Molecular prevalence, co-infection, and phylogenetic characterization of Anaplasma marginale, Anaplasma ovis, and Theileria annulata in Nili-Ravi buffaloes

Nili-Ravi is a major dairy buffalo breed of Pakistan, widely raised in Punjab and valued for its high milk yield. Vector-borne infections pose a serious health threat to this breed, reducing productivity and causing significant economic losses to farmers and the livestock sector. In this study, a total of 250 blood samples from Nili-Ravi buffaloes were collected from District Bahawalnagar, Punjab, Pakistan, and screened for Anaplasma marginale , Theileria annulata , and Anaplasma ovis . Results revealed a significant difference in the infection rates of the three screened pathogens among the enrolled buffaloes (P < 0.001) . A. marginale exhibited the highest prevalence (16%), followed by T. annulata (12%) and A. ovis (4.4%) respectively. Co-infections involving two pathogens were observed, and one buffalo was simultaneously infected with all three screened pathogens. DNA sequencing followed by BLAST analysis confirmed the identity of each detected pathogen in the screened buffaloes. Phylogenetic analysis showed that these isolates are genetically closely related to strains reported from various countries worldwide. None of the evaluated epidemiological factors (age, sex, disease history and ecto-parasite infestation) showed a significant association with the prevalence of any of the screened pathogens in this study. To the best of our knowledge, these findings may represent the first published molecular detection of A. ovis in buffaloes from Pakistan. This study offers important baseline data for developing targeted control strategies against the screened pathogens. It may help improve livestock productivity, which is a major source of income for the population of Pakistan.