Argas persicus and Carios vespertilionis Ticks Infesting Ducks, Domestic Fowls and Bats in Pakistan: First Report on Molecular Survey and Phylogenetic Position of Borrelia anserina

Simple Summary Soft ticks are well-known for vectoring several disease-causing pathogens that are distributed throughout the world. Surveillance of disease-causing agents associated with these ticks is important to avoid any zoonotic consequences. This study reported the epidemiology and molecular characterization of Borrelia anserina in Argas persicus collected from domestic fowls, ducks and their shelters and Carios vespertilionis ticks infesting bats in Khyber Pakhtunkhwa (KP), Pakistan. In the phylogenetic tree, the obtained sequences of A. persicus and C. vespertilionis clustered with the sequences from Pakistan and neighboring countries, while the Borrelial flaB sequence revealed its relationship with the corresponding species belonging to the relapsing fever group. Further studies are encouraged to screen soft ticks for pathogens that affect public and veterinary health. Abstract Argasid ticks have the vectorial potential for transmitting disease-causing pathogens to avian hosts, resulting in economic losses that may not be fully estimated. Borrelia species are the responsible agents of borreliosis in poultry, animals and humans. Our previous studies have reported a high prevalence of Argas persicus infesting domestic fowls in Khyber Pakhtunkhwa (KP), Pakistan. However, molecular screening and genetic characterization of Borrelia spp. in A. persicus have been neglected in Pakistan. In this study, we focused on the molecular epidemiology and genetic characterization of Borrelia spp. associated with A. persicus ticks infesting domestic fowls and ducks, and Carios vespertilionis infesting bats in selected districts of KP. Overall, 1818 ticks, including females (415; 23%), males (345; 19%), nymphs (475; 26%) and larvae (583; 32%), were collected from 27 locations in nine districts (Peshawar, Mardan, Swabi, Charsadda, Chitral, Lakki Marwat, Bannu, Bajaur and Hangu) from domestic fowls, ducks and their shelters, and bats. A subset of 197 ticks was selected for DNA extraction and PCR to amplify fragments of the cytochrome c oxidase (cox) gene for ticks and flagellin B (flaB) for the detection and genetic characterization of associated Borrelia spp. Among these, only Borrelia anserina DNA was detected in 40 ticks (27.2%) of different life stages, where highest prevalence was found in female ticks (18; 45%), followed by nymphs (12; 30%), larvae (7; 17.5%) and males (3; 7.5%). Tick infestation in shelters (1081; 77%) was higher than on hosts (323; 23%). The resultant cox amplicons of A. persicus showed 100% identity with the same species reported from Pakistan, China, Iran, Kenya, Kazakhstan, Algeria and Egypt and C. vespertilionis show 100% identity with the species reported from Pakistan, China, Japan, Kenya, Vietnam, Spain, Netherlands, the United Kingdom and Hungry, and clustered with the aforementioned species in the phylogenetic tree. The obtained Borrelia sequences showed 100% identity with B. anserina and revealed a close resemblance to the relapsing fever group and clustered in a monophyletic clade with B. anserina from India, Iran and Brazil in a phylogenetic tree. These results establish the first molecular characterization of B. anserina in A. persicus infesting domestic fowls and ducks in the region, as well as their shelters. To effectively control zoonotic consequences, country-wide surveillance research should be encouraged to screen soft ticks infesting various birds for associated pathogens.

Simple Summary: Soft ticks are well-known for vectoring several disease-causing pathogens that are distributed throughout the world.Surveillance of disease-causing agents associated with these ticks is important to avoid any zoonotic consequences.This study reported the epidemiology and molecular characterization of Borrelia anserina in Argas persicus collected from domestic fowls, ducks and their shelters and Carios vespertilionis ticks infesting bats in Khyber Pakhtunkhwa (KP), Pakistan.In the phylogenetic tree, the obtained sequences of A. persicus and C. vespertilionis clustered with the sequences from Pakistan and neighboring countries, while the Borrelial flaB sequence revealed its relationship with the corresponding species belonging to the relapsing fever group.Further studies are encouraged to screen soft ticks for pathogens that affect public and veterinary health.
Abstract: Argasid ticks have the vectorial potential for transmitting disease-causing pathogens to avian hosts, resulting in economic losses that may not be fully estimated.Borrelia species are the responsible agents of borreliosis in poultry, animals and humans.Our previous studies have reported a high prevalence of Argas persicus infesting domestic fowls in Khyber Pakhtunkhwa (KP), Pakistan.However, molecular screening and genetic characterization of Borrelia spp. in A. persicus have been neglected in Pakistan.In this study, we focused on the molecular epidemiology and genetic characterization of Borrelia spp.associated with A. persicus ticks infesting domestic fowls and ducks, and Carios vespertilionis infesting bats in selected districts of KP.Overall, 1818 ticks, including females (415; 23%), males (345; 19%), nymphs (475; 26%) and larvae (583; 32%), were collected from 27 locations in nine districts (Peshawar, Mardan, Swabi, Charsadda, Chitral, Lakki Marwat, Bannu, Bajaur and Hangu) from domestic fowls, ducks and their shelters, and bats.A subset of 197 ticks was selected for DNA extraction and PCR to amplify fragments of the cytochrome c oxidase (cox) gene for ticks and flagellin B (flaB) for the detection and genetic characterization of associated Borrelia spp.Among these, only Borrelia anserina DNA was detected in 40 ticks (27.2%) of different life stages, where highest prevalence was found in female ticks (18; 45%), followed by nymphs (12; 30%), larvae

Introduction
Soft tick Argas persicus is the most common ectoparasite of birds that harbors diseasecausing agents, including Borrelia spp.[1][2][3], causing diseases and un-estimated economic losses to the poultry industry.The occurrences and re-occurrences of infections triggered by bat-associated pathogens have considerably increased during the last decades and has attracted attention towards the screening of ectoparasites such as the bat tick Carios vespertilionis [4].
Argas persicus ticks infest birds such as domestic fowls (Gallus gallus domesticus), ducks (Anas platyrhynchos), turkeys (Meleagris gallopavo), geese (Anser anser domesticus), peacocks (Pavo cristatus), pigeons (Columba livia) and other wild birds [3,[5][6][7].The Gram-negative bacterium Borrelia anserina has been detected in A. persicus ticks that cause avian borreliosis in domestic fowls, doves, turkeys, geese, pheasants and canary birds in tropical and subtropical regions [3,[7][8][9].Borreliosis caused by B. anserina was reported for the first time in Russia in a widespread outbreak in geese [10].After a few years, the role of Argas ticks as a natural vector for borreliosis was confirmed in other continents [11].Subsequently, this pathogen attracted attention to its molecular epidemiology and the role of Argas ticks as vector.The role of C. vespertilionis as a carrier of pathogens such as viruses, bacteria and protozoans has been documented previously [12].Among bacterial pathogens, this tick is a competent vector for Borrelia burgdorferi which causes Lyme disease in humans [12,13].
The members of Borrelia spp.complex are recognized as causative agents of numerous human and animal diseases, such as Lyme disease (LD) and relapsing fever group (RFG) [7].The genus Borrelia is comprised of about ~52 species with worldwide distribution [14,15].These species are transmitted to the host by tick species belonging to different genera, such as Argas, Ornithodoros, Ixodes, Amblyomma, Hyalomma, Rhipicephalus and Bothriocroton, while one species is known to be transmitted by Pediculus humanus corporis (human body lice) [16,17].Borreliosis is categorized into different well-defined groups: LD, RFG borreliosis and reptile-associated group (REP) [18,19].The accurate identification of Borrelia spp. at a species level is complicated by using microscopy.However, some reports differentiate the isolates of Borrelia spp.by using different techniques such as serological tests, immunological assays and molecular approaches [20][21][22].
Pakistan is one of the world's largest poultry production industry, providing about 1163 million broilers.The poultry sector offers employment to more than 1.5 million people, and the investment is about Rs 750 billion to the country (Pakistan Economic Survey, Ministry of Finance, Government of Pakistan, 2022).The poultry sector faces severe issues due to vector-borne diseases, including borreliosis.Argas persicus ticks are reservoir hosts for disease-causing pathogens such as Borrelia spp. that cause borreliosis worldwide [23,24].To reduce the economic losses of the poultry sector, regular surveillance and genetic characterization of ticks and tick-borne Borrelia spp.are of great importance.To our knowledge, nine soft tick species have been reported from Pakistan.Among which five species (Argas sp."rousetti", Argas persicus, C. vespertilionis and Ornithodoros sp.) have been molecularly characterized, while four species (Argas abdussalami, Argas lahorensis, Ornnithodoros papillipies and Ornithodoros tholozani) were identified morphologically [2,[25][26][27][28].Several studies have shown a wide range of pathogens associated with A. persicus [3,9].In a previous study [2], we reported the life cycle and molecular phylogeny of the fowl tick A. persicus.The hard ticks and associated pathogens have been investigated in Pakistan [29][30][31][32][33].However, studies have neglected to characterize Borrelia spp.associated with the soft tick.This study was designed to investigate the molecular epidemiology and genetic characterization of A. persicus infesting domestic fowls and ducks, C. vespertilionis infesting bats and their associated Borrelia spp. in Pakistan.

Collection Sites
The current study was carried out in nine districts of Khyber Pakhtunkhwa (KP), Pakistan, including Peshawar (34  E).These regions were selected based on their different climatic and geographic conditions.Three regions were selected for collection in each district.Google maps were used to collect the geographic coordinates, and the collected data were arranged in a Microsoft Excel (Microsoft Corp., Redmond, WA, USA) worksheet to construct a distribution map for the study area using ArcGIS 10.3.1 (ESRI, Redlands, CA, USA) (Figure 1).

Ethical Approval
Prior approval for this research was received from the members of Advance Studies and Research Board, Abdul Wali Khan University, Mardan, under the approval no (Dir/A&R/AWKUM/2022/9395). Written/oral consent was taken from the holders of domestic fowls and ducks before tick collection from the shelters.

Tick Collection, Preservations and Identification
Ticks were collected from shelters of domestic fowls and ducks from different collection spots of the selected districts.Bats were captured by local farmers in their gardens using hand nets and kept in separate perforated bags.Ticks were removed carefully from the bat's body by using fine sterile tweezers.Collected ticks were kept in sterile bottles and labeled with related information (host, collection date, coordinates, temperature and humidity).Before further analysis, ticks were washed with distilled water followed by 70% ethanol and preserved in 100% ethanol in 1.5 mL tubes.Morphological identification was carried out under the stereomicroscope (StereoBlue-euromex, Arnhem, The Netherlands) using available morphological keys [34,35].

Molecular Analyses
Among the collected ticks, 147 partially fed A. persicus ticks (16 ticks from each district; 4 ticks from each life stage, 2 from a host and 2 from a shelter) and 3 ticks from each (F/M/N) of the ducks were randomly selected for further molecular analysis.In case of the bat collected ticks, we selected ten C. vespertilionis ticks from each district for the extraction of genomic DNA via standard phenol-chloroform method [36].Sterile needles were used to make holes in ticks before DNA extraction in the 1.5 mL tube and dried in the incubator to evaporate the ethanol.The quantification of extracted DNA was performed by a NanoDrop (Nano-Q, Optizen, Daejeon, Republic of Korea).

Ethical Approval
Prior approval for this research was received from the members of Advance Studies and Research Board, Abdul Wali Khan University, Mardan, under the approval no (Dir/A&R/AWKUM/2022/9395). Written/oral consent was taken from the holders of domestic fowls and ducks before tick collection from the shelters.

Tick Collection, Preservations and Identification
Ticks were collected from shelters of domestic fowls and ducks from different collection spots of the selected districts.Bats were captured by local farmers in their gardens using hand nets and kept in separate perforated bags.Ticks were removed carefully from the bat's body by using fine sterile tweezers.Collected ticks were kept in sterile bottles and labeled with related information (host, collection date, coordinates, temperature and humidity).Before further analysis, ticks were washed with distilled water followed by 70% ethanol and preserved in 100% ethanol in 1.5 mL tubes.Morphological identification was carried out under the stereomicroscope (StereoBlue-euromex, Arnhem, The Netherlands) using available morphological keys [34,35].The extracted DNA was subjected to PCR for the molecular identification of ticks using the cox and 16S rRNA gene fragment.The PCR mixture (20 µL) was comprised of 1 µL each primer (10 µM), 4 µL PCR grade water, 2 µL template DNA (50-100 ng/µL) and 12 µL of DreamTaq PCR MasterMix (2×) (Thermo Scientific, Waltham, MA, USA).PCR for the cox and 16S rRNA gene was performed according to the previously described conditions [2,32].The electrophoreses of PCR products was performed in 1.5% agarose gel stained with ethidium bromide and visualized under UV in the Gel Documentation System (UVP BioDoc-It Imaging System, Upland, CA, USA).
Each of the extracted DNA samples were screened for tick-associated Borrelia spp.by targeting the amplification of the flaB partial gene.In the initial PCR, the primer pair (Fla LL, and Fla RL) was followed by a nested PCR using 0.5 µL PCR product as the template and primer (Fla SS and Fla RS) to amplify 665 bp and 354 bp, respectively (Table 1).The PCR conditions for both reactions were set according to Stromdahl et al. [37].In the PCR reactions, a Borrelia spp. of Amblyomma gervaisi, Rickettsia massiliae of Rhipicephalus microplus, Ehrlichia spp.and distilled water were taken as a positive and negative control, respectively.

Sequence and Phylogenetic Analysis
The amplified PCR products showing the expected size were purified using a commercial NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Duren, Germany) following the manufacturer's directions.The purified PCR amplicons were sent to a commercial company for bidirectional sequencing (Macrogen, Inc., Seoul, Republic of Korea).The obtained sequences were subjected to trimming and assembled to remove the primer's contamination and poor reading regions in SeqMan v. 5.0 (DNASTAR, Inc., Madison, WI, USA).Trimmed sequences were subjected to BLAST (Basic Local Alignment Search Tool) at NCBI (National Center for Biotechnology Information) [41] to download identical sequences.Identical sequences were downloaded and aligned by ClustalW Multiple alignments [42] in BioEdit alignment editor v. 7.0.5 (Raleigh, NC, USA) [43].The phylogenetic tree, based on cox for ticks and flaB sequence for Borrelia spp., was constructed using the Maximum Likelihood method based on the Kimura 2-parameter in MEGA-X (Molecular evolutionary genetics analysis), aligned by MUSCLE [44] using 1000 bootstrap replicons [45].

Statistical Analyses
The data such as tick infestation on hosts and shelters in the different regions were assembled and arranged in spreadsheets on Microsoft Excel v. 2016.The tick infestation of each life stage on host and shelters, and prevalence of B. anserina between different regions, was analyzed in GraphPad Prism v. 5.00 (GraphPad Software Inc., San Diego, CA, USA).

Detection of Borrelia anserina
Among a subset of 197 screened ticks for Borrelia spp., 40 (27.2%) were found positive for B. anserina based on flaB fragment (long and short sequences).Among the 40 positive samples, only 5 showed faint bands in the initial PCR (long fragments), while 35 were positive in nested PCR (short fragments) and sequencing.In 40 (27.2%)Borrelia positive ticks, the highest prevalence was found in female ticks (18/40; 45%), followed by nymphs (12/40; 30%), larvae (7/40; 17.5%) and males (3/40; 7.5%).The prevalence of ticks, infested shelters, life stages and details of B. anserina detection on host or shelter-collected ticks from selected districts are shown in Table 2.In the case of C. vespertilionis, all the screened ticks were found to be negative for B. anserina.Both A. persicus and C. vespertilionis were found to be negative for the presence of Rickettsia and Ehrlichia DNA.
The amplified flaB fragments showed 100% identity to the sequences of B. anserina from India-MK989712, MK128989, MK128990, Iran-KY438930 and Brazil-DQ849626.Among the 40 B. anserina sequences (5 long and 35 short), both long and short sequences showed identity to each other.Thus, one consensus sequence from each long and short sequence of B. anserina was uploaded to GenBank (OP326592, ON148464).The obtained sequence of B. anserina was clustered with the RFG species in the phylogenetic tree (Figure 3).
the United Kingdom-MF510174 and Hungary-KX431955.In the phylogenatic tree, the ob tained sequences were clustered with mentioned sequences (Figure 2).The consensus se quences of A. persicus and C. vespertilionis were uploaded to GenBank (OP692725 OR614351).showed identity to each other.Thus, one consensus sequence from each long and short sequence of B. anserina was uploaded to GenBank (OP326592, ON148464).The obtained sequence of B. anserina was clustered with the RFG species in the phylogenetic tree (Figure 3).

Discussion
Soft ticks A. persicus and C. vespertilionis are the common ectoparasites of several birds and bats, respectively, that have been morphologically and genetically characterized in several countries, including Pakistan [2,25,28,[46][47][48].However, knowledge of the associated Borrelia spp.has not been molecularly explored in the region.To fill this knowledge gap, the collected ticks were morphologically and molecularly examined and confirmed as A. persicus and C. vespertilionis infesting domestic fowls, ducks and bats in nine districts of KP, Pakistan.To our knowledge, this is the first report on the molecular epidemiology, genetic characterization, and comparison of Borrelia spp. on host-(domestic fowls and ducks) and shelter-collected A. persicus ticks in Pakistan.Ticks were screened for spirochetes, and B. anserina DNA was detected in A. persicus ticks while C. vespertilionis ticks were found negative for Borrelia DNA.The phylogenetic analysis of tick cox sequences revealed their closest relationship with the same species reported from Pakistan [2,28].The phylogenetic tree of Borrelia flaB revealed that the aforementioned species belongs to the RFG of Borrelia species.The collected ticks were also screened for the presence of Rickettsia and Ehrlichia; however, DNA of none of these agents was detected.
The climatic conditions are mostly intricate with the prevalence and diversity of ticks in a region [49,50].The highest prevalence of A. persicus was reported in hot atmospheric and humid regions such as Peshawar and Mardan, while the lowest prevalence was in low temperature, high altitude and less humid regions like Bajaur and Chitral, as previously described [2], while C. vespertilionis was most prevalent in the Mardan district and least prevalent in the Swabi district.The variation in prevalence with environmental temperature, high altitude, and rainfall indicates that the ticks prefer high temperature and rich humid regions where they can survive and reproduce favorably [3,32,50].The annual increase in the climatic temperature facilitates the tick's survival in the least prevalent and tick-free regions [51].
Complexities in the morphological identification of Argasid ticks still exist for taxonomists [52].Thus, molecular approaches are used for accurate identification and genetic characterization of different species, especially Argasid ticks [23,[53][54][55][56].The host-(domestic fowls) and shelter-collected ticks were molecularly confirmed as a species, A. persicus, with the bat-collected ticks as C. vespertilionis.The results revealed that the ticks inhabiting shelters may pose severe health threats to animals and humans due to accidental infestation and the capability of transmitting pathogens [57][58][59].The molecular data showed a close identity of the collected ticks with the previously reported A. persicus and C. vespertilionis from the same region.In the phylogenetic tree, the obtained cox sequences of ticks clustered with the same species reported from Pakistan and neighboring countries which is in agreement with the previous studies [2,28,54].
Argas persicus ticks are commonly involved in the transmission of Borrelia spp. in various domestic and wild birds [3].The DNA of flaB for B. anserina was detected in all life stages, like larvae, nymphs, males and females of A. persicus, indicating their transovarial transmission [17].The host-and shelter-collected ticks were comparatively screened and found positive for B. anserina, while in a similar screening, C. vespertilionis was found negative for any Borrelia spp.In the phylogenetic tree, B. anserina based on flaB sequences grouped with Borrelia species belongs to RFG.The outcomes of our study are consistent with the findings of previous reports that demonstrated B. anserina falls within a monophyletic clade with the species of the RFG [16,55,56,[60][61][62][63][64][65].The presence of Borrelia spp.has also been reported in several birds such as domestic fowls, ducks, geese, king penguins, blackbirds and nightingales in different regions of the world [52,53].Birds should be investigated for Borrelia spp. to restrict any Borrelia epidemics and zoonosis consequences.Since this study represents a limited number of analyzed specimens from selected regions, detailed large-scale molecular surveillance of soft tick-associated pathogens is regularly needed to avoid zoonotic threats to birds and humans.

Figure 1 .
Figure 1.Map showing the collection sites of ticks in Khyber Pakhtunkhwa Pakistan.

Figure 1 .
Figure 1.Map showing the collection sites of ticks in Khyber Pakhtunkhwa Pakistan.

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree based on cox sequences of Argas persicus and Cario vespertilionis.Alveonasus lahorensis was taken as an outgroup.GenBank accession numbers are fo lowed by species (italic) and country names.The obtained A. persicus and C. vespertilionis sequence are represented in bold and underlined.

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree based on cox sequences of Argas persicus and Carios vespertilionis.Alveonasus lahorensis was taken as an outgroup.GenBank accession numbers are followed by species (italic) and country names.The obtained A. persicus and C. vespertilionis sequences are represented in bold and underlined.

Figure 3 .
Figure 3. Maximum likelihood phylogenetic tree based on flaB sequence of Borrelia anserina.Borrelia turcica and "Candidatus Borrelia tachyglossi" were taken as outgroups.GenBank accession numbers are followed by species (italic) and country names (in the case of B. anserina).The obtained B. anserina sequence are represented in bold and underlined.

Table 1 .
List of primers used for the amplification of ticks and their associated pathogens.

Table 2 .
Borrelia anserina detected in different life stages of the Argas persicus collected in nine districts, KP, Pakistan.