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Emerging spotted fever group rickettsiae in ticks, northwestern China
Ticks and Tick-borne Diseases 7 (2016) 1146–1150
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Emerging spotted fever group rickettsiae in ticks, northwestern China Li-Ping Guo a,1 , Su-Hua Jiang a,b,1 , Dan Liu c , Shi-Wei Wang d , Chuang-Fu Chen c , Yuan-Zhi Wang a,∗ a
School of Medicine, Shihezi University, Shihezi 832000, Xinjiang Uygur Autonomous Region, China Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 Hubei, China School of Animal Science and Technology, Shihezi University, Shihezi 832000, Xinjiang, China d School of Animal Science and Technology, Tarim University, Aral 843300, Xinjiang, China b c
a r t i c l e
i n f o
Article history: Received 9 March 2016 Received in revised form 5 August 2016 Accepted 15 August 2016 Available online 16 August 2016 Keywords: Rickettsia conorii Candidatus Rickettsia barbariae Rhipicephalus turanicus Northwestern China
a b s t r a c t We report Rickettsia conorii subsp. indica, Candidatus R. barbariae and R. massiliae in Rhipicephalus turanicus from sheep around the Taklamakan desert, northwestern China. The topology of the phylogenetic trees produced from the maximum likelihood (ML) analyses of the ompA-gltA-rrs-geneD-ompB concatenated sequence data was very similar to that of the neighbor joining (NJ) tree, and with total support of 69%–100% bootstrap values for the inclusion of the rickettsiae in Rh. turanicus within the clade that contained R. conorii subsp. indica; Candidatus R. barbariae and Rickettsia sp. Tselentii; R. massiliae str. AZT80; and R. massiliae MTU5, respectively. Studies suggest that the co-existence of these spotted fever group rickettsiae is a threat to public health in China. Work is important in exploring novel and emerging pathogens. © 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Infections with spotted fever group (SFG) rickettsiae have been reported in various parts of the world over the past decades. Tick bite events are common and cases are globally increasing (Jones et al., 2008). However, reports of tick-borne rickettsial disease as well as the rickettsia species involved are limited in China, especially in less developed communities. To date, in China, seven validated SFG rickettsial species have been detected in ticks: Rickettsia heilongjiangii, R. sibirica, R. raoultii, R. slovaca, R. felis, R. aeschlimannii and R. massiliae, and cases of human infection with rickettsiae such as R. raoultii have been reported in the last years (Wei et al., 2015; Jia et al., 2014). The diagnosis and treatment of the rickettsiosis are difficult in the absence of molecular analysis because the commercial serological assays do not distinguish among the SFG species (Myers et al., 2013). As the largest province in China, Xinjiang Uygur Autonomous Region (XUAR) covers over one-sixth of the country, includes the
∗ Corresponding author at: Department of pathogen biology and immunology, School of Medicine, Shihezi University, Xinjiang Uygur Autonomous Region, Shihezi 832000, China. E-mail addresses: [email protected] (L.-P. Guo), [email protected] (S.-H. Jiang), [email protected] (D. Liu), [email protected] (S.-W. Wang), [email protected] (C.-F. Chen), [email protected] (Y.-Z. Wang). 1 These authors contributed equally to this work.
majority of the arid areas in the country, and is abundant in tick species (Zheng et al., 2006). Tick-associated pathogens and diseases, nevertheless, are underestimated because of the tense political environment and limited research, especially around the Taklamakan desert, in the southern region of XUAR. Here, an investigation was carried out to identify the ticks and Rickettsia spp. in this region. 2. Materials and methods During 2013–2014, a total of 133 adult ticks were collected from sheep from six sites around the Taklamakan desert (Table 1). All of the ticks were identified morphologically according to previous reports and were submitted to molecular analysis based on partial mitochondrial 16S rDNA gene sequences (Dantas-Torres et al., 2013). The genomic DNA was extracted from each individual tick using the TIANamp Genomic DNA Kit (TianGen, Beijing, China). Six PCR targets, 17 kilodalton antigen (17-kDa), 16S rRNA (rrs), citrate synthase (gltA), cell surface antigen 1 (sca1), outer membrane protein A (ompA) and ompB (named ompB1 in this study) were assessed within each sample to investigate the presence of SFG rickettsiae (Anstead and Chilton, 2013a,b). Two additional genetic markers were used to confirm the presence of the rickettsiae in ticks. Another part of the ompB gene (named ompB2 in this study; 1063 bp) was amplified
http://dx.doi.org/10.1016/j.ttbdis.2016.08.006 1877-959X/© 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).
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Table 1 Tick species and PCR results of rickettsiae from questing adult ticks in study sites, northwestern China. Location, coordinates
Tick species
No.
Rickettsia species, No. (%) positive
Yecheng 37◦ 89 N–77◦ 42 E
Rh. turanicus
6
Qira 36◦ 59 N–80◦ 48 E Tumxuk 39◦ 51 N–79◦ 03 E
Rh. turanicus Rh. turanicus
16 30
Pishan 37◦ 37 N–78◦ 16 E
D. marginatus Rh. turanicus
3 62
Kuqa 41◦ 43 N–82◦ 57 E
D. marginatus Rh. turanicus
2 3
Atux 39◦ 42 N–76◦ 09 E Total (133)
Hy. asiaticum D. marginatus Rh. turanicus
2 9 117
D. marginatus Hy. asiaticum
14 2
R. conorii 3 (50) Candidatus Rickettsia barbariae 2 (33.33) Candidatus Rickettsia barbariae 3 (18.75) R. conorii 6 (20) Candidatus Rickettsia barbariae 12 (40) R. massiliae 1 (3.33) 0 R. conorii 10 (16.13) Candidatus Rickettsia barbariae 18 (29.03) R. massiliae 1 (1.61) 0 R. conorii 2 (66.67) Candidatus Rickettsia barbariae 1 (33.33) 0 0 R. conorii 21 (17.95) Candidatus Rickettsia barbariae 36 (30.77) R. massiliae 2 (1.71) 0 0
using primers ompB-out1 (5 -ACAGCTACCATAGTAGCCAG-3 ) and ompB-out2 (5 -TGCAGTATAGTTACCACCG-3 ) for the first phase, and ompB-inter1 (5 -TGCTGCGGCTTCTACATT-3 ) and ompB-inter2 (5 -ACCGCCAGCGTTCCCTAT-3 ) for the second phase. The PCR conditions consisted of an initial 5-min denaturation at 95 ◦ C, followed by 35 cycles at 95 ◦ C for 45 s, 56 ◦ C for 45 s, and 72 ◦ C for 2 min 30 s, with a final extension at 72 ◦ C for 5 min (first phase), and an initial 5-min denaturation at 95 ◦ C, followed by 35 cycles at 95 ◦ C for 45 s, 56 ◦ C for 45 s, and 72 ◦ C for 1 min 10 s, with a final extension at 72 ◦ C for 5 min. Part of the PS120-proteinencoding gene (gene D; 920 bp) fragment was amplified using primers gene D-F (5 -CGGTAACCTAGATACAAGTGA-3 ) and gene DR (5 -TATAAGCTATTGCGTCATCTC-3 ) according to the sequences available in GenBank, and samples were amplified as recommended (Sekeyova et al., 2001). The PCR conditions consisted of an initial 5-min denaturation at 95 ◦ C, followed by 35 cycles at 95 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 8 min R. aeschlimannii from Rhipicephalus turanicus and double distilled water were used, respectively, as positive and negative controls (Wei et al., 2015). The amplification products were purified using the TIANgel Midi Purification Kit, cloned into the pGEM-T Easy vector (TianGen) and then subjected to sequencing (BGI, Shenzhen, China). Phylogenetic trees were constructed using the maximum likelihood (ML) and neighbor joining (NJ) algorithms with MEGA 6.0 (Tamura et al., 2013). Bootstrap analyses (500 replicates for ML analyses and 1000 replicates for the NJ analyses) were conducted to determine the relative support for clades in the consensus trees (Pattengale et al., 2010; Anstead and Chilton, 2013a,b). 3. Results A total of 117 Rh. turanicus ticks (80 males, 37 females), 14 Dermacentor marginatus (9 males, 5 females) and two Hyalomma asiaticum (females) were collected in questing areas. Phylogenetic analyses based on the partial mitochondrial 16S rDNA gene sequences are shown in Technical Appendix A in Supplementary material. The ticks were screened first by ompA PCR, and positive samples were analyzed for all targeted genes. The results showed that 59 (44.36%) of the 133 ticks analyzed were positive for Rickettsia spp. Of these, 36 (30.76%) were confirmed as Candidatus R. barbariae, 21 (17.95%) as R. conorii subsp. indica, and two (1.71%) as R. massiliae in 117 Rh. turanicus ticks (Table 1). The R. conorii subsp. indica showed 100%, 100%, 100%, 99.60%, and 99.84% pair-
wise nucleotide sequence identity to R. conorii subsp. indica rrs (L36107), gltA (U59730), ompA (U43794), ompB (AF123726) and R. conorii subsp. conorii sca1(AE006914), respectively. The R. massiliae species had 99.74%–100% pairwise nucleotide sequence identity to genome sequences of the reference strains R. massiliae MTU5 (accession no. CP000683) for all six genes analyzed. For Candidatus R. barbariae, however, the amplified regions of the gltA and ompB1 genes in this study were different from the existing sequences and no sequences of the sca1 gene were available in GenBank, it showed 99.54%, 99.47% and 99.52% similarity with R. sibirica subsp. sibirica (accession no. KM288711), R. parkeri str. Portsmouth (accession no. CP003341) and R. africae ESF-5 (CP001612) for the gltA, ompB1 and sca1 genes, respectively. The rest of the genes of Candidatus R. barbariae had 100% pairwise nucleotide sequence identity to the reference sequences (EU272186, JF803896 and EU272189 for ompA, 17-kDa and rrs, respectively; Table 2). For the ompB2 (1063 bp) and gene D (920 bp) fragments, the BLAST results showed that the R. conorii subsp. indica identified in this study had 100% pairwise nucleotide sequence identity while the Candidatus R. barbariae had 99.89%–100% similarity with reference sequences availiable in GenBank (Table 2). None of the ticks belonging to the species D. marginatus and Hy. asiaticum was positive for rickettsiae. All the sequences obtained in this study were deposited in GenBank under accession nos. KU364354–KU364359, KU364361–KU364367, KU364369–KU364378, and KU757300–KU757306. The topology of the phylogenetic trees produced from the ML analyses of the ompA, gltA, gene D, and ompB2 gene sequences, as well as the ompA-gltA-rrs-geneD-ompB concatenated sequence data, were very similar to the NJ trees, and with total support of 69%–100% bootstrap values for the inclusion of the rickettsiae in Rh. turanicus within the clade that contained R. conorii subsp. indica; Candidatus R. barbariae and rickettsia sp. Tselentii; R. massiliae str. AZT80 and R. massiliae MTU5, respectively (Technical Appendix A in Supplementary material, Fig. 1). 4. Discussion We report the presence of R. conorii subsp. indica in Rh. turanicus ticks for the first time, and identify Candidatus R. barbariae and R. massiliae in Rh. turanicus from sheep in China. The co-circulation of these rickettsial species around the Taklamakan desert increases the known range of vectors and reservoirs for SFG rickettsiae and provides a basis for assessing the risk of infection in humans.
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Table 2 Most closely related sequences to the partial 17-kDa, 16S, gltA, ompA, ompB1 , ompB2 , sca1 and gene D genes, sequences of Rickettsia conorii (A), Candidatus Rickettsia barbariae (B) and Rickettsia massiliae (C) detected in Rhipicephalus turanicus ticks, in northwestern China. (A) Gene
Rickettsia (GenBank accession No.)
% Sequence similarity (bp)
17-kDa (KU364361)
Rickettsia conorii (M28480) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia rickettsii str. Morgan (CP006010) Rickettsia rickettsii str. R (CP006009) Rickettsia conorii subsp. indica (L36107) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii ITT-597 (U12460) Rickettsia conorii seven (U59730) Rickettsia conorii (HM050292) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia sibirica subsp. sibirica (KM288711) Rickettsia conorii subsp. indica (U43794) Rickettsia conorii isolate ROD (JN944636) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii (U43806) Rickettsia conorii subsp. indica (AF123726) Rickettsia conorii seven (AF123721) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii (AF149110) Rickettsia conorii subsp. indica (AF123726) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii seven (AF123721) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii subsp. caspia A-167 (AY502117) Rickettsia slovaca str. D-CWPP (CP003375) Rickettsia conorii strain ATCC VR-597 (AF163005) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia slovaca str. D-CWPP (CP003375)
100 (410/410) 100 (410/410) 99.75 (409/410) 99.75 (409/410) 100 (1185/1185) 100 (1185/1185) 100 (1185/1185) 100 (1076/1076) 100 (1076/1076) 100 (1076/1076) 99.62 (1072/1076) 100 (451/451) 100 (451/451) 99.11 (447/451) 99.11 (447/451) 99.60 (763/766) 100 (766/766) 100 (766/766) 99.73 (764/766) 100 (1000/1000) 99.70 (997/1000) 99.70 (997/1000) 99.84 (625/626) 99.68 (624/626) 99.52 (623/626) 100 (897/897) 99.88 (896/897) 99.33 (891/897)
16S (KU364355)
gltA (KU364366)
ompA (KU364365)
ompB1 (KU364371)
ompB2 (KU757301)
sca1 (KU364363)
gene D (KU757303)
(B) Gene
Rickettsia (GenBank accession No.)
% Sequence similarity (bp)
17-kDa (KU364358) 16S (KU364356)
Rickettsia sp. Tselentii (GU353184) Candidatus Rickettsia barbariae (EU272189) Rickettsia raoultii isolate BL029-2 (KJ410261) Rickettsia sibirica subsp. sibirica (KM28871) Rickettsia sibirica 246 (U59734) Rickettsia sp. BJ-90 citrate synthase (AF178035) Rickettsia parkeri str. Portsmouth (CP003341) Rickettsia sp. Tselentii (EU194445) Candidatus Rickettsia barbariae (EU272186) Rickettsia sp. PoTiRb169 (DQ423366) Candidatus Rickettsia barbariae (JF700253) Rickettsia parkeri str. Portsmouth (CP003341) Rickettsia parkeri (AF123717) Rickettsia sp. BJ-90 (AY331393) Candidatus Rickettsia barbariae (EU272187) Rickettsia mongolitimonae (DQ097083) Rickettsia sibirica (HM050273) Rickettsia africae ESF-5 (CP001612) Candidatus Rickettsia amblyommii strain Aca3 (AY502116) Rickettsia parkeri str. Portsmouth (CP003341) Candidatus Rickettsia barbariae (EU272188) Candidatus Rickettsia barbariae (JQ480840)
100 (399/399) 100 (1174/1174) 99.82 (1172/1174) 99.54 (1096/1101) 99.54 (1096/1101) 99.54 (1096/1101) 99.45 (1095/1101) 100 (436/436) 100 (436/436) 100 (436/436) 100 (425/425) 99.47 (751/755) 99.47 (751/755) 99.20 (749/755) 99.89 (998/999) 99.29 (992/999) 99.19 (991/999) 99.52 (622/625) 99.36 (621/625) 99.20 (620/625) 100 (917/917) 99.77 (873/875)
gltA (KU364367)
ompA (KU364364)
ompB1 (KU364369)
ompB2 (KU757300)
sca1 (KU364362)
gene D (KU757302)
(C) Gene
Rickettsia (GenBank accession No.)
% Sequence similarity (bp)
17-kDa (KU364359)
Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae strain CABA (KT032120) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae strain Mtu1 (NR 025919) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae MTU1 (U59719) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia sp. Bar 29 (U59720) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae clone 04(KR401146) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae (AF123714) Rickettsia rhipicephali strain HJ#5 (CP013133) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae strain Mtu1(AY355364) Rickettsia massiliae str. AZT80(CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae (AF163003) Rickettsia rhipicephali strain HJ#5 (CP013133)
99.74 (391/392) 99.23 (389/392) 99.23 (389/392) 100 (1182/1182) 100 (1182/1182) 99.91 (1181/1182) 100 (1118/1118) 100 (1118/1118) 99.82 (1116/1118) 99.82 (1116/1118) 100 (491/491) 100 (491/491) 99.59 (489/491) 99.86 (765/766) 99.86 (765/766) 99.08 (759/766) 100(611/611) 100(611/611) 99.18(606/611) 100 (900/900) 99.88 (899/900) 99.33 (894/900)
16S (KU364357)
gltA (KU364354)
ompA (KU757305)
ompB1 (KU364370)
sca1 (KU757304)
gene D (KU757306)
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Fig. 1. Phylogenetic analysis of spotted fever group Rickettsia spp. in Rhipicephalus turanicus in northwestern China. The tree was constructed on the basis of the ompA-gltArrs-geneD-ompB concatenated sequence of Rickettsia species by maximum likelihood (ML) and neighbor-joining (NJ) methods, using MEGA6.0. Sequences of the spotted fever group Rickettsia spp. obtained in this study are shown as “”. Sequences of R. bellii were used as the outgroup in the concatenated sequence data. The relative support for clades in the tree produced from the ML and NJ analyses are indicated above and below branches, respectively. The analysis involved 28 nucleotide sequences. The tree is drawn to scale, with branch lengths representing the number of substitutions per site.
Pathogens in the R. conorii complex comprise four subspecies: R. conorii subsp. conorii, R. conorii subsp. indica, R. conorii subsp. israelensis, and R. conorii subsp. Caspia (Zhu et al., 2005). They are known to cause Mediterranean spotted fever (MSF), Indian tick typhus (ITT), Israeli spotted fever (ISF), and Astrakhan fever (AF), respectively (Zhu et al., 2005; Torina et al., 2012). ITT is characterized by sudden onset of moderate to high-grade fever, malaise, deep muscle pain, headache, and conjunctival suffusion, together with gangrene, and severe manifestations of sepsis and multiorgan dysfunction syndrome such as acute kidney injury, liver dysfunction, delirium, seizures, and sometimes purpura fulminans (Tirumala et al., 2014). R. sanguineus, Boophilus microplus, and Haemaphysalis leachii are the main voctors of R. conorii subsp. indica, which can transmitted and spread to humans by tick bites (Zhu et al., 2005; Tirumala et al., 2014). Chochlakis et al. suggested that changing climate and geographical location (for example, in a wintering area for migratory birds) may be responsible for the possible dispersal of ticks and tick-borne pathogens (Chochlakis et al., 2014). Therefore, Rh. turanicus poses a risk for transmission of such bacteria and serves as a bridge-vector to humans. In addition, our findings suggest that Candidatus R. barbariae may become the eighth rickettsial species in China, following on from our previous work with the first detection of R. massiliae in Rh. turanicus in the northern region of XUAR (Wei et al., 2015). Furthermore, the high infection rate of ticks (17.95% and 30.76% for R. conorii subsp. indica and Candidatus R. barbariae, respectively) is strong evidence that questing Rh.
turanicus ticks from sheep are potential and emerging carriers for human rickettsiosis. Consideration should been given in China to prevention of the outbreak of such diseases, especially in shepherds because they could be the most vulnerable group. Reports showed that R. conorii subsp. indica has been identified in India, Pakistan and Laos (Zhu et al., 2005; Phongmany et al., 2006). China, the neighbor of these countries, seems to be a likely location for this pathogen, but it is unknown whether certain areas, especially the Taklamakan desert, have a connection with the endemic area of R. conorii subsp. indica or not. However, the other two rickettsial species have rarely been described in Asia. Candidatus R. barbariae was first confirmed in Rh. turanicus from Italy in 2008 and since then has been detected only in various European countries and in Israel (Mura et al., 2008; Waner et al., 2014). R. massiliae, one of the agents of human spotted fever, is distributed mainly in Europe, North and Central Africa, and the United States (Fernández de Mera et al., 2009). Our studies suggest that the coexistence of these SFG rickettsiae with public health relevance may favor transmission to humans and that work in this region may be important in exploring novel and emerging pathogens. 5. Conclusions R. conorii subsp. indica, Candidatus R. barbariae, and R. massiliae co-circulate around the Taklamakan desert. Rickettsial agents in ticks (particularly in the genus Rhipicephalus) from birds, livestock,
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wildlife and human beings should be further investigated on a large scale. Novel or emerging Rickettsia spp. may be found in this special habitat. Competing interests The authors declare that there is no conflict of interest in this study. Acknowledgments This research was supported in part by grants from the National Natural Science Foundation of China (grant Nos. 81560338 and U1503283) and the Co-innovation Center for the High Incidence of Zoonotic Disease Prevention and Control in Western China (grant No. 2013-179). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ttbdis.2016.08. 006. References Anstead, C.A., Chilton, N.B., 2013a. A novel Rickettsia species detected in vole ticks (Ixodes angustus) from Western Canada. Appl. Environ. Microbiol. 79, 7583–7589. Anstead, C.A., Chilton, N.B., 2013b. Detection of a novel Rickettsia (Alphaproteobacteria: Rickettsiales) in rotund ticks (Ixodes kingi) from Saskatchewan, Canada. Ticks Tick Borne Dis. 4, 02–06. Chochlakis, D., Ioannou, I., Papadopoulos, B., Tselentis, Y., Psaroulaki, A., 2014. Rhipicephalus turanicus: from low numbers to complete establishment in Cyprus: its possible role as a bridge-vector. Parasite Vectors 7, 1. Dantas-Torres, F., Latrofa, M.S., Annoscia, G., Giannelli, A., Parisi, A., Otranto, D., 2013. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the new and old worlds. Parasite Vectors 6, 213. ˜ Fernández de Mera, I.G., Zivkovic, Z., Bolanos, M., Carranza, C., Pérez-Arellano, J.L., Gutiérrez, C., de la Fuente, J., 2009. Rickettsia massiliae in the canary islands. Emerg. Infect. Dis. 15, 1869–1870.
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Emerging spotted fever group rickettsiae in ticks, northwestern China Li-Ping Guo a,1 , Su-Hua Jiang a,b,1 , Dan Liu c , Shi-Wei Wang d , Chuang-Fu Chen c , Yuan-Zhi Wang a,∗ a
School of Medicine, Shihezi University, Shihezi 832000, Xinjiang Uygur Autonomous Region, China Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 Hubei, China School of Animal Science and Technology, Shihezi University, Shihezi 832000, Xinjiang, China d School of Animal Science and Technology, Tarim University, Aral 843300, Xinjiang, China b c
a r t i c l e
i n f o
Article history: Received 9 March 2016 Received in revised form 5 August 2016 Accepted 15 August 2016 Available online 16 August 2016 Keywords: Rickettsia conorii Candidatus Rickettsia barbariae Rhipicephalus turanicus Northwestern China
a b s t r a c t We report Rickettsia conorii subsp. indica, Candidatus R. barbariae and R. massiliae in Rhipicephalus turanicus from sheep around the Taklamakan desert, northwestern China. The topology of the phylogenetic trees produced from the maximum likelihood (ML) analyses of the ompA-gltA-rrs-geneD-ompB concatenated sequence data was very similar to that of the neighbor joining (NJ) tree, and with total support of 69%–100% bootstrap values for the inclusion of the rickettsiae in Rh. turanicus within the clade that contained R. conorii subsp. indica; Candidatus R. barbariae and Rickettsia sp. Tselentii; R. massiliae str. AZT80; and R. massiliae MTU5, respectively. Studies suggest that the co-existence of these spotted fever group rickettsiae is a threat to public health in China. Work is important in exploring novel and emerging pathogens. © 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Infections with spotted fever group (SFG) rickettsiae have been reported in various parts of the world over the past decades. Tick bite events are common and cases are globally increasing (Jones et al., 2008). However, reports of tick-borne rickettsial disease as well as the rickettsia species involved are limited in China, especially in less developed communities. To date, in China, seven validated SFG rickettsial species have been detected in ticks: Rickettsia heilongjiangii, R. sibirica, R. raoultii, R. slovaca, R. felis, R. aeschlimannii and R. massiliae, and cases of human infection with rickettsiae such as R. raoultii have been reported in the last years (Wei et al., 2015; Jia et al., 2014). The diagnosis and treatment of the rickettsiosis are difficult in the absence of molecular analysis because the commercial serological assays do not distinguish among the SFG species (Myers et al., 2013). As the largest province in China, Xinjiang Uygur Autonomous Region (XUAR) covers over one-sixth of the country, includes the
∗ Corresponding author at: Department of pathogen biology and immunology, School of Medicine, Shihezi University, Xinjiang Uygur Autonomous Region, Shihezi 832000, China. E-mail addresses: [email protected] (L.-P. Guo), [email protected] (S.-H. Jiang), [email protected] (D. Liu), [email protected] (S.-W. Wang), [email protected] (C.-F. Chen), [email protected] (Y.-Z. Wang). 1 These authors contributed equally to this work.
majority of the arid areas in the country, and is abundant in tick species (Zheng et al., 2006). Tick-associated pathogens and diseases, nevertheless, are underestimated because of the tense political environment and limited research, especially around the Taklamakan desert, in the southern region of XUAR. Here, an investigation was carried out to identify the ticks and Rickettsia spp. in this region. 2. Materials and methods During 2013–2014, a total of 133 adult ticks were collected from sheep from six sites around the Taklamakan desert (Table 1). All of the ticks were identified morphologically according to previous reports and were submitted to molecular analysis based on partial mitochondrial 16S rDNA gene sequences (Dantas-Torres et al., 2013). The genomic DNA was extracted from each individual tick using the TIANamp Genomic DNA Kit (TianGen, Beijing, China). Six PCR targets, 17 kilodalton antigen (17-kDa), 16S rRNA (rrs), citrate synthase (gltA), cell surface antigen 1 (sca1), outer membrane protein A (ompA) and ompB (named ompB1 in this study) were assessed within each sample to investigate the presence of SFG rickettsiae (Anstead and Chilton, 2013a,b). Two additional genetic markers were used to confirm the presence of the rickettsiae in ticks. Another part of the ompB gene (named ompB2 in this study; 1063 bp) was amplified
http://dx.doi.org/10.1016/j.ttbdis.2016.08.006 1877-959X/© 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).
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Table 1 Tick species and PCR results of rickettsiae from questing adult ticks in study sites, northwestern China. Location, coordinates
Tick species
No.
Rickettsia species, No. (%) positive
Yecheng 37◦ 89 N–77◦ 42 E
Rh. turanicus
6
Qira 36◦ 59 N–80◦ 48 E Tumxuk 39◦ 51 N–79◦ 03 E
Rh. turanicus Rh. turanicus
16 30
Pishan 37◦ 37 N–78◦ 16 E
D. marginatus Rh. turanicus
3 62
Kuqa 41◦ 43 N–82◦ 57 E
D. marginatus Rh. turanicus
2 3
Atux 39◦ 42 N–76◦ 09 E Total (133)
Hy. asiaticum D. marginatus Rh. turanicus
2 9 117
D. marginatus Hy. asiaticum
14 2
R. conorii 3 (50) Candidatus Rickettsia barbariae 2 (33.33) Candidatus Rickettsia barbariae 3 (18.75) R. conorii 6 (20) Candidatus Rickettsia barbariae 12 (40) R. massiliae 1 (3.33) 0 R. conorii 10 (16.13) Candidatus Rickettsia barbariae 18 (29.03) R. massiliae 1 (1.61) 0 R. conorii 2 (66.67) Candidatus Rickettsia barbariae 1 (33.33) 0 0 R. conorii 21 (17.95) Candidatus Rickettsia barbariae 36 (30.77) R. massiliae 2 (1.71) 0 0
using primers ompB-out1 (5 -ACAGCTACCATAGTAGCCAG-3 ) and ompB-out2 (5 -TGCAGTATAGTTACCACCG-3 ) for the first phase, and ompB-inter1 (5 -TGCTGCGGCTTCTACATT-3 ) and ompB-inter2 (5 -ACCGCCAGCGTTCCCTAT-3 ) for the second phase. The PCR conditions consisted of an initial 5-min denaturation at 95 ◦ C, followed by 35 cycles at 95 ◦ C for 45 s, 56 ◦ C for 45 s, and 72 ◦ C for 2 min 30 s, with a final extension at 72 ◦ C for 5 min (first phase), and an initial 5-min denaturation at 95 ◦ C, followed by 35 cycles at 95 ◦ C for 45 s, 56 ◦ C for 45 s, and 72 ◦ C for 1 min 10 s, with a final extension at 72 ◦ C for 5 min. Part of the PS120-proteinencoding gene (gene D; 920 bp) fragment was amplified using primers gene D-F (5 -CGGTAACCTAGATACAAGTGA-3 ) and gene DR (5 -TATAAGCTATTGCGTCATCTC-3 ) according to the sequences available in GenBank, and samples were amplified as recommended (Sekeyova et al., 2001). The PCR conditions consisted of an initial 5-min denaturation at 95 ◦ C, followed by 35 cycles at 95 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 8 min R. aeschlimannii from Rhipicephalus turanicus and double distilled water were used, respectively, as positive and negative controls (Wei et al., 2015). The amplification products were purified using the TIANgel Midi Purification Kit, cloned into the pGEM-T Easy vector (TianGen) and then subjected to sequencing (BGI, Shenzhen, China). Phylogenetic trees were constructed using the maximum likelihood (ML) and neighbor joining (NJ) algorithms with MEGA 6.0 (Tamura et al., 2013). Bootstrap analyses (500 replicates for ML analyses and 1000 replicates for the NJ analyses) were conducted to determine the relative support for clades in the consensus trees (Pattengale et al., 2010; Anstead and Chilton, 2013a,b). 3. Results A total of 117 Rh. turanicus ticks (80 males, 37 females), 14 Dermacentor marginatus (9 males, 5 females) and two Hyalomma asiaticum (females) were collected in questing areas. Phylogenetic analyses based on the partial mitochondrial 16S rDNA gene sequences are shown in Technical Appendix A in Supplementary material. The ticks were screened first by ompA PCR, and positive samples were analyzed for all targeted genes. The results showed that 59 (44.36%) of the 133 ticks analyzed were positive for Rickettsia spp. Of these, 36 (30.76%) were confirmed as Candidatus R. barbariae, 21 (17.95%) as R. conorii subsp. indica, and two (1.71%) as R. massiliae in 117 Rh. turanicus ticks (Table 1). The R. conorii subsp. indica showed 100%, 100%, 100%, 99.60%, and 99.84% pair-
wise nucleotide sequence identity to R. conorii subsp. indica rrs (L36107), gltA (U59730), ompA (U43794), ompB (AF123726) and R. conorii subsp. conorii sca1(AE006914), respectively. The R. massiliae species had 99.74%–100% pairwise nucleotide sequence identity to genome sequences of the reference strains R. massiliae MTU5 (accession no. CP000683) for all six genes analyzed. For Candidatus R. barbariae, however, the amplified regions of the gltA and ompB1 genes in this study were different from the existing sequences and no sequences of the sca1 gene were available in GenBank, it showed 99.54%, 99.47% and 99.52% similarity with R. sibirica subsp. sibirica (accession no. KM288711), R. parkeri str. Portsmouth (accession no. CP003341) and R. africae ESF-5 (CP001612) for the gltA, ompB1 and sca1 genes, respectively. The rest of the genes of Candidatus R. barbariae had 100% pairwise nucleotide sequence identity to the reference sequences (EU272186, JF803896 and EU272189 for ompA, 17-kDa and rrs, respectively; Table 2). For the ompB2 (1063 bp) and gene D (920 bp) fragments, the BLAST results showed that the R. conorii subsp. indica identified in this study had 100% pairwise nucleotide sequence identity while the Candidatus R. barbariae had 99.89%–100% similarity with reference sequences availiable in GenBank (Table 2). None of the ticks belonging to the species D. marginatus and Hy. asiaticum was positive for rickettsiae. All the sequences obtained in this study were deposited in GenBank under accession nos. KU364354–KU364359, KU364361–KU364367, KU364369–KU364378, and KU757300–KU757306. The topology of the phylogenetic trees produced from the ML analyses of the ompA, gltA, gene D, and ompB2 gene sequences, as well as the ompA-gltA-rrs-geneD-ompB concatenated sequence data, were very similar to the NJ trees, and with total support of 69%–100% bootstrap values for the inclusion of the rickettsiae in Rh. turanicus within the clade that contained R. conorii subsp. indica; Candidatus R. barbariae and rickettsia sp. Tselentii; R. massiliae str. AZT80 and R. massiliae MTU5, respectively (Technical Appendix A in Supplementary material, Fig. 1). 4. Discussion We report the presence of R. conorii subsp. indica in Rh. turanicus ticks for the first time, and identify Candidatus R. barbariae and R. massiliae in Rh. turanicus from sheep in China. The co-circulation of these rickettsial species around the Taklamakan desert increases the known range of vectors and reservoirs for SFG rickettsiae and provides a basis for assessing the risk of infection in humans.
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Table 2 Most closely related sequences to the partial 17-kDa, 16S, gltA, ompA, ompB1 , ompB2 , sca1 and gene D genes, sequences of Rickettsia conorii (A), Candidatus Rickettsia barbariae (B) and Rickettsia massiliae (C) detected in Rhipicephalus turanicus ticks, in northwestern China. (A) Gene
Rickettsia (GenBank accession No.)
% Sequence similarity (bp)
17-kDa (KU364361)
Rickettsia conorii (M28480) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia rickettsii str. Morgan (CP006010) Rickettsia rickettsii str. R (CP006009) Rickettsia conorii subsp. indica (L36107) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii ITT-597 (U12460) Rickettsia conorii seven (U59730) Rickettsia conorii (HM050292) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia sibirica subsp. sibirica (KM288711) Rickettsia conorii subsp. indica (U43794) Rickettsia conorii isolate ROD (JN944636) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii (U43806) Rickettsia conorii subsp. indica (AF123726) Rickettsia conorii seven (AF123721) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii (AF149110) Rickettsia conorii subsp. indica (AF123726) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii seven (AF123721) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia conorii subsp. caspia A-167 (AY502117) Rickettsia slovaca str. D-CWPP (CP003375) Rickettsia conorii strain ATCC VR-597 (AF163005) Rickettsia conorii str. Malish 7 (AE006914) Rickettsia slovaca str. D-CWPP (CP003375)
100 (410/410) 100 (410/410) 99.75 (409/410) 99.75 (409/410) 100 (1185/1185) 100 (1185/1185) 100 (1185/1185) 100 (1076/1076) 100 (1076/1076) 100 (1076/1076) 99.62 (1072/1076) 100 (451/451) 100 (451/451) 99.11 (447/451) 99.11 (447/451) 99.60 (763/766) 100 (766/766) 100 (766/766) 99.73 (764/766) 100 (1000/1000) 99.70 (997/1000) 99.70 (997/1000) 99.84 (625/626) 99.68 (624/626) 99.52 (623/626) 100 (897/897) 99.88 (896/897) 99.33 (891/897)
16S (KU364355)
gltA (KU364366)
ompA (KU364365)
ompB1 (KU364371)
ompB2 (KU757301)
sca1 (KU364363)
gene D (KU757303)
(B) Gene
Rickettsia (GenBank accession No.)
% Sequence similarity (bp)
17-kDa (KU364358) 16S (KU364356)
Rickettsia sp. Tselentii (GU353184) Candidatus Rickettsia barbariae (EU272189) Rickettsia raoultii isolate BL029-2 (KJ410261) Rickettsia sibirica subsp. sibirica (KM28871) Rickettsia sibirica 246 (U59734) Rickettsia sp. BJ-90 citrate synthase (AF178035) Rickettsia parkeri str. Portsmouth (CP003341) Rickettsia sp. Tselentii (EU194445) Candidatus Rickettsia barbariae (EU272186) Rickettsia sp. PoTiRb169 (DQ423366) Candidatus Rickettsia barbariae (JF700253) Rickettsia parkeri str. Portsmouth (CP003341) Rickettsia parkeri (AF123717) Rickettsia sp. BJ-90 (AY331393) Candidatus Rickettsia barbariae (EU272187) Rickettsia mongolitimonae (DQ097083) Rickettsia sibirica (HM050273) Rickettsia africae ESF-5 (CP001612) Candidatus Rickettsia amblyommii strain Aca3 (AY502116) Rickettsia parkeri str. Portsmouth (CP003341) Candidatus Rickettsia barbariae (EU272188) Candidatus Rickettsia barbariae (JQ480840)
100 (399/399) 100 (1174/1174) 99.82 (1172/1174) 99.54 (1096/1101) 99.54 (1096/1101) 99.54 (1096/1101) 99.45 (1095/1101) 100 (436/436) 100 (436/436) 100 (436/436) 100 (425/425) 99.47 (751/755) 99.47 (751/755) 99.20 (749/755) 99.89 (998/999) 99.29 (992/999) 99.19 (991/999) 99.52 (622/625) 99.36 (621/625) 99.20 (620/625) 100 (917/917) 99.77 (873/875)
gltA (KU364367)
ompA (KU364364)
ompB1 (KU364369)
ompB2 (KU757300)
sca1 (KU364362)
gene D (KU757302)
(C) Gene
Rickettsia (GenBank accession No.)
% Sequence similarity (bp)
17-kDa (KU364359)
Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae strain CABA (KT032120) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae strain Mtu1 (NR 025919) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae MTU1 (U59719) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia sp. Bar 29 (U59720) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae clone 04(KR401146) Rickettsia massiliae str. AZT80 (CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae (AF123714) Rickettsia rhipicephali strain HJ#5 (CP013133) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae strain Mtu1(AY355364) Rickettsia massiliae str. AZT80(CP003319) Rickettsia massiliae MTU5 (CP000683) Rickettsia massiliae (AF163003) Rickettsia rhipicephali strain HJ#5 (CP013133)
99.74 (391/392) 99.23 (389/392) 99.23 (389/392) 100 (1182/1182) 100 (1182/1182) 99.91 (1181/1182) 100 (1118/1118) 100 (1118/1118) 99.82 (1116/1118) 99.82 (1116/1118) 100 (491/491) 100 (491/491) 99.59 (489/491) 99.86 (765/766) 99.86 (765/766) 99.08 (759/766) 100(611/611) 100(611/611) 99.18(606/611) 100 (900/900) 99.88 (899/900) 99.33 (894/900)
16S (KU364357)
gltA (KU364354)
ompA (KU757305)
ompB1 (KU364370)
sca1 (KU757304)
gene D (KU757306)
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Fig. 1. Phylogenetic analysis of spotted fever group Rickettsia spp. in Rhipicephalus turanicus in northwestern China. The tree was constructed on the basis of the ompA-gltArrs-geneD-ompB concatenated sequence of Rickettsia species by maximum likelihood (ML) and neighbor-joining (NJ) methods, using MEGA6.0. Sequences of the spotted fever group Rickettsia spp. obtained in this study are shown as “”. Sequences of R. bellii were used as the outgroup in the concatenated sequence data. The relative support for clades in the tree produced from the ML and NJ analyses are indicated above and below branches, respectively. The analysis involved 28 nucleotide sequences. The tree is drawn to scale, with branch lengths representing the number of substitutions per site.
Pathogens in the R. conorii complex comprise four subspecies: R. conorii subsp. conorii, R. conorii subsp. indica, R. conorii subsp. israelensis, and R. conorii subsp. Caspia (Zhu et al., 2005). They are known to cause Mediterranean spotted fever (MSF), Indian tick typhus (ITT), Israeli spotted fever (ISF), and Astrakhan fever (AF), respectively (Zhu et al., 2005; Torina et al., 2012). ITT is characterized by sudden onset of moderate to high-grade fever, malaise, deep muscle pain, headache, and conjunctival suffusion, together with gangrene, and severe manifestations of sepsis and multiorgan dysfunction syndrome such as acute kidney injury, liver dysfunction, delirium, seizures, and sometimes purpura fulminans (Tirumala et al., 2014). R. sanguineus, Boophilus microplus, and Haemaphysalis leachii are the main voctors of R. conorii subsp. indica, which can transmitted and spread to humans by tick bites (Zhu et al., 2005; Tirumala et al., 2014). Chochlakis et al. suggested that changing climate and geographical location (for example, in a wintering area for migratory birds) may be responsible for the possible dispersal of ticks and tick-borne pathogens (Chochlakis et al., 2014). Therefore, Rh. turanicus poses a risk for transmission of such bacteria and serves as a bridge-vector to humans. In addition, our findings suggest that Candidatus R. barbariae may become the eighth rickettsial species in China, following on from our previous work with the first detection of R. massiliae in Rh. turanicus in the northern region of XUAR (Wei et al., 2015). Furthermore, the high infection rate of ticks (17.95% and 30.76% for R. conorii subsp. indica and Candidatus R. barbariae, respectively) is strong evidence that questing Rh.
turanicus ticks from sheep are potential and emerging carriers for human rickettsiosis. Consideration should been given in China to prevention of the outbreak of such diseases, especially in shepherds because they could be the most vulnerable group. Reports showed that R. conorii subsp. indica has been identified in India, Pakistan and Laos (Zhu et al., 2005; Phongmany et al., 2006). China, the neighbor of these countries, seems to be a likely location for this pathogen, but it is unknown whether certain areas, especially the Taklamakan desert, have a connection with the endemic area of R. conorii subsp. indica or not. However, the other two rickettsial species have rarely been described in Asia. Candidatus R. barbariae was first confirmed in Rh. turanicus from Italy in 2008 and since then has been detected only in various European countries and in Israel (Mura et al., 2008; Waner et al., 2014). R. massiliae, one of the agents of human spotted fever, is distributed mainly in Europe, North and Central Africa, and the United States (Fernández de Mera et al., 2009). Our studies suggest that the coexistence of these SFG rickettsiae with public health relevance may favor transmission to humans and that work in this region may be important in exploring novel and emerging pathogens. 5. Conclusions R. conorii subsp. indica, Candidatus R. barbariae, and R. massiliae co-circulate around the Taklamakan desert. Rickettsial agents in ticks (particularly in the genus Rhipicephalus) from birds, livestock,
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wildlife and human beings should be further investigated on a large scale. Novel or emerging Rickettsia spp. may be found in this special habitat. Competing interests The authors declare that there is no conflict of interest in this study. Acknowledgments This research was supported in part by grants from the National Natural Science Foundation of China (grant Nos. 81560338 and U1503283) and the Co-innovation Center for the High Incidence of Zoonotic Disease Prevention and Control in Western China (grant No. 2013-179). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ttbdis.2016.08. 006. References Anstead, C.A., Chilton, N.B., 2013a. A novel Rickettsia species detected in vole ticks (Ixodes angustus) from Western Canada. Appl. Environ. Microbiol. 79, 7583–7589. Anstead, C.A., Chilton, N.B., 2013b. Detection of a novel Rickettsia (Alphaproteobacteria: Rickettsiales) in rotund ticks (Ixodes kingi) from Saskatchewan, Canada. Ticks Tick Borne Dis. 4, 02–06. Chochlakis, D., Ioannou, I., Papadopoulos, B., Tselentis, Y., Psaroulaki, A., 2014. Rhipicephalus turanicus: from low numbers to complete establishment in Cyprus: its possible role as a bridge-vector. Parasite Vectors 7, 1. Dantas-Torres, F., Latrofa, M.S., Annoscia, G., Giannelli, A., Parisi, A., Otranto, D., 2013. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the new and old worlds. Parasite Vectors 6, 213. ˜ Fernández de Mera, I.G., Zivkovic, Z., Bolanos, M., Carranza, C., Pérez-Arellano, J.L., Gutiérrez, C., de la Fuente, J., 2009. Rickettsia massiliae in the canary islands. Emerg. Infect. Dis. 15, 1869–1870.
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