Bacteria and protozoa with pathogenic potential in Ixodes ricinus ticks in Viennese recreational areas

From April to May 2019 questing ticks were collected at Deutschordenswald (48°12′02.6″N 16°13′38.1″E), Donauinsel (48°11′14.3″N 16°28′14.2″E), Grüner Prater (48°11′30.7″N 16°26′11.8″E), Lainzer Tiergarten (48°10′18.5″N 16°14′58.4″E), Nationalpark Lobau (48°11′37.0″N 16°28′19.9″E), Schottenwald (48°14′05.3″N 16°16′04.4″E) and Steinhofgründe (48°12′38.2″N 16°16′32.9″E) (Fig. 1). From May to July 2020 questing ticks were collected again in the same areas with the exception of Schottenwald (due to SARS-CoV‑2 limitations in time and resources). All ticks were collected by dragging a white cotton cloth through the sampling area. The collection sites showed similar features like mixed forests with strong undergrowth, meadows, and wildlife. For tick collection sunny to cloudy, but dry days were chosen and the temperature during sampling varied from 17 °C to 24 °C in 2019 and from 14 °C to 29 °C in 2020.

Prior to DNA extraction, the I. ricinus ticks were morphologically identified using a current identification key [32]. No additional molecular identification of the collected ticks was carried out and we considered all screened I. ricinus ticks to belong to the I. ricinus complex which includes I. inopinatus, due to the recently observed inability to distinguish I. ricinus from I. inopinatus molecularly [33]. In 2019, from all locations 30 ticks were tested. In 2020, 40 ticks were tested from every location except from Schottenwald where no sampling took place in 2020. Our study was focused on nymphal ticks as this is the life stage of the highest medical relevance to humans. In case not enough nymphal ticks were flagged we added adult ticks to reach a minimum number of 30 ticks per location. In 2020 exclusively nymphs were investigated. In 2019 we included some adult ticks for the locations Donauinsel (n = 10), Schottenwald (n = 2), Steinhof (n = 2) and Deutschordenswald (n = 10) to reach 30 ticks per collection site.

The DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Briefly, individual ticks were washed in 70% ethanol, air-dried and cut into halves using a sterile surgical blade. The lysis step was performed by overnight incubation at 56 °C in 180 µL ATL buffer and 20 µL proteinase K. The following DNA extraction steps were performed according to the manufacturer’s protocol. DNA was eluted with 100 µL pre-warmed (70 °C) AE buffer and stored at −20 °C until further use.

Genus-specific PCR targeting the Anaplasma/Ehrlichia/Neoehrlichia 16S rRNA gene, the Babesia/Theileria 18S rRNA gene, the Borrelia 23S-5S intergenic spacer (IGS), the Rickettsia 16S rRNA gene and the Rickettsia 23S-5S IGS were performed for each tick DNA extract using a 5’biotin-labeled primer per PCR reaction as described elsewhere [5, 34]. The RLB was performed according to a protocol described previously [35]. Briefly, the PCR products were combined for each tick individually in 2x SSPE buffer (Thermo Scientific, Vienna, Austria) consisting of 0.3M NaCl, 0.02 NaH₂PO_4, 0.002 M EDTA and additionally added 0.1% sodium dodecyl sulphate (SDS, Applichem, Darmstadt, Germany). The combined PCR products were denatured and the single-stranded PCR products were then hybridized to a nylon membrane (Biodyne C, Pall Laboratories, Crailsheim, Germany) with a set of covalently linked genus-specific (catch-all) and species-specific oligonucleotide probes (Eurofins Genomics GmbH, Ebersberg, Germany) allowing the detection of several Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma and Ehrlichia spp. (individual probes used are listed by Schötta et al. 2017 [5]). After incubation with 1:10,000 diluted horseradish peroxidase-labelled streptavidin (Roche Diagnostics GmbH, Mannheim, Germany) visualization of the hybridized PCR products was enabled by the Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Vienna, Austria) and the ChemiDoc touch imaging system (Bio-Rad, Hercules, CA, USA). For quality control of the PCR step, DNA extracts of bacterial cultures (B. burgdorferi s.s., B. afzelii, R. raoultii) or DNA extracts of confirmed-positive ticks (A. marginale, B. canis, C. N. mikurensis) were used. For the quality control of the blotting step, pooled biotinylated PCR amplicons of confirmed-positive ticks (A. phagocytophilum, T. (B.) microti, B. afzelii, R. monacensis, R. raoultii, R. slovaca) were used.

To detect B. miyamotoi DNA, we used a real-time PCR targeting the glycerophosphodiester phosphodiesterase gene (glpQ) as published by Reiter et al. 2015 [29]. The PCR reaction (20 µL total volume) contained 10 µl 2 × GoTaq® Probe qPCR Master Mix (Promega), 0.5 µM of each primer, 0.25 µM probe (Sigma-Aldrich, Vienna, Austria) and 4 µl of DNA. The reaction volume was adjusted with PCR-grade water (Sigma-Aldrich, Vienna, Austria). A two-step real-time PCR was run in an Applied Biosystems™ (Thermo Fisher Scientific, Vienna, Austria) QuantStudio 5 Real-Time PCR system with the following conditions: 95 °C for 10 min followed by 50 cycles of 95 °C for 15 s and 60 °C for 1 min. One reaction using a plasmid containing the target sequence and one reaction using PCR-grade water instead of DNA were included for quality control of the PCR run.

IBM SPSS Statistics 25.0 was used to analyze the collected dataset by descriptive statistics. Differences within nominal categories (e.g., tick life stage, infection rate) and the significance of co-infections were calculated using Fisher’s exact test or Pearson’s χ²-test. Two-tiered p-values of < 0.05 were considered significant.

In 2019, of the 210 ticks 60 (28.6%) and in 2020 of the 240 ticks 51 (21.3%) were positive for at least 1 member of the B. burgdorferi s. l. complex by RLB. The most common genospecies detected in 2019 as well as in 2020 was B. afzelii (16.2% in 2019; 15.0% in 2020), followed by B. burgdorferi s. s. (6.7% in 2019; 3.8% in 2020; Table 1).

We detected B. garinii/B. bavariensis DNA in 9 of the 210 (4.3%) ticks from 2019 and in 3 of the 240 ticks (1.3%) collected in 2020. B. spielmanii was detected in 3 of the 210 ticks (1.4%) from 2019 and was not detected in any of the ticks collected in 2020, 5 of the 210 ticks (2.4%) from 2019 and 5 of the 240 ticks (2.0%) from 2020 were positive for B. valaisiana. B. spielmanii was only found in three ticks collected at Donauinsel in 2019. The highest number of B. afzelii positive ticks was found in both years at the location Lainzer Tiergarten. In 2019, no other genospecies of the Borrelia burgdorferi s. l. complex was detected at Lainzer Tiergarten; in 2020 we also detected B. valaisiana in 2 of the 40 ticks. In ticks collected at Steinhof we detected only the genospecies B. afzelii and B. burgdorferi s. s. in both years. Ticks from Schottenwald were only collected in 2019 and we detected B. afzelii in 2 out of 30 ticks. Co-infections with multiple Borrelia burgdorferi s. l. species occurred in four ticks, of which two were detected in nymphs (Table 2).

Of the 210 ticks from 2019, 24 (11.4%) ticks were adult ticks of which 15 (62.5%) were positive for Borrelia burgdorferi s. l., hence, contributing to a significant number of positive ticks. A detailed overview with respect to the tick life stage is shown in Table 1.

Of the 210 ticks 29 (13.8%) collected in 2019 and 11 of the 240 ticks (4.6%) from 2020 were positive for Rickettsia species (Table 1); in both years only the species R. helvetica (13.3% in 2019; 3.8% in 2020) and R. monacensis (0.5% in 2019; 0.8% in 2020) were detected. No tick individual was co-infected with two Rickettsia species. In 2019, we detected R. helvetica DNA in ticks from every location and in 2020 we detected R. helvetica at Deutschordenswald, Donauinsel, Grüner Prater, Lainzer Tiergarten and Lobau, but in none of the 40 ticks collected at Steinhof.

Of the 210 ticks 12 (5.7%) collected in 2019 and 10 of 240 ticks (4.2%) collected in 2020 were positive for Babesia spp. (Table 1). All positive ticks from the 2019 collection and 7 positive ticks from the 2020 collection were infected with the species T. (B.) microti; 3 positive ticks from 2020 were infected with B. venatorum. Out of the 12 Babesia positive ticks 7 (58.3%) from 2019 and 5 out of the 10 Babesia positive ticks (50%) from 2020 were collected at the location Lainzer Tiergarten. We did not detect B. venatorum in the 210 ticks collected in 2019. In 2020 we detected B. venatorum in 1 of each of the 40 nymphs collected at Donauinsel, Grüner Prater and Lainzer Tiergarten. Among ticks in the nymphal life stage the Babesia spp. infection rate was 5.9% (11/186) in 2019 and 4.1% (10/240) in 2020; among adult ticks collected in 2019 the infection rate was 4.3% (1/23).

In 1 of the 210 ticks (0.5%) collected in 2019 and in 3 of the 240 ticks (1.3%) collected in 2020 we detected A. phagocytophilum (Table 1). In both years one positive tick was collected at the location Grüner Prater and in 2020 one additional positive tick was collected each at the location Lainzer Tiergarten and at Deutschordenswald. Among the nymphs the infection rate was 0.5% in 2019 and no adult tick from 2019 tested positive for A. phagocytophilum. For Cand. N. mikurensis 8 of the 210 ticks (4.0%) from 2019 and 14 of the 240 ticks (5.8%) from 2020 were positive. Among the nymphs from 2019 the infection rate was 6.0% and among adult ticks the infection rate was 12.5% in 2019.

We detected B. miyamotoi DNA by real-time PCR in 7 out of the 210 ticks (3.3%) collected at the locations Deutschordenswald, Donauinsel, Lainzer Tiergarten and Lobau in 2019 and in 4 of the 240 ticks (1.7%) collected at the locations Lainzer Tiergarten, Lobau and Steinhof in 2020 (Table 1). We collected the highest number of B. miyamotoi positive ticks in 2019 at Lainzer Tiergarten where 4 out of 30 ticks (13.3%) were positive.

Among nymphs, an infection rate of about 3.2% was calculated in 2019 and 1.6% in 2020. In adult ticks collected in 2019, the infection rate was 4.2%.

Of the 210 ticks 97 (46.2%) collected in 2019 and 71 of the 240 ticks (29.6%) collected in 2020 tested positive for at least 1 pathogenic species (Table 2). In 2019, 72 ticks (34.8%) were infected with 1 pathogen, 20 ticks (9.5%) showed a dual infection, 4 ticks (1.9%) harboured 3 and one 1 (0.5%) harbored 4 pathogenic species; from the 2020 collection 58 of the 240 ticks (24.2%) were positive for 1 species, 10 (4.2%) for 2 species, 2 (0.8%) for 3 species and in 1 nymph (0.4%) we detected 5 species (Table 2). Co-occurrences of tick-borne pathogens were investigated with the Fisher’s exact test. In both years, detection of the co-occurrence was significant for Babesia spp. and Cand. N. mikurensis (2019: P = 0.036; 2020: P = 0.003) and B. burgdorferi s. l. and Babesia spp. (2019: P = 0.005, 2020: P = 0.038). In 2020, the co-occurrence of B. burgdorferi s. l. and B. miyamotoi was also significant (P = 0.031).