Development and application of a dot-ELISA test for the detection of serum antibodies to Fasciola hepatica antigens in llamas.
A microenzyme-linked immunosorbent assay (dot-ELISA) was developed to detect serum antibodies against Fasciola hepatica antigens in llamas. Sera from five F. hepatica-infected and 11 non-infected llamas were used in initial test development. Nitrocellulose filter disks containing F. hepatica excretory-secretory product were placed in 96-well microtiter plates, washed, blocked with Tween-20, then incubated with four-fold serial dilutions of llama sera. After incubation with rabbit anti-llama IgG followed by peroxidase-conjugated goat anti-rabbit IgG, addition of precipitable substrate resulted in purple dots on white background (positives) easily read by eye. The technique was further evaluated at titers of 1:512 using an additional six known positive and eight known negative llamas. Test results showed 6/6 known positive as positive and 8/8 known negative as negative. Sera were collected, at approximately weekly intervals, from three llamas experimentally infected with F. hepatica. The dot-ELISA detected antibodies to F. hepatica as early as the second week post-infection in all llamas. In a serologic survey of 256 llamas from an F. hepatica endemic area, the dot-ELISA detected antigen-specific serum antibodies to F. hepatica in 42 (16%) of the llamas. Although no difference was noted in antibody prevalence between sexes, prevalence increased in llamas over 6 months of age.
Rickard
L G
LG
College of Veterinary Medicine, Oregon State University, Corvallis 97331, USA.
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood*; D000947:Antigens, Helminth / Q000276:immunology*; D002161:Camelids, New World / Q000276:immunology / Q000469:parasitology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005210:Fasciola hepatica / Q000276:immunology*; D005211:Fascioliasis / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary*; D005243:Feces / Q000469:parasitology; D005260:Female; D008297:Male; D009922:Oregon / Q000453:epidemiology; D015995:Prevalence; D012680:Sensitivity and Specificity
D000909:Antibodies, Helminth; D000947:Antigens, Helminth