Rabbit anti-deer polyclonal antibody applied to the diagnosis of Mycobacterium avium subsp. paratuberculosis in red deer (Cervus elaphus)

Abstract

The diagnosis of Johne's Disease by isolating Mycobacterium avium subsp. paratuberculosis from feces requires 6 months and high-cost reagents, and therefore an indirect technique like ELISA, is often being used to avoid these obstacles. The aim of this work was the production of a rabbit anti-deer polyclonal antibody (anti-deer pAb) for use in an in-house ELISA, applying protoplasmic antigen paratuberculosis (PPA) as antigen. Two rabbits were immunized subcutaneously with three doses of deer semipurified gamma globulin emulsified with incomplete Freund's adjuvant. Rabbit serum was purified with protein A. Titers of anti-deer pAb against deer serum and cross-reaction against sera from other species were studied by ELISA and immunoblot. Samples of feces and sera from 16 deer were evaluated by fecal culture and the in-house PPA-ELISA. The results were analyzed statistically using ROC curves and the Kappa index to estimate sensitivity (Se), specificity (Sp) and the agreement level between both techniques. Subsequently, sera from three to five years old stags (n = 155) from a herd with reports of Johne's Disease were evaluated using the in-house PPA-ELISA. Anti-deer pAb showed a specific titer of 2.56 × 105. Cross-reaction against cattle, sheep, llama and goat was detected by ELISA and immunoblot. The in-house PPA-ELISA showed a Se = 85.71 % and Sp = 88.89 %. The agreement level with fecal culture was substantial (κ = 0.62 ± 0.19) and the assay was able to identify 76.77 % of the deer in the suspected herd as positive. The anti-deer pAb obtained was not only useful in the development of the in-house PPA-ELISA, which allows us to reduce the costs of Johne's Disease diagnosis, but it could also be used in other diagnosis tests in deer as well as other ruminant species.