Ticks and tick-borne pathogens in popular recreational areas in Tallinn, Estonia: an underestimated risk of tick-borne diseases

For over several decades ticks are noticed to be widely present in the green spaces of urban environments. Rodents that provide essential blood-meals for subadult ticks and serve as natural reservoirs for many known tick-borne diseases, such as Lyme borreliosis and tick-borne encephalitis, might be quite abundant in green areas within urban settlements. In that way, the improvement of green infrastructures within cities possess not only to better human well-being but might also increase the risk of being bitten by a tick and having a tick-borne disease. This study aimed to provide a rst insight into ticks and tick-borne pathogen presence and prevalence in popular recreational green areas in Tallinn, Estonia. were agging May-June, 2018. Tick species identication was on morphological Tick-borne pathogens was and qPCR Two main aspects were considered when choosing study areas: suitable habitats for ticks and popularity among visitors. Satellite imagery from Google Earth and Estonian Land Board Web Map application (xgis.maaamet.ee/maps), as well as personal observations and data on recreational areas from the ocial Tallinn webpage (www.tallinn.ee) were used for initial identication of potentially suitable study areas which were then visited to determine specic agging sites. Urban areas were selected according to the presence of bushes, broad-leaf or temperate forested area and a litter layer. Peri-urban collection sites were situated mostly in deciduous forests or their edges, with known recreational popularity among visitors. According to observations, 17 urban and 3 peri-urban areas were selected in collaboration with the respective authorities where applicable (Fig. 1). Each transect was located along trails or their closest proximity to imitate visitors´ behavior as closely as possible. The observations and variables recorded at each site included date, time, transect length passed and air temperature. Habitat and vegetation type as well signs of any host presence by visualizing an actual animal or any direct evidence (e.g. tracks, birdsongs, nests, faeces) were also noted. Based on climate and weather observations from previous years, the month of May was predicted to be the most suitable time for the survey. Sites were surveyed for ticks from 9th May to 1st June, 2018, preferably during morning hours from 9 to 11 am, as ticks are active and dew is not heavy on the vegetation, which might inuence tick attachment to the collection cloth. Each site was surveyed once. tick Ticks AF497993) MK118761).

within the city and in the nearest peri-urban surroundings.

Study areas selection and tick collection sites
Two main aspects were considered when choosing study areas: suitable habitats for ticks and popularity among visitors. Satellite imagery from Google Earth and Estonian Land Board Web Map application (xgis.maaamet.ee/maps), as well as personal observations and data on recreational areas from the o cial Tallinn webpage (www.tallinn.ee) were used for initial identi cation of potentially suitable study areas which were then visited to determine speci c agging sites. Urban areas were selected according to the presence of bushes, broad-leaf or temperate forested area and a litter layer. Peri-urban collection sites were situated mostly in deciduous forests or their edges, with known recreational popularity among visitors. According to observations, 17 urban and 3 peri-urban areas were selected in collaboration with the respective authorities where applicable (Fig. 1). Each transect was located along trails or their closest proximity to imitate visitors´ behavior as closely as possible. The observations and variables recorded at each site included date, time, transect length passed and air temperature. Habitat and vegetation type as well signs of any host presence by visualizing an actual animal or any direct evidence (e.g. tracks, birdsongs, nests, faeces) were also noted.
Based on climate and weather observations from previous years, the month of May was predicted to be the most suitable time for the survey.
Sites were surveyed for ticks from 9th May to 1st June, 2018, preferably during morning hours from 9 to 11 am, as ticks are active and dew is not heavy on the vegetation, which might in uence tick attachment to the collection cloth. Each site was surveyed once.

Tick collection and species identi cation
Tick collections were performed using the agging technique with a 1 m 2 light-colored annel cotton cloth, attached to a wooden T-shaped handle, which was dragged over the vegetation and observed for tick presence at every 5 meters passed. The minimum transect was 300 m 2 per collection. However, no unique transect sizes nor standardized agging was used as the terrain, weather and vegetation conditions at every chosen area varied and no speci c focus on tick density evaluation had been planned. Ticks were removed from the cloth with tweezers, placed into separate glass vials according to stage and sex, and stored at + 4°C prior to species identi cation. The presence of larvae was noted but these were not collected, counted nor included in any analysis of the study. Adults and nymphal ticks were identi ed individually using a stereomicroscope according to morphological keys [15]. Ambiguous specimens were additionally identi ed by molecular keys using PCR based on internal transcribed spacer 2 (ITS2) and partial 16S rRNA gene as described previously [10,16]. Mean abundance (number of nymphal and adult ticks per 100 m), as well as index of abundance were calculated as described [17].
Nucleic acid isolation and TBPs screening All ticks were individually processed for DNA and RNA isolation by blackPREP Tick DNA/RNA kit (Analytik Jena, Germany). The initial tick lysis step was increased twice in time and the homogenization step was performed twice according to manufacturers' recommendations.
Homogenization was performed using MixerMill MM301 (Retsch, Haan, Germany). Mixing mill cassettes with vials, containing tick homogenate, were ipped over between homogenization steps to assure better milling performance. DNA and RNA solutions were then stored at -20C and − 70C, respectively, prior to further individual screening for the presence of TBPs.
All ticks' nucleic acids extracts were analyzed individually. Positive ticks' samples and deionized PCR-grade water were used as positive and negative controls, respectively, at every ampli cation step. To reduce contamination risk, every procedure, including tick pre-extraction washes, RNA/DNA extraction, PCR reaction mix preparation, DNA/RNA adding step, PCR reaction and gel-electrophoresis were performed in separate rooms.
The initial screening of TBEV RNA, based on the ampli cation of 3´ non-coding region, was performed with primers F-TBE1, R-TBE1 and probe TBE-W [18]. For further sequencing and genotyping, all positive samples were ampli ed additionally by nested RT-PCR for the partial E protein gene with SuperScript III Reverse Transcriptase kit (ThermoFisher Scienti c, USA) and primers described elsewhere [13,19].
Nested PCR reactions were used for screening and genotyping of BBSL, B. miyamotoi, Anaplasmataceae as well as Rickettsia species identi cation prior initial screening.
The detection of BBSL and B. miyamotoi DNA was performed by ampli cation of 245-256 bp long 5S-23S transcribed internal spacer region and 532 bp fragment of p66 gene, respectively [20,21]. To con rm B. miyamotoi species, p66-positive samples were additionally ampli ed for 379 bp long glpQ gene fragment.
Identi cation of Anaplasmataceae DNA in the tick samples was performed by amplifying 524 bp gene fragment of 16S rRNA gene [10]. For further sequencing analysis, all positive samples were subjected to ampli cation of 1350 bp long Anaplasmataceae 16S rRNA gene [10] under modi ed cycling conditions (Supplementary Table 1). Samples positive after initial screening, but negative for 1350 bp long 16S rRNA fragment, were additionally ampli ed for a 1300 bp long fragment of heat shock operon groESL gene with primers described elsewhere [22], under modi ed cycling conditions (Supplementary Table 1).
Rickettsia spp. screening in tick DNA samples was performed with qPCR amplifying 74 bp region of gltA gene [11]. Genotyping of Rickettsia species in positive qPCR samples was performed by sequencing of 667 bp long region of gltA gene, ampli ed by nested PCR [23]. Samples positive for qPCR, but negative for gltA region, were additionally subjected to PCR ampli cation of a 769 bp long ompB gene region as described by Roux and Raoult [24] and an 843 bp long region of sca4 gene as described by Igolkina et al. [23].
Detailed information on all PCR reactions, including targets, product size, primer/probe sets and ampli cation conditions used in the current study are presented in Supplementary Table 1. All nal nested PCR products were visualized by 1% agarose gel electrophoresis, stained with ethidium bromide.
For genotyping, all nal PCR products were subjected to direct Sanger sequencing, performed at Core Facility, Institute of Genomics, University of Tartu.

Tick sampling
From the 20 sites visited during the study, ticks were found at 13 sites within the city, and at all 3 peri-urban sites. There were no ticks found in the city central parks Hirvepark and Toompark, von Glehni park, Järve health trails and Sanatooriumi park (sites 5, 6, 14 and 16, respectively) ( Figure   1). As tick sampling was not standardized for the collection area and time, the collection results could not be extrapolated to questing tick density in the surveyed regions. However, mean tick abundance and abundance index were calculated for all study sites.
A total of 186 adults and 669 nymphs were collected from over 12 000 m 2 of vegetation screened at 17 urban and 3 peri-urban sites ( Table 1). All ticks were identi ed by morphological criteria and by ITS2 based PCR as I. ricinus except one I. persulcatus collected in Sütiste park.
Among all visited sites, signi cantly higher numbers of ticks were collected at Estonian Open Air Museum, Tallinn Zoo and Pirita forest park that accounted for 26.4%, 24.7%, and 14.3% of the total number of collected ticks, respectively. Concordantly, the estimated mean abundance of ticks at the urban sites was also the greatest at Estonian Open Air Museum and Tallinn Zoo (18.8 and 17.6, respectively), followed by Pirita forest park (9.8). Among the peri-urban sites, the highest number of collected ticks, as well as the highest abundance rate was observed in the Männiku forest (Table 1).
The total prevalence of ticks with at least one pathogen was 34.3% (293/855) ( Table 2). Due to non-standardized collections, estimated prevalence rates were calculated only for sites with over 50 adult and nymphal ticks collected and analyzed, that are Pirita forest park, Estonian Open Air Museum, Tallinn Zoo and Männiku. Among these, the site-speci c prevalence of TBP-positive ticks was the highest at Estonian Open Air Museum and Tallinn Zoo -43.8% and 42.2%, respectively, followed by Pirita forest park (31.1%) and peri-urban Männiku forest (18.9%) ( Table 2). Borrelia afzelii was detected in 11 of 13 urban and all sub-urban sites. It was the most prevalent genospecies with rates up to 24.8% and 21.8% at Open Air Museum and Tallinn's Zoo, respectively, followed by Pirita forest park (4.9%) and Männiku (4.1%) ( Table 2). According to the phylogenetic analysis, all B. afzelii 5S-23S spacer region sequences obtained in this study had nucleotide similarity rates within 77.4% to 99.5% between each other and were 100% identical to those previously found to be circulating in Estonian questing and passerine-attached ticks (GenBank accession no. KX418639, KX418638, KX418640), and to other sequences reported from France (acc. no. KY273112, KY273113), Italy (acc. no. MT038899), Slovakia (acc. no. KX906933, KX906945), Taiwan (acc. no. JX649207) and Russia (acc. no. MK118750, AB178349).

Borrelia miyamotoi
Borrelia miyamotoi, belonging to the relapsing-fever group Borrelia, was detected in 2.5% of all analyzed ticks (21/855). This genospecies was found mostly in Estonian Open Air Museum (10/226, 4.4%) and Tallinn Zoo (8/211, 3.8%), followed by peri-urban Männiku forest (2/74, 2.7%). A single B. miyamotoi-positive I. ricinus was also collected from the surroundings of Tallinn Zoo ( Table 2). Analysis of the B. miyamotoi partial p66 gene showed that nucleotide sequences of this study are identical to each other and sequences revealed previously in the Estonian tick population [9].

Rickettsiales
Rickettsia sp. were the second most prevalent bacterial TBP after BBSL: its presence was detected in 13.8% (118/855) of analyzed tick samples. Prevalence in the study sites ranged between 10.8% (8/74) in peri-urban Männiku and 18% (22/122) at Pirita forest park (Table 2). According to phylogenetic analysis of partial gltA gene nucleotide sequences, all Rickettsia positive samples belonged to the R. helvetica species. Sequences were identical to each other and sequences previously reported in Estonian Ixodes ticks Katargina et al. [11]. Samples that were sequenced for partial sca4 and ompB genes were also classi ed as R. helvetica species and were identical to each other within each gene fragment.
A total of 6.0 % (51/ 855) single analyzed ticks collected at 4 urban and 2 peri-urban sites tested positive for the presence of Anaplasmataceae DNA according to partial 16S rRNA PCR results. The highest prevalence was observed among ticks collected at Tallinn Zoo (24/211, 11.4%) and Estonian Open Air Museum (18/226, 8.0%), followed by Pirita forest park with a 4.9% prevalence (6/122). Single Anaplasmataceae-positive ticks were also collected in the Tallinn's Zoo surrounding area and sub-urban Vääna-Jõesuu and Jägala areas. The analysis of Anaplasmataceae 16S rRNA sequences revealed the presence of two species: A. phagocytophilum (0.6%, 5/855) and N. mikurensis (5.4%, 46/855) ( Table 2). At Pirita forest park two ticks out of 122 tested positive for the presence of A. phagocytophilum DNA, as did single ticks from three other locations: Open Air Museum, Tallinn Zoo and the sub-urban area of Jägala. 16S rRNA partial nucleotide sequences of A. phagocytophilum obtained in this study were 99.7% -99.9% similar to each other. The comparison to previously reported sequences from Estonian questing ticks (acc.no HQ629920, HQ629922, HQ629920) and sequences reported from Russia (acc.no HQ629911), Sweden (acc.no AY527213) and Austria (acc.no JX173652) showed 99.7% -100% similarity The highest prevalence of N. mikurensis was observed in questing ticks collected from Tallinn Zoo (10.9%, 23/211), followed by Estonian Open Air Museum (7.5%, 17/226) and Pirita forest park (3.3%, 4/122). Single N. mikurensis-positive ticks were also found in the surrounding area of Tallinn Zoo and peri-urban Vääna-Jõesuu. Sequences of N. mikurensis partial 16S rRNA, retrieved in this study, showed 98.2% -99.6% similarity to GenBank sequences reported previously from Estonian ticks (acc. no KU535862) and 98.1% -99.4% similarity to sequence from Germany (acc. no KU865475) and Russian Western Siberia (acc.no MN736126). TBEV According to qRT-PCR results, TBEV was the least common of detected TBPs, detected with in 4 I. ricinus nymphs of all 855 examined individual ticks found at Pirita river valley, Ilmarise health trails, Estonian Open Air Museum and Männiku forest (total prevalence of 0.5%). Two samples were successfully sequenced and genotyped ( Table 2).
According to the analysis of the partial E gene sequence obtained from an I. ricinus tick sample from the Estonian Open Air Museum, it clustered with TBEV-Sib sequences previously detected in Estonian I. persulcatus ticks collected in Eastern Estonia (TBEV isolates Est222 and Est221, accession numbers KT748749 and KT748748, respectively) at an identity rate of 99.8%, and belonging to the Baltic lineage within TBEV-Sib [13,26]. Another TBEV partial E gene sequence, retrieved from an I. ricinus sample collected at Ilmarise health trails, clustered within the TBEV-Eu subtype with 98.7% similarity to previously reported Estonian strain Est3476 (acc.no GU183383) and 99.6% similarity to TBEV strain Latvia-8110 (acc.no. AJ319583).

Discussion
More than 30 years have passed since the urbanization of arthropod vectors and the occurrence of tick-borne pathogens within cities and industrial regions were rst reported [27,28]. Since then, continuous ecologic and climate changes along with socio-demographic drivers have altered tick-associated natural environments resulting in reports of an increase in tick abundance and various tick-borne pathogens in cities, parks, outdoor leisure areas and other urbanized regions across Europe [4,5,29,30,31]. Furthermore, the northward expansion of I. ricinus and the occurrence of I. persulcatus to the west and north of its main distribution area may also drive the spread of tick-borne pathogens into new areas, giving rise to new foci as well as affecting public health [32,33]. This study con rms the occurrence of Ixodes tick species in popular recreational, outdoor sports and leisure areas in the capital and largest city of Estonia, with abundance rates compatible or even exceeding those detected previously in the most endemic foci in the natural environments [34].
The number of ticks collected at different urban and peri-urban locations during this study might not emulate the actual abundance as this study was not focused on tick density and agging was not highly standardized. It is, however, noteworthy, that a large number of ticks was found in larger, less fragmented forest-type parks with needle-and broad-leaved trees, and underwood with rich litter, as well as signs of the presence of an ample variety of urbanized small and medium-sized mammals such as Apodemus and Myodes rodents, shrews, hedgehogs, ground nesting and feeding birds, foxes and roe deer. Such an environment is a prerequisite for tick survival, development and maintenance. As seen in this study, Zoo, Open Air Museum and Pirita forest park, which are situated in large urban parks with areas similar to natural environments with a variety of small-and medium-sized animal species living there -showed signi cantly higher tick abundance rates compared to those, similar in vegetation but poorer in animal species and more fragmented in size (Sanatooriumi park, Järve forest health trails, Harku-Nõmme and von Glehni park). As a contrast, the carefully managed Kadriorg park, the largest and most popular park in Tallinn with many mowed open areas, but also rich in landscapes and plant communities with oak and chestnut trees, where rodents, hedgehogs, squirrels and various migratory bird species are abundant, as well as small, well-maintained parks with regularly mowed open grass, such as Hirve and Toompark located in the city center, were extremely poor habitats for Ixodes ticks. These are highly fragmented areas with a high anthropogenic habitat disturbance which might negatively affect tick presence and maintenance. While not focusing on tick density and lacking statistical analysis, our study results are generally in agreement with other European studies, pointing out the negative correlation of tick density towards urbanization rather than in relation to natural hosts [35]. As seen in Zoo and Open Air Museum, and also in the peri-urban Männiku forest, the presence of urbanized synanthropic carnivores, i.e. foxes, or roe-deer together with small animals, suitable environment and vegetation is essential for the maintenance of tick populations.
It is well known that the circulation of TBEV in natural foci is maintained by small rodents, which are competent reservoir hosts, and ticks, that are both hosts and vectors [36]. As many rodent species are well-adapted to a human-affected and urbanized environment, the presence of TBEV foci and therefore, the occurrence of autochthonous human TBE cases even within large cities is possible [37]. According to our earlier studies, TBEV was found in questing ticks at prevalence rates varying from 0.2 to 0.8% in the I. ricinus allopatric area and up to 4.9% in the areas of I. persulcatus co-circulation [13] which is in line with the results of this study as well as with prevalence rates of TBEV in I. ricinus in European foci [38]. According to epidemiological data of Estonian Health Board in about 18% of TBE patients in Harju county had a tick bite history from Tallinn [7]. The results of this study not only con rm the presence of TBEV foci in green areas within the city but also indicate circulation of European and Siberian subtypes of TBEV in I. ricinus ticks within urban and peri-urban areas. The presence of TBEV-Sib in I. ricinus ticks in locations with no I. persulcatus co-circulation had also been previously shown [13]; thus, it may be assumed that TBEV-Sib might be potentially spread into I. ricinus distribution areas without the presence of its principal vector, I. persulcatus.
The epidemiological reports of the Estonian Health Board showed that in 2014-2018 up to 19% of all con rmed Lyme borreliosis patients in Harju county with the known geographical origin of a tick bite, had been bitten by ticks within Tallinn [7]. The presence of four Lyme borreliosis associated species -B. afzelii, B. garinii, B. valaisiana, B. bavariensis -is in correspondence with previous results conducted in Estonia, however positivity rates differed signi cantly, being two to three times higher than detected earlier [8]. The results of this study are compatible with BBSL overall prevalence rates calculated for Scandinavia, the Balkan peninsula, and Central Europe (15.5%, 18.5% and 19.3%, respectively) [39]. Similar results have also been shown in urban and suburban areas in Switzerland (18%) and the urban Lazienki Park in Warsaw, Poland (17.3%) [35,40].
As seen in Europe and our previous studies, B. afzelii and B. garinii were the most prominent species found in I. ricinus ticks. Noteworthy, that in natural sylvatic areas B. afzelii and B. garinii constituted 56.1% and 20.3%, respectively, of all Borrelia sp. -positive I. ricinus ticks [8], while in urban and peri-urban ticks of this study the same indicators constituted 84.7% and 7.3%, respectively. A similar disproportion has also been observed in Poland [35], Switzerland [40] and Belgium [41]. Such a divergence might be explained by pathogen dilution-ampli cation effects in natural vs fragmented urban environments as well as by differences in host availability. Natural forests and other sylvatic areas with little anthropogenic disturbance, fragmentation and transformation are inhabited or visited during migration stops by large amounts and varieties of avian species which might serve as sources of avian-associated TBPs, such as B. garinii and B. valaisiana [42]. Thus, in the terms of anthropopressure, the contact of ticks with birds and a prevalence of avian-associated TBPs is signi cantly decreased in comparison to natural areas and may lead to a lower presence of B. garinii and B. valaisiana in urban ticks. In contrast, rodents, which are highly adapted to an urbanized environment, smaller agglomeration-surrounded areas and human interruption, promote sub-adult tick population maintenance, facilitate the frequency of tick-host contacts and trigger an increase and ampli cation of rodent-associated Borrelia, such as B. afzelii [41].
The presence of Borrelia miyamotoi in questing ticks has also been shown for several European countries, including Estonia [9,43]. In this study, the prevalence rate of 2.5% in all analyzed I. ricinus ticks and a site-speci c prevalence from 2.7% to 4.4% is in line with prevalence described for suburban France [44], Switzerland [40] and also with our previous results for Valgamaa and Võrumaa counties, where I. ricinus co-circulates with I. persulcatus and the highest positivity rates of B. miyamotoi (2.8%) in ticks were reported so far [9]. Rodents, especially Myodes and Apodemus, are known reservoirs for B. miyamotoi in Europe and a study of Jahfari et al. [29] also indicated European hedgehogs (Erinaceus europaeus) as reservoirs. As the population density of peridomestic mice and voles is even higher in the urban and peri-urban regions due to favorable breeding and survival factors [45] and as B. miyamotoi, being related to relapsing fever Borrelia, is transovarially transmitted, the higher infection rates in the urban areas versus natural wooded sites may be due to higher ampli cation rates of this pathogen within urban landscape fragments compared to larger natural woodlands and pastures.
Although Anaplasmataceae species are known to cause a variety of human diseases, only single sporadic cases of human infections have been reported in Estonia so far. Our previous studies indicated the circulation of at least four medically important species within the Anaplasmataceae family in Estonian I. ricinus populations, i.e. A. phagocytophilum (average 1.7%-2.6%) [9], N. mikurensis (1.3%) [10], followed by R. helvetica and R. monacensis [11]. The detection of Rickettsia spp. in urban and suburban I. ricinus ticks has been reported from Germany, the Czech Republic, Poland and Slovakia at rates from 1 to 47% [46, 47,48,49,50]. The results of this study showed almost three times higher R. helvetica overall positivity rate compared to those observed in questing ticks in natural environments (6.6%) [11] ; however, the prevalence rate in sub-urban site Männiku corresponds to that of natural tick habitats in Harjumaa county, as reported by Katargina et al. [11]. It is generally agreed upon that I. ricinus is the main vector and natural host of R. helvetica [30] and as a spotted-fever group Rickettsia, it is transmitted transstadially and transovarially among ticks. Thus, these factors along with high tick abundance in urban green areas may contribute to higher infection rates of this pathogen. Also, urban populations of European hedgehogs, as proposed by a study by Jahfari et al. [29], may play role in a pathogen maintenance in natural cycles in areas in uenced by anthropogenic pressure within cities.
Neoehrlichia mikurensis appeared to be also slightly more prevalent in urban I. ricinus ticks compared to those found in the natural I. ricinus allopatric areas with a site-speci c prevalence of 3.3%-10.9% (average 5.4%) versus 1.0% -9.1% (average 0.9%), respectively. The data of this study are in line with those reported from urban and sylvatic areas in Austria, Sweden and Germany [51,52,53] with prevalence rates from 4.2% to 6.4%. Such a widespread of the pathogen may be connected not only to arthropod vectors but also to reservoir hosts -bank voles and yellownecked mice -which are largely spread and have well-established populations within city shrubbery parks, cemeteries, outdoor activity and recreational places. Some studies also claim that non-rodent species such as hedgehogs, but not insectivores, may also contribute to N. mikurensis maintenance in urban and peri-urban green zones and human dwellings [29,54]. Since N. mikurensis has been associated with human clinical cases with symptoms including fever, malaise, septicemia and weight loss in immunocompromised as well as in healthy persons [55,56,57], the possible emerging status of this pathogen should be considered as a potential risk for public health in sylvatic and urban habitats.
In vector-borne diseases, an infection may only occur if human activity coincides with the activities of animal reservoirs and vectors. Climate change and urbanization, which both lead to environmental and microclimatic landscape composition and land-use changes, may impact every pathogen-host-vector system stage in different ways, thus affecting the whole system. Green infrastructure improvements within the cities support human population welfare. However, even patchy urban green areas may provide suitable environmental and microclimatic conditions for ticks, tick-borne pathogens, and their hosts, which in turn, may lead to an increased incidence of tick-borne diseases within the cities. On the other hand, urban heat island effects have a negative impact on tick survival and activity periods, as well as dense fragmentation and human disturbance lead to reduced biodiversity. Although the risk of acquiring a tick bite and being infected with a tick-borne disease in urban recreational sites may vary signi cantly between locations, it should not be ignored and proper information about the precautions might be considered at least in the most popular outdoor locations.

Conclusion
The risk of getting a tick bite, bacterial or viral tick-borne disease must not be underestimated even in urban environments as this study showed.
Proper precaution measures might be taken into consideration by local authorities as well as by citizens and tourists as those might get a tickborne disease even in green urban recreational zones without going far in the woods.

Availability of data and materials
All additional data associated with this study can be obtained from the corresponding author on reasonable request. Unique nucleotide sequences obtained during this study were submitted to GenBank database under the following accession numbers: MW916612 -MW916613 for TBEV, MW924118 -MW924135 for B. burgdorferi s.l. species, MW924974 -MW924983 for B. miyamotoi, MW924984 -MW925050 for R. helvetica, MW922752 -MW922756 for A. phagocytophilum and MW922757-MW922793 for Neoehrlichia mikurensis. Due to number of identical sequences, especially within B. burgdorferi s.l. and R. helvetica, only unique sequences were deposited and duplicate sequences were omitted from submission. Samples which nucleotide sequences allowed to perform pathogen genotyping but with partially poor chromatogram or with possible mixed infections of several pathogen species strains were also excluded from depositing. * -L -larvae, N -nymphs, M -male, F -female; the presence of larvae has been noted without exact count ** number of ticks per 100 m 2 ; *** index of abundance = no. of ticks / all minutes of collection by all collectors x 60 (one-hour reduction index).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TalTickSupplementarytable1methods.docx