Ticks Infesting Dogs in Khyber Pakhtunkhwa, Pakistan: Detailed Epidemiological and Molecular Report.

Ticks and tick-borne diseases are considered a major challenge for human and animal health in tropical, sub-tropical, and temperate regions of the world. However, only scarce information is available on the characterization of tick species infesting dogs in Pakistan. In this study, we present a comprehensive report on the epidemiological and phylogenetic aspects of ticks infesting dogs in Pakistan using the mitochondrial markers i.e. Cytochrome c oxidase subunit 1 () and 16S ribosomal RNA () nucleotide sequences. A total of 300 dogs were examined and 1150 ixodid ticks were collected across central Khyber Pakhtunkhwa, Pakistan. The morpho-molecular characterization of hard ticks revealed the presence of two ixodid tick genera on dogs, i.e., and , including six tick species viz. (15.9%) (3%), (41.3%) (28.7%), (10.2%), and (2%). The total prevalence of tick infestation in dogs was 61%. The district with the highest tick prevalence rate in dogs was Mardan (14.7%), followed by Peshawar (13%), Swabi (12%), Charsadda (11%), and Malakand (10.3%), respectively. Risk factors analysis indicated that some demographic and host management-associated factors such as host age, breed, exposure to acaricides treatment, and previous tick infestation history were associated with a higher risk of tick infestation on dogs. This is the first molecular report confirming the infestation of and tick species in the dog population from the study area. The present study also reported a new tick-host association between , , and dogs. Phylogenetic analysis revealed that partial nucleotide sequences of in our dataset were 100% identical to similar tick specimens identified in Turkey, and those of were identical to tick specimens from Iran. Whereas, and partial nucleotide sequences were identical to sequences previously published from Pakistan. 's isolates from the present study were 99.8-100% identical to Pakistani-reported isolates, and those of were 100% identical to Chinese specimens. Results on the genetic characterization of ticks were further confirmed by partial nucleotide sequences analysis, which revealed 100% identity between the tick isolates of this study and those of reported from Turkey; specimens reported from Senegal; , , and , previously published from Pakistan, and , published from China. Furthermore, phylogenetic analysis showed that the isolates of this study clustered with specimens of the tropical lineage with 7.7-10% nucleotide divergence from the specimens of the temperate lineage. Further molecular works need to be performed throughout Pakistan to present a more detailed map of tick distribution with information about dog host associations, biological characteristics, and pathogen competence.