Molecular Survey of Rickettsia raoultii in Ticks Infesting Livestock from Pakistan with Notes on Pathogen Distribution in Palearctic and Oriental Regions

Simple Summary Ticks are chelicerate arthropods that feed on blood and infest all vertebrates except fish and transmit different disease-causing agents including Rickettsia spp. to domestic and wild animals as well as humans. In the present study, we aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats from five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra) of Khyber Pakhtunkhwa province, Pakistan. A total of 8/148 (5.4%) ticks, including four Hyalomma turanicum, two Haemaphysalis cornupunctata, one Haemaphysalis montgomeryi, and one Haemaphysalis bispinosa, were found positive for Rickettsia sp. The phylogenetic analysis of detected Rickettsia sp. based on three genetic markers (gltA, ompA, and ompB) revealed 100% identity with Rickettsia raoultii, clustered with its corresponding species reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Further comprehensive studies on molecular and serosurveillance of various Rickettsia spp. in different ticks should be conducted in the region to understand the zoonotic threats due to these pathogens. Abstract Ticks are hematophagous ectoparasites that transmit different pathogens such as Rickettsia spp. to domestic and wild animals as well as humans. Genetic characterizations of Rickettsia spp. from different regions of Pakistan are mostly based on one or two genetic markers and are confined to small sampling areas and limited host ranges. Therefore, this study aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats. All the collected tick specimens were morphologically identified, and randomly selected tick species (148) were screened molecularly for the detection of Rickettsia spp. by amplifying three rickettsial DNA fragments, namely, the citrate-synthase gene (gltA), outer-membrane protein A (ompA), and outer-membrane protein B (ompB). After examining 261 hosts, 161 (61.7%) hosts were found infested by 564 ticks, including 287 (50.9%) nymphs, 171 (30.3%) females, and 106 (18.8%) males in five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra). The highest occurrence was noted for Hyalomma dromedarii (number = 72, 12.8%), followed by Haemaphysalis sulcata (n = 70, 12.4%), Rhipicephalus turanicus (n = 64, 11.3%), Rhipicephalus microplus (n = 55, 9.7%), Haemaphysalis cornupunctata (n = 49, 8.7%), Hyalomma turanicum (n = 48, 8.5%), Hyalomma isaaci (n = 45, 8.0%), Haemaphysalis montgomeryi (n = 44, 7.8%), Hyalomma anatolicum (n = 42, 7.5%), Haemaphysalis bispinosa (n = 38, 6.7%), and Rhipicephalus haemaphysaloides (n = 37, 6.6%). A subset of 148 ticks were tested, in which eight (5.4%) ticks, including four Hy. turanicum, two Ha. cornupunctata, one Ha. montgomeryi, and one Ha. bispinosa, were found positive for Rickettsia sp. The gltA, ompA, and ompB sequences revealed 100% identity and were phylogenetically clustered with Rickettsia raoultii reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Additionally, various reports on R. raoultii from Palearctic and Oriental regions were summarized in this study. To the best of our knowledge, this is the first report regarding genetic characterization and phylogenetic analysis of R. raoultii from Pakistan. Further studies to investigate the association between Rickettsia spp. and ticks should be encouraged to apprise effective management of zoonotic consequences.


Introduction
Ticks carry and transmit a wide range of pathogens comprising viruses, fungi, protozoans, and bacteria [1][2][3].The genus Rickettsia is comprised of obligate Gram-negative bacteria, is distributed worldwide, and can cause rickettsiosis in hosts including domestic and wild animals, as well as humans [2,4].Arthropod vectors such as ticks, fleas, mites, and lice may transmit Rickettsia spp.; however, the competent vectors for its propagation are mostly Ixodid ticks [5].Tick-borne rickettsiosis is a known vector-borne zoonotic disease [2], and the majority of the tick-borne Rickettsiae belong to the spotted fever group (SFG) of Rickettsia [6].So far, almost 33 different Rickettsia spp.and 19 different Candidatus (Ca) Rickettsia spp. in the SFG group have been identified globally [7][8][9][10][11].
Pakistan is an agricultural country, and livestock play an important role in its economy, as different animals are a major source of income for rural inhabitants.However, in Pakistan, different ticks including Hyalomma spp., Rhipicephalus spp., Amblyomma spp., Ixodes spp., Ornithodoros spp., Nosomma spp., and Haemaphysalis spp.have been reported infesting livestock and wild animals, and these ticks can transmit different pathogens such as Anaplasma spp., Rickettsia spp., Babesia spp., Theileria spp., and Coxiella spp.[37][38][39][40][41][42][43][44][45].In Pakistan, some Rickettsia spp.have been reported in ticks infesting equids, bovines, and wild animals [37,39,46,47], and these Rickettsia species were reported based on targeting only one or two rickettsial markers.Hence, there is a dearth of information regarding the genetic characterization of Rickettsia spp. in different tick species infesting camels, sheep, and goats.Novel Rickettsia species of undetermined pathogenicity are continuously detected in ticks, necessitating effective tools to infer their phylogenetic relationships.The present study aims to molecularly characterize the Rickettsia spp. in hard ticks infesting camels, sheep, and goats by using three genetic markers in Khyber Pakhtunkhwa (KP), Pakistan.

Ethical Approval
This study was approved by the members of graduate study committee and Advance Studies Research Board (AWKUM/CE/SC/2022/12041) of the Zoology Department, Abdul Wali Khan University Mardan, Pakistan.Verbal and written permission was obtained from livestock owners before examining their animals for the collection of ticks.

Study Area
The current study was performed in five districts of the KP province, including Kohat (33  11 56.1 E).Different hosts including camels, sheep, and goats were examined for the collection of ticks from July 2020-June 2021.The latitudes and longitudes of the tick collection sites were collected via the Global Positioning System (GPS) and imported to Microsoft Excel V. 2013 for processing.The study area map (Figure 1) was designed in ArcGIS V. 10.3.1 (ESRI, Redlands, CA, USA).

Ticks Collection and Identification
Tick collection was performed on camels, sheep, and goats in the selected study area.Ticks were collected from the aforementioned hosts by examining the entire body, and they were found regardless of the specific locations and times within the targeted survey districts in various farms, open fields, and freely moving animals in pastures.Collection was performed only once for each host when tick infestation was detected.Host-based collected ticks were separately stored in micro-tubes labelled with the collection sites and specified host.Tick specimens were washed with distilled water and preserved in 100% ethanol for further processing.

Molecular Screening of Rickettsia spp.
A total of 148 (74N, 74F) tick specimens were subjected to DNA extraction for molecular analyses.Individual ticks were crushed and their genomic DNA was extracted through a standard method of phenol-chloroform [53].Genomic DNA was not extracted from all the morphologically identified ticks because we selected representative ticks of each tick species from their respective host in each district.The extracted DNA was quantified using Nanodrop (Nano-Q, Optizen, Daejeon, Republic of Korea) and stored at −20 • C for further molecular experimentation.The extracted DNA of each individual tick was tested for the presence of Rickettsia spp.through a conventional PCR targeting the amplification of fragments of three genes including citrate-synthase (gltA), outer-membrane protein A (ompA), and outer-membrane protein B (ompB).The PCR reaction was performed in 25 µL, having 1 µL of each primer (forward and reverse) (10 µM), 2 µL of genomic DNA (50-100 ng), 12.5 µL of DreamTaq MasterMix (2×) (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 8.5 µL of PCR water (nuclease free).The primers and PCR conditions used for the amplification of the aforementioned fragments are provided in Table 1.After PCR, the amplified products were run on 1.5% agarose gel and the results were visualized under UV light through the Gel Doc system (UVP BioDoc-It imaging system; Analytik Jena AG, Jena, Germany).

Ticks Collection and Identification
Tick collection was performed on camels, sheep, and goats in the selected study area.Ticks were collected from the aforementioned hosts by examining the entire body, and they were found regardless of the specific locations and times within the targeted survey districts in various farms, open fields, and freely moving animals in pastures.Collection was performed only once for each host when tick infestation was detected.Host-based collected ticks were separately stored in micro-tubes labelled with the collection sites and specified host.Tick specimens were washed with distilled water and preserved in 100% ethanol for further processing.

Molecular Screening of Rickettsia spp.
A total of 148 (74N, 74F) tick specimens were subjected to DNA extraction for molec-

Sequences and Phylogenetic Analyses
The manufacturer's protocol was adopted for the purification of all the amplified PCR products using the GeneClean II Kit (Qbiogene, Illkirch, France), and products were submitted for bidirectional sequencing (Macrogen Inc., Seoul, Republic of Korea) through the Sanger-based method.All the obtained bidirectional sequences were trimmed in Seq-Man v. 5.0 (DNASTAR) by removing poor sequencing reads and primer contaminations.The consensus sequences for each fragment (gltA, ompA, and ompB) were obtained from all the identical trimmed sequences, which were separately subjected to the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI).Sequences with high identity were downloaded and aligned in BioEdit alignment editor v 7.0.5 along with selected outgroups [57].Separately, the phylogenetic trees of gltA, ompA, and ompB were constructed in Molecular Evolutionary Genetic Analysis software [58] by following the neighbor-joining method and Tamura-Nei model [59], in which 1000 bootstrap replicates were used for tree reliability [58].

Literature Search and Selection Criteria
We performed a literature search using databases including Science Direct, Web of Science, Google Scholar, and PubMed.Various keywords including tick(s), small and large ruminant(s), livestock, livestock-holder(s), farmer(s), worker(s), human(s), R. raoultii, Ca.R. raoultii, R. conorii subsp.raoultii, molecular characterization, phylogenetic analysis, and the specific country names were used in the aforementioned databases.Combinations of keywords were used to download research publications, review articles, short communications, and case-reports regarding R. raoultii.A minimum of one report of R. raoultii from each country, as well as all the previously reported human cases of R. raoultii from Palearctic and Oriental regions, were included in the current study.All this literature-based data was retrieved in July 2023 (Table 2).
Hy. anatolicum  Rh. haemaphysaloides Ha. sulcata       Phylogeny was inferred based on rickettsial ompB fragments using the neighbor-joining method.GenBank accession numbers are followed by species and country names at each terminal taxon.A group of sequences of Rickettsia lusitaniae (MN629485), Rickettsia australis (CP003338), and Rickettsia akari (AF123707) were taken as outgroups using 1000 bootstrap values at each node.The present gltA sequence of (accession no.OR400637) R. raoultii is marked with bold and underlined font.

Discussion
Ticks pose health threats to humans and animals, as they can transmit numerous pathogens including SFG Rickettsia spp.[1][2][3].Previously, potential health risks to humans posed by rickettsial agents have been worsened by some Rickettsia spp.including R. raoultii, which can cause rickettsiosis in humans in different regions of the world [22,32,74,91].Camels, sheep, and goats are considered as human companions, and these animals have been identified as a notable reservoir hosts for many Rickettsia spp., which might play an important role in the natural transmission cycle and dispersal of different Rickettsia spp.[2,92,112].Hence, a regular surveillance of various rickettsial agents carried by ticks infesting the aforementioned hosts is essential to minimize public health risks.Herein, R. raoultii was molecularly analyzed via standard genetic markers in eleven morphologically identified tick species infesting camels, sheep, and goats in five districts of KP, Pakistan, and R. raoultii was identified in four tick species including Hy. turanicum, Ha. cornupunctata, Ha. montgomeryi, and Ha.bispinosa.
Host density in the herds ultimately increases the chances of tick infestation compared to those animals kept alone due to the possibility of infestation by detached host-questing ticks in the herd area [46].In current study, ticks of different genera including Haemaphysalis, Rhipicephalus, and Hyalomma were found infesting the aforementioned hosts, which were sharing their habitats, thus enhancing the possibilities for an available wide host range.Here, Hy. dromedarii (n = 72/564, 12.8%) was the most prevalent tick compared to other tick species because the highest number of camels (n = 99/261, 37.9%) was examined compared to other hosts.Previous reports regarding the tick abundance on camels have shown that Hy. dromedarii is the most prevalent tick species of dromedary camels because this tick is not influenced by any season, and hence shows a preponderance on camels during both dry and wet seasonal conditions [113,114].Previous studies have shown that Hyalomma ticks can survive successfully in harsh desert regions [115,116]; therefore, the Hy.dromedarii ticks were most prevalent because the larger proportion of the study area was composed of desertic plains, arid plains, and arid hilly areas that are suitable for the survival of Hy. dromedarii ticks.Additionally, these ticks may act as vectors for the transmission of infectious agents to livestock owners [117,118].Some Hyalomma, Rhipicephalus, and Haemaphysalis tick species infesting humans have been recorded from this region in Pakistan [119].
In Pakistan, different Rickettsia spp.have been detected in various tick species including Ix. kashmiricus, Ornithodoros sp., Rh. turanicus, Rh. haemaphysaloides, Rh. microplus, Hy. dromedarii, and Hy.anatolicum [37,39,41,46,47].Rickettsia raoultii was detected in four tick species including Hy. turanicum, Ha. sulcata, Ha. cornupunctata, and Ha.montgomeryi, and it was previously reported in various tick genera including Hyalomma, Rhipicephalus, Dermacentor, and Ixodes in various regions of the world [14,17,81,95].Rickettsia raoultii was detected in different tick species collected from camels, sheep, and goats in the current study.Similarly, R. raoultii has been detected in various tick species collected from the aforementioned hosts in different countries including Slovakia, Malaysia, China, Greece, Mongolia, India, Iran, and Turkey [14,25,81,89,92,99,100,108,110].Our findings provide the first molecular evidence regarding the genetic characterization of R. raoultii in Hy. turanicum infesting camels, which suggests the possible role of this tick in the dispersal of R. raoultii in the specified region.Since adult female and nymph ticks were found positive for R. raoultii, there is a possibility that the detected R. raoultii was ingested through the blood from infected camels, as this pathogen has been previously detected in the blood of dogs in Germany [77] and Iran [30].Hence, there is a need to conduct comprehensive serosurveillance and molecular studies on different rickettsial agents in different animals to know the factors responsible for the transmission of these bacteria in the region.
Molecular characterization of Rickettsia spp.through the ompB gene relies on its outermembrane locality and the presence of protein epitopes that are common to both typhus and SFG Rickettsiae [7,55].Additionally, it has been previously stated that three rickettsial genes including gltA, ompA, and ompB may be used and are known for the detection of rickettsial agents specifically to investigate the presence of SFG Rickettsiae [13,21] and to provide a significant phylogenetic relationship in the Rickettsiae [120].Hence, R. raoultii has been previously globally detected in different ticks including De. marginatus, De. nuttalli, De. silvarum, De. reticulatus, Am. testudinarium, Am. helvolum, Ha. bispinosa, Rh. microplus, and Rh.sanguineus by using three genetic markers: gltA, ompA, and ompB [21,29,79,81,102,121].The obtained gltA, ompA, and ompB sequences of R. raoultii in this study revealed a close evolutionary relationship and were hence phylogenetically clustered with their corresponding species reported in China, Russia, USA, Italy, Turkey, Denmark, and Austria.
Camels, sheep, and goats are considered as human companions and share their household environment, resulting in close contact with each other.R. raoultii was identified in different tick species collected from the aforementioned animals; thus, these ticks and their specified hosts may play a role as a source of human infection.Therefore, further serological and molecular studies in the region should be encouraged to understand the zoonotic threats due to these infectious agents.

Conclusions
Rickettsia raoultii has been previously reported in different ticks infesting camels, sheep, and goats globally.Hence, this study genetically characterized R. raoultii in four tick species including Ha. bispinosa, Ha. cornupunctata, Ha. montgomeryi, and Hy.turanicum infesting camels, sheep, and goats in Pakistan.Additionally, this is the first report regarding the detection of R. raoultii in Hy. turanicum ticks collected from camels, which suggests that camels may serve as reservoir hosts for R. raoultii in the region.Due to close contact between livestock holders and camels, sheep, and goats in the region, there are possibilities for transmission of these bacteria to humans.Thus, surveillance strategies should be adopted to properly investigate these bacteria to minimize any health threats.Further comprehensive studies on molecular and serosurveillance of Rickettsia spp. in different ticks should be conducted in the region to understand the zoonotic threats due to these pathogens.

20 Figure 1 .
Figure 1.Map showing collection sites in the study area where ticks infesting camels, sheep, and goats were collected for the detection of Rickettsia spp.

Figure 1 .
Figure 1.Map showing collection sites in the study area where ticks infesting camels, sheep, and goats were collected for the detection of Rickettsia spp.

Figure 2 .
Figure 2.Phylogeny was inferred based on rickettsial gltA fragments using the neighbor-joining method.GenBank accession numbers are followed by the species and country names at each terminal taxon.Rickettsia canadensis (CP000409) was taken as an outgroup using 1000 bootstrap values at each node.The present gltA sequence (accession no.OR400635) for R. raoultii is marked with bold and underlined font.

Figure 2 .
Figure 2.Phylogeny was inferred based on rickettsial gltA fragments using the neighbor-joining method.GenBank accession numbers are followed by the species and country names at each terminal taxon.Rickettsia canadensis (CP000409) was taken as an outgroup using 1000 bootstrap values at each node.The present gltA sequence (accession no.OR400635) for R. raoultii is marked with bold and underlined font.

Figure 3 .
Figure3.Phylogeny was inferred based on rickettsial ompA fragments using the neighbor-joining method.GenBank accession numbers are followed by the species and country names at each terminal taxon.Rickettsia canadensis (CP000409) was taken as an outgroup using 1000 bootstrap values at each node.The present ompA sequence (accession no.OR400636) for R. raoultii is marked with bold and underlined font.

Figure 3 .
Figure3.Phylogeny was inferred based on rickettsial ompA fragments using the neighbor-joining method.GenBank accession numbers are followed by the species and country names at each terminal taxon.Rickettsia canadensis (CP000409) was taken as an outgroup using 1000 bootstrap values at each node.The present ompA sequence (accession no.OR400636) for R. raoultii is marked with bold and underlined font.

Figure 4 .
Figure 4.Phylogeny was inferred based on rickettsial ompB fragments using the neighbor-joining method.GenBank accession numbers are followed by species and country names at each terminal taxon.A group of sequences of Rickettsia lusitaniae (MN629485), Rickettsia australis (CP003338), and Rickettsia akari (AF123707) were taken as outgroups using 1000 bootstrap values at each node.The present gltA sequence of (accession no.OR400637) R. raoultii is marked with bold and underlined font.

Figure 4 .
Figure 4.Phylogeny was inferred based on rickettsial ompB fragments using the neighbor-joining method.GenBank accession numbers are followed by species and country names at each terminal taxon.A group of sequences of Rickettsia lusitaniae (MN629485), Rickettsia australis (CP003338), and Rickettsia akari (AF123707) were taken as outgroups using 1000 bootstrap values at each node.The present gltA sequence of (accession no.OR400637) R. raoultii is marked with bold and underlined font.

Table 1 .
List of primers used for the amplification of rickettsial fragments in various ticks collected from camels, goats, and sheep in the present study.

Table 2 .
A summary of some previously published reports on Rickettsia raoultii in ticks, fleas, humans, vegetation, and animals in Palearctic and Oriental regions.

Table 3 .
Table showing the number of various inspected hosts in different localities, collected tick species and their life stages, and molecularly analyzed ticks for the detection of Rickettsia raoultii through gltA, ompA, and ompB fragments.