COMPARISON OF COPROANTIGEN DETECTION WITH NECROPSY I N STRAY DOGS A N D RED FOXES FROM NORTHERN J O R D A N

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DIAGNOSIS OF CANINE ECHINOCOCCOSIS:

COMPARISON OF COPROANTIGEN DETECTION WITH NECROPSY I N STRAY DOGS A N D RED FOXES FROM NORTHERN J O R D A N

EL-SHEHABI F.S.*, KAMHAWI S.A.*, SCHANTZ P.M.**, CRAIG P.S.*** & ABDEL-HAFEZ S.K.*

Summary:

The sandwich enzyme linked immunosorbent assay (ELISA) was used as a diagnostic test for Echinococcus granulosus infection by detecting coproantigens in 94 stray dogs Corn's familiaris and eight red foxes (Vu/pes vulpes) from northern Jordan. The results were analyzed in relation to actual helminth infection as revealed by necropsy. The infection rate of dogs with E. granulosus was 1 3.8 % with a worm load ranging between 3 - > 10,000 per infected dog. In contrast, eight of 1 3 E. granulosus infected dogs were coproantigen positive (overall sensitivity 61.5 %). The sensitivity increased to 87.5 % and 100 % in dogs harboring

> 20 and > 100 worms/dog, respectively. The specificity of coproantigen-ELISA was 91 %. The greatest cross-reactivity was found in dogs infected with Dipylidium caninum. The positive and negative predictive values for the coproantigen-ELISA test were 50 % and 94.2 %, respectively. Thus, a coproantigen negative dog is most probably truly negative for E. granulosus. In contrast, a coproantigen positive dog may not be truly positive for

E. granulosus, except if it has a high worm burden of > 1 00 worms/animal.

KEY WORDS : Echinococcus granulosus, canine echinococcosis, coproantigens, Jordan.

Résumé :

DIAGNOSTIC DE L'ECHINOCOCCOSE CANINE : COMPARAISON ENTRE LA DÉTECTION DE COPROANTIGÈNES ET LA NÉCROPSIE CHEZ DES CHIENS ERRANTS ET DES RENARDS ROUGES DU NORD DE LA JORDANIE

La méthode ELISA de type sandwich a été utilisée pour le diagnostic de l'infection à Echinococcus granulosus par la

détection de coproantigènes chez 94 chiens errants et huit renards rouges (Vulpes vulpes) provenant du nord de la Jordanie. Les résultats ont été comparés avec ceux obtenus par nécropsie. Chez les chiens, le taux d'infection par E. granulosus était de 13,8 % avec une charge parasitaire de trois à plus de 10000 vers par animal. En revanche, sur les 13 chiens infectés, huit étaient coproantigène positifs (sensibilité de 61,5 %). La sensibilité croît à 87,5 % et 100 % chez les chiens porteurs respectivement de plus de 20 et de plus de 100 vers. La spécificité du test ELISA- coproantigène était de 91 %. La réaction croisée la plus importante a été observée chez les chiens porteurs de Dipylidium caninum. Les valeurs prédictives positives et négatives pour le test ELISA-coproantigène étaient respectivement de 50 et 94,2 %.

Ainsi, un chien coproantigène négatif est très probablement non infecté par E. granulosus. A contrario, un chien coproantigène positif n'est peut-être pas réellement infecté par E. granulosus, sauf si sa charge parasitaire est élevée, supérieure à 100 vers.

MOTS CLES :

Echinococcus granulosus, échinococcose canine, coproantigène, Jordanie.

INTRODUCTION

U

nilocular hydatidosis or cystic e c h i n o c o c c o s i s (CE) is a c o s m o p o l i t a n c y c l o z o o n o t i c disease c a u s e d b y the taeniid c e s t o d e Echinococcus granulosus and is o n e o f the major parasitic diseases o f public health i m p o r t a n c e (Mattosian et al., 1977;

Schantz & Kramer, 1 9 9 5 ; Schantz et al, 1 9 9 5 ) . T h e disease is e n d e m i c or highly e n d e m i c in Middle Eas- tern countries including L e b a n o n , Syria, Palestinian Authority, Israel, and J o r d a n ( S e e review by Abdel- Hafez & K a m h a w i , 1 9 9 7 ) . T h e stability o f CE in J o r d a n

* Department of Biological Sciences, Yarmouk University, Irbid, Jordan.

** Division of Parasitic Diseases, National Center for Infectious Diseases, Centers of Disease Control and Prevention, Atlanta, Georgia 30341, USA.

*** Department of Biological Sciences, University of Salford, Salford.

M5 4WT, UK.

Correspondence: Sami K. Abdel-Hafez.

Fax: +962 2 7246575 - e-mail: skhafez@yu.edu.jo

is multi-factorial as pertaining to improper slaughtering and h u m a n practices as well as to the a b u n d a n c e o f stray dogs (Abdel-Hafez et al, 1986; Abdel-Hafez &

Kamhawi, 1 9 9 7 ) .

T h e e p i d e m i o l o g y o f CE requires the consideration o f three host c o m p o n e n t s : the ungulate herbivore intermediate hosts, h u m a n s as accidental hosts and dogs as definitive hosts. Determination o f p r e v a l e n c e and i n c i d e n c e in these hosts should p r e c e d e any planning o f a control program. Direct identification o f hydatid cysts in various organs o f slaughtered intermediate hosts c a n determine the level o f endemicity o f the disease in a given area ( G e m m e l l , 1 9 9 7 ) . B o t h serological a n d imaging t e c h n i q u e s are u s e d to diagnose h u m a n CE infections and to determine the prevalence and incidence in e n d e m i c countries (Schantz & Kramer, 1 9 9 5 ) . Direct and indirect m e t h o d s c a n determine the p r e v a l e n c e o f c a n i n e e c h i n o c o c c o s i s . Direct methods rely o n t h e e x a m i n a t i o n o f purgative s a m p l e s o r contents o f small intestine following n e c r o p s y as well as o f fecal s p e c i m e n s to identify w h o l e w o r m s , pro- glottids a n d / o r eggs (Eckert et al., 1 9 8 4 ; Allan et al., Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2000072083

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EL-SHEHABI F.S;, KAMHAWI S.A., SCHANTZ P.M., CRAIG P.S. & ABDEL-HÁFEZ S.K.

1992; Craig, 1 9 9 3 ) . T h e s e m e t h o d s not only are time- c o n s u m i n g , but also suffer from l o w sensitivity and specificity (Craig, 1 9 9 7 ) . Eggs are not r e l e a s e d during the pre-patent period and their release is irregular during p a t e n c y ( N o n a k a et al, 1 9 9 6 ) . Morphological similarities a m o n g eggs o f all the taeniid s p e c i e s that m a y infect dogs simultaneously limit the specificity o f direct methods. In addition, these tests are quite hazar

dous to b o t h animals and examiners. Indirect m e t h o d s are b a s e d o n the identification o f copro-DNA, anti-adult w o r m s antibodies in the serum and feces as well as by c o p r o a n t i g e n s detection. Serodiagnosis is a c c o m panied with false negative results at the c o m m e n c e ment o f infection and false positive o n e s due to cross reactivity with other helminth infections (Jenkins & Ric- kard, 1 9 8 5 and 1 9 8 6 ; G a s s e r et al, 1 9 8 8 and 1 9 9 3 ) . T h e sandwich e n z y m e linked i m m u n o s o r b e n t assay (ELISA) has recently b e e n applied for diagnosis o f E. granulosus and E. multilocularis in c a n i n e s through t h e d e t e c t i o n o f c o p r o a n t i g e n s (Allan et al, 1 9 9 2 ; D e p l a z e s et al, 1992 and 1 9 9 4 ; Craig et al, 1 9 9 5 ; N o n a k a et al, 1 9 9 6 ; Malgor et al, 1997; A h m a d &

Nizami, 1 9 9 8 ) . T h e c o p r o a n t i g e n test has the advan

tage o f early detection o f the infection during prepa- tency, in 4 - 1 0 days post-infection ( D e p l a z e s et al, 1 9 9 2 ) . Moreover, positive results indicate current infec

tion b e c a u s e c o p r o a n t i g e n s are derived from adult w o r m s and w o u l d disappear as s o o n as the parasites are eliminated.

In this study, the E. granulosus c o p r o a n t i g e n test w a s used to assess the sensitivity, specificity and the posi

tive and negative predictive values o f this test under field conditions in stray d o g s and red f o x e s from nor

thern J o r d a n .

MATERIALS AND METHODS

N

inety four stray dogs (Cants familiaris Linnaeus, 1 7 5 8 ) and eight red foxes (Vulpes vulpes Lin

naeus ( 1 7 5 8 ) ) from Irbid and Mafraq Gover- norates, northern J o r d a n w e r e shot in the field b e t w e e n J u n e , 1994-July, 1 9 9 5 .

T h e n e c r o p s y o f the animals w a s carried out in the field as described b y El-Shihabi et al. ( 1 9 9 9 ) . Briefly, an abdominal cut was m a d e in e a c h animal and the intestine was tied from the pyloric and anal ends and c o l l e c t e d in a bag. B a g s w e r e stored in an i c e b o x and carried to the laboratory within three hours. T h e car

c a s s e s w e r e b u r n e d in the field to ensure n o conta

mination o f the environment. In the laboratory, e a c h intestine was divided into four p i e c e s o f equal length.

Each p i e c e was cut longitudinally and s o a k e d in 0.15 M p h o s p h a t e buffer saline (PBS, pH 7 . 2 ) for five minutes.

T h e m u c o s a l lining was gently s c r a p e d with a spatula

84

into c l e a n glass dishes and the c o l l e c t e d intestinal contents w e r e allowed to settle in 1,000 ml conical Nal- g e n e graduates (Nalge C o m p a n y , R o c h e s t e r , USA).

Following several washes with PBS, aliquots w e r e e x a m i n e d under a dissecting m i c r o s c o p e . T h e intestinal helminth parasites w e r e identified as described earlier b y El-Shihabi et al. ( 1 9 9 9 ) .

E. GRANULOSUS ADULT WORM CRUDE SOMATIC ANTIGEN EXTRACT

E. granulosus adult w o r m c r u d e s o m a t i c a n t i g e n (EgACSA) extract w a s p r e p a r e d as d e s c r i b e d earlier (Allan et al, 1 9 9 2 ) . Briefly, about 5 0 0 - 6 0 0 adult E. gra

nulosus w o r m s w e r e isolated from the small intestines o f infected animals. T h e y w e r e w a s h e d three times with 0.15 M Streptomycin containing p h o s p h a t e buffer saline ( P B S , pH 7 . 2 ) for 3 0 minutes. Thereafter, they w e r e frozen at - 20° C, thawed twice and h o m o g e n i z e d using 2 ml capacity glass h o m o g e n i z e r in ice b u c k e t for five minutes. Finally, the h o m o g e n a t e w a s centrifuged at 1,500 g for 2 0 minutes at 4 ° C and the pro

tein c o n t e n t o f the supernatant w a s determined b y Bradford m e t h o d (Bradford, 1 9 7 6 ) .

COPROANTIGEN PREPARATION

Fresh fecal materials w e r e collected from e a c h dog and fox and mixed in 1:1 w / v ratio with 0.15 M PBS (pH 7 . 2 ) containing 0.3 % T w e e n 2 0 . T h e mixture was s h a k e n vigorously using a V o r t e x s h a k e r and then centrifuged at 2,000 g for 3 0 minutes at 4 ° C . T h e supernatants w e r e frozen at - 20° C and stored until further use.

COPROANTIGEN-ELISA

Hyperimmune rabbit anti EgACSA antiserum was pre

pared as d e s c r i b e d previously (Allan & Craig, 1 9 8 9 ) . T h e IgG fraction w a s purified using protein A as a ligand in affinity chromatography o f protein A-Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, S w e d e n ) . Conjugation o f the IgG fraction with horseradish per

oxidase type VI enzyme (Sigma, USA) and the IgG cap

ture ELISA w e r e carried out as described previously (Allan and Craig, 1 9 8 9 ) with modification. Briefly, wells o f microtiter plates ( G r e i n e r F ELISA plates, Fric- kenhausen, Germany) were coated overnight with 100 ul o f rabbit anti EgACSA IgG at a dilution o f 1:800 in 0.05 M c a r b o n a t e buffer, pH 9-6 at 4 ° C using rocking p l a t e s h a k e r ( D e n l e y , E n g l a n d ) . T h e w e l l s w e r e w a s h e d three times with 0.1 % T w e e n 20 in 0.15 M PBS, pH 7.2 and b l o c k e d with 4 % b o v i n e serum albumin (fraction V, PARK, U K ) or 12 % s k i m m e d milk (Regi- lait instant dried s k i m m e d milk, F r a n c e ) in 0.15 M P B S , pH 7.2 for o n e h o u r at r o o m temperature ( R T ) . Fol

lowing washing the wells three times, the fecal super

natants w e r e m i x e d individually with heat inactivated

Mémoire P a r a s i t e , 2 0 0 0 , 7, 8 3 - 9 0

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D I A G N O S I S O F C A N I N E E C H I N O C O C C O S I S I N N O R T H E R N J O R D A N

Fig. 1. - Detection of E. granulosus coproantigens in dogs and foxes by IgG capture ELISA. The cut-off point value ( — ) was determined by calculating the mean optical density (O. D.) value at A.490 nm for 21 fecal samples of helminth free dogs + 3 S. D.

* Number in parenthesis indicates the worm burden of E. granulosus in infected dogs as determined by necropsy.

** Number in parenthesis denotes number of animals examined in each group.

fetal calf serum ( F C S ) at 1:1 v/v ratio (Sigma, USA), and 1 0 0 pi o f e a c h sample w a s a d d e d per well and i n c u b a t e d for o n e hour at RT. After three washings, 100 pi o f horseradish p e r o x i d a s e ( H R P ) conjugated with anti EgACSA IgG at a dilution o f 1:200 w a s added and i n c u b a t e d for o n e hour. After three washings, 100 pi o f substrate solution consists o f H202 in 0.1 M citrate buffer, pH 4.5 containing ortho-phenyldiamine ( O P D ) c h r o m o g e n (Sigma, USA) was incubated for 30 minutes at RT in dark. Finally, the optical density o f e a c h well was read at a wavelength o f 4 9 0 nm using Micro-ELISA auto-reader ( D y n a t e c h , Virginia, USA).

Positive control wells c o n t a i n e d 5 p g / m l EgACSA ins

tead o f fecal sample. Negative control wells c o n t a i n e d p o o l e d f e c a l m a t e r i a l s from h e l m i n t h free d o g s . External marginal wells o f e a c h micro-titer plate w e r e e x c l u d e d . T h e cut-off point w a s calculated b y measu

ring the m e a n optical density ( O D ) at 4 9 0 nm o f hel

minth free dog fecal s a m p l e s ( n o = 2 1 ) + 3 SD. All fecal s a m p l e s w e r e tested in triplicate.

EVALUATION OF COPROANTIGEN-ELISA TEST T o assess the efficacy o f the c o p r o a n t i g e n ELISA, the sensitivity, specificity, positive predictive and negative predictive values o f the test w e r e determined using n e c r o p s y data as a g o l d e n standard (Schantz, 1 9 9 7 ) .

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EL-SHEHABI F.S;, KAMHAWI S.A., SCHANTZ P.M., CRAIG P.S. & ABDEL-HAFEZ S.K.

RESULTS

NECROPSY DATA

T

able I s h o w s that 13 o f 9 4 ( 1 3 . 8 % ) stray dogs w e r e infected with E. granulosus either as single or concurrent infections with o t h e r helminthic s p e c i e s . Single infection with E. granulosus a c c o u n t e d for 5.3 % o f the d o g s ( 3 8 . 5 % o f infected d o g s ) . None o f eight f o x e s was found infected with E. granulosus.

Worm burden with E. granulosus ranged from 3-> 10,000 w o r m s per dog, with 4 6 . 2 % o f infected d o g s having a w o r m burden o f < 100 w o r m s / a n i m a l ( T a b l e II). All eight foxes and 7 7 . 7 % o f the dogs w e r e infected with at least o n e intestinal helminth s p e c i e s ( T a b l e III).

M o r e o v e r , s i n g l e or c o n c u r r e n t c e s t o d e infections a c c o u n t e d for 7 1 / 7 3 ( 9 7 . 3 % ) o f the infected dogs and six out o f the eight foxes. T h e most predominant hel

minth s p e c i e s e n c o u n t e r e d in stray dogs was Dipyli- clium caninum a l o n e or in c o m b i n a t i o n with other Dipylidids a n d / o r other c e s t o d e s . T h e infection rate

N o . o f i n f e c t e d

I n f e c t i o n m o d e d o g s %

S i n g l e E. granulosus i n f e c t i o n 5 5.3

C o n c u r r e n t i n f e c t i o n with:

Taenia s p p .( a ) 2 2.1

D i p y l i d i d s " " 1 l . l

Taenia s p p .( a l a n d D i p y l i d i d s " " 2 2.1 D i p y l i d i d s " " a n d N e m a t o d e s ' " 1 1.1 Taenia s p p .< a ), D i p y l i d i d s " " a n d N e m a t o d e s ' " 1 1.1 Taenia s p p ., a l, D i p y l i d i d s " " a n d Mesucestoides s p . 1 1.1

T o t a l i n f e c t i o n 1 3 1 3 . 8

( a ) Taenia s p p . i n c l u d e d T. pisiform is. T. taeniaeformis a n d o t h e r uni

d e n t i f i e d Taenia s p e c i e s .

( b )D i p y l i d i d s i n c l u d e d Dipylidium caninum, Diplopylidium s p . a n d

Joyeuxiella s p .

(c) N e m a t o d e s i n c l u d e d Toxocara s p . o r Toxascaris s p .

T a b l e 1. - R a t e s o f d o g i n f e c t i o n with E. granulosus e i t h e r a l o n e ( s i n g l e i n f e c t i o n ) o r c o n c u r r e n t i n f e c t i o n s w i t h o t h e r h e l m i n t h s in 9 4 d o g s from n o r t h e r n J o r d a n .

N o . a n d p e r c e n t a g e o f i n f e c t e d d o g s

W o r m b u r d e n o f E. granulosus N o . %

< 2 0 5 3 8 . 5

2 0 - < 1 0 0 1 7.7

1 0 0 - < 5 0 0 1 7.7

5 0 0 - < 1,000 3 0 . 7

l , 0 0 0 - < 5 , 0 0 0 I 7 . 7

> 1 0 , 0 0 0 1 7.7

T o t a l 13 1 0 0 . 0

T a b l e II. - W o r m b u r d e n o f E. granulosus in i n f e c t e d d o g s from n o r t h e r n J o r d a n .

D o g s F o x e s2

C o p r o -

N e c r o p s y ELISA N e c r o p s y T y p e o f i n f e c t i o n1 N o . % N o . % N o . %

H e l m i n t h free 21 2 2 . 3 0 0 . 0 i i 0 . 0

E. granulosus a l o n e 5 5.3 2 2.1 0 0 . 0

E. granulosus & o t h e r c e s . 6 6 . 4 5 5 . 3 0 0 . 0 E. granulosus, o t h e r c e s . & n e m . 2 2.1 1 1.1 0 0 . 0

O t h e r c e s . 5 3 5 6 . 4 7 7 . 4 3 3 7 . 5

C e s t o d e s , n e m . & / o r a c a n t h o . 5 5.3 1 1.1 3 3 7 . 5 O t h e r n e m . & / o r a c a n t h o . 2 2.1 0 0 . 0 2 2 5 . 0 T o t a l i n f e c t i o n 9 4 1 0 0 . 0 16 1 7 . 0 8 1 0 0 . 0

1 Abbreviations: ces., cestodes; nem., nematodes; acanth., acantho- cephalans.

- Faecal specimens of all foxes were copro-ELISA negative.

Table III. - Intestinal helminth fauna and infection rates in dogs and foxes from northern Jordan. Necropsy data were used to assess the diagnosis of E. granulosus coproantigens by capture ELISA.

with this species was 51.1 % ( 4 8 out o f total 9 4 necrop- sied d o g s ) and 6 5 . 8 % o f the total n u m b e r o f dogs infected with helminths. All foxes w e r e found infected with o n e o r m o r e s p e c i e s o f c e s t o d e s , n e m a t o d e s a n d / o r a c a n t h o c e p h a l a n s but n e v e r with either E. gra

nulosus or Taenia s p e c i e s ( T a b l e I I I ) . Five o f the foxes w e r e found infected with Dipylidids, particularly Diplopylidium and Joyeuxiella s p e c i e s . T h r e e f o x e s w e r e infected concurrently with Macracanthorhyn-

cbus acanthocephalan and other cestodes and/or nema

todes.

SENSITIVITY, SPECIFICITY, POSITIVE AND NEGATIVE PREDICTIVE VALUES OF COPRO ANTIGEN-ELI S A

T a b l e III c o m p a r e s the coproantigen-ELISA results with n e c r o p s y data o f dogs and foxes. W h i l e n o n e o f the fox fecal samples w e r e c o p r o a n t i g e n positive, 16 o f 94 ( 1 7 . 0 % ) o f the d o g fecal samples w e r e c o p r o a n t i g e n positive. Eight o f 13 E. granulosus positive dogs as revealed b y n e c r o p s y w e r e also c o p r o a n t i g e n positive.

In contrast, out o f 8 9 c a n i n e s ( d o g s and f o x e s ) w h i c h w e r e negative for E. granulosus infections b y necropsy, eight w e r e positive in the c o p r o a n t i g e n assay. In this way, t h e sensitivity o f c o p r o a n t i g e n - E L I S A test for 9 4 dogs and eight foxes was 6 1 . 5 %, while the specifi

city o f this test was 9 1 . 0 % ( T a b l e IV). T h e positive pre

dictive value o f this test was 5 0 % while the negative predictive value was as high as 9 4 . 2 %. All o f the five false-coproantigen negative samples w e r e for dogs har

boring an E. granulosus w o r m burden o f < 100 worms (Fig. 1). Moreover, s e v e n out o f eight dogs harboring

> 2 0 E. granulosus w o r m s w e r e c o p r o a n t i g e n positive.

This increases the sensitivity values o f the test for dogs with E. granulosus burden o f > 2 0 and > 100 w o r m s

86 Mémoire P a r a s i t e , 2 0 0 0 , 7, 8 3 - 9 0

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Fig. 2. - Detection of E. granulosus coproantigens by IgG capture ELISA in 8 1 fecal samples from 7 3 stray dogs and eight red foxes, all of which were infected with at least one species of intestinal helminths. The cut-off point value ( ) was calculated as in Figure 1.

DIAGNOSIS OF CANINE ECHINOCOCCOSIS IN NORTHERN JORDAN

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P a r a m e t e r C o p r o a n t i g e n - E L I S A %

S e n s i t i v i t y1 8 / 1 3 6 1 . 5

S p e c i f i c i t y2 8 1 / 8 9 9 1 . 0

P o s i t i v e p r e d i c t i v e v a l u e3 8 / 1 6 5 0 . 0 N e g a t i v e p r e d i c t i v e v a l u e4 8 1 / 8 6 9 4 . 2

1 Sensitivity of coproantigen-ELISA was calculated as % E. granulosus copro-positive animals/all positives by necropsy.

2 Specificity of coproantigen-ELISA was calculated as % E. granulosus copro-negative animals/all negatives by necropsy.

3Positive predictive value of coproantigen-ELISA was calculated as

% E. granulosus positive animals by both necropsy and coproan- tigen-ELISA/all copro-positives by coproantigen-ELISA alone.

4Negative predictive value of coproantigen-ELISA was calculated as

% E. granulosus negative animals by both necropsy and coproan- tigen-ELISA/all copro-negatives by coproantigen-ELISA alone.

Table IV. - Sensitivity, specificity, positive and negative predictive- values of coproantigen-ELISA test for E. granulosus detection in 94 stray dogs and eight red foxes from northern Jordan.

to 8 7 . 5 % and 1 0 0 . 0 %, respectively. Figure 2 depicts the ELISA O D readings for c o p r o a n t i g e n s in the fecal samples o f all dogs and f o x e s infected with intestinal helminths as c o m p a r e d with the n e c r o p s y data. Evi

dently, higher O D values w e r e mostly s e e n in dogs infected with high E. granulosus b u r d e n s w h e t h e r singly or concurrently with o t h e r helminth parasites.

Out o f the eight dog fecal samples that w e r e false c o p r o a n t i g e n positive, six w e r e for dogs infected with D. caninum a l o n e o r with other Dipylidid s p e c i e s (i.e.

Joyeuxiella a n d / o r Diplopylidium s p e c i e s ) . O n e o f the other t w o s a m p l e s was infected with Taenia sp. and the other with both D. caninum and Taenia spp. (Fig. 2).

In this way, out o f 4 8 dogs w h i c h w e r e D. caninum positive as revealed by necropsy, 41 ( 8 5 . 4 % ) w e r e c o p r o a n t i g e n negative.

DISCUSSION

I

n the present study, the IgG capture ELISA for the detection o f coproantigens was used in conjunction with n e c r o p s y to determine the infection rate o f E. granulosus and other intestinal helminths in stray dogs and red foxes from northern J o r d a n . T h e infec

tion rate with E. granulosus as determined by necropsy was 13-8 %. This conforms with our earlier report (El- S h e h a b i et al., 1 9 9 9 ) , but w a s l o w e r than what had b e e n reported over 15 years a g o in w h i c h 19.6 % o f stray dogs from Irbid Governorate w e r e found infected (Ajlouni et al., 1 9 8 4 ) .

T h e sensitivity o f coproantigens ELISA was found to b e highly dependent on the E. granulosus worm burden.

T h e overall sensitivity o f the test was relatively low at 61.5 % regardless o f the worm load. However, the sen

sitivity reached as high as 8 7 . 5 % and 100.0 % for dogs harboring > 2 0 and > 100 worms/dog, respectively. It

is this category o f animals, which is most important epidemiologically as a source o f livestock and h u m a n infection. Seven out o f 13 (53.8 % ) E. granulosus infected dogs had a worm burden o f > 100 worms/dog (Table II).

Thus, the test is quite useful to monitor dog infection rates in surveillance and control program. It is simple, safe, c h e a p and less time-consuming than the o t h e r standard t e c h n i q u e s that determine c a n i n e e c h i n o c o c cosis by n e c r o p s y or purgation with arecoline hydro- bromide.

T h e positive predictive value o f the test, as calculated here, appears to b e quite l o w ( T a b l e IV) and denotes a reflection o f the detectability level o f the w o r m load.

Lower E. granulosus infections (i.e. < 100 wonns/animal) yielded mostly negative coproantigen-ELISA results.

T h e positive predictive value increased significantly with higher w o r m burdens. T h e d e t e c t a b l e w o r m load by coproantigens-ELISA has influenced the positive predictive value o f t h e test as only eight o f total 16 c o p r o a n t i g e n positive dogs w e r e actually E. gra

nulosus infected animals. S e v e n out o f these eight ani

mals had a w o r m burden > 100 w o r m s / d o g . Thus, o n e c a n not rely o n coproantigen-ELISA a l o n e to diagnose low worm burden o f E. granulosus in dogs. In contrast, the negative predictive value was high (Table IV). Thus, a negative result confirms that a dog is either truly non- infected or harbors a very l o w w o r m burden infection, b o t h conditions are epidemiologically not significant.

T h e specificity o f this test was high ( 9 1 . 0 % ) and is c o m p a r a b l e to what has b e e n reported earlier (Allan et al, 1992; D e p l a z e s et al., 1992; Craig et al., 1 9 9 5 ) . In the present investigation, most o f the cross reacti

vity was noted in dogs infected with D. caninum alone or with o t h e r Dipylidid w o r m s . This contrasts earlier observations in w h i c h most false coproantigens-ELISA positive samples w e r e s e e n in Taenia bydatigena infec

tions (Allan et al, 1992; D e p l a z e s et al, 1992; Molgar et al, 1 9 9 7 ) Here, o n e o f the eight false c o p r o a n t i g e n s positive samples was for a dog infected with T. byda

tigena alone. Indeed, T. bydatigena. infection was lower than D. caninum in the present series. This reflects the strayhood o f the dogs in contrast to h o m e o w n e d dogs that are routinely d o s e d for fleas and other e c t o p a r a sites. Thus, the consideration o f coproantigens as being genus-specific (Craig et al, 1 9 9 5 ) must b e re-examined.

Apparently, s o m e somatic antigens may b e shared bet

w e e n Dipylidid and Taeniid w o r m s including E. gra

nulosus. T h e use o f excretory/secretory ( E / S ) antigens instead o f adult somatic crude antigens to hyperim- munize rabbit for the preparation o f reagents to b e used in coproantigens-ELISA may result in m o r e genus- specific antibodies and diminish the cross-reactivity bet

w e e n different g e n e r a o f c e s t o d e s ( D e p l a z e s et al, 1 9 9 2 ) . H o w e v e r , t h e true cross-reactivity b e t w e e n D. caninum and E. granulosus can only b e proven b y

s s P a r a s i t e , 2 0 0 0 , 7, 8 3 - 9 0

EL-SHEHABI F.S„ KAMHAWI S.A., SCHANTZ P.M., CRAIG P.S. & ABDEL-HAFEZ S.K.

Mémoire

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DIAGNOSIS OF CANINE ECHINOCOCCOSIS IN NORTHERN JORDAN

experimental infection studies with the two-helminth species. It should b e p o i n t e d out that only 14.6 % o f 4 8 D. caninum infected d o g s w e r e coproantigen posi

tive. O n e c a n not rule out that such dogs might have had recent E. granulosus infection that had b e e n cleared out. Alternatively, a very small w o r m load o f E. gra

nulosus might have b e e n missed during n e c r o p s y o f s o m e o f these dogs a n d thus resulted in c o p r o a n t i g e n positivity.

The ultimate c o n c l u s i o n o f this study is that c o p r o a n - tigen-ELISA is a suitable test that c a n b e used in epi

demiological surveys. Its sensitivity is very high in dogs harboring > 2 0 w o r m s / a n i m a l a n d is absolute in those with > 100 w o r m load.

Currently, this test h a s b e e n adopted as a routine tech

nique to investigate canine e c h i n o c o c c o s i s prevalence in various parts o f the country.

ACKNOWLEDGEMENTS

T

h e authors wish to thank the technical assistance Mr. J . Hawari. This w o r k received financial sup

port from NIAID-NIH (Grant No. A I - 4 5 1 9 4 ) , European Commission (EC Contract I C 1 8 - C T 9 8 - 0 3 5 4 ) , and Y a r m o u k University Research Council.

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COMPARISON OF COPROANTIGEN DETECTION WITH NECROPSY I N STRAY DOGS A N D RED FOXES FROM NORTHERN J O R D A N

DIAGNOSIS OF CANINE ECHINOCOCCOSIS: