The molecular genotyping of Babesia caballi

Abstract

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis (EP). Three genotypes (A, B, and C) have been identified based on the 18S ribosomal ribonucleic acid (rRNA) and rhoptry-associated protein-1 (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WHOA-recommended serological assays used in declaring horses free of the disease. The spherical body protein 4 (SBP4) was recently identified as a potential antigen for serological detection of B. caballi, however, it remains uncertain whether it can effectively detect the various geographical strains of this parasite. The molecular distinction between variant B. caballi parasite genotypes is limited and therefore, this study aimed to develop sbp4 gene-based quantitative real-time polymerase chain reaction (qPCR) assays for the rapid detection and differentiation between B. caballi parasite genotypes. Retrospective DNA samples from horses and zebras were screened for the presence of B. caballi using an established 18S rRNA-based multiplex equine piroplasmosis qPCR assay. Phylogenetic analysis of sbp4 and 18S rRNA gene sequences confirmed the groupings of the South African isolates into either B. caballi genotypes B or C. Conserved regions in the sbp4 gene were identified through alignment with genotype A reference sequences, enabling the design of three genotype-specific qPCR assays. The B. caballi typing qPCR assays were shown to be efficient and specific in the detection and differentiation of the respective B. caballi genotypes. The 95% detection limit of Babesia caballi spherical body protein 4 gene (Bcsbp4)-based typing qPCR assays (Bcsbp4-A, Bcsbp4-B, and Bcsbp4-C) were determined as 2.67 x 103, 4.4 x 102, and 39 plasmid copies/μl, respectively. The developed B. caballi typing qPCR assays will contribute to the control and diagnosis of EP when used in conjunction with the existing Babesia caballi spherical body protein 4-based indirect enzyme-linked immunosorbent assay (BcSBP4-iELISA) to detect the variant B. caballi genotypes. Thus, preventing the spread of novel genotypes into new areas.