Novel spotted fever group rickettsiae in Haemaphysalis qinghaiensis ticks from Gansu, Northwest China

Rickettsia spp. are obligate intracellular bacteria and well known as transmitted by arthropods. These pathogens have a broad geographic distribution and a high degree of biological and clinical diversity. This study was conducted to determine the prevalence and molecular characterization of Rickettsia spp. in ticks collected from Gansu, where Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum were previously reported in ticks and ruminants. A total of 1,583 questing Haemaphysalis qinghaiensis ticks were collected and tested for the presence of Rickettsia spp. gltA gene by PCR. Samples positive for gltA were examined by specific primers targeted for the ompA gene of SFG rickettsiae. The infections were further validated by sequencing and positive samples were genetically characterized based on the gltA and ompA genes. In total, Rickettsia spp. infection was found in 179 (18.5 %) H. qinghaiensis tick pools by using PCR and primers specific for the gltA gene. Of those, 157 (16.3 %) tick pools were positive for SFG rickettsiae by PCR based on ompA gene. Amplification and molecular analysis of the nucleotide sequences of gltA and ompA genes indicated three potential novel spotted fever group rickettsiae in H. qinghaiensis ticks. These three potential novel spotted fever group rickettsiae were clustered together in a subgroup, which represents a sister taxon to and separates from other known four SFG rickettsiae subgroups. This study revealed a high infection rate of SFG rickettsiae in H. qinghaiensis ticks in northwest China. Three potential novel spotted fever group rickettsiae classified into a novel SFG rickettsiae subgroup were identified and named “Candidatus Rickettsia gannanii” related strains in recognition of the location where it was first detected.


Background
Rickettsiae are highly specialized obligate intracellular gram-negative microorganisms that can cause disease in human and/or in other vertebrate and invertebrate hosts with diverse clinical presentations, from asymptomatic to severe [1]. Recently, rickettsioses have been viewed as emerging or reemerging diseases with an almost worldwide distribution [2,3]. Rickettsia was shown to be a large genus encompassing at least 30 recognized species, 19 of which were considered to be human pathogens [4,5]. A number of other putative species of Rickettsia have also been reported on the basis of phylogenetic analyses of different gene loci [5][6][7][8]. Rickettsia spp. are usually associated with arthropods, and ticks, louse, mites and fleas had been recognized as competent vectors of rickettsial agents [9][10][11][12]. Recently, mosquitoes were considered as potential vectors for R. felis [13]. In addition, numerous Rickettsia spp. or specific DNA have been detected in arthropods other than the aforementioned vectors [2], such as booklice, true bugs, white fly, etc.
Historically, most of the recently discovered pathogenic Rickettsia spp. were first identified in arthropods and much later were reported in human cases, such as R. parkeri and R. slovaca. The former was identified in Amblyomma maculatum in 1937, and it was not until 60 years later that the first human case of R. parkeri infection was described [14]; the latter was described in Dermacentor marginatus in 1968, but the first documented case was reported several years later [1]. Recently, with the development of molecular techniques, new Rickettsia spp. have been identified in places where no rickettsioses had been reported, and some of these have been recognized as human pathogens [15]. The novel identified rickettsial species have enriched our understanding of rickettsioses. The objective of this study was to identify the rickettsial species in ticks collected from a specific area of Qing-Tibetan Plateau that had not been previously described for rickettsioses.

Study site and tick collection
The study site is located on the northeast edge of the Qing-Tibetan Plateau with an important forest zone and the main pasturing area in Gannan Tibetan Autonomous Prefecture (33°06"~36°10"N, 100°46"~104°44"E) that relies heavily on sheep, goat and yak farming for protein and local economy. Its average altitude is over 3,000 m. The annual average temperature here is 3.1°C and annual precipitation is 582.7 mm. A total of 1,583 questing ticks were collected monthly between March and May 2011 by flagging the undergrowth with a flannel cloth. All ticks were identified as Haemaphysalis qinghaiensis based on the taxonomic key and morphological criteria [16].

DNA extraction
Tick larvae and nymphs were pooled before DNA extraction; each tick pool consisted of 10 larvae or 5 nymphs, or a single adult tick. DNA was extracted by using the Puregene DNA purification kit (Qiagen, Beijing, China) according to the protocols.

Sequencing and phylogenetic analyses
The PCR products of the partial gltA and ompA gene were purified using the TaKaRa Agarose Gel DNA purification Kit Ver.2.0 (TaKaRa, Dalian, China), ligated into pGEM-T Easy vector (Promega, USA), and transformed into Escherichia coli JM109 competent cells. Two recombinant clones were selected for sequencing using BigDye Terminator Mix (Sangon, Shanghai, China). The sequences obtained in this study were deposited in the GenBank (not including identical sequences) under accession nos. KT921891-KT921896. Sequences were analyzed by a BLASTn search in GenBank or by using the Clustal W method in the MegAlign software (DNAStar, Madison, WI). Phylogenetic trees were then based on the sequence distance method using the neighbor-joining (NJ) algorithm with the Kimura twoparameter model of the Mega 4.0 Software [19].

Statistical analysis
The infection rates of Rickettsia infection in H. qinghaiensis ticks of larval and nymphal stages were estimated using the minimum infection rate [MIR, (the number of positive pools/the total number of ticks tested) × 100 %]. The results were analyzed using a Chi-square test in Predictive for Analytics Software (PASW) Statistics 18. A difference was considered statistically significant at P < 0.05.

Ethical approval
This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Results
The DNA of Rickettsia spp. was found in 179 (18.5 %) H. qinghaiensis tick pools by using PCR and primers specific for the gltA gene ( Seventy-one sequences (36 for gltA and 35 for ompA) were obtained in this study. Nucleotide sequence identities ranged from 97.0 % to 98.4 % for the gltA gene and from 87.8 % to 96.7 % for the ompA gene. After BLAST and CLUSTAL W alignment, 36 sequences of the gltA gene fragment (770 bp) were classified into three sequence types (ST), representing three different Rickettsia spp. isolates. ST1-3 had the highest nucleotide sequence similarities (99.0 %, 97.7 % and 97.4 %) to R. raoultii (GenBank accession no. KT261764). PCR amplification and sequencing showed the target fragments of the ompA gene were 617, 680 and 641 base pair for Rickettsia ST1-3, respectively. The sequence similarity of the ompA gene sequences of ST1-3 with that of R. raoultii was 79.4 %, 81.1 % and 81.4 %, respectively, which did not meet the threshold for R. raoultii [20].

Discussion
Rickettsia spp. have been recognized as emerging or reemerging pathogens of public health relevance [2]. With the completion of the complete genome sequences, new perspectives on rickettsial evolution have been acquired. Apart from the traditional classification based on their morphological, antigenic, and metabolic characteristics, phylogenomic studies showed that the genus Rickettsia was classified into four different groups, including the well-defined SFG and TG, the R. belli group and the R. canadensis group [2,22]. Until now, 27 characterized and dozens of as yet uncharacterized strains had been recognized worldwide [2]. The availability of specific and sensitive molecular tools used for taxonomic purposes have allowed for the identification of new species of Rickettsia in places where no rickettsioses had been reported. In the current study, the prevalence and molecular characterization of Rickettsia spp. was determined in H. qinghaiensis ticks, which was the dominant tick species and mainly recorded in northwestern China [23,24].
In China, several Rickettsia spp. had been identified in ticks from different geographical locations, i.e. R. sibirica subsp. sibirica in D. silvarum and D. sinicus in northern China [8]; R. sibirica subsp. mongolitimonae in H. asiaticum in Inner Mongolia [25]; R. heilongjiangensis in D. silvarum in Heilongjiang and Yunnan [26,27]; R. japonica in H. longicornis in Zhejiang [3]; R. slovaca and R. raoultii in D. silvarum in Xinjiang [28]; R. monacensis in I. persulcatus in central China [29]. Those studies indicate that numerous tick species maintain or transmit Rickettsia. In this study, we first reported the Rickettsia infections in H. qinghaiensis ticks, and high prevalences of Rickettsia (18.5 %) and SFG rickettsiae (16.3 %) infections were observed in study site. The hard ticks H. qinghaiensis, a distinctive species common in the Qing-Tibetan Plateau, is a three-host tick and preferentially infests domestic animals, such as sheep, goats, yaks and cattle [16,23,24]. Previous studies have  [23,24]. Recently, it has also been shown to be naturally infected with Borrelia burgdorferi (s.l.) and Anaplasma phagocytophilum, which are well-known human pathogens in the world [30,31]. Taken together, our results suggest the potential role of H. qinghaiensis ticks as a reservoir host and carrier for piroplasmosis, Lyme disease, anaplasmosis and rickettsioses in this region. The gltA and ompA genes were considered sufficiently variable to determine more reliable phylogenetic relationships within the genus Rickettsia [17,20]. Previous report have demonstrated that SFG rickettsiae could be subdivided into four subgroups: R. rickettsii, R. massiliae, R. helvetica and R. akari [4]. In this study, phylogenetic tree inferred from gltA and ompA gene sequences revealed that three clusters of SFG rickettsiae were identified in H. qinghaiensis and these three different SFG rickettsiae clustered together in a subgroup separate from other known four SFG rickettsiae subgroups (Figs. 1 and 2). on the data presented here, we believe that the subgroup should be strongly considered as a new SFG rickettsiae subgroup, and we formally propose that these be named "Candidatus Rickettsia gannanii" related strains in recognition of the area where it was detected. In this subgroup, the cluster one may be the most dominant SFG rickettsiae distributed in study site (Figs. 1 and 2). Some rickettsiae have been reported to be specifically associated with tick species, which are highly dependent on their biotopes [2]. Previous reports showed that R. conorii conorii seems to associate mainly with Rhipicephalus sanguineus sensu lato, Haemaphysalis leachi and Rhipicephalus simus in the Mediterranean region and Sub-Saharan Africa [2,32]. In this study, three different SFG rickettsiae were identified in H. qinghaiensis ticks. The geographical distribution and associations between the "Candidatus Rickettsia gannanii" related strains and arthropod species should be further investigated.
In summary, our results showed discrepant SFG rickettsiae were circulating in northwest China. While the SFG rickettsiae identified in the H. qinghaiensis ticks have not been linked to human cases in the area, additional studies are therefore needed to determine if these Rickettsia spp. has any public health significance.

Conclusions
This study represents the first published record of the detection of Rickettsia spp. in H. qinghaiensis ticks. Furthermore, on the basis of the sequence and phylogenetic data, it represents a putative novel SFG rickettsiae subgroup in H. qinghaiensis ticks from northwest China.
performed the experiments, data analysis, and drafted the manuscript. All authors read and approved the final manuscript.