The availability of survival resources (i.e., shelter, food, and water) affects the dog population in a region and directly correlates with the human population (1). Stray dogs are highly vulnerable to vector-borne diseases (VBDs) of zoonotic concern and, consequently, pose threats to human and animal health (2). Although the exact number globally is unknown, 75% out of 700 million dogs are considered stray dogs (3). They are the first farmed animals and are closely associated with humans on cultural, social, and economic levels (4). Pakistan is a developing country, and contemporary social issues have imposed an epidemic of stray dogs that may affect both the welfare of the animals and the public’s health, especially in urban and peri-urban areas (5). The exact number of these free-roaming stray dogs in the country is unknown, and the estimated stray dog population may have reached up to 3 million (6).

Tick species infesting stray dogs in the urban and peri-urban areas include Rhipicephalus sanguineus senso lato (s.l.), Haemaphysalis elliptica, Rhipicephalus microplus, Haemaphysalis erinacei, and Haemaphysalis parva (7, 8). Knowledge of tick identification is vital for surveillance to minimize the zoonotic consequences associated with tick-borne pathogens (9). Despite the morphological identification, genetic characterization using various genetic markers such as 16S rDNA and cox1 is a suitable approach to determine the taxonomic status of a tick species (10–16).

Stray dogs being important reservoirs for tick-borne zoonotic agents may pose a threat to public health by causing anaplasmosis, rickettsiosis, babesiosis, query fever (Q fever), and ehrlichiosis (17–19). The genus Ehrlichia has seven determined species: Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ruminantium, Ehrlichia muris, Ehrlichia ewingii, Ehrlichia ovina, and Ehrlichia minasensis (20, 21). Ticks responsible for the transmission of Ehrlichia spp. include species of the genera Rhipicephalus, Hyalomma, Dermacentor, Amblyomma, and Ixodes (20, 22). Canine monocytic ehrlichiosis (CME) is the most common tick-borne zoonotic disease that affects dogs globally (23). The brown dog tick, Rh. sanguineus, primarily vectors the etiological agent of CME (23). Hepatozoon canis causes canine hepatozoonosis, which is widely distributed in South America, Africa, Asia, and South Europe (24). Q fever is a tick-borne infection caused by Coxiella burnetii (25). Tick species of the genera Rhipicephalus, Hyalomma, Amblyomma, Dermacentor, and Ixodes transmit this pathogen (26). Anaplasmataceae, consisting of different genera, such as Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia, contains vector-borne bacteria that are mainly transmitted by ticks (27). Tick species belonging to the genera Amblyomma, Dermacentor, Haemaphysalis, Ixodes, and Rhipicephalus transmit Anaplasma spp. to various hosts, including dogs (8, 28). Anaplasma platys and Anaplasma phagocytophilum are the most commonly detected species in dogs responsible for zoonotic diseases (2). Dogs are reservoir hosts for various spotted fever group agents (SFG) transmitted by Rh. sanguineus ticks (20). Rickettsial infections transmitted by Rh. sanguineus include the spotted fever group rickettsiosis caused by R. massiliae (20, 29), Mediterranean spotted fever (MSF) caused by Rickettsia conorii (30), and Rocky Mountain spotted fever (RMSF) caused by Rickettsia rickettsii (23). Dogs and their associated ticks play a major role in the ecology and epidemiology of several Rickettsia species, including Rickettsia aeschlimannii (23, 30).

The percentage of stray or semi-domesticated dogs is inversely correlated with the gross domestic product (GDP) per capita. Additionally, the burden of canine zoonotic parasites has been correlated with poverty, but in some cases, accurate and updated estimates regarding this data are lacking (31). Raising community knowledge, raising awareness of unhealthy practices, and ensuring preventive measures against disease-causing agents are crucial for effectively controlling ticks and tick-borne pathogens (32). Stray dogs survive through edible wastes from humans, especially from butchered surroundings in urban and peri-urban areas, mostly in developing countries. An overabundance of butcher trash appears to result from the lack of proper waste management and disposal, mostly in low-income countries (33). The abundance of discarded meat trash that is readily available for feed mostly results in the presence of large numbers of butcher-associated stray dogs (BASDs) close to butcher shops. Pakistan is a low and middle-income country (LMIC) that is heavily populated and socioeconomically underdeveloped (6, 11, 29). Due to the lack of veterinary supervision and management of butcher waste, it attracts hungry stray dogs who feed upon it. In Pakistan, butcher shops on road and street sides are major reasons for the spread of ticks that infest BASDs. No previous study has assessed public health risks and the epidemiological status of ticks and tick-borne pathogens on BASDs. This study aimed to identify the ticks infesting BASDs and to molecularly detect associated tick-borne pathogens that pose public health threats in urban and peri-urban areas.

A survey in the selected districts resulted in an overall collection of 575 ticks (392 and 183 from symptomatic and asymptomatic BASDs, respectively) from 117 BASDs (63 symptomatic and 54 asymptomatic), comprising four hard tick species. Among the identified ticks, Rh. sanguineus was the most prevalent tick species (n = 292/575, 50.78%), followed by Rh. haemaphysaloides (n = 127/575, 22.08%), Rh. turanicus counting (n = 87/575, 15.13%), and Rh. microplus clade C (n = 69/575, 12.00%). The spatial distribution of the collected ticks revealed that Mardan, Lakki Marwat, and Charsadda had the highest tick counts of 58, 53, and 51 ticks, respectively. The Malakand district (27) had the least number of collected ticks among the selected districts.

Of the 575 tick specimens, 271 (47.13%; 193 and 78 from symptomatic and asymptomatic BASDs, respectively) were subjected to PCR for the molecular screening of various tick-borne pathogens, including Ehrlichia, Anaplasma, Rickettsia, Coxiella and Hepatozoon species (Supplementary Table S1). According to the PCR-based screening and subsequent sequencing of targeted bacterial and protozoal agents, Anaplasma spp. were the most prevalent (n = 54/271, 19.92%), followed by Ehrlichia spp. (n = 40/271, 14.76%), Rickettsia spp. (n = 33/271, 12.17%), Coxiella spp. (n = 23/271, 8.49%), and Hepatozoon spp. (n = 9/271, 3.32%) in the ticks infesting BASDs (Table 1). None of the ticks was found positive for multiple pathogens.

The results of the prevalence of symptomatic and asymptomatic BASDs in ticks, as well as the molecular screening of tick-associated bacterial and protozoal pathogens, are presented in Table 1.

BASDs are commonly exposed to vector-borne pathogens due to the lack of adequate protection against ectoparasites in different regions. Different bacterial and protozoal infections have been reported in ticks infesting dogs, depending on the status of reservoir hosts, geographical variations in the exposure to tick vectors, detection methods, and the time of testing (2). The epidemiological situations of ticks and tick-borne pathogens associated with BASDs near butcher shops have not been previously investigated. A combination of morphological and molecular characterization resulted in the identification of several tick species infesting BASDs, including Rh. sanguineus, Rh. haemaphysaloides, Rh. turanicus, and Rh. microplus. The examined ticks were found positive for a variety of pathogenic and undetermined microorganisms, including species of the genera Ehrlichia, Coxiella, Hepatozoon, Anaplasma, and Rickettsia, which can be transmitted and pose health threats to humans, as well as wild and domestic animals. The different detected pathogenic and undermined species included E. minasensis, Ehrlichia sp., H. canis, Coxiella sp., C. burnetii, A. capra, A. platys, Anaplasma sp., R. massiliae, “Ca. R. shennongii” and R. aeschlimannii. This is the first investigation of Ehrlichia spp., Hepatozoon spp., Coxiella spp., Anaplasma spp., and Rickettsia spp. in ticks infesting BASDs. In Pakistan, E. minasensis, H. canis, C. burnetii, A. capra, A. platys, “Ca. R. shennongii” and R. aeschlimannii were detected for the first time, and they pose zoonotic threats to public health.

Abandoned stray dogs are frequently exposed and particularly vulnerable to the high risks of tick infestation and dispersal (5). Several tick species infest stray dogs in urban and peri-urban regions, and various pathogens are transmitted from dogs to other animals and humans (28). Surveillance requires a thorough understanding of tick dentification to reduce losses associated with tick-borne disease-causing agents (9, 16, 40). Morphological identification notwithstanding, the best way to determine the taxonomic status of the tick species is through molecular characterization utilizing 16S rDNA and cox1 genetic markers (10, 15, 41, 42). The 16S rDNA and cox1 sequences for Rh. sanguineus, Rh. turanicus, Rh. microplus, and Rh. haemaphysaloides in the present study revealed the closest identities of these ticks to the corresponding species from the Oriental and Palearctic regions.

BASDs freely roam outdoors, which increases their exposure to ticks and leads to uncontrolled breeding. They are mostly spotted in urban and rural localities. In general, stray dogs tend to carry a number of ticks, most commonly the dog tick Rh. sanguineus, which is a vector for several pathogens (43). Two Ehrlichia species (E. minasensis and Ehrlichia sp.) were detected in ticks in BASDs. In previous studies, E. minasensis has been detected in ticks infesting different hosts, such as cattle, horses, goats, and dogs (44, 45). For the genetic characterization of Ehrlichia spp., the 16S rDNA and dsb partial fragments have been identified as accurate genetic markers at the species level (46, 47). The 16S rDNA fragments of Ehrlichia spp. were closely related to the Ehrlichia sp. and E. minasensis species. In the 16S rDNA-based phylogeny, Ehrlichia sp. formed a sister clustered with E. ewingii and E. ruminantium, suggesting that this species may be of zoonotic concern. In the case of dsb fragments, the present E. minasensis sequences were phylogenetically clustered with the same species reported from the Palearctic, Neotropical, Australian, and Oriental regions. In both phylogenetic trees based on the 16S rDNA and dsb, the appearance of E. minasensis as a sister clade to E. canis might suggest that both are potentially canine pathogens (45, 47). Considering the detection of Ehrlichia spp. in ticks infesting free-roaming BASDs, further investigations are critical to disclose its association between stray dogs (BASDs) and humans.

Hepatozoon canis is a tick-borne apicomplexan parasite infecting canids and has long been known to be transmitted by ingesting infected Rh. sanguineus and Rh. turanicus ticks (48). The available phylogeny of H. canis is based on the 18S rDNA sequence, which has been demonstrated to be useful for inferring phylogenetic analyzes at the species level (48). Phylogenetic analysis based on the 18S rDNA sequence of this species suggests its close resemblance with the same species circulating in the Palearctic and Oriental regions.

More than 40 Ixodid (Rhipicephalus, Dermacentor, Haemaphysalis, Amblyomma, and Ixodes) and Argasid (Ornithodoros and Argas) tick species are known to harbor bacterial microbes related to C. burnetii and other Coxiella-like endosymbionts, which have been commonly detected in Rhipicephalus spp., particularly Rh. sanguineus (25, 49). Rhipicephalus spp. may play an important role in the transmission of C. burnetii among the hosts. Except for C. burnetii, there is no evidence that Coxiella spp. cause disease in the host; however, the pathogenic roles of these species have remained undetermined (49). Q fever is a vector-borne zoonotic disease caused by C. burnetii that affects a wide range of hosts globally. Coxiella spp. have been detected in Rh. sanguineus (s.l.) based on the groEL sequence (26). Since its successful detection in various ticks, the groEL sequences are potentially specific for Coxiella-like endosymbionts (18, 25). Likewise, the groEL partial sequence of Coxiella sp. and C. burnetii was detected in Rhipicephalus ticks of BASDs, which were closely related to the corresponding species.

Several tick species have been reported as vectors of Anaplasma, particularly in the genus Rhipicephalus (14, 50). Although Anaplasma spp. are prevalent in ixodid ticks, relatively limited studies have been conducted to investigate its detection, especially in BASD ticks (2). Anaplasma spp. were detected in high abundance in BASD ticks, which is nearly comparable to prior studies (2). Anaplasma platys and A. capra have mostly been considered to be transmitted by the dog tick Rh. sanguineus, and dogs have been confirmed as the hosts for A. capra in China (27). For the genetic characterization of Anaplasma spp., the highly conserved 16S rDNA marker has been historically employed (14, 27, 50). Likewise, the 16S rDNA fragments of Anaplasma spp., such as A. capra and A. platys, and an undermined Anaplasma sp. detected in Rhipicephalus spp. align with previous reports (14, 50). The detection of Anaplasma spp. in ticks infesting BASDs in Pakistan might suggest its geographic expansion, highlighting the need for further comprehensive studies on its pathogenicity to screen for the epidemiological and evolutionary status, which was previously underestimated due to the lack of sufficient data.

In Pakistan, earlier epidemiological investigations on the detection of Rickettsia spp. were mostly performed on ticks infesting small and large ruminants, equine, and wild hosts (11, 15, 16, 29, 51). Despite this gap, we used molecular screening of Rickettsia spp. in ticks infesting BASDs. Genetic markers, such as gltA, ompA, and ompB, have been utilized to determine a high degree of intraspecific variation and are extensively used for reliable characterization at the species level (42). We detected and characterized for the first time three rickettsial agents, Rickettsia massiliae, “Ca. Rickettsia shennongii” and Rickettsia aeschlimannii in ticks infesting BASDs. Rickettsia massiliae and Rickettsia aeschlimannii have been detected in ticks of multiple hosts in several districts of Pakistan (19, 52). The detection of “Ca. R. shennongii” represents the first record of any host or tick in Pakistan. Recently, this species was detected in Rh. haemaphysaloides ticks in China as a novel spotted fever group (SFGR) (25). The detection of “Ca. R. shennongii” in Rh. sanguineus, Rh. haemaphysaloides, and Rh. turanicus suggests its diverse host and distribution range and enforces the need to investigate its zoonotic threat to other hosts, including humans. These findings might advance our knowledge of the diversity of circulating tick-borne pathogens in the region, highlighting the need for further comprehensive surveillance studies to properly monitor ticks for potential zoonotic threats.

To effectively manage ticks and tick-borne infections, the community must be educated regarding the associated risks and prevention strategies. The current endeavor highlights the risks of ticks infesting BASDs to public health. A comprehensive analysis was undertaken to determine which bacterial and protozoal pathogens might be carried by ticks infesting BASDs that inhabit butcher shops. This study will provide fundamental knowledge about the risks associated with these free-roaming BASDs, convincing health policymakers to ensure the control of any zoonotic consequences associated with these ticks infesting dogs near butcher shops. To minimize the risks of ticks and tick-borne diseases related to BASDs, proper disposal of meat and butcher waste should be adopted, and open-street butcher shops should be properly managed. Further surveillance studies are essential to understand the status of ticks and tick-borne pathogens in BASDs in different regions.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal studies were approved by the current study was approved by the Advance Studies Research Board (ASRB: Dir/A&R/AWKUM/2022/9396) committee members of the Faculty of Chemical and Life Sciences, Abdul Wali Khan University, Mardan Khyber Pakhtunkhwa (KP), Pakistan. Butchers were informed verbally about their observations and collection of ticks. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

AiA designed the study. AiA, SU, and MN collected the ticks and carried out phylogenetic and statistical analysis. AiA, AdA, MA, and TT performed the experiments. All authors contributed to the article and approved the submitted version.