Genetic Differentiation of Chinese Isolates of Rickettsia sibirica by Partial ompA Gene Sequencing and Multispacer Typing

Current data on rickettsiae and rickettsial diseases in Chinaremain limited. Using partial ompA gene sequencing and multispacertyping, we identified 15 rickettsial isolates from China. Allisolates were found to belong to Rickettsia sibirica subsp.sibirica. Four isolates from Dermacentor sinicus collected inBeijing, China, were fully identical to strain BJ-90, previouslydemonstrated to belong to R. sibirica subsp. sibirica despiteantigenic and genotypic specificities. All 11 remaining isolateswere similar to the R. sibirica subsp. sibirica type strain,246. These were widely distributed in China in humans and differenttick species. We emphasize the importance of surveying the distributionof R. sibirica in China.

INTRODUCTION

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Introduction

Spotted fever rickettsioses are a group of tick-, flea-, ormite-borne infections caused by spotted fever group (SFG) rickettsiae.The distribution of each rickettsiosis is directly dependenton that of its arthropod vector or vectors (16). A large-scaleepidemiological investigation of rickettsioses has been performedin China over the past 50 years. This survey has demonstrateda modification of the geographic distribution of SFG rickettsiae,from a limited focus in northern China at the beginning of thesurvey to a wide area expanding from the Xinjiang province inthe west to the Heilongjiang province in the east and from InnerMongolia in the north to Hainan island in the south and finallyto virtually all areas investigated to date (3, 4). Currently,there are three SFG rickettsiae present in China: Rickettsiasibirica subsp. sibirica, R. sibirica subsp. mongolotimonae,and R. heilongjiangensis (7). The majority of Chinese SFG isolatesbelong to the R. sibirica species.

R. sibirica contains two subspecies (12), i.e., R. sibiricasubsp. sibirica, the agent of North Asian tick typhus, and R.sibirica subsp. mongolotimonae, the agent of "lymphangitis-associatedrickettsiosis." The former subspecies was first isolated inRussia but it has subsequently been found in northern China(19). In contrast, R. sibirica subsp. mongolotimonae was firstisolated in Inner Mongolia and then found in southern Europeand Africa (9, 12). R. heilongjiangensis, first isolated fromDermacentor silvarum ticks in the Heilongjiang province of China(22), was subsequently demonstrated to cause a human spottedfever in China and the Russian Far East (15). R. hulinii wasfirst isolated from Haemaphysalis concinna ticks in the Heilongjiangprovince of China, and its pathogenic role for humans is suspectedbut has not been demonstrated as yet (22).

From 1974 to 2000, 16 rickettsial strains identified as R. sibiricawere isolated in the Chinese Center for Rickettsial Diseases,Beijing, China. Herein, in order to precisely identify these16 strains, we used partial ompA sequencing and multispacertyping (11), both tools being described as highly discriminantamong Rickettsia isolates (10, 17).

MATERIALS AND METHODS

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Materials and Methods

R. sibirica strain cultivation and DNA extraction. The 16 studied isolates were obtained from patients or ticksfrom different locations at different times (Fig. 1; Table 1).Rickettsiae were inoculated in L929 cell monolayers in Eagle'sminimal essential medium (Seromed, Berlin, Germany) supplementedwith 4% fetal bovine serum and 2 mM glutamine. They were thencultivated at 32°C in a 5% CO₂-enriched atmosphere. Bacteriawere harvested after Gimenez stain was heavily positive, andthe total genomic DNA was extracted from culture using a QIAamptissue kit (QIAGEN, Hilden, Germany) according to the manufacturer'sinstructions. Two hundred microliters of elution buffer wasused to resuspend each extracted DNA. Genomic DNAs were thenstored at –20°C until further processing.

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FIG. 1. Detailed geographic distribution of studied isolates.

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TABLE 1. Rickettsial strains studied in the present report

PCR amplification and sequencing. The primers used to amplify the ompA gene (17) and the threevariable intergenic spacers are listed in Table 2. PCR amplificationswere performed with a PE 9600 thermal cycler (Applied Biosystems).For each specimen, the 25-µl reaction mixture containedthe following: 1 to 5 µl of the appropriate DNA template,10 pM of each primer, 0.2 mM of deoxynucleoside triphosphatemixture (dATP, dCTP, dGTP, and dTTP), 0.5 U of Hot Star TaqDNA polymerase (QIAGEN), 2.5 µl of 10x Taq buffer, and1 µl of 25 mM MgCl_2. The following conditions were usedfor amplification: predenaturation for 15 min at 94°C, followedby 39 cycles of denaturation for 30 s at 94°C, annealingfor 30 s at 54°C for amplifying intergenic spacer sequencesand 53°C for the ompA gene, and extension for 1 min at 72°C.Amplification was completed by holding the reaction mixturefor 5 min at 68°C for final extension of the PCR products.Positive and negative controls were used to confirm the reagentquality and avoid manipulation errors and contamination (DNAextracted from R. sibirica strain 246 was used as the positivecontrol and sterile water as the negative control). PCR productswere purified using a QIAquick Spin PCR purification kit (QIAGEN)as described by the manufacturer. PCR products were sequencedin both directions by using PCR primers and a D-rhodamine Terminatorcycle sequencing ready reaction kit (Applied Biosystems) asdescribed by the manufacturer. Sequencing products were resolvedusing an ABI 3100 automated sequencer (Perkin-Elmer). Sequenceassembly was performed using ABI Prism DNA sequencing analysissoftware, version 3.4 (Perkin-Elmer), and multisequence alignmentwas done using CLUSTAL W software, version 1.81 (18).

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TABLE 2. List of primers used in the study

Nucleotide sequence accession numbers. The sequences for DQ008262, DQ008247, and DQ008283 have beensubmitted to GenBank.

RESULTS

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Results

Partial ompA gene sequencing. A 602-bp fragment of ompA was obtained for all strains exceptstrain NH-98, which could not be cultivated. Nucleotide sequencealignment showed that all 15 isolates were divided into twotypes (Fig. 2). Four strains, i.e., BJ-90, BJ-91, BJ-93, andBJ-95, isolated from Dermacentor sinicus ticks collected inBeijing in 1990, 1991, 1993, and 1995, respectively, had anidentical ompA nucleotide sequence (GenBank accession numberAF179365). All 11 remaining strains had a nucleotide sequenceidentical to that of R. sibirica strain 246 (GenBank accessionnumber U43807). The degrees of nucleotide and derived aminoacid sequence similarity between BJ-type isolates and all otherisolates described in this paper were 99.8 and 100%, respectively.

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FIG. 2. Unrooted tree showing the relationships among the 15 Chinese R. sibirica strains studied.

Multispacer typing. PCR amplification of the three intergenic spacers in the 15strains we tested yielded products of the expected sizes. Foreach spacer, all 15 strains exhibited identical sequences. Thenucleotide sequences of the three intergenic spacer sequenceswere found to be identical to those of R. sibirica subsp. sibirica(GenBank accession numbers DQ008262 for dksA-xerC, DQ008247for rpmE-tRNA^fMet, and DQ008283 for mppA-purC).

DISCUSSION

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Discussion

Using partial ompA gene sequencing and multispacer typing, wedemonstrated that 15 rickettsial isolates obtained from humanor ticks over a 26-year period in various areas of China belongto R. sibirica subsp. sibirica. In 1993, Yu et al., by usingPCR-restriction fragment length polymorphism, sodium dodecylsulfate-polyacrylamide gel electrophoresis, and Western blotting,demonstrated that Chinese rickettsial isolates could be classifiedwithin three types (19). Type 1 was antigenically and genotypicallyidentical to R. sibirica and was represented by isolate To-85,previously isolated from tick ova of Dermacentor nuttallii inHumeng county, Inner Mongolia, in 1985 (5). Type 2 was genotypicallyidentical to but antigenically slightly different from R. sibirica.The representative isolate was BJ-90, isolated from D. sinicusticks in the Changping district of Beijing in 1990 (7). Type3 exhibited unique genotypic and antigenic characteristics.Its representative strain, HA-91, was first isolated from Hyalommaasiaticum kozlovi olenew ticks collected from Inner Mongoliain 1991 (14). Since that work, strains BJ-90 and HA-91 havebeen demonstrated to belong to the R. sibirica species (8) andthe creation of two subspecies within R. sibirica has been proposed(12). Strain HA-91 is the type strain of R. sibirica subsp.mongolotimonae, whereas strain BJ-90 belongs to R. sibiricasubsp. sibirica. Herein, we found a great genetic homogeneityamong BJ-type isolates from the Beijing area (BJ-90, BJ-91,BJ-93, and BJ-95). These differed from the other R. sibiricaisolates, which themselves were highly homogeneous. It is importantto note that BJ-type isolates have been obtained from only onetick species, that is, D. sinicus, in a single location overdifferent years (2, 20, 21). However, it is not clear whetherthe Changping area of Beijing is the only area of distributionof this rickettsia, as few other rickettsia isolates from D.sinicus ticks from other locations are available. In addition,the pathogenic status of BJ-type strains is currently unknown.

Another significant outcome of this report is that R. sibiricasubsp. sibirica is distributed nationwide in China, from theXinjiang province in the west to the Heilongjiang province inthe east and from Inner Mongolia in the north to the Guangdongprovince in southern China (13). This rickettsia is vectorizedby at least four tick species. We emphasize the fact that fourof the studied isolates were obtained from patients, thus demonstratingthe potential risk of North Asian tick typhus in many areasof China (1, 6). Further surveillance of spotted fever grouprickettsiae and rickettsioses in China will be conducted toestimate the prevalence of the disease.

ACKNOWLEDGMENTS

We thank Guy Vestris and Annick Abeille for their technicalsupport.

This paper was supported by the National Key Technologies R& D program (2003BA712A02), Ministry of Science and Technologyof China.

FOOTNOTES

* Corresponding author. Mailing address: Department of Rickettsiology, National Institute of Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping P.O. Box 5, Beijing 102206, China. Phone: 861061731692. Fax: 861061731692. E-mail: lijuanzhang2006{at}263.net.

REFERENCES

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