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v e t e r i n a r yp a r a s i t o l o g yE L S E V IE R Veter inary Paras itology 61 (1996) 151-156

S h o r t C o m m u n i c a t i o n

C om p arat ive eva lu a t ion o f agar ge l p rec ip ita tion ,c o u n t e r i m m u n o e l e c tr o p h o r e s i s a n d p a s s i v e

h aem agg lu t in a t ion t e st s for th e d iagn o s i so f Dicroco e l ium dendr it icum in fect ionin s h eep an d goa t s

K . P . J i t h e n d r a n * , J . V a i d *, L . K r i s h n aDisease Investigation Laboratory, Indian Veterinary Research Institute, Regional Station,Palampur, H.P., India

Received 18 O ctober 1994; accepted 9 F ebruary 1995

A b s t r a c t

T h e s e n s i ti v i t y a n d s p e c i f i ci t y o f t h e a g a r g e l p r e c i p i t a ti o n te s t ( A G P T ) , c o u n t e r i m m u n o e l e c -t r o p h o r e s is (C I E P ) a n d p a s s i v e h a e m a g g l u t i n a t io n t e st ( P H T ) w e r e e v a l u a t e d fo r t h e d i a g n o s i s o fDicrocoelium dendriticum in fec t ion in na tura l ly in fec ted sheep and goa t s . Two hundred and for tyf ive sheep and goa t s e ra s ample s we re t e s ted us ing phospha te buf fe red sa l ine , pH 7 .2 ex t rac teda d u l t fl u k e a n t ig e n . C I E P d e t e c t e d 6 9 .8 % o f t h e i n f e c t e d a n im a l s a n d w a s f o u n d t o b e t h e m o s ts e n s i t i v e , f o l l o w e d b y P H T w h i c h d e t e c t e d 5 0 . 0 % o f t h e i n f e c t e d s h e e p a n d g o a t s . A G P T w a sfound to be the l ea s t s ens i t ive , de tec t ing only 23 .8% of the in fec ted an ima ls . The spe c i f i c i ty ofA G P T , C I E P a n d P H T w e r e 9 3 . 3% , 8 4 . 0 % a n d 9 3 . 3 % , r e s p e c t iv e l y . C I E P w a s f o u n d t o b e th em o s t s e n s i ti v e , s p e c i fi c a n d r a p i d t e s t f o r t h e s e r o e p i d e m i o l o g i c a l s u r v e y o f d i c r o c o e l i o s i s i n s h e e pand goa t s .Keywords: Sheep-Trematoda; Go at; Dicrocoelium dendriticum; Diagnosis-Trematoda

1 . I n t r o d u c t i o nD i c r o c o e l i o s i s c a u s e d b y Dicrocoelium dendriticum i s a t r e m a t o d e i n f e c t i o n t h a t

o c c u r s i n s h e e p , g o a t s , c a t tl e a n d b u f f a l o ( S o u l s b y , 1 9 8 2 ) . A n i m a l s s u f f e r i n g f r o m i t

* Corresponding author.t Deceased.0304-4017/96/$15.00 1996 Elsevier Science B.V. All rights reservedSSDI 0 3 0 4 - 4 0 1 7 ( 9 5 ) 0 0 8 0 8 - X

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152 K. P . ,l thendran e t a l. / Ve te r inary Paras ito log3 ' 61 (199 0) 1 51 -1 56

s h o w c l in i ca l s y m p t o m s co n s i s ti n g o f a n a e m i a , o e d e m a , e m a c i a t i o n a n d , i n a d v a n c e dc a s e s , e x te n s iv e c i r rh o s is , s c a r r in g o f th e l iv e r s u r fa c e a n d m a rk e d d i s t e n s io n o f b i l ed u c t s . Th o u g h th e d i s e a s e i s l e s s s e v e re th a n fa s c io l io s i s , e c o n o mic lo s s e s a s a r e s u l t o fl i v e r c o n d e m n a t i o n a r e h i g h i n th e N o r t h T e m p e r a t e H i m a l a y a n R e g i o n o f th e I n d i a ns u b c o n t in e n t . Th e fa i lu re to d e te c t p re p a te n t in fe c t io n s b y fa e c a l e x a min a t io n i s o n e o fth e c o n t r ib u t in g fa c to r s wh ic h h in d e rs th e c h e mo th e ra p e u t i c c o n t ro l o f th i s d i s e a s ea mo n g l iv e s to c k . Co rb a e t a l . (1 9 8 7 ) a n d S a n z e t a l . (1 9 8 7 ) h a v e re p o r te d th a t e v e n a tl o w d o s e s, l u x a b e n d a z o le ( 1 0 m g k g 1 b o d y w e i g h t ) a n d n e t o b i m i n ( 1 5 - 2 0 m g k g ]b o d y w e i g h t ) r e s p e c ti v e l y , a r e h i g h ly e f f i c a c i o u s ( 9 8 - 9 9 % ) a g a i n st t h is p a r a si te . T h ed ia g n o s t i c v a lu e o f s e ro lo g ic a l t e s t s fo r d e te c t io n o f d ic ro c o e l io s i s in s h e e p a n d g o a t sh a s n o t b e e n c r i t i c a l ly a d d re s s e d . Th e p re s e n t s tu d y wa s th e re fo re c o n d u c te d to e v a lu a teth e s u i ta b i l i ty o f s o m e s e ro lo g ic a l t e s t s fo r th e d ia g n o s i s o f d ic ro c o e l io s i s in mig ra to rys h e e p a n d g o a t s a n d th e p o s s ib i l i ty o f i t s c o n t ro l in th e s e f lo c k s u s in g s u c h d ru g s .

2 . M a t e r ia l s a n d m e t h o d s

2.1. Ant igenA d u l t f l u k e s o f D i c r o c o e l i u m d e n d r i t i c u m we re c o l l e c te d f ro m th e b i l e d u c t s o f

n a tu ra l ly in fe c te d s h e e p a n d g o a t s . Af te r r e mo v a l f ro m th e b i l e d u c t s , t h e f lu k e s we rewa s h e d in se v e ra l c h a n g e s o f c h il l e d p h o s p h a te b u f fe re d s a l in e (P BS , p H 7 .2) , h o -m o g e n i s e d i n a g l a s s h o m o g e n i s e r a n d k e p t a t 4 C . T h e h o m o g e n a t e w a s c e n t r i f u g e d a t1 0 0 0 0 g fo r 3 0 m in a t 4 C . T h e s u p e rn a ta n t f lu id wa s c o l l e c te d a n d u s e d a s a d u l tf lu k e a n t ig e n (ALVA). Th e p ro te in c o n c e n t ra t io n o f th e a n t ig e n w a s d e te rm in e d b y th em e t h o d o f L o w r y e t a l . ( 1 9 5 l ) .2.2. Sera

B l o o d s a m p l e s w e r e c o l l e c t e d f r o m 2 - 6 y e a r o ld s h e e p a n d g o a t s b e f o r e s l a u gh t e r a tth e lo c a l a b a t to i r a n d th e i r l iv e r s we re s u b s e q u e n t ly e x a min e d fo r D . d e n d r i t i c u mi n f e c t i o n . S e r a s a m p l e s f r o m 7 0 s h e e p a n d 5 6 g o a t s , w h i c h w e r e c o n f i r m e d t o b ep o s i t iv e fo r th i s in fe c t io n , we re s e le c te d fo r th e s tu d y . S e ra s a mp le s f ro m 4 7 s h e e p a n d5 4 g o a t s w h i c h w e r e f r e e f r o m D . d e n d r i t i c u m i n fe c t io n we re u s e d a s c o n t ro l s e ra .Ad d i t io n a l ly , 1 8 F a s c i o l a g i g a n t i c a i n fe c te d re fe re n c e s e ra f ro m s h e e p a n d g o a t s we ree mp lo y e d fo r t e s t in g a p p a re n t c ro s s r e a c t iv i ty . S e ra s a mp le s , c o n ta in in g 0 .2 % s o d iu ma z id e (1 : 1 0 0 ) we re s to re d a t -2 0 C u n t i l fu r th e r u s e . P r io r to th e i r u s e in s e ro lo g ic a lt e s t s th e s e s e ra s a mp le s we re in a c t iv a te d a t 5 6 C fo r 3 0 min .2 . 3 . l m m u n o d i a g n o s t i c t e s t s

T h e a g a r g e l p r e c i p i t a t i o n t e s t ( A G P T ) w a s p e r f o r m e d a s d e s c r i b e d b y E d w a r d s(1 9 7 9 ) . On e p e r c e n t No b le a g a r (D i fc o La b o ra to r i e s , De t r io t , MI , US A) in P BS , p H 7 .2w a s u s e d i n t he p r e p a r a t i o n o f g el . T h e d i lu t ed a n t ig e n ( p r o t e in c o n t e n t 4 - 6 m g m l - ] )wa s u s e d a g a in s t th e u n d i lu te d s e ra a n d d i f fu s io n wa s a l lo we d to t a k e p la c e in a mo is t

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K.P. J i thendran e t a l. / Ve te r inary Paras i to logy 61 (1996) 15 1-1 56 153

c h a m b e r a t 3 7 C f o r 4 8 h . T h e p r e c i p i t at i o n l i ne s w e r e e x a m i n e d a f t e r w a s h i n g i nn o r m a l s a l i n e a n d t h e s l i d e s w e r e s t a i n e d w i t h a m i d o b l a c k .

C o u n t e r i m m u n o e l e c t r o p h o r e s i s ( C I E P ) w a s c a r r i e d o u t a c c o r d i n g t o t h e m e t h o d o fE d w a r d s ( 1 9 7 9 ) u s i n g 1 % a g a r o s e ( L o b a C h e m i e , V i e n n a ) i n 0 . 0 2 M b a r b i t a l b u f f e r , p H8 .6 . T h e e l e c t r o p h o r e s i s w a s c o n d u c t e d a t r o o m t e m p e r a t u r e a t 1 0 m A p e r s l id e f o r 9 0m i n . K n o w n p o s i t i v e a n d n e g a t i v e c o n t r o l s e r a w e r e a l s o r u n w i t h e a c h t e s t . T h e s l i d e sw e r e w a s h e d a n d s t a i n e d a s f o r A G P T .

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 s ( P H T ) w e r e c a r r i e d o u t a c c o r d i n g t o t h e m e t h o dd e s c r i b e d b y S u n d i c k a n d R o s e ( 1 9 8 0 ) . T h e t e s t s w e r e s t a n d a r d i s e d u s i n g t h e a n t i g e nc o n t a i n i n g 1 m g m l - ~ p r o t e i n a n d d i lu t e d t en t i m e s i n P B S . I n b r ie f , s h e e p r e d b l o o dc e l l s ( S R B C ) w e r e c o l l e c t e d a s e p t i c a l l y i n A l s e v e r ' s s o l u t i o n ( 1 : 1 , v / v ) a n d w a s h e dt h r e e ti m e s w i t h 0 . 15 M P B S , p H 7 . 2 b y s e d i m e n t a t i o n ( 8 0 0 X g ) . T h e w a s h e d S R B Cw e r e i n c u b a t e d f o r 1 5 m i n a t 3 7 C w i t h a n e q u a l v o l u m e o f P B S c o n t a i n i n g 0 . 0 0 5 %t a n n ic a c i d . T h e t a n n e d c e l l s w e r e c o n c e n t r a t e d b y c e n t r i f u g a ti o n a t 8 00 g f o r 2 0 m i n ,w a s h e d t h r e e t i m e s a n d d i v i d e d i n t o t w o v o l u m e s . Dicrocoelium dendriticum AFAd i lu t e d i n P B S ( 1 m g m l - ~ p r o t e i n c o n c . ) w a s a d d e d t o o n e a l iq u o t o f t h e c e l l s an d t h em i x t u r e w a s i n c u b a t e d f o r 3 0 m i n a t 3 7 C . T h e s e n s i t i s e d c e l l s w e r e w a s h e d t h r e e t i m e sa n d r e s u s p e n d e d t o 1 % s u s p e n s i o n i n P B S c o n t a i n i n g 1 % i n a c t i v a t e d n o r m a l r a b b i ts e r u m . T h e o t h e r a l i q u o ts o f c e l ls s e r v e d a s u n s e n s i ti s e d c o n t r o l s . T w o - f o l d d i l u t i o n s o ft e st s e r u m i n 0 .2 5 m l P B S w e r e m a d e i n a p e r s p e x p la t e a n d 0 .2 5 m l o f se n s i ti s e d S R B Cw e re a d d e d to e a c h d i lu t io n . In a d d i t io n , e a c h t e s t s a m p le wa s t e s t e d a g a in s t u n s e n s i t i s e dc e l l s fo r h e te ro p h i l i c a n t ib o d ie s . To c o mp a re th e t e s t r e s u l t s , 0 .2 5 ml o f s e n s i t i s e d S RBCw e r e a l s o a d d e d t o 0 . 2 5 m l P B S . T h e p l a t e s w e r e i n c u b a t e d a t 3 7 C a n d r e a d a f t e r 2 h .T h e a g g l u t i n a t i o n p a t t e r n o f s e n s i t i z e d S R B C i n t e s t d i l u t i o n s w a s c o m p a r e d w i t ha p p r o p r i a t e c o n t r o l s a n d t h e a b s e n c e o f a b u t t o n f o r m a t i o n w a s t a k e n a s p o s i t iv e e n dpoin t .

Th e s e n s i t iv i ty a n d s p e c i f i c i ty o f th e s e t e s t s we re c a lc u la te d a s fo l lo wsT r u e P o s i t i v e s - F al se N e g a ti v esS e n s i t iv i t y ( % ) = X 1 0 0T r u e P o s i t i v e sT r u e N e g a t i v e s - F al se P o si ti v esS p e c i f i c i ty ( % ) = 1 0 0T r u e N e g a t i v e s

3 . R e s u l t s

Th e re s u l t s o f th e s e ro lo g ic a l t e s t s fo r th e d ia g n o s i s o f D. dendriticum i n fe c t io n ins h e e p a n d g o a t s b y A G P T , C I E a n d P H T a r e p r e s e n t e d i n T a b l e s 1 a n d 2 , r e s p e c t i v e l y .

I n A G P T , s h a r p p r e c i p i t a t i o n l i n e s w e r e e v i d e n t i n 1 6 o u t o f 7 0 k n o w n p o s i t i v e s e r as a m p l e s f r o m s h e e p a n d 1 4 o u t o f 5 6 s u c h s a m p l e s f r o m g o a t s . T h e s e n s i t iv i t y o f A G P Tw a s f o u n d t o b e l o w e r t h a n t h a t o f t h e o t h e r t e st s a s it g a v e 2 2 . 9 % f o r s h e e p a n d 2 5 . 0 %fo r g o a t s .U s i n g t h e r e s o l v i n g p o w e r o f C I E P , i t w a s p o s s i b l e to d e t e c t 7 1 .4 % i n f e c ti o n i n s h e e pa n d 6 7 . 9 % i n g o a t s w i t h a r e a s o n a b l y h i g h d e g r e e o f s p e c i f ic i t y o f 8 5 . 5 % a n d 8 2 . 8 %

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154 K . P . J i t h e n d r a n e t al . / V e t e r i n a r y P a r a s i t o l o g y 6 1 ( 1 9 9 6 ) 1 5 1 - 1 5 6Table 1Comparative sensitivity and specificity of different immunodiagnostic tests using D . d e n d r i t i c u m adult flukeantigen in sheepSerum samp les Total sera tested No. positive (%)

A G P T CI EP P H TD . d e n d r i t i c u m infected 70 16 50 35

(22.9) (71.41 (50.0)Non -infected 47 2 4 3

(4.3) (8.5) (6.4)F. g igan t ica infected 8 2 4 4

(25.0) (50.111 (50.0)Total non-specific reaction 55 4 8 7

(7.3) (14.5) (12.71Specificity (%) 92.7 85.5 87.3

r e s p e c t i v e l y . T h e t e st , h o w e v e r , g a v e 8 . 5 % a n d 1 3 . 0 % f a l s e r e a c t i o n in n o n i n f e c t e ds h e e p a n d g o a t s e r a , r e s p e c t i v e l y .

I n P H T , t h e c o n t r o l ( u n i n f e c t e d ) s e r a s a m p l e s g a v e t i t re s r a n g i n g f r o m 1 : 2 t o 1 : 3 2,w h i l e i t r a n g e d f r o m 1 : 2 t o 1 : 1 0 2 4 i n i n f e c te d s h e e p a n d g o a t s. S i n c e 9 6 . 1 % o f t h eu n i n f e c t e d s e ra s h o w e d t it re s r a n g i n g f r o m 1 :2 to 1 : 1 6 , t it re s o f o v e r 1 : 3 2 w e r ec o n s i d e r e d a s s i g n i f i c a n t t e s t t i t r e f o r P H T i n t h e p r e s e n t s t u d y .

Table 2Comparative sensitivity and specificity of different immunodiagnostic tests using D . d e n d r i t i c u m adult flukeantigen in goatsSerum samp les Total sera tested No. positive (%)

A G P T CIE P P H TD . d e n d r i t i c u m infected 56 14 38 28

(25.0) (67.9) (50.0)Non -infected 54 0 7 1

(0.0) (13.111 (1.9)F. g igan t ica infe cted 111 4 4 0

(40.0) (40.0) (0.0)Total non-specific reaction 64 4 I 1 1

(6.3) (17.2) (1.6)Specificity ( % ) 93.8 82.8 98.4

Table 3Overall sensitivity and specificity of different immunodiagnostic tests using D . d e n d r i t i c u m adult flukeantigen in sheep and goatsImm unodiagn ostic tests Sensitivity (%) Specificity (%)AG PT 23.8 93.3CIEP 69.8 84.0PHT 50.0 93.3

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K.P. J i thendra n e t a l. / Ve ter inary Para si to logy 61 (1996) 15 1-1 56 1 5 5

The comp ara t ive sens i t iv i ty and spec i f ic i ty o f d i f f e r en t imm unod iagn os t ic t es ts u sedin the p resen t s tudy a re p resen ted in Tab le 3 . The overa l l s ens i t iv ity o f AG PT, CIEP andPH T w ere 23 .8%, 69 .8% and 50 .0%, r espec t ive ly . How ever , the spec i f ic ity o f the tes t swas var iab le w i th 93 .3%, 84 .0%, and 93 .3% in AGPT, CIEP and PHT, r espec t ive ly .

The c ross r eac t iv ity o f these tes t s w i th F. gigantica in f ec ted sheep and goa t r e f e rences e ra i nd i c a te d 2 5 - 4 0 % , 4 0 - 5 0 % a n d 0 - 5 0 % p o s it i ve r e a c ti o n i n A G P T , CI E P a n d P H T ,respec t ive ly .

4. D iscuss ionImm unod ia gnos t ic t es ts have an impor tan t ro le to p lay in the se roep idem io logy o f

he lmin thos i s and se rve as a va luab le ad junc t to ex is t ing d iagnos t ic methods . Thespec i f ic i ty and sens i t iv i ty o f the th ree se rod iagnos t ic tes t s were eva lua ted in the p resen ts tudy . The A GP T show ed the low es t sens i tiv i ty o f the th ree test s as i t gave a r a te o f22 .8% fo r sheep se ra and 25 .0% fo r goa t se ra . The low sens i t iv i ty o f th i s t es t wasp rev ious ly r epo r ted by Pa thak and Gaur (1986) in po rc ine cys t ice rcos i s and by Swampet a l . (1987) in bovine fascio l ios is . In contras t , CIEP has been repor ted to be h ighlysens i t ive in the se rod iagnos is o f var ious paras i t i c in fec t ions inc lud ing F. gigantica insheep (Pa thak et a l ., 1986) a nd buffalo es (Sw arup et a l . , 1987), and cyst icerco sis in p igs(Ku m ar and Gaur , 1989) . PH T sho wed a h igher sens i t iv i ty than AG PT bu t was les sspec i f ic than CIEP . How ever , the spec i f ic i ty was found to be r easonab ly h igh in PH Tfo l lowe d by A GP T and leas t in CIEP . In the p resen t s tudy , 1 :32 was cons idered themin imum d iagnos t ic t i t r e fo r PHT. Recen t ly , Somvansh i e t a l . ( 1993 ) a l so r epo r ted theu s e o f P H T a n d CI E P f o r t h e d i a g n o s i s o f D. dendriticum in goa ts and ind ica ted tha tCIEP was more sens i t ive than PHT, wh ich i s in ag reemen t w i th the p resen t observa-t ions. The backg rou nd t i t r e a t the low er d i lu t ions o f the con t ro l se r a p robab ly r e f lec t s theapparen t l ack o f spec i f ic i ty a n d / o r i s due to over t c ro ss r eac t iv ity w i th o ther parasi tes .In the p resen t s tudy , CIEP w as fou nd to be m ore sens i t ive , qu i te spec i f ic and a r ap id tes tfo r the d iagnos is o f d ic rocoe l io s i s in sheep and goa ts . However , pu r i f ica t ion o f f lukean t igens ma y qua l i ta t ive ly improve the d iagnos t ic va lue o f the tes ts , wh ich needs fu r there luc ida t ion .

A c k n o w l e d g e m e n tThe au tho r s a r e thank fu l to the D i rec to r , I nd ian Vete r inary Research In s t i tu te ,

Izatnagar , (U.P.) for provid ing faci l i t ies .

R e f e r e n c e sC o r b a , J. , H o v o r k a , J ., S p a l d o n o v a , R . , S t o f f a , P ., L e g e n y , J . a n d A n d r a s k o , H . , 1 9 8 7 . E f f i c a c y o f

l u x a b e n d a z o l e ( H o e 2 1 6 V ) s u s p . 5 % i n s h e e p n a t u r a l l y i n f e c t e d w i t h t h e m o s t i m p o r t a n t h e l m i n t h s .H e l m i n t h o lo g i a , 2 4 : 2 2 7 - 2 3 5 .

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