A study of fractions from Brucella organisms

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A 3OTDY Of FRACTIONS FROM BK0C3LLA. OROAiilSMS

By J o h n W# F o s t e r

’T h e s i s s u b m i t t e d t o t h e F a c u l t y o f th e G r a d u a t e S c h o o l o f th® U n iv e r s ity o f Maryland i n p a r t i a l f u lf illm e n t o f t h e r e q u i r e m e n t s f o r t h e degree o f B o o t o r o f P h i l o s o p h y

19S0

UM! Number: DP70352

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A.0 kno w l e d m e n 1 8 ;

I 'wish t o a c k n o w le d g e w i t h d e e p e s t a p p r e c i ­

a t i o n t h e w i l l i n g and a b l e a s s i s t a n c e and h e l p f u l com ments I h a v e r e c e i v e d fro m t h e members o f t h e s t a f f a n d my f e l l o w s t u ­ d e n t s a t t h e D e p a rtm e n t o f B a c t e r i o l o g y a n d t h e L iv e S t o c k S a n ita ry S e rv ic e L a b o ra to rie s .

S p e c i f i c a l l y I w is h t o e x p r e s s

my s i n c e r e t h a n k s t o my a d v i s o r s , D r. F . A rn e H an sen and D r. Jo h n B. F a b e r J r . and t o D r s . C o r n e l i a C. C o t t o n , F o r r e s t Woods, F l e t c h e r P . V e i t c h , Leo F . P o e lm a , A r t h u r L* B r u e c k n e r a n d Mr* C h a r l e s 1 . S u i t f o r d i r e c t a s s i s t a n c e on in u m e ra b l© o cc asio n s.

i

TABLE OF CONTENTS

Page I II

................................................

1

HISTORICAL.................................................................................................

2

INTRODUCTION

K i l l i n g c e l l s b y p h y s i c a l a g e n t s . . ............................ .....

.

3

1 . H e a t .....................................................................................................

3

2. U l t r a v i o l e t l i g h t ............................

*1-

3- S o n ic t r e a t e d o r g a n i s m s .....................................................

M -

4* M e c h a n ic a l d i s i n t e g r a t i o n

.......................................

h

............................................

5

..............................................................................

5

..................................................................................

5

3 . E t h e r ................................................................................................

5

F o r m a l i n ............................................................................................

5

.

K i l l i n g b y c h e m ic a l a g e n t s 1. T o l u o l . 2. P h e n o l .

.

5. P h e n y l m e rc u ric n i t r a t e

. . . . .

.............................

6

6 . A l c o h o l ...........................................

6

7 . C h l o r o f o r m ...........................................

6

O t h e r m e th o d s

. . . . . .

F r a c t i o n a t i o n ............................................................................................

6

7

Use o f l i v i n g v a c c i n e .............................................................................. 11 Use o f a d j u v a n t s ................................................................................. III

EXPERIMENTAL

12

....................................................................................1^

R a t i o n a l e ...................................... 1. I s o e l e c t r i c p o i n t ....................................................................

1^ 15

2. I o n i c s t r e n g t h ................................................................................... 16 3 . T e m p e r a t u r e ................................................

C o n c e n tra tio n o f o rg a n ic s o lv e n ts . . . . . . .

17 17

11

5 . C o n c e n t r a t i o n o f p r o t e i n ............................................... . . M e th o d s.

. . . . . . . . .

...........................................................

C u l t i v a t i o n and c h a r a c t e r i s t i c s o f B r u c e l l a s p e c ie s used . . . . . . . . . P r e p a r a t i o n o f a n t i g e n s ......................................................... T e m p e r a tu r e c o n t r o l .

19 19

.

22

......................................................

24-

P r e p a r a t i o n o f f r a c t i o n s .....................................................

.

25

F r a c t i o n I ...............................

26

F r a c t i o n s I I a n d I I I ............................

27

F r a c t i o n IV .......................................................................................

29

F r a c t i o n s V - l and V -2 ...............................................................

3^

F r a c t i o n f l ..............................................................

32

F r a c t i o n ¥ 1 1 ..................................................................................

33

M e th a n o l p r e c i p i t a t i o n ......................................................... C r ite ria fo r s t e r i l i t y o f fra c tio n s .

.

33

.........................

3 I4.

C are o f a n im a l® ................................................

3^

In o c u la tio n o f r a b b its

35

............................................

I n o c u l a t i o n a n d p o s t mortem e x a m i n a t i o n o f g u i n e a p i g s .........................................................

35

I n f e c t i o n c o n t r o l s .................................

39

E s t i m a t e o f c h a l l e n g e d o s e .....................................................

4o

A g g l u t i n a t i o n t e s t ® . ........................

4l

P r e c i p i t i n t e s t s ......................................

42

C h e m ic a l s t u d i e s

44

. . . . . . .

Changes. I n m o r p h o lo g y IV

17

.

45

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

47

Xmm&nization o f g u i n e a p i g s ..........................................................

47

F r a c t i o n I .............................................................................

^7

F r a c t i o n X I.

. .

. ..........................................................

52

F ra c tio n I I I

........................................................................

5*

F r a c t i o n I V ................................................ ............................

58

F r a c t i o n V - l ........................................................................

61 .

64

F r a c t i o n V I.............................................................................

69

F r a c t i o n VII ........................................................................

73

I n V ivo a n t i b o d y p r o d u c t i o n ............................................

77

In V itro s e r o lo g ic a l t e s t s

............................................

95

C h e m ic a l n a t u r e o f th e f r a c t i o n s .............................

105

F r a c t i o n V - 2 ..............................................................

.

lo g

U re a s e a c t i v i t y ........................ ..... R e s u l t s from m o r p h o l o g i c a l s t u d i e s ........................

log

P r e g n a n c y e,nd a b o r t i o n s

' . .

112

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

115

SUMMARY. . . .

.........................................................................

126

CONCLUSIONS..................................................................................

129

LITERATURE CITED ....................................................................

131

APPENDIX .......................................................................................

139

C o m p o s itio n o f C u l t u r e M edia U s e d .............................

139

Dye I n h i b i t i o n T e s t s ..........................................................

l4o

U re a s e S o l u t i o n ........................................................................

l4 o

C h e m ic a l M ethods . ...............................................................

l4 i

B u f f e r s .................................. ....

.................................................

144

C a l c u l a t i o n o f Mean V a lu e s ............................................

146

iT

PROTOCOL number I 11

page Schedule o f I n o c u la tio n s o f V arious f r a c t io n s in Guinea f ig s *

37

I n f e c t io n C o n tr o ls.

40

.............................

V

F I (HIKES Humber T i t l e 1

Page

S u o t i o n A p p a r a tu s f o r C o l l e c t i o n o f S e l l s fro m B la k e B o t t l e s ............................

23

2

P r e p a r a t i o n o f B r u c e l l a F r a c t i o n s ............................. . .

26a

3

P r o d u c t i o n o f A g g l u t i n i n s fro ® I n o c u l a t i o n o f W a te r S o l u b l e F r a c t i o n s o f B r u c e l l a a b o r t u s ,

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%

P r o d u c t i o n o f A g g l u t i n i n s fro ® I n o o u l a t i o n o f A c e to n e T r e a t e d F r a c t i o n s o f B r u e e l l a a b o r t u s .

66

5 6

C o m p a r a tiv e C hro m ato gram s o f U n h y d ro ly E e d B r u c e l l a F r a c t i o n s . . . .................... . . . . . . . C o m p a riso n o f P r o t e c t i o n P r o d u c e d by F r a c t i o n s .

107 .

117

Vi

PLATES Humber T i t l e

Page

1

B r u c e l l a i n f e c t e d s p l e e n s ........................

2

P h o t o m ic ro g r a p h o f B r . a b o r t u s 2 3 0 & sh o w in g c a p s u l e s .......................

109

3

S w e llin g o f c e l l w a lls a f t e r ac e to n e e x t r a c ti o n .

110

^

C e l l s e x t r a c t e d w i t h w a t e r .............................................................110

5

C e l l e x t r a c t e d w i t h b u f f e r a t pH 9 . ^ .................................... I l l

6

C e l l s e x t r a c t e d w i t h a c e t o n e and r e f r i g e r a t e d 2 w eeks i n w a t e r ................................................

*

36

I ll

Til

TABLES T able T i t l e

F ag#

1

B r u c e l l a S p e c i e s S t u d i e d and S o u r c e ..................................

19

2

Number o f M in u te s R e q u ir e d by B r u c e l l a S p e c i e s t o H y d r o l y s e U r e a , .................................

21

P l a t e C o u n ts o f S u s p e n s i o n s o f B r. a b o r t u s 2 JOB Crown 5 Bays a t 37 C and S t a n d a r d i z e d t o 9 ^ p e r c e n t L i g h t T r a n s m i t t a n c e ............................................

^1

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s i n G roup I-B G u in e a P i g s ............................

M-g

M a c r o s c o p ic P a t h o l o g y a n d C u l t u r a l R e s u l t s i n Group X-C G u in e a P i g s ...............................................................

50

C u l t u r a l and P a t h o l o g i c a l R e s u l t s i n G u in e a P i g s I n o c u l a t e d w i t h C h a lle n g e Used f o r G roups I - C an d I I - C ............................................................................................

51

M a c r o s c o p ic P a t h o l o g y an d C u l t u r a l R e s u l t s i n Group XI-A G u in e a P i g s . ...........................................

.

53

C u l t u r a l a n d P a t h o l o g i c a l R e s u l t s i n G u in e a P i g s I n o c u l a t e d w i t h C h a l l e n g e U sed f o r G rou p s 11-A a n d 311 - A ............................................................................................

5^

3

5 6

7 g

9

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s i n G roup IX-B G u in e a P i g s ........................... 55

10

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s i n G roup XI-C G u in e a P i g s ........................... 57

11

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s I n Group I I I - A G u in e a P i g s ........................... 59

12

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s i n G roup I I I - C G u in e a P i g s . . . . . . . . . . . . .

13

M a c r o s c o p ic P a t h o l o g y a n d C u l t u r a l R e s u l t s I n G roup IV-B G u in e a P i g s ........................... 62

1^

M a c r o s c o p ic P a t h o l o g y a n d C u l t u r a l R e s u l t s i n Group V -l-B G u in e a P i g s ........................... 63

15

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s i n G roup V -l-C G u in e a P i g s . .. . . . . . . . . . .

60

65

Till

T a b le T i t l e 16 17 IS 19

20

21 22

23 2^

25

26 27 2g

Page

M a c r o s c o p ic P a t h o l o g y an d C u l t u r a l R e s u l t s i n G roup V- 2 - S G u in e a P i g s ..........................................................

Si

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s i n Group V- 2 -C G u in e a P i g s ..........................................................

SB

M a c r o s c o p ic P a t h o l o g y and C u l t u r a l R e s u l t s i n Group VI-A G u in e a P i g s ..........................................................

70

C u l t u r a l a n d P a t h o l o g i c a l R e s u l t s i n G u in e a P i g s I n o c u l a t e d w i t h C h a l l e n g e U sed f o r G ro up s V -1 -B , V -2-B an d V I - A ..............................................................................

71

C u l t u r a l and P a t h o l o g i c a l R e s u l t s i n G u in e a P i g s I n o c u l a t e d w ith C h a l l e n g e Used f o r G ro u ps V -l-C a n d V- 2 - 0 ............................................................................................

72

M a c r o s c o p ic P a t h o l o g y a n d C u l t u r a l R e s u l t s i n G roup VI-B G u in e a P i g s ..........................................................

7^

C u l t u r a l and P a t h o l o g i c a l R e s u l t s i n G u in e a P i g s I n o c u l a t e d w i t h C h a lle n g e U sed f o r G ro u p s VI-B an d V I I - B . .......................................................................................

75

M a c r o s e o p ie P a t h o l o g y and C u l t u r a l R e s u lt s I n Group V II-B G u in e a P i g s ..........................................................

J&

T i t e r s P r o d u c e d fro © 1 I n o c u l a t i o n o f F r a c t i o n VI-A a n d a C h a l l e n g e Bose i n G u in e a P i g s .............................

JB

A n tib o d y T i t e r s i n G u in ea P i g s 30 Bays F o l l o w i n g I n o c u l a t i o n o f F r a c t i o n I -B a n d 30 d a y s F o l l o w i n g C h a l l e n g e Bose o f 1 0 0 ,0 0 0 B r . a b o r t u s PJOB . . .

79

T i t e r s P r o d u c e d fro © 1 I n o c u l a t i o n o f F r a c t i o n V - l- R an d a C h a l l e n g e Bose i n G u in ea P i g s . . . .

SO

T i t e r s P r o d u c e d fro m 1 I n o c u l a t i o n o f F r a c t i o n V -c-B and a C h a l l e n g e Bose I n G u in e a P i g s . .

.

.

A g g l u t i n i n R e sp o n se F o l l o w i n g 1 I n o c u l a t i o n o f F r a c t i o n I I - A an d o f C h a l l e n g e .......................................

29

T i t e r s P r o d u c e d fro © 1 I n o c u l a t i o n o f F r a c t i o n I I - B a n d a C h a lle n g e Bose i n G u in e a P i g s .............................

30

T i t e r s P r o d u c e d fro © 3 I n o c u l a t i o n s o f F r a c t i o n I-C an d a C h a lle n g e Bose i n G u in e a P i g s .................. SB

31

T i t e r © P r o d u c e d fro m 3 I n o c u l a t i o n s o f F r a c t i o n VI-B an d a C h a l l e n g e B ose I n G u in e a P i g s ................. ....... .

gl 63

B$

T itle

P ag e

T i t e r e P r o d u c e d from 3 I n o c u l a t i o n s o f F r a c t i o n V II-B a n d a C h a l l e n g e Dose I n G u in e a P i g s . . .

$0

T i t e r s P ro d u c e d from 3 I n o c u l a t i o n s o f F r a c t i o n V -l-C r i d a C h a lle n g e Bose i n G u in e a P i g s . . .

91

T i t e r s P ro d u c e d from 3 I n o c u l a t i o n s o f F r a c t i o n V-P-C an d a C h a lle n g e Bose i n G u in e a P i g s . . .

92

T i t e r s P r o d u c e d from 3 I n o c u l a t i o n s o f F r a c t i o n IV-B and s. C h a l l e n g e Bose i n G u in e a P i g s . . .

93

T i t e r s P r o d u c e d from 3 I n o c u l a t i o n s o f F r a c t i o n I I - C and a C h a l l e n g e Bose i n G u in ea P i g s . . .

9^

A g g l u t i n i n T i t e r s Found I n Homologous and H e t e r ­ o l o g o u s S e r a (AMS) U s in g BAI A n t i g e n ........... 96 A g g l u t i n i n T i t e r s o f B r u c e lla S p e c ie s w ith H e te r­ o lo g o u s an d Homologous A n t i s e r a ......................... 9& P r e c i p i t i n T i t e r s o f Homologous a n d H e t e r o l o g o u s A n ti s e r a w i t h F r a c t i o n s I - A and I I - A ........... 99 A b s o r p t i o n o f A g g l u t i n a t i n g A n t i b o d i e s from Homolo­ g ous an d H e t e r o l o g o u s A nti s e r a w ith F r a c t i o n s I - A and I I - A ........................................... 99 P r e c i p i t i n T i t e r s P ro d u ced by P r e p a r a t i o n s o f F r a c t i o n s I an d XI w i t h Homologous a n d H e t e r ­ o l o g o u s B e r n ................................................................................... 101 A g g l u t i n i n A b s o r p t i o n from Homologous a n d H e t e r ­ o lo g o u s S t r a i n A n tis e r a by F r a c t i o n s I I - P ^ , I I - B 3 ...............................................................

102

A g g l u t i n i n A b s o r p t i o n from Homologous a n d H e t e r ­ o l o g o u s S t r a i n A n ti sera, b y F r a c t i o n IX-B . . . IO 3 A g g l u t i n i n R e sp o n se o f I n f e c t i o n C o n t r o l A n im a ls A b o r t lo n s O c c u r r i n g i n F em ale G u in e a P i g s I n o c u l a t e d w i t h V a r io u s F r a c t i o n s an d C h a l l e n g e d w i t h B r. a b o r t u s 2J0&....................................................... 113 P r o t e c t i o n o f G u in e a P i g s b y V a r io u s B r u e e l l a E x t r a c t s ....................................................................................116 D e v i a t i o n i n T jim s o f S ta n d a r d E r r o r ..................... 121

IOM-

X XMfB0 BUCTXOH The p r o b le m o f Im m u n ity t o t h e g r o u p o f d i s e a s e s w h ich h as not y ie ld e d r e a d ily to a a n ’ s p ro p h y la x is i s a te a s in g one.

I t h a d b e e n t h o u g h t by many t h a t s u c h d i s e a s e s a s t u ­

b e r c u l o s i s a n d b r u c e l l o s i s w ere e n t i r e l y d e p e n d e n t u p o n m fa v o r a b le b a la n c e o f th e i n f e c t i o u s a g e n t w ith in th e h o s t . I t i s n o t d e n i e d t h a t s u c h s t a t e s e x i s t b u t fro m r e c e n t d e ­ velopm ents i n te c h n iq u e s o f s e p a r a tio n o f b i o l o g i c a l l y a c tiv e m a t e r i a l s t h e r e have a l r e a d y a r i s e n new a g e n t s w h ic h may b e used f o r im m u n iza tio n .

P u r i f i e d Im m u n iz in g a g e n t s may r e s u l t

i n r e d u c t i o n o f s i d e e f f e c t s and a v o i d a n c e o f s e n s i t i z i n g doses;

s u c h a g e n t s c o n t r i b u t e to t h e s a t i s f a c t i o n o f o n e ’ s

c u r i o s i t y ( o r p e r h a p s add t o i t )

a® t o t h e n a t u r e o f an a g e n t

( o r a g e n t s ) w hich s o p o w e r f u l l y lay® i t s

i m p r i n t on t h e t i s s u e s

o f h i g h e r a n i m a l s t h a t f o r l o n g p e r i o d s a f t e r t h e i m p r i n t one r e s i s t s th e advances of th e p a r t i c u l a r d is e a s e . T h is work h a s n o t p r e t e n d e d t o d e l v e d e e p l y i n t o t h e chem­ i c a l n a t u r e o f t h e f r a c t i o n s w h ic h w e r e s e p a r a t e d .

I t ha® a t ­

tem pted to r e l a t e th e s o l u b i l i t y o f p r o t e i n s t o th e e x t r a c t i o n o f an e f f e c t i v e

im m u n iz in g a g e n t i n t h e n a t i v e s t a t e f r om

B ru c e lla ab o rtus.

II HISTORICAL The d e s i r a b i l i t y o f o b t a i n i n g r e l a t i v e l y p u r e a n d b i o ­ l o g i c a l l y a c t i v e s u b s t a n c e s from t h e b a c t e r i a l c e l l h a s b e e n e m p h a s iz e d b y t h e r e c e n t p a p e r s o f s e v e r a l w o rk e rs * fo il, er,

P illem er,

and B a d g e r ( 1 9 ^ 7 )* P i l l e m e r a n d Moore ( 1 9 ^ $ ) and P i l l e m ­

w ittier,

B u r r e l l # an d O r o s s b e r g ( 1 9 ^ 6 ) h a v e d e s c r i b e d

t h e i s o l a t i o n and is u s u n o c h e m is tr y o f hom ogeneous d i p t h e r i a t o x ­ o i d and c r y s t a l l i n e t e t a n a l t o x i n ;

Lamma n a and d-la s sman (1 9 ^ 7 )

h a v e d e s c r i b e d t h e i s o l a t i o n o f t y p e B C l o s t r l c l i um b o t u l l n u a to x in ; Baker,

Sommer, L. E. F o s t e r , 1 . Meyer and K , F. Meyer

( 1 9 ^ 7 ) r e p o r t e d the i s o l a t i o n o f a c r y s t a l l i n e a n t i g e n from P asteu rella pest i s ;

and B e v i l a q u a and M c C a r t e r (19^-g) f r a c t i o n '

a t e d an d c h a r a c t e r i z e d t h e co m p o n e n ts from t u b e r c l e c u l t u r e filtrates*

A l l o f t h e s e w o r k e r s h a v e I s o l a t e d s u b s t a n c e s w hich

a p p e a r t o be homogeneous an d a r e b i o l o g i c a l l y a c t i v e I n p r o d u c ­ i n g a n t i b o d i e s a n d / o r im m u n i t y , i n e x p e r i m e n t a l a n i m a l s .

The

m e th o d s e m p lo y ed f o r th e i s o l a t i o n o f v a r i o u s compounds a r e b a s e d on t h e m u l t i v a r i a b l e s y s te m d e v e l o p e d by Cohn, S t r o n g , H u g h es, M u lf o r d , A s h w o rth , M eli n , and T a y l o r ( 1 9 ^ 6 ) and t h e i r c o - w o r k e r s f o r t h e f r a c t i o n a t i o n o f p r o t e i n co m p o n en ts o f b lo o d plasm a in h i g h l y p u r i f i e d s t a t e . A s t u d y o f t h e l i t e r a t u r e on i m m u n i z a t i o n a g a i n s t b r u c e l ­ l o s i s shows t h a t t h e m e thods o f t r e a t i n g t h e c e l l s o r p r o d u c t s u s e d f o r i m m u n i s a t i o n may r o u g h l y b e g r o u p e d a s f o l l o w s ; t r e a t m e n t o f whole c e l l s p r i m a r i l y b y p h y s i c a l a g e n t s , t r e a t -

3

merit o f w h o le c e l l s p r i m a r i l y b y c h e s t e a l a g e n t s , e x t r a c t i o n o f the c e l l s ,

u s e o f a d j u v a n t s and a t t e n u a t i o n ,

K illin g c e l l s by p h y s ic a l a g e n ts 1.

H eat.

M e a t - k i l l e d b a o t e r i n was f i r s t e m p loyed by

S l r t and Lamb ( 1 6 9 9 ) ^ho i n j e c t e d h e a t - k i l l e d B r. m e l i t e n e i e i n an a t t e m p t t o im m unise a man a g a i n s t l i v e c u l t u r e s o f t h e same o r g a n i s m - s u b s e q u e n t l y i n j e c t e d .

The man becam e i n f e c t e d .

Shaw ( 1 9 0 7 ) a n d E yre ( 1 9 0 6 ) i n j e c t e d h e a t - k i l l e d B r . me11t e n sis

i n t o t h e monkey a n d man r e s p e c t i v e l y w i t h o u t o b t a i n i n g

sig n ific a n t p ro tectio n .

N l c o l l e , B u r n e t t , and C o n s c i l , work­

i n g on man., ( 1 9 2 3 ) , A s c o l i and S a n f i i i p o (1 2 9 ) , Z a m i t t and Dabono ( 1 9 3 0 ) , on g o a t s , a n d Dubois .and S o l H e r ( 1 9 3 0 ) on man, a l l c la im e d e v i d e n c e o f p r o t e c t i o n f o l l o w i n g t h e i n j e c t i o n o f h e a t - k i l l e d B r. m e l i t e n g i s .

T h i s work h a s n o t b e e n s u b s t a n ­

t i a t e d a n d Huddle s o n (19**-2 a) s t a t e s t h a t im m u n iz a tio n o f human b e i n g s h a s n o t been s u c c e s s f u l .

h f u r th e r f a i l u r e o f vaccin a­

t i o n o f man a g a i n s t b r t s e e l l o s l s i s r e p o r t e d by Howe, M i l l e r , K e lly , B rookw alter,

and. E l l i n g t o n ( 1 9 ^ 7 ) i** a r e p o r t o f 17 e a s e s

o f b r u c e l l o s i s among a g r o u p o f l a b o r a t o r y w o r k e r s i n j e c t e d w i t h a k i l l e d vaccine.

Mo d e t a i l s a s t o how t h e v a c c i n e was mad©

w ere g i v e n . H e a t - k i l l e d B r. a b o r t u s b a n t e r i n g h a v e b e e n u s e d w i t h e q u a l l a c k o f s u c c e s s by Bang ( 1 9 0 6 ) i n h e i f e r s , M&cfadyean and S to e k m an ( 1 9 0 9 ) i n co w s, A s c o l i (1 9 1 5 ) i n g u i n e a p i g s , Z e l l e r , K r a g e , a n d O m lnder (1 9 2 0 ) and S m ith and L i t t l e i n p r e g n a n t and n o n - p r e g n a n t cows.

Z w ick, (I923)

Smith and L i t t l e f e l t some

k

p r o t e c t i o n was e v i d e n c e d .

F a i l u r e s to im m unize g u i n e a p i g s

b y i n j e c t i o n o f b e a t - k i l l e d B r . a b o r t u s c e l l s a r e r e c o r d e d by A s c o l i ( 1 9 1 5 )# S t a f a e t h ( 1 9 B 0 ) , Hagan ( 1 9 ? £ b ) , S c h r o e d e r and C o t t o n ( 1 9 2 ‘4.}, C a t k i n

( 1 9 3 1 ) , H o l t h ( 1 9 3 3 ) , KcEwen and R o b e r t s

( I 9 3 6 ) , a n d H uddle so n ( 1 9 ^ 7 ) •

CSw&tkin a l s o u s e d p h e n o l i z e d

h e a t —k i l i e d ce l i e * The o n l y a p p a r e n t s u c c e s s w i t h h e a t - k i l l e d B r . a k o r t u a a s f a r a s i s know n, was i m m u n i z a t i o n o f r a t a r e p o r t e d by H o l t h (1911).

However, t h e same w o r k e r f o u n d no p r o t e c t i o n

f o r m ic e

t e s t e d a t t h e same t i m e . 2.

U ltrav io let l i g h t .

H uddle so n (19*4-7) r e p o r t e d some

p r o t e c t i o n t o be e v i d e n t i n g u i n e a p i g s i n j e c t e d w i t h i r r a d i ­ ated c e lls

and s u b s e q u e n t l y c h a l l e n g e d w i t h 100(3 t o 1 0 0 , 0 CO

o r g a n i s m s o f hom ologous a n d h e t e r o l o g o u s s p e c i e s . 3.

Sonic t r e a t e d o r g a n is m s .

L ive, S p erlin g ,

and S tu b b s

(19*4*5) d i s i n t e g r a t e d whole c u l t u r e s o f B r. a b o r t u s w ith h i g h f r e q u e n c y so u n d w a v e s.

The c e l l f r e e e x t r a c t , p r e c i p i t a t e d

and p r e s e r v e d w i t h e t h y l e t h e r , was g i v e n i n a s e r i e s o f 3 i n ­ j e c t i o n s a t 3 day i n t e r v a l s a n d Im m unized 16 o f 145 g u i n e a p i g s w h i l e a l l b u t one o f t h e c o n t r o l a n i m a l s was i n f e c t e d b y c h a l ­ l e n g e d o s e s r a n g i n g from ?.JO t o 1*500 B r . *4-.

M echanical d i s i n t e g r a t i o n .

a b o rtu s organism s.

Huddle son (19*4-0) ( 1 9 *+TD)

h a s u s e d an e t h e r e x t r a c t o f B r . s u l s a n d B r . a b o r t u s crushe d, i n a wet c r u s h i n g m i l l a t low t e m p e r a t u r e .

This p ro d u ced a

p r o d u c t w h ic h e n g e n d e r e d some p r o t e c t i o n when u s e d as a v a c c i n e i n g u i n e a p i g s c h a l l e n g e d w i t h IpB t o

o r g a n i s m s a n d was

i m p o r t a n t i n b r i n g i n g o u t t h e n e e d f o r u s i n g low t e m p e r a t u r e s

5

when se p a r a tin g p r o t o p l a s m i c c o m p o n e n t s . I C l l l l n g b y c h e m i c a l &&e a t a I*

Ifoluol*

I m m u n i z a t i o n o f cows b y © e l l s o f Br* a b o r t u s

k i l l e d w i t h t o l u e n e was u n s u c c e s s f u l l y a t t e m p t e d by Bang (1 9 0 b )* Mo o t h e r a t t e m p t s w i t h t o l u o l - k i l l e d organ lasts w e r e n o t e d i n t h e l i t « ra t u r e S*

s u rva y e d *

P henol*

O w atk iu ( 1 9 3 1 ) u s e d c u l t u r e s o f B r . a b o r t u s

k i l l e d w i t h 0*5 p e r c e n t p h e n o l I n a t t e m p t i n g t o immunize

guinea p i g s a g a i n s t c h a lle n g e d o s e s w h ic h m u s t h a v e b e e n q u it® l a r g e a s t h e y c o n s i s t e d o f 1 * 0 ml o f a s u s p e n s i o n , o f mm e n t i r e 4 8 h o u r a g a r s l a n t o f B r * a b o r t u s rem ove d i n a few ml o f s a ­

Bud&leeon ( 1 9 4 ? ) g o t no p r o t e c t i o n f r o m p h e n o l i z e d

lin e*

c u l t u r e s o f Br* a b o r t u s u s i n g c o n s i d e r a b l y l o w e r c h a l l e n g e doses* 3* by A s c o l i

Ith e r*

S t h e r - k l l l a d c e l l s were u se d in g u in e a p i g s

( 1 9 1 5 ) who f o u n d no p r o t e c t i o n *

L iv e , S p e r l i n g an d

S t u b b s ( 1 9 4 5 ) , L i v e ( 1 9 4 9 ) , a n d L a r s e n (1949) h a v e f o u n d s i g ­ n i f i c a n t p r o t e c t i o n u s i n g c u l t u r e s o f Br* a b o r t u s k i l l e d w ith e th y l ether*

L arsen h a s , in a d d i t i o n , found c e r t a i n a d ju v a n ts

enhance t h e e f f e c t *

The e a r l i e r f a i l u r e , r e p o r t e d b y A s c o l i

may h a v e b e e n d u e t o t h e s i z e o f t h e c h a l l e n g e dose*

5Bi© l a t ­

e r , more s u c c e s s f u l , r e s u l t s e m p loyed c h a l l e n g e d o s e s r a n g i n g f ro m £50 t o 1800 o r g a n i s m s .

Sonic r u p tu r e d c e l l e x t r a c t p r e ­

s e r v e d w i t h e t h y l e t h e r b y L i v e a t a l i n t h e work c i t e d a b o v e seem ed t o b® o f a b o u t e q u a l e f f i c a c y * 4*

F o rm alin *

F o r m 1 i n - k i l l e d c e l l s of B r * a b o r tu s have

0

b e e n u s e d , w i t h o u t s u c c e s s , b y C M atkin (1 9 3 1 ) t an d i&s’Rwen a n d R o b e r t s (1 9 3 6 ) i n a t t e m p t s t o Immunize g u i n e a p i g s *

On t h e

o t h e r h a n d , u s i n g c h a l l e n g e d o se s o f c o n s i d e r a b l y l e s s m agni­ t u d e H u d d le son (194"?) f o u n d some p r o t e c t i v e o f f s e t e x i s t e d when c e l l s k i l l e d w i t h n o t o v e r 0*25 p e r c a n t f o r m a l i n w ere used*

! l i # t e r c o n c e n t r a t i o n s d e s t r o y e d t h e immunogen!o compo­

n en t*

F o s t e r (1948) f o u n d no o r g a n i s m s o n p o s t - m o r t e m exam­

i n a t i o n on ono o f two g u i n e a p i g s i n j e c t e d w i t h Br* a b o r t u s c o l l s k i l l e d by s u s p e n s i o n i n o o l d 1 * 0 p a r c e n t f o r m a l i n a t 4 -6 C c h a lle n g e d w ith 1 0 ,0 0 0 ,0 0 0

B r. a b o r tu s *

i m a l was i n f e c t e d b u t t h e

was s m a l l e r t h a n t h a t i n c o n ­

t r o l an im als*

sp le e n

Uhe s e c o n d a n ­

A n im a ls g i v e n f o r m l i n - k l l l e d c e l l s a n d no

c h a l l e n g e d o s e showed no e v i d e n c e o f i n f e c t i o n o r t o x i c r e a e tio n * S*

P h e n y l merourlo n i t r a t e - k i l l e d c e l l ® *

Phenyl m ercuric

n i t r a t e - k i l l e d c e l l s w e r e shown by H u d d le s o n (1 9 4 7 ) t o be i n e f ­ f e c t i v e Im m u n is in g a g e n t s * 6*

A lcohol*

cohol f a ile d

B r * a b o r t u s k i l l e d b y c o l d 50 p e r c e n t a l ­

t o p r o t e c t two g u i n e a p i g s f r o m c h a l l e n g e d o s e s

o f 1 0 , 0 0 0 , 0 0 0 o r '■ei I s a s

7*

(F o ster 1948).

Chloroform. O w atkin (1931)

f o u n d no immunizing e f f e c t

f r o m B r * a b o r t u s k i l l e d w i t h 2*0 p e r c e n t c h l o r o f o r m . B* . O t h e r m e t h o d s *

S c a l e s a n t H u t d i e s o n (1 9 3 6 ) u s e d g r o u n d

up s p l e e n s t h a t w ere I n f e c t e d w i t h B r u c e l l a a n d t r e a t e d with, e i t h e r 2*0 p e r c e n t f o r m a lin o r 8*0 p e r c e n t c h lo ro fo rm to k i l l t h e o r g a n is m s *

fh e s e p r e p a r a t i o n s d id n o t p r o t e c t g u in e a p ig s

fro m I n f e c t i o n f o l l o w i n g a d m i n i s t r a t i o n o f c h a l l e n g e d o s e s o f

7

l n t e r p l & c e n t a l exudate t r e a t e d w ith 2 .0 p e r c e n t ch lo ro fo rm . F ra c tlo n a tio n The u s e o f e x t r a c t © o r f r a c t i o n s o f b a c t e r i a l c e l l s wag e a r l y shown t o be o f v a l u e I n t h e c a s e o f t h e

exotoxin o f

Co r y neb a c t e r 1 um d i p t h e r l a e by von B e h r i n g a n d K l t a s a t o , a s c i t e d b y Z i n s s e r , Under® a n d F o t h e r g i l l ( 1 9 ^ 5 ) wbo s u c c e s s f u l l y Im­ m unized a n i m a l s a g a i n s t t h a t o rg an ism by I n j e c t i o n o f c u l t u r e flitra te s. T r e a t m e n t o f c u l t u r e f i l t r a t e s o f B r u c e l l a c e l l ® was f i r s t e m p loyed b y H o l t h i n hi® p r e v i o u s l y c l t e r ( 1 9 1 1 ) work on h e a t k i l l e d an tig en .

Both a l c o h o l and ammonium s u l f a t e w e r e u s e d t©

p r e c i p i t a t e an a n t i g e n i c m a t e r i a l from B r . a b o r t u s c u l t u r e f i l ­ trate .

A® w i t h t h e h e a t

t r e a t e d c e l l s , r a t s showed some e v i ­

d e n c e o f i m m u n i z a t i o n w h i l e m ice showed n o n e .

Ctwatkln ( 1 9 ^ 1 )

( 1 9 3 5 ) u t i l i z e d b o t h h e a t e d and u n h e a t e d c u l t u r e serv ed w ith 0.5 p er cen t phenol.

f i l t r a t e s pre­

I n a l a t e r p u b l i c a t i o n 0-wat-

k l a ( 1 9 3 5 ) i n u n s u c c e s s f u l I m m u n i z a t i o n a t t e m p t s i n g u i n e a pig® r e p o r t e d f u r t h e r f a i l u r e ® t o Immunize u s i n g a l c o h o l p r e c i p i ­ ta te d B r. ab o rtu s f i l t r a t e . natu re, h ighly to x ic ,

T h is m a t e r i a l was c a r b o h y d r a t e I n

s e r o lo g ic a lly a c tiv e , but f a i l e d to p ro ­

t e c t guinea, p i g s from i n f e c t i o n a g a i n s t r a t h e r h e a v y c h a l l e n g e dose®.

The f i l t r a t e

resid u e

l e f t a f t e r p r e c i p i t a t i o n was

d r i e d , t o x i c i t y and im m u n o lo g ic t e s t s s h o w in g i t t o x i c b u t o f no p r o t e c t i v e e f f e c t . ta tio n

to be l e s s

Ammonium s u l f a t e p r e c i p i ­

a p p a r e n t l y p r o d u c e d c o m p o n e n ts s i m i l a r t o t h o s e o b t a i n e d

by e th a n o l p r e c i p i t a t i o n

from p r e c i p i t a t e and s u p e r n o t e .

A

t o l u o l - e t h e r and s a l i n e e x t r a c t o f d r i e d and g r o u n d B r . a b o r t u s

B

and. an a l c o h o l i c p r e c i p i t a t e

of c u lt are f i l t r a t e

fro® a ro u g h

s t r a i n o f B r . a b o r t u s l i k e w i s e g a v e no e v i d e n c e o f p r o t e c t i v e ab ility . F e n n e l l and Hud d i e so n

(1S3&) e nd H uddle son (19^-0)

s t u d i e d t h e e f f e c t o f e n d o - a n t i g e n a s an im m u n isin g a g e n t . T h i s s u b s t a n c e was a n a l b u m i n o i d f r a c t i o n w h ich p r o d u c e s a n t i ­ b o d i e s and was t o x i c on i n t n f c p e r l t o n e a l i n o c u l a t i o n i n g u i n e a p ig s.

An S f r a c t i o n l i n k e d t o t h e a l b u m i n o i d f r a c t i o n and o f

c a rb o h y d ra te n a tu r e did not p ro d u ce a n t i b o d i e s .

The n i t r o g e n

c o n te n t o f th e e n d o -a n tig e n a o f t h e v a r io u s s p e c ie s o f B ru c e lla d i d n o t a l w a y s s e e s t o b e i n t i m a t e l y conn a c t e d ? d t h t h e a b i l i ­ t y to p r e c i p i t a t e s p e c i f i c a n tib o d ie s .

I n I m m u n iz a tio n e x p e r i ­

m e n ts e n d o - a n t i g e n f a i l e d t o p r o t e c t guinea, p i g s . S t a h l , P e n n e l l , and. K u d d le s o n (19395 and F w l d le s o n ( 1 9 *4-0 ) r e p o r t e d a p r o t e i n n u c l e a t e f r a c t i o n e x t r a c t e d by 3 and p p e r c e n t B&OH, s p l i t w i t h sod ium c a r b o n a t e , and s e p a r a t e d w ith c h l o r o f o r m , t o be a n t i g e n i c i n 19 o f 20 g u i n e a p i g s when g i v e n i n 10 mg d o s e s .

The t o x i c i t y

o f t h i s f r a c t i o n was low b u t i t

t o o f a i l e d t o p r o t e c t g u i n e a p ig * fro® i n f e c t i o n .

The n u c l e i c

a c i d p o r t i o n o f p r o t e i n n u c l e a t e was o n l y f e e b l y a n t i g e n i e by itse lf.

P r e c i p i t i n s t u d i e s o f t h e f r a c t i o n w i t h smooth s t r a i n

a n t i s e r a o f B r.

a b o r t u s , S r . a u l a , a n d B r . m e l l t e n s l a were a l l

p o s i t i v e i n d i l u t i o n s o f from. 1 : 1 0 0 0 t o 1; 3 2 , OCX), g i v i n g n o n ­ s p e c i f i c c r o s s r e a c t i o n s w i t h i n the g ro u p .

I n te r m e d i a te rough

and r o u g h s t r a i n s gove no p r e c i p i t i n r e . a c t i o n s .

A llerg ic re­

a c t i o n s o c c u r r e d f o l l o w i n g i n o c u l a t i o n i n d i l u t i o n s o f 1 :2 0 0 t o 1 : £ 0 0 0 w i t h c r o s s - s e n s i t i z a t i o n i n d i l u t i o n s o f I s 1000 w i t h

9

rough s t r a i n s . P o p , Bamboviee&nu, B a r b e r and M a rin o u ( 1 9 3 ^ ) &nd Baittbov i c e a n u , B a r b e r , P op, M arin o n ( 1 9 3 ^ ) r e p o r t e d t h a t an a n t i g e n from c e l l s e x t r a c t e d i n t h e c o l d w i t h 15 p e r c e n t t r i c h l o r a c e t i c a c i d , a f t e r t h e t e c h n i q u e o f BoI v i n and M esro b e an u (193*0 > p r o ­ duced a n t i b o d i e s in the r a b b i t ,

and was q u i t e t o x i c f o r t h e

mouse t h o u g h l e s s so f o r t h e g u i n e a p i g . bohydrate in n a tu re .

The a n t i g e n was c a r ­

D&voli ( 1 9 3 9 ) p r e p a r e d a s i m i l a r f r a c t i o n

w i t h e s s e n t i a l l y t h e same t e c h n i q u e w h ic h was n i t r o g e n f r e e , also to x ic

f o r m i c e , and p r o d u c e d a n t i b o d i e s i n r a b b i t s .

T his

f r a c t i o n p r e c i p i t a t e d h o m o lo g o u s a n t i s e r u m . The e t h y l e t h e r e x t r a c t s p r e p a r e d by L i v e ,

S p e r l i n g and

S t u b b s ( 1 9 ^ 5 ) * L iv e ( 1 9 ^ 9 ) , a n d .Larsen ( 1 9 ^ 9 ) were b r i e f l y d is c u s s e d e a r l i e r and w i l l n o t b e d is c u s s e d f u r t h e r . M iles (1 9 3 9 ), M iles and P l r l e

( 1 9 3 9 ab od e % and P a t t e r s o n ,

X -irie a n d S t a b l e f o r t h ( 1 9 ^ 7 ) h a v e i s o l a t e d a h i g h l y a n t i g e n i e com­ p o n e n t by a c o m b i n a t i o n o f d i f f e r e n t i a l c e n t r i f u g a t i o n and e x ­ t r a c t i o n o f H r. m e l i t e n s i s an d B r . phenol in th e co ld .

a b o rtu s w ith 3 .0 p e r cent

T h is m a t e r i a l p r o d u c e d a n t i b o d i e s upon i n ­

j e c t i o n i n t o g u i n e a p i g s an d was com posed o f p o l y s a c c h a r i d e , a m i n o f end p h o s p h o l i p i n r e s i d u e s .

The l a t e s t p u b l i c a t i o n c i t e d

f o u n d p r o t e c t i o n e q u i v a l e n t t o t h a t p r o d u c e d by B r . a b o r t u s s t r a i n 19 v a c c i n e , t o b e p r o d u c e d upon i n o c u l a t i o n i n t o g u i n e a pigs*

M i l e s and P l r i e ( 1 9 3 9 c ) made t h e i m p o r t a n t o b s e r v a t i o n

t h a t t h e i r compound was p r o b a b l y s i m i l a r t o some i s o l a t e d b y o t h e r w o r k e r s and t h e s u p e r i o r e f f i c a c y o f t h e a n t i g e n p r o b ­ a b l y was due t o t h e g e n t l e r t r e a t m e n t e m ployed i n p r e p a r i n g

10

the a n tig e n .

Indeed th ey u r s

a b l e t o show a p r o g r e s s i v e l o s s

In t i t e r c o r r e la te d w ith degree o f h y d ro ly s is of th e a n tig e n . I n a r e c e n t work L a r s e n ( 1 9 ^ 9 ) h a s u s e d e t h e r e x t r a c t i o n o f c e l l s p r e v i o u s l y t r e a t e d w i t h p h e n o l i z e d s a l i n e and s e p a ­ r a t e d f r o m t h e l i q u i d a f t e r *4* w e e k s 1 s t o r a g e a t ^ 0 .

The c e l l s

w ere re s u s p e n d e d I n 0 . S 5 p e r c e n t s a l i n e a n d , w i t h two v o lu m e s o f e t h y l e t h e r a d d e d , v i g o r o u s l y s h a k e n end a l l o w e d t o s t a n d 2 d a y s a t room t e m p e r a t u r e .

The a q u e o u s p h a s e , c e n t r i f u g e d

f r e e o f c e l l s , was u s e d f o r i n j e c t i o n . S ero lo g ical

s t u d i e s on B n i c e l la f r a c t i o n s h a v e e m p h a s i z e d

t h e w orks o f Evans ( 1 9 1 8 ) ( 1 9 2 3 ) # F e u s l e r an d Meyer { 1 9 2 0 ) , en d F r a n c i s (1931)

show ing t h e c l o s e r e l a t i o n s h i p o f t h e B r u c e l l a

s p e c i e s a n d s e p a r a t i n g them i n t o s e r o l o g i c a l g r o u p s B r . a b o rti* * s u i s and B r . m e l i t e n s i s . b e e n made.

In f a c t,

Ho s i m p l e s e p a r a t i o n b e y o n d t h i s h a s

the a n t i g e n i c s t r u c t u r e a c c o rd in g to th e

work o f W i ls o n an d M i l e s a s c i t e d by M i l e s ( 1 9 3 9 ) and M i l e s and P irie

(19 3 9 e b c d e ) o f t h e two g r o u p s i s p r o b a b l y d e p e n d e n t upon

a p re p o n d e ra n c e of e i t h e r A ( a b o r tu s - s u i s ) s u b s t a n c e o r M ( m eli t e n s i s ) substance.

A b s o r p t i o n t e c h n i q u e s can a b s o r b t h e a n t i ­

b o d i e s f o r one o r t h e o t h e r from a n t i serum to p r o d u c e a n a n t i s eru© sp ecific

fo r e i t h e r the A o r th e E a n tig e n .

( 1939 b) w ere n o t a b l e t o s e p a r a t e t h e

M i l ’s a n d P i r i e

a n t i g e n s by f r a c t i o n a t i o n .

S t u d i e s on h y p e r s e n s i t i v i t y and. p r e c i p i t i n t e s t s by S t a h l , F e n ­ n e l 1 a n d H uddle son (1 9 3 9 )* H u d d le s o n ( 1 9 ^ 0 ) , Fop e t a l ( 1 9 3 ^ ) a n d D am bo viceanu o t a l (193&) u s i n g p r e v i o u s l y m e n t i o n e d f r a c t i o n s have f u r t h e r em phasized t h e d i f f i c u l t y cies se ro lo g ically .

o f s e p a r a tin g th e s e spe­

Mazzl i n 1922 and N ln n i I n 1 9 3 °

cite d by

11

G w atk in {1952} f o u n d i a a u a e s e ru m t o g i v e p r o t e c t i o n a g a i n s t b o t h h o m o lo g o u s and h e t e r o l o g o u s s p a d e s * Tie© o f l . l v t uk t o c c i n e W ith t h e w o rk by F v a n s (1 9 1 8 ) {1923} e s t a b l i s h i n g t h e r e l a t l o n s h i p o f 3 r * a b o r t s o f b o v i n e and p o r c i n e o r i g i n w i t h B r * m a l l t e n s l s an d d e m o n s t r a t i o n t h a t B r , a b o r t u s c o u l d be i s o l a t e d f ro m m i l k i t m s r e c o g n i s e d t h a t t h e e a r l i e r p r a c t i c e o f ^ Im m u n isin g * c a t t l e w i t h l i v i n g v a c c i n a a g a i n s t a b o r t i o n m s a h e a lth hazard*

I h e work bp $1 1 1 n o r , H u d d le s o n , C l a r k ,

a n d O chlingM an ( 1 9 2 9 ) o n t h e v a c c i n a t i o n o f c a t t l e w i t h a s t r a i n o f l i v e Sr.* a b o r t u s n o n - i n f e c t i o u s f o r g u i n e a p i g s a n d p r e g n a n t and n o n - p r e g n a n t c a t t l e

(H u d d le s o n 19 2 2 ) show ed su c h

o r g a n i s m s c o u l d b e i n j e c t e d w i t h o u t a p p a r e n t harm t o c a t t l e * S uck (1 9 3 0 ) s c r e e n e d s e v e r a l s t r a i n s o f Br# a b o r t u s a rut f o u n d on e o f m o d e r a te v i r u l e n c e , s t r a i n 1 9 , w h ic h d i d p r o t e c t c a l v e s a g a i n s t s u b s e q u e n t i n f e c t i o u s doses*

t h i s v a c o in s h as sin c e

b e e n w i d e l y u s e d i n c a t t l e b y many w o r k e r s i n t h i s c o u n t r y a n d H u d d le s o n ( 1 9 4 2 a ) h a s a d e q u a t e l y r e v i e w e d t h e u s a g e *

M a n th e i

{1948} fo u n d s t r a t a 19 t o p r o d u c e im m u n ity o f s h o r t d u r a t i o n u p o n i n o c u l a t i o n i n t o 4 t o 6 m onth g i l t s .

Metwen (1 9 3 9 ) h a s

r e p o r t e d on a n o t h e r a v i r u l e n t s t r a i n a n d t h e u s e o f a v i r u i e r t t s t r a i n s o f B r. m i l g In em ulsions o f l a n o l i n a r e m entioned in th e fo llo w in g s e c t i o n .

H u d d le s o n (1 9 4 7 ) f o u n d m u c o id d i s s o c i -

a n t s o f B r * s u i s a n d Br# m c l l t e n s l g t o b e e f f e c t i v e i n p r o t e c t % i n g g u in e a p i g s a g a i n s t c h a l l e n g e d o s e s o f I x 1 0 B r * s u i s and 5 x XG4 o r 1 x 10s Br* a e l l t e n s i s o r B r . a b o r t u s . F o l l o w i n g

i n j e c t i o n o f 5 i 1 0 ^ t o 1 i 10^® v i a b l e o r g a n i s m s wops demon­ s t r a b l e 3 b u t ' n o t 4 w eeks f o l l o w i n g , v a c o t a a t i o n *

C h a lle n g e

was mad# 4 w e e k s f o l l o w i n g v a e o l n u t i o n . U n f o r t u n a t e l y t h e us© o f t h e 11 t o v a c c i n e s d e s c r i b e d i s n o t w ith o u t hasard*

C r a n f o r d {1948) - a n d l i l a a n ( 1 9 4 4 ) © i t s

d e f i n i t e o a s e s i n w h ich human b e l u g a h a v e b e e n I n f e c t e d w i t h s t r a i n 19 v a c c i n e *

L e v in # a n d W ils o n {1949} hav© r e p o r t e d t h e

I s o l a t i o n o f s t r a i n 19 fro m t h e m i l k o f 3 v a c c i n a t e d cow s an d a hog*

P e r s i s t e n t t i t e r s f o u n d w i t h some f r e q u e n c y i n v a c c i n ­ nmm.

a te d c a t t l e f u rth e r su g g est p o ss ib le la te n t in f e c tio n s , an d C r o s s

(1 9 4 5 ) h a v e s u g g e s t e d t h e © t l o l o g l c r e l a t i o n s h i p o f

s t r a i n 19 t o c a s e s o f a b o r t i o n i n c a t t l e .

Us® ©f ad ju van ts D u b o is ( 1 9 3 6 a ) (1 9 3 9 b ) f o u n d mn a v i r u l e n t s t r a i n o f B r . s u l a s u s p e n d e d i n e m u l s i o n s o f l a n o l i n and p e t r o l a t u m m a rk e d ­ l y re d u c e d a b o r t i o n s in o v in e , c a p r i n e , and b o v in e s p e c i e s and no u n t o w a r d r e s u l t s upon v a c c i n a t i o n o f p r e g n a n t cows*

T e lu

a n d £ o t t n e r ( 1 9 3 8 ) , C o r d i e r ( 1 9 3 8 ) , and B i o h o r n , S i n g l e , a n d Murdock ( 1 9 4 0 ) f o u n d t h a t s u c h e m u l s i o n s w e re s t e r i l i z e d u p o n stan d in g in v i t r o b u t s t i l l possessed co n sid e ra b le a n tig e n ­ ic ity .

H ow ever l l e h o r n and h i s c o - w o r k e r s (1 9 4 0 ) wore a b l e

t o I s o l a t e v i a b l e B r u c e l l a from th e o r i g i n a l s it© o f in o c u l a ­ t i o n I n t h e cow up t o 85 day© a f t e r I n o c u l a t i o n .

X olaer,

B o n d i , a n d B u ie (1 9 3 9 ) f o u n d t h a t m i x t u r e s o f h e a t - k i l l e d c e l l s o f I S b e r t h e l l a typho© a and B r * a b o r t u s w e r e more e f f i c a c i o u s I n p r o d u c i n g p r o t e c t i v e a n t i b o d i e s f o r m ic e u p o n i n o c u l a t i o n I n t o

15

r a b b i t s th a n b e a t - k i l l e d B r. a b o r tu s c e l l s u se d a lo n e ,

A

f u r t h e r i n t e r e s t i n g f i n d i n g w as a h i g h e r d e g re e o f p r o t e c t i o n a g a i n s t c h a l l e n g e by B r . m e l i t e n s i s t h a n by Br* a b o r t u s .

Ih «

p h e n o l - k i l l e d c e l l s a n d e t h e r e x t r a c t s p r e p a r e d fro m them h a v e b e e n show n b y L a r s e n (1 9 4 9 ) t o h a v e a n e n h a n c e d immuno­ g e n i c e f f e c t when u s e d w i t h F r e u n d ’ s a d j u v a n t , c o n t a i n i n g d r i e d , h e a t - k i l l e d M. t u b e r c u l o s i s b u t n o t w i t h o u t th e m . F reu n d ’s a d ju v a n t i s

a w a t e r and m i n e r a l o i l e m u l s i o n .

L iv e

(1 9 4 9 ), on th e o t h e r h a n d , fo u n d e t h e r - k i l l e d B r. a b o r t u s v a c c in e to be d e f i n i t e l y enhanced by e m u ls io n s in l a n o l i n and m in e ra l o il* e ffe c tiv e .

Alum p r e c i p i t a t e d e t h e r - k i l l e d c e l l s w e re l e s s

XXX m>aBTinarPATr

She a n tig e n ic m a te r ia ls to be sep a ra ted ware eo n sid ered to t o l a b i l e complex p r o te in s p o s s ib ly lin k ed to carbohydrate and l i p i d c o n s titu e n ts *

Hence, the te ch n iq u e s wore th o se s p -

p lle a b le to p r o te in s and th ey s o r e based on r e s e n t te ch n iq u e s which have shown so n sid e r a b le su c c e ss in I s o la t in g b i o l o g i c a l ­ l y s a t i r e m a teria l* As p o in ted o u t by th e p h y s ic a l c h e m ists, Cotin e t a l (1 9 4 0 }t M ulford {194?}* Brand and B d a sll {194?}* s o lu b ility o f a p r o te in may be c a r e f u lly re g u la te d by v a ry in g c o n d itio n s o f pH* Io n ic stren g th * tem perature* and c o n c e n tr a tio n o f o r ­ gan ic s o lv e n t s and p r o te in *

Ih ese f a c t o r s may be so chosen

th a t th e d e s ir e d component has* f o r example* a h igh degree* and th e unde s i red components have a low degree o f s o l u b i li t y * o r v i c e versa*

*Bm s im p le s t procedure and one which had n o t been p r e ­ v io u s ly attem p ted in s o fa r a s * search o f th e l i t e r a t u r e cou ld rev ea l* was to u se th e u n trea ted c u ltu r e f i l t r a t e ; th a t is * aqueous e x tr a c t*

Huddleson {1945} had found th e ca p su la r

su b stan ce o f B r u c e lla t o be s o lu b le in h o t w ater and a n tig en * ie«

During th e course o f t h i s work i t was d isc o v e r e d t h a t

C otton (1943) a ls o had dem onstrated l o s s o f c a p su la r m a te r ia l during treatm ent o f th e c e l l and dem onstrated (1949) i t s a n t i­ g en ic i t y i n guinea p igs*

15

C o n c e n t r a t e d a c e t o n e was s e l e c t e d i n l i g h t o f t h e s u c c e s s o f Esher e t a l.

( 1 9 ^ 7 ) i n i s o l a t i n g an iss a u n o g e n ic a c e t o n e

s o l u b l e m a t e r i a l from P . p e s t l e .

A ls o H o r r e c k e r (19**-$) h a d

m e n t i o n e d I t s o c c a s i o n a l u s e f u l n e s s i n e x t r a c t i n g enzymes. I n t h e 1’l r s t e x t r a c t i o n s i t was n o t e d t h a t w a t e r s o l u b l e e x t r a c t s c o n t a i n e d t h e enzyme u r e a s e .

H y p o th e siz in g a p o s s i b l e

r e l a t i o n s h i p i n n a tu re o f th e B ru ce1 la u re a s e to t h a t of th e u r e a s e o f J a c k b e a n m e a l , s i m i l a r c o n d i t i o n s w ere c h o s e n t o t h o s e a p p l i e d by Sumner ( 1 ^ 2 6 ) who u s e d 3° p e r c e n t a c e t o n e , r e f r i g e r a t o r t e m p e r a t u r e s , a n d a pH o f 5* 2 i n c r y s t a l l i z i n g u rease. I t was t h e I n t e n t o f t h i s work t o e x t r a c t a n t i g e n i c ma­ t e r i a l by s o l u t i o n i n t h e e x t r a c t a n t ,

ta k in g advantage o f

s e v e r a l t h e o r e t i c a l f a c t o r s b ro u g h t o u t by p h y s i c a l chem ical s t u d i e s on p r o t e i n s : I.

Iso ele ctric p o in t.

T hat i s o e l e c t r i c p o i n t a f f e c t s

t h e s o l u b i l i t y o f l a r g e m o l e c u l e s i s w e l l known.

In fra c tio n ­

a t i o n , e x t r a c t s o f w a t e r s o l u b l e m a t e r i a l w ere made n e a r t h e n e u t r a l zone ( f l o w s h e e t f r a c t i o n I ) , t h e pH o f a p l a i n a q u e ­ o u s e x t r a c t b e i n g a b o u t 6.9*

E x t r a c t i o n was a l s o a p p r o a c h e d

from t h e a c i d ( f r a c t i o n ¥11) and a 3k a l i n e ( f m e t i o n VI) s i d e s o f t h a t zone.

O t h e r I m p o r t a n t c o n s i d e r a t i o n s were t h e e f f e c t

pH h a s on s t a b i l i t y o f c e r t a i n corapoiands and on enz ym es.

It

was c o n s i d e r e d t h a t a much h i g h e r o r l o w e r pH m i g h t I n h i b i t t h e a c t i v i t y o f a u t o l y t i c enzymes s h o u l d s u c h b e p r e s e n t . The p o s s i b i l i t y

t h a t a g i v e n component m i g h t ,

in I t s e l f ,

more - s t a b l e i n a d i f f e r e n t pH was a l s o c o n s i d e r e d .

be

pH was a

16

f a c t o r r e c o r d e d i n a l l c a s e s b u t f u r t h e r a d j u s t m e n t was n o t c o n s i d e r e d n e c e s s a r y i n t h e c a s e o f f r a c t i o n s l f I I , an d I f , t h e pH o f a b o u t 6 . 9 o f t h e medium I t s e l f b e i n g u s e d .

Frac­

t i o n s ¥ - 1 a n d ¥ - 2 were p r o d u c e d from a c e l l s u s p e n s i o n b u f ­ f e r e d t o pH 5*2. 2.

Io n ic stre n g th .

Low c o n c e n t r a t i o n s o f i o n s h a v e b e e n

d e m o n s tr a te d t o have g r e a t e r s p e c i f i c i t y o f a c t i o n f o r g iv e n p ro tein s.

Combined w i t h t h e p r o p e r pH, s h a r p e r s e p a r a t i o n o f

v a r i o u s p r o t e i n s can be o b t a i n e d .

The I o n i c s t r e n g t h was n o t

v a r i e d i n most o f t h e s e s e p a r a t i o n s b u t o r i g i n a l e x t r a c t o f medium.

fcept a t t h a t o f t h e

I t may be r e c a l l e d t h a t t r y p t o s e

a g a r h a d 0 . 5 gm o f so d iu m c h l o r i d e p e r l i f e r .

The i o n i c

s t r e n g t h b a s e d on m o l a r s o l u t i o n s w ould b e :

r Where C i i s t h e c o n c e n t r a t i o n and

the v alen ce o f

t h e i f t h s p e c i e s o f i o n I n m o le s p e r l i t e r o r t h e io n ic co n cen tratio n .

T h i s i s a b o u t t w i c e t h e mo-

l a l I o n i c s t r e n g t h (]U) a s c a l c u l a t e d by L ew is a n d B a n d a l l ( c i t e d by D a n i e l s 1 9 ^ $ ) •

And s i n c e t h e

s a l t w o uld be . 0 0 5 6 m o l a r :

r

* ( . 0 0 5 6 x 12) - ( . 0 0 ^ 6 x 1 2 ) s .0 1 7 2

o r p/*

.0 1 7 2 * . 0 0 5 6 ~ V-

T h i s w ould b e t h e i o n i c s t r e n g t h i n t h e medium.

H ow ever, t h e

c o n c e n t r a t i on i n a n a q u e o u s e x t r a c t o f g r o w t h from a g a r medium

w o u ld b© c o n s i d e r a b l y l e s s .

I t was n o t m e a s u r e d * b u t s h o u l d

be re p r o d u c a b le f o r t h e g i v e n medium*

O th e r s a l t s were a s -

s u n e d t o h© p r e s e n t i n n e g l i g i b l e am ount s .

I n %h a c a s e o f

s e p a r a t i o n o f a acill re s id u a w ith su b s e q u e n t r e c o n s t it u t i o n i n a n o t h e r s o l v e n t i t w as a s s u m e d t h a t the r e s i d u a l i o n i c

b t r e u g t h from c o l l u l a r d e b r i s m s n e g l i g i b l e a n d i o n i c s t r e n g t h woo os s a d on t h a t of t h e f l u i d u s e d f o r r e c o n s t i t u t i o n * u>

T em perature *

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d s s i g n e d t o m i n i m i s e d e n a i u r a t i o n and w as p r a c t i c e d w i t h a l l fractio n s*

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t e m p e r a t u r e i s l o w e r e d a n d t h i s , c om bined w i t h t h e s p e c i f i c s a l t a f f e c t , no d o u b t h a d some e f f e c t on s o l u b i l i t y . 4*

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t r o a t m a r i i o f f r a c t i o n s XI a n d IV a n d i n 30 p e r c e n t c o n c e n t r a ­ t i o n f o r t r e a t m e n t o f f r a c t i o n s ¥ - 1 and ¥ - £ .

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s t r a i n s c o u l d b© d e t e c t e d b y f a i l u r e t o a g g l u t i n a t e upo n h e a t ­ i n g when w a t e r w as s u b s t i t u t e d f o r s a l i n e i n d i l u t i o n s o f a c r i f l a v i n © w h e r e a s sm ooth c u l t u r e s a g g l u t i n a t e d *

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b a s i s a l l o f t h e c u l t u r e s em p lo y ed a r e i n t e r m e d i a t e s t r a i n s . A l l o f t h e s e s t r a i n s were u se d f o r c o m p a ra tiv e s e r o l o g i ­ cal stu d ies.

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o f i m m u n i s i n g a n t i g e n s and c h a l l e n g e d o s e s .

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w e r e i n o c u l a t e d on t r y p f o s e a g a r s l a n t s ( a p p e n d i x I ) u p o n r e ­ c e i p t a n d k e p t i n the r e f r i g e r a t o r a f t e r 48 h o u r s * g r o w t h a t 37 C .

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a g a r s l a n t s w i t h 48 h o u r s g r o w t h f o r t h e v a r i o u s t e s t s b y 0 . 9 par cent sa lin e or,

l a the case o f in te rm e d ia te s t r a i n t e s t s ,

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f o r u s e o f t h e c e l l s i n a g g l u t i n a t i o n a n d o t h e r t e s t s a n d was b a s e d o n t h e a v e r a g e o f s e v e r a l s e p a r a t e r e a d i n g s made ©a B u r e a u o f A n im a l I n d u s t r y

(BAI) p h e a o l i s e d a n t i g e n *

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'S n r ic h e d t r y p t o s e a g a r ( a p p e n d i x I ) was u s e d i n grow ­

i n g a n t i g e n s f o r p r e p a r a t i o n o f f r a c t i o n s Y - l an d V - 2 .

3eed

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s p r e a d o v e r t h e a g a r s u r f a c e i n B l a k e b a t t l e s w h i c h w ere t h e n i n c u b a t e d f o r 48 t o 72 h o u r s a t 3 ? Gs t h e g r o w t h b e i n g u s e d fo r th e v ario u s e x tra c tio n s .

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f o r a n i m a l i n o c u l a t i o n a n d s e r o l o g i c a l t e s t s war® p r e p a r e d f r o m 48 h o u r g r o w th on a g a r s l a n t s , t h e c e l l s b e i n g w ashed f r e e by 0 . 9 p e r o m it s a l i n e .

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t o 61 p e r c e n t l i g h t t r a n s m i t t a n c e and u s e d w i t h o u t f u r t h e r treatm en t.

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^ w a l k - i n " r e f r i g e r a t o r w as u se d *

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b a t h o f a l c o h o l o r a c e t o n e and d r y i c e was c o n v e n i e n t .

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k e e p i n g one th e r m o m e te r i n t h e i c e b a t h a n d o n e i n th e s p e c i ­ men l a r g e d i f f e r e n c e s i n t e m p e r a t u r e w e re a v o i d e d .

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o f s m a l l , 1 * 0 t o 2 .0 cm, p i e c e s o f d r y i c e m a i n t a i n e d te m p e r ­ a t u r e w i t h i n a d e g r e e o f t h a t w hich w as d e s i r e d .

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o f a b o u t - 6 0 t i o C w ere o b t a i n e d i n a d r y i c e box*

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as

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f e r s t o t h a t a l r e a d y p r e s e n t i n t h e medium. D esignation of f r a c tio n s !

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n n

n n

B n

liv e b irth e a r l y p ro g

PM m

n n few a b s n n abs y ello w abs* mum a b s * abs mbs e n l* mb© few ab s n n

n n n n n n

n s l cong mod cong s l cong pnouao cong

n n ■n B n n

s l cong te s te © s mod cong t e s t e s s l con g t e s t e s cong u t e r u s 2 wks p r e g

m pm

n n n n » n n n

cong n cong n n n n* pneumo

n n n u n n n n

s l cobs t e s t e s e a r l y prmg* cong; t e s t e s n n e a rly preg 3~4 wks p r e g cons, t e s t e s

Keys s l * slig h t abs - a b s c e s s a a r - marked

weSk of ae a th

mod - m oderate p reg - pregnant n - n o m tl

nod - n o d u la r t h - th ic k e n e d cong - c o n g e s te d

pm

kd la PM PM PM m PM pm PM PM la /sh 1.%

50

d u e e d s e r o l o g i c a l a c t i v i t y vmm e v i d e n t th o u g h a n t i b o d i e s w a re pro d u ced i n g u in e a p ig s ?

U reas© a c t i v i t y w as a b s e n t *

T h ree i n o c u l a t i o n s a t 10 d ay i n t e r v a l s o f f r a c t i o n XI-C i n 20 g u i n e a p i g s , 10 o f w h ic h w ore s u b s e q u e n t l y c h a l l e n g e d w i t h 700 B r * a b o r t u s £300 p r o d u c e d v a r i a b l e r e s u l t s b o t h i n p r o d u c t i o n o f a g g l u t i n i n t i t e r s an a i n im m u n is in g c a p a c i t y * F o u r w eek s f o l l o w i n g t h e c h a l l e n g e d o se t h e s t e r i l i t y c o n t r o l a n i m a l s s t i l l m a i n t a i n e d a mean t i t e r o f 1 .5 w h i l e c h a l l e n g e d t e s t a n i m a l s a n d i n f e c t i o n c o n t r o l s showed i n c r e a s e d m ean t i ­ t e r s o f I s SO an d 1 : 5 0 r e s p e c t i v e l y * c o n t r o l s ( t a b l e 6} w as n o t i n f e c t e d

One o f t h e i n f e c t i o n ( a n i m a l 0} a n d I t l a c o n ­

s i d e r e d l i k e l y t h a t b o t h t i t e r s and i n f e c t i o n o f t h e c o n t r o l s w o u ld h a v e b e e n m ore s e v e r e i n t h e a b s e n c e o f t h e s u l f a g u a n i d in e th e s e a n im a ls had been r e c e iv in g * c o n tro ls

Two o f t h e I n f e c t i o n

( a n i m a l s IS and T ) fr o m w h ic h o r g a n i s m s w a rs r e c o v e r e d

showed no e v i d e n c e o f t i t e r e v e r, in s p ite

l a I s £5 d i l u t i o n s o f serum*

How­

o f t h e v e r y low c h a l l e n g e d o s e 5 o f t h e a n i m a l a

g i v e n XI-C a n d c h a l l e n g e d show ed d i s t i n c t r i s e s i n t i t e r s a n d B r u c e l l a w e re r e c o v e r e d from t h e s p l e e n s , w h ic h h a d a mean w e i g h t o f 5 . 9 gm, a n d some o f t h e o t h e r o r g a n s a s shown i n ta b le 10.

G u in ea p i g nu m b er €29 showed B r u c e l l a i n c u l t u r e s

fro m t h e s p l e e n a n d l i v e r th o u g h no t i t e r w as e v i d e n t *

The

a b s e n c e o f o r g a n i s m s o n c u l t u r e co m b in ed w i t h n e g a t i v e t i t e r s i n d i c a t e d fre e d o m fro m i n f e c t i o n i n t h e r e m a i n i n g 4 c h a l l e n g e d an im als*

"These b ad a mean s p l e e n w e i g h t o f 1 . 0 3 c o m p a re d w i t h

1*22 ipa f o r t h e i n f e c t i o n c o n t r o l s u n c h a lle n g e d s t e r i l i t y

c o n tro ls *

{t a b l e 6 ) an d 0 . 9 i n t h e IShe s p l e e n s i n b o th c h a l l e n g e d

Table 1 0 M acroscopic P a th o lo g y and C u ltu ra l R e su lts in Oroup II«C uuiaea R ig s g u in ea s p le e n p ig sex c h a l ­ number le n g e d wt.gms . p a th o lo g y s l t h nod* a a r th nod* s l th nod* n m x t h nod abs* 1*3 s l th nod 416 y es s l nod 41? 1*0 M yes no s l t h nod 0 .8 418 M BO s l t h nod 0 .9 S 419 0 .8 n 420 M no no s l nod 421 M s l th f 422' no 1 .3 no s l t h nod F 423 1 .1 s l th f no 1*7 424 s l th •F no 1 .2 425 s l t h nod 426 M yes 0 .5 F n 42? bo 0 .9 428 M y es n 1*0 s l t h nod* 429 M y es s l th 1*5 430 r %- g M y es — on c u l t u r e . ru es a rk e d a l l e r re a c tio n h ip 411 41£ 413 414 415

yes yes yes m yes

M F M P M

1 .0 1 0 .0 0 .? 0 .6 6*9

f

0

. 6

1

i H

I f f

a

~

r

e

c

o

g

v

. 0

e

i c

r

e

d

o

M acroscopic P a th o lo g y week liv e r h e a r t lu n g k l d n e y r e p x o d u o t i v e of d e a th o rg a n s inm abs few abs few

am a b s n mod oong nm mod cong a n l* tk s l cong abs 11 mod cong mod cong abs n

n a n n

few a b s n n 1 abs n a n n n n a

n

n n *

a*

n

n A

mod cong n s l cong s l cong n n s l cong n s l nod n s l nod n a tr o p h y n a tr o p h y n nod cong a mod co ng B mod cong n s l cong a o rg a n s c u l t u r e d

l l

o

t b

e

r

a n* n n n

mar 6-7 mod a! mar

n n n n n n n n n a n n n n

3 -3 wks o re g s l eon eg mod eong t e s t e s mod cong t e s t e s it s l oong t e s t e s £ -3 wks p r e g liv e b irth 2 -3 wks e a rly preg n 3-4 wks p r e g t e s t e s s l cong r

e

m

m lie.

m

Vu\ V£ 2

m m *»

g

m m

k t *

a

nod cong n

e

Key:

nod - nodular thickened cong - congested

th -

cong t e s t e s

p

n

s l ~ s lig h t Mod - moderate abs - abscess p r o g - pregnant mar - marked n - norm al ru® - numerous am - Mia11

PM1 FM m £ m

1*3

n

and

cong t a s t e s wks p re g ^ cong t a s t e s

g

a

t i v

e

t

e

fo r

s

t b

e r

ktt

s u

o

e

l l a

58

a n d u n c h a l l e n g e d a n i m a l s eh owed soma no d u l a t i o n . F ra c tio n I I I *

A ll a n i m a l s i n o c u l a t e d w ith f r a c t i o n s XXX-A

a n d XIX-C show ed h i g h t i t e r s and c u l t u r a l a n d p a th o lo g ic a l @vi~ dene© o f i n f a c t i o n , showing t h a t B r . a b o r t u s 2308 s u r v iv e ® p r o ­ l o n g e d t r e a t m e n t w ith a c e t o n e a t low tem peratures*

T a b l e s 11

a n d 12 show th e r e s u l t s o b t a i n e d i n t h e E g r o u p s o f a n i m a l s . I t h a d b e e n w r o n g l y assumed th a t B r u c e l l a w o uld n o t su rv iv e a c e t o n e treatm ent an d t h e r e f o r e t h e r e w ere n o c u l t u r e s mad® o f th® c e l l r e s i d u e a f t e r su c h t r e a t m e n t .

T h re e w eeks f o l l o w i n g

I n j e c t i o n o f f r a e t i o n IXX-A, h a l f o f th© 10 -{guinea p i g s r e c e i v ­ i n g I t were g iv e n 1 . 0 m l a m o u n ts o f th© same c h a l l e n g e d o s e t h a t w as g i v e n g r o u p I I - A , o r 8 , 2 0 0 B r . a b o r t u s 2 3 0 8 .

Th© o t h e r

5 an im als w h ic h h a d r e c e i v e d th e f r a c t i o n w e re s a c r i f i c e d a n d c u l t u r a l exam ination show ed B r u c e l l a t o b e p r e s e n t .

The s p l e e n s

o f th e se h a d a mean w e i g h t o f 3 . 1 4 goi an d p a t h o l o g y a s shown i n t a b l e 11 w as t y p i c a l o f B r u c e l l a i n f e c t i o n .

Mean a g g l u t i n i n

tite rs

f o r t h e g r o u p w e r e a b o u t 1*370 a t t h i s t i m e .

Four w eeks

la te r,

f o l l o w i n g c h a l l e n g i n g , th© t i t e r s i n th® 5 r e m a i n i n g

a n i m a l s a v e r a g e d 1 * 25 00 a n d th© s p l e e n s ta v e r a tp d 5 . 1 gm.

The

c h a l l e n g e d o s e was o m i t t e d I n th© 10 g u i n e a p i g s I n o c u l a t e d

w ith I I I - C a n d o n l y th e t i t e r 8 w eeks fo llo w in g I n o c u l a t i o n w i ta k e n .

A l l 10 a n i m a l s h a d a t i t e r o f more t h a n I s 2 0 0 0 ,

t h e mean, s p l e e n w e i g h t w as 5 . 9 5 gm a n d c u ltu r e s d e m o n s t r a t e d B r u c e l l a i n a l l o f t h e a n im a l® . F r a c t i o n IV.

A c h a l l e n g e dos© o f a b o u t 1 0,000 organism s

was g i v e n .10 d a y s a f t e r t h e 3 i n j e c t i o n ® o f f r a c t i o n IT -B a t 9 a n d 7 d a y i n t e r v a l s to h a l f o f th® 20 g u i n e a pig® in o c u la te d

Table 11 M acroscopic P ath ology and C u ltu ra l R esu lts in Group III-*. Guinea r i g s guinea

~

p ig se x ®\m 1 number longed 11 IB

M M

BO

13

^ ^^o'sacsnlIc1 T ,^

n ^ rlT lu iii' '

" 'in m i"'

.. "...................: ~

k i d n c y ^ e p T O d u c t l f e ' w eeF’o:

p a th o lo g y _________

organs

i.i death

abs* n pun abs* n'm

cong* n* mod cong abs*

a b s* m r th nod*

abs*

2 .8

a&p t h nod

m m a b s* n

s a r cong* n so d cong a

s l cong t e s t e s sw o lle n p u s PM f i l l e d ta ste s * cong t e s t e s ^ ' i*3£ .V 1 e a r l y p re g

5.0

m at th nod

m m a b s* ii

cong

ab s*

s l cong t e s t e s * PM

nuj& a b s B mua a b s * n

oong cong

n n

cong t e s t e s cong t a s t e s *

surf aba n ab s* m i ab s * a

mar m u i f abs* ab s e n l t e s t e s * m

yas

1 .6 6.5

m>& t h nod* m&T th nod

M

DO

3.7

14

f

yes

*?f-j

II

no

n*

a b s* a b s*

16 17

M

m

M

yes

1 .1 4 .6

mod t h sod* mar e n l a b s

m m

t h nod* 18

M

yes

9 .9

19

M

yes

1 .7

BO

11

no

4.3

mar t h a b s

nod* mod th* mar t h nod abs*

a b s*

cong cong

n n

s l co ng t e s t e s cong t e s t e s

*$y$$ k%k

* B n i c e l l a r e c o v e re d on c u l t u r e . 411 o t h e r o rg a n s c u l t u r e d and mere n e g a t i v e f o r ~"" lr u 'a e lla ~LefV forepaw s w o lle n . %*arge s u p u r a t l n g sub derm al a b s c e s s e s i p a x i l l a r y f o l d s . Keys

s l - slig h t

th - thickened

mod * m o d e ra te nod - noclular abs - ab scess p reg - p regnant

rnkd • marked n * n o rr;al cong - c o n g e s te d s u r f - su rface

Itebl& 12 M acroscopic P ath ology and C u ltu ra l ii©s u i t s in Group IIJ-C Guinea P ig s guinea P is

mx

nuisber

sp le e n w i.g& s. p a th o lo g y

week k id n e y r e p r o d u e t i v e of organs death

Macroscopic Pathology heart lung liv e r

1 mod cong t^stee^ F M early p ro g * FM

6.4

sl th nod* m x t h sod abs*

n* Bill m m

n n

cong consol u consol* n*

f

12.4

'mar th nod abs*

mbs* sal mum abs*

a

cong

n*

8 -5 **ks p reg *

404

M

S .?

mar th -mad abs*

n

n

n

mod cong t e s t e e PM

405

f

2 .3

mar th mod abs*

e n l mam mbs* nuia abs*

n

sl cong

33

aborted once early preg at

406 40?

M f

8 .5

■mar m d .th abs* mar th nod abs*

n n

mod cong

408

F S M

mum abs* mum mbs enl* num. abs* nua mbs* n urn abs*

n* ©ml*

cong cong* eomg

401 402

U f

1 .1

403

400 410

1 0 .2 6 .0

6 .2 2 .0

mar th mod abs* mar th mod abs* th sal nod*

♦B r u o e l la r e c o v e r e d on c u l t u r e * 2-Left f o r e paw a r t h r i t i c . % bsccss a t s i t e o f in o c u la tio n

n

cong

e l . e n l xi n aborted once n n B*

Hi

PM

fM

early preg at

PM

4 wks preg* e l cong t e s t e s

PM

s l cong testes

PM PM

411 o t h e r o rg a n s c u l t u r e d and n e g a t i v e f o r B r u c e l l a .

Ho a n im a ls in t h i s group c h a l l e n g e d .

Hays s l - slig h t mod - m oderate nod - n o d u la r abs * abscess p reg * preg n an t

t h - th ic k e n e d akd - marked n - norm al cong - c o n g e s te d

ai

an d 4 i n f e c t i o n c o n t r o l s *

F o u r w eeks f o l l o w i n g c h a lle n g e s a l l

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

i n e x c e s s o f 1; EDO an d 3

o f , th® 4 i n f e c t i o n c o n t r o l s show ed t i t e r s - o f a t l e a s t 1;B 000* Hi@ moan w e i g h t o f t h e s p l e e n s o f t h e c h a l l e n g e d a n i m a l s ( t a b l e 13) « s

£*35 up com p ared to a mean w e i g h t o f 4*03 gm

i n t h e i n f a c t i o n , c o n t r o l s u s e d f o r t h i s g ro u p a n d g r o u p s VX-B and 1TXI-B*

Among t h e u n c h a l l e n g e d a n i m a l s t h e mean w e i g h t o f

t h e s p l e e n s w as 1*1 g® •

Some i i o d u l a t i o n w as a p p a r e n t i n § o f

t h e s p l e e n s o f t h e u n c h a l l e n g e d g ro u p b u t 4 a p p e a r e d q u i t e n o rm a l#

R e s u l t s on i n f e c t i o n c o n t r o l s a r e i n t a b l e 2 2 *

F ra c tio n ¥ -1 . ^

F r a c t i o n V -l-B a p p a r e n t l y p r o t e c t e d 3 o f

g u i s e s p i g s fro m i n f e c t i o n w h i l e f r a c t i o n V- 1 - 0 may h a v e

p r o t e c t e d 7 o f 10 a n i m a l s *

I n t h e f o r m e r c a s e , a l l o f 5 in*'

f a c t i o n c o n t r o l s w e re i n f e c t e d w h i l e i n th® l a t t e r c a s e one o f 5 I n f e c t i o n c o n t r o l s w as n o t i n f e c t e d *

Bone o f SO u n c h a l ­

l e n g e d a n i m a l s , h a l f g iv e n e a c h f r a c t i o n b u t n o t c h a l l e n g e d , w ere i n f e c t e d * A s i n g l e i n o c u l a t i o n o f f r a c t i o n Y - l - B w as g i v e n i n - t r a p e r i t o n e a l l y a n d 4 w e e k s l a t e r 7 ,5 0 0 o r g a n i s m s w e r e g i v e n by t h e same ro u t® a s a c h a l l e n g e dose#

Fro® 3 a n i m a l s , n u m b e rs

3 5 2 , 3 5 4 , a n d 357 a s shown- i n t a b l e 1 4 , no o r g a n i s m s w e re r e ­ c o v e re d *

A bsence o f a g g l u t i n a t i n g a n t i b o d i e s in t h e i r s e r a

su p p o rte d th e r e s u l t * f r a c t i o n ¥ - 1 - 0 w as .given 20 g u i n e a pig® i n t h r e e I n t r a p e r i t o n e a 1 i n j e c t i o n s s p a c e d 10 d a y s a p a r t ,

1 ml

a c h a lle n g e

d o s e o f 1 4 , 0 0 0 o r g a n i s m s w a s g i v e n h a l f o f th© a n i m a l s 1 0 d a y s a f t e r th© l a s t i n j e c t i o n o f f r a c t i o n *

O rg a n is m s w e re r e c o v e r e d

Table 13 Macros oop ic Pathology- and C u ltu ra l He s u i t a in Group X¥~B Guinea P ig s © unaa sex e h a l s p le e n P ig number leisged w t ^ a s * p s t i a l o g y f Fee 1 .0 t h nod ab s* 861 502 6 ,1 1! Fes t h nod mbs* 503 504

I M

fe n ye b

1 ,3 1 .6

505 505 50?

II F M

yes yes yes

4 .0 1 .9 2 .0

508 509 510 511 512 515 514 515 51S 51? 518 519 520

s l t h nod* mod th nod*

a a r th nod* mod t h nod* sod th nod abs* yes th aod abs* F 1 .8 M yem 1 .5 s l t h nod* ym decomposed U M no s l nod 1 .2 M no s l th nod 1 .0 M no n 1 .3 F n 0 .6 no no f s l nod 1 .0 f no s l nod 1 .0 M no n 1 .0 no F 0 .7 n r s l nod no 1 .1 M no 0 .9 n i #*»treat ttet .4 ^Wt ¥I-**** ru a 8tntXi »laa '''re'ooTered on c u l t u r e , a D e a W c s i i sad b j h em o rrhage.

y« tviOftOpi liv e r h e a r t lu n g

mmk k id n e y r e p r o d u c t i v e o f organs d e a th n n s l cong n 3 wka preg* PM num sm abs* a c o n s o l adh* a b s e n l * mod cong PM te ste s * n n m m aba 2*3 m s p r e g B! n tmm a b s enl mod cong* t e s t e s FM n consol areas enl s l cong mod cong t e s t e s PM num abs B enl ab s s l cong n abs* 5 -7 wks p r e g BM ab s a E n mod cong t e s t e s PM abs* ab s n o t done n n a n n n n n n n A ll o t h e r

n n

s o d cong so d oo ag

n n

2 wks p r e g s l cong t e s t e s

ill PM

n n s l -cong a PM ssOd cong n n s l co ng t a s t e s PM n s l cong n n pM a XI 5*0 wks p r e g PM n n n a 5*6 wks p r e g PM n n n n PM n n s i c o n g t e s t e s I'M a n s l cong a preg f u l l t e r n Bf s l cong n B 3 wks p r e g PM 13 a n a 3a o rg a n s e n l t u r i T 'a i i d n e g-«#ait 4«t i4 v'STe f o r dP r' Su ,»es n o l d a b s ’" a n

ii

ft

n n n

ft

ii a a ft a a n e ii n ft ft a a s i oong n n n n s i eo n s few a b s s c a r tin : r* abs ft a a ft ft a ii. s a n a n a 1 sm abs ft n aha a s i cong few abs* ii ft s i cong n cong u ii AL Xl7,u'o'€HS?~*^or‘i,:an J'aoiT -tir n

n u n ft

D n i* n a n n n a V*.

ti

’

Inr'm mi i inmimm i» III IimiIiinrirrfr1- 1IIn inrniri rrruin nnr- m iniwi

oF re]p ro d u c tiv e we re o rg a n s dea th 2 m s n r eg e a rly p res I'Ifi PM e a rly p re s e a r l y p ra g s i cong t e s t a e PM PM e a rly preg » •-f 0 if*4' 0 r1**

I H

2? © H H O; 0 ■43 © ti ©: ©5 9 0 S4' 43 © ©J »:

ri m

H

£3 Q © go 0 0 o X2

m &

H O* Q S lS flC S O fJffl® H

t

Hi

gj £3 £t

. 1 HI 4 3 © £ 3 0 * 0

ft

i % D

©

) 3 « «© c o

a *© a

g

© 1

0

m m «o

d

0 0 44

©

89 43

89. 43

SO *0

c

£

*&

1 H 5 ri ■0 © 4 3 «Si ■>» © Hi

©&

WH © •

«3«O^fitf)O^

§

1

«30

*» P* ©

§0 ft tsD 8

§ N3 99 as

d ©

S'ii N O

H

© .E j m p* o ►

o >g © *HJg 22

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m

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r*4 9£

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m

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*# £0 lj@ & S3 S3 f l Q © © o © si ©©©©g *©*©*©'© •© es H i ? * * d

8

j2 © m •H ©I © W

mm

2+1:50 1+1:25 1+1:25 + 1 :2 5 2+lslOO 2+1:50 * + 1:50 1+1:100 1+1:25 3+1:100

H i g h e s t IT S ?

-* a ^

mm

mm

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T a b le 44 A g g l u t i n i n R e sp o n se o f I n f e c t i o n C o n t r o l A n im a ls G ro u p s I -C an d I I - C two w e e k s g u i n e a in f-so t e d a f t e r c h a lle n g e c o m p lete p a r t i a l - piiS.. ......... • I yes « yes F G no H yes I yes nie s u b s t a n c e s w e r e p r e s e n t , a s shown by

p r e c i p i t i n t e s t , a n d a n t i b o d y p r o d u c t i o n , t h i s m a te ria l w as

f a b le 4*6 P r o te c tio n o f Guinea P ig* by Various B r u c e lla E x tr a c ts

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Guinea pigs used Guinea pig® used for each batch for each fraction to tal no. ChallengedAInfected^ Total no. Challenged Infected Uninfected ................... - .................... 3G*t-3°6. B oyd , !>. M. and Casman# E. P / ' ^ I n h i b i t i o n o f a s t r a i n o f B r . a b o r t u s b y medium f i l t e r e d t h r o u g h c o t t o n ( u n p u b 1. wor k ) .

132

Brandy E. and E & s a ll# J . T. 19^7 ^he chem istry o f th e p ro ­ t e i n s a n d amino a c i d s . Ann. Rev. Biochem . 1 6 22^272. Braun# W. an d Hauge, S. 19**& S t u d i e s on i n f e c t i o n w i t h B r u cella. a b o r tu s v a r i a n t s . J . Immunol. 6o# M*3-**57* Breed# H. S. , M u r r a y , E. G. D. and H i t c h e n s # A. P . 19**£> B e r g e y 1s m a n u al o f d e t e r m i n a t i v e b a c t e r i o l o g y . W i l l i a m s s.nd W i l k i n s Co. B altim ore, B u c k , J . M. 1930 S t u d i e s o f v a c c i n a t i o n d u r i n g c & l f h o o d t o p r e v e n t bovine i n f e c t i o u s a b o r t i o n . J . A g r. R e s e a r c h **1, 6 6 7 - 6B9'

Cohn# E. J . , S t r o n g , L. E . , H u g h e s , W. L. # J r . , M u l f o r d , D. J . , Ashworth# , MeXin, M ., a n d T a y l o r # H. L. 1 9 ^ 6 The f r a c t i o n a t i o n of th e plasm a n r o t e i n s . J . Am. Chem. S o c . , 6a, *5 9 - ^ 7 5 . J

.

O o r d i e r , G. # 193^ E f f e t s d e e i n o c u l a t i o n s d e c a r t e l n e s s o u c h e s de B r u c e l l a a v e c ou s a n s e x c i p i e n t i r r e s o r b a b l e s c h e z d i v e rse s especes anim ales. Rev. Med. V e t . , 9 0 # 4 ^ 1 - 5 0 3 . Got t o n , C o r n e l i a M.

19^3

F h . D. T h e s i s , U n i v e r s i t y o f M a r y l a n d .

_______

19^9

P e r s o n a l comm u n i c a t i o n u n p u b l i s h e d

d aliu Craw ford,

A. B.

19**$

P e r s o n a l com m unication.

D&mbovlceanu# A ., B e r b e r , C . , Pop, A . , and M a r i n o u , I . 197$ A n t i g e n e comp l e t d e s B r u c e l l a . C h a r a c t e r * * c h l m i q u e s . Corapt. r e n d . s o c . B i o l . , 1P77 77*>*77$. D a v o l i , R. 1939 O s s e r v a z i o n i e r i c h e r c h e s u l l e s o s t a n y a s p e c i f i c h e s o l u b i l l d e l gen. B ru c e lla .. B o ll. I n s t . S ieroterap . M i l a n e s e , 1 $ , 97*6*'. ( T T aT lan w i t h E n g l i s h su ® a r y ). D a n i e l s , F. 194$ O u t l i n e s o f p h y s i c a l c h e m i s t r y . and S o n s , Mew York.

J o h n W iley

Deem# A. W. and C r o s s , F. 19**5 A b o r t i o n f o l l o w i n g t h e v a c c i n a t i o n o f p r e g n a n t c o w s w i t h s t r a i n 1 9 . J . .Am. V et. Med. A ssn . 1 0 6 , 213-21**. D ubois, 0. I93$& I n n o c u l t e # che z la, b r e b i s , l a c h e v r e e t l a v a c h e , du v a c c i n p r e p a r e a v e c d e s g e r m e s v i v a n t s a v i r e ­ l e n t s , de fcr. a b o r t u s - s u i s , en e m u l s i o n d a n s un e x c i p i e n t i r r e s o r b a b l e . ~~WuX± . Acad. V e t . F r . # 1 1 , 3 $ 3 - 4 o 4 . 1 9 3 ^"D T r a i t e m e n t de l a b r u e e l l o s e a n l m a l e p a r le s g e rm e s v i v a n t s , a v i r u l e n t s , de B r . a b o r t u s - a u l 8 e n e m u l s i o n d a n s un e x c i p i e n t i r r e & o r b a b l e . ~BuTT. Acad. V e t. F r . , 11, **2-51. *

133

D ubois# 0 . a n d S o l l i a r # F. 1938 Re s u i t e , t s a c q u i s ,u p r e s h u i t an® d e p r a c t i q u e d e l a v a c c i n a t i o n p r e v e n t i t i v e c o n t r a l a f le v r e ondu lants. A rc h . I n t e r n a t . F r u e e l l o s e s , jL, 7 2 1 - 7 3 4 . E i c h h o r n # A . , F i n a l e , C. I t . # and Murdock# F. M. 19^0 The b e h a v i o r o f Br, a b o r t u s v a c c in e i n v a r i o u s e x c i p i e n t s . Jm. J . V e t . R e s . , T, d'-l?'. E v a n s , A. C. 191$ F u r t h e r r t u d i e s on B a cte r i u m a b o r t u s and re la ted bac t e r i a . I I . A compa r i e o n o f Ba c t e r i um'TiIIo r t u s v l t h Ba c t e r i \m b r o n eh 1 s e p t i c n s and a / i t h r g R T n Tim w hich c a u s e s K&T£a"~fever. I n f e c t i o u s D i s e a s e s # 2 2, 580-597. 19 FI S e r o l o g i c a l C l a s s i f i c a t i o n o f B r u c e l l a © e l i t e n s i s from v a r i o u s s o u r c e s . P ub. H e a l t h R e p t s . im g = W 6 J T E y r e , J . T. H. 190$ V a c c i n a t i o n o f man f o r M e d i t e r r a n e a n fever, L a n c e t , ^1, Fabyan# J£. 1912 A c o n t r i b u t i o n t o t h e p a t h o g e n e s i s o f ? r u e e l l a ab o rtu s. J . Med. He s . 26, M4l~4$9. F e u s i e r # M. L. and Meyer# K. F. 1920 P r i n c i p l e s i n t h e s e r o ­ l o g i c a l g r o u p i n g o f B r u c e l l a a f c o r t u s and B r u c e 1 1 a m e l l t e n sis. C o r r e l a t i o n h e e en a b s o r p t i o n a n d a g g l u T i nafTon te sts. S t u d i e s on t h e g e n u s B r u c e l l a . Kov. Gen. I I . 1. I n f . B i s . 2^# l $ 5 - 2 0 6 . F o s t e r , J* Eg 19**-$ Some s t u d i e s on im m unity i n B r u c e l l o s i s . M a s t e r 1s T h e s i s , U n i v e r s i t y o f M a r y l a n d . F r a n c i s , E. 1911 A g g l u t i n i n a b s o r p t i o n i n u n & u la n t f e v e r (B ru cello sis). Pub. H e a lth R e p ts. 46, ? 4 l6 -2 4 3 7 . G ilm a n , H. L. 1944 H n d u l n n t f e v e r c a u s e d b y 8 r . a b o r t u s g t r a i n 19. Th® Corne 1 1 V e t e r i n a r i a n . T 53C i l t n e r # -V. # H u d d le so n, I . F. , CIs. r b , L. T. and S c h l i n g i a a n , A. S. 1929 R e s u l t s from t h e u s e o f H uddle s o n ’ s v a c c i n e f o r B«ngf s d i s e a s e . J . Am. V e t . Bed. A s s o c . , 7 4 , $ $ 5 -8 9 1 . G w a t k l n # R. 1931 The e f f e c t o f k i l l e d c u l t u r e s and f i l t r a t e s o f E r . a. bo r t u s i n t h e p r e v e n t i o n o f I n f e c t i o n i n g u i n e a p i g s . ' J T T n T T D i e . ^ 8 , 381-4-03. —_____ , guinea immune —

1932 The p r e v e n t i o n o f E r . a b o r t u s i n f e c t i o n i n p ig s. The e f f e c t o f convaTe s c e n t an d o f h y p e r ­ serum . J, I n f . D is. , 5 0 * 111-11$.

_ 1935 A s t u d y o f v a r i o u s f r a c t i o n s o f E r . CaST^T. R e s e a r c h # 1 2 , l i p - 155*

ab o rtu s.

H aga n, W. A. 1922a t o B. a b o r t u s .

S t u d i e s on t h e d i s e a s e o f g u i n e a p i g s J . Exp, Med, , 3 6 « 697~7(!9*

due

_ _______ 1922b The v a l u e o f h e a t - k i l l e d c u l t u r e s f o r t h e prev erT F io n o f t h e B a c i l l u s a b o r t u s i n o c u l a t i o n d i s e a s e of guinea p ig , J . Exp. M e d ., ■3 5 , 7 1 1 - 7 2 5 . H o l t h , K. 1911 U n t e r e u c h e n u b e r d i e B i o l o g ! e d e s A b o r t u s b a s i l l u s und d i e I m m i m i t a t e v e r h a l t n i s s e d e s I n f e k t i o s e n A bortus d e r H inder. Z eitsch r. fu r In rek tio n sk . P a r a s it. K ra n k h . H y g ie n e d e r K a u s t i e r e . 10, 207-277* _______ 1933 l a m u n l za.%1 on e x p e r i m e n t s and v a c c i n e t r e a t ­ m ent i n i n f e c t i o u s a b o r t i o n . S a e r t r y c k 3 > a n d . V e t. , 23, 5 5 7 - &6 2. ( E n g l i s h a uam ar y ) . H o r r e c k e r , B. L.

19^-S

P e r s o n a l C o m m u n ic a tio n .

Howe, 0. , M i l l e r , E. S . , K e l l y , E. H. , B r o o k w a l t e r , H. L, a n d ■ E l l i n g s t o n , ii. V. 19^*7 A c u te b r u c e l l o s i s among l a b o r a ­ to ry w orkers. Hew Eng la n d J . Med. 2 3 6 , 7 ^ 1 -7 ^ 7 « K o y e r , 3 . H. press.

19^9

The p h y s i o l o g y o f B r u c e l l a o r g a n i s m s .

In

H uddle s o n , I . F. 1922 The c o m p a r a t i v e p a t h o g e n i c i t y o f s e v e r ­ a l s t r a i n s o f E r . a b o r t u s . T e c h . B u l l . Wo. 55# Mich. E xpt. S ta . , 193^ D i f f e r e n t i a t i o n o f t h e s p e c i e s o f t h e u o e I I a . Am. J . P ub. H e a l t h , J j l , U>91 - ^ 9 8 . _____ 19^0 The i i b s u n i t i n g ; v a l u e o f c h e m i c a l f r a c ­ t i o n s b?"~Bruc e l l a c e l l s a g a i n s t b r u c e l l o s i s and a g a i n s t th e to x ic '^ e fT e e ts of the en d o an tig en . Hep. P r o c . I n t e r n a l . Cong. M i c r o b i o l . , 6 0 7 . New Y o r k , 1 9 ^ 0 . 19^2a •

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I nunu n i t y i n b r u c e l l o s i s .

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F /"rri^p rs7 19^ 3& B r u c e l l o s i s i n man a n d a n i m a l s . “ - SFrk7""^HF'7£Jomiaonwea 1 th Fund.

New

1 9 ^ 3^ 2he I m m u n i z a t i o n o f g u i n e a p i g s a g a l h s t _ d r u e e l l o s i s . T ec h . B u l l . Mo. 182, M ic h . Agr. E x p t . S t a . 182#

_ _ _ _ _ _ _ _ _ 1 9l4'7 The i m m u n i s a t i o n o f g u i n e a p i g s w i t h l u c o i d oha i e & o f B r u c e l l a . Am. J o u r . V e t . R e s . , 8 , 37^-379K a b a t i E. A. snd M *yer, M. K. 19^8 E x p e r i m e n t a l Imraunochem istry. Ch&s. C. T h o s . , S p r i n g f i e l d .

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l o c h , F* C* and jNnnhe, M. 19^3 P r a c t i c a l Methods I n B i o chem istry* The L i l l i a n s and W i l k i n s C o , , B a l t i m o r e * K o l m e r , J. A ., B o n d i , A. and Rule, A. A. 1939 S t u d i e s In a c t i v e i m m u n is a t io n a g a i n s t unci u l ant fe v e r * J* Immunol* , 3 1 , 4-89-506. Lamraana, 0 . and G l a s s ra&n, H. N. 19^7 The I s o l a t i o n o f ty p e B b o t u lin u m t o x i n . J1 E « c t. 5 4 , 575~52>4. L a r s e n , C. L. 191-9 Im munlration o f t'uinea p i g s w ith a. s o l u b l e a n t i g e n o b t a i n e d from Era e e l l a a b o r t u s . J . Immunol. 6 3 , 4 7 1- 47 g . ' “ “ L e v i n e , H. B* an d F l i e o n , J . B. 19^9 The i & e n t i f t e n t i o n o f F r u e e l l a a b o r t u s s t r a i n 19 by d y b & e t e r l o s t s s i s . J. I n f , L ive,

I . , Ep e r i l me, FA S . , and Dtutb*- , I-.* L. 194-5 S t u d i e s on t h e Im m unisation of r u l r e a r-i^ % a r n l n e t B r u c e l l a i n f e c t i o n . J . I n f . "Die., 21» —

L ive, I. 1949 E f f e c t o f a d j u v a n t s upon t h e im m u n isin g q u a l i t y o f e t h e r - k i l l e d 1 r u c e l l a a b o r t u e . Am. J . V e t . R es. 1 0 , 347-330. “ “ ~~ “ II an t h e ! , 0 . A. 1 S'4b R ese a rc h on s w in e b r u e e l l o e i e by the Bureau o f Animal I n d u s t r y ( 1 9 4 1 - 4 7 ) Am. J* V e t . He#. 9 , 40-45. H & rtin , A. J. P. 21- OS.

1947

The p r i n c i p l e s o f ch ro ma to g r a p h y , 6, ~ ~

McPwen, A. £• 1937 E x p e r i m e n t s on c o n t a g i o u s a b o r t i o n I I . F i e l d e x p e r i m e n t s w i t h a. v a c c i n e p r e p a r e d from s t r a i n 4-5, V e t . R ecord , 4-9, 15S5-1596* MeEwan, A. D. and R o b e r t s , R. 5. 1 9 36 B o v i n # c o n t a g i o u s ab o rtio n . 'The us# o f g u i n e a p i g s i n i m m u n i s a t i o n s t u d i e s . J . Comp. P a t h . T h e r a p . , 4 $ , 9 7 - 1 1 7 . K cFs&yean, J . and S to c k m a n , S. 1909 Report o f th e D ep a rt­ m e n t s ! C o m m itt e e o f t h e B o a rd o f A g r i c u l t u r e and F i s h e r i e s t o I n q u i r e i n t o ©pi zoo t i c a b o r t i o n . P a r t s I and I I ep i­ zo o tic a b o rtio n in c a t t l e . L o n d o n , Kyre and S p o t t l s w o o d e . M i l e s , A. A. 1939 The a n t i g e n i c s u r f a c e o f sm o o th B r u c e l l a a b o r t u s a n d a e l i t c n s i s . ' " B r i t . J . Exs* L a t h . 2