Molecular characterization of Rhipicephalus microplus and tick-borne pathogens in cattle in Nepal

Ticks and tick-borne pathogens (TBPs) have a significant economic impact on livestock. In Nepal, only limited information is available for the molecular identification of ticks and TBPs. This study aims to address this major challenge by providing data on the diversity of TBPs in Nepalese cattle, along with molecular sequences to facilitate accurate identification. A total of 145 cattle blood samples were collected from Pokhara (n = 70) and Madi (n = 75). The cattle were also examined for tick infestations, and the detected ticks were subjected to morphological identification, followed by molecular characterization using the cytochrome c oxidase subunit I (COI) gene. The heat shock protein (groEL) and citrate synthase (gltA) genes were targeted for screening Anaplasmataceae. Piroplasmida infections were initially screened using nested polymerase chain reaction (PCR) targeting the 18S ribosomal RNA gene. Theileria orientalis and Anaplasma marginale were further characterized by the major surface protein 4 (MSP4) gene. Rhipicephalus microplus was the only tick species detected in the examined cattle. Overall, 29.7% (43/145) of the cattle examined were infected with at least one TBP, with 3.4% (5/145) being co-infected with more than one TBP. The detection rate of TBPs was 20% (n = 29) for A. marginale, 3.4% (n = 5) for Anaplasma bovis, 6.2% (n = 9) for T. orientalis, and 4.1% (n = 6) for Theileria annulata. The phylogenetic analysis showed that two genotypes of A. marginale and T. orientalis (types 5 and 7) were present in cattle. This study provides preliminary baseline data on the prevalence and genetic diversity of ticks and TBPs in cattle in Nepal.