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Article

First Report of Rickettsia conorii in Hyalomma kumari Ticks

by
Shafi Ullah
1,
Abdulaziz Alouffi
2,
Mashal M. Almutairi
3,
Nabila Islam
4,
Gauhar Rehman
1,
Zia Ul Islam
5,
Haroon Ahmed
6,
Itabajara da Silva Vaz Júnior
7,
Marcelo B. Labruna
8,
Tetsuya Tanaka
9,* and
Abid Ali
1,*
1
Department of Zoology, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan
2
King Abdulaziz City for Science and Technology, Riyadh 12354, Saudi Arabia
3
Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
4
Department of Chemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan
5
Department of Biotechnology, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan
6
Department of Biosciences, COMSATS University Islamabad (CUI), Islamabad 44000, Pakistan
7
Centro de Biotecnologia and Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre 91501-970, Brazil
8
Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine, University of São Paulo, Sao Paulo 05508-060, Brazil
9
Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan
*
Authors to whom correspondence should be addressed.
Animals 2023, 13(9), 1488; https://doi.org/10.3390/ani13091488
Submission received: 1 February 2023 / Revised: 29 March 2023 / Accepted: 20 April 2023 / Published: 27 April 2023
(This article belongs to the Special Issue Detection of Parasites Using Traditional and Advanced Molecular Techniques)
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Abstract

:

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.

1. 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].
Subtropical and harsh semi-arid climatic conditions are ideal for the development and growth of many Hyalomma species [17,18]. Located in subtropics, the majority of the rural population in Pakistan relies on livestock, particularly small ruminants, for dairy and meat products. Pakistan has a huge population of small ruminants that consists of approximately 78.2 million goats and 31.2 million sheep [19]. The list of Hyalomma ticks infesting small ruminants in Pakistan includes Hyalomma anatolicum, Hyalomma dromedarii, Hyalomma isaaci, Hyalomma hussaini, Hyalomma scupense, Hyalomma asiaticum, Hyalomma kumari, and Hyalomma turanicum [2,12,13,20,21,22].
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 classification [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.

2. 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).

2.1. Hosts and Spatial Survey of Ticks

Overall, 409 H. kumari ticks were collected from 163 infested out of 295 examined hosts with a prevalence rate of 55.25%. A total of 204 females, 158 males, and 47 nymphs were collected (Table 1). Goats were highly infested, with an infestation rate of 98/168 (58.33%) counting for 224 ticks, while sheep were less infested with a rate of 65/127 (51.18%) and counting for 185 ticks. Gender-wise, data showed a high prevalence of ticks on female goats (126), followed by female sheep (104), male goats (98), and male sheep (81) (Table 1). Hyalomma kumari ticks were collected from the four districts (Nowshera, Mardan, Shangla, and Mohmand) of northern Pakistan. Among the selected districts, the highest number of ticks was collected in Mohmand, 120 (57 females, 49 males, 14 nymphs), followed by Nowshera, 118 (58 females, 44 males, 16 nymphs), Shangla, 91 (48 females, 32 males, 11 nymphs), and Mardan, 80 (41 females, 33 males, 6 nymphs).

2.2. 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 insufficient ITS-2 sequences of Hyalomma species, we opted to not construct the ITS-2 based phylogenetic tree.

2.3. Rickettsia spp. Prevalence and Phylogenetic Analyses

Among the collected ticks, 83 samples were subjected to PCR (Table 1) for the characterization of Rickettsia using rickettsial primers (Table 2). The overall detection rate of Rickettsia was 9.6% (8/83), being highest in Nowshera with 12.5% (3/24), followed by Mardan 10.5% (2/19), Mohmand 9.5% (2/22), and Shangla 5.5% (1/18). The spatial survey of Rickettsia in each district is shown in Table 2.
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).

3. 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.

4. Materials and Methods

4.1. Study Area and Tick Collection

Ticks were collected from herds of grazing goats and sheep in four districts including Nowshera (34.0105° N, 71.9876° E), Mardan (34.1986° N, 72.0404° E), Shangla (34.8872° N, 72.7570° E), and Mohmand (35.2227° N, 72.4258° E), Pakistan. Nowshera district consists mainly of sandy plains surrounded by low elevated hills, comprised of a semi-arid zone. The district Mardan can be divided into two parts: a hilly area in the northeast and a plain area in the southeast. The hills run the length of the district’s northern border. The southwestern part is largely made up of fertile plains with minor scattered hills. District Shangla comprises small valleys surrounded by forest covered high mountains. Natural geographic borders delineate the district Swat, which is centered on the Swat River. Mountains surround the valley on all sides, the high valleys and alpine meadows of Swat-Kohistan, a place where several glaciers originated, are the northernmost area of Swat district. Coordinates of collecting sites in each district were retrieved from Google Map and spread on a Microsoft Excel sheet to design a map using ArcGIS 10.3.1 (Figure 7).

4.2. 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 sheep). The selected districts where hosts were examined included Mohmand 48/85 (26/47 goats, 22/38 sheep), Nowshera 43/81 (24/44 goats, 19/37 sheep), Shangla 38/74 (21/40 goats, 17/34 sheep), and Mardan 34/55 (19/31 goats, 15/24 sheep). 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.

4.3. 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) 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).
Table
Table 2. Primers used to amplify partial sequences of tick and rickettsial genes.
Table 2. Primers used to amplify partial sequences of tick and rickettsial genes.
Organisms and GenesPrimers Sequences (5′-3′)Amplicon Size (bp)Annealing TemperatureReference
Ticks
16S rRNAF-CCGGTCTGAACTCAG
R-CAATGATTTTTTAAA		46054 °C[36]
cox IF-GGAACAATATATTTAATTTTTGG
R 5-ATCTATCCCTACTGTAAATATATG		80155 °C[46]
ITS-2F-CCATCGATGTGAAYTGCAGGACA
R-GTGAATTCTATGCTTAAATTCAGGGGGT		90055 °C[57]
Rickettsia
gltAF-GCAAGTATCGGTGAGGATGTAAT
R-GCTTCCTAAAATTCAATAAATCAGGAT		40156 °C[58]
ompAF-ATGGCGAATATTTCTCCAAAA
R-GTTCCGTTAATGGCAGCATCT		53255 °C[59]
ompBF-CCGCAGGGTTGGTAACTGC
R-CCTTTTAGATTACCGCCTAA		86250 °C[60]

4.4. 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].

4.5. 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].

5. 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani13091488/s1, Figure S1: PCR amplified cox I DNA. 1; molecular marker, 2; control negative (PCR water), 3; Hyalomma kumari cox I, 4; control positive (Hyalomma anatolicum cox I). Figure S2: PCR amplified 16S rDNA. 1; molecular marker, 4; control negative (PCR water), 2-3,5-7; Hyalomma kumari 16S rDNA, 8; control positive (Hyalomma anatolicum 16S rDNA). Figure S3: PCR amplified ITS-2 DNA. 1; molecular marker, 5; control negative (PCR water), 2-3; Hyalomma kumari ITS-2 DNA, 4; control positive (Hyalomma anatolicum ITS-2). Figure S4: PCR amplified Rickettsial ompA. 1; molecular marker, 4; control negative (PCR water), 2; Rickettsia conorii ompA, 3; control positive (Rickettsia massiliae ompA). Figure S5: PCR amplified Rickettsial ompB. 1; molecular marker, 4; control negative (PCR water), 2-3,5-6; Rickettsia conorii ompB, 7; control positive (Rickettsia massiliae ompB). Figure S6: PCR amplified Rickettsial gltA. 1; molecular marker, 4; control negative (PCR water), 2; Rickettsia conorii gltA, 3; control positive (Rickettsia massiliae gltA).

Author Contributions

A.A. (Abid Ali) designed the study. A.A. (Abid Ali) and S.U. collected the tick samples and conducted experimentation. A.A. (Abid Ali), S.U., A.A. (Abdulaziz Alouffi), M.M.A., N.I., Z.U.I., G.R., H.A., I.d.S.V.J., T.T. and M.B.L.; methodology, writing—review and editing, A.A. (Abid Ali); supervision, A.A. (Abid Ali) and S.U.; project administration, A.A. (Abdulaziz Alouffi), M.M.A. and T.T.; funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

The researchers supporting project number (RSP2023R494), King Saud University, Riyadh, Saudi Arabia. This work was supported by JSPS KAKENHI Grant Numbers JP20KK0154 and JP22H02522, Heiwa Nakajima Foundation, and Kieikai Research Foundation.

Institutional Review Board Statement

The design of the present study was approved by the Advanced Study and Research Board (ASRB: Dir/A&R/AWKUM/2022/9396) and ethical committee of the Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan, Pakistan. All animal owners were informed verbally, and approval was taken before observing their hosts.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This study was carried out under the financial support driven by Higher Education Commission of Pakistan, Pakistan Science Foundation. The researchers supporting project number (RSP2023R494), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Animals 13 01488 g001 550
Figure 1. Hyalomma kumari, (A) male dorsal (I: cervical groove, II: lateral side punctation, III: posteromedian groove); (B) male ventral (I: adanal plates, II: accessory anal plate); (C) female dorsal (I: cervical field, II: scutum shape); (D) female ventral (I: genital aperture).
Figure 1. Hyalomma kumari, (A) male dorsal (I: cervical groove, II: lateral side punctation, III: posteromedian groove); (B) male ventral (I: adanal plates, II: accessory anal plate); (C) female dorsal (I: cervical field, II: scutum shape); (D) female ventral (I: genital aperture).
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Animals 13 01488 g002 550
Figure 2. Phylogenic tree based on cox I partial sequences of Hyalomma spp. using maximum likelihood method. Hyalomma aegyptium (KY548846) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of H. kumari from Pakistan is highlighted by bold font and marked with black circle.
Figure 2. Phylogenic tree based on cox I partial sequences of Hyalomma spp. using maximum likelihood method. Hyalomma aegyptium (KY548846) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of H. kumari from Pakistan is highlighted by bold font and marked with black circle.
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Animals 13 01488 g003 550
Figure 3. Phylogenic tree based on 16S rRNA gene partial sequences of Hyalomma spp. using maximum likelihood method. Nosomma monstrosum (KU130406) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of H. kumari from Pakistan is highlighted by bold font and marked with black circle.
Figure 3. Phylogenic tree based on 16S rRNA gene partial sequences of Hyalomma spp. using maximum likelihood method. Nosomma monstrosum (KU130406) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of H. kumari from Pakistan is highlighted by bold font and marked with black circle.
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Animals 13 01488 g004 550
Figure 4. Phylogenic tree based on ompA partial sequences of Rickettsia spp. using maximum likelihood method. Rickettsia australis (AF149108) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of R. conorii (OP957009) from Pakistan is highlighted by bold and underline font.
Figure 4. Phylogenic tree based on ompA partial sequences of Rickettsia spp. using maximum likelihood method. Rickettsia australis (AF149108) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of R. conorii (OP957009) from Pakistan is highlighted by bold and underline font.
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Animals 13 01488 g005 550
Figure 5. Phylogenic tree based on ompB sequences of Rickettsia spp. using maximum likelihood method. Rickettsia felis (MN817154, MN817153) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of R. conorii (OP957007) from Pakistan is highlighted by bold font and marked by an underline.
Figure 5. Phylogenic tree based on ompB sequences of Rickettsia spp. using maximum likelihood method. Rickettsia felis (MN817154, MN817153) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of R. conorii (OP957007) from Pakistan is highlighted by bold font and marked by an underline.
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Animals 13 01488 g006 550
Figure 6. Phylogenic tree based on gltA partial sequences of Rickettsia spp. using maximum likelihood method. Rickettsia canadensis (CP000409) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of R. conorii (OP957008) from Pakistan is highlighted by bold font and marked by an underline.
Figure 6. Phylogenic tree based on gltA partial sequences of Rickettsia spp. using maximum likelihood method. Rickettsia canadensis (CP000409) was used as an outgroup, using 1000 bootstrap value at each node. The number of substitutions per site has been shown by scale bar. The present sequence of R. conorii (OP957008) from Pakistan is highlighted by bold font and marked by an underline.
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Figure 7. Elevation based map showing collection points of Hyalomma kumari in north of Pakistan.
Figure 7. Elevation based map showing collection points of Hyalomma kumari in north of Pakistan.
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Table
Table 1. Number of Hyalomma kumari ticks according to geographic districts of Pakistan, host species, and gender, and the results of these ticks for detection of Rickettsia conorii DNA by molecular analyses.
Table 1. Number of Hyalomma kumari ticks according to geographic districts of Pakistan, host species, and gender, and the results of these ticks for detection of Rickettsia conorii DNA by molecular analyses.
DistrictsHostsHost GenderNo. of TicksTick Life StagesMolecularly Analyzed TicksRickettsia Positive (%)p-Value *
FemaleMaleNymph
NowsheraGoatmale271310411 F, 9 M, 4 N1 F, 1 M, 1 N (12.5)0.0058
female3920136
Sheepmale231193
female2914123
MardanGoatmale2010828 F, 6 M, 5 N1 F, 1 N (10.5)
female2412111
Sheepmale15861
female211182
ShanglaGoatmale22136310 F, 5 M, 3 N1 M (5.5)
female2816111
Sheepmale18972
female231085
MohmandGoatmale29151229 F, 9 M, 4 N1 F, 1 M (9.1)
female3514138
Sheepmale251582
female3113162
Total4092041584738 F, 29 M, 16 N3 F, 3 M, 2 N (9.6)
* Statistic test: chi-square. F: females; M: males; N: nymphs).
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Ullah, S.; Alouffi, A.; Almutairi, M.M.; Islam, N.; Rehman, G.; Ul Islam, Z.; Ahmed, H.; Júnior, I.d.S.V.; Labruna, M.B.; Tanaka, T.; et al. First Report of Rickettsia conorii in Hyalomma kumari Ticks. Animals 2023, 13, 1488. https://doi.org/10.3390/ani13091488

AMA Style

Ullah S, Alouffi A, Almutairi MM, Islam N, Rehman G, Ul Islam Z, Ahmed H, Júnior IdSV, Labruna MB, Tanaka T, et al. First Report of Rickettsia conorii in Hyalomma kumari Ticks. Animals. 2023; 13(9):1488. https://doi.org/10.3390/ani13091488

Chicago/Turabian Style

Ullah, Shafi, Abdulaziz Alouffi, Mashal M. Almutairi, Nabila Islam, Gauhar Rehman, Zia Ul Islam, Haroon Ahmed, Itabajara da Silva Vaz Júnior, Marcelo B. Labruna, Tetsuya Tanaka, and et al. 2023. "First Report of Rickettsia conorii in Hyalomma kumari Ticks" Animals 13, no. 9: 1488. https://doi.org/10.3390/ani13091488

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