Genetic diversity of tick-borne zoonotic pathogens in ixodid ticks collected from small ruminants in Northern Pakistan

Mapping tick distribution and pathogens in unexplored areas sheds light on their importance in zoonotic and veterinary contexts. In this study, we performed a comprehensive investigation of the genetic diversity of tick and tick-borne pathogens (TBPs) detection infesting/infecting small ruminants across northern Pakistan. We collected 1587 ixodid ticks from 600 goats and sheep, an overall tick infestation rate of 50.2 %. Notably, genderbased infestation rates were higher in female goats and sheep compared to their male counterparts. Age-wise analysis showed that the tick infestation rate was higher in older animals. This study identified 11 ixodid tick species within three genera: Hyalomma, Haemaphysalis, and Rhipicephalus, which were taxonomically classified using 16S rRNA and cytochrome oxidase I (cox1) molecular markers. Sequence analysis indicated that reported ticks are similar to ixodid species found across various Asian and African countries. Tick-borne pathogens were detected by amplifying 16S rRNA and citrate synthase (gltA) for bacterial pathogens and 18S rRNA for apicomplexan parasites. The present study reported a diverse array of TBPs in ticks from the study area, with Rickettsia massiliae (24.5 %) and Theleria ovis (16.4 %) as the most prevalent bacterial and apicomplexan pathogens. Phylogenetically, detected TBPs shared evolutionary relatedness with identical TBPs from old and new world countries. These findings highlight the presence of zoonotic TBPs in ixodid ticks from Pakistan. In addition, it also provides a foundation for future epidemiological research on ticks and TBPs, emphasizing their relevance in both zoonotic and veterinary contexts.