The first isolation of Borrelia burgdorferi sensu stricto in Russia

18 Borrelia isolates obtained from adult ticks of the Ixodes ricinus species, collected from different plants in April 2000 in the Khostinsk region of the Krasnodar Territory in the vicinity of the health resort of Matsesta not far from Sochi, were identified by means of PCR and the analysis of polymorphism of the restriction fragments of ribosomal rrf-rrl spacer amplicon. Among them, in addition to Borrelia species, found in Russia earlier (B. garinii, B. afzelii, B. valaisiana and B. lusitaniae), the classical causative agent of Lyme borreliosis (B. burgdorferi sensu stricto) was detected for the first time. The isolated strain (lr-4721) is now kept in the collection of the Borreliosis Center of the Ministry of Health of Russia (the Laboratory of Infections Vectors at the Gamaleya Research Institute of Epidemiology and Microbiology). The probable role of this infective agent in infectious pathology in Russia is discussed.     

Bacteriolytic activity of selected vertebrate sera for Borrelia burgdorferi sensu stricto and Borrelia bissettii

An in vitro assay to evaluate the bacteriolytic activity of the complement pathway was applied to 2 strains of Borrelia bissettii, CO501 and DN127, and compared with that of B. burgdorferi sensu stricto B31. Sera from mule deer (Odocoileus hemionus) and the Western Fence lizard (Sceloporus occidentalis) were completely borreliacidal for B. burgdorferi and for both strains of B. bissettii. Serum from Bobwhite quail (Colinus virginianus) was nonlytic for B. burgdorferi and partially lytic for B. bissettii strains, CO-501 and DN127. Serum from a New Zealand White rabbit (Oryctolagus cuniculus) was partially lytic for all 3 strains of Borrelia, whereas serum from white-footed mice (Peromyscus leucopus) were nonlytic for all 3 Borrelia strains. The spectrum of complement sensitivity of B. bissettii appears to be similar to that of European B. afzelii in that tested rodent serum is not lytic to these 2 genospecies. Interestingly, both B. bissettii and B. afzelii have been found to be closely associated with rodents. Complement sensitivity demonstrated in these experiments may suggest and possibly predict specific reservoir-host associations.     

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.     

Acylated cholesteryl galactoside as a novel immunogenic motif in Borrelia burgdorferi sensu stricto

Borrelia burgdorferi sensu lato is the causing agent of Lyme disease, an infectious disease frequently occurring in the United States, Europe, and Northern Asia. Currently, diagnosis of and vaccination strategies against this pathogen are exclusively based on proteinaceous structures. Here we report on a novel class of immunogenic glycolipids purified from B. burgdorferi sensu stricto B31. Employing a butanol/water extraction procedure with subsequent Bligh/Dyer extraction of the organic phase, thin layer chromatography analysis revealed the presence of three distinct glycolipids, which were chemically analyzed employing combined gas-liquid chromatography/mass spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and NMR. We identified acylated cholesteryl galactoside (ACG) next to cholesteryl galactoside and alpha-monogalactosyl-diacylglycerol. After extensive purification, the glycolipids investigated failed to cause proinflammatory responses in human cells transfected with human toll-like receptor (TLR)-2 or -4. However, we observed a marked recognition of ACG by sera derived from patients suffering from Lyme disease. These data indicate that newly described ACG is involved in developing host immunity during Lyme disease and thus may be useful for diagnosis and vaccination.     

Interaction and transmission of two Borrelia burgdorferi sensu stricto strains in a tick-rodent maintenance system

In the northeastern United States, the Lyme disease agent, Borrelia burgdorferi sensu stricto, is maintained by enzoonotic transmission, cycling between white-footed mice (Peromyscus leucopus) and black-legged ticks (Ixodes scapularis). B. burgdorferi sensu stricto is genetically variable and has been divided into three major genotypes based on 16S-23S ribosomal DNA spacer (RST) analysis. To better understand how genetic differences in B. burgdorferi sensu stricto may influence transmission dynamics in nature, we investigated the interaction between an RST1 and an RST3 strain in a laboratory system with P. leucopus mice and I. scapularis ticks. Two groups of mice were infected with either BL206 (RST1) or B348 (RST3). Two weeks later, experimental mice were challenged with the opposite strain, while control mice were challenged with the same strain as that used for the primary infection. The transmission of BL206 and B348 from infected mice was then determined by xenodiagnosis with uninfected larval ticks at weekly intervals for 42 days. Mice in both experimental groups were permissive for infection with the second strain and were able to transmit both strains to the xenodiagnostic ticks. However, the overall transmission efficiencies of BL206 and B348 were significantly different. BL206 was more efficiently transmitted than B348 to xenodiagnostic ticks. Significantly fewer double infections than expected were detected in xenodiagnostic ticks. The results suggest that some B. burgdorferi sensu stricto strains, such as BL206, may be preferentially maintained in transmission cycles between ticks and white-footed mice. Other strains, such as B348, may be more effectively maintained in different tick-vertebrate transmission cycles.     

Vector competence of Ixodes angustus (Acari: Ixodidae) for Borrelia burgdorferi sensu stricto

The vector competence of Ixodes angustus for Borrelia burgdorferi sensu stricto (s.s.) was investigated in the laboratory. The larval progeny of female ticks from Washington State were placed on Swiss-Webster mice that had been inoculated intravenously with 10⁸ spirochetes each of a Californian isolate of B. burgdorferi. Spirochetes were detected in 6 (12%) of 50 nymphs derived from larvae that had fed on these animals. Ten nymphs from the same cohort of experimentally infected ticks were placed on each of 4 naive deer mice (Peromyscus maniculatus). One of the mice seroconverted to B. burgdorferi and spirochetes were isolated from its ear tissues 4 weeks after exposure to ticks. Further vector competence trials were conducted with I. angustus ticks from California. Larvae were fed on deer mice that had been inoculated intradermally with B. burgdorferi along with larvae of I. spinipalpis as a comparison group. There was no significant difference in the prevalence of infection in nymphs of I. angustus (8.2%) versus those of I. spinipalpis (12.1%). We conclude that I. angustus is a competent experimental vector of B. burgdorferi s.s. and its efficiency for acquiring and transstadially passing such spirochetes is similar to that of I. spinipalpis.     

Comparative genome hybridization reveals substantial variation among clinical isolates of Borrelia burgdorferi sensu stricto with different pathogenic properties

Clinical and murine studies suggest that there is a differential pathogenicity of different genotypes of Borrelia burgdorferi, the spirochetal agent of Lyme disease. Comparative genome hybridization was used to explore the relationship between different genotypes. The chromosomes of all studied isolates were highly conserved (>93%) with respect to both sequence and gene order. Plasmid sequences were substantially more diverse. Plasmids lp54, cp26, and cp32 were present in all tested isolates, and their sequences and gene order were conserved. The majority of linear plasmids showed variation both in terms of presence among different isolates and in terms of sequence and gene order. The data strongly imply that all B. burgdorferi clinical isolates contain linear plasmids related to each other, but the structure of these replicons may vary substantially from isolate to isolate. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many isolates. There is a strong correlation between complete genome hybridization profiles and other typing methods, which, in turn, also correlate to differences in pathogenicity. Because there is substantially less variation in the chromosomal and circular plasmid portions of the genome, the major differences in open reading frame content and genomic diversity among isolates are linear plasmid driven.     

Preparation and purification of recombinant outer surface protein A (rOspA) of Borrelia burgdorferi sensu stricto and Borrelia afzelii

The recombinant Outer surface protein A (rOspA) from Borrelia burgdorferi is a possible immunogen for protection of infected humans and animals against development of Lyme borreliosis (Lyme disease), a chronic tick-borne disease characterised by diverse dermatologic, neurologic, rheumatic, and cardiac manifestations. For several years, research and development have been directed towards a vaccine for the prevention of this debilitating disease. Numerous animal studies demonstrate that pre-existing antibodies against the outer surface proteins of B. burgdorferi can prevent infection and disease caused by this organism. In this communication, using recombinant DNA technology, genes from B. burgdorferi sensu stricto and B. afzelii were inserted into E. coli-expression vectors and the rOspA were produced. Our aim was to obtain rOspA protein in a purity and quantity desirable for immunization of experimental animals. rOspA is currently the most developed, molecularly-defined vaccine candidate for the prevention of Lyme borreliosis.     

Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.

We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.     

Microarray analyses of inflammation response of human dermal fibroblasts to different strains of Borrelia burgdorferi sensu stricto

In Lyme borreliosis, the skin is the key site of bacterial inoculation by the infected tick, and of cutaneous manifestations, erythema migrans and acrodermatitis chronica atrophicans. We explored the role of fibroblasts, the resident cells of the dermis, in the development of the disease. Using microarray experiments, we compared the inflammation of fibroblasts induced by three strains of Borrelia burgdorferi sensu stricto isolated from different environments and stages of Lyme disease: N40 (tick), Pbre (erythema migrans) and 1408 (acrodermatitis chronica atrophicans). The three strains exhibited a similar profile of inflammation with strong induction of chemokines (CXCL1 and IL-8) and IL-6 cytokine mainly involved in the chemoattraction of immune cells. Molecules such as TNF-alpha and NF-κB factors, metalloproteinases (MMP-1, -3 and -12) and superoxide dismutase (SOD2), also described in inflammatory and cellular events, were up-regulated. In addition, we showed that tick salivary gland extracts induce a cytotoxic effect on fibroblasts and that OspC, essential in the transmission of Borrelia to the vertebrate host, was not responsible for the secretion of inflammatory molecules by fibroblasts. Tick saliva components could facilitate the early transmission of the disease to the site of injury creating a feeding pit. Later in the development of the disease, Borrelia would intensively multiply in the skin and further disseminate to distant organs.     

Lectin-binding characteristics of a lyme borreliosis spirocheteBorrelia burgdorferi sensu stricto

Borrelial glycoconjugates were localized by labeled lectins on ultrathin cryosections and on surfaces of intact negatively stained bacteria. Protein-saccharide complexes in these glycoconjugates were partially characterized by means of enzyme deglycosylation and mild alkali pretreatment of cryosections. The results of labeling were examined by transmission electron microscopy. Statistically evaluated results (relative labeling index, chi2 test) of gold labeling indicated that surfaces of Borrelia burgdorferi strain B31 and external (outer) membrane vesicles (MVs) were covered with glycoconjugates containing O-glycosidically linked N-acetyl-D-galactosamine (GalNAc) and N-glycosidically linked N-acetyl-D-glucosamine (GlcNAc). The presence of N-linked GalNAc, sialic acid, mannose and fucose on the surfaces of outer membranes and MVs was probably due to an adherence of BSK-H medium components, especially rabbit serum, to Borrelia surfaces.     

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.     

Borrelia burgdorferi sensu lato infection in mosquitoes from Szczecin area

The aim of the study was to determine the level of infection in mosquitoes with spirochetes Borrelia burgdorferi sensu lato in the woody areas of Szczecin. The mosquitoes were collected from May to September 2003. The spirochetes, Borrelia burgdorferi s. l., present in mosquitoes were detected in mosquitoes with indirect immunofluorescence assay (IFA) using rabbit anti-Borrelia burgdorferi antibodies and goat anti-rabbit IgG marked with fluorescein isocyanate (FITC). A total of 1557 females and 58 males were collected. They represented the genera Aedes (63%) and Culex (37%). The infection level of the mosquitoes from the area studied amounted to 1.7%. The results of the present study confirm the potential of these arthropods to spread Lyme borreliosis.     

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.     

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.     

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named "Borrelia finlandensis."     

Analysis of the bmp gene family in Borrelia burgdorferi sensu lato

BmpA, BmpB, BmpC, and BmpD are homologous Borrelia burgdorferi lipoproteins of unknown functions, encoded by the bmp genes of paralogous chromosomal gene family 36. At least some of the Bmp proteins are immunogens in infected vertebrate hosts. The genetic organization of the bmp region has been characterized for a variety of B. burgdorferi sensu lato strains by Southern hybridization, PCR amplification, and DNA sequencing. All four bmp genes were present in the same relative order in all B. burgdorferi sensu lato low- and high-passage-number isolates. While there were no differences in the relative orders of the bmp genes in these species, variations in DNA sequence in the bmpD-bmpC and bmpC-bmpA intergenic regions were significantly more common than in the corresponding 3' bmpD and bmpC coding regions. The genetic structure of the chromosomal region containing the bmp genes thus appears to be well conserved across different species of B. burgdorferi, but variations in DNA fine structure that prevent PCR primer annealing may occur in this region and make Southern hybridization much more reliable than PCR for detection of the presence of these genes. Our results also suggest that bmp gene products may be used as reagents in the preparation of vaccines and diagnostic assays to protect against and diagnose Lyme disease produced by B. burgdorferi sensu lato.     

Host association of Borrelia burgdorferi sensu lato--the key role of host complement

Borrelia burgdorferi sensu lato (s.l.), the tick-borne agent of Lyme borreliosis, is a bacterial species complex comprising 11 genospecies. Here, we discuss whether the delineation of genospecies is ecologically relevant. We provide evidence that B. burgdorferi s.l. is structured ecologically into distinct clusters that are host specific. An immunological model for niche adaptation is proposed that suggests the operation of complement-mediated selection in the midgut of the feeding tick. We conclude that vertebrate hosts rather than tick species are the key to Lyme borreliosis spirochaete diversity.