Nationwide Investigation of Eukaryotic Pathogens in Ticks from Cattle and Sheep in Kyrgyzstan Using Metabarcoding

Ticks are major vectors of pathogens, including bacteria, viruses, and protozoa, posing serious risks to public health and agriculture. In Kyrgyzstan, tick-borne diseases are an emerging threat with significant health and economic implications. Although bacterial and viral tick pathogens are well-studied, the eukaryotic microbiota, particularly protozoa, remains understudied. This study examined the eukaryotic microbiota of ticks in Kyrgyzstan, focusing on protozoan pathogens, such as Babesia and Theileria. A total of 472 tick samples were collected from cattle and sheep across seven regions of Kyrgyzstan between March and July 2022. Tick species were identified through Sanger sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) gene. The eukaryotic microbiota was analyzed using next-generation sequencing (NGS) of the V9 region of the 18S rRNA gene. Sanger sequencing identified five genera and 11 tick species, while NGS revealed 266 eukaryotic taxa, with Babesia (13.3%) and Theileria (12.7%) being the most prevalent protozoan pathogens. Babesia was more common in sheep and nymph-stage ticks, whereas Theileria was detected across a broader range of tick species and hosts, with less variation across life stages. Geographic variation was observed, with the Osh region exhibiting the highest detection rates of Babesia in both sheep and cattle ticks. This study presents the first comprehensive analysis of the eukaryotic microbiota of ticks from Kyrgyzstan, focusing on the distribution of pathogenic protozoa. The findings highlight how regional and host-specific factors influence pathogen prevalence, providing a basis for future research and tick management strategies.