Babesia bovis msa-1 and msa-2c genes belong to the variable merozoite surface antigen gene family. These genes code for antigenic proteins present on the merozoite surface (MSA) and are involved in the parasite invasion to the bovine erythrocyte. Previous studies carried out on MSA-1 have evidenced antigen allelic variation in B. bovis isolates from similar endemic regions, as well as in isolates from different geographic regions of the world (Argentina, Australia, Israel). Studies conducted on MSA-2c, however, have shown that this antigen is widely conserved on isolates from distinct geographic regions. In this study, it was hypothesized that MSA-1 and MSA-2c antigens would contain common epitopes despite the presence of nucleotide sequence differences found in 13 B. bovis isolates and strains collected in geographically distant regions of Mexico. Bioinformatics analysis of the primary structure from DNA fragments derived from PCR amplification, cloning, and sequencing of msa-1 and msa-2c genes from the 13 B. bovis populations revealed that the msa-1 gene product present in the various isolates tested is less conserved among isolates obtained within a similar geographic region in Mexico (51-99.7% sequence identity). Results obtained by immunoblot analysis of B. bovis protein extracts reacted with a monoclonal antibody to MSA-1 42-kDa antigen, conclusively showed cross-reactive common epitopes only in Mexican isolates having high sequence identity (>/=99%, eight isolates). Sequence analysis and multiple alignment of deduced MSA-2c demonstrated a high degree of sequence identity (90-100%) among the Mexican B. bovis isolates and strains. Immunoblot results using a polyclonal antibody to MSA-2c reacted against the protein extracts recognized conserved epitopes in at least nine of the B. bovis isolates. The results obtained in this study agree with those previously reported by other researchers and confirm that, based in sequence identity conservation in Mexican B. bovis isolates and strains so far collected and analyzed, MSA-2c represents an ideal antigen worth evaluating as a vaccine candidate.

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2), and msa-2b). Small subunit ribosomal (ssr)RNA gene is subject to evolutive pressure and has been used in phylogenetic studies. To determine the phylogenetic relationship among B. bovis Mexican isolates using different genetic markers, PCR amplicons, corresponding to msa-1, msa-2c, msa-2b, and ssrRNA genes, were cloned and plasmids carrying the corresponding inserts were sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 12 geographically different B. bovis isolates and a reference strain. Overall sequence identities of 47.7%, 72.3%, 87.7%, and 94% were determined for msa-1, msa-2b, msa-2c, and ssrRNA, respectively. A robust phylogenetic tree was obtained with msa-2b sequences. The phylogenetic analysis suggests that Mexican B. bovis isolates group in clades not concordant with the Mexican geography. However, the Mexican isolates group together in an American clade separated from the Australian clade. Sequence heterogeneity in msa-1, msa-2b, and msa-2c coding regions of Mexican B. bovis isolates present in different geographical regions can be a result of either differential evolutive pressure or cattle movement from commercial trade.

The purpose of this research was to evaluate the recombinant proteins MSA-1, MSA-2c and 12D3 as a combined immunogen for cattle. Fifteen steers were randomly assigned into three groups of five animals each (I, II and III). On day 0, cattle in group I were injected with 50 microg each of rMSA-1, rMSA-2c and r12D3 with the adjuvant Montanide 75; cattle in Group II received adjuvant-PBS, and Group III were untreated controls. On day 14, cattle in Group I received a second injection of the three recombinant proteins in adjuvant and cattle in Group II again received adjuvant alone. On day 28, all groups of cattle were challenged with a field strain of Babesia bovis. After challenge, the experimental cattle were clinically and serologically monitored. Three of the five steers immunized with the combined recombinant B. bovis proteins seroconverted on day 14 post-immunization (P.I.) and the maximum titre was 1 : 1600. All five immunized steers presented strong seropositivity to B. bovis antigens at day 21 P.I. The prepatent periods of vaccinated cattle were delayed until day 10 post-challenge exposure versus 8 and 7 days in Groups II and III, respectively. Cattle in all groups had fever above 41 degrees C; the reduction in packed cell volume was not significantly different (P > 0.05) in vaccinated group I compared with Groups II and III (29% versus 26% and 31%, respectively). Treatment was required for one steer in the control group. During the period of the study, the weight of cattle in Groups I and II increased an average of 9 and 7 kg, whereas the weight of the control cattle was reduced on average 4 kg. Immunization with rMSA-1-rMSA-2c-r12D3 proteins was not sufficient to prevent clinical symptoms against challenge, but the immunologic response was sufficient to protect steers against a mild virulent strain of B. bovis.