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.     

Identification of a new Borrelia species among small mammals in areas of northern Spain where Lyme disease is endemic

The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.     

Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

Borrelia burgdorferi sensu lato detected in skin of Norwegian mountain hares (Lepus timidus) without signs of dissemination

The mountain hare (Lepus timidus) population in southern Norway appears to be in decline. Necropsy and laboratory examinations of 36 hares found dead or diseased during 2007-2009 in Vest- and Aust-Agder counties showed that disease and deaths were attributed to multiple causes, with no specific etiology emerging as a cause for population decline. To investigate whether Borrelia burgdorferi sensu lato (s.l.) infection is associated with mortality in mountain hares, tissues and ticks collected from hares were investigated for infection with the spirochete. Borrelia burgdorferi s.l. DNA was not detected in samples from internal organs, whereas Borrelia afzelii, B. burgdorferi sensu stricto (s.s.), and the not-yet-defined Borrelia sp. SV1 were found in skin samples from hares and in adult and nymphal Ixodes ricinus feeding on hares. Only B. burgdorferi s.s. and Borrelia sp. SV1 were detected in larvae feeding on hares. Our results indicate that disseminated Borrelia infection in hares rarely occurs and, presumably, does not play a central role in the suspected population decline. The results also suggest that the mountain hare to some degree functions as a transmission host for B. burgdorferi s.s. and Borrelia sp. SV1.     

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.     

Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes.

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.     

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.     

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.     

The presence of Borrelia valaisiana-related genospecies in ticks and a rodent in Taiwan

A field survey was conducted to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) in six counties of Taiwan. Spirochetes were successfully isolated from one rodent ear sample out of 485 rodent ears and 53 live, fed tick (Ixodes granulatus) samples. The spirochetes were confirmed to be B. burgdorferi s.l. by real-time PCR. In addition, 23 of 113 tick samples were tested positive for Borrelia DNA according to real-time PCR. The Borrelia isolate from the rodent and the 23 Borrelia DNA samples from the ticks were identified as B. valaisiana-related genospecies by phylogenetic analysis based on flagellin gene sequences. These findings suggest that the Borrelia valaisiana-related strains are maintained in a zoonotic cycle between tick vectors and reservoir hosts in Taiwan.     

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.     

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.     

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.     

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.     

Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa

Background

In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and miscarriage. The closely related Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis and Borrelia hispanica are rarely diagnosed at the species level, hampering refined epidemiological and clinical knowledge of the relapsing fevers. It would be hugely beneficial to have simultaneous detection and identification of Borrelia to species level directly from clinical samples.

Methodology/Principal Findings

We designed a multiplex real-time PCR protocol targeting the 16S rRNA gene detecting all four Borrelia, the glpQ gene specifically detecting B. crocidurae, the recN gene specifically detecting B. duttonii/B. recurrentis and the recC gene specifically detecting B. hispanica. Compared to combined 16S rRNA gene and flaB gene sequencing as the gold standard, multiplex real-time PCR analyses of 171 Borrelia-positive and 101 Borrelia-negative control blood specimens yielded 100% sensitivity and specificity for B. duttonii/B. recurrentis and B. hispanica and 99% sensitivity and specificity for B. crocidurae.

Conclusions/Significance

The multiplex real-time PCR developed in this study is a rapid technique for both molecular detection and speciation of relapsing fever borreliae from blood in Africa. It could be incorporated in point-of-care laboratory to confirm diagnosis and provide evidence of the burden of infection attributed to different species of known or potentially novel relapsing fever borreliae.     

Columbian black-tailed deer (Odocoileus hemionus columbianus) as hosts for Borrelia spp. in northern California

The prevalence of infection of Columbian black-tailed deer (Odocoileus hemionus columbianus) with Borrelia spp. was evaluated in an area of northwestern California (USA) where Lyme disease is endemic and the relapsing-fever group spirochete Borrelia coriaceae is enzootic, and in a far-removed comparison area having a disparate climate and lower density of vector ticks. Blood samples collected from both deer herds in 1987, 1988, and from 2000-02 were assayed for borrelial infection with microscopic and molecular methods. Serum specimens from two (5%) of 39 deer from the Dye Creek Preserve in Tehama County versus 13 (20%) of 64 animals from the Hopland Research and Extension Center (HREC) in Mendocino County, California were polymerase chain reaction (PCR) test positive for B. burgdorferi sensu lato. DNA sequencing analyses revealed that eight animals were infected with B. bissettii, six with three unclassified genotypes, and one with B. burgdorferi sensu stricto. One serum sample (2%) from HREC was positive for a relapsing-fever group spirochete that had a 16S rRNA sequence homology of 99% with the C053 type strain of B. coriaceae. Spirochetes undetermined to geno-species were detected in thick-blood drops prepared from three (8%) of 36 deer from the HREC by direct immunofluorescence. Adults of the hippoboscid flies Lipoptena depressa (n=73) and Neolipoptena ferrisi (n=24), the Pacific Coast tick (Dermacentor occidentalis) (n=22), and the western black-legged tick (Ixodes pacificus) (n=1) that had been removed from deer from both study areas in 2002 were PCR test negative for borreliae. The occurrence of diverse borreliae in deer from northern California confounds and, consequently, reduces the utility of borrelial serosurveys for detecting specific genospecies, unless they are complemented by more specific assays (e.g., immunoblotting, PCR/sequencing analysis).     

The cell-mediated immune response to Borrelia afzelii, garinii and burgdorferi in C57BL/6 mice is dependent on antigen specificity

Inbred C57BL/6 mice were challenged with Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen-specific T-cell response in vitro. The sonicated preparations of in vitro grown spirochetes were capable of stimulating polyclonal proliferation and specific cell-mediated response, depending on duration of the cell culture. Murine splenocytes previously sensitized to B. burgdorferi sensu lato (s.l. ), but not those from control mice, could be induced for antigen-specific proliferation in vitro. Moreover, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies as revealed by the [(3)H]thymidine incorporation assay. The current study considers that the strict B. burgdorferi s.l. antigen-specific response may also be expected in infections in humans and contributes to the explanation of the frequently poor antibody- and cell-mediated immune response observed in patients diagnosed with Lyme disease.     

Molecular evidence of coinfection of Borrelia burgdorferi sensu lato,human granulocytic ehrlichiosis agent,and Babesia microti in ticks from northwestern Poland

To assess the potential risk for tick-borne agents, Ixodes ricinus were collected from 2 sites in northwestern Poland. The ticks were tested by polymerase chain reaction for coinfection with Borrelia burgdorferi sensu lato (s. l.), human granulocytic ehrlichiosis (HGE) agent, and Babesia microti. Of the 533 processed ticks, 16.7% were positive for B. burgdorferi s. l., 13.3% for B. microti, and 4.5% for the HGE agent. Twenty ticks were coinfected with 2 or 3 of the pathogens.     

Comparative cryo-electron tomography of pathogenic Lyme disease spirochetes

Spirochetes of the Borrelia burgdorferi sensu lato group, the causative agents of Lyme borreliosis, exhibit a complex biology evolved in its zoonotic cycle. Cryo-electron tomography was used to investigate structural features of three species, B. burgdorferi , B. garinii and B. afzelii , known to cause different clinical manifestations in humans. All three organisms revealed an overall similar architecture and showed different numbers of periplasmic flagellar filaments, polar periplasmic void regions, vesicles budding from the outer membrane sheath, which was covered by an amorphous slime layer. The latter was shown to be distinct in its density when comparing the three human-pathogenic Lyme disease spirochetes and Borrelia hermsii , a species causing relapsing fever. Tomograms of dividing bacteria revealed vesicles near the site of division and new basal bodies that were attached at each end of newly establishing cytoplasmic cylinder poles, while periplasmic flagellar filaments still passed the impending site of division. Two different kinds of cytoplasmic filaments showed similarities to MreB or FtsZ filaments of other bacteria. The similar and distinct structural features of Borrelia and the previously investigated pathogenic and non-pathogenic Treponema species emphasize the importance of further studying phylogenetically distant spirochetes.     

DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.