Ornithodoros (Pavlovskyella) ticks associated with a Rickettsia sp. in Pakistan

Soft ticks (Ixodida: Argasidae) are medically important ectoparasites that mainly feed on birds and mammals, which play a key role in their geographic distribution and dispersion. Despite their importance, studies on soft ticks are scarce for many regions and countries of the world, including Pakistan. In this study, 2330 soft ticks—179 larvae (7.7%), 850 nymphs (36.4%), 711 males (30.5%) and 590 females (25.3%)—were collected from animal shelters in 18 locations within five districts of Khyber Pakhtunkhwa, Pakistan. A subset of the collected ticks was processed for DNA extraction and polymerase chain reaction (PCR) for the amplification of tick 12S ribosomal DNA (rDNA), 16S rDNA and cytochrome c oxidase subunit I (cox1), and rickettsial 16S rDNA gene fragments. The obtained sequences were used for the construction of a phylogenetic tree. All the specimens were morphologically identified as Ornithodoros, and were morphologically similar to Ornithodoros tholozani. The genus was confirmed by sequencing partial 12S rDNA, 16S rDNA and cox1 gene fragments. Additionally, a Rickettsia sp. was detected in some of the collected ticks by PCR targeting 16S rDNA. The morphological relatedness of the tick specimens with O. tholozani was confirmed by phylogenetic analysis, in which the Ornithodoros sp. clustered with Ornithodoros tholozani and Ornithodoros verrucosus, both of which belong to the subgenus Pavlovskyella and have been previously reported from Israel, Ukraine and Iran. The phylogenetic tree also indicated that the Ornithodoros sp. from Pakistan corresponds to an undetermined species. Furthermore, the associated Rickettsia sp. grouped with the limoniae group of Rickettsia species previously reported from Argas japonicus ticks from China. This is the first molecular study of an Ornithodoros species from Pakistan. Further studies are essential to confirm its identity and possible pathogenicity with regard to its associated microorganisms in the studied region.

The family Argasidae comprises 218 species, of which approximately 133 have not been assigned accurately to genera [8,9]. Ornithodoros is the most diverse genus within the Argasidae, with approximately 130 known species [2,[10][11][12][13]. The phylogeny and taxonomy of argasid ticks are still controversial, and many species have been assigned to more than one genus [14]. In this study, we follow the nomenclature proposed by Mans et al. [15] who, among others, showed that phylogenetic reconstructions using tick mitogenomes indicate that the genus Ornithodoros (Pavlovskyella) is paraphyletic [15]. Ornithodoros tholozani has been described from specimens collected in Persia [16], a geographical region that does not include Pakistan. Only two Ornithodoros species, Ornithodoros tholozani [17,18] and Ornithodoros papillipes [19], which were identified morphologically, have been reported in Pakistan thus far. Ornithodoros tholozani mainly infests livestock [17], birds and pigs [4]. A similar tick, Alveonasus lahorensis, also parasitizes cattle, camels and sheep, although the Asiatic mouflon is its primary host [4]. In Pakistan, O. tholozani is widely distributed, whereas the distribution of A. lahorensis is limited to the western part of the country [20]. Currently, no genetic data are available for Ornithodoros spp. from Pakistan.
The traditional method of tick identification is based on morphological traits and requires a specialist's expertise, particularly for soft ticks. The morphological identification of argasid ticks is particularly difficult in the case of closely related species, immature stages, engorged stages and damaged specimens [30]. The identification of argasid ticks can be facilitated by the use of genetic markers, such as 12S ribosomal DNA (rDNA), 16S rDNA and cytochrome c oxidase subunit I (cox1) mitochondrial genes [31][32][33]. Moreover, there is a lack of data on the Rickettsia species harbored by soft ticks in Pakistan [17,31,[34][35][36]. Therefore, the aims of this study were to genetically identify soft ticks collected in the mountainous region of Khyber Pakhtunkhwa (KP) in Pakistan, and to detect the presence of Rickettsia DNA in these ticks.

Tick collection and preservation
Ticks were collected manually from crevices, cracks, burrows and debris in 60 different animal shelters (housing cows, buffaloes, goats, sheep, and equids) in a total of 18 sampling locations in the selected districts ( Fig. 1). Tick collection was performed during the day and at night between June and October 2019. The tick samples were stored in Eppendorf tubes and transported to the Department of Zoology, Abdul Wali Khan University, Mardan. Some of the collected ticks were subjected to low intensity ultrasonic vibration for 5 min with a Qsonica sonicator Q700, LLC (Newtown, CT), and washed with distilled water and 70% ethanol to remove contaminants. The specimens were subjected to morphological identification and individually preserved in 100% ethanol in 2-ml Eppendorf tubes for further molecular examination.

Morphological identification
The collected soft ticks were morphologically identified to genus level using a standard identification key [37]. Representative specimens were photographed at 50-200× magnification using a Keyence VHX 900F microscope (Keyence, Itasca, IL).

DNA extraction and PCR
A total of 54 specimens, i.e. two nymphs and one female from each of the 18 sampling locations, were subjected to DNA extraction for subsequent genetic identification. Each specimen was cut in half using a sterile blade and the DNA of one half was extracted using the Nucleospin Tissue Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's guidelines. Extracted DNA was stored at −20 °C until further analysis.
Extracted DNA samples were examined by PCR amplification of partial fragments of the 12S rDNA, 16S rDNA and cox1 tick mitochondrial genes. A 25-µl PCR reaction mixture was prepared for 16S rDNA and cox1 genes; the mixture consisted of 12.75 µl water, 5 µl of 5X Fusion HF Buffer (Mobidiag), 2.5 µl of 2 mM deoxyribose nucleoside triphosphates (dNTPs), 1 µl of 10 µM primers, 0.25 S7 Fusion polymerase and 2.5 µl template DNA. The primers 16S+1 (5'-CCG GTC TGA ACT CAG ATC AAGT-3') and 16S-1 (5'-GCT CAA TGA TTT TTT AAA TTG CTG T-3') were used for the amplification of 16S rDNA fragments [38]. Universal cox1 gene primers HC02198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') and LCO1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') were used for the amplification of fragments of the cox1 gene [39]. Cycling conditions were as follows: 98 °C for  The PCR reactions contained Argas persicus DNA as the positive control in the case of tick species, and Rickettsia massiliae DNA as the positive control for Rickettsia species, while PCR-grade water was used as the negative control. The PCR amplified products were run on a 2% agarose gel and observed using gel documentation (Bio-Doc-It Imaging Systems). Amplicons were purified using the DNA Clean & Concentrator Kit (Zymo Research, Irvine, CA) and sequenced in both directions by LGC Genomics (Berlin, Germany).

Sequencing and phylogenetic analysis
The obtained sequences were trimmed using Seq-Man version 5.0 (DNASTAR) to remove primers and poor-quality sequence reads, and subjected to a Basic Local Alignment Search Tool [BLASTn; National Center for Biotechnology Information (NCBI)] search. Trimmed sequences were imported together with sequences from related species in BioEdit alignment editor v 7.0.5 [42] and subjected to ClustalW Multiple alignment [43]. The phylogenetic trees for tick mitochondrial 12S rDNA, 16S rDNA, and cox1 sequences, and for Rickettsia 16S rDNA sequences, were created separately, in accordance with the neighbor-joining method in Molecular evolutionary genetics analysis (MEGA-X) software [44], using align by MUSCLE [45] with a bootstrapping value of 1000 [44].

Identified ticks
In total, 2330 soft ticks were collected (Table 1) and morphologically identified as Ornithodoros (Pavlovskyella) sp. from the following traits: nymphs and adults had small cheeks; the dorsal idiosome had faint dorsal disks and small mammillae; a dorsoventral groove was present; a pre-anal groove, which completely intersected the transverse postanal groove, was present (Fig. 2).
Abundance and percentage of the Ornithodoros sp. collected from various locations in the examined districts are summarized in Table 2.  In the phylogenetic tree, the 16S rDNA gene sequence of Rickettsia sp. (MW308520) from the present study clustered with rickettsial sequences of the limoniae group, including those from Rickettsia limoniae (AF322442) and uncultured Rickettsia species (MG827265, MG827266 and MG827267) detected in A. japonicus (Fig. 6).

Discussion
Like in many other countries, and regions of the world, the diversity of soft ticks in Pakistan has barely been explored. To the best of our knowledge, only three species of soft ticks from Pakistan have been molecularly identified, namely Argas persicus [33], Argas sp. "rousetti" and Carios vespertilionis [46]. In addition, five other species of soft ticks have been reported from Pakistan based on morphological identification: Argas abdussalami [4], Argas reflexus [4], O. tholozani [17,18], Argas lahorensis [18,19] and O. papillipes [19]. There is no genetic information on Ornithodoros spp. from Pakistan, and this lack of information needs to be addressed. Therefore, we aimed to molecularly characterize Ornithodoros ticks by using three genetic markers to delineate the species of this genus collected in the KP region of Pakistan. The next objective was to molecularly detect Rickettsia spp.
in the collected ticks. The phylogenetic analysis revealed that the collected ticks are related to O. verrucosus and O. tholozani reported from Ukraine, Iran and Israel [21,[47][48][49]. The phylogenetic analysis of the Rickettsia sp. showed that it is closely related to the limoniae group, a basal group of Rickettsia species.
Many argasids, including Ornithodoros species, are nidicolous and live in sheltered habitats such as burrows, crevices, nests and old man-made shelters [6,48,50], and have a long lifespan of up to 20 years [51]. Ornithodoros tholozani, a member of the subgenus Pavlovskyella within the genus Ornithodoros, has been reported from stables, barns, stone walls, recording studios and human dwellings [4], whereas O. verrucosus has been reported to mainly inhabit burrows and/or crevices inhabited by reptiles and small mammals [3,4]. These tick species are found in deserts, semi-deserts and dry areas of the Palearctic zoogeographical region, and chiefly feed on rodents of the family Cricetidae [4,47]. In the present study, Ornithodoros ticks were collected from burrows, crevices, and cracks in the walls and from the ground debris of domestic animal shelters in northern and southern districts of KP. These districts generally have cold winters, mild summers and rainfall in all the seasons. The collected ticks were morphologically comparable to Ornithodoros (Pavlovskyella), and their genetic identity confirmed this similarity since the sequences of their loci clustered with those of Ornithodoros (Pavlovskyella) specimens from Iran, Israel, and Ukraine in the phylogenetic tree (Figs. 3, 4, 5). However, it is important to note that, on the basis of the topology of the phylogenetic tree and genetic comparisons with other Ornithodoros ticks, the collected ticks might represent separate species to O. tholozani or O. verrucosus. Ornithodoros tholozani was originally described from Persia [16], an antique territory that does not include Pakistan, and the sequences presently available for this species are from Israel. Ornithodoros verrucosus occurs mainly in the Caucasus and eastern Europe, thus the available sequences from Ukraine are likely representative of this species [21]. In  (Fig. 4). However, it is noteworthy that the sequences of the ticks identified as O. verrucosus from Iran did not cluster with those of O. verrucosus from Ukraine, and thus are thought to correspond to another  showed maximum percentage identity with O. tholozani (88.9%), O. verrucosus (93.5%) and O. verrucosus (90.2%) reported from Israel, Ukraine and Iran, respectively. As the study of larvae has proved useful for the differentiation of Ornithodoros spp. [4], a taxonomic approach that considers this life stage should be used to elucidate the status of O. verrucosus from Iran, and also that of the Ornithodoros sp. collected in this study.
The nidicolous lifestyle of Ornithodoros species makes it challenging to develop potential mathematical distribution models for these soft ticks using correlative niche modeling [52]. It is also difficult to judge the actual relationships between their life history traits and external environment [3]. However, it has been observed that the geographical distribution of species of the subgenus Pavlovskyella within the genus Ornithodoros, which include O. tholozani and O. verrucosus, is influenced by humidity and precipitation [3,53]. These ticks have been mostly reported from areas with microclimatic conditions marked by temperature and relative humidity ranges of 17-20 °C and 70-80%, respectively [48,54]. Similarly, in the present study, the Ornithodoros specimens were collected from regions with high relative humidity, high precipitation, and high temperatures. The monthly mean temperature, relative humidity and annual precipitation in the studied districts in the summer were as follows: Bajaur-25 °C, 68%, and 700 mm; Lower and Upper Dir-22 °C, 64% and 750 mm; Shangla-20 °C, 72% and 850 mm; and Orakzai-25 °C, 61%, and 600 mm (climate-data.org). Interestingly, favorable conditions for the survival and growth of most argasid ticks, including Ornithodoros species, were reported to be provided by the heat and respiration generated by their host animals inside the caves in which they were found [3,48]. Additionally, temperature has a profound effect on the activity of argasids [3], including Ornithodoros species [55]. We collected Ornithodoros ticks in the summer, from June to September, and even in this season, the specimens were never found out of their microhabitats during the daytime, which agrees with previous findings that these soft ticks are mainly nocturnal [50]. According to Hopla et al. [56], few Ornithodoros species that feed on livestock and poultry also feed on wild animals. Accordingly, the inspected districts in the present study were mostly rural, with an abundance of livestock (buffaloes, other cattle, equids and small ruminants).
Being an agricultural country, inhabitants of several regions of Pakistan rear livestock and poultry in animal shelters, and within or outside their houses. Soft ticks have been documented as vectors and reservoirs for several disease-causing agents throughout the world [57]. Thus, Ornithodoros species might pose a threat to livestock, poultry, and to people who rear livestock, in Pakistan. To the best of our knowledge, to date, there is no published report on the molecular detection of Rickettsia spp. associated with Ornithodoros species in Pakistan. Despite the fact that soft ticks are not considered natural vectors of Rickettsia spp., several rickettsiae have been detected in them [10,51,[58][59][60]. In several regions of the world, Ornithodoros species are vectors and reservoirs of causal agents of tick-borne relapsing fever (TBRF) [54,57]. In the present study, a Rickettsia sp. was recorded for the first time in Ornithodoros specimens from Pakistan. The pathogenicity of the Rickettsia sp. detected in the present study needs to be investigated, given the relevance of the rickettsiae bacterial group as causal agents of emerging infectious tick-borne diseases [24].

Conclusions
The present study reports on an Ornithodoros (Pavlovskyella) sp. tick and an associated Rickettsia sp. of the limoniae group for the first time from Pakistan. The morphological and phylogenetic analyses revealed that the Ornithodoros sp. is an undetermined sister species of O. verrucosus and O. tholozani; formal identification of this Ornithodoros sp. is pending. The Rickettsia sp. detected in the Ornithodoros specimens clustered within the limoniae group of Rickettsia species reported from China. Further studies on soft ticks, especially Ornithodoros species, are essential to explore their diversity, associated pathogens, and any economic losses that they may cause.