Expanded diversity among Californian borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127).
ABSTRACT: Up to now, the only species in the complex Borrelia burgdorferi sensu lato known to cause Lyme borreliosis in the United States has been B. burgdorferi sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of B. burgdorferi sensu lato group DN127, we analyzed the nucleotide sequences of the rrf-rrl intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American B. burgdorferi sensu stricto strains (6 from California). The spacer region sequences from the entire B. burgdorferi sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B. andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from B. burgdorferi sensu stricto. Within this cluster, the deep branches expressing the distances between the rrf-rrl sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by B. burgdorferi sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the rrs sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American B. burgdorferi sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species, B. bissettii sp. nov., and that other related but distinct strains, which require further characterization, be referred to as Borrelia spp.
Project description:Approximately 118 Borrelia isolates were cultured from a variety of rodents, birds, and ticks collected in the southern United States. In addition to a highly diverse group of Borrelia bissettii strains and a homogenous group of Borrelia burgdorferi sensu stricto strains, a group of 16 isolates with unusual characteristics was found. The isolates were cultured from ear biopsy samples of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina. A multilocus sequence analysis of the rrf-rrl intergenic spacer, 16S rRNA, fla, ospA, and p66 genes were used to clarify the taxonomic status of the new group of B. burgdorferi sensu lato isolates. Thirteen species of the B. burgdorferi sensu lato complex were used as controls. Unique restriction fragment length polymorphism patterns of the rrf-rrl intergenic spacer region and fla gene were recognized. Unique signature nucleotides were also found in the 16S rRNA gene. A phylogenetic analysis shows that the 16 new isolates cluster together but separately from the other species in the B. burgdorferi sensu lato complex. Our data strongly support the recognition of the 16 isolates as a new B. burgdorferi sensu lato species. We propose to name this genospecies "Borrelia carolinensis" with respect to the place of its currently known geographic location.
Project description:Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of rrf-rrl intergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in the B. burgdorferi sensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related to Borrelia garinii. The MOK-3a group separated into a distinct deep branch in the B. andersonii lineage. PCR-RFLP analysis results and the results of sequence analyses of the rrf-rrl intergenic spacer confirm that greater genetic heterogeneity exists among B. burgdorferi sensu lato strains isolated from the southern United States than among strains isolated from the northern United States. The B. andersonii genospecies and its MOK-3a subgroup are associated with the I. dentatus-cottontail rabbit enzootic cycle, but I. scapularis was also found to harbor a strain of this genospecies. Strains that appear to be B. bissettii in our study were isolated from I. minor and the cotton mouse, cotton rat, and wood rat. The B. burgdorferi sensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.
Project description:To date Borrelia lusitaniae is the only genospecies of Borrelia burgdorferi sensu lato isolated from Ixodes ricinus ticks collected in Portugal and Tunisia. This suggests that the genospecies diversity of B. burgdorferi sensu lato decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of B. burgdorferi sensu lato from this region, 55 I. ricinus and 27 Hyalomma marginatum questing adults, collected during the spring of 1998 from a sylvatic habitat south of Lisbon, Portugal, were analyzed. Infection prevalences of 75% in I. ricinus ticks and 7% in H. marginatum ticks were detected by a nested PCR that targets the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. Restriction fragment length polymorphism (RFLP) analysis of the I. ricinus-derived amplicons showed that the sequences in the majority of samples were similar to those of B. lusitaniae type strains (76% for strain PotiB1, 5% for strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samples. The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic analysis of rrf-rrl spacer sequences revealed a diverse population of B. lusitaniae in questing adult I. ricinus ticks (the sequences did not cluster with those of any other genospecies). This population consisted of 10 distinct sequence types, suggesting that multiple strains of B. lusitaniae were present in the local I. ricinus population. We hypothesize that B. lusitaniae has a narrow ecological niche that involves host species restricted to the Mediterranean Basin.
Project description:Previous work described Borrelia burgdorferi sensu lato group DN127 as a new genospecies, Borrelia bissettii, and prompted the present study to identify the Borrelia spp. that exist in northern Colorado. To determine the genospecies present, we analyzed two specific intergenic spacer regions located between the 5S and 23S and the 16S and 23S ribosomal genes. Phylogenetic analysis of the derived sequences clearly demonstrated that these isolates, originating from rodents captured in the foothills of northern Colorado, diverged from B. burgdorferi sensu stricto by 5 to 5.5% and were members of the new genospecies B. bissettii.
Project description:We genotyped 102 Borrelia burgdorferi sensu lato strains isolated from ticks, animals, and patients in 11 provinces in China by PCR-restriction fragment length polymorphism (PCR-RFLP) amplification of 5S (rrf)-23S (rrl) rRNA gene spacer amplicons and multilocus sequence analysis (MLSA). The results showed that Borrelia garinii was the main genotype in China (65/102) and that it was distributed mainly in northern China. Borrelia afzelii was the second most frequently found species (22/102), and it was distributed in both northern and southern China. All Borrelia valaisiana strains were isolated from Guizhou Province. Additionally, one B. burgdorferi strain was isolated from Hunan Province. Our results show the diversity and wide distribution of B. burgdorferi sensu lato in China.
Project description:A group of 16 isolates with genotypic characteristics different from those of known species of the Borrelia burgdorferi sensu lato complex were cultured from ear biopsies of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina, USA, and from the tick Ixodes minor feeding on N. floridana. Multilocus sequence analysis of members of the novel species, involving the 16S rRNA gene, the 5S-23S (rrf-rrl) intergenic spacer region and the flagellin, ospA and p66 genes, was conducted and published previously and was used to clarify the taxonomic status of the novel group of B. burgdorferi sensu lato isolates. Phylogenetic analysis based on concatenated sequences of the five analysed genomic loci showed that the 16 isolates clustered together but separately from other species in the B. burgdorferi sensu lato complex. The analysed group therefore represents a novel species, formally described here as Borrelia carolinensis sp. nov., with the type strain SCW-22(T) (=ATCC BAA-1773(T) =DSM 22119(T)).
Project description:Borrelia garinii spirochete was detected for the first time in Ixodes ovatus tick ectoparasitized on stray cat in Taiwan. The genetic identity of this detected spirochete was determined by analyzing the gene sequence amplified by genospecies-specific polymerase chain reaction assays based on the 5S-23S intergenic spacer amplicon (rrf-rrl) and outer surface protein A (ospA) genes of B. burgdorferi sensu lato. Phylogenetic relationships were analyzed by comparing the sequences of rrf-rrl and ospA genes obtained from 27 strains of Borrelia spirochetes representing six genospecies of Borrelia. Seven major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. Phylogenetic analysis based on rrf-rrl gene revealed that this detected spirochete (strain IO-TP-TW) was genetically affiliated to the same clade with a high homogeneous sequences (96.7 to 98.1% similarity) within the genospecies of B. garinii and can be discriminated from other genospecies of Borrelia spirochetes. Interspecies analysis based on the genetic distance values indicates a lower level (<0.022) of genetic divergence (GD) within the genospecies of B. garinii, and strain IO-TP-TW was genetically more distant ( >0.113) to the strains identified in I. ovatus collected from Japan and China. Intraspecies analysis also reveals a higher homogeneity (GD<0.005) between tick (strain IO-TP-TW) and human (strain Bg-PP-TW1) isolates of B. garinii in Taiwan. This study provides the first evidence of B. garinii isolated and identified in an I. ovatus tick in Asia, and the higher homogeneity of B. garinii between tick and human strain may imply the risk of human infection by I. ovatus bite.
Project description:We determined the nucleotide sequences (329 bp) of the rpoB DNAs from 22 reference strains of Borrelia. No insertions or deletions were observed. Deduced amino acid sequences of amplified rpoB DNA comprised 109 amino acid residues (N(450) to M(558) [Escherichia coli numbering]). All amino acid sequences were identical with the exception of those of Borrelia lusitaniae PotiB2 (T(461)-->A) and B. bissettii DN127 (I(498)-->V). Each species of B. burgdorferi sensu lato was differentiated as a distinct entity in the phylogenetic tree constructed by a neighbor-joining method. B. burgdorferi sensu lato could be distinguished from B. turicatae and B. hermsii, which are associated with relapsing fever. Seventeen Korean isolates could be identified by PCR-linked direct sequencing and restriction analysis of the rpoB DNA. These results suggest that rpoB DNA is useful for identification and characterization of Borrelia. In addition, we developed the rapid species identification method using the species-specific primer sets based on rpoB gene sequences.
Project description:To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species.
Project description:The molecular and antigenic variabilities of BmpA (P39) among European isolates of Borrelia burgdorferi were analyzed. The bmpA sequences of 12 isolates representing all three species of B. burgdorferi sensu lato pathogenic for humans were amplified by PCR, cloned, and sequenced. The BmpA protein of Borrelia garinii is heterogeneous, with an amino acid sequence identity ranging from 91 to 97%, whereas the BmpA proteins of Borrelia afzelii and B. burgdorferi sensu stricto strains appear to be highly conserved (>98.5% intraspecies identity). The interspecies identities ranged from 86 to 92%. Cluster analysis of BmpA reflected the subdivision of B. burgdorferi sensu lato isolates into the three species as well as a considerable heterogeneity among B. garinii strains. The BmpA protein of each species of B. burgdorferi sensu lato was recombinantly expressed in Escherichia coli, purified, and used to generate monoclonal antibodies. Seven BmpA-specific antibodies were identified; six of them recognized conserved epitopes of all three species, whereas one was specific for BmpA of B. afzelii and B. garinii. A monoclonal antibody (H1141) recommended by the Centers for Disease Control and Prevention for use in the standardization of immunoblots showed strong reactivity with BmpA of B. burgdorferi sensu stricto but no or only weak reactivity with BmpA of B. garinii and B. afzelii, respectively. Sera from 86 European patients with Lyme borreliosis in different stages and 73 controls were tested in immunoglobulin G (IgG) and IgM immunoblots with the recombinant BmpA proteins of the three species, revealing specificities of 98.6 to 100%. IgM antibodies against recombinant BmpA were only rarely detected (1.1 to 8.1%). With the BmpA proteins of B. afzelii and B. garinii, sensitivities for the IgG test (sera from stages I to III) were 36.0 and 34.9%, respectively, in contrast to 13.9% with BmpA of B. burgdorferi sensu stricto. Therefore, we recommend that recombinant BmpA of B. afzelii or B. garinii should be used solely, or in addition to B. burgdorferi sensu stricto BmpA, in serodiagnostic tests for Lyme borreliosis in Europe.