Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus ticks collected in nature

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31-35 kDa and 18-25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.     

Infection rate of Ixodes ricinus ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto in Slovenia

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.     

Occurrence of Borrelia spirochaetes in ticks (Acari, Ixodidae) collected in the forest areas in Olsztyn province (north central Poland)

In 1993, 2178 out of 3816 Ixodes ricinus and 45 out of 82 Dermacentor reticulatus collected from vegetation at 35 sites and removed from hunter-killed deer in Olsztyn province (N. C. Poland) were examined individually for the presence of Borrelia spirochaetes. Detection of spirochaetes was carried out by the routine indirect immunofluorescence assay (IFA) using polyclonal antibody anti-B. burgdorferi, strain 1 B 29. Borreliae, presumably B. burgdorferi, were evident in 192 (11.5%) I. ricinus (nymphs and adults) collected from vegetation (n = 1666) and in 6 (1.2%) of those removed from hosts (n = 512). Among the first ones, infection rate in nymphs (7.5%) was 2.5 times lower than in adults (18.8%) but was similar in females (18.7%) and males (18.9%). The calculated minimal and maximal infection rates of ticks collected from different locations were 2.9% and 35.7%, respectively. No spirochetes were observed in D. reticulatus tested.     

Tick infestation patterns and prevalence of Borrelia burgdorferi in ticks collected at a veterinary clinic in Germany

A total of 4803 domestic and wild animals which were presented for examination at a veterinary clinic in north Baden, Germany over a period of 1 year were examined for tick infestation. A total of 434 nymphal and adult ticks were collected from 175 hosts. Ticks found belonged to the species Ixodes ricinus (385), Ixodes hexagonus (48), and Ixodes ventalloi (one). The polymerase chain reaction was used to examine 132 I. ricinus and 21 I. hexagonus for the presence of Borrelia burgdorferi. Twenty-two per cent of adult I. ricinus were infected as were one female and one larval I. hexagonus.     

Ultrastructural nucleolar alterations induced by an ametantrone--poly r(A-U) complex

Several studies have described changes in the expression of proteins, especially of OspA and OspC, of B. burgdorferi sensu stricto during tick feeding. In this study, the expression of OspA and OspC of B. afzelii in unfed and feeding I. ricinus nymphs and in the subsequent adults was followed by means of the immunofluorescence test. Spirochaetes expressing OspA and OspC were observed in 70% and 80%, respectively of the unfed nymphs. In feeding and in fully engorged ticks, spirochaetes expressed OspC, while OspA disappeared 24 hours after the beginning of the blood meal. Spirochaetes expressing OspC in salivary glands were observed in one engorged tick. After molting, in unfed adults spirochaetes again expressed OspA and OspC but did so less frequently (6% and 13%, respectively). The mouse strain (AKR/N or BALB/C) on which ticks had their infectious blood meal influenced OspC expression in the following tick stage. In the skin of AKR/N mice, at the tick feeding site, B. afzelii expressed OspC only, as was shown by immunostaining.     

Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California

Previous studies describing the occurrence and molecular characteristics of Lyme disease spirochetes, Borrelia burgdorferi, from California have been restricted primarily to isolates obtained from the north coastal region of this large and ecologically diverse state. Our objective was to look for and examine B. burdorferi organisms isolated from Ixodes pacificus ticks collected from numerous regions spanning most parts of California where this tick is found. Thirty-one isolates of B. burgdorferi were examined from individual or pooled I. pacificus ticks collected from 25 counties throughout the state. One isolate was obtained from ticks collected at Wawona Campground in Yosemite National Park, documenting the occurrence of the Lyme disease spirochete in an area of intensive human recreational use. One isolate from an Ixodes neotomae tick from an additional county was also examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, agarose gel electrophoresis, Southern blot analysis, and the polymerase chain reaction were used to examine the molecular and genetic determinants of these uncloned, low-passage-number isolates. All of the isolates were identified as B. burgdorferi by their protein profiles and reactivities with monoclonal and polyclonal antibodies, and all the isolates were typed by the polymerase chain reaction as North American-type spirochetes (B. burgdorferi sensu stricto). Although products of the ospAB locus were identified in protein analyses in all of the isolates, several isolates contained deleted forms of this locus that would result in the expression of chimeric OspA-OspB proteins. The analysis of OspC demonstrated that this protein was widely conserved among the isolates but was also quite variable in its molecular mass and the amount of it that was expressed.     

Individual pharmacodynamics assessed by antilymphocyte action predicts clinical cyclosporine efficacy in psoriasis

The vector competence of 2 tick species, Ixodes ricinus (L.) and Ixodes scapularis Say, was determined and compared for 3 genospecies of Borrelia burgdorferi. The 3 genospecies of B. burgdorferi used in the following experiments were Borrelia burgdorferi sensu stricto (B-31 and B-31.D1 clone), Borrelia afzelii (strain Pgau. C3), and Borrelia garinii (strain VS286 and VSBP). Spirochetes from all 5 strains were inoculated intradermally into outbred mice; larval ticks of both species were subsequently fed on those mice and replete larvae were assayed for infection by culture in BSK-H media every 7 d for 4 wk. Infection frequencies in I. scapularis exposed to the 5 strains were as follows: B-31 (90%), B-31.D1 (83%), Pgau.C3 (87%), VS286 (10%), and VSBP (5%). The comparable infection frequencies for I. ricinus were B-31 (3%), B-31.D1 (3%), Pgau.C3 (90%), VS286 (5%), and VSBP (3%). Resultant nymphal I. scapularis successfully transmitted B-31, B-31,D1, Pgau.C3, and VS286 to outbred mice. I. ricinus nymphs transmitted Pgau.C3 and VS286. Both species failed to transmit strain VSBP.     

Transmission of Borrelia burgdorferi s.l. from mammal reservoirs to the primary vector of Lyme borreliosis, Ixodes ricinus (Acari: Ixodidae), in Sweden

Factors regulating prevalence of Borrelia burgdorferi s.l. Johnson, Schmid, Hyde, Steigerwalt & Brenner in Ixodes ricinus (L.) were examined during 1991-1992 at Bogesund, near Stockholm in south-central Sweden. Nine species of small and medium-sized mammals (Sorex araneus L., S. minutus L., Neomys fodiens Pennant, Clethrionomys glareolus [Schreber], Microtus agrestis [L.], Apodemus sylvaticus [L.], A. flavicollis [Melchior], Lepus europaeus Pallas, L. timidus L.) were found to infect feeding tick larvae with B. burgdorferi s.l., whereas two species of large mammals (Capreolus capreolus L., Alces alces L.) failed to infect feeding tick larvae with this spirochete. The most important mammalian reservoirs at the study locality were S. araneus and rodents, accounting for 91% of all I. ricinus larvae infected. In view of the great number of potentially effective reservoirs for B. burgdorferi s.l. in Sweden, control of Lyme disease by reduction of abundance of reservoir hosts will be difficult to achieve. We also found that infectivity of a rodent species is related to the number of infesting, potentially infective nymphal I. ricinus. Insectivores and rodents were the most important hosts of larval I. ricinus, whereas most nymphal ticks fed on hares and cervids. Adult I. ricinus were frequently found on all species of hares and cervids examined but never on insectivores and rodents. No single species seemed to be of paramount importance as a source of blood for female ticks. Therefore, control of Lyme disease by reduction of abundance of mammal hosts available for female tick engorgement will probably require massive reductions of numbers of both C. capreolus and L. timidus.     

Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes

We developed a rapid and reliable method for the identification Borrelia burgdorferi sensu lato species in ticks. We used the DNA sequence polymorphism of the spacer region between 5S and 23S rRNA genes, which has been shown to be able to discriminate between eight genomic groups of B. burgdorferi sensu lato (D. Postic, M. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). Spacer DNA was amplified by PCR and was then hybridized to five membrane-bound oligonucleotides. The oligonucleotides were specific for B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and group VS116. A probe which reacted with all genomic groups of B. burgdorferi sensu lato was also used. Ninety-six ticks collected in the field were destructed by bead beating, and the supernatant was used directly in a PCR. B. burgdorferi sensu lato DNA was detected in 6 of 57 adult ticks (11%) and 9 of 39 nymphs (23%). B. garinii was found in three nymphs and four adults, three nymphs carried B. afzelii, and one adult and one nymph carried group VS116. Double infections with B. afzelii and group VS116 were found in two nymphs and one adult. Thus, our method can simultaneously identify three genomic groups of B. burgdorferi sensu lato in ticks collected in the field. This technique provides new ways to study the association of genomic groups present in ticks and the risk of Lyme borreliosis.     

A method and device for measuring force transfers between the deep flexors in the musician''s hand

The efficacy of passive immunisation against tick-transmitted Lyme disease spirochaetal infection was determined in relation to the duration of previous feeding of infected vector ticks. Thus, mice challenged with spirochaete-infected unfed or partially fed nymphal ticks were passively immunised with monoclonal and polyclonal antibodies against the Lyme disease spirochaete (Borrelia burgdorferi) at various intervals after tick attachment. Spirochaetal infection in challenged mice and engorged ticks was verified by xenodiagnosis and indirect immunofluorescent antibody assay, respectively. Although tick-transmitted spirochaetal infection could be aborted by anti-OspA antibodies and hyperimmune antiserum, nearly all immunised mice challenged with infected ticks that had previous 36-h attachment became infected. More than 72% of the nymphal ticks used in this challenge retained their B. burgdorferi infection after engorgement on mice immunised with anti-spirochaete antibodies, and their subsequent infectivity to mice remained effective. It is concluded that a higher efficiency of transmission by partially fed infected nymphs and a lower efficacy of passive immunisation against infection result from an effect of previous feeding of infected ticks that activates antigenic change and enables the spirochaetes to circumvent OspA-based humoral immunity.     

Screening and chemoprophylaxis for tuberculosis infection in college populations

Lyme borreliosis is the most frequent tick-borne disease in the Northern hemisphere. Here we describe the first isolation of Borrelia burgdorferi sensu lato in Bulgaria: the midguts of 47 Ixodes ricinus obtained by flagging from the Central park in Sofia, Bulgaria were cultivated for borreliae in BSK medium. The eight isolates obtained from the ticks and one skin isolate from a Bulgarian patient with erythema migrans were subjected to phenotypic [outer surface protein A (OspA) serotyping] and genotypic analysis (pulsed-field gel electrophoresis typing followed by large restriction fragment pattern analysis after MluI digestion, polymerase chain reaction with 16S rRNA-directed oligonucleotide probes, and restriction fragment length polymorphism analysis of rrf-rrl intergenic spacer amplicons). The skin isolate was B. burgdorferi sensu stricto, as were four of the tick isolates; the other four tick isolates were B. garinii representing three different OspA serotypes (types 3, 5 and 7). These findings confirm the wide geographic distribution of the different B. garinii-associated OspA serotypes in Europe (shown here for the first time for the Southeastern part of Europe) and of B. burgdorferi sensu stricto in the Western hemisphere. These findings have implications for development of diagnostic tests and a borrelia vaccine in Southeastern Europe.     

Borrelia burgdorferi sensu lato in Russia and neighbouring countries: high incidence of mixed isolates

A total of 365 isolates of Borrelia burgdorferi sensu lato from 12 major administrative territories of Russia (from St. Petersburg in the west to South Sakhalin in the east) and from the Czech Republic, Estonia, Lithuania, Byelorussia, Moldavia, Ukraine and Kirghizia were identified by analysis of restriction polymorphism of ribosomal rrf-rrl spacer amplicons. The isolates were obtained mainly from ixodes persulcatus and I. ricinus ticks. Other sources included small mammals, human patients and I. trianguliceps ticks. The results showed that B. garinii (two variants) together with B. afzelii circulated throughout the territories studied. The distribution of the variant NT29 of the species B. garinii, the most frequently isolated, was associated with that of I. persulcatus ticks. B. burgdorferi sensu stricto, and the species B. valaisiana and B. lusitaniae (formerly the genomospecies VS116 and PotiB2, respectively) were isolated only from I. ricinus ticks in the western part of the studied territories. None of these three species were found in 327 isolates from Russia where I. persulcatus is the most frequently distributed vector. This work also provides evidence for a high incidence of mixed Borrelia infections within vectors and hosts (9.3% of isolates were mixtures of Borrelia species). A detailed analysis of Borrelia species distribution over the territories studied is presented.     

Immunization with a polyvalent OspA vaccine protects mice against Ixodes ricinus tick bites infected by Borrelia burgdorferi ss, Borrelia garinii and Borrelia afzelii

Sequence variability of the outer surface protein (Osp) A among Borrelia burgdorferi sl species suggests that a monovalent OspA vaccine may not protect against the various Borrelia present in Eurasia. Here, we confirmed that a monovalent recombinant OspA (rOspA) vaccine does not protect mice against Ixodes ricinus mediated infection with B. burgdorferi ss, Borrelia garinii and Borrelia afzelii. However, when mice were vaccinated with a cocktail of various rOspA from these three species, they were protected, and all challenge ticks that fed on them were cleared of their spirochetes. These results showed that a multiple OspA antigens vaccine, compatible with human use, was very efficient at protecting mice against B. burgdorferi ss, B. garinii, and B. afzelii.     

Seasonal variation in the capacity of the bank vole to infect larval ticks (Acari: Ixodidae) with the Lyme disease spirochete, Borrelia burgdorferi

Seasonal variation in the capacity of bank voles, Clethrionomys glareolus (Schreber), to infect larval Ixodes ricinus (L.) ticks with Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner was examined from May through October 1991 at Bogesund, near Stockholm in south-central Sweden. Although larval infestations of bank voles were greatest in June and July, nearly 70% of all larval ticks infected with spirochetes by bank voles at this site became infected during August and September. Seasonality of infectivity was related to the degree of earlier nymphal infestation on voles as well as to the age composition of the vole population. These factors may influence the infectivity of other rodent reservoirs of B. burgdorferi, both in Europe and North America. Moreover, in determining the reservoir potential of tick hosts, a host population's spirochetal infectivity should be determined for the entire period of larval infestation rather than just during the period of peak larval infestation.     

Erythema migrans-like rash illness at a camp in North Carolina: a new tick-borne disease?

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, has never been isolated from a patient thought to have acquired Lyme disease in any southeastern state. OBJECTIVE: To investigate 14 cases of an erythema migrans (EM)-like rash illness that occurred during 2 summers at an outdoor camp in central North Carolina in an effort to determine the etiologic, epidemiological, and clinical aspects of this illness. METHODS: Using active surveillance, we identified cases of clinically diagnosed EM in residents and staff of the camp. We collected clinical and demographic information; history of exposure to ticks; acute and convalescent serum antibodies to B. burgdorferi, Rickettsia rickettsii, and Ehrlichia chaffeensis; and cultures for spirochetes from biopsy specimens of skin lesions. Serum samples from a group of residents and staff who did not develop rashes were tested for the same antibodies. We speciated ticks removed from people and collected from vegetation. RESULTS: We identified 14 cases of EM-like rash illness during the 2 summers. Of the 14 case-patients, 10 had associated mild systemic symptoms and 1 had documented fever. All 14 case-patients had removed attached ticks, and 8 remembered having removed a tick from the site where the rash developed a median of 12 days earlier (range, 2-21 days). One tick removed from the site where a rash later developed was identified as Amblyomma americanum, the Lone Star tick; 97% of ticks collected from vegetation and 95% of ticks removed from people were A. americanum. No spirochetes were isolated from skin biopsy specimens. Paired serum samples from 13 case-patients did not show diagnostic antibody responses to B. burgdorferi or other tick-borne pathogens. CONCLUSIONS: This investigation suggests the existence of a new tick-associated rash illness. We suspect that the disease agent is carried by A. americanum ticks. In the southern United States, EM-like rash illness should no longer be considered definitive evidence of early Lyme disease.     

Accelerated infectivity of tick-transmitted Lyme disease spirochetes to vector ticks

We determined whether the span of infectivity of Lyme disease spirochetes (Borrelia burgdorferi) to vector ticks varies with the mode of infection in laboratory mice. Noninfected larval deer ticks were permitted to feed on two strains of spirochete-infected mice that had been naturally (via tick bite) and parenterally (via needle injection) infected with B. burgdorferi 2, 4, or 8 weeks earlier, and engorged ticks were dissected and examined for spirochetes by direct immunofluorescence microscopy. After initial infection, spirochetal infectivity to ticks was less efficient in needle-infected mice than in mice infected via tick bites. Tick-transmitted spirochetes develop more rapidly from the skin of infected mice and do not induce a strong antispirochete antibody response during the early stage of infection.     

Borrelia burgdorferi erpT expression in the arthropod vector and murine host

The expression of a Borrelia burgdorferi gene, erpT, was investigated throughout the spirochaete life cycle in the arthropod vector and the murine host. Three phage clones from a B. burgdorferi DNA expression library synthesized a 30 kDa antigen that was recognized by antibodies in the sera of B. burgdorferi-infected mice but not mice hyperimmunized with B. burgdorferi lysates. Differential antibody binding suggested that this protein was preferentially expressed in vivo. This antigen was designated ErpT, based upon 99.6% homology with the BBF01 sequence in the B. burgdorferi genome. ErpT was not detected on spirochaetes cultured in BSK II medium by indirect immunofluorescence or in B. burgdorferi lysates by immunoblotting, implying that ErpT is not readily produced in vitro. erpT mRNA was not discernible by Northern blot but was identified by RNA polymerase chain reaction in vitro, indicating that erpT is expressed at low levels by cultured spirochaetes. erpT expression was then investigated in the vector and mice because B. burgdorferi do not normally reside in culture medium. RNA polymerase chain reaction and immunofluorescence studies demonstrated that erpT was expressed by a small minority of B. burgdorferi (11/500, 2.2%) within unfed ticks and then repressed during engorgement. erpT mRNA or ErpT antibodies were first detected in B. burgdorferi-infected mice at 4 weeks, suggesting that erpT was not expressed in the early stages of murine infection. Then, during persistent infection, RNA polymerase chain reaction showed that erpT was expressed by B. burgdorferi within the joints, heart and spleen, but not by spirochaetes in the skin. Immunization of mice with ErpT was antigenic but was not protective. These studies demonstrate that B. burgdorferi erpT is differentially expressed throughout the B. burgdorferi life cycle, in both the vector and the mammalian host, and is primarily expressed in extracutaneous sites during murine infection.     

An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes

The reservoir competence of passerine birds for the Lyme borreliosis spirochetes was studied in an enzootic focus in Switzerland. Skin aspirates and skin biopsies were used to isolate Borrelia spirochetes from Turdus species. B. burgdorferi sensu lato was isolated and/or PCR-detected in BSK medium containing skin biopsy or skin aspirate from 5 blackbirds (T. merula) and one song thrush (T. philomelos). Seven isolates were obtained from 3 different blackbirds. Either B. garinii or Borrelia from the genomic group VS116 was found in bird skin samples. Mixed infection occurred in 2 cases. Tick xenodiagnosis was used to determine whether blackbirds transmitted Borrelia to ticks. Five xenodiagnoses were performed on 3 different blackbirds. Borrelia DNA was detected in BSK medium inoculated with xenodiagnostic ticks from all the passerines tested. Isolates cultured from xenodiagnostic ticks were obtained from 2 blackbirds. Isolates belonged to group VS116 (n = 10) and to B. garinii (n = 1). Our study has shown that Turdus sp. are infected by B. garinii and by Borrelia from group VS116 and that blackbirds are implicated as reservoirs for these 2 genomic groups of Borrelia, as they transmit living borreliae to ticks. An association seems to exist between birds and Borrelia VS116, and to a lesser extent, B. garinii, similar to the association existing between small rodents and B. afzelii. Our observations emphasize the fact that different enzootic cycles maintain Lyme borreliosis spirochetes in nature.     

Risk of contamination by Borrelia burgdorferi S. lato in a forest environment. Survey during 13 months of abundance of the tick Ixodes ricinus and its level of infestation by the Lyme borreliosis agent in Brittany

The authors, in a forest in Brittany previously studied for several years, caught by flagging, each month from April 1992 to May 1993, nymphs of I. ricinus tick, and looked by indirect immunofluorescence, for B. burgdorferi infestation. An amount of 1,506 ticks was thus studied. Infestation frequency was varying from 0 per cent in January and February to 14.4 per cent in August. Standarding of tick collecting method allowed to establish, for each month, a tick, "availability" index, and, according to the spirochete infestation frequency, to do estimation of the risk level, for human visiting the concerned forest, of being infected by B. burgdorferi. Obtained results show that this risk is the highest in August, and quite non-existent in January and February.     

NICE, a new cause of death classification for stillbirths and neonatal deaths. Neonatal and Intrauterine Death Classification according to Etiology

During May 1996, field surveys on Lyme disease spirochetes were conducted in Beijing, Shenyang, Fushun, and Inner Mongolia in northeastern China. The ticks collected consisted of 3 genera and 12 species. Of these, Ixodes persulcatus was dominant in sun-exposed vegetation in forests in Inner Mongolia; 57 Borrelia strains (55/123 unfed adults and 2/5 immature stages fed on a rodent) were obtained from this tick by BSK culture. Additionally, 2/2 Apodemus peninsulae were positive. Ixodes nipponensis, Ixodes pavlovskyi, Haemaphysalis douglasi, and Haemaphysalis megaspinosa, newly recorded in China, and other Haemaphysalis spp. were all negative for Borrelia. Based on a polymerase chain reaction restriction fragment length polymorphism analysis of the 45 strains successfully subcultured, these were classified as 29 Borrelia garinii and 16 Borrelia afelii. These strains seemed to be more closely related to Japanese strains in genetic features than to those from Europe. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested more diversity in both genospecies, but Borrelia burgdorferi sensu stricto was not found.