Ticks and -borne Diseases 7 (2016) 1146–1150

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Ticks and Tick-borne Diseases

j ournal homepage: www.elsevier.com/locate/ttbdis

Emerging group rickettsiae in ticks, northwestern

a,1 a,b,1 c d c

Li-Ping Guo , Su-Hua Jiang , Dan Liu , Shi-Wei Wang , Chuang-Fu Chen , a,∗

Yuan-Zhi Wang

a

School of Medicine, University, Shihezi 832000, Uygur Autonomous Region, China

b

Tongji Medical College, Huazhong University of Science and Technology, 430030 , China

c

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

a r t i c l e i n f o a b s t r a c t

Article history: We report conorii subsp. indica, Candidatus R. barbariae and R. massiliae in Rhipicephalus turan-

Received 9 March 2016

icus from sheep around the Taklamakan desert, northwestern China. The topology of the phylogenetic

Received in revised form 5 August 2016

trees produced from the maximum likelihood (ML) analyses of the ompA-gltA-rrs-geneD-ompB concate-

Accepted 15 August 2016

nated sequence data was very similar to that of the neighbor joining (NJ) tree, and with total support

Available online 16 August 2016

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.

Keywords:

AZT80; and R. massiliae MTU5, respectively. Studies suggest that the co-existence of these spotted fever

Rickettsia conorii

group rickettsiae is a threat to public health in China. Work is important in exploring novel and emerging

Candidatus Rickettsia barbariae .

Rhipicephalus turanicus

Northwestern China © 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 majority of the arid areas in the country, and is abundant in

tick species (Zheng et al., 2006). Tick-associated pathogens and

Infections with spotted fever group (SFG) rickettsiae have been diseases, nevertheless, are underestimated because of the tense

reported in various parts of the world over the past decades. Tick political environment and limited research, especially around the

bite events are common and cases are globally increasing (Jones Taklamakan desert, in the southern region of XUAR. Here, an inves-

et al., 2008). However, reports of tick-borne rickettsial disease as tigation was carried out to identify the ticks and Rickettsia spp. in

well as the rickettsia species involved are limited in China, espe- this region.

cially in less developed communities. To date, in China, seven

validated SFG rickettsial species have been detected in ticks: Rick-

2. Materials and methods

ettsia heilongjiangii, R. sibirica, R. raoultii, R. slovaca, R. felis, R.

aeschlimannii and R. massiliae, and cases of human infection with

During 2013–2014, a total of 133 adult ticks were collected from

rickettsiae such as R. raoultii have been reported in the last years

sheep from six sites around the Taklamakan desert (Table 1). All

(Wei et al., 2015; Jia et al., 2014). The diagnosis and treatment of

of the ticks were identified morphologically according to previous

the are difficult in the absence of molecular analy-

reports and were submitted to molecular analysis based on par-

sis because the commercial serological assays do not distinguish

tial mitochondrial 16S rDNA gene sequences (Dantas-Torres et al.,

among the SFG species (Myers et al., 2013).

2013).

As the largest province in China, Xinjiang Uygur Autonomous

The genomic DNA was extracted from each individual tick using

Region (XUAR) covers over one-sixth of the country, includes the

the TIANamp Genomic DNA Kit (TianGen, , China). Six PCR

targets, 17 kilodalton antigen (17-kDa), 16S rRNA (rrs), citrate syn-

thase (gltA), cell surface antigen 1 (sca1), outer membrane protein

∗ 1

Corresponding author at: Department of biology and immunology, A (ompA) and ompB (named ompB in this study) were assessed

School of Medicine, Shihezi University, Xinjiang Uygur Autonomous Region, Shihezi within each sample to investigate the presence of SFG rickettsiae

832000, China.

(Anstead and Chilton, 2013a,b).

E-mail addresses: [email protected] (L.-P. Guo), [email protected]

Two additional genetic markers were used to confirm the

(S.-H. Jiang), [email protected] (D. Liu), [email protected] (S.-W. Wang),

presence of the rickettsiae in ticks. Another part of the ompB

[email protected] (C.-F. Chen), [email protected] (Y.-Z. Wang).

1 2

These authors contributed equally to this work. gene (named ompB 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/).

L.-P. Guo et al. / Ticks and Tick-borne Diseases 7 (2016) 1146–1150 1147

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 R. conorii 3 (50)

Candidatus Rickettsia barbariae 2 (33.33)

◦ ◦

Qira 36 59 N–80 48 E Rh. turanicus 16 Candidatus Rickettsia barbariae 3 (18.75)

◦ ◦

Tumxuk 39 51 N–79 03 E Rh. turanicus 30 R. conorii 6 (20)

Candidatus Rickettsia barbariae 12 (40)

R. massiliae 1 (3.33)

D. marginatus 3 0

◦ ◦

Pishan 37 37 N–78 16 E Rh. turanicus 62 R. conorii 10 (16.13)

Candidatus Rickettsia barbariae 18 (29.03)

R. massiliae 1 (1.61)

D. marginatus 2 0

◦ ◦

Kuqa 41 43 N–82 57 E Rh. turanicus 3 R. conorii 2 (66.67)

Candidatus Rickettsia barbariae 1 (33.33)

Hy. asiaticum 2 0

◦ ◦

Atux 39 42 N–76 09 E D. marginatus 9 0

Total (133) Rh. turanicus 117 R. conorii 21 (17.95)

Candidatus Rickettsia barbariae 36 (30.77)

R. massiliae 2 (1.71)

D. marginatus 14 0

Hy. asiaticum 2 0

using primers ompB-out1 (5 -ACAGCTACCATAGTAGCCAG-3 ) and wise nucleotide sequence identity to R. conorii subsp. indica rrs

ompB-out2 (5 -TGCAGTATAGTTACCACCG-3 ) for the first phase, (L36107), gltA (U59730), ompA (U43794), ompB (AF123726) and R.

and ompB-inter1 (5 -TGCTGCGGCTTCTACATT-3 ) and ompB-inter2 conorii subsp. conorii sca1(AE006914), respectively. The R. massil-

(5 -ACCGCCAGCGTTCCCTAT-3 ) for the second phase. The PCR con- iae species had 99.74%–100% pairwise nucleotide sequence identity

◦

ditions consisted of an initial 5-min denaturation at 95 C, followed to genome sequences of the reference strains R. massiliae MTU5

◦ ◦ ◦

by 35 cycles at 95 C for 45 s, 56 C for 45 s, and 72 C for 2 min (accession no. CP000683) for all six genes analyzed. For Candidatus

◦ 1

30 s, with a final extension at 72 C for 5 min (first phase), and R. barbariae, however, the amplified regions of the gltA and ompB

◦

an initial 5-min denaturation at 95 C, followed by 35 cycles genes in this study were different from the existing sequences and

◦ ◦ ◦

at 95 C for 45 s, 56 C for 45 s, and 72 C for 1 min 10 s, with no sequences of the sca1 gene were available in GenBank, it showed

◦

a final extension at 72 C for 5 min. Part of the PS120-protein- 99.54%, 99.47% and 99.52% similarity with R. sibirica subsp. sibirica

encoding gene (gene D; 920 bp) fragment was amplified using (accession no. KM288711), R. parkeri str. Portsmouth (accession

1

primers gene D-F (5 -CGGTAACCTAGATACAAGTGA-3 ) and gene D- no. CP003341) and R. africae ESF-5 (CP001612) for the gltA, ompB

R (5 -TATAAGCTATTGCGTCATCTC-3 ) according to the sequences and sca1 genes, respectively. The rest of the genes of Candidatus

available in GenBank, and samples were amplified as recommended R. barbariae had 100% pairwise nucleotide sequence identity to the

(Sekeyova et al., 2001). The PCR conditions consisted of an ini- reference sequences (EU272186, JF803896 and EU272189 for ompA,

◦ ◦ 2

tial 5-min denaturation at 95 C, followed by 35 cycles at 95 C for 17-kDa and rrs, respectively; Table 2). For the ompB (1063 bp)

◦ ◦

30 s, 55 C for 30 s, and 72 C for 1 min, with a final extension at and gene D (920 bp) fragments, the BLAST results showed that the

◦

72 C for 8 min R. aeschlimannii from Rhipicephalus turanicus and R. conorii subsp. indica identified in this study had 100% pairwise

double distilled water were used, respectively, as positive and neg- nucleotide sequence identity while the Candidatus R. barbariae had

ative controls (Wei et al., 2015). The amplification products were 99.89%–100% similarity with reference sequences availiable in Gen-

purified using the TIANgel Midi Purification Kit, cloned into the Bank (Table 2).

pGEM-T Easy vector (TianGen) and then subjected to sequencing None of the ticks belonging to the species D. marginatus and

(BGI, , China). Phylogenetic trees were constructed using Hy. asiaticum was positive for rickettsiae. All the sequences

the maximum likelihood (ML) and neighbor joining (NJ) algorithms obtained in this study were deposited in GenBank under

with MEGA 6.0 (Tamura et al., 2013). Bootstrap analyses (500 repli- accession nos. KU364354–KU364359, KU364361–KU364367,

cates for ML analyses and 1000 replicates for the NJ analyses) were KU364369–KU364378, and KU757300–KU757306. The topology

conducted to determine the relative support for clades in the con- of the phylogenetic trees produced from the ML analyses of the

2

sensus trees (Pattengale et al., 2010; Anstead and Chilton, 2013a,b). ompA, gltA, gene D, and ompB 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%

3. Results

bootstrap values for the inclusion of the rickettsiae in Rh. turanicus

within the clade that contained R. conorii subsp. indica; Candidatus

A total of 117 Rh. turanicus ticks (80 males, 37 females), 14

R. barbariae and rickettsia sp. Tselentii; R. massiliae str. AZT80

Dermacentor marginatus (9 males, 5 females) and two Hyalomma

and R. massiliae MTU5, respectively (Technical Appendix A in

asiaticum (females) were collected in questing areas. Phyloge-

Supplementary material, Fig. 1).

netic analyses based on the partial mitochondrial 16S rDNA gene

sequences are shown in Technical Appendix A in Supplementary

material. 4. Discussion

The ticks were screened first by ompA PCR, and positive sam-

ples were analyzed for all targeted genes. The results showed that We report the presence of R. conorii subsp. indica in Rh. turanicus

59 (44.36%) of the 133 ticks analyzed were positive for Rickettsia ticks for the first time, and identify Candidatus R. barbariae and R.

spp. Of these, 36 (30.76%) were confirmed as Candidatus R. bar- massiliae in Rh. turanicus from sheep in China. The co-circulation of

bariae, 21 (17.95%) as R. conorii subsp. indica, and two (1.71%) these rickettsial species around the Taklamakan desert increases

as R. massiliae in 117 Rh. turanicus ticks (Table 1). The R. conorii the known range of vectors and reservoirs for SFG rickettsiae and

subsp. indica showed 100%, 100%, 100%, 99.60%, and 99.84% pair- provides a basis for assessing the risk of infection in humans.

1148 L.-P. Guo et al. / Ticks and Tick-borne Diseases 7 (2016) 1146–1150

Table 2

1 2

Most closely related sequences to the partial 17-kDa, 16S, gltA, ompA, ompB , ompB , sca1 and gene D genes, sequences of (A), Candidatus Rickettsia barbariae

(B) and (C) detected in Rhipicephalus turanicus ticks, in northwestern China.

(A)

Gene Rickettsia (GenBank accession No.) % Sequence similarity (bp)

17-kDa (KU364361) Rickettsia conorii (M28480) 100 (410/410)

Rickettsia conorii str. Malish 7 (AE006914) 100 (410/410)

Rickettsia rickettsii str. Morgan (CP006010) 99.75 (409/410)

Rickettsia rickettsii str. R (CP006009) 99.75 (409/410)

16S (KU364355) Rickettsia conorii subsp. indica (L36107) 100 (1185/1185)

Rickettsia conorii str. Malish 7 (AE006914) 100 (1185/1185)

Rickettsia conorii ITT-597 (U12460) 100 (1185/1185)

gltA (KU364366) Rickettsia conorii seven (U59730) 100 (1076/1076)

Rickettsia conorii (HM050292) 100 (1076/1076)

Rickettsia conorii str. Malish 7 (AE006914) 100 (1076/1076)

Rickettsia sibirica subsp. sibirica (KM288711) 99.62 (1072/1076)

ompA (KU364365) Rickettsia conorii subsp. indica (U43794) 100 (451/451)

Rickettsia conorii isolate ROD (JN944636) 100 (451/451)

Rickettsia conorii str. Malish 7 (AE006914) 99.11 (447/451)

Rickettsia conorii (U43806) 99.11 (447/451)

1

ompB (KU364371) Rickettsia conorii subsp. indica (AF123726) 99.60 (763/766)

Rickettsia conorii seven (AF123721) 100 (766/766)

Rickettsia conorii str. Malish 7 (AE006914) 100 (766/766)

Rickettsia conorii (AF149110) 99.73 (764/766)

2

ompB (KU757301) Rickettsia conorii subsp. indica (AF123726) 100 (1000/1000)

Rickettsia conorii str. Malish 7 (AE006914) 99.70 (997/1000)

Rickettsia conorii seven (AF123721) 99.70 (997/1000)

sca1 (KU364363) Rickettsia conorii str. Malish 7 (AE006914) 99.84 (625/626)

Rickettsia conorii subsp. caspia A-167 (AY502117) 99.68 (624/626)

Rickettsia slovaca str. D-CWPP (CP003375) 99.52 (623/626)

gene D (KU757303) Rickettsia conorii strain ATCC VR-597 (AF163005) 100 (897/897)

Rickettsia conorii str. Malish 7 (AE006914) 99.88 (896/897)

Rickettsia slovaca str. D-CWPP (CP003375) 99.33 (891/897)

(B)

Gene Rickettsia (GenBank accession No.) % Sequence similarity (bp)

17-kDa (KU364358) Rickettsia sp. Tselentii (GU353184) 100 (399/399)

16S (KU364356) Candidatus Rickettsia barbariae (EU272189) 100 (1174/1174)

Rickettsia raoultii isolate BL029-2 (KJ410261) 99.82 (1172/1174)

gltA (KU364367) subsp. sibirica (KM28871) 99.54 (1096/1101)

Rickettsia sibirica 246 (U59734) 99.54 (1096/1101)

Rickettsia sp. BJ-90 citrate synthase (AF178035) 99.54 (1096/1101)

Rickettsia parkeri str. Portsmouth (CP003341) 99.45 (1095/1101)

ompA (KU364364) Rickettsia sp. Tselentii (EU194445) 100 (436/436)

Candidatus Rickettsia barbariae (EU272186) 100 (436/436)

Rickettsia sp. PoTiRb169 (DQ423366) 100 (436/436)

Candidatus Rickettsia barbariae (JF700253) 100 (425/425)

1

ompB (KU364369) str. Portsmouth (CP003341) 99.47 (751/755)

Rickettsia parkeri (AF123717) 99.47 (751/755)

Rickettsia sp. BJ-90 (AY331393) 99.20 (749/755)

2

ompB (KU757300) Candidatus Rickettsia barbariae (EU272187) 99.89 (998/999)

Rickettsia mongolitimonae (DQ097083) 99.29 (992/999)

Rickettsia sibirica (HM050273) 99.19 (991/999)

sca1 (KU364362) ESF-5 (CP001612) 99.52 (622/625)

Candidatus Rickettsia amblyommii strain Aca3 (AY502116) 99.36 (621/625)

Rickettsia parkeri str. Portsmouth (CP003341) 99.20 (620/625)

gene D (KU757302) Candidatus Rickettsia barbariae (EU272188) 100 (917/917)

Candidatus Rickettsia barbariae (JQ480840) 99.77 (873/875)

(C)

Gene Rickettsia (GenBank accession No.) % Sequence similarity (bp)

17-kDa (KU364359) Rickettsia massiliae MTU5 (CP000683) 99.74 (391/392)

Rickettsia massiliae strain CABA (KT032120) 99.23 (389/392)

Rickettsia massiliae str. AZT80 (CP003319) 99.23 (389/392)

16S (KU364357) Rickettsia massiliae MTU5 (CP000683) 100 (1182/1182)

Rickettsia massiliae strain Mtu1 (NR 025919) 100 (1182/1182)

Rickettsia massiliae str. AZT80 (CP003319) 99.91 (1181/1182)

gltA (KU364354) Rickettsia massiliae MTU5 (CP000683) 100 (1118/1118)

Rickettsia massiliae MTU1 (U59719) 100 (1118/1118)

Rickettsia massiliae str. AZT80 (CP003319) 99.82 (1116/1118)

Rickettsia sp. Bar 29 (U59720) 99.82 (1116/1118)

ompA (KU757305) Rickettsia massiliae MTU5 (CP000683) 100 (491/491)

Rickettsia massiliae clone 04(KR401146) 100 (491/491)

Rickettsia massiliae str. AZT80 (CP003319) 99.59 (489/491)

1

ompB (KU364370) Rickettsia massiliae MTU5 (CP000683) 99.86 (765/766)

Rickettsia massiliae (AF123714) 99.86 (765/766)

Rickettsia rhipicephali strain HJ#5 (CP013133) 99.08 (759/766)

sca1 (KU757304) Rickettsia massiliae MTU5 (CP000683) 100(611/611)

Rickettsia massiliae strain Mtu1(AY355364) 100(611/611)

Rickettsia massiliae str. AZT80(CP003319) 99.18(606/611)

gene D (KU757306) Rickettsia massiliae MTU5 (CP000683) 100 (900/900)

Rickettsia massiliae (AF163003) 99.88 (899/900)

Rickettsia rhipicephali strain HJ#5 (CP013133) 99.33 (894/900)

L.-P. Guo et al. / Ticks and Tick-borne Diseases 7 (2016) 1146–1150 1149

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-gltA-

rrs-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: turanicus ticks from sheep are potential and emerging carriers for

R. conorii subsp. conorii, R. conorii subsp. indica, R. conorii subsp. human rickettsiosis. Consideration should been given in China to

israelensis, and R. conorii subsp. Caspia (Zhu et al., 2005). They are prevention of the outbreak of such diseases, especially in shepherds

known to cause Mediterranean spotted fever (MSF), Indian tick because they could be the most vulnerable group.

(ITT), Israeli spotted fever (ISF), and Astrakhan fever (AF), Reports showed that R. conorii subsp. indica has been iden-

respectively (Zhu et al., 2005; Torina et al., 2012). ITT is character- tified in India, Pakistan and Laos (Zhu et al., 2005; Phongmany

ized by sudden onset of moderate to high-grade fever, malaise, deep et al., 2006). China, the neighbor of these countries, seems to be a

muscle pain, headache, and conjunctival suffusion, together with likely location for this pathogen, but it is unknown whether certain

gangrene, and severe manifestations of sepsis and multiorgan dys- areas, especially the Taklamakan desert, have a connection with the

function syndrome such as acute kidney injury, liver dysfunction, endemic area of R. conorii subsp. indica or not. However, the other

delirium, seizures, and sometimes purpura fulminans (Tirumala two rickettsial species have rarely been described in Asia. Candi-

et al., 2014). R. sanguineus, Boophilus microplus, and datus R. barbariae was first confirmed in Rh. turanicus from Italy in

leachii are the main voctors of R. conorii subsp. indica, which can 2008 and since then has been detected only in various European

transmitted and spread to humans by tick bites (Zhu et al., 2005; countries and in Israel (Mura et al., 2008; Waner et al., 2014). R.

Tirumala et al., 2014). Chochlakis et al. suggested that changing cli- massiliae, one of the agents of human spotted fever, is distributed

mate and geographical location (for example, in a wintering area mainly in Europe, North and Central Africa, and the United States

for migratory birds) may be responsible for the possible dispersal (Fernández de Mera et al., 2009). Our studies suggest that the co-

of ticks and tick-borne pathogens (Chochlakis et al., 2014). There- existence of these SFG rickettsiae with public health relevance may

fore, Rh. turanicus poses a risk for transmission of such and favor transmission to humans and that work in this region may be

serves as a bridge-vector to humans. In addition, our findings sug- important in exploring novel and emerging pathogens.

gest that Candidatus R. barbariae may become the eighth rickettsial

species in China, following on from our previous work with the

5. Conclusions

first detection of R. massiliae in Rh. turanicus in the northern region

of XUAR (Wei et al., 2015). Furthermore, the high infection rate of

R. conorii subsp. indica, Candidatus R. barbariae, and R. massiliae

ticks (17.95% and 30.76% for R. conorii subsp. indica and Candida-

co-circulate around the Taklamakan desert. Rickettsial agents in

tus R. barbariae, respectively) is strong evidence that questing Rh.

ticks (particularly in the genus Rhipicephalus) from birds, livestock,

1150 L.-P. Guo et al. / Ticks and Tick-borne Diseases 7 (2016) 1146–1150

wildlife and human beings should be further investigated on a large Jia, N., Zheng, Y.C., Ma, L., Huo, Q.B., Ni, X.B., Jiang, B.G., 2014. Human infections

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Competing interests

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Rickettsial infections and fever vientiane, Laos. Emerg. Infect. Dis. 12, 256–262.

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