University of Nigeria · certificate ebele uchenna umeh (nge otubelu) , a postgraduate student in the department of microbiology, universlty of iv iglhia, nsukka , nigeria , has satisfactorily

University of Nigeria Research Publications

UMEH, Ebele Uchenna

Aut

hor

1982/1533

Title

An Intracellular Toxic Factor Of Vibrio parahaemolyticus Associated With Its Pathogenicity

Facu

lty

BIOLOGICAL SCIENCES

Dep

artm

ent

Microbiology

Dat

e

MAY, 1990

Sign

atur

e

AN INTIIACYLJJJLAII 'YOXlC B ' A C ~ ~ t ' m o l y t i c u s -

ASSOCIATED WIT11 I T S PATHOGENlClTY

xUdLE UCHUNNA UMLH (nee OTUBELU) B.Sc. (NIG.), M.Sc. (NIG.)

PG/Ph .1>/82/ 1 533

A THESIS SUBMITTEU TO THE DEPARTMENT OF MICROBI~LOGY

UNIVERSITY Oh' NIGERIA, NSUKKA, I N FULE'TLMENT OF

THE REQUIRLMENTS FOR THE DEGRXE OF DOCTOR

OF PHILOSUPHY (Ph.D) I N MEDICAL

MICROBIOLOGY.

SUPERVISOR: PROFESSOR A . N . U . NJOKU-OBI 1'h.D. (Cali] FMC P a t h ; FAS. ( N i g . ) ; FRC P a t h . ( L o n d . : FlSCD ( D e l h i ) ; FWACP Dip . Adv. S e r o l . alter R e e d ) . PROFESSOH O F MEC ROBIOLOGY , COLLEGE OF MLDIClNE, UNIVERSITY OF NIGLRlA, ENUGU CAMPUS, NIGLillA.

HEAD OF UEPA'RTMUNT : DR. (MRS.) J.I. OKAFbR Ph.D. SENIOR LECTURER, DEPARTMENT OF I'JLIC H0H.l (jLOGY, UNIVERSITY OF NlGXR LA, NSUKKA , NIGERIA.

MAY, 1 9 9 0

CERTIFICATE

EBELE UCHENNA UMEH, a P o s t g r a d u a t e s t u d e n t i n t h e

Department o f M i c r o b i o l o a s and w i t h t h e Reg. NO.

PG/Ph.D/82/1533 h a s s a t i s f a c t o r i l y completed t h e

r e q u i r e m e n t s f o r c o u r s e and r e s e a r c h work f o r t h e

degree o f DOCTOR OF PHILOSOPHY (Ph.D) i n

MEDICAL MICROBIOLOGY. The work embodied i n t h i s

t h e s i s i s o r i g i n a l and h a s n o t been s u b m i t t e d i n

p a r t o r f u l l f o r any o t h e r diploma o r degree o f

t h i s o r any o t h e r u n i v e r s i t y .

D r . (Mrs.) J.I. Okafor Prof . (Dr . ) A.N.U. Njoku-Obi

Head o f ' Department S u p e r v i s o r

CERTIFICATE

EBELE UCHENNA UMEH ( n g e OTUBELU) , A POSTGRADUATE

STUDENT I N THE DEPARTMENT OF MICROBIOLOGY, UNIVERSlTY OF

IV IGLHIA, N S U K K A , NIGERIA , HAS SATISFACTORILY COMPLETED THE

REQUIREMENTS FOR RESEARCH WORK FOR TH6 DEGIUE OF DOCTOR OF

PHILOSOPHY ( ~ h .D) I N MEDICAL MICROBIOLOGY. THE WORK

EMBODIED I N THIS THESIS IS ORIGINAL A N U HAS NOT BEXN

SUBMITTED I N PART OR FULL FOR ANY OTHER DIPLOMA OR DEGREE

OF' THIS OR OTHER UNIVERSITY.

Ph.D. ( c a l i f > FMC P a t h . (Lond .

Adv. Se ro l .

HEAD, DEPARTMENT OF MED LCAL MICROBIOLOGX COLLEGE OF MEDICINE, UNIVERSITY OF NIGEFSA, ENUGU CAMPUS, NIGERIA .

D e d i c a t e d t o my b e l o v e d h u s b a n d , CIIIEDU,

a n d c h i l d r e n , ADAEZE a n d TOBECHUKWU.

ACKNOWLEDGEMENT

I wish t u e x p r e s s my profound g r a t i t u d e t o P r o f . A.N.U.

Njoku-Obi f o r h i s i n v a l u a b l e a d v i c e and s u g g e s t i o n s ; and a l s o

t o a h o s t of o t h e r s i n c l u d i n g D r . O.U. Osoagbaka o f t h e

Dept. o f Microbiology, U N N ; D r . A . C . Aneruwa o f t h e Dept. o f L

Microbiology, U.N.N. ; M r ' . Nwegbu o f t h e Dept. o f Animal

S c i e n c e ; D r . S. Anika a n d , D r . Ezeasor b o t h o f F a c u l t y o f

V e t e r i n a r y Medicine; f o r t h e v a r i o u s ways i n which t h e y

have c o n t r i b u t e d t o t h e s u c c e s s f u l comple t ion o f t h i s s t u d y .

LIST FIGURES.. ............................ LIST OF TABLgS ............................ ABSTKACT. . . , . ............................. CHAPTER 1 : 1NTHODUCTION. . . . . . . . . . . . . . . . . CHAPTER 2 : LIg.L'ERATURE REVIEW

V i b r i o ~ar .akiaemolyt icus

H i s t o r y ............................... Occurrence ........................... Epidemiology ......................... C l i n i c a l s i g n i f i c a n c e ................ V i r u l e n c e f a c t o r s . . . . . . . . . . . . . . . . . . . .

TOXINS OF V i b r i o p a r a h a e m o l y t i c u s - -- - - -

Thermostable d i r e c t haemolys ins ...... Thermolab i l e haemolys ins . . ...........

8 8 E n t e r o t o x i n . . . . . . . . . . . . . . . . . . . . . ..... I n t r a c e l l u l - a r haemolys ins ............ Endotoxin. . . . . . . . . . . . . . . . ............ Toxins from o t h e r Vibr ionaceae . . . . . . .

IDEN'P~FICAT~UN CHARACTERISTICS OF 1. p a r a h a e m o l ~ t i c u s . ................

CHAPTER 3: MATElUALS A N D MET1iOL)S

y. p a r a h a e m o l y t i c u s ISOUTE

I s o l a t i o n . . .......................... I d e n t i f i e a t i o n . . . . . . . . . . . . . . . . . . . ..... E v a l u a t i o n o f l e t h a l a c t j v i t y . . . . . . . . Dete rmina t ion o f f i f t y p e r c e n t l e t h a l d o s e (LD .....................

50 E f f e c t o f I r o n on LD ................

50 Adherence a s s a y s . . . . . . . . . . . . . . . . . . . . .

CONTENTS ( c o n t i n u e d )

PAGE

I n v i v o a d h e r e n c e -7 ................. 60 I n v i t r o a d h e r e n c e ................ - 61

INTRACELLULAR TOXIC FACTOR H a r v e s t i n g o f b a c t e r i a l c e l l s ........ 62 D i s r u p t i o n o f b a c t e r i a l c e l l s ........ 62

....... Removal of i n s o l u b l e p r t i c l e s 63 F r a c t i o n a l p r e c i p i t a t i o n wi th Ammonium s u l p h a t e .................... 6 3 Sephadex G-150 g e l f i l t r a t i o n ........ 64

B i o l o a i c a l C h a r a c t e r i s a t i o n L e t h a l a c t i v i t y

I n o c u l a t i o n o f a n i m a l s ............ 65 I n o c u l a t i o n o f c h i c k embryo ....... 66 Necropsy .......................... 67 hl i s to logy o f v i t a l o r g a n a ......... 67

P y r o g e n i c i t y t e s t .................... I 67 Haemolytic a c t i v i t y t e s t .............. 68 C y t o t o x i c i t y t e s t .................... 69

H a r v e s t i n g o f macro phages ......... 69 E f f e c t o f s u b t o x i c d o s e s o f t o x i n on macrophage f u n c t i o n ............ 70

E n t e r o t o x i g e n i c i t y t e s t s Rabbi t i l e a l l o o p t e s t ............ 7 3 S u c k l i n g mouse t e s t ............... 7 3 Sk in r e a c t i o n tes t ................ 74

Immunogenici t y t e s t s Antiserum p r o d u c t i o n .............. 74 Tube a g g l u t i n a h i o n ................ 76 P a s s i v e haemagglu t ina t ion .......... 76 Immunodiffusion t e s t s ............. 77

Immunoelec t r o p h o r e s i s ............. 78

CONTENTS ( c o n t i n u e d )

Antiserum b a c t e r i c i d a l a c t i v i t y .... .............. Mouse p r o t e c t i o n t e s t

.... Antiserum n e u t r a l i s a t i o n ....... Physicoc hemical Charac t e r i s a t i o n

................ UV-absorption spect rum ................. P r o t e i n d e t e r m i n a t i o n

............ Carbohydra te d e t e r m i n a t i o n

................... L i p i d d e t e r m i n a t i o n Po lyac ry lamide g e l e l e c t r o m o r e s i s ....

...... Molecu la r weight d e t e r m i n a t i o n .. ............ Temperature s t a b i l i t y t e s t ............ Temperature a c t i v i t y r ange

E f f e c t o f pH .......................... ......... E f f e c t o f p r o t e o l y t i c enzymes

..... E f f e c t o f m e t a l i o n s ............. CHAPTER 4: RXSULTS . .

............................ I s o l a t i o n ...................... L e t h a l . . a c t i v i t y

LDjOa ................................. I n v i v o adherence .................... -- I n v i t r o adherence .................... -

TOXIC FACTOR

T o x i c i t y o f d i s r u p t e d c e l l s u p e r n a t e s T o x i c i t y o f Ammonium s u l p h a t e f r a c t i o n s ............................ B i o l o g i c a l C h a r a o t e r s

........................ L e t h a l a c t i v i t y . H i ~ t o l o g y o f v i t a l o r g a n s ............

................... Pyrogenic a c t i v i t y

CONTENTS (continued)

.................. Haemolytic a c t i v i t y

C y t o t o x i c a c t i v i t y .................. E f f e c t o f t o x i c f a c t o r on macrophage f u n c t i o n s ............................ E n t e r o t o x i g e n i c a c t i v i t y

........ Rabbi t i l e a l l o o p r e a c t i o n

S u c k l i n g mouse r e a c t i o n ........... ............ S k i n t o x i c i t y r e a c t i o n

Immunogenic a c t i v i t y

.................. Antiserum t i t r e s ............. Immunoelec t rophores is

Immunodiffueion ................... ... Antiserum b a c t e r i c i d a l a c t i v i t y

.................. Mouse p r o t e c t i o n I n v i t r o n e u t r a l i s a t i o n by . a n t i s e r u m .........................

Phys icochemica l C h a r a c t e r s . ............... UV-absorption spect rum

P r o t e i n d e t e r m i n a t i o n ................ ........... Carbahydra te d e t e r m i n a t i o n

L i p i d d e t e r m i n a t i o n .................. ... Polyacry lamide g e l e l e c t r o p h o r e s i s ...................... Molecu la r weight

Temperature a c t i v i t y r a n g e ........... ................ Temperature s t a b i l i t y

pH a c t i v i t y r ange .................... ........ E f f e c t o f p r o t e o l y t i c enzymes

................. E f f e c t o f m e t a l i'ons

CHAPTER 5 . : DISCUSSION

ACKNOWLEDGEMENT

APPENDICES REFERENCES

Flow c h a r t f o r t h e f r a c k i o n a t i o n and .... p u r i f i c a t i o n o f t o x i c f r a c t i o n . .

L i v e r o f mice i n j e c t e d i n t r a v e n o u s l y w i t h t o x i c f a c t o r . . ................ Kidney of mice i n j e c t e d w i t h t o x i c f a c t o r . .............................. Myocardium o f mouse i n j e c t e d w i t h t o x i c factor................... .....

I n t e s t i n e of mouse f e d o r a l l y w i t h t o x i c f a c t o r . . . . . . . . .................

Spleen o f mouse i n j e c t e d w i t h t o x i c f a c t o r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E f f e c t o f t o x i c f a c t o r on v i a b i l i t y o f mouse p e r i t o n e a l macrophages.. ... Cyto tox ic andhaemolyt ic a c t i v i t y cu rves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E f f e c t of s u b l e t h a l d o s e s 6: t o x i c f a c t o r on macrophage phagocy t i c ............................ c a p a c i t y

Immunoe lec t rophores i s ............... Imyunodif fus ion ..................... Absorpt ion spect rum o f t o x i c f a c t o r .

Po lyac ry lamide g e l e l e c t r o p h o r e s i s . . Curve o f s t a n d a r d marker p r o t e i n s used t o de te rmine t h e m o l e c u l a r weight o f t o x i c factor... . . . . . . . . . . . . . . . . . .; . .

Curve of s t a n d a r d p r o t e i n (Bovine serum albumin) used t o de te rmine p r o t e i n concentration...............

ii.

E x t r a c e l l u l a r p r o t e i n p r o f i l e o f x. parahaernolyt icus . . . . . ............ Some b iochemica l t e s t s f o r y. pa rahae- m o l ~ t i c u s . ...........................

... Schedule f o r immunising r a b b i t s . .

Molecu la r w e i g h t s and e l u t i o n volume^ o f s t a n d a r d p r o t e i n markers... . . . . . .

P e r c e n t a g e o f 1. p a r a h a e m o l ~ t i c u s . p o s i t i v e f o r i d e n t i f i c a t i o n tests . .

M o r t a l i t y r a t e s o f mice i n j e c t e d i n t r a - p e r i t o n e a l y wi th 1. p a r a h a e m o l y t i c u s

C a l c u l a t i o n o f LD o f 1. parahaernoly- t i c u a i n mice i n j a e t e d i n b r a p e r i t m e a l y

C a l c u l a t i o n o f LD o f 1. parahaemoly- .. i n mice i n j & t e d p e r o r a l l y . . .

LD o f 1. p a r a h a e m o l y t i c u s I s o l a t e suagended i n J e c t o f e r . . ......... ....

s

LD o f 1. p a r a h a e m o l y t i c u s I s o l a t e suzgendad i n I r o n I1 s u l p h r t e . . ..... I n v i v o adherence t o mouse i leum. . . . -- I n t v i t r b adherence t o mouse p e r i t ; . n e a l -- macrophages................ ......... Haemolytic d o s e s o f Ammonium s u l p h a t e ...... p r e c i p i t a t e d f r a c t i o n s . . . . . . . .

P u r i f i c a t i o n o f t h e t o x i c f r a c t i o n . .

M o r t a l i t y r a t e s o f mice i n j e c t e d through v a r i o u s ' r o u t e s w i t h graded d o s e s o f t o x i c factor. . . . . . . . . . . . . . . . . . . . . . . . 1 07

M o r t a l i t y o f c h i c k embryos i n j e c t e d w i t h . graded d o s e s o f t o x i c f r a c t i o n 103

H i s t o l o g i c a l changes i n b i o p s y o f v i t a l ~ r g a n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

iii.

PAGE

18. R e c t a l t e r n p e r j t u r e s o f r a b b i t i n j e c t e d 1 1 1 w i t h t o x i c f a c t o r . . . . . . .....,.......

19. P e r c e n t a g e haemolys i s o f e r y t h r o c y t e s i n c u b a t e d wi th t o x i c f r a c t i o n . . . . . . . 112

C y t o t o x i c a c t i v i t y on mouse macrophhges 1 1 5

R e l a t i n g haernolyt ic a c t i v i t y w i t h c y t o t o x i c ac t iv i ty . , . . . . . . . . . . . . .... 116

E f f e c t o f s u b t o x i c d o s e s o f t o x i c f r a c - t i o n on macrophage phagocy t i c a b i l i t y 118

E f f e c t o f s u b t o x i c d o s e s o f t o x i c f r a c t i o n on macrophage adherence . . . . 121

E f f e c t o f s u b t o x i c d o s e s o f t o x i c f r a c - t i o n on macrophage m i g r a t i o n . . . . . . . 123

R e a c t i o n o f t o x i c f r a c t i o n on r a b b i t i l e a l loop. . . . . . . . . . . . . . . . . . . . . . .... 124

Reac t ion o f t o x i c f r a c t i o n on s u c k l i n g mouse.................,...,..,...... 125 . . Skin r e a c t i o n test.................. 127

A g g l u t i n a t i o n and h a e m a g g l u t i n a t i o n titres........,..................... 128

~ a c t e r i c i d a l a c t i v i t y o f a n t i - t o x i c f r a c - t i o n serum on l. parahaemoLyt icus c e l l s 132

L y t i c t i t r e o f a n t i - t o x i c f a c t o r serum 133

31 N e u t r a l i s a t i o n t e s t . . . . . . . . . . . . . . . . . 1 34

32 UV-absorption spect rum o f t o x i c f r a c t i o n 135

33. E f f e c t ' o f t e m p e r a t u r e on t o x i c f r a c t i o n 141

34. Temperature a c t i v i t y r ange o f t o x i c f r ac t ion . . . . . . . . . . . . . . . . . ........... 143

35. pH a c t i v i t y r ange o f ' t o x i c f a c t o r .... 1 44

36. E f f e c t of p r o t e o l y t i c enzymes. . . . . . . 145

37. E f f e c t o f some m e t a l ions . . . . . . . . . . . 147

i v .

ABSTRACT

A t o x i c f a c t o r which may b e v i t a l t o t h e p a t h o g e n i c i t y

o f V i b r i o ~ a r a h a e m o l y t i c u s was i s o l a t e d from a v i r u l e n t Kanagawa-

p o s i t i v e 1. p a r a h a e m o l ~ t i c u s i s o l a t e . Homogenised c e l l s o f t h e

mic roo rgan i sm were d i f f e r e n t i a l l y c e n t r i f u g e d , and t h e t o x i c

f a c t o r was p r e c i p i t a t e d from t h e s u p e r n a t a n t w i t h Ammonium

s u l p h a t e (40% s a t u r a t i o n ; 11.9% r e c o v e r y r a t e ; x 8.4 p u r i f i c a t i o n

f o l d ) . The p r e c i p i t a t e was p u r i f i e d by Sephadex G-150 g e l

f i l t r a t i o n (0 .35% r e c o v e r y ra te ; x 28 000 p u r i f i c a t i o n f o l d ) ,

a n d was c h a r a c t e r i s e d b i o l o g i c a l l y and p h y s i c o c h e r n i c a l l y . The

v a l u e s of LD o f t h e t o x i c f a c t o r i n mice were 6 .4 tug o f p r o t e i n 50 ( i n t r a v e n o u s l y ) , 11.8 a g o f p r o t e i n ( i n t r a p e r i t ( : , n e a l y ) , 18.0 a g

o f p r o t e i n ( s u b c u t a n e o u s l y ) and .p 18.0 &g o f p r o t e i n ( p e r 0 s ) .

The l i v e r , k i d n e y and myocardium o f mice i n j e c t e d i n t r a v e n o u s l y

a n d i n t r a p e r i t o n e a l y showed v a s c u l a r c o n g e s t i o n a n d t i s s u e n e c r o s i *

w h i l e t h e i n t e s t i n e o f o r a l l y f e d mice showed desquarna t ion o f

t h e e p i t h e l i u m . R e c t a l t e m p e r a t u r e s o f r a b b i t s i n j e c t e d i n t r a -

v e n o u s l y w i t h . 20.0 f ig o f p r o t e i n i n t h e t o x i c f a c t o r i n c r e a s e d

by 0 . 4 5 ~ ~ two h o u r s a f t e r i n j e c t i o n . F i f t y p e r c e n t h a e m o l y t i c

d o s e s ( H D ) o f t h e f a c t o r a c t i v e a g a i n s t mammalian e r y t h r o c y t e s 50 were 0 . 1 5 m g o f p r o t e i n f o r man, 0.30 f i g o f p r o t e i n f o r r a b b i t ,

0.70 u g o f p r o t e i n f o r s h e e p , and 5.OOtug o f p r o t e i n f o r ra t .

The c y t o t o x i c 'dose a g a i n s t mouse p e r i t o n e a l macro phages (MPM)

r a n g e d from 0.07 - 5.00'mg o f p r o t e i n . The p h a g o c y t i c i n d e x ,

a d h e r e n c e r a t e , and m i g r a t i d n r a t e of MPM r e d u c e d markedly a f t e r

exposure o f t h e c e l l s t o s u

I

b t o x i c d o s e s o f t h e t o x i c f a c t o r . 1 I

The g u t weight / remaining body weight r a t i o s s u g g e s t f l u i d

accumula t ion i n s u c k l i n g mice fo rce - fed w i t h 0.07 - 25.00 mg o f 1

p r o t e i n of t h e t o x i c f a c t o r . The f a c t o r produced in f l ammatory

r e s p o n s e on g u i n e a - p i g s i n j e c t e d i n t r a d e r m a l l y . The a n t i - t o x i c

f a c t o r serum a g g l u t i n a t e d Y. p a r a h a e m o l y t i c u s c e l l s ( a g g l u t i n a t i o

t i t r e = 1 i n 128) and s h e e p e r y t h r o c y t e s ( h a e m a g g l u t i n a t i o n t i t r e

= 1 i n 2 048) , and l y s e d Y. parahaemol.yt icus c e l l s i n t h e p resenc

o f complement ( l y t i c t i t r e - 1 i n 6 4 ) . The mouse p r o t e c t i o n

t e s t was p o s i t i v e . %''he t o x i c f a c t o r c o n t a i n e d p r o t e i n b u t n o t

c a r b o h y d r a t e s o r l i p i d s . S i n g l e band o f p r o t e i n was s e e n on

PAGE and on immunoe lec t rophore t i c g e l s . The m o l e c u l a r weight

was 6 3 095, and maximal a b s o r p t i o n o f UV-rays o c c u r r e d a t 280nm.

The f a c t o r h e a t e d a t 6 0 ' ~ r e t a i n e d haemolyt ic a c t i v i t y , whi le s I

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

a c t i v i t y . Toxic a c t i v i t y w a s maximal a t 37 '~ and a t pH 7.0.

I n c u b a t i o n w i t h a n t i - t o x i c f a c t o r serum o r p r o t e o l y t i c enzymes I I

( P e p s i n o r T r y p s i n ) a b o l i s h e d t o x i c a c t i v i t i e s . Toxic a c t i v i t i e s

were s t i m u l a t e d by Calcium, Copper, Manganese, and Zinc i o n s .

A r o l e f o r t o x i c f a c t o r i n t h e p a t h o g e n i c i t y o f 1. pafahaemoly-

t i c u s and i n t h e h o s t d e f e n c e mechanism a g a i n s t t h e microorganism - was sugges ted .

Chapter 1

I N T R O D U C T I O N

Vibr io parahaemolyt icus i s a pleomorphic, h a l o p h i l i c ,

Gram-negative rod found most ly i n c o u n t r i e s w i t h w a r m c o a s t a l

waters. F i r s t i s o l a t e d by D r . T. F'ujino fol lowing an outbreak

of foodpoisoning i n Osaka, Japan i n 1950, t h e microorganism

h a s s i n c e been a common cause of foodpoisoning and g a s t r o w ' &

e n t e r i t i s i n many c o a s t a l r e g i o n s o f the world, i n A f r i c a

(Sklarow e t a l . , 1973; Bubb, 1975; David-Prince e t a l . , 1980;

El Sahn e t al. , 1982; and Mhalu e t al., 1982), America

( C h a t t e r j e e and Neogy, 1972; and Kaneko & Colwell , 1975), Asia

( ~ r a i g , 1972; and Kr i s tensen , 1974), A u s t r a l i a (Pan Urai, 1974;

Muic, 1974; Bockehuehl & Triemer, 1975; C a t t a n i a n i e t a l . , 1978;

Mihaj lovic e t a l . , 1982), Ind i a (Baross e t a l . , 1975; Mhalu e t

a l . , 1982; and Kourany e t al. , 1984), and i n o t h e r p a r t s of t h e m

world (Neumann e t a l . , 1972; P e f f e r s e t a l . , 2973; Sakazaki,

1973; Barker , 1974; Chun e t al., 1974; Wagatsuma, 1974; Bonange

e t a l . , 1975; 'Cabassi & Mori, 1976; Ciufecu e t a l . , 1977;

Mazumder e t al., 1977; Lam and Goh, 1977; Hughes e t a le , 1978;

Lawrence e t a l . , 1979; S r i r a t anaban & Reinprayoon, 1979; Nair

e t a l . , I980;.Ducklow e t al . , 1980; and S r i r a t anaban & Reinprayoon,

1982).

Eco log ica l ly , 1. parahaemolyt icus i s a n e s t u a r i n e bacter ium /

t h a t occurs as p a r t o f t h e normal f l o r a of most e s t u a r i n e and

c o a s t a l wate rs of t h e world. P t s d i s t r i b u t i o n i n n a t u r e

appears t o be r e s t r i c t e d by such environmental parameters

as s a l i n i t y and tempera ture . The v i b r i o o c c u r s ma in ly i n ,

c o a s t a l w a t e r s where t h e sa l t c o n c e n t r a t i o n i s h i g h , Its

d i s t r i b u t i o n , t h e r e f o r e , i s found t o d e c r e a s e w i t h d i s t a n c e

from s h o r e (Colwel l , 1975). I n t empera te r e g i o n s such a s

J a ~ a n ( ~ i w a t a n i & Takeda, 1976) , Dorea (Chun e t al . , 1974) and

t h e Uni ted S t a t e s (Kaneko & Colwel l , l 9 7 3 ) , l a r g e numbers o f t h e

microorganism can be i s o l a t e d from s e a w a t e r i n summer, b u t n o t

i n win te r . At tached t o b e n t h i c organisms, t h e v i b r i o p a s s e s

t h e w i n t e r i n w a t e r s e d i m e n t s and becomes r e l e a s e d as t h e

t e m p e r a t u r e i n c r e a s e s i n s p r i n g . The r e l e a s e d microorganism

then becomes a t t a c h e d t o zooplankton f o r p r o l i f e r a t i o n

(Kaneko & Colwel l , 1975).

y. p a r a h a e m o l v t i c u s d e m o ~ t r a t e c b i o l o g i c a l a s s o c i a t i o n s

w i t h p l a n k t o n i c and b e n t h i c organisms. Adsorbing on t o c h i t i n

and o n t o copepodn (minute c r u s t a c e & ? s ) (Kaneko & Colwel l , 1975),

i t a p p e a r s t o be a commensal n o t o n l y o f p lank ton b u t a l s o o f

c r a b , t h e r e b y , p o s i n g a p o t e n t i a l h e a l t h h a z a r d ( ~ o l w e l l , 1975).

Transmiss ion i s u s u a l l y th rough i n g e s t i o n o f con tamina ted

wa te r , u n r e f r i g e r a t e d and improper ly cooked s e a f o o d s , a s w e l l

a s t h r o u g h ' c o n t a m i n a t e d k i t c h e n u t e n s i l s ( S i r k a e t a l . , 1976).

Secondary s p r e a d between p a t i e n t and household c o n t a c t s as w e l l

a s long-term asymptomatic c a r r i e r s t a t e have n o t been r e p o r t e d ,

Few e x t r a i n t e s t i n a l i n f e c t i o n s o f t h e eye , e a r , and wound have

been d e s c r i b e d and most of t h e s e have a h i s t o r y o f p r e v i o u s

exposure t o s e a w a t e r (von. - . G r a e v e n i t z & C a r r i n g t o n , 1973; B o r r e s

& Fuchs, 1975; Bowner e t a l . , 1977; Tay & Yu, 1978; O l s e n ,

1978; Mautner & Halnoe , 1979; and B lake & Weaver, 1980). The

o n l y r e p o r t e d c a s e o f d i a r r h o e a and s e p t i c a e m i a (Tay & Yu, 1978)

was l i k e l y t o hav.e been i n f e c t e d by t h e o r a l r o u t e .

I n f e c t i o n s by t h e mic roo rgan i sm seem n o t t o show a n y

p r e d i l e c t i o n f o r s e x o r a g e , s i n c e i n d i v i d u a l s o f b o t h sexes

a n d a g e are a f f e c t e d . The d i s e a s e i s c h a r a c t e r i s e d by sudden

o n s e t o f a c u t e g a s t r o e n t e r i t i s w i t h n a u s e a , v o m i t i n g , &bdominal

c ramps , l ow g r a d e f e v e r and c h i l l s . The d i a r r h o e a i s w a t e r y b u t

somet imes b loody. The d i s e a s e , u s u a l l y m i l d and l a s t i n g f o r a

few d a y s is s e l f - l i m i t e d , even though s e v e r e c a s e s r e q u i r i n g

h o s p i t a l i z a t i o n some t imes do o c c u r . I n J a p a n , f o r i n s t a n c e ,

o n l y 0.04% d e a t h s o u t o f 81 534 c a s e s have been r e c o r d e d , w h i l e

no d e a t h h a s b e e n r e p o r t e d i n t h e Un i t ed S t ~ t e s (Miwatan i , B a r k e r a

& Gangarosa , 1974; and Miwatan i & , T a k e d a , 1976) . The i n c u b a t i o n

p e r i o d i s s h o r t and l a s t s f o r 10 t o 20 h o u r s .

V. p a r a h a e . m o l y t i c u s i s r e s i s t a n t t o p e n i c i l l i n a n d a m p i c i l l i n , - b u t is s u s c e p t i b l e t o t e t r a c y c l i n e , g e n t a m i c i n , a n d c h l o r a m p h e n i c o l

(Farmer e t a l . , 1985) . The mechanism o f p a t h o g e n i c i t y o f 1. g a r a h a e r n o l y t i c u s h a s

been e x t e n s i v e l y ~ t u d i e d , y e t , i t i s n o t u n d e r s t o o d how t h e

v i b r i o c a u s e s i n f e c t i o n i n humans. Fo rmer ly , t h e a b i l i t y o f

t h e mic roo rgan i sm t o c a u s e h a e m o l y s i s on i'dagatsuma a g a r ( t h e

Kanagawa phenomenon) was a s s o c i a t e d w i t h g a s t r o i n t e s t i n a l i l l n e s s

( S a k a z a k i e t a l . , 1 9 6 8 ) ; a b o u t 96.5% of' i s o l a t e s f r m p a t i e n t s

w i t h d i a r r h o e a were haemoly t i c (Kanagawh-posi t ive) i n c o n t r a s t

t o o n l y 1 % of i s o l a t e d from s e a f o o d s and s e a w a t e r . However,

t h e r e s u l t s o f Cherwonogrodzky and C l a r k (198%) and o f lfonda

e t a l . (1983) showed t h a t accumula t ion o f f l u i d s induced by

V. p r a h a e m o l y t i c u s i s n o t d i r e c t l y r e l a t e d t o t h e Kanagawa- - phenomenon haemolysin o r t h e t h e r m o s t a b l e d i r e c t haemolysin

(TUH. ) , s i n c e n e i t h e r anti-TDH n o r a n t i - c h o l e r a t o x i n was a b l e

t o w e v e n t accumula t ion o f f l u i d caused by l i v i n g c e l l s of x. p a r a h a e m o l y t i c u s (Honda e t al., 1983). Hackney e t a l . (1980)

observed t h a t even though Kanagawa-posi t ive s t r a i n s o f t h e

microorganism a r e u s u a l l y c o n s i d e r e d more v i r u l e n t , a

s i g n i f i c a n t p o r t i o n o f 1. p a r a h a e m o l y t i c u s foodpo i son ings

a p p e a r t o be caused by Kanagawa-negative s t r a i n s . Moreso, most

i s o l a t e s from e x t r a i n t e s t i n a l i n f e c t i o n s t e s t e d a t CUC were *

found t o b e Kanagawa-negative l lake.' Weaver & H o l l i s , 1980).

The c l i n i c a l impor tance of t h e TDH eemains unknown.

The exis . te ,nce o f an e n t e r o t o x i n i n 1. p a r a h a e r n o l ~ t i c u s ' 8

i s n o t y e t c o n c l u s i v e l y proven, a l t h o u g h C r a i g and a s s o c i a t e s

(1972) a t t r i b u t e d a x. p r a h a e m o l y t i c u s - a s s o c i a t e d d i a r r h o e a

t o a n e n t e r o t o x i n e l a b o r a t e d i n t h e small i n t e s t i n e . honda

e t a l . (1976) a l s o d e t e c t e d a t h e r m o l a b i l e f a c t o r i n c u l t u r e

f i l t r a t e s o f a kanagawa-posi t ive x. parahaemoly t i cus . The

f a c t o r produced a r e a c t i o n s i m i l a r t o t h a t produced by c h o l e r a

t o x i n i n C h i i e s e Hamster Ovar'y (CHO) c e l l s .

A t h e r m o l a b i l e haemolys in , found i n c u l t u r e s u p e r n a t e s

o f bo th Kanagawa-posi t ive and Kanagawa-negative s t r a i n s

( S a k u r a i e t al., 1974) i s a n o t h e r e x t r a c e l l u l a r p r o d u c t of t h e

v i b r i o though t t o be a v i r u l e n t f a c t o r . I ts r o l e i n t h e

p a t h o g e n i c i t y o f 1. parahaemol.yt icus h a s n o t been e v a l u a t e d .

Pew r e p o r t s a r e a v a i l a b l e on i n t r a c e l l u l a r haemolysins.

I i d a and Takagi (1979) i d e n t i f i e d a t h e r m o s t a b l e i n t r a c e l l u l a r

haemolysin c o n t a i n i n g 6 p e r c e n t n e u t r a l l i p i d s which i s weakly

t o x i c t o mice and is e x t r a c t a b l e w i t h a c e t o n e . I i d a and

coworkers (1982) fdund t h a t mo~i t of the l y t i c act;.i.vity

i n t h e i n t r a c e l l u l a r t h e r m o s t a b l e haelnolyt ic f r a c t i o n ( ITHF)

was p r e s e n t i n t h e f ree f a t t y a c i d f r a c t - . d n r a t h e r t h a n i n

o t h e r l i p i d s .

S t u d i e s w i t h b a c t e r i a l c e l l s have shown t h a t t h e i n v a s i v e I

c a p a c i t y o f t h e microorganism i s n o t w e l l e s t a b l i s h e d . Although

bo th Kanagawa-posi t i v e and Kanagawa-negative s t r a i n s c a n i n v a d e

human i n t e s t i n a l e p i t h e l i u m c a u s i n g u l c e r a t i o n o f t h e , colon

rec tos igmoid , (Bol len , Zamiska & Greenough, 1974), b o t h s t r a i n s

a r e s e r e n y t es t s n e g a t i v e (Johnson & C a l i a , 1975). N e v e r t h e l e s s ,

bacteremqe have r e s u l t e d f o l l o w i n g i n t r a g a s t r i c c h a l l e n g e

and t r a n s i l e a l i n o c u l a t i o n of s u c k l i n g mice w i t h booth c u l t u r e s

o f e i t h e r strains ( J o s e p h e t a l . , 1978).

Both Kanagawa-positiGe anu Kanagawa-negative s t r a i n ,

adhered t o human f o e t a l i n t e s t i n a l (HFI) c e l l s in v i t r o ,

a l t h o u g h Kanagawa-negative s t r a i n s adhered a t a much . s l o w e r

r a t e ( C a r r u t h e r s , 1977; and Hackney e t a l . , 1380).

D e s p i t e t h e e x t e n s i v e s t u d i e s c a r r i e d o u t on l i v i n g c e l l s

o f 1. parahaemol .y t icus and on b o t h t h e e x L r a c e l l u l a r and

i n t r a c e l l u l a r p r o d u c t s of t h e micoorganisrn, t h e pa thogen ic

mechanism of t h e v i b r i o i s y e t t o be e l u c i d a t e d . The v a r i e d

symptoms a s s o c i ~ t e d wi th t h e i n f e c t i o n have l e d t o t h e

s u g g e s t i o n t h a t one f a c t o r o r t o x i n may n o t be r e s p o n s i b l e f o r

t h e p a t h o g e n i c i t y b u t a combinat ion o f f a c t o r s (Sochard &

Colwel l , 1977).

I n t h i s s t u d y , t h e r e f o r e , a n i n t r a c c - l l u l a r t o x i c f a c t o r

o b t a i n e d from a v i r u l e n t Kanagawa-positive l. p a r a h a e m o l ~ t i c u s

was c h a r a c t e r i s e d b i o l o g i c a l l y and phys icochemica l ly , and i ts

p o s s i b l e roles_* i n t h e pa t h o g e r l i c i t y o f 1. p a r a h a e m o l u t i c u s

i n f e c t i o n s were a s s e s s e d and d i s c u s p e d . rn

CHAP'l'X13 2

LITEWi'l'URE: l?EVIUW

HISTORY

I n Oc tober 1950, a n o u t b r e a k o f foodpo i son ing o c c u r r e d

i n Osaka, Japan . Out o f t h e two hundred a n d seventy-two ~ e o ~ l e

who developed a c u t e g a s t r o e n t e r i t i s accompanied wi th watery

and smetimes bloody s t o o l , twenty p e r s o n s d ied . An e x t e n s i v e

i n v e s t i g a t i o n was t h e r e f o r e c a r r i e d o u t t o f i n d t h e c a u s e o f

t h e ou tb reak . I n 1951, D r . T. F u j i n o and h i s a s s o c i a t e s t r a c e d

t h e cause t o s h i r a s u , a small s a r d i n e b o i l e d i n s a l t e d w a t e r

a n d e a t e n when p a r t i a l l y d r i e d . A homogenate o f f i s h was

p r e p a r e d i n s a l i n e and s t r e a k e d o n t o TGC, S a l m o n e l l a - S h i g e i l a ,

and n u t r i e n t a g a r p l a t e s . The p l a t e s were i n c u b a t e d a e r o b i c a l l y

and a n a e r o b i c a l l y a t 37 '~. The homogenate was a l s o i n j e c t e d

i n t o mice and gu inea -p igs , and a t a u t o p s y samples o f b lood,

s p l e e n and i n t e s t i n a l c o n t e n t s were c o l l e c t e d and c u l t u r e d .

Both f a t and s l e n d e r Gram-negative r o d s were i s o l a t e d . A f t e r

r e p e a t e d i n o c u l a t i o n i n t o mice and t h e subsequen t p l a t i n g o u t o f

e a c h i s o l a t e ' , t h e f a t , rod-shaped bac te r ium was found t o be

haemoly t i c and e x t r e m e l y pa thogen ic f o r mice. I t was p l a c e d

under t h e P a s t e u r e l l a because of i t s b i p o l a r s t a i n i n g , l u c k

o f c u r v a t u r e and n o n - r e a c t i v i t y w i t h 1. c h o l e r a e a n t i s e r a .

F u j i n o and h i s ' a s s o c i a t e s named i t P a s t e u r e l l a haemolu t i ca .

I n 1955, f o l l o w i n g Another o u t b r e a k o f foodpo i son ing i n

Yokohama Hospi ta l . , ~ a k i k a w a (1956) obse rved t h e h a l o p h i l i c

n a t u r e of t h e microorganism; b u t he named h i s i s o l ~ t e

Pseudomonas e n t e r i t i s . He w a s a b l e t o d e s c r i b e t h e g e n e r a l

c h a r a c t e r i s t i c s o f t h e mic roo rgan i sm a n d t o e s t a b l i s h i t s

p a t h o g e n i c i t y a n d m e d i c a l i m p o r t a n c e .

Subsequen t r e p o r t s o f f o o d p o i s o n i n g c a u s e d by t h e

mic roo rgan i sm were t r a c e d t o t h e i n g e s t i o n o f some s e a f o o d s

s u c h as s q u i d , h o r s e m a c k e r e l , and c r a b .

A f t e r a s t u d y o f many gram-negat ive s h o r t r o d s , S a k a z a k i

e t a l . (1963) s u g g e s t e d t h a t t h e m i c r o o r g s n i s m be c l a s s i f i e d

as V i b r i o , and t h e name V i b r i o p a r a h a e m o l y t i c u s was p roposed .

Zen-Yoji e t a l . (1965) c a r r i e d o u t a n u m e r i c a l taxonomy

s t u d y o f 1. p a r a h a e m o l y t i c u s and i t was c o n c l u d e d t h a t b i o t y p e

2 s h o u l d be s e p a r a t e d from 1. p a g a h a e m o l y t i c u s . A l so , t h e

i n c i d e n c e o f i s o l a t i o n o f b i o t y p e 2 f ror~i p i ~ t i e n t s was e x t r e m e l y

low compared w i t h t h e h i g h i n c i d e n c e o f b i o t y p e 1 i n and on ' .

f i s h . s h e l l f i s h , and u t e n s i l s u s e d i n c o o k i n g f i s h . B i o t y p e 2

was named 1. a l g i n o l y t i c u s a n d o n l y b i o t y p e 1 i s r e g a r d e d as

V p a r a h a e m o l y t i c , ~ . I n 1967. S a k a z a k i a l s o a r r i v e d a t t h e -*

same c o n c l u s i o n .

Ano the r s e p a r a t i o n o f g. p a r a h a e m o l y t i c u s must b e c o n s i d e r e d .

C u l t u r e s o f t ,he mic roo rgan i sm i s o l a t e d from human d i a r r h o e a 1

s t o o l s d i f f e r from c u l t u r e s i s o l a t e d from s e a f i s h and s e a w a t e r

w i t h r e s p e c t t o h a e m o l y t i c a c t i v i t y on Knga tsuma medium.

'The h a e d y t i c r e a c t i o n was c a l l e d t h e Kanar;nwa r e a c t i o n o r

phenomenon ( ~ a k a z s k i e t a l . , 1968) . Ep ide r f l i o log ica l d a t a

c o l l e c t e d b ~ them showed tllat a b o u t 96% o f s t r a i n s i s o l a t e d

from human p a t i e n t s gave a p o s i t i v e r e a c t i o n when t e s t e d on

Wagatsurna medium whereas on ly 1% e f t h e s t r a i n s i s o l a t e d from

s e a f i s h and s e a w a t e r gave p o s i t i v e r e s u l t s . Although t h e

Kanagawa phenomenon i s though t t o be a s s o c i a t e d w i t h e n t e r o -

p a t h o g e n i c i t y of ' 1. p a r a h a e m o l y t i c u s , t h e r o l e o f t h e

haemolysin i n t h e p a t h o g e n i c i t y o f t h e microorganism is still

n o t c o n c l u s i v e l y proven.

OCCURRENCE

Vibr io parahaemolyt icus is widely d i s t r i b u t e d , and h a s

been found i n many p a r t s of t h e world, i n A f r i c a ( ~ k l a r o w e t

a l . , 1975; Bubb, 1975; David-Prince e t al., 1980; E l ~ a h n e t

al., 1982; and M h l u e t a l e , 1982), America ( C h a t t c r j e o & Neogy,

1972; Kaneko & Colwel l , 1975; and Tacket e t al. , 1982), Asia

( ~ r a i g , 1972; and Kr i s t ensen , l974) , A u s t r a l i a (Pan Urai, l974) ,

Europe (Libinzon & Domaradsky, 1974; Mackowiak, 1974; Muic,

1974; Bockemuehl & Triemer, 1975; C a t t a b i a n i e t al . , 1978;

and Miha j lov ic e t a l . , 1982), and i n I n d i a (Kourany e t a l . ,

1974; Baross e t al . , 1975; and Mhalu e t a l . , 1982).

I t o c c u r s as p a r t o f t h e normal f l o r a of e s t u a r i n e and

c o a s t a l wa te rs , anu h a s been i s o l a t e d from many c o a s t a l r e g i o n s m

o f t h e world i n c l u d i n g t h e Natal a n d m E a s t e r n Cape c o a s t a l

wa te r s o f South ~ f r d c a (Bubb, 1975), t h e A d r i a t i c c o a s t o f

C r o a t i a (Muic e t a l e , 1974), and t h e sho re o f t h e Black Sea

(Libinzon & Domaradsky, 1974). In. t h e A t l a n t i c c o a s t a l r e g i o n s ,

a DNA-bacteriophage s p e c i f i c f o r Y. p r a i m e m o l ~ t i c u s was

i s o l a t e d (Sklarow e t a l e , 1973). Although i s o l a t i o n from

of f - shore l o c a t i o n s have been r e p o r t e d (Hhame & Werner, 1974),

f o r example, ir-f s eawate r o f f t h e P a c i f i c c o a s t l i n e (Kourariy

e t a l . , 1974), i t s inc idence shows marked d e c l i n e with d i s t a n c e

from sho re (,Colwell, 1975). ,

The inc idence o f 2. parahaemolyt icus depends on such

e c o l o g i c a l f a c t o r s as t e m p e r a t u r e , s a l i n i t y , anti hydrogen i o n

c o n c e n t r a t i o n o f t h e envi ronment , and on b i o t i c f a c t o r s such

as t h e p r e s e n c e o f some mar ine organisms i n t h e environment .

I n w i n t e r when t h e t e m p e r a t u r e i s c o l d , t h e v i b r i o becvmes

a b s e n t from w a t e r columns, b u t c o u l d be d e t e c t e d i n o c e m

sediments . The microorganism, t h e r e f o r e , a p p e a r s t i ) p a s s t h e

w i n t e r i n ocean sed-iments (Co lwel l , 1975). and may t h u s a c c o u n t

f o r t h e p r e s e n c e of a DXA-containing b a c t e r i o p h a g e s p e c i f i c

f o r y. pa rahaemoly t i c u s i n sendiment samples o f A t l a n t i c c o a s t a l

r e g i o n s (Sklarow el; a l . , 1973). The mo: t s t a b l e e c o l o g i c a l

n i c h e f o r t h e s u r v i v a l o f v i b r i o s , t h e r e f o r e , seems t o be

ocean s e d i m e n t s ( N a t a r a j a n , 1979).

I n s p r i n g and i n e a r l y summer, when t h e t e m p e r a t u r e

r i s e s , t h e microorganism i s r e l e a s e d from t h e bottom o f t h e *

ocean and becomes a t t a c h e d t o zooplankton (e.g., a d u l t copepod) a

from where i t p r o l i f e r a t e s ( N a i r e t a l . , 1980). Durin g t h i s

p e r i o d , t h e i n c i d e n c e o f L. p a r a h a e m o l y t i c u s i n c r e a s e s and

c o u l d a c c o u n t ' f o r up t o 50% o f c a s e s o f g i \ s t r o e n t e r i t i s

(Rhame & Werner, 1974; Nair e t a l . , 1980; and E l Sahn e t al.,

1982). Out of t h e t o t a l b a c t e r i a l p o p u l a t i o n a s s o c i a t e d w i t h

zooplankton, o f which i s V i b r i o s p e c i e s , 9.5% a r e 1.

S a l i n i t y a l s o p l a y s a r o l e i n t h e d i s t r i b u t i o n o f t h e

microorganism. The r e s t r i c t i o ;. of t h e v l b r i o t o c o a s t a l

w a t e r columns c o r r e l a t e s wi th t h e s a l i n i t y o f t h e a r e a , 15 t o

20% s a l i n i t y b e i n g o p t i m a l ( N a t a r a j a n e t a l . , 1979). The

d i s t r i b u t i o n o f 1. parahaemolu t i cuq i n w a t e r s e d i m e n t s and

p lank ton , however, show marked d e c l i n e wi th d i s t a n c e from

s h o r e , and some r e s e a r c h e r s i n d i c a t e d t h a t t h e microorganism

i s i s o l a t e d o n l y from e s t u a r i e s and n e a r c o a s t a l w a t e r s

( C o l w e l l , 1975).

V p a r a h a e m o l y t i c u s a s s o c i a t e s s y m b i o t i c a l l y wi th a l a r g e

p e r c e n t a g e o f s e a o rgan i sms ( e . g . , c r a b s , o y s t e r s , s h e l l - f i s h ,

e t c , ) a d s o r b i n g o n t o t h e i r c h i t i n p a r t i c l e s ( ~ i b i n z o n &

Domaradsky, 1974; Muic e t al., 1974; Pan Urai, 1974; Rhame &

Werner, 1974; C o l w e ~ l , 1975; Kaneko & Colwel l , 1975; N a t a r a j a n

e t al . , 1979; B i n t a e t a l , , 1982; and Huq e t a l , , 1983). The

e f f i c i e n c y o f a d s o r p t i o n depends on t h e pH o f t h e environment

and on t h e c o n c e a t r a t i o n o f sodium ~ h l o r i d e and o t h e r s a l t s

found i n s e a w a t e r , Adsorp t ion is one o f t h e f a c t o r s which

de te rmine t h e d i s t r i b u t i o n and annua l c y c l e o f t h e microorganism

i n t h e e s t u a r i n e s,stea (Kaneko & Colwel l , 1975). The micro-

organism h a s ' a l s o been i s o l a t e d from cold-blooded a n i m a l s of

non-marine o r i g i n such as c r a b s , earthworms, prqwns, s h r i m p s ,

s n a i l s , and small v e r t e b r a t e f i s h e s ( C h a t t e r j e e % Neogy, 1972).

A s t u d y on t h e changes o f m i c r o b i a l populat.iori i n s e a w a t e r

and i n s e a w a t e r ' c m t a i n i n g s a n d (Yamamoto e t al,, 1982) showed

t h a t mic roorgar i i s r~ such a s amoebae, b d e l l o v i b r i o ,

myxobacter ia and p l ~ q u e - f o r m i n g microorganisms i n c r e a s e d . i n

number f o l l o w i n g a d e c l i n e i n t h e v i a b l e coun t o f y. parahae-

molyt icus; but the e f f e c t i v e n e s s o f predation decreased i n

the samples containing sand. The dec l ine o f Y. parahaemolu-

Ijicus, therefore , can be a t t r i b u t e d t o indigenous predat iors

and p a r a s i t e s i n the s ea .

EPIDEMIOLOGY

G a s t r o e n t e r i t i s caused by x. p a r a h a e m o l y t i c u s i s common

i n many c o u n t r i e s ( ~ a c k o w i a k , 1974), and h a s been r e p o r t e d i n

A f r i c a , Nor th America, C e n t r a l America, Europe, . and . Asia. I n

Japan , t h e v i b r i o accoun ted f o r 24% of c a s e s o f g a s t r o e n t e r i t i s ;

1 1 % o f c a s e s i n C a l c u t t a , I n d i a ; 10% i n T h a i l a n d ; 8.5% t o 15%

i n Vietnam; 2.6% t o 3.7% i n I n d o n e s i a ; and 1.5% i n Korea

(Blake , Weaver, & H o l l i s , 1980). A c a r r i e r r a t e o f 0.3%

was d e t e c t e d i n h e a l t h y p e r s o n s i n Japan . The i n f e c t i o n i s

a p p a r e n t l y a b s e n t from some c o u n t r i e s l i k e t h e U S A , because

most l a b o r a t o r i e s do n o t u s e c u l t u r e media a p p r o p r i a t e f o r

i s o l a t i n g t h e v i b r i o from s t o o l s . Moreso, many p h y s i c i a n s

a r e unaware o f i t s e x i s t e n c e t h e r e b y , making t h e d i a g n o s i s o f I

t h e i n f e c t i o n t o - b e a rare phenomenon ( Z a l e s k i e t al., 1975).

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

o u t b r e a k s 6 d c 8 s i o n a l l y o c c u r (Lam &, T a i , 1977).

E x t r a i n t e s t i n a l i n f e c t i o n s caused by t h e v i b r i o have

been r e p o r t e d i n Nonth America, Asia, A u s t r a l i a , and Europe.

V p a r a h a e m o l y t i c u s i n f e c t i o n s a r e s e a s o n a l w i t h o u t b r e a k s -*

o c c u r r i n g a l m o s t e x c l u s i v e l y i n t h e w a r m summer and e a r l y

f a l l months ( B a r k e r , 1975; Nair e t a l . , 1980; and E l Sahn

e t al., 1982). I n T h a i l a n d , t h e p r o p o r t i o n o f p e r s o n s w i t h

V. parahaemol~ticus-associated g a s t r o e n t e r i t i s i n c r e a s e d

from 3.9% i n t h e c o o l month o f J a n u a r y t o 22.6% i n t h e w a r m

month o f September ( S r i r a t a n a b a n & Reinprayoon, 1982) . The

s e a s o n a l i t y o f t h e i n f e c t i o n may be r e f l e c t i n g t h e enhanced

o p p o r t u n i t y o f t h e microorganism t o m u l t i p l y i n u n r e f r i g e r a t e d

s e a f o o d s d u r i n g t h e summer months, as w e l l a s t h e i n c r e a s e d

p r e v a l e n c e o f t h e v i b r i o i n t h e environment d u r i n g t h e w a r m

weather . E x t r a i n t e s t i n a l i n f e c t i o n s a l s o o c c u r d u r i n g t h e

w a r m months.

V. parahaemo1yticu~-associated g a s t r o e n t e r i t i s a p p e a r s t o - be e x c l u s i v e l y t r a n s m i t t e d by food, e s p e c i a l l y saw a q d . l m p r o p e r l y

cooked seafood. Raw s e a f o o d becomes s o u r c e o f i n f e c t i o n when

i t i s h e l d u n r e f r i g e r a t e d l o n g enough t o a l l o w t h e v i b r i o , a

n a t u r a l c o n t a m i n a t o f s e a f o o d s , t o m u l t i p l y t o l a r g e i n f e c t i v e

numbers. The s e a f o o d s can a l s o c ross -con tan imate o t h e r

t y p e s o f food. (ahame & Werner, 1974). m

Improper ly p r e p a r e d s e a f o o d s are a n o t h e r s o u r c e o f i n f e c t i o n

( B a r k e r e t a l . , 1974; Sizemore e t a l . , 1975; and S i n g &

J e g a t h e s a n , ' l 9 7 7 ) , e s p e c i a l l y when t h e f o als are n o t b o i l e d

a t t e m p e r a t u r e s h i g h enougb t o k i l l t h e v i b r i o . Cooked

s e a f o o d contanin&tt&by raw s e a f o o d may be i n a d e q u a t e l y

r e f i g e r a t e d t h e r b y a l l o w i n g t h e b a c t e r i a t o p r o l i f e r a t e i n

t h e food. I n a d e q u a t e r e f r i g e r a t i o n i s a n i m p o r t a n t f a c t o r h e r e ,

s i n c e t h e v i b r i o grows w e l l a t a t e m p e r a t u r e r ange o f 12.8-

t o 44.0 '~ (Broadshaw e t a l . , J974 ; and Blake , Weaver & H o l l i s ,

1980). The s h o r t g e n e r a t i o n t ime o f t h e microorganism ( 9 m i n u t e s )

(Kato, 1965) enable^ i t t o p r o l i f e r a t e r a p i d l y i n mishandled

foods (Sanya l e t a1 . , 1974).

Poor p e r s o n a l hyg iene as w e l l as i n a d e q u a t e s a n i t a r y

c o n d i t i o n e s p e c i a l l y i n p l a c e s where food i s p r e p a r e d ( ~ h a l u ,

1982) are o t h e r f a c t o r s t h a t enhance Y. p a r a h a e m o l y t i c u s

i n f e c t i o n s .

Exposure t o seawater a p p e a r s t o be t h e s o u r c e o f e x t r a -

i n t e s t i n a l i n f e c t i o n s . P e r s o n s w i t h wound i n f e c t i o n s a l l had

h i s t o r y of p r e v i o u s exposure t o seawater. Cases o f t i s s u e

i n v a s i o n s were a s s o c i a t e d w i t h r e c r e a t i o n a l expoplure i n f r e s h

water andseawate r ( Z i d e e t al., 1975), and w i t h ' d c c i d e n t a l

exposure ( a p l a n e c r a s h t h a t o c c u r r e d n e a r a sea c o a s t (Rhame

& Werner, 1974). A c a s e of p a n o p h t h a l m i t i s r e s u l t e d

from c o n t a m i n a t i o n o f wound through con tamina ted w a t e r

( ~ a c k e t e t al . , 1982). There was no h i s t o r y o f exposure f o r

t h e pe r son wi th s e p t i c a e m i a , and he seems l i k e l y t o have been

i n f e c t e d b): the o r a l r o u t e (Tay & K, 1978).

Seafood and s e a w a t e r i n t h e c o - a s t a l a r e a s seem t o be t h e

r e s e r v o i r f o r y. parahaemoly t i cus . However, t h e microorganism

has been i s o l a t e d from r i v e r wa te r , pond w a t e r and from f r e s h

w a t e r f i s h i n I n d i a (De e t a l . , 1977). I t i s l i k e l y t h a t

deep oceans a r e con tamina ted by sewage ( B a r o s s e t a l . , 1975) .

There i s no e v i d e n c e t h a t asymptomatic c a r r i e r s ( food

h a n d l e r s ) a r e a s o u r c d o f t h e i n f e c t i o n i n any o u t b r e a k s (Blake , Weaver & H o l l i s , 1980). The m a j o r i t y o f t h e c a r r i e r s

were, however, found t o excrete serotypes d i f f erent from those

o f t h e i r index cases ( ~ i r c a e t a l . , 1979). I n any case,

no long-term carr iers of the microorganism have ever

been reported.

C L I N I C A L S I G N I F I C A N C X

G a s t r o e n t e r i t i s

y. parahaemolyticus-associated g a s t r o e n t e r i t i s i s a v e r y

common d i s e a s e a c c o u n t i n g f o r a t l e a s t two-percent o f food-

borne g a s t r o e n t e r i t i s i n some c o u n t r i e s (Mackowiak, 1974).

The c l i n i c a l syndromes are d e s c r i b e d f o r p a t i e n t s w i t h ' the

i n f e c t i o n . The first and t h e most commonly encoun te red

symptom is wate ry d i a r r h o e a , w h i l e t h e second is a d y s e n t e r i c

syndrome w i t h bloody s t o o l s ,

The c l i n i c a l m a n i f e s t a t i o n s o f t h e i n f e c t i o n i n e i g h t

o u t b r e a k s i n Uni ted S t a t e s i n c l u d e d d i a r r h o e a ( 9 8 % ) ,

abdominal cramps (82$), nausea (71%), v o m i t i n g (52%), headache

(42%), f e v e r (27%), and c h i l l s (24%). Temperature r a r e l y

exceeded 38.9'~ (Blake , Weaver & ~ o l ' l i s , 1980). The i l l n e s s

is u s u a l l y s e l f - l i m i t e d w i t h a median d u r a t i o n o f t h r e e days.

I n swvere c a s e s , d e h y d r a t i o n , hypo tens ion , and a c i d o s i s may

o c c u r ( ~ a z u m d e r e t a l . , 1977).

A d y s e n t e r i c syndrome w i t h mucoid o r sangu inous (b loody)

s t o o l s have been d e s c r i b e d i n s e v e r a l c o u n t r i e s . I n India,a

s t u d y showed' t h a t 25% o f s i x t y (60) h o s p i t a l i s e d p a t i e n t s w i t h

V parahaemolyticus-associated g a s t r o e n t e r i t i s had b lood and -*

mucus i n t h e i r s t o o l s ( ~ ~ r k a e t a l . , 1976). E i g h t p e r s o n s

i n v o l v e d i n a n o u t b r e a k i n Bangladesh (Hughes e t a l . , 1378)

had l i q u i d s t o o l s , s e v e r e abdominal cramps, nausea and vomit ing .

The s i x most s e v e r e l y ill p e r s o n s had g r o s s b lood i n t h e i r

s t o o l s . These s t o o l s c o n t a i n e d numeruus b lood c e l l s and

polymorphonuclear l e u c o c y t e s on mic roscop ic examinat ion . The

median d u r a t i o n o f i l l n e s s was 2.5 days.

Although 1. p a r a h a e m o l y t i c u s i n f e c t i o n s a r e u s u a l l y mi ld

o r moderate, s e v e r e c a s e s which r e q u i r e h o s p i t a l i s a t i o n do

occur . Deaths have n o t been r e p o r t e d i n some c o u n t r i e s l i k e

t h e Uni ted S t a t e s , b u t m o r t a l i t y r a t e o f 0.04% o u t o f 81 534

c a s e s i n J a p a n between 1965 and 1974 was recorded . Rehydra t ion

i s u s u a l l y t h e o n l y t r e a t m e n t needed, b u t i n s e v e r e c a s e s a n

a n t i m i c r o b i a l t h e r a p y may be b e n e f i c i a l , and t e t r a c y c l i n e

a p p e a r s t o be t h o d r u g o f c h o i c e .

The i n c u b a t i o n p e r i o d r a n g e s from 4 t o 96 h o u r s i n c a s e s

o f H. parahaerno lv t i cus g a s t r o e n t e r i t i s wi thou t bloody s t o o l s

(Barker , 1974; and Miwatani & Takeda, 1976), b u t i t i s s h o r t e r

i n g a s t o e n t e r i t i s w i t h b loody s t o o l and r a n g e s from 20 minu tes

t o 9 hours . The r e a s o n f o r t h e s h o r t e r i n c u b a t i o n p e r i o d i s . .

n o t y e t kxiown. The i n c u b a t i o n p e r i o d i s u s u a l l y ene t o two

days i n e x t r a i n t e s t i n a l i n f e c t i o n s .

E x t r a i n t e s t i n a l I n f e c t i o n s

The r o l e o f Y. p a r a h a e m o l y t i c u s i n i n f e c t i o n s o u t s i d e t h e

i n t e s t i n a l t r a c t i s u n c l e a r . Many a u t h o r s . r e f e r t o r a p i d l y

f a t a l sep t i cae rn ia o r wound i n f e c t i o n due t o t h e microor&mism,

b u t i t i s b e i n g s p e c u l a t e d t h a t t h e microorganism i n q u e s t i o n

may b e a c t u a l l y y . v u l n i f i c u s which was fo rmer ly i n c o r r e c t l y i d e n t i f i e d as x. parahaemoly t i cus . Twedt e t a l . (1969) were

t h e first t o r e p o r t t h a t 1. parahaemolyt icus might be t h e

cause of some e x t r a i n t e s t i n a l i n f e c t i o n s , bu t Zen-Yoji e t a l e

(1973) l a t e r found t h a t s i x o u t of t h e fou r t een organisms were

indeed Y. pa rahaeeo l s t i cug , Between 1970 and mid 1979, about

n ine c a s e r e p o r t s of e x t r a i n t e s t i n a l i n f e c t i o n s a s s o c i a t e d

wi th i s o l a t i o n o f 1, parahaemols t icus were publ i shed (Roland,

1970; von Graeveni tz & Carr ing ton , 1973; Zide, Davis &

- McSweeny & Forgan-Smith, 1977; Olsen, 1978; and Tay & Eta, 1978).

Hbwever, two of t h e s e were subsequent ly r epo r t ed t o be caused

by Y. v u l n i f i c u s (Roland, 1970; Zide, Davis & Ehrenkranz, 1974;

Weaver &. Ehrenkranz, 1975; and H o l l i s e t a l e , 1976). Tay

and Yu (1978) i s o l a t e d t h e microorganism from t h r e e blood I

c u l t u r e s taken from a p a t i e n t admi t ted i n h o s p i t a l f o r l i v e r

c i r r h o s i s and d ia r rhoea . Olsen (.1978) i s o l a t e d 1, parahaemolu-

t i c u s and ye a l a i n o l s t i c u ~ from t h e e a r o f two p a t i e n t s . I . .

S t e i n k u l l e k e t a l e (1980) r e p o r t e d t h e c a s e o f a hea l thy c h i l d

who developed x. parahaemolyt icus endophtha lmi t i s a f t e r

r e c e i v i n g a p e r f o r a t i n g c o r n e a l i n j u r y while swimming i n

t h e Gulf of ' ~ e x i c o .

Tison and- Kel ly (1 984) r epo r t ed two c a s e s of eye

i n f e c t i o n s whereas Farmer e t a l . ( 1985) r epo r t ed two c a s e s

i s o l a t e d from blood and e leven from wounds, The pathogenic

s i g n i f i c a n c e of . the microorganism i n some o f t h e c a s e s i s

u n c e r t a i n , b u t i t p robab ly caused some of t h e i l l n e s s e s ,

For i n s t a n c e , t h e microorganism was i s o l a t e d from t h e p u s

from a n i n f e c t e d f o o t l a c e r a t i o n i n p u r e c u l t u r e (Mc~weeny

& Forgangsmith, 1977) and from t h e s y n o v i a l f l u i d from a

p a t i e n t w i t h s y n o v i t i s o f t h e knee who had s u f f e r e d a punc tu red

wound n e a r t h e p a t e l l a ( P o r r e s & Fuchs, 1975).

g v e r s i n c e 1. p a r a h a e m o l y t i c u s was i n c r i m i n ~ t e d a s a n e

o f t h e m a j o r c a u s e s o f g a s t r o e n t e r i t i s i n some p a r t s o f t h e

wor ld , i t s mechanism o f p a t h o g e n i c i t y h a s been e x t e n s i v e l y

s t u d i e d u s i n g l i v e c e l l s and t o x i n s e l a b o r a t e d by t h e micro-

organism. However, i t i s n o t y e t u n d e r s t ~ j o d how t h e micro-

o r g a n i s m c a u s e s g a s t r o e n t e r i t i s ' i n humans. S t u d i e s have shown

t h a t p a t h o g e n i c mechanism c a n n o t b e a s c r i b e d t o a s i n g l e

t o x i n o r f a c t o r , b u t p e r h a p s t o a number o f f a c t o r s .

U n l i k e most g ram-negat ive . .. b a c t e r i a members o f t h e f a m i l y

Vib rdonaceae p ruduce s e v e r a l e x t r a c e l l u l a r enzymes and t o x i n s ,

some o f which are v i r u l e n c e f a c t o r s , d u r i n g g rowth ( T a b l e 1 ) .

S t r a i n s o f y. p a r a h a e m o l ~ t i c u s p roduce amylase ( i n d u c e d by

t h e p r e s e n c e o f s t a r c h , d e x t r i n , o r m a l t J s e i n t h e growth *

medium), b e t a - l a c t a m a s e , catalase,.DNAse, i n d o p h e n o l o x i d a s e ,

p r o t e a s e s , a n d p h o s p h o l i p a s e s (Tanaka e t a l . , 1969; and J o s e p h ,

C o l w e l l & Kaper , 1982) . Acid anu a l k a l i n e p h o s p h o l i p a s e s are . ,

produced. - I n d i v i d u a l s t r a i n s may p roduce b o t h t y p e s o f

p h o s p h a t a s e s s i g n i f y i n g e x t e n s i v e p h o s p h a t a s e a c t i v i t y by t h e

mic roo rgan i sm ( ~ a k a z a k i e t al. , 1972) .

Some s t r a i n s p r o d u c e c h i t i n a s e ( J o s e p h , C o l w e l l a n d Kaper ,

i 9 8 2 ) , s u p e r o x i d e d i s m u t a s e ( ~ a i l y , DeBel l & J o s e p h , l 982 ) ,

a s p a r t o k i n a s e (Baumann & Baumannk 1973) , a n d g l y c e r o p h o s p h o r ~ l

c h o l i n e e s t e r a ~ e (Yanagese e t a l . , 1370) . O t h e r s t r a i n s c a n

h y d r o l y s e Tween 80 ( C h a k r a b a r t y , Adhy & Pramanik, 1970; a n d

Table 1 , E x t r a c e l l u l a r P r o t e i n P r o f i l e o f V. parahaemolgticus. -

Enzymes

Amylase

C a t a l a s e

C h i t i n a s e *

Entero tox in *

Haernolysins

Lipase

Nuclease

Phosphatases . Phosphol ipases

P ro t eases

. , Superoxide dismutase

* Those a s s o c i a t e d with t o x i c a c t i v i t i e s .

J o s e p h , C o l w e l l & Kaper , 1982) .

T h e r m o s t a b l e a ~ l d t h e r m o l a b i l e h a e w l y s i n s have been

c h a r a c t e r i s e d i n 1. parahaernol .y t icus , b u t t h e e x i s t e n c e o f a n

e n t e r o t c l x i n h a s n o t been c o n c l u s i v e l y p roven . Kanagawa-

p o s i t i v e s t r a i n s o f t h e v i b r i o y i e l d b o t h t h e r m o s t a b l e and

t h e r m o l a b i l e t o x i n s from c u l t u r e s u p e r r i a t ~ n t s arkas as-~imsley,

S a k a z a h i a n d c u w o r k e r s (1968) were t h e f i r s t t,, a s s o c i a t e t h e

p a t h o g e n i c i t y o f 1. p r ; l r a h a e m o l ~ t i c u s w i t 1 1 t h e a b i l i t y o f t h e

~ n i c r o o r g a n i s m t o c a u s e h a e m o l y s i s on Wagatsuna a g a r ( i . e . t h e

Kanagawa phenmenon). They had o b s e r v e d t r l a t a b d u t 98% of

t h e s t r a i n s i s o l a t e d from p a t i e n t s w i t h g a s t r o e n t e r i t i s were

Kanagawa-pos i t i ve , whcreas o n l y o n e - p e r c c n t i s o l a t e d from raw

s e a f o o d s anu f r ..m seawater were Kanagawa-posi t i v e . S i ~ n i l a r

o b s e r v a t i o n s were l a t e r made by Cra'ig (1072) and by

Cherwonogrodzky and C l a r k ( 1 9 8 1 ) . Most i s o l a t e s from e x t r a -

i n t e s t i n a l , i n f e c t i o n s . , t e s t e d a t CDC were howiiver, found t o b e

Kmagawa-nega t ive e x c e p t t h e i s o l ; t e f r c , i ~ ~ a p a l i e n t w i t h

s e p t i c a e m i a wl-ose i l l n e s s began w i t h d i ~ r r h o e a (Tay & Lti, 1478).

S t u d i e s w i L h l i v e b a c t e r l . ~ ( S a k a z a k i e t a i . , 13'71) showed

t h a t a l a r g e r p e r c e n t a g e o f Kanagawa-po:-:i. t i v e s t r a i n : ; ( 8 8 6 )

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

t h e Kanagawa-negs t ive s t r a i n s (22';6), t i l e reby sugc;est . lng a

g r e a t e r v i r u l e n c e p o t e n t i a l ' f o r Kariagawa-posi t i v e st r a i n s .

C u l t u r e f i l t r a t e a n d whole -ce l l l y s a t e s from bo th s t r a i n s gave

n e g a t i v e r a b b i t i l e a l l o o p ( R I L ) t e s t ( Johnson & C a l i a , 1976).

Only c e l l - f r e e cuS.ture filtrate^ from b o t h Kanagwa-positive

anu Kanagawa-negative s t r a i n s c o n c e n t r a t e d by l y o p h i l i s a t i o n

produced p o s i t i v e HIL t e s t , b u t t h i s r e s p o n s e was a t t r i b u t e d

t o t h e h i g h ( l e s s t h a n 20%) c o n c e n t r a t i o n o f sodium c h l o r i d e

produced by t h e c o n c e n t r a t i o n method (Johnson 1L Calia, 1976).

L ive c e l l s of 1. p a r a n a e m o l y t i c u s do not. i n d u c e i n t e s t i n a l

f l u i d accumula t ion i n i n f a n t r a b b i t s ( C a l i a & Johnson, 19')5).

The r e s u l t s ofHonda e t a l . (1983) show t h a t accumula t ion

o f f l u i d induced by 1. p a r a h a e m o l y t i c u s i s n o t d i r e c t l y r e l a t e d

t o t h e TDH. N e i t h e r anti-TDH n o r a n t i - c h o l e r a t o x i n (CT) was

a b l e t o p r e v e n t accumula t ion of f l u i d caused by l i v i n g c e l l s

o f t h e v i b r i o ( a p p r o x i m a t e l y 5 x lo8 co lony forming u n i t s - c.f .u.) . S i m i l a r r e s u l t s had been o b t a i n e d by Cherwonogrodzky

& C l a r k (1981 and 1982). S e c t i o n s of t h e g u t l o o p o f a

p o s i t i v e t e s t r e v e a l e d mucosal d i s a r r a y w i t h n e c r o s i s ,

u l c e r a t i o n n a n d haemorrhage. . . The Karlagawa phenomenon i s caused by a t h e r m o s t a b l e d i r e c t

haemolysin (TDH) which p o s s e s s e s such v i r u l e n t f a c t o r s as

l e t h a l a c t i v i t y t o mice ( Z e n - ~ o j i e t a l . , 1971; and Monda e t

a l . , 1976) , c y t o t o x i c i t y t o human f o e t a l l u n g (FL) c e l l s i n

c e l l c u l t u r e (Honda e t a l . , 1976) , and c ~ r d i o t o x i c a c t i v i t y

i n mice in y i v o and v d t r o (Ilonda e t a l . , 1976). The TDH

i s a p r o t e i n f r e e of o r g a n i e , phosphorouc, c a r b o h y d r a t e s and

26. l i p i d s (Zen-Yoji e t a l . , 1971; and M i y a m t o e t a l . , 1981) and

w i t h a m o l e c u l a r weight o f a b o u t 42 000 d a l t o n s (Honda e t a l . ,

1976; and Miyaltloto e t a l . , 1981). I t i s haemoly t i c t o e r y t h r o -

c y t e s from some m a m m a l s , namely, ra ts , dogs , humans, monkeys,

g u i n e a - p i g s , c k i c k e n s . r a b b i t s , mice , and sheep . Horse e r y t h r o -

c y t e s a r e n o t l y s c d (Zen-Yoji e t a l . , 1971 ) . The t o x i n h a s

a minimal l e t h a l d o s e o f a b o u t 5.0 ug p e r mouse, k i l l i n g t h e

a n i m a l w i t h i n one minu te (Honda e t a l . , 1976). Both t o x i c

and h a e m o l y t i c p r o p e r t i e s a r e t h e r r n o s t a d e a t 100'~ f o r t e n

minutes .

D e s p i t e t h e h i s t o l o g i c a l changes obse rved i n a c u t e l y

ill p a t i e n t s , t h e c l i n i c a l i m p o r t a n c e of t h e TUH is n o t y e t

under s tood (Honda, Takeda & Miwatani , 19'77).

The e x i s t e n c e of' a n e n t e r o t o x i n i n x. p a r a n a e m o l y t i c u s i s n o t rn

y e t c o n c l u s i v e l y proven, even though a y. parahae rno ly t i cus -

a s s o c i a t e d d i a r r h o e a was a t t r i b u t e d t o a n e n t e r o t o x i n e l a b o r a t e d

i n t h e small i n t e s t i n e ( C r a i g e t a l . , 19'?2). H e a t - l a b i l e f a c t o r & , ,

have been d e t e c t e d which c a u s e d d i l c l t i o n o f t h e r a b b i t g u t

l o o p , and in f l ammatory changes i n t h e i l l - t e s t i n a l mucosa

i r e e s p e c t i v e o f t h e Kariagawa r e a c t i o n oi' t h e stra n ( S a k a z a k i .

e t a l . , 197i+), and which produced a r e ~ c t i o n in Chinese Hamster

Ovary (CMO) ce-11s similar t o t h a t produced by t h e c h o l e r a

t o x i n (CT) ( ~ o n d a e t al.,, 1976). These r e p o r t s sup;gest t h e

p r e s e n c e of e n t e r o t d x i n - l i k e s u b s t a n c e / s produced by t h e v i b r i o

27. r e g a r d l e s s o f t h e Kunagawa r e a c t i o n , a n d which may p l a y a

r o l e i n i n d u c i n g g a s t r o e n t e r i t i s anu d i a r r h o e a . Mowevcr,

s t u d i e s have shown absence o f e n t e r o t o x i n i n c u l t u r e f i l t r a t e s

o f t h e microorganism i s o l a t e d from p a t i e n t s w i t h g a s t r o -

e n t e r i t i s (Donta & Smith, 1974).

I n v a s i v e n e s s

There i s ev idence t h a t 1. p a r a h a e m o l y t i c u s i n v a d e s t h e i n t e s t i n a l

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

(Bolen , Zamiska & Greenough, 1974) and l e u k o c y t e s (Hughes, Boyce

& A l l e n , 1978). Live c e l l s , however, a r e r e q u i r e d f o r e n t e r o -

p a t h o g e n i c i t y as non-v iab le c e l l s have no e f f e c t on t i s s u e s

( C a r r u t h e r s , 1975).

Although b o t h Kanagawa-posi t ive and Kanagawa-negative

s t r a i n s a r e Sereny t e s t n e g a t i v e ( ~ a l i a & Johnson, 1975),

b r o t h c u l t u r e s o f bo th s t r a i n s can p e n e t r a t e t h e i n t e s t i n a l I

e p i t h e l i u m ( ~ a l i a & Johnson, 1975; C a r r u t h e r s , 1975; Bout in

e t a l . , 1978,; and Joseph e t a l . , 1978) l e a d i n g t o bac te remia

and d i a r r h o e a ( J o s e p h e t a l . , 1978) and i n some c a s e s wi thou t

d i a r r h o e a ( C a r r u t h e r s , 1975). C a l i a and Johnson (1975) obse rved

t h a t Kanagawa-positive s t r a i n s , b u t n o t Kanagawa-negative

s t r a i n s can p e n e t r a t e t h e i n t e s t i n a l e p i t h e l i u m o f i n f a n t

r a b b i t s . Both s t r a i n s a r e , however, c y t o t o x i c tc, lleLa c e l l

c u l t u r e s , a l t h o u g h Kanagawa-positive s t r a i n s a c t more r a p i d l y

( C a r r u t h e r s , 1975). His f i n d i n g s s u g g e s t t h u t t h e p r e s e n c e o f

l i v e microorganism may b e r e q u i r e d f o r i t s e n t e r o p a t h o g e n i c i t y

28.

a s non-viable c e l l s have no e f f e c t or1 HeLa c e l l s .

D i r e c t t r a n s d c r m a l i n v a s i o n o l' t i s s u e s o t h e r than t h e

i n t e s t i n e have been obse rved and a r e a s s o c i a t e d wi th r e c r e a t i o n a i

ext;osure i n f r e s h o r s a l t w a t e r ( Z i d e e t a l , , 1974). I t i s

n o t known whether i n v a s i o n was due t o unique h o s t s u s c e p t i -

b i l i t y , t h e i n g e s t i o n of a l a r g e number o f microorganism o r

due t o an u n c o m o n l y v i r u l e n t s t r a i n .

R e s u l t s have c u n s i s t e n t l y shown t h a t k e r a t o c o n j u c t i v i t i s

( S e r e n y - t e s t ) i s n o t induced (Barrow 6 Miller, 1974; Sakazaki

e t al . , 1974; anci Twedt, Brown & Zink, 19U1). However,

bac te remia was o b t a i n e d a f t e r i n t r a g a s t r i c c h a l l e n g e of

s u c k l i n g r a b b i t s wi th Kanagawa-posi t ive b r o t h c u l t u r e s o f t h e

microorgani .m. The bac t e r e m i a was preccbued by pene t ra t : iun

o f t h e i n t e s t i n a l l . e p i t h e l i u m a t a n unknown s i t e . No d i r a r r h o e a w

was no ted d u r i n g t h e p e r i o d o f o b s e r v a t i o n . Joseph e t a l , w

( 1978) demonst ra ted bac t e r e m i a and d i a r r h o e a 5.5 h o u r s a f t e r

t r a n s i l e a l . i n 'ocu la t ion o f a b r o t h c u l t u r e of a Karlagawa-

n e g a t i v e s t r a i n s c m p e n e t r a t e t h e l amina p r o p r i a o f r a b b i t

i n t e s t i n e (Bout in e t a l . , 1978).

Adhesion .

Kanagawa-positive s t r a i n s o f 1. pc;\rbk!acln~:iyi;icus a r e u c u u i l y

c o n s i d e r e d v i r u l e n t ; y e t a s i g n i f i c a n t p o r t l o n o f 1. purahaemo-

I s t i c u s - a s s o c i a t e d f o o d p ~ i s ~ ~ n i n g s seem L o be caused by

Kanagawa-negative s t r a i n s a s w e l l (Hackney e t a l . , 1980).

Kanagawa-negative s t r a i n s do n o t a d h e r e a t a l l t o HeLa c e l l s

29

( C a r r u t h e r s , lYJ)'7).

The d e g r e e o f v i t r o adherence i s r e l s t e d t o a g e of, t h e

c u l t u r e , t h e Kanagawa r e a c t i o n and s o u r c e of t h e s t r a i n , l e n g t h

o f t ime o f exposure t o t h e t i s s u e c u l t u r e c e l l s , and t o t h e

compos i t ion o f t h e growth medium. Cells h a r v e s t e d d u r i n g

t h e l a t e l o g phase of growth adhered more i n t e n s e l y t h a n t h o s e

ha rves ted . from l a t e s t a t i o n e r y phase. S t r a i n s i s o l a t e d from

p a t i e n t s w i t h foodpo i son ings ( i . e . Kanagawa-positive o r

Kanagawa-negative s t r a i n s ) adhered more i n t e n s e l y t h a n Kanagawa-

n e g a t i v e s t r a i n s i s o l a t e d from sea foods . Converse ly , Kanagawa-

p o s i t i v e s t r a i n z i s o l a t ' e d from s e a f o o d s adhered s t r o n g l y t o

t h e HFI c e l l s .

The p r e s e n c e o f f e r r i c i o n i n t h e growth medium i n c r e a s e d

t h e i n t e n s i t i e s o f t h e v i r u l e n t s t r a i n s , because d e c r e a s i n g

t h e n e g a t i v e s u r f a c e c h a r g e ( a s dofle by t h e f e r r i c i o n s )

enhances adherence ( C a r r u t h e r s & Aaderson, 19'79). Adherence

o f t h e microorganism t o t i s s u e c u l t u r e c e l l s depcnds on t h e . ,

v i a b i l i t y o'f t h e b a c t e r i a l c e l l s , and i s improved by t h e

m o t i l i t y o'f t h e a d h e r e n t c e l l s ( J o s e p h & Merre l , 1979; and

Shinoda e t a l . , 1979).

P u r i f l e d TDH b i n d s t o a wide v a r i e t y o f c u l t u r e d c e l l s ,

b u t p r e i n c u b a t h n o f haemoly t i c s t r a i n s wi th a n t i s e r u m t o

TDH do n o t b l o c k t h e i r adherence . TDH, t h e r e f o r e , i s n o t an

adherence f a c t o r , I n a d d i t i o n , Kanagawa-negative s t r a i n s

which do n o t produce TDH do b ind t o humm arid i n t e s t i n a l c e l l s

go . a n d t o HeLa c e l l s ( C a r r u t h e r s , 1977; J o s e p h e t a l e , 1978;

G i n g r a s & Howard, 1980; a n d I i j ima, Yamoda & Sh inoda , 1981) .

S p e c i f i c a d h e s i n s have s o f a r n o t been d e m o n s t r a t e d i n

V . p a r a h a e m o l y t i c u s even t hough n o n - f i m b r i a l h a e m a g g l u t i n i n s - have been o b c e r v e d i n v i b r i o strams r e l a t e d t o 1. parahaemo-

l y t i c u s ( O i s h i e L a l e , 1979) . E l e c t r o n ni icroscopy r e v e a l e d

t h e p r e s e n c e o f f i m b r i a e on t h e v i b r i o c e l l s ( F a r i s e t a l e ,

1982) . V i b r i o h a e m a g g l u t i n i n s were found t o be e a s i l y d e t a c h e d

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

h y d r o p h o b i c i t y d e c r e a s i n g s i m u l t a n e o u s l y ( J o s e p h e t a l . , 1978) .

A l though a d h e s i v e p r o c e s s e s c o u l d be i n v o l v e d i n d i s e a s e

c a u s e d by ye p a r a l i a e m o l y t i c u s , no c o n c l u s i v e r e s u l t have been

o b t a i n e d on i t s r u l e as a p r e d i c t o r of p a t h o g e n i c i t y

(Reyes e t a l e , 1903) . B

D e s p i t e t h e e x t e n s i v e s t u d i e q on t h e p a t h o g e n i c

mechanisms o f ye p a r a h a e m o l y t i c u s , i t i s n o t y e t u n d e r s t o o d . ,

how i t c a u s b s g a s t r o e n t e r i t i s i n humans. Again, t h e r e l a t i o n s h i p

be tween e n k e r o p a t h o g e n i c i t y and haemolys in p r o d u c t i o n h a s n o t

been d e t e r m i o e d . Moreover , i t i s n o t l i k e l y t h a t b o t h

e n t e r o t o x i c . and h a e m o l y t i c a c t i v i t i e s s h o u l d r e s i d e i n t h e

same molecule , . tliat i s , on t h e 'TDH mo lecu le .

THERMOSTABLE DIRECT HAEMOLYSLNS

The a b i l i t y of Y, parahaemoly t icus t o produce a haemolysin

was f i r s t de sc r ibed by Fu j ino and h i s a s s o c i a t e s i n 1953. The

haemolysin, which could be d e t e c t e d from c u l t u r e f i l t r a t e s i s

thermostaet 'e, f r e e of pwlysacchar ides and l i p i d s , and i s l e t h a l . . .- .

- . f o r mice (MLD 5 ug o f t b x i n per -mouse) (Kato e t a l . , 1966;

Sakura i e t a l e , 1973; Honda e t a l e , 1976; and Miyamoto e t a l e ,

1980). The molecu la r weight r anges f r m 42 000 t o 44 000

d a l t o n s (Honda . .. e t a l e , 1976; Takeda e t a l e , 1978; and Miyamoto

e t a l e , 1980), a l t hough i n a n e a r l i e r s t u d y 118 000 d a l t o n s

( s a k u r a i e t a l e , 1973) were recorded, The haemolysin i s a

s ing1e .pro te i .n (Sakura i e t a l . , 1973) made up of two p r o t e i n

s u b u n i t s (Hyamoto e t al., 1980) and c o n t a i n i n g a c i d i c amine

a d i d s (43%) and b a s i c amino a c i d s (11%)(Sakura i e t a l . , 1973). u

The N-terminal amino a c i d is phenyla lan ine wi th e s o e l e c t r i c

p o i n t o f 4.2 o r 4.9 (Zen-Yoji e t a l e , 1975; Honda e t a l e , 1976; . ,

Takeda e t el. , 1978; and Miyamoto e t a l , , 1980). The p u r i f i e d A

haemolysin is i n a c t i v a t e d . by chymotrypsin and pepsin ( p r o t e o l y t i l

enzymes), bu t c o n t r a d i c t o r y ' r e s u l t s ' r egard ing i n a c t i v a t i o n by

t r y p s i n a r e ob ta ined ,

The producttm o f t h e haemolysin i n pH dependent. Maximal

p roduc t ion o c c u r s a t a pH range of 5,5 t o 6.5, 6.2 be ing op t imal

Mannitol , t h e s u g a r used i n Wagatsuma a g a r , lowere t h e pH of t h e

c u l t u r e medium to t h i s range. Other fermentable s u g a r s , g lucose

and mannose, lower t h e pH below t h i s range and, t h e r e f o r e ,

i n h i b i t growth anu haemolysin p r o d u c t i o n (Cherwonogrodzky

& C l a r k , 1981).

High c o n c e n t r a t i o n o f s a l t i n c o r p o r a t e d i n t o c u l t u r e media

promotes i n c r e a s e d haemolysin p r o d u c t i o n . C l e a r l y d e f i n e d

haemolys i s c o u l d be obse rved i n b l ~ o d a g a r wi th a h i g h s a l t

c u n t e n t , b u t n o t i n t h e same medium c o n t a i n i n g a low c o n c e n t r a t i o

o f sa l t ( s a y 3% NaCl)(Chun e t m t a l . , 1975).

Kanagawa-negative s t r a i n s O ' d o n o t e x p r e s s d e t e c t a b l e

haemolysin under t h e s e c o n d i t i o n s .

B i o l o ~ < i c a l a c t i v i t i e s

Haemolysis

Haemolysis was t h e f i r s t of t h e b i o l o g i c a l a c t i v i t i e s o f t h e

t h e r m o s t a b l e d i r e c t haemolysin (TDH) t o be r e c o f n i s e d (Zen-

Y o j i e t al. , 1971). S e n s i t i v i t y q f t h e d i f f e r e n t mammalian

c e l l s t o t h e haemolys in v a r i e s , t h e most s e n s i t i v e b e i n g rat

e r y t h r o c y t e ~ s , ' f o l l o w e d by dog, mouse, monkey, man, r a b b i t and

guinea-pig. Horse e r y t h r o c j t e s were n o t l y s e d due t o t h e

p resence ofsome g a n g l i o s i d e s .

The h a s m o l y t i c a c t i v i t y is temperatue-dependent wi th

maximal l y s i s o c c u r r i n g a t 37'~. A t low t e a p e r a t u e s (0 - ~ O C ) ,

t h e haemolysin does n o t c a u s e l y s i s , b u t b i n d s t o e r y t h r o c y t e

membrane ( ~ a k u r a i e t a l . , 1975). The b i n d i n g i s s t i m u l a t e d

i n t h e p r e s e n c e o f some d i v a l e n t c a t ~ o n s , namely, ca lc ium,

manganese, arximagnesium i o n s , b u t i s i n h i b i t t e d by z i n c

(Avigard & Bernheimer, 1976; anu Takeda, Ogiso & M i w a k n i , 1977).

Haemolysis i s a c h i e v e d i n two s t e p s , namely a d s o r p t i o n

fo l lowed by l y s i s ( S a k u r a i e t a l . , 1975). Anti-haemolysin

serum i n h i b i t e d t h e haemoly t i c a c t i v i t y of t h e TDH by

p r e v e n t i n g a d s o r p t i o n o f t h e haemolysin t o t h e e r j l t h r o c y t e

membrane, However, t h e i n h i b i t o r y e f f e c t o f t h e a n t i s e r u m

d imin i shed (as shown by p a r t i a l l y s i s ) w i t h d e l a y i n t h e t ime

o f a d d i t i o n o f t h e a n t i s e r u m . The i n h i b i t o r y e f f e c t o f t h e

a n t i s e r u m a l s o d e c r e a s e d w i t h i n c r e a s e i n t h e c o n c e n t r a t i o n

o f d i v a l e n t c a t i o n s , i n c r e a s e i n * i n c u b a t i o n p e r i o d , and w i t h

t h e d e c r e a s e i n pH.

C y t o t o x i c i t y

The c y t o t o x i c e f f e c t s o f TDH h a s been s t u d i e d i n s e v e r a l c e l l

sys tems such as H i L a c e l l s (Sakazak i e t a l . , 1974). mouse

melanoma c e l l s , B-16 c e l l s , human Bpidermoid carcinoma, KB c e l l s ,

f o e t a l mouse myocard ia l c e l l s , (Goshima e t a l . , 1978; and Goshim&

e t a l . , 197,8),, human i n t e s t i n a l CCL6 c e l l s ( ~ a k e d a , Honda &

Takeda, 1980) , and i n f o e t a l l u n g c e l l s ( S a k u r a i e t a l . , 1975).

Exposure o f FL c e l l s t o 5.ug o f TDH caqoed t h e comple te

d i s a p p e a r a n c e o f m i c r o v i l l i w i t h i n 10 minu tes o f exposure .

A f t e r 60 minu tes , . t h e c e l l s u r f a c e had d e g e n e r a t e d , and more

t h a n 95% o f t h k c e l l s had l o s t v i a b i l i t y as measured by t r y p a n

b l u e s t a i n i n g and release o f a l k a l i n e phospha tase from t h e c e l l s .

S i m i l a r changes were oTbserved i n t h i n s e c t i o n s o f t h e FL

c e l l s as w e l l . Within 5 minutes o f exposure , t h e morphology

o f t h e m i c r o v i l l i had changed compared t o c o n t r o l c e l l s , b u t

t h e cytoplasm was u n a f f e c t e d . A f t e r 30 minu tes , t h e m i c r o v i l l i

had d i s a p p e a r e d and t h e n u c l e u s had d i s i n t e g r a t e d . By 60

minutes , t h e cytoplasm had d e g e n e r a t e d and t h e n u c l e u s was no

l o n g e r v i s i b l e .

Goshima e t a l . (1978) s t u d i e d t h e morpho log ica l changes

i n t h e cytoplasm o f mouse myocard ia l c e l l s . Exposure o f t h e

c e l l s t o j a g m l - I o f TDH induced r a p i d f o r m a t i o n o f small

b a l o o n s a l o n g t h e c e l l b o r d e r which g r a d u a i l y e n l a r g e d and

fused. The c e l l s t h e n were condensed i n some c e l l s . S i y i l a r

changes were obse rved i n mouse melanoma c e l l s whereas t h i c k

myocardia l c e l l s were a f f e c t e d t o a l e s s e r e x t e n t .

Exposure o f the c e l l s t o t h e ionophore A23187 induced

morpho log ica l ly i d e n t i c a l changes i n myocard ia l as w e l l as i n

m8lanoma c e l l s (Goshima e t a l . , 1978) . The p r e s e n c e o f 1 0 - 6 ~

++ C a i n t h e c e l l medium was e s s c n t P a 1 f o r TDH-induced c e l l

d e g e n e r a t i o n . Exposure o f t h e c e l l s t o TUII ( o r ionophore . ,

A23187) caused an e x c e s s u p t a k e o f 4 5 ~ a + + , b u t t h e r a t e o f

B 5 ~ a + + e f f l u x was n o t a f f e c t e d .

S i m i l a r morpho log ica l changes i n myocard ia l c e l l s

o c c u r r e d when t h e c o n c e n t r a t i o n of ~ a + + i n t h e c e l l medium

i n c r e a s e d f r o a I O - ~ M t o normal c o n c e n t r a t i o n (1.8 x 10-%I)

(Goshima e t a l . , 1978). , C u l t u r e d myocard ia l c e l l s c o n t r a c t e d

f u l l y i n t h e p r e s e n c e of TDHjl aa a n e f f e c t o f e x c e s s u p t a k e o f

ca++ The c o n c l u s i o n t h e r e f o r e , is t h a t TUIi i n c r e a s e s t h e p e r

p e r m e a b i l i t y o f t h e c e l l membrane of s u s c e p t i b l e c e l l s t o c a t + ;

and t h e morpho log ica l changes i n t h e cy top lasm b e i n g caused by

e x c e s s up take o f ~ a + + i o n s . O n t h e c o n t r a r y , t h e changes

observed i n t h e n u c l e i o c c u r r e d a l s o i n c a t + d e p l e t e d medium.

Non-muscular c e l l s such as HeLa, KB, and B-16 c e l l s responded

t o ionophore ~ 2 3 1 8 7 and t o TDH w i t h morpho log ica l cha1:ges i n

t h e cytoplasm, b u t i n t h e absence o f i n c r e a s e d c o n c e n t r a t i o n

o f cat+ i n t h e medium..

C a r d i a t o x i c e f f e c t

F u j i n o and h i s coworkers (1953) observed t h a t e x p e r i m e n t a l

V . p a r a h a e m o l y t i c u s i n f e c t i o n was l e t h a l f o r mice. Subsequent - s t u d i e s showed t h a t t h e p u r i f i e d TDH is l e t h a l t o small a n i m a l s

when a d m i n i s t e r e d i n t r a v e n o u s l y , i n t r a p e r i b n n e a l y , o r o r a l l y

(Obara e t al . , 1974; Zen-Yoji e t al:, 19,74; and Honda e t alms

1976). Death o c c u r r e d one minute a f t e r i n t r a v e n o u s i n j e c t i o n

o f 5 .ug o f TDH. E l e c t r o c a r d i o g r a m and e lec t roencepha logram . ,

r e c o r d i n g ,dur ing an exper iment i n which 7.5 rug o f TDH was i n j e c t e

i n t o a rat . weighing 450 g showed i n c r e a s e i n wid th and h e i g h t o f

t h e P wave a f t e r 15 seconds . A f t e r 17 t o 18 seconds , t h e PQ

i n t e r v a l s became- longer . A f t e r 41 seconds , t h e r a t e of h e a r t

b e a t d e c r e a s e d r a p i d l y fo l lowed by v e n t r i c u l a r f l u t t e r . The

h e a r t s t o p p e d b e a t i n g a f t e r 148 seconds . Some changes were

a l s o r e c o r d e d i n b o t h P and Q waves i n p a t i e n t s w i t h a c u t e 1.

p a r a h e e m o l v t i c u s g a s t r o e n t e r i t i s (Honda e t a l . , 1976). I t i s

l i k e l y t h a t TDII, a f t e r i t i s absorbed from t h e i n t e s t i n e ,

c i r c u l a t e s i n t h e b lood s t r e a m , and may c a u s e t h e r e c o r d e d

changes i n e l e c t r o c a r d i o g r a m s .

. . The e f f e c t o f TDH on c u l t u r e d m y o c a r d i a l c e l l s was a l s o

s t u d i e d (Honda e t a l . , 1976). When added I n low doses , t h e

TDH is i n a c t i v a t e d i n t h e medium d u r i n g t h e i n c u b a t i o n p e r i o d .

When 0.0 - 0.20 wg rnl- ' o f t h e TDH was added t o t h e myocardia l

c e l l s , t h e b e a t i n g o f t h e c e l l s i n c r e a s e d r a p i d l y b u t s topped

suddenly a f t e r one m.inute. A f t e r a b o u t f i v e mvnutes, t h e c e l l s

s t a r t e d t o c o n t r a c t a g a i n a t a normal r a t e . Larger d o s e s

( abou t 1 d g ) on t h e o t h e r hand, cause r a p i d d i s i n t e g r a t i o n

o f t h e myocardia l c e l l s . The e f f e c l ; o f t h e TDH on t h e c o n t r a c t i l i t

o f myocard ia l c e l l s is, however, r e v e r s i b l e (Goshima e t al . , 1978).

The p u r i f i e d TD1.I c a u s e s d e g e n e r a t i o n o f c e l l shape as

evidenced by b l e b and Baloon forrnat ian o f mouse myocard ia l and n

mouse melanoma c e l l s i n c u l t u r e (Goshima e t a l . , 1978). The

p resence o f e x t r a c e l l u l a r ca lc ium i o n s ( c a t + ) wi th c o n c e n t r a t i o n . .

g r e a t e r t h a n q 1 0 - 6 ~ is n e c e s s a r y f o r t h e d e g e n e r a t i o n o f c e l l

shape . The p r e s e n c e o f e x t r a c e l l u l a r magnesium (Mg), sodium

(Na) , and potass lum (K) i o n s , on t h e o t h e r hand, do n o t have

any e f f e c t on c e l l shape . However, t h e d e p o l a r i s a t i o n of t h e

maximal d i a s t o l i c p o t e n t i a l and t h e r e d u c t i n of membrane

r e s i s t a n c e i n r a b b i t h e a r t c e l l s caused by t h e TDH may be due

t o i n c r e a s e d p e r m e a b i l i t y o f t h e c e l l membrane t o Na i o n s

(Seyama e t a l , , 1977; and Goshima e t a l . , 1978).

I n t h e p resenc e o f C a i o n s , TDH c a u s e s f u l l c o n t r a c t i o n

o f m y o f i b k i l s o f mouse myocard ia l c e l l s as w e l l as t h e r e d u c t i o n

o f bo th a c t i n c a b l e s and t u b u l i n ne tworks of mouse melanoma

c e l l s . The TDH, however, does n o t have any e f f e c t on c h i c k

myocard ia l c e l l s which n e i t h e r shows d e g e n e r a t i o n o f c e l l shape

n o r f u l l c o n t r a c t i o n o f m y o f i b r i l s as a r e s u l t o f t h e i r i n a b i l i t y

t t a k e up e x c e s s 4 5 ~ a i o n s . TDH-induced d e g e n e r a t i o n o f

c e l l shape o f mouse myocard ia l c e l l s and mouse melanom c e l l s

i s due t o t h e e x c e s s u p t a k e o f C a i o n s (Goshima e t a l . , 1978) .

En te ropa thoaen ic e f f e c t

The e n t e r o p a t h o g e n i c i t y o f 1. p a r a h a e m o l ~ t i c u s i c w e l l e s t a b l i s h e s

(Ljungh & Wadstrom, 1 9 8 3 ) ( u n p u b l i ~ h e d d a t a ) ; haemoly t i c s t r a i n s

7 o f t h e microorgunibm.-(about 2 x lo5 t o 3 x 10 c . f a ) induced

9 a d i a r r h o e a whereas non-haemolytic s t r a i n a ( I @ - t o 0'' c . f .u. )

d i d n o t ( ~ a k a z a k i e t a l . , 1974; a n d ' s a n y a l & Sen, 1974) . 8

Exper imenta l d i a r r h o e a was induced i n monkeys as w e l l a s mice

a f t e r o r a l c h a l l e n g e . . w i t h p u r i f i e d .TDH (Obara . . . e t a l . , 1974; and

Miyamoto e t ' a l . , 1980). H i s t o p a t h o l o g i c a l examina t j on o f t h e

i n t e s t i n a l wall o f a n i m a l s w i t h d i a r r h o e a showed oedema o f lamina

p r o p i a , and w i t h l a r g e r d o s e s , d e s t r u c t i v e changes o f t h e

endoplasmic r e t i c u l u m , d e g e n e r a t i o n o f m i c r o v i l l i , and s w e l l i n g

o f mitonhondri'a. Vacuoles were obse rved .in t h e cytoplasm,

and i n some e p i t h e l i a l c e l l s t h e n u c l e i showed d e g e n e r a t i v e

changes. A n a l y s i s o f t h e i r i t e s t ~ n e o f a s u r v i v i n g an imal

showed s t r o n g mucosalconges t ion w i t h procounced in f l ammatory

r e a c t i o n (Obara e t a l . , 1974).

I n o c u l a t i o n o f TDH i n l i g a t e d r a b b i t i n t e s t i n a l l o o p

i n d x e s f l u i d accumula t ion w i t h mucosal i n j a r y l i k e c h o l e r a

t o x i n . I n j e c t i o n o f l a r g e d o s e s (125 t o 500 ug ), however,

caused accumula t ion o f t u r b i d , s a n g u i n o l e n t f l u i d , accompanied

by e r o s i v e l e s i o n s i n t h e mucosa and n e u t r o p h i l i n f i l t r a t i o n

of t h e i n t e s t i n ~ l w a l l (Zen-Yoji e t a l . , 1974; unu Miyamoto

e t a l . , 1980).

S k i n P e r m e a b i l i t y E f f e c t

Miyamoto e t a l . (1980) obse rved t h a t p u r i f i e d TDH induced

i n d u r a t i o n and b l u i n g i n t h e gu inea -p ig s k i n wi th rnax4mal

ef fec l , a f t e r 3 and 4 h o u r s r e s p e c t i v e l y . The e f f e c t was n o t

n e u t r a l i s e d by a n t i - c h o l e r a g e n , b u t by a n t i s e r u m t o T U H .

C e l l Membrane Recep to r f o r TDH

The haemoly t i c a c t i v i t y o f TDH was d e c r e a s e d by p r i o r 9

i n c u b a t i o n o f TDII wsth g a n g l i o s i d e GT1 an t i t o a l e s s e r e x t e n t

by g a n g l i o s i d e GDla; b u t n o t by g a n g l i o s i d e s GM1 o r GM2 . ,

(Takeda e t a i . , 1975; and Takeda e t a l . , 1976). H o r s e ' e r y t h r o -

c y t e s l a c k g a n g l i o s i d e GT1 and G D I a , hence t h e y were n o t l y s e d

by TDH. P r i o r i n c u b a t i o n o f t h e g a n g l i o s i d e s w i t h neuramini -

d a s e i n h i b i t e d t h e i r e f f e c t on TDH. I t w a s t h u s concluded t h a t

the. c e l l rnernbrqne r e c e p t o r f o r TDH i s a n e u r a m i n i d a s e - s e n s i t i v e

g a n g l i o s i d e r e l a t e d t o GT1 (Takeda e t a l . , 1975). The l e t h a l

e f f e c t of TDH i n mice was l i k e w i s e i h h i b i t e d by p r i o r i n c u b a t i o n

o f TaH. w i t h GT1 (Takeda e t al . , 1975). Chick m y o c a r d i a l c e l l s

were l e s s s u s c e p t i b l e t o TDH t h a n r a b b i t myocardia l c e l l s which

39

c o u l d be due t i , t h e p r e s e n c e of few r e c e p t o r s on t h e s u r f a c e ,

o r t o t h e presc n c e o f r e l a t e d g a n g l i o s i d e s w i t h l o w e r a f f i n i t y

f o r TDH (Goshima e t a l . , 1978).

Ph.ysicochemica1 19-0 p e r t i e s o f TIM

Miyarnoto e t a l . ( 1980) d e s c r i b e d a s i m p l i f i e d p u p i f i c a t i o n

p r o c e d u r e f o r 'TDH which i n v o l v e s t h e f o l l o w i n g :

1 . a d s o r p t i o n o n t o DGAE-sepharose

2. a c i d p r e c i p i t a t i o n

3 . chromatograph on sephadex G-200, and

The m o l e c u l a r weight of TDII was found t o be 42 000 o r 44 000.

The molecu le i s composed o f two i d e n t i c a l s u b u n i t s (Honda e t

a l . , 1976; Takeda e t a l . , 1978; and Miyamoto e t a l . , 1980) . m

The amino a c i d cdrrlposit ion was r e ~ o r t e d by t h r e e g r o u p s w i t h

similar r e s u l t s en-Yoji e t al . , 1975; .I.Ionda e t a l . , 1976;

Takeda e t al., 1978; and Miyamoto e t a l . , 1980). The

N- terminal a c i d i c p h e n y l a l a n i n e . The i s o e l e c t r i c p o i n t o f

t h e molecu le i s 4.2 o r 4.9.

P u r i f i e d '1'1)li is i n a c t i v a t e d by p e p s i n and chyanotrypsin

b u t c o n t r a d i c t o r y r e s u l t s regarding i n a c t i v a t i o n by

t r y p s i n were b b t n i n e d .

, 4 0 A r r h e n i u s Ef f e c L

Takeda e t a l . (19'74) d e t e c t e d a f a c t o r i n 1. p u r a h a e m o l y t i c u s

which i n a c t i v a t e d t h e h a e m o l y t i c a c t i v i t y of a p u r i f i e d '1'Dli.

The i n a c t i v a t i n g f a c t o r was a s s o c i a t e d w i t h t h e haemolys in ,

b u t c o u l d b e s e p a r a t e d from i t by d i e t h y l - a m i n o - e t h y l ( D L A ~ )

c e l l u l o s e column chromatography. The i n a c t i v a t i n g f a c t o r was

s c t l v a t e d by h e a t i n g a t 50 '~ t o 60°c, b u t was i t s e l f thermo-

l ~ b i l e and l o s t a c t i v i t ~ on h e a t i n g a t 70 - 1 0 0 ~ ~ . Ohta (1975)

o b s e r v e d a p e c u l i a r t l i c . r ~ n ~ s t a b i l i t y w i t h a h i g h l y p u f i f i e d

TUH, t h a t is, h e a t i n g o f t h e p u r i f i e d t o x i n a t 6 0 ' ~ r e s u l t e d

i n complex i n a c t i v a t i o n o f h a e m o l y t i c a c t i v i t y , l e t r i a l e f f e c t

i n mice, gu inea -p ig s k i n r e a c t i v i t y , and r e a c t i v i t y w i t h

a n t i s e r a bo th by s i n g l e r a d i a l and d o u b l e irnrnunoaiffusion, b u t

h e a t i n g a t 1 OO'C e x h i b i t e d no s u c h e f f e c t s . Farkas-IIimsley

(1978) d e t e c t e u a h e a t - s e n s i t i v e + n h i b i t o r i n y. p ~ r a i ~ a e m o - *

1 - y t i c u s t o x i n p r e p a r a t i o n .

Takeda and coworke r s (1975) i s o l a t e d a n i n a c t i v a t i n g

f a c t o r frm th,e c u l t u r e f i l t r a t e s o f 1. p a r a i ~ a e m o l y t i c u s

which d e s t r o y e d t h e TDH a c t i v i t y when b o t h a r e h e a t e d a t

55 - 60°c, t h u s p r e v e n t i n g t h e h a e m o l y t i c a c t i v i t y . The

i n a c t i x a t i n g f a c t o r i s t h e r m o l a b i l e and p o s s e s s e s p r o t e o l y t i c

a c t i v i t y . It l o o s e s i t s a c t i v i t y on h e a t i n g w i t h t h e haemolys in

a t 95 - 1 0 0 ~ ~ ' f o r 15 minu tes . The i n a c t i v a t i n g f a c t o r was

s t i m u l a t e d by t h e p r e s e n c e o f N a C l o r MgC12 and showed

maximal a c t i v i t y a t a round p H 8.0.

These r e s u l t s show t h a t t h e A r r h e n i u s e f i ' e c t o b s o r v c d ,

w i t h t h e c r u d e haernolysin o f 1. p a r a h a e m o l y t i c u s i s d u s t o

t h e p r e s e n c e o f a t e m p e r a t u r e - d e p e n d e n t i n a c t i v a t i n g f a c t o r .

The i n a c t i v a t i n g f a c t o r i s a c t i v a t e d on h e a t i n g a t 50 -60°c,

b u t i s i t s e l f i n n c L i v a t e d a t 70 - 10oOc, t h e r e b y e x p l a i n i n g

t h e A r r h e n i u s e f f e c t o f c r u d e haemo1ysi.n o f 1. psrahaemo-

l y t i c u s .

TIILRMOLABILL HAEMOLYSINS

F'ujino and coworkers (1969) no ted t h e p resence o f two

d i s t i n c t haemolys ins of Y. p a r a h a e m o l u t i c u s i n c u l t u r e

s u p e r n a t a n t s o f t h e microorganism. The second haemolysin

i s i n a c t i v a t e d by h e a t i n g a t 5 6 ' ~ f d r 10 minu tes ( D a i l y ,

DeBell & Joseph , 1978). It i s produced by both Kanagawa-

p o s i t i v e and Kanagawa-negative s t r a i n s , and was shown t a be

a n t i g e n i c a l l y d i s t n c t from TDH ( ~ a k u r a i e t a l . , 1974).

The e f f e c t of t h e t h e r m o l a b i l e haemolysin on e r y t h r o c y t e s

was enhanced by l e c i t h i n , b u t t h e haemolysin i s n o t a

phospho l ipase A ( D a i l y , DeBell & Joseph , 1978). S t r a i n s

producing o n l y t h e t h e r m o l a b i l e haemolysin and n o t TDH have

been i s o l a t e d from p a t i e n t s wi th d ' iarrhoea, b u t t h e i r r o l e

i n p a t h o g e n i c i t y h a s n o t been evalu 'ated q a a k u r a i e t a l . , 1974).

ENTEROTOXIN

The e x b t e n c e o f a n e n t e r o t o x i n which i n d u c e s f l u i d I

a ccumula t ion i n r a b b i t i l e a l l o o p t e s t h a s n o t been

c o n c l u s i v e l y proven. However, f l u i d accumula t ion i n t h e R I L

t e s t h a s been r e c o r d e d w i t h bao th c u l t u r e s o f b o t h haemoly t i c

and non-haemolytic s t r a i n s o f 1. p a r a h a e m o l y t i c u s (Sakazak i

e h a l . , 1974; and Twedt e t a l . , 1980). The accumula t ion o f

f l u i d i n r a b b i t i l e a l l o o p t e s t caused by non-haemolytic s t r a i n s

o f t h e v i b r i o c o u l d be due t c , i n c r e a s e d c o n c e n t r a t i o n o f

N a C 1 , s i n c e no e x t r a c e l l u l a r f a c t o r h a s been shawn t o i n d u c e

a p o s i t i v e RIL t e s t s o f a r ( ~ a k a z a k i e t a l . , 1974; and Johnson

& Calia, 1976).

Honda and coworkers (1976) i s o l a t e d a t h e r m o l a b i l e non-

haemoly t i c f a c t v r from c u l t u r e f i l t r a t e s o f haemoly t i c s t r a i n s

which induced changes i n Ch inese ~ ' a ~ s t e r Ovary c e l l s s i m i l a r

t o t h a t caused by c h o l e r a t o x i n . , c u l t u r e f i l t r a t e s from s t r a i n s

i s o l a t e d from p a t i e n t s w i t h d i a r r h o e a i n Seden induced t . .

c h o l e r a - l i k e changes i n a d r e n a l Y1 c e l l s . S e r a o b t a i n e d

from t h e s e p a t i e n t s e x e r t e d some n e u t r a l i s i n g e f f e c t on c h o l e r a

t o x i n . These d i f f e r e n t s t u d i e s i n d i c a t e t h a t c t r a i r l s o f

V . pa rahaemoly t i cus may produce an e x t r a c e l l u l d r e n t e r o t o x i n - - l i k e f a c t o r which i s d i s t i n c t from TDH.

INTHACELLULAR HAEMOLYSlNS

Two ce l l -bouna haernolysins were i d e n t i f i e d i n s t r a . . i n s

of y. pa-ahaemo1,yticug ( ~ a k a z a k i e t a l . , 1975) . One o f t h e s e

may be i d e n t i c a l t o t h e haemolysin p u r i f i e d from d r i e d c e l l s

( i i d a & Takagi, 1979). The haemolysin was h e a t - s t a b l e ,

e x t r a c t a b l e w i t h a c e t o n e and weakly t o x i c t o mice a f t e r

i n t r a p e r i t o n e a l i n j e c t i o n . It c o n t a i n e d a b o u t 6% n e u t r a l

l i p i d e and h a s no c o r r e l a t i o n w i t h t h e Kanagawa phenomenon.

I i d a e t a l . (1982) s t u d i e d t h e haemoly t i c a g e n t s o f

i n t r a c e l l u l a r t h e r m o s t a b l e haemoly t i c f r a c t i o n ( ITHF) u s i n g

two s t r a i n s o f t h e foodpo i son ing microorganism. Each ITHF

p r e p a r a t i o n was s e p a r a t e d i n t o seven p o r t i o n s by L i p i d

e x t r a c t i o n and column and t h i n - l a y e r chromatography. Most

o f t h e a c t i v i t y was d e t e c t e d i n t h e free f a t t y a c i d f r a c t i o n . r a t h e r t h a n i n o t h e r l i p i d s .

Only D-tryptophan added t o a s y n t h e t i c medium indbced

t h e producCio'n of Kanagawa haemolyein, and t h a t was a t pH

v a l u e s below 6.5, b u t a d d i t i o n o f lOOmM o f D-tryptophan t o

e a r l y log phaee c u l t u r e s caused a n i n h i b i t i o n o f growth and

o f s u b s t r a t e - u t i l i s a t i o n , i n s t e a d haemolys in was produced o n l y

i n t r a c e l l u l a r l y . The haemolys in appeared i n t h e s u p e r n a t a n t

o n l y when growth recommenced.

ENDOTOXIN

The 0 an t igen of 1. parahaemolyt icus is a l ipopoly-

s accha r ide c o n t a i n i n g glucose, glucosamine, heptose , pho$pho-

rous , f a t t y a c i d e s t e r , and n i t r o g e n compounds ( T o r r i e e t al.,

4969). The t o x i c i t y of t h e s e LPS's h a s no t been determined,

bu t may be low f o r v e r t e b r a t e s . Endotoxin from v a r i o u s

souruea as wel l as his tamine, were shown t o enhance pathogeni-

c i t y o f caude haemolytic c u l t u r e f i l t r a t e .

Sero ton in and h i s t amin were shown t o enhance and

aggrava te t h e symptoms of Y. parahaemo1uticu~-foodpoisoning.

The endotoxins o f 1. garahaemolvt icug c o n t a i n s a l a r g e

amount o f p r o t e i n s (sochard & Colwell , 1977).

About 12 0-ant igens (thermostgible somatic a n t i g e n s ) and

59 K-antigens ( c a p s u l a r o r envelope a n t i g e n s ) have been

i d e n t i f i e d (Miwatani & Takeda, 1976). Many d i f f e r e n t

s e r o t y p e s are ,found throughout t h e world, and t h e most

common ones vary from p lace t o place . No p a r t i c u l a r s e r o t y p e s

have been s t r o n g l y a s s o c i a t e d wi th t h e Kanagawa phenomenon,

o r wi th human i l l n e s s .

TOXINS FROM OTHER VIBRIONACEAE

You e t al . (1986) desc r ibed a new type o f haemolysin

produced by non-01 1, c h o l e r a e which i s similar, bu t not

i d e n t i c a l t o t h e thermos tab le d i r e c t haemolysin o f 1.

g a r a h a e m o l ~ t i c u s . Haemolysin from 1. c h o l e r a e E l Tor h a s

been shown t o s h a r e s e v e r a l p r o p e r t i e s i n common wi th Y. parahaemols t icus TDH such as haemolytic spectrum, c y t o t o x i c i t y

and i n h i b i t i o n o f haemolytic a c t i v i t y by crude g a n g l i o s i d e s

(Honda e t a l . , 1976). S i m i l a r results were ob ta ined i n s t u d i e s

o f a haemolysin from a peychroWophic V ib r io s p e c i e s ( P e t e r s

e t al., 1982). Th i s haemolysin w a s no t n e u h r a l i s e d by

an t i se rum t o TDH.

S t r a i n s o f 1, f l u v i a l i & wereashown t o produce one

haemolytic and sne non-haemolytic c i t o l ~ s i n , a p r o t e a s e and

an e n t e r o t o x i n caus ing p o s i t i v e RIL t e s t and e longa t ion i n

Chinese Hamster Ovary c e l l s (Lockwood e t al., 1982). One

haemolysin from t h e f i s h pathogen 1. anrruillarum h a s been

p u r i f i e d and charac t e r i s e d (Munn, 1978).

Non-01 Y. cho le rae i n c r i ~ n i n a t e d i n a wide range o f d i s e a s e s

were shown t o produce haemolysin as wel l a s an e n t e r o t o x i n

(Blake e t a1 . , '1980) . No s t u d i e s on e x t r a c e l l u l a r t o x i n s from

v i b r i o s of p o t e n t i a l human pa thogen ic i ty , l i k e 1. a l ~ i n o l ~ t i c u s

V. v u l n i f i c u s , Y . d m i c u a , Y 8 aamselaL y. metschnikovi i , and - V. h o l l i s a have s o f a r been publ ished. -

Plesiomonas s h i n e l l o i d e s i s t h e only Speciesof t h e family

Vibrionaceae which is c o n s i s t e n t l y non-haemolytic. The

microorganism h a s been i s o l a t e d i n pure c u l t u r e from p a t i e n t s

wi th unexplained d i a r rhoea . An e n t e r o t o x i n h a s been desc r ibed

(Sanua e t al., 1980.) and some s t r a i n s are ,known t o produce

phospholipase and high titres of elastase r e s p e c t i v e l y .

S t r a i n s from p a t i e n t s wi th d i a r r h o e a were f imbr i a t ed and t h e y

g e n e r a l l y expressed* a hydrophobic su r f ace .

En te ro tox ins from y. c h o l e r a e and non-01 1. c h o l e r a e a r e

an t igen ic ra l ly r e l a t e d ( ~ p i r a & Daniel , 1978). Although t h e

e f f e c t s of Aeromonas e n t e r o t o x i n appea r s t o be CAMP-mediated

l i k e c h o l e r a t o x i n and Esche r i ch i a c o l i t he rmolab i l e haemolysin

(Dubey e t al., 1981; Ljungh & K r W , . - 1982; Ljungh e t a l . ,

1982; and Ljungh e t al . , 1982). A~romonas e n t e r o t o x i n d i f f e r s

from t h e s e e n t e r o t o x i n i n many r e spec t s . So f a r , no conc lus ive

s t u d y h a s demonstrated a n t i g e n i c r e l a t i o n s h i p between Aeromonag

e n t e r o t o x i n ' a n d o t h e r en t e ro tox ins . I n view of r e c e n t s t u d i e s

on a n t i g e n i c h e t e r o g e n e i t y o f c h o l e r a tomin s u b u n i t s and g.

c o l i t he rmolab i l e haemolysin o f human and porc ine o r i g i n - (Clement8 & F i n k e l s t e i n , 1972; and Robb e t a l . , 1982), a s wel l

as t h e f i n d i n g o'f a the rmolab i l e e n t e r o t o x i n i n &. c o l i .sr. -

which was not neu t r a l i s edvby an t i se rum t o E. c o l i t he rmolab i l e

haemolysin, t h e r e may e x i s t deve ra l e n t e r o t o x i n s wi th similar

b i o l o g i c a l a c t i v i t y and vary ing degree of a n t i g e n i c -.

re latedness .

Future s t u d i e s with DNA t o x i n probes may revea l the

extent o f an t igen ic r e l a t i o n s h i p between enterotox ins

a s wel l a s haemolysins within Vibrionaceae.

IDENTIFICATION CHARACTERISTlCS

;*no t y p i c c h a r a c t e r s

Y. parahaemolvt icus i s a gram-negative, non-sporing, non- - c a p s u l a t e b l ende r rods , a c t i v e l y m o t i l e wi th one p o l a r

f lagel lum. In young c u l t u r e s t h e microorganism appea r s curved

l i k e a comma w i t h rounded and po in ted ends, bu t i n o l d c u l t u r e s

pleomorphic and i n v o l u t i o n forms a r e common. I n l i q u i d

media, t h e c e l l s occur i n p a i r s o r i n s h o r t cha ins . Sometimes

undivided s p i r a l s may occur. The v i b r i 0 o r comma appearance

may become l e s s obvious a f t e r f r equen t subcu l tu r ing .

E lec t ron microscopy s t u d i e s shows some s i m i l a r i t y

between t h e swarming o f x. parahaemolvt icus and P r o t e u s

s p e c i e s (Belas & Colwell , 1982). The microorganism produces u

f r e e s l ime i n c u l 4 u r e s ( C h a t t e r j e e & Neogy, 1973).

Unlike most known b a c t e r i a , t h e v i b r i o shows d e v i a t i o n

o f f l a g e l l a ' morphology which means t h a t f l a g e l l a arraqgement

ranges from the t y p i c a l p o l a r t ypes t o t h e p e r i t r i c h ~ u s t y p e s

w i t h wavy, c u r l y , s t r a i g h t and l i n e a r p a t t e r n s ( C h a t t e r j e e , 1974)

Biochemical c h a r a c t e r s

V. parahaemolvt icus belongs t o t h e l y s i n e - p o s i t i v e , a rg in ine - - nega t ive group o f v i b r i o s . It u t i l i s e s g lucose o x i d a t i v e l y

and f e rmen ta t ive ly w i t h t h e product ion o f a c i d bu t n o t gas.

No o t h e r suga r i s fermented. The d i f f e r e n t i a l c r i t e r i a f o r

i d e n t i f i c a t i o n i n c l u d e t h e a b i l i t y t o grow a t 4 2 ' ~ and t h e

i n a b i l i t y t o produce a c e t o i n from g lucose b r o t h ( Pan-Urai,

1974). The presence o f t h e enzyme oxidase i s used as a tes t .-'-. - -'

f o r s c r een ing non-lac t o s e ferment ing c o l o n i e s from s t o o l

specimens (Oberhofer & Podgore, 1982).

The microorganism i s h a l o p h i l i c and can wi ths tand 7%

s a l i n i t y . Biochemical t es t media are t h e r e f o r e supplemented

w i t h I k C l (1%) f o r more r e l i a b l e r e s u l t s (Bot tone & Robin, 1978).

Some biochemical c h a r a c t e r s u s e f u l f o r l a b o r a t o r y

i d e n t i f i c a t i o n o f t he microorganism a r e shown i n Table 2.

S u l t u r e media and charac t e r i s t i c a

The microorganism forms l a r g e , smooth, and dome-shaped c o l o n i e s

wi th opaque c e n t r e s on thiosulphate-citrate-bile s a l t s - s u c r o s e

(TCBS) a g a r when grown a e r o b i c a l l y a t 37'~. The v i b r i o

r e q u i r e s 6 - 9% salt f o r growth, ;;rd t h e s t u d i e s o f Pa lasuntheran

(1981) show t h a t t h e microorganism can grow e q u a l l y we l l i n

media c o n t a i n i n g o t h e r sa l t s o t h e r than NaCl s i n c e i t h a s no

e p e c i f i c requirement f o r ~ a * o r ~ 1 ' ions .

N e w methods o f i s o l a t i o n and i d e n t i f i c a t i o n a r e being

i n v e s t i g a t e d , and some modified media have been devised.

TCBS, ~ ~ i c h ' i s commercially a v a i l a b l e and easy t o p repare is

used t o d i s t i n g u i s h c o l o n i e s of suspected 1. cho le ra and y.

parahaemols t icus (Morr is e t al., 1979). Another medium

con ta in ing t r y p t i c a s e s , l Y a g a r tiupplemented w i t h N a C l pe rmi t s

b e t t e r d i f f e r e n t i a t i o n of Y, parahaemolyt icus from 1, a lg ino lu -

Table 2. Some b iochemica l t e s t s f o r 1. parahaemoly t i cus (Farmer e t a l e , 1985).

T e s t Reac t ion

Gram-reac t i o n k ~ o t i l i t y

I n d o l e

Voges-Proskauer

Oxidase Urease G e l a t i n l i q u e f a c t i o n 15s p r o d u c t i o n

T r i p l e Sugar I r o n

A r g i n i n e d e h y d r o l a s e

Lys ine deca rboxy lase

Hydrolase

Mal tose T r e h a l o s e

Sucrose

Halophi l i sm

1 % t r y p t o n e + 0% N a C l 1 1 + 3% "

1 1 t 8% I I + 10% ' I

Haemolysis

Human blood a g a r

Sheep blodd a g a r

Wagat suma blood agar (Kanagawa r e a c t i o n )

Growth a t 3 7 ' ~ Growth a t 4 2 ' ~

t i c u s and o t h e r b a c t e r i a (Kourany, 1983). BTB-teepol and - Ammonium su lpha te -cho la t e a g a r were i nco rpo ra t ed wi th

ant iserum ( o r IgG) a g a i n s t TDH and used f o r t h e i s o l a t i o n

and i d e n t i f i c a t i o n of Kanagawa-positive s t r a i n s of 1.

parahaemolyticus.

A n t i b i o t i c s e n s i t i v i t y

v -. parahaemolyt icus i s s e n s i t i v e t o polymyxinB ( C h a t t e r j e e

& NeOgy, 1973), chloramphenicol a n d , t e t r a c y c l i n e ,but i s

r e s i s t a n t t o a m p i c i l l i n (Joseph e t al., 1978). The presence

of beta-lactamase i n t h e microorganism i s r e s p o n s i b l e f o r

t h e r e s i s t a n c e . The a d d i t i o n o f N a C l t o growth medium

diminished t h e s e n s i t i v i t y ts gentamicin.

Chapter 3

METHODS

ISOLATION OF L, parahaemolyt icua

Source C l a m s ( seafood) ob ta ined from t h e P o r t - b r c o u r t

c o a s t of t he A t l a n t i c ocean were used.

I s o l a t i o n ~ r o c e d u r e The method used was as desc r ibed by

Vanderzant and Nickel (1972) and Farmer e t al. (1985). Each

seafood (50 g ) was blended f o r 2 minutes i n 450 m l o f 7%

S o d i m c h l o r i d e (NaC1.) . Tenfold s e r i a l d i l u t i o n s of t h e

homogenate was made i n 7% NaC1, and 0 , l m l o f each d i l u t i o n

was spread ove r t h e s u r f a c e o f TCBS agar (Oxoid) and on MT

medium ( See Appendix 1 ) . The p l a t e s were incuba ted a e r o b i c a l l y

a t 37 '~ f o r 24 t o 48 hours. White t o creamy, c i r c u l a r , amooth, I

amylase-posi t ive c o l o n i e e on MT medium; and l a r g e , smooth,

dome-shaped c o l o n i e e .- w i t h opaque c e n t r e and green c o l o u r on

TCBS a g a r were picked as suspec ted c o l o n i e s o f L. 5 . ,

Prepar ing inoculum f o r i d e n t i f i c a t i o n To exclude t h e

p o s s i b i l i t y of contaminat ion w i t h admixture of ano the r bacter iun

each suspec ted colony was p l a t e d o u t on an urnselective c u l t u r e

medium ( N u t r i e n t a g a r (Oxoid) and incuba ted f o r 24 hours a t 37'~

A wel l i s o l a t e d colony on t h e secondary p l a t e was subcu l tu red

i n Bra in-hear t - in fus ion ( B H I ) bro th supglamented w i t h 3% NaCB.

The b r o t h c u l t u r e was used f o r subsequent t e s t s .

I d e n t i f i c a t i o n ~ r o c e d u r e

Gram-staininq The method used was t h e Hucker mod i f i ca t ion

technique. A smear o f t h e 4-hour s u b c u l t u r e on a microscope

s l i d e was hea t - f ixed and f looded wi th 1 % aqueous s o l u t i o n o f

c r y s t a l v i o l e t mixed with 5% Sodium hydrogen carbona te (NaHCO ) 3 s o l u t i o n . The smear was al lowed t o s t a n d f o r 2 t o 3 minutes.

The primary s t a i n was washed o f f w i t h Lugol ' s i o d i n e s o l u t i o n ,

b l o t t e d , and w a s deco lou r i s ed wi th acetone. A c o u n t e r s t a i n ,

2% s o l u t i o n o f S a f r a n i n was a p p l i e d and al lowed t o s t a y f o r

10 seconds b e f o r e washing o f f . The prepared s l i d e was

observed under t h e microscope msing t h e o i l immersion l e n s . I

M o t i l i t y t e s t The hanging d r o i method was used. A l o o p f u l

o f t h e s u b c u l t u r e was gnocula ted i n t o Peptone water (Oxoid)

and incubated. f o r 4 hohr s a t 37'~. A drop o f t h e b r o t h

c u l t u r e w& p laced on a c o v e r s l i p , i n v e r t e d over a p l a s t i c i n e

r i n g on a g l a s s s l t d e , and viewed under t h e h igh power l e n s

of t h e microscope f o r m o t i l i t y .

Indole t e s t A few drops of Kovact s reagent were added t o

18- t o 24-hour b r o t h c u l t u r e s o f each i s o l a t e on Peptone

water. The c u l t u r e was observed f o r t h e product ion o f rose-

pink c o l o u r s i g n i f y i n g i n d o l e product ion.

Voges-Proskauer t e s t About 3 m l o f 5% a l c o h o l i c a lpha-

n a p h t h o l s o l u t i o n and 3 m l o f 40% Pa tass ium hy.droxide (KOH)

s o l u t i o n were added t o 2- t o 4-day b r o t h c u l t u r e o f each i s o l a t c

The medium was obse rved f o r p ink c o l o u r a t i o n s i g n i f y i n g

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

Oxidase t e s t Each i s o l a t e was grown on a n u t r i e n t agar

s l o p e a t 3 7 ' ~ f o r 24 houre , Few d r o p s o f f r e s h l y p repared

1% aqueous s o l u t i o n o f para-aminodimethylaniline o x a l a t e and

1% a lpha-naph tho l i n e t h a n o l were added and a l lowed t o r u n

o v e r t h e growth. The c u l t u r e was observed f o r a deep, b l u e

c o l o u r i n d i c a t i n g a p o s i t i v e r e a c t i o n .

Urease t e s t A l o o p f u l o f each s u b c u l t u r e was h e a v i l y

i n o c u l a t e d on a g a r s l a n t c o n t a i n i n g u r e a a g a r base . A f t e r

i n c u b a t i n g a t 3 7 ' ~ f o r 5 hours , t h e u r e a a g a r *observed f o r

p i n k c o l o u r a t i o n s i g n i f y i n g h y d r o f y s i s .

G e l a t i n l i a u e f a c t i o n A s t a b c u l t u r e o f each i s o l a t e was

made on n u t r i e n t b r o t h c o n t a i n i n g 15% g e l a t i n and was

i n c u b a t e d o v e r n i g h t a t 37'~. To t e s t f o r g e l a t i n h y d r o l y s i s ,

t h e c u l t u r e was h e l d a t 4 ' ~ f o r 30 minu tes , and was t h e n

observed f o r h y d r o l y s i s shown by t h e i n a b i l i t y o f t h e

c u l t u r e t o s o l i d i f y .

C a t a l a s e t e s t A s u b c u l t u r e o f each i s o l a t e was seeded

o n t o a n u t r i e n t a g a r s l o p e , a n d i n c u b a t e d o v e r n i g h t a t 37'~.

S e v e r a l d r o p s o f hydrogen p e r o x i d e were poured down t h e s l o p e .

56.

C u l t u r e was obse rved f o r f o r m a t i o n o f b u b b l e s o f g a s

s i g n i f y i n g p r e s e n c e o f c a t a l a s e .

Sugar f e r m e n t a t i o n and hydrogen s u l p h i d e p r o d u c t i o n t e s t s - T r i p l e s u g a r i r o n (TSI) a g a r (Oxoid) p r e p a r e d as a s l o p e w i t h

deep b u t t and s e r v i n g as a n i n d i c a t o r f o r g l u c o s e , l a c t o s e

and s u c r o s e f e r m e n t a t i o n was used. A heavy inoculum o f each

i s o l a t e was s t a b - i n o c u l a t e d deep down i n t o t h e b u t t and

a l o n g t h e s u r f a c e o f t h e s l o p e . A f t e r o v e r n i g h t i n c u b a t i o n ,

t h e s l a n t was obse rved f o r g a s p r o d u c t i o n ( b u b b l e s ) ,

p r o d u c t i o n o f H2S ( b l a c k e n i n g o f t h e medium a l o n g t h e l i n e o f

s t a b i n o c u l a t i o n ) , y e l l o w i n g o f t h e b u t t ( g l u c o s e f e r m e n t a t i o n )

and ye l lowing o f t h e s l a n t ( s u c r o s e and l a x t o s e f e r m e n t a t i o n ) .

Oxida t ion-Fermenta t ion t e s t Two t u b e s c o n t a i n i n g Hugh and

Le i f son medium were s teamed b e f o r e use. The c u l t u r e was s t a b - *

i n o c u l a t e d w i t h a s t r a i g h t w i r e i n t o each o f t h e t u b e s . One

o f t h e t u b e s was o v e r l a i d w i t h approx imate ly 2 m l o f s t e r i l e

p a r a f f i n o i l ,and i n c u b a t e d a t 37'~. The t u b e s were observed I .

d a i l y f o r y e l l o w i n g o f b o t h t u b e s ( f e r m e n t a t i v e microorganisms) ,

o r f o r ye l lowing i n t h e open t u b e ( o x i d a t i v e mic roorgan i sms) .

Amylase t e s t S t a r c h h y d r o l y s i s was de te rmined on corn-

s t a r c h medium ( i . c . MT medium). A c u l t u r e o f esct l i s o l a t e on

t h e mddium was obse rved f o r zones o f c l e a r i n g around c o l o n i e s

a f t e r f l o o d i n g each p l a t e w i t h i o d i n e s o l u t i o n .

Lys ine decarbox.ylase t e s t Falkow rnediurii c o n t a i n i n g 0.5$

l y s i n e was i n o c u l a t e d w i t h tes t microorganism and i n c u b a t e d

f o r 24 h o u r s a t 37'~. The c u l t u r e was observed f o r change o f

c o l o u r from y e l l o w t o p u r p l e i n d i c a t i n g p o s i t i v e r e a c t i o n .

Arg in ine deca rboxy laee t e s t Same method as above was used

excep t t h a t 0.5% a r g i n i n e i n s t e a d o f l y s i n e -*as i n c o r p o r a t e d

i n t o t h e medium.

H a l o ~ h i l i s m T e s t microorganism was i n o c u l a t e d i n t o BHI

b r o t h c o n t a i n i n g 0, 3.0, 8.0, and 10% NaC1. A f t e r i n c u b a t i o n

o v e r n i g h t a t .37Oc, t h e medium wae obse rved f o r growth.

Kanaaawa r e a c t i o n T e s t microorganism was i n o c u l a t e d on

Wagatsumats b lood a g a r p l a t e (Appendix 2 ) , i n c u b a t e d a t 3 7 ' ~

f o r 2 4 t o 48 hours . Each p l a t e was obse rved f o r zones o f

c l e a r i n g around t h e c o l o n i e s . ' s

Growth t e m p e r a t u r e MT medium i n o c u l a t e d w i t h t h e t e s t

m i c r o o r g a n i ~ m was i n c u b a t e d a t 370, 420, o r 48 '~ f o r 24 t o

48 hours; ,The medium was obse rved f o r growth.

PRELIMINARY STUDIES WITH LIVING CELLS OF V. p a r a h a e m o l ~ t i c u s ISOLATES

E v a l u a t i o n o f l e t h a l a c t i v i t y

Microoraanism M v e Kanagawa-posi t ive i s o l a t e s showing

wider z o n e s o f haemolys i s on Wagatsuma blood a g a r , and f i v e

Kanagawa-negative i s o l a t e s randomly p icked from t h e same medium

were used. One l o o p f u l o f a 4-hour b r o t h c u l t u r e o f each

i s o l a t e i n BHI-3'$1#uC1 medium was t r a n s f e r r e d i n t o 10 m l o f

BHI-3% N a C l b r o t h and i n c u b a t e d a t 37°~ . After 24 h o u r s ,

t h e c u l t u r e was c e n t r i f u g e d a t 3 000 r e v o l u t i o n s p e r minu tes

( r e v . min-' ) f o r 3 0 minutes . The b a c t e r i a l c e l l s ( s e d i m e n t )

were washed by r e s u s p e n d i n g t h e sed iment i n phosphate b u f f e r e d

s a l i n e , PBS (pH 7 .2) and c e n t r i f u g i n g i t . The c e l l s were *

washed t w i c e and were s e r i a l l y d i l 6 t e d 10-fold f o r v i a b l e coun t .

C u l t u r a l c o n d i t i o n s .... V p a r a h a e m o l y t i c u ~ i s o l a t e s were

main ta ined ; in BHI medium supplemented w i t h 3% N a C l and

c o n t a i n i n g 0.3% a g a r ( I i jima e t a l . , 1981 1.

Animal4 Four-week o l d w h i t e mice weighing 17 t o 20 g and

i n g roups o f f i v e a n i m a l s were used.

I n o c u l a t i o n of a n i m a l s A c e l l s u s p e n s i o n o f each i s o l a t e i n P.t!

c o n t a i n i n g 1 x c . f , u . was i n j e c t e d i n t r a p e r i t o n e a l y ( i . p.)

i n 0.5 m l p o r t i o n s i n t o each o f f i v e mice i n a group,

I n o c u l a t e d a n i m a l s were obse rved f o r 96 h o u r s f o r d e a t h , and

t h e p e r c e n t a g e m o r t a l i t y was c a l c u l a t e d .

Dete rmina t ion o f f i f t y p e r c e n t l e t h a l dose (LD,,)

M i c r o o r ~ a n i s m The i s o l a t e showing t h e h i g h e s t p e r c e n t a g e

m o r t a l i t y i n mice was used.

I n o c u l a t i o n o f a n i m a l s Tenfo ld s e r i a l d i l u t i o n s o f t h e

washed c e l l s of t h e i s o l a t e were made i n PBS (pH 7.2). Each

d i l u t i o n was i n j e c t e d i n t o each o f t e n mice i n a group, e i t h e ~

i n t r a p e r i t o n e a l y o r o r a l l y , I n t r a p e r i t o n e a l i n j e c t i o n s were

made i n 0.5 m l p o r t i o n s , whereas o r a l i n o c u l a t i o n were made

through p l a s t i c t u b i n g s i n 0.2 m l amounts. I n o c u l a t e d a n i m a l s

were obse rved f o r d e a t h f o r 96 hours. The LD50 was c a l c u l a t e d

a c c o r d i n g t o t h e method o f Reed and Muench (1938).

E f f e c t o f I r o n on LD,,

I r o n compounds J e c t o f e r and I r ~ n I1 s u l ' p h a t e were t h e

i r o n compounds used.

P r e p a r i n g t h e s u s p e n s i o n s f o r i n o c u l a t i o n . ,

One-ml o f J e c t o f e r

s o l u t i o n c o n t a i n i n g 5 000 mg o f i r o n w a s p i p e t t e d i n t o e a c h o f

t e n t e s t - t u b e s , and t h e q u a n t i t y was made up t o 9 m l w i t h PBS.

One-ml o f , b a c t e r i a l s u s p e n s i o n c o n t a i n i n g 2 x lo8 c. f ,u. was t h e ' .

added t o n f i r s t t e s t - t u b e , s o t h a t 0.5 m l o f b a c t e r i a l s u s p e n s i o n

i n J e c t o f e r c o n t a i n e d 250 mg o f i r o n . Tenfo ld s e r i a l d i l u t i o n s

were p r e p a r e d from t h e . f i r s t tube . S i m i l a r l y , b a c t e r i a l

s u s p e n s i o n i n I r o n I1 s u l p h a t e s o l u t i o n was p r e p a r e d t o

c o n t a i n 700 mg o f i r o n i n 0.5 m l o f b a c t e r i a l suspens ion .

I n o c u l a t i o n o f a n i m a l s Tenfo ld s e r i a l d i l u t i o n s o f washed

b a c t e r i a l c e l l s i n e i t h e r o f t h e i r o n compounds were e a c h

i n j e c t e d i m p . i n t o each o f t e n mice i n a group. !!!he LD5-, o f

t h e b a c t e r i a l c e l l s i n e a c h i r o n compound was c a l c u l a t e d as

d e s c r i b e d by Reed and Muench (1938).

Adherence a s s a y s

harvest in^ o f m a c r o ~ h a ~ e g P e r i t o n e a l macrophages from

mice were h a r v e s t e d as d e s c r i b e d below, were pooled and

suspended a t a c o n c e n t r a t i o n o f 1 x l o 4 c e l l s m l - ' i n Medium

199 supplemented w i t h 3% Bovine serum albumin (BSA).

Kanagawa-posi t ive i s o l a t e s , K V , , KV3, and KV4;

and Kanagawa-negative i s o l a t e s , V6 and V I 0 were used. a

I n v i v o adherence assay The mice used were f a s t e d f o r two

days and a n a e s t h e s i s e d ( I i j i m a e t a l . , 1981). The abdomen was

opened an,d t h e small i n t e s t i n e t i e d 3 cm d i s t a l t o t h e stomach.

The b a c t e r i a l s u s p e n s i o n ( 1 x lo8 c . f .u . ) o f e a c h i s o l a t e was

i n j e c t e d i n 0.5 m l p o r t i o n s i n t o t h e i n t e s t i n e immedia te ly

below t h e t i e , and t h e abdomen was c l o s e d and s u t u r e d . A f t e r

6 hours , each an imal used was s a c r i f i c e d and t h e small

i n t e s t i n e removed and d i v i d e d i n t o 4 s e c t i o n s o f e q u a l l e n g t h

( I i j i m a e t a l . , 1981); Each s e c t i o n was homogenised i n 3% N a C l

s o l u t i o n and t h e number o f b a c t e r i a i n each homogenate was

determined by t h e s t a n d a r d method o f M i l e s and Mizra (1938)

u s i n g TCBS a g a r . Three a n i m a l s were used f o r each b a c t e r i a l

i s o l a t e , and t h e a v e r a g e v i a b l e c o u n t taken.

I n v i t r o adherence A s u s p e n s i o n o f each b a c t e r i a l i s o l a t e

i n PBS ( c d n t a i n i n g 2 x lo4 c.f.u. m1-') was added t o a n e q u a l

1 volume o f macrophage s u s p e n s i o n c o n t a i n i n g 1 x lo4 c e l l m l - , o r t o b u f f e r ( c o n t r o l ) . The m i x t u r e was i n c u b a t e d w i t h

a g i t a t i o n i n a 37 '~ water -bath f o r 60 m i n u t e s and was c e n t r i -

fuged a t 1 000 rev . min-l. The s u p e r n a t a n t was d i l u t e d

s e r i a l l y f o r v i a b l e count .

% a t t a c h e d b a c t e r i a =, No - Nt x 100 *o

where, No I v i a b l e c o u n t o f c o n t r o l a f t e r 6 0 minute i n c u b a t i o n .

N t = v i a b l e c o u h t o f tes t microorganism a f t e r 60 minute i n c u b a t i o n .

R e l a t i n g b a a t e r i a l a d h e r e n c e t o l e t h a l a c t i v i t y

A s u s p e n s i o n o f each a d h e r e n t o r non-adherent i s o l a t e (concen-

t r a t i o n 1 x l o 7 c.f.u.) was i n j e c t e d i n t r a p e r i t o n e a l y i n t o

each o f f i v e mice.

% m o r t a l i t y = number o f dead mice

-S

62.

ISOLATION AND PURIFICA'RION OF AN INTRACELLULAR TOXIC FACTOR

Mic r o o r ~ a n i s m A v i r u l e n t Kanagawa-posi t ive i s o l a t e (LD

7 50

1 x 10 c . f e u . ; adherence r a t e t o mouse p e r i t o n e a l macrophages,

85%), and a Kanagawa-negative i s o l a t e (LD 1 x 10'' c . f.u. ) 50 were used.

C u l t u r a l c o n d i t i o n s The b a c t e r i a l i s o l a t e s were main ta ined

i n B H I (Oxoid) medium c o n t a i n i n g 0 , a g a r a n d supplemented

wi th 3% N a C l ( I i j i m a e t a l . , 1981).

H a r v e s t i n g o f b a c t e r i a l c e l l g One-ml o f a 4-hour b r o t h

c u l t u r e o f each i s o l a t e i n BHI-3aaC1 was poured o n t o each o f

s e v e r a l agar s l o p e s kn Roux b o t t l e s . A f t e r i n c u b a t i o n a t 3 7 ' ~

f o r 24 h o u r s , t h e b a c t e r i a l growth on each s l o p e was washed

o u t w i t h PBS (pH 7.2) and c e n t r i f u g e d a t 3 000 rev . min-' f o r

30 minutes . The b a c t e r i a l c e l l s ( p e l l e t ) were washed t w i c e

i n PBS t o r i d t h e c e l l s o f media components. P u r i t y o f c e l l s

was checked by i n o c u l a t i n g a s u s p e n s i o n o f t h e c e l l s o n t o

TCBS a g a r . ' The washed c e l l s were k e p t frozem a t - 4 ' ~ i n

a r e f r i g e r a t o r .

D i s r u p t i o n . o f b a c t e r i a l c e l l s A t h i c k s u s p e n s i o n o f

b a c t e r i a l c e l l s c o n t a i n i n g approx imate ly 0.7 g o f c e l l s m l - '

was s u b j e c t e d t o s o n i c v i b r a t i o n a t maximum speed i n a V i r s o n i c

c e l l d i s r u p t o r f o r 9 0 seconds . The p r o c e s s was r e p e a t e d f i v e

times t o e n s u r e maximum breakage o f c e l l s . A l o o p f u l o f t h e

r e s u l t a n t suspension was gram-stained t o ensure e x t e n t o f

complete d i s i n t e g r a t i o n o f c e l l s . The suspension was k e p t

coo l du r ing o p e r a t i o n i n an ice -ba th .

Removal o f i n s o l u b l e p a r t i c l e s C e l l wall components and

o t h e r i n s o l u b l e p a r t i c l e s were removed by c e n t r i f u g a t i o n

(Gallenkamp high-speed e n t r i f u g e ) a t 2 3 000 rev. min-' f o r

30 minutes.

Assay f o r t o x i c a c t i v i t y The r e s u l t a n t s o l u t i o n s from t h e

d i s r u p t e d c e l l s o f t h e Kanagawa-positive i s o l a t e and t h e

Kanagawa-negative i s o l a t e were t e s t e d f o r b i o l o g i c a l a c t i v i t y .

One-ml o f each s o l u t i o n , o r b u f f e r ( c o n t r o l ) was added t o an

equa l volume o f 1 % d e f i b r i n a t e d e r y t h r o c y t e suspension.

The mixture was incuba ted f o r 1 hour i n a 3 7 ' ~ water-bath. m

Erythrocyte l y s i s s i g n i f i e d ac t iv i t ' y .

Fkac t iona l p r e c i p i t a t i o n w i t h Ammonium s u l p h a t e P r o t e i n

was f r a c t i o n e t e d acco rd ing t o t h e method desc r ibed by Suther land

(1979). Ammonium s u l p h a t e ((NH4)2S04) c r y s t a l s (4.46 g ) were

slowly added t o t h e c o l d supe rna t an t w i t h g e n t l e s t i r r i n g t o

g i v e a 20% s a t u r a t e d s o l u t i o n i n 100 m l s o l u t i o n . The s a t u r a t e d

s o l u t i o n was c h i l l e d a t 4 ' ~ f o r 4 t o 24 hours , and any

p r e c i p i t a t e formed was removed by c e n t r i f u g a t i o n a t 500 rev.

mino' f o r 10 minutes , i h i l e t h e s u p e r n a t a n t was r e t a i n e d f u r

f u r t h e r a d d i t i o n o f (NH ) SO The p r e c i p i t a t e was d i s so lved 4 2 4 ' i n a small volume (2 ml) of a z i d e s a l i n e ( p r e s e r v a t i v e ) and

d i a l y s e d a g a i n s t d i s t i l l e d w a t e r a t 4 ' ~ . The a z i d e s a l i n e

was p r e p a r e d by a d d i n g sodium a z i d e t o form a 0.08% s o l u t i o n

i n PBS. The d i a l y s i s t u b e k n o t t e d a t b o t h ends and

c o n t a i n i n g t h e t e s t s u s p e n s i o n was c o m p l e t e l y immersed i n

a beaker c o n t a i n i n g d i s t i l l e d water . The b e a k e r was p laced

i n a magnet ic s t i r r e r . A s o l u t i o n o f Barium c h l o r i d e i n

d i s t i l l e d w a t e r was added t o a sample o f t h e d i a l y s i n g f l u i d

p r e c i p i t a t e o f Barium s u l p h a t e s i g n i f i e d absence o f SO4 i o n s .

S e r i e s o f s a t u r a t i o n s t e p s , 20-, 40-, 60-, 80-, and 100% were

o b t a i n e d i n t h e same manner, and t h e i r p r o t e i n c o n t e n t s

de termined by t h e method o f Lowry (1951) . The amount o f

( N H q ) 2 ~ ~ 4 c r y s t a l s added were deduced from a monogram f o r

Ammonium s u l p h a t e s o l u t i o n s by Dixon (1953) . s

Assay f o r t o x i c a c t i v i t i e s To t e s t f o r haemoly t i c a c t i v i t y ,

e a c h Ammonium s u l p h a t e p r e c i p i t a t e d f r a c t i o n was d i l u t e d , . .

t w o f o l d s i n PBS i n s i l i c o n i s e d t e s t - t u b e s . One-percent o f

d e f i b r i n a t e d e r y t h r o c y t e s i s p e n s i o n was added t o a n e q u a l

volume o f each d i l u t i o n , o r t o b u f f e r e d s a l i n e ( c o n t r o l l .

The m i x t u r e s were i n c u b a t e d f o r 1 h o u r w i t h o c c a s i o n a l s h a k i n g

i n a 3 7 ' ~ wat-er-bath. The 50% haemoly t i c dose was t h e s m a l l e s t

amou*. o f p r o t e i n i n e a c h f r a c t i o n which caused v i s i b l e

e r y t h r o c y t e l y s i s .

To t e s t f o r l e t h a l a c t i v i t y , 3 mg o f p r o t e i n i n e a c h f r a c t i o n was i n j e c t e d i n t r a v e n o u s l y i n t o each o f f i v e mice i n

a group.

The i n j e c t e d a n i m a l s were obse rved f o r 96 h o u r s f o r d e a t h .

Sephadex G-150 g e l f i l t r a t i o n The f r a c t i o n (40% s a t u r a t i o n

s t e p ) which had t h e l o w e s t haemoly t i c dose (HD ) and which 50 k i l l e d t h e h i g h e s t number o f mice was produced i n l a r g e

q u a n t i t y and s u b j e c t e d t o gel f i l t r a t i o n . One-ml o f t h e c7t?l~ f .mwr)

f r a c t i o h w a s a p p l i e d t o a 2.5- by 50 cm Sephadex (3-150 column

which had been e q u i l i b r a t e d w i t h PBS (pX 7.2) . E l u t i o n was

w i t h 500 m l o f t h e b u f f e r c o l l e c t e d i n 5 m l f r a c t i o n s a t a

f low r a t e o f 15 m l p e r hour. The e l u t e d f r a c t i o n s were

a s s a y e d f o r haemoly t i c a c t i v i t y . The haemoly t i c f r a c t i o n s

were pooled and c o n c e n t r a t e d by p e r c o l a t i o n i n a d i a l y s i s

bag o v e r n i g h t . The p r o t e i n c o n t e n t o f t h e pooled e l u a t e was

determined as d e s c r i b e d by Lowry ( 1 9 5 1 ) , and was d e s i g n a t e d *

t o x i c f r a c t i o n .

BIOLOGICAL CHARACTERISATION

L e t h a l a c t i v i t y

Animals Four- t o five-week o l d w h i t e mice, weighing 17 t o

20 g i n g roups o f f i v e a n i m a l s were used.

I n o c u l a t i o n o f a n i m a l g . To o b t a i n v a r y i n g d o s e s o f t h e t o x i c

f a c t o r , a s o l u t i o n o f t h e f r a c t i o n was s e r i a l l y d i l u t e d tyrg@lds

i n PBS (pH 7.2). Each9dose was i n j e c t e d n t o a g roup o f mica'

e i t h e r i n t r a v e n o u s l y (i .v. )', i n t r a p e r i t o n e a l y ( i . p. ) , subcu ta -

n e o s l y ( s a c . ) o r o r a l l y . I n j e c t i o n s were g i v e n i n 0.1 m l

volumes f o r t h e i .v . r o u t e , 0.5 m l f o r t h e i . p . r o u t e , and i n

0.2 r n l volumes f o r t h e S.C. and t h e o r a l r o u t e s . I n o c u l a t e d

anima.16 were obse rved f o r 96 h o u r s f o r d e a t h , d i a r r h o e a ,

l e t h a r g y , r u f f l e d f u r , c l o s e d eyes , and f o r any o t h e r r e a c t i o n s .

I n t r a v e n o u s i n j e c t i o n s were made. th rough a t a i l v e i n n e a r t h e

r o o t o f t h e t a i l . The v e i n was du-a ted by p l a c i n g t h e t a i l

o f t h e mouse i n water a t a t e m p e r a t u r e o f 45 '~ . The i n t r a p e r i -

t o n e a l i n j e c t i o n s were made on one s i d e o f t h e middle l i n e i n

t h e l o w e r h a l f o f t h e abdomen. F o r t h e subcu taneous i n j e c t i o n s ,

s o l u t i o n s o f t h e t o x i c f r a c t i o n s were i n t r o d u c e d umder t h e s k i n

n e a r t h e r o o t o f t h e t a i l . I n j e c t i o n s i t e s were swabbed w i t h

70% a l c o h o l b e f o r e o p e r a t i o n s . F r a c t i o n s o l u t i o n s were

i n t r o d u c e d o r a l l y th rough t h i n p l a s t i c tubes . The LDW a t I

each r o u t e o f i n j e c t i o n was c a l c u i a t e d a c c o r d i n g t o t h e method

o f Reed and Muench (1938) .

Xnocula t ion o f c h i c k embryo Seven-day embryonated eggs i n

g r o u p s o f f i v e eggs were each i n o c u l a t e d th rough t h e yo lk

s a c w i t h 0.2 m l o f t o x i c f r a c t i o n o f v a r y i n g d o s e s (0.2 - 25.0 rot& \

m g ) . The eggs were i n c u b a t e d f o r 4 8 b u r s , c a n d l e d and c u t

open t o de te rmine t h e number o f dead and s u r v i v i n g embryos.

The LDW was c a l c u l a t e d .

Nec r o ~ s v P o s t mortem examina t ion was c a r r i e d o u t on dead

a n i m a l s immedia te ly a f t e r d e a t h . Each c a r c a s s was d i s i n f e c t e d by c o m p l e t e l y immersing i t i n 3% l y s o l , and f i x e d o n t o a

d i s s e c t i n g board . A l o n g median i n c i s i o n was made through

t h e s k i n o f abdomen and c h e s t . The p e r i t o n e a l c a v i t y was opened wa and t h e abdominal wall r e f l e c t e d , careA8aken n o t t o a l l o w t h e

s k i n - con tamina te t h e i n t e r n a l o rgans . V i t a l o r g a n s , namely,

t h e l i v e r , k idney , s p l e e n , h e a r t , and t h e i n t e s t i n e were

removed w i t h s t e r i l e i n s t r u m e n t s and p l a c e d i n s t e r i l e P e t r i

d i shea . The appearance o f t h e o r g a n s were noted . Heart b lood

was i n o c u l a t e d o n t o TCBS and BHI-3WaC1 a g a r , and i n c u b a t e d

a t 3 7 ' ~ f o r 24 t o 48 houre.

C u l t u r e o f v i t a l organg! The l i v e r , k idney , h e a r t , s p l e e n ,

i n t e s t i n e , and t h e p e r i t o n e a l f l u i d were c u l t u r e d . A small

p o r t i o n o f each o rgan was c u t o u t w i t h a s t e r i l e i n s t r u m e n t

and smeared o n t o TCBS a g a r . A smear o f t h e p e r i t o n e a l f l u i d . was made w i t h a s t e r i l e swab. T h e - i n o c u l a t e d a g a r p l a t e s

were i n c u b a t e d f o r 24 t o 48 hours .

H i s t o l o a y o f v i t a l o r ~ a n s S e c t i o n s o f t h e l i v e r , s p l e e n ,

k idney, hea ' r t , and t h e i n t e s t i n e were p repared on microscope

s l i d e s and s t a i n e d . H i s t o p a t h o l o g i c a l o b s e r v a t i o n s were made

by microscopy ( x4, x10, o r x40 m a g n i f i c a t i o n ) . Same t x a e u e s

from u n i n o c u l a t e d a n i m a l s were used as c o n t r o l s .

P y r o g e n i c i t y t e s t The method used was ac d e s c r i b e d i n t h e

B r i t i s h Pharmacopeia (1$l80). A s o l u t i o n o f t h e t o x i c f r a c t i o n

c o n t a i n i n g 20 ug o f p r o t e i n was i n j e c t e d i n t r a v e n o u s l y th rough

t h e e a r v e i n o f an a d u l t r a b b i t weighing 3.0 kg. The

t e m p e r a t u r e o f t h e rabb i t was t a k e n i m m e d i a t e l y b e f o r e i n j e c t i o n

and 30 m i n u t e s a f t e r i n j e c t i o n . The mean o f t h e two t e m p e r a t u r e s

were r e c o r d e d as t h e i n i t i a l t e m p e r a t u r e o f t h e r a b b i t .

Subsequent t e m p e r a t u r e s were t aken ' a t 30 minute i n t e r v a l s f o r

3 hours . To t a k e t h e t e m p e r a t u r e , a c l i n i c a l thermometer w a s

i n s e r t e d 5 cm deep i n t o t h e rec tum o f t h e a n i m a l f o r 2 minutes .

The r e s p o n s e was t a k e n as t h e d i f f e r e n c e between t h e i n i t i a l

t e m p e r a t u r e and t h e maximum t e m p e r a t u r e o f t h e r a b b i t .

Haemolyt ic a c t i v i t y t e s t The method d e s c r i b e d by Miyamoto

e t al . (1980) was m o s i f i e d and used. Toxic f r a c t i o n c o n t a i n i n g

10.0 ug o f p r o t e i n m l - ' was d i l u t e d t w o f o l d s i n PBS (pH 7 .2)

t u o b t a i n graded l o s e s o f t h e f r a c t i o n . One-percent o f a

a

d e f i b r i n a t e d e r y t h r o c y t e s u s p e n s i o n was added t o a n e q u a l

volume (2.0 m l ) o f e a c h d i l u t i o n i n a t e s t - t u b e and t o b u f f e r

( n e g a t i v e c o n t r o l ) . The t e s t - t u b e s were k e p t f o r 1 h o u r i n

a 3 7 ' ~ water -bath w i t h p e r i o d i c a g i t a t i o n ( C a v a l i e r i & Snyder,

1982). Unlysed c e l l s were p e l l e t e d by c e n t r i f u g a t i o n a t 1 000

r e v . min-' f o r minutes , and t h e o p t i c a l d e n s i t y (O.D.) o f t h o

s u p e r n a t a n t was r e a d o f f a t 545 nm wavelength u s i n g t h e

n e & a t i v e c o n t r o l as b lank . One hundred-percent l y s i s was

e s t a b l i s h e d by t h e a d d i t i o n o f 0.1% sodium c a r b o n a t e (Na$03)

s o l u t i o n t o e r y t h r o c y t e s u s p e n s i o n (Cherwonogrodzky & Clark, 1981)

P e r c e n t a g e haemolys i s was de te rmined calorimetrically.

One haemoly t i c dose (HD ) i s t h e s m a l l e s t amount o f p r o t e i n 50 i n t h e t o x i c f r a c t i o n which caused 50% e r y t h i o c y t e l y s i s .

C y t o t o x i c l t u t e s t

H a r v e s t i n g o f macroahaaeg Macrophages were h a r v e s t e d from . .

t h e p e r i t o n e a l e x u d a t e o f mice by t h e method d e s c r i b e d by

S u t h e r l a n d (1979). Mouse used was k i l l e d by r a p i d d i s l o c a t i o n

o f t h e neck. The s k i n on t h e a n t e r i o r abdominal wall was

dampened w i t h 70% a l c o h o l and l i f t e d w i t h f o r c e p s . A m i d l i n e

i n c i s i o n , a b o u t 50 mm l o n g was made on t h e abdominal s k i n

and t h e f u r was s t r i p p e d hack, c a r e t a k e n n o t t o touch t h e

abdomen. Medium -\@$$f)- 3 HI.) was i n j e c t e d i n t o t h e

p e r i t o n e a l c a v i t y . A f t e r prodding, t h e p e r i t o n e a l f l u i d was

a s p i r a t e d . b y P l a t e r a l t r a c t i o n on t h e l e f t f l a n k o f t h e an imal .

Macrophagee t h u s o b t a i n e d were washed t w i c e i n Medium 199-

3%BSA, pooled , and coun ted i n a haemocytometer, and a d j u s t e d

t o t h e d e s i r e d c o n c e n t r a t i o n .

Assay To measure t h e e f f e c t o f t h e t o x i c f r a c t i o n on

macrophage v i a b i l i t y , t h e method d e s c r i b e d by C a v a l i e r i and

Snyder (1982) 'was used.. Graded d o s e s o f t h e t o x i c f r a c t i o n

was ob ta ined by 2- fo ld s e r i a l d i l u t i o n 6 o f a 10 wg m l m l ( s S ?r0t4$

s o l u t i o n o f t h e f r a c t i o n . One-ml of macrophage suspens ion

( 1 x lo4 c e l l s ml - l ) was added t o each d i l u t i o n i n s i l i c o n i s e d

tubes . The t u b e s were k e p t i n a 3 7 ' ~ water-bath w i t h shak ing

f o r 60 minutes. Cont ro l t ube con ta ined macrophage suspens ion

and PBS only. Viable c e l l s were counted by add ing 0.4 m l o f

0.4% Trypan b l u e t o 2.0 m l o f c e l l suspension. Unstained c e l l s

( v i a b l e c e l l s ) were counted i n a haemacytometer and t h e

percen tage o f dead c e l l s c a l c u l a t e d as shown below.

% Dead c e l l s = No - Nt x 100 ' No

where, No = number o f v i a b l e c e l l s i n t h e c o n t r o l

Nt = number o f v i a b l e c e l l s i n t h e t e s t s o l u t i o n . I

One c y t o t o x i c dose (CD ) is t h e > e a s t amount o f p r o t e i n i n 50 t o x i c f r a c t i o n which k i l l e d 50% of mouse p e r i t o n e a l

macro phages . . (MPM) . E f f e c t o f sub tox i c doses o f t o x i c f r a c t i o n on v i t r o Y a c r o ~ h a ~ e f u n c t i o n

Treatment of m a c r o p h a ~ e s wi th sub tox i c doses A s o l u t i o n

o f t o x i c f r a c t i o n c o n t a i n i n g 1 CD50 was s e r i a l l y d i l u t e d 2-fold

i n PBS (.pH 7.2) and p laced i n 1.5 m l p o r t i o n s i n s i l i c o n i s e d

t e s t - t u b e s . An equa l 'volume of macrophage suspens ion

( 1 x lo4 c e l l s r n l - I ) i n ~ e d i u m 199 was added t o each t e s t - t u b e

and t o a t e s t - t u b e c o n t a i n i n g b u f f e r f o r c o n t r o l . The mix tu re s

were incuba ted f o r

The macrophages i n

30 minutes i n a 3 7 ' ~ water-bath w i t h shaking.

each mixture were harves ted by c e n t r i f u g a t i o n

(500 rev. min- ' ), washed once i n c o l d Medium 199, and then

resuspended i n t h e same medium ( C a v a l i e r i &. Snyder, 1982).

Macrophages from mice immunised with 3CD o f t o x i c f r a c t i o n 50

were s i m i l a r l y t r e a t e d wi th sub tox ic doses. Trypan b l u e was

used t o determine v i a b l e c e l l s .

Opsonisat ion o f b a c t e r i a l c e l l s One-ml of washed c e l l s

6 o f y. parahaemols t icus (1 x 10 c.f.u. n l - I ) suspended i n

Medium 199 were added t o 2.0 m l o f f r e s h human serum and

incuba ted f o r 30 minutes a t 37'~. The c e l l s were washed

once i n Medium 199 and were resuspended i n t h e same medium

( C a v a l i e r i & Snyder, 1982). - s

Phagocytic and adherence assays The t e s t s were c a r r i e d ou t

as desc r ibed by C a v a l i e r i and Snyder (1981). A suspension of 6 b a c t e r i a l , c e l l s (1 x 10 c. f .u. m l - ' ) was added t o an equa l

4 volume of t o x i c - f a c t o r - t r e a t e d macrophages ( 1 x 10 c e l l s m l - l )

i n each t e s t - t u b e and a l s o t o b u f f e r - t r e a t e d macrophages,

The c o n t r o l t e s t - t u b e conta ined opsonised b a c t e r i a only . The

tubes were incuba ted i n a 3 7 ' ~ water-bath w i t h shaking. For

phagocytic a s say , 0.1 m l of t h e suspension was withdrawn a f t e r

60 minutes of i ncuba t ion , added t o 9.9 m l of s t e r i l e d i s t i l l e d

water , and blended i n a b l ende r f o r 1 minute t o l y s e t h e

macrophages. The r e s u l t i n g suspension was d i l u t e d s e r i a l l y

f o r v i a b l e ' c o u n t .

$ P h a g o c y t o s i s (Phagocj t i c i n d e x ) = N ~ , - Nt x 100 No

where, No = v i a b l e c o u n t o f 0.1 m l o f c o n t r o l c o n t a i n i n g b a c t e r i a o n l y .

Nt = v i a b l e c o u n t o f 0.1 m l o f s u s p e n s i o n a f t e r i n c u b a t i o n w i t h t o x i c f a c t o r - t r e a t e d MPM.

To measure b a c t e r i a l adherence , 1.0 m l o f t h e s u s p e n s i o n was

c e n t r i f u g e d a t 500 rev. min-l. The s u p e r n a t a n t c o n t a i n i n g

unadhered b a c t e r i a was d i l u t e d s e r i a l l y and p l a t e d o u t on

TCBS a g a r f o r v i a b l e

% Adherence

where, No

coun t .

= v i a b l e c o u n t o f 1.0 m l o f c o n t r o l c o n t a i n i r i g o p s o n i s e d b a c t e r i a on ly .

I

= v i a b l e c o u n t o f 1.0 m l o f s u s p e n s i o n a f t e r i n c u b a t i o n w i t h t o x i c f a c t o r - t r e a t e d MPM. -.

E f f e c t on macrophaae chemotax i s S i l i c o n i s e d c a p i l l a r y

t u b e s (1.2 x 60.0 mm) h a l f - f i l l e d w i t h 1 % t r y p t o n e (chemoatkrac-

t a n t ) ( F r e t e r & O t B r i e n , 1981) and s e a l e d a t empty ends wi th

p l a s t i c i n e were d ipped i n t o 1.0 m l o f t o x i c f a c t o r - t r e a t e d

o r b u f f e r - t r e a t e d macrophage s u s p e n s i o n , and i n t o b u f f e r

s o l u t i o n ( c o n t r o l ) . The t u b e s were i n c u b a t e d o v e r n i g h t a t

3 7 ' ~ (Hudson & Hay, 1980) . M i g r a t i o n was marked by i n c r e a s e i n t h e h e i g h t o f f l u i d i n t h e c a p i l l a r y t u b e s .

Migrat ion (mm) = H f - Hi where, Hf = f i n a l h e i g h t of f l u i d

Hi = i n i t i a l he igh t o f f l u i d

Migrat ion r a t e = Migration by t o x i n - t r e a t e d MPM x 101

Migrat ion by b u f f e r - t s e a t e d MPM

E n t e r o t o x i a e n i c i t y t e s t a

Rabbit i l e a l loo^ t e s t I n t e s t i n a l l o o p t e s t i n r a b b i t was

c a r r i e d ou t as desc r ibed by Reyes e t a l . (1983) .TRew Zealand 7

white r abb iQweigh ing 4.0 kg were a n a e s t h e s i s e d by i n h a l i n g

chloroform. An i n c i s i o n was made a long t h e mid-antdrior l i n e

of t h e hbdomen and loops (10 cm) of t h e jejumum were l i g a t e d .

Graded amounts o f t o x i c f r a c t i o n ,(20 - 200 ag) i n 1.0 m l po r t i on ;

were i n j e c t e d i n t o a l t e r n a t e loops: BHI-3gNaC1 was used as

a p o s i t i v e c o n t r o l , while s a l i n e was used as a n e g a t i v e c o n t r o l .

The abdomen was su tured . The animal was s a c r i f i c e d 18 hours

l a t e r , a n d ' t h e l o o p s obwerved f o r accumulation o f sanguinolen t

f l u i d .

Suckl ing mouse assay, The s u c k l i n g mouse t e s t was c a r r i e d

o u t as desc r ibed by Dean et al . (1972). I n f a n t mice 1 - 4

days o l d were s epa ra t ed from t h e i r mothers and used immediately.

One-tenth m l of graded doses o f t o x i c f r a c t i o n (0.04 - 25.00 .ug)

was in t roduced o r a l l y through t h i n p l a s t i c tub ing i n t o groups

of mice. The an imals were s a c r i f i c e d 4 hours l a t e r , and t h e - ....

f l u i d accumula t ion r e s p o n s e q u a n t i f i e d by d e t e r m i n i n g t h e r a t i o

g u t weinht = mouse u n i t remaining body weight

R a t i o s l ess t h a n 0.070 were c o n s i d e r e d n e g a t i v e . t h o s e i n t h e

r a n g e O.070 - 0.090 q u e s t i o n a b l e p o s i t i v e , and t h o s e g r e a t e r

than 0.090 s t r o n g l y p o s i t i v e , S a l i n e was used as n e g a t i v e

c o n t r o l .

Sk in r e a c t i o n t e s t A s l i g h t m o d i f i c a t i o n o f t h e method

d e s c r i b e d by Miyamoto e t el . (1989) was used. Two guinea-

p i g s and one r a b b i t were each i n j e c t e d i n t r a d e r m a l l y w i t h 0.2 ml

o f g raded d o s e s o f t h e t o x i c f r a c t i o n (0.8 - 20.0 lug). The

s k i n a t t h e s i t e s o f i n j e c t i o n was- observed f o r 48 h o u r s f o r

inf lammatory r e s p o n s e , S a l i n e was i n j e c t e d f o r c o n t r o l . . I m m u n o ~ e n i c i t ~ t e s t g

Antiserum p r o d u c t i o q N e w Zealand w h i t e r a b b i t weighing 3 k g . ,

was i n t r a v e n o u s l y i n j e c t e d w i t h t h e u n p u r i f i e d f r a c t i o n

f o l l o w i n g t h e s c h e d u l e shown i n T a b l e 3. The an imal was b l e d

on t h e t h i r t e e n t h day by c a r d i a c p u n c t u r e , and wholm blood

c o l l e c t e d i n s t e r i l e u n i v e r s a l b o t t l e s was a l lowed t o c l o t a t

room tempera tu re . The c l e a r s u p e r n a t a n t was s e p a r a t e d from

t h e c l o t by c e n t r i g u a t s o n , c o l l e c t e d i n 2.0 m l p o r t i o n s i n

B i j o u b o t t l e s , and k e p t f r o z e n a t - 4 ' ~ i n a r e f r i g e r a t o r .

M i g r a t i o n (mm) = Hf - Ha where, H f = f i n a l h e i g h t o f f l u i d

Hi = i n i t i a l h e i g h t o f f l u i d

M i g r a t i o n r a t e = Migra t ion by t o x i n - t r e a t e d MPM x 101

M i g r a t i o n by b u f f e r - t s e a t e d MPM

E n t e r o t o x i a e n i c i t y t es t s

Rabbi t i l e a l loo^ tes t I n t e s t i n a l l o o p t es t i n r a b b i t was

c a r r i e d o u t as d e s c r i b e d by Reyes e t a l . (1983) .?Mew Zealend \

w h i t e r a b b i $ weighing 4.0 k g were a n a e s t h e s i s e d by i n h a l i n g

chloroform. An i n c i s i o n was made a l o n g t h e m i d - a n t a i o r l i n e

o f t h e !abdomen and l o o p s ( 1 0 cm) o f t h e jejumum were l i g a t e d .

Graded amounts o f t o x i c f r a c t i o n d20 - 200 a g ) i n 1 -0 r n l p o r t i o n ,

were i n j e c t e d i n t o a l t e r n a t e loops: BHI-3%NaCl was used a s

a p o s i t i v e c o n t r o l , w h i l e s a l i n e was used as a n e g a t i v e c o n t r o l .

The abdomen was s u t u r e d . The an imal was s a c r i f i c e d 18 h o u r s

l a t e r , and t h e l o o p s obmerved f o r accumula t ion o f s a n g u i n o l e n t

f l u i d .

S u c k l i n ~ mouse assag The s u c k l i n g mouse t e s t was c a r r i e d

o u t as d e s c r i b e d by Dean e t a l . (1972) . I n f a n t mice 1 - 4

days o l d were s e p a r a t e d from t h e i r mothers and used immedia te ly ,

One-tenth m l o f g raded d o s e s o f t o x i c f r a c t i o n (0.04 - 2 5 . O O . u g )

was i n t r o d u c e d o r a l l y th rough t h i n p l a s t i c t u b i n g i n t o c o u p s

o f mice. The a n i m a l s were s a c r i f i c e d 4 h o u r s l a t e r , and t h e - ..

Day of inmunisation ;.lose o f un : iu r i l ':~.ed t o x i c f ~ : c t o r i i ~ j e c t : . . d (ii~g)

................... ._.. ..- .. -.- .... -. -.- ................ - I.. ......... .--..

1 = r e s t

Tube a g g l u t i n a t i o n The a n t i s e r u m o b t a i n e d above was

d i l u t e d t w o f o l d s i n PBS (pH 7.2) . One-ml s u s p e n s i o n o f 6 I s o l a t e KV ( o r KV1 o r V6) i n PBS c o n t a i n i n g 1 x 10 c . f .u . 3

m l - ' was added t o 2 .8 m l o f each a n t i s e r u m d i l u t i o n i n '

s i l i c o n i s e d t e s t - t u b e s , t o normal r a b b i t serum ( c o n t r o l ) , o r

t o PBS ( c o n t r o l ) . The t u b e s were k e p t i n a 3 7 ' ~ r a t e r - b a t h br 1 hour. Each m i x t u r e was obse rved m a c r o s c o p i c a l l y ( x 10

m a g n i f i c a t i o n ) f o r clumping o f b a c t e r i a l c e l l s .

P a s s i v e h a e m a g g l u t i n a t i o n t e s t

To d e t e c t t h e p reeence of a n t i b o d y i n t h e rabb i t immune

serum, t h e p a s s i v e h a e m a g g l u t i n a t i o n t e s t was c a r r i e d o u t as

d e s c r i b e d by Cruickshank e t a l . ( 1 975) .

Tanning o f r e d b lood c e l l s To make t h e e r y t h r o c y t e s more

a g g l u t i n a b l e , t a n n i c a c i d s o l u t i o n ( 1 i n 20 000 d i l u t i o n )

p repared by J i s s o l v i n g 12.5 (ug o f t a n n i c a c i d ( a n a l y t i c a l

g r a d e ) i n , 2 2 0 m l o f PBS was u s e d . Thir ty-ml o f 6% s h e e p

e r y t h r o c y t e s s u s p e n s i o n i n PBS, pH 7.2, were i n c u b a t e d w i t h

a n e q u a l volume o f t h e t a n n i c a c i d s o l u t i o n f o r 1 5 m i n u t e s

a t 37'~. The e r y t h r o c y t e s were t h e n washed twkcesand

r e c o n a t i t u t e d i n PRS a t t h e same c o n c e n t r a t i o n .

S e n s i t i s a t i o n / c o a t i n ~ o f t anned e r y t h r o c y t e s Five-ml o f

t h e t anned e r y t h r o c y t e s were suspended i n a n e q u a l volume o f

t o x i c f r a c t i o n and i n c u b a t e d f o r 30 m i n u t e s w i t h o c c a s i o n a l

s h a k i n g a t 37'~. The now s e n s i t i s e d ( o r c o a t e d ) e r y t h r o c y t e s

were washed t h r e e times i n PBS c o n t a i n i n g 1 % no rma l r a b b i t

serum, and r e c o n : ; t i t u t e d i n t h e PBS t o 1 % e r y t h r o c y t e

s u s p e n s i o n . Untanned e r y t h r o c y t e s were p r e p a r e d i n t h e same

way a n d u s e d f o r c ' l n t r o l .

I l a e m a a g l u t i n a t i o n Doubl ing d i l u t i o n s o :f a n t i s e r u m h e a t e d

f o r j O m i n u t e s a t ~ G O C was made i n 0,1 m l volumes o f PBS i n

m i c r o t i t r e wells. One-percent s e n s i t i s e d t a n n e d e r y t h r o c y t e s ,

o r u n s e n s i t i s e d e r y t h r o c y t e s ( c o n t r o l ) was added t o e q u a l

volume o f e a c h d i l u t i o n and t h e m i x t u r e i n c u b a t e d f o r 2 h o u r s

a t 3 7 O ~ . A c o n t r o l w e l l c o n t a i n e d b u f f e r and s e n s i t i s e d t a n n e d

e r y t h r o c y t e s i n e q u a l volumes. The a n t i s e r u m t i t r e was

t a k e n as t h e r e c i p r o c a l o f t h e h i g h e s t d i l u t i o n w i t h a g g l u t i n a -

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

c r e n a t e d r i m . s

Immunodi f fus ion t e s t s Immunodif f u s i o n t e s t s were c a r r i e d

o u t by t h e s t a n d a r d method o f O u c h t e r l o n y (1958) . A P e t r i

d i s h ( o r a m i c r o s c o p e s l i d e ) was e v e n l y c o a t e d w i t h 10.0 m l

( o r 3.0 ml) o f 1.5% (w/v) a g a r no. 1 s o l u t i o n i n 0 . 2 m l

p h o s p h a t e b u f f e r e d s a l i n e , pH 7.2. Sodium a z i d e (0.1%) was

added t o p r e v e n t m i c r o b i a l g rowth , a n d t h c a g a r was a l l o w e d

t o h a r d e n f o r 30 m i n u t e s . A m a t r i x o f f o u r wells e a c h 3.5 mm

and s p a c e d a t a d i s t a n c e o f 7.0 mrn was made on t h e P e t r i d i s h

(2 .0 mm w e l l s s p a c e d a t a d i s t a n c e o f 4 , O mm on t h e m i c r o s c o p e

s l i d e ) . O u t e r wells were f i l l e d w i t h t h e t o x i c f r a c t i o n s o l u t i o

(20.0 ug) o r w i t h t h e u n p u r i f i e d f r a c t i o n (1 i n 10 d i l u t i o n ) .

The c e n t r a l w e l l s were f i l l e d w i t h t h e a n t i s e r u m ( 1 i n 20

d i l u t i o n ) . The g e l p r e p a r a t i o n s were i n c u b a t e d o v e r n i g h t i n

a m o i s t chamber f o r p r e c i p i t a t i o n bands t o d e v e l o p where

p o s s i b l e .

J m m u n o e l e c t r o ~ h o r e s i ~ The tes t was c a r r i e d o u t on a

microscope s l i d e o v e r l a i d w i t h a g a r g e l ( a s d e s c r i b e d above)

by a m o d i f i c a t i o n o f t h e m i c r o t e c h n i q u e o f Sche idegger ( 1955)

(Sochard & C 0 1 w e l l . i 1977). An u p p e r w e l l c o n t a i n e d t h e

t o x i c f r a c t i o n , w h i l e t h e lower one c o n t a i n e d t h e u n p u r i f i e d

f r a c t i o n . The s l i d e was p l a c e d between two b u f f e r r e s e r v o i r s

wi th t h e p o l a r i t y as i n d i c a t e d i n , the diagram below i n an

e l e c t r o p h o r e s i s a p p a r a t u s , and was 'connected a t b o t h e n d s t o

t h e b u f f e r by means o f m o i s t f i l t e r p a p e r wicks. A d i r e c t

c u r r e n t power , supp ly was connec ted t o a p p l y 45- t o 50 v o l t s

P o l a r i t y on a m i c r o s c 0 ~ 6 s l i d e p l a c e d i n a n e l e c t r o - - p h o r e t i c a p p a r a t u s

a c r o s s t h e s l i d e s and was d i s c o n n e c t e d a f t e r 45 minutes.

Antiserum a g a i n s t t h e u n p u r i f i e d t o x i c f r a c t i o n was poured

i n t o t h e midd le t r o u g h , The s l i d e was k e p t i n a m o i s t chamber

f o r 24 h o u r s t o a l l o w p r e c i p i t a t i o n bands t o d e v e l o p ,

- S t a i n i n a t e c h n i o u e The a g a r on t h e immunod$ffusion and

i m m u n o e l e c t r o p h o r e t i c s l i d e s was washed by s e v e r a l c h a n g e s o f t

t h e b u f f e r and c o v e r e d w i t h m o i s t f i l t e r p a p e r a n t i 1 t h e a g a r

d r i e d , The d r i e d a g a r was s t a i n e d i n 2% Ponceau S i n 3%

t r i - c h l o r o a c e t i c a c i d (TCA) f o r m i n u t e s , and was washed

i n 5% A c e t i c a c i d ,

Ant i serum b a c t e r i c i d a l a c t i v i t y t e s t A +hour c u l t u r e o f

y, p a r a h a e m o l y t i c u s I s o l a t e KV i n B H I - % N a ~ 1 b r o t h was 3 used , The b a c t e r i a l c e l l s were h a r v e s t e d , washed t w i c e , and

8 r e suspended i n Medium 199 a t a c o n c e n t r a t i o n o f 1 x 10 c . f .urn

m l - ' , Each r e a c t i o n m i x t u r e i n a s i l i c o n i s e d t e s t - t u b e I

compr ised one-ml o f b a c t e r i a l s u s p e n s i o n , one-ml o f a t w o f o l d

s e r i a l d i l u t i o n o f a n t i s e r u m , and one-rnl o f complement ( f r e s h

guinea-pfg serum) . The c o n t r o l t e s t - t u b e s c o n t a i n e d e i t h e r

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

s u s p e n s i o n , complement and b u f f e r . The c o n t e n t s o f t h o t u b e s

were mixed by f l i p p i n g and i n c u b a t e d i n a 3 7 ' ~ wa te r -ba th .

Samples '

60-,

o f Miles

d i l u t i o n

were removed from e a c h t u b e a t i n t e r v a l s o f 0-, 30-,

120 m i n u t e s f o r v i a b l e c o u n t a c c o r d i n g t o t h e methods

and Mizra (1938) . The l y t i c t i t r e i s t h e h i g h e s t

o f a n t i s e r u m which c a u s e d more t h a n 50% c e l l d e a t h .

Mouse p r o t e c t i o n t e s t To e v a l u a t e t h e p r o t e c t i v e power

o f t h e a n t i - t o x i c f a c t o r serum, one-ml o f a n t i s e r u m ( u n d i l u t e d )

was i n j e c t e d i n t r a p e r i t o n e a l y i n t o each o f two mice. C o n t r o l

a n i m a l s were i n j e c t e d w i t h normal r a b b i t serum. A f t e r one

hour , each o f t h e c o n t r o l and t es t a n i m a l s was c h a l l e n g e d i n t r a -

p e r i t o n e a l y w i t h a l e t h a l dose (10 LD i . p . , i . e . 118.0.ug) 50 o f t h e t o x i c f r a c t i o n . The a n i m a l s were obse rved f o r 96 h o u r s

f o r d e a t h o r s u r v i v a l .

Antiserum n e u t r a l i s a t i o n t e s t Two-ml o f a n t i s e r u m o r

normal r a b b i t serum ( c o n t r o l ) were added t o each of two

t e s t - t u b e 6 c o n t a i n i n g 2.0 m l o f t o x i c f r a c t i o n . The t u b e s

were k e p t i n a 3 7 ' ~ water -bath f o r 1 5 minu tes ( C a v a l i e r i &

Snyder, 1982) a f t e r which a n a l i q u o t was removed from each * *

t u b e and a s s a y e d f o r haemoly t i c o r c y t o t o x i c a c t i v i t y .

PHYSICOCHEMICAL CHARACTERISATION

Absorp t ion spect rum The o p t i c a l d e n s i t y r e a d i n g s o f t h e

t o x i c f r a c t i o n ( 10.0 ug m l - ' ) were determined a t 350-, 320-,

300-, 280-, 250- and 200 nm wavelength i n a UV-spectro-

photometer (SP6-400 PYE U N I C A M ) .

P r o t e i n d e t e r m i n a t i o n *o te in was e s t i m a t e d by t h e method

of Lowry e t a l . (1941) . s o v i n e serum albumin (BSA) a t

c o n c e n t r a t i o n s o f 10.0 t o 100.Omg m l - ' i n 10.0 a g m l - 1

i n c r e m e n t s were used as s t a n d a r d p r o t e i n c o n c e n t r a t i o n s

(Miyamoto e t a l . , 1980) . The r e a g e n t s o l u t i o n c o n t a i n e d a

m i x t u r e of 2% sodium c a r b o n a t e (Na CO ) i n 0 . 1 N sodium 2 3

hydroxide (NaoH), and 0.5% CuSO .5H20 i n 1 % sodium t a r t a r a t e 4 ( 1 i n 50 p a r t s ) . The r e a g e n t was added t o 0.2 m l o f sample

and t h e m i x t u r e a l lowed t o s t a n d f o r 10 minu tes a t room

tempera tu re . One-tenth m l o f F o l i n - C i o c a l t e a u ' s phenol

r e a g e n t d i l u t e i ; twofo ld i n d i s t i l l e d w a t e r was added. The

m i x t u r e was r a p i d l y mixed and a l lowed t o s t a n d f o r 30 m i n u t e s

a t room t e m p e r a t u r e a f t e r which t h e o p t i c a l d e n s i t y (O.D.)

was measured s p e c t r o p h o t o m e t r i c a l l y a t 280 nm. The p r o t e i n

c o n c e n t r a t i o n o f t h e t e s t sample was r e a d o f f from a

s t a n d a r d c o n c e n t r a t i o n o f BSA p l o t t e d a g a i n s t t h e i r O.D.

r e a d i n g s . I

Carbohydra te d e t e r m i n a t i o n Carbohydra te was a s ~ a y e d by

t h e a n t h r o n e r e a g e n t (Mor r i s , 1948) u s i n g s u c r o s e a s a

s t a n d a r d i n 10 ug inc rements . A m i x t u r e of 1.0 m l o f t e s t . .

. ,

a m p l e and '4.0 m l o f a n t h r o n e r e a g e n t (0.2% i n conc. H2S04)

in"a r u b b e r s t o p p e r e d t e s t - t u b e was b o i l e d f o r 10 minu tes i n

a b o i l i n g water -bath , and c o o l e d r a p i d l y i n a n i c e - b a t h .

Co lour ( b l u e ) development was measured s p e c t r o p h o t o m e t r i c a l l y

a t 620 nm. The b lank c o n t a i n e d a m i x t u r e o f t h e r e a g e n t and

water t r e a t e d as d e s c r i b e d above.

L i ~ i d debermina t ion L i p i d was determined by t h e method of

I i d a and Takagi (1977) . A 0.5 m l o f t o x i c f r a c t i o n was added

t o 2.0 m l o f ch loroform-methanol -water (1:%:0.8) m i x t u r e and

c e n t r i f u g e d a t 1 000 r ev . min-' f o r 5 minu tes . The s u p e r n a t a n t

was c o l l e c t e d , e v a p o r a t e d a t 5 0 ' ~ i n a wa te r -ba th , and

obse rved f o r t h e p r e s e n c e o f i n s o l u b l e p a r t i c l e s .

P o l y a c r y l a m i d e g e l e l c t r o ~ h o r e s i s (PAGE) PAGE was c a r r i e d

o u t as d e s c r i b e d by Dav i s (1964) . G i l c o n t a i n i n g 7.5%

a c r y l a m i d e , 0.2 &is-acrylamide and ammonium p e r s u l p h a t e ( a s

i n i t i a t o r ) was used . E l e c t r o p h o r e s i s was per formed i n T r i s -

g l y c i n e b u f f e r (pH 8.6) a t a c o n s t a n t c u r r e n t o f 5 m i l l i a m p e r e

(mA) p e r t u b e f o r 3 h o u r s u n t i l t h e t r a c k i n g dye went o f f t h e

bot tom o f t h e columns. A l l g e l s were s t a i n e d w i t h

bromophenol b l u e .

A c e t a t e p a p e r e l e c t r o ~ h o r e s i s S t r i p s o f a c e t a t e p a p e r were

mois t ened w i t h phospha te b u f f e r , pH 7.2, and t h e spec imen 8

a p p l i e d toward t h e c a t h o d e end o f t h e s t r i p . E l e c t r o p h o r e s i s

was c a r r i e d o u t i n 0.02M phospha te b u f f e r , pH 7.2, a t a

c o n s t a n t c u r r d n t o f 40 mA f o r 30 minu tes . The p a p e r s t r i p s

were s t a i n e d w i t h brvmophenol b l u e .

Molecu la r w e i ~ h t d e t e r m i n a t i o n The methods o f P e t e r s e t a l .

(1980) and Andrews (1964) were used. The m o l e c u l a r we igh t of protein

o f t h e t e s t s o l u t i o n ( 1 0 . 0 ~ @ was approx ima ted w i t h Sephadex

GI50 i n a 2.5- by 50 cm columns. S t a n d a r d marker p r o t e i n s ,

Myoglobulin, T r y p s i n , Bovine serum a lbumin (BSA), L a c t a t e

dehydrogenase, and P y r u v a t e k i n a s e w i t h known m o l e c u l a r prctek 1

w e i g h t s were used a t c o n c e n t r a t i o n s o f 5.0 rug m l i n 0.02M A

phosphate b u f f e r , pH 7.2. E l u t e d f r a c t i o n s were measured

s r e c t r o p h o t o m e t r i c a l l y a t 280 nm. The t e s t s o l u t i o n was

i d e n t i f i e d by i t s haemoly t i c a c t i v i t y . The m o l e c u l a r w e i g h t s

and e l u t i o n volumes o f t h e s t a n d a r d marker p r o t e i n s a r e

g i v e n i n Tab le !+. Temperature s t a b i l i t y Two-ml o f t o x i c f r a c t i o n ( 1 0 . 0 4 g prdW

* m l - ' ) were p l a c e d i n each o f t h r e e t e s t - t u b e s . One t u b e was

h e a t e d a t 60°c f o r 30 minu tes , and t h e o t h e r a t 1 0 0 ~ ~ f o r 15

minutes. The h e a t e d samples were c o o l e d r a p i d l y by immersing

t h e t e s t - t u b e s i n a n i c e - b a t h l m m e d i a t e ' l y a f t e r h e a t i n g (Snyder

& Koch, 1966; and C a v a l i e r i & Snyder, 1982). For c o n t r d l , t h e

t h i r d t e s t - t u b e was k e p t a t 37 '~ irl a water-bath. The

u n p u r i f i e d t o x i c f r a c t i o n , i . e . t h e 40% (NH4)$04 p r e c i p i t a t e

n o t s u b j e c t e d t o g e l f i l t r a t i o n , was s i m i l a r l y t r e a t e d . A l l

samples and t h e i r c o n t r o l s were a s s a y e d f o r haemoly t i c and

c y t o t o x i c a c t i v i t i e s .

Temperature a c t i v i t y r a n a e Serial d i l u t i o n s o f t h e t o x i c

f r a c t i o n t o which 1% e r y t h r o c y t e s were added were i n c u b a t e d

a t d i f f e r e n t t e m p e r a t u r e s of loOc, 2 8 O ~ , 3 7 ' ~ . . . , o r 6 0 ' ~ f o r

1 hour. The haemoly t i c dose a t each t e m p e r a t u r e was determined

( P e t e r s , Baross , and Mor i t a , 1982).

Optimum p H Serial d i l u t i o n s o f t h e sample s o l u t i o n were

Toxic f ~ l c t o r 63 950 250

O~t imum DH S e r i a l d i l u t i o n s o f t h e sample s o l u t i o n

were p repared i n 0.OZM phosphate b u f f e r of pH v a l u e s 5.8, 6.4,

..., o r 8.4. Washed e r y t h r o c y t e s suspended i n t h e same

b u f f e r a t t h e v a r i o u s pH v a l u e s were used f o r haemoly t i c

a s s a y s . Washed macrophages were a l s o used f o r c y t o t o x i c a s s a y s .

E f f e c t o f p r o t e d l y t i c enzymes One-ml o f t o x i c f r a c t i o n

was added t o a n equa l volume o f 1 % t r y p s . i n o r p e p s i n , o r t o

b u f f e r ( . c o n t r o l ) pH 7.2, i n a t e s t - t u b e . Each t u b e was

i n c u b a t e d a t 3 7 ' ~ f o r 1 h o u r (Miyamoto e t al . , 1980). Samples

o f each m i x t u r e were t a k e n and a s s a y e d f o r haemoly t i c ancl

c y t o t o x i c a c t i v i t i e s .

E f f e c t of added meta l i o n @ One m i l l i m o l a r ( 1 m M ) solutions

* +t ++ +t f t o f C a , Mg, Mn, Hg, a n , C< and Fe i o n s were p r e p a r e d i n PBS

pH 7.2 u s i n g t h e i r c o r r e s p o n d i n g s a l t s (CaC12, MgS04, MnS04,

HgC12, ZnC12, CuSO and FeS04). Each c a t i o n s o l u t i o n 4'

p repared was 'added t o e q u a l volumes o f twofo ld s e r i a l d i l u t i o n s

o f t o x i c f r a c t i o n . E r y t h r o c y t e s u s p e n s i o n was added f o r t h e

a s s a y o f haemoly t i c a c k i v i t y , whi le macrophage s u s p e n s i o n

was added f o r c y t o t o x i c i t y a s s a y .

I s o l a t i o n o f y. p a r a h a e m o l v t i c u s One hundred l a r g e ,

smooth, dome-shaped c o l o n i e s w i t h opaque c e n t r e and g r e e n

c o l o u r were i s o l a t e d on thiosulphate-citrate-bile salts-

s u c r o s e (TCBS) a g a r .

I d e n t i f i c a t i o n o f i s o l a t e s T a b l e 5 shows t h e s t a n d a r d

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

o f p o s i t i v e r e s p o n s e s o f each tes t . A l l t h e i s o l a t e s were

s h o r t , curved, gram-negative r o d s , m o t i l e w i t h p o l a r

f l a g e l l a t i o n . A l l produced t h e enzymes c a t a l a s e , o x i d a s e ,

amylase, and l y s i n e deca rboxy lase . They l i q u e f i e d g e l a t i n

and u t i l i s e d g l u c o s e o x i d a t i v e l y and f e r m e n t a t i v e l y . The

Voges-Proskauer t e s t was n e g a t i v e l a a l l t h e i s o l a t e s .

None of t h e i s o l a t e s produced hydrogen s u l p h i d e (HZS) from

T S I a g a r , n o r t h e enzymes u r e a s e and . a r g i n i n e d i h y d r o l a s e .

Most i s o l a t e s ' grew i n n u t r i e n t b r o t h s u p ~ l e m e n t e d w i t h 1%,

3%. 5%, and 8% N a C 1 , b u t none grew on media supplemented w i t h

10% N a C l . Twenty-five p e r c e n t o f t h e i s o l a t e d c o l o n i e s

produced t h e Kanagawa haemolysin as shown by zones o f

c l e a r i n g around t h e c o l o n i e s on t h e Wagatsuma b lood agar.

~ 1 1 i s o l a t e s grew w e l l a t t e m p e r a t u r e r a n g e o f 3 7 ' ~ t o 4 2 ' ~ .

S u c r o s e and l a c t o s e were n o t fermented.

L e t h a l a c t i v i t y Tab le 6 shows t h e m o r t a l i t y r a t e of mice

L y s i n c decarboxylase

Percent ; ge n ~ o r t a l i t y nu i~ lbcr 01 (lead n i c e x 100 ----

i n j e c t e d i n t r a p e r i t u n e a l y w i t h t h e same dose (1 x lo8 c . f a u r n )

o f d i f f e r e n t Y. p a r a h a e m o l ~ t i c u s i s o l a t e s . The m o r t a l i t y r a t e

was 80% i n mice i n j e c t e d w i t h I s o l a t e K V b u t was lower i n 3'

mice i n j e c t e d w i t h o t h e r i s o l a t e s . For example, i t was 60%

i n t h o s e i ~ j e c t e d w i t h I s o l a t e s K V l and V 1 , and was 40%

i n t h o s e i n j e c t e d w i t h I s o l a t e s KV4. KV,-, V3, V4, and V5.

I s o l a t e s K V 2 and V2 each k i l l e d o n l y 20% o f ' m i c e .

The LDg0 o f I s o l a t e KV i n j e c t e d 3 l i n t r a p e r i t m e a l y i n t o mice was 1' x lo7 c . f .u . , b u t was

8e6 c.f.u. when i n t r o d u c e d o r a l l y ( T a b l e s 7 and 8 ) . 1 x 10.

I n j e c t i n g b a c t e r i a l c e l l s suspended i n i r o n compound reduced

t h e LD 50 ' The LD50 was 1 x 10 3*7 c . f e u . when b a c t e r i a l

s u s p e n s i o n s i n 250 mg of J e c t o f e r .were i n j e c t e d i .p . , and

6 was 1 x 1 0 c . f .u. when b a c t e r i a l $uspens ions i n 700 mg o f

I r o n I1 s u l p h a t e were i n j e c t e d i .p . ( T a b l e s 9 and 10) .

I n v i v o adherence Tab le 1 1 shows t h e v i a b l e c l . )un t s of some

V p a r a h a e m o l v t i c u s i s o l a t e s s i x h o u r s a f t e r each i s o l a t e was

i n j e c t e d i n t o mouse i leum. 'The v i a b l e c o u n t s o f I s o l a t e s KV, J

and KVh i n c r e a s e d from t h e i n i t i a l i n j e c t e d dose of 1 x lo8 c . f e u

t o 1 x 1012 c.f.u. and t o 1 x 10" c.f .u. r e s p e c t i v e l y , i n d i c a t i n

b a c t e r i a l adherence t o i l e a l wall. The v i a b l e c o u n t s o f V 1 0

8 and V6 i n c r e a s e d o n l y s l i g h t l y t o 1 x lo9 c . f .u. and 2 x 10 c . f .u

r e s p e c t i v e l y , and were c o n s i d e r e d non-adherent .

J 0 f . u . * I * o r t a l i t y

in j cc t ed D(?:.$. Livcd D S I - rite (%) -- ,A -----... . --- me.-- -

.y

= Dead

X * = S u r v i v e d

= 8.65

There f [;re, LLg0 = 1 x 10 8 * 6 5 c . f . u . . . . . . . . . . . . . . . . . . . -

Table 10. C a l ~ u l . ~ , t i o n of :r,?..jO of 1so l s . t e KV, o f V_. yr.aal\a.mg- 1

lxt-&r,uLx i n j e c t e d i .?. !,:i.th Ir;.;n EI s u 1 p h r . t ~ (700m.g).

AccuLiiul;.ted v: lues .......... - .....-.. -- ... .- . -.- . . . . . . . . . . . . . .

C.f .u . N o r ' k d i t y

i n j e c t e d 9eaa L i v e d D ,. ,-I j D/I)+S r a t e PA) .--.- ..... --.-..--. . . . . . . -- .-. . -- ........... --.. . . . ............. --.-. . . -

Table 1 1 . " J i s b l e coun t s ol? V. l~~raa~F-a.epl.o.l$t.tii~Cu,s_ i s o l ~ ~ t e s s i x hou r s a f t e r 1 i: 1;' c . f .u . o f each i s o l ~ t e we;x

i n j e c t e d i n t o mouse i l e u i ~ .

coun t s (c:f .u. )

I n v i t r o adherence T h t adherence o f g. p a r a h a e m o l ~ t i c u s

i s o l a t e s t o muuse p e r i t o n e a l macrophages (MPM) in v i t r o was

de termined. A s shown i n Tab le 12, 80% o f I s o l a t e KV c e l l s 3

adhered , whereas o n l y 60% of I s o l a t e KV and 27% o f V10 c e l l s 4

adhered . The adherence r a t e o f I s o l a t e V6 w a s o n l y 15%.

Tab le 12 a l s o shows t h e m o r t a l i t y r a t e s o f mice i n j e c t e d w i t h

t h e i s o l a t e s t e s t e d f o r adherence . The m o r t a l i t y r a t e was

80% i n mice i n j e c t e d wi th ' I s o l a t e KV3, t h e i s o l a t e w i t h t h e

h i g h e s t ra te o f adherence , b u t w a s l o w e r i n t h e o t h e r i s o l a t e s

showing l o w e r rates o f adherence .

ISOLATION AND PURIFICATION OF INTRACELLULAR TOXIC FACTOR

T o x i c i t y o f s u p e r n a t a n t s fcom s o n i c a t e d c e l l s o f Kanagawa-

p o s i t i v e i s o l a t e and Kartagawa-neaative i s o l a t e . The haemoly t i c a c t i v i t y o f t h e sup ' e rna tan t from s o n i c a t e d

c e l l s o f t h e Kanagawa-posi t ive i s o l a t e and t h e haemoly t i c

a c t i v i t y ,of a similar sample from t h e Kanagawa-negative i s o l a t e

were determined. Haemolysis , shown m i c r o s c o p i c a l l y by e r y t h r o -

c y t e l y s i s , w a s obse rved i n t h e t e s t - t u b e c a n t a i n i n g t h e

s u p e r n a t a n t from t h e Kanagawa-posi t ive c e l l s , b u t n o t i n t h e

tubs c o n t a i n i n g t h e s u p e r n a t a n t from t h e Kanagawa-negative

c e l l s . The s u p e r n a t a n t showing t o x i c a c t i v i t y was p u r i f i e d

by p r e c i p i t a t i o n w i t h Ammonium s u l p h a t e c r y s t a l s .

T a b l e 12.. The a d h e r e n c e r e t e s o f some 1. pg~_a1-agrgg.1.&icus i s o l s t e s t o mouse per i tonec.1 -c;acrophzges, and t h e m o r t c l i t y y a t e s of' inice i n j e c t e d w i t h t h e i s o l a t e s ,

Bac t e r i . c l Adherent e K o r t a l i t y

i s o l a t (2s ($1 ( 7 3 - - . -.-....----- .-.-.-.- -- ---- -.--- .-----.-.-- - --.-...----

T o x i c i t y o f Ammonilcm s u l p h a t e p r e c i p i t a t e d f r a c t i o n 6 The

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

a r e shown i n 'Fable 13. One h a e m o l y t i c d o s e (HD ) i s t h e 50

smallest amount o f p r o t e i n i n a f r a c t i v n which c a u s e d l y s i s

o f 50% o f e r y t h r o c y t e s . The 40% p r e c i l i t a t e d f r a c t i o n showing

t h e ..&owest HD (0 .05 mg) was t h e most a c t i v e f r a c t i o n 50 f o l l o w e d by t h e u n p r e c i p i t a t e d s u p e r n a t a n t (HD 0.42 mg), and 50 t h e 20% p r e c i p i t a t e d f r a c t i o n (HD 3.50 mg). Haemolys i s was 50 n o t o b s e r v e d i n t h e o t h e r p r e c i p i t a t e d f r a c t i o n s .

me m o r t a l i t y r a t e s o f mice i n j e c t e d i n t r a v e n o u s l y w i t h

3.0 mg o f p r o t e i n i n e a c h f r a c t i o n are s l~own i n T a b l e 13. The

m o r t a l i t y r a t e was 80% i n mice i n j e c t e d w i t h t h e 40% p r e c ~ p i -

t a t e d f r a c t i o n , b u t was 40% i n t h o s e i n j e c t e d w i t h t h e 20% 1

1

p r e c i p i t a t e d f r a c t i o n . The m o r t a l i t y r a t e s were l o w e r i n mice

i n j e c t e d w i t h t h e o t h e r p r e c i p i t a t e d f r a c t i o n s .

P a r t i a l l y ~ u r i f i e d t o x i c f a c t o r F i g u r e 1 i s a f l o w c h a r t

o f t h e p u r i f i c a t i o n s teps -0.f 40% Ammonium s u l p h u t e p r e c i p i t a t e d

f r a c t i o n which i s t h e b i o l o g i c a l l y mos t a c t i v e f r a c t i o n .

The a c t i v i t y o f e a c h p u r i f i c a t i o n s t e p i s shown i n T a b l e 14.

The h a e m o l y t i c d o s e o f t h e unprecipitated-soni~ated~~~11~ s u p e r -

n a t a n t was 420.0 mg, w h i l e t h a t o f t h e g e l f i l t r a t i o n e l u a t e

was 0.15 rug. P u r i f i c a t i o n f o l d o f t h e Ammonium s u l p h a t e

p r e c i p i t a t e d f r a c t i o n was x8.4, whereas t h a t o f t h e gel f i l t r a t i o

e l u a t e ( t o x i c f a c t o r o r t o x i c f r a c t i o n ) was x2 800.

, : . .

T a b l e 13. H a e a o l y t i c d o ~ e s (XI),. ) o f Ai.dtii01~iun sul:,:hu'te ~0 7. , . .

p r c c i ; i t a t e d frat t i o n o , nnci t h e m o r t d i t y r~ i ' t es ..

of iilice i n j e c t e d b ' , i th th,: f ' r ~ c t i o n s .

( $ s a t u r a t i o n ) dose (mg) (7;) --...-. . ...--- -..-... -*.-- . - - . - . - . .-- ... --..-- , -- .. .-.a. . --4 -.-

F i g u r e 1 , Flow c h a r t f o r t h e f r a c t i o n a t i o n and

p u r i f i c a t i o n methods,

BACTERIAL CELLS ( t h i c k s u s p e n s i o n ) : h a r v e s t e d from 18-hour b r o t h c u l t u r e o f V , p a r a h a e m o l s t i c u s on B H I - . ~ ~ N a C l

U l t r a s o n i c d i s r u p t i o n o f c e l l s

I CELL SUSPENSION I D i f f e r e n t i a l c e n t r i f u g - -B t i o n ( 2 3 000 6ev. min ; 30 min. a t -4 C),

J PELLET

C e l l wall components N 'I'

INTRACELLULAR COMPONENTS I

d i s c a r d e d P r e c i p i t a t i o n w i t h Ammonium s u l p h a t e (40% s a t u r a t i o n ; 5 000 rev . mine

SUPERNATANT I r e t a i n e d f o r f u r t h e r p r e c i p i t a t i o n w i t h Ammonium s u l p h a t e .

1 PELLET i n PBS I

1 Dia lysed a g a i n s t d h s t i l l e d wa te r a 4 C u n t i l a l l (NH4)2S04 was c o m p l e t e i y removed.

1 c o n c e n t r a t e d by p e r c o l a t i o n 1 Sephadex G-150 g e l f i l t r a t i o n

p a r t i a l l y p u r i f i e d

T a b l e 14. P u r i f i c a t i o n o f t h e t o x i c f r a c t i o n .

P u r i f i - T o t c i T o t n l S p e c i f i c T o t d :,Juri f.1- c a t i o n .;)rotcAn volume a c t i v i t y r e c o v e r y c n t i o n

s t e p (ms> (ml) ( S > -,.- . -..-_. . ... ...- ...- - -.., . - .,... . ... -- -.... .. _- .. , . . . f o l d

a . ---.-,,

u n p r e c i p i - t a t e d s u p e r n n t e 25 000 1 000 420.0 0.002 1 00 x 1

Gel I

f i l t r ~ ~ t i o n e l u e t e 0.05 5 0.15 5.67 0.35 x 2 8GO.O

'ID o f y u r i f i c c t i o n s t o p x 100 T o t a l r e c o v e r y = 5 0 _ , - - ---- . -- HD o f u n p r e c i p i tzttcd s u p r n ~ . t e 50 FD o f u n p r e c i > i t ? . t e d supe rn t i t e

' I : 'u r i f icu t ion f ~ l d = > .--. - - . - - - . - - - - . - ._ .- *----- -,

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

BIOUGICAL CHARACTERS

L e t h a l a c t i v i t y The l e t h a l a c t i v i t y o f t h e t o x i c f r a c t i o n

( o r t h e g e l f i l t r a t i o n e l u a t e ) was measured by t h e m o r t a l i t y

r a t e o f a n i m a l s g i v e n g raded d o s e s o f t h e f r a c t i o n th rough

d i f f e r e n t r o u t e s (Tab le 15) . The l e t h a l dose 0.f t h e t o x i c Cpmteh content)

f r a c t i o n & mice i n j e c t e d i n t r a v e n o u s l y ( i . v . ) ranged from

18.0 - 2.2 hg,' w h i l e i t ranged from 18.0 - 4.5 f i g i n mice

i n j e c t e d i n t r a p e r i t n e a l y ( i .p .1 , and from 18.0 - 9 . 0 4 g i n

mice i n j e c t e d subcu taneous ly . Higher d o s e s ~ h o w e d more

a c t i v i t y t h a n l o w e r doses . For example, t h e m o r t a l i t y r a t e

was - .. 80% ..,. i n mice i n j e c t e d i . v . w i t h 18.0 41g o f t o x i c f r a c t i o n ,

whereas i t was o n l y 20% i n mice i n j e c t e d i .v . w i t h 2 . 2 A g .

The l e t h a l dose o f t h e t o x i c f r a c t i o n on c h i c k embryo g i v e n

th rough t h e yo lk s a c i n o c u l a t i o n Fanged from 25.0 -- 0.2 a g e n

The h i g h e r dose k i l l e d 100% o f t h e embryos, w h i l e t h e l o w e r

dose k i l l e d 20% o f t h e c h i c k embryos (Tab le 16). The

c a l c u l a t e & L D ~ ~ ~ S , t h e r e f o r e , are 6.4 . u g i n t r a v e n o u s l y , 1 1.8 ~ l g

i n t r a p e r i t o n e a l y , and 18.0 rug subcu taneous ly . I n c h i c k

embryos, t h e LD is 1 . 9 ~ g . 50

Within f i v e minu tes o f i n t r a v e n o u s i n j e c t i o n s o f t h e

f r a c t i o n i n t o mice, t h e a n i m a l s became l e t h a r g i c wi th e y e s

c l o s e d , r u f f l e d f u r , and l o s s o f a p p e t i t e . Only a few o f t h e

a n i m a l s f e d o r a l l y developed watery a n d ye l low d i a r r h o e i c s t o o l .

Recovery o f s u r v i v o r s o c c u r r e d between 24 ail: 48 h o u r s a f t e r

t h e i n j e c t i l J n . C u l t u r e o f t h e v i t a l o r g a n s (e.g., t h e l i v e r ,

Table 15. M o r t z l i t y r a t e s 01 mice i n j c c t c d i n t r s v e n o u s l y ,

i n t r : . p c r i t o n e a l y , s u b c u t n ~ ~ c ~ ~ ~ e l y , o r i n t r o d u c e d

I n j e c t e d

dose h g ) i . p . o r2.l

Table 16. N o r ~ ~ i i t y r a t e of c h i c k ecibrjros; i n j e c t e d vic, t h e yolk s;:c u i t h grc.ded doses of' t h e tox ic

kidney , h e a r t - b l o o d , s p l e e n , and t h e p e r i t o n e a l f l u i d from

dead a n i m a l s ) on 'llCBS a g a r , and on BI-II-3%NaCl agar showed no

b a c t e r i a l growth i n d i c a t i n g t h a t d e a t h o f mice was n o t as a

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

H i s t o l o g y o f t h e v i t a l o r g a n s Biopsy o f t h e v i t a l o r d a n s

were t a k e n from dead and d is ,eased a i f m a l s , and t h c h i s t o l o g i c a l

changes observed a r e shown i n Tab le 17. Nochanges were

obse rved i n t h e s p l e e n .

Pyroaen ic ac t i v i t y Tab le 18 shows t h o t e m p e r a t u r e changes

i n t h e r a b b i t i n j e c t e d i . v . w i t h t h e t ~ x i c f r a c t i o n . The

t e m p e r a t u r e o f t h e r a b b i t i n c r e a s e d from ~ 8 . 3 5 ~ ~ t o 3 8 . 8 0 ~ ~

a f t e r t h e t o x i c f r a c t i o n was i n j e c t e d . The c a l c u l a t e d r e s p o n s e

i s 0.45 '~, and i t l i e s w i t h i n t h e l i m i t f o r a c c e p t a n c e

( B r i t i s h ~harmacopoeia, 1980). v

Haemolvtic a c t i v i t y The p e r c e n t a g e haemolys i s o f some

mammalian e r y t h r o c y t e s i n c u b a t e d wi th t h e t o x i c f a c t o r a r e shown

i n Tab le 19. The haemoly t i c d o s e s ranged from 5.00 - 0.03 rug ?rotel

or: hupjan e r y t h r o c y t e s , f r o 5.00 - - 0.07 ~ l g on r a b b i t e r y t h r o -

c y t e s , from 5.00 - 0.15 'ug on s h e e p e r y t h r o c y t e s and from

5.00 - 1.20 a g on r a t e r y t h r o c y t e s . Higher d o s e s o f t h e f a c t o r

l y s e d more e r j t h r o c y t e s t h a n lower doses . For example, 5.00 a g prc

o f t h e t o x i c f a c t o r lyse'd 100% o f human e r y t h r o c y t e s , whereas

0.03 a g o f t h e t o x i c f a c t o r ' lysed on ly 12% o f t h e same c e l l s .

'T~:b le 17. H i s t l o g i c a l c h m g e s o b s e r v e d i a b i o y s y o f v i t a l o r g m s of dend u i c e ,- f t e r i n o c u l e t i o n w i t h t h e t o x i c f x c t i o n f r o ~ n 1. ~ ~ ~ J 1 < L ~ A ~ i o - l y t i c u s .

L i v e r

Kidney

: :ongcs t ion o f b l o o d v e s s e l s I n f i . l . . t ~ ~ t i o n o f tlic: 7'2:::.11 o f tlic: :,lul 2 f t h e b i l e d u c t i j i t h rou.iid ( I n o c u l a t i o n i ~ t o n o n u c l e ~ . r and fc:i polyuiorphon- i n t r a v e n o u s ) n u c l e n r c c l l s ; T h i c k e n i n g of t h L ~ ,x l l s , a d I ' i u l t i f o c z l ~ . . ~ e c : s or' r i e c r o ~ i s i n psrerlchyiac ( F i g u r e 2 )

Eiarkcd c o n g e ~ t i c ~ n o f b lood v e s s c l s p : . r t i c u l ~ r l y i n t h e ~ i e d u l l ~ . ; ( d i t t o ) h i l d t u b u l z r de[<cner,. t i o n o f t h e c o r t e x ( F i g u r e 3 ).

I

, iyocc.rdium .Cor?gest ion o f t l i ~ b lood v e s s e l s S i g n i f i ~ ~ n t l e s i o n s ( O C ~ C I X . C J I ~ ( d i t t o ) :~lyocc rdi.cl.1 d e g e n s r ~ t i o n ) ( F i g . 4 ) .

I n t e s t i n e Desqu~.;;zc.t:i.ori oi' t l i c iri-L5s'ti.ni'.l ( I n o c u l a t i o n e p i t h e l i u m ( F i g u r e 5 ) . 0s)

f i g u r e 2. A . L i v e r o f an imal i n j e c t e d i n t r a v e n o u s l y

w i t h Toxic f a c t o r : c o n g e s t e d b lood v e s s e l s , i n f i l t r a t i o n o f b i l e d u c t w i t h mononucllear and polymorphonuclear c e l l s , t h i c k e n i n g o f

walls, and m u l t i f o c a l a r e a s of n e c r o s i s i n

parenchyma.

B. L i v e r o f u n t r e a t e d animal ( c o n t r o l ) .

Figure 2.

F i g u r e 3 . A . Kidney o f mouse i n j e c t e d i n t r a v e n o u s l y

wi th Toxic f a c t o r : marked c o n g e s t i o n o f blood v e s s e l s , t u b u l a r d e g e n e r a t i o n o f c o r t e x .

B. Kidney o f u n t r e a t e d mouse ( c o n t r o l ) .

Figure 3

F i g u r e 4. A . Myocardium of mouse i n j e c t e d i n t r a - venous ly wi th Toxic f a c t o r : c o n g e s t i o n o f b lood v e s s e l s , oedema and myocard ia l d e g e n e r a t i o n .

B. Myocardium o f u n t r e a t e d mouse ( c o n t r o l ) .

Figure 4

Figure 5, A, I n t e s t i n e o f mouse f ed o r a l l y wi th Toxic f a c t o r : desquamation of i n t e s t i n a l ep i the l ium,

B, I n t e s t i n e o f u n t r e a t e d animal

( . con t ro l ) ,

Figure 5

F i g u r e 6. A . Sp leen o f mouse i n j e c t e d

i n t r a v e n o u s l y w i t h Toxic f a c t o r :

no s i g n i f i c a n t l e s i o n i n t h i s

organ.

Figure 6

Averqge Tcible 18., ~ e c t c l t o m p s r ~ t u r c c l i x L g e s l n r c b b i t S i i ~ j o c t c d \;.ith

t h e t o x i c m a t e r i ~ l .

T h e r e f o r e , response = (38.8 - 3 8 . ~ 5 ) ' ~

- ~ ~ 4 5 ' ~ ( thc ; i nc rease l i e s wi th in t h u l i n i t f o r accept ; .nce) .

Tzble 19. P e r c entagr;. hcemolys i s o f m ~ . . i i i ~ ~ ~ , l i c . n e r y t h r o c y t e s i n c u b a t e d w i t h t h e t o x i c f r r , c t i o n .

Wzemolytic dose b g )

k o f t o x i n x 100 Yercent 'ge h~ c l ao lys i s = 540 - -- " 540 oP 1 0 0 3 c u n t r o l

The f i f t y - p e r c e n t haemoly t i c dose (HD ) was 0.15 tug on hwman 50

e r y t h r o c y t e s , 0.30 rug on r a b b i t e r y t h r o c y t e s , 0 . 7 4 g on

s h e e p e r y t h r o c y t e s , and 5.00 .ug on rat e r y t h r o c y t e s .

C y t o t o x i c a c t i v i t y The e f f e c t of t h e t o x i c f r a c t i o n on t h e

v i a b i l i t y o f mouse p e r i t o n e a l macrophages (MPM) was determined

by t h e d e a t h r a t e o f MPM i n c u b a t e d w i t h graded d o s e s o f t h e

t o x i c f a c t o r . C e l l d e a t h , marked by u p t a k e o f Trypan b l u e ,

i m p l i e s c y t o t o x i c a c t i v i t y . A s shown i n F i g u r e ? , t h e d e a t h

r a t e o f MPM i n c r e a s e d w i t h i n c r e a s e i n t h e dose o f t o x i c f a c t o r .

Larger d o s e s o f t h e f a c t o r k i l l e d more MPM t h a n s m a l l e r d o s e s

(Tab le 2 0 ) . For example, t h e d e a t h r a t e o f MPM e x p o ~ e d t o

5 .00mg o f t h e t o x i c f a c t o r was 90%, whereas i t w a s o n l y 10%

i n t h e c e l l s exposed t o 0 . 0 3 r ~ g o f t h e f a c t o r . . The haemoly t i c a c t i v k y o f t h e t o x i c f r a c t i o n was

compared w i t h i t s c y t o t o x i c a c t i v i t y (Tab le 21) . Both

c y t o t o x i c an,d haemoly t i c d o s e s ranged from 5.00 - 0.03 h g ,

t h e minimumpoint o f a c t i v i t y b e i n g t h e same f o r b o t h ( F i g u r e 8

E f f e c t o f t o x i c f a c t o r on m a c r o ~ h a ~ e functions The e f f e c t

o f s u b t o x i c dc,ses o f t o x i c f r a c t i o n on MPM f u n c t i o n was

de te rmined by t h e p e r c e n t a g e of b a c t e r i a l c e l l s phagocytosed

by MPM t r e a t e d w i t h s u b t o x i c d o s e s o f t h e t o x i c f a c t o r . A s

shown i n Tab le 22, b u f f e r - t r e a t e d MPM ( c o n t r o l ) phagocytosed a

h i g h e r p e r c e n t a g e o f b a c t e r i a t h a n t o x i c f a c t o r - t r e a t e d MPM.

B u f f e r - t r e a t e d MPll phagocytosed 85% o f b a c t e r i a l c e l l s , whereas

Figure 7 . E f f e c t o f graded doses o f Toxic f ac tor on v i a b i l i t y o f mouse per i tonea l macrophages (MPM).

Table 20. The dea th r ~ ~ t c o f a1c;crophcgss sxposed t o g rndcd d o s e s o f t o x i c f r a c t i o n .

Dose of D m t h r~.tf: l o g o f t o x i n k g ) ( +') / J d w d c t l l s - -. ------..-

5.00

2.50

1 .20

0.60

0.30

0.15

0.07

0.03

Cont ro l

where, No = v i z b l ~ c e l l s

c e l l s

c o n t r o l

Con t ro l 0 0

F i g u r e 8. Cyto tox ic and haemoly t i c a c t i v i t y c u r v e s .

0-----0 C y t o t o x i c i t y c u r v e

p----U Haemolysis c u r v e

Twofold d i l u t i o n s o f t o x i c f r a c t i o n (10.0 ag o f p r o t e i n ) .

T a b l e 22. Perccnt,-.ge o f bc.ct6ri.i; L l l i c g o c y t o s ~ d by ~ic .c rophnges t r c s t e d w i t h lo\;! d o s e s o f t o x i c fr.c.cl;ion.

Dosu o f t o x i n

(4%

Bac to r i c .1 c o u n t

(c . f .u . )

N' = b c . c t s r i ~ ; l c o u n t i n t h o tub^ ccl-atc.ining o o n l y b r , c t c r i a l c c l l ~ .

N t = b2,ctur ic . l c \ jun t i n t h e t u b e contc i ln ing bzc to r i c .1 c e l l s 2nd t r e z . t e d nncrophages .

MPM t ~ e a t e d -. w i t h 1.20 tug t o x i c f a c t o r phagocytosed o n l y 10% o f

b a c t e r i a l c e l l s . Fur thermore , t h e p e r c e n t a g e of phagocytosed

b a c t e r i a d e c r e a s e d l i n e a r l y ( F i g u r e 9 ) a s t h e t r e a t m e n t dose o f

MPM i n c r e a s e d . For example, MPM t r e a t e d w i t h 0.03 h g o f t o x i c

f a c t o r phagocytosed 62% o f b a c t e r i a , w h i l e MPM t r e a t e d w i t h 1 .20/

o f t o x i c f a c t o r phc.,gocytosed o n l y 10% o f b a c t e r i a l c e l l s .

The e f f e c t o f s u b t o x i c d o s e s on t h e a d h e r e n t c a p a c i t y o f

MPM w a s measured by t h e adherence r a t e o f b a c t e r i a l c e l l s t o

MPM t r e a t e d w i t h s u b t o x i c d o s e s o f t h e f r a c t i o n . A s shown i n

Tab le 23, 85% o f b a c t e r i a adhered t o b u f f e r - t r e a t e o MPM ( c o n t r o l )

whereas on ly 15% adhered t o MPM t ~ e a t e d w i t h 1 .20mg o f t o x i c

f a c t o r . The adherence r a t e was h igh i n MPM t r e a t e d wi th lower

duses , b u t was low i n MPM t r e a t e d w i t h h i g h e r d o s e s , imply ing 8

t h a t more b a c t e r i a l c e l l s adhered t o MPM t r e a t e d w i t h low d o s e s

o f f r a c t i o n , i . e . , t r e a t m e n t of MPM wi th lower d o s e s i n c r e a s e d

adherence o f b a c t e r i a l c e l l s . For i n s t a n c e , 60% o f ) . b a c t e r i a . ,

adhered t o 'MPM t r e a t e d wi th 0.03 Alg, but o n l y 15% adhered t o

MPM t r e a t e d w i t h 1 .20 ag.

A l a r g e r p e r c e n t a g e o f b a c t e r i a i ~ d h e r e d t o M F M from mice

imrnunised w i t h -&LD o f t o x i c f r a c t i o n t h a n t o MPM from non- 50 ilnrnunised an imals . For example, 50% o f b a c t e r i a l . c e l l s

adhered t o immune MPM t r e a t e d w i t h 1 . 2 0 ~ g o f t o x i c f r a c t i o n ,

whereas o n l y 15% a d h e r e d to,non-immune BPM t r e a t e d wi th t h e

same dose o f toxin , , A s w i t h t h e non-iuirnune MPM, t r e a t m e n t

wi th h i g h e r d o s e s o f t o x i n reduced t h e p e r c e n t a g e o f adherence

F i g u r e 9. E f f e c t o f s u b l e t h a l d o s e s o f Toxic

f a c t o r on mouse p e r i t o n e a l macrophages

f u n c t i o n s measured by t h e p e r c e n t a g e

o f b a c t e r i a phagocytosed by t r e a t e d

macrophages. I

Phagocy-

t o sed b a c t e r i a

(%>

Fiaure 9.

D i l u t i o n s of suble thc .1 do;:ss of' Toxic f c n c t i o n

Table 23. P ~ r c c n t c , g t : o f b z c t e r i c . 1 c c l l s a d h 3 r i n g tL m c r o p h n g e s t r c n t c d ~ ! i t h lo l l doses o f t o x i c f r z . c t i o n .

Dose o f t o x i n

No = b c c t o r i n l cl.,unt i n t h e t u b e c o n t a i n i n g o n l y bt,c t erj-c.1 c e l l s .

N,t = b a c $ e r i a l c o u n t i i i t h e t u b e c L , n t c i n i n g

b n c t b r i a l c e l l s 2nd t r e c t e d n n c r o p h c g e s .

more t h a n lower doses . P e r c e n t a g e adherence was 50% i n immune

MPM t e e a t e d w i t h 1.20 rug o f t h e t o x i c f a c t o r , b u t was 87%

i n t h o s e t r e a t e d w i t h 0 . 0 3 d g o f t h e t o x i n .

Tab le - 24 shows t h e m i g r a t i o n p a t t e r n o f MPM t r e a t e d w i t h

s u b t o x i c d o s e s o f t o x i c f r a c t i o n . P e r c e n t a g e m i g r a t i o n was

based on t h e d i s t a n c e moved by b u f f e r - t r e a t e d MPM. Macrophages

t r e a t e d w i t h l a r g e r s u b t o x i c d o s e s showed l o w e r p e r c e n t a g e o f

m i g r a t i o n t h a n t h o s e t r e a t e d w i t h lower s u b t o x i c doses .

P e r c e n t a g e m i g r a t i o n was 10% i n MPM t r e a t e d w i t h 1 . 2 0 d g o f

t o x i c f r a c t i o n , b u t was 60% i n t h o s e t r e a t e d w i t h O.O7/ug o f

t o x i c f r a c t i o n .

E n t e r o t o x i a e n i c a c t i v i t y

Rabbit i l e a l l o o p r e a c t i o n No r e a c t i o n s i g n i f i e d by ' I

d i l a t a t i o n and accummulat ion o f s a a g u i n o l e n t f l u i d i n t h e

l i g a t e d r a b b i t i l e u m w a s o b s e r v e d - 1 8 h o u r s a f t e r v a r y i n g d s s e s

o f t o x i c f r a c t i o n were i n j e c t e d i n t o a l t e r n a t e segments o f t h e

i l e d (Tab le 2 5 ) ,

S u c k l i n g mouse r e a c t i o n Tab le 2 6 shows t h e r a t i o o f g u t

weight t o r emain ing body w e i g h t o f s u c k l i n g mice f o u r h o u r s

a g t e r t h e y were o r a l l y g i v e n v a r y i n g d o s e s o f t o x i c f r a c t i o n .

Boses r a n g i n g from 1.00 - 2 5 . 0 0 ~ 1 g gave r a t i o s g r e a t e r t h a n

0.08 and were c u n s i d e r e d p o s i t i v e , w h i l e l o w e r d o s e s , 0.04 - 0 . 2 0 4 g gave r a t i o s l ess t h a n 0.08 and were c o n d i d e r e d n e g a t i v e .

Tcble 24, M i g r - t i o n r a t e of i : l ac roph~ges exposed t o low

dcjses of t o x i c f r c c t i o n .

0

Cont ro l tube c o n t s i n c d buf fer- trct : tcd ix.cro?hc.ges

25. The r c z c t i ~ n of l i g a t e d r z b b i t i lec.1 l oops i n j e c t e d wi th graded closes o f t h e t o x i c f r n c t i o n .

o f t z x i n R e x t i d n

,%line (negz t ive c u n t r c l )

p o s i t i v e

nega t ive .

Table 2 6 . The g u t n e i g h t / r e n n i n i n g body w i g h t r z t i o s o f s u c k l i n g mice force-fctd 1vith g raded diises o f

t o x i c f r n c t i o n .

R c t i o s a b ~ v e 0.08 1:Jerc c s n s i d e r e d p o s i t i v e

$kin t o x i c i t y r e a c t i o n Areas o f in f l ammat ion and redden ing

measur ing 3.0 mm and 1.0 mm i n d i a m e t e r were obse rved a t t h e

s i t e s where 20.0.mg and 4.0 a g o f t o x i c f a c t o r were i n j e c t e d

i n t r a d e r n l a l l y i n t o r a b b i t s k i n (Tab le 27). No r e a c t i o n was

observed where 0.8 d g o f t h e f a c t o r w a s i n j e c t e d i n t o

gu inea -p ig s k i n .

Antiserum t i t r e s The a g g l u t i n a t i n g c a p a c i t y o f t h e a n t i -

t o x i c f a c t o r serum was determined. Haemagglut ina t ion , s i g n i f i e d

by a m a t t e d d e p o s i t o f r e d c e l l s wi th c r e n a t e d r i m , was obse rved

i n - t h e - m i x t u r e o f a n t i s e r u m and t o x i c f a c t o r - c o a t e d e r y t h r o c y t e s .

The h a e m a g g l u t i n a t i o n t i t r e was 2 01+8 (Tab le 2 8 ) . I n c u b a t i o n

o f a n t i s e r u m wi th b a c t e r i a l c e l l s o f a 1. parahaemol.vt icus

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

f y i n g b a c t e r i a l a g g l u t i n a t i o n . ~ h k . a n t i s e r u m a g g l u t i n a t e d

t h e y. p a r a h a e m o l r t i c u s i s o l a t e s KV KV and V6 t e s t e d . 3' 4 The a g g l u t i n a t i o n t i t r e w i t h KV c e l l s was 128 (Tab le 2 8 ) .

. , 3

Immunoelec t r o p h o r e s i s One p r e c i p i t a t i o n band developed

between t h e w e l l c o n t a i n i n g t h e t o x i c f a c t o r and t h e t r o u g h

c o n t a i n i n g t h e a n t i - t o x i c f a c t o r serum ( F i g u r e 1 0 ) .

Immunodif f u s i o n A s i n g l e p r e c i p i t a t i o n l i n e developed

between t h e a n t i s e r u m c o n t a i n i n g w e l l and t h e well c o n t a i n i n g

t h e t o x i c f a c t o r . On t h e o t h e r hand, a b o u t f o u r p r e c i p i t a t i o n

l i n e s were obse rved between t h e a n t i s e r u m w e l l and t h e w e l l

c o n t a i n i n g t h e 40% Ammonium s u l p h a t e p r e c i p i t a t e d f r a c t i o n ( F i g u r e 11).

T a b l e 28. Ag. : lu t inut ion u f s h e s ? u r y t h r o c 3 t e s coz-tcd w i t h t o x i c f r a c t i o n (hz .emagglu t in~ . t ion) 2nd o f y. p.?ra?cel.~olyticug c e l l s by m t i - t c ~ x i c f r c c t i u n serum.

+ = p o s i t i v e ' r c c . c t i m , i. C. q g l u t i n c z t i 6 n .

- = n b g c t l v e re;.ctiun, i. c . c:bsencc o f c g g l u t i n a t i o n .

F i g u r e 10. Immunoelec t rophores is .

A . Trough c o n t a i n i n g a n t i - t o x i c f a c t o r serum.

B. Well c o n t a i n i n g u n p u r i f i e d f a c t o r .

C. Well c o n t a i n i n g t h e t o x i c f a c t o r . . .

figure 11. Immunodif f u s i o n .

A . C e n t r a l w e l l c o n t a i n i n g a n t i s e r u m .

B. Peripheral well c o n t a i n i n g Toxic

f a c t o r .

1 go.

f igure 11

Antiserum b a c t e r i c i d a l a c t i v i t y The v i a b l e coun t o f a

m i x t u r e compr i s ing I s o l a t e KV c e l l s , a n t i - t o x i c f a c t o r serum, 3 and complement i s shown i n T a b l e 29. The v i a b l e c o u n t which

8 7 was 1 x 10 c.f .u . b e f o r e i n c u b a t i o n d e c r e a s e d t o 3.2 x 10 c.f.1

6 a f t e r 60 min of i n c u b a t i o n and t o 2.4 x 10 c . f .u . a f t e r 120 min

The v i a b l e coun t d e c r e a s e d onnly s l i g h t l y i n t h e m i x t u r e s

c o n t a i n i n g b a c t e r i a l c e l l s and a n t i s e r u m , o r b a c t e r i a l c e l l s

and complement. The l y t i c t i t r e which i s t h e h i g h e s t a n t i s e r u m

d i l u t i o n t h a t caused more t h a n 50% r e d u c t i o n i n v i a b l e c o u n t

i s on Tab le 30.

Mouse ~ r o t e c t i o n The p r o t e c t i v e c a p a c i t y o f t h e a n t i - t o x i c

f a c t o r serum was determined. A l l mice i n j e c t e d wi th t h e

a n t i s e r u m b e f o r e c h a l l e n g i n g t h e same a n i m a l s w i t h t h e t o x i c I

f a c t o r s u r v i v e d , On t h e o t h e r .harid, t h o s e i n j e c t e d w i t h normal

r a b b i t serum b e f o r e c h a l l e n g i n g wi th t o x i c f r a c t i o n d i e d .

I n v i t r o n e u t r a l i s a t i o n o f a n t i s e r u m The t o x i c f r a c t i o n

i n c u b a t e d w i t h a n t i s e r u m f o r 15 min f a i l e d t o l y s e d e f i b r i n a t e d

e r y t h r o c y t e s , b u t t h e t o x i c f r a c t i o n i n c u b a t e d w i t h normal

r a b b i t serum caused e r y t h r o c y t e l y s i s (WD 0.15 ug) and 50 d e a t h of MPM (81% d e a t h r a t e ) . The t o x i c f a c t o r i n c u b a t e d

w i t h b u f f e r a l s o r e t a i n e d b o t h a c t i v i t i e s as shown i n T a b l e 31.

T c b l e 29, V i ~ b l e c o u n t s of 1. p.Crt.ih,~ll'.ii,1.yt~c~ I s o l a t e KV 3

i n c u b a t e d i n a a i i x t u r e o f c n t i s u r u n C ~ L co~. ip lenent f o r vckrying l e n g t h s o f t i u e .

Viable c ~ u n t ( c . f .u. )

Uactoric? +

P e r i o d o f An t i se ru r i B c c t e r i c B r ~ c t eri:, Bnc t c-rick + + 4- 4- incubatiyn C o ~ l p l e u a n t Antiserui.: Cunplu , .~ent Hu f f e r

( ~ ~ i n u t o s I - - _ I l -

Tab le 30. Death r a t e 6 o f V , p : . r ~ h : , ~ ~ . ~ 1 y ~ t ~ $ ~ s _ c e l l s i ncube t t ld --.

i n m i x t u r e s c): a n t i s e r u i ; d i l u t i o n s and co1.1pletient.

A n t i s k r u . : ~ d i l u t i ~ n Vi:.ble c o u n t D w t h r = t e ( 7 ; )

Lytic t i t r e = t h e r e c i p r d c c l c f t h c h i g h e s t d i l u t i o n o f c n t i s e r u u s h o ~ r i n g i:l~)re t h m 50,;; r e d u c t i o n i n v i a b l e c ~ ~ u n t .

= 64 - Death mte = ---- 'o - *t x , 100

No

u h e r e , M = vit:ble c o u n t a t 0 i l : U

N t = v i a b l e c o u n t n f t b r 120 n i n .

T z b l e 31 . Toxic :..c t i v i t i e s (mc;.qsured 'by t h e h:,.dmlyt:ic doaes

2nd t h e dc.r.th r r . t c of i,:iPPI) o f t i j x i c f r : . c t im incub:-ted w i t h z n t i - t o x i c f r x t l o n seru1.i.

Toxic f r x t i o n +

Ankiserurr

Toxic f r a c t i o n +

Norrml r c b b i t s e rui.1

Toxic f r a c t i b n +

B u f f e r

Tnble 32. UV z b s o r p t i o n cpec t r u n of t ~ i c fr:..c t i o n i . w s u r e d by t h e o p t i c z l a e n s i t i c s o f t h o f r a c t i o n ct d i f f e r e n t wavelengths.

F i g u r e 12. Absorp t ion spect rum o f Toxic f a c t o r .

F i g u r e 13. Po lyac ry lamide g e l e l e c t r o p h o r e s i s .

A . The p r o t e i n band.

Figure 13

PHYSICOCIiMICAL CIIARACTERS

UV-absorpt ion UV-absorpt ion s p e c t r u m o f t h e t o x i c f r a c t i o n

measured by i t s o p t i c a l d e n s i t i e s a t d i f f e r e n t wave leng th was

shown i n T a b l e 32. As shown i n F i g u r e 1 2 , t h e t o x i c f r a c t i o n

a b s o r b e d maximal ly (O.D. 0 .064) a t 880 nm, and g a v e a c u r v e

w i t h s i n g l e peak.

Pro t e i n d e t e r m i n a t i o n The p r e s e n c e o f p r o t e i n i n t h e

t o x i c f r a c t i o n was s i g n i f i e d by t h e deve1~;pment o f b l u e c o l o u r

i n t h e r e a c t i o n s o l u t i o n . P r o t e i n concentration was r e a d o f f

from t h e BSA s t a n d a r d c u r v e .

C a r b o h y d r a t e d e t e r m i n a t i o n The O.D. r e a d i n g o f t h e t o x i c

f r a c t i o n t r e a t e d w i t h t h e a n t h r o n e r e a g e n t was z e r o a t 620 nm.

T h i s r e a c t i o n i n d i c a t e s a b s e n c e o f c a r b o h y d r a t e . I

L i p i d d e t e r m i n a t i o n E v a p o r a t i o n o f t h e m i x t u r e c o m p r i s i n g

t o x i c f r a c t i o n and water -methanol -c h l o r o f o r m s o l u t i o n y i e l d e d

no p r e c i p i t a t e , and i n d i c a t e s a b s e n c e o f l i p i d .

Po lvac rv l a rn ide ge l e l e c t r o p h o r e s i s (FUGE) The t o x i c f r a c t i o n

showed a s i n g l e band o f p r e c i p i t a t e a f t e r e l e c t r o p h o r e s i s

on p o l y a c r y l a m i d e g e l ( F i g u r e 13 ) . M o l e c u l a r we igh t The t o x i c f r a c t i o n i d e n t i f i e d by i t s

h a e m o l y t i c a c t i v i t y was e l u t e d a t 250 m l from t h e g e l f i l t r a t i o n

column. The e l u t i o n volume c o r r e s p o n d s t o a m o l e c u l a r w e i g h t

o f 63 095 s r e a d o f f from t h e c u r v e of s t a n d a r d marker p r o t e i n s ( F i g u r e 1 4 ) . Temperature s t a b i u The u n p u r i f i e d t o x i c f r a c t i o n

(40% Ammonium s u l p h a t e p r e c i p i t a t e d f r a c t i o n ) h e a t e d a t 3 7 ' ~

had a haemoly t i c o f 50 ag, and was c y t o t o x i c t o 75% o f

MPM Table j j ) , b u t t h e same eample h e a t e d a t 60°c f o r 30 min

f a i l e d t o l y s e e r y t h r o c y t e s , a n d ' w a s c y t o t o x i c t o o n l y 5% o f

MPM. On t h e c o n t r a r y , t h e t o x i c f r a c t i o n ( p a r t i a l l y p u r i f i e d )

h e a t e d a t 6 0 ' ~ had a haemoly t i c dose o f 0 . 1 5 u g and was

c y t o t o x i c t o 80% o f MPM. However, t h e same sample h e a t e d a t

1 0 0 ~ ~ f o r 15 min had a haemoly t i c dose o f 0.30 ~g and was

c y t o t o x i c t o 50% MPM. The p e r c e n t a g e a c t i v i t y r e t a i n e d a f t e r

h e a t i n g a t t h i s t e m p e r a t u r e was 50%.

T e m ~ e r a t u r e a c t i v i t y r a n n e The t e m p e r a t u r e ( s ) t h a t f a v o u r s . maximal a c t i v i t y of t h e t o x i c f r a c t i o n was determined. The

HD50 of t h e f r a c t i o n i n c u b a t e d a t 10°c W a s 2.50 A[:, w h i l e t h a t

o f t h e f r a c t i o n i n c u b a t e d a t 2 8 ' ~ was 1 . 2 0 a g ( T a b l e 3 4 ) .

I n c r e a s e i n i n c u b a t i o n t e m p e r a t u r e i n c r e a s e d t o x i c a c t i v i t y

as shown by t h e lowered HD 50 ' For example, t h e HD50 o f t h e

f r a c t i o n i n c u b a t e d a t 3 7 ' ~ was 0 . 1 5 4 g and was same f o r f r a c t i o n s

i n c u b a t e d a t 42 - 57'~. E r y t h r o c y t e l y s i s o c c u r r e d a t 60°c

making hamolys i s t e s t i m p o s s i b l e a t h i g h e r t empera tu res .

Similar r e s u l t s were o b t a i n e d when t h e c y t o t o x i c a c t i v k t y was

t e s t e d a t d i f f e r e n t t e m p e r a t u r e s . H i g h d e a t h r a t e s o f MPM

F i g u r e 14. Curve o f s t a n d a r d marker p r o t e i n s used t o d e t e r m i n e t h e m o l e c u l a r weight o f Toxic f a c t o r .

I a

4OC Elu t io r vo lu~ i~e

( m u

Elu t i o ~

volurne

( n l )

l o g , Xioldculor w i g h t

Tcble 33. '.i'ei;i:~,c-ir;:.ture s t c .b i l . i ty r.!~c.surc.d by thi;. h a e x ) l y t i c d...::;es u f 2nd d ~ ~ i t h rctes o f !'!PI1 exposed tcl t o x i c f r e c t i u n o r u n p r i f i o d t . ,xic f r n c t i ~ n huctcd z t 60 C o r ? t 190 C.

Toxic frt:.ctiLin ( p n r t i a l l g p u r i f i e d )

Toxic frcict i"n ( u n p u r i f i e d ) 100 .oo

no h~:c;;.~oly:;is

n o t t e s t u d

75

5

n o t t e s t e d

s i g n i f y i n g maximal a c t i v i t y o c c u r r e d a t temperature^

between 3 7 ' ~ amd 5 7 ' ~ (Tab le 34) .

pH a c t i v i t y r ange The pH which enhances maximal a c t i v i t y

o f t h e t o x i c f r a c t i o n was determined. E r y t h r o c y t e l y s i s was

n o t obse rved i n t h e f r a c t i o n samples i n c u b a t e d a t pH 5.8 and

6.4 b u t i t o c c u r r e d i n t h o s e i n c u b a t e d a t h i g h e r pH. A t pH

7.0, t h e HDSO was 0.15 rug, b u t i t i n c r e a s e d t o 0.60 f i g a t

pH 7.6 s i g n i f y i n g reduced a c t i v i t y . A t pH 8.0, t h e HD50

remained 0.6 mg, b u t i n c r e a s e d a g a i n t o 5.0 ~ 1 g a t PI-1 8.4.

A similar p a t t e r n o f a c t i v i t y .was observed when t h e

c y t o t o x i c i t y o f t h e t o x i c f r a c t i o n t o MPM a t d i f f e r e n t pH was

t e s t e d . The d e a t h r a t e of MPM were 5% and 7% a t pH 5.8 and

6.4 r e s p e c t i v e l y . A t pH 7.0, t h e de'ath rate i n c r e a s e d and .

was 75%, b u t i t d e c r e a s e d t o 30% a t pH 7.6 and f u r t h e r t o 20%

a t pH 8.4 (Tab le 3 5 ) .

E f f e c t o f p r o t e o l y t i c enzymes The t o x i c f r a c t i o n i n c u b a t e d

w i t h p r o t e o l y t i c enzymes was t e s t e d f o r haemoly t i c and c y t o t o x i c

a c t i v i t i e s . , A s shown i n Tab le 36, t h e HD50 o f t h e f r a c t i o n

i n c u b a t e d w i t h e i t h e r T r y p s i n o r P e p s i n was n i l i n d i c a t i n g t h a t

e r y t h r o c y t e s were n o t l y s e d . Likewise , t h e d e a t h r a t e o f MPM

exposed t o t h e f r a c t i o n i n c u b a t e d wi th Tryps in was 0% and

was 5% f o r MPM exposed t o f r a c t i o n i n c u b a t e d wi th Peps in . The

c o n t r o l compr i s ing t h e t o x i c f r a c t i o n i n c u b a t e d wi th b u f f e r

had an HD50 o f 0.15 u g , and caused 80% MPM d e a t h rate.

Tc..ble 34. I I c e ~ ~ o l y t i c do=;;.:2 of t o x i c f r c . c t i o n st d i f f k r e n t i n c u b n t i o n t e m p e r e t u r e o , and ,the ( i ~ c t h r s t e o f I W M i n c u b a t cd lviith thc tcxS.c fr:.l.c ti m z t dif f e r m t t o n p e r c t u r e s .

. . " - = nbcence o f t c x i c ~ c t i v i t y .

Tab le 35. Haei.:olytic dLJ.es o f t o x i c f r c . c t i o n zit d i f f e r e n t pH, cLnd t h e dez.th rate of ' %PI< i n c u b a t e d w i t h t o x i c f r c : c t i o n a t d i f f e r e n t p H vr . lucs .

Maeno ly t i c dase i.iPZ1 dec.th r n t e

LL - - - absence o f hce:. iolysis

Table 36. Hae::iolytic d o s e s o f t o x i c f r c . c t ion i n c u b c t e d w i t h P e p s i n o r T r y p s i n , a n d t h e d e a t h x t e o f i.;PiJI

exposzd t o t o x i c f r a c t i o n i n c u b n t ed w i t h P e p ~ i n o r T r y p s i n ( p r a t e ~ l y t i c e n a y n ~ s ) .

H a c ~ i o l y t i c d o s e i m 1 d e a t h 1 r a t e (75)

B u f f e r + t d x i c f r c c t i o n

T r y p s i n + t o x i c f r c c t i o n

P e p s i n 4.. ,

t o x i c f r c c t i o n

E f f e c t o f added m e t a l i o n s The e f f e c t o f d i v a l e n t c a t i o n s

on t h e t o x i c f r a c t i o n was measured by t h e HD 1 s o f f r a c t i o n 50 samples c o n t a i n i n g d i v a l e n t c a t i o n s ( T a b l e 3 7 ) . The HDW o f

t h e sample c o n t a i n i n g M ~ * + i o n s and o f t h a t c o n t a i n i n g ~ e + +

i o n s were each n i l i n d i c a t i n g absence o f haemolys is . On t h e

c o n t r a r y , t h e samples c o n t a i n i n g o t h e r c a t i o n s showed haemolyt ic

a c t i v i t y . The IdD501s o f t h e sample c o n t a i n i n g cat+, c u t + ,

and ~ n + + i o n s were each 0 . 0 1 6 ~ 1 g o f p r o t e i n s i g n i f y i n g enhanced

t o x i c a c t i v i t y when compared wi th t h e c o n t r o l which had a n

HD50 0 . 1 5 m g of p r o t e i n . Less a c t i v i t y was observed i n t h e

sample c o n t a i n i n g H ~ + + i o n s (HD50 O . 3 a g o f p r o t e i n ) . The

sample c o n t a i n i n g ~ n + + i o n s showed t h e same HD ( 0 . 1 5 ~ g o f I .

50 p r o t e i n ) as t h e c o n t r o l .

Tzbl.e 37. The h~ie~:.:r>lytic dose s o f t o x i c f r u c t i o n d i s s o l v e d i n b u f f e r s o l u t i o n s contcAning ciiv,:lent c c t i o n s .

Me

Fe

Hg

?In

Zn

cu

Ca

Cont ro l

no h ~ ~ e n o l y s i s

0 . goo

0,150

0,016

0.016

0.016

0.150

A t o x i c m a t e r i a l which may be v i t a l t o t h e pa thogen ic i ty

o f 1, p a r a h a e m o l v t i c u ~ was i s o l a t e d f r o m a v i r u l e n t Kanagawa-

p o s i t i v e i s o l a t e of t h e microorganism obta ined l o c a l l y . The

s o n i c a j a d c e l l supe rna t e o f a non-v i ru len t Kanagawa-negative

i s o l a t e o tud ied f a i l e d t o l y s e any e ry th rocy te , t h e r e a c t i o n

i n d i c a t i n g t h a t t h e t o x i c f a c t o r i s n o t present- i n t h i s

i s o l a t e . Its absence from t h e non-virulent i s o l a t e s u g g e s t s

t h a t t h e t o x i c f a c t o r may be e s s e n t i a l t o pa thogenic i ty . The

t o x i c f a c t o r can a l s o be d i f f e r e n t i a t e d from t h e i n t r a c e l l u l a r

haemolytic f a c t o r (ITHF) and from t h e the rmolab i l e f a c t o r

which were d e t e c t e d from both Kanagawa-positive and Kanagawa- *

n e g a t i v e s t r a i n s of t h e v i b r i o (Cra ig e t al. , 1972; Sakazaki

e t a l . , 1975; Daily, DeBell & Joseph, 1978; and I i d a & Takagi,

The f r a c t i o n , p r e c i p i t a t e d by t h e 40% Ammonium s u l p h a t e

s a t u r a t i o n , e x h i b i t t e d g r e a t e r t o x i c a c t i v i t y t han t h e

f r a c t i o n s from o t h e r f r a c t i o n a t i o n s t e p s . Th i s r e s u l t

shows t h a t t h e t o x i c m a t e r i a l was p r e c i p i t a t e d ou t a t t h i s

s a t u r a t i o n s-tep. The t o x i c f a c t o r is , t h e r e f o r e , d i f f e r e n t

from any o t h e r t o x i c product o f x. parahaernolyticus none of

which was obta ined a t t h i s s a t u r a t i o n s t e p of Ammonium

su lpha te .

The t o x i c m a t e r i a l i s l e t h a l t o mice and ch i ck embryos

as shown by t h e dea th o f t h e an imals I n J e c t e d w i t h c e r t a i n dose

o f t h e tox in . C u l t u r e s o f the. v i t a l o r g a n i s of t h e deceased

animals on TCBS a g a r y i e l d e d no bac . t e r i a1 growth, thereby ,

conf i rming t h a t dea th o f t h e an imals r e s u l t e d from t h e l e t h a l

e f f e c t o f t h e t o x i c f a a t o r and n o t from any b a c t e r i a l

i n f e c t i o n . T h i s r e s u l t a g r e e s wi th t h e r e p o r t s which showed

t h a t some b a c t e r i a l t o x i n s and haemolysins e x h i b i t l e t h a l

a c t i v i t y t o exper imenta l animals. Included i n t heee produc ts

a r e t h e thermos tab le d i r e c t haemolysin from K. partahaemoly-

t i c u a (Kato e t a l . , 1966; Sakura i e t al., 1973; Honda e t a l . ,

1976; and Mlyamoto e t a l . , 1980), t h e haemqlysins o f & c o l i

(Mathi.er e t a l . , 1969) and Bac i l lus a l v e i ( ~ l o u f . .e t a l . , 19771,

t h e t ype C t o x i n o f Clos t r id ium p e r f r i n ~ e n s (Dohms & Cloud,

1982), b a c t e r i a l enda tox ins and exotox ins (Truszczynski e t

al., 1969; end Kono & Yoshicka, 1972).

The l e t h e 1 e f f e c t o f t h e t o x i c f a c t o r appea r s t o be

dependent on t h e dose o f t h e t o x i n adminis te red and on t h e

r o u t e of t o x i n i n o c u l a t i o n . Grea t e r percentage of mice and

ch i ck embryos were k i l l e d by high doses of t h e t o x i n than

by low doees. For i n s t a n c e , t h e dea th r a t e o f mice i n j e c t e d

i n t r a v e n o u s l y (1.v.) w i t h 1 8 . 0 0 ~ g o f t h e p r o t e i n was 80$,

whereas i t was 20% i n t hose i n a e c t e d w i t h 2.20 mg. The dea th

r a t e i n c r e a s e d as t h e dose o f t o x i n increased . In t ravenous

i n j e c t i o n o f t h e t o x i c f a c t o r i n t o mice r e s u l t e d i n a h i g h e r

r a t e o f m o r t a l i t y than i n j e c t i o n through o t h e r rou te s . When

18.00 ug o f t o x i c f a c t o r were given, t h e m o r t a l i t y r a t e was

80% i n i n t r a v e n o u s l y i n j e c t e d mice, whereas i t was 60% and

50% i n mice i n j e c t e d i n t r a p e r i t o n e a l y and subcutaneously

r e s p e c t i v e l y , Furthermore, t h e was lowes t (6.40 ~ g ) i n

i n t r a v e n o u s l y i n j e c t e d an imals than i n mice i n j e c t e d i n t r a -

p e r i t n n e a l y (LD50 11.80A1g) o r subcutaneously (LD50 18.00mg).

The in t r avenous r o u t e , t h e r e f o r e , i s t h e most e f f e c t i v e means

o f t o x i n i n o c u l a t i o n followed by t h e i n t r a p e r i t o n e a l r o u t e

and t h e subcutaneous r o u t e , These r e s u l t s a g r e e wi th t h e

f i n d i n g s o f Bergdol (1982) t h a t t h e main b a c t e r i a l t o x i n s

a r e most e f f e c t i v e when admin i s t e r ed i n t r a v e n o u s l y and i n t r a -

p e r i t o n e a l y , and with f i n d i n g s o ) o ohms and Cloud ( 1982) t h a t

subcutaneous i n o c u l a t i o n o f l a b o r a t o r y an imals i s more

e f f e c t i v e in, induc ing i n f e c t i o n than o r a l i n o c u l a t i o n ,

Apart from demons t ra t ing t h e n e c r o t i c e f f e c t of t h e

t o x i c f a c t o r , t h e h i s t o p a t h o l o g i c a l chan@s observed on t h e

v i t a l organ o f deceased an imals show t h a t d i a r rhoea may no t

be t h e on ly s i g n i f i c a n t symptomin Y. parahaemolyt icus

i n f e c t i o n , and may e x p l a i n why o r a l a d m i n i s t r a t i o n o f t h e

t o x i n was o f no s e r i o u s consequence. Similar h i s t o l o g i c a l

changes i n c l u d i n g haemorrh'agic n e c r o s i s as were observed i n

t h i s s tudy were induced by t h e thermos tab le d i r e c t haemolysin

(Miyamoto e t a l e , 1980), b a c t e r i a l t o x i n s and endotoxins

(Cra ig , 1972; Goshima e t a l . , 1979; C i f u e n t e s de Cas t ro &

Alexander, 1979; and Miyamoto e t a l . , 1980), and by t h e p r o t e i n s

o f Aeromonas l i a u e f a c i e n s (Shimizu, 1969). I n t h e s e c a s e s ,

t h e organs u s u a l l y a f f e c t e d were t h e myocardia l and i n t e s t i n a l

c e l l s exposed t o TDH (Miyamoto e t a l . , 1980), t h e l i v e r o f

animals in jeckhd w i t h t h e t o x i n from Pseudomonas ae rua inosa

(Lutz , lg79) , o r t h e l ungs o f mice i n j e c t e d w i t h t h e kappa-

t o x i n o f =, p e r f r i n ~ e s (Kameyama & Akama, 1971).

The r i s e i n t h e r e c t a l t empera tures of t h e r a b b i t s i n j e c t e d

wi th t h e t o x i c m a t e r i a l shows t h a t t h e f a c t o r i s pyrogenic.

Although py rogen ic i ty i s u s u a l l y a s s o c i a t e d w i t h b a c t e r i a l

endotoxins , some b a c t e r i a l exo tox ins and haemolysins as wel l

have been shown t o posses s t h i s q;ality. Included i n t h i s

c l a s s are t h e haemolysin o f L i s t e r i a monocyto~enes ( ~ i d d i q u e

e t al . , 1960), t h e e n t e r o t o x i n of g. c o l i (Truszymski & . ,

P i l a s z e k i , '1970) and t h e exotox ins from group A s t r e p t o c o c c a l

f i l t r a t e s ( K i m & Watson, 1970).

The i n t r a c e l l u l a r t o x i c f a c t o r i s haemolytic caus ing

l y s i s of c e r t a i n mammalian e r y t h r o c y t e s . I n t h i s r e s p e c t , i t

resembles some t o x i c p roduc t s o f Y. parahaemolyticus, namely,

t h e TDH ( Z e n - ~ j i e t al . , 1971 ; and Ronda e t a l e , 1976), t h e

ITHF ( I i d a & Takagi, 1977) and t h e ther inolabi le f a c t o r (Da i ly

e t a l e , 1978). Other b a c t e r i a l t o x i n s which e x h i b i t t h i s

c h a r a c t e r i n c l u d e t h e haemolys ins o f y. a n ~ u i l l a r u m (Munn,

1978), o f &. c o l i ( C a v a l i e r i & Snyder, 1968), P r o t e u s mirabi l is

(Peerbooms e t a l . , 19831, a. a e r u a i n o s a (Johnson & C a l i a ,

1978), and o f L. m o n o c s t o ~ e n e s . ( S i d d i q u e e t a l . , 1969); Lhe S

and 0 t o x i n s o f s t e e p t o c o e c u s , t h e a lpha- and beta-haemolys ins

of s t r a p h y l o c o c c n s . The t o x i c f a c t o r , however, is d i s s i m i l a r

t o c h o l e r a t o x i n (Tu & Toom, 1971) and t h e t o x i n o f B a c i l l u s

c e r e u a (Ezepch&k & F l u e r , 1971) b o t h o f which are non-haemoly-

t i c .

The haemoly t i c a c t i v i t y o f t h e t o x i c f a c t o r depends on

t h e s o u r c e o f e r y t h r o c y t e s . High d o s e s o f t h e t o x i c f a c t o r

l y s e d g r e a t e r p e r c e n t a g e o f e r y t h r o c y t e s t h a n low d o s e s , t h e

p e r c e n t a g e o f l y s i s i n c r e a s i n g as t h e dose o f t o x i n i n c r e a s e d .

For example, 0 .03 a g o f t o x i n ly&d 1296 o f human e r y t h r o c y t e s ,

whereas 5.00 ug l y s e d 100%. Also, 81% o f s h e e p e r y t h r o c y t e s

were l y s e d .by 5.00 fig o f t h e t o x i c f a c t o r , w h i l e o n l y 1q%

were l y s e d by 0 . 1 5 ~ g .

The haemoly t i c spect rum o f o u r t o x i c p roduc t r e s e m b l e s

that o f t h e TDH i n t h a t human, r a b b i t , s h e e p and rat e r y t h r o c y -

tes were l y s e d . Unl ike t h e TDH (Zen-Yoji e t a l . , 1971),

however, humah e r y t h r o c y t e s w i t h t h e l o w e s t f i f t y p e r c e n t

haemoly t i c dose (HD ) 'of 0.15 ~ t g were most s e n s i t i v e t o 50 t h e haemoly t i c a c t i v i t y o f ' the t o x i c m a t e r i a l . L y s i s o f

e r y t h r o c y t e s frm c e r t a i n s o u r c e s may mean g r e a t e r s u s c e p t i -

b i l i t y of t h e an imals whose e r y t h r o c y t e s were l y s e d t o

i n f e c t i o n by t h e microorganism producing t h e haemolysin. I n

a d d i t i o n , s i n c e haemolysin produc t ion i s a v i r u l e n c e a t t r i b u t e

o f some microorganisms (Johnson & Calla, 1978; Munn, 1978;

Van den Bosch, 1980; and Peerbooms e t a l . , 1983), t h e t o x i c

f a c t o r is l i k e l y t o play a r o l e i n t h e pa thogen ic i ty o f x. parahaernolyticus.

Like some b a c t e r i a l t o x i n s (Asnani & J h a n j e e , 1982;

Bernheimer, 1964 and 1967; and Emeruwa, l986) , t h e t o x i c

f a c t o r had d i f f e r e n t HDwle t o e r y t h r o c y t e s from d i f f e r e n t

sources . The v a r i e d H D t j O t 5 s u g g e s t s v a r i e d s u s c e p t i b i l i t y o f

t h e animal sourcea t o y. p a r a h a e m o l u t i c u ~ i n f e c t i o n s . T h i s

may e x p l a i n why man wi th t h e most s e n s i t i v e e r y t h r o c y t e s i s

more s u s c e p t i b l e t o Y. parahaemol '~$icnq i n f e c t i o n s than o t h e r

animals.

The d e a t h oT mouse p e r i t o n e a l macrophages (MPM) on

exposure to' t h e t o x i c m a t e r i a l shows t h a t t h e t o x i c f a c t o r ,

l i k e t h e d i p h t h e r i a l t o x i n (Ubysz-Jergamanowska, 1972) and

t h e & c o l i l i popo lysaccha r ide (Davis e t al . , 1980), is

cy to tox ic t o MpM. T h a t t h e t o x i c f a c t o r causes dea th i n

animale by damaging t i s s u e s of v i t a l o rgans s u p p o r t s t h e

view o f Siddique & al. (1969) t h a t c y t o t o x i c a c t i v i t y p l a y s - 0 a s i g n i f i c a n t r o l e i n t h e thog genesis o f a d i s e a s e process .

By i ts c y t o t o x i c n a t u r e , t h e t o x i c f a c t o r resembles some

b a c t e r i a l t o x i n s which a l s o e x h i b i t c y t o t o x i c a c t i v i t y

a l t h o u g h t o o t h e r c e l l l i n e e . These i n c l u d e t h e TDH (Zen-

Y o j i e t al., 1971), t h e haemolys in o f h. m o ~ ~ c ~ t o ~ e n e s

( S i d d i q u e e t a l . , 1969), t h e e x o t o x i n from Group A s t r e p t o c o c c a l

f i l t r a t e s (Kim & Watson, l g 8 0 ) , t h e a l p h a - t o x i n from L e g i o n e l l a

s p e c i e s (Heolund, 1981 ), and t h e s c ~ n i c e x t r a c t s frmn B a c i l l u s

act inomycetemcomitans (Baehni e t al . , 1981). f

The c y t o t o x i c a c t i v i t y o f t h e t o x i c f a c t o r i s dose-

dependent . The p e r c e n t a g e of c e l l d e a t h i n c r e a s e d p r o g r e s s -

i v e l y as t h e dose of t h e t o x i c f a c t o r exposed t o t h e c e l l s

i n c r e a s e d . S i m i l a r r e s u l t s were o b t a i n e d w i t h t h e a c t i o n o f

alpha-haemolysin o f g. g o l i on human l e u k o c y t e s ( C a v a l i e r i &

Snyder, 1982), of s t r e g t o l y s i n 0 on human n e u t r o p h i l ( ~ n d e r s o n . & Duncan, 1980), o f TDH on f o e t a l n l u n g c e l l s ( S a k u r a i e t al.,

1975), and o f g. c o l i l i p o p o l y s a c c h a r i d e on human a l v e o l a r

macrophages (Dav i s e t a l . , 1980). . ,

P r e - t r e a t i n g MPM w i t h s u b t o x i c d o s e s o f t o x i c f a c t o r

i m p a i r e d t h e f u n c t i o n s o f t h e MPM as ev idenced by t h e d e c r e a s e d

r a t e of MPM m i g r a t i o n and t h e d e c r e a s e d number o f b a c t e r i a

a d h e r i n g f o o r phagocytosed by p r e - t r e a t e d MPM. A similar

r e a c t i o n c h a r a c t e r i s e d by d e p r e s s e d f n n c t i o n and r e s p o n s i v e n e s s

w a s obse rved i n macropbages, polymorphonuclear l e u k o c y t e s

and n e u t r o p h i l s from v a r i o u s s o u r c e s t r e a t e d w i t h s u b t o x i c d o s e s

d o s e s o f botu l inum t o x i n (Mierze jewsk i , 1968), s t r e p t o l y s i n s

S and 0 )Ofek e t al., l 9 7 2 ) , s t a p h y l o c o c c a l a l p h a - t o x i n

(Klhutec & Precech te l , 1972; and Schmeling e t a l . , 1981),

E. c o l i l i p o p o l y s a c c h a r i d e s ( I s s e k u t z & Biggar, 1978; and - - Davis e t al., 1980), d i p h t h e r i a l exo tox in (Sovetova, Kravehendo,

& Dzagurov, 1979 ; and Ubysz-Jergamanowska, 1979), Lea ione l l a

t o x i n (Meolund, 1981) and t h e E. c o l i alpha-haemolysin

( ~ a v a l i e k i & Snyder, 1982). I t i s thought t h a t t h e in vivo

product ion o f small amounts of t h e s e t o x i n s may c o n t r i b u t e t o

i nvas iveness and pa thogen ic i ty o f t h e microorganism producing

them by i n h i b i t t i n g t h e phagocyt ic and chemotact ic a c t i v i t i e s

chemotact ic response i n r a b b i t l eukocy te s (.Walker e t al. , 1969)

and t h e migra t ion o f monocytes (Ogawa e t a l . , 1983) a r e enhanced

by some t o x i c b a c t e r i a l products . . The f a i l u r e o f t h e t o x i c f a c t b r t o induce accumulation of

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

t h e t o x i c , . f ac to r from o t h e r t o x i n s o f 1. parahaemolyt icus ,

namely, t h e TDH .and t h e e n t e r o t o x i n - l i k e f a c t o r ; t h e chole ra -

t ox in , and t h e e n t e r o t o x i n of K l e b s i e l l a pneumonia0 a l l of which

a r e r a b b i t i l e a l l o o p (RIL) t e s t p o s i t i v e . The p o s i t i v e mouse

t o x i c i t y t e s t and t h e s k i n r e a c t i o n t e s t shows t h a t t h e t o x i c

f a c t o r i s e n t e r o t o x i c , andsugges t s t h a t d i a r rhoea may n o t

n e c e s s a r i l y be t h e on ly symptom manifes ted du r ing 1.

parahaemolyt icus i n f e c t i o n s .

The t o x i c f a c t o r i s immunogenic s t i m u l a t i n g an t ibody product ion. The r e s u S t s o f g e l d i f f u s i o n , immunoelectrophoresis

and h a e m a g g l u t i n a t i o n t es t s are i n d i c a t i o n s t h a t anigen-

a n t i b o d y r e a c t i o n s have t a k e n p lace . T h i s means t h a t t h e

immune serum c o n t a i n s c i r c u l a t i n g a n t i b o d y a g a i n s t t h e t o x i c

f a c t o r . The immune r e s p o n s e a g a i n s t t h e f a c t o r . is , t h e r e f o r e ,

humoral.

A g g l u t i n a t i o n o f v i b r i o c e l l s by t h e a n t i s e r u m shows t h e

p r o t e c t i v e power o f t h e a n t i - t o x i c f a c t o r serum a g a i n s t 1.

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

t i t r e was low p r o b a b l because o f t h e c r o s s - r e a c t i o n between Y t h e b a c t e r i a l c e l l s and a non-homologous a n t i g q r u . A h i g h e r

h a e m a g g l u t i n a t i o n t i t r e , on t h e o t h e r hand, s i g n i f i e s a

r e a c t i o n between a n a n t i g e n and i ts homologous a n t i b o d y .

The b a c t e r i c i d a l e f f e c t o f t h e a n t i - t o x i c f a c t o r serum a I

shown by t h e reduced v i a b l e c o u n t o f t h e m i x t u r e c o n t a i n i n g

v i b r i o c e l l s , a n t i s e r u m andcomplement s u g g e s t s complement-

media ted h o s t * . d e f e n s e mechanism a g a i n s t 1. p a r a h a e m o l y t i c u s

i n f e c t i o n s . P r e v i o u s s t u d i e s have shown t h a t immune s e r u m

e x e r t s a b a c t e r i c i d a l e f f e c t on t h e homologous microorganism

(Gower e t a l e , 1972) , and t h a t immune b a c t e r i c i d a l a c t i v i t y

can be obse rved 2 v i t r o as w e l l as in v i v o (Audran, 1972).

The a n t i - t o x i c f a c t o r serum p o s s e s s e s some p r o t e c t i v e

power. T h i s i s shown by t h e a b i l i t y o f a n i m a l s i n j e c t e d w i t h

t h e a n t i s e r u m t o s u r v i v e a l e t h a l dose o f t h e t o x i n . The

p r o t e c t i v e c a p a c i t y o f t h e a n t i - t o x i c f a c t o r serum is a l s o made e v i b n t by t h e a b i l i t y o f t h e a n t i s e r u m t o n e u t r a l i s e bo th

t h e haemolytic and c y t o t o x i c a c t i v i t i e s of t h e t o x i c f a c t o r .

Like most p r o t e i n s , t h e t o x i c f a c t o r abso rbs maximally uv- a t 280 nm. The s i n g l e peak on t h e curve of abso rp t ion spectrum 4

i l l u s t r a t e s t h e chemical p u r i t y o f t h e t o x i c f a c t o r ,

Add i t i ona l ly , t h e s i n g l e band o f p r o t e i n on PAGE and t h e s i n g l e

a r c of p r e c i p i t a t i o n on immunoelec t r o p h o r e s i s a r e i n d i c a t i o n s

t h a t t h e t o x i c f a c t o r i s both chemical ly and immunologically

pure.

The r e s u l t s of biochemical t e s t s show t h a t t h e t o x i c

f a c t o r i s composed p r i m a r i l y o f p r o t e i n , l i k e t h e TDH

(Sakura i e t al. , 1973)' and t h e t o x i n from t h e c u l t u r e f i l t r a t e

of a managma-negative s t r a i n of 1. parahaemolyt icus (Sochard

& Colwell , 1977). The absence of , ca rbohydra tes and l i p i d s

d i s t i n g u i s h e s o u r product from t h e ITHF which h a s f r e e f a t t y

a c i d s as its a c t i v e f r a c t i o n ( I i d a & Takagi, 1977; and 1979;

and Iida . e t a le , '1982), and from b a c t e r i a l endotoxins which

a r e complexes o f l i p i d , p r o t e i n and pb lyeacchar idee

The e s t ima ted molecu la r weight of t h e t o x i c f a c t o r is

63 095 showing t h a t i t i s a l a r g e r molecule than t h e TDH

(molecular W i g h t 42 000 - 44 000)(Honda e t a l e , 1978;

Takeda e t a l . , 1978; and Miyamoto e t al., 1980).

The t o x i c f a c t o r i s thermostable . Heat ing a t 1 0 0 ~ ~

d i d no t cause t h e f a c t o r t o l o s e i t s t o x i c i t y . A f t e r being

hea ted , t h e t o x i c f a c t o r r e t a i n e d t h e a b i l i t y t o l y s e e ry th ro -

c y t e s , a l t h o u g h o n l y 9% a c t i v i t y was r e t a i n e d . The HD50

o f t h e t o x i c f a c t o r b e f o r e t h e h e a t t r e a t m e n t was O . l 5 h g ,

whereas i t was O.3Oag a f t e r t h e h e a t t r e a t m e n t . The

u n p u r i f i e d t o x i c f a c t o r , on t h e o t h e r hand, i s t h e r m o l a b i l e

hav ing l o s t a l l t o x i c a c t i v i t y ( i . e . , t h e B b i l i t y t o l y s e

e r y t h r o c y t e s and t o c a u s e MPM d e a t h ) a f t e r i t w a s h e a t e d a t

60°c. L ike t h e TDH W h t a , 1975) and t h e s t a p h y l o c o c c a l a l p h a -

haemolysin ( S t e w a r t , 1968), t h e t o x i c f a c t o r e x h i b i t s t h e

A r r h e n i u s e f f e c t which means t h a t w h i l e t h e p a r t i a l l y p u r i f i e d

f a c t o r is t h e r m o s t a b l e a t 10oOc, i ts u n p u r i f i e d form loses

a c t i v i t y a t 60'~. A t h e r m o l a b i l e i n h i b i t o r which combines w i t h

t h e t o x i n a t 60°c w a s found r e s p o n s i b l e f o r s u c h p a r a d o x i c a l

b e h a v i o u r s (,Takeda e t al., 1974; and Farkas-Himsley, 1978) . . The i n c u b a t i o n t e m p e r a t u r e a f f e c t s t h e t o x i c i t y o f t h e

t o x i c f a c t o r . Maximal a c t i v i t y o c c u r r e d w i t h i n a nange o f

t o 5 7 O ~ , i n d i c a t i a g .. , t h a t t h e s e t e m p e r a t u r e s a r e o p t i m a l f o r

t o x i c a c t i v i t y o f t h e f a c t o r . The H D 5 0 ' ~ ( 0 . 1 5 f i g ) were lowest

and t h e MPM d e a t h r a t e s h i g h e s t ( 8 0 t o 90%) a t t h e s e t empera tu re [

A t l ower t e m p e r a t u r e s , however, t o x i c i t y w a s reduced. For

i n s t a n c e , a t IO'C, t h e HD50 W a s 2 . 5 0 4 3 and t h e MPM d e a t h

r a t e was 20%: The r e p o r t o f S a k u r a i e t a l . (1975) which s t a t e s

t h a t b i n d i n g o f haemolysin t o e r y t h r o c y t e s ( a p r o c e s s which

p r e c e e d s e r y t h r o c y t e s l y s i s 9 t a k e s p l a c e a t low t e m p e r a t u r e s

may e x p l a i n t h e d e c r e a s e i n a c t i v i t y a t l o w e r t e m p e r a t u r e s ,

The pH o f bile s u s p e n d i n g tnediurn h l s o a f f ec t : ; t h e t o x i c i t y

o f t h e t o x i c f a c t o r . L i k e most p r o t e i n s , a c t i v i t y was maximal

a t pH 7.0. A t t h i s pH, t h e )ID v a l u e was l o w e s t (0 .15 rug p r o t e i 5 0

and t h e MPM d e a t h r a t e was h i g h e s t (85%). The pH optimum f o r

t h e t o x i c f a c t a r i s t h u s c h a r a c t e r i s t i c o f most p r o t e i n s . On

t i i e c o n t r a r y , t h e pneumococcal be t a -hae rno lys in ( L o r i a n e t a l e ,

19'73) a n d t h e &. - c o l i a l p h a - h a e m o l y s i n ( p a l k i n a e t a l . , 1975)

were i n h i b i t e d a t t h i s pH, a n d t h e TDH, a p r o t e i n , a c t s

o p t j m a l l y a t a h i g h e r pH o f 8 .0 ( ~ o n d a e t a l . , 1976; and

Miyamcto e t a l . , 1 9 8 0 ) . A s t h e pl1 i n c r e , " s e d above n e u t r a l i t y

t o a l k a l i n i t y , t h e t o x i c i t y d e c r e a s e d a s shown by t h e h i g h e r

iIL)501s a n u l o w e r MPH d e a t h r a t e s . A s t h e pH d e c r e a s e d t o

a c i d i t y (6.4 a n d 5 . 8 ) , t o x i c i t y was v i r t u a l l y a b s e n t a s shown

by t h e f a i l u r e o f t h t t o x i c f a c t o r t o 1y:;e c r .y t i i r~ocybes arid t o

k i l l MPM.

I n c u b a t i o n w i t h P e p s i n o r T r y p s i n a b o l i s h e d t h e t o x i c

a c t i v i t y o f t h e t o x i c f a c t o r . Haemolys i s was n o t o b s e r v e d . ,

and t h e MPM d e a t h r a t e was v e r y low (0% and 5%) i n t h e t o x i c

f a c t o r i n c u b a t e d w i t h e i t h e r o f t h e p r o t e o l y t i c enzymes.

S e n s i t i v i t y t o p r o t e o l y t i c enzymes, e s p e c i a l l y T r y p s i n , h a s

been r e p o r t e d f o r o t h e r b a c t e r i a l t o x i n s i n c l u d i n g S t a p h y l o -

c o c c u s a u r e u s d .ermonecrotoxin ( P e t e r , 1 9 7 2 ) , g. c e r e u s

t o x i n ( F l u e r & Ezepchuk, 1 9 7 3 ) , t h e haemolys in o f M o r a x e l l a

b o v i s (Sandhu Xc White , 1977) , , a n d t h e D-toxin from CJ.

b o t u l i n u m (Nakane, Oguma and I i d a , 1 9 7 9 ) .

S e n s i t i v i t y t o p r o t e o l y t i c enzymes shows t h a t t h e a c t i v e

p o r t i o n o f t h e t o x i c f a c t o r is a p r o t e i n . The t o x i c f a c t o r is ,

thus , d i s t i n g u i s h e d from t h e i n t r a c e l l u l a r haemolytic f a c t o r

which has f r e e f a t t y a c i d s as i ts a c t i v e p a r t ( I i d a & Takagi,

1977). Secondly, s e n s i t i v i t y t o g r o t e o l y t i c enzymes may

e x p l a i n t h e reduced m o r t a l i t y o f t h e mice fed o r a l l y w i t h

t h e t o x i c f a c t o r , s i n c e t h e presence o f t h e enzymes i n t h e &ut

where t hey occur n a t u r a l l y may i n h i b i t t o x i c i t y o f t h e f a c t o r ,

Toxic a c t i v i t y o f t h e t o x i c m a t e r i a l was enhanced by

some me ta l i o n s , as shown by t h e lowered o f t h e t o x i c

f a c t o r when t h e s e i o n s were p r e s e n t i n t h e r e a c t i o n mixture .

The s t u d i e s o f Takeda e t a l . (1976) and of Ogiso and Miwatani

(1977) showed t h a t t h e b ind ing o f haemolysins t o e r y t h r o c y t e s

seem t o be s t i m u l a t e d by t h e presence of some d i v a l e n t c a t i o n s . I

Some c a t i o n s were, however, more e f f e c t i v e t han o t h e r s i n

enhancing t o x i c i t y . For example, t h e was lowest (0.016 a g )

when cat: C< or ~ n " i o n s was inc luded i n t h e r e a c t i o n mixture.

I n o t h e r c a s e s , t h e t o x i c f a c t o r l o s t i ts t o x i c i t y as shown by

t h e absence of haemolysis when some i o n s (e,g., Mn and Fe i o n s )

were added. O t h e r s l i k e t h e Mn produced no observable e f f e c t s

s i n c e t h e t o x i c f a c t o r s o l u t i o n c o n t a i n i n g e i t h e r of t h e i o n s

had t h e same HD as t h e c o n t r o l . The a b i l i t y o f d i v a l e n t 50 i o n s t o s t i m u l a t e haemolysins of v a r i o u s microorganisms have

been r e p o r t e d ( ~ a k u r a i e t al., 1975; Honda e t al. , 1976;

Palk ina , 1976; Takeda, Ogiso & Miwatani, 1977; Goshima e t al . ,

1978; and Emeruwa, 1986).

From t h e r e s u l t s ob t a ined i n t h i s s tudy , t h e t o x i c

f a c t o r i s d i s t i n c t from o t h e r t o x i c p roduc ts o f 1.

parahaemolyt icus h i t h e r t o desc r ibed , and appea r s t o be

r e spons ib l e f o r some symptoms encountered i n g. parahaemolyt icus

i n f e c t i o n s . The t o x i c m a t e r i a l a l s o appea r s t o play a

s i g n i f i c a n t r b l e no t on ly i n t h e pa thogen ic i ty o f x. p a r a h a e m o l ~ t i c u s bu t a l s o i n t h e defence of h o s t a g a i n s t

V. parahaemolyt icus i n f e c t i o n s . -

APPENDIX 1

Composit ion of MT medium

Peptone

Yeast e x t r a c t

Corn s t a r c h 1.0%

Sodium c h l o r i d e (NaC1) 7 00%

Agar (Oxo id ) 1.5%

APPENDIX 2

Compos i t i on o f Wagatsuma's medium

Yeast e x t r a c t 003%

Bac t o p e p t o n e 1.0%

Sodium c h l o r i d e 7.0%

Dipo ta s s ium p h o s p h a t e (K2HPq+) 005%

Agar (Oxoid) 1.5%

M a n n i t o l 0.1%

C r y s t a l v i o l e t i n a l c o h o l 0.1%

Human b lood 5.0% ' *

A l l made u p t o 1 l i t r e o f s o l u t i o n .

F i g u r e 15. Curve o f a s t a n d a r d p r o t e i n (Bovine serum

albumin) used t o d e t e r m i n e p r o t e i n

c o n c e n t r a t i o n s . , .

Optical

D e n s i t y a t 280 nm

20 4.0 60

P r o t e i n c,onczntrc.t ion (a$ ml-' )

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University of Nigeria · certificate ebele uchenna umeh (nge otubelu) , a postgraduate student in the department of microbiology, universlty of iv iglhia, nsukka , nigeria , has satisfactorily