First detection and molecular identification of Rickettsia massiliae, a human pathogen, in Rhipicephalus sanguineus ticks collected from Southern Taiwan

The Rickettsia massiliae was firstly detected and identified in Rhipicephalus sanguineus ticks infested on dogs in Taiwan. A total of 1154 Rh. sanguineus ticks collected from 158 dogs of four districts of Tainan city were examined for Rickettsia infection by nested-PCR assay targeting the citrate synthase (gltA) and outer membrane protein B (ompB) genes of Rickettsia. The Rickettsia infection was detected with a general infection rate of 2.77%, and was detected in male, female and nymphal stage with an infection rate of 2.77%, 3.22% and 1.32%, respectively. Phylogenetic relationships were analyzed by comparing the gltA and ompB sequences obtained from 9 Taiwan strains and 16 other strains representing 13 genospecies of Rickettsia. Results revealed that all Taiwan strains were genetically affiliated to the same clades of R. massiliae (spotted fever group) and R. felis (transitional group), and can be discriminated from other genospecies of Rickettsia. This study provides the first evidence of R. massiliae, a pathogenic spotted fever Rickettsia, identified in Rh. sanguineus ticks and highlight the potential threat for the regional transmission of Rickettsia infection among humans in Taiwan.


Introduction
The Rhipicephalus sanguineus tick is a haematophagus arthropod and is observed as the most common ectoparasite of dogs around the world [1,2]. Previous investigations demonstrated that the existence of at least two distinguished groups (tropical vs temperate lineage) of Rh. sanguineus sensu lato ticks based on the genetic comparison of 16S mitochondrial rRNA gene [3,4]. In addition, Rh. sanguineus has been attributed to the main vector for the transmission of Babesia, Ehrlichia, and Rickettsia among humans and animals [5][6][7]. Due to the increasing detection of B. vogeli, B. gibsoni, Anaplsma platys, R. felis and canine babesiosis in Taiwan [8][9][10][11][12], the medical and veterinary importance of Rh. sanguineus ticks raise the research attention on this tick species. Although the Rh. sanguineus ticks had been identified as the potential vector ticks for a variety of pathogens in Taiwan, there has no research confirming the genetic identification of Rickettsia massiliae, a pathogenic strain for human infection, in this tick species in Taiwan.
The genus Rickettsia composed of approximately 27 species of obligate intracellular gramnegative bacteria that can be classified into four major groups and the spotted fever group (SFG) is responsible for the most Rickettsia infections in humans [13][14][15]. Various arthropods including tick, flea, mite and louse, may serve as vector for Rickettsia transmission. However, the Ixodid ticks may serve as the primary vectors and reservoirs of amplifying hosts for Rickettsia agents [16]. Except the flea-borne R. felis and mite-borne R. akari, the SFG rickettsiae are mainly transmitted by vector ticks and some of these ticks can transmit the Rickettsia agents through the transovarial and transstadial pathways [17]. During the past decades, Rickettsia infections become a global threat of emerging tick-borne diseases [18,19], and the R. massiliae and R. felis have been identified as the pathogenic strains for infecting humans [20][21][22][23][24][25][26]. In addition, many validated SFG rickettsial species have been discovered in Australia, Central and South America, and Asia [27][28][29][30][31][32][33][34][35][36][37]. In Taiwan, human serosurvey of Rickettsia infections had been attributed to the agent of R. felis in southern Taiwan [38]. However, there is no confirming evidence of human isolate and vector tick that is responsible for the transmission of Rickettsia infections in Taiwan. Thus, epidemiological survey on tick-borne rickettsiae in Rh. sanguineus ticks is crucial to understand the potential threat of emerging tick-borne Rickettsia infections in Taiwan.
Molecular approach based on the genetic variance at the individual base-pair level gives much more direct pathway for measuring the genetic diversity between and within species of Rickettsia [39,40]. Previous studies based on the molecular marker of citrate synthase (gltA) and outer membrane protein B (ompB) genes have concluded that it is sufficiently informative for the analysis of evolutionary relationships between the genetic diversity of Rickettsia species among various vectors and hosts [31][32][33][34][35][36][37][38][39][40]. Thus, molecular detection and genetic analysis based on the phylogenetic analysis of gltA and ompB genes have made possible in facilitating the identification and discrimination of Rickettsia species within ticks.
It may be that the Rickettsia infection in Rh. sanguineus ticks of Taiwan is genetically distinct genospecies, as compared with the existing common genospeciess of Rickettsia around the world. Thus, the objectives of this study intend to investigate the prevalence of Rickettsia infection in Rh. sanguineus ticks collected from Taiwan and to determine the phylogenetic relationships between and within the genospecies of Rickettsia in these ticks. The genetic affiliation of Rickettsia strains detected in Rh. sanguineus ticks of Taiwan was analyzed by comparing their differential nucleotide composition with other Rickettsia strains identified from various biological and geographical sources which have been documented in GenBank.

Tick collection and species identification
All specimens of Rh. sanguineus ticks used in this study were collected from 158 dogs of four districts of Tainan city that include Yong-Kang (YK), Ren-De (RD), East District (ED) and South District (SD) in southern Taiwan (Fig 1). All these dogs were handled by veterinary practitioner for collecting the attached ticks. All these ticks were subsequently cleaned and stored in separate glass vials containing 75% ethanol. All tick specimens of Rh. sanguineus were identified to the species level on the basis of their morphological characteristics [2,41] and genetic identification was also performed based on the mitochondrial 16S rRNA gene, as described previously [9]. Briefly, the external features of the Rh. sanguineus ticks were recorded by using a stereo-microscope (SMZ 1500, Nikon, Tokyo, Japan) equipped with a fiber lamp and photographed for species identification.

DNA extraction from tick specimens
Total genomic DNA was extracted from individual tick specimens used in this study. Briefly, tick specimens were cleaned by sonication for 3-5 min in 75% ethanol solution and then washed twice in sterile distilled water. Afterwards, the individual tick specimen was homogenized in a microcentrifuge tube filled with 180-μL lysing buffer solution (DNeasy Blood &Tissue Kit, catalogue no. 69506, Qiagen, Taipei, Taiwan) and then homogenized with a TissueLyser II apparatus (catalogue no. 85300, Qiagen, Germany), instructed by the manufacturer. The homogenate was centrifuged at room temperature and the supernatant fluid was further processed by a DNeasy Blood & Tissue Kit (catalogue no. 69506, Qiagen, Taipei, Taiwan), as instructed by the manufacturer. After filtration with the kit, the filtrated fluid was collected and the DNA concentration was determined spectrophotometrically with a DNA calculator (Epoch, Biotek, USA) and the extracted DNA is stored at -80˚C for further investigations [9].

DNA amplification by nested polymerase chain reaction
DNA samples extracted from each tick specimens were used as a template for PCR amplification. Two primer sets based on the citrate synthase gene (gltA) were used for amplification. Initially, the primer set of RpCS.877p (5'-GGGGGCCTGCTCACGGCGG-3') and RpCs.1258n (5'-ATTGCAAAAAGTACAGTGAACA-3') was used to amplify the primary product of gltA. Nested PCR was then performed using the species-specific primer sets: RpCS.896p (5'-GGCTAATGAAGCAGTGATAA-3') and RpCS.1233n (5'-GCGACGGTATACCCATAGC-3') for amplifying a product approximately 338-bp [42]. All PCR reagents and Taq polymerase were obtained and used as recommended by the supplier (Takara Shuzo Co., Ltd., Japan). Briefly, each 25-μl reaction mixture containing 3-μl DNA template, 1.5-μl forward and reverse primers, 2.5-μl 10X PCR buffer (Mg 2+ ), 2-μl dNTP mixture (10 mM each), 1 unit of Taq DNA polymerase and filled-up with adequate volume of ddH 2 O. In contrast, adequate amounts of sterile distilled water were added for serving as a negative control. PCR amplification was performed with a thermocycler (Veriti, Applied Bioosystems, Taipei, Taiwan) and was denaturation at 95˚C for 5 min and then amplified for 35 cycles with the conditions of denaturation at 95˚C for 30 sec, annealing at 54˚C for 30 sec, extension at 72˚C for 1 min, and followed by a final extension step at 72˚C for 3 min. For the nested-PCR, the following conditions were used: denaturation at 95˚C for 5 min and then amplified for 40 cycles with the conditions of denaturation at 95˚C for 30 sec, annealing at 50˚C for 30 sec, extension at 72˚C for 1 min, and followed by a final extension step at 72˚C for 3 min.
All amplified PCR products were electrophoresed on 1.5% agarose gels in Tris-Borate-EDTA (TBE) buffer and visualized under ultraviolet (UV) light after staining with ethidium bromide. A 100-bp DNA ladder (GeneRuler, Thermo Scientific, Taiwan) was used as the standard marker for comparison. A negative control of distilled water was included in parallel with each amplification.

Sequence alignments and phylogenetic analysis
Approximately 10-μl of each selected samples with clear bands on the agarose gel was submitted for DNA sequencing (Mission Biotech Co., Ltd., Taiwan). After purification (QIAquick PCR Purification Kit, catalog No. 28104), sequencing reaction was performed with 25 cycles under the same conditions and same primer set of nested amplification by dye-deoxy terminator reaction method using the Big Dye Terminator Cycle Sequencing Kit in an ABI Prism 377-96 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The resulting sequences were initially edited by BioEdit software (V5.3) and aligned with the CLUSTAL W software [43]. Thereafter, the aligned sequences of Rickettsia gltA gene from 9 Taiwan strains were analyzed by comparing with other 16 strains of Rickettsia sequences from the different biologiical and geographical origin that are available from GenBank. Further analysis based on ompB gene of 8 Taiwan strains belonging to the SFG Rickettsia was performed by comparing with other 15 strains of Rickettsia sequences documented in GenBank. Phylogenetic analysis was performed by neighbour-joining (NJ) compared with maximum likelihood (ML) methods to estimate the phylogeny of the entire alignment using MEGA X software package [44]. The genetic distance values of inter-and intra-species variations were also analyzed by the Kimura two-parameter model [45]. All phylogenetic trees were constructed and performed with 1000 bootstrap replications to evaluate the reliability of the construction, as described previously [46].

Ethical approval
The collection of ticks from dogs was assistant by veterinary practitioners and approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (IACUC Approval No: 106142).

Detection of Rickettsia infection in Rh. sanguineus ticks
The existence of Rickettsia in Rh. sanguineus ticks was detected by nested PCR assay targeting the gltA and ompB genes (Fig 2). In general, a total of 2.77% (32/1154) Rh. sanguineus ticks were detected with Rickettsia infection by targeting the gltA gene. Further analysis of 32 gltA positive specimens by targeting the ompB gene revealed 19 positive Rickettsia detection and only 8 specimens were positive detected by both gltA and ompB genes. According to the life stage of ticks, the Rickettsia infection was detected in males, females and nymphs of Rh. sanguineus ticks with an infection rate of 2.77%, 3.22% and 1.32%, respectively ( Table 1). The highest geographical prevalence of Rickettsia infection was detected in Yong-Kang (4.26%) and East District (3.87%).

Sequence alignment and genetic analysis of Rickettsia in Rh. sanguineus ticks
To clarify the genetic identity of Rickettsia in Rh. sanguineus ticks of Taiwan, the sequences of gltA and ompB gene fragments from 9 Taiwan strains of Rickettsia performed by this study were aligned and compared with the downloaded sequences of 16 other Rickettsia strains from different biological and geographical origin documented in GenBank. Results indicate that all these Rickettsia strains detected in Rh. sanguineus ticks of Taiwan were genetically affiliated to the genospecies of R. massiliae and R. felis with the highly sequence similarity of 99.01-99.68% and 99.01-99.67% respectively ( Table 2). In addition, intra-and inter-species analysis based on the genetic distance (GD) values of gltA gene indicated a lower levels (GD<0.013 and <0.059 for R. massiliae and R. felis) of genetic divergence within the Rickettsia strains of Taiwan, as compared with the type strain of R. massiliae and R. felis, respectively (Table 3). Further analysis based on the GD values of ompB gene indicated a lower levels (GD<0.013 and <0.052 for R. massiliae and R. felis) of genetic divergence within the Rickettsia strains of Taiwan, as compared with the type strain of R. massiliae and R. felis, respectively ( Table 3).

Nucleotide sequence accession numbers
The nucleotide sequences of PCR-amplified gltA gene of 9 Rickettsia strains from Rh. sanguineus ticks of Taiwan determined in this study have been registered and assigned the following    Table. Phylogenetic analysis of Rickettsia detected in Rh. sanguineus ticks Phylogenetic relationships based on the sequence alignment of gltA and ompB genes were performed to demonstrate the genetic relationships among 28 and 23 strains of Rickettsia investigated in this study, respectively. Phylogenetic trees constructed by neighbour-joining (NJ) and maximum likelihood (ML) methods were used to analyze the phylogenetic relationships of Rickettsia strains. Bootstrap analysis was used to analyze the repeatability of the clustering of specimens represented in phylogenetic trees. Results showed congruent basal topologies with nine major clades of Rickettsia that can be easily distinguished by gltA analysis (Fig 3A and 3B) and were congruent by ompB analysis (Fig 4A and 4B). In general, all these Rickettsia strains from Taiwan constitute a monophyletic clade closely affiliated to the genospecies of R. massiliae and R. felis, respectively. These results reveal a lower genetic divergence within the same genospecies of Rickettsia detected in Rh. sanguineus ticks from Taiwan, but a higher genetic variations from other group of Rickettsia detected in different biological and geographical origins.

Genetic identification of Rh. sanguineus ticks collected from southern Taiwan
Molecular analysis of Rh. sanguineus ticks based on the mitochondrial 16S rRNA gene was performed to demonstrate the genetic relationships among 12 Tainan strains of Rh. sanguineus Results showed that all the Tainan strains of Rh. sanguineus ticks are genetically affiliated with the tropical lineage of Rh. sanguineus sensu lato and can be distinguished from other tick species (Fig 5). The submitted sequences of 16S mitochondrial gene were assigned the GenBank accession numbers: ON951615-620 and ON951652-657.

Discussion
This study provides the first molecular detection and genetic identification of R. massiliae in Rh. sanguineus ticks of Taiwan. In previous studies, the R. massiliae was firstly isolated from a patient in Sicily of Italy [20] and further identified in patients from southern France and Argentina [21,22]. In addition, R. massiliae was first isolated from Rhipicephalus ticks in Marseilles [47] and is commonly found in Rh. sanguineus or Rh. turanicus ticks from France, Greece, Spain, Portugal, Switzerland, Central Africa, and Mali [48,49]. In this study, Rickettsia species detected in Rh. sanguineus ticks from Taiwan are genetically affiliated to the genospecies of R. massiliae and R. felis, and these detections are different from the previous studies which described a R. honei-like organism in Ixodes granulatus ticks identified from Thailand, Taiwan and Japan [31,32,37]. Indeed, our study provides the first evidence of R. massiliae detected in Rh. sanguineus ticks of Taiwan (Figs 3 and 4) which is considered as an emerging tick-borne pathogen of Spotted Fever Group Rickettsia. However, the role of R. massiliae in public health needs to be further investigated. Thus, our study provides the first molecular evidence and convincing sequences of R. massiliae in Rh. sanguineus ticks of Taiwan based on gltA (GenBank accession numbers: ON093122-093130) and ompB (GenBank accession numbers: ON093131-093138) genes. The actural mechanism for the transmission of flea-borne R. felis into Rh. sanguineus ticks remains elusive. It is possible that the horizontal transmission of Rickettsia was occurred between tick-flea interaction. Indeed, ticks may accidentally feed on dogs that were previously fed by infected fleas, and these ticks may acquire the Rickettsia infection through feeding blood from the same parasitized dogs [50]. Another possible mode of transmission by co-feeding mechanism may also contributed to the ticks feeding closely with another infected flea on the same host that may facilitate the transmission of pathogens from an infected vector to a new vector [51]. In addition, detection of Rickettsia DNA in ticks may not actually demonstrate the viable bacteria in these ticks and also unable to discriminate the source of Rickettsia from infested ticks or from blood of dogs. Because of the close contact of dogs with humans, these observations may highlight the epidemiological significance of dogs serving as carrier host for the Rickettsia transmission to human population.
Phylogenetic relationships among Rickettsia in Rh. sanguineus ticks can be determined by analyzing the sequence homogeneity of the gltA and ompB genes of Rickettsia. Indeed,

PLOS NEGLECTED TROPICAL DISEASES
First detection of Rickettsia massiliae in Rhipicephalus ticks of Taiwan sequence analysis based on the gltA and ompB genes of Rickettsia strains among various species from different origins had been shown to be useful for evaluating the genetic relatedness of Rickettsia detected from various biological and geographical sources [13,[28][29][30][31][32][33][34][35][36][37]. In this study, the phylogenetic analysis based on the sequences of gltA and ompB genes from Rh. sanguineus ticks of Taiwan demonstrated a highly genetic homogeneity affiliated to the genospecies of R. massiliae and R. felis (Figs 3 and 4). The R. massiliae strains from Taiwan are mainly associated with the Rickettsia strains identified from human patients (GenBank accession no. KJ663741 and KJ663753) and the R. felis strains are mainly affiliated to the Rickettsia strains from cat flea and lice (GenBank accession no. MG893575 and MG818715). In addition, the genetic analysis based on gltA also revealed the discrimination of R. massiliae from the clades composed by R. honei, R. conorii and R. rickettsia as well as by R. japonica, R. parkeri and R. sibirica. The phylogenetic trees constructed by either NJ or ML analysis strongly support the discrimination between the Rickettsia strains in Rh. sanguineus ticks collected from Taiwan and other genospecies of Rickettsia from different geographic and biological origins. Thus, the genetic identities of Rickettsia strains detected in Rh. sanguineus ticks of Taiwan were verified as a monophyletic group affiliated to the spotted fever (R. massiliae) and transitional (R. felis) groups of Rickettsia.
The global climate change may also increase the geographical expansion of ticks that will enhance the transmission of tick-borne pathogens [52]. Indeed, a previous 10-year study on rickettsias conducted in Germany demonstrated that the Rickettsia infection rate was significantly increased over the years from 33.3% in 2005 to 50.8% in 2015 [53]. In addition, the previous study also discover that the I. ricinus tick is reported to have spread into the previously unidentified northern areas of Sweden, Finland and Norway [54]. Because the Rh. sanguineus ticks are mainly parasitized on dogs that are living closely with residential area of humans. There is a serious concern regarding whether the Rickettsia species within this tick species can be transmitted to humans. Thus, further studies focused on the geographical identification of vector ticks and genetic diversity of tick-borne Rickettsia may help to illustrate the spread of vector ticks and the risk of transmission of tick-borne rickettsial infections in Taiwan.