Molecular detection and phylogenetic characterization of tick-borne pathogens in Anatolian water buffalo (Bubalus bubalis)

Tick-borne pathogens (TBPs) represent a major constraint to livestock health and productivity worldwide. Water buffaloes (Bubalus bubalis) are an important livestock species in Türkiye; however, molecular data on TBPs in this host remain limited. This study aimed to investigate the prevalence and molecular diversity of Babesia, Theileria, Anaplasma, and Ehrlichia species in water buffaloes from Iğdır Province, Eastern Türkiye. A total of 200 blood samples were analysed using genus-specific PCR assays targeting the 18S and 16S rRNA genes, followed by Reverse Line Blot (RLB) hybridisation and Sanger sequencing. Genus-level prevalence was 28.0% for Theileria spp., 6.0% for Babesia spp., and 2.5% for Anaplasma spp., while Ehrlichia spp. were not detected. Six species were identified: Theileria annulata (22.0%), T. ovis (5.0%), T. buffeli (1.0%), Babesia canis (5.0%), B. ovis (1.0%), and Anaplasma phagocytophilum (2.5%). This study provides the first molecular evidence of B. canis and B. ovis DNA in water buffaloes. However, the detection of parasite DNA alone does not confirm active infection and likely reflects incidental or spillover events associated with shared tick vectors in multi-host grazing systems. Phylogenetic analyses based on 18S and 16S rRNA genes revealed high sequence similarity with isolates from neighbouring regions, supporting regional circulation of closely related strains. These findings highlight the exposure of Anatolian water buffaloes to a broad spectrum of TBPs and underline the need for integrated studies combining vector surveillance, multilocus genotyping, and serological approaches to clarify host competence and transmission dynamics.