First report on tick-borne pathogens detected in ticks infesting stray dogs near butcher shops

Public health is a major concern for several developing countries due to infectious agents transmitted by hematophagous arthropods such as ticks. Health risks due to infectious agents transmitted by ticks infesting butcher-associated stray dogs (BASDs) in urban and peri-urban regions have been neglected in several developing countries. To the best of the authors’ knowledge, this is the first study assessing public health risks due to ticks infesting BASDs in Pakistan’s urban and peri-urban areas. A total of 575 ticks (390 from symptomatic and 183 from asymptomatic BASDs) were collected from 117 BASDs (63 symptomatic and 54 asymptomatic); the ticks belonged to 4 hard tick species. A subset of each tick species’ extracted DNA was subjected to polymerase chain reaction (PCR) to amplify the 16S rDNA and cox1 sequences of the reported tick species, as well as bacterial and protozoal agents. The ticks’ 16S rDNA and cox1 sequences showed 99–100% identities, and they were clustered with the sequence of corresponding species from Pakistan and other countries in phylogenetic trees. Among the screened 271 ticks’ DNA samples, Anaplasma spp. was detected in 54/271 (19.92%) samples, followed by Ehrlichia spp. (n = 40/271, 14.76%), Rickettsia spp. (n = 33/271, 12.17%), Coxiella spp. (n = 23/271, 4.48%), and Hepatozoon canis (n = 9/271, 3.32%). The obtained sequences and phylogenetic analyzes revealed that the pathogens detected in ticks were Ehrlichia minasensis, Ehrlichia sp., Hepatozoon canis, Coxiella burnetii, Coxiella sp., Anaplasma capra, Anaplasma platys, Anaplasma sp., Rickettsia massiliae, “Candidatus Rickettsia shennongii” and Rickettsia aeschlimannii. Tick-borne pathogens such as E. minasensis, H. canis, A. capra, A. platys, and R. aeschlimannii, were detected based on the DNA for the first time in Pakistan. This is the first report on public health risks due to ticks infesting BASDs. These results not only provided insights into the occurrence of novel tick-borne pathogens in the region but also revealed initial evidence of zoonotic threats to both public health and domestic life.


Introduction
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).
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 tickborne 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 periurban areas.

Study model
This study was designed to detect infectious agents harbored by ticks infesting BASDs.Stray dogs were observed in or near butcher shops.To test the hypothesis that meat buyers in the study area might be at risk due to tick-borne pathogens, ticks were observed on BASDs mostly in or near butcher shops.Ticks were collected from these BASDs and subjected to molecular detection for various bacterial and protozoal pathogens (Figure 1).

Tick collection and preservation
Ticks were collected from BASDs in or near butcher shops from June 2021 to May 2022 in 14 selected districts.They were carefully isolated from the host body using tweezers to avoid external damage to the specimens.The specimens were rinsed in distilled water, followed by 70% ethanol, and preserved in 100% ethanol.
The examined BASDs consisted of both symptomatic and asymptomatic dogs.Symptomatic dogs were characterized by accompanying symptoms such as diarrhea, vomiting, limb swelling, rashes, itching, seizures, fatigue, scabs or skin lesions, and hair loss.

Morphology of ticks
The collected tick specimens were identified morphologically at the species level under a stereomicroscope (SZ61, Olympus Corporation, Tokyo, Japan) using standard morphological identification keys (29,34).

DNA extraction and PCR
A total of 271 ticks (193 and 78 from symptomatic and asymptomatic BASDs, respectively) were randomly selected for molecular analyzes.Before DNA extraction, the specimens were washed in distilled water, followed by absolute ethanol.Then, they were kept in an incubator (30-45 min) until dry (20-30 min).Tick specimens were cut with sterile scissors and homogenized in phosphate-buffered saline using a micro pestle, and DNA was extracted by employing the phenol-chloroform method (35).A NanoDrop (Nano-Q, Optizen, Daejeon, South Korea) was used to quantify the DNA, and the DNA samples were stored at −20°C for further examination.
The extracted DNA was utilized for screening various tick-borne pathogens based on different partial genes, such as 16S rDNA (Anaplasma/Ehrlichia spp.), groEL (Coxiella spp.), dsb (Ehrlichia spp.), 18S rDNA (Hepatozoon spp.), and gltA, ompA, and ompB (Rickettsia spp.).The nested PCR was used in the case of the groEL partial gene.Each PCR experiment contained a negative control (PCR water instead of the template DNA) and a positive control (the DNA of Rh. microplus for ticks, R. massiliae for bacterial species, and Theileria annulata for protozoal species).The primers used in the current study and their thermocycler conditions are given in Supplementary Table S1.
The PCR products were electrophoresed on 1.5% agarose gel, and the amplified samples were visualized under ultraviolet (UV) light using a gel documentation system (BioDoc-IT™ Imaging Systems UVP, LLC, Upland, United States).The PCR products that showed the expected size results were purified using a commercial PCR clean-up kit (Macherey-Nagel, Duren, Germany) following the manufacturer's instructions.

DNA sequencing and phylogenetic analysis
PCR products of 16S rDNA and cox1 for ticks, as well as 16S rDNA, groEL, dsb, 18S rDNA, gltA, ompA, and ompB for associated tick-borne pathogens, were sent for capillary bidirectional sequencing (Macrogen Inc., Seoul, South Korea).The obtained sequences were trimmed and assembled using SeqMan v. 5 (DNASTAR, Inc., Madison/WI, Map showing the collection sites of tick samples in the selected districts.United States) to remove poor reading sequences.The obtained multiple identical sequences for each gene were considered a consensus sequence and subjected to the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (NCBI).Sequences with maximum identities were downloaded in FASTA format from the NCBI.The obtained sequences were aligned with the downloaded sequences using ClustalW multiple alignments in BioEdit Sequence Alignment Editor v. 7.0.5 (36).The coding nucleotide sequences were aligned using MUSCLE (37).Phylogenetic trees were constructed individually for each partial sequence of tick and associated tick-borne pathogens, by employing the maximum likelihood (ML) method with Kimura 2-parameter in Molecular Evolutionary Genetics Analysis (MEGA-X), accompanied by a bootstrapping value of 1,000 (38).

Statistical analyzes
Tick data and associated pathogen information from all 14 districts were subjected to descriptive statistical analysis, and graphs were designed in Microsoft Excel v. 2016 (Microsoft Office 365 ® ).The minimum infection rate (MIR) was used to estimate the prevalence of infected ticks.To accomplish this, the number of PCR product ticks was divided by the total number of collected ticks (39).
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.

DNA sequences and phylogeny of ticks
Sequencing by BLAST analysis of the 16S rDNA for Rh.sanguineus, Rh. turanicus, Rh. microplus, and Rh.haemaphysaloides showed 99.24-100% maximum identities to the corresponding species.In the phylogenetic tree, the 16S rDNA sequences of Rh. sanguineus, Rh. turanicus, Rhipicephalus microplus (clade C), and Rhipicephalus haemaphysaloides were clustered with the corresponding species (Figure 3A).

Prevalence of pathogens in ticks
Altogether, 271 out of 575 collected ticks were screened for detecting bacterial and protozoal pathogens.Among the four tested tick species, Rh. sanguineus was the most infected tick comprising 81 positive samples with a minimum infection rate of 29.88% (n = 81/271), followed by Rh. haemaphysaloides with a minimum infection rate of 12.91% (n = 35/271), Rh. turanicus with a minimum infection rate of 10.70% (n = 29/271), and Rh.microplus with a minimum infection rate of 5.16% (n = 14/271).All these minimum infection rates were statistically significant.Sequencing of the PCR-based amplified DNA resulted in the detection of eight pathogens-E.minasensis, H. canis, C. burnetii, Anaplasma capra, A. platys, R. massiliae, "Candidatus Rickettsia shennongii" and R. aeschlimannii.Three undetermined species, Ehrlichia sp., Coxiella sp., and Anaplasma sp. were also detected.All pathogens and undetermined species were detected in the collected four tested tick species, except R. aeschlimannii, which was detected only in Rh. sanguineus, whereas A. capra, A. platys, and "Ca.R. shennongii" were detected in Rh. sanguineus, Rh. haemaphysaloides, and Rh.turanicus (Figure 4).

Sequence and phylogenetic analyzes of tick-borne pathogens
Ehrlichia spp.
According to the BLAST results, the 16S rDNA sequences of Ehrlichia sp. and E. minasensis showed 100% identity with the corresponding species.In the phylogenetic tree, the 16S rDNA sequences of Ehrlichia sp. and E. minasensis were clustered with the corresponding sequences (Figure 5A).In the case of the dsb sequence, the BLAST results of the dsb sequence of Ehrlichia sp.showed 100% identity with the E. minasensis sequence.In the phylogenetic tree, the dsb sequence of E. minasensis was clustered with the same species (Figure 5B).The obtained 16S rDNA and dsb sequences of Ehrlichia sp. and E. minasensis were deposited to the GenBank (Supplementary Table S2).

Hepatozoon canis
The obtained 18S rDNA sequence of Hepatozoon sp.showed 99-100% identity with the H. canis sequence.In the phylogenetic tree, frontiersin.orgthe 18S rDNA sequence of H. canis was clustered with the same species (Figure 5C).The obtained 18S rDNA sequence of H. canis was deposited to the GenBank (Supplementary Table S2).

Coxiella spp.
The present groEL sequence of Coxiella sp.showed 97-98% maximum identity with the Coxiella sp.sequence, followed by 96.55% identity with C. burnetii.Other Coxiella sp.groEL sequences showed 100% identity with C. burnetii.In the phylogenetic tree, the groEL sequence of Coxiella sp. was clustered with the Coxiella sp.endosymbiont sequences, while the sequence of C. burnetii was clustered with the corresponding species (Figure 6A).The obtained groEL sequences of Coxiella sp. and C. burnetii were deposited to the GenBank (Supplementary Table S2).

Anaplasma spp.
According to the BLAST results, 16S rDNA Anaplasma spp.sequences showed 99.12% maximum identity with A. capra, 100% maximum identity with A. platys sequences, and 99.13% maximum identity with an undermined Anaplasma sp.In the phylogenetic tree, the 16S rDNA sequences of A. capra, A. platys, and undetermined Anaplasma sp. were clustered with the corresponding species (Figure 6B).The obtained 16S rDNA sequences of A. capra, A. platys, and Anaplasma sp. were deposited to the GenBank (Supplementary Table S2).

Rickettsia spp.
A rickettsial gltA sequence showed 100% identity with R. massiliae, and in the phylogenetic tree, it was clustered with the corresponding species (Figure 7A).Another obtained gltA sequence showed 100% identity with "Ca.R. shennongii, " and it was phylogenetically clustered with the corresponding species (Figure 7A).A third rickettsial gltA sequence showed 100% identity with R. aeschlimannii, and in the phylogenetic tree, it was clustered with the corresponding species (Figure 7A).
The obtained ompA sequence showed 100% identity with R. massiliae and was phylogenetically clustered with the same species (Figure 7B).Another ompA sequence showed 100% identity with "Ca.R. shennongii" and in the phylogenetic tree, it was clustered with the same species (Figure 7B).A third ompA fragment showed 99.8-100% identity with R. aeschlimannii sequences, and in the phylogenetic tree, it was clustered with the same species (Figure 7B).
In the case of ompB, the obtained sequences showed 100% identity with R. massiliae.In the phylogenetic tree, the obtained R. massiliae sequence was clustered with the same species.Another ompB sequence showed 100% identity with "Ca.R. shennongii" and in the phylogenetic tree, this sequence was clustered with "Ca.R. shennongii" (Figure 7C).A third ompB sequence showed 100% identity with R. aeschlimannii, and it was phylogenetically clustered with the corresponding species (Figure 7C).The obtained gltA, ompA, and ompB sequences of R. massiliae, "Ca.R. shennongii" and R. aeschlimannii were deposited to the GenBank (Supplementary Table S2).

Discussion
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     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 freeroaming 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.

Conclusion
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.

FIGURE 1
FIGURE 1 Study model describing the possible risks related to butcher-associated stray dogs.Parts of the figures were drawn by using pictures from Servier Medical Art (http://smart.servier.com/),licensed under a Creative Commons Attribution 3.0 Unported License (accessed 02 May 2023).

FIGURE 3
FIGURE 3 Maximum likelihood phylogenetic trees based on 16S rDNA (A) and cox1 (B) partial sequences of Rhipicephalus spp.The Rhipicephalus glabrosscutatus sequence was used as an outgroup.The obtained sequences are shown in bold underline font.

FIGURE 4
FIGURE 4Molecular prevalence of detected pathogens in butcher-associated stray dog ticks.

FIGURE 7
FIGURE 7 Maximum likelihood phylogenetic trees based on gltA (A), ompA (B), and ompB (C) partial sequences of Rickettsia spp.The Rickettsia canadensis gltA, Rickettsia akari and Rickettsia australis ompA, and Rickettsia akari and Rickettsia australis ompB sequences were used as an outgroup.The obtained sequences are shown in bold underline font.

FIGURE 6
FIGURE 6 Maximum likelihood phylogenetic trees based on groEL (A) and 16S rDNA (B) partial sequences of Coxiella spp.and Anaplasma spp., respectively.The Legionella jordanis groEL and Ehrlichia canis 16S rDNA sequences were used as an outgroup.The obtained sequences are shown in bold underline font.

TABLE 1
Distribution pattern of butcher-associated stray dog ticks and the detection of bacterial and protozoal pathogens in these ticks.

TABLE 1 (
Continued) 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. of