Sequence diversity of a gene encoding a putative primary sigma factor among Borrelia burgdorferi sensu lato strains

Twenty-six strains of Borrelia burgdorferi sensu lato were subjected to polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis for assessing the sequence divergence of rpoD gene encoding the primary sigma factor. Four and five RFLP patterns were observed from two fragments of rpoD gene. Sequence analysis of a subgenic fragment covering region 1 through 4 from 13 strains of Borrelia burgdorferi s. l. revealed that 21 of 450 deduced amino acid residues were diverged. These results indicate that the sequence heterogeneity of rpoD is present in different strains of Borrelia burgdorferi s. l., and agreed well with the current classification of genospecies.     

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.     

Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones: Relevance for Lyme vaccine design

Lyme borreliosis is the most important vector‐borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological, and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a one dimensional gel electrophoresis followed by LC‐MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild‐type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify upregulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT‐PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. All MS data have been deposited in the ProteomeXchange with identifier PXD000876 ( http://proteomecentral.proteomexchange.org/dataset/PXD000876 ).     

Borrelia burgdorferi sensu lato in the female cement plug of Ixodes persulcatus ticks (Acari,Ixodidae)

Borrelia burgdorferi sensu lato was detected in one out of five cement plugs of female Ixodes persulcatus ticks. The spirochetes were found by dark field microscopy as early as 18 h after attachment of the ticks to the skin of a white mouse. The relevance of this finding is discussed in relation to the epidemiology of Lyme Borreliosis.     

Detection of Borrelia burgdorferi sensu lato by PCR in questing Ixodes ricinus larvae from the Dutch North Sea island of Ameland

In May 1995, 61 Ixodes ricinus larvae were collected from vegetation on the Dutch North Sea island of Ameland. Fifty-seven lysates (94%) were analysed for the presence of Borrelia DNA by the polymerase chain reaction (PCR), which amplified the intergenic spacer region between the 5S and 23S rRNA genes. Three samples (5%) were positive and hybridization of the PCR product to genomic group-specific probes revealed that two larvae were infected with Borrelia afzelii and group VS116, respectively and one larval tick carried a mixed infection of B. afzelii and Borrelia garinii. Previously, we showed that these three genomic groups were present in adult and nymphal ticks collected on Ameland. Transovarial transmission may be an important factor in the establishment of these genomic groups in the local tick population.     

Detection of Lyme disease spirochete,Borrelia burgdorferi sensu lato,including three novel genotypes in ticks (Acari: Ixodidae) collected from songbirds (Passeriformes) across Canada

Lyme disease is reported across Canada, but pinpointing the source of infection has been problematic. In this three‐year, bird‐tick‐pathogen study (2004–2006), 366 ticks representing 12 species were collected from 151 songbirds (31 passerine species/subspecies) at 16 locations Canada‐wide. Of the 167 ticks/pools tested, 19 (11.4%) were infected with Borrelia burgdorferi sensu lato (s.l.). Sequencing of the rrf‐rrl intergenic spacer gene revealed four Borrelia genotypes: B. burgdorferi sensu stricto (s.s.) and three novel genotypes (BC genotype 1, BC genotype 2, BC genotype 3). All four genotypes were detected in spirochete‐infected Ixodes auritulus (females, nymphs, larvae) suggesting this tick species is a vector for B. burgdorferi s.l. We provide first‐time records for: ticks in the Yukon (north of 60° latitude), northernmost collection of Amblyomma americanum in North America, and Amblyomma imitator in Canada. First reports of bird‐derived ticks infected with B. burgdorferi s.l. include: live culture of spirochetes from Ixodes pacificus (nymph) plus detection in I. auritulus nymphs, Ixodes scapularis in New Brunswick, and an I. scapularis larva in Canada. We provide the first account of B. burgdorferi s. l. in an Ixodes muris tick collected from a songbird anywhere. Congruent with previous data for the American Robin, we suggest that the Common Yellowthroat, Golden‐crowned Sparrow, Song Sparrow, and Swainson's Thrush are reservoir‐competent hosts. Song Sparrows, the predominant hosts, were parasitized by I. auritulus harboring all four Borrelia genotypes. Our results show that songbirds import B. burgdorferi s.l.‐infected ticks into Canada. Bird‐feeding I. scapularis subadults were infected with Lyme spirochetes during both spring and fall migration in eastern Canada. Because songbirds disperse millions of infected ticks across Canada, people and domestic animals contract Lyme disease outside of the known and expected range.     

The role of fat dormouse (Glis glis L.) as reservoir host for spirochete Borrelia burgdorferi sensu lato in the region of Gorski Kotar, Croatia

To determine whether some of the B. burgdorferi sensu lato genospecies associate with fat dormouse as a reservoir host, we investigated the prevalence of infection in questing animals. A total of 45 adult fat dormice (30 female and 15 male) were captured by hunters during their hunting season in the region of Gorski Kotar, Croatia. Dead animals were aseptically dissected, and the urinary bladder tissue was used for isolation attempt and for deoxyribonucleic acid (DNA) extraction. Out of 45 DNA samples extracted from urine bladder tissue, we found four (8.88%) to be polymerase chain reaction (PCR) positive. The RFLP analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: B. burgdorferi sensu stricto, B. garinii and B. afzelii. All positive samples were typed as B. afzelii with a unique DraI or MseI pattern. The results of the analysis of urinary bladder tissue samples culture for the presence of Borrelia were negative. Results showed that a prevalence of the Borrelia infection among population of fat dormice indicated their epizootiological involvement as a reservoir of Borrelia spirochetes. Furthermore, this work is an initial step in the investigation of the molecular epidemiology/epizootiology of Lyme borreliosis in Croatia.     

Risk indicators for the tick Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden

The distributional area of the tick Ixodes ricinus (L.), the primary European vector to humans of Lyme borreliosis spirochaetes (Borrelia burgdorferi sensu lato) and tick‐borne encephalitis virus, appears to be increasing in Sweden. It is therefore important to determine which environmental factors are most useful to assess risk of human exposure to this tick and its associated pathogens. The geographical distribution of I. ricinus in Sweden was analysed with respect to vegetation zones and climate. The northern limit of I. ricinus and B. burgdorferi s.l. in Sweden corresponds roughly to the northern limit of the southern boreal vegetation zone, and is characterized climatically by snow cover for a mean duration of 150 days and a vegetation period averaging 170 days. The zoogeographical distribution of I. ricinus in Sweden can be classified as southerly–central, with the centre of the distribution south of the Limes Norrlandicus. Ixodes ricinus nymphs from 13 localities in different parts of Sweden were examined for the presence of B. burgdorferi s.l. and found to be infected with Borrelia afzelii and Borrelia garinii. Tick sampling localities were characterized on the basis of the density of Borrelia‐infected I. ricinus nymphs, presence of specific mammals, dominant vegetation and climate. Densities of I. ricinus nymphs and Borrelia‐infected nymphs were significantly correlated, and nymphal density can thus serve as a general indicator of risk for exposure to Lyme borreliosis spirochaetes. Analysis of data from this and other studies suggests that high densities of Borrelia‐infected nymphs typically occur in coastal, broadleaf vegetation and in mixed deciduous/spruce vegetation in southern Sweden. Ixodes ricinus populations consistently infected with B. burgdorferi s.l. can occur in: (a) biotopes with shrews, rodents, hares and birds; (b) biotopes with shrews, rodents, hares, deer and birds, and (c) island locations where the varying hare (Lepus timidus) is the only mammalian tick host.     

Borrelia burgdorferi sensu lato in ticks (Acari: Ixodidae) from two different foci in Spain

The prevalence of Borrelia burgdorferi sensu lato in several tick species was studied over a 2 year period in two ecologically different areas in Spain. One area was an endemic area for Lyme disease, with a number of autochthonous human cases and supported large populations of Ixodes ricinus on cattle and birds; the second area was characterized by the absence of I. ricinus together with the presence of foxes and their associated tick species. While I. ricinus was the main vector of B. burgdoreri in the endemic area (with a mean prevalence of 14% in adults and 51% in nymphs), adults of both Ixodes canisuga and Ixodes hexagonus had high rates of B. burgdorferi prevalence (30 and 28%, respectively) in the zone where I. ricinus was absent. Immatures of Ixodes frontalis were found to be carriers of the spirochete only in those zones where I. ricinus is present, suggesting evidence for reservoir competence in a tick-bird cycle.     

Borrelia burgdorferi sensu lato infection pressure shapes innate immune gene evolution in natural rodent populations across Europe

Although parasite-mediated selection is assumed to be the main driver of immune gene evolution, empirical evidence that parasites induce allele frequency changes at host immune genes in time and/or space remains scarce. Here, I show that the frequency of a protective gene variant of the innate immune receptor Toll-like receptor 2 in natural bank vole (Myodes glareolus) populations is positively associated with the strength of Borrelia burgdorferi sensu lato infection risk across the European continent. Thereby, this study provides rare evidence for the role of spatially variable infection pressures in moulding the vertebrate immune system.     

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.     

Loss of plasmids of Borrelia burgdorferi sensu lato during prolonged in vitro cultivation

In the present study we analyzed stability of plasmid content in 34 Borrelia strains of three different species (13 Borrelia afzelii, 10 Borrelia garinii and 11 Borrelia burgodorferi sensu stricto) using pulse field gel electrophoresis (PFGE). During long-term in vitro cultivation consisting of 50 passages, plasmid loss was established in 46% of B. afzelii, 40% of B. garinii and 36% of B. burgdorferi sensu stricto strains. Loss of plasmids occurred as early as between the 5th and 10th passage, affected only plasmids in the range 9-41 kb but not plasmids in the range 50-68 kb and manifested with the loss of one to up to three plasmids.     

Expression and sequence of outer surface protein C among North American isolates of Borrelia burgdorferi

Abstract The expression of outer surface protein C (OspC) was determined for North American Borrelia burgdorferi isolates HB19, DN127c19-2, 25015 and both low and high culture passage B31. A monoclonal antibody detected the presence of OspC protein in only two isolates, while polyclonal antiserum identified this protein in all five isolates. The ospC gene was cloned and sequenced for isolates HB19, DN127c19-2 and 25015, and compared with the published ospC sequences of other Lyme disease spirochetes. Bothe the nucleotide and amino acid sequences were found to vary as much among isolates from the same geographic area as between isolates of different species.     

Adaptation of Borrelia burgdorferi in the tick and the mammalian host

Borrelia burgdorferi, the causative agent of Lyme disease, shows a great ability to adapt to different environments, including the arthropod vector, and the mammalian host. The success of these microorganisms to survive in nature and complete their enzootic cycle depends on the regulation of genes that are essential to their survival in the different environments. This review describes the current knowledge of gene expression by B. burgdorferi in the tick and the mammalian host. The functions of the differentially regulated gene products as well as the factors that influence their expression are discussed. A thorough understanding of the changes in gene expression and the function of the differentially expressed antigens during the life cycle of the spirochete will allow a better control of this prevalent infection and the design of new, second generation vaccines to prevent infection with the spirochete.     

Molecular characterization of a flagellar/chemotaxis operon in the spirochete Borrelia burgdorferi

A chemotaxis gene cluster from Borrelia burgdorferi, the spirochete that causes Lyme disease, was cloned, sequenced, and analyzed. This cluster contained three chemotaxis gene homologs (cheA, cheW and cheY) and an open reading frame we identified as cheX. Although the major functional domains for B. burgdorferi CheW and CheY were well conserved, the size of cheW was significantly different from the homolog of other bacteria. Phylogenetic analysis of CheY indicated that B. burgdorferi constitutes a distinct branch with Treponema pallidum and is closely associated with Archea and Gram-positive bacteria. RT-PCR analysis indicated that the chemotaxis genes and the upstream flagellar gene flaA constitute an operon. Western blot analysis using antibody to Escherichia coli CheA resulted in two reactive proteins in the cell lysates of B. burgdorferi that is consistent with two cheA homologs being present in this organism. The results taken together suggest both similarities and differences in the chemotaxis apparatus of B. burgdorferi compared to those of other bacteria.     

First report of Anaplasma phagocytophilum and its co-infections with Borrelia burgdorferi sensu lato in Ixodes ricinus ticks (Acari: Ixodidae) from Republic of Moldova

We examined 198 questing Ixodes ricinus ticks collected in Chisinau City, Republic of Moldova by PCR assays for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and co-infection of both pathogens, which were detected in 9%, 25.2% and 2.5% of tested ticks, respectively. B. burgdorferi s.l. genotyping revealed the presence of five genospecies with dominance of B. garinii. Our preliminary study provides evidence about occurrence of both pathogens in this populated area, which represent a potential health risk for inhabitants.     

Ixodes (Pholeoixodes) hexagonus, an efficient vector of Borrelia burgdorferi in the laboratory

Borrelia burgdorferi Johnson et al. was first isolated from the midgut of Ixodes dammini Spielman et al. in the U.S.A. and from the midgut of I.ricinus (L.) in Europe. I.ricinus was considered to be the only tick vector of this borrelia, in Europe, until I.hexagonus Leach, the hedgehog tick, was found to harbour spirochaetes. This paper reports an evaluation of the vector competence of I.hexagonus for the spirochaete B.burgdorferi. Transovarial and trans-stadial survival were demonstrated and the spirochaete was transmitted to laboratory mice via the bites of trans-stadially infected I.hexagonus females.     

Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen

Abstract The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyne disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum .