Genomic Analysis of Borrelia japonica Sp. Nov. Isolated from Ixodes ovatus in Japan

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.     

Detection and typing of Borrelia burgdorferi sensu lato genospecies in Ixodes persulcatus ticks in West Siberia, Russia

The prevalence of Borrelia burgdorferi sensu lato (s.l.) genospecies in West Siberia as well as in many other regions of Russia remains insufficiently investigated. In the present study a total of 151 adult female ticks Ixodes persulcatus Schulze, collected at three localities in eastern regions of West Siberia, where Lyme disease is endemic, were examined for the presence of the spirochete B. burgdorferi s.l. by polymerase chain reaction targeting the 23S-5S rRNA intergenic spacer regions. Spirochetal DNA was detected in on average 15.2+/-3.0% of the ticks examined. The infection rate of adult ticks with B. burgdorferi s.l. at various localities ranged from 8.6+/-3.4% to 29.0+/-7.6%, being greatest in the northernmost site studied and decreasing southwards. The restriction patterns obtained after MseI digestion of the 23S-5S rRNA intergenic spacer amplicons assigned 23 DNA samples to the following genomic groups: 19 to B. garinii (12 to group NT29 and seven to group 20047(T)), three to B. afzelii, and one to mixed B. afzelii and B. garinii NT29. We have not detected other genospecies, which were found in ticks in Europe, the Russian Far East and Japan. Thus, the ticks examined were associated only with two genospecies of Borrelia burgdorferi s.l. pathogenic to humans (B. garinii and B. afzelii), and B. garinii was the major genospecies infecting adult I. persulcatus in eastern regions of West Siberia.     

Molecular characterization of Borrelia isolates from ticks and mammals from the southern United States

Fifty Borrelia isolates from ticks and rodents from several geographic regions of the southern United States were analyzed by genomic macrorestriction analysis. Significant genetic diversity was observed among them. These isolates segregated into 4 major clusters and 10 subclusters, which are correlated with the genospecies distribution. Nineteen pulsed-field gel electrophoresis (PFGE) types were recognized among the isolates. The genospecies Borrelia andersonii and Borrelia bissettii consisted of 5 and 2 subclusters, respectively. Two subclusters comprised the Borrelia burgdorferi sensu stricto (s. s.) strains. These results indicated that PFGE is a suitable molecular typing method for B. burgdorferi at both the genospecies and strain levels. Seventeen representative isolates from different PFGE groups were analyzed by restriction fragment length polymorphism (RFLP) and sequence analysis of flaB. Twenty-three AluI, 3 CelII, and 11 DdeI RFLP patterns were found among strains from the B. burgdorferi sensu lato (s. l.) complex and the relapsing fever borreliae complex. Three genospecies in the B. burgdorferi s. l. complex and 1 species in the relapsing fever borreliae complex were recognized. Phylogenetic analysis based on nucleotide sequences of flaB indicated that all the Borrelia strains analyzed here could be divided into 2 parts, i.e., B. burgdorferi s. l. complex and the relapsing fever borreliae complex. The flaB appears to be a useful target gene to screen and identify strains from both B. burgdorferi s. l. and relapsing fever borreliae complexes.     

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis

Abstract This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii . To assess specificity, patient sera were statistically ( χ ², P ≤ 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30–33, 36–37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally isolated strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.     

Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis

In Europe, Borrelia burgdorferi genospecies causing Lyme borreliosis are mainly transmitted by the tick Ixodes ricinus. Since its discovery, B. burgdorferi has been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies of I. ricinus ticks infected with B. burgdorferi sensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection of I. ricinus with Borrelia in Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection of I. ricinus with Borrelia genospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection of I. ricinus were found in countries in central Europe. B. afzelii and B. garinii are the most common Borrelia species, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection by Borrelia species was found for B. garinii and B. valaisiana.     

Characterization and identification of Borrelia isolates as Borrelia valaisiana in Taiwan and Kinmen Islands

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.     

Evaluation of a genotyping method based on the ospA gene to detect Borrelia burgdorferi sensu lato in multiple samples of lyme borreliosis patients

In this study we have developed a new Restriction-Fragment-Length-Polymorphism (RFLP) genotyping method for rapid detection and identification of Borrelia genospecies present as unique species or as co-infection in multiple specimens obtained simultaneously from 29 individual patients affected by early or late Lyme borreliosis (LB). The target of the RFLP-genotyping was the heterogeneous plasmid located ospA gene, thus we developed a method able to detect and differentiate between six clinically relevant Borrelia genospecies circulating in Europe, B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, B. bissettii and B. spielmanii. In this study Borrelia DNA could be detected by PCR in at least one specimen of each patient, except in one case of neuroborreliosis (NB); blood samples gave the highest sensitivity in all patient groups. The genotyping indicated that B. afzelii was present in 8 patients with skin involvement, B. garinii in 2 cases of NB and 4 cases with skin involvement, B. burgdorferi sensu stricto was detected in one patient with skin involvement and another with Lyme arthritis. Different Borrelia species in distinct specimens were identified in one patient with EM. The RFLP analysis of 11 patients revealed mixed patterns, which suggested pluri-infection with different Borrelia species.     

Molecular characterization of Borrelia spp. isolates from greater metropolitan Chicago reveals the presence of Borrelia bissettii. Preliminary report

Lyme Disease in the US is concentrated in three endemic areas: the Northeast, the upper mid-West, and the Pacific coast. In the mid-West, the range of Lyme disease has expanded to include large parts of Wisconsin and Minnesota. Despite its proximity to the mid-Western focus, Illinois, so far, has not been considered an endemic area. However, more recent data suggest that this situation may be changing. Also, the extent of borrelial diversity in the mid-West remains largely unexplored. Here, we present preliminary results on the molecular characterization of Borrelia isolates from rodents captured in Cook and Lake Counties, both of which are parts of the greater metropolitan Chicago area in Illinois. We investigated the rodent reservoir present in forested areas of suburban Chicago in order to determine the frequency of infection with the Lyme disease agent(s) by culture isolation of Borrelia spirochetes (Picken et al., unpublished). Rodent isolates of Borrelia were identified to the species level by genetic characterization. In total, 19 isolates were obtained over 3 years from NW Cook Co. and Lake Co. Pulsed-field gel electrophoretic analysis of Mlul digested DNA from these isolates showed macrorestriction patterns similar to that of the Californian isolate, strain DN127 (PF type I), New York isolate strain 25015 (PF type II), or a variant of the latter (PF type III). Sequence data generated from the rrf(5S)-rrl(23S) intergenic spacer region of the ribosomal RNA gene cluster confirmed the identity of all the Chicago isolates studied to date as B. bissettii. These strains are unlike our previous Borrelia isolates from NW Illinois and Wisconsin. In addition, there was a predominant association of B. bissettii infection with pratal rodent species such as Microtus pennsylvanicus and Zapus hudsonius. The relationship of this novel enzootic focus to the established mid-Western endemic focus of Lyme disease remains to be elucidated. The geographic range and reservoir diversity of this organism may have hitherto been underestimated.     

Genetic variants of Borrelia garinii, a widely spread Eurasian pathogen of ixodic tick borreliosis

As a result of PCR-RFLP analysis and the degree of similarity between the nucleotide sequences analysis of the rrfA-rrlB intergenic spacer DNA of 227 primary isolates of Borrelia garinii and 71 isolates/ amplicons from GenBank database in different regions of Eurasia revealed significant intraspecific heterogeneity among those of Borrelia. It was shown that genospecies B. garinii had within the two genetic subgroups (20047 and NT29) 16 genetic variants, whose geography was likely to be different.     

Prevalence of IgG reactivity in Lyme borreliosis patients versus Borrelia garinii and Borrelia afzelii in a restricted area of Northern Italy

Abstract This survey evaluates the antibody band patterns of sera taken from clinically defined cases of Lyme borreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii , by means of Western blot. The sera were taken from patients resident in a limited area of Friuli Venezia Giulia (FVG) region. The data indicated that, besides a different feature of the band reactivity which correlated to the different stages of Lyme borreliosis, there was a preferential reactivity to the species Borrelia afzelii and Borrelia garinii . An immunodominant band at 51 kDa, corresponding to a protein visible in the electrophoretic profile of strain BL3 ( B. afzelii ), behaved like a marker of an early infection, because it was present exclusively in the sera of patient with ECM. The overall findings would indicate that B. afzelii and B. garinii are the prevalent genospecies in the FVG area, even if strains belonging to B. sensu stricto have been also isolated in this area. Consequently strains representative of these two species must be used as antigens in Western blot.     

Polymerase Chain Reaction Analysis of Borrelia Species Isolated in Japan

Primer reactivities of 25 Borrelia burgdorferi sensu lato isolates from the ticks, Ixodes persulcatus and I. ovatus, in Japan and 10 isolates in Europe and North America were investigated. The methods used in this study were the polymerase chain reaction (PCR) on the flagellin structural gene (fla), the outer surface protein A gene (osp A) and the outer surface protein B gene (osp B), and the restriction fragment length polymorphism (RFLP) analysis of PCR products from osp A and osp B, The flagellin PCR primer set reacted with all the Borrelia strains tested. Four genospecies, B. burgdorferi sensu stricto, B. garinii, B. afzelii and B. japonica, were differentiated by PCR using osp A and osp B primers combined with RFLP analysis. Some Japanese isolates from I. persulcatus were identified as B. garinii or B. afzelii. The other isolates from I. persulcatus did not fit in any of the 4 genospecies. These results suggested that Japanese isolates from I. persulcatus are highly heterogeneous in their osp A and osp B structures. Furthermore, PCR primers targeting fla are applicable to the gene diagnosis for Lyme disease in Japan and osp A and osp B primers can be used to classify B. burgdorferi sensu lato isolates into genospecies by PCR and RFLP analyses.     

Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

DNA homology comparison between American and European Borrelia burgdorferi strains

Seven strains of Borrelia burgdorferi isolated from ticks and from human beings in Europe and U.S.A. were analyzed for DNA restriction patterns with several enzymes and for DNA homology in Southern blot hybridizations. The restriction patterns showed a moderately high variability. In Southern blot hybridization, strain B31 (U.S.A.) DNA gave a strong signal with itself, strain Bsf (U.S.A.) and Alcaide (isolated in Italy but presumably contracted in Venezuela). Strain B45 (F.R.G.) hybridized to itself, strain BITS (Italy) and to strain D.A. (Italy). Strain Nancy (Italy) gave a signal only when hybridized to itself, although it was classified as Borrelia on the basis of the clinical manifestations, SDS-PAGE protein pattern and antigenic determinants. No hybridization differences were observed for strains isolated from different hosts in the same continental geographical area.     

Comparison of the spirochete Borrelia burgdorferi S. L. isolated from the tick Ixodes scapularis in southeastern and northeastern United States

Thirty-five strains of the Lyme disease spirochete Borrelia burgdorferi sensu lato (B. burgdorferi s. l.) were isolated from the blacklegged tick vector Ixodes scapularis in South Carolina, Georgia, Florida, and Rhode Island. They were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. PCR-RFLP analysis indicated that the strains represented at least 3 genospecies (including a possible novel genospecies) and 4 different restriction patterns. Thirty strains belonged to the genospecies B. burgdorferi sensu stricto (B. burgdorferi s. s.), 4 southern strains were identified as B. bissettii, and strain SCCH-5 from South Carolina exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Complete sequences of rrf-rrl intergenic spacers from 14 southeastern and northeastern strains were determined and the phylogenetic relationships of these strains were compared. The 14 strains clustered into 3 separate lineages on the basis of sequence analysis. These results were confirmed by phylogenetic analysis based on 16S rDNA sequence analysis.     

Comparison of Borrelia isolated from UK foci of Lyme disease

Abstract Restriction endonuclease digestion of linear borrelial chromosomal DNA showed that three isolates of UK Lyme disease spirochaetes differed markedly from each other and from published data for other isolates from North America and continental Europe. Analysis of linear plasmid bands revealed that UK isolates differed from each other in the number and sizes of the plasmids in isolates from different foci of UK Lyme disease. Fatty acid analysis (of fatty acid methyl ester (FAME) profiles) showed the UK isolates clustering together with the relapsing fever spirochaetes, Borrelia turicatae and Borrelia parkeri . These data are discussed in respect of current knowledge of Lyme borreliosis in the UK.     

Characterization of Monoclonal Antibodies for Identification of Borrelia japonica,Isolates from Ixodes ovatus

Monoclonal antibodies for identification of Borrelia japonica isolated from tick, Ixodes ovatus and long-tailed shrew, Sorex unguiculatus in Japan and Borrelia related to Lyme disease (Borrelia burgdorferi sensu lato) were prepared and characterized. All isolates belonging to B. japonica and isolates from I. dentatus and cottontail rabbit in North America reacted with MAb O1441b against flagellin which was prepared from immunized mice with strain HO14, type strain of B. japonica, but isolates from I. persulcatus, patient, and wood mouse, Apodemus speciosus ainu, in Japan, and isolates belonging to B. burgdorferi, B. garinii and B. afzelii from North America and Europe did not. Strains used in this study reacted with MAb P62 against common antigen which was prepared from immunized mice with strain NT24 isolated from I. persulcatus in Japan, but B. japonica did not. These MAbs are useful for identification and differentiation of B. japonica and B. burgdorferi sensu lato in Japan.     

Occurrence of multiple infections with different Borrelia burgdorferi genospecies in Danish Ixodes ricinus nymphs

The pathogen Borrelia burgdorferi causes Lyme Borreliosis in human and animals world-wide. In Europe the pathogen is transmitted to the host by the vector Ixodes ricinus. The nymph is the primary instar for transmission to humans. We here study the infection rate of five Borrelia genospecies: B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae in nymphs, by IFA and PCR. 600 nymphs were collected in North Zealand of Denmark. Each nymph was first analysed by IFA. If positive for spirochaetal infection, the genospecies was determined by PCR. The infection rate of B. burgdorferi sensu lato was 15.5%, with the primary genospecies being B. afzelii (64.3%), B. garinii (57.1%), and B. lusitaniae (26.8%). It is the first time B. lusitaniae is documented in Denmark. Even though, the highest infection rate was discovered for B. afzelii and B. garinii, mixed infections are more common than single infections. Fifty-one percent (29/56) of these were infected with two genospecies, 7.1% (4/56) with three, and 5.3% (3/56) with four. We try to explain the high infection rate and the peculiar number of multiple infections, with a discussion of changes host abundance and occurrence of different transmission patterns.     

A mutagenic PCR identifies isolates of Borrelia garinii responsible for Lyme borreliosis

Borrelia garinii is one of the three major Borreliae responsible for Lyme borreliosis in Europe. We have characterized a protein of B. garinii (VS102) and a genomic fragment from the gene encoding this protein was cloned. The DNA sequence of the fragment showed high homology with a known gene of B. burgdorferi sensu stricto. The protein encoded by this gene in B. burgdorferi sensu stricto is a phosphocarrier protein (histidine-containing protein). A mutation T to G polymorphism at codon 57 was found to be specific to B. garinii. A PCR-based approach that allows the rapid detection of this mutation made it possible to specifically discriminate B. garinii from other B. burgdorferi genospecies with high sensitivity and specificity.     

Controlled activation of the Cpx system is essential for growth of Yersinia enterocolitica

Until recently, three spirochete genospecies were considered to be the causative agents of Lyme borreliosis (LB) in Europe: Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii . However, the DNA of Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii and Borrelia bissettii has already been detected in samples of human origin, or the spirochetes were isolated from the patients with symptoms of LB. Molecular analysis of 12 selected serum samples collected in the regional hospital confirmed the presence of B. bissettii DNA in cases of single and multiple infection in patients with symptomatic borreliosis or chronic borrelial infection. The presence of B. bissettii as a single strain in patients provides strong support of the fact that B. bissettii might be a causative agent of the disease. After the first isolation of B. bissettii from the samples of human origin in Slovenia, following the detection of this species in cardiac valve tissue of the patient with endocarditis and aortic valve stenosis in the Czech Republic, here we present additional molecular data supporting the involvement of B. bissettii in LB in Europe.     

Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in ixodes ricinus ticks from the Czech Republic

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.