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Abstract Tick-borne bacteria of the genera Rickettsia and Coxiella cause several emerging veterinary and human infectious diseases. Ticks of the genus Hyalomma are medically important vectors due to their potential role in the transmission of pathogens to vertebrate hosts. There is an inadequate knowledge on tick-borne Rickettsia spp. and Coxiella spp. in ticks infesting transhumant camels in Pakistan. In this study, we conducted a molecular survey for screening of Rickettsia spp. and Coxiella spp. in ticks infesting camels. Seven hard tick species including Hyalomma dromedarii, Hyalomma anatolicum, Hyalomma scupense, Hyalomma isaaci, Hyalomma turanicum, Hyalomma asiaticum, and Rhipicephalus sanguineus s.l were confirmed on camels in three distinct physiographic regions of Khyber Pakhtunkhwa, Pakistan. A subset of morphologically identified ticks were subjected to molecular assays for the genetic characterization of ticks and the detection and genetic characterization of Rickettsia and Coxiella species using standard genetic markers. Ticks screened for pathogens resulted in the detection of Rickettsia aeschlimannii and Candidatus Rickettsia shennongii and Coxiella burnetii. The molecular analysis further reveals the presences of an undetermined Rickettsia aeschlimannii-like species, that is making a distinct phylogenetic clade with R. aeschlimannii. The detection of pathogens in camel ticks poses potential health hazards as these ticks frequently bites humans. Molecular screening of Rickettsia spp. and Coxiella spp. associated with camel ticks is a preliminary step toward the surveillance of evaluating their zoonotic threats in the region.

This study aimed to detect Hepatozoon spp. in ticks infesting asymptomatic domestic animals and to provide insight into their potential spillover from wild to domestic animals. In total, 537 tick specimens were collected in Khyber Pakhtunkhwa, Pakistan, and morphologically identified. The most prevalent tick species was Haemaphysalis cornupunctata (69; 12.8%), followed by Haemaphysalis kashmirensis (62; 11.5%), Rhipicephalus microplus (58; 10.8%), Haemaphysalis montgomeryi (51; 9.5%), Rhipicephalus sanguineus (49; 9.1%), each Haemaphysalis bispinosa and Haemaphysalis sulcata (43; 8.0%), each Hyalomma anatolicum and Rhipicephalus turanicus (37; 6.9%), Rhipicephalus haemaphysaloides (33; 6.1%) Hyalomma scupense (30; 5.6%), and Hyalomma isaaci (25; 4.7%). The extracted DNA from a subset of each tick species was subjected to PCR to amplify 18S rRNA fragments of Hepatozoon spp. By BLAST analysis, the Hepatozoon sp. detected in Hy. anatolicum infesting cows and in Ha. sulcata infesting sheep showed 99.7% maximum identity with Hepatozoon colubri. Similarly, the Hepatozoon sp. detected in R. haemaphysaloides infesting goats shared 99.49% maximum identity with Hepatozoon ayorgbor, and the Hepatozoon sp. detected in R. sanguineus infesting dogs exhibited 99.7% identity with Hepatozoon canis. Having an overall infection rate (9.3%; 16/172), the highest infection rate was recorded for each H. canis, and H. colubri (3.5%; 6/172), followed by H. ayorgbor (2.3%; 4/172). In the phylogenetic tree, H. colubri clustered with corresponding species from Iran, H. ayorgbor clustered with the same species from Croatia, Ghana, and Portugal, and H. canis clustered with the conspecifics from Iran, Israel, Romania, and Zambia. Regarding the potential spillover of Hepatozoon spp. from wildlife through ticks, free ranging animals was at higher risk compared to confined animals (RR = 3.05), animals consuming food from wildlife habitats were at higher risk compared to those consuming domestic food (RR = 3.06), and animals residing in farm buildings located in wildlife habitats were at higher risk compared to those residing in farm buildings located in villages (RR = 3.28). In addition to the first report on H. canis in R. sanguineus in Pakistan, this is the earliest data showing H. ayorgbor in R. haemaphysaloides and H. colubri in Ha. sulcata and Hy. anatolicum. These preliminary findings suggest a potential spillover of Hepatozoon spp. from wild to domestic animals via ticks under certain risk factors.