| Summary: | <i>Blastocystis</i> is a common intestinal protist in humans and animals worldwide. Wild and domestic animals are thought to be reservoirs of <i>Blastocystis</i> subtypes that also infect humans. There are limited studies on the prevalence and subtype distribution of <i>Blastocystis</i> in horses. In this study, 185 fecal samples were collected from horses (1 month to 17 years of age) in four regions of Colombia (Sabana de Bogotá, Costa Atlántica, Llanos Orientales, and Bogotá D.C.). <i>Blastocystis</i> presence and subtypes were determined by PCR and next generation amplicon sequencing. Eighty-one (43.8%) horses were positive for <i>Blastocystis</i>, with positive horses in all four regions. Molecular characterization identified 12 <i>Blastocystis</i> subtypes, 10 known subtypes (ST1, ST3–ST6, ST10, ST14, ST25, ST26), and 2 novel subtypes (ST33 and ST34). The validity of the novel subtypes was confirmed via phylogenetic and pairwise distance analyses of the full-length <i>SSU</i> rRNA gene sequences. Mixed subtype infections were common (55.6% of <i>Blastocystis</i>-positive horses). ST10 was the most prevalent subtype, present in 82.8% of <i>Blastocystis</i>-positive horses. Potentially zoonotic subtypes were identified in 88.9% of the <i>Blastocystis</i>-positive horses. This constitutes the most comprehensive study of <i>Blastocystis</i> in horses. Our findings indicate that horses harbor potentially zoonotic subtypes and could contribute to the transmission of <i>Blastocystis</i> to humans.
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