Occurrence of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in the Ixodes ricinus ticks from Eastern Slovakia

A total of 2816 unfed adults nymphs of Ixodes ricinus ticks were collected from vegetation in Koice (Eastern Slovakia) from 1994 to 1997. Prevalence of Borrelia burgdorferi s. 1. in I. ricinus ticks, detected by dark field microscopy, varied and depended upon the year and the habitat of the collected ticks. The lowest prevalence was observed in 1994 (4.8%). During 1995 it increased to 17.2% and during the next two years decreased to 15.5% and 14.2%. The rate of infection varied from 2.1 to 23.3% within 10 examined habitats of the Koice area. A different value of relative density of ticks was observed in various habitats. It ranged from 9–212 ticks per collecting hour within one flagged area (600m²) which is 1.5–35.5 ticks per 100m². Eight isolates were obtained from the infected ticks. Electrophoresis and immunoblotting with 6 monoclonal antibodies were used for the identification of Borrelia strains. Three tick isolates were identified as B. burgdorferi s. s. and the other three isolates were found to be B. garinii. One strain reacted as a mixed culture of B. burgdorferi s. s., and B. garinii. The strain originated from the Vihorlat Mountains habitat and was detected by PCR-SSCP as B. burgdorferi s. s. with a small amount of B. afzelii. The obtained results emphasize the epidemiological importance not only of B. garinii and B. afzelii but also of B. burgdorferi s. s. in Central Europe.     

Predicting distributions of blacklegged ticks (Ixodes scapularis), Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) and human Lyme disease cases in the eastern United States

Lyme disease is the most commonly reported vector-borne disease in the United States (US), with approximately 300,000 -to- 40,000 cases reported annually. The blacklegged tick, Ixodes scapularis, is the primary vector of the Lyme disease-causing spirochete, Borrelia burgdorferi sensu stricto, in high incidence regions in the upper midwestern and northeastern US. Using county-level records of the presence of I. scapularis or presence of B. burgdorferi s.s. infected host-seeking I. scapularis, we generated habitat suitability consensus maps based on an ensemble of statistical models for both acarological risk metrics. Overall accuracy of these suitability models was high (AUC = 0.76 for I. scapularis and 0.86 for B. burgdorferi s.s. infected-I. scapularis). We sought to compare which acarological risk metric best described the distribution of counties reporting high Lyme disease incidence (≥10 confirmed cases/100,000 population) by setting the models to a fixed omission rate (10%). We compared the percent of high incidence counties correctly classified by the two models. The I. scapularis consensus map correctly classified 53% of high and low incidence counties, while the B. burgdorferi s.s. infected-I. scapularis consensus map classified 83% correctly. Counties classified as suitable by the B. burgdorferi s.s. map showed a 91% overlap with high Lyme disease incidence counties with over a 38-fold difference in Lyme disease incidence between high- and low-suitability counties. A total of 288 counties were classified as highly suitable for B. burgdorferi s.s., but lacked records of infected-I. scapularis and were not classified as high incidence. These counties were considered to represent a leading edge for B. burgdorferi s.s. infection in ticks and humans. They clustered in Illinois, Indiana, Michigan, and Ohio. This information can aid in targeting tick surveillance and prevention education efforts in counties where Lyme disease risk may increase in the future.     

Host-utilization differences between larval and nymphal deer ticks in northeastern U.S. sites enzootic for Borrelia burgdorferi sensu stricto

In the northeastern U.S., Borrelia burgdorferi sensu stricto, the agent of Lyme disease, is maintained between vertebrate hosts and subadult deer ticks (the northern clade of Ixodes scapularis, formerly known as Ixodes dammini). Theoretical arguments suggest that the force of transmission would be greatest when infected nymphal ticks focus their bites on the same host as the uninfected larvae. Stage-specific differences in host utilization would reduce the force of transmission, but to date such differences remain understudied. We determined the host utilization differences of larval and nymphal deer ticks using bloodmeal analysis of host-seeking nymphs and adults collected from 5 field sites in New England. Matched cohorts of ticks (nymphs=506, adults=451), i.e. ticks that had fed during the same summer season, were used to control for yearly host population variations. Infection status of all ticks was determined by real time PCR. Nymphal deer ticks were more likely to have fed on birds and sciurids (13% vs 3%, and 41% vs 9%, respectively p<0.001) and larvae were more likely to have fed on shrews (26% vs 3%, p<0.001). Similarly, ticks that had fed on a mouse or a shrew as larvae were likely to become infected (OR= 3.195, 95% CI [1.9, 5.1] and OR=2.5[1.6,3.8] respectively), and they were positively associated with infection prevalence at our sites. However, very few nymphs fed on shrews, and they were not associated with infection, raising the question of how uninfected shrews acquire infection each year. Sciurids did not appear to contribute to the enzootic cycle at our sites, which may be due to the low numbers of larvae that fed on them. Sciurid-fed ticks of either stage were not associated with infection. Both stages of ticks were less likely to be infected if they had fed on deer (OR=0.08 [0.02.0.3] and OR=0.4 [0.2,0.7] tested as nymphs and adults, respectively) and thus deer likely served to reduce the force of transmission at our sites. Site-specific analysis of differential host utilization by subadult deer ticks may contribute to appropriate targeting of interventions and thereby promote reducing risk of acquiring Lyme disease and the other deer tick-transmitted infections.     

Borrelia burgdorferi sensu lato infection in Ixodes ricinus ticks in urban green areas in Prague

Ixodes ricinus ticks are considered as the vector of the Borrelia burgdorferi sensu lato complex in urban areas, including city parks and green recreational areas. The aim of the present study was to determine the prevalence of B. burgdorferi s. l. in urban areas in the city of Prague, Czech Republic. In selected public green areas in Prague, a total of 2819 I. ricinus ticks were collected in spring, from April to June, in 2014-2020. Quantitative real time PCR revealed 28.1% of ticks (31% of males, 33.7% of females and 25.8% of nymphs) to be positive for B. burgdorferi s. l. The prevalence varied significantly (p?0.01) between collection sites, with the highest numbers of infected ticks found in the central city areas. The places serving people for recreational and sport activities in urban areas are characterized by a lower diversity of reservoir hosts, provide opportunity for exposure to Borrelia infected ticks, and pose a higher infection risk. We have detected seven Borrelia species in ticks: B. garinii, B. afzelii, B. bavariensis, B. burgdorferi sensu stricto, B. valaisiana, B. spielmanii, and B. finlandensis. Most positive ticks were infected by B. garinii (35%) and B. afzelii (36.9%). Our results show that the Borrelia transmission cycle occurs within urban biotops and highlight the need for surveillance of tick-borne pathogens in public green areas.     

Antigenic and genomic analysis of a Borrelia burgdorferi sensu stricto strain isolated from Ixodes ricinus ticks in Alto Adige-South Tyrol, Italy

A Borrelia burgdorferi sensu lato strain isolated from Ixodes ricinus ticks in Alto Adige-South Tyrol (Northern Italy) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins, Western immunoblotting analysis (WBA) with polyclonal and monoclonal antibodies, and pulsed-field gel electrophoresis (PFGE). The isolate named BZ6 was identified as belonging to the genospecies B. burgdorferi sensu stricto on the basis of its protein profile and its reactivity with monoclonal and polyclonal antibodies. The PFGE study performed with the two rare-cutting restriction enzymes MluI and SmaI confirmed the SDS-PAGE and WBA characterizations, but showed a genetic diversity between the isolate and two out of the three B. burgdorferi sensu stricto strains used in this study as controls, the American type strain B31 and the locally isolated strain BZ1. No difference in the PFGE patterns between the isolate BZ6 and the Swiss strain IRS was noted. Our findings show the value of PFGE analysis for classifying B. burgdorferi sensu lato isolates and for revealing their genetic diversity, and its usefulness for epidemiological investigations.     

Anaplasma phagocytophilum, Babesia microti, and Borrelia burgdorferi in Ixodes scapularis, southern coastal Maine

Ixodes scapularis (deer ticks) from Maine were tested for multiple infections by polymerase chain reaction and immunofluorescence. In 1995, 29.5%, 9.5%, and 1.9% of deer ticks were infected with Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti, respectively. In 1996 and 1997, the number of A. phagocytophilum-infected ticks markedly declined. In 1995 through 1996, 4 (1.3%) of 301 were co-infected.     

Distribution and prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks of canton Ticino (Switzerland)

Free-living Ixodes ricinus ticks were collected from 12 different sites of canton Ticino, south of the Alps (Switzerland). Each tick was examined for the presence of Borrelia burgdorferi sensu lato (sl), the etiologic agent of Lyme borreliosis using direct fluorescent antibody assay, and isolation of the bacteria. Borreliae were characterized by PCR followed by RFLP. The abundance and infection rates of I. ricinus ticks varied greatly between the areas. Two localities were found free of Borrelia. The prevalence of infected ticks ranged from 5 to 19%. Most ticks (96%) were found infected by <50 spirochetes. Three B. burgdorferi sl species were successfully isolated: B. garinii dominated, followed by B. lusitaniae and B. valaisiana. Additionally, a mixed infection with B. garinii and B. valaisiana was observed. The distribution of the various Borrelia species in the different areas was heterogeneous. This is the first report of the presence of B. lusitaniae in I. ricinus in Switzerland.     

Inter-annual variation in prevalence of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum in host-seeking Ixodes scapularis (Acari: Ixodidae) at long-term surveillance sites in the upper midwestern United States: Implications for public health practice

The geographic range of the blacklegged tick, Ixodes scapularis, and its associated human pathogens have expanded substantially over the past 20 years putting an increasing number of persons at risk for tick-borne diseases, particularly in the upper midwestern and northeastern United States. Prevention and diagnosis of tick-borne diseases rely on an accurate understanding by the public and health care providers of when and where persons may be exposed to infected ticks. While tracking changes in the distribution of ticks and tick-borne pathogens provides fundamental information on risk for tick-borne diseases, metrics that incorporate prevalence of infection in ticks better characterize acarological risk. However, assessments of infection prevalence are more labor intensive and costly than simple measurements of tick or pathogen presence. Our objective was to examine whether data derived from repeated sampling at longitudinal sites substantially influences public health recommendations for Lyme disease and anaplasmosis prevention, or if more constrained sampling is sufficient. Here, we summarize inter-annual variability in prevalence of the agents of Lyme disease (Borrelia burgdorferi s.s.) and anaplasmosis (Anaplasma phagocytophilum) in host-seeking I. scapularis nymphs and adults at 28 longitudinal sampling sites in the Upper Midwestern US (Michigan, Minnesota, and Wisconsin). Infection prevalence was highly variable among sites and among years within sites. We conclude that monitoring infection prevalence in ticks aids in describing coarse acarological risk trends, but setting a fixed prevalence threshold for prevention or diagnostic decisions is not feasible given the observed variability and lack of temporal trends. Reducing repeated sampling of the same sites had minimal impact on regional (Upper Midwest) estimates of average infection prevalence; this information should be useful in allocating scarce public health resources for tick and tick-borne pathogen surveillance, prevention, and control activities.     

Update on the presence of Ixodes ricinus at the western limit of its range and the prevalence of Borrelia burgdorferi sensu lato

It is often suggested that due to climate and environmental policy changes, the risk from tick-borne disease is increasing, particularly at the geographical limits of the vector distribution. Our project aimed to determine whether this was true for the risk of Lyme borreliosis in Ireland which is the western-most limit of Ixodes ricinus, the European vector of Borrelia burgdorferi sensu lato. The availability of a historical data set of tick infection rates compiled in the 1990s represented a unique opportunity as it provided a baseline against which current data could be compared.Following construction of a spatial predictive model for the presence and absence of I. ricinus based on data from 491 GPS locations visited between 2016 and 2019, 1404 questing nymphs from 27 sites were screened for the presence of Borrelia spp. using a TaqMan PCR aimed at the 23S rRNA gene sequence. All positive ticks were further analysed by nested PCR amplification and sequence analysis of the 5 S–23 S intergenic spacer.The model indicated that areas with the highest probability of tick presence were mostly located along the western seaboard and the Shannon and Erne river catchments, coinciding with historical high incidence areas of bovine babesiosis, while the infection rate of questing nymphs with B. burgdorferi s.l. and the prevalence of the various genospecies have remained surprisingly stable over the last 3 decades. Clear communication of the potential disease risk arising from a tick bite is essential in order to allay undue concerns over tick-borne diseases among the general public.     

Genetic diversity of Borrelia burgdorferi sensu lato isolates obtained from Ixodes ricinus ticks collected in Slovakia

In Europe, Borrelia burgdorferi sensu lato is diverse, including B.burgdorferi s.s., B.garinii, B.afzelii, B.valaisiana and B.lusitaniae. In this study, we focused on the distribution of the different B.burgdorferi species among Ixodes ricinus adult ticks collected in an endemic area within Slovakia. We compared results of prevalence of B.burgdorferi infection in ticks obtained by immunofluorescence (IF) and by isolation. Isolates were characterized by restriction fragment length polymorphism (RFLP) of the rrf-rrl intergenic spacer genes using MseI. Using immunofluorescence we observed that 56/114 (49%) ticks were infected by B.burgdorferi s.l. Males were found to be more often infected (32/57, 56%) than females (24/57, 42%) but the difference was not significant (p=0.1895). From the same 114 ticks a total of 37 isolates were obtained: 19 from males (33%) and 18 from females (32%). The RFLP identification revealed 25 B.afzelii (68%), 5 B.garinii (14%), 5 B.valaisiana (14%) and 2 B.lusitaniae (5%). The infection in ticks was more often detected by IF than by isolation (p=0.0153) and isolation success was higher when the infection degree in ticks was high (p=0.0397). The infection prevalence observed in this area is among the highest observed in Europe.     

Nonspecific symptoms following infection with Borrelia burgdorferi sensu lato: A retrospective cohort study

Whether infection with Borrelia burgdorferi sensu lato (sl) can cause nonspecific symptoms is a matter of controversy. We investigated whether individuals infected with B. burgdorferi sl develop unspecific symptoms more frequently than non-infected controls. Eighty-nine persons having presented with B. burgdorferi sl infection between 2015 and 2019 were asked to participate. The infection was defined as erythema migrans diagnosed either in the course of a previous study, during medical visits in the outpatient department, or as seroconversion in asymptomatic subjects. The control group consisted of 85 seronegative individuals without erythema migrans in the past. About two and a half years later, participants were asked to fill out a questionnaire with a list of nonspecific symptoms. The data of 37 persons with previous Borrelia infection and 49 uninfected controls were available for analysis. Muscle pain was significantly (P = 0.040) more frequent in the control group. Fatigue occurred more often in the infected group, but this was not statistically significant (P = 0.109). Likewise, the distribution of other symptoms did not differ considerably. The analysis revealed no difference in the frequency of symptoms of persons who had EM or asymptomatic Borrelia infection 2 years prior assessment in comparison to persons without Borrelia infection.     

Transmission risk of Borrelia burgdorferi sensu lato from Ixodes ricinus ticks to humans in southwest Germany.

The risk of Borrelia burgdorferi infection and the value of antibiotic prophylaxis after tick bite are controversial. In this study, performed in two areas of southwestern Germany, ticks were collected from 730 patients and examined by the polymerase chain reaction (PCR) for B. burgdorferi. To assess whether transmission of B. burgdorferi occurred, the patients were clinically and serologically examined after tick removal and during follow-up examinations. Data from all tick bites gave a total transmission rate of 2.6% (19 patients). Eighty-four ticks (11.3%) were PCR positive. Transmission occurred to 16 (26.7%) of 60 patients who were initially seronegative and could be followed up after the bite of an infected tick. These results indicate that the transmission rate from infected ticks in Europe is higher than previously assumed. Examination of ticks and antibiotic prophylaxis in the case of positivity appears to be indicated.     

Persistence of Borrelia burgdorferi sensu lato in patients with Lyme borreliosis

In 18 patients with Lyme borreliosis the authors proved the persistence of Borrelia burgdorferi sensu lato by detection of the causal agent by immune electron microscopy or of its DNA by PCR in plasma or cerebrospinal fluid after an interval of 4-68 months. Clinical manifestations common in Lyme borreliosis were present in only half the patients, in the remainder non-specific symptoms were found. In nine subjects with confirmed Borrelia burgdorferi sensu lato in the cerebrospinal fluid the cytological and biochemical finding was normal. Examination of antibodies by the ELISA method was negative in 7 of 18 patients during the first examination and in 12 of 18 during the second examination. In all negative examinations the specific antibodies were assessed by the Western blot or ELISA method after liberation from the immunocomplexes. In the authors' opinion it is advisable to examine repeatedly plasma and other biological material from potentially affected organs by PCR and subjects with persisting or relapsing complaints after the acute form of Lyme borreliosis as well as to examine cerebrospinal fluid in case on non-specific symptoms and concurrent pathic EEG or MR findings.     

Mapping distributions of the Lyme disease vector,Ixodes scapularis,and spirochete,Borrelia burgdorferi,in Kentucky using passive and active surveillance

Lyme disease is the most common tick-borne illness in the United States and is becoming more prevalent each year. It is transmitted to humans and animals through the bites of Ixodes scapularis ticks infected with Borrelia burgdorferi in the eastern United States, I. pacificus in the western U.S, and I. ricinus in Europe and Asia. In Kentucky, where Lyme disease is non-endemic, the number of reported human cases in 2010 totaled five. In 2019, that number had increased by over 300%. Identifying the distribution of I. scapularis populations infected with B. burgdorferi is important data for effective prevention strategies and an important first step in monitoring disease spread. In collaboration with the Kentucky Department for Public Health, we performed surveillance for I. scapularis throughout the state of Kentucky using both active and passive surveillance methods. Diagnostic testing for the identification of B. burgdorferi (sensu stricto) was also conducted. We identified 457 I. scapularis ticks from March 2019 to December 2020 from 32 counties in Kentucky. B. burgdorferi was detected in I. scapularis populations collected from 14 different counties. These results add to the little data that exists in Kentucky on I. scapularis and B. burgdorferi distribution.     

Synchronous phenology of juvenile Ixodes scapularis,vertebrate host relationships,and associated patterns of Borrelia burgdorferi ribotypes in the midwestern United States

To elucidate features of enzootic maintenance of the Lyme disease bacterium that affect human risk of infection, we conducted a longitudinal study of the phenology of the vector tick, Ixodes scapularis, at a newly invaded site in the north-central United States. Surveys for questing ticks and ticks parasitizing white-footed mice and eastern chipmunks revealed that I. scapularis nymphal and larval activity peaked synchronously in June and exhibited an atypical, unimodal seasonality. Adult seasonal activity was bimodal and distributed evenly in spring and fall. We discuss implications of these phenology data for the duration of the I. scapularis life cycle. Densities of Borrelia burgdorferi-infected, questing nymphs were comparable to those found in endemic areas elsewhere in the midwestern and northeastern U.S. Molecular genetic diversity of B. burgdorferi infecting these ticks and rodents was assessed by analysis of the ribosomal spacer types (RSTs). RST 1, a clade that includes strains with highly pathogenic properties, was relatively uncommon (3.4%) in contrast to the northeastern U.S., whereas less pathogenic ribotypes of the RST 2 and 3 clades were more common. These features of the ecology of this midwestern Lyme disease system likely contribute to the lower incidence of Lyme disease in humans in the Upper Midwest compared with that of the Northeast owing to reduced exposure to pathogenic strains of B. burgdorferi.     

Outer surface protein E (OspE) mediates Borrelia burgdorferi sensu stricto strain-specific complement evasion in the eastern fence lizard,Sceloporus undulatus

In North America, Lyme disease is primarily caused by the spirochetal bacterium Borrelia burgdorferi sensu stricto (Bb), which is transmitted between multiple vertebrate hosts and ixodid ticks, and is a model commonly used to study host-pathogen interactions. While Bb is consistently observed in its mammalian and avian reservoirs, the bacterium is rarely isolated from North American reptiles. Two closely related lizard species, the eastern fence lizard (Sceloporus undulatus) and the western fence lizard (Sceloporus occidentalis), are examples of reptiles parasitized by Ixodes ticks. Vertebrates are known to generate complement as an innate defense mechanism, which can be activated before Bb disseminate to distal tissues. Complement from western fence lizards has proven lethal against one Bb strain, implying the role of complement in making those lizards unable to serve as hosts to Bb. However, Bb DNA is occasionally identified in distal tissues of field-collected eastern fence lizards, suggesting some Bb strains may overcome complement-mediated clearance in these lizards. These findings raise questions regarding the role of complement and its impact on Bb interactions with North American lizards. In this study, we found Bb seropositivity in a small population of wild-caught eastern fence lizards and observed Bb strain-specific survivability in lizard sera. We also found that a Bb outer surface protein, OspE, from Bb strains viable in sera, promotes lizard serum survivability and binds to a complement inhibitor, factor H, from eastern fence lizards. Our data thus identify bacterial and host determinants of eastern fence lizard complement evasion, providing insights into the role of complement influencing Bb interactions with North American lizards.     

Failure of the Asian longhorned tick,Haemaphysalis longicornis,to serve as an experimental vector of the Lyme disease spirochete,Borrelia burgdorferi sensu stricto

The invasive, human-biting Asian longhorned tick, Haemaphysalis longicornis, was detected in New Jersey in the eastern United States in August of 2017 and by November of 2018 this tick had been recorded from 45 counties across 9 states, primarily along the Eastern Seaboard. The establishment of H. longicornis in the United States has raised the questions of how commonly it will bite humans and which native pathogens may naturally infect this tick. There also is a need for experimental vector competence studies with native pathogens to determine if H. longicornis can acquire a given pathogen while feeding, pass it transstadially, and then transmit the pathogen in the next life stage. In this experimental study, we evaluated the vector competence of a population of H. longicornis originating from the United States (New York) for a native isolate (B31) of the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.). In agreement with a previous experimental study on the vector competence of H. longicornis for Borrelia garinii, we found that uninfected H. longicornis larvae could acquire B. burgdorferi s.s. while feeding on infected Mus musculus mice (infection prevalence >50% in freshly fed larvae) but that the infection was lost during the molt to the nymphal stage. None of 520 tested molted nymphs were found to be infected, indicating that transstadial passage of B. burgdorferi s.s. is absent or rare in H. longicornis; and based on the potential error associated with the number of nymphs testing negative in this study, we estimate that the upper 95% limit for infection prevalence was 0.73%. An Ixodes scapularis process control showed both effective acquisition of B. burgdorferi s.s. from infected mice by uninfected larvae and transstadial passage to the nymphal stage (infection prevalence of 80–82% for both freshly fed larvae and molted nymphs). We also observed that although H. longicornis larvae could be compelled to feed on mice by placing the ticks within feeding capsules, attachment and feeding success was minimal (<0.5%) when larvae were placed freely on the fur of the mice. We conclude that H. longicornis is unlikely to contribute more than minimally, if at all, to transmission of Lyme disease spirochetes in the United States.     

Prevalence of tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks in Lower Bavaria and Upper Palatinate,Germany

Lyme borreliosis and tick-borne encephalitis (TBE) are the most common tick-borne diseases in Germany. We collected Ixodes ricinus ticks from 16 high-risk and four low-risk sites distributed in Lower Bavaria and Upper Palatinate based on the number of human TBE cases recorded at the Robert Koch Institute from 2001 to 2009. A total of 8805 questing ticks (8203 nymphs, 602 adults) were collected in 2010 and examined in pools for the presence of tick-borne encephalitis virus (TBEV) using real-time RT–PCR. Overall TBEV prevalence evaluated as the minimum infection rate (MIR) was 0.26 % (23 positive pools/8805 ticks in 1029 pools). TBEV was detected at seven of the 16 high-risk sites, where MIR ranged from 0.16 to 2.86 %. A total of 3969 ticks were examined by PCR for infection with Borrelia burgdorferi sensu lato (s.l.) targeting the 5 S–23 S rRNA intergenic spacer (IGS) region. IGS nucleotide sequences were used to determine genospecies. Selected positive Borrelia samples were subjected to PCR and sequencing targeting the OspA gene, providing 46 sequences for molecular phylogenetic analysis. Of the 3969 questing ticks, 506 (12.7 %) were positive for B. burgdorferi s.l. Seven B. burgdorferi s.l. genospecies were identified: B. afzelii (41.3 %), B. garinii (19 %), B. valaisiana (13.8 %), B. burgdorferi sensu stricto (11.1 %), B. spielmanii (0.4 %), B. lusitaniae (0.2 %), and Candidatus B. finlandensis (0.6 %). Mixed infections were identified in 13.6 % of the ticks. The rate of infection in questing ticks varied among sites from 5.6 % (72 examined, four positive) to 29.5 % (88 examined, 26 positive). B. burgdorferi s.l. occurred at all 20 sites, whereas TBEV was detected only at the high-risk sites where more human TBE cases were reported compared to low-risk sites.