First Molecular-Based Confirmation of Dermacentor marginatus and Associated Rickettsia raoultii and Anaplasma marginale in the Hindu Kush Mountain Range

Simple Summary Dermacentor ticks have a wide geographic range with an uneven distribution in the globe. They are not scientifically well known because they survive in hard topographic and harsh climatic regions along with elevated mountains. Many mammals serve as a primary host for Dermacentor ticks, like many other tick species. The present study aimed to provide the first morphological and molecular confirmation of Dermacentor marginatus and its related pathogens like Anaplasma marginale and Rickettsia raoultii in Pakistan. In this study, a total of 26 specimens (19 males and 7 females) were collected from goats and morphologically identified. A subset of 18 specimens were subjected for the molecular characterization of ticks and associated pathogen detection. In the BLAST and phylogenetic analyses, D. marginatus and their associated pathogen sequences showed close resemblance with their corresponding species. In the present study, we reported the first genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan. Due to the difficult access and harsh climate, it is important to investigate the ticks and related pathogens in the northern parts of Pakistan due to their zoonotic threats. Abstract Ticks of the genus Dermacentor Koch, 1844 (Acari: Ixodidae) are poorly known systematically due to their habitation in harsh topographic environments and high mountains. Dermacentor ticks are diversely distributed in the Palearctic, Nearctic, and Oriental regions. There is no available information on the occurrence of Dermacentor marginatus in Pakistan; thus, the current investigation aimed the first morphological and molecular confirmation of this species and associated Anaplasma marginale and Rickettsia raoultii. Ticks were collected from goats (Capra hircus) and morphologically identified. Genomic DNA was extracted from 18/26 (69.23%) tick specimens, including 11 males and 7 females (1 unfed and 6 fed females). Extracted DNA was subjected to PCR for the amplification of genetic markers like 16S rDNA and cox1 for ticks, 16S rDNA for Anaplasma spp., and gltA and ompB for Rickettsia spp. A total of 26 D. marginatus ticks composed of 19 males (73.07%) and 7 females (26.9%) [1 (3.84%) unfed and 6 (23.07%) fed females] were collected from goats. According to amplicons via BLAST analysis, the 16S rDNA sequence showed 97.28–98.85% identity and the cox1 sequence showed 95.82–98.03% identity with D. marginatus. Additionally, the 16S rDNA sequence for Anaplasma sp. was detected in D. marginatus that showed 100% identity with Anaplasma marginale. Rickettsial gltA and ompB sequences for Rickettsia sp. showed 100% identity with Rickettsia raoultii. In phylogenetic analysis, ticks’ 16S rDNA and cox1 sequences clustered with the same species. In phylogenetic analysis, A. marginale based on 16 rDNA clustered with A. marginale, while gltA and ompB sequences clustered with R. raoultii. This is the first study on the genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan. The northern areas of Pakistan, which need to be explored in terms of ticks and associated pathogens due to their zoonotic threats, have been neglected due to the inaccessible climatic conditions.

Simple Summary: Dermacentor ticks have a wide geographic range with an uneven distribution in the globe.They are not scientifically well known because they survive in hard topographic and harsh climatic regions along with elevated mountains.Many mammals serve as a primary host for Dermacentor ticks, like many other tick species.The present study aimed to provide the first morphological and molecular confirmation of Dermacentor marginatus and its related pathogens like Anaplasma marginale and Rickettsia raoultii in Pakistan.In this study, a total of 26 specimens (19 males and 7 females) were collected from goats and morphologically identified.A subset of 18 specimens were subjected for the molecular characterization of ticks and associated pathogen detection.In the BLAST and phylogenetic analyses, D. marginatus and their associated pathogen sequences showed close resemblance with their corresponding species.In the present study, we reported the first genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan.Due to the difficult access and harsh climate, it is important to investigate the ticks and related pathogens in the northern parts of Pakistan due to their zoonotic threats.
Abstract: Ticks of the genus Dermacentor Koch, 1844 (Acari: Ixodidae) are poorly known systematically due to their habitation in harsh topographic environments and high mountains.Dermacentor ticks are diversely distributed in the Palearctic, Nearctic, and Oriental regions.There is no available information on the occurrence of Dermacentor marginatus in Pakistan; thus, the current investigation aimed the first morphological and molecular confirmation of this species and associated Anaplasma marginale and Rickettsia raoultii.Ticks were collected from goats (Capra hircus) and morphologically identified.Genomic DNA was extracted from 18/26 (69.23%) tick specimens, including 11 males and 7 females (1 unfed and 6 fed females).Extracted DNA was subjected to PCR for the amplification of genetic markers like 16S rDNA and cox1 for ticks, 16S rDNA for Anaplasma spp., and gltA and ompB for Rickettsia spp.A total of 26 D. marginatus ticks composed of 19 males (73.07%) and 7 females (26.9%) [1 (3.84%) unfed and 6 (23.07%) fed females] were collected from goats.According to amplicons via BLAST analysis, the 16S rDNA sequence showed 97.28-98.85%identity and the cox1 sequence showed 95.82-98.03%identity with D. marginatus.Additionally, the 16S rDNA sequence for

Introduction
Dermacentor Koch, 1844 (Acari: Ixodidae) ticks have a wide geographical range with an uneven distribution, and not a single species is reported from poles and remote islands.Like many other tick species, mammals act as a common host for Dermacentor ticks [1,2].The adult ticks mainly feed on medium-to large-sized mammals, while the larvae feed on small mammals [3].Dermacentor species are important from veterinary and public health perspectives, parasitizing artiodactyl, carnivores, rodents, and insectivores [4] and transmitting different protozoans and bacteria like Rickettsia spp.and Anaplasma spp.[1,5].The greatest diversity of these ticks is confined to the Palearctic, Nearctic, and Oriental regions, while only a single species Dermacentor steini has been reported from the Australasian zoogeographical region [6].
Difficulties persist in the morphological separation of Dermacentor ticks in species complex [7,8].Recently, Dermacentor atrosignatus has been reinstated as Dermacentor tricuspis, and D. atrosignatus has been considered as a synonym of D. tricuspis [9,10].A single species of the genus Dermacentor, i.e., Dermacentor raskemensis, has been confirmed and reported from Pakistan based on the collection of immature and adult stages [11][12][13].
Dermacentor marginatus was named by German vulgars as 'Schafzecke' (sheep tick) due to its infestation with domestic sheep, but it has been also recorded from domestic and wild hosts including cattle, goat, dog, horse, hedgehog, hare, deer, wild boar, and hare [14,15].This tick generally inhabits steppes, alpine steppes, forest steppes, and semi-desert steppes, particularly open sheep meadows [16].D. marginatus is an ornate tick, geographically distributed from southern Europe to China [15].The life cycle takes 1-2 years, and its developmental stages are interrupted by low temperatures or by snow cover in winter [17].Previously, Pakistan was considered out of the adaptation range for D. marginatus [6].
These species are important vectors of microorganisms that cause diseases in domestic and wild animals and humans [3].D. marginatus ticks act as potential vectors for the rickettsial group causing zoonotic diseases, which has an important role in the eco-epidemiology of rickettsiosis [18].The Rickettsia species are divided on the basis of genotype and phenotypic characteristics into four major groups, namely spotted fever, typus, bellii, and limoniae groups [19].Rickettsia species have been reported from D. marginatus, which include Rickettsia felis, Rickettsia monacensis, Rickettsia raoultii, Rickettsia slovaca, and "Candidatus Rickettsia rioja" [20][21][22].
There are insufficient data available on Dermacentor ticks and their associated pathogens in Pakistan.To the best of our knowledge, this is the first study to report D. marginatus and their associated pathogens by using genetic markers to spot the occurrence of this tick in the Hindu Kush Mountain Range in the northern areas of Khyber Pakhtunkhwa (KP), Pakistan.

Ethical Statement
For the current study, ethical approval was obtained from the Advanced Studies and Research Board of the Faculty of Chemical and Life Sciences, with notification number Dir/A&R/AWKUM/2022/9674, Abdul Wali Khan University Mardan, KP, Pakistan.Oral permissions were taken from the animal owners.

Study Area
This study was conducted in the border region of the following districts: Dir Upper (35 •  of this tick in the Hindu Kush Mountain Range in the northern areas of Khyber Pakhtunkhwa (KP), Pakistan.

Ethical Statement
For the current study, ethical approval was obtained from the Advanced Studies and Research Board of the Faculty of Chemical and Life Sciences, with notification number Dir/A&R/AWKUM/2022/9674, Abdul Wali Khan University Mardan, KP, Pakistan.Oral permissions were taken from the animal owners.

Study Area
This study was conducted in the border region of the following districts: Dir Upper (35°30′00.1″N, 72°20′43.0″E) and Swat (35°34′27.3″N 72°22′07.7″E)-Badgoi pass of the Hindu Kush Mountain Range, the northern part of the KP province of Pakistan.The Badgoi pass is the link between districts Dir Upper and Swat, with an altitude of nearly 3520 m above sea level.The physiography of the area is composed of gently steep, rocky mountains covered with snow in winter and turned into green pastures in summer.The summer season is from moderate to warm, and temperatures rapidly fall in November to February.The average temperature ranges from −7 °C to 22 °C (climate-data.org).The geographical coordinates of the collection sites were obtained by using a Global Positioning System (GPS), and the map was designed via ArcGIS V. 10.3.1 (Figure 1).

Ticks Collection and Morphological Identification
Tick specimens were collected in June 2022 from goats (Capra hircus) in the study area.Ticks were washed with distilled water followed by 70% ethanol to remove the contaminants and extra tissues from the body surface.Ticks were morphologically identified under a stereo-microscope (StereoBlue-euromex SB.1302-1, Arnhem, The Netherlands), and their morphological characters were compared with the standard identification keys [23,24].

DNA Extraction and PCR
A total of 18/26 (69.23%)Dermacentor specimens comprising 9 specimens from each locations: Dir Upper (6 males and 1 unfed and 2 fed females) and Swat (5 males and 4 fed females) were subjected to genomic DNA extraction.The specimens were washed and crushed in a sterilized 1.5 mL Eppendorf tube.The genomic DNA was extracted by the phenol-chloroform method [25].The DNA pellet was hydrated by adding 30 µL "nuclease-free" PCR water and quantified with NanoDrop (Optizen, Daejeon, South Korea).
A conventional PCR (GE-96G, BIOER, Hangzhou, China) was performed to amplify the16S rRNA and cox1 genes for tick identification (Table 1).All genomic DNA samples were utilized to test for associated pathogens through the amplification of the 16S rRNA partial gene for Anaplasma species and the gltA and ompB partial genes for Rickettsia species (Table 1).Each PCR mixture was prepared in 25 µL, composed of 2 µL genomic DNA (100 ng/µL), 1 µL each (forward and reverse) primer (10 µM), 8.5 µL PCR water "nuclease-free", and 12.5 µL DreamTaq green MasterMix (2×) (Thermo Fisher Scientific, Inc., Waltham, MA, USA).Each of the PCRs contained a positive control (DNA of Rickettsia massiliae and Anaplasma marginale for pathogens, and DNA of Rhipicephalus microplus for ticks) as well as a negative control (PCR water that was "nuclease-free" rather than DNA).The PCR products were run on a 2% agarose gel electrophoresis and observed via Gel documentation system (BioDoc-It™ Imaging Systems, UVP, LLC., Upland, CA, USA).

DNA Sequencing and Phylogenetic Analysis
The amplicons were purified by using a Gene Clean II Kit (Qbiogene, Il-lkirch, France) as per the manufacturer's protocol.The PCR-amplified products were sent for bidirectional sequencing to Macrogen, Inc. (Seoul, Republic of Korea).The obtained sequences were trimmed to remove the contaminated and poor reading regions via SeqMan v. 5 (DNAS-TAR, Inc., Madison, WI, USA).The trimmed sequences were subjected to the Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI).The high-percentage identity sequences were downloaded in FASTA format from the NCBI for phylogenetic analyses.The downloaded sequences were aligned with the obtained sequences and an outgroup using ClustalW multiple alignments [31] in BioEdit Sequence Alignment Editor v. 7.0.5 [32].The coding sequences were aligned by using MUSCLE statistical algorithms [33].The phylogenetic trees were constructed through the maximum likelihood method and the Tamura-Nei model with a 1000 bootstrapping value in MEGA-X [34].

Tick Record
Goat herds were observed for tick collection in 11 different localities in Badgoi pass Dir Upper and Swat.Among these goat herds, 11 goats (5 males and 6 females) were infested by D. marginatus ticks, counting 26 (19 males and 1 unfed and 6 fed females) ticks.Among these, 16 ticks (13 males and 1 unfed and 2 fed females) were from Dir Upper and 10 ticks (6 males and 4 fed females) from Swat.The Dermacentor-infested goats were of three age groups, namely 4 adults, 4 young, and 3 kids.Besides D. marginatus ticks, these goats were co-infested by Haemaphysalis montgomeryi.A total of 45 H. montgomeryi ticks including 23 males and 22 females (6 unfed and 16 fed females) were collected.Environmental and topographic conditions as well as the host records are mentioned in Table 2. M: male, F *: unfed female and F: fed female.

D. marginatus Male
Idiosoma: The body is medium-sized with an elongated oval shape.Conscutum is inornate, dark reddish with irregular black patches.Cervical grooves are short, deep, and comma-shaped converging anteriorly and posteriorly.Punctuations are deep, not uniform, and densely distributed in the anterior (Figure 2A(I)).The marginal groove does not enclose the posterior festoon and starts from the last festoon and ends at the point of 2nd coxae (Figure 2B(I, II)).The width of the basis capitulum is greater than its length with moderately pointed broad cornua (Figure 2C(I)).Legs: dorsal side segments are with pale yellow color spots.Coxa I is small with paired spurs equal in size (Figure 2E

D. marginatus Female
Body idiosoma: The body is dark reddish and oval-shaped.The scutum is moderatesized and sub-ovate in shape.The scutum posteriorly diverges at mid-position and gradually converges to a narrow-rounded position (Figure 3A(I)).The cervical groove is

D. marginatus Female
Body idiosoma: The body is dark reddish and oval-shaped.The scutum is moderatesized and sub-ovate in shape.The scutum posteriorly diverges at mid-position and gradually converges to a narrow-rounded position (Figure 3A(I)).The cervical groove is deep and curved inside.The alloscutum has inner deep grooves, with posterior-marginal depressions.Capitulum width is greater than length.The basis capitulum is broader as compared to the length.Punctuations are deep, not uniform, and irregularly distributed (Figure 3A).Coxa I is small with a paired spur (Figure 3B(I)).
deep and curved inside.The alloscutum has inner deep grooves, with posterior-marginal depressions.Capitulum width is greater than length.The basis capitulum is broader as compared to the length.Punctuations are deep, not uniform, and irregularly distributed (Figure 3A).Coxa I is small with a paired spur (Figure 3B(I)).
Animals 2023, 13, x 11 of 16 followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR647516).and phylogenetically as D. marginatus.This is the first confirmed host record and the preliminary phylogenetic position of D. marginatus from Pakistan.Despite being widely distributed throughout the Palearctic region, the D. marginatus tick has not yet been identified in Pakistan [6,24].This raises concerns about D. marginatus whether it is considered an exotic or native species in the country.Environmentalists and public health professionals face an increasing concern about the spread of exotic ticks into novel ecosystems [37].D. marginatus ticks are difficult to morphologically identify, as Dermacentor nuttalli, Dermacentor ushakovae, Dermacentor niveus, and Dermacentor silvarum were considered as synonyms; in this case, [6] these species were confirmed as D. marginatus species complex.The morphological description of the male and female D. marginatus has revealed that both sexes have white enamel on the scutum and conscutum [4].The collected male tick during this study has reddish-black pigmentation and with no ornamentation on the conscutum; this phenomenon has been previously reported in Dermacentor ticks, i.e., Dermacentor albipictus [38].
Together with morphological identification, the genetic characterization of ticks has been considered as an important component in understanding tick systematic and evolutionary history [39].In this study, we employed DNA barcoding for the precise identification of D. marginatus.The 16S rRNA and cox1 genes are important in marking tick phylogenies due to the availability of the dataset in GenBank for these sequences as well as containing hypervariable regions [40].In the phylogenetic trees, the obtained 16S rDNA and cox1 sequences found a close resemblance with the sequences of D. marginatus reported from different countries.
Exponential rates of ticks and tick-borne diseases due to climate change have threatened global health.In Pakistan, serious attention to regular surveillance of ticks and tick-borne pathogens has not been paid yet.However, previous studies have highlighted various ticks as zoonotic risks regarding the detection of pathogens like "Candidatus Rickettsia shennongii", Rickettsia massiliae, Rickettsia aeschlimannii, Rickettsia conorii, Rickettsia hoogstraalii, Coxiella burnetii, Borrelia sp., A. marginale, and Theileria annulata [41][42][43][44][45].Although the occurrence of D. marginatus in Pakistan and the detection of A. marginale and R. raoultii in this tick are a serious concern, as previously mentioned, this species has been found to be involved in the transmission of various pathogens like Rickettsia slovaca, A. marginale Coxiella spp., Rickettsia spp., Shigella spp., and Francisella spp.[46].To assist the public and veterinary health surveillance, the current study monitored the abundance and distribution of the utmost important goat-associated Dermacentor ticks in the highlands of the northern region of Pakistan.Moreover, the molecular detection of pathogens like R. raoultii and A. marginale highlights the zoonotic risks due to these parasites.Future research must address the gaps regarding the presence of D. marginatus in various topographic regions of Pakistan in order to understand whether this species is established or is invasive.

Conclusions
This study presents a comprehensive report on D. marginatus ticks and associated A. marginale and R. raoultii for the first time in Pakistan.Molecular identification and host record confirmed that the ticks of the genus Dermacentor mostly prefer cold climatic conditions.Furthermore, pathogens were detected that have a zoonotic threat to the livestock-holders of the study areas.Moreover, this study may assist in understanding the epidemiology and systematic history of Dermacentor species.
Institutional Review Board Statement: For the current study, ethical approval was obtained from the Advanced Studies and Research Board of the Faculty of Chemical and Life Sciences, with notification number: Dir/A&R/AWKUM/2022/9674, Abdul Wali Khan University Mardan, KP, Pakistan.Oral permissions were taken from the owners of animals or herds.
Informed Consent Statement: Not applicable.
30 00.1 N, 72 • 20 43.0 E) and Swat (35 • 34 27.3 N 72 • 22 07.7 E)-Badgoi pass of the Hindu Kush Mountain Range, the northern part of the KP province of Pakistan.The Badgoi pass is the link between districts Dir Upper and Swat, with an altitude of nearly 3520 m above sea level.The physiography of the area is composed of gently steep, rocky mountains covered with snow in winter and turned into green pastures in summer.The summer season is from moderate to warm, and temperatures rapidly fall in November to February.The average temperature ranges from −7 • C to 22 • C (climate-data.org).The geographical coordinates of the collection sites were obtained by using a Global Positioning System (GPS), and the map was designed via ArcGIS V. 10.3.1 (Figure 1).Animals 2023, 13, x 3 of 16

Figure 1 .
Figure 1.Tick collection site in the border region (Badgoi pass) of districts Dir Upper and Swat of the Hindu Kush Mountain Range.Figure 1. Tick collection site in the border region (Badgoi pass) of districts Dir Upper and Swat of the Hindu Kush Mountain Range.

Figure 1 .
Figure 1.Tick collection site in the border region (Badgoi pass) of districts Dir Upper and Swat of the Hindu Kush Mountain Range.Figure 1. Tick collection site in the border region (Badgoi pass) of districts Dir Upper and Swat of the Hindu Kush Mountain Range.
(II)).A single external spur is present on coxae II, III, and IV.Coxa IV is large and broader anteriorly while becoming narrow posteriorly (Figure 2D(I)).The coxa size progressively increases from coxae I to IV. Hypostome is club-shaped accompanied by 3/3 dentation (Figure 2E(I)).Spiracular plates are broad at the upper side and gradually pointed posteriorly.Goblet cells are around the macula.Macula is parallel with the lateral margin (Figure 2F(I)).
yellow color spots.Coxa I is small with paired spurs equal in size (Figure2E(II)).A single external spur is present on coxae II, III, and IV.Coxa IV is large and broader anteriorly while becoming narrow posteriorly (Figure2D(I)).The coxa size progressively increases from coxae I to IV. Hypostome is club-shaped accompanied by 3/3 dentation (Figure2E(I)).Spiracular plates are broad at the upper side and gradually pointed posteriorly.Goblet cells are around the macula.Macula is parallel with the lateral margin (Figure2F(I)).

Figure 4 .
Figure 4. Maximum-likelihood phylogenetic analysis of nucleotide sequences of 16S rDNA partial sequences for Dermacentor spp.The sequences are represented by their GenBank accession number followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR647518).

Figure 4 .
Figure 4. Maximum-likelihood phylogenetic analysis of nucleotide sequences of 16S rDNA partial sequences for Dermacentor spp.The sequences are represented by their GenBank accession number followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR647518).

Figure 5 .
Figure 5. Maximum-likelihood phylogenetic analysis of nucleotide sequences of cox1 partial sequences for Dermacentor spp.The sequences are represented by their GenBank accession number followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR647517).

Figure 5 .
Figure 5. Maximum-likelihood phylogenetic analysis of nucleotide sequences of cox1 partial sequences for Dermacentor spp.The sequences are represented by their GenBank accession number followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR647517).

Figure 6 .
Figure 6.Maximum-likelihood phylogenetic analysis of nucleotide sequences of 16S rDNA partial sequences for Anaplasma spp.The sequences are represented by their GenBank accession number

Figure 6 .
Figure 6.Maximum-likelihood phylogenetic analysis of nucleotide sequences of 16S rDNA partial sequences for Anaplasma spp.The sequences are represented by their GenBank accession number followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR647516).

Figure 7 .
Figure 7. Maximum-likelihood phylogenetic analysis of nucleotide sequences of gltA partial sequences for Rickettsia spp.The sequences are represented by their GenBank accession number followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR659582).

Figure 7 .
Figure 7. Maximum-likelihood phylogenetic analysis of nucleotide sequences of gltA partial sequences for Rickettsia spp.The sequences are represented by their GenBank accession number followed by the name of species and countries (when applicable).The obtained sequence in the present study is indicated in bold and underlined (accession number: OR659582).

Table 1 .
List of primers utilized for the amplification of target DNA sequences.

Table 2 .
Table describing the comprehensive data regarding the collection of D. marginatus in Hindu Kush Mountain Range.