Immunochemical characterization of in vitro culture-derived antigens of Babesia bovis and Babesia bigemina

Cross-reactivity between Babesia bovis and B. bigemina becomes a problem in discrimination of the two infections in endemic areas where the two species usually occur in association. With the aim of identifying candidate proteins for use as specific diagnostic tools, culture-derived components of three geographically different stocks of B. bovis (Lismore, Kwanyanga and Mexico) and one of B. bigemina (Mexico) were analyzed by immunoprecipitation using acrylamide gel electrophoresis. The approach taken was based on the analysis of S-35-methionine-labelled parasite antigens released into culture supernatant. A variety of serum samples were tested, including a panel of calf sera experimentally produced against the different stocks of Babesia, serum samples from cattle naturally infected in the field in Brazil, and a panel of anti-B. bovis monoclonal antibodies, previously characterized by the indirect fluorescent antibody test, ELISA and Western immune-blotting. Approximately 28 and 23 bands (with molecular weights ranging from 200 to 14 kDa) were detected in total protein profiles of B. bovis and B. bigemina culture supernatants, respectively, whereas no bands were seen in the uninfected red blood cell culture supernatant (negative control). The immunoprecipitation analysis showed antigenic diversity amongst the stocks of B. bovis and resulted in identification of at least five B. bovis specific antigens common to the three stocks (molecular weights of 80, 72, 58, 38 and 24 kDa) and four B. bigemina specific antigens (molecular weights of 240, 112, 50 and 29 kDa).