Identification of IgG2-specific antigens in Mexican Anaplasma marginale strains

To identify novel antigens with immunoglobulin G2 (IgG2) specificity and immunostimulant properties for bovine Th1 cells, humoral and cellular responses were studied in cattle inoculated with initial bodies from a Mexican isolate of Anaplasma marginale and challenged with a heterologous strain. Analysis of post-immunization sera by ELISA and assaying of in vitro cellular responses in peripheral blood mononuclear cells (PBMCs) cultured in the presence of protein extracts from three Anaplasma marginale strains showed positive values of optical density ELISA readings and stimulation indices in the immunized but not control cattle. Post-immunization and post-challenge sera recognized in Western blots several proteins with molecular weights ranging from 15 to 209 kDa, twelve of which were recognized by IgG2 in the three Anaplasma marginale strains. Seven of these are novel and have not been previously reported for their IgG2 specificity; three are confirmed to be major surface proteins (MSP-1a, MSP-2, and MSP-5); and the others correspond to other well-studied MSPs but were not confirmed. Partially purified fractions of protein extracts of the Mex-17 strain were tested against PBMCs cultured in vitro. One out of the seven novel proteins induced detectable lymphoproliferation (LP) of PBMCs, and interferon-gamma was detected in supernatants of PBMC cultured in the presence of two protein fractions, including the one that caused LP. It is concluded that novel antigens, particularly the 28-kDa protein, played an additional role in the protection of immunized cattle and should be considered vaccine candidates after in vivo immunization experiments are concluded.