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Andrze j K . S iwick ia , D oug las P. A nderso n * 'b , G ary L . R um sey calnland Fisheries Institute, Ichthyopathology an d Im m un olo gy Laboratory, 05-500 Piaseczno, Po land

bUS Fish a nd Wildlife Service, Nationa l Fish H ealth Research L aboratory, I mm uno logy Section,Bo x 700, Leetown, Kearneysville, W V2 54 30 , USA

cU S Fish and Wildlife Service, Tunison Laboratory o ff is h Nutrition, 3075 G racie Road, Cortland,NY 13045, USA

(Ac cepte d 8 Ju ly 1993 )

b s t r a c t

I m m u n o s t i m u l a n t p r e p a ra t io n s M a c r o g a rd®, Candida utilis, Saccharomyces cerevisiae,Eve tsel ®,C h i t o s a n ® , o r F i n n S t i m ® w e r e m i x e d i n t o s e m i p u r i fi e d d i e ts a n d f e d to g r o u p s o f r a i n b o w t r o u t f o r1 week . F i sh were b l ed by non- l e tha l cauda l pun c tu re and b loo d sam ples a s sayed fo r changes in non-

s p e ci fi c c e ll u l a r i m m u n i t y a n d h u m o r a l p r o t e i n l e v el s. I n t h e i m m u n o s t i m u l a t e d f is h , h e m a t o c r i t l e v -e ls and lym phocy te coun t s r em a ined r e l a t ive ly s t ab le; however, e l eva t ions were obse rved in ox ida t iverad ica l r e l ease , m ye lop e rox idase ac t iv i ty, phago cy t i c i ndexes , and po ten t i a l k i l li ng ac t iv i t ie s o f phag-ocy t i c ce l ls i nc lud ing neu t roph i l s . To ta l p l a sm a p ro te in and to t a l imm uno g lob u l in l eve l s were e l e-va ted by the d i e t a ry imm uno s t im ulan t s . A cha l l enge wi th the v i ru l en t pa thogen tha t causes fu runcu-losis ,Aero mo nas salmonicida,s h o w e d t h a t t h e i m m u n o s t i m u l a t e d g r o u p s o f f i s h w e r e m o r e r e si s ta n tto the d i sease , conf i rm ing the po ten t i a l u se o f t hese subs t ances in f i sh cu l tu re fo r the p reven t ion o fdisease .

b b r e v i a t i o n s

M P O , m y e l o p e r o x id a s e ; N B T, n i t ro b l u e t e t r a z o l iu m .

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T h e u s e o f i m m u n o s t i m u l a n t s i n f i s h c u lt u r e fo r th e p r e v e n t i o n o f d is e a se si s a p r o m i s in g n e w d e v e l o p m e n t A n d e r s o n , 1 9 92 ; O n a r h e i m , 1 9 92 ; R a a eta l ., 1992 ) . A t p re sen t , subs t a nces fo r t r ea t i ng fi sh d i s ea se s i n c lud e an t ib io t i c s ,d r u g s a n d c h e m i c a l s s u c h a s t h o s e u s e d f o r s t e r i l i z i n g f i s h - h o l d i n g p o n d s

S tos kop f , 1993 ) . W hi l e each the r apeu tan t i s a t l e a s t pa r t i a l ly e f f ec t i ve i n thet r e a t m e n t o f a p a r t ic u l a r d i s e a s e , p r o b l e m s a r i s e w i t h a c c u m u l a t i o n o f th e s e

* C o r r e s p o n d i n g a u t h o r.

S S D I 0 1 6 5 - 2 4 2 7 ( 9 3 ) 0 5 2 3 1 - 3



126 A.K. Siwic ki et aL / Veterinary Im mu nolo gy and lmrnuno pathology 41 (1994) 125 -139

s u b s t a n c e s i n th e e n v i r o n m e n t a s w e l l a s t h e e m e rg e n c e o f r e s is t a n t p a t h o -gen i c st r a i n s w h e n u s i n g a n t i b i o t i c s .

I n t h e p a st , t h e i m m u n o l o g i c a l a p p r o a c h t o p r e v e n t i n g fi sh d i se a s es ha s

be en by vac c in a t io n aga ins t spec i fi c pa th og ens (A nde rso n , 1974; E l li s, 1988 ) .S o m e t im e s , t h e v a c c in e s h a v e c o n t a i n e d a d j u v a n t s w h i c h b o o s t t h e i m m u n er e s p o n s e . A d j u v a n t s a r e r e c o g n i z e d a s e f fe c t iv e i n e l e v a t in g p o t e n c y a n d e f-f ic a c y o f b a c t e r i n s i n f is h. F r e u n d ' s c o m p l e t e a n d i n c o m p l e t e a d j u v a n t s , m u r -a m y l d ip e p ti d e , F K - 5 6 5 a n d n e w e r im m u n o s t im u l a n t s s u c h a s l ev a m i so l e h a v eb e e n a d d e d t o v a c c i n e s w i t h p r o m i s i n g r e s u lt s ( O l i v i e r et a l., 1 98 5; K i t a o e ta l ., 1987; G ra y so n e t al ., 1 9 8 7 ; S iw ick i , 1987; S iw ick i an d K orw in -K ossa -k o w s k i , 1 98 8; J e n e y a n d A n d e r s o n , 1 9 9 3 ) .

I n g e ne ra l , i m m u n o s t i m u l a n t s c o m p r i se a g r o u p o f b io l o g ic a l a n d s y n t h e ti c

c o m p o u n d s t h a t e n h a n c e t h e n o n - s p e c if ic d e f e n se m e c h a n i s m s i n an i m a l s .I m m u n o s t i m u l a n t s s u c h as t h e b e t a -g l u c an s , t ra c e m i n e r a l a n d v i t a m i n c o m -b i n a t io n s , a n d v a r io u s p r o d u c t s d e r i v e d fr o m m a n y p l a n t a n d a n i m a l s o u rc e sa r e e f f e c t i v e i n p r e v e n t i n g d i s e a s e s w h e n g i v e n a l o n e , w i t h o u t b e n e f i t o f av a c c i n e . S e v e ra l t y p e s o f b e t a - g l u c a n s s e e m e s p e c ia l ly p r o m i s i n g f o r s ti m u l a t -i n g t h e n o n - s p e c i f ic i m m u n e r e s p o n s e s o f f is h. Ya n o e t a l. ( 1 9 8 9 ) r e p o r t e don a s e r ie s o f b e t a - ( 1,3 ) d e r i ve d g lucan s f romS c h iz o p hy ll u m c o m m u n e(af u n g u s ) ,Sclero t ium g lucanicum( a f u n g u s ) a n dLent inus edodes( a n e d i b l em u s h r o o m ) t h a t e n h a n c e d re si st an c e o f c o m m o n c a rp ( C y p ri n u scarpio) toi n f e c t io n s o fEdwardsiella tarda.M - g l u c a n i s o l a t e d f r o m t h e c el l w a l l s o fSac-cha rom yces cerevisiaew a s u s e d b y R o b e r t s e n e t al. ( 1 9 9 0 ) t o e n h a n c e r es is -t a n c e t o Yersinia ruckeri, Vibrio anguil laru ma n d Aeromonas sa lmonic idainA t l a n ti c s a l m o n(Sa lm o sa l ar ) .N ik l e t a l. ( 1991 ) a l so fo u nd a be ta -g lu can( l e n t i n a n ) f r o mLent inus edodese f fe c t iv e w h e n u s e d t o p o t e n t i a t e i n j e c t e dAerom onas s a lmon ic idab a c t e r i n s . C h e n a n d A i n s w o r t h ( 1 99 2 ) u s e d i n je c -t i o n s o fSaccha rom yces cerevis iae (bake rs 'yeas t ) to enh an ce r e s i s tance t oEd-wardsiella ictalurii n c h a n n e l c a t fi s h( Ic ta luruspunc ta tusR a f i n e s q u e ) .

W h e n a f is h i n i ti a l ly e n c o u n t e r s a p a t h o g e n i c m i c r o o rg a n i s m , th e n o n - s p e -c if ic d e f e n se m e c h a n i s m s a re m o r e i m p o r t a n t t h a n t h e sp e ci fi c i m m u n e r e-s p o n s e, a s th e l a t te r r e q u i r e s a l o n g e r t im e f o r a n t i b o d y b u i l d - u p a n d s p ec i fi cce l l u l a r a c t i v a t i o n (A n d e r son , 1992 ) . I n gene ra l , f ish hav e sho r t l i fe spans andm o s t l i ve i n c o o l w a t e r e n v i r o n m e n t s w h i c h sl o w s d e v e l o p m e n t o f t h e s pe c if ici m m u n e r e s p o n se . T h e s e f a ct o rs m a y n o t a ll o w fi sh to d e v e l o p t h e c o m p l e xp h y s io l o g ic a l p a t h w a y s t h a t a r e c e n tr a l to d e v e l o p m e n t o f a n a d a p t i v e i m -m u n e r e sp o n s e . T h e n o n - s p e ci fi c de f e n se m e c h a n i s m s , w h i c h i n cl u d e p h ag o -c y t o s is a n d t h e p r o d u c t i o n o f o x i d a t i v e r a d ic a l s, a r e q u i c k l y a c t iv a t e d b y t h ei m m u n o s t i m u l a n t s a n d a r e ra p i d ly p r e p a r e d t o p r o t e c t t h e f is h a g a in s t p a th -o g e n s. I n p r e v i o u s s t u d ie s , w e m o d i f i e d a n d d e v e l o p e d a s sa y s to d e t e c t a c ti -

va t i on o f neu t roph i l s an d o t h e r p hag ocy t i c c e ll s i n f ish (S iw ick i e t a l. , 1990;A n d e r s o n a n d J e n ey, 1 9 9 2 ). T h e s e te c h n i q u e s h a v e b e e n i n c o r p o r a t e d t om o n i t o r t h e e f fe c ts o f t h e i m m u n o s t i m u l a n t s i n t h e r es e a rc h r e p o r te d h e r e .



A.K. Siwicki et aL / Veterinary Immunology and Immunopathology 41 1994) 125-139 12 7

S i m i l a r t o m a m m a l i a n m o d e l s , se v e ra l d r u g s a n d c h e m i c a ls h a v e b e e ns h o w n t o b e e f f e c t iv e in s t i m u l a t i n g n o n - s p e c i f ic d e f e n s e m e c h a n i s m s i n fi sh(S iw ick i e t a l ., 1 9 9 0 ) . Be c a u se t he se subs t anc es a r e t o be u sed i n f ish de s t i n e d

f o r h u m a n c o n s u m p t i o n , a d h e r e n c e t o g o v e r n m e n t a l s a fe ty r eg u l a ti o n s is n ec -e s sa ry fo r a p p ro va l .

I n t h e fo l lo w i n g s tu d i e s w e e v a l u a t e d six n e w i m m u n o s t i m u l a n t s w h i c h w e r ec h o s e n p r i m a r i l y b e c a u s e t h e y a r e d e r i v e d f r o m s u b s t a n c e s t h a t a r e c l o s e l yr e l a te d to n a t u r a l p r o d u c t s. R e p l i c a t e d g r o u p s o f ra i n b o w t r o u t ( O n c o r h y n -chus mykiss)w e r e f e d t h e i m m u n o s t i m u l a n t - c o n t a i n i n g d ie ts fo r a p e r i o d o f7 d a y s , a f t e r w h i c h t im e t h e y w e r e s a m p l e d a n d a s s a y e d f o r c h a n g e s i n n o n -s p e c i f i c d e f e n s e m e c h a n i s m s a n d p r o t e c t i o n a g a i n s t f u r u n c u l o s i s d i s e a s echa l lenge .

a t e r i a l s a n d m e t h o d s

Exp erim ental protocol

R a i n b o w t r o u t m e a n b o d y w e i g h t 2 0 0 g , f r o m a d i s e a s e - f r e e p o p u l a t i o nm a i n t a i n e d a t t h e U S F i sh a n d Wi l d li fe S e rv ic e , Tu n i s o n L a b o r a t o r y o f F i shN u t r it i o n , w e r e u s e d i n b o t h e x p e r i m e n t s o f t h is s tu d y. B o t h e x p e r i m e n t s h a da p o s i ti v e c o n t r o l t re a t m e n t w h i c h a l o n g w i th t h e e x p e r i m e n t a l t re a t m e n t sw e r e r a n d o m l y a s s i g n e d to d u p l i c a t e 19 1 P l e x ig l as t a n k s c o n t a i n i n g 1 0 f is he a c h ( 2 0 f is h p e r t r e a t m e n t ). T h e t a n k s w e r e i n d i v i d u a l l y su p p l i e d w i th a e r -a t e d c o n s t a n t t e m p e r a t u r e ( 9 .2 ° C ) w e l l w a t e r a t a f l o w r a t e o f 4 .5 1 m i n - ~.T h e f i sh w e r e f e d to s a t i a ti o n t w i c e d a i ly, 7 d a y s a w e e k . D u r i n g t h e 1 w e e ka c c l i m a t i o n p e r i o d a n d a f t er th e 7 d a y p e r i o d o f f e e d i n g t h e d i e t s c o n t a i n i n gt h e i m m u n o s t i m u l a n t s , t h e f is h w e r e f e d t h e b a s a l c o n t r o l d i et . F i s h w e r e o b -s e rv e d d a i ly f o r u n u s u a l b e h a v i o r , m o r p h o l o g i c a l c h a n g e s a n d m o r t a li ty. F o rt h e i m m u n o l o g i c a l as sa y s a n d c h a l le n g e t es ts p e r f o r m e d w i th t h e g r o u p s f r o mt h e s e c o n d e x p e r i m e n t , 1 0 f is h ( f i v e f r o m e a c h re p l ic a t e t a n k ) w e r e r a n d o m l yse l e c t ed fo r e a c h a s s a y o r t e s t.

Diets

T h e f o r m u l a s f o r t h e b a sa l c o n t ro l d ie t in t o w h i c h t h e im m u n o s t i m u l a n t sw e r e i n c o r p o r a t e d a r e g i v e n i n Ta b l e 1. A l l d i e ts w e r e fo r m u l a t e d t o b e n u t r i-t i o n a l l y c o m p l e t e ( N a t i o n a l R e s e a r c h C o u n c i l , 1 98 1 ) c o n t a i n i n g fi sh m e a l a st h e o n l y s o u r c e o f p r o t e i n , f i sh o i l f o r l ip i d , d e x t r i n a n d g l u c o se f o r c a r b o h y -d r a t e a n d v i t a m i n - m i n e r a l p re m i x e s. T h e a c t u a l c h e m i c a l a n a ly s e s o f th e b a s a lcon t ro l and expe r imen t a l d i e t s we re p ro t e in 41 .9%, l i p id 15 .4%, f i be r 5 . 1%,

a s h 9.3 % a n d m o i s t u r e 10.0% ; t h e m o d e s t i m m u n o s t i m u l a n t a d d it i o n s w e r en o t d e t e c t a b l e b y t h e s e c h e m i c a l a n a ly s e s .

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1 2 8 A.K. Siwi eki et a l. / Veterinary Im mu nolo gy and lmm unop atho logy 41 (1994) 125- 139

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A.K. S iwick i e t a l. / Ve te rinary Im mun ology an d lmmuno patho logy 41 (1994) 125-139 12 9

f o r e t h e e x p e r i m e n t s a n d s t o r e d i n p l a s ti c b a gs a t 0 ° C u n t i l u s ed , a s d e s c r i b e db y R u m s e y ( 1 99 2 ) .

I m m u n o s t i m u l a n t s

I n t h e f i rs t e x p e r i m e n t , t h e f o l lo w i n g d i e t a r y s u p p l e m e n t s w e r e t e st e d: M a -cro ga rd ®,Ca ndida u t il isyeas t , Saccharomyces ce rev i s iaey e a s t . T h e M a c r o -g a r d ® p r e p a r a t i o n ( K S B i o t e c -M a c k z y m a l , Tr o m s o , N o r w a y ) is d e sc r ib e d b yt h e m a n u f a c t u r e r a s a fe e d - g r a d e b e t a - l , 3 / 1 , 6 - g l u c a n ( b e t a - l i n k e d g l u c o sep o l y m e r ) d e r i v e d f ro m t h e c ell w a l ls o f S .cerevis iaeyeas t . TheC. utilisa n dS. cerevisiaey e a st s w e r e o b t a i n e d f r o m l o c al c o m m e r c i a l so u r c es a n d w e r ew a t e r - w a s h e d a n d f r e e z e - d r i e d b e f o r e b e i n g i n c o r p o r a t e d i n t o t h e d i e ts . T h e

d i e t a r y le v e l o f y e a st g lu c a n u s e d w a s b a s e d o n t h e r e c o m m e n d a t i o n s o f t h em a n u f a c t u r e r ( M a c k z y m a l , Tr o m s o , N o r w a y ) w h i l e t h e l ev e ls o f y e a s t u s e dw e r e p r e d i c a t e d o n t h e o b s e r v a t i o n t h a t a b o u t 13 g o f y e a s t ce ll s y i e l d a b o u tl g o f c ru de ce l l- w a l l g luc a n ( R u m sey e t a l. , 1992 ) .

I n t h e s e c o n d e x p e r i m e n t , s ix s u p p l e m e n t s , a l o n g w i t h a c o n t r o l , w e r e e v a l-u a t e d : M a c r o g a r d ®,C. utilis, S. cerevisiae,Ch i to sa n ® , Eve t s e l ® , F inn S t im ® .T h e f ir st t h r e e a d d i t i v e s w e r e id e n t i c a l to t h o s e u s e d i n t h e f ir st e x p e r i m e n t .C h i t o s a n ® i s a d e a c y l a t e d c h i ti n ( b e t a - l i n k e d p o l y m e r o f N - a c e ty l - D - g lu c o s -a m i n e ) f r o m s h r im p c a r a p a c es t h a t w a s p r o d u c e d b y t h e S e a F is h e ri e s I n st i-t u t e i n G d y n i a , P o l a n d . E v e t s e l ® i s a p r o p r i e t a r y ( P o l f a , K r a k o w , P o l a n d )v e t e r i n a r y n u t r i t i o n a l s u p p l e m e n t c o n s i s ti n g o f v i t a m i n C , v i t a m i n E a n d s e-l e n i u m t h a t c a n b e a d m i n i s t e re d b y i n j e c t io n o r p e r o s. F i n n S t i m ® ( F i n n su -g a r B i o p r o d u c t s , H e l si n k i , F i n l a n d ) is a p r o p r i e t a r y 9 7 3 m i x t u r e o f a n h y -d r o u s b e t a i n e a n d a m i n o a c id s w h i c h w h e n u s e d a s a d i e ta r y s u p p l e m e n tr e p la c e s c h o l i n e ( R u m s e y, 19 91 ) , e n h a n c e s f e ed a c c e p t a b i li t y ( R u m s e y,1 98 6 ) a n d f a c il it a te s s e a w a t e r a d a p t a t i o n o f s a l m o n i d f is h es ( Vi r t a n e n e t a l.,1 98 9 ) . T h e r a t i o n a l e f o r i n c l u d i n g F i n n S t i m ® w a s t h a t b e t a i n e o r t r im e t h y l -g l y c in e , i ts p r i m a r y c o n s t i t u e n t , is m e t a b o l i z e d i n t h e f is h 's l i v e r t o d i m e t h y l -g ly c i ne w h i c h h as b e e n r e p o r t e d t o e n h a n c e b o t h h u m o r a l a n d c e l l- m e d i a t e di m m u n e r e s p o n s e s i n h u m a n s a n d d o m e s t i c a n i m a l s ( G r a b e r e t a l . , 1 9 8 1 ;C h a r e s t e t a l. , 1 98 8; R e a p a n d L a w s o n , 1 9 9 0 ) .

Im m u n o l o g i c a l a s s a ys a n d c h a l lenge te s ts

F o r t h e f ir st e x p e r i m e n t , t h e f o l lo w i n g a s sa y s w e r e r u n : b l o o d h e m a t o c r i tl ev e ls a n d l e u k o c y t e n u m b e r s , o x i d a ti v e r a d ic a l p r o d u c t i o n ( a s m e a s u r e d b yn i t ro b l u e t e tr a z o l i u m ( N B T ) r e d u c t i o n u s i ng a s p e c t r o p h o t o m e t e r ) , p h a go -c y t ic in d e x r e a d in g s , m y e l o p e r o x i d a s e p r o d u c t i o n o f n e u tr o p h i ls a n d m o n o -

c y te s, t o ta l p l a s m a p r o t e in , a n d t o ta l p l a s m a i m m u n o g l o b u l i n . F o r t h e s e c o n de x p e r i m e n t , t h e s a m e a s sa y s w e r e ru n p l u s a n a d d i t i o n a l a s sa y t o m e a s u r e t h ep o t e n t i a l k i l li n g a c t i v i ty o f n e u t r o p h i l s a n d m o n o c y t e s .



130 A.K. Siw icki et aL / Veterinary lmrnuno logy and lrnmunopathology 41 (1994) 1 25-1 39

Chal lengeA d i s e a s e c h a l l e n g e t e s t u s i n gAerom onas s a lmon ic idaw a s c o n d u c t e d in t h e

s e c o n d e x p e r i m e n t u s in g g r o u p s o f 1 0 fi sh ( fi v e f ro m e a c h r e pl ic a t e t a n k ) f o r

e a c h t re a t m e n t t o d e t e r m i n e i f p r o t e c t i o n w a s p r o v i d e d b y t he d i et a ry i m -m u n o s t i m u l a n t s ag a in s t fu r u n c u l o s is . Te n f is h fr o m e a c h tr e a t m e n t w e ret r a n s p o r t e d f r o m t h e Tu n i s o n L a b o r a t o ry, C o r t l a n d , N Y, to t h e N a t i o n a l F i s hH e a l t h R e s e a r c h L a b o r a t o r y, K e a r n e y s v i l l e , W V, 2 d a y s a f t e r t h e 7 d a y p e -r i o d o f fe e d i n g t h e i m m u n o s t i m u l a n t s . A f t er 1 w e e k o f a d j u s t m e n t to t h e n e wc o n d i t i o n s , t h e f is h w e r e g i v e n s i n gl e i n t r a p e r i t o n e a l i n je c t i o n s o f a 4 8 h g r o w t ho f A . sa lmonic ida (1 X 10 7) b a c t e ri a s u s p e n d e d i n g ro w t h m e d i u m . M o r t a li -t i es w e r e t a b u l a t e d a n d c o l le c te d a n d t h e p r e se n c e o f p a t h o g e n w a s c o n f i r m e db y i s o l a ti o n f r o m t h e f i s h 's k i d n e y s .

A ssa y proceduresF o r b o t h e x p e r i m e n t s , 1 0 f is h ( f i v e f r o m e a c h re p l ic a t e t a n k ) w e r e s a m p l e d

f r o m e a c h o f t h e t r e a t m e n t g r o u p s , i n c lu d i n g th e c o n t ro l s. F i s h w e r e i m m o -b i l i z e d b y a s h a r p b l o w t o t h e c r a n i u m , a n d a b o u t 2 m l o f b l o o d c o ll ec te df r o m t h e c a u d a l v e i n u s i n g a h e p a r i n - t r e a t e d ( 1 50 u n i t s m l - 1 s y ri ng e . A f t e rw i th d r a w a l , t w o m i c r o h e m a t o c r i t c a p il la r y tu b e s w e r e f il le d d ir e c tl y fr o m t h es y r in g e f o r h e m a t o c r i t o b s e r v a t io n s . H e m a t o c r i t v a l u e s w e r e m e a s u r e d a f te rc e n t r i f u g a t i o n a t 5 4 0 0 X g f o r 1 m i n . T h e r e m a i n i n g b l o o d w a s d i s p e n s e d i n t oa 1.5 m l p l a st ic tu b e a n d i m m e d i a t e l y a 0 .1 m l s a m p l e w a s p i p e t t e d i n to a n -o t h e r tu b e c o n t a i n i n g 0.9 m l E a g le 's m i n i m a l e s se n tia l m e d i u m w i t h 2 % c a l fs e r u m f o r c ell c o u n t s u s in g a h e m o c y t o m e t e r .

T h e N B T - s p e c t r o p h o t o m e t r i c o x id a t iv e r ad ic a l p r o d u c t i o n a ss ay u s e d int h is s tu d y w a s a m o d i f i c a t i o n o f t h e m e t h o d o u t l i n e d b y S i g m a ( S ig m a C h e m -i ca l C o m p a n y , 1 9 7 3 ) a n d a d a p t e d t o f i s h ce il s b y S i w i c k i et a l. ( 1 9 8 5 ) . O n e -t e n t h m l o f a 0 .2 % N B T in p h o s p h a t e b u f f e r e d sa l in e ( P B S ) s o l u t io n w a sa d d e d t o 0 .1 m l o f b l o o d i n a m i c r o t i t e r p l a te w e l l a n d i n c u b a t e d f o r 3 0 m i n .T h e n , a 0 .0 5 m l s a m p l e w a s ta k e n f r o m t h is m i x t u r e , a d d e d to 1 .0 m lo f N , N -d i m e t h y l f o r m a m i d e , c e n t r if u g e d f o r 5 m i n a t 3 0 0 0 X g , a n d r e a d i n a s p e c tr o -p h o t o m e t e r a t 54 5 r im .

F o r t h e p h a g o c y t i c i n d e x a s sa y, 0 .1 m l o fStaphylococcus aureus( 1 X 108m l - 1 ) s u s p e n d e d in P B S w a s a d d e d t o 0 .1 m l o f b l o o d i n t h e m i c r o t i t e r w e ll.T h e c o m b i n a t i o n w a s i n c u b a t e d at r o o m t e m p e r a t u r e f or 20 m i n a n d m i x e dt h o r o u g h l y u s in g a p i p e t t e to e n s u r e c o n t a c t o f t h e b a c t e r ia a n d l e u k o c y t e s.F i ft y m i c r o l it e r s w e r e p l a c e d o n a g la ss s li de to m a k e a s m e a r s i m i la r to t h em e t h o d o f m a k i n g a b l o o d s m e a r. T h e s li de w a s a ir d r i ed , f ix e d w i th 9 5 %e t h a n o l , r e d r ie d , a n d t h e n s t a i n e d w i t h 0 .1 5 % s a f r a n in s o l u t io n f o r 10 m i n .T h e p h a g o c y t i c c ells w e r e o b s e r v e d a n d e n g u l f e d b a c t e r i a w e r e c o u n t e d f o r

c a l c u l a t io n o f a p h a g o c y t i c in d e x .M y e l o p e r o x i d a s e p r o d u c t i o n w a s m e a s u r e d b y s m e a r i n g 5 0/~ 1 o f b lo o d o n

a g la s s s li d e, a l lo w i n g t o a i r d r y a n d f i x i n g w i t h 9 5 % e t h a n o l f o r 5 m i n . T h e n



A.K. Siwicki et al. / Veterinary Immunology and lmmunopathology 41 1994) 125-139 131

a 9 5 % e t h a n o l p e r o x i d a s e i n d i c a t o r re a g e n t w i t h t r iz m a l b u f f e r ( S i g m a K i t3 9 0- A ; S i g m a , St. L o u i s , M O ) w a s u s e d t o i m m e r s e a n d i n c u b a t e th e s li de f o r1 5 m i n a t r o o m t e m p e r a t u r e . F i n a l l y, t h e s l i d e s w e r e w a s h e d w i t h d i s t i l l e d

w a t e r a n d o b s e r v e d f o r d e g r e e o f s ta i n i n g i n o r d e r t o o b t a i n a g r a d e o f n e u t r o -p h i l a c t iv i ty. A ' 0 ' w a s a s s i g n e d i f n o a c t i v i t y w a s o b s e r v e d , a ' 1 ' w a s g iv e nf o r l i g h t l y s t a i n i n g p o s i t i v e c e l l s , a n d a ' 2 ' w a s a s s i g n e d w h e n t h e c e l l w a sd e e p l y c o l o r e d . O n e h u n d r e d c e lls w e r e g r a d e d f r o m e a c h f is h a n d t h e m e a na n d s t a n d a r d d e v i a t i o n ( S D ) w e r e d e ri v e d fo r e a c h g r o u p .

A m o d i f i c a t i o n o f a t e c h n i q u e b y R o o k e t al. ( 1 98 5 ) w a s u s e d to m e a s u r ep o t e n t i a l k i l li n g a c t i v i t y o f b l o o d p h a g o c y t i c ce lls . F ir s t, 0 .1 m l o f b l o o d w a sa d d e d t o 0 .1 m l o f E a g le 's m i n i m a l e s se n t ia l m e d i u m w i th 0 .1 % f e ta l c a l fs e r u m i n a m i c r o t i t e r p l a te w e ll. T h e c o m b i n a t i o n w a s in c u b a t e d f o r 1 h a t

r o o m t e m p e r a t u r e t o a l lo w a d h e r e n c e o f c ells t o t h e p l a s ti c su r fa c e s. T h e n ,t h e s u p e r n a t a n t a n d n o n - a d h e r e n t c e lls w e r e r e m o v e d u s i ng a p i p e tt e a n d a0 .2 % N B T i n P B S s o l u t i o n c o n t a i n i n g l iv e 1 X l 0 sA. salmonicida ce l l s wasa d d e d . T h i s s u s p e n si o n , w h i c h c o n t a i n e d 1 0% h o m o l o g o u s s e r u m t o e n h a n c ep h a g o c y t o s i s, w a s c e n t r i f u g e d fo r 5 m i n a t 1 5 0 X g t o b r i n g th e b a c t e r i a i n t oc o n t a c t w i th t h e a d h e r e n t ce lls . A f t e r i n c u b a t i o n f o r 3 0 - 6 0 m i n a t r o o m t e m -p e r a tu r e , t h e s u p e r n a t a n t w a s p i p e t t e d o f f a n d 2 0 0 / A o f a b s o l u te m e t h a n o la d d e d t o f ix th e c ells . A f t e r in c u b a t i o n f o r 2 - 5 m i n , t h e s u p e r n a t a n t w a s re -m o v e d a n d t h e ce lls r i n s e d t h r e e to f o u r t i m e s w i th 7 0 % m e t h a n o l . T h e m i -c r o t i te r p la t e w a s d r i e d b y p l a c i n g i t i n a w a r m i n c u b a t o r f o r 4 0 r a i n a n d t h e n1 2 0 /t l o f 2 M K O H a n d 1 40 /z l o f d i m e t h y l s u lf o x id e ( D M S O ) w e r e a d d e d tos o l u b il iz e t h e f o r m a z a n . T h i s m i x t u r e w a s i n c u b a t e d f o r 5 m i n , a n d t h e p l a te sw e r e t h e n p l a c e d i n th e m i c r o t i te r r e a d e r s et fo r re a d in g s a t 6 2 0 n m ; t h e K O H -D M S O c o m b i n a t i o n w a s u s e d a s t h e b l a nk . T h e s p e c t r o p h o t o m e t r ic r e ad i ng sf r o m e a c h f is h w e r e a v e r a g e d a n d S D c a l cu l a te d . D u p l i c a t e s a m p l e s w e r e r u nf o r c o n f i r m a t i o n .

To t a l p l a s m a p r o t e i n w a s d e t e r m i n e d b y t h e L o w r y e t a l. ( 1 95 1 ) c o l o ri -m e t r ic m e t h o d , c o m b i n i n g 5 / A o f p l a sm a , 2 5 / z l o f r e a ge n t A a n d 2 0 0 /z l o fr e a g e n t B ( B i o - R a d , H e r c u l e s , C A ) i n a m i c r o t i t e r w e ll. T h e c o m b i n a t i o n w a sm i x e d b r ie f ly u s i n g a p ip e t t e , a n d a f te r 15 m i n m e a s u r e m e n t s w e r e ta k e nu s i n g t h e m i c r o t i t e r re a d e r a t 6 5 0 n m .

To t a l p l a s m a i m m u n o g l o b u l i n w a s a ls o m e a s u r e d , u s in g t h e L o w r y m e t h o d ,w i t h m o d i f i c a t i o n b y S i w i ck i a n d A n d e r s o n ( 1 9 94 ) b y f i rs t p r e c ip i t a t in g th ei m m u n o g l o b u l i n o u t o f t h e p l a s m a w i th p o l y e t h y l e n e g ly c o l ( 10 0 0 0 k D a ) .F o r t h is p r o c e d u r e , 1 00 /z l o f th e p l a s m a w e r e m i x e d w i t h a n e q u a l a m o u n t o f1 2% p o l y e t h y l e n e g ly c o l a n d i n c u b a t e d f o r 2 h u n d e r c o n s t a n t a g i t a t io n a tr o o m t e m p e r a t u r e . A f t e r c e n t r i f u g a t i o n a t 3 0 0 0 × g f o r 1 5 m i n , t h e su p e r n a -t a n t w a s r e m o v e d a n d t h e r e m a i n i n g p r o te i n w a s d e t e r m i n e d a ft er it w a s s u b -

t ra c t e d f r o m t h e t o ta l s e r u m p r o t e i n c o n c e n t r a t io n .F o r s t a t i s t i c a l a n a l y s i s , m e a n s a n d s t a n d a r d d e v i a t i o n s f o r a l l t e s t v a l u e s

w e r e c a l c u l a t e d a n d S t u d e n t ' s t -t es t w a s u s e d t o d e t e r m i n e w h e t h e r d i ff e r-



1 3 2 A.K. Siwicki et al. / Veterinary Immunology and Immunopathology 41 (1994) 125-139

e n c e s e x i s te d b e t w e e nt w o o r m o r e g r o u p s . T h e s ig n i fi c a n ce l e v e lu s e d w a sP < 0 . 0 5 .

R e s u l t s

I n t h e f i rs t e x p e r i m e n t , 1 w e e k a f te r f e e d i n g t h e d i e ts c o n t a i n i n g th e y e a s tg l u c a n p r e p a r a t i o n M a c r o g a r d ® , a n d t h e d r i e d y e a s t sC . u t i l i s a n d S. ce rev i s -i a e t h e h e m a t o c r i t l e v e l s s h o w e d n o s i g n i f i c a n t d i f f e r e n c e s b e t w e e n d i e t a ryt r e a t m e n t s a n d th e c o n t r o l Ta b l e 2 ) . B y c o n tr a s t, t h e r e w a s a m a r k e d a n ds i g n i f ic a n t e l e v a t i o n i n l y m p h o c y t e n u m b e r s i n f i sh f e d t h e t w o y e a s t s u p p l e -m e n t s . N e u t r o p h i l o x i d a t i v e r a d ic a l p r o d u c t i o n N B T t e s t) w a s n o t i ce a b l ya n d s i g n i f i c a n t l y h i g h e r, a s w e r e t h e p h a g o c y t i c i n d e x e s a n d n e u t r o p h i l m y e -l o p e r o x i d a s e l e v e l s o f f i sh f e d t h e t h r e e d ie t a r y i m m u n o s t i m u l a n t s . A g a i n , t h e

Ta b l e 2E f f ec t s o f d i e t s c o n t a i n i n g d i f f e r e n ty e a s t / g l u c a n p r e p a r a t i o n s o n n o n - s p e c i f i c s e ro l o g i ca l a n d i m -m u n o l o g i c a l a s sa y s in r a i n b o w t r o u t ( E x p e r i m e n t 1 )

I . H e m a t o c r i t s a n d l y m p h o c y t e le v e ls

D i e t H e m a t o c r i t L e u k o c y t e s( % ) ( X 1 0 3 )

C on t ro l 43 .2 -+7 .4 32 .1 +_3.5 aM a c r o g a r d ® 4 1 . 2 - + 5 .6 3 5 .8 + 2 . 6 aC. u t i l i s 46 .6 -+6 .4 42 .2 -+4 .4 bS . c e r e v i s i a e 4 2 . 8 - + 3 .5 4 4 . 4 + 3 . 7 b

I I. N e u t r o p h i l o x i d a t i v e r a d i c a l p r o d u c t i o n ( N B T t e s t ) , p h a g o c y t i c i n d e x , a n d m y e l o p e r o x i d a s ea c ti v it y ( M P O ) i n p o l y m o r p h o n u c l e a r l e u k o c y te s

D i e t N B T t e s t P h a g o c y t i c i n d e x M P O a c t iv i t y( m g m l - ~ ) ( t o t a l)

C o n t r o l 1 . 1 8 0 . 1 a 5 . 9 8 0 . 9 a 1 0 1 . 6 + - 4 . 8 a

M ac ro ga rd ® 1 .79 -+ 0 .13 b 9 .32 _+ 0 .8 b 123 .2 _+ 5 .5 bC. u t i l i s 2 . 1 0 + 0 . 1 2 ~ 1 0 . 54 - + 0 .9 ¢ 1 2 7 . 8 + 8 . 6 bS . c e r e v i s i a e 1 . 98 + 0 . 12 c 11 .26_+ 1 .1 c 136 _+9.8 b

I II . To t a l s e r u m p r o t e i n a n d i m m u n o g l o b u l i n ( I g ) le v e ls

D i e t To t a l p r o t e i n To t a l I g( m g m l - l ) ( m g m l - l )

Co nt ro l 60 .5 _+4.0 18 .5 _+2.5 aM a c r o g a r d ® 6 2 . 8 -+ 5 . 4 2 2 . 4 -+ 2 . 5 bC. u t i l i s 61 .4 -+4.2 24.5 _+2.5 b

S . c e r e v i s i a e 61 .0 _+3.5 25 .0 _+3.58

Ea ch va lue i s a m ea n ( -+_S D ) o f t w o g r o u p s o f f iv e f is h ( n = 1 0 ) . M e a n s w i t h i n a c o l u m n n o t s h a r i n ga c o m m o n s u p e r s c r i p t l et t e r a r e s i g n if i c a n tl y d i ff e r e n t ( P < 0 .0 5 ).



A.K. Siwicki et al. / Veterinary Immu nology and lmmunopathology 41 (1994) 125 -139 13 3

Table 3Effects of diets containing imm unos ti mul ant s on non-specific serological and immunological assaysin ra inbow t rout (Exper iment 2)

I. Hematocr its and lymphocyte levels

Diet Hematocr it ( ) Leukocytes( X 10 3)

Co nt ro l 40.8 + 5.5 a 29.6 + 7.7 abMa cr og ar d ® 41.2 + 3.4 a 26.8 _+ 6.5 aC. u t i l i s 38.0 + 4.6 a 24.2 _+ 5.7 aS . c e r e v i s i a e 35.4 + 2.6 b 27. 4 _+ 4.9 abEve tse l ® 39.4 + 2.8 ~ 26.4_+ 6.9 ~Ch it os an ® 42.0 + 3.7 ~ 25.2 ___4.6 aFi nn St im ® 35.0 + 1.8 b 34.2 ___9.4 b

II. Neutrophil oxidative radical production (NB T), myeloperoxidase activity ( MPO ), phagocyticindex and potential neutr ophil kil l ing activity in polymo rphonu clear leukocytes

Diet NBT test MP O activity Phagocytic index Potential killing(mg ml - ~ activity

Co nt ro l 1.29 + 0. I 1 a 97.2 + 3.7 a 5.5 + 0.45 a 0.37 + 0.10 aMacrogard ® 1.65+0. 24 ~ 110.0+2.7 b 9.3+ 0.50 c 0.58+ 0.06 ~C. u t i l i s 1.51 +0. 17 b 106. 6+2. 5 b 9. 8+0 .70 cd 0.5 3+0 .10 ~S . c e r e v i s i a e 1.87 +0. 13 ¢ 121. 0+6. 7 ¢ 10.1 +0. 54 a 0.6 2+0 .12 ¢Eve tse l ® 1.64 + 0.09 bc 113.5 + 3.4 b 8.9 + 0.40 c 0.46 + 0.10 ~b

Ch it os an ® 1.71 + 0.13 ¢ 124.2 + 3.8 ¢ 10.4 + 0.65 a 0.59 + 0.08 cFi nn St im ® 1.48 + 0.10 b 110.0 + 3.0 b 6.6 + 0.48 b 0.42 + 0.08 ab

III. Total serum protein and total imm unogl obuli n (Ig) levels

Diet Total prote in Total Ig( m g m l - l ) ( m g m 1 - 1 )

Co nt ro l 56.1 + 3.7 a 20.9 + 1.9 aMa cr og ar d ® 61.5 + 5.2 b 22.7 + 1.0 bC. u t i l i s 60.4 + 4.8 a 26.5 + 2.7 ¢S . c e r e v i s i a e 62. 7+2 .4 b 28. 5+ 1.0 c

Eve tse l ® 60.5 + 2.6 b 27.5 + 1.7 cCh it os an ® 64.3 + 2.7 b 28.6 + 1.1 cFi nn St im ® 64.8 _+ 2.1 b 26.5 + 2.0 c

Each value is a mean + SD of two gr oups of five fish (n = 10).Means ( + SD) within a col umn not sharing a commo n superscript letter are significantly different(P < 0 .05) .

f i s h fe d t h e y e a s t s s h o w e d g r e at e r s t i m u l a t i o n t h a n t h o s e f e d M a c r o g a r d ® .W h e r e a s to t al p l a s m a p r o t e in w a s n o t a f f e c te d b y th e d i et a ry i m m u n o s t i m u -

l a n ts u s e d in t h is e x p e r i m e n t t h e to t a l s er u m i m m u n o g l o b u l i n w a s s i g n if i-c a n tl y el e v a te d b y i m m u n o s t i m u l a n t fe e d in g .I n t h e se c o n d e x p e r i m e n t w h e r e i n s ix i m m u n o s t i m u l a n t s w e r e f e d f o r 1



134 A.K. S iwick i e t a l. / V e te rina ry lmm unolo gy and lmmun opa tho logy 41 (1994) 125-139

1 0 0

'~ /30

0fl 6o

-~ 40

E 2o

0

~ 1 1 . . . . . . . . • . . . . . . . . • . . . . . . . . . Q

/

/ / / o . . . . c ~ . . . . ~ > . . . . . c >

, ,~ / , / / - - - o - - - C o n t r o l- - ~ - - E v e t s e l/ /

g / / - - + - - F i n n S t i m, ' / ~ Y ~ - 0 C a n d i d a

, / / - - ~ - - S a e c h a r o .

- - - ~ - - - C h i t o s a n

I I i i I

3 5 7 9 I I 13

D a y s a f t e r c h a l l e n g e

15

Fig. 1. Percentage cum ulative m ortality after cha llenge w ithA. salmonicidain grou ps of rain-bow trout fed different immunostimulants: Control, regular diet (O); Evetsel® ([]) ; Finn-Stim® ( L ;C. utilis ( ~ ) ; S. cerevisiae ( •); M acrogard® (O ); Chitosan ® ( * ). G roups o f fishgiven the E vetsel® and FinnStim ® had the same m ortality pattern.

w e e k , h e m a t o c r i t l e ve ls w e r e u n a f f e c t e d e x c e p t fo r f is h f e dS . c e r ev i s i a ea n dF i n n S t i m ® (Ta b l e 3 ) . T h e n u m b e r s o f le u k o c y t e s s e e m e d to b e u n a f f e c t e d b ya ll d ie t a r y t re a t m e n t s c o m p a r e d w i t h t h e c o n t ro l . H o w e v e r , f is h fe d th e

F i n n S t i m ® a d d i t iv e s h o w e d e l e v a te d l e u k o c y t e n u m b e r s . P r o d u c t i o n o f o x y-g e n ra d i ca l s b y n e u t r o p h i l s a s m e a s u r e d b y t h e N B T t es t w a s s i g n if ic a n tl yh i g h e r fo r all i m m u n o s t i m u l a n t - f e d f is h ; f is h fe dS . c e r ev i s i a ea n d C h i t o s a n ®s h o w e d h i g h e r o x y g e n r a d i ca l p r o d u c t i o n t h a n t h o s e f e d M a c r o g a r d ®,C. uti l is ,E v e t s e l ® , o r C h i t o s a n ® M y e l o p e r o x i d a s e le v el s a n d p h a g o c y t i c i n d e x e s w e r ea l s o s i g n i f i c a n t l y e l e v a t e d f o r t h e s t i m u l a t e d f is h ; f is h f e dS. ce rev i s iaea n dC h i t o s a n ® a g a i n s h o w e d t h e g r e a t e st i n c re a s e s . N e u t r o p h i l k i ll in g a c t i v it y w a ss i g n if i ca n t l y a f f e c t e d b y a ll d i e t a r y t r e a t m e n t s e x c e p t f o r E v e t s e l ® a n dF i n n S t i m ®. F i s h f e d M a c r o g a r d ®,C. u t i l i s , S . ce rev i s iaea n d C h i t o s a n ®

s h o w e d t h e h i g h e s t l ev e ls o f k i ll in g a c ti v it y. To t a l p r o t e i n a n d i m m u n o g l o b -u l i n l e v el s w e r e s i g n i fi c a n t ly e l e v a t e d o v e r t h e c o n t r o l s f o r a l l t r e a t m e n t s , e x -c e p t f o r t o t a l p r o t e i n i n t h e f is h f e dC. ut i l i s .

T h e v u l n e r a b i l it y o f ra i n b o w t r o u t t o c h a ll en g e w i t h th e v i ru l e n t b a c t e r i u mA . s a l m o n i c i d a ,a n d t h e r e s u l ta n t p r o t e c t i o n i n d u c e d b y e a c h o f t h e se p o t e n -t ia l i m m u n o s t i m u l a n t s a r e sh o w n in F ig . 1. C u m u l a t i v e m o r t a l i t y w a s lo w e s ti n f i sh f e d th e d i e t a r y t r e a t m e n t s c o n t a i n i n g M a c r o g a r d ®,S . c e r ev i s i a ea n dC h i to s a n ® ; t h e l e a st p r o t e c t io n w a s p r o v i d e d b y t h e d i e ts s u p p l e m e n t e d w i thF i n n S t i m ® a n d E v e t s e l ®.

i s c u s s i o n

D i e t a r y i n t a k e o f i m m u n o s t i m u l a n t s b y f is h fo r th e p r e v e n t i o n o f d i se a se sh a s d e f i n i t e a d v a n t a g e s i n l a rg e - s c a le f i sh c u l t u r e . I n f is h f a r m s a n d h a t c h e r i e s



A.K. S iwick i e t a l. / Ve te rinary Im mun ology and lmm unopatho logy 41 (1994) 125-139 13 5

c o n t a i n i n g m i l li o n s o f y o u n g f is h, i n d i v i d u a l i n j e c t io n s a r e v e r y e x p e n si v e ;i m m e r s i o n , b a t h i n g a n d s h o w e r te c h n i q u e s a r e a ls o c o s tl y a n d i n v o l v e h a n -d l in g , c a u s i n g Stress. I n c o r p o r a t i n g a n i m m u n o s t i m u l a n t i n t h e f e e d m a y b e

t h e b e s t w a y o f p r e s e n t i n g it to f i sh . T h i s m e t h o d c o u l d a ls o i n t r o d u c e p r o b -l em s , h o w e v e r , s u ch a s h o w t o e n s u r e p r e s e rv a t i o n o r p r e v e n t d e n a t u r a t i o no f t h e i m m u n o c h e m i c a l s tr u c tu r e o f th e i m m u n o s t im u l a n t s f r o m p ro t ea s esa n d o t h e r a n t i n u t r i t i o n a l f a c to r s , n a t u r a l l y o c c u r r i n g in m a n y f i sh f e e d i n g re -d i en t s. Q u e s t io n s m a y a r is e c o n c e r n i n g h o w l o n g f e ed c a n b e s to r e d w i t h o u tt h e i m m u n o s t i m u l a n t s l o s i n g e f fi c a cy a n d p o t e n c y ( i. e. s h el f- li fe ) , a s w e l l a sh o w t o m i x t h e s u b s t a n c es i n t h e f e e d fo r c o n s i s te n c y o f d o s e . T h e s e p r o b l e m sw i l l b e o v e r c o m e , f o l lo w i n g e x a m p l e s s et b y t h e d e v e l o p m e n t s i n f is h n u t ri -t i o n a n d f e e d p ro c e s s i n g t e c h n o l o g y d u r i n g t h e l a st f o u r d e c a d e s , w h i c h h a v e

a l l o w e d fi sh c u l t u r is t s to r e ly c o m p l e t e l y o n c o m m e r c i a l l y a v a i l a b l e f is h f e e d( R u m s e y, 1 9 9 3 ). I n d e e d , s o m e e x p e r ie n c e h a s a l re a d y b e e n g a i n e d in m i -c r o n u t r i e n t a d d i t i v e s f o r d is e a s e p r e v e n t i o n ( B l a z er , 1 99 2 ) .

To t h e b e s t o f o u r k n o w l e d g e , t h is i s t h e f ir st t im e f i sh g i v e n im m u n o s t i -m u l a n t s i n f e e d h a v e b e e n t e s t e d f o r c h a n g e s i n n o n - s p e c if i c d e f e n s e m e c h a -n i s m s a n d t h e c h a n g e s in t h e s e m e c h a n i s m s c o r r e la t e d w i t h p r o t e c ti o n a g a i n s td is ea s e. We h o p e t h e s e r e su l ts p r o v i d e i n s ig h t as t o h o w i m m u n o s t i m u l a n t sc a n b e u s e d i n f is h fe e d s f o r t h e p r e v e n t i o n o f d is e a se s . I n m a n y f is h f a r m i n gope ra t i ons , ou tb r ea k s o f d i se a s e s a r e qu i t e p r ed i c t ab l e . Fo r i n s t ance , a s wa t e r sw a r m u p i n t h e s p r in g , d i se a s e s su c h a s f u r u n c u l o s i s m a y o c c u r. I f a c u l t u r i stc o u l d f e ed a n i m m u n o s t i m u l a t o r y d i e t in a n t i c ip a t i o n f o r t h e p r e d i c te d t i m eo f t h e e p i z o o t ic , h ig h n u m b e r s o f m o r t a li ti e s m i g h t b e a v o i d e d .

F r u s t r a t i n g i n c o n s i s te n c i e s m a y o c c u r in t h e d a t a o f n o n - sp e c i f ic d e fe n s em e c h a n i s m s w h e n s e v e r a l d i f f e r e n t p a r a m e t e r s a r e m e a s u r e d , e x a m p l e s o fw h i c h c a n b e s e e n i n Ta b l e s 2 a n d 3 . P a r t o f t h e d i ff i cu l t y w h e n m e a s u r i n gt h e n o n - s p e c i fi c d e f e n s e m e c h a n i s m s o f fi sh i s t h e n o r m a l b i o l o g ic a l v a ri a b i l-i t y ( O l iv i e r e t a l ., 1985 ; M i c h e l e t a l ., 1990 ) . Th i s p r e d i c am en t is e spec i a l lyf ru s t ra t in g b e c a u s e th e i m m u n i z a t i o n a n d a ss ay s m e a s u r i n g sp e ci fi c i m m u n er e sp o n s e ( h u m o r a l a n t i b o d y o r p l a q u e - f o r m i n g c el ls ) a r e m u c h m o r e s ta tis -t ic a l ly p r e d i c ta b l e . T h i s v a r i a b i l i ty i n t h e n o n - s p e c i fi c d e fe n s e m e c h a n i s m r e-s p o n s e s b eg s t h e q u e s t i o n o f w h e t h e r a s i n gl e sp e c if ic a ss a y c a n p r o v i d e a na c c u r a t e i n d e x o f i m m u n e c o m p e t e n c e . Ir re s p e c ti v e o f t h e a n s w e r to t h a tq u e s t io n , o u r d a t a s h o w t h a t w h e n t h e m u l t ip l e n o n - sp e c if ic d ef e n se m e c h a -n i s m a s sa y s a r e u s e d , c r o s s -c o r r e la t i o n s c a n b e m a d e t h a t a l lo w s u b s t a n ti a -t i o n o f d i f fe r e n c e s b e t w e e n e x p e r i m e n t a l t r e a t m e n t s .

T h e h e m a t o c r i t l e v el s o b t a i n e d i n t h e s e e x p e r i m e n t s y i e l d e d li tt le i n f o r-m a t i o n a b o u t a d i e t a r y t r e a t m e n t - i m m u n o s t i m u l a t o r y e ffe ct; n e v e rt h el es s ,h e m a t o c r i t l e v el s a r e r o u t i n e l y u s e d i n f is h h e a l t h s u r v e y s, a n d d i f fe r e n c e s

s e e m t o a p p e a r w h e n fi sh a r e h ig h l y s t re s s ed ( A n d e r s o n , 1 9 9 0 ). T h e n u m b e r so f c i r c u l a t o r y l e u k o c y t e s i n c r e a s e d s i g n i f ic a n t ly i n t h e f ir st e x p e r i m e n t i n a l lg r o u p s g iv e n th e i m m u n o s t i m u l a t o r y d i et s; h o w e v e r , r e su l ts f ro m t h e s e c o n d



136 A.K. Siwicki et a l . / Veterinary lmm uno logy and lmm uno path olog y 41 (1994) 125- 139

e x p e r i m e n t d i d n o t c o n f i r m t h o s e c h a n g e s. C o u n t i n g le u k o c y t e s b y h e m o c y -t o m e t e r i s r e la t iv e l y e a s y b u t v e r y t e d i o u s , a n d w e s u s pe c t t h a t m o r e o b s e r-v a t io n s p e r s a m p l e w o u l d m i n i m i z e v a r ia b i li ty.

T h e a ss ay s u s in g N B T r e d u c t i o n t o d e t e c t o x i d a t i v e ra d i c a l p r o d u c t i o n w e r er e li a b le i n b o t h e x p e r i m e n t s a n d s h o w e d o x i d a t iv e r a d i c a l p r o d u c t i o n t o b ec o n s i st e n tl y h ig h e r i n th e i m m u n o s t i m u l a t e d f is h. Tw o m e t h o d s t o a ss ay t h eox ida t i ve r ad i c a l r e l e a s e w e re u sed . Th e f i r st r e l ied on a dhe renc e to g l a ss byt h e N B T - p o s i t i v e c e ll s, a n d t h e s e c o n d u s e d p l a s t ic m i c r o t i t e r p l a te s f o r a d -h e r e n c e. I n th e s e c o n d m e t h o d , w e m e a s u r e d N B T r e d u c t i o n b y s p e c tr o p h o -t o m e t e r a f te r re s o lu b i l iz i n g t h e f o r m a z a n w i th N , N - d i m e t h y l f o r m a m i d e . T h er e su l t s f r o m t h e t w o v a r i a t i o n s o f t h e N B T a s sa y s w e r e s im i la r.

T h e p h a g o c y t i c in d e x a l s o s e e m e d t o b e a r e li a b l e in d i c a t o r o f th e h e i g h t-

e n e d i m m u n o s t i m u l a t o r y r e s po n s e to t h e fe e d s u p p l e m e n t s . T h e e n g u l fm e n to f t h eS t a p h y lo c o c c u s a u r e u sb a c t e ri n w a s o b s e rv e d b y m i c ro s c o p e f ro m b l o o ds m e a r s , b u t s o m e e x p e r i e n c e i s r e q u i r e d i n t h e o b s e r v a t i o n a n d t h e d e t e c t i o no f eng u l f ed b a c t e r i a l p a r t i c le s .

T h e r es u lt s f r o m t h e m y e l o p e r o x i d a s e s t a in i n g p r o c e d u r e c o r r el a t ed w i tht h e N B T a n d p h a g o c y t o s i s a s sa y s. T h i s te c h n i q u e a l s o r e q u i r e d m i c r o s c o p i co b s e r v a t i o n o f b l o o d s m e a r s , b u t d e t e c t i n g t h e p o s i ti v e - st a i n in g c e ll s w a s e a s-i e r t h a n m a k i n g t h e r e a d i n g s f o r t h e p h a g o c y t i c in d e x a s s ay.

To t a l p r o t e in i n t h e p la s m a w a s n o t r a i s e d in th e i m m u n o s t i m u l a t e d f is h i nthe f i rs t expe r ime n t ; h o w e ve r, i n the s ec ond exp e r im en t , a l l f ish show ed h ighe rl e ve l s w i t h t h e e x c e p t i o n o f th e g r o u p f e dC. util is.

To t a l i m m u n o g l o b u l i n w a s s ig n if ic a n tl y e l e v a te d a n d m a y b e a g o o d i n d i-c a t o r f o r t h e a c t i o n o f th e i m m u n o s t i m u l a n t s . T h e s e t es ts w e r e in n o v a t i v e fo rt h e f i sh u s e d i n o u r s t u d i es , b e c a u s e w e l o w e r e d t h e p o l y p r o p y l e n e g ly c o l c o n -c e n t ra t io n f o r m a x i m u m p r e c ip i ta t io n o f r a in b o w t ro u t i m m u n o g l o b u l i n s( S iw i c k i a n d A n d e r s o n , 1 9 9 4 ). M e a s u r i n g t o ta l p r o t e i n o r i m m u n o g l o b u l i nb y t h i s m e t h o d , w h i c h u s e s m i c r o t i t e r p l a t e s f o r a n a l y s i s b y a m i c r o r e a d e rs p e c t r o p h o t o m e t e r , is r a p i d a n d r e l ia b l e .

I n t h e s e c o n d e x p e r i m e n t t h e g r o u p s f e d C h i t o s a n ®,C. ut i l is and t he S .cerevis iaeh a d s i m i l a r p r o t e c t i o n l e ve l s w h e n c h a l l e n g e d w i t hA . s a l m o n i c i d a .T h e g r o u p s f e d E v e t s el ® a n d F i n n s t i m ® h a d f e w e r s u r v iv o r s t h a n t h e g r o u pf e d M a c r o g a r d ® . W e w e r e l i m i t e d b y t h e n u m b e r o f f is h a v a il a b l e f o r t h e e x -p e r i m e n t s ; l a rg e r s a m p l e s iz e s a re n e e d e d t o d e t e r m i n e e x a c t l ev e ls o f e f fi c a cya n d p o t e n c y o f t h e i m m u n o s t i m u l a n t s . C h a l l e n ge b y i n j e c ti o n o fA .s a l m o n i -c ida i n r a i n b o w t r o u t is a l w a y s d i f fi c u l t b e c a u s e o f t h e p r o b l e m s i n f i x in gdose s , such a s e s t ab l i sh ing L Ds0 va lue s (M iche l et a l. , 1990 ) . N eve r th e l e s s ,p r o t e c t i o n t e s ts o n t h e g r o u p s s h o w e d m o r t a l i t y p a t t e r n s t h a t c o r r e la t e d w i t ht h e r e s u lt s f r o m t h e n o n s p e c i f i c d e f e n s e m e c h a n i s m s a ss ay s .

M u c h r e m a i n s to b e a c c o m p l i sh e d in i m p r o v i n g t h e t e c h n iq u e s a n d r el ia -b i l it y o f t h e a s sa y s f o r d e t e c t i n g a c t i v a t i o n o f n o n - s p e c i f ic d e f e n s e m e c h a -n i s m s . H o w e v e r, w e c o u l d c o n s i st e n t ly d e t e c t d i f fe r e n c e s u s i n g t h e n e u t r o p h i l



A.K. S iwick i e t a l. / Ve te r ina ry Imm unolo gy and lmmu nopa th o logy 41 (1994) 125-139 13 7

a n d p h a g o c y t i c i n d e x a s sa y s b e t w e e n t h e c o n t r o l f i sh a n d t h e f is h fe d t h e d i e t sw i th i m m u n o s t i m u l a n t s . M o r e o v e r , t h e d a t a f r o m t h e f is h f ed th e tw o y e a s tp r e p a r a t i o n s(S. cerevisiaea n d C. utilis) a n d t h e C h i t o s a n ® s h o w e d t h a t t h es e

w e r e a s g o o d a s o r b e t t e r t h a n t h e M a c r o g a r d ® i n i n d u c i n gi m m u n o s t i m u l a t i o n .In t he f i r s t e x p e r im e n t , t h e h i ghe s t c e l lu l a r a c t iv i t i e s we re i n t he f i sh fed C .

uti l is a n d S. cerevisiae.I n t h e s e c o n d e x p e r i m e n t , t h e s t i m u l a t o r y a c t iv i ty o fS. cerevis iaew a s c o n f i r m e d b y t h e c h a l l e n g e t e st a ls o . T h e y e a s tS. cerevisiaeh a s b e e n s h o w n to h a v e a n t i v i ra l p r o p e r t ie s w h e n f e d t o m i c e ( F a t t a l- G e r -m a n an d B i zz in i , 1992 ) . I t a l so ha s b een u sed succe s s fu ll y i n f ish fe eds a s ap r o t e i n s o u r c e , su b s t i t u t in g f o r e x p e n s i v e f is h m e a l p r o t e i n ( R u m s e y e t a l.,1990, 1991, 1992 ) ; t h u s i t c o u ld s e rve tw o pu rpo se s . E v ide nce is a cc um ula t -

i n g t h a t n u c l e i c a c i d s f ro m y e a s t s o u r c es , p r e v i o u s l y c o n s i d e r e d n u t r i t i o n a l l yn o n - e s s e n t i a l f o r g r o w t h a n d r e p r o d u c t i o n , a r e e s se n t ia l fo r i m m u n e r e a c t i v -i ty in m a m m a l s ( R u d o l p h e t a l., 1 99 0; C e r r a e t a l., 1 9 91 ) . I n d e e d , t h e r e m a yb e a n u t r i t i o n a l r e q u i r e m e n t f o r y e a s t R N A a n d i t s c o n s t i t u e n t p r e f o r m e dp u r i n e s a n d p y r i m i d i n e s f o r o p t i m a l f u n c t i o n i n g o f l y m p h o c y t e s a n d m a c r o -p h a g e s ( K u l k a r n i e t a l ., 1 9 8 7 ) . I n t h e s e c o n d e x p e r i m e n t , C h i t o s a n ® a l sop r o v e d t o b e a g o o d i m m u n o s t i m u l a t o r a n d i n d u c e r o f p r o te c ti o n i n r a in b o wt rou t , w he r e a s Eve t s e l ® w a s i nco ns i s t en t i n t h e ce l l u l a r a ss ays and gave t hel ea s t p ro t ec t i o n t o d i s e a s e cha l lenge .

I n o u r e x p e r i m e n t s , a l l f is h s h o w e d s a t is f a c to r y f e ed i n t a k e , s e e m i n g l y u n -i n f lu e n c e d b y t h e p r e s e n c e o f th e i m m u n o s t i m u l a n t s . F u t u r e s tu d i e s w i ll i n-c l u d e d e t e r m i n i n g o p t i m a l d o s e s a n d p r o t o c o l s f o r f e e d i n g t h e s e s u b s ta n c e st o m a x i m i z e p r o t e c t i o n , g i v e n t h e c o n s t r a i n t s o f f is h c u l t u r e a n d e c o n o m i c s .B a s e d o n t h e re s u lt s o f o u r e x p e ri m e n t s , t h e u s e o f i m m u n o s t i m u l a n t s f o r t h ep r e v e n t i o n o f d i se a s e s in f i sh c u l t u r e s e e m s to h o l d g r e a t p r o m i s e . T h e f a c tt h a t w e c a n u s e s pe c if ic n u t r i t io n a l s u p p l e m e n t a t i o n t o m a n i p u l a t e t h e i m -m u n e r e s p o n s e o v e r a n d a b o v e s i m p l y p r o v i d i n g s u f f i c i e n t v i t a m i n s , p r o -t ei n s, a n d c a l o ri e s i n t h e d i e t o p e n s a w h o l e n e w v i st a fo r i m m u n o l o g y - n u t r i -t i o n c o l l a b o r a t i v e r e s e a r c h .

cknowledgments

We t h a n k t h e U S D e p a r t m e n t o f A g r ic u l tu r e , O f fi ce o f In t e r n a t io n a l C o -o p e r a t io n a n d D e v e l o p m e n t a n d t h e M a r i a S k l o d o w s k a - C u ri e J o i nt F u n d I I( M R / U S D A - 9 1 - 6 6 ) f o r s u p p o r t in g D r. A . K . S i w i ck i 's r e se a r ch a t t h e N a -t io n a l F i s h H e a l t h R e s e a r c h L a b o r a t o r y i n K e a r n e y s v il le , W V, U S A . R o b i nO w e n s ' w o r k o n m a n u s c r i p t p r e p a r a t i o n a n d D a v e L e m a r i6 ' s a s si st a nc e w i tht a b l e s a n d s ta t is ti c s a r e a p p r e c i a t e d . U s e o f t r a d e n a m e s d o e s n o t i m p l y U SG o v e r n m e n t e n d o r s em e n t o f c o m m e r c ia l p ro d u cts .



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