HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Masuzawa, T. Articles by Imai, Y. Search for Related Content PubMed PubMed Citation Articles by Masuzawa, T. Articles by Imai, Y. Agricola Articles by Masuzawa, T. Articles by Imai, Y.

New genomospecies related to Borrelia valaisiana, isolated from mammals in Okinawa archipelago, Japan

¹Department of Microbiology, and COE Program in the 21st Century, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan ²Department of Parasitology, Aichi Medical University, Aichi 480-1195, Japan ³Okinawa Prefectural Institute of Health and Environment, Okinawa 901-1202, Japan ⁴Department of Bacteriology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan

Correspondence Toshiyuki Masuzawa masuzawa{at}u-shizuoka-ken.ac.jp

Received June 3, 2003
Accepted December 22, 2003

In 2000 and 2001, a survey was conducted of Borrelia isolated from various mammals in the southernmost islands of Japan, including Okinawa (main island), Izena, Iriomote and Ishigaki. Isolates obtained from the ear tissues of Suncus murinus (10 isolates), Mus calori (four isolates), Rattus norvegicus (one isolate) and Crocidura watasei (one isolate), were characterized by RFLP of the 5S–23S rDNA intergenic spacer and sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene. While these isolates showed identical RFLPs to Borrelia valaisiana found in Korea, Taiwan and the southern and central parts of China, their RFLP patterns differed from those of B. valaisiana found in European countries, and strain Am501 isolated from Ixodes columnae in Japan. It was found that these isolates clustered with each other on a phylogenetic tree based on flagellin gene and 16S rDNA sequences, but were relatively divergent from the European B. valaisiana and strain Am501. These findings suggest that these isolates found in East Asia should be classified as a new genomospecies of Borrelia burgdorferi sensu lato.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Lyme borreliosis (Burgdorfer et al., 1982) is the most prevalent tick-borne zoonotic disease in Europe, North America and Far Eastern countries (Steere, 2001; Yanagihara & Masuzawa, 1997). The causative bacteria, Borrelia burgdorferi sensu lato, are classified into 11 genomospecies. Three genomospecies are pathogenic to humans (Wang et al., 1999). B. burgdorferi sensu stricto has been isolated in North America and Europe, while Borrelia garinii (Baranton et al., 1992) and Borrelia afzelii (Canica et al., 1993) have been isolated in Europe and East Asian countries. In Europe, these human pathogens are transmitted by Ixodes ricinus, ticks also known to carry Borrelia valaisiana (Wang et al., 1997) and Borrelia lusitaniae (Le Fleche et al., 1997), spirochaetes whose pathogenicities are still unknown (Wang et al., 1999). Furthermore, in Japan, Borrelia japonica, Borrelia tanukii and Borrelia turdi have been isolated from Ixodes ovatus, Ixodes tanuki and Ixodes turdi, respectively (Fukunaga et al., 1996a; Kawabata et al., 1993).

In Japan, the geographical distribution and enzootic transmission cycles of B. garinii and B. afzelii transmitted by Ixodes persulcatus ticks in Hokkaido and the northern half of Honshu have been well characterized (Masuzawa et al., 1991; Miyamoto & Masuzawa, 2002; Nakao et al., 1994a, c), and B. japonica carried by I. ovatus is found in Hokkaido and most of Honshu (Masuzawa et al., 1995; Nakao et al., 1994b). Furthermore, the Japanese borrelia strain Am501, which has only been isolated once, from the rare tick species Ixodes columnae (Fukunaga et al., 1996b), has been identified as B. valaisiana, a spirochaete common in Europe (Wang et al., 1997).

It was of interest to us that a Borrelia isolate obtained from a mouse (Mus calori) captured on Okinawa, the subtropical, southernmost island in Japan, had been identified by Takada et al. (2001) as a member of B. valaisiana. This was because we had previously found similar Borrelia in small, wild mammals captured in Taiwan and China (Masuzawa et al., 2000, 2001), and Ixodes nipponensis and Ixodes granulatus ticks from Korea (Masuzawa et al., 1999) and China (Masuzawa et al., 2001), respectively. The distribution, prevalence, vector ticks and reservoir hosts for Borrelia isolated in Okinawa have yet to be elucidated. The aim of this study was to determine the ecological characterization of Okinawa isolates, and the genetic relationships between Okinawa isolates and B. valaisiana-related borreliae found in China, Korea and Taiwan.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Survey sites and Borrelia isolation.

The small mammals used in this study were captured on Okinawa, Izena, Iriomote and Ishigaki using live traps (Fig. 1). The surveys were carried out in October 2000 and November 2001. Borreliae that were isolated (Takada et al., 1998) from earlobe tissues and bladder of the animals, and the midguts of fed ticks (I. granulatus) were cultivated at 30 °C in Barbour-Stoenner-Kelly II medium (Barbour, 1984). The pure cultures obtained were subjected to further characterization as described below.

View larger version (26K): [in this window] [in a new window]   Fig. 1. The surveyed area in the Okinawa islands, Japan. The following numbers indicate trap sites on Okinawa: 1, Kunigami; 2, Ogimi; 3, Higashi; 4, Nago; 5, Gushikawa; 6, Okinawa; 7, Ozato; 8, Naha.

5S–23S rDNA intergenic spacer, 16S rDNA and flagellin gene sequences.

Primers used for PCR (listed in Table 1) were synthesized by a custom oligonucleotide synthesis service (Lifetec Oriental). For DNA sequence analysis, the same sequence primer, labelled with Texas red (Proligo), was used. PCR was performed as described previously (Masuzawa et al., 1996; Postic et al., 1994) and the amplicons were purified on DNA purification columns (Microcon-PCR; Millipore). DNA cycle sequencing was performed using the Thermo Sequenase pre-mixed cycle sequencing kit (Amersham Biosciences) according to the manufacturer's instructions. DNA sequences were determined using a model SQ5500EL DNA-sequencer (Hitachi). RFLP analysis of the 5S–23S rDNA intergenic spacer was performed on the basis of the DNA sequences obtained using Genetyx-Mac software (Hitachi). CLUSTAL X software (Thompson et al., 1997) was used to align the sequences, and phylogenetic distances were calculated using the neighbour-joining method (Saitou & Nei, 1987). Phylogenetic trees were drawn using N-J plot software.

Nucleotide sequence accession numbers.

The nucleotide sequences of the 5S–23S rDNA intergenic spacer, flagellin gene and 16S rDNA determined in this study, have been submitted to the DDBJ, EMBL and GenBank databases (see Table 3 and Figs 2 and 3).

View larger version (37K): [in this window] [in a new window]   Fig. 2. Phylogenetic tree based on flagellin gene sequences. Okinawa isolates are indicated in bold type. Isolates from China, Taiwan and Korea, indicated in parentheses, are abbreviated as C, T and K, respectively. Numbers in parentheses show accession numbers. The bar indicates a sequence divergence of 1.0 %.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Phylogenetic tree based on 16S rDNA sequences. Okinawa isolates are indicated in bold type. Isolates from China and Korea, indicated in parentheses, are abbreviated C and K, respectively. Numbers in parentheses show accession numbers. The bar indicates a sequence divergence of 0.2 %.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Isolation of Borrelia

An examination of 217 mammals belonging to four rodent species, two insectivore species and a small carnivore species for the presence of Borrelia revealed a total of 16 positive culture samples under dark-field microscopic observation (Table 2). All Borrelia isolates were obtained from ear tissues, and not from the bladder. The overall prevalence rate was 7.4 %. Borrelia were cultured from 10 of 111 Suncus murinus from Izena and Okinawa, four of 44 Mus calori from Okinawa, one of 13 Rattus norvegicus and one of two Crocidura watasei from Izena. The Borrelia isolates were detected by dark-field microscopy within 4 weeks following inoculation of the cultures. Furthermore, two Borrelia strains were isolated from fed I. granulatus ticks. One Borrelia isolate (strain OG1/01) was cultured from larvae that fed on a C. watasei that was confirmed to be spirochaete-positive, while the other isolate (strain OG45/01) was obtained from nymphal I. granulatus that fed on culture-negative S. murinus (Table 3).

RFLP analysis and sequencing of the 5S–23S rDNA intergenic spacer

Sixteen of the 18 spirochaete-positive cultures obtained from I. granulatus ticks and small mammals were subjected to RFLP analysis. In addition, 250 bp DNA fragments from all of the cultures were amplified by the intergenic spacer PCR system. The results of RFLP analysis of the 5S–23S rDNA intergenic spacer sequences are summarized in Table 3. Exact fragment sizes generated after digestion with the restriction enzymes DraI and MseI were determined by sequence analysis of the amplicons. All the strains tested generated fragment patterns identical to those of B. valaisiana-related isolates obtained from Korea, China and Taiwan. In addition, these RFLP patterns differed from the B. valaisiana type strain VS116^T found in Europe and Japanese Am501 isolated from I. columnae. It was also found that the Borrelia strains from Okinawa were more closely related (91.5–99.6 %) to strains belonging to the B. valaisiana-related genomic group isolated in East Asia or to B. valaisiana VS116^T (91.9–94.5 %), than to Am501 (89.1–91.2 %).

Borrelia isolated from a larval I. granulatus (strain OG1/01) that fed on the insectivore C. watasei showed an identical RFLP profile to the isolates (strain OC1/01) obtained from the host animal. Furthermore, strain OG45/01 from a nymphal I. granulatus fed on culture-negative S. murinus showed similar RFLP patterns to most of the Okinawa isolates.

Phylogenetic analysis based on flagellin gene sequences

A phylogenetic tree was constructed on the basis of a partial sequence (about 600 bp) similarity matrix of the flagellin gene (Fig. 2). The sequences of the Okinawa isolates showed similarity values ranging from 97.0 to 100 % to each other and to the East-Asian isolates. It was also found that B. valaisiana VS116^T and Am501 were more closely related to B. tanukii and B. afzelii than to the Okinawa isolates and the East-Asian isolates.

Phylogenetic analysis of 16S rDNA sequences

To confirm the phylogenetic relationships among the Okinawa isolates, the Asian isolates and the type strain of B. valaisiana, VS116^T, a phylogenetic tree (Fig. 3) was constructed on the basis of a sequence similarity matrix of the 16S rDNA (about 1270 bp). The highest level of sequence similarity to East-Asian isolates was exhibited by the Okinawa isolates. Furthermore, B. valaisiana VS116^T and Am501 were more closely related to B. afzelii than the East-Asian isolates on the 16S rDNA tree.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
From a survey performed in 2000 and 2001 on 217 captured small mammals, 16 Borrelia isolates were obtained from ear tissues, but not from the bladder. In a previous survey, while nine of 11 borreliae were also isolated from ear tissue of wild rodents captured in Taiwan, two isolates were obtained from bladder (Masuzawa et al., 2000). From the survey performed in China, all the isolates belonging to the B. valaisiana-related genomic group were isolated from ear tissue of wild rodents (Masuzawa et al., 2001). All isolates were identified by RFLP analysis of the 5S–23S rDNA intergenic spacer as members of the B. valaisiana-related genomic group, a group distributed in South Korea and Taiwan, as well as the Zhejiang and Sichuan provinces in China (Masuzawa et al., 1999, 2000, 2001). Further sequence analysis of 16S rDNA and the flagellin gene lead us to conclude that the Okinawa isolates are members of the B. valaisiana-related genomic group.

S. murinus found in South-East Asia (i.e. Taiwan and China) is an insectivore that prefers human habitats and fields. M. calori lives near rice and sugarcane fields in Okinawa, Taiwan, southern China and the Malay Peninsula. To our knowledge, in Taiwan, S. murinus has been identified as a reservoir host for one B. burgdorferi sensu lato isolate (Shih et al., 1998). Mus formosanus and Rattus losea are also prominent reservoir hosts in Taiwan (Masuzawa et al., 2000; Shih et al., 1998). In this study, we also recovered one isolate from R. norvegicus. It is tempting to speculate that the same genus of wild rodents might play a role for maintenance and transmission of Borrelia in Okinawa.

Blackbirds and pheasants have been identified as reservoir hosts for both B. valaisiana and B. garinii in Europe (Kirstein et al., 1997; Kurtenbach et al., 1998b). It is of interest to note that there is a correlation between the sensitivities of Borrelia for the sera of their reservoir hosts: i.e. rodent serum lyses B. valaisiana while bird serum does not (Kurtenbach et al., 1998a, 2002). The finding that B. valaisiana-related groups in East Asia are found in wild rodents and insectivores further emphasizes the phenotypic differences between B. valaisiana isolates found in Europe and Asia.

In this survey, we also found two isolates from I. granulatus ticks that had fed on small mammals, that were either positive (in C. watasei) or negative (in S. murinus) for spirochaetes at the time the cultures were initiated. No unfed I. granulatus ticks were obtained by flagging the vegetation, since all developmental stages of this tick species prefer to live near the ground where they are able to infest rodents. While this method presented some difficulties in collecting unfed ticks, we have previously successfully isolated six Borrelia strains belonging to the B. valaisiana-related group from I. granulatus, collected by flagging vegetation in Hangzhou, Zhejiang province, China (Masuzawa et al., 2001). Focusing on Okinawa, we found that our Borrelia isolates preferred I. granulatus ticks that fed on small mammals and reservoir animals, as we have previously discovered for other tick isolates found in China. Because it had been reported that this tick also inhabits the southern islands of Japan, Korea and South-East Asian countries, where the B. valaisiana-related group was found (Yamaguti et al., 1971), we speculated that the Okinawa isolates carried by I. granulatus would also be related to B. valaisiana. This is further suggested from enzootic transmission cycles established between this tick species and small mammals such as Mus, Apodemus and Rattus, and the insectivores Suncus and Crocidura.

In conclusion, Borrelia isolated from Okinawa appeared to be genetically and phenotypically more closely linked to the B. valaisiana-related genomic group that is found in East Asia than they are to those found in Europe. The classification of this group of Borrelia remains to be determined.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We thank Katsuya Taira (Okinawa Prefectural Institute of Public Health and Environment) and Hiromi Fujita (Ohara Research Laboratory), for help with the survey in Okinawa. We also thank Dennis J. Grab (Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD, USA) for reading the revised manuscript. This study was supported by Health and Labour Sciences Research Grants for Research on Emerging and Reemerging Infectious Disease (2000–2002) from the Ministry of Health, Labour and Welfare of Japan.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES