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 m2 of vegetation screened at 17 urban and 3 peri-urban sites (Table 1). All ticks were identified by morphological criteria and by ITS2 based PCR as I. ricinus except one I. persulcatus collected in Sütiste park.
Among all visited sites, significantly 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).
All adults (n=186) and nymphal (n=669) ticks were individually screened for the presence of followed tick-borne pathogens: TBEV, B. burgdorferi s.l., B. miyamotoi, Anaplasmataceae (Anaplasma, Ehrlichia and Neoehrlichia) and Rickettsia spp. Overall, TBPs were detected at every site where ticks were found, except Harku-Nõmme, although their taxonomic composition and prevalence varied.
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-specific 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). For other sites, only the presence of TBPs and the number of ticks tested positive have been noted.
Borrelia burgdorferi (sensu lato)
B. burgdorferi s.l. was the most prevalent detected TBP. In total, 150 ticks tested positive for the presence of BBSL DNA by 5S-23S based PCR, indicating a 17.5% prevalence among all analyzed ticks, and 51.2% among ticks with at least one TBP. BBSL was found in ticks collected at almost every collection site, except Pirita river valley, and Harku-Nõmme. The highest site-specific prevalence was observed at Estonian Open-Air Museum and Tallinn’s Zoo – 25.2% and 22.7%, respectively. Surprisingly, a significantly lower rate was detected in ticks collected in the peri-urban Männiku forest.
Sequence analysis of the 5S-23S intergenic spacer region revealed the presence of three B. burgdorferi s.l. genospecies: B. afzelii (127/150; 84.7%), B. garinii (11/150; 7.3%), B. valaisiana (7/150; 4.7%) and B. bavariensis (1/150; 0.7%), while four samples remained unspecified at the genospecies level.
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).
The second most prevalent BBSL genospecies detected in urban and peri-urban ticks, although at significantly lower rates, was B. garinii, which was detected in 11 ticks, collected at 5 urban sites, with the majority from Pirita forest park (6/122), followed by Tallinn’s Zoo (2/211). Single B. garinii -positive ticks were detected also at Kadrioru, Ilmarise health trails, and Estonian Open Air Museum (Table 2). The B. garinii 5S-23S IGS sequences of this study showed similarity rates from 79.0% to 99.5% between each other and clustered with sequences reported from Estonia (acc. no. KX418634 and KX418637) as well as from Taiwan (acc. no. JX649205), Italy (acc. no. MT038900) Belarus (acc. no. AY772205), Sweden (acc. no. JX909934), Czech (acc. no. AF497993) and Russia (acc. no. MK118761).
Borrelia valaisiana and B. bavariensis genospecies were also found, albeit at overall prevalence rates of <1%. B. valaisiana was detected at overall prevalence of 0.8% (7/855) in ticks collected at Pirita forest park (2/122), Ilmarise health trails (1/37), Stroomi (1/38), Nõmme-Mustamäe (1/30) and Sütiste (TalTech) park (2/21). Borrelia bavariensis was detected in a single tick from the suburban site Jägala (Table 2). According to the phylogenetic analysis of 5S-23S sequences of B. valaisiana obtained in this study, they were identical to each other and to B. valaisiana isolate 122 (acc. no. KX418641) previously detected in Estonian I. ricinus, removed from the Common Blackbird , and also to B. valaisiana strains reported from Spain (acc. No. MG245790), Czech Republic (acc.no AF497989) and Italy (acc.No MT038902). The B. bavariensis 5S-23S rRNA sequence retrieved from I. ricinus collected in Jägala was identical to that found in I. ricinus from the Estonian county Läänemaa  and also to strain Ir-4370, reported from Stavropol, Russia (acc.no KU672534) and B. bavariensis prototype strain PBi (acc. no FJ546494).
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 .
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. . Samples that were sequenced for partial sca4 and ompB genes were also classified 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).
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).
In all, 15.0 % (44/293) of all TBP-positive ticks contained double infections and 4 tick samples tested positive for three tick-borne pathogens (4/293, 1.4%). The most frequently detected TBP combination in double infected ticks was B. afzelii with N. mikurensis (18/44) or R. helvetica (14/44) and these originated mainly from Open Air Museum, Tallinn Zoo and Pirita-Pirita forest park. It is noteworthy that of the 4 TBEV-positive tick samples, 2 were co-infected with bacterial TBP: one tick sample from Estonian Open Air Museum was positive for TBEV-Sib and N. mikurensis, and the TBEV-Eu – positive sample from Männiku was also positive for unspecified BBSL. Of the four tick samples with triple infections, three tested positive for the presence of B. afzelii, R. helvetica and N. mikurensis, and one for B. afzelii, R. helvetica and B. miyamotoi.