First Molecular Detection of Human Pathogen Rickettsia Raoultii in Ticks from Republic of Korea

Background: Rickettsial diseases, associated with the spotted fever group (SFG), constitute a growing number of newly identied rickettsia pathogens and their tick vectors, in various parts of the world. At least 15 distinct tick species owing to six genera have shown the presence of Rickettsia raoultii. Here, we report the detection of R. raoultii in ticks from the Republic of Korea (ROK). Methods: A total of 35 ticks, collected from patients of tick bites in Gwangju Metropolitan City, Jeollanam Province, ROK. The ticks were were identied through their molecular, morphological, and taxonomic characteristics. All samples were screened by nested polymerase chain reactions of their outer membrane protein (ompA) and citrate synthase (gltA) genes. The amplied products were sequenced and their phylogenetic analyses were carried out. Results: Sequencing data showed that the DNA sequences of R. raoultii found in the three H. longicornis ticks. All 3 tick samples were 99.4-100% analogous to the previously reported partial sequences of ompA of R. raoultii strains CP019435 and MF002523, forming a single clade with the reference strains. Conclusions: Present study provides the rst description and molecular identication of R. raoultii detected in H. longicornis ticks in ROK. This observation extends the geographical distribution of R. raoultii. Screening of human samples for this pathogen will provide information about the prevalence of rickettsial infection in this region.


Background
Tick-borne diseases are a growing medical concern worldwide. Ticks are considered as the main reservoir and vectors of rickettsia, a set of obligately intracellular bacteria, and responsible for transmission of rickettsial diseases to humans. This zoonosis represents both the oldest known and recently identi ed vector-borne infectious disease [1]. The causative agents belong to the genus Rickettsia and are presently classi ed into four groups: the spotted fever group (SFG), the typhus group, the Rickettsia bellii, and the Rickettsia canadensis group [2]. SFG rickettsiae constitutes of newly identi ed Rickettsia species around the world. In the last few decades, numerous species of tick-borne rickettsiae, previously thought to be non-pathogenic, are recognized as human pathogens [2].
In 1999, three novel rickettsial genotypes, RpA4, DnS14, and DnS28 were recognized in ticks from Russia [3]. Consequently, with genotypic and phenotypic analysis of their characteristics, these bacteria were recognized as novel species of SFG rickettsiae, and in 2008, these were named Rickettsia raoultii [4].The major clinical manifestations of R. raoultii include scalp eschar and neck lymphadenopathy (SENLAT). Initially, these were called as dermacentor borne necrosis erythema and lymphadenopathy (DEBONEL) or tick-borne lymphadenopathy (TIBOLA) [2]. To date, R. raoultii has been identi ed in many Asian and European countries [5][6][7][8]. In 1999, Dermacentor nuttalli and Rhipicephalus pumilio ticks collected from the southern part of the former Soviet Union were shown to carry these bacteria [3], and thereafter, other species of Dermacentor ticks (i.e., D. reticularis, D. marginatus, D. silvarum, and D. niveus) from various parts of the former Soviet Union, France, Spain, and Germany, were also reported to harbor them [5,9]. Subsequently R. raoulttii were detected in other hard ticks too, such as Haemaphysalis, Rhipicephalus, Hyalomma, and Ambylomma, predominantly in Europe and Asia [10].
The aim of this study was to determine the existence of R. raoultii in ticks, and to examine the presence and circulation of this pathogen in tick populations in ROK. We found R. raoultii in Haemaphysalis longicornis ticks. To the best of our knowledge, this is the rst molecular evidence of R. raoultii in ticks from the ROK.

DNA extraction
Total genomic DNA was extracted from 150 µL of the tick homogenate, using a QIAamp Tissue & Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, and eluted in 50 µL volume.
The samples were stored at -20 °C until their polymerase chain reaction (PCR) ampli cation.

PCR ampli cation
To analyze the presence of Rickettsia species in ticks, the bacterial DNA samples were subjected to nested PCR (N-PCR), designed to target the outer membrane protein A (ompA) and citrate synthase (gltA) genes. The primers used for the reactions and the product sizes are shown in Table 1. The reactions were carried out in 20 µL volume, comprised of 16 µL of distilled water, 1 µL of each primer (10 pmol/µL), and 2 µL of genomic DNA template using AccuPower® PCR PreMix (Bioneer Corp., Korea). PCR was carried out using AB thermal cycler (Applied Biosystems, Foster City, CA, USA). A positive control with R. conorii DNA, and negative control with distilled water instead of template DNA were also included in each set of PCR. The ampli ed products were visualized by electrophoresis on a 1.2% agarose gel containing ethidium bromide.

Results
The molecular, morphological, and taxonomic characteristics revealed that 4 of the 35 ticks were Ixodes nipponensis, 14 were Amblyomma testudinarium, and 17 were Haemaphysalis longicornis ( Table 2). PCR products were ampli ed from 35 ticks for the identi cation of SFG rickettsial disease, targeting the ompA and gltA genes. Sequencing data of positive samples, targeting the ompA gene, revealed one distinct rickettsial species in three of the H. longicornis ticks, which were identi ed as R. raoultii. Morphological and taxonomic characteristic showed the these ticks were adult female. Conversely, the results of PCR targeting the gltA gene did not detect any distinct rickettsial species. Even though these three ticks were harvested from patients, PCR results of blood samples obtained from respective patients did not show the R. raoultii infection nor did they show any symptoms speci c to such an infection. R. raoultii sequence of the ompA gene from all 3 tick samples were 99.4-100% homologous to the previously reported partial sequence of ompA from R. roultii IM-16 strain isolated from CP019435 and MF002523. In the phylogenetic analyses, neighborhood-joining tree of rickettsial species indicated that the present isolates formed a single clade with R. raoultti reference strains (Fig. 1). The bootstrap analyses statistically supported the main clustered sequence.

Discussion
Tick-borne rickettsiosis results from a member of genus Rickettsia, of the family Rickettsiaceae and the order Rickettsiales [2]. Clinically, SFG rickettsioses are associated with headache, pyrexia, myalgia, localized lymphadenopathy, characteristic eschar, and rash. Following the tick bite, R. raoultii has known to be presents with a clinical syndrome characterized by scalp eschars and neck lymphadenopathy. In 2010, to collectively explain this clinical manifestation, the syndrome was termed as "SENLAT" [14].  [15,16]. A report has also shown its presence in Melophagus ovinus, a louse y or sheep ked [17]. Dermacentor ticks are considered as the main hosts and natural reservoirs of R. raoultii all over Europe and in a few countries of Asia, including China and Mongolia [18].
In the ROK, the rst evidence of the existence of SFG rickettsia in ticks was reported in 2003, followed by the rst case of SFG rickettsioses (Japanese spotted fever) in a patient in 2005 [19,20]. Over the period of 16 years, various species of SFG rickettsial agents from ticks (R. japonica, R. monacensis, and R .rickettsii) and humans (R. japonica and R. monacensis) have been identi ed in the ROK [19][20][21][22][23][24][25] Till date, R. raoultti was not identi ed in this region, and the present study reports the rst detection of R. raoultii belonging to the genus H. longicornis. Previously, only one study from China indicated the presence of R. raoultii in H. longicornis ticks [16]. The strains of R. raoultii, including Marne, 8/9 Karaganda, Khabarovask T , Shayman, and IM 16, have been documented in Europe, Russia, and China [4,9]. The present phylogenetic tree showed that the positive samples formed a distinct clade with high (100) bootstrap value with R. raoultii IM 16 strain found in China. The nucleic acids of R. japonica, R. monacensis, and R. rickettsii have been identi ed in H. longicornis from ROK [19,23]. According to a recent study, H. longicornis ticks are the most prevalent species in ROK (88.9%) with its geographical distribution nationwide [25]. Despite its identi cation in multiple tick species, reports on human infections are still lacking. The infection of R. raoultii in patients has been reported in Europe, the Far East of Russia, and a few cases in China [26]. Another study from China [18] identi ed R. raoultii DNA in clinical samples, besides the positive serological reports in patients from other countries. Based on these ndings, R. raoultii has been considered as a human pathogen [27].
Present observations indicate the emergence of R. raoultii in ticks from ROK and warrant additional survey for this pathogen in a broader range of ticks. Furthermore, public health providers and physicians should use diagnostic tests of R. raoultii in patients of suspected rickettsioses. A special drive should be undertaken to investigate the presence of this emerging pathogen in human cases

Conclusions
This study con rms the rst identi cation of R. raoultii in ticks from the ROK. Detection of Rickettsial specie in H. longicornis ticks, suggests that these ticks may act as a potent vector for this pathogen in ROK. This observation broadens our knowledge of the geographical spread of R. raoultii. Although no human clinical infection has been reported, its relatively strong pathogenicity is a substantial threat and a major concern for public health in this region. More extensive research and surveillance in this regard are essential. Availability of data and materials:

List Of Abbreviations
The datasets analyzed during the current study are available in the GenBank-database with the accession numbers CP019435 and MF002523.
Competing interests: The authors declare that they have no competing interests. Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or nonpro t sectors.
Authors' contributions: Dong-Min Kim designed and coordinated the study and contributed to drafting and reviewing the manuscript during the course of submission. Misbah Tariq and Jun-Won Seo collected the data, wrote the manuscript, and revised the draft during the course of submission. You Mi Lee constructed the phylogenetic tree and performed the molecular analysis. Choon-Mee Kim carried out the molecular analysis, wrote the manuscript, and also revised the draft during the course of submission. Na Ra Yun and Da-Young Kim helped to draft the manuscript. All the authors read and approved the nal version of the manuscript.