10.1186/1756-3305-5-19Parasites and Vectors511

Liu, Guang-Yuan

Luo, Jin

Shen, Hui

Tian, Mei-Yuan

Tian, Zhan-Cheng

Xie, Jun-Ren

Parasites & Vectors

English

PubMed

Abstract
          
            Background
            Rickettsioses are among both the longest known and most recently recognized infectious diseases. Although new spotted fever group rickettsiae have been isolated in many parts of the world including China, Little is known about the epidemiology of Rickettsia pathogens in ticks from Xinjiang Autonomous Region of China.
          
          
            Methods
            In an attempt to assess the potential risk of rickettsial infection after exposure to ticks in Xinjiang Uygur Autonomous Region of China, a total of 200 Dermacentor silvarum ticks collected in Xinyuan district were screened by polymerase chain reaction based on the outer membrane protein A gene.
          
          
            Results
            22 of the 200 specimens (11%) were found to be positive by PCR. Phylogenetic analysis of OmpA sequences identified two rickettsial species, Rickettsia raoultii (4.5%) and Rickettsia slovaca (6.5%).
          
          
            Conclusions
            This study has reported the occurrence of Rickettsia raoultii and Rickettsia slovaca in Xinjiang Autonomous Region of China and suggests that Dermacentor silvarum could be involved in the transmission of rickettsial agents in China. Further studies on the characterization and culture of rickettsial species found in Dermacentor silvarum should be performed to further clarify this. Additionally, the screening of human specimens for rickettsial disease in this region will define the incidence of infection.
          </jats:sec

Zhan-Cheng Tian

Guang-Yuan Liu

Hui Shen

Jun-Ren Xie

Jin Luo

Mei-Yuan Tian

Crossref

First report on the occurrence of Rickettsia slovaca and Rickettsia raoultii in Dermacentor silvarum in China

Springer - Publisher Connector

RESEARCH Open Access
First report on the occurrence of Rickettsia
slovaca and Rickettsia raoultii in Dermacentor
silvarum in China
Zhan-Cheng Tian, Guang-Yuan Liu*, Hui Shen, Jun-Ren Xie, Jin Luo and Mei-Yuan Tian
Abstract
Background: Rickettsioses are among both the longest known and most recently recognized infectious diseases.
Although new spotted fever group rickettsiae have been isolated in many parts of the world including China, Little
is known about the epidemiology of Rickettsia pathogens in ticks from Xinjiang Autonomous Region of China.
Methods: In an attempt to assess the potential risk of rickettsial infection after exposure to ticks in Xinjiang Uygur
Autonomous Region of China, a total of 200 Dermacentor silvarum ticks collected in Xinyuan district were screened
by polymerase chain reaction based on the outer membrane protein A gene.
Results: 22 of the 200 specimens (11%) were found to be positive by PCR. Phylogenetic analysis of OmpA
sequences identified two rickettsial species, Rickettsia raoultii (4.5%) and Rickettsia slovaca (6.5%).
Conclusions: This study has reported the occurrence of Rickettsia raoultii and Rickettsia slovaca in Xinjiang
Autonomous Region of China and suggests that Dermacentor silvarum could be involved in the transmission of
rickettsial agents in China. Further studies on the characterization and culture of rickettsial species found in
Dermacentor silvarum should be performed to further clarify this. Additionally, the screening of human specimens
for rickettsial disease in this region will define the incidence of infection.
Background
Tick-transmitted diseases are a focus of increasing med-
ical interest worldwide. Ticks are the main vectors and
reservoirs of rickettsial pathogens responsible for
spotted fever. Rickettsioses are among both the longest
known and most recently recognized infectious diseases.
The clinical features include fever, headache, eruption,
and incidental eschar formation at the site of tick bites
[1]. The etiological agents belonging to the genus Rick-
ettsia are currently divided into two groups: the typhus
group and the spotted fever group. The latter group
includes an increasing number of newly identified
species.
In China, many spotted fever group (SFG) rickettsiae
belong to R. sibirica, including 2 subspecies, i.e., R. sibir-
ica sibirica, the agent of North Asian tick typhus
detected in Dermacentor silvarum and D. sinicus in
northern China, and R. sibirica mongolotimonae, the
agent of lymphangitis-associated rickettsiosis isolated
from Hyalomma asiaticum in Inner Mongolia [2,3].
Rickettsia heilongjiangensis, first isolated from D. sil-
varum ticks in Heilongjiang Province, can cause spotted
fever in humans [4,5]. Rickettsia hulinii was first isolated
from Haemaphysalis concinna in Heilongjiang Province,
but its pathogenic role in humans has not been demon-
strated [6]. However, there is limited information on the
epidemiology of rickettsial species in ticks from the Xin-
jiang Uygur Autonomous Region (XUAR), China, apart
from a case report of a SFG rickettsia from a patient in
XUAR [7].
In the present study, we assessed the prevalence of
rickettsial pathogens in D. silvarum from Xinyuan dis-
trict, XUAR using molecular techniques. Identification
and characterization of these circulating agents is crucial
for the development of preventive measures in response
to the gradually increasing exposure of humans to tick
vectors.
* Correspondence: liuguangyuan2002@sina.com
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of
Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research
Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province
730046, People’s Republic of China
Tian et al. Parasites & Vectors 2012, 5:19
http://www.parasitesandvectors.com/content/5/1/19
© 2012 Tian et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Methods
Ticks and DNA extraction
A total of 200 adult female ticks were identified as D.
silvarum based on morphological characteristics [8].
Briefly, the ticks were disinfected in 70% ethanol for 10
min, rinsed with sterilized distilled water, placed in a
microtube, and mechanically disrupted with sterile scis-
sors in 50 μl of DNA extraction buffer (10 mM Tris pH
8.0, 2 mM EDTA, 0.1% sodium dodecyl sulfate, and 500
μg of proteinase K per ml). The sample was incubated
at 56°C for 4 hr, then boiled at 100°C for 10 min to
inactivate the proteinase K. After centrifugation, the
supernatant was transferred to a fresh microtube and
DNA was purified by extracting twice with an equal
volume of phenol-chloroform, precipitated in ethanol
and the DNA resuspended in 20 μl elution buffer, which
was then stored at -20°C until used.
PCR amplification and sequence analysis of ompA
PCR reactions were performed using primers Rr190.70p
and Rr190.602n (5’-ATGGCGAATATTTCTCCAAAA-
3’; 5’-AGTGCAGCATTCGCTCCCCCT-3’) designed to
amplify the outer membrane protein A (ompA) gene of
rickettsial species as described previously [9]. Distilled
water instead of tick DNA template was used as a nega-
tive control. PCR products were purified and sequenced.
These were compared with previously published
sequences deposited in GenBank using BLAST. Partial
ompA sequences of rickettsial species were aligned with
that of 27 rickettsial species by the Clustal W program
with default parameter settings (DNAStar version 4.01,
Madison, WI, USA). Outer membrane protein P44 from
Anaplasma phagocytophila (AF412830) was used as an
outlier group in the alignments of nucleotide sequences
of ompA. A phylogenetic tree was constructed using the
Kimura 2-parameter model and the neighbour-joining
algorithm of MEGA 4.0 software [10].
Results
PCR products of the rickettsial ompA gene with
expected size (530-533 bp) were amplified from D. sil-
varum ticks. Sequencing data of the 22 positive samples
indicated two distinct rickettsial species from the 200
ticks screened. Nine of these were identified as R. raou-
tii and the remaining 13 were R. slovaca. Six of the R.
raoutii samples were 100% identical to each other but
exhibited 99.1-99.8% (530/530) variability with the
remaining 3 R. raoutii samples. However, all 9 samples
of R. raoutii were 99.8-100% and 99.2-99.4% (511/511)
homologous with the R. raoultii Marne and Khabarovsk
strains respectively. Of the 13 R. slovaca samples identi-
fied, 11 were 100% identical, while the remaining 2 were
slightly divergent with a 99.6 and 99.8% (533/533)
homology. Collectively all the R. slovaca samples were
99.8-100% (533/533) homologous with the R. slovaca
(HM161798.1) strain. The variability between the two
rickettsial species identified from the ticks was 5.5-5.8%.
All of the unique sequencing data (not including identi-
cal sequences) were deposited in GenBank with acces-
sion numbers JN400401-JN400407.
Phylogenetic analysis indicated the formation of 3
clades within the rickettsial neighbour-joining tree
(Figure 1). All of R. slovaca like samples formed a single
clade with R. slovaca while the R. raoutii like samples
formed two clades. One of these clades was closely
related to the R. raoutii reference strains and the other
formed a unique R. raoutii clade which is possibly a
new strain.
Discussion
The present study reports for the first time the occur-
rence of R. raoultii and R. slovaca in D. silvarum ticks
in China. Rickettsia raoultii has been reported in Der-
macentor ticks in Europe and Russia, including strains
KhabarovskT, Marne, Shayman, 8/9 Karaganda and
Elanda-23/95 [11]. Until the present study R. raoultii
had not been definitely reported in China and only two
ompA gene sequences from two isolates from Jilin pro-
vince had previously been deposited in GenBank
(AY093696.1 and DQ188831.1), however, this was not
reported in the literature. The rickettsial strains identi-
fied in this study were defined by using published phylo-
genetic classifications of rickettsial species [3,6]. PCR
and sequencing of rickettsial ompA genes, identified R.
raoultii in 4.5% of D. silvarum ticks collected in XUAR.
Furthermore, phylogenetic analysis revealed that the
positive samples detected formed a distinct clade with R.
raoultii with a high (91) bootstrap value, which included
strains KhabarovskT and Marne. Of the nine R. raoutii
samples identified in this study, eight were from
Xinyuan and one was from Gansu province. Together
with the two samples from Jilin province (AY093696.1
and DQ188831.1). This suggests that R. raoultii has a
geographical spread that encompasses a large area of
China.
Rickettsia slovaca as a human pathogen [12] was first
isolated in 1968 from a Dermacentor marginatus tick in
Slovakia [13]. Since then, it has been found in both D.
marginatus and D. reticulatus ticks from Western Eur-
ope and central Asia [14-18]. The rickettsial disease
caused by R. slovaca is called tick-borne lymphadenopa-
thy (TIBOLA) or Dermacentor-borne necrosis-
erythema-lymphadenopathy [19,20] and its epidemiolo-
gical pattern and clinical features in patients from
France, Hungary and Spain have been investigated
[18,21]. It therefore seems to closely follow the
Tian et al. Parasites & Vectors 2012, 5:19
http://www.parasitesandvectors.com/content/5/1/19
Page 2 of 4
distribution of its main host tick, D. marginatus, which
can be found throughout Europe and as far as the wes-
tern border of China [15]. However, in the present
study, we detected R. slovaca by PCR in 6.5% of D. sil-
varum ticks collected from XUAR. The results of the
present study identified R. slovaca in the D. silvarum
ticks which could be transmitted to humans and cause
disease. Further studies on the characterization and cul-
ture of rickettsial endosymbionts found in D. silvarum
collected in XUAR should be performed.
Figure 1 Constructed phylogenetic tree based on partial ompA gene sequences from rickettsial positive tick samples using
neighbour-joining method with 1,000 bootstraps and a cut off value of 50%.
Tian et al. Parasites & Vectors 2012, 5:19
http://www.parasitesandvectors.com/content/5/1/19
Page 3 of 4
Conclusions
This is the first report of R. raoultii and R. slovaca in
China and suggests that D. silvarum could be involved
in the transmission of R. slovaca in China. Further stu-
dies on the characterization and culture of rickettsial
endosymbionts found in D. silvarum from XUAR should
be performed.
Acknowledgements
The authors thank Prof. Una Ryan and Prof. Xing-Quan Zhu for improving
the manuscript. We also thank the anonymous reviewers for their helpful
comments and suggestions. This work was supported, in part, by the Natural
Science Foundation of Gansu Province, China (No.096RJZA128) and Hi-Tech
Research and Development Program of China (No. 2009AA10Z402).
Authors’ contributions
LGY and TZC conceived and designed the study, and critically revised the
manuscript. TZC, SH and LJ performed the experiments, analysed the data
and drafted the manuscript. XJR and TMY helped in study implementation
and data collection. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 21 September 2011 Accepted: 19 January 2012
Published: 19 January 2012
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doi:10.1186/1756-3305-5-19
Cite this article as: Tian et al.: First report on the occurrence of
Rickettsia slovaca and Rickettsia raoultii in Dermacentor silvarum in
China. Parasites & Vectors 2012 5:19.
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Dermacentor-borne necrosis erythema and lymphadenopathy: clinical and epidemiological features of a new tick-borne disease. Clin Microbiol Infect

Estimation of prokaryote genomic DNA G+C content by sequencing universally conserved genes.

Fournier PE: Rickettsia raoultii sp. nov., a spotted fever group rickettsia associated with Dermacentor ticks in Europe and Russia.

Fournier PE: Spotless rickettsiosis caused by Rickettsia slovaca and associated with Dermacentor ticks. Clin Infect Dis

Genetic classification of “Rickettsia heilongjiangii“ and “Rickettsia hulinii,” two Chinese spotted fever group rickettsiae.

Genomic identification of Rickettsia slovaca among spotted fever group rickettsia isolates from Dermacentor marginatus in Armenia. Acta Virol

Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China.

HW: Isolation of a Spotted Fever Group Rickettsia from a Patient and Related Ecologic Investigations in Xinjiang Uygur Autonomous Region of China.

L: Analysis of immunoprotectivity of the recombinant OmpA of Rickettsia heilongjiangensis.

Letaief A: Characterization of rickettsial diseases in a hospitalbased population in central Tunisia.

M: MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol

PE: Genetic differentiation of Chinese isolates of Rickettsia sibirica by partial 154 ompA gene sequencing and multispacer typing.

Plikaytis BD: Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

Proteinic and genomic identification of spotted fever group rickettsiae isolated in the former USSR.

Raoult D: First isolation of Rickettsia slovaca from a patient, France. Emerg Infect Dis

Raoult D: First isolation of Rickettsia slovaca from Dermacentor marginatus in France. Am J Trop Med Hyg

Raoult D: Identification of spotted fever group rickettsiae isolated from Dermacentor marginatus and Ixodes ricinus ticks collected in Switzerland. Am J Trop Med Hyg

Rickettsia slovaca, the organism and its ecology.

Sexual dimorphism of the nymphal phase of ixodid tick species in the genus Dermacentor. Parazitologiia

Tick-borne lymphadenopathy (TIBOLA)–a new, probably rickettsial infection. Inf Dis Rev

Una nueva enfermedad transmitida por garrapatas? Enferm Infecc Microbiol Clin

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First report on the occurrence of Rickettsia slovaca and Rickettsia raoultii in Dermacentor silvarum in China

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