Molecular Survey and Spatial Distribution of Rickettsia spp. in Ticks Infesting Free-Ranging Wild Animals in Pakistan (2017–2021)

Rickettsia spp. associated with ticks infesting wild animals have been mostly neglected in several countries, including Pakistan. To address this knowledge gap, ticks were collected during 2017 to 2021 from wild animals including cats (Felis chaus), Indian hedgehogs (Paraechinus micropus), and wild boars (Sus scrofa). The collected ticks were morpho-molecularly identified and screened for the detection of Rickettsia spp. Morphologically identified ticks were categorized into four species of the genus Rhipicephalus: Rhipicephalus haemaphysaloides, Rh. turanicus, Rh. sanguineus sensu lato (s.l), and Rh. microplus. Among 53 wild animals examined, 31 were infested by 531 ticks, an overall prevalence of 58.4%. Adult female ticks were predominant (242 out of 513 ticks collected, corresponding to 46%) in comparison with males (172, 32%), nymphs (80, 15%) and larvae (37, 7%). The most prevalent tick species was Rh. turanicus (266, 50%), followed by Rh. microplus (123, 23%), Rh. sanguineus (106, 20%), and Rh. haemaphysaloides (36, 7%). Among the screened wild animals, wild boars were the most highly infested, with 268 ticks being collected from these animals (50.4%), followed by cats (145, 27.3%) and hedgehogs (118, 22.3%). Tick species Rh. haemaphysaloides, Rh. turanicus, and Rh. sanguineus were found on wild boars, Rh. haemaphysaloides, and Rh. microplus on cats, and Rh. turanicus on hedgehogs. In a phylogenetic analysis, mitochondrial cytochrome C oxidase 1 (cox1) sequences obtained from a subsample (120) of the collected ticks clustered with sequences from Bangladesh, China, India, Iran, Myanmar, and Pakistan, while 16S ribosomal DNA (16S rDNA) sequences clustered with sequences reported from Afghanistan, Egypt, India, Pakistan, Romania, Serbia, and Taiwan. Among Rickettsia infected ticks (10/120, 8.3%), Rh. turanicus (7/10, 70%), and Rh. haemaphysaloides (3/10, 30%) were found infesting wild boars in the districts Mardan and Charsadda. The obtained rickettsial gltA gene sequences showed 99% and ompA gene sequences showed 100% identity with Rickettsia massiliae, and the phylogenetic tree shows ompA clustered with the same species reported from France, Greece, Spain, and USA. This study emphasizes the need for effective surveillance and control programs in the region to prevent health risks due to tick-borne pathogens, and that healthy infested wild animals may play a role in the spread of these parasites.


Introduction
Interactions between domestic and wild animals have increased due to urbanization, deforestation, and anthropogenic activities which enhance the risk of emergence of zoonotic diseases [1]. Ticks' harmful effects are not only restricted to livestock and humans, but are also a major threat to wild animals and thus are important from a conservation point of view [2]. Moreover, wild animals serve as bridges for pathogen transmission between wildlife and humans. Tick-infested wild and domestic animals exchange ticks, and thus pathogenic organisms, upon sharing habitats [2].

Ticks' Morphological Description
The collected ticks were identified according to their distinguishing features, e.g., basis capituli of male Rh. haemaphysaloides bear slightly sharp and pointed cornua and sickle-shaped adanal plates. The female Rh. haemaphysaliodes scutum has nearly the same length as width, with a slightly sinuous posterior margin. The genital opening is narrowly U-shaped on the ventral side. The width of capituli in male Rh. sanguineus sensu lato (s.l) is greater than its length with acutely-curved lateral angles of the basis capituli. Adanal plates Pathogens 2022, 11, 162 3 of 14 are subtriangular or rounded posteriorly. The length of scutum in female Rh. sanguineus is more than its width, having a sinuous posterior margin. The posterior lip genital aperture is broad and U-shaped. Basis capituli of male Rh. turanicus have sharp and pointed cornua with comma-shaped cervical grooves. The adanal plates may be broad and shortened or sharp and longer posteriorly. The posterior margin of the scutum in female Rh. turanicus is distinctly sinuous, having a small genital aperture and U-shaped to broadly V-shaped posterior lip. Male Rh. microplus has a distinct cornua, and ventrally the coxa 1 bears long and distinct spurs. In female Rh. microplus the scutum is pear shaped, with a broader U-shaped genital aperture ( Figure 1).

Ticks' Morphological Description
The collected ticks were identified according to their distinguishing feature basis capituli of male Rh. haemaphysaloides bear slightly sharp and pointed cornu sickle-shaped adanal plates. The female Rh. haemaphysaliodes scutum has nearly th length as width, with a slightly sinuous posterior margin. The genital opening is na U-shaped on the ventral side. The width of capituli in male Rh. sanguineus sensu la is greater than its length with acutely-curved lateral angles of the basis capituli. A plates are subtriangular or rounded posteriorly. The length of scutum in female R guineus is more than its width, having a sinuous posterior margin. The posterior lip aperture is broad and U-shaped. Basis capituli of male Rh. turanicus have shar pointed cornua with comma-shaped cervical grooves. The adanal plates may be and shortened or sharp and longer posteriorly. The posterior margin of the scu female Rh. turanicus is distinctly sinuous, having a small genital aperture and U-s to broadly V-shaped posterior lip. Male Rh. microplus has a distinct cornua, and ve the coxa 1 bears long and distinct spurs. In female Rh. microplus the scutum is pear s with a broader U-shaped genital aperture ( Figure 1).
BLAST analysis of the obtained gltA gene (Rickettsia) sequences from Rh. turanicus an Rh. haemaphysaloides infesting wild boars showed 99% identity with R. massiliae sequence from China. On the other hand, ompA gene sequences detected in Rh. turanicus and Rh. haem aphysaloides infesting wild boars showed 99-100% identities with previously reported se quences of R. massiliae from France, Greece, Spain, and the USA. In a phylogenetic tree, th obtained sequences clustered with R. massiliae from France (CP000683), Greece (MG521363 Spain (KR401146), and USA (CP003319, DQ212707) ( Figure 4). The resulting gltA and ompA sequences for R. massiliae were deposited in GenBank under accession numbers: (OM066912 and (MZ540775 and OM174266), respectively.
BLAST analysis of the obtained gltA gene (Rickettsia) sequences from Rh. turanicus and Rh. haemaphysaloides infesting wild boars showed 99% identity with R. massiliae sequences from China. On the other hand, ompA gene sequences detected in Rh. turanicus and Rh. haemaphysaloides infesting wild boars showed 99-100% identities with previously reported sequences of R. massiliae from France, Greece, Spain, and the USA. In a phylogenetic tree, the obtained sequences clustered with R. massiliae from France (CP000683), Greece (MG521363), Spain (KR401146), and USA (CP003319, DQ212707) ( Figure 4). The resulting gltA and ompA sequences for R. massiliae were deposited in GenBank under accession numbers: (OM066912) and (MZ540775 and OM174266), respectively.  Rickettsia canadensis was used as an outgroup. Bootstrap values are presented at each node (1000). Accession numbers are followed by species and country name. Sequences obtained in the present study were labeled with black circles.

Discussion
Climate change, urbanization, and other anthropogenic activities have led to the destruction of wildlife habitats, which in turn has increased the chances of interaction between wild and domestic animals [19]. Diverse geographical regions comprising mountainous ranges and agro-wildlife localities serve as habitats for several wildlife species in Pakistan. Studies have been conducted on ticks infesting domestic animals, but research has often neglected ticks infesting wild animals in Pakistan. In this study, we inspected cats, hedgehogs, and wild boars for tick infestation in four districts of KP, Pakistan. The collected ticks were morpho-molecularly identified as Rh. haemaphysaloides, Rh. turanicus, Rh. sanguineus and Rh. microplus and screened for tick-associated Rickettsia species. Rickettsia massiliae was detected in Rh. turanicus and Rh. haemaphysaloides infesting wild boars in the Charsadda and Mardan districts.
Cats infested by ticks can bolster the dispersion of ticks and tick-borne pathogens to predisposed owners and other domestic animals [4]. In the current study, we observed the infestation of Rh. microplus and Rh. haemaphysaloides on cats. Cats and other wild animals generally acquire ticks from natural habitats and their inside access may create a risk of tick infestation

Discussion
Climate change, urbanization, and other anthropogenic activities have led to the destruction of wildlife habitats, which in turn has increased the chances of interaction between wild and domestic animals [19]. Diverse geographical regions comprising mountainous ranges and agro-wildlife localities serve as habitats for several wildlife species in Pakistan. Studies have been conducted on ticks infesting domestic animals, but research has often neglected ticks infesting wild animals in Pakistan. In this study, we inspected cats, hedgehogs, and wild boars for tick infestation in four districts of KP, Pakistan. The collected ticks were morpho-molecularly identified as Rh. haemaphysaloides, Rh. turanicus, Rh. sanguineus and Rh. microplus and screened for tick-associated Rickettsia species. Rickettsia massiliae was detected in Rh. turanicus and Rh. haemaphysaloides infesting wild boars in the Charsadda and Mardan districts.
Cats infested by ticks can bolster the dispersion of ticks and tick-borne pathogens to predisposed owners and other domestic animals [4]. In the current study, we observed the infestation of Rh. microplus and Rh. haemaphysaloides on cats. Cats and other wild animals generally acquire ticks from natural habitats and their inside access may create a risk of tick infestation to indoor domesticated animals, pets, and humans [4,6]. In this study, Rh. turanicus ticks were found infesting hedgehogs, and other tick species including Rh. haemaphysaloides, Rh. sanguineus, and Rh. turanicus were found infesting wild boars. Rh. turanicus has been previously reported as infesting hedgehogs in Iran [14] and Turkey [13]. Accordingly, Rh. turanicus infestation in hedgehogs, as observed in this study, provides evidence that hedgehogs are not accidental hosts for this tick. In wild boar, Rh. sanguineus and Rh. turanicus infestation has been previously reported in Sri Lanka [19], and Rh. sanguineus in KP, Pakistan [20]. The variety of ticks found infesting wild boar may be due to the free movement of this host and contact with other wild and domestic animals.
Morphological identification of the tick species was confirmed by sequencing fragments of mitochondrial genes (cox1 and 16S rRNA). Using morphology alone is insufficient for the precise identification of tick species due to morphological similarities, the presence of engorged as well as immature stages, and damaged specimens [20,[35][36][37]. In several studies, both morphological and molecular identification of ticks have been implemented to achieve accurate taxonomic classification [34,36]. Molecular markers, including mitochondrial cox1 and 16S rRNA, have been reported in the successful determination of the evolution and phylogeny of ticks [37]. Among genetic markers, 16S rRNA and cox1 are useful for understanding interspecific phylogenetic and intraspecific genetic variabilities among ticks [20,37]. In this study, phylogenetic analysis of the identified Rhipicephalus species was performed using cox1 and 16S rDNA partial sequences, which revealed close evolutionary relationship with ticks of the same species reported from Afghanistan, Bangladesh, China, Egypt, India, Iran, Myanmar, Pakistan, Romania, and Taiwan.

Ethical Approval
The experimental design of the present study was approved by the Advance Studies Research Board members of Abdul Wali Khan University, Pakistan (Dir/A&R/AWKUM/2018/1410).

Study Area
The rural areas of the Charsadda, Mardan, Peshawar, and Swabi districts were selected for the collection of wild animals, including cats, Indian hedgehogs, and wild boars, during 2017-2021. The study area comprising selected districts in the KP northern province have their highest (33.4 • C) and lowest (11.7 • C) mean temperatures in July and December, respectively (climate-data.org) accessed on 27 May 2021. The exact geographical coordinates of sample locations were obtained using Global Positioning System (GPS) and added to the attribute table for tagging on the study area map using ArcGIS v. 10.3 ( Figure 5).

Tick Collection and Morphological Identification
Wild animals including cats, hedgehogs, and wild boars found dead on highways, killed or captured by local farmers to secure their crops, were screened for ticks. Ticks found on the host body were carefully collected to avoid any damage to the specimens. All collected ticks were preserved in 100% ethanol. Morphological identification of the collected ticks was done using morphological features under Stereozoom microscope (BIOBASE, Jinan, China), by comparing with standard available morpho-taxonomic keys [44,45].

DNA Extraction and PCR
All ticks were morphologically identified, and 120 ticks comprising 10 specimens (different life stages) of each species from all districts were further processed for genomic DNA extraction (Table 1). Ticks were washed with distilled water followed by 70% ethanol and PBS for the removal of any surface contaminants. Washed ticks were individually kept in 1.5 ml tubes and dried in an incubator. Holes were made with needles, and the whole body of each tick was cut into small pieces using sterile scissors and homogenized by micro pestle for DNA extraction using phenol chloroform method [46]. The concentration of extracted DNA was measured using NanoQ (Optizen, Daejeon, South Korea), and samples were maintained at -20 ℃ for further analysis.
Mitochondrial cytochrome C oxidase 1 (cox1) and 16S ribosomal RNA (16S rRNA) genes' partial sequences were amplified for the molecular identification of ticks. The PCR was performed in a total volume of 25 µL reaction mixture comprised of 1 µL each forward and re-

Tick Collection and Morphological Identification
Wild animals including cats, hedgehogs, and wild boars found dead on highways, killed or captured by local farmers to secure their crops, were screened for ticks. Ticks found on the host body were carefully collected to avoid any damage to the specimens. All collected ticks were preserved in 100% ethanol. Morphological identification of the collected ticks was done using morphological features under Stereozoom microscope (BIOBASE, Jinan, China), by comparing with standard available morpho-taxonomic keys [44,45].

DNA Extraction and PCR
All ticks were morphologically identified, and 120 ticks comprising 10 specimens (different life stages) of each species from all districts were further processed for genomic DNA extraction (Table 1). Ticks were washed with distilled water followed by 70% ethanol and PBS for the removal of any surface contaminants. Washed ticks were individually kept in 1.5 ml tubes and dried in an incubator. Holes were made with needles, and the whole body of each tick was cut into small pieces using sterile scissors and homogenized by micro pestle for DNA extraction using phenol chloroform method [46]. The concentration of extracted DNA was measured using NanoQ (Optizen, Daejeon, South Korea), and samples were maintained at -20°C for further analysis.
Mitochondrial cytochrome C oxidase 1 (cox1) and 16S ribosomal RNA (16S rRNA) genes' partial sequences were amplified for the molecular identification of ticks. The PCR was performed in a total volume of 25 µL reaction mixture comprised of 1 µL each forward and reverse primers (10 µM), 2 µL template DNA (50 ng), 8.5 µL PCR water, and 12.5 µL DreamTaq PCR Master Mix (2×) (Thermo Scientific, Waltham, MA, USA). Primers used in the present study are given in Table 2, and thermocycling conditions were set as previously described [47,48].

Detection of Rickettsia
All extracted genomic DNA samples were screened for the presence of any Rickettsia spp. targeting the amplification of rickettsial citrate synthase (gltA) and outer membrane protein (ompA) partial genes. The PCR reaction was performed in a total volume of 25 µL reaction mixture comprised of 1 µL each forward and reverse primers (10 µM), 2 µL template DNA (50 ng), 8.5 µL PCR water, and 12.5 µL DreamTaq PCR Master Mix (2×) (Thermo Scientific, Waltham, MA, USA). Primers used in the present study are given in (Table 2), and thermocycling conditions were set as previously described [49,50]. All genomic DNA samples that yielded visible amplicons for gltA PCR were subjected to second PCR assay for the amplification of ompA gene. The amplified PCR products were electrophoresed on 1.5% agarose gel and results were visualized under UV light using a GelDoc (UVP BioDoc-It imaging system, Upland, CA, USA).

DNA Purification and Sequencing
Prior to sequencing, the positive PCR products were purified with GeneClean II DNA purification Kit (Qbiogene, Illkirch, France) following the protocol provided by the manufacturer. All 120 purified PCR products for each cox1 and 16S rRNA gene of ticks, and 10 positive samples for each gltA and ompA of Rickettsia spp. were sent for bidirectional sequencing (Macrogen Inc., Seoul, South Korea).

Phylogenetic Analysis
The obtained sequences were trimmed in SeqMan V. 5.00 (DNASTAR) for the removal of unnecessary nucleotides and primer contamination. Redundant sequences (100% identity) were excluded from further analysis. Sequences with maximum identities were retrieved from NCBI (National Center for Biotechnology Information) using BLAST (Basic Local Alignment Search Tool) [51]. The obtained sequences were aligned in BioEdit V. 7.0.5 [52]. Phylogenetic trees were constructed in MEGA X [53], and different phylogenetic methods (Maximum likelihood, Neighbor-Joining, Minimum-Evolution, Parsimony, and UPGMA) were tested for consistency, efficiency, and robustness. The Maximum likelihood method was used for the phylogenetic tree, with bootstrap 1000 replicates, and an outgroup was used for estimating tree stability and validity, respectively. Finally, the sequences of cox1, 16S rDNA, gltA and ompA were submitted to NCBI.

Statistical Analysis
The recorded data was organized in spreadsheets using Microsoft Excel V. 2016 (Microsoft). A chi-square test was performed using GraphPad prism software V. 5.00 (Graph-Pad Software Inc) considering a significant p value < 0.05.

Conclusions
The present study reported tick infestation in wild animals in KP, Pakistan, and for the first-time detected R. massiliae in Rh. turanicus and Rh. haemaphysaloides ticks infesting wild boars in Charsadda and Mardan. These results improve our knowledge of the circulation of R. massiliae in Rhipicephalus ticks infesting both domestic and wild animals. These findings reinforce the need to further understand the diversity of ticks infesting wild animals, tick-associated Rickettsia spp. and other pathogens across the country.

Conflicts of Interest:
The authors declare no conflict of interest.