Abstract

An enzyme-linked immunosorbent assay (ELISA) has been previously described for detection and quantification of antibody specific for the bacterium Erysipelothrix rhusiopathiae in Atlantic bottlenose dolphin (Tursiops truncatus). In this assay, extracted (Frasch method) E. rhusiopathiae antigens were coated to the wells of a polystyrene microtiter plate (as capture antigen) to bind dolphin antibodies reactive with the bacterial antigens. Bound dolphin antibodies were then quantified using an indicator system consisting of biotin-labeled rabbit anti-T. truncatus IgG, avidin-labeled alkaline phosphatase and p-nitrophenyl phosphate. In the development of this assay, it is desirable to identify an E. rhusiopathiae capture antigen preparation that has minimal cross-reactivity with other bacterial antigens. Ideally this goal would be accomplished by assaying serum samples from individuals of the target species (T. truncatus) that had been immunized with antigen preparations derived from bacterial species of concern, or were known to contain antibodies against these bacterial species. Since such cetacean serum samples were unavailable, a mouse model was used to evaluate the ability of the ELISA to discriminate between antibodies in animals exposed to E. rhusiopathiae,, to other bacterial species closely related to E. rhusiopathiae, or two bacteria (unrelated to E. rhusiopathiae) commonly found in the environment of cetaceans. Seventeen groups of three mice each were immunized subcutaneously with 30 µl of a 50:50 solution of heat-killed bacteria (1.0 x 10⁶ cells/ml) and TiterMax® Gold Research Adjuvant (CytRx Corporation). Each of the seventeen groups were injected with one of the following bacterial preparations: one of the four field isolates of E. rhusiopathiae (designated NRaD, Shedd 2, Mystic, MWA) cultured from deceased cetaceans, one of three ATCC strains of E. rhusiopathiae (Bergey's Manual reference strain, serotype 1a, or serotype 2), E. tonsillarum, Listeria monocytogenes, Corynebacterium renale, C. bovis, Brochothrix thermosphacta, B. campestris, Kurthia gibsonii, K. zopfii, Staphylococcus aureus, Escherichia coli. The serum from these mice was collected twenty-one days post immunization and assayed using a slightly modified form of the ELISA (biotin-labeled rabbit anti-mouse IgG was used instead of biotin-labeled rabbit anti-T. truncatus IgG). For serum from each group of mice, titers were determined against all bacterial antigens used in the study.