Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes.

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source: https://doi.org/10.7892/boris.118665 | downloaded: 1.2.2022

Parasitology Research

9 Springer-VerIag 1992

Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes*

P. Deplazes 1, B. Gottstein l, J. Eekert 1, D.J. Jenkins

2,

D. Ewald 1, and S. Jimenez-Palacios 3 i Institute of Parasitology, University of Z/irich, Winterthm'erstrasse 266a, CH-8057 Z/irich~ Switzerland 2 Hydatid Control Campaign, P.O. Box 112, Queanbeyan NSW 2620, Australia

Consejeria de Salud, Consumo y Bienstar Social, C/Villamedina 17, 26071-Logrono (La Rioja), Spain Accepted February 1, 1992

Abstract. An enzyme-linked immunosorbent assay (EL- ISA) was developed for the detection of

Echinococcus

coproantigens in fecal samples from dogs, dingoes or foxes infected with either

E. granuIosus

or

E. multilocu- Iaris.

The ELISA was based on protein-A-purified poly- cIonal antibodies [anti-E.

granulosus

excretory/secretory (E/S) antigens]. The specificity of the assay as determined in 155 samples derived from carnivores that were free of helminth infection (n = 37) or infected with non-

Echinococcus

cestodes (n=76) or with various nematodes (n--42) was found to be 98% overall. The diagnostic sensitivity was strongly dependent on the homolo- gous worm burden. All 13 samples from foxes harboring

> 1,000

E. multilocularis

worms and 13 of 15 (87%) samples from dogs or dingoes containing > 200

E. granulo- sus

worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring < 1,000

E. multilocularis

and 9 of 10 samples from dogs or dingoes bearing <200

E. granulosus

tested negative. Experimental prepatent infections of dogs with

E. granulosus

revealed positive ELISA reactions within the prepatent period (1~20 days post-infection) for six animals bearing > 1,000 E.

granulosus

each; a low worm burden ( < 1,000 tapeworms/animal) resulted in ELISA positivity in only 2 of 3 animals at 30 days post-infection at the earliest.

All five dogs that had been experimentally infected with

E. multilocularis

tested positive in the coproantigen EL- ISA as early as on day 5 post-infection.

The established methods for the diagnosis of intestinal

Echinococcus

infections in carnivores include (a) the identification of proglottids or whole parasites elimi- nated spontaneously or after purging (of dogs) with are- coline hydrobromide or (b) the examination of the small intestine at necropsy (Eckert et al. 1984). The detection

* Dedicated to Prof. K.T.F. Friedhoff on the occasion of his 60tb birthday

Offprint requests to:

P. Deplazes

of eggs on the skin of carnivores using the Scotch-tape technique (Deplazes and Eckert 1988) or in fecal samples by flotation techniques is indicative of infection with

Taenia

or

Echinococcus,

but the eggs of these genera cannot be differentiated by microscopy.

Serodiagnosis has been alternatively proposed in re- cent reports, as definitive hosts infected with intestinal stages of cestodes obviously exhibit the potential to de- velop a parasite-specific serum antibody response. Ex- perimental investigations have been performed for the diagnosis of

E. granulosus

infections in dogs (Jenkins and Rickard 1985, 1986; Gasser et al. 1988) of

E. multi- tocularis

infections in foxes (Gottstein et al. 1991 a). Un- fortunately, serological studies demonstrated that antibody detection did not always reflect a current intestinal

E. granulosus

or

7". hydatigena

infection in carnivores (Jenkins et al. 1990, 1991). In areas endemic for

E. multi- locularis,

prevalence rates of serum antibodies against Em2-antigen (Gottstein 1985) in fox populations re- flected previous or continuing exposure to

E. muItilocu- laris

antigen, most likely related to contact with eggs/

oncospheres (Gottstein et al. 1991 a), but did not enable the determination of the actual intestinal infection status in individual animals. However, on a population basis, the antibody prevalence showed a close correlation to the parasitological prevalence of

E. multilocularis

(Gott- stein etal. 1991a).

Two alternatives have been proposed for the diagnosis of intestinal echinococcosis in definitive hosts; both methods involve the detection of parasites on a molecular level. One was defined as an antibody-sandwich enzyme-linked immunosorbent assay (ELISA; Deplazes et al. 1990, 1991) after it had been demonstrated that coproantigens were detectable by precipitation reactions with hyperimmune rabbit serum (Babos 1962). Such an ELISA used affinity-purified polyclonal antibodies raised against excretory/secretory (E/S) antigens of adult

T. hydatigena

or

T. saginata.

C0Proantigens were relia- bly assayed and detected in fecal samples from dogs infected with

T. hydatigena.

Diagnostic genus specificity was underlined by the absence of cross-reactions related

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to i n f e c t i o n s w i t h Echinococcus s p p . o r o t h e r c e s t o d e s o r n e m a t o d e s . S e q u e n t i a l a n a l y s e s r e v e a l e d t h a t T. hyda- tigena c o p r o a n t i g e n s w e r e d e m o n s t r a b l e d u r i n g p r e p a - t e n c y b e g i n n i n g a t d a y 18 p o s t - i n f e c t i o n (p.i.). A n o t h e r a s s a y e n a b l e d the r e l i a b l e d i a g n o s t i c d e t e c t i o n o f T. sa- ginata c o p r o a n t i g e n s in 8 5 % o f 34 fecal s a m p l e s f r o m 23 p a t i e n t s w i t h t a e n i o s i s , w h e r e a s d i a g n o s i s b y egg de- t e c t i o n w a s o n l y p o s s i b l e in 6 2 % o f t h e s a m e i n d i v i d u a l s ( D e p l a z e s et al. 1991). I n this respect, the p o t e n t i a l o f d e t e c t i n g c o p r o a n t i g e n s h a s b e c o m e a g e n e r a l focus in t h e d i a g n o s i s o f i n f e c t i o n w i t h i n t e s t i n a l c e s t o d e s t a g e s ( A l l a n a n d C r a i g 1989; A l l a n et al. 1990; D e p l a z e s et al.

1990, 1991). A l t e r n a t i v e l y , i n f e c t i o n b y a d u l t c e s t o d e s c o u l d also b e d e m o n s t r a t e d a n d i d e n t i f i e d b y the d e t e c - t i o n o f p a r a s i t e - s p e c i f i c D N A f r a g m e n t s o r i g i n a t i n g ei- t h e r f r o m p a r a s i t e eggs o r f r o m cells o r tissue d e b r i s o f a d u l t t a p e w o r m s . T h i s a p p r o a c h h a s r a p i d l y a t t r a c t e d a t t e n t i o n , e s p e c i a l l y since t h e a d v e n t o f h i g h l y sensitive t e c h n i q u e s such as t h e p o l y m e r a s e c h a i n r e a c t i o n , a n d e x a m p l e s in this r e s p e c t h a v e b e e n d e s c r i b e d e l s e w h e r e ( G o t t s t e i n a n d M o w a t t 1991 ; G o t t s t e i n et al. 1991 b).

T h i s p a p e r d e s c r i b e s a n E L I S A d e s i g n e d f o r t h e de- t e c t i o n o f Echinococcus c o p r o a n t i g e n s in fecal s a m p l e s o b t a i n e d f r o m d e f i n i t i v e h o s t s i n f e c t e d w i t h E. granulo- sus o r E. multilocularis.

Materials and methods E x p e r i m e n t a l design

A sandwich ELISA was developed for the diagnostic determination of Echinococcus antigens in fecal samples obtained from dogs, dingoes and foxes. The test, which uses protein-A-purified polyclonal hyperimmune antibodies, was evaluated in the following steps:

1. Determination of an ELISA threshold value, which enables the reliable discrimination between "positive" (detectable Echino- coccus coproantigens) and ', negative" (no detectable Eehinococcus coproantigens) reactions; this step included the determination of the test's specificity against heterologous intestinal helminth infections (groups I and III for dogs and dingoes and group IV for foxes; see below)

2. Determination of the test's ability to detect coproantigens during the prepatent period of experimental E. granulosus or E.

multilocularis infections in dogs (group lI; see below)

3. Determination of the test's diagnostic sensitivity in fecal samples from dogs, dingoes or foxes naturally infected with E. granulo- sus or E. multilocularis (groups IlI and IV)

4. Assessment of specificity parameters based on samples obtained from uninfected animals or from animals showing experimentally or naturally acquired heterologous helminth infections (from groups I-IV)

A n i m a l s : f e c a l samples and intestinal contents

Fecal samples (FS) or intestinal contents (IC) from the rectum were collected from the following groups of animals as described below. Group I" consisted of t3 dogs (Niederlaufhunde; 7 females and 6 males aged between 1 month and 6 years) kept under cestode- free conditions (13 FS). This group also included 7 dogs experimentally infected with Taenia hydatigena (3-6 adult T. hydatigena tapeworms were recovered per dog at necropsy); 2 fecal samples were collected per animal (on different days) during patency (14 FS).

All samples were positive at the same time in a T. hydatigena coproantigen ELISA that was performed as previously described by Deplazes et al. (1990). In addition, this group contained 28 dogs of various ages and breeds, all of which showed no indication of an infection with Taenia or Eehinococcus spp. However, 5 were infected with Mesocestoides corti; 4, with DipyIidium caninurn; 5, with Triehuris vulpis; 6, with Toxocara canis; 3, with Toxascaris leonina; 3, with hookworms; 1, with T. leonina and hookworms;

and 1, with T. canis and T. leonina (28 FS).

Group II consisted of 9 dogs (Niederlaufhunde; 4 males and 5 females aged between 1 and 2 years) raised under helminth-free conditions. The dogs were experimentally infected with approximately 0.6 ml sedimented (1 g) E. granulosus protoscolex suspen- sion per os (infection mode according to Eckert et al. 1989). The protoscoleces were isolated from the following sources: a liver cyst from a naturally infected Swiss horse (6 dogs), a liver cyst from a naturally infected pig from Poland (2 dogs), and a lung cyst from a naturally infected Swiss cow (1 dog). All dogs were necrop- sled on day 30 p.i. This group also contained 5 female dogs (Nieder- laufhunde) aged 2 years that were raised under helminth-free conditions. Each dog was experimentally infected with 20 g minced E.

multilocularis metacestode tissue (obtained from Meriones unguieu- latus experimentally infected with the parasite isolate SLI-144) con- taining approximately 0.5 z 106 protoscoleces. All dogs were necropsied on day 25 p.i. Fecal samples were collected at different intervals during prepatency (93 FS). The recovery and counting of (live) E. granulosus and E. multilocularis tapeworms were done according to Eckert et al. (1989).

Group III consisted of 50 stray dogs from the Logrono area of Spain. Parasitological examination of the intestine and the recovery and counting of E. granulosus tapeworms were performed after necropsy using conventional parasitological methods. The respective results are shown in Fig. 4 (50 IC). This group also included 16 dingoes shot in the Bondo State Forest in central southeastern New South Wales, Australia, where E. granulosus is known to be prevalent (Jenkins and Morris 1991). The parasitological results are shown in Fig. 5 (16 IC). In addition, this group contained 12 foxes free of E. granulosus infection. These foxes were shot in Taralga, New South Wales, Australia, all were infected with Uncinaria stenocephala, and some were additionally infected with T. canis (n=4) and D. caninum ( n = l ) . E. granulosus has been found in foxes in the Blue Mountains National Park adjacent to this area (Jenkins, personal communication). The parasitological examination of the dingo and fox intestines and the recovery and counting of E. granulosus tapeworms (12 IC) were done according to Jenkins et al. (1990).

Group IV consisted of 100 red foxes shot in the Canton of Ziirich, Switzerland, over a 5-month period in 1990. Necropsy was performed after the carcasses had been frozen at - 8 0 ~ for 5 days (a procedure that kills Echinococeus eggs; Eckert et al. 1991).

The recovery and counting of E. multilocularis tapeworms 000 IC) were done according to Eckert et al. (1989). This group also included 40 Swiss red foxes free of E. muItiIocularis infection; 14 were parasitologically free of helminths at necropsy, 5 were infected with other non-Echinoeoccus cestodes, 12 were infected with intestinal nematodes, and 9 displayed simultaneous infections with non- Echinoeoccus cestodes and nematodes (40 IC). In addition, this group contained 60 E. multilocularis-infected Swiss red foxes grouped according to the numbers of E. multilocularis tapeworms recovered per animal (28 foxes harboring 1-100, 18 containing 101-1,000, and 14 bearing > 1,000 adult-stage E. multilocularis).

Among these 60 foxes, 21 harbored no other intestinal helminths, 4 additionally bore other intestinal non-Echinococcus cestodes, 14 had intestinal nematodes, and 21 harbored non-Echinococcus cestodes and nematodes simultaneously (60 IC).

From all animals in groups ~IV, fresh FS or IC were collected and mixed at a 1 : 4 (v/v) ratio with a buffer solution [phosphate- buffered saline (PBS) containing 0.04% NAN3, 0.05% bovine he- moglobin (Fluka) and 0.3% Tween-20]. All samples were stored or transported at - 2 0 ~ until further processed. The samples were thawed and the fecal suspensions were ultrasonicated for 30 s

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(40 W) and subsequently sedimented at 3,000 g for 10 min prior to their use in ELISA.

Echinococcus granulosus antigens

Living immature adult E. granulosus tapeworms were recovered from the intestines of six dogs (experimentally infected with a horse parasite isolate) on day 30 p.i. (methodology according to Eckert et al. 1989). Approximately 2,000 E. granulosus tapeworms were cultivated in a 30-ml plastic tissue-culture flask (Falcon) containing 30 ml Eagle's minimal essential medium (EMEM ; Gibco, catalogue number 072 1100) supplemented with D-glucose (4 mg/ml), genta- mycin (200 #g/ml) and Fungizone (250 ng/ml) at pH 7.2. The medium was replaced after 4, 14, 22 and 34 h in vitro maintenance and then every 2nd day until day 14. The aliquots of tissue-culture medium (TCM) collected from day 3 until day 14 were stored at - 20 ~ C until further processed. The viability of the worms was judged by their motility and morphology as viewed with an inverted microscope (Laborvert FS, Leitz). The sterility of the cultures was tested on day 3 of maintenance according to standard bacteriologi- cal procedures. E/S antigens were dialysed and concentrated from the collected and pooled TCM to a volume containing 0.1 mg protein/ml PBS using an Amicon ultrafiltration unit with a YM-10 membrane. All protein concentrations were assessed by a Bio-Rad protein assay using bovine serum albumin as the standard. The E. granulosus E/S antigens were used for the elaboration of the Echinococcus coproantigen-detecting ELISA as described below.

Anti-E. granulosus hyperimmunoglobulins

Anti-E. granulosus hyperimmunoglobulins were generated by the immunization of a rabbit with E. granulosus E/S antigens (100 l-tg/

injection) emulsified in Ribi adjuvants according to the manufacturer's recommendations (Ribi Immunochem Research Inc., Ha- milton, Montana, USA). Immunoglobulins were purified on a protein A-Sepharose CL-4B column according to the manufacturer's instructions (Pharmacia Fine Chemicals, Uppsala, Sweden). Serum from a non-immunized rabbit was processed in exactly the same way to obtain "irrelevant" negative control antibodies. All further steps, including the preparation of rabbit anti-E, granulosus E/S conjugate (alkaline phosphatase-labeled), were performed according to the procedure of Baumann and Gottstein (1987).

Enzyme-linked immunosorbent assay

The concept for the development of a sandwich ELISA for the detection of fecal Echinococcus antigens in dogs, dingoes and foxes was based (with few modifications) on that previously decribed for the detection of T. hydatigena coproantigens in dogs (Deplazes et al. 1990) and can be summarized briefly as follows. Protein-A- purified rabbit anti-E, granulosus E/S IgG was used in the solid phase as a coproantigen-specific catching antibody (at 20 p.g protein/mt coating solution), with an irrelevant rabbit (solid-phase) IgG being included in parallel as a negative control. Parasite-specific antibodies enabled the complexing of coproantigens from diagnostic samples; visualization of the immune reaction was obtained by using liquid-phase (protein-A-purified) rabbit anti-E, granulosus E/S IgG labeled with alkaline phosphatase and the corresponding chromogenic substrate solution (4-nitrophenyl phosphate). The re- suits were expressed in corrected A4o 5 ~ values (the value obtained for the specific reaction minus that found for the control reaction using the irrelevant antibody) and were monitored in reference to standard positive and negative control samples. When the results obtained using the irrelevant control IgG exceeded 30% of the positive A4o 5 nm value found for the specific IgG reaction, the sample run in question was excluded.

A4o5 nm 0.5

0.4 9

0 . 3 -

0.2- 0.1

0

Dogs: A

Echinococcus granutosus

9 cut-off point ~ + 3 SD

, . : : : . . . ~ ; : : . . " - - .

B 0 D

Taenia Non-Echinococcus Helminth- hydatigena helminths free controls Fig. 1. Arbitrary determination of a discriminating threshold in the ELISA for Echinococcus coproantigen detection in dogs. The cut-off point (--) was determined by calculating the mean A4o snm value +3 SD for group I animals (B, C, D). A, Preselected E.

granulosus-positive reference controls (5 naturally infected dogs from Spain); B, 14 samples from 7 dogs with patent Taenia hydati- gena infections; C, 28 samples from dogs with other non-Echino- coccus helminth infections; D, samples from 13 helminth-free con- trot dogs

Results

T h e o r d e r o f the results r e p o r t e d b e l o w c o r r e s p o n d s to the s c h e m e d e s c r i b e d in E x p e r i m e n t a l d e s i g n a n d a r e s u m m a r i z e d in Figs. 1-5.

Determination o f a diagnostic cut-off value

I n the E L I S A , a c u t - o f f v a l u e w a s d e t e r m i n e d b y c a l c u - l a t i n g the m e a n A4o s ,m v a l u e + 3 S D f o r a n i m a l g r o u p s w i t h n o Echinococcus i n f e c t i o n a n d f o r t h o s e w i t h h e t e r - o l o g o u s h e l m i n t h i n f e c t i o n s c a p a b l e o f p r o d u c i n g c r o s s - r e a c t i n g a n t i g e n s . T h e results a r e s h o w n in Fig. 1. I n all d o g s t h a t e x h i b i t e d n o h e l m i n t h i n f e c t i o n o r w e r e i n f e c t e d w i t h n o n - t a e n i i d h e l m i n t h s the f i n d i n g s w e r e c l o s e l y g r o u p e d in the s a m e r a n g e o f l o w A4o 5 nm values.

U s i n g these results t o g e t h e r w i t h t h e v a l u e s o b t a i n e d for d o g s i n f e c t e d w i t h Taenia hydatigena, the r e s p e c t i v e c u t - o f f v a l u e w a s c a l c u l a t e d , r e s u l t i n g in n e g a t i v e test i n t e r p r e t a t i o n s f o r all n o n - t a e n i i d s a m p l e s . O n e o f t w o s a m p l e s f r o m a d o g i n f e c t e d w i t h 7 a d u l t T. hydatigena r e a c t e d a b o v e the c u t - o f f v a l u e ( p o s i t i v e r e a c t i o n ) .

Antigen detection in prepatent infections o f E. granulosus or E. multilocularis

T h e results o b t a i n e d for d o g s e x p e r i m e n t a l l y i n f e c t e d w i t h E. granulosus o r E. multilocularis ( g r o u p I I a n i m a l s ) a r e s h o w n in Fig. 2. A n a l y s i s o f t h e s m a l l i n t e s t i n e a f t e r n e c r o p s y ( o n d a y 30 p.i.) r e v e a l e d a w i d e r a n g e o f w o r m b u r d e n s ( b e t w e e n 200 a n d > 10,000 w o r m s / d o g f o r E.

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A )oo

1 ,o lO,OOO

4 0 , 0 0 0

1 0 , 0 0 0 1 1

0,9 2 1

3 1

1 0 , 0 0 0 4 1

0 , 8 5 1

6 1

7 1

0 , 7 8 f

9 1

10 2

0 . 6 t l 2

6 , 5 7 0 1 2 3

t 3 3

0 . 5 14 5

15 5

16 1

0 . 4 17 1

18 1

19 1

0.3 20 1

21 2

22 3

N'o 23 3

v.~ 1 , 4 0 0 24 3

3 5 0 25 6

0.1 800 2 6 7

2 2 4 27 7

2 8 8

29 8

0 30 11

0 5 10 15 20 25 30 31 1 3

d a y s a f t e r e x p e r i m e n t a l i n f e c t i o n

Fig. 2. Detection of coproantigens by ELISA during the prepatent

period of experimental Echinococcus granulosus ( I ) or E. rnultilocu- laris ( x ) infections in 9 and 5 dogs, respectively (group II animals).

Right-hand numbers indicate recovered worm burden. The cut-off point ( - ) was determined as described in Fig. l

granulosus and between 10,000 and 100,000 worms/dog for E. muttiloeularis). Reliable detection of coproanti- gens was correlated to the number of worms detected in the dogs. The first positive reactions were detected at 10 days p.i. in dogs showing burdens of >10,000 E. granulosus worms and at 5 days p.i. in all dogs harbor- ing burdens of > 10,000 E. multiloeularis. By 20 days p.i., all dogs with worm burdens of > 1,000 E. granulosus had become positive. Dogs harboring < 1,000 E. granu- losus exhibited only weakly positive (n = 2) or negative (n = 1) reactions at 30 days p.i.

Antigen detection in natural infections with E. granulosus or E. multiIocularis

The intestines of necropsied Spanish dogs and Austra- lian dingoes and foxes (all animals came from areas that are endemic for E. granulosus and are not known to be endemic for E. multilocularis) were parasitologically examined (group III animals) and the intestinal content was tested in parallel by ELISA.

A positive reaction could be detected in 1 of 31 sam- ples from Spanish stray dogs that were infected with different numbers of T. hydatigena, T. pisiformis and Dipylidiurn caninum but harbored no E. granulosus, dem- onstrating the high specificity of the test (Fig. 3). All 9 dogs containing >_200 E. granulosus worms showed a positive reaction in the ELISA; on the other hand, coproantigens could be detected in only 1 of 8 samples t?om dogs infected with 3-70 E. granulosus.

Dog N u m b e r of c e s t o d e s f o u n d a f t e r n e c r o p s y

Taenia hydatigena T. hydatigena T. hydatigena

T. hydatigena, Dipylidium caninum T. hydatigena

T. hydatigena, 1 T. plsiformis 72 hydatigena

T. hydatigena

T. hydatigena, 13, caninum 7-. hydatigena, 1 7". pisiformis T. hydatigena, 1 T. pisiformis T. hydatigena

T. hydatigena, D. caninum T. hydatigena

T. hydatigena, 2 7". pisiformis T. pisiformis

72 pisiformis

T. pisiformis, D. caninum 72 pisiformis, D. caninum 7-. pisiformis, D. caninum T. pisiformis

T. pisiformls T. pisiformis T. pisiformis 72 pisiforrni$

T. pisiforrnis 7-. pisiformis T. pisiformis 7-. pisiformis 7-. pisiformis T. pisiformis 3 2 3 Echinococcu$ granulosus 3 3 4 E. granulosus, I D. cat~inum 3 4 6 E. granulosus, 1 T. hydatigena 3 5 10 E. granulosus, 7 72 pisiformls 3 6 25 E. granulosus

3 7 60 E. granulosus, 5 72 pisiformis 3 8 70 E. granulosus, Taenia spp.

3 9 70 E. granulosus, Taenia spp.

4 0 200 E. granulosus, 2 7-. hydatigena 4 1 >500 E. granulosus, D. caninum 42 >500 E. granulosus

43 >500 E. granutosus 4 4 >500 E. granulosus

45 >500 E. granutosus, Taenia spp.

4 6 >500 E. granulosus, Taenia spp.

4 7 >500 E. granulosus 48 >500 E. granulosus

A 405 nm

0 0.1 0 . 2 0 . 3 0 . 4 0 . 5

I i i i

I cut-off

~

^int

I 1

1

II 1

[ I

1

|

| 1

1

|

I

1

Fig. 3. Detection of Echinococcus granulosus coproantigens by EL- ISA in samples obtained from stray Spanish dogs (from group III). The cut-offpoint ( - ) was determined as described in Fig. t

The results obtained for samples from dingoes (Fig. 4) again demonstrated the dependence of a positive ELISA reaction on the number of E. granulosus worms recovered. Cross-reactions occurred in one sample from a dingo with a T. hydatigena infection; all other samples showing no E. granulosus infection were negative. Aus- tralian foxes (n= 12) were negative for E. granulosus in parasitological examinations as well as in the ELISA.

Frequently occurring heterologous helminth infections underlined the high specificity of the ELISA.

Figure 5 shows the results of investigations of samples from foxes that were selected on the detection of adult- stage E. multilocularis in the intestine (group IV ani- mals). The findings, grouped according to the worm bur- den of the animals, confirmed both the strong correla- tion between ELISA positivity and the number of worms recovered and the high specificity (100%) of the test for samples from animals showing no Echinococcus in- fection.

The overall diagnostic sensitivity of the ELISA for

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D i n g o N u m b e r of c e s t o d e s f o u n d a f t e r n e c r o p s y

1 1 Taonia pisiformis

2 negative

3 4 T. pisiformis, 1 T. senalis 4 1 T. pisiforrnis

5 2 1 7". pisiformis

6 1 4 7". pisiformis, 8 T. serialis, 2 T. hydatigena

7 1 6 72 pisiformis, 4 7-. serialis

8 4 7-. hydatigena

9 30 Echinococcus 9ranulosus, 50 T . seriafis, 10 T. pisiformis 1 0 48 E. granulosus

1 1 300 E. granulosus 1 2 725 E. granulosus 1 3 800 E. granulosus

1 4 2,100 E. granulosus, 2 7-. pisiforrnis 1 5 t 2,1 2 0 E. granulosus, 27 72 pJsiformis

97 72 serialis

1 6 4 6 , 7 5 0 E. granulosus, 156 7-. serialis, 2 72 pisiformis

A

4 0 5 n m 0 0.1 0 . 2 0 . 3 0 . 4

r , i i

c u t - o f f point I

I i m

M e

i l l

i n

Fig. 4. Detection of Echinococcus granulosus coproantigens by EL- ISA in samples obtained from Australian dingoes (from group III).

The cut-off point (--) was determined as described in Fig. t

A4o5 nm

0.8-

0.7- 9

0.6-

:~

0 . 5

0 . 4

0 . 3 g ^"

0 . 2 9

9 o 9

c u t - o f f p o i n t ~ + 3 S D 9 9 "

o -" ::

.%...

Foxes: A 81 82 B3

without with E. m u l t i l o c u l a r i s E. m u l t i l o c u l a r i s

Fig. 5. Detection of Echinococcus multiIocularis coproantigens by ELISA in samples obtained from Swiss foxes. Evaluation of the sensitivity and specificity of the assay in samples obtained from group IV animals : A, foxes (n = 40) with no E. multilocularis infection; B1, foxes (n=28) with 1-100 E. multilocularis; B2 foxes (n=

18), with 101-1000 E. multilocularis; B3 foxes (n= 13) with > 1000 E. muItilocularis. The cut-off point ( - - ) was determined by calcu- lating the mean A4o s nm value + 3 SD for group A

the detection ofEchinococcus spp. was 4 6 % for E. granu- losus infections in dingoes a n d 56% for infections in dogs; for animals h a r b o r i n g >_ 200 worms, the sensitivity was 87%. The diagnostic sensitivity was 4 2 % for E.

multilocularis infections in foxes (18% for animals harboring < 100 worms, 39% for those bearing 100 1,000 w o r m s and 100% for those containing > 1,000 worms).

Assessment o f the overall test specificity

The overall specificity of the E L I S A as determined using 155 samples obtained f r o m carnivores that were free o f infection or h a r b o r e d experimentally or naturally acquired heterologous helminth infections (group I, II, I I I and IV) was found to be 98%. The samples were collected f r o m animals that were n o t infected with Echinococ- cus spp., animals showing no helminth infection (n = 37) and animals that were infected with non-Echinococcus cestodes (n = 76) or with various n e m a t o d e species (n = 42). Cross-reactions were detected in two samples taken f r o m dogs (Figs. 1, 3) and in one obtained f r o m a dingo (Fig. 4); the animals h a d been infected with 7, 4 and i T. hydatigena, respectively. O f the total o f 324 samples used in the present study, only 3 could not be accurately assessed due to a strong reaction with the control I g G ; these samples had been collected f r o m a fox infected with > 1,000 E. multilocularis and f r o m two stray dogs that showed no Echinococcus infection.

Discussion

This report addresses the diagnostic potential of an EL- I S A for the detection o f soluble metabolic, intestinal- stage antigens of Echinococcus granulosus or E. multilo- cularis in feces. The use o f h y p e r i m m u n e antibodies enabled the development o f a test with high specificity (98%), which represents a m a r k e d i m p r o v e m e n t as com- p a r e d with c o p r o s c o p y , in which eggs o f taeniid cestodes are morphologically indistinguishable. The test is easier to p e r f o r m t h a n other immunological approaches for the identification o f the generic origin of taeniid eggs via the use o f m o n o c l o n a l antibodies directed against oncospheres hatched in vitro (Craig et al. 1986). In contrast to serological methods, whereby specific antibodies persist after the elimination of the parasite, coproantigens are excreted only during or shortly after the termi- nation o f a current infection. The potential danger to investigators working with feces c o n t a m i n a t e d with Echinococcus eggs is minimal in c o m p a r i s o n with that to workers engaged in coproscopy. Risks can be further reduced if the eggs are first killed by freezing of fecal samples at - 70 ~ to - 80 ~ C for at least 48 h (Blunt et al.

1991; Eckert et al. 1991). We especially addressed the logistical a p p r o a c h to the detection of E/S E. granulosus antigens, as we assumed that these would be m o r e con- stantly released t h a n somatic antigens. F o r this purpose, w o r m s with i m m a t u r e eggs were cultivated in a defined m e d i u m in vitro. As described by T h o m p s o n and Eckert (1982), no apolysis o f proglottids was observed during the in vitro cultivation, suggesting that the parasite antigens in the s u p e r n a t a n t were mostly metabolic products.

The diagnostic sensitivity o f this E L I S A was found to be closely d e p e n d a n t on the Echinococcus w o r m burden in natural and experimental infections, which pres- ents problems for the sensitivity of the test that have not yet been solved. In this context, it has been shown that Taenia hydatigena coproantigens remain stable for at least 5 days in feces at 25 ~ C (Deplazes et al. 1990).

(6)

Our experimentally infected dogs were fed daily and reg- ular defecation was observed. In contrast, wild carnivores are k n o w n to have irregular and very different feeding habits; this m a y indirectly affect the digestive activity and the time required for passage o f the ingested meal through the gut. Such physiological factors could reduce the stability of E/S antigens and m a y influence the sensitivity o f the test system.

In experimental infections with high numbers o f worms, coproantigens could first be detected on day 5 p.i. for E. multilocularis and on day 10 p.i. for E. granu- losus. At this time, the length o f E. granuIosus does not exceed 0.7 m m ( T h o m p s o n 1975) and the lateral excretory canals become conspicuous with the first visible signs o f proglottid development ( T h o m p s o n 1986). Little is k n o w n a b o u t the metabolic activity o f Echinococcus at different stages o f its development in the gut. During the prepatent period (up to day 35 p.i.), a steady growth o f the worms has been described ( T h o m p s o n 1975).

However, there is no information on w o r m growth or the rate of proglottisation as indicators o f metabolic activity during the patent period. In one study, after the first appearance o f E. granulosus eggs in the feces o f experimentally infected dogs, the subsequent release o f eggs was delayed by 4 6 weeks (Yamashita et al. 1956).

Such latent periods involving a lack of egg or proglottid excretion have also described in experimental infections of dogs with T. hydatigena (Deplazes and Eckert 1988).

In the same investigation, the patent periods were reduced after reinfection and were often followed by latent periods during which no eggs and/or proglottids were excreted for several months.

The high specificity o f the present E L I S A suggests good diagnostic potential, provided that technical devel- opments improve its relatively low sensitivity. This m a y be a p p r o a c h e d by the molecular identification and purification o f the crucial antigenic parasite components, which could then be followed by the generation of the respective monoclonal antibodies to be used in the assay.

Acknowledgements. We thank Mrs. J.S. Skaggs, Mrs. I. Tanner and Mr. A. Rfidemann for their technical assistance and Dr. K.

Wolff for providing routine diagnostic samples.

References

Allan JC, Craig PS (1989) Coproantigens in gut tapeworm infections : Hymenolepis diminuta in rats. Parasitol Res 76: 68-73 Allan JC, Avila G, Garcia Noval J, Flisser A, Craig PS (1990)

Immunodiagnosis of taeniasis by coproantigen detection. Par- asitology 101 : 473-477

Babos S (1962) Untersuchungen fiber die Serodiagnostik der Echin- okokkose. Angew Parasitol 3 : 2M

Baumann D, Gottstein B (1987) A double-antibody sandwich EL- ISA for the detection of Entamoeba histolytica antigen in stool samples of humans. Trop Med Parasitol 38:81-85

Blunt DS, Gubrud JA, Hildreth MB (1991) Lethal effects of freezing Echinoeoceus multilocularis eggs at a low temperature. Ab- stract 205, 66th Annual Meeting of the American Society of Parasitology, Madison, Wisconsin, August 4-8

Craig PS, Macpherson CNL, Nelson GS (1986) The identification of eggs of Echinoeoeeus by immunofluorescence using a specific

antioncospheral monoclonal antibody. Am J Trop Med Hyg 35:152-158

Deplazes P, Eckert J (1988) Untersuchungen zur Infekfion des Hundes mit Taenia hydatigena. Schweiz Arch Tierheilkd 128:307-320

Deplazes P, Gottstein B, Stingelin Y, Eckert J (1990) Detection of Taenia hydatigena coproantigens by ELISA in dogs. Vet Par- asitol 36:91-103

Deplazes P, Eckert J, Pawlowski ZS, Machowska L, Gottstein B (1991) An ELISA for diagnostic detection of Taenia saginata coproantigens in humans. Trans R Soc Trop Med Hyg 85:391- 396

Eckert J, Gemmell MA, Soulsby EJL, Matyas Z (eds) (1984) Guidelines for surveillance, prevention and control of echinococcosis/hydatidosis. World Health Organization, Geneva, pp 1-147

Eckert J, Thompson RCA, Michael SA, Kumaratilake LM, E1- Sawah HM (1989) Eehinoeoceus granulosus of camel origin:

development in dogs and parasite morphology. Parasitol Res 75 : 53(~544

Eckert J, Deplazes P, Ewald D, Gottstein B (1991) Parasitologische und immunologische Methoden zum Nachweis yon Echinococ- cus multilocularis bei Ffichsen. Mitt 13sterr Ges Tropenmed Par- asitol 13:25-30

Gasser RB, Lightowlers MW, Obendorf DL, Jenkins DJ, Rickard MD (1988) Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens. Aust Vet J 65: 369-373

Gottstein B (1985) Purification and characterisation of a specific antigen from Echinococcus multilocularis. Parasite Immunol 7:201-212

Gottstein B, Mowatt MR (1991) Sequencing and characterization of an E. multilocularis DNA probe and its use in the polymerase chain reaction. Mol Biochem Parasitol 44: 183-194

Gottstein B, Deplazes P, Eckert J, Mfiller B, Schott E, Helle O, Boujon P, WolffK, Wandeler A, Schwiete U, Moegle H (1991 a) Serological (Em2-ELISA) and parasitological examinations of fox populations for Echinococcus multilocularis infections. Zen- tralbl Veterinfirmed [B] 38:161-168

Gottstein B, Deplazes P, Tanner I, Skaggs JS (1991b) Diagnostic identification of Taenia saginata with the polymerase chain reaction. Trans R Soc Trop Med Hyg 85:248-249

Jenkins DJ, Rickard MD (1985) Specific antibody response to Taenia hydatigena, Taenia pisiformis and Eehinococcus granulo- sus infection in dogs. Aust Vet J 62: 7~78

Jenkins DJ, Rickard MD (1986) Specific antibody responses in dogs experimentally infected with Echinococcus granulosus. Am J Trop Med Hyg 35:345 349

Jenkins D J, Morris B (1991) Unusually heavy infections of Echino- coccus granulosus in wild dogs in south-eastern Australia. Aust Vet J 68 : 36-37

Jenkins D J, Gasser RB, Zeyhle E, Romig T, Macpherson CNL (1990) Assessment of a serological test for the detection of Echinococcus granulosus infection in dogs in Kenya. Acta Trop (Basel) 47: 245~48

Jenkins DJ, Gasser RB, Romig T, Zeyhle E (1991) Antibody response against natural Taenia hydatigena infection in dogs in Kenya. Int J Parasitol 21:251-253

Thompson RCA (1975) Studies on equine hydatidosis in Great Britain. PhD Thesis, University of London

Thompson RCA (1986) Biology and systematics of Echinococcus.

In: Thompson RCA (ed) The biology of Echinococcus and hydatid disease. Allen & Unwin, London, pp 5-43

Thompson RCA, Eckert J (1982) The production of eggs by Echin- ococcus multilocularis in the laboratory following in vivo and in vitro development. Z Parasitenkd 68 : 227-234

Yamashita J, Ohbayashi M, Konno S (1956) Studies on echinococcosis: III. On experimental infections in dogs, especially on the development of Echinocoecus granulosus (Batsch, 1786). Jpn J Vet Res 4:113-122