Molecular evidence of Borrelia theileri and closely related Borrelia spp. in hard ticks infesting domestic animals

Ticks pose significant threats to hosts by transmitting Borrelia spp., which are grouped into Lyme borreliae, relapsing fever borreliae (RF), and reptiles- and monotremes-associated borreliae. The RF borreliae encompass a group of Borrelia species predominantly transmitted by soft ticks, but some of its members can also be transmitted by hard ticks. Information on the detection and genetic characterization of tick-borne RF borreliae, including Borrelia theileri, is notably rare in Asia, particularly in Pakistan. Herein, we employed molecular techniques to detect borreliae in hard ticks collected from domestic animals in Khyber Pakhtunkhwa, Pakistan. Ticks were subjected to morphological analysis, followed by DNA extraction and PCR amplification of partial fragments of borrelial 16S rRNA and flaB genes. A total of 729 ticks were collected from 264 hosts, with Haemaphysalis cornupunctata (12.9%; 94/729) being the most prevalent, followed by Hyalomma anatolicum (11.7%; 85/729), Rhipicephalus microplus (10.0%; 73/729), Haemaphysalis kashmirensis (9.1%; 66/729), Haemaphysalis bispinosa (8.5%; 62/729), Rhipicephalus sanguineus (8%; 58/729), Haemaphysalis montgomeryi (6.2%; 45/729), Rhipicephalus turanicus (5.5%; 40/729), Hyalomma dromedarii and Ixodes kashmirensis (4.4%; 32/729 each), Rhipicephalus haemaphysaloides (4.1%; 30/729), Haemaphysalis sulcata and Hyalomma scupense (3.8%; 28/729 each), Haemaphysalis danieli (2.9%; 21/729), Hyalomma kumari (2.6%; 19/729), and Hyalomma isaaci (2.2%; 16/729). Based on 16S rRNA detection of Borrelia spp., only R. turanicus yielded positive results, resulting in an overall infection rate of 0.3% (2/160), while using flaB-based detection, four tick species including R. microplus, R. turanicus, Ha. sulcata, and Ha. cornupunctata showed positive results, yielding an overall infection rate of 6.9% (11/160). The amplified DNA fragments of borrelial 16S rRNA and flaB in R. turanicus from goats shared maximum identities of 100 and 99.40% with Borrelia theileri, respectively. Amplified borrelial flaB fragments in R. microplus from cows and sheep displayed 100% identity with B. theileri, while flaB fragments in Ha. cornupunctata and Ha. sulcata from goats revealed identities of 99.32 and 99.75% with undetermined RF Borrelia spp., respectively. Phylogenetic analysis revealed clustering of B. theileri from R. microplus and R. turanicus with the same species, while Borrelia spp. from Ha. cornupunctata and Ha. sulcata with undetermined RF Borrelia spp. Notably, this research marks the first documentation of B. theileri in R. turanicus and the identification of RF Borrelia spp. in Ha. cornupunctata and Ha. sulcata.

The pathogenicity of B. theileri in domestic animals is known (36, 37), while there is a notable diversity and abundance of their vertebrate hosts and tick vectors in Pakistan (38)(39)(40)(41)(42). Concern exists regarding the negative impacts of this pathogen on the country's livestock industry, though this impact has yet to be determined.In continuation with our previous studies, which detected REP Borrelia sp. and Borrelia anserina (43,44) in ticks, the objective of this study was to genetically characterize RF borreliae in hard ticks infesting domestic animals.

Study area
In toto, 15 districts of Khyber Pakhtunkhwa, including Abbottabad, Bajaur, Buner, Charsadda, Dir Lower, Dir Upper, Haripur, Malakand, Mardan, Mohmand, Nowshera, Peshawar, Shangla, Swabi, and Swat, were included in this study.Google Maps was utilized to determine the precise geographical coordinates of the collection points in the study area, and these information were organized in Microsoft Excel 2016.The land-cover map of the study area was created using ArcGIS version 10.3.1 (ESRI, Redlands, CA, United States; Figure 1).

Tick collection, preservation, and identification
The study was conducted from March to September 2022, covering three seasons in Pakistan: spring (March-May), summer (June-August), and early autumn (September).Different domestic animals were screened for ticks in farms and grazing fields, when found, ticks were collected using tweezers.Essential information, including the host type, collection date, and collection site coordinates, was recorded during fieldwork.Before preservation in 70% ethanol, all collected ticks were washed with distilled water followed by 70% ethanol.Tick specimens were morphologically identified up to the species level using a stereomicroscope (Stemi 508, Zeiss, Germany) and standard taxonomic identification keys and morphological descriptions (45)(46)(47)(48)(49)(50)(51).

DNA extraction and polymerase chain reaction
A subset of 160 specimens, consisting of at least 1 female per tick species per district and 3 nymphs per tick species, underwent DNA extraction.Tick homogenization was performed individually using sterile scissors in 1.5 mL Eppendorf tubes.DNA extraction was carried out using the phenol-chloroform method with minor modifications (52).The extracted DNA was then hydrated by adding 20-30 μL of "nuclease-free" water, and its quantification was analyzed using a NanoDrop spectrophotometer (Nano-Q, Optizen, South Korea).
All extracted genomic DNA samples were subjected to a conventional PCR (BIOER, China).Partial fragments of borrelial 16S rRNA were amplified through standard PCR, while borrelial flaB amplification was achieved through nested-PCR.For 16S rRNA and the first round of flaB PCR, the reaction mixture had a total volume of 25 μL and included components: 2 μL of extracted DNA (50-100 ng/ μL), 1 μL of each forward and reverse primer (Table 1) at a concentration of 10 pmol/μL, 8.5 μL of nuclease-free PCR water, and 12.5 μL of DreamTaq green MasterMix (2X).The second round of flaB PCR was carried out with 1 μL of the first round PCR product and 9.5 μL of PCR water.All PCRs were conducted under experimental conditions, as previously described (43).PCR water (nuclease-free) was used as a negative control, while DNA of Borrelia sp. from A. gervaisi ticks as a positive control.The amplified fragments were resolved on a 2% agarose gel, visualized using a Gel Documentation system (BioDoc-It™ Imaging Systems, Upland, CA, United States), and purified using the GeneClean II Kit (Qbiogene, Illkirch, France).

DNA sequencing and phylogenetic analysis
The amplicons were sent for DNA sequencing (Macrogen, Inc., Seoul, South Korea) using the Sanger sequencing method with an ABI 373XL system.The raw sequences obtained were then visualized and analyzed using SeqMan version 5.0 (DNASTAR, Inc., Madison, WI, United States) to obtain clean sequences.These clean sequences were subsequently subjected to BLAST (Basic Local Alignment Search Tool) analysis at the NCBI (National Center for Biotechnology Information) to identify the closest matches with sequences already deposited in the GenBank.
The obtained sequences, homologous sequences (downloaded from the BLAST results), and an appropriate outgroup were imported and aligned using the BioEdit alignment editor version 7.0.5 (55) with the ClustalW multiple alignment method (56).The tree topology was adapted from previous studies (10,13,16,20,43).The alignments were then used in Molecular Evolutionary Genetics Analysis (MEGA-X) (57) to construct phylogenetic trees, utilizing the Maximum Likelihood method with 1,000 bootstrap replicates.

Tick and host description
Of the total collected specimens (729), Haemaphysalis ticks were the most abundant comprising 43

Sequences analyses
The partial fragments of borrelial flaB were amplified in 11 ticks, while partial fragments of borrelial 16S were amplified in 2 flaB-positive ticks.The attempts to amplify 16S rRNA in the remaining 9 flaB-positive ticks were unsuccessful.Overall, 4 sequences (1 forward and 1 reverse per positive tick sample) were obtained for 16S rRNA, while 22 longer sequences (1 forward and 1 reverse per positive tick sample) were obtained for flaB.A subset of sequences of 16S rRNA obtained from the genomic DNA of R. turanicus from goats and cattle were found to be identical, which resulted in consensus sequences of 644 bp.Similarly, all long sequences of flaB within each subset were identical in the following ways: (a) sequences obtained from R. microplus from sheep and cattle were identical, (b) sequences obtained from R. turanicus from goats were identical, (c) sequences obtained from Ha. cornupunctata from goats were identical, and sequences obtained from Ha. sulcata from goats were identical.These four subsets resulted in consensus sequences of 587, 522, 547, and 559 bp, respectively.Besides the long sequences for flaB, 11 short sequences (1 forward per positive tick sample) were obtained from their corresponding positive PCR samples.

Detection of Borrelia spp. in ticks
The consensus sequences of borrelial 16S rRNA obtained from genomic DNA of R. turanicus from goats shared a maximum identity of 100% with B. theileri.The long consensus sequences of borrelial flaB obtained from the same samples also showed a maximum identity of 99.40% with B. theileri.Other long consensus sequences of borrelial flaB obtained from tick's genomic DNA showed their BLAST identities in the following ways: (a) Borrelia sp.detected in R. microplus from cows and sheep showed a maximum identity of 100% with B. theileri, (b) Borrelia sp.detected in Ha. cornupunctata from goats depicted a maximum identity of 99.32% with a Borrelia sp., and (c) Borrelia sp.detected in Ha. sulcata from goats displayed a maximum identity of 99.75% with Borrelia sp.Among the determined RF borreliae, all these sequences were found close to B. theileri followed by B. lonestari.
Considering 16S rRNA-based detection of Borrelia spp., only R. turanicus was positive, resulting in an overall infection rate of 0.3% (2/160).When considering flaB-based detection of Borrelia spp., four ).Notably, within the overlapped region, the short and long sequences of flaB were identical, confirming the BLAST identities of each other.However, only long sequences were used in phylogenetic analyses.Table 2 gives details about Borrelia spp., including the associated hosts and the corresponding geographical sites.The obtained sequences were submitted to GenBank under the following accession numbers: 16S rRNA OR561043 (B.theileri haplotype detected in R. turanicus from goats); and flaB OR574987 (B.theileri haplotype detected in R. turanicus from goats), OR574986 (B.theileri detected in R. microplus from cows and sheep), OR574984 (Borrelia sp.detected in Ha. cornupunctata from goats), OR574985 (Borrelia sp.detected in Ha. sulcata from goats).

Phylogenetic analysis
A phylogenetic tree was obtained based on 16S rRNA, in which B. theileri detected in R. turanicus from goats in the present study clustered with the same species from Egypt and Zambia (Figure 2A).Furthermore, this species appeared in a monophyletic group alongside B. lonestari and B. miyamotoi.Another phylogenetic tree was obtained based on flaB (Figure 2B), revealing the clustering of Borrelia spp.detected in the current study in the following manner.The haplotype of B. theileri found in R. microplus from sheep and cows, as well as another haplotype of B. theileri found in R. turanicus from goats clustered with the corresponding species from Brazil.The Borrelia sp.found in Ha. sulcata from goats clustered with undetermined Borrelia sp. from Brazil, while the Borrelia sp.found in Ha. cornupunctata from goats clustered with undetermined species from Portugal.Additionally, these species also formed a monophyletic group along with B. lonestari and B. miyamotoi.

Discussion
Compared to LB borreliae, RF borreliae have received less attention globally (26).Similarly, despite reported RF cases (58), the association of borreliae with ticks in in Asia in general and in Pakistan in particular is poorly known.Neglecting RF borreliae can have significant adverse consequences for both public and animal health.To address this knowledge gap, we conducted Borrelia spp.detection in 16/160 hard ticks collected from six different domestic animals across different geographical areas.In addition to detecting B. theileri in R. microplus, our study represents, to the best of our knowledge, the first report of B. theileri in R. turanicus, as well as the detection of Borrelia spp. in Ha. cornupunctata and Ha.sulcata.
In Pakistan, previous studies suggest that Haemaphysalis spp., Hyalomma spp., and Rhipicephalus spp.are commonly associated with domestic animals (38,40,42,45,(59)(60)(61)(62)(63).Considering their distribution in previous studies, it can be inferred that Haemaphysalis spp.are prevalent in humid and vegetated areas, Hyalomma species prevalent in dry and desert areas, and Rhipicephalus spp.are abundant in humid and warm areas.Given that the current study area primarily consists of rangeland, cropland, and forested land surfaces, Haemaphysalis spp.and Rhipicephalus spp.were found the most abundant ticks in this study, in contrast to Hyalomma spp.Moreover, the higher abundance of Haemaphysalis and Rhipicephalus ticks could also be associated with their broader host range and greater number of main hosts.
Regarding HTRF borreliae, it is well studied that B. theileri is associated with the genus Rhipicephalus, especially R. microplus (26,31).Along with R. microplus, the current study also detected this pathogen in R. turanicus, marking the earliest such finding.In contrast, the association of HTRF borreliae with the genus Haemaphysalis is poorly understood.This study and previous related reports (64,65) suggest that there could be a considerable association between HTRF borreliae and Haemaphysalis ticks, which needs further investigation.Furthermore, apart from being found in hard ticks, previous studies have also detected B. theileri and other closely related undetermined Borrelia spp. in vertebrate hosts, including cattle, goats, and sheep (6, 31, 36).Given the known pathogenicity of B. theileri in domestic animals (26, 36, 37, 66), hard ticks could pose threats to domestic animals in the region.Several other factors in the studied region, including a high tick abundance, a large population of cattle and small ruminants, and their combined farming practices and unmonitored movements, could further exacerbate health threats.
Different analysis based on molecular data are considered powerful tools for the identification of biological species (67,68).Furthermore, unlike the traditional systematic and taxonomy of TBRF borreliae, which was based on co-speciation of ticks and borreliae (26, 69), the advanced approach relies on molecular evidence (10,26,(67)(68)(69)(70)(71).It is also studied that different molecular markers, including 16S rRNA and flaB are compatible in the case of identification and phylogenetic analysis of TBRF (67,72).Therefore, 16S rRNA and flaB based molecular data was obtained for Borrelia spp. in the current study, which was subsequently subjected to phylogenetic analysis.The genetic variations in the detected Borrelia spp.could be associated with the difference in their tick hosts in general.Despite their mutual genetic variations, in the BLAST and phylogenetic analysis, these Borrelia spp.exhibited proximity to B. theileri, B. lonestari and B. miyamotoi.The observed closeness could be attributed to their shared niche, while most RF borreliae are associated with soft ticks, these are associated with hard ticks.Candidatus Borrelia texasensis, although considered to be transmitted by hard ticks (26, 73), did not cluster in the mentioned group.

Conclusion
This study not only confirmed the presence of B. theileri in R. microplus but also provided the first documented evidence of B. theileri in R. turanicus collected on cattle and sheep, along with the detection of RF Borrelia spp. in Ha. cornupunctata and Ha.sulcata collected on goats.The study contributes to the expansion of the geographical and tick host range of RF borreliae, which, in turn, could support future research efforts focusing on veterinary health.Further studies should be encouraged to investigate the ticks-borne bovine borreliosis in order to reduce potential risks in the region.

TABLE 1
Primers used for amplification of targeted DNA of Borrelia spp.by conventional PCR.
*Used in a second round/nested reaction.

TABLE 2
Information related tick species, their hosts and collection sites, as well as PCR results of associated Borrelia spp.