Molecular detection and genetic diversity of blood borne pathogens infecting domestic donkeys (Equus asinus).

Donkey population in Pakistan was estimated to be 5.9 million during 2023-24 and despite this huge population, donkeys remained unexplored for the presence of blood-borne parasites. This study aimed to report the molecular prevalence and phylogenetic evaluation of Toxoplasma gondii, Anaplasma marginale and Theileria annulata in the donkey blood samples (N = 272) that were collected during May till November 2024 from nine districts (Rajanpur, Khanewal, Dera Ghazi Khan, Lodhran, Bahawalnagar, Pakpattan, Faisalabad, Muzaffargarh and Kasur) in Pakistan. Molecular analyses revealed that the prevalence of T. gondii, A. marginale and T. annulata in screened donkey blood samples was 41%, 4% and 6% respectively. Co-infection with two parasites was also observed but none of the enrolled donkeys from any district were found infected with all three pathogens. DNA sequencing and BLAST analysis confirmed the presence of all three parasites. Phylogenetic analysis of these pathogens showed their genetic similarities with the sequences deposited from worldwide countries. Toxoplasma gondii (P < 0.001) and T. annulata (P = 0.004) prevalence varied between the sampling sites. While donkey breeds, age or sex was not associated with any of the parasitic infection (P > 0.05 for all). In conclusion, we are reporting the presence of T. gondii, A. marginale and T. annulata among Pakistani donkeys and we recommend similar large-scale studies in various geo-climatic regions of Pakistan to further explore the epidemiology, genetic diversity, host-parasite interactions, and effective control of these pathogens among the local donkey breeds.