First Report of Rickettsia conorii in Hyalomma kumari Ticks

Simple Summary Ticks are blood-feeding ectoparasites that transmit life-threatening pathogens to humans and animals. Only 10% of all identified tick species have been screened for different tick-borne pathogens. Hyalomma ticks are associated with a wide range of pathogens including Theileria species, Babesia species, Anaplasma species, Ehrlichia species, and Rickettsia species. Moreover, ticks of genus Hyalomma are vectors for the Crimean-Congo hemorrhagic fever (CCHF), a serious threat endemic in Pakistan. In Pakistan, different tick species have been found positive for rickettsial agents; however, Hyalomma kumari ticks have never been investigated for any potential pathogens. In this work, H. kumari ticks were collected from goats and sheep, and morphologically and molecularly identified using different genetic markers. The identified ticks were screened for rickettsial agents using genetic markers that resulted in the detection of Rickettsia conorii for the first time in this tick. A proper surveillance program should be designed to effectively avoid any zoonotic consequences associated with these rickettsial pathogens. Abstract As a vector of wide range of pathogenic agents, ticks pose health threats to wild and domestic animals, and humans. Information is unavailable about the prevalence and spatial survey of Hyalomma kumari ticks and associated Rickettsia spp. in Pakistan. Concerning this knowledge gap, the present study aimed to molecularly detect Rickettsia species associated with H. kumari infesting small ruminants in Khyber Pakhtunkhwa (KP), Pakistan. A total of 409 H. kumari ticks were collected from 163/295 infested hosts with an infestation rate of 55.25%. A total of 204 females, 158 males, and 47 nymphs were collected. Goats were heavily infested by 224 ticks having an infestation rate of 58.33% (98/168), whereas sheep were infested by 185 ticks having a lesser infestation rate of 51.18% (65/127). Genomic DNA extracted from ticks was used for the amplification of tick (cox I, 16S rRNA, ITS-2) species and Rickettsia (gltA, ompA, and ompB) partial genes. Eighty-three ticks were subjected to PCR, and 8/83 (9.6%) were found positive for rickettsial agents. The cox I and 16S rRNA sequences of H. kumari showed 98.90–99.74% identity with H. kumari sequences reported from Pakistan, and phylogenetically clustered to the corresponding species reported from Pakistan and India. The obtained rickettsial gltA, ompA, and ompB sequences showed 100% identity with Rickettsia sp. of the Rickettsia conorii reported from Pakistan. In the phylogenetic trees, rickettsial sequences clustered with uncharacterized Rickettsia sp. from Pakistan and R. conorii from Israel, Russia, South Africa, and India. The present molecular based detection of H. kumari-associated R. conorii will facilitate effective surveillance in the region.


Introduction
In the tropical and subtropical climates, the ixodid ticks of the genus Hyalomma are of great medical and veterinary importance in terms of health risks and economic burden [1,2]. Hyalomma ticks are associated with a wide range of pathogens including Theileria spp. [3,4], Babesia spp. [5], Anaplasma spp., Ehrlichia spp. [6], and Rickettsia spp. [7]. Moreover, ticks of genus Hyalomma are vectors for the Crimean-Congo hemorrhagic fever (CCHF), a serious threat endemic in Pakistan [8]. To date, 27 species of Hyalomma have been identified in Palearctic, Oriental, and African regions [9], of which 13 have been reported in Pakistan [2,[10][11][12][13][14][15]. The genus Hyalomma has been classified into two subgenera, i.e., Euhyalomma and Hyalommina. The most diversified subgenus Euhyalomma is abundant in Africa, Asia, and Europe while species of subgenus Hyalommina are divided in to two groups based on specificity to their geographies. Hyalomma punt, Hyalomma rhipicephaloides, and Hyalomma arabica have been distributed in the Arabian Peninsula, northeast Africa, and the Near East, while Hyalomma hussaini, Hyalomma brevipunctata, and Hyalomma kumari are restricted to South Asia [16].
The Rickettsiaceae represents a broad group of Gram-negative intracellular bacteria that can serve as symbionts or pathogens and infect a broad range of hosts. The genus Rickettsia, which was first recognized as the agents responsible for spotted fever and other rickettsioses in vertebrate hosts, which spread through ticks, lice, fleas, and mites, is perhaps the best-known group of Rickettsiaceae [23]. Globally, Rickettsia conorii has been detected in various species including Rhipicephalus sanguineus, Rhipicephalus turanicus, Rhipicephalus bursa, Rhipicephalus pumilio, Rhipicephalus evertsi, Rhipicephalus simus, Rhipicephalus mushamae, Haemaphysalis leechi, and Haemaphysalis punctaleechi in several countries such as France, Bulgaria, Turkey, India, African, and Caspian countries [24]. So far, 25 species of Rickettsia are known to have pathogenic potential causing rickettsioses, including spotted fever group (SFG) rickettsioses, as for example, Mediterranean spotted fever (Rickettsia conorii conorii) in Europe and Africa, African tick bite fever (Rickettsia africae) in Africa, and Rocky Mountain spotted fever (Rickettsia rickettsii) in the Americas [25]. In addition, Rickettsia conorii israelensis is the causative agent of Israeli spotted fever in the Mediterranean region, Rickettsia conorii caspia is the etiological agent of Astrakhan fever in Caspian Sea region, northern Africa, and some Mediterranean countries, and Rickettsia conorii indica is the agent of Indian tick typhus in the Oriental region and southern Europe [26,27]. In Pakistan, there is a single earlier record of R. conorii in ticks using a toxin neutralization test (TNT) [28].
The advancement of molecular approaches as well as the increasing number of tick sequences annotated in GenBank enable a complementary categorization strategy based on genetic characteristics rather than only morphology [29][30][31]. In recent years, this methodology was adopted to identify and classify various tick groups, allowing for a re-definition of species distribution as well as detailed studies on tick-host preferences and population dynamics [32,33]. Mitochondrial genes are helpful as genetic markers because of the strict maternal inheritance and in some conditions, a quicker evolutionary change rate [34,35]. These markers have been used to classify and investigate tick populations, resulting in a huge amount of data on tick mitochondrial markers and improved field sample classifi-cation [36]. This tick has never been reported for rickettsial agent, although several other tick species have been observed as positive for different Rickettsia spp. in the region. The molecular and phylogenetic characterization of Hyalomma ticks, especially H. kumari and their associated Rickettsia, has been neglected in Pakistan. Keeping in view, this study was focused on investigating the molecular surveillance of rickettsial agents associated with H. kumari parasitizing small ruminants in Pakistan.

Results
Morphologically, males of H. kumari were identified by two pairs (adanal and accessory adanal) of anal plates which differentiate them from the rest of Hyalomma (three pairs of anal plates) species. Conscutum is yellowish brown in colour with medium and small sized punctations, distributed mostly on anterior, lateral, and caudal fields. The females of H. kumari were identified by the yellowish brown colour scutum, as in males. The scutum is slightly longer than its breadth, with a small size, and moderately sparse punctations. The genital aperture is narrow and U-shaped ( Figure 1). the strict maternal inheritance and in some conditions, a quicker evolutionary chan [34,35]. These markers have been used to classify and investigate tick populations, ing in a huge amount of data on tick mitochondrial markers and improved field s classification [36]. This tick has never been reported for rickettsial agent, although s other tick species have been observed as positive for different Rickettsia spp. in the r The molecular and phylogenetic characterization of Hyalomma ticks, especially H. and their associated Rickettsia, has been neglected in Pakistan. Keeping in view, this was focused on investigating the molecular surveillance of rickettsial agents asso with H. kumari parasitizing small ruminants in Pakistan.

Results
Morphologically, males of H. kumari were identified by two pairs (adanal and sory adanal) of anal plates which differentiate them from the rest of Hyalomma (thre of anal plates) species. Conscutum is yellowish brown in colour with medium and sized punctations, distributed mostly on anterior, lateral, and caudal fields. The fe of H. kumari were identified by the yellowish brown colour scutum, as in males. Th tum is slightly longer than its breadth, with a small size, and moderately sparse p tions. The genital aperture is narrow and U-shaped ( Figure 1).

Sequence and Phylogenetic Analyses of Tick
The PCR amplified cox I, 16S rRNA, and ITS-2 products have been shown in Supplementary Files (Figures S1-S3), respectively. The obtained identical sequences for each cox I, 16S rRNA, and ITS-2 fragment were considered as a single consensus sequence. In NCBI BLAST, the obtained trimmed cox I (755 bp) showed 98.90-99.47% identity with H. kumari sequences reported from Pakistan (KU130608 and KU130607) and India (MW587126, MW587125, MW587123, and MW587124). The phylogenetic tree was designed by downloading 28 sequences of various Hyalomma species. The obtained sequence was clustered with the sequences reported from Pakistan and India ( Figure 2). The obtained cox I sequence was uploaded to the GenBank under accession number OP453967. The ITS-2 (778 pb) partial sequence was uploaded to the GenBank under the accession number OP454037. Similarly, the obtained trimmed 16S rRNA gene fragment (384 bp) showed 98.96-99.74% identity with H. kumari sequences reported from Pakistan (KU130442 and KU130443). Twenty-six sequences of different Hyalomma species were downloaded to design a phylogenetic tree, taking Nosomma monstrosum as an out group. In the phylogenetic tree, the obtained sequence clustered with H. kumari from Pakistan ( Figure 3). Our obtained 16S rRNA gene partial sequence was uploaded to the GenBank under accession number OP452898. Due to Animals 2023, 13, 1488 5 of 18 insufficient ITS-2 sequences of Hyalomma species, we opted to not construct the ITS-2 based phylogenetic tree.
OP454037. Similarly, the obtained trimmed 16S rRNA gene fragment (384 bp) showed 98.96-99.74% identity with H. kumari sequences reported from Pakistan (KU130442 and KU130443). Twenty-six sequences of different Hyalomma species were downloaded to design a phylogenetic tree, taking Nosomma monstrosum as an out group. In the phylogenetic tree, the obtained sequence clustered with H. kumari from Pakistan ( Figure 3). Our obtained 16S rRNA gene partial sequence was uploaded to the GenBank under accession number OP452898. Due to insufficient ITS-2 sequences of Hyalomma species, we opted to not construct the ITS-2 based phylogenetic tree.
The PCR amplified ompA, ompB, gltA products have been shown in Supplementary  Files (Figures S4-S6), respectively. The obtained Rickettsia DNA partial sequences for each gene (ompA, ompB, gltA) were assembled and considered as a single consensus sequence. The BLAST result for the ompA (466 bp) showed 100% identity with the reported sequence of the Rickettsia sp. From Pakistan (MN548863), 99.60% with R. conorii israelensis from Israel (U43797), 99.36% with R. conorii caspia from Russia (U43791), 97.01% with R. conorii indica from India (U43794), and 96.90% identity with R. conorii conorii from South Africa (U43806). The phylogenetic tree was designed based on the ompA gene by downloading 29 ompA sequences of different Rickettsia species, including all four subspecies of R. conorii. The obtained ompA sequence in the phylogenetic tree clustered with Rickettsia sp. From Pakistan, while grouped in a sister clade with R. conorii israelensis from Israel among all subspecies of the R. conorii (Figure 4). Our ompA sequence has been uploaded to the GenBank under the accession number (OP957009). Similarly, the obtained trimmed ompB (773 bp) partial sequence showed 100% identity with the Rickettsia sp. Reported from Pakistan (MN581992), 99.61% with R. conorii israelensis from Israel (AF123712), 99.36% with R. conorii caspia from Russia (AF123708), 96.64% with R. conorii indica from India (AF123726), and 96.51% with R. conorii conorii from South Africa (AF123721). The phylogenetic tree was designed by downloading 32 sequences of various Rickettsia species along with subspecies of R. conorii. The obtained sequence clustered with the Rickettsia sp. reported from Pakistan and R. conorii israelensis from Israel appeared on the sister clade ( Figure 5). Our obtained ompB sequence was uploaded to the GenBank under accession number (OP957007). The trimmed gltA partial sequence (359 bp) showed 100% identity with the Rickettsia sp. reported from Pakistan (MN581988), 99.72% with R. conorii israelensis from Israel (U59727), 99.44% with R. conorii caspia from Russia (U59728), and 99.44% identity with R. conorii conorii from South Africa (U59730). The phylogenetic tree was designed by downloading 27 sequences of various Rickettsia species as well as subspecies of R. conorii. The obtained sequence was clustered with the sequences reported from Pakistan while grouped in a sister clade with R. conorii israelensis from Israel, among the subspecies of R. conorii ( Figure 6). Our obtained gltA sequence was uploaded to the GenBank under accession number (OP957008).

Discussion
The significance of Hyalomma ticks as potential vectors for Rickettsia is under debate. Several studies have revealed the detection of different Rickettsia species in Hyalomma ticks. A limited number of studies have been conducted on the prevalence and spatial distribution of Hyalomma ticks, specifically H. kumari in Pakistan [12]. To the best of our understanding, this is the first survey investigating the spatial distribution and host-wise prevalence of H. kumari infesting small ruminants. The knowledge regarding the detection of Rickettsia spp. in H. kumari infesting small ruminants is inadequate. This study contributes to the neglected knowledge about R. conorii in H. kumari ticks in Pakistan.
Within a specific zoogeographic region, the prevalence and distribution of ticks have been influenced by climatic and environmental factors [37,38], with Hyalomma species as the top successful flourisher of harsh desert climatic conditions [9,16]. Unlike other Hyalomma species, H. kumari favors areas receiving sufficient rainfall (greater than desert regions) [12,39], as the current study was done in a region having prescribed climatic and metrological conditions. Hyalomma kumari is a three-host tick that infests nearly all types of domestic and some wild hosts [16]. Herein we report H. kumari collected from goats and sheep inhabiting less elevated hill stations in the selected Pakistan Districts, as this tick species prefers such topography [12,16]. Rhipicephalus turanicus and Rhipicephalus haemaphysaloides have been reported infesting goats and sheep in the current study region [30]. However, these two tick species were only found positive for Rickettsia massiliae [40,41]. The fact that no rickettsial DNA was previously detected in H. kumari ticks [42], motivated our work to search for H. kumari-associated Rickettsia species.
For taxonomic purposes and to identify the vectors of a wide range of pathogens, accurate classification of related tick species is essential [9]. Validating the morphological identification, recent investigations have been focused on the taxonomy and evolutionary history of ticks and tick-borne Rickettsia species utilizing different genetic markers [24,[43][44][45].
Understanding phylogenetic relationships at the species level, in several studies the mitochondrial genes 16S rRNA and cox I have been implemented as appropriate genetic markers [36,[45][46][47]. In 16S rRNA and cox I based phylogenetic trees, the H. kumari of the current study clustered with the same species reported from Pakistan and India. In contrast to lower identities with other countries, the Indian sequences had the highest nucleotide identity and a similar phylogeny, indicating a high genetic conservation among H. kumari from Pakistan and India. Before the political separation in 1947, both countries were part of the same Indo-Pak subcontinent, where animals were moving freely. The transmigration of tick-infested hosts across the borders and similar agro-ecological conditions are the reasons for such situations [48]. The grouping of H. kumari as a sister clade to H. hussaini coincides with the morphological similarities between H. kumari and H. hussaini, as both belong to the H. kumari group of the subgenus Hyalommina [16].
Rickettsia species are mostly zoonotic pathogens that are harbored by a wide range of arthropod vectors, especially ticks [27]. The diagnosis of Rickettsia in ticks is of great importance, not only to identify the infected ticks, but also to determine the risk of transmission to humans [49,50]. The occurrence of SFG Rickettsia is common in Hyalomma ticks globally [23,51], though in Pakistan, SFG Rickettsia have been detected in Rhipicephalus turanicus and Rhipicephalus haemaphysaloides [40,41]. To evaluate the taxonomy and phylogeny of Rickettsia spp., the gltA, ompA, and ompB genes are among the most suitable genetic markers [52]. All four subspecies of R. conorii (R. conorii israelensis, R. conorii caspia, R. conorii conorii, R. conorii indica) were analyzed in the phylogenetic trees based on ompA, ompB, and gltA [53]; however, the absence of a specific gltA sequence of R. conorii indica in the GenBank compelled us to include the gltA based aligned sequence from the whole genome shotgun (WGS) sequence of the R. conorii indica [54]. Herein, we detected R. conorii that was associated with H. kumari collected from goats and sheep, which validates and confirms the previous knowledge on TNT based detection of R. conorii in Pakistan [28]. The obtained sequences based on the three rickettsial genes (gltA, ompA, and ompB) showed 100% identity with unpublished Rickettsia sp. detected in Rhipicephalus turanicus collected in 1963 from goats in Pakistan [28], which may be due to the expansion of Rickettsia associated with tick vectors infesting migrant hosts [25]. All the Rickettsia phylogenetic trees clustered with reported sequences of Pakistan and formed a sister clade with R. conorii israelensis, revealing the evolutionary closeness.
The presence of R. conorii in ticks feeding on sheep and goats represents a zoonotic concern. Notably, sheep and goats are among the commonly kept animals in Pakistan [15,22] because of their high fertility, short generation period, tolerance to severe climatic conditions, and status as a source of income for rural households [55]. They are typically kept as free-ranging animals and are frequently tethered at night close to human settlements. This creates a favorable condition for both interactions with disease carriers such as ticks, enabling the spread of pathogens like R. conorii to humans. Further studies should evaluate the transmission of R. conorii israelensis by these ticks to humans and animals, to understand any zoonotic consequences related to these parasites.

Study Area and Tick Collection
Ticks were collected from herds of grazing goats and sheep in four districts including Nowshera (34.

Collection, Preservation and Morphological Identification
Ticks were collected manually from 20 grazing herds of goats and sheep in the study region. Among them, 163 hosts (86 goats, 77 sheep) were infested out of 295 examined hosts (164 goats and 131  Collected ticks were put in safe lock Eppendorf tubes containing 100% ethanol, which were labeled with information regarding host type, gender, and area of collection. Before identification, ticks were cleaned in order to compare and clarify each taxonomic character with previous standard identification keys [12,33]. After morphological identification, only H. kumari ticks were considered for further analysis.

DNA Extraction and Polymerase Chain Reaction (PCR)
Prior to the DNA extraction, the ticks were washed with distilled water and 70% ethanol to remove the contaminants and dried on sterile filter paper. Onward, these ticks were ground in the sterile Eppendorf using scalps and scissors. Whole genomic DNA was extracted manually from 83 randomly selected ticks (unfed) by using the phenol-chloroform method (Table 1) [56]. The purity and quantity of the extracted genomic DNA was checked by a NanoDrop (NanoQ, Optizen, Korea). For the genetic characterization of the H. kumari, partial fragments of the tick mitochondrial cytochrome c oxidase subunit 1 (cox I) and 16S rRNA genes, and tick nuclear second internal transcribed spacer (ITS-2) were amplified using conventional PCR assays Table 2. PCR reagents were prepared in a 25 µL containing 2 µL genomic DNA (60 ng), 8.5 µL nuclease free water, 1 µL each forward and reverse primer (10 µM), and 12.5 µL DreamTaq green 2x PCR MasterMix (Thermo Scientific, Waltham, MA, USA). Hyalomma kumari-associated Rickettsia was targeted and characterized through the amplification of partial sequences of the rickettsial citrate synthase (gltA), outer membrane protein A (ompA), and outer membrane protein B (ompB) genes (Table 1)

DNA Extraction and Polymerase Chain Reaction (PCR)
Prior to the DNA extraction, the ticks were washed with distilled water and 70% ethanol to remove the contaminants and dried on sterile filter paper. Onward, these ticks were ground in the sterile Eppendorf using scalps and scissors. Whole genomic DNA was extracted manually from 83 randomly selected ticks (unfed) by using the phenolchloroform method (Table 1) [56]. The purity and quantity of the extracted genomic DNA was checked by a NanoDrop (NanoQ, Optizen, Korea). For the genetic characterization of the H. kumari, partial fragments of the tick mitochondrial cytochrome c oxidase subunit 1 (cox I) and 16S rRNA genes, and tick nuclear second internal transcribed spacer (ITS-2) were amplified using conventional PCR assays Table 2. PCR reagents were prepared in a 25 µL containing 2 µL genomic DNA (60 ng), 8.5 µL nuclease free water, 1 µL each forward and reverse primer (10 µM), and 12.5 µL DreamTaq green 2x PCR MasterMix (Thermo Scientific, Waltham, MA, USA). Hyalomma kumari-associated Rickettsia was targeted and characterized through the amplification of partial sequences of the rickettsial citrate synthase (gltA), outer membrane protein A (ompA), and outer membrane protein B (ompB) genes (Table 1) using the aforementioned reagents mixture. Hyalomma anatolicum DNA and Rickettsia massiliae DNA were taken as positive controls, while PCR water as a negative control. The amplified PCR products were run on 1.8% agarose gel electrophoresis and visualized through Gel Documentation (LGD-101, Labocon, Hampshire, UK). The resultant amplicons were cleaned and purified through a DNA purification Kit (Invitrogen™JetFlex™, Invitrogen, Waltham, MA, USA).

Sequencing and Phylogenetic Analyses
Twelve amplicons, four of each 16S rRNA, cox I, and ITS-2, and portions of all amplified rickettsial genes (gltA, ompA, ompB) of the positive samples mentioned in Table 1, were sequenced bi-directionally (Macrogen, Seoul, Republic of Korea). The obtained sequences were assembled and trimmed to eliminate the poor nucleotide regions using SeqMan V. 5 (DNASTAR, Madison, WI, USA). Trimmed sequences were subjected to BLAST (Basic Local Alignment Search Tool) at NCBI (National Center for Biotechnological Information) to download identical sequences [61]. Downloaded sequences, along with obtained sequences, and out group were aligned using ClustalW multiple alignment in BioEdit V.7.0.5 [62]. Aligned sequences were used to construct phylogenetic trees through the Tamura-Nei model and maximum-likelihood statistical method, keeping bootstrap value 1000 in MEGA-11 [63].

Statistical Analysis
Microsoft Excel V.2016 (Microsoft 365 ® ) was used for the descriptive analyses of the obtained data. Chi-square multiple variance test was accomplished in the GraphPad Prism version 5, considering p-value < 0.05 as significant. This test was carried out to find the significance of the prescribed data as well as to find the difference between variables. The tested variables were male, female and nymphs infesting different genders of the reported hosts [22].

Conclusions
This is the first extensive analysis based on the prevalence of H. kumari in the KP, Pakistan. Furthermore, R. conorii detection in H. kumari represents the first report of R. conorii in this tick. Further studies should be evaluated regarding the prevalence, genetic characterization of H. kumari and their associated Rickettsia.