INTRODUCTION

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Lyme disease is an emerging tick-borne spirochetal infection (13) that can cause multisystem human illness with various degrees of clinical symptoms among infected persons, ranging from a relatively benign skin lesion to severe arthritic, neurologic, and cardiac manifestations (36, 37). The etiologic agent of Lyme disease, Borrelia burgdorferi sensu lato, is transmitted mainly by ticks of the Ixodes ricinus complex in North America and Europe (25, 35) and by Ixodes persulcatus and Ixodes ovatus ticks in the countries of Far East Asia (2, 19, 26). Although the first laboratory-confirmed case of human Lyme disease had been reported in Taiwan (33), the strain of spirochetes and the tick vector responsible for transmission in Taiwan remain undefined.

The diversity of molecular and immunological characteristics among isolates of B. burgdorferi sensu lato from different regions of endemicity has been demonstrated previously (1, 7, 20, 21, 24, 38). On the basis of immunoreactivity with B. burgdorferi-specific monoclonal antibodies (MAbs), plasmid profiles, and the clinical manifestations of the patient, the causative agents of Lyme disease can be classified into three major genospecies, i.e., B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii (group VS461) (5, 17). In addition, analysis of genetic similarities among isolates by PCR with species-specific primer sets has been proven to be useful for the typing or species identification of Borrelia isolates from new geographical areas (22, 23, 29).

The prevalence of spirochetal infection among small mammals had been surveyed in Taiwan, and spirochetes can be isolated from six species of rodents (31). However, the protein and genetic similarities of these isolates have not been compared with those of the known species of Lyme disease spirochetes. Thus, the intent of the present study was to characterize the antigenic determinants of Taiwan isolates by analyzing the protein profiles and reactivities with MAbs against outer surface proteins (Osps) of B. burgdorferi, and attempts to identify the genospecies of Taiwan isolates were also made by investigating their differential primer reactivities in a PCR assay.

	MATERIALS AND METHODS

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Isolation of spirochetes. For the isolation of spirochetes, rodents from the northern (Taipei and Ilan counties), southern (Chiayi County), western (Taichung County), eastern (Hualian and Taitung County), and offshore island (Kimmen County) areas of Taiwan were trapped from May to December 1996. Ear tissues from each rodent including 55 brown country rats (Rattus losea Swinhoe), 31 black rats (Rattus rattus Linnaeus), 67 brown rats (Rattus norvegicus Erxleben), 22 house shrews (Suncus murinus Linnaeus), 74 bandicoot rats (Bandicota indica Hodgson), and 22 Formosan field mice (Apodemus semutus Thomas) were collected, stored at 4°C, and subsequently transferred to the laboratory for cultivation of spirochetes. Briefly, ear tissues were washed in 70% ethanol and were rinsed in sterile phosphate-buffered saline (PBS) before transfer to a culture tube (D51588; Sarstedt, Nümbrecht, Germany) containing 5 ml of BSK-H medium (catalog no. B3528; Sigma Chemical Co., St. Louis, Mo.) supplemented with 6% rabbit serum (catalog no. R7136; Sigma) as described previously (32). After incubation at 34°C in a humidified incubator (Nuaire, Inc., Plymouth, Minn.) with 5% CO₂, all tissue cultures were examined weekly for 8 weeks for evidence of spirochetes by dark-field microscopy (model BX-60; Olympus Co., Tokyo, Japan).

Purification of spirochetes. For purification of cultivable spirochetes, spirochete-positive cultures were transferred to new culture tubes by serial dilution. One week after passage, the spirochete cultures were further filtered with a 0.45-µm-pore-size syringe filter (Sartorius, Göttingen, Germany) and were diluted in several tubes of fresh BSK-H medium as described previously (16). Axenic cultures of spirochetes were examined every 3 days for 3 weeks by dark-field microscopy. When spirochetes were observed in the medium without bacterial contaminants, pure isolates were subcultured and were used for further analysis. A total of seven isolates were purified from three species of rodents captured on Kimmen Island of Taiwan (Table 1). Additional isolates from North America, Europe, and Japan were included for comparison (Table 2).

MAbs. Five murine MAbs were obtained as undiluted hybridoma supernatants from Alan G. Barbour (Department of Microbiology and Medicine, University of Texas Health Science Center, San Antonio). MAbs H5332 and H3TS are specific for OspA of B. burgdorferi (6, 8), MAbs H6831 and H614 are specific for OspB (7), and MAb H9724 reacts with a protein of the periplasmic flagella of the genus Borrelia (9). In addition, an MAb against the p39 protein (30) was obtained from Tom G. Schwan (Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Mont.) and was used to identify the positions of the respective antigens.

SDS-PAGE. For protein analysis, whole-cell lysates of cultured spirochetes were prepared from Borrelia isolates from Taiwan (isolates TWKM1 to TWKM7), B. burgdorferi sensu stricto (strains B31 and JD1), B. garinii (strain K48), and B. afzelii (strain VS461). All Taiwan isolates used in this study were used after only three to five serial passages following the original isolation. Briefly, spirochetes were cultured in BSK-H medium supplemented with 6% rabbit serum and were grown to a density of ~2 × 10⁸ cells per ml of medium. After proper centrifugation, harvested cells were washed three times with PBS (pH 7.2) containing 5 mM MgCl₂ and were resuspended in sodium dodecyl sulfate (SDS)-sample buffer (62.5 mM Tris-Cl [pH 6.8], 2% SDS, 50 mM dithiothreitol, 10% glycerol, 0.004% bromophenol blue) as described previously (12). The prepared samples were boiled for 5 min, and the aliquoted antigens were stored at 20°C. For protein electrophoresis, each lane was loaded with 5 to 10 µg of protein antigens and was subjected to continuous SDS-polyacrylamide gel electrophoresis (PAGE) on 12.5% gels (PhastGel; Pharmacia Biotech, Taipei, Taiwan) with a minigel electrophoresis apparatus (PhastSystem; Pharmacia Biotech) according to the instructions of the manufacturer. The protein profiles of the major spirochetal protein bands were stained and visualized by Coomassie brilliant blue staining (PhastGel Blue R; Pharmacia Biotech). The molecular masses of the major spirochetal protein bands were calculated by comparing their electrophoretic mobilities with those of molecular mass standards (14- to 94-kDa marker; Pharmacia Biotech).

Western blot analysis. Electrophoresed protein antigens were transferred from the SDS-polyacrylamide gels to nitrocellulose blotting membranes (Sartorius) with a semidry electroblotter (PhastTransfer electrode cassette; Pharmacia Biotech). The transferred membranes were blocked for 2 h with 3% gelatin in Tris-buffered saline (TBS; pH 7.5) containing 20 mM Tris and 500 mM NaCl and were then incubated for 2 h at room temperature with a 1:40 dilution of hybridoma supernatant containing MAbs against OspA (MAbs H5332 and H3TS), OspB (MAbs H6831 and H614), or flagellin (MAb H9724) proteins of B. burgdorferi. An MAb against the p39 protein was incubated at a dilution of 1:20. After being washed with buffer solution, the membranes were immersed for 2 h in horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G (catalog no. NA931; Amersham, Little Chalfont, Buckinghamshire, England) diluted 1:500 with 0.05% Tween 20 in TBS (T-TBS) as described previously (4, 26). The reacted membranes were then washed twice with T-TBS, a substrate solution (10 ml of methanol containing 30 mg of 4-chloro-1-naphthol and 25 µl of 30% hydrogen peroxide was mixed with 50 ml of TBS) was added to develop the color for 5 to 10 min, and the reacted membranes were washed with distilled water and air dried for analysis of spirochetal protein bands.

DNA extraction and plasmid analysis. Total genomic DNA from all Borrelia strains was extracted as described previously (15, 27). Briefly, samples (3 ml) of cultured spirochetes were grown to a density of ~2 × 10⁸ cells per ml of medium and were centrifuged for 10 min at 12,000 × g to pellet the spirochetes. The pellets were washed twice with PBS (pH 7.2) containing 5 mM MgCl₂, resuspended in 150 µl of distilled water, and boiled for 10 min. After centrifugation at 10,000 × g for 10 s, the supernatant was collected and the DNA concentrations were determined spectrophotometrically by using a DNA calculator (GeneQuant II; Pharmacia Biotech).

PCR analysis. DNA samples extracted from the Taiwan isolates and other spirochetes representative of the three genospecies of B. burgdorferi sensu lato were used for PCR analysis to identify the genospecies of Taiwan isolates, as described previously (28). Five sets of PCR primers were synthesized and used in the present study (Table 3). Primer sets a and c were designed to amplify DNA of the North American type (B31) and all species (universal) of Lyme disease spirochetes, respectively (29). Other primer sets, BB, BG, and VS461, were designed to amplify the DNAs of B. burgdorferi sensu stricto, B. garinii, and B. afzelii (group VS461), respectively (22). All PCR reagents and TaqGold DNA polymerase were obtained from the GeneAmp kit and were used as recommended by the supplier (Perkin-Elmer Cetus, Taipei, Taiwan).

	RESULTS

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The protein profiles of spirochetal isolates from Taiwan were compared with those of other genospecies of B. burgdorferi and were demonstrated by SDS-PAGE. Although the seven Taiwan isolates (isolates TWKM1 to TWKM7) had protein bands of various sizes ranging from 24 to 67 kDa, the protein profiles of these isolates were consistent with that for B. burgdorferi sensu lato (Fig. 1). Two dominant stained protein bands with molecular masses of approximately 31 and 41 kDa were observed for all Taiwan isolates and were presumptively identified as the OspA and flagellin proteins of Lyme disease spirochetes, respectively. The protein profiles of isolates TWKM5 to TWKM7 are identical to that of the North American B31 strain of B. burgdorferi. However, one major protein band with a molecular mass of approximately 34 kDa (OspB) was distinctive when compared with the bands for isolates TWKM1 to TWKM4 (Fig. 1). All of the Taiwan isolates also contained the other two dominant protein bands with molecular masses of approximately 39 and 65 to 67 kDa. These results indicate that the spirochetal isolates from Taiwan are closely related to the causative agent of Lyme disease, B. burgdorferi sensu lato.

View larger version (82K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of whole-cell lysates of Borrelia isolates. The 12.5% gel was revealed by Coomassie brilliant blue staining. Lane B, American type strain B31 (control); lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii. Molecular mass standards (M) are provided on the left (in kilodaltons). Arrows identify the OspA, OspB, and flagellin proteins of B. burgdorferi sensu lato.

Western immunoblot analysis was also performed to determine whether the identities of these Taiwan isolates could be confirmed by the presence of several antigenic proteins that were known to be correlated with B. burgdorferi sensu lato from different geographical areas. Thus, spirochetal isolates from Taiwan were assayed with B. burgdorferi-specific MAbs, and the B31 strain as well as other strains of spirochetes belonging to the three major genospecies were used for comparison. All of the Taiwan isolates have protein bands that react intensely with MAbs H5332 and H9724, which correspond to the OspA and flagellin proteins of B. burgdorferi, respectively (Fig. 2). Only isolates TWKM5 to TWKM7 and B31 reacted with MAb H6831, which reacts with the OspB protein of B. burgdorferi. Immunoreactivities with MAbs H3TS, H614, and anti-P39 were also observed in all of the Taiwan isolates. These results reveal that the spirochetal isolates from Taiwan are serologically related to the B31 and JD1 strains of spirochetes and they are presumptively identified as B. burgdorferi sensu lato.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Western immunoblot analysis of Borrelia isolates with MAbs against OspA (H5332 and H3TS), OspB (H6831 and H614), flagellin (H9724), and p39 (anti-p39) proteins of B. burgdorferi sensu lato. Lane B, American type strain B31; lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii.

Plasmid profile analysis of the Taiwan isolates also revealed that the Taiwan isolates have profiles consistent with the profile of B. burgdorferi sensu lato, and variable numbers and sizes of these extrachromosomal elements were detected in the different isolates from various geographical sources and genospecies (Fig. 3). Two distinct groups of plasmid profiles were evident among the seven Taiwan isolates. The plasmid profiles of isolates TWKM1 to TWKM4 contained six major plasmid elements ranging from 15 to 48.5 kb and were consistent with the plasmid profiles of JD1 spirochetes. In contrast, the plasmid profiles of isolates TWKM5 to TWKM7 contain only four major plasmid elements and were consistent with the plasmid profiles of B31 spirochetes. When compared with the B31 type strain, four dominant stained plasmid bands (approximately 15, 16, 18.2, and 48.5 kb) were observed in all of the Taiwan isolates. These results indicate that the plasmid profiles of Taiwan isolates can be related to the protein profiles and Western immunoblot analysis results for Taiwan isolates and that these Taiwan isolates are closely related to the genospecies of B. burgdorferi sensu stricto.

View larger version (90K): [in this window] [in a new window]   FIG. 3.   Plasmid profiles of Borrelia isolates from Taiwan and three genospecies of Lyme disease spirochetes. Lane B, American type strain B31; lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii; lanes M, the high-molecular-mass DNA markers (Gibco BRL).

PCR analysis with the genospecies-specific primers was also performed to confirm the identities of the seven Taiwan isolates. Additional isolates of spirochetes of different geographical origins and genospecies were examined by using the PCR primers for the type strains (Table 2). All of the Taiwan isolates and type strains (strains B31, JD1, K48, and VS461) can be amplified with primer set c (universal primers), with a DNA fragment of approximately 127 bp being found on a 1.5% agarose gel (Fig. 4a). With primer set a (B31 primers), only Taiwan isolates and the genospecies of B. burgdorferi sensu stricto (B31 and JD1 strains) were amplified, with a DNA fragment of approximately 374 bp being found (Fig. 4b). In addition, all of the Taiwan isolates and spirochetes of B. burgdorferi sensu stricto (strains B31 and JD1) were also amplified by primer set BB, with a DNA fragment of approximately 575 bp being found (Fig. 5a). However, primer sets BG and VS461 amplified DNA only from the genospecies of B. garinii (strain K48) and B. afzelii (strain VS461), with DNA fragments of approximately 575 and 590 bp, respectively, being found (Fig. 5b and c, respectively). These results indicate that all of the Taiwan isolates were identified as B. burgdorferi sensu stricto.

View larger version (58K): [in this window] [in a new window]   FIG. 4.   Amplification specificities of the universal-type (a) and B31 (b) primer sets with Borrelia isolates from Taiwan and three genospecies of Lyme disease spirochetes. Lane B, B31 isolate; lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii; lanes M, 100-bp DNA ladder (GIBCO BRL). The amplification products for the universal-type (a) and B31 (b) primers were DNA fragments with size of 127 and 374 bp, respectively.

View larger version (54K): [in this window] [in a new window]   FIG. 5.   Amplification specificities of the primer sets of BB (a), BG (b), and VS461 (c) with Borrelia isolates from Taiwan and three genospecies of Lyme disease spirochetes. Lane B, B31 isolate; lanes 1 to 7, Taiwan isolates TWKM1 TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii; lanes M, 100-bp DNA ladder (Gibco BRL). The expected amplification products for primer sets BB, BG, and VS461 were DNA fragments with sizes of 575, 575, and 590 bp, respectively.

	DISCUSSION

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Our report describes the first identification and characterization of the Lyme disease spirochete, B. burgdorferi sensu lato, isolated from rodents in Taiwan. In our previous investigations, spirochetes could be cultured from six species of wild and peridomestic rodents, and the prevalence of spirochetal infection ranged from 4.5 to 36.4% among those captured rodents (31). Thus, the evidence of zoonotic transmission of Lyme disease spirochetes in Taiwan was proved. However, the perpetuation of Lyme disease spirochetes in nature would require a competent tick vector for maintenance of the natural cycle of transmission. Further investigations on the isolation and characterization of spirochetes from the possible tick vectors would help to elucidate the enzootic transmission cycle of Borrelia spirochetes in Taiwan.

The identity of a Borrelia isolate can be classified according to the protein profiles and immunoreactivities with B. burgdorferi-specific MAbs. Although the diversity of major Osps had been demonstrated in Lyme disease isolates from various geographical areas (8, 38), almost all Borrelia isolates reacted with a genus-specific antiflagellar MAb (MAb H9724) (9), and seroreactivity with an MAb against OspA (MAb H5332) was also evident in most of the spirochetal isolates that cause Lyme disease (6). In the present study, all of the Taiwan isolates reacted intensely with MAbs against the OspA (H5332) and flagellin (H9724) proteins of B. burgdorferi. On the basis of serological similarity, all of the Taiwan isolates had profiles that were consistent with that of the causative agent of Lyme disease, B. burgdorferi sensu lato.

The heterogeneity among major protein bands in various spirochetal isolates may be correlated with their biological and geographical origins. Different compositions of the Osps proteins had been documented among spirochetal isolates derived from various hosts and geographical origins (4, 18). Indeed, even an isolate from a rabbit kidney differed from isolates derived from the larval tick vector that had fed on that particular rabbit (3). In addition, the antigenic component of the OspB protein represents more variability among both the European and the North American isolates of B. burgdorferi (7, 8). In our study, spirochetal isolates TWKM1 to TWKM4 from R. losea differed markedly from isolates TWKM5 to TWKM7 derived from R. norvegicus and S. murinus in the protein component of OspB (which reacted with MAb H6831). Thus, the heterogeneity among dominant the protein bands of OspB in these Taiwan isolates may be attributed to the diverse animal hosts from which spirochetes are isolated.

Although the identities of Lyme disease spirochetes can be related by the protein profiles and immunoreactivities with B. burgdorferi-specific MAbs, determination of heterogeneity in plasmid content was considered the most discriminating method for differentiating one isolate from another (10, 34). It had been documented that various sizes and conformations of linear and circular plasmids may create a complex gel profile for Lyme spirochetes (14). Indeed, a 49-kb linear plasmid molecule had been identified as the Osp-bearing plasmid of the Lyme disease spirochete, B. burgdorferi sensu lato (10, 11, 18). In the present study, two distinct patterns of plasmid contents were observed among the seven Taiwan isolates, and all Taiwan isolates contained two dominant plasmid elements with fragments of approximately 16 and 48.5 kb, respectively. On the basis of the genetic similarities of the plasmid profiles, the profiles of all of the Taiwan isolates were consistent with that of B. burgdorferi sensu lato.

The genospecies of Lyme disease spirochetes can be identified by their differential reactivities with genospecies-specific PCR primers. Although molecular and immunological characteristics had been used for the typing or species identification of Lyme disease isolates, the validity of the methods used was not fully satisfied (4, 18). On the other hand, genetic analysis based on the genospecies-specific PCR primers provides a rapid and distinguishable assay for the species identification of Lyme disease spirochetes, regardless of the biological and geographical origins (15, 18, 22, 23, 28, 29). In our study, we used two typing schemes based on five sets of oligonucleotide primers to distinguish and identify the genospecies of Borrelia isolates from Taiwan. All of the Taiwan isolates were genetically identified as B. burgdorferi sensu stricto. Further application of these genospecies-specific PCR primers to the clinical and tick specimens would help to identify the new foci of endemicity and heterogeneity of Borrelia isolates in Taiwan.

In conclusion, our report describes the first identification and characterization of Borrelia spirochetes isolated from rodents in Taiwan. On the basis of their seroreactivities and genetic similarities, all of the Taiwan isolates were genetically related to B. burgdorferi sensu stricto. Further investigations of the abundance of animal reservoirs and possible tick vectors responsible for transmission may help to determine the risk of acquiring spirochetal infection by the human population in Taiwan.