Identification, quantitation and immunogenicity of culture-derived soluble Babesia bigemina antigens

Soluble Babesia bigemina antigens were prepared from short-term in vitro cultures utilizing a microaerophilous stationary phase culture system. In two pilot immunization experiments involving 6 cattle, 3 doses of 25 ml-equivalent (838 EIA units) and 2 doses of 10 ml-equivalent (529 EIA units), respectively, of the crude culture-derived antigens administered with 1 mg of saponin (Quil-A) adjuvant, evoked primary and secondary antibody responses as detected and measured by the indirect fluorescent antibody (IFA) test and immunodiffusion tests.Immunoglobulin G (IgG) isolated from bovine hyper-immune serum was utilized to develop and standardize a sandwich enzyme immunoassay (EIA) for the quantitation of B. bigemina antigens. Polyethylene glycol at a 2% concentration was used to amplify the sensitivity of the assay. The specificity of the system was validated by the inhibition tests utilizing B. bigemina immune serum and by the lack of reactivity with other common hemoparasites. The assay was sensitive for detection of low concentrations of antigen in crude culture supernatants (28 ug total protein) and sera of acutely infected animals.Three antigens were demonstrated in the 24 h culture supernatant by analytical electrophoretic techniques and Western blots. The molecular weights of these antigens as estimated by SDS-PAGE and molecular sieve chromatography were in the range of 53-64 kd. Enzyme sensitivity and thermostability studies indicated the antigens were proteinaceous in nature.The three soluble antigens were also identified in the serum of acutely infected animals and in erythrocytes parasitized by B. bigemina. Partial purification of these antigens was achieved by anion-exchange and size exclusion high performance liquid chromatography.U of I OnlyETDs are only available to UIUC Users without author permissio