IDENTIFICATION OF BABESIA-BOVIS MEROZOITE ANTIGENS SEPARATED BY CONTINUOUS-FLOW ELECTROPHORESIS THAT STIMULATE PROLIFERATION OF HELPER T-CELL CLONES DERIVED FROM B-BOVIS-IMMUNE CATTLE

To characterize Babesia bovis merozoite antigens that stimulate anamne stic T helper (Th) cell responses from B. bovis-immune cattle, B. bovi s-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun; 60:4364-4372, 1992), were tested in p roliferation assays against fractionated merozoite antigens. The antig enic groups were determined by the patterns of response of Th clones t o different parasite isolates and soluble or membrane forms of merozoi te antigen. Soluble antigen fractionated by anion-exchange chromatogra pby or gel filtration by using fast-performance liquid chromatograpby resolved two or three antigenic peaks, respectively. To enable fractio nation of membrane-associated proteins and to resolve more precisely t he proteins present in homogenized inerozoites, a novel technique of c ontinuous-flow electrophoresis was employed. Merozoite membranes or wh ole merozoites were homogenized and solubilized in sodium dodecyl sulf ate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual f ractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel el ectrophoresis and tested for the ability to stimulate Babesia-specific CD4(+) T-cell lines and clones. CD4(+) Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous pe aks of antigenic activity, ranging from <14 to 76 kDa Th-cell clones p reviously categorized into different antigenic groups detected antigen ic peaks unique for clones representative of a given group. Antigens o f 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 2b kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VI I) were identified in the stimulatory fractions. Immunization of rabbi ts with selected fractions produced a panel of antisera that reacted s pecifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate an tigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clone s representative of five different antigenic groups. These antisera ma y be useful for isolating recombinant proteins that are immunogenic fo r Th cells of immune cattle and therefore potentially useful for vacci ne development.