Molecular survey of piroplasm species from selected areas of China and Pakistan

Background: Piroplasmosis is an important animal disease that is a major constraint to the development of the livestock industry, often resulting in significant economic losses. Therefore, there is an urgent need to further understand the etiology of this and other tick-borne infections. Methods: Blood samples were collected from tick-infested animals from the Chakwal, Jhang, and Faisalabad districts of Punjab, Pakistan and from peri-urban areas around Hohhot, Inner Mongolia, China to investigate the presence of Babesia and Theileria species. In total, 450 blood samples were collected with FTA cards from cattle of the study areas of Pakistan; the genomic (g)DNA of one hundred and twenty samples from cattle in Inner Mongolia were provided by the Lanzhou Veterinary Research Institute, China. Following the extraction of gDNA, the 185 rRNA gene (V4 hypervariable region) of piroplasms was amplified in all samples using semi-nested PCR. Positively identified samples were sequenced for the identification of Theileria and Babesia species. The partial full-length sequence of 185 rDNA was amplified for species confirmation of Theileria-positive samples, whereas the RAP-1c gene was amplified for Babesia bigemina-positive samples. Results: Semi-nested PCR results revealed that 144 (2526%) samples were positive for piroplasms. Theileria annulata was the most prevalent species (115/144; 20.17%), followed by Theileria orientalis (16/144; 2.80%). Among Babesia, the only species recorded was Babesia bigemina (13/144; 2.28%). Conclusion: The present study reveals new data on the prevalence of piroplasm species in bovine populations of selected areas of China and Pakistan and their phylogenetic relationships. It is also the first detailed report of T. orientalis from native animals in Pakistan.