First report of spotted fever group Rickettsia aeschlimannii in Hyalomma turanicum, Haemaphysalis bispinosa, and Haemaphysalis montgomeryi infesting domestic animals: updates on the epidemiology of tick-borne Rickettsia aeschlimannii

Tick-borne Rickettsia spp. have long been known as causative agents for zoonotic diseases. We have previously characterized Rickettsia spp. in different ticks infesting a broad range of hosts in Pakistan; however, knowledge regarding Rickettsia aeschlimannii in Haemaphysalis and Hyalomma ticks is missing. This study aimed to obtain a better understanding about R. aeschlimannii in Pakistan and update the knowledge about its worldwide epidemiology. Among 369 examined domestic animals, 247 (66%) were infested by 872 ticks. Collected ticks were morphologically delineated into three genera, namely, Rhipicephalus, Hyalomma, and Haemaphysalis. Adult females were the most prevalent (number ₌ 376, 43.1%), followed by nymphs (303, 34.74%) and males (193, 22.13%). Overall, genomic DNA samples of 223 tick were isolated and screened for Rickettsia spp. by the amplification of rickettsial gltA, ompA, and ompB partial genes using conventional PCR. Rickettsial DNA was detected in 8 of 223 (3.58%) ticks including nymphs (5 of 122, 4.0%) and adult females (3 of 86, 3.48%). The rickettsial gltA, ompA, and ompB sequences were detected in Hyalomma turanicum (2 nymphs and 1 adult female), Haemaphysalis bispinosa (1 nymph and 1 adult female), and Haemaphysalis montgomeryi (2 nymphs and 1 adult female). These rickettsial sequences showed 99.71–100% identity with R. aeschlimannii and phylogenetically clustered with the same species. None of the tested Rhipicephalus microplus, Hyalomma isaaci, Hyalomma scupense, Rhipicephalus turanicus, Hyalomma anatolicum, Rhipicephalus haemaphysaloides, Rhipicephalus sanguineus, Haemaphysalis cornupunctata, and Haemaphysalis sulcata ticks were found positive for rickettsial DNA. Comprehensive surveillance studies should be adopted to update the knowledge regarding tick-borne zoonotic Rickettsia species, evaluate their risks to humans and livestock, and investigate the unexamined cases of illness after tick bite among livestock holders in the country.


Introduction
Ticks are important ectoparasites due to their capacity to feed on animals including humans (De la Fuente et al., 2017) and transmit various pathogens including viruses, bacteria, and protozoans to their hosts (Boulanger et al., 2019).Among bacteria, Rickettsia species are transmitted by ticks, mites, and fleas, causing rickettsioses (Fournier and Raoult, 2009;Labruna, 2009).The main tick genera that are the potential vectors for Rickettsia spp.are Amblyomma, Dermacentor, Ixodes, Hyalomma, Rhipicephalus, and Haemaphysalis (Fournier and Raoult, 2009).Several studies have revealed the extensive diversity of the spotted fever group rickettsiae in different tick species and geographic locations (Socolovschi et al., 2009).Approximately 34 species in the genus Rickettsia have been validated, and several others are yet to be determined to the species level (El Karkouri et al., 2022).Spotted fever group Rickettsia spp.are distributed in Pakistan and have recently been reported in different ticks including Rh. microplus, Rh. haemaphysaloides, Rh. turanicus, H. kashmirensis, and H. cornupunctata infesting different animals (Ali et al., 2021(Ali et al., , 2022;;Khan et al., 2023).
Earlier, we characterized pathogenic and undetermined Rickettsia spp.associated with different ticks parasitizing a wide range of vertebrate hosts in Pakistan (Karim et al., 2017;Ali et al., 2021Ali et al., , 2022Ali et al., , 2023;;Numan et al., 2022;Aneela et al., 2023;Shehla et al., 2023;Ullah et al., 2023).However, there is a paucity of information regarding the presence of R. aeschlimannii in different ticks in the country, and its potential risks to the public and animal's health.Updated knowledge regarding the epidemiology, association with ticks infesting different hosts, and phylogenetic position of spotted fever group (SFG) R. aeschlimannii is essential for effective management of infection.This study aimed to molecularly characterize tick-borne Rickettsia spp. in selected districts of Pakistan and update the information about the global epidemiology of uncharacterized Rickettsia spp.(35.3356°N,72.0468°E),71.8097°E).Data regarding the host, ticks, climate, and location were noted.Climatic information was taken from the website climate-data.org.Geo-coordinates of location sites were obtained from the global positioning system and were entered into a spreadsheet, and the study map was designed in ArcGIS10.8.1.3(ESRI, Redlands, CA, USA) (Figure 1).

Ethical statement
The design of the current study has received approval from the members of the Advanced Study and Research Board (Dir/A&R/ AWKUM/2023/0014) and the Faculty of Zoology Department, Abdul Wali Khan University Mardan, Pakistan.Permission was obtained from the owner of the animal before collecting ticks from their animals.

Collection and morphological identification of ticks
Tick specimens were collected conveniently from March 2020 to February 2021 from domestic animals in seven districts of Khyber Pakhtunkhwa, Pakistan.The collection was opportunistic occurring whenever tick-infested animals were found within the survey regions.The whole body of the animal was examined for ticks.With the help of curved forceps, ticks were collected carefully so that the morphological features of ticks were not damaged.Every sample was tagged with its location of collection, date, and species of the animal.

Genomic DNA extraction
All ticks were identified morphologically, and genomic DNA was individually extracted from a subset of 223 (122N, 86F, and 15M) ticks.Ticks were cleaned with 70% ethanol, followed by distilled water and phosphate-buffered saline for the elimination of surface contaminants.Each rinsed tick was separately kept in a 1.5 mL tube and subjected to drying within an incubator for 30 min.Using a sterilized scalpel, the samples were cut into pieces inside the Eppendorf tube.The phenol-chloroform method was used for the extraction of genomic DNA from the ticks (Sambrook et al., 1989).The DNA concentration in each extracted sample was quantified with Nanodrop (OPTIZEN, Daejeon, South Korea).

Amplification of targeted rickettsial DNA
The extracted DNA was used in conventional PCR (Thermo Fisher Scientific, and Walham, MA, USA), to amplify fragments of three genes of Rickettsia, namely, gltA (citrate synthase gene), ompA (outer membrane protein A), and ompB (outer membrane protein B).PCR was conducted in a 25 μL reaction mixture, containing 1 μL of Map showing locations in seven districts of Khyber Pakhtunkhwa, where ticks were collected from domestic animals.1, and thermocycling conditions were set as previously used (Regnery et al., 1991;Roux and Raoult 2000;Labruna et al., 2004).Positive and negative control samples were Rickettsia massiliae DNA and "nuclease-free" water, respectively (Shehla et al., 2023).The amplified products of each PCR were observed by electrophoresis on 2% agarose gel and stained with ethidium-bromide, and the results were visualized on the Gel Doc system (BioDoc-It™ Imaging Systems, Upland, CA, USA).

Sequencing and phylogenetic analysis
The amplified DNA fragments were purified using the GENECLEAN II Kit (Qbiogene, Illkirch, France) and sequenced using the Sanger sequencing (Macrogen, Inc., Seoul, South Korea) in both forward and reverse directions.Poor-quality sequences were removed by trimming all the obtained sequences using SeqMan v 5.00 (DNASTAR, Inc.), and a consensus sequence was generated.Sequences with the highest identity were selected from GenBank using the Basic Local Alignment Search Tool (BLAST) on the user interface of National Center for Biotechnology Information, and these sequences were aligned with the obtained sequences by BioEdit v. 7.0.5 using CLUSTALW multiple alignments (Thompson et al., 1994), followed by MUSCLE alignment (Edgar, 2004).The Neighbor-Joining method was applied for obtaining phylogenies with 1,000 bootstrap replicates in Molecular Evolutionary Genetic Analysis (MEGA-X) software (Kumar et al., 2018).The sequences that were obtained made up the final positions in the dataset.

Data analysis
The recorded data of tick-infested hosts and tick distribution were described with frequency and percentage using descriptive statistics.Fisher's exact test was used to determine the association between host, tick species, rickettsial species, and locations in GraphPad Prism software (V 5.0).p-value <0.05 was considered as significant standard.

Literature-based search
The literature-based search was carried out by using different databases including ScienceDirect, PubMed, Web of Sciences, and Google Scholar to collect published data regarding Rickettsia aeschlimannii in different tick species, wild and domestic animals, humans, environment, and vegetation.The search was conducted by using some keywords, such as ticks, tick-borne pathogens, domestic animals, small ruminants, zoonosis, livestock, and Rickettsia aeschlimannii.Complete research articles, short communication, review papers, and conference articles were downloaded by using a combination of the above mentioned keywords.Lists of references from downloaded studies were examined to relevant articles (Table 2).

Sequence and phylogenetic analysis
Assembled contigs of direct and reverse sequence reads for each PCR-amplified fragment were analyzed.Due to a single haplotype, consensus sequences were generated for each partial gene.The consensus sequence of gltA (348 bp) showed 100% identity with R. aeschlimannii from Russia, Senegal, and Kazakhstan, followed by 99.71% identity with R. aeschlimannii-type strain from Morocco.Similarly, 100% identity was shown by the obtained ompA (467 bp) consensus sequence with R. aeschlimannii from Russia, Spain, Turkey, and Kazakhstan, followed by 99.79% identity with R. aeschlimannii-type strain.Similarly, the ompB (764 bp) consensus sequence also showed 100% identity with R. aeschlimannii from Kazakhstan, Russia, Italy, and Portugal, followed by 99.74% identity with R. aeschlimannii-type strain.In all cases, the query coverage was 100%.The obtained rickettsial gltA sequence (accession number: OR351959), ompA sequence (accession number: OR351960), and ompB sequence (accession number: OR351961) were submitted to GenBank.

Discussion
Previous studies have recorded the presence of Rickettsia spp. in different tick-infesting hosts in Pakistan.However, there was a paucity of information regarding R. aeschlimannii in the region.This study presents the first report on molecular detection of R. aeschlimannii in H. bispinosa, H. montgomeryi, and Hy.turanicum ticks collected from sheep and goats in Pakistan.The obtained sequences showed maximum identity and phylogenetically clustered with Rickettsia aeschlimannii, which confirms the occurrence of Rickettsia aeschlimannii in the regions.Rickettsia aeschlimannii, an emerging pathogen with zoonotic potential, has been observed to cause infections in humans across various countries such as Morocco (Raoult et al., 2002), Tunisia (Znazen et al., 2006), Algeria (Mokrani et al., 2008), Greece (Germanakis et al., 2013), andRussia (Igolkina et al., 2022).The detection of R. aeschlimannii in tick-parasitizing domestic animals suggests a high exposure of livestock holders to this pathogen.Small ruminants (goats and sheep), domestic dogs, and cattle were found infested by the ticks of genera Rhipicephalus, Hyalomma, and Haemaphysalis.Among the collected ticks, Rh. microplus, Hy. anatolicum, H. bispinosa and H. montgomeryi were the dominant tick species.These findings mirror the pattern observed in other previous studies conducted in same region (Karim et al., 2017;Ali et al., 2019;Alam et al., 2022;Khan Z. et al., 2022;Tila et al., 2023) underscoring the regional significance of these tick species.Furthermore, it emphasizes the need for further research to comprehensively investigate their prevalence, distribution, and potential implications for public health (Ali et al., 2023).Ticks of different genera have been reported as carriers for various Rickettsia spp. in Pakistan (Ali et al., 2021(Ali et al., , 2022;;Khan M. et al., 2022;Khan Z. et al., 2022;Numan et al., 2022;Ullah et al., 2023).Herein, Rickettsia aeschlimannii was detected in H. bispinosa, H. montgomeryi, and Hy.turanicum ticks using three genetic markers, namely, gltA, ompA, and ompB.To date, there has been a lack of information regarding the detection of R. aeschlimannii in H. bispinosa and H. montgomeryi tick-infesting domestic animals, such as goats and sheep.There is also a possibility that the rickettsial DNA is detected in the ticks may be due to ingesting rickettsemic host blood.Literature search revealed that R. aeschlimannii is associated with a variety of tick species belonging to six genera of hard ticks, namely, Hyalomma, Rhipicephalus, Haemaphysalis, Amblyomma, Dermacentor, and Ixodes (Parola et al., 2001;Fernández-Soto et al., 2003;Shpynov et al., 2004;Tomassone et al., FIGURE 4 Phylogenetic analysis based on the sequences of the ompB genes of R. aeschlimannii.The obtained sequences in the present study are indicated in bold and underlined fonts.Rickettsia australis used as an out-group.2010; Karasartova et al., 2018).Furthermore, there are limited reports, which have detected this pathogen in Asia (Wei et al., 2015;Satjanadumrong et al., 2019).This study found its association with two new ticks, expanding its known host and geographical range.The detection of R. aeschlimannii in Hyalomma and Haemaphysalis ticks suggests a potential threat to livestock holders.Additionally, the rate of R. aeschlimannii was observed highest in Haemaphysalis ticks, which are the primary ticks that infest goats and sheep.This enhances the feasibility of public health risks as these ticks may occasionally infest humans (Guglielmone and Robbins, 2018).
Rickettsia aeschlimannii has been detected in all life stages of ticks, such as adult females, males, larvae, and nymphs (Raoult et al., 2002;Shpynov et al., 2009;Germanakis et al., 2013;Orkun et al., 2014;Wallmenius et al., 2014;Tosoni et al., 2016).This study presents the first molecular evidence of R. aeschlimannii in H. bispinosa and H. montgomeryi ticks, which suggests that other tick species could also serve as competent vectors for this pathogen in the region.Molecular methods are considered faster and more accurate for the genetic characterization and phylogenetic analysis of Rickettsia spp.(Fournier et al., 1998;Roux and Raoult, 2000).The gltA, ompA, ompB, and sca4 DNA sequences have been used as suitable genetic markers to discriminate different Rickettsia spp (Roux and Raoult, 2000;Fournier et al., 2003;Labruna et al., 2004).Herein, these sequences were targeted for molecular characterization and phylogenetic analysis of R. aeschlimannii which revealed the close relatedness with corresponding species of the SFG.We assume that human infection caused by rickettsial agents of SFG maybe underreported due to the lack of epidemiological information among health practitioners and laboratory technicians and the lack of diagnostic procedures in Pakistan, given the relevance of the occurrence of these agents in the region.Further studies in the region should be encouraged to obtain information on zoonotic outcomes due to these infectious agents.

Conclusion
This study for the first time contributes to the neglected knowledge and genetic characterization of tick-borne R. aeschlimannii in H. bispinosa, H. montgomeryi, and Hy.turanicum ticks in Pakistan.The results of this study also indicated that goats and sheep are exposed to R. aeschlimannii.Further molecular studies are important to screen R. aeschlimannii in livestock and livestock holders who have close contact with domestic animals.

FIGURE 2
FIGURE 2Phylogenetic analysis based on the gltA sequences of Rickettsia aeschlimannii.The obtained sequences of the present study are indicated in bold and underlined fonts.Rickettsia akari and Rickettsia australis were used as out-group.

FIGURE 3
FIGURE 3Phylogenetic analysis based on the ompA sequences of R. aeschlimannii.The sequences obtained in this study are indicated in bold and underlined fonts.Rickettsia australis is used as an outgroup.

TABLE 1
Primers used for the amplification of rickettsial DNA in the current study.

TABLE 2
Global epidemiology of Rickettsia aeschlimannii detected in different ticks and vertebrate hosts including human.

TABLE 2
Rickettsia spp.by the amplification of three rickettsial markers, namely, gltA, ompA, and ompB gene fragments.Positive ticks for rickettsial gltA were also positive when tested with the primers of ompA and ompB.Rh.sanguineus (23), H. cornupunctata(7), and H. sulcata  (23).Information regarding the prevalence of Rickettsia spp. in various ticks is shown in Table3.

TABLE 3
Occurrence of ticks infesting various domestic animals and detection of rickettsial DNA associated with ticks.Adult females; F*, Engorged females; M, males.*Ticks subjected to PCR to screen for Rickettsia spp.