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 noninfected llamas were use d in initial test development. Nitrocellulose filter disks containing F. hepatica excretory-secretory product were placed in 96-well microti ter plates, washed, blocked with Tween-20, then incubated with four-fo ld serial dilutions of llama sera. After incubation with rabbit anti-l lama IgG followed by peroxidase-conjugated goat anti-rabbit IgG, addit ion of precipitable substrate resulted in purple dots on white backgro und (positives) easily read by eye. The technique was further evaluate d at titers of 1:512 using an additional six known positive and eight known negative llamas. Test results showed 6/6 known positive as posit ive and 8/8 known negative as negative. Sera were collected, at approx imately weekly intervals, from three llamas experimentally infected wi th F. hepatica. The dot-ELISA detected antibodies to F. hepatica as ea rly as the second week post-infection in all llamas. In a serologic su rvey of 256 llamas from an F. hepatica endemic area, the dot-ELISA det ected antigen-specific serum antibodies to F. hepatica in 42 (16%) of the llamas. Although no difference was noted in antibody prevalence be tween sexes, prevalence increased in llamas over 6 months of age.