Fasciola hepatica:Detection of Antigenemia and Coproantigens in Experimentally Infected Rats

Abstract

The presence of eggs in stool is the standardmethod for a fascioliasis diagnosis. However, itlacks sensitivity since parasite eggs do not ap-pear during acute fascioliasis and are intermit-tently shed during a chronic disease. As anti-body titers persist after curing, serological testsare also of limited diagnostic value (Garci´a-Rodri´guezet al. 1985; Espino et al. 1992). Alack of specificity has been observed due tocross-reactions between Fasciola and otherparasites (Hanna and Hillyer 1984; Hillyer andSoler de Galanes 1988). We have developed amonoclonal antibody (MAb-ES78, mouseIgG2a) sandwich ELISA to detect circulatingexcretory–secretory (ES) antigens (Espinoet al.1990) and coproantigens (Espino et al. 1994) inpatients with fascioliasis. In the present paperwe compare this technique with a conventionalantibody test during the course of an experimen-tal primary infection in rats.Twenty female, outbred Wistar strain rats,aged 6–8 weeks, were infected orally with 25metacercariae of Fasciola hepatica. Blood andstool samples were collected immediately be-fore infection and weekly for 16 weeks, afterwhich the rats were sacrificed and flukes col-lected from the liver. Parasitological diagnosiswas performed by direct wet mount examina-tion (Ash and Orihel 1987). Stool suspensionswere prepared from each specimen by mixing 1g with 3 ml of PBS containing 0.05% Tween20. These suspensions were centrifuged at 900 gfor 3 min and the supernatants collected andtested to measure coproantigen levels. Bloodsamples were centrifuged at 3000 g for 3 min at4°C. The sera were collected and tested bysandwich ELISA to detect circulating free anti-gens. MAb-ES78 sandwich ELISA was per-formed essentially as previously reported for thehuman model (Espino et al. 1994). Antibodylevels to F. hepatica ES antigens were mea-sured in an indirect ELISA following a proce-dure similar to that described previously(Espino et al. 1987) using a sheep peroxidase-labeled anti-rat conjugate.Prior to infection, antibodies to F. hepaticaES antigens were not detected (Fig. 1). Anti-bodies were detected in 80% of the rats 2 weeksafter infection and in all animals at 3 weekspostinfection, reaching absorbance value peaksbetween 6 and 10 weeks, while the mean absor-bance value in controls was significantly differ-ent (P < 0.01; Student t test). Antigenemia wasobserved from 1 to 3 weeks postinfection (Fig.2), which corresponds to the time when juvenileworms are actively migrating through the liverparenchyma excreting significant amounts ofantigens directly into the blood stream. At thistime absorbance values were significantly in-creased relative to control sera and to those ob-tained 5 to 16 weeks postinfection (P < 0.001).To determine approximate amounts of ES an-tigens in sera and stools of infected rats, absor-