Genetic diversity and co-circulation of tick-borne pathogens in Haemaphysalis spp. from Pakistan

Haemaphysalis is the second largest genus of hard ticks (Acari: Ixodidae), both in terms of diversity and global distribution. Despite their wide occurrence, genetic information on this genus remains scarce. This study investigated the genetic diversity, phylogenetic relationships, and associated pathogens of Haemaphysalis ticks infesting small ruminants across northern Pakistan. A total of 613 ticks were collected from 95 infested goats and sheep. Four species were identified based on morphological and molecular analyses, including Haemaphysalis (Ha.) cornupunctata, Ha. danieli, Ha. montgomeryi, and a distinct Haemaphysalis sp. closely related to Ha. sulcata. Species identification was confirmed using mitochondrial 16 S rDNA and cox1 markers. Molecular confirmation of unidentified Haemaphysalis sp. was detected across multiple districts of northern Khyber Pakhtunkhwa, indicating close relation to Ha. sulcata. Pathogen screening revealed Rickettsia hoogstraalii (gltA and ompB) in Haemaphysalis sp. and Ha. danieli, Theileria sinensis-like (18 S rDNA) in Ha. montgomeryi and Ha. danieli, and Anaplasma bovis (16 S rDNA) in Ha. cornupunctata and Ha. montgomeryi. This study provides the first molecular evidence of T. sinensis-like in Ha. montgomeryi and Ha. danieli, R. hoogstraalii in Ha. danieli and Haemaphysalis sp., and A. bovis in Ha. cornupunctata and Ha. montgomeryi from Pakistan. Phylogenetic analyses supported species-level relationships and revealed substantial genetic divergence within Haemaphysalis. These findings highlight the importance of locality-based molecular surveillance for resolving cryptic diversity and improving understanding of tick–pathogen associations in mountainous regions of Pakistan.