Identification of 23 kD Immunogen from Native Antigens of Babesia bigemina in Splenectomized Calf

Primers were designed to amplify 18S RNA sequences of Babesia bigemina and B. bovis. Blood samples were collected from naturally infected calves. PCR products of sizes 321 bp and 269 bp, were obtained for B. bigemina and B. bovis, respectively. Infected RBCs (iRBCs) were then, cultured in in vitro to achieve >10% parasitemia of B. bigemina. Splenectomized calf was infected with 1x10(8) iRBCs. Three intact calves aged 4 to 6 months were infected with iRBCs with B. bigmenia from splenectomized calf. Then, merozoites of B. bigemina were harvested from iRBCs of splenectomized calf and were analyzed through immuno-blotting through homologous and heterologous sera collected from field and experimentally infected calves. SDS-PAGE was performed to analyze the protein bands of native antigens of B. bovis and B. bigemina. The gel was transferred on to nitrocellulose membrane containing native antigens of B. bigemina. The membrane was immuno-blotted with serum sample from B. bigemina-positive animal and it showed a clear band of about 23 kD, while this band was absent on membranes which were incubated with sera from B. bovis-infected calf and negative control animal Further, native antigens of B. bigemina were coated for optimization of indirect ELISA with homologous serum of experimentally infected intact calves. Cut off point >= 0.4893 was considered positive. We obtained antibody titer of 149 +/- 97.8 to 299 +/- 196 in wells of microtiter plate coated with 5 mu g/mL of native antigens while this titer was from 597 +/- 391 to 1195 +/- 782 in wells coated with 10 ,ug/mL of antigens. This putative protein would need to be characterized in future to validate its diagnostic value for screening field samples. (C) 2020 Friends Science Publishers