Rickettsia raoultii in Haemaphysalis erinacei from marbled polecats, China-Kazakhstan border.

We found Rickettsia raoultii DNA in 2 out of 32 (6.25 %) Haemaphysalis erinacei ticks. Result showed that the sequences of five genes (17-kDa, gltA, ompA, rrs, and ompB) were 100 % identity with that of R. Raoultii in GenBank. This study is the first report on the presence of R. raoultii in H. erinacei from wild marbled polecat, Vormela peregusna. Our findings suggest that H. erinacei parasitizing wild marbled polecat may serve as reservoir and carriers for R. raoultii in areas around the China-Kazakhstan border. The transmission of tick-borne diseases originated from wildlife should not be underestimated in border region.

Findings

In China, at least five validated spot fever group (SFG) rickettsial species have been detected in ticks, including Rickettsia heilongjiangii, R. sibirica [1], R. raoultii, R. slovaca [2] and R. felis [3]. Of these five rickettsial species, none has been identified in the tick Haemaphysalis erinacei. Although no published evidence indicates that H. erinacei ticks bites humans, this species is interesting because it coexists with various animal species, including the hedgehog Hemiechinus auritus and the marbled polecat, Vormela peregusna [4], the later is listed as vulnerable globally by the International Union for Conservation of Nature (IUCN) [5]. The marble polecat is distributed from southeast Europe, through southwest and Central Asia, to Mongolia and northern China [6]. In the present study, we determined the presence of R. raoultii in H. erinacei from marbled polecats in wetlands around Ebinur Lake, northwest China.

Thirty-two adult ticks, 21 (14 male and seven female) from two marbled polecats and 11 (seven male and four female) from three hedgehogs, were collected in wetlands around Ebinur Lake (189 m above sea level; 82°48′51E 45°04′22N) in northwest China in 2014. The ticks were identified morphologically as H. Erinacei and the molecular identification of those ticks by using 16S mitochondrial gene results showed that they have a similarity of 90.95 % with that of H. concinna (there are no corresponding 16S mitochondrial gene sequence for H. Erinacei in GenBank). The sequences obtained were deposited in GenBank [GenBank: KR053302-KR053305]. Genomic DNA was extracted from individual specimens by using a TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). A targeting gene fragment (434 bp) from the Rickettsia-specific 17-kDa surface antigen gene was amplified by PCR following a previously published methodology [7]. Another five genetic markers [1332-, 1060-, 488-, 491-, and 812-bp products of the genes encoding 16S rRNA (rrs), citrate synthase (gltA), surface cell antigen 1 (sca1), and outer membrane proteins A and B (ompA and ompB)] were amplified by using primers previously described to detect Rickettsia spp. in H. erinacei [8]. The PCR products were sequenced and phylogenetically analyzed to certify the taxonomic identification of the rickettsial agent.

Rickettsial DNA was detected in two (both female) out of 32 (6.25 %) H. erinacei ticks, which were collected from the same marbled polecat. No rickettsial agent was found in hedgehogs. The sequences BLAST results showed that these two rickettsial sequences of five genes (17-kDa, gltA, ompA, rrs, and ompB) were the same, and 100 % identity with that of R. raoultii. The sca1 sequences obtained were closest to that of R. montanensis str. OSU 85–930 and R. montanensis str. M/5-6, with a sequence similarity of 99.18 % (612 out of 617 bp) (There are no corresponding sequence for R. raoultii in GenBank). All of the obtained sequences were deposited in GenBank [GenBank: KR608783-KR608788]. The phylogenetic tree produced from the Maximum Likelihood and Neighbor-Joining analyses of the sequence data for the six genes (17-kDa-ompA-gltA-rrs-sca1-ompB) revealed that the R. raoultii obtained from H. erinacei was culstered into a clade including “R. Raoultii (Heilongjiang, China)”, “R. raoultii (Russia)”, and “R. raoultii isolate BL029-2 (Xinjiang, China)” (Fig. 1).

Fig. 1

Phylogenetic tree of 17-kDa-ompA-gltA-rrs-sca1-ompB concatenated sequence of Rickettsia raoultii in Haemaphysalis erinacei (◆). The tree was constructed on the basis of Maximum Likelihood (Bootstrap replicates: 1000) and Neighbor-Joining (Bootstrap replicates: 500) analyses of concatenated sequence data of six genes (17-kDa-ompA-gltA-rrs-sca1-ompB) using MEGA6. The sequences of R. bellii were used as the outgroup in the concatenated sequence data. The scale bar represents the inferred substitutions per nucleotide site. The relative support for clades in the tree produced from the ML and NJ analyses are indicated above and below branches, respectively

Based on the information in GenBank, R. raoultii have been detected at least in 13 tick species, namely: Dermacentor nuttallii, D. marginatus, D. reticulatus, D. silvarum, Rhipicephalus pumilio, Rh. turanicus, H. concinna, H. japonica, Ixodes persulcatus, I. ricinus, Amblyomma helvolum, Hyalomma asiaticum, and Hy. lusitanicum [9]. However, this study is the first to report the presence of R. raoultii in H. erinacei. In previous studies, H. erinacei has been found in birds, the desert hedgehog Paraechinus aethiopicus, the North African hedgehog Atelerix algirus, stray dogs, the beech marten Martes foina, and the least weasel Mustela nivalis [10-13]. Here our sampling site, the Ebinur Lake, is widely known to be a station for thousands of wildlife around the China–Kazakhstan border. Approximately 1 million migratory birds arrive here, which is known to be home every year, and more than 160 wild vertebrate species and 230 bird species inhabit and/or migrate at this region [14]. Another several previous studies gave the strong evidence that R. raoultii is common and widespread across wildlife such as wild snakes, rats and Mongolian gazelle [15-17]. Our findings suggest that H. erinacei parasitizing wild marbled polecat may serve as reservoirs and carriers for R. raoultii in areas around the China-Kazakhstan border. In the future, the transmission of tick-borne diseases originated from wildlife should not be underestimated in border region. There is a need for international cooperation to survey this and other tick-borne pathogens in migratory birds and wildlife.