Diversity and population genetic analysis of the Babesia bovis merozoite surface Antigen 1 (MSA-1) gene in South African cattle isolates from field and clinical samples.

Babesia bovis causes significant losses in South African cattle, and antigenic variation in the merozoite surface antigen 1 (msa-1) complicates vaccine and diagnostic development. This study provides the first detailed molecular characterisation of msa-1 genetic diversity in South African cattle populations. Analysis of 18 successfully sequenced isolates (∼960 bp) revealed extensive polymorphism, with pairwise nucleotide sequence identities ranging from 30.1% to 100%, and amino acid similarities ranging from 37.6% to 100%. Population genetic metrics of the highly variable MSA-1 confirmed high nucleotide diversity (π = 0.37) and haplotype diversity (Hd = 0.94). Although these estimations are based on a modest sample size (n = 18), high levels of diversity highlight the extreme polymorphism of the msa-1 gene. A larger sample size in future studies would yield narrower confidence intervals and more precise estimates of these population genetic parameters. Phylogenetic analysis grouped the South African isolates into two of the three global clades (clades I and II). Clades I and II showed high heterogeneity among isolates, both within and between countries, unlike Clade III, which mainly contained Asian isolates. Codon-based Z-test demonstrated that the msa-1 gene undergoes strong positive selection in South Africa (Z = 5.25, p < 0.01), providing evidence of B. bovis diversification into different variants. Genetic differentiation at the continental and country levels showed moderate to very high subdivision (Fst = 0.10-0.48) between South African and global populations, and particularly high subdivision (Fst = 0.38-0.48) with Asian isolates. These findings provide initial evidence of the complex evolutionary dynamics of B. bovis in South Africa and corroborate that the high variability of msa-1 facilitates immune evasion and contributes to vaccine breakthrough. Future research should evaluate more conserved antigenic markers for effective vaccine development.