Molecular investigation of tick-borne and zoonotic pathogen dissemination and host-pathogen interactions in canines.

Dogs are among the most widespread and beloved domestic animals worldwide. As close companions of humans, they are commonly exposed to the environment and to various vectors, making them potential reservoirs for a wide range of vector-borne pathogens. This study was aimed to determine the molecular prevalence and genetic diversity of Anaplasma (A.) ovis, Toxoplasma (T.) gondii and Theileria (T.) ovis among domestic dogs in Pakistan. A total of 275 canine blood samples were collected from five districts in Punjab, Dera Ghazi Khan (n = 57), Vehari (n = 7), Layyah (n = 34), Bahawalpur (n = 38) and Muzaffargarh (n = 139), during March till October 2024. Polymerase Chain Reaction (PCR) based analysis revealed that 8.7% (24/275), 16.7% (46/275) and 11.6% (32/275) dogs were infected with A. ovis, T. gondii and T. ovis, respectively. Co-infections involving two or all three pathogens were also detected. The presence of all three pathogens was confirmed through Sanger sequencing followed by BLAST analysis and the amplified genes were similar to the pathogenic sequences deposited from countries worldwide. Prevalence of all three pathogens varied significantly between the sampling sites. Theileria ovis infection varied significantly between the screened dog breeds. Data analysis revealed that male dogs had a significantly higher of A. ovis infection rate as compared to females (p = 0.008). Overall, our findings indicate moderate infection rates of A. ovis, T. gondii and T. ovis in dogs from Pakistan. These findings highlight the need to include local dog populations in future vector-borne disease surveillance programs across Pakistan, following the One Health approach.