Seasonal Dynamics, Record of Ticks Infesting Humans, Wild and Domestic Animals and Molecular Phylogeny of Rhipicephalus microplus in Khyber Pakhtunkhwa Pakistan

Although ticks prevalent in various agro-systems of Pakistan are associated with economic losses, information is still missing about the tick’s diversity, hosts they infest, seasonal dynamics and molecular phylogeny of Rhipicephalus microplus in Khyber Pakhtunkhwa (KP) Pakistan. This study for the first time enlisted ticks infesting diverse hosts including humans in various regions of KP. A total of 8,641 ticks were collected across the northern, southern and central regions of KP and were morpho-taxonomically categorized into six genera comprising 17 species, R. microplus (n = 3,584, 42%), Hyalomma anatolicum (n = 2,253, 27%), Argas persicus (n = 1,342, 16%), Hya. impeltatum (n = 586, 7%), R. turanicus (n = 161, 2%), R. haemaphysaloides (n = 142, 2%), R. annulatus (n = 132, 2%), Hae. montgomeryi (n = 123, 1.4%), Hya. marginatum (n = 110, 1.3%), R. sanguineus (n = 34, 0.4%), and Hae. longicornis (n = 31, 0.4%). Ticks infesting wild animals included Amblyomma gervaisi, Amb. exornatum, Amb. latum, Dermacentor marginatus, and Hae. indica, while ticks collected from humans included R. microplus, R. annulatus, Hya. anatolicum, Hya. marginatum, and Hae. punctata. The overall prevalence of ticks infesting domestic animals was 69.4% (536/772). Among animal hosts, cattle were found highly infested (87.2%, 157/180) followed by buffalos (79%, 91/114), domestic fowls (74.7%, 112/150), goats (68.3%, 82/120), dogs (66.7%, 32/48), horses (61.3%, 49/80), and sheep (16.3%, 13/80). Analysis revealed that the tick burden significantly differed among domestic animals and was found to be high in cattle, followed by buffalos, goats, sheep, domestic fowl, dogs, and horses. Seasonal patterns of ticks distribution showed highest prevalance in July, August, and September due to the prevailing high temperature and humidity during these months. The phylogenetic analysis of cattle tick R. microplus based on partial mitochondrial cytochrome oxidase subunit I (COX1), 16S ribosomal RNA (16S rRNA) and internal transcribed spacer 2 (ITS2) sequences, revealed that R. microplus prevalent in this region belongs to clade C which include ticks originating from Bangladesh, Malaysia, and India. Further large scale studies across the country are necessary to explore the molecular and cross breeding aspects at the geographical overlapping of various tick species and their associated pathogens to facilitate designing control strategies as well as awareness against tick infestation in the region.


INTRODUCTION
Ticks are hematophagous arthropods, notorious vectors of human and animal pathogens and constitute an emerging economic and health problem in tropical and sub-tropical regions (de la Fuente et al., 2017). As a major challenge to public health and veterinary sector, ticks can harbor several pathogens that cause numerous infectious diseases. The emergence and resurgence of several tick-borne diseases pose increasing public health concerns (Gratz, 1999;Paddock and Childs, 2003;Jones et al., 2008). As a vector reservoir for several emerging and re-emerging infectious pathogens of medical and veterinary importance, ticks transmit a wide variety of arboviruses (Crimean-Congo hemorrhagic fever virus, tickborne encephalitis virus), bacteria (Rickettsia spp.), spirochetes (Anaplasma spp. Borrelia spp. and Ehrlichia spp.) and protozoans (Babesia spp. and Theileria spp.), more than any other blood feeding arthropod (Labruna et al., 2004;Gondard et al., 2017;Schorderet-Weber et al., 2017). These voracious ectoparasites can infest wild, terrestrial, semi-aquatic vertebrates and humans, and are globally distributed from the Arctic to tropical regions (Sherrard-Smith et al., 2012;Yu et al., 2015). Ten percent of the total hard and soft tick species are known to transmit diseases to humans and other animals (Parola and Raoult, 2001;Lew-Tabor and Valle, 2016). Ticks and tick-borne diseases affect approximately 80% of the world's cattle population, particularly in tropical and subtropical countries. Infestation with cattle tick Rhipicephalus microplus economically impact the livestock industry in different regions and annual losses due to only this tick have been estimated 22-30 billion US$ (Jabbar et al., 2015;Lew-Tabor and Valle, 2016).
The prevalence of ticks in different regions is mostly associated with various environmental factors that affect tick distribution and adaptation (Estrada-Peña, 2008). Like other hematophagous arthropods, ticks spend their life cycle in such an environment where they depend on host availability, host lifestyle, host interaction with other animals, vegetation coverage, habitat type and geo-climatic conditions (temperature and humidity) for their survival and development (Estrada-Peña, 2008;Gondard et al., 2017). Climatic changes influence tick distribution, shape the biodiversity of ticks and tick-borne pathogens and increase the risks of transmission of pathogens to humans and other hosts (Leger et al., 2013;Dantas-Torres, 2015). The dispersal of ticks in previously tick free areas is consociated with humans and other animals movements due to environmental changes (Estrada-Peña, 2008;Dantas-Torres, 2015) which has resulted in the emergence and re-emergence of tick-borne diseases.
Ticks that belong to the genus Rhipicephalus are responsible for severe economic losses in tropical and subtropical agrosystems. Previous studies have enlisted intraspecific variations in genus Rhipicephalus based on morpho-taxonomic complications in identification at species level (Lempereur et al., 2010;Barker and Walker, 2014). Morphological variations in Rhipicephaline ticks make it difficult to distinguish these tick species morphologically and to date, several valid species of Rhipicephalus (Boophilus) have been confirmed (Estrada-Peña et al., 2012;Guglielmone et al., 2014;Ali et al., 2016;Coimbra-Dores et al., 2018;Roy et al., 2018). The morpho-taxonomy of R. microplus (cattle tick) has been challenged in the last few years due to a hypothesis suggesting that biogeographical and ecological separations have occurred in Boophilid ticks across continents based on morphological and genetic variations (García-García et al., 1999;de la Fuente et al., 2000;Ali et al., 2016;Lew-Tabor et al., 2017). The genetic assemblage study based on mitogenome (12S rRNA and 16S rRNA) and microsatellites markers of R. microplus from America, Asia, Australia and Africa have confirmed that R. microplus consist of at least two different species (Labruna et al., 2009;Low et al., 2015;Ali et al., 2016;Baron et al., 2018). Mating experiments among these ticks evidenced that reproductive crosses between R. microplus ticks from Australia and Argentina or Mozambique are infertile while crosses between Boophilid ticks from Argentina and Mozambique are fertile. These findings based on genetic divergence and reproductive isolation put forward that the Australian R. microplus strain is different from the American, Asian and African strain. Phylogenetic analysis of the R. microplus mitogenome (COX1 and 16S rRNA) and internal transcribed spacer (ITS) gene revealed that R. microplus is a complex of species. The cryptic diversity of R. microplus complex includes R. annulatus, R. australis and two clades of R. microplus, clade A and B. Clade A R. microplus includes ticks from America and Africa and clade B includes ticks from China (Labruna et al., 2009;Burger et al., 2014;Low et al., 2015;Roy et al., 2018). Recent mitogenome approaches based on COX1 and 16S rRNA haplotypes suggested a distinct genetic assemblage of R. microplus from Malaysia resulting in novel clade C which includes ticks from Pakistan, Bangladesh, Myanmar, India and Malaysia (Low et al., 2015;Baron et al., 2018;Roy et al., 2018).
Pakistan is an agricultural country where livestock plays a crucial role in the national economy. In Pakistan, climatic conditions are extremely favorable for the growth and survival of diverse tick species. These ticks cause severe problems for livestock holders which mainly include low income farmers. The majority of these farmers consider all tick species as one species and are mostly unaware of the capability of pathogens transmission to humans as well as other associated health risks. Tick and tick-borne diseases are prevalent in the province Khyber Pakhtunkhwa (KP) of Pakistan, causing economic loss to livestock holders (Jabbar et al., 2015;Karim et al., 2017;Rehman et al., 2017). However, there is a paucity of documented information about ticks spatial distribution and diversity, their diverse hosts and seasonal dynamics from this region. Therefore, the present endeavor was focused on tick diversity to investigate the present status of tick species infesting various animal hosts, their seasonal dynamics and to obtain the evolutionary information of cattle tick R. microplus which is prevalent in the study area. .0 E), in the northern, southern and central KP (Northwestern geographic state of Pakistan previously known as North-West Frontier Province) were selected to assess the diversity of ticks, their hosts and seasonal dynamics. Nine regions of each aforementioned districts were selected for ticks sampling (Figure 1). A Global Positioning System (GPS) was used to obtain geographic coordinates data and loaded onto a Microsoft Excel sheet to develop a distribution map for the study areas using ArcGIS 10.3.1.

Ethics Statement
The protocol for the present study was approved by the ethical committee and board of study members of the Department of Zoology Abdul Wali Khan University Mardan. Written informed consent was obtained from the owners of animals and individuals infested by ticks prior to tick collection.

Ticks Collection
Tick samples were collected during April 2017 to March 2018, from different domestic animals (cattle, buffalo, horse, sheep, goat, dogs, and domestic fowl) at different localities (villages and towns) in various tehsils (an administrative division within the district) of the aforementioned districts (Figure 1). For ticks collection, a regular visit was made three times a month and sampling was done randomly with the help of fine tweezers from sampling animals reared at animal farms, houses or grazing in the field. Collected specimens were rinsed with distilled water followed by absolute ethanol to remove any surface confined microbes and host contaminating tissues and stored in properly labeled separate plastic tubes until further analyses. The relevant information including age of host, physical status, previous acaricides or any drugs used against ticks, the body region from where ticks were isolated, date and place of collection were recorded and all the samples were shipped to the Parasitology laboratory of the department of Zoology, Abdul Wali Khan University Mardan. Ticks were immediately identified or preserved (100% ethanol or in a mixture of 95% ethanol, 4% distilled water and 1% glycerol) (Walker et al., 2003) for further study.
During collection, ticks were accidentally found infesting humans and wild goats (locally known as markhor, a national animal of Pakistan mostly found on the Himalaya Mountains). Wild rodents (Indian gray mongoose), wild boars (wild pig), and wild reptiles (monitor lizard and Indian python) were captured by local farmers or found dead on highways. In this collection, ticks were also included from districts other than the above-mentioned districts such as Chitral (

DNA Extraction and Molecular Identification
A total of 225 (45 per district) morphologically identified R. microplus from nine locations in each of the five districts were selected for genomic DNA extraction. Tick samples from each district were separately pooled into five groups, each pool containing ticks of a different location within the district. Ticks were cleaned with distilled water, dried and cut into small pieces using a sterile scalpel and homogenized by a sterile micro pestle in separate 1.5 ml Eppendorf tubes. Genomic DNA was separately extracted from each pool of tick samples using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) according to the standard DNA extraction protocol of the manufacturer. DNA concentration in the samples was quantified using a Nanodrop ND-100 (Thermo Fisher Scientific) and stored at −20 • C until further processing. COX1, 16S rRNA and ITS2 gene fragments of R. microplus were amplified by PCR in a thermocycler (HT, ILF, UK) using specific primers ( Table 1). PCR reactions were performed in a total volume of 25 µL reaction mixture with a composition of 1 µL of each forward and reverse primer, 2 µL of template DNA (50 ng), 7.5 µL of deionized water and 13.5 µL of master mix (DreamTaq PCR Master Mix [2X] (Thermo Fisher Scientific). The thermocycling conditions for COX1, 16S rRNA and ITS2 were optimized separately for each reaction. Standardized PCR conditions was, an initial denaturation of 5 min at 95 • C followed by 35 cycles of denaturation at 95 • C for 30 s, annealing at 53.8 • C for COX1, 55.9 • C for 16S rRNA and 58 • C for ITS2 for 30 s, an extension at 72 • C for 30 s and a final extension of 10 min at 72 • C. A negative control (distilled water) was included in each run of the amplification reaction for validation. PCR products were confirmed by running the amplified DNA in 2% ethidium bromide stained agarose gel with a 50 bp DNA marker (Thermo Fisher Scientific). The results were visualized using the GeneDoc (UVP BioDoc-It Imaging System).

DNA Purification and Sequencing
The amplified PCR products were purified using the GeneClean II kit (Qbiogene) according to the manufacturer's instructions. For the sequencing of PCR based amplified product, 45 DNA samples of each district were separately grouped containing fifteen samples for each gene fragment. The purified PCR amplicons (45 PCR product samples, 15 samples for each marker) were sequenced unidirectionally by Macrogen Inc. (Seoul, South Korea). The obtained sequences (nucleotide sequences) were analyzed using BioEdit V. 7.0.5 (Hall et al., 2011) and NCBI BLAST 1 (Altschul et al., 1990). All the relevant library sequences of closely related species and of R. microplus complex available in GenBank were downloaded and saved for downstream analysis to construct a phylogenetic tree. The obtained sequences were trimmed for the removal of unnecessary nucleotides and those with 100% similarity with each other were discarded. Finally, 12 sequences were uploaded to NCBI GenBank including COX1 (4), 16S rRNA (3) and ITS2 (5).

Phylogenetic Analysis
The obtained trimmed sequences were aligned using ClustalW in BioEdit Sequence Alignment Editor V 7.0.5 (Hall et al., 2011). To test molecular phylogeny of the cattle tick R. microplus a   (Kumar et al., 2018) with bootstrapping at 1000 replications (Tamura et al., 2013).

Statistical Analysis
The overall tick prevalence was calculated by the total ticks divided by total hosts, host wise infestation was calculated by the infested hosts divided by total examined. The mean percent of collected ticks and binomial confidence intervals for proportion were calculated ( Table 2). The median tick burden was analyzed on hosts by applying the Kruskal-Wallis test (P < 0.001) among

Tick's Description and Host Population
Morpho-taxonomic analysis categorized collected ticks into six genera including 17 species (Figure 1)

Seasonal Dynamics of Ticks
The tick species included in the seasonal dynamics analysis were; Hya. anatolicum, Hya. marginatum, Hya. impeltatum, R. microplus, R. haemaphysaloides, R. sanguineus, R. turanicus, R. annulatus, Hae. montgomeryi, Hae. longicornis, and A. persicus. Tick species showed spatio-temporal variations in their distribution. Specifically, species of the genus Hyalomma were mainly reported in dry regions while species belonging to the genus Rhipicephalus were reported in semi-arid regions of the study area ( Figure 6B). During ticks collection, adult females (partially fed or nymphs) were highly prevalent followed by males and larvae ( Table 2). Other tick species were not included in the temporal analysis because they were collected by chance from novel hosts (these hosts were not the focus of the present FIGURE 5 | Maximum likelihood tree inferred from ITS2 sequences of the genus Rhipicephalus and using Rhipicentor and Hyalomma sequences as outgrowth. GenBank accession numbers are followed by species name and collection location. Support values (Bootstrapping values) were indicated at each node. The bar represents 0.020 substitutions per site. Sequences obtained in the present study were underlined. study) either from the study area or from other districts within KP Pakistan, during some parts of the year. This included tick species such as Amb. gervaisi, Amb. exornatum, Amb. latum, D. marginatus, Hae. indica, and R. sanguineus from monitor lizards, Indian pythons, markhor, mongoose and wild boars, respectively.
The highest number of ticks were collected in July followed by August and September as an increase in the infestation rate of ticks were recorded during ticks collection and these finding significantly correlated with an increase in mean temperature [r(10) = 0.929, p < 0.01] and relative humidity [r(10) = 0.831, p < 0.01] during these months in the region. As the mean temperature fell and relative humidity decreased, the infestation rate also decreased (Figure 7).

Sequence and Phylogenetic Analysis
Genomic DNA extracted from morphologically identified R. microplus tick samples were amplified by PCR using COX1, 16S rRNA and ITS2 specific primers ( Table 1). Results were observed on agarose gel electrophoresis and the resultant amplicons (COX1, 16S rRNA and ITS2) were 620, 376, and 267 bp, respectively. After sequencing, BLAST analysis of the COX1 nucleotide partial sequences showed 95-100% identity with the same sequence reported from Pakistan (accession no. MG459963), Bangladesh (accession no. MG459961, MG459962) Myanmar (accession no. MG459964) and China (accession no. KC503259). In the case of 16S rRNA nucleotide partial sequences, BLAST analysis revealed that it shares a 98-100% identity with previously reported sequences for R. microplus from India (accession no. KY458969; MG811555) and China (accession no. KU664521; MH208600). ITS2 partial nucleotide sequences showed that it shares 99% identity with the same sequences reported for the same tick species from India  Maximum likelihood (ML) trees were inferred from COX1, 16S rRNA and ITS2 partial sequences of R. microplus to establish its phylogenetic relationship. ML analysis of COX1 nucleotide sequences revealed three clades, clade A includes ticks from America, Malaysia, and China, clade B was comprised of ticks originating from China (Burger et al., 2014;Low et al., 2015) and clade C includes ticks from Pakistan, India, Bangladesh, Myanmar, and Malaysia (Figure 3) (Roy et al., 2018). The COX1 phylogenetic analysis provided better support to resolve the evolutionary relationships of R. microplus. On the other hand, 16S rRNA partial sequences provided little phylogenetic structure of R. microplus complex and divided into two phylogenetic clades, the clade A comprised of the R. microplus tick species from China, India and Pakistan. Clade B includes R. microplus ticks originating from Malaysia, Africa and America and the R. australis (formerly recognized as R. microplus) from Australia, Indonesia and New Caledonia formed a separate subclade along with the aforementioned ticks (Figure 4). Based on ITS2 analysis, all species of the R. microplus complex were clustered together and the ITS2 tree provided support to the monophyly of R. microplus complex (Figure 5).

DISCUSSION
Climatic change and global warming have a vital impact on the distribution of ticks and tick-borne pathogens, as each tick species selects a set of ecological conditions and biotopes that determine its dispersal and outline risk areas for their associated pathogens transmission (Leger et al., 2013). Tick habitat expansion and novel pathogens (protozoans, bacteria, rickettsia, and viruses) are re-emerging in new geographic areas, posing a serious threat to public and veterinary health (Estrada-Peña et al., 2013;Jore et al., 2014). In KP Pakistan, the majority of farmers lack basic knowledge on ticks, their disease causing potential and the wide variety of hosts they infest, mostly resulting in a failure of controlling tick infestations. The present study encompasses the identification of diverse tick species and novel hosts, including humans, along with the seasonal dynamics and molecular phylogeny of R. microplus from north-western regions of Pakistan. The collected ticks were categorized into six genera including 17 species of medical and veterinary concern. We reported Amb. latum, Amb. gervaisi, Amb. exornatum, A. persicus, D. marginatus, Hae. indica, Hae. longicornis, Hae. punctata, Hae. montgomeryi, Hya. impeltatum, H. anatolicum, Hya. marginatum, R. annulatus, R. haemaphysaloides, R. microplus, R. sanguineus, and R. turanicus from the KP province. The majority of these ticks are responsible for the transmission of Babesia bovis, Theileria annulata, Anaplasma marginale, and Anaplasma centrale in the region (Jabbar et al., 2015). Our findings on the tick species prevalent in Pakistan correspond with previously reported studies (Manan et al., 2007;Karim et al., 2017;Rehman et al., 2017). Additionally, these ticks have been shown to be responsible for considerable losses in the livestock industry, causing severe threats to animal hosts and public health (Tadesse et al., 2012;Rehman et al., 2017).
Amblyomma ticks are vectors of several pathogens known to transmit disease causing agents to humans and has never been investigated in Pakistan. Amblyomma species infesting reptiles are vectors of Aeromonas hydrophila that cause bacterial stomatitis, paralysis and pneumonia in snakes (Tenderio, 1953;Stephen and Achyutha, 1979;Marcus, 1981;Stenos et al., 2005;Hanson et al., 2007). D. marginatus were found to infest wild goats (locally known as markhor), which demands further studies to investigate the negative impact of this tick species in markhor which is considered as endangered species. The genus Dermacentor is of public and veterinary health concern as a vector reservoir for several pathogens with unknown potential risks in Pakistan. R. sanguineus, a vector of microorganisms of medical and veterinary health concern such as Hepatozoon canis and Ehrlichia canis (Dantas-Torres et al., 2018;Cabezas-Cruz et al., 2019) was collected for the first time from wild boars in the region.
Studies showing the seasonal pattern of tick distributions have never been reported from KP, Pakistan. During this study, information about the tick burden associated with relative humidity and temperature was recorded which is essential for the timing of potential control measures in tick infestation season. The target study region has high temperature and humidity during July, August and September therefore, high tick infestations were recorded during these months. On the other hand, due to the low temperatures during December, January, and February, several tick species were found to exhibit low infestation rates.
Several studies have used COX1, 16S rRNA and ITS2 as genetic markers for the accurate molecular identification and phylogenetic relationship of various organisms, including hard ticks such as R. microplus (Burger et al., 2014;Lv et al., 2014;Low et al., 2015;Coimbra-Dores et al., 2018). COX1 sequences have been used to show that cryptic species of R. microplus contain and display different population structures in different geographical regions (Burger et al., 2014;Low et al., 2015). Thus, R. microplus appears to consist of a complex of distinct genetic assemblages, namely R. australis, R. annulatus, R. microplus clade A (Burger et al., 2014), R. microplus clade B (Burger et al., 2014), and R. microplus clade C (Low et al., 2015). Burger et al. (2014) showed that R. microplus from clade B (Southern China and Northern India) is more closely related to R. annulatus than to R. microplus from clade A (Asia, South America, and Africa). In accordance with previous studies, phylogenetic trees based on COX1, 16S rRNA and ITS2 of R. microplus were obtained to establish the phylogenetic relationship of this tick (Lv et al., 2014;Low et al., 2015;Csordas et al., 2016;Rehman et al., 2017;Roy et al., 2018). COX1 was found to be an established marker for the phylogenetic analyses and informative for R. microplus phylogeny as compared to 16S rRNA and ITS2 as the latter provides monophyletic support to Boophild ticks (Burger et al., 2014). The COX1 phylogenetic analysis provided support to resolve the evolutionary relationship of R. microplus. Our findings about obtained COX1 sequences and phylogenetic analysis are parallel with previous reports from the region (Roy et al., 2018). ML analysis of COX1 nucleotide sequences revealed that these ticks are closely related to clade C (Low et al., 2015;Roy et al., 2018) (Figure 3). On the other hand, 16S rRNA partial sequences provided support to the monophyly of R. microplus complex and our tick sequences were clustered with clade A (Figure 4) (Brahma et al., 2014;Lv et al., 2014;Low et al., 2015). Based on ITS2 analysis, all species of the R. microplus complex were clustered together on the tree and provided support to the monophyly of R. microplus complex. The phylogenetic relationship in the R. microplus species complex was poor and could not accurately categorized as a sister species since ITS2 is highly conserved and is insufficient in distinguishing these closely related species (Figure 5).

CONCLUSION
This study for the first time explored the tick diversity infesting various hosts in KP, Pakistan. The important tick species of domestic animal hosts are R. microplus, R. annulatus, R. haemaphysaloides, R. sanguineus, R. turanicus, Hae. montgomeryi, Hae. longicornis, Hya. marginatum. Hya. anatolicum, Hya. impeltatum, and A. persicus. The seasonal pattern of several tick species showed high infestation during July, August and September in which high temperature and humidity persists in the region. The collection of tick species parasitizing humans in the study area may facilitate further studies to identify the functional role of these parasites in the transmission of pathogens that cause human diseases.