i o of o p t i c a l

density

a t 280 mu t o t h a t a t 260 mu as de s c r i be d by Warburg and
Christian

(68).

18

RESULTS
A.

V i r u l e n c e Studies
The f i r s t

a toxic

e x pe r i me n t s were designed t o de t e r mi ne

if

substance coul d be o b t a i n e d from c o n c e n t r a t e d c e l l

suspensi ons o f L.

pomona.

were resuspended t o

Washed c e l l s ,

l/lOO the o r i g f n a l

were t hen d i s r u p t e d by t r e a t m e n t f o r
lOkc water-cockl ed,

soni c o s c i l l a t o r .

s t r a i n s LW and LB,

c u l t u r e volume.

They

15 minutes in a Raytheon
Intraperitoneal

t i o n o f hamsters w i t h up t o 2 ml o f t h i s
produced no o b s e r v a b l e si gns o f i l l n e s s .

inocula­

sonic e x t r a c t

(SE)

The ani ma l s were

s a c r i f i c e d and observed f o r gross p a t h o l o g i c a l

changes.

No

l e s i o n s were obser ved.
SE p r e p a r a t i o n s of s t r a i n LW were t hen t e s t e d f o r
their

ability

t o produce a ski n r e a c t i o n f o l l o w i n g

intradernal

inoculation.

Shaved areas on 3 hamsters and 3 gui nea pi gs

were i n j e c t e d

i n t r a d e r m a I Iy wi t h 0 . 1 ,

0 . 2 and 0 . 3 ml o f SE.

No r e a c t i o n was observed over a 72 hour p e r i o d

in e i t h e r

hamsters or gui nea p i g s .
The next approach t o t h e problem was t o compare t he
hematological

changes in hamsters i n f e c t e d wi t h whole c e l l s

wi t h t hose r e c e i v i n g
were employed in t h i s
animals,

cell

extracts.

e x p e r i me n t .

Two groups o f hamsters
Group I c o n s i s t e d o f 25

17 o f which were i n o c u l a t e d i n t r a p e r i t o n e a I Iy wi t h

1 . 0 ml o f a 7 day ol d c u l t u r e o f s t r a i n LW.
served as c o n t r o l s and r e c e i v e d
medium.

Ei g h t ani mal s

1.0 ml o f s t e r i l e c u l t u r e

At 28 and 48 hours a f t e r

i n o c u l a t i o n 3 hamsters

19
were s a c r i f i c e d and a blood sample o b t a i n e d .
t h e s e t i m e s one c o n t r o l

At each o f

hamster was a l s o s a c r i f i c e d .

At 72

hours 5 a ni ma l s were moribund and were s a c r i f i c e d wi t h 2
control

hamst er s.

Ni n e t y s i x hours a f t e r

samples were o b t a i n e d from a l l
Group I I

r emai ni ng hamst er s.

c o n t a i n e d 9 hamst er s,

cardial

in o c u la t io n of

sterile

s a l i n e by t h e same r o u t e .

and I c o n t r o l

normal

6 r e c e i v i n g an i n t r a -

1. 0 ml o f SE and 3 gi v e n
Four hours

1. 0 ml of

later

3 hamsters

were s a c r i f i c e d and a blood sample o b t a i n e d .

The r emai ni ng ani mal s were s a c r i f i c e d
s i gn s o f

i n o c u l a t i o n blood

at

18 hour s.

i l l n e s s were observed in t h i s group.

hamsters were used t o det er mi ne normal

No o v e r t

Twel ve a p p a r e n t l y
he ma t o l og i c a l

vaIues.
The changes observed in t he blood c he mi s t r y va l ue s
and d i f f e r e n t i a l

l e uk o c y t e d e t e r m i n a t i o n s ar e pr esent ed

Table

I.

firs t

48 hours a f t e r

I n f e c t e d hamsters remained normal t hr oughout t he
inoculation.

At 72 hours t h e obvious

i l l n e s s o f t h e a ni ma l s was r e f l e c t e d

in t h e blood p i c t u r e .

Urea v a l u e s were i ncr eased as much as 15 f o l d ,
l e v e l s were i n c r e a s e d ,

determinations

l e uk o c y t e s

lymphocytes.

except t h a t

urea v a l u e s ,

Differential

l eukocyt e

i n d i c a t e d an i nc r e a s e in t h e pe r c e nt a ge o f

poIymorphonucI ear
cr e a s e i n

bilirubin

w h i l e a decrease i n hemoglobin and

h e m a t o c r i t r e a d i n g s was obser ved.

similar

in

(PMN) and a cor respondi ng de­

At 96 hours t he blood p i c t u r e was

bilirubin

l e v e l s were i ncr eased w h i l e

al t hough abnormal ,

v a l ue s o f c o n t r o l

had decr eased.

ani ma l s remained w i t h i n normal

ing t h e e x p e r i m e n t .

Hemat ol ogi cal
l i m i t s dur­

20
The blood c h e mi s t r y va l ue s o f Group I I
mained e s s e n t i a l l y nor mal .
t i o n s demonstr ated
cytes.

Cont r ol

Differential

hamsters r e ­

l euk oc y t e d e t e r m i n a ­

i n c r e a s ed numbers o f PMN and f e we r

lympho­

ani ma l s i n t h i s group remained normal .

No i n d i c a t i o n havi ng been found t h a t a t o x i c substance
was p r e s e n t

i n e x t r a c t s of L. pomona, t h e p o s s i b i l i t y

an a g g r e s s i v e agent was p r e s e n t
gated.

o f s t r a i n W.
wi t h
larly

in t he e x t r a c t was i n v e s t i ­

An exper i ment was performed t o de t e r mi ne i f

o f t h e hamster

lethal

1. 0 ml o f a c u l t u r e o f s t r a i n W.

received

extracts

s t r a i n LW coul d e f f e c t t h e p a t h o g e n i c i t y

Two hamsters were i n o c u l a t e d

i n j e c t e d with

that

i n t r a p e r i t o n e a I Iy

Two ani mal s were s i m i ­

1. 0 ml o f SE o f s t r a i n LW and 2 hamsters

1. 0 ml o f a m i x t u r e o f equal

c u l t u r e and SE of s t r a i n LW.

volumes o f s t r a i n W

The ani mal s were observed f o r

2 weeks w i t h o u t any appearance o f i l l n e s s .

No l es i o n s were

observed in any o f t h e 6 ani mal s when s a c r i f i c e d .
Failing

t o f i n d any evi dence t h a t

c o n t a i n e d a t o x i n or a g g r e s s i v e a ge n t ,

e x t r a c t s of L.

a t t e n t i o n was t u r n e d t o

t h e hemol ysi n found in t h e supernat es o f

leptospiral

cultures.

To a s c e r t a i n what r o l e t h e hemol ysi n might pl ay
leptospirosis
which t h i s

it

seemed a d v i s a b l e t o f i r s t

in

f i n d a method by

substance coul d be s e pa r a t e d from c u l t u r e super nat es

and t hen c o n c e n t r a t e d .
ability

pomona

The t hermal

instability

o f t h e hemol ysi n suggested t h a t

te in nature.

it

and n o n - d i a l y s -

might o f of a pr o­

T h e r e f o r e ammonium s u l f a t e from a weighed con­

t a i n e r was added t o 30 ml o f a c u l t u r e s uper nat e o f s t r a i n
LW.

When a p r e c i p i t a t e f or med, t h e amount of s a l t

d e t e r mi ne d by t h e d i f f e r e n c e

in wei ght o f t h e s a l t

added was
container.

21
After

s t an di n g

15 minut es a t 4°C t h e p r e c i p i t a t e was removed

by c e n t r i f u g a t i o n a t
natent f l u i d

10, 000 RPM f o r

was removed f o r f u r t h e r

ment resuspended t o t h e o r i g i n a l

volume in s a l i n e .

a g a i n s t sheep RBC.

i s o t o n i c i t y o f t he f r a c t i o n s ,

a g a i n s t gui nea pi g RBC.
are presented.

All

e x t r a c t i o n and t he s e d i ­

As a c o n t r o l

of

t he y were a l s o t i t r a t e d

In t a b l e 2 t he r e s u l t s o f t h e t e s t s

of the hemolytic a c t i v i t y

in t h e p r e c i p i t a t e s formed a t Bl
No hemol ysi s in t he c o n t r o l

was pr e s e nt

and 35 per cent s a t u r a t i o n .

t ubes was observed.

T h i s pr ocedur e was then a p p l i e d to
culture f i l t r a t e s

T h i s pr o­

The 12 r e s u l t i n g f r a c t i o n s were t e s t e d

f o r hemolytic a c t i v i t y
t he

The s up e r -

100 per cent s a t u r a t i o n of ammonium

cedure was c on t i n ue d u n t i l
s u l f a t e was r ea c h e d.

i 5 mi nut e s .

as f o l l o w s :

l a r g e volumes of

culture f i l t r a t e s

were brought

t o 36 per cent s a t u r a t i o n wi t h ammonium s u l f a t e and l e f t
s t a n di n g a t 4°C f o r 6 t o

12 hour s.

moved by c e n t r i f u g a t i o n a t

The p r e c i p i t a t e was r e ­

10, 000 RPM f o r

p r e c i p i t a t e was resuspended t o about

15 mi nut e s .

The

l / 20 th t h e o r i g i n a l

volume in s a l i n e or b u f f e r e d s a l i n e pH 7 . 3 .

The r e s u l t i n g

p a l e y e l l o w s o l u t i o n was then d i a l y s e d wi t h d i s t i l l e d

wat er

f o r 2 hours and a g a i n s t s a l i n e o f b u f f e r e d s a l i n e f o r

12

hour s .
fate

The d i a l y s a t e was examined f o r t h e presence o f s u l ­

ions by t h e a d d i t i o n of a few drops o f a 10 per cent

s o l u t i o n o f barium c h l o r i d e ,
p i t a t e o f barium s u l f a t e
Following

t he f o r m a t i o n o f a whiTe p r e c i ­

indicating

i ncompl et e d i a l y s i s .

compl ete d i a l y s i s t h e p r e p a r a t i o n was s t o r e d

either

a t 4°C or f r o z e n a t - 20 ° C.
I n t he f i r s t

exper i ment t o de t e r mi ne t he t o x i c i t y

of

22
t h e he mo l y s i n ,

2 lambs o f about 2 weeks o f age and wei ghi ng

8 t o 9 kgm, were used.

Lamb 771 r e c e i v e d an i n t r a v e n o u s

i n o c u l a t i o n o f 20 ml o f hemolysi n c o n t a i n i n g a t o t a l
8 0 , 0 0 0 HU.
saline.

Lamb 800 was i n j e c t e d wi t h 20 ml o f s t e r i l e

The e f f e c t o f t h e hemol ysi n can be a s c e r t a i n e d f r o m

t h e r e s u l t s pr e s e nt e d

in T a b l e 3.

hematocrit readings of
hour s .
mal

of

lamb 771 dropped r a p i d l y a f t e r

Duri ng t h e f i r s t

20 hours a f t e r

showed no o v e r t si gns o f

was unabl e t o s t a n d ,

Hemoglobin val ues and

illness.

10

in o c u la t io n the a n i ­
At 26 hours t he

lamb

e x c r e t e d a dark red col o r e d u r i n e and

d i s p l a y e d a marked i c t e r u s o f t h e mucous membranes.

The

was comi tose a t 29 hours and di ed 30 hours a f t e r

animal
lation.

The h e m o l y t i c anemia was f u r t h e r man i f e s t e d by t he

red t i n g e d appearance o f t h e plasma from 10 hours u n t i l
Cont r ol

lamb 800 remained e s s e n t i a l l y normal

experiment.

t hr oughout t h e

o f t h e jjn vi vo a c t i o n o f

t h e hemol ysi n was under t aken in t h e next e x p e r i me n t .
lambs of 2 t o 3 weeks o f age were s e l e c t e d f o r
we i g h t s ranged from 10 t o
leptospiral

its

of

dam.

15 kgms.

a n t i b o d f e s except

All

ce i v e d

Their

lamb 32 which had an a nt i bo dy

I 0 " 5 due t o pa s s i v e i mmuni zati on from col ost r um o f
Lamb 24 served as a c o n t r o l

and r e c e i v e d 30 ml o f

was d e t e c t a b l e

in t h i s p r e p a r a t i o n .

12 ml o f washed c e l l s

s t r a i n LW.

were made i n t r a v e n o u s l y .
study.

No h e mo l y t i c
Lamb 20 r e ­

Lambs 25,

and 32 r e c e i v e d v a r i o u s dosages o f hemol ysi n.

this

s t udy .

Si x

lambs were f r e e o f

a c o n c e n t r a t e d c u l t u r e s uper nat e o f s t r a i n J .
activity

death.

,

A more e x t e n s i v e a p p r a i s a l

tite r

i nocu­

All

28,

29

injections

T a b l e 4 summarizes t h e data from

23
Animal

25 r e c e i v e d t he

l a r g e s t dose o f hemol ysi n and

devel oped an a c ut e h e m o l y t i c anemia evi denced by a drop in
hemogl obi n o f about 60 per cent

in 31 hour s.

h e m o g l o b i n u r i a and i c t e r u s were observed.

Weakness,

The animal

unconsci ous a t 31 hours and was s a c r i f i c e d .

was

Lambs 28 and

29 r e c e i v e d s m a l l e r doses o f hemol ysi n and t h e e x t e n t o f t h e
resulting

anemia was c o r r e s p o n d i n g l y

less s e v e r e .

Hemoglobin

and h e m a t o c r i t v a l u e s decreased up t o 73 hours a f t e r
tion.

Hemoglobin l osses of from 30 t o 50 per cent were not ed.

The ani ma l s were p a l e ,

their

plasma was a dark y e l l o w c o l o r

and a n o r e x i a and dyspnea were no t ed .
lethal

inocula­

Lamb 32 r e c e i v e d a

dose o f hemol ysi n but except f o r a 6 per cent decrease

in hemoglobin and a reduced h e m a t o c r i t r ea di ng a t 24 hour s,
no evi dence o f anemia was observed.
a slight

drop i n hemoglobin and h e m a t o c r i t

samples were not
si gns o f
after

Cont rol

l e v e l s but plasma

i c t e r i c nor d i d t h e animal

illness.

lamb 24 showed

show ob s e r v a b l e

Lamb 20 r e c e i v e d washed l e p t o s p i r a e and

an i n c u b a t i o n p e r i o d o f 4 days became weak and i c t e r i c .

Hemoglobin and h e m a t o c r i t
RBC from t h i s

animal

l e v e l s decreased 65 per c e n t .

lysed r a p i d l y at

37°C.

Washed

A f t e r 6 days t he

animal

was comi tose and t h e t e mp e r a t u r e was sub- nor mal .

animal

was s a c r i f i c e d a t t h i s t i m e .

The

The r e l a t i o n s h i p between

t h e course o f t h e anemi a, t h e number of organisms in t he
plasma samples and t h e appearance o f a nt i bo dy
shown in F i g u r e
second day a f t e r
relatively
of

I.

in

lamb 20 i s

Hemoglobin val ues decreased from t he

i n o c u l a t i o n through t h e f o u r t h day a t a

c on s t a n t r a t e .

Duri ng t h i s same t i me t h e number

l e p t o s p i r a e in t he plasma i ncr eased t o

10^ per ml.

Anti body

24
coul d be d e t e c t e d on t h e t h i r d day and i nc r e a s ed t o a t i t e r
of

10*4 on day 5 ^

At

days t h e number o f

l e p t o s p i r a e de­

creased and t h e r a t e o f hemol ysi s was s l i g h t l y
Lambs 771» 24,

increased.

25* 29 and 20 were submi t t ed t o gross

and mi c r o s c o p i c p a t h o l o g i c a l

examination.

In

lambs r e c e i v ­

ing hemol ysi n t he gross changes were r e p o r t e d as c o n s i s t i n g
of generalized

i c t e r u s with the

liver,

s k i n being most markedl y a f f e c t e d .
and s w o l l e n .

Ap pr ox i ma t e l y

observed on each k i d n e y .
and 25 was f i l l e d

disrupted

The l i v e r s were f r i a b l e

10 p e t e c h i a l

hemorrhages were

The u r i n a r y b l a d de r o f

lambs 771

wi t h dark red u r i n e .

Mi c r o s c o p i c
necrosis

mucous membranes and

l e s i o n s c o n s i s t e d o f a r eas o f c e n t r i I o b u I a r

i n many p a r t s o f t h e
in the periph era l

liver.

The h e p a t i c cords were

portions of the a ff e c t e d areas.

In t he k i dn e y t h e r e were numerous p y k n o t i c n u c l e i
pr oxi mal

and d i s t a l

c onvol ut ed t u b u l e s .

Many t u b u l e s con­

t a i n e d a homogenous red s t a i n i n g m a t e r i a l .
markedl y congest ed.
heart,

adr enal

in t he

The spl een was

No l e s i o n s were observed i n t h e b r a i n ,

gl and or s k e l e t a l

muscle o f any o f t hese a n i ­

mal s.
The lamb k i l l e d
infection

duri ng t h e acut e stage o f t h e

showed g r o s s l y a s l i g h t

leptospiral

i c t e r u s wi t h t h e l i v e r a ppear ­

ing copper c o l o r e d and s l i g h t l y f r i a b l e .
markedl y p a l e wi t h numerous p e t e c h i a l

The ki dneys were

hemorrhages.

The spl een

was s wol l e n and congest ed.
M i c r o s c o p i c a l l y t he
observed

in t he

exceptions.

l e s i o n s were s i m i l a r t o t hose

lambs gi ven hemolysi n w i t h t he f o l l o w i n g

The a f f e c t e d a r e a s o f t h e

liver

were not as

25
e x t e n s i v e and t h e r e appeared to be an i ncr eased number o f
lymphocytes i n t h e

i n t e r t u b u l a r a r e a s in t h e k i d n e y .

No gross or mi c r os c opi c
control

lamb.
To f u r t h e r

in

leptospiral

sub-lethal

a s c e r t a i n t h e r o l e pl ayed by t he hemol ysi n

i n f e c t i o n s and t o de t e r mi ne i f t h e hemol ysi n

was immunogenic,

lamb,

l e s i o n s were observed i n t h e

5 lambs wei ghi ng

15 t o 25 kgm were gi ven 3

doses o f hemol ysi n over an 18 day p e r i o d .

number 2 9 , had r ecover ed from a s u b - l e t h a l

One

dose o f

hemol ysi n i n t h e exper i ment p r e v i o u s l y d e s c r i b e d .
The i n j e c t i o n
day

I,

5 , 0 0 0 HU on

1 , 0 0 0 HU on day 5 , and 2 0 , 0 0 0 HU on day 18.

the f i r s t
in

schedul e was as f o l l o w s :

After

i n j e c t i o n a mi l d a n a p h y l a c t i c r e a c t i o n was observed

lamb 29 hut not a f t e r any of t h e subsequent i n o c u l a t i o n s .

T h i r t y t o 40 hours a f t e r t h e

last

i n j e c t i o n one of t h e

lambs

died.

Exami nat i on o f t he cadaver r e v e a l e d e x t e n s i v e i c t e r u s

o f the

liver,

ski n and mucous membranes and p e t e c h i a l

hemor­

rhages on t he k i d n e y .
Serum samples t a ke n from t h e

lambs duri ng t h e s e r i e s

o f hemol ysi n i n j e c t i o n s were t e s t e d f o r hemol ysi n i n h i b i t i o n
titers

(HI)

and a g g l u t i n a t i n g

a nt i bo dy t i t e r s

cr e a s e was observed i n HI t i t e r s
except

in

lamb 29.

Thi s animat

No i n ­

nor were AB t i t e r s observed
demonstrated an i n c r e a s e o f

HI t i t e r

o f from 8 t o 64 and an AB t i t e r

the

injection.

last

( AB).

of

10“ * f o l l o w i n g

Three o f t h e 4 lambs which had r e c e i v e d hemol ysi n
i n j e c t i o n s were c h a l l e n g e d wi t h s t r a i n LW.

A control

lamb

23 which had not r e c e i v e d hemol ysi n i n o c u l a t i o n s was al so

26

inoculated with t h i s

strain.

The c h a l l e n g i n g dose c o n s i s t e d

of an i n t r a v e n o u s i n o c u l a t i o n o f
10^ or gani sms per ml
mai ni ng

10 ml o f washed c e l l s .

were c o n t a i n e d in t h e

lamb which had r e c e i v e d small

challenged with

i nocul um.

10, 0 0 0 HU/kgm o f hemol ysi n.

and serum a n t i b o d y t i t e r s

cr e a s e i n hemoglobin

Lamb 29 which
change

Lambs 31 and 40 demonstrated a de­
l e v e l s which t e r m i n a t e d wi t h t h e appearance

No organisms could be observed i n t he plasma of

t h e s e ani ma l s a t 41 hours a f t e r
thereafter.

inter­

a t t h e t i me o f c h a l l e n g e showed l i t t l e

in hemoglobin v a l u e s .

of antibody.

temperature,

were det er mi ned a t p e r i o d i c

These va l ue s a r e summarized in Tabl e 5.

had an AB t i t e r

The r e ­

doses o f hemol ysi n was

F o l l o w i n g c h a l l e n g e t h e hemoglobin v a l u e s ,

vals.

About

i n o c u l a t i o n or a t any t i me

Lamb 22 which was c h a l l e n g e d wi t h hemol ysin

devel oped onl y a minimal

loss o f hemoglobin which t e r m i n a t e d

w i t h t h e presence o f a n t i b o d y .
Lamb 23 devel oped an a c ut e case o f
abbreviated
tent fe b r ile

A l oss o f 70 per cent of

t h e hemoglobin oc c ur r e d duri ng a p e r i o d o f 3 days.
g l o b i n u r i a was observed a t 96 t hrough
tion

and t h e animal

s t a r t e d on t h e s i x t h

Hemo­

120 hours a f t e r

was weak and i c t e r i c .
day.

An

1^ days wi t h a p e r s i s ­

i n c u b a t i o n p e r i o d o f about
response was not ed.

leptospirosis.

A gradual

inocula­
r ec o v e r y

The hemoglobin changes, t h e number

o f organisms i n t h e plasma and t he appearance o f a nt i bo dy
in t h i s

lamb a r e shown in F i g u r e 2.
Because lambs 29 and 22 developed an AB t i t e r

r e c e i v i n g onl y hemol ysi n

injections,

d e t e r m i n e i f t h e hemol ysi n or anot her

it

was o f

after

i n t e r e s t to

leptospiral

antigen in

27
t h e p r e p a r a t i o n was t h e a n t i g e n i c
3 we e k - ol d

Therefore a

lamb was gi v e n a s e r i e s o f i n t r a v e n o u s

10 ml o f c o n c e n t r a t e d c u l t u r e f i l t r a t e s

tio n s of
J.

stimulus.

No h e m o l y t i c a c t i v i t y

coul d be d e t e c t e d

of strain

in t h i s m a t e r i a l .

After

3 i n j e c t i o n s a t 7 day i n t e r v a l s t h i s animal

tite r

of

10- 5 .

f h e lamb was t hen c h a l l e n g e d wi t h

HU/kgm o f he mol y s i n.

A f t e r 5 hours t h e hemoglobin

decr eased f rom 9 . 0 t o 8 . 2 .
was f ound dead.

had an AB

15*000
level

had

Twel ve hours l a t e r t he animal

Exami nat i on o f t h e cadaver r e v e a l e d no

e v i de n c e o f i c t e r u s .
mortem e x a m i n a t i o n .
but t h e

inocula­

The t i s s u e s were u n s u i t a b l e f o r post
The cause o f death was not det ermi ned

lamb had had a d i a r h e a and appeared b l o a t e d p r i o r

t o chaI I enge.
The next

i n v e s t i g a t i o n was concenred wi t h t he appearance

o f an i n h i b i t o r o f t h e hemol ysi n i n normal

sera.

Lamb 771

was observed a t b i r t h and a blood sample was o b t a i n e d b e f o r e
the

Lamb 800 was bl ed w i t h i n 30

lamb was a l l owe d t o nur s e .

mi nut es a f t e r r e c e i v i n g
titers

The HI t i t e r s

and AB

o f both lambs were then det ermi ned f o r a 48 hour
The r e s u l t s are pr e s e nt e d i n T a b l e 6 .

period.
neither

c ol o s t r um.

lamb was i n h i b i t o r y a t

creased to

birth

16 a f t e r 7 t o 24 hour s.

body coul d be d e t e c t e d i n e i t h e r
I n a s i m i l a r e x p e r i me n t ,

The serum o f

but t h e HI t i t e r s

in­

No a g g l u t i n a t i n g a n t i ­

lamb.
summarized in Tabl e 6 , t wi n

lambs 974 and 975 were t a ke n a t b i r t h and a blood sample
obtained.

Lamb 975 was then a l l owed t o nurse whereas

lamb

974 was kept f rom f e e d i n g f o r 2 hours b e f o r e r e c e i v i n g
colostrum.

It

can be seen from t h e t a b l e t h a t animal

974

28

had an i n c r e a s e
After

i n HI t i t e r

duri ng t h e 2 hour f a s t i n g

period.

r e c e i v i n g c o l o s t r u m which c ont ai ned a g g l u t i n a t i n g a n t i ­

body both

lambs devel oped i nc r e a s ed HI and AB t i t e r s .

shoul d a l s o be noted t h a t a c o n s i s t e n t c o r r e l a t i o n
HI and AB t i t e r s

It

between

was not p r e s e n t .

B e f o r e any f u r t h e r s t u d i e s coul d be c a r r i e d out on
t h e n a t u r e and p r o p e r t i e s o f t h e hemol ysi n i t

was necessary

t o devel op an assay pr ocedur e which was more a c c u r a t e and r a p i d
t han t h e t i t r a t i o n
cated t h a t

wi t h

method.

Pr evi ous o b s e r v a t i o n s had i n d i ­

low d i l u t i o n s o f hemol ysi n some hemol ysi s

t ook p l a c e a f t e r

30 minutes a t

37°C.

A series of d ilu tio n s

o f hemol ysi n were t hen pr epar ed in a t o t a l
and a f t e r t her mal
cent

equilibration,

an equal

suspension o f sheep RBC was added.

volume o f 6 ml
volume o f a 2 per

After

incubation at

37°C f o r 30 mi nut es t h e t e s t s were pl aced in t he r e f r i g e r a t o r

(4°c).

Three ml

samples were withdrawn from t he mi x t u r e s at

i n t e r v a l s and pl aced i n matched c u v e t t e s .

The RBC were

sedimented by c e n t r i f u g a t i o n and t he o p t i c a l

d e n s i t y o f t he

sup e r n a t e measured a t 540 mu in a Bausch and Lomb S p e c t r o n i c

20 c o l o r i m e t e r .
3.
al ly

The r a t e s of hemol ysi s a r e shown i n F i g u r e

A f t e r 60 mi nut es a t 4°C t h e r a t e s appeared t o be e s s e n t i ­
l i n e a r and t h i s t i me was s e l e c t e d f o r t h e assay pr oc e dur e .

The p r o t o c o l

f o r t h e pr ocedur e was as f o l l o w s :

t o be assayed was d i l u t e d

Is 10 and from t h i s master d i l u t i o n

a s e r i e s o f 4 t o 6 d i l u t i o n s from 1:15 t o
a total

volume o f

The hemol ysi n

1:100 were made in

1.5 cc in o p t i c a l l y matched t e s t t u b e s .

The d i l u t i o n s and a 2 per cent suspension o f sheep RBC were
p l a c e d i n a 37°C wa t e r ba t h f o r 5 minutes t o a l l o w f o r t hermal

29
equilibration.

A volume o f

I . 5 ml o f t h e RBC suspension was

t hen added t o each t ube and mixed w e l l .

The t e s t s were i n ­

cubat ed a t 3 7 ° C f o r 30 mi nut es wi t h a g i t a t i o n a t about
mi nut e

intervals.

minutes.

st roma.

i n c u b a t i o n t h e t ubes were c e n t r i f u g e d

1, 500 RPM to sediment t h e

The o p t i c a l

at 540 mu.
1. 5

The t ubes were t hen pl aced a t 4°C f o r 60

Following

f o r 5 mi nut es a t

10

i n t a c t RBC and

d e n s i t y o f t h e super nat es was r ecorded

Wi t h each t e s t was i nc l u de d one t ube c o n t a i n i n g

ml RBC suspensi on and 1. 5 ml o f a saponin s o l u t i o n and

one t u b e w i t h
pension.

I . 5 ml

s a l i n e and an equal

The o p t i c a l

was de s i g n a t e d

volume o f RBC sus­

d e n s i t y o f t h e t ube c o n t a i n i n g saponin

100 per cent h e mol y s i s .

t ube served as a b l a n k .

The o p t i c a l

The o t h e r c o n t r o l

d e n s i t y r e a d i ng s were

c on v e r t e d to per cent hemol ysi s and p l o t t e d a g a i n s t d i l u ­
t i o n s o f h e mo l y s i n .

The r e c i p r o c a l

o f t he d i l u t i o n o f hemolysi n

produci ng hemol ysi s o f 50 per cent o f t he e r y t h r o c y t e s was
de s i g n a t e d as t he number o f h e mo l y t i c u n i t s

(HU) per ml of

hemoIysi n .
To de t e r mi ne i f t he procedure was t r u l y
v a r y i n g amounts o f hemol ysi n were pl aced
to a volume o f

1. 0 ml.

f o r hemolytic a c t i v i t y .
that

in t ubes and brought

Each o f t he s e samples was t hen assayed
The r e s u l t s

t he method was a t r u e

lysin present.

quantitative,

seen in F i g u r e 4 i n d i c a t e d

i n d i c a t i o n of t h e amount o f hemo­

The r e p r o d u c i b i l i t y o f t he procedure as

measured over a 10 day p e r i o d wi t h a c o n c e nt r a t e d c u l t u r e
filtra te

i s summarized in Tabl e 7.

o b t a i n e d wi t h t h e t i t r a t i o n
(assay pr ocedur e I I )

A comparison o f t h e v al ues

pr ocedur e and t h e new method

i s shown in T a b l e 8.

30
The r a t ( o s o f t h e t i t r a t i o n
range f rom about
error

method and procedure I I

18 t o 3 8 , g i v i n g some i n d i c a t i o n o f t he

in the t i t r a t i o n

procedure.

The average r a t i o

t h e two methods was 28 which i s a usef ul

between

f a c t o r f o r convert­

ing r e s u l t s o b t a i n e d by one method t o t h a t o f t he o t h e r .
subsequent s t u d i e s employed pr ocedur e I I

Al l

f o r de t e r mi n i n g

hemolytic a c t i v i t y .
An a c c u r a t e and c o m p a r i t i v e I y r a p i d assay procedure
now a v a i l a b - f e ,

t h e degree o f p u r i f i c a t i o n o f t h e hemol ysi n

a c hi e v e d by ammonium s u l f a t e p r e c i p i t a t i o n was examined.
Further p u rific a tio n

was a l s o at t empt ed by s e l e c t i v e adsorp­

t i o n and e l u t i o n from cal ci um phosphate gel
loss i n a c t i v i t y
cation.

r e s u l t e d wi t h

little

(33).

A l a r ge

or no observed p u r i f i ­

The h i g h e s t degree o f p u r i f i c a t i o n

was achi eved by

d i a l y s i n g t h e p r e c i p i t a t e d hemol ysi n a g a i n s t d i s t i l l e d
A p r e c i p i t a t e formed which was s o l u b l e

in s a l i n e .

i n s o l u b l e f r a c t i o n had a s p e c i f i c a c t i v i t y
t h e water s o l u b l e p o r t i o n ,
culture f i l t r a t e .

2§- t i me s t h a t of

can be f o l l o w e d by t he

The average y i e l d s o f hemolysin

o b t a i n e d by s a l t p r e c i p i t a t i o n
ther p u r i f ic a t io n resulted

The water

and 7^ t i mes t h a t o f t he o r i g i n a l

The p u r i f i c a t i o n

d a t a pr esent ed i n T a b l e 9.

water.

were 30 t o 35 per c e n t .

Fu r ­

in a p p r o x i ma t e l y a 15 per cent

yield.
An a t t e mp t was made t o remove the hemol ysi n i n h i b i t o r
present

in t h e r a b b i t

serum used f o r growing t he c e l l s .

cause p h o s p h o l i p i d s and c h o l e s t e r o l
inhibiting

c o n s t i t u e n t s o f serum,

t r a c t i o n s were pe r f or me d.

are normal

Be­

hemol ysi n-

c hl o r o f o r m and e t h e r ex­

Culture f i l t r a t e s

were shaken f o r

31

10 mi nut es w i t h 2 volumes of e x t r a c t a n t and then t h e e x t r a c t e d
phase removed in a s e p e r a t o r y f u n n e l .

Ammonium s u l f a t e was

added t o 36 per cent s a t u r a t i o n and t h e p r e c i p i t a t e resuspended
and d i a l y s e d o v e r n i g h t a g a i n s t

saline.

D i l u t i o n s of t he pr e pa r a
volume o f a 2

t i o n s were made and a f t e r a d d i t i o n o f an equal
per cent RBC suspensi on,
a t 37 ° C.

t he t e s t s were i ncubat ed f o r

30 minutes

The per cent hemol ysi s was t hen det ermi ned as p r e ­

viously described.
Only a s l i g h t

The r e s u l t s are pr esent ed

removal

in F i g u r e 3.

o f i n h i b i t o r was accompl i shed wi t h e t h e r

and c h l o r o f o r m w h i l e no i n h i b i t o r was removed by ammonium s u l ­
fate

precipitation.
Al though

at 37° C,

has been r e p o r t e d t h a t f o l l o w i n g

incubation at

p l e t e hemol ysi s
of

it

( 57),

low t e mp e r a t u r e s

i s necessary f o r

T h e r e f o r e t he

r a t e s of hemol ysi s

were examined duri ng v a r i o u s

i n c u b a t i o n a t 37°C

and 4° C.

Ex a mi na t i on o f F i g u r e 6 shows t h a t f o l l o w i n g
a lower t e mp e r a t u r e

i n t e r v a l s of

A 1:10 and a I ; I C O d i l u t i o n o f a

hemol ysi n p r e p a r a t i o n which c ont a i ne d 60 Hll/ml

rate.

were used.

incubation at

i nc r e a s es c o n s i d e r a b l y t h e h e m o l y t i c

At t he h i g h e r d i l u t i o n t h e

length of i n c u b a t i o n a t 37°C

seems t o e f f e c t t h e degree of hemol ysi s which w i l l
The next exper i ment concerned t he e f f e c t o f
a n t i s e r a on t h e course of he mol y s i s .
( s he e p)
37° C,

w i t h an AB t i t e r

of

oc c ur .
leptospiral

L. pomona a nt i s e r um

10" ^ was added t o hemol ysi n at

5 mi nut es b e f o r e t h e a d d i t i o n o f RBC suspensi on,

t a n e o u s l y wi t h t h e
suspensi on.

RBC suspension and 5 minutes

The f i n a l

com­

no r e p o r t has been made o f the e f f e c t

low t e m p e r a t u r e s on t he r a t e of he mol ysi s.

37 °C,

incubation

reaction

after

simul-.
t he RBC

v e ssel s c ont ai ned 5 . 0 ml o f

32
h e mol y s i n ,

1. 0 ml o f a n t i s e r u m and 5 . 0 ml o f RBC suspensi on.

Samples were removed a t

15 minute i n t e r v a l s f o r d e t e r m i n a t i o n s

o f per cent h e m o l y s i s .

The r e s u l t s as seen in F i g u r e 7 i n d i ­

cate t h a t

t he

longer t h e a n t i serum was in c o n t a c t wi t h t h e

hemol ysi n be f or e a d d i t i o n o f e r y t h r o c y t e s ,
i n h i b i t i o n o f h e mo l y s i s .
it

t h e g r e a t e r t he

Al though not pr esent ed in t he graph

shoul d be noted t h a t normal

serum shows a s i m i l a r

inhibition

but t o a l e s s e r degr ee.
The next study was made to a s c e r t a i n whether t he hemol ysi n
i s adsorbed by t h e e r y t h r o c y t e s and i f
this

reaction.

so,

t he s p e c i f i c i t y

of

Var yi ng amounts o f packed sheep and gui nea pig

e r y t h r o c y t e s were added t o 2 . 0 ml o f a 1:8 d i l u t i o n

o f hemolysin

c o n t a i n i n g 58 HU/ ml .

The mi x t u r e s were a g i t a t e d f o r 5 minutes

in a 37°C w a t e r b a t h .

The RBC and hemol ysi n were separ at ed by

c e n t r i f u g a t i o n and t h e h e m o l y t i c a c t i v i t y

d e t e r mi ne d .

e r y t h r o c y t e s were washed t w i c e and resuspended t o

The

1.0 ml

in

s a l i n e and i ncubat ed a t 37°C f o r

I hour and 2 hours a t 4 ° C.

The r e s u l t s ,

10,

summarized in Tabl e

s p e c i e s o f RBC removed equal

i n d i c a t e d t h a t both

amounts o f hemol ysi n a l t hough

onl y t h e sheep RBC were l y s e d.

There was no apparent d i f f e r ­

ence i n t h e amount o f hemol ysi n removed by t he 3 d i f f e r e n t
volumes o f e r y t h r o c y t e s ,

i n d i c a t i n g t h a t under t he c o n d i t i o n s

o f t h e exper i ment even 0 . 2 ml was an excess of RBC.
Because gui nea pi g RBC removed amounts of hemol ysi n
equ iv a le n t to the r e a c t i v e

sheep RBC, the p o s s i b i l i t y t h a t

t he a d s o r p t i o n was a ph y s i c a l
c ombi n a t i o n was t e s t e d
(Dow Chemical

Co. )

one r a t h e r t han a chemical

by using p o l y s t y r e n e

as t h e a ds o r b e nt .

latex

spheres

The spheres were

33
examined m i c r o s c o p i c a l l y and
o f about

5

They were,
Hemol ysi n

to

12

jj

ranged in s i z e from a d i a me t e r

w i t h an average di a me t e r of

therefore,

6

to

9

JJ.

a p p r o x i ma t e l y t h e same s i z e as e r y t h r o c y t e s .

( 2 ml ) and 0 . 5 ml and 1. 0 ml o f spheres were i ncubat ed

at 37°C f o r 5 mi n u t e s .

After

removing t h e spheres by c e n t r i f u ­

gation the hemolytic a c t i v i t y

o f t h e supernat es was d e t e r mi n e d .

Ta b l e

10 shows t h a t

larger
used,

1. 0 ml o f spheres which r e p r e s e n t e d a

s u r f a c e a r e a t han any o f t h e amounts o f e r y t h r o c y t e s
removed s l i g h t l y

l ess hemolysi n t han t he RBC.

The 0 . 5

ml volume o f spheres was about one h a l f as e f f e c t i v e as
erythrocytes

in removing hemol ysi n.

The p o s s i b i l i t y t h a t t h e hemol ysi n might be a phosphoI i pase
has been suggested ( 5 6 ) .

If

a s a l i n e e x t r a c t o f egg yol k
l e c i t h o v i t e l I in r e a c t i o n
y ol k

(48).

true,

t he hemol ysi n should r e a c t wi t h

t o produce a t u r b i d suspensi on, t h e
A 5 per cent suspension o f egg

i n s a l i n e was pr epar ed and c l a r i f i e d

passi ng t hrough a S e i t z f i l t e r .
final

and s t e r i l i z e d

D i l u t i o n s o f hemol ysi n in a

volume o f 2 . 0 ml were added t o an equal

egg y ol k e x t r a c t and i ncubat ed a t 37°C.
tained saline

i n p l a c e o f hemol ysi n.

volume o f t he

A control

Optical

were made a t 640 mu and are recorded in T a b l e
seen t h a t no d e t e c t a b l e

by

tube con­

d e n s i t y r eadi ngs
II.

It

can be

i n c r e a s e i n t u r b i d i t y coul d be ob­

served.
B.

Citric

a c i d c y c l e enzymes.

To f u r t h e r a s c e r t a i n t he b i o l o g i c a l

activity

o f ex­

t r a c t s o f L. pomona. SE from s t r a i n LW were t e s t e d f o r t he
pr esence o f f i v e

enzymes o f t h e c i t r i c

acid cycle.

Al though

34
several

d e t e r m i n a t i o n s were made on most o f t h e enzymes,

t he

r e s u l t s o b t a i n e d w i t h t h e most a c t i v e p r e p a r a t i o n s are p r e ­
sented h e r e .
S u c c i n i c dehydrogenase a c t i v i t y
pomona i s shown in F i g u r e 8 .

from L ,

of cell

free extracts

The r a t e o f o x i d a t i o n

was c a l c u l a t e d using t h e s p e c i f i c a b s o r p t i o n c o e f f i c i e n t o f
1. 6

x icAcm^/ mole f o r d i c h I o r o p h e n o I i n d o p h e n o I .

rate of

leptospiral

Protein/hr.

s u c c i n i c dehydrogenase was 7 . 4 juM Og/mgm

The e f f e c t o f mal onat e on t he r a t e o f o x i d a t i o n

i s e v i d e n t from t h e graph.
cent was o b t a i n e d
Isocitric

in t h i s

An i n h i b i t i o n of about 50 per
e x pe r i me n t .

dehydrogenase a c t i v i t y

seen i n F i g u r e 9 .

was demonst r abl e as

The r a t e o f r e d u c t i o n o f TPN t o TPNH was

0 . 7 2 juM TPN/mgm P r o t e i n / h r .
that

The o x i d a t i o n

reported f o r yeast

Thi s val ue i s about 25 per cent

isocitric

dehydrogenase ( 2 9 ) .

The o x i d a t i o n of DPNH t o DPN dur i ng t h e conversi on of
o x a l a c e t a t e t o ma l a t e was evi dence f o r t h e presence o f m a l i c
dehydrogenase ( F i g u r e

10).

tein/hr.

in t h i s

was o b t a i n e d

A r a t e of 9 . 4 2 juM DPNH/mgm Pro­

Considerable d i f f i c u l t y

st udy.
was exper i enced

f umarase and a c o n i t a s e a c t i v i t y .
buffered d ilu e n t
substrates,

P r e p a r a t i o n s made using a

were c o n s i s t e n t l y

f u m a r a t e or c i t r a t e ,

suspending medium t h e c e l l
as seen in F i g u r e s

II

in demonst r at i ng

inactive.

When t he s p e c i f i c

were i n c o r p o r a t e d

e x t r a c t s demonstrated

and 12.

i n t o t he

low a c t i v i t i e s

Because of t he hi gh endogenous

r a t e s o b t a i n e d due t o t he presence o f s u b s t r a t e

in t h e d i l u e n t ,

endogenous v al ues were s u b t r a c t e d from t he r e a d i ng s obt ai ned
after

a d d i t i o n o f more s u b s t r a t e .

35
Duri ng t h e course o f t he s e s t u d i e s t he soni c o s c i l l a t e d
cell

p r e p a r a t i o n s were examined by d a r k f i e l d microscopy t o

d e t e r m i n e t he e f f i c i e n c y o f t h e br eaki ng p r oc e d ur e .
noted t h a t
tions

enzyme a c t i v i t y

in which f ew or no i n t a c t
Enzymat i c a c t i v i t y

r u ption of the c e l l s .
after

was demonst r abl e onl y

It

was

in p r e p a r a ­

l e p t o s p i r a e were observed.

a l s o diminshed r a p i d l y a f t e r

Preparations

l o s t al most a l l

dis­

activity

5 hours e i t h e r when kept a t 4°C or f r o z e n a t - 2 0 ° C .

DISCUSSION
There a r e t h r e e predomi nant a c t i v i t i e s
initiate

in a susc ep tib le host.

t i o n and i c t e r u s .

They a r e ki dney damage, abor ­

The p r e v a l e n c e wi t h which each o f t he s e i s

found v a r i e s w i t h t h e s peci es o f
t h e host s p e c i e s .

l e p t o s p i r a e and

icterohemorrhaqiae

infections

by an a cut e n e p h r i t i s and i c t e r u s .

In

human cases of l.. pomona i n f e c t i o n s r a r e l y show

j a u n d i c e but r e n a l
L.

infecting

For example L.

o f humans a r e t y p i f i e d
contrast,

which l e p t o s p i r a e

pomona w i l l

damage i s p r e v a l e n t .

Swine i n f e c t e d wi t h

f r e q u e n t l y a b o r t and devel op v a r y i n g degrees

o f n e p h r i t i s whereas sheep i n f e c t e d wi t h t h e same organism
will

o f t e n show a l l

three manifestations.

For a compl ete under st andi ng o f t h e v i r u l e n c e o f
l e p t o s p i r a e t h e f a c t o r or f a c t o r s r e s p o n s i b l e f o r t he ki dney
alterations,

a b o r t i o n and i c t e r u s must be e l u c i d a t e d .

st udies reported

i n t h i s t h e s i s have been c on f i n e d t o t he

p o s s i b l e mechanisms by which L.
on r ena l

pomona produces i t s e f f e c t

t i s s u e and t o t he b a s i s of t h e development o f j a u n d i c e .

Our f a i l u r e t o f i n d any t o x i c a c t i v i t y
t r a c t s o f JL. pomona i s not
i ngs using e x t r a c t s o f L.
possible t h a t the t o x i c
effects,

The

therefore,

in soni c ex­

i n accord wi t h t he r e p o r t e d f i n d ­
i c t e r o h e mo r r h a q i a e ( 3 0 ) .

system i s ex t r e me l y

d i f f i c u l t t o r epr oduce.

It

is

l a b i l e and i t s
The di scr epancy

in t h e f i n d i n g s may a l s o be a r e f l e c t i o n o f a d i f f e r e n c e
t h e v i r u l e n c e mechanisms o f t h e two l e p t o s p i r a l
seems

in

species.

un fortunate t h a t the e x t r a c t of L . icterhemorrhagiae

It

37
was not examined f o r
t i s s u e s since skin

its

e f f e c t on o t h e r t han cutaneous

lesions in

l e p t o s p i r o s i s are r e l a t i v e l y

i nfrequent.
The h e m a t o l o g i c a l

changes o c c u r r i n g

i n hamsters i n ­

f e c t e d w i t h L_. pomona g i v e some i n d i c a t i o n o f t he p o s s i b l e
cause o f d e a t h .

On t h e t h i r d day a f t e r

inoculation a l l

the

hamsters examined were moribund and deat h would pr obabl y
have oc c u r r e d i n 2 t o 6 hour s.

Although hemoglobin l e v e l s

were reduced about 50 per cent t h e approxi mat e

10 f o l d

in­

c r e a s e in t h e blood urea va l ue s I n d i c a t e t h a t t h e deaths
would p r o b a b l y be due t o ur aemi a.
infections

L.

icterhemorrhagiae

in humans and gui nea pi gs and L. cani coI a i n f e c t i o n s

in humans and dogs a l s o produce death from uraemia ( 2 ) *
The o b s e r v a t i o n t h a t t he

leptospiral

hemol ysi n coul d

be p r e c i p i t a t e d from c u l t u r e super nat es by ammonium s u l f a t e ,
a f f o r d e d t h e o p p o r t u n i t y t o study t h e t o x i c i t y o f t h i s agent
using c o m p a r a t i v e l y small

volumes.

The c a p a c i t y o f t he

p r e p a r a t i o n t o produce hemol ysi s i n v i v o was i n d i c a t e d by
t h e reduced hemoglobin l e v e l s and h e m a t o c r i t r e a d i ng s i n
lambs 7 71,
the

25,

28 and 29.

inoculum was o f

protective effect

of

That t h e h e mol y t i c a c t i v i t y o f

leptospiral
leptospiral

o r i g i n was e v i d e n t from t h e
a n t i serum (lambs 52 and 2 2 ) .

The amount o f hemol ysi n necessary t o produce a f a t a l
seemed t o be somewhat v a r i a b l e .
dose o f

1 0 , 0 0 0 HU/kgm whereas

s i m i l a r dosages.

Animal

Lamb 771

result

succumbed t o a

lambs 28 and 29 r ecovered f rom

25 r e c e i v e d 1 5 , 000 HU/kgm and de­

vel oped a h e m o l y t i c anemia which would pr obabl y have been
fatal.

Pr ob a bl y t he s e d i s c r e p a n c i e s a r e due i n l a r g e p a r t

38
to the f a c t t h a t t he hemolytic a c t i v i t i e s
d e t e r mi ne d by t h e r e l a t i v e l y

inaccurate t i t r a t i o n

The a pp r o x i ma t e t w o - f o l d e r r o r
by subsequent f i n d i n g s

o f t h e i n o c u l a were

i n t h i s method was i n d i c a t e d

(Table 6 ) .

However, t h i s

a dequat e e x p l a n a t i o n f o r t h e deat h o c c u r r i n g
receiving three

" s u b - l e t h a l " h e m o l y t i c doses.

i n o c u l a t i o n o f about

It

i s do u b t f u l

l e ng t h o f t i m e an a c c u m u l a t i v e e f f e c t

in a lamb a f t e r
The f i n a l

whether a f t e r t h i s
coul d be r e s p o n s i b l e .

lambs r e c e i v e d a s i m i l a r

j e c t i o n s without observable e f f e c t ,
t h a t t h i s animal

i s not an

1 , 0 0 0 HU/kgm was r e c e i v e d 2 weeks a f t e r

a dose o f 200 HU/kgm.

Because t h r e e o t h e r

procedure.

it

series of in­

can onl y be concluded

was u n i q u e l y s u s c e p t i b l e t o t h e hemol ysi n.

The gross and mi c r os c opi c changes i n t he t i s s u e s of
lambs r e c e i v i n g hemol ysi n were s i m i l a r t o t hose found in
lambs r e c e i v i n g

leptospirae.

The l i v e r seemed t o be most

s e v e r l y a f f e c t e d whereas ki dney damage was s l i g h t .
difficu lt

t o d e t e r mi n e t o what e x t e n t t he

to t h e pr i ma r y e f f e c t

It

is

l e s i o n s ar e due

of t h e hemol ysi n or t o secondary e f f e c t s

o f t h e e x i s t i n g anemi a.

There i s no apparent reason why t h e

c y t o t o x i c a c t i o n o f t he hemolysi n should be c o n f i n e d t o
erythrocytes.

However,

i n vi ew o f t h e e x t e n t and r e l a t i v e l y

l o c a l i z e d n a t u r e o f t h e t i s s u e damage,

it

would seem wise

a t t h i s t i m e t o assume t h a t t h e cause o f t h e c e l l u l a r changes
is anoxia.

The p a t h o l o g i c a l

changes observed i n t h e

lambs

r e c e i v i n g hemol ysi n a r e s i m i l a r t o those r e p o r t e d by H a r t l e y
(25)

i n a severe ou t br e a k o f

leptospirosis

i n sheep.

The r e s u l t s o f t h e exper i ment s concerni ng t h e immunog e n l c i t y o f t h e hemol ysin were obscured by an apparent

39
contaminating
tion.

leptospiral

a n t i g e n I n t h e hemol ysi n p r e p a r a ­

T h i s c o n c l u s i o n i s based on t h e f a c t t h a t p r e p a r a t i o n s

f r om c u l t u r e f i l t r a t e s

o f both s t r a i n LW and t h e n o n - h e m o l y t i c

s t r a i n J i n i t i a t e d t h e appearance o f
antibodies.

It

leptospiral

agglutinating

is possible t h a t s t r a i n J contained undetect­

a b l e amounts o f hemol ysi n and t h a t a n t i b o d i e s t o t he hemol ysi n
a r e c a pa b l e o f a c t i n g on t h e s u r f a c e o f
produce a g g l u t i n a t i o n .
serological

leptospiral

c e l l s to

An a t t e m p t was made t o c l a r i f y

the

p r o p e r t i e s o f t h e hemol ysi n by produci ng a n t i b o d y

t o t h e a n t i g e n o f t h e s t r a i n J p r e p a r a t i o n and t hen c h a l l e n g ­
ing t h i s animal

w i t h hemol ysi n.

If

the antigen

t i o n was not t h e hemol ysi n t hen t he animal
h e m o l y t i c anemi a.

U n f o r t u n a t e l y t h e animal

in t h e p r e p a r a ­

should have developed
di ed s h o r t l y a f t e r

c h a l l e n g e o f an a p p a r e n t l y no n - h e mo l y t i c c o n d i t i o n o f u n d e t e r ­
mined o r i g i n .

At t empt s t o demonstrate an a nt i bo dy t o t h e

he mol ysi n i n l e p t o s p i r a l

a n t i s e r a by means o f a t ube p r e c i p i t i n

t e s t have been unsuccessf ul
d i f f u s i o n technique

(50)

(3).

to t h i s

A p p l i c a t i o n o f t h e agar
system might be r e wa r d i n g .

The use o f t h e hemol ysi n i n h i b i t i o n t e s t as a means o f demon­
strating
natural

a n t i b o d y t o t h e hemol ysin i s c ompl i c a t e d by t he
i n h i b i t o r present

i n normal

sera.

The dat a i n T a b l e

6 I n d i c a t e t h a t hemol ysi n I n h i b i t i o n t i t e r s cannot be c o r r e ­
lated with a g g l u t i n a t i n g
had HI t i t e r s
antibody.

of

antibody t i t e r s .

Lambs 771 and 800

16 w i t h o u t any demonstrabl e a g g l u t i n a t i n g

The same HI t i t e r

was observed in lamb 974 wi t h

an a g g l u t i n a t i n g t i t e r

of

doubt t h a t

a n t i s e r u m i n h i b i t s t h e hemol ysi n t h e

leptospiral

I 0 “ 3.

Al though t h e r e

is

little

q u e s t i o n as t o whether t he hemol ysin i d a n t i g e n i c can not
be answered a t t h i s t i m e .

40
At b i r t h
in t h e i r
birth

sera.

lambs have

little

This property

or no a n t l h e m o l y t l c

i nc r e a s e s in a few hours a f t e r

e v i d e n t l y w i t h o u t t h e i n f l u e n c e o f c o l o s t r u m.

absence o f h e m o l y t i c
a s s o c i a t e d wi t h

i n h i b i t i o n may be a f a c t o r

leptospirosis.

i s due t o h e m o l y t i c anemi a,

substance

The

in a b o r t i o n

I f t h e death o f t h e f o e t u s

as has been proposed ( 1 8 ) ,

t he

f o e t u s would seem t o be h i g h l y s u s c e p t i b l e t o t h e a c t i o n o f
leptospiral

hemolysin.

the p l a c e n ta l

Whether t he hemolysi n can pass t hrough

membranes, however, has y e t to be a s c e r t a i n e d .

A l s t o n and Broom ( 2 ) have o b j e c t e d t o d e s c r i b i n g t he
hemol ysi n as a t o x i n .

T h e i r c r i t i c i s m was based on t he f i r s t

r e p o r t o f t h e pr esence o f hemol ysin i n c u l t u r e super nat es ana
not on any study o f t h e i n vi vo e f f e c t s o f t h i s substance.
They reasoned t h a t
not

if

cui nea pi g and human e r y t h r o c y t e s were

lysed by t h e hemol ysi n and i f _L. i cterohemorrhaq i ae c u l ­

t u r e s were not h e m o l y t i c ,
cause o f t h e

i c t e r u s and hemorrhage a s s o c i a t e d wi t h c l a s s i c a l

W e i l ' s disease.
it

t he hemol ysin could not be t he

This

i s o b v i ou s l y a v a l i d c r i t i c i s m al t hough

should be mentioned t h a t

that

some s t r a i n s o f L;.

subsequent r e p o r t s have shown

i ct erohemorrhaq i ae do produce small

amounts o f hemol ysi n and t h a t human RBC ar e moder at el y sus­
c e p t i b l e t o he mol y s i n.
Some r e s o l u t i o n o f t he problem seems p o s s i b l e when
it

i s r e c o g n i z e d t h a t t he i c t e r u s a s s o c i a t e d wi t h acut e

l e p t o s p i r o s i s o r i g i n a t e s from two s e p a r a t e causes.
t h e so c a l l e d t o x i c

j a u n d i c e in which e x t e n s i v e damage t o

h e p a t i c t i s s u e p r e v e n t s t he normal
to c h o l e b i J i r u b i n ,

One i s

conver si on o f h e m o b i I i r u b i n

and h e m o b i I i r u b i n

i s t h e r e f o r e pr esent

in

41
l a r g e amounts i n t he bl ood,
Toxic

j a u n d i c e i s c h a r a c t e r i s t i c of L.

infections

i cterohemorrhaa i ae

in humans and gui nea p i g s .

i c t e r u s a s s o c i a t e d wi t h
Rapi d

e v e n t u a l l y s ta in in g the t i s s u e s .

l y s i s of

The second t ype of

leptospirosis

is hemolytic i c t e r u s .

l a r g e numbers o f e r y t h r o c y t e s r e s u l t s

f o r m a t i o n o f e x c e s s i v e amounts o f h e m o b i I i r u b i n .
the

liv e r tissue

i s f u n c t i o n i n g no r ma l l y

it

such l a r g e amounts and a ga i n h e m o b i I i r u b i n
circulation,

resulting

in i c t e r u s .

and t o a l e s s e r e x t e n t

Although

cannot cope wi t h
i s pr e s e nt

Hemol yt i c

duced i n L. pomona i n f e c t i o n s of c a t t l e

in t h e

(54)

icterus

in t he
i s pr o­

and sheep ( 4 7 )

in i n f e c t i o n s due t o L . cani coI a and

L. q r i p p o t y p h o s a ( 2 ) .
Therefore,

it

would seem best t o c l a s s i f y t he hemol ysi n

as a t o x i n produced by some s t r a i n s o f
responsible f o r the
mal s .

l e p t o s p i r a e which i s

i c t e r u s seen p r i m a r i l y

in rumi nant a n i ­

The cause o f t h e h e p a t i c damage r e s u l t i n g

which i s observed

in o t h e r

assi gned t o anot her
t h a t other

leptospiral

leptospiral

leptosiral

i n f e c t i o n s must be

toxin.

F u r t h e r evi dence

t o x i n s must be a c t i v e ,

t he c h a r a c t e r i s t i c ki dney

lesions of

in i c t e r u s

i s t he f a c t t h a t

l e p t o s p i r o s i s cannot

be produced by i n j e c t i o n s o f hemol ysi n.
I n vi ew o f t h e mechanisms proposed f o r t h e h e mo l y t i c
anemia i n sheep ( 4 7 )

it

in Figures

The hy p o t h e s i s

I and 2.

destroys the

leptospirae

i s i n t e r e s t i n g t o examine t h e data
is t h a t

l y t i c a nt i bo dy

i n t h e blood stream r e s u l t i n g

r e l e a s e o f a h e m o l y t i c endot oxi n or h e mol y t i c a n t i g e n .
both

lambs, 20 and 23, t h e

initiation

in the
In

of hemol ysi s seems t o

be c l o s e l y a s s o c i a t e d wi t h t he presence of

leptospirae

in

42
t h e blood s t r eam.

Wi t h t h e appearance o f a n t i b o d y ,

the

organi sms d i s a p p e a r and t h e r a t e of hemol ysi s seems t o be
increased.

This

latter

t he h y p o t h e s i s t h a t
lysin.

o b s e r v a t i o n would be in accord wi t h

lysis of

l e p t o s p i r a e r e l e a s e s t he hemo­

However, t h e r e s u l t s o f ot h e r exper i ment s do not seem

t o be in agreement w i t h t h i s h y p o t h e s i s .

Lambs 32 and 22

were p r o t e c t e d from t h e hemol ysin by a n t i b o d y , and t h e i n ­
hibitory

e f f e c t o f a n t i s e r u m on in v i t r o hemol ysi s

f rom F i g u r e 7.
spirae

it

Therefore,

i f antibody

should n e u t r a l i z e or

F u r t h e r e vi dence t h a t a n t i b o d y
hemolytic e f f e c t

is

is evident

l y s i ng t h e

lepto­

i n h i b i t t h e r e l e a s e d hemol ysi n.
i s not a s s o c i a t e d wi t h t h e

i s a f f o r d e d by t he f a c t t h a t hemol ysi s pr o­

ceeds b e f o r e a n t i b o d y

is detectable ,

t h e hemol ysi n i s p r e s e n t
maximum l e p t o s p i r a l

and t h a t

in c u l t u r e ,

in g r e a t e s t amounts I t o 3 days a f t e r

growth has occurr ed but does not i n c r e a s e

upon agi ng and a u t o l y s f s o f t h e

leptospirae.

Al t hough t hese r e s u l t s do not show c o n c l u s i v e l y t h a t
h emol ysi s does not r e s u l t from t he
l y s i m by a n t i b o d y

it

l y s i s and r e l e a s e of hemo-

seems wo r t h wh i l e i n vi ew o f t he r e s u l t s

o b t a i n e d t o propose an a l t e r n a t i v e

sequence o f event s which

result

i n a h e m o l y t i c anemia.

A f t e r e s t a b l i s h i n g t hemsel ves

in t h e

l i v e r and l ymphat i c t i s s u e t he l e p t o s p i r a e m u l t i p l y

and r e l e a s e hemol ysi n i n t o t h e c i r c u l a t o r y

system.

leptospirae

in t he blood and

increase

in number t he y appear

hemol ysi n p r o d u c t i o n r eaches a maximum.
adsorbed t o t h e e r y t h r o c y t e s
ceeds.

( Ta b l e

The hemolysi n is

10) and hemol ysi s pr o­

One t o 2 days a f t e r t h e appearance o f

the c i r c u l a t i o n ,

As the

leptospirae

in

a nt i bo dy can be d e t e c t e d and t he m u l t i p l i c a t i o n

43
of

leptospirae

is h a lt e d .

Hemolysin which i s adsorbed t o

the erythrocyts

i s not e f f e c t e d by t h e a n t i bo dy

l i n e s 2 and 3 ) ,

and hemol ysi s c o n t i n u e s .

(Figure 7

The appar ent

i n t h e h e m o l y t i c r a t e about 24 hours a f t e r t h e

increase

leptospirae

have been de s t r o y e d by a nt i bo dy can be e x p l a i n e d by examining
t h e da t a

in Tabl es 3 and 4 .

h e mo l y s i n ,

When lambs a r e

i n j e c t e d wi th

maximum r a t e s of hemol ysi s do not occur u n t i l

about 24 hours a f t e r

inoculation.

Some of t h i s

inhibition

i s pr o b a b l y due t o t h e hemol ysi s i n h i b i t o r s pr e s e nt
sera.

Therefore,

to the f a c t th a t

the

in normal

i ncr eased h e mo l y t i c r a t e would be due

l e p t o s p i r a e were in g r e a t e s t number and

t h e r e f o r e produci ng maximum amounts of hemol ysi n 24 hours
b e f o r e t h e e f f e c t o f t h i s hemol ysi n was m a n i f e s t e d .
It

may be argued t h a t t he

inoculated

into

l a r g e number o f

leptospirae

lambs 20 and 23 do not compare wi t h t he

i noculum i n a n a t u r a l

exposure t o

leptospirae,

and t h a t the

results therefore,

do not r e f l e c t a t r u e p i c t u r e o f t he r e ­

l a t i o n s h i p between

Ieptospiremia,

o f antibody.

i s pr oba bl y t r u e t o some e x t e n t

Thi s

hemol ysi s and appearance
in lamb

23 i n which t h e i n c u b a t i o n p e r i o d was l ess than 2 days.
However,

p r e v i o u s work ( 1 4 ) has i n d i c a t e d t h a t t h e c u l t u r e s

used f o r t h e i n o c u l a were a t a st age o f in v i t r o c u l t i v a t i o n
such t h a t t h e i r LD^q f o r hamsters was about
F u r t h e r m o r e t h e number o f
infection

10^ organi sms.

leptospirae required to

initiate

i n a hamster was found t o be a p p r o x i ma t e l y one.

A f t e r washing and r esuspensi on t h e i n o c u l a f o r t h e
contained a t o t a l
I virulent

o f about

10® l e p t o s p i r a e per ml.

organism was pr e s e nt f o r every

10^ t o t a l

lambs
I f onl y
cells,

44
t he n about
the

10^ v i r u l e n t

inocula.

Ten t o

t ha t the t o t a l
I0^#

Thi s

l e p t o s p i r a e per ml were pr e s e nt

12 ml o f suspension was i n j e c t e d so

number o f v i r u l e n t organisms was a p p r o x i ma t e l y

i s not an unreasonabl e number when i t

nized t h a t

jn

swine u r i n e ,

c o n t a i n a p p r o x i ma t e l y

a frequent

source o f

10? l e p t o s p i r a e per ml

i s recog­

infection,
(44).

It

may
is

t h e a u t h o r ' s o p i n i o n t h a t t h e r e s u l t s o b t a i n e d from lambs
20 and 23 a r e a p p l i c a b l e ,

f o r t he most p a r t ,

to natural

in­

fections.
The assay pr ocedur e devel oped f o r de t e r mi n i n g t h e
hemolytic a c t i v i t y
relatively

o f hemol ysi n p r e p a r a t i o n s seems t o be

a c c u r a t e and r a p i d .

Other

investigators

(I)

have

employed s p e c t r o p h o t o m e t r i c methods f o r assayi ng

leptospiral

hemol ysi n but

The r e s u l t s

i n c u b a t i o n was performed a t 37°C,

shown in F i g u r e 6 i n d i c a t e t h a t wi t h hi gh d u l u t i o n s of hemo­
lysin

i n c u b a t i o n at 37°C should be f o l l o w e d by a p e r i o d at

lower t e m p e r a t u r e s f o r maximum hemol ysi s t o t a k e p l a c e .
T h i s so c a l l e d h o t - c o l d hemol ysi s has a l s o been observed wi t h
t h e al pha t o x i n of C l o s t r i dui m we Ich i i
t o x i n of staphylococci

(22).

The a t t e m p t s t o remove t he
pr e s e n t

in r a b b i t

i n h i b i t o r o f t h e hemol ysi n

serum were not s u c c e s s f u l .

has de s c r i b e d f o u r hemol ysi n i n h i b i t o r s
lecithin,

cholesterol,

indicating that

not t h e p r i n c i p a l
cent

Ponder ( 5 1 )

in normal

al bumi n and g l o b u l i n .

and e t h e r e x t r a c t i o n removed only small
(Figure 5 ) ,

( 6 5 ) and t h e bet a

se r a ;

Chl orof or m

amounts o f i n h i b i t o r

l e c i t h i n and c h o l e s t e r o l

cause o f t h e i n h i b i t i o n .

were

Thirty-five

per

s a t u r a t i o n wi t h ammonium s u l f a t e di d not remove i n h i b i t o r

45
which would
ble.

i n d i c a t e t h a t al bumin was pr obabl y not r e s p o n s i ­

Whether serum g l o b u l i n

leptospiral

i s t he p r i n c i p a l

hemol ysi n cannot be a s c e r t a i n e d at t h i s

The f a c t t h a t t h e hemol ysi n i s p r e c i p i t a t e d
s a t u r a t i o n w i t h ammonium s u l f a t e

g l o b u l i n molecules.

it

i s formed ( T a b l e 9 ) .

has s i m i ­

i s adsorbed t o t he

water a p r e c i p i t a t e

Euglobulin is insoluble

wat er and p r o b a b l y much o f t h e p r e c i p i t a t e

in d i s t i l l e d

i s composed of

Ps e udogl obul i n i s pr oba bl y one o f t he major

components o f t h e wat er s o l u b l e s up e r n a t e .
is e s s e n t i a ll y

it

When t he p r e c i p i t a t e d hemol ysi n i s

d i s s o l v e d and d i a l y s e d w i t h d i s t i l l e d

subst ance.

time.

by 35 per cent

indicates that

l a r p r o p e r t i e s t o a g l o b u l i n or t h a t

this

i n h i b i t o r of

The hemol ysi n

e q u a l l y d i s t r i b u t e d between t h e s e two f r a c ­

t i o n s which may i n d i c a t e t h a t t he hemol ysin i s adsorbed t o
the g l o b u l i n .

However,

d i l u t e d the i n h i b i t o r
t he

inhibitory

can not be d e t e c t e d .

component o f serum then i t

l i k e l y t h a t hemol ysi n
if

when hemolysi n p r e p a r a t i o n s are

will

is the i n h i b i t o r .

it

is

does not seem

i s adsorbed to g l o b u l i n .

hemol ysi n i s adsorbed t o g l o b u l i n

globulin

I f globulin

In c o n t r a s t

is u n lik e ly that

The answers t o t hese q u e s t i o ns

be p o s s i b l e when f u r t h e r p u r i f i c a t i o n o f t he hemolysi n

has been a c h i e v e d .

The numerous methods now a v a i l a b l e f o r

s e p a r a t i o n and p u r i f i c a t i o n

of p r o t e i n s should be a p p l i e d

t o p u r i f i c a t i o n o f t h e hemol ysi n.
of the hemolytic

inhibitor

The problem o f t he i d e n t i t y

can a l s o be approached by f r a c ­

t i o n a t i o n o f serum and t e s t i n g

f o r t he i n h i b i t o r y

component.

The a d s o r p t i o n o f hemol ysi n t o e r y t h r o c y t e s does not
seem t o be a s p e c i f i c

union o f "enzyme” wi t h " s u b s t r a t e "

46
(Table

10).

reactive

Guinea pi g RBC, which e i t h e r do not have t he

s i t e f o r t h e hemol ysin or t he s i t e

t o t h e h e mol y s i n ,
as t h e r e a c t i v e

seem t o adsorb hemol ysi n t o t he same degree

sheep c e l l s .

Polystyrene

adsorb t h e hemol ysi n but t o a somewhat
t h e b a s i s o f t h e a v a i l a b l e dat a i t
t i o n o f hemol ysi n t o e r y t h r o c y t e s
o f a physical

i s not a v a i l a b l e

latex

spheres al so

l esser degr ee.

On

appears t h a t t he adsorp­
i s n o n - s p e c i f i c and pr oba bl y

nature.

The i n h i b i t o r y

e f f e c t o f a nt i s e r u m on h e mol y s i s ,

as

seen i n F i g u r e 7,

is s i m i l a r to that of other t o x i n - a n t i to x in

systems ( 6 ,

If

70).

a nt i s e r u m and hemol ysi n a r e i n c o n t a c t

before the a d d i t i o n of e ry th r oc y te s ,

hemol ysi s i s

When t h e a n t i s e r u m and hemol ysi n ar e in c o n t a c t
sence o f RBC t he

inhibition

is diminished.

inhibited.

in t h e p r e ­

These r e s u l t s

i n d i c a t e t h a t once hemol ysi n and e r y t h r o c y t e have combined
a n t i s e r a has no e f f e c t .
findings

The p o s s i b l e s i g n i f i c a n c e o f t hese

in r e l a t i o n t o in v i vo hemol ysi s has a l r e a d y been

di scussed.
The f a i l u r e t o f i n d any a c t i o n o f t he hemolysi n on
I e c i t h o v i t e l I i n i s not

in agreement wi t h t he suggested

phos phoI i pa s e a c t i v i t y of t he hemolysi n ( 5 6 ) .
t i o n was based on t h e f a c t t h a t
inhibit

t he hemol ysi n o f

T h i s sugges­

l e c i t h i n and o t h e r p h o s p ho l i pi d s

leptospirae.

The f a c t t h a t

lecithin

i s an i n h i b i t o r o f many t ypes o f hemol ysi ns such as saponin
and d i g i t o n i n has been de s c r i be d by Ponder ( 5 1 ) .
t he o b s e r v a t i o n t h a t
does not

seem t o

lecithin

inhibits

leptospiral

indicate necessarily that

s u b s t r a t e f o r t h e hemol y s i n.

Therefore,

lecithin

hemol ysi n
i s t he

However, t h e f a i l u r e of the

47
hemol ysi n t o produce a p o s i t i v e egg y o l k r e a c t i o n does not
n e c e s s a r i l y p r e c l u d e t h a t phosphoI i pase a c t i v i t y
For

i s a bs e n t .

i n s t a n c e snake venom l e c i t h i n a s e does not cause t u r b i d i t y

o f egg y o l k suspensions ( 6 6 ) .

The mode o f a c t i o n o f t he

hemol ysi n shoul d be i n v e s t i g a t e d by examining t h e product s
o f t h e r e a c t i o n o f hemol ysi n wi t h e r y t h r o c y t e s and v a r i o u s
possible substrates.

Such pr ocedur es have been employed

in d e t e r m i n i n g t h e a c t i o n o f t h e hemol ysi n o f mumps v i r u s
(42,

60),

and would be a usef ul

gui de t o t he i n v e s t i g a t i o n

o f o t h e r hemol ysi n r e a c t i o n s .
B.

C itric

Aci d Cycl e Enzymes

The r a t e s o f enzymati c a c t i v i t y
o f L.

observed wi t h e x t r a c t s

pomona were low i n comparison t o enzymes o f o t h e r

mi c r oo r g a ni s ms.

However, a low m e t a b o l i c r a t e would not

be unexpect ed when one c o n s i de r s t he f a c t t h a t t h e g e n e r a t i o n
t i me o f

leptospirae

i s about 8 hours as compared t o 30 t o 40

mi nut es f o r many b a c t e r i a .

Pr obabl y t h e r a t e s observed f o r

fumarase and a c o n i t a s e a r e not a t r u e
activity

o f t he s e enzymes.

bl y r e s u l t e d

Their evident

in c o n s i d e r a b l e

pre pa ratio n of the c ell

instability

loss o f a c t i v i t y

duri ng t he

r e p o r t e d t h a t malonate did

i n h i b i t t h e r e s p i r a t i o n of r e s t i n g c e l l

leptospirae.

proba­

extracts.

F u l t o n and Spooner ( 2 0 )
not

i n d i c a t i o n o f t he

preparations of

In t h e s t u d i e s r e p o r t e d in t h i s t h e s i s ,

malonate

i n h i b i t i o n o f t h e s u c c i n i c dehydrogenase system was r e a d i l y
detectable.

The f a c t t h a t

in c o n t r a s t t o t h e c e l l

t he e a r l i e r

work used whole c e l l s

e x t r a c t s employed in t h i s

study may

48
indicate that

l e p t o s p i r a e are

permeability of resting

bacterial

and i n h i b i t o r s has f r e q u e n t l y
regarding t h e i r

role

However,

c e l l s to various substrates

led t o erroneous c onc l us i ons

o f t he enzymes o f t he c i t r i c

c y c l e were i n v e s t i g a t e d ,

t h e presence o f s u c c i n i c ,

dehydrogenases, fumarase and a c o n i t a s e

that the c i t r i c
it

Im­

in metabol i sm ( 3 5 ) .

Al t hough not a l l

isocitric

impermeable t o mal ona t e .

acid cycle

is pr obabl y o p e r a t i n g

acid

mal i c and

i s evi dence
in l e p t o s p i r a e .

has not been a s c e r t a i n e d whether o t h e r me t a b o l i c

pathways may a l s o be f u n c t i o n i n g .

The g l y o x y l a t e c y c l e

( 28),

i n v o l v i n g t he breakdown o f i s o c i t r a t e to g l y o x y l a t e and
s u c c i n a t e wi t h subsequent combi nat i on of a c e t a t e and g l y o x y ­
l a t e t o f or m m a l a t e ,

may be a second method by which l e p t o ­

spirae metabolize n u t r i e n t s .
P h o s p h o l i p i d s and amino aci ds seem t o be u t i l i z e d

by

leptospirae.

Both s u b s t r a t e s would be e v e n t u a l l y me t a bo l i z e d

in t h e c i t r i c

acid cy cle ,

acids to acetyl

either

by t h e breakdown o f f a t t y

coenzyme A or in t he case o f amino a c i d s by

t h e a p p r o p r i a t e t r ansami nase formi ng

i n t e r m e d i a t e s of t he

eye I e .
If

l e p t o s p i r a e possess a phosphoI i pase f o r p h o s p ho l i pi d

met a bol i s m,

i t s possible

common a r e a o f
studies.

i d e n t i t y wi t h hemol ysin pr ov i d e s a

investigation for

v i r u l e n c e and metaboli sm

At t empt s to c o r r e l a t e t he v a r i ous v i r u l e n c e f a c t o r s

of pathogens w i t h t h e i r m e t a b o l i c enzymes or pr oduct s would
c o n t r i b u t e much t o our concepts o f p a r a s i t i s m .

49
SUMMARY AND CONCLUSIONS
Washed c e l l

suspensions o f L.

pomona were s u b j e c t e d

t o s oni c o s c i l l a t i o n and t h e c e l l - f r e e
toxic

activity.

extracts tested for

N e i t h e r hamsters or guinea pi g s showed e v i ­

dence o f an i n f l a mma t o r y r e a c t i o n f o l l o w i n g
t i o n o f O.l

t o 0 . 5 ml o f e x t r a c t .

1 . 0 ml o f e x t r a c t
alterations

i n t r a d e r ma l

Intracardial

inocula­

i n j e c t i o n of

in hamsters di d not produce s i g n i f i c a n t

in t h e hematol ogy,

i nc r e a s e d urea and b i l i r u b i n

whereas whole c e l l s produced

l e v e l s and decreased hemoglobin

and h e m a t o c r i t v a l u e s .
The hemol ysi n produced by l e p t o s p i r a e was p r e c i p i t a t e d
by 35 per cent s a t u r a t i o n wi t h ammonium s u l f a t e .
hemol ysi n p r e p a r a t i o n s were i n o c u l a t e d i n t o
sulting

lambs wi t h a r e ­

l oss o f hemoglobin o f 20 to 60 per c e n t .

and j a u n d i c e were noted i n
h e mo l y s i n .
the

Concent r at ed

lambs r e c e i v i n g

Weakness

l a r g e doses of

Gross and mi cr oscopi c exami nat i on o f t i s s u e s o f

lambs r e v e l a e d ar e a s o f de g e ne r a t i o n and n e c r o s i s

l i v e r and p e t e c h i a l

hemorrhages on t h e ki dney wi t h small

a r e a s o f d e g e n e r a t i o n o f t h e r enal
j a u n d i c e was promi nent
o f h e mol y s i n .

in t h e

in a l l

tubules.

A generalized

a ni mal s r e c e i v i n g

l a r ge doses

The gross appearance and l e s i o n s o f

lambs r e ­

c e i v i n g washed c e l l s of L. pomona were s i m i l a r except f o r
more e x t e n s i v e r enal

damage.

Lambs which had demonstrabl e

serum a g g l u t i n a t i n g a nt i bo dy were p r o t e c t e d from l e t h a l
o f h e mo l y s i n .

At t empt s t o immunize lambs wi t h small

doses

doses

o f hemol ysi n were hampered by t he development o f a g g l u t i n a t i n g

50
antibody.

Conc e nt r a t e d c u l t u r e f i l t r a t e s

s t r a i n a l s o produced a g g l u t i n a t i n g

o f a no n - h e mo I y t i c

antibody.

Anti body t o

t he hemol ysi n coul d not be demonstrated by use of hemol ysi n
inhibition tests.
spiral

Al though hemol ysi n i s

inhibited

by l e p t o ­

a n t i s e r u m no evi dence coul d be o b t a i n e d t h a t t he

hemol ysi n was a n t i g e n i c .
I n v i t r o s t u d i e s on t h e hemol ysi n demonstrated t h a t
ammonium s u l f a t e p r e c i p i t a t i o n gave y i e l d s of hemol ysi n of
about 30 per c e n t .
precipitating

Maximum p u r i f i c a t i o n was achi eved by

c o n c e n t r a t e d hemol ysi n p r e p a r a t i o n s by d i a l y s i s

with d i s t i l l e d

water.

A 7 to 8 fold

i nc r e a s e in p u r i t y r e s u l t e d

from t h i s p r oc e d ur e .
The e f f e c t

o f t e mp e r a t u r e on t h e r a t e of hemolysi s

was i n d i c a t e d by t h e demonst r at i on o f

i ncr eased r a t e s

hemol ysi n t e s t s were i ncubat ed at 4°C a f t e r

initial

i f t he

incubation

a t 37° C.

Ad s or pt i o n o f hemolysin t o e r y t h r o c y t e s was demon­

strated.

RBC o f both gui nea pi gs and sheep adsorbed t he

hemol ysi n but onl y sheep c e l l s were l ysed.

The n o n - s p e c i f i c i t y

o f t h e a d s o r p t i o n was f u r t h e r shown by t h e a d s o r p t i o n o f t h e
hemol ysi n t o p o l y s t y r e n e spheres.
The e f f e c t o f a n t i serum on hemol ysi s was shown t o be
dependent on t h e sequence in which hemol ysi n,
RBC were mixed.

If

a nt i s e r u m and

hemol ysi n and a nt i s e r u m were in c ont a c t

b e f o r e a d d i t i o n o f RBC, hemol ysi s was i n h i b i t e d .

When a n t i ­

serum was added a f t e r or wi t h t h e a d d i t i o n o f RBC t o t he
hemol ysi n t he

inhibitory effect

was di mi n i s h e d .

At t empt s t o demonstrate phosphoI i pase a c t i v i t y
hemol ysi n p r e p a r a t i o n s by means of t h e
were negat i v e .

IecithovitteIin

wi t h
reaction

51
The r e s u l t s o f jjn v i t r o
p r o p e r t i e s and e f f e c t

studies of the adsorption

o f a n t i s e r u m on t h e hemol ysi n were used

t o a t t e m p t t o d e s c r i b e t h e Jji vi vo processes which produce a
h e m o l y t i c anemia in l e p t o s p i r o s i s .
Wi th t h e de mons t r a t i on o f t h e t o x i c i t y
h e mol y s i n ,
with

of

our under st andi ng of t h e h e mo l y t i c anemia a s s o c i a t e d

l e p t o s p i r o s i s has been g r e a t l y

clarified.

Al though t he

hemol ysi n i s pr oba bl y not o f major s i g n i f i c a n c e
spiral

leptospiral

infections,

in a l l

lepto­

i t s e f f e c t s pr oba bl y c o n t r i b u t e s i g n i f i c a n t l y

t o t h e economic

l osses s u f f e r e d by t h e

leptospirosis.

The evi dence t h a t once t h e hemol ysin has i n i t i ­

at ed

its effect,

h a l t i n g m u l t i p l i c a t i o n of the

does not a r r e s t h e mol y s i s ,

Cel I f r e e e x t r a c t s from L.
acid cycle.

of

pomona were t e s t e d f o r enzymes

V a r i o u s degrees o f a c t i v i t y

genase, m a l i c dehydrogenase,

It

were

i s o c i t r i c dehydro­

fumarase and a c o n i t a s e .

i s concluded t h a t t he c i t r i c

pr ob a bl y o p e r a t i n g

leptospirae

leptospirosis.

demonstr ated f o r s u c c i n i c dehydrogenase,

t h i s evi dence i t

i n d u s t r y from

emphasizes t he need f o r an adequate

v a c c i n a t i o n program f o r t he c o n t r o l

of t h e c i t r i c

livestock

From

aci d c y c l e

is

in l e p t o s p i r a e .

i s hoped t h a t

by use o f some o f the t e chni que s

d e s c r i b e d in t h i s t h e s i s f o r p u r i f i c a t i o n and assaying o f t he
hemol ysin and f o r de mon s t r a t i o n o f enzyme systems, f u r t h e r
vestigations w ill
enzymes o f
wi t h
for

be undertaken concerni ng t he hemol ysin and

leptospirae.

Because of the compar at i ve ease wi t h

l e p t o s p i r a e can be grown j_n v i t r o t hey are

i d e a l l y suited

i n v e s t i g a t i o n s o f t he pat hogeni c s p i r o c h e t e s .

lack o f

i n­

i n f o r m a t i o n concerni ng t he b i o l o g i c a l

Our pr e s e nt

p r o p e r t i e s of

52
this

group o f b a c t e r i a may wel l

by t h e r e s u l t s o f

be c o r r e c t e d

i n v e s t i g a t i o n s o f t he

in t he f u t u r e

leptospirae.

2
1
0

CM-----

vo m —

-f KVO

OtnKN
cor-vo

— 00vo
r- vo vo

r- o —
co r*- m

h-fn<r
ooin m

OJ —-if
mm—

in o vo
oi oi —

OJ Q\hOJ-----

■St vo —
•4- oj —

COVOr<j- m

vo ovvo
vo -sr m

o < t oj

OV1"-VO

m-sj- -=t-

vo o m

0 vo CO
01 —

o o o

— cvi o
•
o

oo o

o o o

COO'* o
OJ-----

— oo in
OJ-----

COVO-sj-

mo o
ojcn-t
fOOJ —

vo co rvo — n-

00 —-sf
. vo m
• • •
t o o o

m r—oj
in<r <j• • •
o o o

vo — oi
min-sr
« • •
o o o

OJO
o vo m
• • «
—o o

o o—
r-— ov
• • .
h-m

in n uw m

01 — 0
m inin

OvoOVO
■tf -st" -sf

invo -st
moi —

vo o
OJ—

HGB
g / 100ml

cor- o
• • •
VOoj o\

co in cvi
• • •
m torn

01— 01
• « «
^ m oi

10. 0
7. 1
6.4

O OI o
• . •
OJCO-s}-

Hi gh
Mean
Low

Group I H i gh
WhdeCells Mean
Strain LW Low
28 h r s .

H i gh
Mean
Low

Hi gh
Mean
Low

Hi gh
Mean
Low

72 h r s .

*
10
L.
.c
vo
ov

U Q
CO E t
,
_J
nt
C
C
Qj E
L.
<U
<*-Q
— <fl \
Q
CO

B i 1i r u b i n
m g / 100 ml

Urea
m g / 100 ml

COi o o o
IxJ

*■>
U

Norma 1
Ranges

POMONA
L.
HAMSTERS INFECTED WITH
WITH CELL EXTRACTS
HEMATOLOGICAL VALUES OF
OR INJECTED

VO KV0J

p
\U19H

Mono

53

•
0)
u
SZ
CO

o o o

O O O

CM VO
m

cm

—

O C O 00
i n n cm

vo *!tKA
in inin

O CM 00
v o tn n

voi^-n

CM O VO

o o o

OOO

04 I O
m CM CM

inco -it

00 CT\-3"

— co-si-

•sT CM 03

CO

O IA O

o o

O J --------

in k>k\
•
•
•
o o o

<t

•

fn

•

•

n n n
•
•
•
oo o

CM

m<j- m

•

•

C M -------

«=t CM

SZ C

a c

CTt (« 5

~

X

Group $

c m --------

a> o
2 -J

om i

•— a> o
X 5 _J

(2
XI
00
LU O T <

•

OV

55

TABLE 2
AMMONIUM SULFATE FRACTIONATION OF CULTURE SUPERNATES
FOR ISOLATION OF HEMOLYSIN

2

3

4

5

6

00

1

r-

Fraction Original

9

10

11

12

%
S a t u r a t i on

0

31

35

39

43

47

50

55

61

66

71

75

100

Hemo1y t i c
Act 1 vi t y
( Un f t s/m 1)

8

4

4

0

0

0

0

0

0

0

0

0

0

56
TABLE 3
TOXICITY OF HEMOLYSIN FOR LAMBS
EXPERIMENT I

LAMB 800 ( c o n t r o 1)
0 HU/kgm**

LAMB 777
1 0 , 0 0 0 HU/kgm*
Temp,

T i me

T emp

Hgb
He ma t o c r i t
g / 1 0 0 cc
%

Pre-Inoculation

9.4

35

Post I noc­
ulation
102. 2

9.0

40

2 .5 hrs.

104. 2

7.8

5 . 5 hrs.

102. 2

10.0 hrs.

Hematocr i t
Hgb
g / 1 0 0 cc
ft

10. 4

39

104. 3

10. 0

38

30

104. 2

9.8

36

7.4

28

103.7

9.4

35

102. 4

6.9

27

102. 5

9.4

34

26.0 hrs.

—

3.0

7

—

9.8

35

30.0 hrs.

98. 1

2.4

—

9.6

34

*HU/kgm = h e m o l y t i c u n i t s per k i l o g r a m body w e i g h t .
Lamb r e c e i v e d 20 ml o f hemol ysi n p r e p a r a t i o n .
**Lamb r e c e i v e d 20 ml o f s t e r i l e
# * * An i ma l

died.

saline.

57

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cr
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CD
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m knkncm i m cu cm oj i oj

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• • • • f • • • • | «
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58

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r -o ~ h - o ~ v o m ^ } - m o j

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59
TABLE 5
EFFECT OF SUBLETHAl DOSES OF HEMOLYSIN ON LAMBS CHALLENGED
WITH L. POMONA. STRAIN LW AND WITH HEMOLYSIN

Lamb 23
No p r e v i o u s
hemol ysi n i n j e c t i o n s
Cha11 eng i ng
P r e p a r a t i on

10 ml washed c e l l s
S t r a i n LW

Time A f t e r
ChaI Ienge

Temp.

Hgb.
g / 1 0 0 cc

0
18
41
72
96
112
120
196

103.3
106*6
105. 6
105. 6
105. 8
106. 2
105. 2
102. 9

12. 8

hrs.
hrs.
hrs.
hrs.
hrs.
hrs.
hrs.
hrs.

Lamb 29
4 sub l e t h a l doses
o f hemol ysi n

- -

1 1. 0
9.0
6.4
3.7
3.6
4.6

10 ml washed c e l l s
S t r a i n LW
AB*

0
0
c
1**
3
4
-

4

Temp.

103. 0
102. 8
102. 6
102. 2
102. 8
—
102. 3
102. 0

Hgb
g / 1 0 0 cc
8.4
—

6.2
8.8
8.3
- -

7.8
8.6

AB

1
1
3
5
5
5
5

*AB = serum a nt i bo dy
* # R e c i p r o c a I o f d i l u t i o n o f serum a g g l u t i n a t i n g 50 per
cent o f t h e a n t i g e n .

60
TABLE 5 ( CONTINUED)

Lamb 31
3 sub l e t h a l doses
o f hemol ysi n

Lamb 40
3 sub l e t h a l doses
o f hemol ysi n

10 ml washed c e l l s
S t r a i n LW

10 ml washed c e l l s
S t r a i n LW

Temp.

Hgb.
g/ 1 0 0 cc

102.2
105.0
102.4
102.6
102. 7

6.8
—

5.9
5.8
6.0

AB

Temp.

0
0
0
2
4

101.8
105. 0
103.0
102.6
102.0

Hgb.
g/ 1 0 0 cc

6.8
mmmm

5.0
5.6
5.6

6.8
7.0

4
4

102. 5
102.0

5.5
6.7

10, 000 HU/kgm

AB

Temp.

Hgb.
g/ 1 0 0 cc

AB

0
0
0
3

101.8
102.0
102. 4
102.5
--

9.8
9.8
9.6
9.2
9.4

0
0
0
1
2

fl
H

103.0
1 02. 7

Lamb 22
3 sub l e t h a l doses
o f hemol ysi n

4
4

61

CD

X

O
O
O

»

—

o
—

vo

OJ
m

<%

o

T3
0)
03

e

cc*
_
1 <->
X

o
o
o
*
o

o

O

in <
hov

OJ X
3

<4o
+J
c
a>
o

C

-Cf <
r"~

o

o

o\

x

AGGLUTININ
LAMBS
TITERS AND
OF NEWBORN
INHIBITION
OF SERA

o
o

0J
Q.

•»

O
in

X

E

OJ
03

cn

OJ

HEMOLYSIN

o

—.

X

-3" X CO
3
c

X

cn
• c
03 —
—

vo

03 05

>> C
o ■*->

X

e

OJ
■*-»
<
qj

3

CD —
•

#

•

♦

X

x

X

X

X

+->
X
—

X

X

X

X

OJ

m

E CD

**■»
K

X

LTJ
OJ
•

o

03

cn
O I (TJ
c
— E
-H 3
— X
X CD
— 03

37

TITERS

CD

X

C

CD

1

O <
O
CO

o

o

X
QJ
<0
U

o

O

X

0)
+-*
—
1—

x

E

c

oj

•

—
I

—

o

1

1

00

vo

—

X

vo
—

<i-

o

X c c
CD O O
f t W* w
•— +J
1— 3 3
—■ —
X X X

c O
o x -+j a»
— 03 03
+J +J Qj QJ
— C X X
J 3 < Ol d )

— CO
r^ <
r-

o

o

X

o

o

O

•r*
X

c

1
—
1

<U

*—*

c

X
3
C

E
(TJ
_j *

o

•— «F
—
X X

c
<j—**-

+j o

03
X

CJ3

1

©

1

vo

VO

vo

X

o

CT5

•*— c --------03 — ctJ aj
>>-*-> o u
— 3 0 0
o — X X
Em a a
OJ 03 — —
x < u u

CD 0J
It
x
QJ
+-> X

V - -M
< x

•
X
X

•

•

•

X
X

X

X

X

X

—

rn

•
X

X

•

«

X

X
X

X

-3"

CO

O) CQ

E
f—

in
•

o

1 "-

OJ

II X X

•-* CD II II
X <
_
*
*

62

TABLE 7
REPRODUCIBILITY OF ASSAY PROCEDURE I I

Det ermi nat i on

HU/ml

Days A f t e r F i r s t
Determi nat i on

1

0

61

2

1

58

3

4

62

4

5

60

5

10

60
Mean

CM
•

Standard D e v i a t i o n

6 0 .2

63

TABLE 8
COMPARISON OF TITRATION AND ASSAY PROCEDURE I I
FOR DETERMINING HEMOLYTIC ACTIVITY

Hemol ysin
Preparation

Titration
Method
HU/ml

Procedure I I
HU/ml

Ratio of
T i t r a t i o n Method
t o pr ocedure I I

150 - 0

512

25

20.5

150- S

2048

58

35.3

15 0 - P

2048

56

36.6

149

1024

54

19. 0

142

512

28

1 8. 3

143

8192

213

37.8
Avg.

28 .0

64

TABLE 9
PREPARATION OF HEMOLYSIN

Fraction

Tr e a t me nt

S t e r i l e Culture
Fi I t r a t e

Volume
(ml)

HU/ml

mgm
Pr ot e i n/ml

1680

25

8.05

3.

100

110

58

6.36

9.1

15.2

NO

56

2.44

23.0

14. 7

Specific Yield
Activity
{%)
HU/m^mP

P r e c i p i t a t e d wi t h
56 per cent s a t u r a ­
t i o n (NH4 ) 2 SO4 .
P r e c i p i t a t e r esus­
pended in s a I i n e
and d i a l y s e d %rsk
di e t f I Ied H2o.
P r e c i p i t a t e re­
moved and r esus­
pended .
F r a c t i o n soluble
i n H20
Insoluble

in H20

65

TABLE 10
ADSORPTION OF HEMOLYSIN TO ERYTHROCYTES
AFTER 5 MINUTES AT 37°C

Adsorbent

1
2
3
4
5

6
7

8
9
JO
i i

ml Adsorbent

Packed Sheep
Erythrocytes

HU/ml a f t e r % Hemolysin Hemol ysi s
Adsorpt i on
o f AdsorbAdsorbed
ant RBC

0

62

0

ii

0.2

14

77

+

ii

0.4

14

77

+

ti

0.6

15

76

+

0

62

0

11

0.2

15

76

-

II

0.4

12

81

-

11

0.6

14

77

-

0

38

0

0.5

23

40

1.0

13

66

Packed Guinea
Pig E r y t h r o c y t e s

Packed P o l y s t y r ene
Spheres
ri
ii

66

TABLE I 1
NON-REACTIVITY OF HEMOLYSIN WITH LECITHOVITELLIN
AS DETERMINED BY CHANGE IN TURBIDITY

Tube

I
2
26HU
58HU
2 ml L e c 2 ml Leci t h o v i t e 11i n i t h o v i t e 11 in

3
84HU
2 ml Leci t h o v i t e l 1 in

4
0 HU
2 ml Leci thov i t e 11i n

Time
0 mi n.

0.06*

0.05

0.06

0.06

10 min.

0.07

0.05

0.06

0.07

20 min.

0.07

0.05

0.06

0.06

30 min.

0.07

0.05

0.06

0.06

60 min.

0.08

0.06

0.08

0.08

^ O p t i c a l D e n s i t y at 650 mjj.

67
Hgb ( g / 1 0 0 m l )
Ant i body T f t e r ( n e g a t i v e exponent to base 10)
Number o f I e p t o s p l r a e / c c ( exponent t o base 10)
to

Vj J

-fc-

Vjn

On

cd

vo

o

Hgb (gms/lOOml)
Ant i body T i t e r ( n e g a t i v e exponent t o base 10)
Number o f L e p t o s p l r a e / c c ( exponent t o base 10)
ro
jy
vn
o - v i o o v o
o
—

ro

69
% Hemolysis
On

03

a
Mf nut es
o :'

ro-

o
ir

VO

HU/ml

70

71

% Hemolysis

D i l ut i on

of

Hemolysin

72

%Hemol ysi s

Mi nut es

73
% H e m o ly s is

0\

Co

i±
:

Ml nut es

O

u

ill

74

Op t i c a l
,0\

a.

a.n~i-

Ml nut es
CO

rvl

id:

Density

75

Opt i cal Densi ty

Mi nut es

76

Opt i c a l Densi ty

Mi nut es

li

77

A Opt i c a l Oensity

Mi nut es

78
A Opt i c a l Densi ty

s

Hi nutes

79

REFERENCES
1.

A l e x a n d e r , A. A . , Smi t h, 0 . H . , H i a t t , C. W. and G l e i s e r ,
C. A. ( 1 9 5 6 )
Presence o f hemol ysi n in c u l t u r e s o f
pa t hoge ni c l e p t o s p i r e s .
Pr oc. Soc. Exp. B i o l , and
Med. ,
2 0 5 - 2 1 1.

2.

A l t s o n , J . M. and Broom, J. C.
(1958)
Leptosp i ros i s
in man and ani maI s .
E. and S. L i v i n g s t o n e L t d . ,
Edi nburgh and London.

3.

Bauer , D. C.

4-.

(1959)

Unpubli shed o b s e r v a t i o n s .

» and Morse, E. V.
( 1 95 8 )
V a r i a t i o n and hemolosin
p r o d u c t i o n in r e l a t i o n t o v i r u l e n c e o f L e p t o s p i r a
pomona.
Proc. Soc. Exp. B i o l , and Med. . 98. 5 0 5 - 5 0 8 .

5.

Beamer, P. D . , Hardenbrook, H. and M o r r i l l , C. C.
( 1 95 3 )
S t u d i e s on l e p t o s p i r o s i s in domestic a ni ma l s .
I.
L e p t o s p i r o s i s in sheep.
V e t . Med., 48, 3 6 5 - 3 6 6 .

6.

Ber nhei mer , A. W.
(1947)
Co mp a r i t i v e k i n e t i c s o f hemo­
l y s i s induced by b a c t e r i a l and o t h e r hemol ysi ns.
J.
Gen. P h y s i o l . ^ 0 , 3 3 7 - 3 5 3 .

7.

Bohl , E. H . , Powers, T. C. and Ferguson, L. C.
( 1954)
Ab o r t i o n in swine a s s o c i a t e d wi t h I e p t o s p i r o s i s .
J.
Am. V e t . Med. Assoc. , 12 4 . 2 6 2 - 2 6 8 .

8.

Bryan, H. S.
( 1955)
L e p t o s p i r o s i s in c a t t l e and swi ne.
C i r c u l a r 750, U n i v e r s i t y o f I l l i n o i s .

9.

Chang, S. L.
( 1947)
S t u d i e s on L e p t o s p i r a i c t e r oha e mor rhaqiae.
III.
The growth r a t e o f , and some bi ochemi cal o b s e r v a t i o n s on, L e p t o s p i r a i c t e r o h a e mo r r h a q i a e
in c u l t u r e .
J. I n f . P i s . 81. 3 5 - 4 7 .

10.

C h o l v i n , N. R . , Morse, E. V. and Langham, R. F . ( 1 95 9 )
Expe r i me nt a l L e p t o s p i r a pomona i n f e c t i o n s in dogs.
J . I n f . D i s . . H W , 92-Too.

11.

Cox,

12.

___ , and Lar son,
I ept ospi r a e .

13.

C z e k a l o ws k i , J . W., McLeod, J. W. and Rodi can, J.
( 1 95 3 )
The growth and r e s p i r a t i o n o f l e p t o s p i r a in s o l i d or
s e m i - s o l i d media w i t h s pe c i a l r e f e r e n c e t o Di nger s
phenomenon.
B r i t . J . Exp. Pat h. ^ 4 , 5 8 8 - 5 9 5 .

C. D.

(1959)

Personal

communication.

A. D.
( 1 95 7 )
J. B a c t . , J jt,

Co l o ni a l
587-589.

growth o f

80

14.

C z e k o I o w s k I , J . W. , McLeod, J . W. and Rodi can, J.
( J954)
O b s e r v a t i o n s on t h e n u t r i t i o n o f l e p t o s p i r a based on
t h e development o f Di nger s phenomenon.
J. Gen. Mi cr ob,
JO, 1 99- 208.

15.

Faine, S . , (1957)
V i r u l e n c e in l e p t o s p i r a .
I.
Re a c t i o ns
o f gui nea pi g s t o ex pe r i me nt a l i n f e c t i o n wi t h L e p t o s p i r a
i cterohaemorrhaq i a e .
B r i t . J. Exp. Pat h. j58, 1 - 7.

16.

. (1957)
V i r u l e n c e in l e p t o s p i r a .
II,
The growth
in v i v o o f v i r u l e n t L e p t o s p i r a i c t e r o h a e m o r r h a q i a e .
B r i t . J. Exp. Pa t h . j58, 8 - 1 4 .

17.

. ( 1 9 5 9 ) I r o n as a growth r equi r ement f o r pat hogeni c
Teptospirae.
J . Gen. M i c r o b . , 20, 2 4 6 - 2 5 1 .

18.

Fer guson, L. C . , Ramge, J. C. and Sanger, V. L,
( 1 95 7 )
Exper i ment al bovine I e p t o s p i r o s i s .
Am. J. V e t . Res.
J 8, 43-49.

19.

Fukushima, B.
and Hosoya, S.
(194-8)
The L e p t o s p i r o s e s .
Lei den.

20.

F u l t o n , J . D.
and Spooner, D. F.
(1956)
The metabol i sm
o f L e p t o s p i r a i c t e r o h a e mo r r h a q i a e in v i t r o .
Exp.
Paras i t . , 5 , 154- 177.

21

.

( 1 92 6 )
In Van T h e i I , P.H.
U n i v e r s i t a i r e Pers L e i d e n.

G e r h a r d t , M. R. and B a l l , M. G.
( 1959)
utilization
by L e p t o s p i r a c a n i c o l a .
17-22.

Amino a c i d
J. B a c t . , 7 7 .

22.

Gl enny, A. T. and St evens, M. F. ( 1 9 3 5 )
St aphyI ococcus
t o x i n s and a n t i t o x i n s .
J. Pat h. B a c t . , ^ 2 , 5 3 - 7 4 .

23.

G o l d b e r t , H. S. and Armstrong, J . C.
( 1 95 9 )
Oxidase
r e a c t i o n wi t h l e p t o s p i r a l c o l o n i e s and i t s a d a p t a t i o n
to a n t i b i o t i c s e n s i t i v i t y t e s t i n g .
J. Bact., 77.
512-513.

24.

Gsell,

25.

H a r t l e y , W. J.
Vet . J . , 28,

26 .

H e l p r i n , J. J . and H i a t t , C. W.
( 1957) The e f f e c t o f
f a t t y a c i d s on t h e r e s p i r a t i o n o f L e p t o s p i r a i c t e r o h e mo r rhagiae.
J . I n f . D i s . , 10 0 . 136- 140. ”

27.

H i g u c h i , S.
(1931)
I n Van T h e i l , P.H. ( 1 9 4 8 )
The
Lept ospi r o s e s .
U n i v e r s i t a i r e Pers L e i d e n .
Le i den.

28.

Ho c h s t e r , R. M. and Ka t ne l s on , H.
( 1 95 8 )
On t h e mechan­
ism o f gl ucose - 6- phosphate o x i d a t i o n in c e l l f r e e ex­
t r a c t s o f Xanthomonous
phaseol i (XP 8 ) .
Can. J. Bi o chem. and P h y s i o l . ^ 6 , 6 6 9 - 6 8 9 .

0.

(1952)

Leptospi r os e n.

Huber,

Bern.

(1952)
Ovine l e p t o s p i r o s i s .
169- 170.

Australian

81

29.

H o r e c k e r , B. L. and Kornberg, A.
(1948)
The e x t i n c t i o n
c o e f f i c i e n t s o f t h e reduced band o f p y r i d i n e nu c l e o ­
tides.
J . B i o l . Chem., J J £ , 3 8 5 - 3 9 0 .

30.

Imanmura, S . , K u r i b a y a s h i , K. and Kameta, M.
( 1 95 7 )
S t u d i e s on t o x i n s o f pat hogeni c l e p t o s p i r a .
Jap. J .
M i c r o b . J_, 4 3 - 4 7 .

31.

I n a d a , R . , I d o , Y . , Ho k i , R . , Kaneko, R. and I t o , H.
(1916)
The e t i o l o g y , mode o f i n f e c t i o n and s p e c i f i c
t h e r a p y o f W e i l ' s di sease ( s p i r o c h a e t o s i s i c t e r o h a e mo r rhagica).
J. Exp. Med. 2£, 3 7 7 - 4 0 2 .

32.

I na da , R.
(1922)
spirochaetosis
2, 189- 194.

33.

K e i l i n , D. and H a r t r e e , E. F.
( 1 93 8 )
On t he mechanism
o f t h e decomposi t i on o f hydrogen p e r ox i d e by c a t a l a s e .
Pr oc. Roy. Soc. (London), 12 4 . 3 9 7 - 4 0 5 .

34.

Kemenes, F.
(1958)
Di e durchpathogene l e p t o s p i r e n
v e r u r s a c h t e hSmoIyse ind i h r e p a t h o I o g i s c h e bedeutung.
Acta V e t e r i n a r i a . 8 , 143- 150.

35.

Lamanna, C. and M a l l e t t e , M. F.
( 1 95 3 )
W i l l i a m s and Wi k i ns C o . , B a l t i m o r e .

36.

Lar son, A. D . , T r e i c k , R. W., Edwards, C. L. and Cox,
C. D.
(1959)
Growth s t u d i e s and p l a t e count i ng of
leptospires.
J. B a c t . , 2Z» 3 6 1 - 3 6 6 .

37.

L i n d q u i s t , K. J . , Morse, E. V.
and Lundberg, A. M. ( 1958)
Exper i ment al L e p t o s p i r a pomona i n f e c t i o n in pregnant
ewes.
Co r n e l l V e t . . 48. 2~/7-290.

38.

Lowry, 0 . H . , Rosebrough, N. J . , F a r r , A. L. and R a n d a l l ,
R. J .
(1951)
P r o t e i n measurement wi t h t h e F o l i n
phenol r e a g e n t .
J . B i o l . Chem. 19 3 . 2 6 5 - 2 7 5 .

39.

M a t s u z a k i , S. and I m a i , I .
( 1 91 7 )
( 1 9 5 7 ) Jap. J. Mi cr ob. J_, 4 3 - 4 7 .

40.

Mayer, M. M , , O s i e r , A. G . , B i e r , 0 . G. and H e i d e l b e r g e r ,
M.
(1946)
The a c t i v a t i n g e f f e c t o f magnesium and
o t h e r c a t i o n s on t h e h e mol y t i c f u n c t i o n s of complement.
J . Exp. Med. , 84, 5 3 5 - 5 4 8 .

41.

M e h l e r , A. H . , Kornberg, A . , G r i s o l i a , S. and Ochoa, S.
(1948)
The enzymati c mechanism of o x i d a t i o n - r e d u c t i o n s
between mal at e or i s o c i t r a t e and p y r u v a t e .
J. B i o l .
Chem., 17 4 . 9 6 1 - 9 7 7 .

P r o p h y l a x i s and serum t r e a t m e n t o f
icterohaemorrhagica.
Jap. Med. Worl d,

Basic B a c t e r i o l o g y .

In Imamura,

et a I .

82
42.

Mo b e r l y , M, L . , M a r i n e t t i , G. V . , W i t t e r , R. F. and
Morgan, H. R.
(1958)
St u d i e s o f t he hemol ysi s o f
red blood c e l l s by mumps v i r u s .
J . Exp. Med., 107,
87-94.
— L

43.

Morse, E. V . , A l l e n , V . , Kroho, A. F. and H a l l , R.
( 1 95 5 )
L e p t o s p i r o s i s in Wi sconsi n.
I.
E p i z o o t i o I o g y and
c lin ic a l features.
J . Am. Ve t . Med. Assoc. , 127, 417421 .

44.

Morse, E. V . , Bauer, D. C . , Langham, R. F . , Lang, R. W.
and U l l r e y , D. E.
(1958)
Experi ment al l e p t o s p i r o s i s .
IV.
Pat hogenesi s o f p o r c i n e L e p t o s p i r a pomona i n ­
fections.
Am. J. V e t . Re s . , 19, 3 5 8 - 3 9 4 .

45.

Morse, E. V. and Langham, R. F.
( 1 58 )
Experi ment al
leptospirosis.
III.
Capri ne L e p t o s p i r a pomona i n ­
fection.
Am. J . V e t . Res. 19, I 3 9 - 144.

46.

Morse, E. V. and M o r t e r , R. L.
( 1955)
Protection afforded
gui nea pi g s by por c i ne gamma g l o b u l i n a g a i n s t Leptosp i ra
pomona i n f e c t i o n .
Co r n e l l V e t . , 45 . 5 2 0 - 5 2 5 .

47.

Morse, E. V . , M o r t e r , R. L . , Langham, R. F . , Lundberg,
A. and U l l r e y , D. E.
( 1 95 7 )
Experi ment al ovine
I e p t o s p i r o s i s , L e p t o s p i r a pomona i n f e c t i o n .
J. I n f .
D i s . , i O i , I 29-136.

48.

N a g l e r , F. P. 0 .
( 1 93 9 )
Obs e r v a t i o ns on t he r e a c t i o n
between t h e l e t h a l t o x i n o f C l o s t r i di urn w e l c h i i (Type
A) and human serum.
B r i t . J., Exp. Pat h. 20, 4 7 3 - 4 8 5 .

49 .

N a t e l s o n , S.
(1957)
Mi c r ot e c hn i qu e s o f c l i n i c a l chemi st r y
f o r the r o u t i n e l a b o r a t o r y .
Chas. C. Thomas, S p r i n g ­
field , I I I .

50.

Ouchterlony, 0.
(1948)
In v i t r o method f o r t e s t i n g t he
t o x i n produci ng c a p a c i t y o f d i p t h e r i a b a c t e r i a .
Acta
Pa t h, e t Mi c r ob. s c a n d i n a v . , 2 £ , 186-191.

51.

Ponder, E.
(1948)
Hemol ysi s and r e l a t e d
and S t r a t t o n .
New York.

52.

R a c k e r , E.
(1950)
S p e c t r op ho t o me t r i c measurements of
t h e enszymati c f or m a t i o n s o f f uma r i c and c i s - a c o n i t i C
acids.
Bi ochi m. et Biophys. Act a, 4, 2 1 1 - 2 1 4 .

53.

Rh e i n h a r d , K. R . , T i e r n e y , W. F. and R o b e r t s , S. J.
( 1950)
A study o f two e n z o o t i c occurrences o f bovine l e p t o s p i r o s i s .
C o r n e l l V e t . , 4 0 , 148- 164.

54 .

phenomena.

Grune

, (1951)
A c l i n i c a l p a t h o l o g i c a l study o f exper i'ment a I l e p t o s p i r o s i s in c a l v e s .
Am. J. V e t . R e s . ,
12 , 2 8 2 - 2 9 1 .

83
55 .

Rh e i n h a r d , K. R.
(1952)
Symposi um on t h e I e pt os p i r o s e s .
Medi cal Sci ence P u b l i c a t i o n No. I . Uni t e d S t a t e s Govern­
ment P r i n t i n g O f f i c e , Washi ngton.

56.

Ro go l s , S . , Fi z e t t e , W. B. and Boh I , E. H.
( 1959)
Studies
on t h e r e l a t i o n s h i p o f a phosphoi i pase t o t h e hemolysin
o f L e p t o s p i r a pomona.
Am. J . V e t . Re s . , 20, 5 9 2 - 5 9 6 .

57.

R u s s e l l , C. M.
leptospirae.

58.

Schnei derman, A . , Greene, M. R . . S c h i e l e r , L . , McCl ure,
L. E. and Dunn, M. S.
(1951)
N u t r i t i o n of Leptospira
cani coI a .
II.
A c h e m i c a l l y de f i ne d basal medium con­
t a i n i n g p u r i f i e d r a b b i t serum al bumi n.
Proc. Soc.
Exp. B i o l . Med. , JQ, 7 7 7 - 7 8 1 .

59.

(1956)
A "hemol ysi n" a s s oc i a t e d wi t h
J . Immun., 77, 4 0 5 - 4 0 9 .

Simmons, J . S. and Gentzkow, C. J.
( 1 95 5 )
Medical and
Pub I i c H e a I t h La b or a t o r y Methods.
Lea and F e b i g e r ,
Phi I adeI phi a.

60.

So u l e , D. W., M a r i n e t t i , G. V. and Morgan, H. R .
( 1959)
St u d i e s o f t h e hemol ysi s o f red blood c e l l s by mumps
v i r u s . IV.
Q u a n t i t a t i v e study o f changes in red blood
c e l l l i p i d e s and o f v i r u s l i p i d e s .
J . Exp. Med.,
I 10. 9 3 - 1 0 2 .

61.

S t a v i t s k y , A. B.
leptospirosis.

62.

__________ , (194-8)
C h a r a c t e r i s t i c s o f pat hogeni c s p i r o c h e t e s
and s p i r o c h e t o s i s wi t h s pe c i a l r e f e r e n c e t o t he mechan­
isms o f host r e s i s t a n c e .
Bact . R e v . , _lj2» 2 0 3 - 2 5 5 .

63.

S t u a r t , R. D.
(1946)
c u l t u r e medium f o r
343-346.

64.

S u t h e r l a n d , A. K . , Simmons, G. C. and Kenny, G. C.
( 1949)
Bovine l e p t o s p i r o s i s :
t h r e e out br eaks in c a l v e s in
Queensl and.
Aust . V e t . J . , 2£, 197- 200.

65.

van Heyni ngen, W. E.
( 1941)
The bi o c he mi s t r y o f t he
gas gangrene t o x i n s .
II.
Partial p u rific a tio n of
t h e t o x i n s of _C|>. we I ch i i . t y pe A.
Se p a r a t i o n o f alpha
and t h e t a t o x i n s .
Biochem. J . ,
1257-1269.

66.
ific
67.

(1945)
S t u d i e s on t h e pat hogenesi s of
J . I n f . D i s . , 2 6* 179- 192.

The p r e p a r a t i o n and use of a si mple
leptospirae.
J . Pat h. B a c t . , 5 8 .

. (1950)
Bacterial
Publ i c a t i o n s , Oxf ord.

Toxins.

Bl a c k we l l

Scient-

Vo I I and, W. and Brede, H. D,
( 1951)
Zur f r a g e der
h y a l u r o n i d a s e b i I dung durch L e p t o s p i r e n .
Med. M s c h r . ,
£ , 698-700.

84
68.

Warburg, 0 . and C h r i s t i a n n , W.
(1936)
P y r i d i n , der
w a s s e r s t o f f i i b e r t r o g e n d e B e s t a n d t e i I von Garungs
fermenten.
Biochem.
Z . , 287. 291-328.

69.

Webst er , W. M. and Reynol ds, B. A.
(1955)
I mmunizati on
a g a i n s t L e p t o s p i r a pomona.
New Zeal and V e t . J . f 3»
47-59.

70.

Zamecni k, P. C. and Lipmann, F. A.
( 1 94 7 )
A study o f
t h e c o m p e t i t i o n o f l e c i t h i n and a n t i t o x i n f o r Cl .
we Ich i i l e c i t h i n a s e .
J. Exp. Med. , 8£, 395-40T T