Inactivation of Genes for Antigenic Variation in the Relapsing Fever Spirochete Borrelia hermsii Reduces Infectivity in Mice and Transmission by Ticks

Borrelia hermsii, a causative agent of relapsing fever of humans in western North America, is maintained in enzootic cycles that include small mammals and the tick vector Ornithodoros hermsi. In mammals, the spirochetes repeatedly evade the host’s acquired immune response by undergoing antigenic variation of the variable major proteins (Vmps) produced on their outer surface. This mechanism prolongs spirochete circulation in blood, which increases the potential for acquisition by fast-feeding ticks and therefore perpetuation of the spirochete in nature. Antigenic variation also underlies the relapsing disease observed when humans are infected. However, most spirochetes switch off the bloodstream Vmp and produce a different outer surface protein, the variable tick protein (Vtp), during persistent infection in the tick salivary glands. Thus the production of Vmps in mammalian blood versus Vtp in ticks is a dominant feature of the spirochete’s alternating life cycle. We constructed two mutants, one which was unable to produce a Vmp and the other was unable to produce Vtp. The mutant lacking a Vmp constitutively produced Vtp, was attenuated in mice, produced lower cell densities in blood, and was unable to relapse in animals after its initial spirochetemia. This mutant also colonized ticks and was infectious by tick-bite, but remained attenuated compared to wild-type and reconstituted spirochetes. The mutant lacking Vtp also colonized ticks but produced neither Vtp nor a Vmp in tick salivary glands, which rendered the spirochete noninfectious by tick bite. Thus the ability of B. hermsii to produce Vmps prolonged its survival in blood, while the synthesis of Vtp was essential for mammalian infection by the bite of its tick vector.     

Real-time PCR for simultaneous detection and quantification of Borrelia burgdorferi in field-collected Ixodes scapularis ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.     

Genetic variation at the vlsE locus of Borrelia burgdorferi within ticks and mice over the course of a single transmission cycle

The Lyme disease spirochete, Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete. One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens. The vlsE locus represents the best-studied example of antigenic variation in B. burgdorferi. During vertebrate host infection, recombination between the active vlsE locus and silent, partial vlsE copies leads to gene conversion events and the generation of novel alleles at the expression site. In the present study, we followed a population of B. burgdorferi organisms moving through vertebrate host and tick stages to complete one transmission cycle. The major goal of the study was to determine if the vlsE locus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle. We report here that the vlsE genetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages. Therefore, naturally infected ticks are likely to transmit spirochete populations with multiple vlsE alleles into naive vertebrate hosts. Although vlsE genetic diversity in mice was maintained through tick stages, the dominant vlsE alleles were different between tick stages as well as between individual ticks. We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominant vlsE alleles. Although vlsE genetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector.     

Glycerol-3-phosphate acquisition in spirochetes: distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species

Relapsing-fever spirochetes achieve high cell densities (>10(8)/ml) in their host's blood, while Lyme disease spirochetes do not (<10(5)/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.     

Isolation and genetic characterization of a relapsing fever spirochete isolated from Ornithodoros puertoricensis collected in central Panama

Tick-borne relapsing fever (TBRF) spirochetes are likely an overlooked cause of disease in Latin America. In Panama, the pathogens were first reported to cause human disease in the early 1900s. Recent collections of Ornithodoros puertoricensis from human dwellings in Panama prompted our interest to determine whether spirochetes still circulate in the country. Ornithodoros puertoricensis ticks were collected at field sites around the City of Panama. In the laboratory, the ticks were determined to be infected with TBRF spirochetes by transmission to mice, and we report the laboratory isolation and genetic characterization of a species of TBRF spirochete from Panama. Since this was the first isolation of a species of TBRF spirochete from Central America, we propose to designate the bacteria as Borrelia puertoricensis sp. nov. This is consistent with TBRF spirochete species nomenclature from North America that are designated after their tick vector. These findings warrant further investigations to assess the threat B. puertoricensis sp. nov. may impose on human health.     

Prevalence of the Lyme disease spirochete in populations of white-tailed deer and white-footed mice

The prevalence of the Ixodes dammini spirochete (IDS) in white-tailed deer (Odocoileus virginianus) and white-footed mice (Peromyscus leucopus) was studied on the eastern end of Long Island, New York. Both species commonly occur in a variety of habitats, are preferred hosts of Ixodes dammini, and can harbor the spirochetes in the blood. Each animal was examined for spirochetemia, tick infestation, and IDS infection rates in the ticks that were removed from it. The results obtained suggest that in winter deer can be infected by questing adult I. dammini. Adult ticks apparently are infected through transtadial transmission of spirochetes from subadult ticks which had fed earlier in their life history on infected animals. Deer are important hosts of adult ticks and the IDS in winter and probably are a reservoir host in other seasons. The patterns of spirochete prevalence suggest that deer and mice are reservoirs of the organism and thus are fundamental to the ecology of Lyme disease on Long Island.     

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.     

Transmission Dynamics of Borrelia turicatae from the Arthropod Vector

Background

With the global distribution, morbidity, and mortality associated with tick and louse-borne relapsing fever spirochetes, it is important to understand the dynamics of vector colonization by the bacteria and transmission to the host. Tick-borne relapsing fever spirochetes are blood-borne pathogens transmitted through the saliva of soft ticks, yet little is known about the transmission capability of these pathogens during the relatively short bloodmeal. This study was therefore initiated to understand the transmission dynamics of the relapsing fever spirochete Borrelia turicatae from the vector Ornithodoros turicata, and the subsequent dissemination of the bacteria upon entry into murine blood.

Methodology/Principal Findings

To determine the minimum number of ticks required to transmit spirochetes, one to three infected O. turicata were allowed to feed to repletion on individual mice. Murine infection and dissemination of the spirochetes was evaluated by dark field microscopy of blood, quantitative PCR, and immunoblotting against B. turicatae protein lysates and a recombinant antigen, the Borrelia immunogenic protein A. Transmission frequencies were also determined by interrupting the bloodmeal 15 seconds after tick attachment. Scanning electron microscopy (SEM) was performed on infected salivary glands to detect spirochetes within acini lumen and excretory ducts. Furthermore, spirochete colonization and dissemination from the bite site was investigated by feeding infected O. turicata on the ears of mice, removing the attachment site after engorment, and evaluating murine infection.

Conclusion/Significance

Our findings demonstrated that three ticks provided a sufficient infectious dose to infect nearly all animals, and B. turicatae was transmitted within seconds of tick attachment. Spirochetes were also detected in acini lumen of salivary glands by SEM. Upon host entry, B. turicatae did not require colonization of the bite site to establish murine infection. These results suggest that once B. turicatae colonizes the salivary glands the spirochetes are preadapted for rapid entry into the mammal.     

Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.     

TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.     

Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.     

Vector-host interactions in disease transmission

Tick-borne spirochetes include borreliae that cause Lyme disease and relapsing fever in humans. They survive in a triangle of parasitic interactions between the spirochete and its vertebrate host, the spirochete and its tick vector, and the host and the tick. Until recently, the significance of vector-host interactions in the transmission of arthropod-borne disease agents has been overlooked. However, there is now compelling evidence that the pharmacological activity of tick saliva can have a profound effect on pathogen transmission both from infected tick to uninfected host, and from infected host to uninfected tick. The salivary glands of ticks provide a pharmacopoeia of anti-inflammatory, anti-haemostatic and anti-immune molecules. These include bioactive proteins that control histamine, bind immunoglobulins, and inhibit the alternative complement cascade. The effect of these molecules is to provide a privileged site at the tick-host interface in which borreliae and other tick-borne pathogens are sheltered from the normal innate and acquired host immune mechanisms that combat infections. Understanding the key events at the tick vector-host interface, that promote spirochete infection and transmission, will provide a better understanding of the epidemiology and ecology of these important human pathogens.     

Arthropod- and host-specific Borrelia burgdorferi bbk32 expression and the inhibition of spirochete transmission

Antisera to BBK32 (a Borrelia burgdorferi fibronectin-binding protein) and BBK50, two Ags synthesized during infection, protect mice from experimental syringe-borne Lyme borreliosis. Therefore, B. burgdorferi bbk32 and bbk50 expression within Ixodes scapularis ticks and the murine host, and the effect of BBK32 and BBK50 antisera on spirochetes throughout the vector-host life cycle were investigated. bbk32 and bbk50 mRNA and protein were first detected within engorged ticks, demonstrating regulated expression within the vector. Then bbk32 expression increased in mice at the cutaneous site of inoculation. During disseminated murine infection, bbk32 and bbk50 were expressed in several murine tissues, and mRNA levels were greatest in the heart and spleen at 30 days. BBK32 antisera protected mice from tick-borne B. burgdorferi infection and spirochete numbers were reduced by 90% within nymphs that engorged on immunized mice. Moreover, 75% of these ticks did not retain spirochetes upon molting, and subsequent B. burgdorferi transmission by adult ticks was impaired. Larval acquisition of B. burgdorferi by I. scapularis was also inhibited by BBK32 antisera. These data demonstrate that bbk32 and bbk50 are expressed during tick engorgement and that BBK32 antisera can interfere with spirochete transmission at various stages of the vector-host life cycle. These studies provide insight into mechanisms of immunity to Lyme borreliosis and other vector-borne diseases.     

Why Lyme disease is common in the northern US,but rare in the south: The roles of host choice,host-seeking behavior,and tick density

Lyme disease is common in the northeastern United States, but rare in the southeast, even though the tick vector is found in both regions. Infection prevalence of Lyme spirochetes in host-seeking ticks, an important component to the risk of Lyme disease, is also high in the northeast and northern midwest, but declines sharply in the south. As ticks must acquire Lyme spirochetes from infected vertebrate hosts, the role of wildlife species composition on Lyme disease risk has been a topic of lively academic discussion. We compared tick–vertebrate host interactions using standardized sampling methods among 8 sites scattered throughout the eastern US. Geographical trends in diversity of tick hosts are gradual and do not match the sharp decline in prevalence at southern sites, but tick–host associations show a clear shift from mammals in the north to reptiles in the south. Tick infection prevalence declines north to south largely because of high tick infestation of efficient spirochete reservoir hosts (rodents and shrews) in the north but not in the south. Minimal infestation of small mammals in the south results from strong selective attachment to lizards such as skinks (which are inefficient reservoirs for Lyme spirochetes) in the southern states. Selective host choice, along with latitudinal differences in tick host-seeking behavior and variations in tick densities, explains the geographic pattern of Lyme disease in the eastern US.

Lyme disease is common in the northeastern United States, but rare in the southeast, even though the tick vector is found in both regions. This study shows that this is largely because the tick vectors attach abundantly to rodents (which are good hosts for the Lyme bacteria) in the north, and to lizards (which are relatively poor hosts for Lyme bacteria) in the south.     

Suppression of Th2 cytokines reduces tick-transmitted Borrelia burgdorferi load in mice

Previous work has indicated that both Borrelia burgdorferi and the process of tick feeding (saliva) modulate the host immune response. Molecules have been identified in tick saliva that effect T cell proliferation by binding to specific cytokines, thereby promoting a Th2 cytokine response that does not afford protection against tick-transmitted B. burgdorferi in mice. Moreover, reconstitution of a Th1-biased T cell response prior to spirochete challenge effectively neutralizes tick modulation of host immunity and affords protection against tick transmission of spirochetes. The current studies were undertaken to determine the effect of neutralizing specific Th2 cytokines prior to tick feeding and subsequent transmission of B. burgdorferi. The results indicate that suppression of both IL-4 and IL-5 prior to the feeding of B. burgdorferi-infected ticks significantly decreased spirochete load in target organs such as joint, bladder, heart, and skin of the Lyme disease-susceptible host.     

Genetic transformation of the relapsing fever spirochete Borrelia hermsii: stable integration and expression of green fluorescent protein from linear plasmid 200

Tick-borne relapsing fever (TBRF) is a spirochetal disease caused by at least 15 different Borrelia species. It is a serious human health concern in regions of endemicity throughout the world. Transmission to humans occurs through the bites of infected Ornithodoros ticks. In North America, the primary Borrelia species associated with human disease are B. hermsii and B. turicatae. Direct demonstration of the role of putative TBRF spirochete virulence factors in the disease process has been hindered by the lack of a genetic manipulation system and complete genome sequences. Expanding on recent developments in these areas, here we demonstrate the successful generation of a clone of B. hermsii YOR that constitutively produces green fluorescent protein (GFP) (B. hermsii YOR::kan gfp). This strain was generated through introduction of a kan-gfp cassette into a noncoding region of the 200-kb B. hermsii linear plasmid lp200. Genetic manipulation did not affect the growth rate or trigger the loss of native plasmids. B. hermsii YOR::kan gfp retained infectivity and elicited host seroconversion. Stable production of GFP was demonstrated both in vitro and in vivo. This study represents a significant step forward in the development of tools that can be employed to study the virulence mechanisms of TBRF spirochetes.     

Pathobiology of Borrelia theileri in the tropical cattle tick, Boophilus microplus

The development of Borrelia theileri infections in the tick Boophilus microplus was studied during all stages of the tick developmental cycle. Light microscopical examination of hemolymph and ovary smears from ovipositing females allowed identification and separation of infected and uninfected ticks. A Borreli-free tick colony was established. Small numbers of spirochetes were present in larvae, with numbers increasing through the nymphal and adult tick stages. Borreliae occurred in hemolymph, hypodermis, midgut, Malpighian tubules, ovary, Gené's organ, and the central ganglion of engorging and ovipositing females and their eggs. The ovary, central ganglion, and hemolymph seemed to be preferred sites for the spirochete, with extensive multiplication occurring in hemocytes. No measurable effect of spirochete multiplication upon feeding and reproductive performance of ticks could be detected. Infections in cattle caused fever of short duration which coincided with the presence of spirochetes in blood smears. Morphology and size of blood and tick forms were consistent with those of B. theileri reported by other authors. B. theileri is important because infections of invertebrate and vertebrate hosts may interfere with the interpretation of data in various experimental designs, and because it is probably endemic in populations of one or more tick species and their hosts throughout the world.     

The diguanylate cyclase, Rrp1, regulates critical steps in the enzootic cycle of the Lyme disease spirochetes

Rrp1 is the sole c-di-GMP-producing protein (diguanylate cyclase) of Borrelia burgdorferi. To test the hypothesis that Rrp1 regulates critical processes involved in the transmission of spirochetes between ticks and mammals, an rrp1 deletion mutant (B31-Δrrp1) and a strain that constitutively produces elevated levels of Rrp1 (B31-OV) were constructed. The strains were assessed for progression through the enzootic cycle using an Ixodes tick/C3H-HeJ mouse model and tick immersion feeding methods. B31-Δrrp1 infected mice as efficiently as wild type but had altered motility, decreased chemotactic responses to N-acetylglucosamine (NAG) and attenuated ability to disseminate or colonize distal organs. While this strain infected mice, it was not able to survive in ticks. In contrast, B31-OV displayed normal motility patterns and chemotactic responses but was non-infectious in mice. Using immersion feeding techniques, we demonstrate that B31-OV can establish a population in ticks and survive exposure to a natural bloodmeal. The results presented here indicate Rrp1, and by extension, c-di-GMP, are not strictly required for murine infection, but are required for the successful establishment of a productive population of B. burgdorferi in ticks. These analyses provide significant new insight into the genetic regulatory mechanisms of the Lyme disease spirochetes.     

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%.