Molecular detection and diversity of piroplasmids in cattle from Jiri, Nepal

Babesia and Theileria species are tick-borne protozoan parasites that cause piroplasmosis in cattle, a disease associated with substantial economic losses in the cattle industry. This study aimed to detect piroplasmid infections in cattle in Nepal, using microscopic and molecular methods. Eighty blood samples were collected from cattle herds at Jiri, Nepal. Blood smears were examined microscopically for the presence of piroplasmids, and samples were assayed using nested PCR (nPCR) targeting the 18 S rRNA gene. All nPCR positive samples underwent direct sequencing and representative positive samples were further analyzed through cloning and sequencing. Selected blood samples were used for hematological analysis; and microscopic examination revealed 23.8% (19/80) positivity for piroplasmids, whereas corresponding nPCR detected 77.5% (62/80) positive samples. Direct sequencing identified infections with Babesia bovis and B. bigemina , and with Theileria annulata and T. orientalis . Of 27 selected samples subjected to cloning and sequencing, 23 (85.2%) represented single infections, 3 (11.1%) were mixed infections containing both B. bovis and B. bigemina , while a single sample (0.04%) revealed the presence of Colpoda , a free-living protozoan. Phylogenetic analysis showed that most of the detected sequences clustered into species-specific clades, although one sample formed a distinct branch from the nearby B. bovis clade. There was no significant correlation between the presence of blood parasites and alterations of hematological parameters. This study highlights the importance of combining microscopic and molecular methods for the detection of piroplasmids and provides crucial baseline data on the prevalence and diversity of these parasites in cattle herds in Nepal.