In vivo binding of immunoglobulin M to the surfaces of Babesia bigemina-infected erythrocytes.

Babesia bigemina infection of mature bovine erythrocytes results in new proteins specifically exposed on the parasitized cell surface. Monoclonal antibody (MAb) 64/32 binds a protein, designated p94, on B. bigemina-infected erythrocytes but not on either uninfected or B. bovis-parasitized erythrocytes. However, p94 was not encoded by B. bigemina and was not a parasite-modified erythrocyte membrane protein. In contrast, we showed that p94 could be eluted from the infected erythrocyte surface and was identified as specifically bound immunoglobulin M (IgM) heavy chain for the following reasons: (i) MAb 64/32 bound a reduced molecule of 94 kDa in both infected erythrocyte lysates and normal bovine serum; (ii) MAb 64/32 bound a 94-kDa molecule in reduced preparations of purified IgM; (iii) an anti-bovine mu heavy-chain MAb, BIg73, reacted specifically with the surface of infected erythrocytes and bound the 94-kDa molecule in lysates of infected erythrocytes, normal bovine serum, and purified IgM; and (iv) immunoprecipitation of infected erythrocyte lysates with MAb 64/32 depleted the 94-kDa antigen bound by anti-mu MAb BIg73 and vice versa. Binding of IgM to the infected erythrocyte surface was detected in vivo early in acute parasitemia and occurred during both the trophozoite and merozoite stages of intraerythrocytic parasitism. The common feature of IgM binding to the parasitized erythrocyte surface among otherwise genetically and antigenically distinct B. bigemina strains is suggestive of an advantageous role in parasite survival in vivo.

Viability after thawing and dilution of simultaneously cryopreserved vaccinal Babesia bovis and Babesia bigemina strains cultured in vitro.

A live, frozen experimental vaccine containing Babesia bovis and Babesia bigemina multiplied in vitro was stored in liquid nitrogen after simultaneous cryopreservation using glycerol as cryoprotectant. The viability of the vaccine was tested by inoculating (subcutaneously) three groups of seven steers each, 2, 12 and 24 h after thawing at 40 degrees C and dilution to obtain a dose of 2 x 10(7) of each organism. All vaccinated cattle developed detectable parasitaemia in thin and/or thick blood smears. No statistically significant differences in the prepatent period were detected amongst the cattle groups (analysis of variance). This prepatent period was 12.3 days for B. bovis and 8.4 days for B. bigemina. Vaccinal organisms derived from in vitro culture systems may replace antigens obtained from in vivo culture to produce vaccine against cattle babesiosis.

Live and soluble antigens for cattle protection to Babesia bigemina.

Attenuated Babesia bigemina were multiplied in vivo and in vitro to vaccinate two groups (Groups 2 and 3) of Holstein Friesian heifers. Another group (Group 1) of heifers was vaccinated twice with purified soluble antigens obtained from the supernatant of in vitro culture combined with saponin. All these heifers plus controls (Group 4) were inoculated with heterologous pathogenic B. bigemina 5 months later. Heifers vaccinated with live organisms (Group 2 and 3) were able to stand the challenge without specific treatment whereas the opposite occurred in heifers vaccinated with soluble antigens (Group 1) and controls (Group 4). Antibody titres were higher in heifers inoculated with soluble antigens than in heifers inoculated with live B. bigemina multiplied in vivo, indicating that antibody titres may not be a proper indicator of protection. In vitro culture of this protozoan is probably a better source of live antigens for vaccine production than in vivo culture. These kind of immunogens can fill the gap until improved vaccines are available.