Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks in Austria

Unfed ticks of all instars (Ixodes ricinus, n=853; Haemaphysalis concinna, n=11) collected in all nine federal states of Austria were individually examined for the presence of Borrelia burgdorferi sensu lato (s.l.) using PCR. The mean overall infection rate was 14.4%. Infection rates were 24.5% in adult ticks, 16.1% in nymphs, and 1.6% in larvae. Four genospecies were detected, including B. valaisiana which was detected for the first time in Austria. The most common B. burgdorferi s.l. genospecies was B. garinii (66.9%), followed by B. valaisiana (13.7%), B. afzelii (11.3%), and B. burgdorferi sensu stricto (s.s.) (6.5%). Two specimens (1.6%) could not be identified to the genospecies level. Geographically, the highest infection rates were detected in the federal state of Vorarlberg (33.3%), B. garinii and B. afzelii being the most prevalent genospecies. B. valaisiana occurred most often in the federal state of Lower Austria, and B. burgdorferi s.s. was focally distributed in the Tyrol, in the surroundings of Imst.     

Detection of three genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in different regions of Poland

Ixodes ricinus ticks, collected in 1996-1998 in different Polish woodlands, were examined to assess the frequency of the occurrence of Lyme borreliosis-associated genospecies. A total of 568 samples of individual adults and 162 samples of individual (n =48) and pooled (of 2 to 7) samples of nymphs were analysed by the polymerase chain reaction (PCR) for Borrelia burgdorferi sensu lato. Spirochetes were detected in 130 adult ticks (22.9%) and in a minimum of 32 (5.3%) nymphs. Further identification of 153 B. burgdorferi s.l.-positive samples by nested PCR using three species-specific primers revealed the occurrence of B. afzelii, B. burgdorferi sensu stricto and B. garinii. Both single-species and mixed infections were noted. Single-species infections were observed in the majority of samples (n = 83/153; 54.2%). Within this group B. afzelii was found in 38/153 samples (24.9%), followed by B. burgdorferi sensu stricto (n = 23/153; 15.0%) and B. garinii (n = 22/153; 14.4%). Dual infections with B. burgdorferi s.s. and B. afzelii were detected in 17/121 (14.0%) adults, while both B. burgdorferi s. s./B. garinii and B. afzelii/B. garinii coinfected 11/121 (9.1%) adult ticks. Triple infection with B. burgdorferi s.s., B. afzelii and B. garinii was noted twice (1.6%). In general, B. afzelii was found in 72/153 (47.1%) tick samples and was the predominant species. B. burgdorferi s. s. and B. garinii were detected in a total of 60/153 (39.2%) and 51/153 (33.3%) samples, respectively. Although, 21 (13.7%) samples were infected by B. burgdorferi s.l. genospecies undetectable by the primers used, results of our study confirm that Lyme borreliosis pathogenic genospecies are well established in tick populations throughout Poland.     

Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks

A total of 301 adult questing Ixodes ricinus ticks were collected at 15 different locations along the south and east coasts of Sweden to determine the Borrelia genospecies diversity. Thirty-two ticks (11%) were found to be positive by nested PCR with Borrelia burgdorferi sensu lato-specific primers. Species determination was based on partial sequencing of the 16S rRNA gene and the flagellin gene. Five different Borrelia species were found. The nucleotide sequence of the Borrelia DNA found in two ticks differed extensively from the nucleotide sequences of the Borrelia DNA found in the other ticks, and analysis revealed that they were closely related to the relapsing fever borrelia species Borrelia miyamotoi. This is the first report of a B. miyamotoi-like borrelia in I. ricinus and in Europe. Moreover, the Borrelia DNA of two ticks (6%) clustered within the B. valaisiana complex. B. valaisiana has not previously been reported in Sweden. B. afzelii DNA was found in 14 ticks (44%), and B. garinii DNA was found in 10 ticks (31%). B. burgdorferi sensu stricto DNA was found in four ticks (13%). We conclude that all of the known human-pathogenic species (B. garinii, B. afzelii, and B. burgdorferi sensu stricto) and B. valaisiana found elsewhere in Europe are also present in the Swedish host-seeking tick population and that a B. miyamotoi-like Borrelia species seems to be present in I. ricinus ticks in Europe.     

Preventive effect of permethrin-impregnated clothing to Ixodes ricinus ticks and associated Borrelia burgdorferi s.l. in Germany

The protective effectiveness of a factory-based, permethrin-impregnated military battle dress uniform (BDU) trouser using a new high-residual, polymer-coating technique has been evaluated by field testing against Ixodes ricinus, a vector of Borrelia burgdorferi s.l. During 36 h exposure of test subjects walking in known tick-infested habitats in the Kühkopf mountain area, Koblenz, Germany, between June and October 2006, 6 I. ricinus were found crawling with a visible excitatory effect on those legs covered with fabric impregnated with 1200 mg permethrin/m², whereas 132 ticks were collected from the negative control legs. B. burgdorferi s.l. infection was detected in 33% (2/6) adult male, 56% (9/16) adult female, 11% (6/56) nymphal, and 0% (0/46) larval I. ricinus sampled from the negative control legs. The few ticks collected from the impregnated fabric tested all negative for B. burgdorferi s.l. The mean tick infestation rate on the negative control legs of test subjects was 3.6±2.7 (mean±SD; range 0–12) per hour exposure. Permethrin-impregnated clothing conveyed a high mean protection rate of 95.5% against questing I. ricinus ticks, making it an excellent tool for the prevention of associated tick-borne diseases.     

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in urban recreational areas of Helsinki

Lyme borreliosis, an infection caused by the tick-borne spirochete Borrelia burgdorferi, is a major health problem for populations in areas of endemicity in the Northern Hemisphere. In the present study we assessed the density of ticks and the prevalence of B. burgdorferi sensu lato among ticks in popular urban recreational areas of Helsinki, Finland. Altogether 1,688 Ixodes ricinus ticks were collected from five areas located within 5 km of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory analysis. The midguts of the ticks were divided into three pieces, one for dark-field microscopy, one for cultivation in BSK-II medium, and one for PCR analysis. Ticks were found in all the study areas; their densities varied from 1 to 36 per 100 m along which a cloth was dragged. The rate of tick infection with B. burgdorferi sensu lato varied from 19 to 55%, with the average being 32%. Borellia afzelii was the most predominant genospecies in all the areas, and no B. burgdorferi sensu stricto isolates were detected. Only two ticks were concurrently infected with both B. afzelii and Borrelia garinii. Dark-field microscopy gave more positive results for B. burgdorferi than did cultivation or PCR analysis. However, the agreement between all three methods was fairly good. We conclude that Lyme borreliosis can be contracted even in urban environments not populated with large mammals like deer or elk. The disease should be taken into account in the differential diagnosis of certain symptoms of patients from these areas, and the use of measures to improve the awareness of the general population and health care officials of the risk of contracting the disease is warranted.     

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe

Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0–11%), 10.8% (2–43%) and 17.4% (3–58%) for larvae (n = 5699), nymphs (n = 48 804) and adults (n = 41 666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17–20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed. Received: 18 August 1997 / Accepted: 12 September 1997     

Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus and I. lividus ticks collected from wild birds in the Republic of Moldova

Wild birds are important reservoirs of Borrelia burgdorferi sensu lato, the bacterial species complex comprising the agents of Lyme disease in North America and Eurasia. We studied the prevalence of B. burgdorferi s.l. in Ixodes ricinus and I. lividus ticks collected from wild birds in the Republic of Moldova. Wild birds were captured in Kodri forest reserve and in biocenoses near the Dnestr River bank and examined for infestation with ticks. A total of 123 I. ricinus and 54 I. lividus ticks were analyzed for Borrelia spp. B. burgdorferi s.l. was detected in 14% of I. ricinus and 5.5% of I. lividus. Borreliae were most prevalent in I. ricinus ticks collected from blackbirds (17%). BLAST analysis of sequenced PCR products confirmed that I. lividus was infected with both B. burgdorferi s.s. and B. garinii and that I. ricinus was infected with B. burgdorferi s.s. only. This is the first record of the Lyme disease agent in I. lividus ticks.     

Prevalence of granulocytic Ehrlichia and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from Southwestern Finland and from Vormsi Island in Estonia

Altogether, 343 adult and 111 nymphal Ixodes ricinus ticks collected from parks in Turku and suburban and rural islands of the Turku archipelago, Finland, and 100 adult I. ricinus ticks collected from Vormsi Island, Estonia, were included in this study. Using the polymerase chain reaction the ticks were examined for 16S rDNA of the Ehrlichia phagocytophila genogroup and for Borrelia burgdorferi sensu lato recA and flagellin genes. None of the Finnish ticks was found to be infected with E. phagocytophila, whereas 3% of the Estonian ticks were positive for this organism. The rate of Finnish ticks infected with B. burgdorferi sensu lato varied from 0% to 11.6% (mean 5%; 9% for adult and 4% for nymphal ticks). The corresponding rate for Estonian ticks was 15%. Borrelia afzelii was the most common genospecies in both Finnish (2.6%) and Estonian (12%) ticks. B. burgdorferi sensu stricto was detected in 2.0% of the Finnish ticks, but in none of the Estonian ticks. These results suggest that the E. phagocytophila genogroup is very rare in Finnish ticks, although the ticks were collected from an area endemic for Lyme borreliosis. In Estonia, E. phagocytophila is found in ticks and may cause disease.     

Diversity in prevalence and genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and rodents in Lithuania and Norway

A total of 1679 questing Ixodes ricinus ticks collected in Lithuania and 535 I. ricinus ticks collected in Norway from locations with different habitats were investigated for the presence of Borrelia burgdorferi sensu lato. In Lithuania, 223 ticks (13.3%) were infected with B. burgdorferi s.l., and in Norway, 28 ticks (5.2%). The highest prevalence of B. burgdorferi s.l was found in deciduous and mixed forests (19.4% in Lithuania, 8.6% in Norway). A lower prevalence was determined in pine forests (8.6% in Lithuania) and costal zones (4.3% in Norway), and the least prevalence was found in grasslands (2.5% in Lithuania, 1% in Norway). A total of 398 rodents belonging to 9 species were live-captured in Lithuania and Norway. Prevalence of B. burgdorferi s.l. in rodents varied between species and sampling sites in both countries. In Lithuania, the prevalence of infection was higher in Microtus arvalis (range 25–57% in different sampling sites) and in Myodes glareolus (range 14–71%) than in Apodemus flavicollis (range 0–37%) and in A. agrarius (range 11–33%). In Norway, the prevalence of B. burgdorferi s.l. in rodents was lower (range from 5% in A. sylvaticus to 6% in A. flavicollis). B. afzelii was the predominant genospecies in ticks and rodents in Lithuania and Norway. In Lithuania, B. afzelii was found in 76%, B. garinii in 10%, B. burgdorferi sensu stricto in 7%, and Borrelia spp. in 6% of infected ticks. Double infections were observed in 1% of the infected ticks. In Norway, B. afzelii was found in 68%, B. garinii in 21%, and B. burgdorferi s.s. in 11% of infected ticks. All infected rodents from both countries hosted B. afzelii genospecies. Only the red squirrel (Sciurus vulgaris) harbored both B. afzelii and B. burgdorferi s.s.     

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.     

Borrelia burgdorferi sensu lato infection in larval Ixodes ricinus (Acari: Ixodidae) feeding on blackbirds in northwestern Italy

Birds belonging to 59 species (n = 1,206) were live captured in Piemonte, northwestern Italy, in 2001. Ixodes ricinus (L.) larvae were collected from 59 birds belonging to nine species, and nymphs were recovered on 79 birds belonging to 10 species. Eurasian blackbirds, Turdus merula L., had significantly higher levels of infestation by ticks than other passerine species. Larval I. ricinus of blackbirds peaked in summer, when prevalence was 39% (95% confidence interval 24.2-55.5) and mean number of ticks per host was 3.3 (1.6-7.2), whereas nymphs peaked in spring, when prevalence was 72.2% (54.8-85.8) and mean number of ticks per host was 6.9 (4.4-10.7). Immature I. ricinus were coincidentally aggregated on blackbirds, with 15 blackbirds feeding 67.4% of nymphs and 40.3% of larvae, and coinfestation by both stages was relatively high in summer: Kappa = 0.64 (0.40-0.88). Borrelia burgdorferi sensu lato was identified by polymerase chain reaction (PCR) in 58.3% (35.9-78.5) of larvae with engorgement ratio > or = 3 that were collected from blackbirds. Larvae that were collected from other passerine species gave negative PCR results. Sixteen of 21 PCR-positive samples belonged to B. garinii (76.2%), and five (23.8%) were Borrelia valaisiana. Results of this study suggest that blackbirds play an important role as hosts for immature I. ricinus and as reservoir of Borrelia garinii in northwestern Italy.     

Prevalence of Borrelia burgdorferi and Granulocytic and Monocytic Ehrlichiae in Ixodes ricinus Ticks from Southern Germany

A total of 287 adult Ixodes ricinus ticks, collected in two regions of southern Germany (Frankonia and Baden-Württemberg) where Borrelia burgdorferi infections are known to be endemic, were examined for the presence of 16S ribosomal DNA specific for the Ehrlichia phagocytophila genogroup, E. chaffeensis, E. canis, and B. burgdorferi by nested PCR. Totals of 2.2% (6 of 275) and 21.8% (65 of 275) of the ticks were positive for the E. phagocytophila genogroup and B. burgdorferi, respectively. Two ticks (0.7%) were coinfected with both bacteria. Of 12 engorged I. ricinus ticks collected from two deer, 8 (67%) were positive for the E. phagocytophila genogroup and one (8%) was positive for B. burgdorferi. There was no evidence of infection with E. canis or E. chaffeensis in the investigated tick population. The nucleotide sequences of the 546-bp Ehrlichia PCR products differed at one or two positions from the original sequence of the human granulocytic ehrlichiosis (HGE) agent (S.-M. Chen, J. S. Dumler, J. S. Bakken, and D. H. Walker, J. Clin. Microbiol. 32:589-595, 1994). Three groups of sequence variants were detected; two of these were known to occur in other areas in Europe or the United States, whereas one has not been reported before. Thus, in the German I. ricinus tick population closely related granulocytic ehrlichiae are prevalent, which might represent variants of E. phagocytophila or the HGE agent.     

Prevalence of Borrelia burgdorferi s.l. in ticks feeding on humans in Thuringia/Germany

In 2004, a total of 618 Ixodes ricinus ticks fed on humans were collected by physicians throughout Thuringia. The prevalence rates of Borrelia burgdorferi sensu lato (s.l.) genotypes were determined by nested PCR and restriction fragment length polymorphism, targeting a 0.8-kb fragment of the ospA gene. The total prevalence of B. burgdorferi s.l. was 6.1%. B. afzelii was found in 67%, B. valaisiana in 15% and B. garinii in 15% of the positive ticks (3% could not be determined). In one tick, a double infection with B. valaisiana type II and B. garinii OspA type V was detected. Female adult ticks had the highest infection rate (11.7%), followed by nymphs (4.5%) and larvae (3.4%). The overall prevalence increased from spring (4.0%) to autumn (10.0%). Nevertheless, the risk of infection was maximal in summer, because of a much higher infestation and consequently a higher absolute number of infected ticks. The predominance of B. afzelii probably results from its resistance against human serum. The unexpectedly low total prevalence is possibly caused by immune defence mechanisms (e.g. complement) effective against less resistant B. burgdorferi s.l. strains in the tick.     

High Prevalence of Granulocytic Ehrlichiae and Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks from Bulgaria

Bulgarian Ixodes ricinus ticks were examined for Ehrlichia and Borrelia coinfection: 34 and 32% of adult ticks and at least 2 and 10% of nymphs were positive for these infections, respectively. Coinfections and dual or triple Borrelia infections were frequent, although Ehrlichia phagocytophila heterogeneity was minimal. Multiple tick-borne bacteria coexist in I. ricinus ticks in southeastern Europe.     

Identification of host bloodmeal source and Borrelia burgdorferi sensu lato in field-collected Ixodes ricinus ticks in Chaumont (Switzerland)

To evaluate the importance of vertebrate species as tick hosts and as reservoir hosts in two endemic areas for Lyme borreliosis in Switzerland, we applied molecular methods for the analysis of bloodmeal source and Borrelia infection in questing Ixodes ricinus L. ticks. In total, 1326 questing ticks were simultaneously analyzed for Borrelia and for blood meal remnants by using reverse line blot. An overall infection prevalence of 19.0% was recorded for Borrelia sp., with similar rates in both sites. Using a newly developed method for the analysis ofbloodmeal targeting the 12S rDNA mitochondrial gene, identification of host DNA from field-collected ticks was possible in 43.6% of cases. Success of host identification at the genus and species level reached 72%. In one site, host identification success reached its maximum in spring (93% in May), decreasing in summer (20% in July) and rising in autumn (73% in October). In the other site, identification rate in ticks remained low from April to July and increased in autumn reaching 68% in October and November. The most prevalent identified host DNA was artiodactyls in both sites. Red squirrel DNA was significantly more frequently detected in ticks collected in one site, whereas insectivore DNA was more frequent in ticks in the other site. DNA from more than one vertebrate host was detected in 19.5% of nymphs and 18.9% of adults. Host DNA was identified in 48.4% of the Borrelia infected ticks. Although DNA from all Borrelia species was found in at least some ticks with DNA from mammals and some ticks with DNA from birds, our results confirm a general association of B. afzelii and B. burgdorferi sensu stricto with rodents, and B. valaisiana and B. garinii with birds.     

Purification and characterization of Borrelia burgdorferi from feeding nymphal ticks (Ixodes scapularis)

Here we describe a protocol for purifying Borrelia burgdorferi from feeding ticks by velocity centrifugation and Percoll density gradient centrifugation. The purified spirochetes were motile and 10- to 20-fold purer than the bacteria in crude tick homogenates. The purified bacteria were present in sufficient quantity for protein and gene expression studies. In comparison to culture-grown bacteria, tick-borne spirochetes had several proteins that were upregulated and a few that were downregulated. When the levels of B. burgdorferi outer surface proteins OspA and OspC were measured, OspC protein and mRNA levels were lower in cultured bacteria than in bacteria purified from ticks. Although differences in OspA mRNA levels were observed between cultured and tick-borne bacteria, no differences were observed at the protein level. These experiments demonstrate that tick-transmitted borreliae display a gene expression and antigen profile different from that of spirochetes cultured in vitro.     

Distribution of Borrelia burgdorferi sensu lato in Ixodes ricinus (Acari: Ixodidae) ticks from the Basque Country, Spain

Borrelia burgdorferi was found widespread in ixodid ticks from the Basque Country (Spain) during a two-step study. In the first part, a total of 7,835 ixodids of eight different species was collected from vegetation, classified, and processed using polymerase chain reaction (PCR) for detection of B. burgdorferi ospA DNA. B. burgdorferi DNA wasdetectedin < or = 12.5% of adults and > or = 0.6% of Ixodes ricinus (L., 1758) nymphs (mean 1.5 and 0.05%, respectively), and in < or = 14.3% of adult Hemaphysalis punctata (Canestrini & Fanzago, 1877) analyzed (mean 1.2%). The second part of the study was undertaken 2 yr later to characterize B. burgdorferi distribution by focusing on the areas where L. ricinus was the predominant species. Ten areas were selected from which 1,535 nymphs and adults of I. ricinus were collected and processed by PCR and culture techniques. Infected ticks were found in all zones. B. burgdorferi DNA was detected in a mean of 9.3 and 1.5% of adults and nymphs, respectively. Nine isolates of B. burgdorferi were obtained, belonging to four different genospecies (B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. lusitaniae). The results indicate that some areas of Spain have a potential risk for Lyme disease agent exposure and that B. borgdorferi appears to have an increasing occurrence in ticks in the Basque Country.     

Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans

A total of 131 Ixodes ricinus (51 females, 1 male and 79 nymphs) removed from persons living in Southern Germany were investigated by immunofluorescence assay for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and monoclonal antibodies specific for outer surface proteins (Osp) A or C. Borreliae were detectable in 48 (36.6%) of the ticks. Infection rates of these adults and nymphs were significantly higher than infection rates of unfed ticks from Southern Germany. Borreliae in 31.3% (n = 15) of the infected ticks expressed solely OspA, solely OspC in 12.5% (n = 6), and both OspA and OspC in 39.6% (n = 19) of ticks, while in 16.7% (n = 8) of ticks neither were expressed. Presentation of OspC by B. burgdorferi in I. ricinus was correlated with tick weight: in females, OspC was detectable only in ticks with a minimum weight of about 3.5 mg, and in nymphs weighing at least 1 mg. These results indicate that in I. ricinus removed from humans OspC is up-regulated during the blood meal of the tick, but in most ticks OspA is still detectable and might even be present in the absence of OspC expression in the midgut and salivary glands of nearly fully engorged nymphal ticks. Furthermore, we found strong evidence that borreliae expressing solely OspA while in the salivary glands can cause Lyme borreliosis. Our findings indicate that during tick feeding, humans are exposed to borreliae that may express either OspA or OspC or both, or lack both OspA and C. These findings suggests that, at the minimum, both OspA and C should be considered as vaccine candidates for prophylaxis of Lyme borreliosis in Europe. Received: 28 July 1998     

Genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from the Autonomous Province of Trento, Italy

Sequences of the variable intergenic spacer region 5S (rrfA) 23S (rrlB) rRNA were used to identify Borrelia genospecies present in Ixodes ricinus nymphs collected from the Lamar Lakes area of the Province of Trento, Italy (overall prevalence=6.3%). Four genospecies were identified, one for the first time in this Province (B. valaisiana), and three which have been noted previously (B. afzelii, B. garinii, and B. burgdorferi s.s.). In order to compare the genetic variability of these genospecies in Trento with that at a European level, our 21 sequences (15 new haplotypes) and all appropriate European Borrelia sequences registered in GenBank (up to the end of 2004) were subjected to a phylogenetic analysis (for a total of 73 sequences and 43 haplotypes). Clusters of sequences representing the five main European genospecies (afzelii, garinii, burgdorferi s.s., valaisiana, lusitaniae) are well-supported. At least two other groups of haplotypes (genospecies) are suggested by our analysis; moreover, divergent evolution may be occurring in several genospecies. The maximum uncorrected pairwise differences between sequences within genospecies ranges from 1.5% (B. burgdorferi s.s.), to 2.3% (B. garinii and B. valaisiana) to 4.7% (B. afzelii), and are not correlated with geographical distribution. Within the Province of Trento, these values for the same genospecies are 1.5%, 2.3%, 0.9%, 1.9%, respectively. These high mutation rates within genospecies suggest that the sequencing of haplotypes should continue if we are to fully understand and monitor the evolution and epidemiology of Borrelia.