title: An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3).
abstract: A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.
authors.affiliation: Centro de Investigaciones Agrarias, Mabegondo, PO Box 10, 15080 A Coruña, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Mezo
authors.forename: Marta
authors.initials: M
authors.lastname: González-Warleta
authors.forename: Carmen
authors.initials: C
authors.lastname: Carro
authors.forename: Florencio M
authors.initials: FM
authors.lastname: Ubeira
mesh_terms: D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000947:Antigens, Helminth / Q000032:analysis* / Q000378:metabolism; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000276:immunology; D002846:Chromatography, Affinity / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards / Q000662:veterinary; D005210:Fasciola hepatica / Q000276:immunology* / Q000302:isolation & purification; D005211:Fascioliasis / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary*; D005243:Feces / Q000469:parasitology; D015151:Immunoblotting / Q000662:veterinary; D007700:Kinetics; D008099:Liver / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D010270:Parasite Egg Count / Q000662:veterinary; D011817:Rabbits; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000276:immunology
chemical_list: D000909:Antibodies, Helminth; D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth