Taxonomy of the Lyme disease spirochetes.

Morphology, physiology, and DNA nucleotide composition of Lyme disease spirochetes, Borrelia, Treponema, and Leptospira were compared. Morphologically, Lyme disease spirochetes resemble Borrelia. They lack cytoplasmic tubules present in Treponema, and have more than one periplasmic flagellum per cell end and lack the tight coiling which are characteristic of Leptospira. Lyme disease spirochetes are also similar to Borrelia in being microaerophilic, catalase-negative bacteria. They utilize carbohydrates such as glucose as their major carbon and energy sources and produce lactic acid. Long-chain fatty acids are not degraded but are incorporated unaltered into cellular lipids. The diamino amino acid present in the peptidoglycan is ornithine. The mole % guanine plus cytosine values for Lyme disease spirochete DNA were 27.3-30.5 percent. These values are similar to the 28.0-30.5 percent for the Borrelia but differed from the values of 35.3-53 percent for Treponema and Leptospira. DNA reannealing studies demonstrated that Lyme disease spirochetes represent a new species of Borrelia, exhibiting a 31-59 percent DNA homology with the three species of North American borreliae. In addition, these studies showed that the three Lyme disease spirochetes comprise a single species with DNA homologies ranging from 76-100 percent. The three North American borreliae also constitute a single species, displaying DNA homologies of 75-95 percent. Lyme disease spirochetes and Borrelia exhibited little or no DNA homology (0-2 percent) with the Treponema or Leptospira. Plasmids were present in the three Lyme disease spirochetes and the three North American borreliae.     

Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete

The Lyme disease spirochetes, comprised of at least three closely related species, Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii, are fascinating and enigmatic bacterial pathogens. They are maintained by tick-mediated transmission between mammalian hosts, usually small rodents. The ability of these bacteria, which have relatively small genomes, to survive and disseminate in both an immunocompetent mammal and in an arthropod vector suggests that they have evolved elegant and indispensable strategies for interacting with their hosts. Recognition of specific mammalian and tick tissues is likely to be essential for successful completion of the enzootic life cycle but, given the historical difficulties in genetic manipulation of these organisms, characterization of factors promoting cell adhesion has until recently largely been confined to either the manipulation of host cells or the analysis of potential bacterial ligands in the form of recombinant proteins. These studies have led to the identification of several mammalian receptors for Lyme disease spirochetes, including glycosaminoglycans, decorin, fibronectin and integrins, as well as a tick receptor for the bacterium, and also candidate cognate bacterial ligands. Recent advances in our ability to genetically manipulate Lyme disease spirochetes, particularly B. burgdorferi, are now providing us with firm evidence that these ligands indeed do promote bacterial adherence to host cells, and with new insights into the roles of these multifacted Borrelia-host cell interactions during mammalian and arthropod infection.     

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.     

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.     

Ultrastructural features of Borrelia garinii in naturally infected voles, Clethrionomys rufocanus, with special reference to the relationship with peripheral nerves

The ultrastructural features of Borrelia garinii found in the ear tissues of the vole, Clethrionomys rufocanus, are described. The spirochetes were observed in the interstitium of connective tissue or in contact with fibroblasts and were occasionally situated in the endoneurium of the peripheral nerves. The spirochetes did not injure or enter into the fibroblasts or Schwann cells. The cytotoxicity and migration of the spirochetes are also discussed.     

New World relapsing fever Borrelia found in Ornithodoros porcinus ticks in central Tanzania

Ticks were collected from 8 houses in Mvumi Mission village, near Dodoma, Tanzania. All ticks were examined for Borrelia infestation by flagellin gene-based nested polymerase chain reaction. All houses were highly infested with ticks, and all ticks collected were of the Ornithodoros porcinus species. Fifty-one out of 120 ticks were infected with spirochetes, and a flagellin gene sequence comparison showed that most of the spirochetes belonged to Borrelia duttonii, which is the causative agent of tick-borne relapsing fever in East Africa. The rest of the spirochetes were quite different from B. duttonii and instead resembled the New World tick-borne relapsing fever borreliae. Phylogenetic analysis using 16S ribosomal RNA gene sequences also supported the interpretation that the spirochete was a Borrelia species distinct from previously described members of the genus.     

Molecular phylogenetic analysis of ixodid ticks based on the ribosomal DNA spacer, internal transcribed spacer 2, sequences

An internal transcribed spacer (ITS2) sequence between the 5.8S and 28S rRNA genes was used to estimate the phyletic relationships among Ixodes spp. tick vectors of Lyme disease-causing Borrelia spirochetes. Analysis indicates that Borrelia burgdorferi sensu lato species associated with Lyme disease are found mainly in ticks of the Ixodes ricinus species complex. Other closely related tick species are not known to transmit the Borrelia-that cause Lyme disease in humans, but they appear to have a specific association with other closely related Borrelia species. There is a high degree of concordance in the phylogenetics of Borrelia taxa and the phylogenetic relationships among Ixodes ticks.     

Borrelia pathogenesis research in the post-genomic and post-vaccine era

In the two years after publication of the genome sequence of Borrelia burgdorferi and reports on human field trials of a vaccine against Lyme borreliosis, there has been further progress in understanding of host-parasite interactions during Lyme borreliosis and relapsing fever. Some mechanisms that Borrelia spirochetes use to avoid elimination and to persist in the host are novel. In addition, the recent discovery of antigenic variation in the Lyme disease agent B. burgdorferi adds to the complexity of the possible virulence properties of this human pathogen.     

First report of Borrelia burgdorferi sensu lato in two threatened carnivores: the Marbled polecat, Vormela peregusna and the European mink

ABSTRACT: BACKGROUND: Lyme disease is a widespread cosmopolitan zoonosis caused by species belonging to the genus Borrelia. It is transmitted from animal reservoir hosts to humans through hard - ticks of genus Ixodes which are vectors of the disease. CASE PRESENTATION: Borrelia burgdorferi sensu lato infection was identified in a marbled polecat, Vormela peregusna, and two European minks, Mustela lutreola, from Romania, by PCR. RFLP revealed the presence of a single genospecies, Borrelia burgdorferi sensu stricto. CONCLUSIONS: This is the first report of the Lyme disease spirochetes in the two mentioned hosts.     

The bacterial nucleoid visualized by fluorescence microscopy of cells lysed within agarose: comparison of Escherichia coli and spirochetes of the genus Borrelia.

The nucleoids of Escherichia coli and the spirochetes Borrelia burgdorferi and Borrelia hermsii, agents of Lyme disease and relapsing fever, were examined by epifluorescence microscopy of bacterial cells embedded in agarose and lysed in situ with detergent and protease. The typical E. coli nucleoid was a rosette in which 20 to 50 long loops of DNA emanated from a dense node of DNA. The percentages of cells in a population having nucleoids with zero, one, two, and three nodes varied with growth rate and growth phase. The borrelia nucleoid, in contrast, was a loose network of DNA strands devoid of nodes. This nucleoid structure difference correlates with the unusual genome of Borrelia species, which consists primarily of linear replicons, including a 950-kb linear chromosome and linear plasmids. This method provides a simple, direct means to analyze the structure of the bacterial nucleoid.     

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.     

Interactions between Borrelia burgdorferi and mouse fibroblasts

Borrelia burgdorferi spirochetes are an infectious agent of Lyme borreliosis. The aim of our studies was to investigate the fate of engulfed B. burgdorferi cells in L-929 mouse fibroblasts and to observe development of intracellular infection in vitro after 2 and 48 h. Electron microscopic studies reveal consecutive stages of B. burgdorferi spirochetes penetration to mouse fibroblasts in vitro. It has been observed, as a first step attachment and engulfment of spirochetes followed by formation of vacuoles. After 48 hours of infection, vacuoles of fibroblastic cells have been seen full of B. burgdorferi bacteria and latter they have been released from infected cells to extracellular space. It can be the evidence that B. burgdorferi multiply intracellulary.     

Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

Phylogenetic analysis of the spirochetes.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.     

The flagellar cytoskeleton of the spirochetes

The recent discoveries of prokaryotic homologs of all three major eukaryotic cytoskeletal proteins (actin, tubulin, intermediate filaments) have spurred a resurgence of activity in the field of bacterial morphology. In spirochetes, however, it has long been known that the flagellar filaments act as a cytoskeletal protein structure, contributing to their shape and conferring motility on this unique phylum of bacteria. Therefore, revisiting the spirochete cytoskeleton may lead to new paradigms for exploring general features of prokaryotic morphology. This review discusses the role that the periplasmic flagella in spirochetes play in maintaining shape and producing motility. We focus on four species of spirochetes: Borrelia burgdorferi, Treponema denticola, Treponema phagedenis and Leptonema (formerly Leptospira) illini. In spirochetes, the flagella reside in the periplasmic space. Rotation of the flagella in the above species by a flagellar motor induces changes in the cell morphology that drives motility. Mutants that do not produce flagella have a markedly different shape than wild-type cells.     

Sequence Analysis and Serological Responses against Borrelia turicatae BipA,a Putative Species-Specific Antigen

Background

Relapsing fever spirochetes are global yet neglected pathogens causing recurrent febrile episodes, chills, nausea, vomiting, and pregnancy complications. Given these nonspecific clinical manifestations, improving diagnostic assays for relapsing fever spirochetes will allow for identification of endemic foci and expedite proper treatment. Previously, an antigen designated the Borrelia immunogenic protein A (BipA) was identified in the North American species Borrelia hermsii. Thus far, BipA appears unique to relapsing fever spirochetes. The antigen remains unidentified outside of these pathogens, while interspecies amino acid identity for BipA in relapsing fever spirochetes is only 24–36%. The current study investigated the immunogenicity of BipA in Borrelia turicatae, a species distributed in the southern United States and Latin America.

Methodology/Principal Findings

bipA was amplified from six isolates of Borrelia turicatae, and sequence analysis demonstrated that the gene is conserved among isolates. A tick transmission system was developed for B. turicatae in mice and a canine, two likely vertebrate hosts, which enabled the evaluation of serological responses against recombinant BipA (rBipA). These studies indicated that BipA is antigenic in both animal systems after infection by tick bite, yet serum antibodies failed to bind to B. hermsii rBipA at a detectable level. Moreover, mice continued to generate an antibody response against BipA one year after the initial infection, further demonstrating the protein''s potential toward identifying endemic foci for B. turicatae.

Conclusions/Significance

These initial studies support the hypothesis that BipA is a spirochete antigen unique to a relapsing fever Borrelia species, and could be used to improve efforts for identifying B. turicatae endemic regions.     

Relationships of a novel Lyme disease spirochete, Borrelia spielmani sp. nov., with its hosts in Central Europe

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for Borrelia afzelii, because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies, Borrelia spielmani sp. nov., one of several that cause Lyme disease in people.     

Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in ixodes ricinus ticks from the Czech Republic

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.     

Ability of Ixodes persulcatus, Haemaphysalis concinna and Dermacentor silvarum ticks to acquire and transstadially transmit Borrelia garinii

In China, the ability of Ixodes persulcatus, Haemaphysalis concinna and Dermacentor silvarum to transmit Borrelia spirochetes was determined under laboratory conditions. Results showed that all three tick species can acquire spirochetes by feeding on infected mice. However, the capability of the three species to maintain spirochetes was different. Only I. persulcatus is able to maintain spirochetes alive during molting, and subsequent tick stages transmitted the infection to naive mice. In H. concinna and D. silvarum ticks, spirochetes could not survive after the digestion period of blood and after the molting process was finished, spirochetes were no longer detected. Contrary to H. concinna and D. silvarum, I. persulcatus should be considered the principal vector of Lyme disease in north China.