Whole-genome sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii

It has been known for decades that human Lyme disease is caused by the three spirochete species Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. Recently, Borrelia valaisiana, Borrelia spielmanii, and Borrelia bissettii have been associated with Lyme disease. We report the complete genome sequences of B. valaisiana VS116, B. spielmanii A14S, and B. bissettii DN127.     

Diversity of Borrelia burgdorferi sensu lato in Russian Far East

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full-length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well-defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.     

Borrelia burgdorferi sensu lato, the agent of lyme borreliosis: life in the wilds

In Europe, Borrelia burgdorferi sensu lato (sl) the agent of Lyme borreliosis circulates in endemic areas between Ixodes ricinus ticks and a large number of vertebrate hosts upon which ticks feed. Currently, at least 12 different Borrelia species belonging to the complex B. burgdorferi sl have been identified among which seven have been detected in I. ricinus: B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. valaisiana, B. spielmanii and B. bissettii. A few dozens of vertebrate hosts have been identified as reservoirs for these Borrelia species. Specific associations were rather early observed between hosts, ticks and borrelia species, like for example between rodents and B. afzelii and B. burgdorferi ss, and between birds and B. garinii and B. valaisiana. The complement present in the blood of the hosts is the active component in the Borrelia host specificity. Recent studies confirmed trends toward specific association between Borrelia species and particular host, but also suggested that loose associations may be more frequent in transmission cycles in nature than previously thought.     

Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia

The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii ) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).     

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.     

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.     

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.     

Evolution of the Borrelia burgdorferi outer surface protein OspC.

The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n = 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n = 9) Borrelia garinii strains. The ratio of the mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.     

Phenotypic and genotypic analysis of<Emphasis Type="Italic">Borrelia</Emphasis> spp. isolated from<Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks by using electrophoretic chips and real-time polymerase chain reaction

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.     

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.     

Borrelia burgdorferi inhibits human neutrophil functions

Outer surface proteins OspA and OspB are among the most prominent Borrelia burgdorferi surface molecules. We constructed OspAB and OspA complementation mutants of B. burgdorferi Osp-less strain B313 and investigated the role of these surface proteins in the interactions of B. burgdorferi, human neutrophils and the complement system. We found that (1) OspB inhibits the phagocytosis and oxidative burst of human neutrophils at low serum concentrations, whereas OspA induces the oxidative burst in neutrophils; (2) OspB may have an inhibiting role in serum sensitivity and complement activation; (3) all studied strains inhibit the chemotaxis of human neutrophils specifically towards fMLP but not towards C5a, regardless of their Osp expression. These results suggest that although OspA and OspB are co-ordinately transcribed, they differ in their effects on human neutrophil functions. Our findings suggest that B. burgdorferi exploits a wide variety of immune evasion mechanisms, besides previously documented complement resistance, to survive in the vertebrate host.     

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.     

Lysine-dependent multipoint binding of the Borrelia burgdorferi virulence factor outer surface protein E to the C terminus of factor H

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18-20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala(151)-Lys(166)) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.     

Comparative analysis of Borrelia isolates from southeastern USA based on randomly amplified polymorphic DNA fingerprint and 16S ribosomal gene sequence analyses

Fifty-three southern USA Borrelia isolates were characterized using randomly amplified polymorphic DNA fingerprinting analysis (RAPD). Twenty-nine types were recognized among 37 B. andersonii strains, seven types among eight B. bissettii strains, and seven types among seven B. burgdorferi sensu stricto strains. Strain TXW-1 formed a separate RAPD type. Nearly complete sequences of the rrs genes from 17 representative southern Borrelia were determined. The similarity values were found to be 96-100% within the B. burgdorferi sensu lato (s.l.) complex, 94-99% among the relapsing fever borreliae, and 93-99% between the two complexes. Phylogenetic analysis indicated that all the Borrelia strains we analyzed could be divided into two parts: the B. burgdorferi s.l. complex and the relapsing fever borreliae complex. TXW-1 segregated with the North American relapsing fever borreliae and formed a separate subbranch.     

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Refractoriness of the western fence lizard (Sceloporus occidentalis) to the Lyme disease group spirochete Borrelia bissettii

The western fence lizard, Sceloporus occidentalis, is refractory to experimental infection with Borrelia burgdorferi sensu stricto, one of several Lyme disease spirochetes pathogenic for humans. Another member of the Lyme disease spirochete complex, Borrelia bissettii, is distributed widely throughout North America and a similar, if not identical, spirochete has been implicated as a human pathogen in southern Europe. To determine the susceptibility of S. occidentalis to B. bissettii, 6 na?ve lizards were exposed to the feeding activities of Ixodes pacificus nymphs experimentally infected with this spirochete. None of the lizards developed spirochetemias detectable by polymerase chain reaction for up to 8 wk post-tick feeding, infected nymphs apparently lost their B. bissettii infections within 1-2 wk after engorgement, and xenodiagnostic L. pacificus larvae that co-fed alongside infected nymphs did not acquire and maintain spirochetes. In contrast, 3 of 4 na?ve deer mice (Peromyscus maniculatus) exposed similarly to feeding by 1 or more B. bissettii-infected nymphs developed patent infections within 4 wk. These and previous findings suggest that the complement system of S. occidentalis typically destroys B. burgdorferi sensu lato spirochetes present in tissues of attached and feeding I. pacificus nymphs, thereby potentially reducing the probability of transmission of these bacteria to humans or other animals by the resultant adult ticks.     

Whole-genome sequences of two Borrelia afzelii and two Borrelia garinii Lyme disease agent isolates

Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.     

Acylated cholesteryl galactosides are ubiquitous glycolipid antigens among Borrelia burgdorferi sensu lato

Lyme disease (LD) is the most common tick-borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato, in particular, B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. However, other genospecies have been implicated as causative factors of LD as well. Borrelia burgdorferi exhibits numerous immunogenic lipoproteins, but due to strong heterogeneity, the use of these proteins for serodiagnosis and vaccination is hampered. We and others have identified acylated cholesteryl galactosides (ACGal) as a novel glycolipid present in B. burgdorferi sensu stricto, B. afzelii, and B. garinii. ACGal is a strong antigen and the majority of patients display anti-ACGal antibodies in the chronic stages of LD. However, it is unknown whether ACGal is present in other presumably pathogenic B. burgdorferi genospecies. Therefore, we performed an analysis of the total lipid extracts of a wide spectrum of genospecies of B. burgdorferi sensu lato using thin-layer chromatography as well as Western blot and dot-blot assays. We show that ACGal is present in substantial quantities in all B. burgdorferi genospecies tested. Therefore, this molecule might improve the serological detection of rarely pathogenic genospecies, and may be used as a protective vaccine regardless of the prevailing genospecies.