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1 Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA 30460-8056, USA
2 Laboratory of Molecular Parasitology and Medical Microbiology, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, USA
Correspondence
James H. Oliver, Jr
Joliver{at}GeorgiaSouthern.edu
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and flaB gene sequences of TXW-1 are AF467976 and AF264901.
A scanning electron micrograph of a cell of TXW-1 is available as supplementary material in IJSEM Online.
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). Two major groups of Borrelia are the Borrelia burgdorferi sensu lato species complex and the relapsing fever borreliae. To date, 11 genospecies in the B. burgdorferi complex and more than 20 species of relapsing fever borreliae have been reported. Only three of the 11 genospecies of B. burgdorferi sensu lato are proven agents of Lyme disease in humans. Borrelia recurrentis, the agent of louse-borne relapsing fever, and several species of tick-borne spirochaetes are included among the relapsing fever borreliae.
Specific relationships are often found among borreliae and vector species. Although several different genospecies of B. burgdorferi sensu lato can be transmitted by several species of ticks, the ticks are closely related and most are in the Ixodes ricinus species complex. The relapsing fever borreliae and their tick vectors have an even stricter relationship. Some borreliae are reported to be transmitted by a single tick species (Kelly, 1976; Schwan & Piesman, 2002); however, this tick–spirochaete specificity theory needs reinvestigation using molecular analysis and experimental transmission experiments.
Most relapsing fever borreliae are transmitted by soft-bodied ticks (Ornithodoros or Argas species), but there are exceptions. For example, B. recurrentis is transmitted by the human biting louse, Pediculus humanus, and Borrelia theileri is vectored by the hard-bodied ticks Rhipicephalus (several species) and Boophilus microplus (Anderson & Magnarelli, 1993). Moreover, ‘Borrelia lonestari’ has been detected and cultured in the hard tick Amblyomma americanum from the US states of Texas, New Jersey, New York, Missouri and Georgia (Barbour et al., 1996; Varela et al., 2004), and a Borrelia species genetically close to Borrelia miyamotoi has been detected in Ixodes scapularis from Rhode Island, Connecticut, New York and New Jersey (Scoles et al., 2001). Also, a B. miyamotoi-like Borrelia species appears to be present in Ixodes ricinus ticks in Europe (Fraenkel et al., 2002). In Japan, B. miyamotoi is vectored by the closely related Ixodes persulcatus, which also is a vector of the Lyme borreliosis spirochaetes Borrelia garinii and Borrelia afzelii (Fukunaga & Koreki, 1995).
The American tick-borne relapsing fever spirochaetes Borrelia hermsii, Borrelia parkeri and Borrelia turicatae were successfully cultured 30 years ago (Kelly, 1971, 1976, 1984). Although investigation of Lyme borreliosis stimulated improvements in the cultivation of some of the most notable borreliae, such as B. recurrentis and Borrelia duttonii (Cutler et al., 2000), some species remain uncultivated, such as the borreliae from New England transmitted by Ixodes scapularis (Scoles et al., 2001).
The aim of this study was to characterize the genetic and phenotypic features of isolate TXW-1. By using different genotyping and phenotyping methods, we were able to classify this isolate and clarify its taxonomic status. Our results indicate that TXW-1 represents an undescribed species in the relapsing fever borreliae complex. Although we were able to culture this spirochaete, we are currently unable to revive the frozen cultures and thus can not meet the requirements of the Bacteriological Code to deposit viable type material at two different culture collections. Therefore we propose that TXW-1 should be described as ‘Candidatus Borrelia texasensis’.
TXW-1 was isolated in BSK-H medium (Sigma-Aldrich) in March 1998 from an adult male Dermacentor variabilis feeding on a coyote from Webb county, Texas. Cultures were incubated at 34 °C for 1–2 weeks until the cell density reached about 2x106 cells ml–1. Spirochaete genomic DNA was extracted by methods described previously (Zingg et al., 1993).
A 348 bp fragment of the flagellin B gene (flaB) (for RFLP analysis) was amplified by using a pair of primers, FlaLS (5'-AACAGCTGAAGAGCTTGGAATG-3'; positions 438–459) and FlaRS (5'-CTTTGATCACTTATCATTCTAATAGC-3'; 791–766) (Barbour et al., 1996). Another fragment of flaB (for sequence analysis) was amplified by using primers FlaLL (5'-ACATATTCAGATGCAGACAGAGGT-3'; 301–324) and FlaRL (5'-GCAATCATAGCCATTGCAGATTGT-3'; 942–965) (Barbour et al., 1996). A 1532 bp segment of the 16S rRNA gene (rrs) was amplified by using a pair of primers fD3 (5'-AGAGTTTGATCCTGGCTTAG-3'; 8–27) and UniB (5'-TACAAGGAGGTGATCCAGC-3'; 1539–1522) (Le Flèche et al., 1997). The outer surface protein C gene (ospC) was amplified by using primers ospC3 (5'-AAGTGCAGATATTAATGACTTTA-3') and ospC4 (5'-TTTTTTGGACTTTCTGCCACA-3') (Marti Ras et al., 1997).
The 348 bp fragment of flaB was digested with AluI (Gibco-BRL, Life Technologies) as recommended by the manufacturer. Ten microlitres of the PCR product was digested with 4 U CelII (Amersham Pharmacia Biotech) and DdeI (Gibco-BRL) for 1 h at 37 °C. The AluI, CelII and DdeI digests were respectively separated in 3·8, 3·0 and 3·8 % (w/v) agarose gels (NuSieve GTG; FMC) containing 0·5 µg ethidium bromide ml–1 in 1x TBE for 3, 2·5 and 3·5 h at a constant voltage of 100 V. The molecular marker pBR322/HaeIII (Sigma-Aldrich) was used. Gels were photographed using the Eagle Eye II System (Stratagene) and a Polaroid GelCam. The RFLP patterns were measured and analysed by using EagleSight software (version 3.2) in the Eagle Eye II System as recommended by the manufacturer.
RFLP of rrs was carried out with restriction enzyme BfaI (Le Flèche et al., 1997).
The PCR products were purified by QIAquick gel extraction kit (Qiagen). The DNA sequences of flaB and rrs genes were determined by using an ABI Prism model 377 sequencer. Sequences were aligned manually and by using CLUSTAL W software (Thompson et al., 1994). Phylogenetic trees were constructed by neighbour-joining (Saitou & Nei, 1987) using PAUP (Swofford, 2001).
RAPD-PCRs were performed by using previously published primers 1254, 1283 and AP13 (Wang et al., 1998). Reactions were performed as described previously (Wang et al., 1998) in a GeneAmpPCR System 9700 (PE Biosystems). The amplified DNA fragments were separated in 1 % agarose (w/v) gels in 0·5x TBE containing 0·5 µg ethidium bromide ml–1. Gels were photographed using the Eagle Eye II System (Stratagene) and PCR-RAPD patterns were analysed as described above for RFLP.
Phenotypic characters of TXW-1 and reference strains were analysed by SDS-PAGE and Western blot. Briefly, reference strains and TXW-1 were incubated in BSK-H medium (Sigma-Aldrich) with 6 % rabbit serum at 34 °C for 1–2 weeks until the cell density reached about 2x106 cells ml–1. Ten millilitres of culture was washed with three cycles of washing with 0·01 M PBS (pH 7·2) with 5 mM MgCl2 and centrifugation at 10 000 r.p.m. (12 096 g) for 10 min. Washed pellets of the spirochaetes were then lysed by adding loading buffer. Twenty microlitres of cell lysate was heated at 100 °C for 10 min and then separated by SDS-16·5 % PAGE. A low-molecular-mass marker (Sigma-Aldrich) was used to determine molecular masses. The gel was then stained with Coomassie brilliant blue R250. Western blotting was carried out by electrotransferring the proteins from the SDS-PAGE gel to a nitrocellulose membrane (Bio-Rad). The membrane was blocked by immersing in 5 % dry milk for 1 h at room temperature. The whole membrane was reacted with mAb H9724 (1 : 100) for 1 h at room temperature and washed three times in Tris-buffered saline (TBS) with 0·1 % Tween 20 for 5 min each at room temperature. The membrane was then incubated in horseradish peroxidase-labelled anti-mouse second antibody (Kirkegarrd & Perry Laboratories, Inc.) at 1 : 1000 dilution for 1 h at room temperature and subsequently washed three times in TBS with 0·1 % Tween 20 and once in distilled water, 5 min for each washing. The membrane was then incubated in TMB substrate solution at room temperature. The reaction was stopped by immersing the membrane in distilled water for 10–20 s when a suitable colour intensity was observed.
Measurement of DNA–DNA hybridization was performed by dot-blot analysis of samples with 32P-labelled probes. DNAs were extracted and purified from TXW-1 and reference strains by the method described previously (Lin et al., 2002). Aliquots of 2·5 µl total genomic DNA purified from the various bacterial strains were spotted at four different concentrations (25, 5, 1·25 and 0·25 ng DNA per dot) onto Hybond-XL nylon membrane (Amersham) and cross-linked with a Stratalinker 1800 UV cross-linker (Stratagene). Subsequently, membranes were prehybridized for 1 h at 60 °C in ExpressHyb solution (Clontech) followed by 1 h hybridization at 60 °C in ExpressHyb solution with [32P]dCTP-labelled probe made from 100 ng TXW-1 or B. parkeri genomic DNA by using an oligolabelling kit (Amersham). After hybridization, membranes were washed with 2x SSC, 0·05 % SDS at room temperature for 40 min with five changes of wash solution. This was followed by two high-stringency washes at 50 °C for 20 min each with 0·1x SSC, 0·1 % SDS, according to the manufacturer's instructions (Clontech). Hybridization signal was detected by phosphorimager analysis using a Storm840 PhosphorImager system (Molecular Dynamics). Quantification of hybridization signal was performed by the NIH Image 1.62 software (http://rsb.info.nih.gov/nih-image/) and data are given as percentages relative to the homologous probe for four independent hybridizations (mean±SD).
B. burgdorferi sensu lato complex strains and relapsing fever borreliae consisted of 21 AluI RFLP types, two CelII RFLP types and 11 DdeI RFLP types. TXW-1 formed the unique AluI RFLP type E1 and DdeI RFLP type F3 (Fig. 1; Table 1). The same strains contained 11 BfaI RFLP types; TXW-1 formed pattern I (Fig. 2; Table 2).
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). Similar results were found for rrs nucleotide sequences. The rrs nucleotide sequence similarities among TXW-1 and B. turicatae and B. parkeri were 99 %. Nucleotide identities among many species were 99 % (Table 4).
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). Similarly, the rrs phylogenetic tree revealed a separate branch for TXW-1 within the relapsing fever cluster. The genetic distance (indicated by branch length) between TXW-1 and B. turicatae (0·002) is greater than that between B. parkeri and B. turicatae (0·001) (Fig. 5).
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). Major proteins including 31 kDa OspA, 34 kDa OspB and 22 kDa OspC were clearly resolved for strain B31T on a Coomassie brilliant blue-stained 16·5 % polyacrylamide gel (Fig. 6a). mAb H9724, which is specific for Borrelia flagellar protein, was used as a probe for Western blotting. Flagellar proteins of 41 kDa were found in B. burgdorferi sensu lato strains B31T, 20047T, DN127, 25015 and 21038; however, a 38-kDa flagellar protein was found in TXW-1, similar to those found in relapsing fever borreliae HS1, RML and OZ-1 based on protein profiles and reactivity with mAb 9724 (Fig. 6a, b).
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). The ad hoc committee on reconciliation of approaches to bacterial systematics recommended that the level of DNA relatedness for separate species should be less than 70 % (Wayne et al., 1987). DNA–DNA hybridization experiments revealed levels of DNA reassociation between TXW-1 and previously described relapsing fever borreliae of 38·64 % (B. turicatae), 38·40 % (B. parkeri), 7·39 % (B. hermsii) and 18·30 % (B. coriaceae). However, the level of DNA relatedness between B. parkeri and B. turicatae was 78·78 %. Strain TXW-1 exhibited lower levels of DNA relatedness with B. parkeri and B. turicatae than that between B. parkeri and B. turicatae. Based on these results, TXW-1 is different from other described borreliae and therefore represents an undescribed species. Its closest relationship is to B. parkeri and B. turicatae. It is not known whether the coyote is a natural host for TXW-1 and whether D. variabilis can maintain and transmit the spirochaete. The tick may have become infected during the larval or nymphal stages when feeding on unknown hosts. Thus, only limited data are available regarding the vector, vertebrate reservoir host and complete geographical distribution of TXW-1; nothing is known about its infectivity or pathogenicity. Nevertheless, our study provides the description of a novel spirochaete species among the relapsing fever borreliae. The results also enhance understanding of the diversity among borreliae and may be useful for future studies of the evolution of relapsing fever borreliae.
Description of ‘Candidatus Borrelia texasensis’
‘Candidatus Borrelia texasensis' (te.xas.en'sis. N.L. fem. adj. texasensis of Texas, USA, where the organism was isolated).
Cells are actively motile with reversal, rotational and translational movements. Structurally, they consist of an outer membrane that surrounds a protoplasmic cylinder complex and flagella. They have six to ten flexible helical coils and are 0·21–0·24 µm wide and 9·41–11·23 µm long. The wavelengths are 1·12–1·53 µm (a scanning electron micrograph of TXW-1 is available as supplementary material in IJSEM Online). Cells have tapered ends. Microaerophilic spirochaete that can be cultured in BSK-H medium at 34 °C. Gram-negative and stains well with Giemsa's stain. Stained cells are visible by bright-field microscopy but, to observe unstained cells, dark-field microscopy is required. Pathogenicity and infectivity remain to be determined.
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ACKNOWLEDGEMENTS |
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