Background: Vector-/tick-borne pathogens (V/TBPs) pose a potential threat to human and animal health globally. Information regarding canine V/TBPs is scarce and no specific study has been conducted so far to explore the microbial diversity within ticks infesting dogs from Pakistan. Herein, this knowledge gap is addressed by assessing the genetic diversity and prevalence pattern of V/TBPs in ixodid ticks with special implications for public and canine health.Methods: A total of 1150 hard ticks were collected from 300 dogs across central Khyber Pakhtunkhwa (KP), Pakistan. After morpho-molecular identification, 120 tick samples were screened for the presence of V/TBPs by amplifying16S rRNA/gltA(Rickettsia/EhrlichiaandWolbachiasp.),18S rRNA(Theileriasp.) andcox1(Dirofilariasp.) genes through PCR followed by sequencing and phylogenetic study.Results: In toto, 50 ixodid ticks (50/120, 41.7%) were found positive for V/TBPs DNA. The detected V/TBPs were categorized into five genera and eight species, viz.Ehrlichia(E. canisandEhrlichiasp.),Rickettsia(R. massiliae, R. raoultiiandRickettsiasp.),Theileria(T. annulata),Dirofilaria(D. immitis) andWolbachia(Wolbachiasp.). The pathogen prevalence patterns showed thatR. massiliaewas the most prevalent zoonotic V/TBP (19.5%), followed byE. canis(10.8%),Rickettsiasp. (7.5%), R. raoultii(6.7%),T. annulata(5.8%),D. immitis(5.8%),Wolbachiasp. (4.2%) andEhrlichiasp. (3.3%), respectively. Among the screened tick species, mostRhipicephalus sanguineussensu lato samples were found positive for V/TBP DNA (20/20,100%) followed byRh. turanicussensu stricto (13/20, 65%),Hyalomma dromedarii(8/20, 40%),Rh. haemaphysaloides(6/20, 30%),Hy. excavatum(2/20, 10%) andRh. microplus(1/20, 5%). Co-occurrence of V/TBP was also detected in tick specimens (single V/TBP infection: 32 ticks; double and triple: 13 and 5 tick samples). The detected pathogens shared a phylogenetic relationship with similar isolates published in NCBI GenBank from Old and New World countries.Conclusion: Ixodid ticks infesting dogs harbor a diverse array of V/TBPs including zoonotic agents from Pakistan. Furthermore, the presence ofD. immitisin ticks that infest dogs raises the possibility that this parasite has either attained its dead-end host (i.e. the tick) while feeding on dogs or has expanded its range of intermediate/paratenic hosts. Further research work is needed to investigate the epidemiology and confirm the vector competence of screened tick species for these pathogens from Pakistan.Graphical

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 (cox1) and 16S ribosomal RNA (16S rRNA) 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., Hyalomma and Rhipicephalus, including six tick species viz. Hyalomma dromedarii (15.9%), Hyalomma excavatum (3%), Rhipicephalus sanguineus s.l. (41.3%), Rhipicephalus turanicus s.s. (28.7%), Rhipicephalus haemaphysaloides (10.2%), and Rhipicephalus microplus (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 Hyalomma and Rhipicephalus tick species in the dog population from the study area. The present study also reported a new tick–host association between Hy. excavatum, Hy. dromedarii, and dogs. Phylogenetic analysis revealed that cox1 partial nucleotide sequences of Hy. excavatum in our dataset were 100% identical to similar tick specimens identified in Turkey, and those of Hy. dromedarii were identical to tick specimens from Iran. Whereas, Rh. haemaphysaloides and Rh. microplus’ cox1 partial nucleotide sequences were identical to sequences previously published from Pakistan. Rhipicephalus turanicus s.s. ‘s cox1 isolates from the present study were 99.8–100% identical to Pakistani-reported isolates, and those of Rh. sanguineus s.l. were 100% identical to Chinese specimens. Results on the genetic characterization of ticks were further confirmed by 16S rRNA partial nucleotide sequences analysis, which revealed 100% identity between the tick isolates of this study and those of Hy. excavatum reported from Turkey; Hy. dromedarii specimens reported from Senegal; Rh. haemaphysaloides, Rh. microplus, and Rh. turanicus s.s., previously published from Pakistan, and Rh. sanguineus s.l., published from China. Furthermore, phylogenetic analysis showed that the Rh. sanguineus s.l. 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.

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, gender-based 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.