Perpetuation of the Lyme Disease Spirochete Borrelia lusitaniae by Lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

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.     

Introduced Siberian chipmunks (Tamias sibiricus barberi) harbor more-diverse Borrelia burgdorferi sensu lato genospecies than native bank voles (Myodes glareolus)

Little attention has been given in scientific literature to how introduced species may act as a new host for native infectious agents and modify the epidemiology of a disease. In this study, we investigated whether an introduced species, the Siberian chipmunk (Tamias sibiricus barberi), was a potentially new reservoir host for Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. First, we ascertained whether chipmunks were infected by all of the B. burgdorferi sensu lato genospecies associated with rodents and available in their source of infection, questing nymphs. Second, we determined whether the prevalence and diversity of B. burgdorferi sensu lato in chipmunks were similar to those of a native reservoir rodent, the bank vole (Myodes glareolus). Our research took place between 2006 and 2008 in a suburban French forest, where we trapped 335 chipmunks and 671 voles and collected 743 nymphs of ticks that were questing for hosts by dragging on the vegetation. We assayed for B. burgdorferi sensu lato with ear biopsy specimens taken from the rodents and in nymphs using PCR and restriction fragment length polymorphism (RFLP). Chipmunks were infected by the three Borrelia genospecies that were present in questing nymphs and that infect rodents (B. burgdorferi sensu stricto, B. afzelii, and B. garinii). In contrast, voles hosted only B. afzelii. Furthermore, chipmunks were more infected (35%) than voles (16%). These results may be explained by the higher exposure of chipmunks, because they harbor more ticks, or by their higher tolerance of other B. burgdorferi sensu lato genospecies than of B. afzelii. If chipmunks are competent reservoir hosts for B. burgdorferi sensu lato, they may spill back B. burgdorferi sensu lato to native communities and eventually may increase the risk of Lyme disease transmission to humans.     

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Loss of Lyme disease spirochetes from Ixodes ricinus ticks feeding on European blackbirds.

To determine whether blackbirds (Turdus merula), the most abundant and most abundantly tick-infested ecotonal bird of Central Europe, may contribute to the transmission of the Lyme disease spirochete (Borrelia burgdorferi), we compared the infectivity to ticks of naturally as well as experimentally infected blackbirds and rodents. European blackbirds experience intense exposure to Ixodes ricinus ticks and to the pathogens that they transmit. In nature, subadult I. ricinus ticks found feeding on these birds generally contain no spirochetes, although infection is universal in those found on black-striped mice (Apodemus agrarius). Those found on yellow-necked mice (A. flavicollis) are less frequently infected. Ticks lose infection in the course of feeding on blackbirds and fail to infect them. Subadult I. ricinus ticks readily feed on blackbirds, black-striped mice, and jirds (Meriones unguiculatus), but engorge less fully on the bird than on the rodents. Although birds may burden human health by establishing new infestations of I. ricinus ticks, our observations indicate that particular birds may benefit health by locally diminishing transmission of the Lyme disease spirochete.     

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.     

Refractoriness of the western fence lizard (Sceloporus occidentalis) to the Lyme disease group spirochete Borrelia bissettii

The western fence lizard, Sceloporus occidentalis, is refractory to experimental infection with Borrelia burgdorferi sensu stricto, one of several Lyme disease spirochetes pathogenic for humans. Another member of the Lyme disease spirochete complex, Borrelia bissettii, is distributed widely throughout North America and a similar, if not identical, spirochete has been implicated as a human pathogen in southern Europe. To determine the susceptibility of S. occidentalis to B. bissettii, 6 na?ve lizards were exposed to the feeding activities of Ixodes pacificus nymphs experimentally infected with this spirochete. None of the lizards developed spirochetemias detectable by polymerase chain reaction for up to 8 wk post-tick feeding, infected nymphs apparently lost their B. bissettii infections within 1-2 wk after engorgement, and xenodiagnostic L. pacificus larvae that co-fed alongside infected nymphs did not acquire and maintain spirochetes. In contrast, 3 of 4 na?ve deer mice (Peromyscus maniculatus) exposed similarly to feeding by 1 or more B. bissettii-infected nymphs developed patent infections within 4 wk. These and previous findings suggest that the complement system of S. occidentalis typically destroys B. burgdorferi sensu lato spirochetes present in tissues of attached and feeding I. pacificus nymphs, thereby potentially reducing the probability of transmission of these bacteria to humans or other animals by the resultant adult ticks.     

Controlled activation of the Cpx system is essential for growth of Yersinia enterocolitica

Until recently, three spirochete genospecies were considered to be the causative agents of Lyme borreliosis (LB) in Europe: Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii . However, the DNA of Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii and Borrelia bissettii has already been detected in samples of human origin, or the spirochetes were isolated from the patients with symptoms of LB. Molecular analysis of 12 selected serum samples collected in the regional hospital confirmed the presence of B. bissettii DNA in cases of single and multiple infection in patients with symptomatic borreliosis or chronic borrelial infection. The presence of B. bissettii as a single strain in patients provides strong support of the fact that B. bissettii might be a causative agent of the disease. After the first isolation of B. bissettii from the samples of human origin in Slovenia, following the detection of this species in cardiac valve tissue of the patient with endocarditis and aortic valve stenosis in the Czech Republic, here we present additional molecular data supporting the involvement of B. bissettii in LB in Europe.     

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.     

Comparative Studies on Borrelia afzelii Isolated from a Patient of Lyme Disease,Ixodes persulcatus Ticks,and Apodemus speciosus Rodents in Japan

The genospecies Borrelia afzelii was isolated from a patient of Lyme disease in Hokkaido, Japan, for the first time, by culturing the minced erythema lesion in BSK II medium. Two analytical methods, rRNA gene restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) using the specific primer set to amplify the 16S rRNA gene, revealed that this clinical isolate belongs to the group of B. afzelii. In our culture collection of spirochetes, part of the isolates from Ixodes persulcatus ticks, and from Apodemus speciosus rodents, were also classified as B. afzelii. These results strongly suggest that the agent pathogenic to humans is maintained in “rodent-tick” transmission cycle.     

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.     

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.     

Diversity of European Lyme Disease Spirochetes at the Southern Margin of Their Range

We determined whether the genospecies diversity of Lyme disease spirochetes in vector ticks questing on a subtropical island is as broad as that in Central Europe. Although spirochetes infected <1% of the ticks sampled on Madeira Island, these infections included all three genospecies implicated in human disease. Therefore, spirochetal diversity is as great at the southern margin as it is in the center of this pathogen’s range.     

Occurrence of multiple infections with different Borrelia burgdorferi genospecies in Danish Ixodes ricinus nymphs

The pathogen Borrelia burgdorferi causes Lyme Borreliosis in human and animals world-wide. In Europe the pathogen is transmitted to the host by the vector Ixodes ricinus. The nymph is the primary instar for transmission to humans. We here study the infection rate of five Borrelia genospecies: B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae in nymphs, by IFA and PCR. 600 nymphs were collected in North Zealand of Denmark. Each nymph was first analysed by IFA. If positive for spirochaetal infection, the genospecies was determined by PCR. The infection rate of B. burgdorferi sensu lato was 15.5%, with the primary genospecies being B. afzelii (64.3%), B. garinii (57.1%), and B. lusitaniae (26.8%). It is the first time B. lusitaniae is documented in Denmark. Even though, the highest infection rate was discovered for B. afzelii and B. garinii, mixed infections are more common than single infections. Fifty-one percent (29/56) of these were infected with two genospecies, 7.1% (4/56) with three, and 5.3% (3/56) with four. We try to explain the high infection rate and the peculiar number of multiple infections, with a discussion of changes host abundance and occurrence of different transmission patterns.     

Seasonal Prevalence of Lyme Disease Spirochetes in a Heterothermic Mammal,the Edible Dormouse (Glis glis)

In Europe, dormice serve as competent reservoir hosts for particular genospecies of the tick-borne agent of Lyme disease (LD) and seem to support them more efficiently than do mice or voles. The longevity of edible dormice (Glis glis) and their attractiveness for ticks may result in a predominance of LD spirochetes in ticks questing in dormouse habitats. To investigate the role of edible dormice in the transmission cycle of LD spirochetes, we sampled skin tissue from the ear pinnae of dormice inhabiting five different study sites in south western Germany. Of 501 edible dormice, 12.6% harbored DNA of LD spirochetes. Edible dormice were infected most frequently with the pathogenic LD spirochete Borrelia afzelii. The DNA of B. garinii and B. bavariensis was detected in ca. 0.5% of the examined individuals. No spirochetal DNA was detectable in the skin of edible dormice until July, 6 weeks after they generally start to emerge from their obligate hibernation. Thereafter, the prevalence of spirochetal DNA in edible dormice increased during the remaining period of their 4 to 5 months of activity, reaching nearly 40% in September. Males were more than four times more likely to harbor LD spirochetes than females, and yearlings were almost twice more likely to be infected than adults. The seasonality of the prevalence of LD spirochetes in edible dormice was pronounced and may affect their role as a reservoir host in respect to other hosts.     

Differential Transmission of the Genospecies of Borrelia burgdorferi Sensu Lato by Game Birds and Small Rodents in England

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest prevalences found for Borrelia garinii and Borrelia valaisiana. B. burgdorferi sensu stricto was rare (<1%) in all tick stages. Borrelia afzelii was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly B. garinii and/or B. valaisiana. Although 19% of the rodents harbored B. burgdorferi sensu stricto and/or B. garinii in internal organs, only B. burgdorferi sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.     

Correlation of Borrelia burgdorferi sensu lato prevalence in questing Ixodes ricinus ticks with specific abiotic traits in the western palearctic

This meta-analysis of reports examining ticks throughout the Western Palearctic region indicates a distinct geographic pattern for Borrelia burgdorferi sensu lato prevalence in questing nymphal Ixodes ricinus ticks. The greatest prevalence was reported between the 5°E and 25°E longitudes based on an analysis of 123 collection points with 37,940 nymphal tick specimens (87.43% of total nymphs; 56.35% of total ticks in the set of reports over the target area). Climatic traits, such as temperature and vegetation stress, and their seasonality correlated with Borrelia prevalence in questing ticks. The greatest prevalence was associated with mild winter, high summer, and low seasonal amplitude of temperatures within the range of the tick vector, higher vegetation indices in the May-June period, and well-connected vegetation patches below a threshold at which rates suddenly drop. Classification of the target territory using a qualitative risk index derived from the abiotic variables produced an indicator of the probability of finding infected ticks in the Western Palearctic region. No specific temporal trends were detected in the reported prevalence. The ranges of the different B. burgdorferi sensu lato genospecies showed a pattern of high biodiversity between 4°W and 20°E, partially overlapping the area of highest prevalence in ticks. Borrelia afzelii and Borrelia garinii are the dominant species in central Europe (east of ～25°E), but B. garinii may appear alone at southern latitudes and Borrelia lusitaniae is the main indicator species for meridional territories.     

Inhibition of Borrelia burgdorferi-tick interactions in vivo by outer surface protein A antibody

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.     

Lyme disease Borrelia spp. in ticks and rodents from northwestern China.

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis

In Europe, Borrelia burgdorferi genospecies causing Lyme borreliosis are mainly transmitted by the tick Ixodes ricinus. Since its discovery, B. burgdorferi has been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies of I. ricinus ticks infected with B. burgdorferi sensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection of I. ricinus with Borrelia in Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection of I. ricinus with Borrelia genospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection of I. ricinus were found in countries in central Europe. B. afzelii and B. garinii are the most common Borrelia species, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection by Borrelia species was found for B. garinii and B. valaisiana.