Molecular characterization of Babesia ovis in an enzootic focus in eastern Bosnia and Herzegovina

Abstract Purpose Babesia ovis , the agent of malignant ovine babesiosis, is transmitted by Rhipicephalus bursa within discrete enzootic foci. Whether the parasite varies genetically within a single focus and between transmission seasons is unknown. We examined 18S rRNA gene diversity within the enzootic focus of Rudo, eastern Bosnia and Herzegovina. Methods Blood samples were collected from 41 clinically or subclinically infected sheep from 15 flocks in seven villages (approximately 36 km²) during four transmission seasons (2020, 2021, 2025 and 2026). A 549 bp fragment of the 18S rRNA gene was amplified by PCR and sequenced bidirectionally. Diversity indices, a TCS haplotype network and a UPGMA dendrogram were computed, and the association of haplotype with village and season was tested with chi-square and Fisher–Freeman–Halton exact tests. Results Six haplotypes (H1–H6) with eight polymorphic sites were recovered. Genetic diversity was low (haplotype diversity 0.313 ± 0.093; nucleotide diversity 0.00098). H1 occurred in all villages and seasons and was identical to 23 of 48 reference sequences from eight countries. H4 matched the Turkish isolate MN611759. Each derived haplotype (H2–H6) was found in a single village and season, but neither association was statistically significant (exact test, P = 0.82 and P = 0.62, respectively). Conclusion The 18S rRNA gene of B. ovis is not uniform within an enzootic focus. Rare haplotypes appeared sporadically, so repeated sampling across seasons is needed to capture local diversity, and higher-resolution markers are required to resolve the genetic structure of B. ovis within foci.