This study aimed to serologically and molecularly survey Babesia caballi and Theileria equi in thoroughbred horses from racecourses in Chile. Additionally, the genetic diversity of the positive samples was assessed. A total of 286 thoroughbred horses from the Santiago and Valparaíso racecourses had their serum samples submitted to an ELISA for B. caballi and T. equi, and 457 samples (from the Santiago, Valparaíso, and Concepción racecourses) were tested with nested PCRs for the B. caballi 48 KDa rhoptry protein (RAP-1) and T. equi 18S rRNA genes. Selected RAP-1 and 18S positive products were sequenced to perform phylogenetic and haplotype analyses. An overall seroprevalence of 35.6% was observed for these Chilean racecourses: 23.7% for T. equi, 8.4% for B. caballi, and 3.5% for both agents. Overall, a 53.6% occurrence by nPCR was detected for the three Chilean racecourses: 44.2% for T. equi, 5.4% for B. caballi, and 3.9% for both agents. Phylogenetic analysis of T. equi and B. caballi showed genetic proximity with sequences previously detected in other countries. Haplotype analysis revealed a low diversity among the Chilean sequences, which may have originated from those reported in Brazil, Israel, or Cuba. Babesia caballi and T. equi were detected for the first time in Chilean thoroughbred horses.

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