In vitro cultivation of Babesia caballi genotype B2 and its genetic, growth, and morphological characterization.

The genotypic diversity of Babesia caballi, comprising three genotypes, namely A, B1, and B2, presents challenges for managing equine piroplasmosis. Therefore, research on individual B. caballi genotype is essential, but has been constrained by the lack of in vitro cultures representing genotypes other than the widely studied genotype A. In the present study, we established in vitro cultures of two B. caballi isolates from donkeys in Sri Lanka. Based on 18S rRNA and rhoptry associated protein-1 (rap-1) gene sequences, the isolates were identified as genotypes B2 and A, and designated NRCPD-B2 and NRCPD-A, respectively. NRCPD-B2 represents the first successful in vitro culture of a genotype B2 strain. Genetic characteristics, growth dynamics, and morphological features of NRCPD-B2 were evaluated in comparison with NRCPD-A and the long-term culture-adapted USDA strain (genotype A). Analyses of nuclear and mitochondrial gene sequences revealed substantial divergence between genotype B2 and genotype A, suggesting a deeper evolutionary separation than previously recognized. While the NRCPD-B2 and NRCPD-A grew similarly in vitro, both had lower parasitemia than the USDA strain, likely reflecting long-term adaptation of the latter to in vitro conditions. Microscopic examination revealed that the dimensions of paired and single pyriforms, as well as ring forms, were comparable among all three strains. The establishment of NRCPD-B2 in the present study provides the first in vitro platform for genotype B2 and will support improved diagnostics, genotype-specific drug testing, and refinement of B. caballi taxonomy.