Genetic Characterization and Phylogenetic Analysis of Babesia bigemina Isolates in Cattle from South Africa Based on BgRAP-1, BgAMA-1 and BgβTUB Genes

<i>Babesia bigemina</i> is one of the most important etiological agents of bovine babesiosis, a tick-borne disease posing a major threat in the livestock industry globally, including South Africa. Despite the huge economic impact of cattle babesiosis in South Africa, antigenic variation observed among <i>B. bigemina</i> strains worldwide has impeded the successful development of a single vaccine with the potential to eliminate the disease. As such, there is still a dearth of information regarding the conservation of <i>B. bigemina</i> genes encoding functionally important proteins that play a crucial role during the invasion of bovine erythrocytes by merozoites. Fifty blood samples previously collected from cattle in eight provinces of South Africa were genetically tested for the presence of <i>B. bigemina</i> DNA fragments using four nested PCR-based assays. The genes targeted coded for <i>Spe</i>I-<i>Ava</i>I restriction fragment, rhoptry-associated protein 1 (<i>BgRAP-1</i>), apical membrane antigen 1 (<i>BgAMA-1</i>) and β-tubulin (<i>BgβTUB</i>). PCR-generated fragments of randomly selected samples were sequenced. BLAST searches in GenBank were performed with newly determined sequences to search for homologous sequences. Neighbor-joining phylogenies were inferred from aligned, contiguous sequences of <i>BgRAP-1</i>, <i>BgAMA-1</i> and <i>BgβTUB</i> genes. Nested PCR assays generated single fragments of 170 bp, 472 bp, 765 bp and 302 bp for <i>Spe</i>I-<i>Ava</i>I, <i>BgRAP-1</i>, <i>BgAMA-1</i> and <i>BgβTUB</i> fragments, respectively. Of the 50 bovine samples tested by nested PCR, 82% (42/50; 95% CI = 69.2-90.2%), 68% (34/50; 95% CI = 54.2-79.2%), 50% (25/50; 95% CI = 36.6-63.4%) and 46% (23/50; 95% CI = 33.0-59.6%) possessed <i>B. bigemina</i>-specific <i>Spe</i>I-<i>Ava</i>I, <i>BgRAP-1</i>, <i>BgAMA-1</i> and <i>BgβTUB</i> DNA fragments, respectively. The <i>BgRAP-1</i>, <i>BgAMA-1</i> and <i>BgβTUB</i> sequences of South African <i>B. bigemina</i> isolates shared 98-100% similarity with previously reported sequences of strains originating from cattle in countries other than South Africa. The high genetic conservation observed among geographical isolates of <i>B. bigemina</i> suggests the conserved functional role of BgRAP-1 and BgAMA-1 proteins as potential candidates that could be incorporated in recombinant subunit vaccines.