The first detection of Rickettsia aeschlimannii and Rickettsia massiliae in Rhipicephalus turanicus ticks, in northwest China

Background Rickettsia spp. belonging to the spotted fever group (SFG) cause infections in humans, domestic animals and wildlife. At least five SFG rickettsial species have been reported in China, but the occurrence of Rickettsia aeschlimannii and R. massiliae in ticks has not been characterized to date. Findings A total of 114 adult ticks were collected from sheep in Yining County, Xinjiang Uygur Autonomous Region, in northwest China. The ticks were identified from morphological and molecular characteristics. All samples were examined by polymerase chain reaction (PCR), and six genetic markers were used to determine the Rickettsia spp. in the ticks. The ticks collected were identified as Rhipicephalus turanicus. Three different lineages of Rh. turanicus from Yining County were discovered on phylogenetic analysis of 16S rDNA and cox1. Twenty-one of the 114 samples (18.42%) were positive for rickettsial agents. Phylogenetic analysis based on six genetic sequences showed that three rickettsial species were present, namely: R. aeschlimannii (19.05%, 4/21), R. massiliae (19.05%, 4/21) and R. sibirica variant (61.90%, 13/21), which is clustered in the clade of R. sibirica subsp. sibirica. Conclusions This is the first description of R. aeschlimannii and R. massiliae in China. R. massiliae, R. aeschlimannii and R. sibirica variant co-circulate in the region of the China-Kazakhstan border, in northwest China. Rickettsial agents in ticks of the genus Rhipicephalus from migrant birds, transported livestock, wildlife and human beings should be investigated further in the region of the China–Central Asian border. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1242-2) contains supplementary material, which is available to authorized users.


Findings
Background Rickettsia spp. belonging to the spotted fever group (SFG) cause infections in animals and humans worldwide [1,2]. To date, at least five validated SFG rickettsial species have been detected in ticks in China, including R. heilongjiangii, R. sibirica, R. raoultii, R. slovaca and R. felis [3]. Molecular evidence of the first four species was reported in northeastern and northwestern China, mainly in Dermacentor and Haemaphysalis ticks [4][5][6], and the last was found in Rhipicephalus sanguineus from Jiangsu Province [7].
Xinjiang Uygur Autonomous Region (XUAR), the largest province in China, occupies one-sixth of China, borders eight countries with a 5,600-km frontier, and there are 29 trading ports. In the present study, we assessed the occurrence of rickettsial agents in Rh. turanicus ticks in Yining County, the location of Yining Port, which is adjacent to Kazakhstan. 558182′E). All of the ticks were identified morphologically according to previous reports, and 23 representative ticks underwent molecular analysis based on partial mitochondrial (16S rDNA and cox1) gene sequences [8].

Ethical approval
This study was approved by the Animal Ethics Committee of Shihezi University (Approral No. AECSU2014-6).

PCR amplification and sequence analysis
For genetic detection of Rickettsia spp., the genomic DNA of all the ticks was extracted from individual specimens using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China). All samples were examined by polymerase chain reaction (PCR), and six genetic markers [434-, 1332-, 1060-, 488-, 491-, and 812-bp products of the genes encoding the 17 kilodalton antigen (17-kDa), 16S rRNA (rrs), citrate synthase (gltA), surface cell antigen 1 (sca1), and outer membrane proteins A and B (ompA and ompB)] were amplified using previously described primers [3]. The amplication products were purified using the TIANgel Midi Purification Kit (TIANGEN, Beijing, China) and then cloned into the pGEM-T Easy vector and subjected to sequencing. A phylogenetic tree was constructed using the maximum likelihood and neighbor-joining algorithms with MEGA 6.0 software [9].

Results
The ticks were identified morphologically as Rh. turanicus. Sequencing data from the 23 representative ticks indicated three different lineages of Rh. turanicus from Yining County on the basis of phylogenetic analysis of 16S rDNA and cox1 (shown in Additional file 1). Six nucleotide sequences from our study have been deposited in the GenBank database (16S rDNA: KF547984, KF547987, and KF547989; cox1: KF188136-KF188138).

Discussion
R. massiliae, R. rhipicephali and R. aeschlimannii are grouped phylogenetically into a clade in the family Rickettsiaceae [10]. R. massiliae was first isolated in 1990 from a Rh. turanicus tick in an area near Marseille, France [11]. Since then, this pathogen has been identified from other Rhipicephalus ticks in regions of Europe, North and Central Africa, and the United States [12]. Furthermore, cases showed that it can cause human infection. R. aeschlimannii was first described from Hyalomma marginatum in Morocco in 1997 [13]. The presence of R. aeschlimannii has been demonstrated in Hyalomma ticks from Europe (e.g. France, Croatia, Spain, Italy), Asia (e.g. Israel, Turkey) and Africa (e.g. Mali, Algeria, Egypt) [14][15][16] and from Haemaphysalis ticks in Spain and Kazakhstan [17]. Furthermore, Ixodes ricinus, H. punctata, Rh. bursa, and Rh. sanguineus isolated from human Spanish patients were shown to contain DNA from R. aeschlimannii [14], and there is a report of R. aeschlimannii from Rh. turanicus infecting a man in Greece in 2013 [18]. In this study, we report the first molecular evidence that R. aeschlimannii and R. massiliae are present in Rh. turanicus from sheep in the region of the China-Kazakhstan border, in the northwest of China.
To date, R. sibirica is known to contain two subspecies [19], R. sibirica subsp. sibirica and R. sibirica subsp. mongolotimonae. The former was first isolated in Russia but it has subsequently been found in northern China [5]. In contrast, R. sibirica subsp. mongolotimonae was first isolated in Inner Mongolia and then found in Europe and Africa [20,21]. Here, the R. sibirica variant found in the region of the China-Kazakhstan border appeared divergent in the ompA, ompB and sca1, used to differentiate Rickettsia species, although it was closest to R. sibirica subsp. sibirica, on the basis of the gltA gene and the phylogenetic tree of the 17-kDa-ompA-gltA-rrs-sca1-ompB concatenated sequence. Further genomic analysis should be carried out to confirm the classification of the R. sibirica variant found in this study.
The Rh. turanicus tick is widely distributed throughout the Mediterranean subregion, Africa, and Asia, including China, especially in XUAR [22], and it has been implicated as a vector of several human and veterinary pathogens, including Rickettsia spp. [18]. Here, R. massiliae, R. aeschlimannii and R. sibirica variant were found in the same area, Yining County, which suggests that several SFG Rickettsia spp. co-circulate in Rh. turanicus as a potential vector near the China-Kazakhstan border.
In 2004, Shpynov et al detected R. aeschlimannii in the Alma-Ata region, east of Kazakhstan [17]. Here we found that Rh. turanicus in the region of the China-Kazakhstan border showed genetic divergence in the loci of 16S rDNA and cox1, which indicates that these ticks collected from sheep may come from different lineages. At present, it is unknown whether these ticks are imported from the Chinese hinterland or abroad through migrant birds, or with internationally transported livestock. This topic needs to be further investigated.

Conclusions
This is the first report of the molecular analysis of R. aeschlimannii and R. massiliae in China. The findings of the study suggest that R. massiliae, R. aeschlimannii and R. sibirica variant co-circulate in Rh. turanicus in the region of the China-Kazakhstan border, in northwest China. The origin of the Rhipicephalus genus (such as migrant birds, transported livestock, wildlife and human beings) and the epidemiology of tick-borne pathogens should be further investigated in the region of the China-Central Asian border.