Detection of Rickettsia raoultii in Dermacentor reticulatus and Haemaphysalis inermis ticks in Slovakia

Ticks are vector arthropods responsible for the transmission of several pathogenic agents that affect both human and animal health worldwide. In this study our objective was to analyse, using molecular tools, the bacterial community of Dermacentor reticulatus and Haemaphysalis inermis ticks collected in south-eastern Slovakia. Using real-time PCR, we identified the presence of Rickettsia spp. DNA at levels of 14/59 (23.72 %) and 29/173 (16.76 %) in D. reticulatus and H. inermis, respectively. In addition, using standard PCR and sequencing, we identified the presence of Rickettsia raoultii DNA in 13 ticks belonging to the two investigated species. Rickettsia raoultii blast results revealed an average identification percentage of 99.62 %. Following the results of this molecular study there is a possibility that D. reticulatus and H. inermis play a potential role in the transmission of R. raoultii. To prove the possibility of validity of this hypothesis, we suggest performing experimental models in future studies. Our results can serve as preliminary data for future transmission models.


Introduction
Bacteria belonging to the genus Rickettsia are known as causative agents of various vector-borne zoonotic diseases. They are responsible for mild to severe diseases in humans (Parola et al. 2013;Eldin and Parola 2018).
The diversity of pathogens in ecosystems also depends on the species diversity of vectors and hosts and the diversity of habitats (de la Fuente et al. 2008). Many ticks in Europe, including Dermacentor reticulatus Fabricius, 1794 are known to act as carriers, reservoirs and/or vectors of different pathogenic Rickettsia species e.g., Rickettsia slovaca (Sekeyová et al. 1998;Garcia-Vozmediano et al. 2020) and Rickettsia raoultii (Boldiš et al. 2008;Földvári et al. 2013).
Revising the role of another tick species, Haemaphysalis inermis Birula, 1895 in the spread of rickettsiae continues to be challenging as few data have been previously published (Portillo et al. 2008;Špitalská et al. 2018). In a study carried out in Hungary, the authors detected the presence of R. helvetica in H. inermis and suggested that this tick species could be a potential vector of this pathogen (Hornok et al. 2010).
Recent epidemiological studies of Rickettsia in Slovakia were completed in suburban, natural and rural habitats (Minichová et al. 2017), or in an urban temperate forest (Chvostáč et al. 2018) in the western part of the country. A dry forest-steppe biotope is rarely referenced in literature except, in the past century, by Řeháček et al. (1976a, b). The authors indicated that: "The biotopes of the spotted fever group rickettsiae occurring in east Slovakia-namely, cultivated and meadow steppes with sparse forests-share some of the characteristics of the biotopes of Rickettsia sibirica. Thus, besides the new species of spotted fever group rickettsiae, the circulation of R. sibirica and perhaps of Rickettsia conorii in east Slovakia should not be excluded". However, none of them were ever confirmed in south-eastern Slovakia afterwards (Řeháček et al. 1976a, b).
Our objective was to investigate the possibility of detecting rickettsiae in D. reticulatus and H. inermis, two relatively abundant tick species in the steppe habitats of south-eastern Slovakia.

Materials and methods
Tick collection and sampling site

DNA extraction and molecular detection of bacteria in ticks using real time PCR
Half of the tick body without legs was selected for DNA extraction using EZ1 DNA tissue kit (Qiagen, Hilden, Germany) following a protocol previously elaborated in our laboratory (Diarra et al. 2017).
After extraction, the DNA of each sample was screened with the CFX96 real-time PCR (Bio -Rad, Marnes-la-Coquette, France) and the LightCyclerR 480 Probes Master Mix (Roche Diagnostics, Indianapolis, USA) for the presence of the following bacterial microorganisms (Rickettsia spp., Bartonella spp., Borrelia spp., Coxiella burnetii and Anaplasma spp.) using specific primers and probes listed in Diarra et al. (2020) and Ouarti et al. (2020).
The qPCR reaction mixture for each plate has been used according to the manufacturer's protocol. Negative controls were used in each qPCR and consisted of DNA extracted from uninfected Rhipicephalus sanguineus ticks from the laboratory colony of IHU -VITROME (Marseille, France). Positive controls included DNA extracted from a dilution of cultured strains of Borrelia crocidurae, Bartonella henselae, Rickettsia montanensis, Ehrlichia canis and Coxiella burnetii (Socolovschi et al. 2012b;Aouadi et al. 2017). Results were deemed positive if the cycle threshold (Ct) value obtained by CFX96 was lower than ≤ 35 (Ouarti et al. 2020).

Bacterial species identification using sequencing
All ticks tested positive for Anaplasma spp., Borrelia spp. and Rickettsia spp. in qPCR were subjected to amplification using standard PCR prior to sequencing for identification of the bacterial species (Dahmani et al. 2015b;Diarra et al. 2017). In standard PCR the primers used to amplify the ompA gene were (f-70 and r-701). For the sequencing we used the following primers (f-70, f-180 and r-701) which amplify (629 to 632 bp) of protein A (Table 1) (Socolovschi et al. 2012a).

Results
A total of 232 adult ticks (63 H. inermis males and 110 females, 31 D. reticulatus males and 28 females) were collected from vegetation in the protected zone of the Slovak Karst National Park. The proportion of ticks examined are detailed in (Table 2).
The 43 ticks positive for Rickettsia in qPCR were then subjected to standard PCR amplification.
A total of 13/43 (30.23 %) ticks were successfully sequenced. BLAST analysis of these 13 sequences obtained showed an identity percentage ranging from 97.98 to 100 % with the R. raoultii sequence. Details are given in Table 3. No Borrelia spp. or Anaplasmataceae sequences could be amplified.

Discussion
The investigated locality (Slovak karst near the village of Hrhov) is specific and unique due to the joint occurrence of seven tick species, Ixodes ricinus (Linnaeus, 1758), I. trianguliceps Birula, 1895, I. frontalis (Panzer, 1795), Dermacentor marginatus Sulzer, 1776, D. reticulatus, Haemaphysalis concinna C. L. Koch, 1844 and H. inermis (Černý 1972;Nosek 1972;Bona and Stanko 2013;Heglasová et al. 2020), which is an exceptional phenomenon in the conditions of Central Europe. Haemaphysalis punctata Canestrini & Fanzago, 1878 which was the dominant species in tick communities in the area of Slovak karst in the last century, has not been confirmed there in recent decades. The high concentration of potential vectors (seven tick species) and hosts (cattle, free-living ungulates, several species of rodents and shrews) in the studied locality makes the Slovak karst a unique model for studying the circulation of several species of pathogens in their natural habitat (Nosek 1971;Heglasová et al. 2020). Uniqueness of the site (hereby presented) is from the point of view of the common occurrence of several species of ticks (see above). In terms of uniqueness or differentiation of pathogens in ticks compared to other sites, confirmation of a variety of bacteria in Haemaphysalis spp. which live on a common site with both species the genus Dermacentor is exclusive. In Slovakia, the presence of D. reticulatus has been identified along the rivers in southwestern and south-eastern Slovakia (Nosek 1972). Ticks of the genus Haemaphysalis (H. inermis, H. concinna, H. punctata) are known to have a more focal distribution (Černý 1972;Nosek 1973). In previously published studies from the Slovak karst area, we confirmed several species of rickettsiae (R. slovaca, R. helvetica, Rickettsia felis, Rickettsia sp.) and Borrelia miyamotoi in small mammals (especially in rodents), from rodent-attached ticks, as well as from questing ticks sampled from vegetation (Radzijevskaja et al. 2015;Heglasová et al. 2018). There is a characteristically high concentration of deer in the area, as well as cattle grazing on the pastures and both vertebrate groups are hosts for adult ticks. These factors increase the significance of the study area from an epidemiological point of view (Černý 1972).
Here, we demonstrated the circulation of R. raoultii in the studied area. Although R. raoultii has been already detected in D. reticulatus and in patients bitten by D. reticulatus (Parola et al. 2009), the vector competence of D. marginatus and H. inermis to transmit R. raoultii cannot be confirmed, because the detection of this bacterium in the latter could be the consequence of a bacterial blood meal of these ticks. Therefore, additional epidemiological studies and experimental models will be needed.
Both Dermacentor and Haemaphysalis ticks from which confirmed positive cases of Rickettsia originate are typical species for forest-steppe zones (D. marginatus, H. inermis), alluvial forests and wet meadows, which are commonly seen biotopes in eastern Slovakia (D. reticulatus, H. concinna). The joint occurrence of steppe landscape and xerothermic tick species together with ticks that prefer humid habitats in the studied area is probably caused by the presence of ponds and canals in the karst area as well as by the high density of hosts (birds, small mammals, wild ungulates, cattle) for all life stages of ticks. Dermacentor reticulatus was described as harbouring more bacteria than D. marginatus (Zhang et al. 2019). Rickettsia raoultii is usually associated with Dermacentor spp. (Špitalská et al. 2012; Švehlová et al. 2014). Sequencing partial ompB genes revealed the presence of R. raoultii in the larvae and nymphs of D. reticulatus ticks in Germany (Schmuck et al. 2020). In recent decades, the spread of D. reticulatus to new territories has been confirmed, having been reported in, for example, in Poland (Kiewra and Czulowska 2013;Mierzejewska et al. 2016), Germany (Dautel et al. 2006) as well as other European countries (Földvári et al. 2016;Kjaer et al. 2019;Capligina et al. 2020). One study carried out in Latvia detected the presence of R. raoultii in D. reticulatus, their results corroborate with our study. Experimental models will therefore be necessary to understand the role of D. reticulatus in the appearance of R. raoultii (Capligina et al. 2020).

Conclusions
This molecular study supports the data that R. raoultii could be a source of lymphadenopathy in Slovak populations. Further studies, including experimental models, will be needed to assess the role of D. reticulatus and H. inermis in the transmission of R. raoultii. Nevertheless, based on the positive detection of R. raoultii, we assume that Haemaphysalis and Dermacentor spp. should be considered as potential reservoirs of R. raoultii in Slovakia.
Acknowledgements The authors thank Ladislav Mošanský for his help with tick collection.
Author Contributions ZS, PP and OM conceived and designed the research. BO, BE and ML conducted the experiments. MS, and ZS contributed analytical tools. BO and BE analysed the data. BO and ZS wrote the manuscript. All authors read and approved the manuscript.

Declarations
Conflict of interest The authors have no conflicts of interest to disclose.
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