Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal

Medhavi Dhakal; Tulsi Ram Gompo; Prakash Devkota; Sharmila Chapagain Kafle; Janak Raj Subedi; Haiyan Gong; Hiroaki Arima; Richard Culleton; Masahito Asada; Kishor Pandey

doi:10.3390/pathogens12081045

Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal

Pathogens. 2023 Aug 15;12(8):1045. doi: 10.3390/pathogens12081045.

Affiliations

¹ Central Department of Zoology, Institute of Science and Technology, Tribhuvan University, Kathmandu 44601, Nepal.
² Central Veterinary Laboratory, Kathmandu 44600, Nepal.
³ Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
⁴ Department of International Health and Medical Anthropology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.
⁵ Division of Molecular Parasitology, Proteo-Science Centre, Ehime University, Ehime, Matsuyama 791-0295, Japan.
⁶ Research Unit for Global Infection Control, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan.

Abstract

Background: Tick-borne protozoan parasites (TBPPs) cause significant problems for domestic animals' health in Nepal. TBPPs are routinely diagnosed by labor-intensive blood smear microscopy. In Nepal, there are some reports of Babesia and Theileria in cattle, although species identification is rarely performed. Therefore, we performed conventional nested PCR (nPCR) followed by sequence analysis to identify TBPP species infecting cattle in Nepal.

Methods: One hundred and six blood samples were collected from cattle in the Kathmandu Valley. Thin blood smears were prepared for microscopic examination. Parasite DNA was extracted from the blood, and nPCR and sequencing were performed to identify the TBPPs present.

Results: Among the 106 samples, 45 (42.5%) were positive for piroplasm (Babesia spp. and Theileria spp.) via microscope observation and 56 (52.8%) samples were positive via nPCR. The obtained PCR products were used for direct sequencing, and we identified the species as B. bigemina, B. bovis, T. annulate and T. orientalis. Phylogenetic analyses showed that the B. bovis, B. bigemina and T. orientalis sequences from this study belonged to each species clade. On the other hand, T. annulate was divided into two clades in the analysis, and our T. annulate sequences were also divided in these two clades. The piroplasm-positive cattle showed lower hemoglobin and red blood cells than healthy cattle.

Conclusions: To the best of our knowledge, this study is the first to apply molecular detection and species determination of TBPPs in cattle in Nepal. The results of this study may be used as a starting point for the development of successful TBPP surveillance and prevention programs in Nepal.

Keywords: Babesia; Nepal; PCR; Theileria; cattle.

Grants and funding

This research was funded by the Higher Education Reforms Project (HERP DLI-7B), Nepal to K.P. and the University Grant Commission Nepal grant (MRS-78/79-S&T-132) to M.D. This research was also supported by the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine (NRCPD-OUAVM) joint research in FY2023-2024 to K.P.