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Prevalence of tick-borne pathogens in questing Ixodes ricinus ticks in urban and suburban areas of Switzerland

Parasites & Vectors volume 10, Article number: 558 (2017) Cite this article

Abstract

Background

Throughout Europe, Ixodes ricinus transmits numerous pathogens. Its widespread distribution is not limited to rural but also includes urbanized areas. To date, comprehensive data on pathogen carrier rates of I. ricinus ticks in urban areas of Switzerland is lacking.

Results

Ixodes ricinus ticks sampled at 18 (sub-) urban collection sites throughout Switzerland showed carrier rates of 0% for tick-borne encephalitis virus, 18.0% for Borrelia burgdorferi (sensu lato), 2.5% for Borrelia miyamotoi, 13.5% for Rickettsia spp., 1.4% for Anaplasma phagocytophilum, 6.2% for "Candidatus Neoehrlichia mikurensis", and 0.8% for Babesia venatorum (Babesia sp., EU1). Site-specific prevalence at collection sites with n > 45 ticks (n = 9) significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis", but were not related to the habitat type. Three hundred fifty eight out of 1078 I. ricinus ticks (33.2%) tested positive for at least one pathogen. Thereof, about 20% (71/358) were carrying two or three different potentially disease-causing agents. Using next generation sequencing, we could detect true pathogens, tick symbionts and organisms of environmental or human origin in ten selected samples.

Conclusions

Our data document the presence of pathogens in the (sub-) urban I. ricinus tick population in Switzerland, with carrier rates as high as those in rural regions. Carriage of multiple pathogens was repeatedly observed, demonstrating the risk of acquiring multiple infections as a consequence of a tick bite.

Background

Ixodes ricinus is the most frequent tick species throughout Europe. Its life-cycle proceeds through three developmental stages, larvae hatching from eggs, nymphs, and adult males or females. Ixodes ricinus may act as a parasite on more than 200 different species, including humans. It serves as a vector for numerous human and animal pathogens of bacterial, viral, or protozoic origin [1, 2].

Tick-borne encephalitis virus (TBEV) causes disease of variable severity, ranging from subclinical infections to severe disease with neurological involvement and potentially fatal outcome. TBEV is taxonomically classified into European, Siberian and Far Eastern subtypes; I. ricinus is the principal vector for the European subtype of the virus [3, 4]. Multiple species of rodents, insectivores and carnivores serve as reservoir hosts of TBEV [5, 6]. Although the virus is transmitted transovarially in I. ricinus ticks, this transmission is not effective enough in sustaining viral circulation in nature [7]. Co-feeding is essential for TBEV maintenance in natural foci [8]. Mean prevalence in endemic regions ranges from < 0.1 to 5% in Europe and 4 to 39% in Asia [9]. In Switzerland, 38/165 rural sites screened for the presence of TBEV in I. ricinus ticks were shown to harbor natural foci, with a mean virus prevalence of 0.46% [10].

Lyme borreliosis is a multisystemic disease that causes local infections in the skin or disseminates to various tissues, including joints, the central nervous system and the heart [11]. It is prevalent in North America, Europe, parts of North Africa, and northern Asia. Within the B. burgdorferi (sensu lato) complex, B. afzelii, B. burgdorferi and B. garinii are confirmed agents of localized, disseminated and chronic manifestations of Lyme borreliosis, whereas B. spielmanii, B. bissettii and B. valaisiana have only been associated with few cases of Lyme borreliosis [11]. Ixodes ricinus is the predominant vector of B. burgdorferi (s.l.) in Europe and small mammals and ground-foraging birds serve as reservoir hosts [2, 12]. Transovarial transmission of B. burgdorferi (s.l.) in I. ricinus is limited [13]. Mean carrier rates are higher in adults (18.6%) than in nymphs (10.1%), and highest carrier rates are found in central Europe [14]. In questing I. ricinus ticks in (sub-) urban areas of Europe, carrier rates range between 2 and 40.8% [2]. In rural areas of Switzerland, prevalence ranges between 9 and 40% for nymphs and from 22 to 47% in adults [15].

Borrelia miyamotoi may cause a febrile illness possibly presenting as relapsing fever. In immunocompromised patients, it may cause severe disease including meningoencephalitis. The prevalence of B. miyamotoi in I. ricinus ticks in Europe ranges between 0 and 4%. In urban areas of France, a prevalence of 4% was found, whereas the carrier rate was much lower (2/428) in a study conducted in peri-urban and urban areas in southern England [16,17,18,19,20]. Potential reservoir hosts include species of rodents and birds. Different tick species such as Ixodes scapularis and I. ricinus transmit B. miyamotoi transovarially [13, 18, 21,22,23].

Various Rickettsia species are transmitted by hard ticks in Europe, including R. helvetica, R. monacensis, R. conori and R. slovaca implicated in human disease [2, 24, 25]. In Switzerland, R. helvetica and R. monacensis appear to be of particular importance [26, 27]. Clinical signs of infections with R. helvetica include fever, headache and myalgia [28]. Rickettsia monacensis may cause Mediterranean spotted-fever like illness [29]. The prevalence of R. helvetica and R. monacensis in I. ricinus ticks in Europe ranges from 0.5 to 66%, or 0.5 to 34.5%, respectively [2, 30,31,32]. In Germany and Slovakia, prevalence of Rickettsia spp. in urban sites ranged between 2.2 and 30.1% [30, 31, 33, 34]. Ticks serve as both the vector and main reservoir of Rickettsia spp., with transstadial and transovarial transmission being documented [2].

Anaplasma phagocytophilum causes disease in domestic ruminants and horses [35], but may also infect other mammalian species, including humans [36]. Clinical manifestation in humans ranges from mild self-limiting febrile illness to fatal infections [36,37,38,39]. Anaplasma phagocytophilum is not transmitted transovarially in I. ricinus ticks [40]. Its epidemiological cycles involving mammalian hosts and vectors are complex and comprise different bacterial ecotypes. Carrier rates of I. ricinus in Europe range between < 1% and about 20% [36]. At urban sites (Austria, France, Slovakia, Hungary), carrier rates between 0.7 and 8.8% have been documented [20, 41,42,43,44].

"Candidatus Neoehrlichia mikurensis" has been detected in I. ricinus ticks in various European countries, with carrier rates ranging from 0.95 to 23.5% [42, 45,46,47,48]. The reservoir role of several rodent species has been proven [49,50,51,52]; transovarial transmission in I. ricinus has not yet been reported [2]. In urban habitats in Slovakia, "Ca. N. mikurensis" has been detected in both I. ricinus ticks and rodents, with prevalence in I. ricinus ranging between 1.0–2.4% [44, 53]. Only a limited number of severe human disease cases associated with fever, septicemia, malaise and weight loss have been described so far, most often but not exclusively affecting patients with immune deficiency [54,55,56,57].

Babesia spp. are best known to cause animal illness. Three species are currently recognized to be involved in human disease in Europe: B. divergens, B. venatorum (Babesia sp., EU1), and B. microti, with the bovine parasite B. divergens being thought to be responsible for most cases. Clinical signs of babesiosis such as flu-like symptoms or hemolytic anemia are usually but not exclusively limited to immunocompromised patients [2, 58, 59]. Carrier rates of I. ricinus ticks in Europe range around 0.2 to 3.0% for B. divergens and 0.4 to 1.3% for B. venatorum [2, 60,61,62]. There is evidence for circulation of B. divergens and B. venatorum in urban areas, given that the respective host species (cattle, ungulates) are present [2, 30, 63]. In Germany, Poland and Slovakia, prevalence in urban habitats ranges from 0.4 to 4.5% [33, 64, 65]. In rural areas of Switzerland, a prevalence of 1.9% has been documented [27]. Babesia spp. are generally known to be transmitted both transstadially and transovarially in ticks [66]. However, transovarial transmission could so far not be experimentally demonstrated for B. microti [67].

In Switzerland, several studies on the prevalence of all of the above-described tick-borne pathogens in questing ticks have been performed [10, 27, 68,69,70,71,72,73]. However, data on the carrier rate of ticks in suburban areas of Switzerland are scarce [60, 74], and data on tick-borne pathogens in questing ticks in urban areas were not available to date. In this study, we analyzed 1078 questing I. ricinus ticks sampled at (sub-) urban collection sites throughout Switzerland for the presence of TBEV, B. burgdorferi (s.l.), B. miyamotoi, Rickettsia spp., A. phagocytophilum, "Ca. N. mikurensis" and Babesia spp. Additionally, we analyzed ten tick DNA samples using next generation sequencing (NGS), including two positive samples as well as eight randomly selected samples negative for the investigated pathogens. In these latter eight samples, we searched for pathogens potentially missed using specific screening PCRs as well as for members of the tick microbiota.

Methods

Tick sampling

A total of 45 (sub-) urban study areas were defined in collaboration with the respective authorities. Within the areas, similar collection sites of at least 100 square meters were chosen. Collection sites in urban parks, river sides, cemeteries or open air swimming pool areas were characterized by the presence of bushes or trees and some kind of litter layer. Within urban forests surrounded by built-up areas and within suburban forests located at the border of the city, collection sites were situated at the edge of deciduous forest with high recreational frequentation. Sampling was performed between 10:00 am and 16:00 pm but not on rainy days. Most collection sites were visited only once in June 2016, with a monthly average temperature of about 16 °C. At 11 collection sites in the city of Zürich, ticks were collected throughout the year at 6 different time points (June, July, September and November 2015, April and May 2016). These sites were selected to be visited several times in the framework of another study, where the presence of ticks was related to the number of registered tick bites (unpublished data). Temperature at the collection days for these sites ranged between 13–30 °C, with a relative humidity ranging between 50–85%. Ticks were collected by flagging low vegetation using a terry towel of 1 m of width and length fixed to a wooden stick. Time invested for tick collection at one collection site ranged between 3 and 5 h. Tick collection was not standardized, since this study did not focus on the tick density in the investigated sites, but rather on the pathogen prevalence found within the analyzed ticks. Collected ticks were kept alive at 4 °C. Following identification based on morphological characteristics [75, 76], ticks were individually sorted into collection microtubes (Qiagen, Hilden, Germany) and stored at -20 °C.

Sample preparation

Tick samples were homogenized in 600 μl of pre-cooled PBS using the TissueLyser system (Qiagen, Hilden, Germany). After a short centrifugation step, 400 μl of the supernatant were transferred to a Deepwell plate (Eppendorf, Hamburg, Germany), 60 μl of glycerin were added per well and the plates stored at -80 °C for further use. 100 μl of the supernatant were used for nucleic acid extraction.

Nucleic acid (NA) extraction

100 μl of tick homogenate supernatant were lysed in 400 μl of AVL buffer supplemented with InhibitEX Tablets (Qiagen, Hilden, Germany) in a 96-well MagNA Pure processing cartridge (Roche, Penzberg, Germany). NA extraction was performed with the MagNA Pure 96 instrument and the MagNA Pure 96 DNA and Viral NA Large Volume kit, using the Pathogen Universal LV 2.0 protocol, a sample volume of 500 μl and an elution volume of 100 μl. NA quality was randomly controlled using the Agilent 2100 Bioanalyzer system with the Agilent High Sensitivity DNA Kit (Agilent Technologies Inc., Santa Clara, California, USA).

Real-time (reverse transcription-) PCR

The real-time (RT-PCR) systems used for screening the tick samples on the presence of TBEV, Borrelia spp., B. miyamotoi, Rickettsia spp., A. phagocytophilum, "Ca. N. mikurensis" and Babesia spp. are summarized in Table 1. For Rickettsia spp. and Babesia spp., two screening systems were used.

Table 1 Real-time (RT-PCR) systems used for screening tick samples for the presence of various pathogens
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Sanger (capillary electrophoresis) sequencing

Samples positive for Borrelia spp., Rickettsia spp. and Babesia spp. were further examined by sequence analyses to identify the respective species. A subset of samples, where tick species identification based on morphological characteristics was unclear (mainly larvae, n = 75), were analyzed by Sanger sequencing as well. Nested PCR amplifications and sequence analyses were done by Microsynth (Balgach, Switzerland) using the primers and annealing temperatures summarized in Table 2. First-step PCR reactions were run with 2.5 μl of template DNA in a total volume of 12.5 μl including 0.5 μM of each primer, 200 μM dNTPs, 1.5 mM MgCl2 and 0.02 U/μl KAPA2G Robust polymerase (Axon Lab, Baden, Switzerland). Fourty cycles were run for each PCR (denaturation: 20 s, 95 °C; annealing: locus-specific temperatures, 20 s; elongation: 100 s, 72 °C; final elongation step: 45 s, 72 °C). First-step PCR products were diluted 1:100 for the second-step PCRs, which were run under the same PCR conditions as described for the first-step PCR using the nested primers described in Table 2. Successful amplification was verified on a 1.5% agarose gel. PCR products were purified and uni-directionally Sanger sequenced. Sequences were quality-trimmed and manually edited, then locus-wise subjected to alignment and phylogenetic analysis using the Phylogeny.fr website [77]. Species identification was done using BLASTn comparison (NCBI nucleotide database) [78, 79]. The sequences obtained from this study have been deposited in the GenBank database (MF121944–MF121977).

Table 2 Primer sequences and annealing temperatures of Sanger sequencing reactions
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Reverse line blot (RLB)

Samples positive for Borrelia spp. yielding mixed sequences in Sanger sequencing, indicating the presence of multiple Borrelia species, were additionally analyzed using RLB as described before [80,81,82,83]. The variable spacer region between 2 repeated copies of the 23S and 5S ribosomal genes was amplified by PCR with primers 23S-Bor and B-5S-Bor [80]. For species identification, PCR products were hybridized to 15 oligonucleotide probes [81,82,83] and blotted on an active Biodyne C membrane using a Miniblotter 45 (Immunetics, Boston, Massachusetts, USA). Hybridization was visualized by incubating the membrane with enhanced chemiluminescence detection liquid and by exposing the membrane to an X-ray film.

NGS and bioinformatics pipeline

Eight randomly chosen samples negative in all pathogen screening PCRs (samples 3–8), 1 sample positive in Borrelia spp., R. helvetica and A. phagocytophilum screening PCRs (sample 1), and 1 sample positive for R. helvetica (sample 2) were subjected to NGS. With these analyses, we aimed to (i) demonstrate the congruency of detecting known pathogens using NGS and real-time screening PCR, (ii) investigate whether some pathogens may potentially be missed using specific screening PCR, and (iii) analyze the microbiota of our I. ricinus tick samples. The NGS workflow as well as the bioinformatics pipeline used for data evaluation are described in Additional file 1.

Pathogen prevalence

Individual carrier rates were assessed for collection sites with more than 45 collected ticks (n = 9). Furthermore, since the carrier rates did not significantly differ between habitat types (see statistical analysis), overall prevalence was calculated. Larvae were only included for calculation when the respective pathogen is transmitted transovarially (B. miyamotoi, Rickettsia spp., Babesia spp.). As the samples size for the different dates and collection sites was small, a statistical evaluation of pathogen prevalence in dependence of collection dates was not possible.

Statistical analysis

The stats package of the R software (version 3.3.2) [84] was used to assess differences in pathogen prevalence between collection sites, habitats (cemetery, urban park, urban forest, suburban forest), developmental stages (larvae, nymphs, adults) and gender (male, female). A generalized linear model (GLM) using the logit link function under the binomial distribution was applied. Chi-square tests were performed to assess significance levels. Pathogen prevalence with respect to developmental stages were analyzed using collection site as an interacting explanatory variable and the Chi-square test was used to compare the main (Stage/Gender*Collection site) model to the reduced model (only collection site). For all analyses, larvae were only included when the respective pathogen is transmitted transovarially (B. miyamotoi, Rickettsia spp., Babesia spp.). To evaluate the frequency of certain pathogen combinations, a GLM was applied comparing the prevalence of each pathogen in either mono- or multi- (≥ 2) infected ticks. Since only R. helvetica were found in larvae, developmental stage was included as an interacting explanatory variable. Finally, Chi-square significance testing was used to compare total count of collected ticks according to season (spring or fall) for sites where multiple collections had been performed. For this purpose a GLM under the Poisson distribution was applied using collection site as an interacting explanatory variable. After applying the Bonferroni correction for multiple comparisons (n = 5), a P-value < 0.05 was regarded as significant.

Results

Tick sampling and species identification

A total of 1,079 ixodid ticks (66 larvae, 740 nymphs, 138 adult males and 135 adult females) were collected at 18 collection sites (Fig. 1, Table 3); at 27 sites, no ticks were found. Tick collection was not standardized with respect to collection time and area, with exception of the sites where flagging was done at multiple time points. Therefore, the collection success in this study must not be equated to questing tick density in the sampling regions. At the collection sites where flagging was done at six different time points, collection was significantly more successful in spring (June 2015, April and May 2016) than in summer or fall (July, September, November 2015) (Chi-square test with Bonferroni correction, χ 2 = 52.62, df = 2, P < 0.0001) (Table 4). Except one female Ixodes hexagonus, all ticks were identified as I. ricinus based on morphological criteria or Sanger sequencing results of the cytochrome c oxidase locus.

Fig. 1
figure 1

Urban areas in Switzerland analyzed for the presence of pathogens in questing I. ricinus ticks. Tick collection was successful at 18 collection sites: (a) Basel (2 sites), (b) Bern (1 site), (c) Geneva (2 sites), (d) Lausanne (1 site), (e) Lugano (1 site), (f) Luzern (1 site), (g) Neuchâtel (2 sites), (h) Sion (1 site), (i) St. Gallen (1 site), (j) Winterthur (1 site), and (k) Zürich (5 sites)

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Table 3 Overview of ticks collected at the different collection sites
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Table 4 Tick collection success with respect to collection dates for sites with multiple collection attempts
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Pathogen prevalence

Table 5 summarizes the number of positive I. ricinus ticks found per collection site. Pathogen prevalence was not significantly different between collection sites belonging to different habitat types (i.e. cemetery, urban park, urban forest, suburban forest) (P-values with Chi-square test using Bonferroni correction > 0.1 for B. burgdorferi (s.l.), B. miyamotoi, A. phagocytophilum and B. venatorum, > 0.05 for "Ca. N. mikurensis"). We therefore calculated overall prevalence, which was 0% for TBEV, 18.0% for B. burgdorferi (s.l.) (8.2% for B. afzelii, 1.3% for B. burgdorferi (sensu stricto), 2.8% for B. garinii, 0.9% for B. valaisiana, 2.3% for multiple Borrelia spp., see below), 2.5% for B. miyamotoi, 13.5% for Rickettsia spp. (13.2% for R. helvetica, 0.3% for R. monacensis), 1.4% for A. phagocytophilum, 6.2% for "Ca. N. mikurensis" and 0.8% for B. venatorum. In addition to overall prevalence, we calculated individual carrier rates for collection sites where more than 45 I. ricinus ticks had been collected (Table 6). Site-specific carrier rates were was significantly different for B. burgdorferi (s.l.) (χ 2 = 50.04, df = 8, P < 0.0001), Rickettsia spp. (χ 2 = 56.85, df = 8, P < 0.0001) and "Ca. N. mikurensis" (χ 2 = 27.86 df = 8, P = 0.006). Pathogen carrier rates did not significantly differ in relevance to tick developmental stages (P-values Chi-square test with Bonferroni correction > 0.08). Larvae were exclusively found to be positive for Rickettsia spp. at a percentage of 32.8%.

Table 5 Pathogen screening results of I. ricinus ticks from 18 urban collection sites
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Table 6 Pathogen prevalence in I. ricinus ticks collected at 9 urban or suburban collection sites
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Samples with multiple pathogens

Out of 1078 ticks, 358 (33.2%) were carrying at least one pathogen. 287 ticks (26.6%) were infected with one, 64 (5.9%) with two, and seven (0.7%) with three different pathogens (Figs. 2, 3). For this analysis, we regarded samples with mixed sequences for B. burgdorferi (s.l.) in the respective Sanger sequencing reaction as being infected with two different B. burgdorferi (s.l.) species. The prevalence of R. helvetica in mono-infected ticks was significantly higher than the prevalence in multi-infected ticks (Chi-square test with Bonferroni correction, χ 2 = 9.34, df = 2, P = 0.023) (Fig. 2).

Fig. 2
figure 2

Number of ticks positive for different tick-borne pathogens. The overall height of the bars represents the percentage of infected ticks tested positive for the respective pathogen. The proportions at which the pathogens were detected alone or in combination with one or two others are shown in light gray, dark gray, and black, respectively. Abbreviations: B.g., B. garinii; B.a., B. afzelii; B.b.(s.s.), B. burgdorferi (sensu stricto); B.va., B. valaisiana; B.m., B. miyamotoi; R.h., R. helvetica; R.m., R. monacensis; A.p., A. phagocytophilum; B.ve., B. venatorum (Babesia sp., EU1); N.m., "Candidatus N. mikurensis"; B.b.(s.l.), two (or more) different B. burgdorferi (sensu lato) species. R. helvetica was significantly more often detected alone than in association with another pathogen (GLM with developmental stage as a dependent variable; Chi-square test with Bonferroni correction, P = 0.023)

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Fig. 3
figure 3

Correlation-plot showing the pathogen combinations observed in urban I. ricinus ticks in Switzerland. The more frequent a pathogen combination, the bigger the respective circle in the plot. In addition, the absolute counts of ticks with the particular pathogen combination are given in numbers. Abbreviations: B.g., B. garinii; B.a., B. afzelii; B.b.(s.s.), B. burgdorferi (sensu stricto); B.va., B. valaisiana; B.m., B. miyamotoi; R.h., R. helvetica; R.m., R. monacensis; A.p., A. phagocytophilum; B.ve., B. venatorum (Babesia sp., EU1); N.m., "Candidatus N. mikurensis"; B.b.(s.l.), two (or more) different B. burgdorferi (sensu lato) species, not distinguishable. Other combinations of three different pathogens are not shown in this plot; these were 1× B. afzelii + R. helvetica + A. phagocytophilum and 1× B. afzelii + B. venatorum (Babesia sp., EU1) + "Ca. N. mikurensis"

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RLB for samples with suspected carriage of multiple B. burgdorferi (s.l.) species

Carriage of multiple B. burgdorferi (s.l.) species, indicated by mixed sequences in Sanger sequencing analyses, was found in 23 I. ricinus ticks (13 nymphs, four males, six females). Using RLB, however, only five of these samples were found to be positive for B. garinii and one sample was found to be positive for B. afzelii. The remaining 17 samples were negative in RLB analysis. Carriage of multiple Borrelia spp. could not be confirmed in any of the samples using RLB.

NGS

NGS was done with a total of ten samples. Although most of the taxonomically classified reads (Kraken output) were assigned to ixodid ticks (96.5–99.9%), which is expected in untreated metagenomics samples of eukaryotes, the read numbers of the pathogens previously identified by screening PCRs were clearly distinguishable from the background noise in sample 1 and 2. The reads assigned to R. helvetica denoted 76.3 and 82.6%. The reads of sample 1 classified to A. phagocytophilum and B. afzelii represented 1.7 and 0.1%, respectively (Fig. 4a). In the remaining eight samples (3–8), no known pathogens could be detected using NGS, which is in agreement with the negative screening PCRs. However, a total of 8 samples (2 adult female, 1 adult male and 5 nymphal ticks) were positive for the tick endosymbiont "Candidatus Midichloria mitochondrii" [85] (Fig. 4a, b). In the adult female ticks, the reads classified to "Ca. M. mitochondrii" represented 74 and 92% of all bacterial reads. For the male and nymphal ticks, the percentages of bacterial reads classified to this endosymbiont were 0.1% or 0.5–25%, respectively. In addition, every sample contained variable proportions of organisms known to be residents of soil and water, plant associated bacteria, or normal human microbiota (Fig. 4b)

Fig. 4
figure 4

NGS results for 10 I. ricinus tick samples. Two samples positive in one or more pathogen screening PCR (a) and 8 samples negative in all screening PCRs (b) were analyzed. Whole genome amplified samples were sequenced on an Ion S5™, Kraken was used for taxonomic profiling of trimmed reads, and species with low read support were filtered out. Species pathogenic for humans, i.e. R. helvetica (R.h.), A. phagocytophilum (A.p.), B. afzelii (B.a.), as well as the tick endosymbiont "Candidatus Midichloria mitochondrii" (C.M.m.), are represented individually. The remaining species are grouped in Pseudomonas spp. (P.spp.), other residents of soil and water, commensals, human microbiota, and plant associated bacteria. The bars indicate the percentages of reads assigned to the respective species or groups in a logarithmic scale

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Discussion

Throughout Europe, I. ricinus transmits numerous human and animal pathogens. Its widespread distribution includes urbanized areas. Most wildlife species found in urbanized areas in Europe act as maintenance hosts for I. ricinus, but may also serve as reservoirs of tick-borne pathogens. In urban sites, these hosts may be rodents, hedgehogs, shrews, birds, lizards, dogs and cats. In peri-urban areas, larger animals such as foxes, roe deer, and wild boars may act as tick-maintenance and pathogen reservoir hosts. As a consequence of increasing urbanization and the behavior of humans increasingly encroaching on their peri-urban surroundings, the exposition of humans to vector ticks and tick-transmitted pathogens is increasing [2, 86]. Whereas several studies on the prevalence of various tick-borne pathogens in questing I. ricinus ticks have been done in Switzerland so far [10, 27, 68,69,70,71,72,73], only limited research has focused on ticks collected in suburban areas [60, 74]. Here, we analyzed questing I. ricinus ticks collected at (sub-) urban sites for the presence of various pathogens. Furthermore, we analyzed ten DNA samples using NGS, thereby detecting true pathogens, tick symbionts, as well as organisms of environmental or human origin.

Tick collection was successful in about 40% (18/45) of the areas flagged during this study. As the focus of our study was the investigation of pathogen prevalence rather than tick density, our tick collection method was not highly standardized. Therefore, the number of collected ticks at the different collection sites does not necessarily reflect the tick density in these areas. As an exception, tick collection effort was approximately standardized at eleven collection sites in the city of Zürich, where tick sampling was done throughout the year at six different time points (June, July, September and November 2015, April and May 2016) (Table 4). When comparing collection success between collections done in spring (April, May, June) to collections done in summer or fall (July, September, November), we found that collection was significantly more successful in spring than in summer or fall (P < 0.0001). These findings are in agreement with the results of a study focusing on seasonality of I. ricinus ticks on the vegetation in two regions in Switzerland, where a significant decline of questing activity in June was observed [87]. Also, they are explained by the conditions for tick activity (temperature and humidity), which are more likely fulfilled in spring than in summer or fall.

TBEV-infected ticks are distributed in a patchy manner in so-called natural foci. In Europe, within these foci, carriage rates of I. ricinus ticks range between < 0.1% and 5% [9] (Switzerland: 0.46% [10]). In the present study focusing on urban areas, we could not detect any TBEV-positive I. ricinus ticks. However, given the low expected carrier rates, the sample sizes per collection site are too small to allow for a reliable estimation of TBEV prevalence. Accordingly, the prevalence of 0% has to be interpreted with caution, and more extensive studies are needed to precisely estimate the carrier rate of (sub-) urban I. ricinus ticks with TBEV in Switzerland. In studies focussing on urban or peri-urban regions of other European countries (Germany, Poland), TBEV has been detected with carrier rates of 0.31% or 0.1%, respectively [88, 89]. On the other hand, other authors estimate the risk for contracting TBE in urban areas to be low [4].

Four different species belonging to the B. burgdorferi (s.l.) complex were detected in questing I. ricinus ticks in our study: B. afzelii (8.2% of ticks), B. garinii (2.8%), B. burgdorferi (s.s.) (1.3%) and B. valaisiana (0.9%). All of them are confirmed agents of Lyme borreliosis [11] and have already been detected in I. ricinus ticks in other studies in Switzerland [15, 27]. In agreement with previous observations, we found that B. afzelii and B. garinii are the most prominent species, and that adult ticks are more often infected with B. burgdorferi (s.l.) than nymphs. The latter observation is explained by the fact that adult ticks had two blood meals with the possibility of acquiring B. burgdorferi (s.l.), whereas nymphs only had one [14]. The overall prevalence of B. burgdorferi (s.l.) in the present study was 18.0% (11.7% for nymphs, 25% for adults), with site-specific prevalence being significantly variable (7.2–35.8%, P < 0.0001). These observations are in agreement with a study realized in rural areas of Switzerland, where carriage rates ranged between 9–40% for nymphs and from 22 to 47% in adults [15]. The overall prevalence of B. burgdorferi (s.l.) in questing I. ricinus ticks in our study is highly comparable to carriage rates found in urban areas of neighboring countries, ranging from 2.4 to 26.6% in Germany, 10 to 30% in France, and 10.4% in Italy [2, 20, 90,91,92]. In Sanger sequence analyses of the 5S-23S intergenic spacer region, mixed sequences indicating the presence of multiple B. burgdorferi (s.l.) species were obtained for 23 samples (13 nymphs, four males, six females). In confirmatory analyses using RLB, however, only 6 of these samples gave positive results (five B. garinii, one B. afzelii), and carriage of multiple Borrelia spp. could not be confirmed in any of the samples. Since many of these samples contained only very small amounts of Borrelia DNA with cycle threshold values in screening PCR ranging between 36–40 (data not shown), false-negative results in RLB cannot be excluded. In Sanger sequencing, we were able to raise the sensitivity of the test by adding a second-step PCR using nested primers. Since this was not possible in RLB, we expect this test to have a slightly lower sensitivity, accounting for the discrepancy between RLB and Sanger sequencing results. Therefore, the 23 samples, representing 14.9% of ticks positive for B. burgdorferi (s.l.), are regarded as being infected with more than one B. burgdorferi (s.l.) species despite the negative RLB results. This proportion is in agreement with the percentage of carriage of multiple B. burgdorferi (s.l.) found previously [14].

Human disease cases caused by B. miyamotoi, usually presenting as febrile illness have been reported in Russia, USA, the Netherlands and Japan [18]. In I. ricinus, the pathogen is found at a prevalence ranging between 0–3.5% in Europe [17,18,19]. Borrelia miyamotoi has been shown to be present in I. ricinus ticks in Switzerland in rural areas at a prevalence of about 1% [27]. In our study 2.5% of I. ricinus ticks (2.7% of nymphs, 2.6% of adult ticks) were infected with B. miyamotoi, which is slightly less than the prevalence described for urban I. ricinus ticks in France (4%) [20], but higher than the number of B. miyamotoi positive ticks (2/428) reported in a study focusing on urban and peri-urban areas in southern England [16]. Thus, although no disease cases have been reported so far, there is a potential of acquiring such an infection, in urban as well as in rural regions in Switzerland.

Studies investigating I. ricinus ticks collected from vegetation or animals in Switzerland revealed Rickettsia spp. carriage rates of 7.3 to 14% [21, 26, 93]. In accordance with these results and with the detection of Rickettsia spp. in urban areas in other studies in Germany and Slovakia at carrier rates ranging between 2.2–30.1% [30, 31, 33, 34], we found R. helvetica-positive I. ricinus ticks at a prevalence of 13.2% in urban areas of Switzerland. We observed significant differences in site-specific carrier rates (2.6–24.5%, P < 0.0001), which is in agreement with a study in Germany, where prevalence of Rickettsia spp. in I. ricinus ticks ranged between 0–50% [31]. Unlike the frequent detection of R. helvetica in I. ricinus, the documentation of human infection with this agent in different countries, including Switzerland, remains rare [25]. In addition to R. helvetica, three samples were found to be positive for R. monacensis, which has been detected for the first time in Switzerland in 2009 [26] and is known to be present in I. ricinus ticks in at least 18 European countries [25]. R. monacensis has already been discovered in I. ricinus ticks in some urban and peri-urban sites in different European countries [2], which is in accordance with our findings.

Anaplasma phagocytophilum has been detected in I. ricinus ticks in Europe at a prevalence between < 1% and about 20%. In Switzerland, carrier rates between 1.2–2% have been found [27, 36, 93,94,95,96,97]. Corresponding to these findings we found a carrier rate of 1.4% in urban I. ricinus ticks. This rate is in agreement with carrier rates found in urban areas of Austria and France (1.0 and 0.7%, respectively) [20, 41], but is rather low compared to the prevalence found in Slovakia or Hungary (4.5–5.5% and 8.8%, respectively) [42,43,44]. In Switzerland, human granulocytic anaplasmosis (HGA) is a rarely diagnosed disease so far. However, considering the repeated detection of the causative agents in ticks and knowing that the seroprevalence in humans bitten by I. ricinus ticks is 17.1% [98], HGA may increasingly be included in the diagnostic workup of patients with a history of a tick bite. In our study, we merely focused on the detection of A. phagocytophilum, without considering the four different ecotypes. So far, all human cases clustered in ecotype I. The different ecotypes are known to have significantly different host ranges, with ecotype I hosts including numerous urban species [2, 50]. We would therefore expect many of the A. phagocytophilum isolates detected by real-time PCR in our study to belong to ecotype I. However, the respective analyses have not been done so far.

Neoehrlichiosis is a rare human disease. In Switzerland, a close geographic association of disease cases with I. ricinus populations carrying "Ca. N. mikurensis" has been shown for the region of Zürich, where pool carrier rates of 0–8% were found [99]. In our study we could confirm the presence of "Ca. N. mikurensis" in I. ricinus ticks in the region of Zürich, focusing on (sub-) urban areas. In addition, we could show the pathogen to be present in the cities of Basel, Bern, Geneva, and Neuchâtel, with an overall prevalence of 6.2%. This is a higher rate of carriage compared to findings from urban habitats in Slovakia, where prevalence ranged between 1–2.4% [44, 53]. Site-specific carrier rates for "Ca. N. mikurensis significantly differed in our study, ranging from 0 to 10.6% (P < 0.006). This is in agreement with the variation found in the Swiss study in the rural region of Zürich (pool carrier prevalence between 0–8%) [99]; variable carriage rates ranging between 1.1–4.5% were also found in (sub-) urban habitats in a study conducted in Slovakia, the Czech Republic and Austria [42].

Three Babesia species, B. divergens, B. venatorum and B. microti are currently known to cause human disease, and all of them have been found to circulate in urban areas [2, 30, 63]. In 2012 other authors found Babesia spp. to be present in 1.9% of I. ricinus ticks collected in deciduous forests in Western Switzerland. Thereof, 64.3% were identified as B. venatorum and 17.9% as B. divergens [27]. Here, we found a carriage rate of questing urban I. ricinus ticks of 0.83%. All positive samples were classified as B. venatorum using Sanger sequencing. The prevalence of 0.8% is in accordance with I. ricinus carrier rates with this parasite in different urban regions in European countries (Germany, Poland and Slovakia), ranging from 0.4% to 4.5% [33, 64, 65]. Since B. divergens is a bovine parasite, it would only be expected in areas where cattle are found concurrently with I. ricinus ticks [2]. To our knowledge, none of the collection sites of our study represent areas where cattle are present, wherefore the absence of B. divergens is plausible. Human babesiosis is a rare but possibly emerging disease in Europe, with about 50 disease cases reported so far [2, 100].

Site-specific pathogen prevalence significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis" (P < 0.0001 for B. burgdorferi (s.l.) and Rickettsia spp., < 0.006 for "Ca. N. mikurensis"). However, these differences were not attributable to the habitat type (i.e. cemetery, urban park, urban forest, suburban forest) (P > 0.1 for B. burgdorferi (s.l.), B. miyamotoi, A. phagocytophilum, and B. venatorum, > 0.05 for "Ca. N. mikurensis"). When comparing the carrier rates from our study focusing on (sub-) urban areas to carrier rates found in rural areas of Switzerland, no obvious differences were found for most pathogens (prevalence in urban vs rural regions for B. burgdorferi (s.l.) 18.0 vs 9.0–47.0% [15], for Rickettsia spp. 13.5 vs 7.3–14.0% [21, 26, 93], for A. phagocytophilum 1.4 vs 1.2–2.0% [27, 36, 93,94,95,96,97], and for "Ca. N. mikurensis" 6.2 vs 0–8.0% [99]). For B. miyamotoi, the overall prevalence was 2.5%, which is higher than the prevalence of about 1% assessed in a study focusing on rural areas of Switzerland. For Babesia spp., the overall prevalence assessed in our study focusing on (sub-) urban areas was lower than the prevalence found in the rural area (0.8 vs 1.9%) [21]. This latter finding is in agreement with a study comparing the carrier rates between urban and natural habitats in Slovakia [64] and might be in association with the presence of competent reservoir hosts. Altogether, the potential of pathogen transmission as a consequence of a tick bite is highly comparable between urban and rural areas.

In our study, 358 I. ricinus ticks (33.2%) were carrying at least one potentially disease-causing agent: 287 (26.6%) were infected with one, 64 (5.9%) with two, and seven (0.7%) with three different pathogens (Figs. 2, 3). In a study investigating about 270 female I. ricinus ticks in the French Ardennes, 45% of infected ticks were carrying multiple pathogens [101]. In our study involving I. ricinus ticks of all developmental stages, about 80% of infected ticks were positive for only one pathogen, giving a lower proportion of multiple carriage rates. Nevertheless, carriage of multiple pathogens by ticks and therewith co-transmission of pathogens to humans might have important consequences with respect to disease severity and treatment [101,102,103,104]. The most frequent pathogen combinations in our study were B. afzelii + R. helvetica (n = 11) and B. afzelii + "Ca. N. mikurensis" (n = 8). Interestingly, the same pathogens have been found to be predominantly involved in coinfections in a study focusing on mixed deciduous forests in the western part of Switzerland. In both, the present and the previous study, B. afzelii and R. helvetica were the pathogens with the highest prevalence, possibly accounting for the frequent combination of these two bacteria within ticks. B. afzelii and "Ca. N. mikurensis" share common reservoir hosts, which might account for their concurrent detection in individual I. ricinus ticks [27, 105, 106].

Using NGS, we could confirm the presence of all pathogens previously detected by screening PCRs in 2 samples (Fig. 4a, b). In the eight samples negative in all pathogen screening PCRs (samples 3–8), we did not identify any known pathogen using NGS. However, in six of these samples as well as in samples 1 and 2, we could detect the tick endosymbiont "Ca. M. mitochondrii", a member of the order Rickettsiales (Fig. 4a, b). This bacterium is localized in the mitochondria of ovarian cells in I. ricinus female ticks and is transmitted to all offspring. It has been shown to be highly prevalent in I. ricinus ticks, with a mean carrier rate of females of 95%, but a lower prevalence in other developmental stages [85, 107]. Our results agree with these findings with both female, but only five out of seven nymphal I. ricinus ticks being positive for "Ca. M. mitochondrii". Also, the number of reads was much higher in female ticks than in male or nymphal ticks, which is in agreement with the described lower bacterial load in male than in female I. ricinus ticks [107].

Besides known pathogens (R. helvetica, A. phagocytophilum, B. afzelii) and tick endosymbionts, we detected various organisms known to be residents of soil and water, plant associated organisms or members of the normal human microbiota in NGS analyses of ten tick samples (Fig. 4a, b). Since we did not wash the surface of the collected ticks prior to sample preparation and nucleic acid extraction, these findings are easily explainable by the presence of these organisms on the exterior of the ticks. While plant, soil and water organisms originate from the collection sites, members of the human microbiota were transmitted to the tick surface during the collection and sorting procedure.

Conclusions

In this study we documented the presence of B. burgdorferi (s.l.), B. miyamotoi, R. helvetica, R. monacensis, A. phagocytophilum, "Ca. N. mikurensis" and B. venatorum in the (sub-) urban I. ricinus tick population in Switzerland. The pathogen prevalence was as high as the one in rural regions and thus there is a risk of contracting tick-transmitted diseases in urban areas of Switzerland. Carriage of multiple pathogens was observed in about 20% of infected I. ricinus ticks, and therefore there is a true risk of acquiring multiple infections as a consequence of a tick bite.

Abbreviations

HGA:

human granulocytic anaplasmosis

NGS:

next generation sequencing

RLB:

reverse line blot

TBE:

tick-borne encephalitis

TBEV:

tick-borne encephalitis virus

References

  1. Gray JS. Biology of Ixodes species ticks in relation to tick-borne zoonoses. Wien Klin Wochenschr. 2002;114(13-14):473–8.

    PubMed  Google Scholar 

  2. Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubalek Z, Foldvari G, et al. Ixodes ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: New hazards and relevance for public health. Front Public Health. 2014;2(251)

  3. Ecker M, Allison SL, Meixner T, Heinz FX. Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia. J Gen Virol. 1999;80(Pt 1):179–85.

    Article  CAS  PubMed  Google Scholar 

  4. Lindquist L. Tick-borne encephalitis. In: Tselis ACB, editor. Handbook of Clinical Neurology, vol. 123, 1 edn. Amsterdam, Netherlands: Elsevier; 2014. p. 531–59.

    Google Scholar 

  5. Kozuch O, Gresikova M, Nosek J, Lichard M, Sekeyova M. The role of small rodents and hedgehogs in a natural focus of tick-borne encephalitis. Bull World Health Organ. 1967;36:61–6.

    PubMed  PubMed Central  Google Scholar 

  6. Suss J. Epidemiology and ecology of TBE relevant to the production of effective vaccines. Vaccine. 2003;1(21):S19–35.

    Article  Google Scholar 

  7. Mansfield KL, Johnson N, Phipps LP, Stephenson JR, Fooks AR, Solomon T. Tick-borne encephalitis virus - a review of an emerging zoonosis. J Gen Virol. 2009;90(Pt 8):1781–94.

    Article  CAS  PubMed  Google Scholar 

  8. Randolph SE. The shifting landscape of tick-borne zoonoses: tick-borne encephalitis and Lyme borreliosis in Europe. Philos Trans R Soc Lond B Biol Sci. 2001;356(1411):1045–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Suss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia - an overview. Ticks Tick Borne Dis. 2011;2(1):2–15.

  10. Gaumann R, Muhlemann K, Strasser M, Beuret CM. High-throughput procedure for tick surveys of tick-borne encephalitis virus and its application in a national surveillance study in Switzerland. Appl Environ Microbiol. 2010;76(13):4241–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Stanek G, Reiter M. The expanding Lyme Borrelia complex - clinical significance of genomic species? Clin Microbiol Infect. 2011;17(4):487–93.

    Article  CAS  PubMed  Google Scholar 

  12. Anderson JF. Mammalian and avian reservoirs for Borrelia burgdorferi. Ann NY Acad Sci. 1988;539:180–91.

    Article  CAS  PubMed  Google Scholar 

  13. van Duijvendijk G, Coipan C, Wagemakers A, Fonville M, Ersoz J, Oei A, et al. Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors. 2016;9:97.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Rauter C, Hartung T. Prevalence of Borrelia burgdorferi (sensu lato) genospecies in Ixodes ricinus ticks in Europe: a meta analysis. Appl Environ Microbiol. 2005;71(11):7203–16.

  15. Jouda F, Perret JL, Gern L. Density of questing Ixodes ricinus nymphs and adults infected by Borrelia burgdorferi (sensu lato) in Switzerland: spatio-temporal pattern at a regional scale. Vector Borne Zoonotic Dis. 2004;4(1):23–32.

  16. Hansford KM, Fonville M, Gillingham EL, Coipan EC, Pietzsch ME, Krawczyk AI, et al. Ticks and Borrelia in urban and peri-urban green space habitats in a city in southern England. Ticks Tick Borne Dis. 2017;8(3):353–61.

    Article  PubMed  Google Scholar 

  17. Kiewra D, Stanczak J, Richter M. Ixodes ricinus ticks (Acari, Ixodidae) as a vector of Borrelia burgdorferi (sensu lato) and Borrelia miyamotoi in Lower Silesia, Poland - preliminary study. Ticks Tick Borne Dis. 2014;5(6):892–7.

    Article  PubMed  Google Scholar 

  18. Krause PJ, Fish D, Narasimhan S, Barbour AG. Borrelia miyamotoi infection in nature and in humans. Clin Microbiol Infect. 2015;21(7):631–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Platonov AE, Karan LS, Kolyasnikova NM, Makhneva NA, Toporkova MG, Maleev VV, et al. Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg Infect Dis. 2011;17(10):1816–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Reis C, Cote M, Paul RE, Bonnet S. Questing ticks in suburban forest are infected by at least six tick-borne pathogens. Vector Borne Zoonotic Dis. 2011;11(7):907–16.

    Article  PubMed  Google Scholar 

  21. Lommano E, Dvorak C, Vallotton L, Jenni L, Gern L. Tick-borne pathogens in ticks collected from breeding and migratory birds in Switzerland. Ticks Tick Borne Dis. 2014;5(6):871–82.

    Article  PubMed  Google Scholar 

  22. Rollend L, Fish D, Childs JE. Transovarial transmission of Borrelia spirochetes by Ixodes scapularis: a summary of the literature and recent observations. Ticks Tick Borne Dis. 2013;4(1-2):46–51.

  23. Wagemakers A, Jahfari S, de Wever B, Spanjaard L, Starink MV, de Vries HJ, et al. Borrelia miyamotoi in vectors and hosts in The Netherlands. Ticks Tick Borne Dis. 2017;8(3):370–4.

    Article  PubMed  Google Scholar 

  24. Blanco JR, Oteo JA. Rickettsiosis in Europe. Ann NY Acad Sci. 2006;1078:26–33.

    Article  CAS  PubMed  Google Scholar 

  25. Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. Update on tick-borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev. 2013;26(4):657–702.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Boretti FS, Perreten A, Meli ML, Cattori V, Willi B, Wengi N, et al. Molecular Investigations of Rickettsia helvetica infection in dogs, foxes, humans, and Ixodes ticks. Appl Environ Microbiol. 2009;75(10):3230–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lommano E, Bertaiola L, Dupasquier C, Gern L. Infections and coinfections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in western Switzerland. Appl Environ Microbiol. 2012;78(13):4606–12.

  28. Fournier PE, Allombert C, Supputamongkol Y, Caruso G, Brouqui P, Raoult D. Aneruptive fever associated with antibodies to Rickettsia helvetica in Europe and Thailand. J Clin Microbiol. 2004;42(2):816–8.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Jado I, Oteo JA, Aldamiz M, Gil H, Escudero R, Ibarra V, et al. Rickettsia monacensis and human disease, Spain. Emerg Infect Dis. 2007;13(9):1405–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Overzier E, Pfister K, Thiel C, Herb I, Mahling M, Silaghi C. Diversity of Babesia and Rickettsia species in questing Ixodes ricinus: a longitudinal study in urban, pasture, and natural habitats. Vector Borne Zoonotic Dis. 2013;13(8):559–64.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Silaghi C, Hamel D, Thiel C, Pfister K, Pfeffer M. Spotted fever group rickettsiae in ticks, Germany. Emerg Infect Dis. 2011;17(5):890–2.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sprong H, Wielinga PR, Fonville M, Reusken C, Brandenburg AH, Borgsteede F, et al. Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species. Parasit Vectors. 2009;2(1):41.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Schorn S, Pfister K, Reulen H, Mahling M, Silaghi C. Occurrence of Babesia spp., Rickettsia spp. and Bartonella spp. in Ixodes ricinus in Bavarian public parks, Germany. Parasit Vectors. 2011;4:135.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Špitalská E, Boldiš V, Derdáková M, Selyemová D, Rusňáková Taragelová V. Rickettsial infection in Ixodes ricinus ticks in urban and natural habitats of Slovakia. Ticks Tick Borne Dis. 2014;5(2):161–5.

    Article  PubMed  Google Scholar 

  35. Foggie A. Studies on the infectious agent of tick-borne fever in sheep. J Pathol Bacteriol. 1951;63(1):1–15.

    Article  CAS  PubMed  Google Scholar 

  36. Stuen S, Granquist EG, Silaghi C. Anaplasma phagocytophilum - a widespread multi-host pathogen with highly adaptive strategies. Front Cell Infect Microbiol. 2013;3(31)

  37. Bakken JS, Dumler JS. Clinical diagnosis and treatment of human granulocytotropic anaplasmosis. Ann NY Acad Sci. 2006;1078:236–47.

    Article  CAS  PubMed  Google Scholar 

  38. Bakken JS, Dumler JS, Chen SM, Eckman MR, Van Etta LL, Walker DH. Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? Jama. 1994;272(3):212–8.

    Article  CAS  PubMed  Google Scholar 

  39. Dumler JS, Madigan JE, Pusterla N, Bakken JS. Ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment. Clin Infect Dis. 2007;15(45):S45–51.

    Article  Google Scholar 

  40. Ogden NH, Bown K, Horrocks BK, Woldehiwet Z, Bennett M. Granulocytic Ehrlichia infection in ixodid ticks and mammals in woodlands and uplands of the U.K. Med Vet Entomol. 1998;12(4):423–9.

    Article  CAS  PubMed  Google Scholar 

  41. Glatz M, Mullegger RR, Maurer F, Fingerle V, Achermann Y, Wilske B, Bloemberg GV. Detection of "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (sensu lato) genospecies and Anaplasma phagocytophilum in a tick population from Austria. Ticks Tick Borne Dis. 2014;5(2):139–44.

    Article  PubMed  Google Scholar 

  42. Derdáková M, Václav R, Pangrácova-Blaňarová L, Selyemová D, Koči J, Walder G, Špitalská E. "Candidatus Neoehrlichia mikurensis" and its co-circulation with Anaplasma phagocytophilum in Ixodes ricinus ticks across ecologically different habitats of central Europe. Parasit Vectors. 2014;7:160.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Hornok S, Meli ML, Gonczi E, Halasz E, Takacs N, Farkas R, Hofmann-Lehmann R. Occurrence of ticks and prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi s.l. in three types of urban biotopes: forests, parks and cemeteries. Ticks Tick Borne Dis. 2014;5(6):785–9.

    Article  PubMed  Google Scholar 

  44. Pangrácova L, Derdáková M, Pekárik L, Hviščová I, Vichová B, Stanko M, et al. Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban areas of Eastern Slovakia. Parasit Vectors. 2013;6:238.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Andersson M, Zaghdoudi-Allan N, Tamba P, Stefanache M, Chitimia L. Co-infection with 'Candidatus Neoehrlichia mikurensis' and Borrelia afzelii in an Ixodes ricinus tick that has bitten a human in Romania. Ticks Tick Borne Dis. 2014;5(6):706–8.

    Article  PubMed  Google Scholar 

  46. Michelet L, Delannoy S, Devillers E, Umhang G, Aspan A, Juremalm M, et al. High-throughput screening of tick-borne pathogens in Europe. Front Cell Infect Microbiol. 2014;4:103.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Moniuszko A, Dunaj J, Czupryna P, Zajkowska J, Pancewicz S. Neoehrlichiosis - a new tick-borne disease - is there a threat in Poland? Przegl Epidemiol. 2015;69(1):23–6.

    PubMed  Google Scholar 

  48. Richter D, Matuschka FR. "Candidatus Neoehrlichia mikurensis", Anaplasma phagocytophilum, and Lyme disease spirochetes in questing european vector ticks and in feeding ticks removed from people. J Clin Microbiol. 2012;50(3):943–7.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Burri C, Schumann O, Schumann C, Gern L. Are Apodemus spp. mice and Myodes glareolus reservoirs for Borrelia miyamotoi, "Candidatus Neoehrlichia mikurensis", Rickettsia helvetica, R. monacensis and Anaplasma phagocytophilum? Ticks Tick Borne Dis. 2014;5(3):245–51.

    Article  CAS  PubMed  Google Scholar 

  50. Jahfari S, Fonville M, Hengeveld P, Reusken C, Scholte EJ, Takken W, et al. Prevalence of Neoehrlichia mikurensis in ticks and rodents from north-west Europe. Parasit Vectors. 2012;5:74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Silaghi C, Woll D, Mahling M, Pfister K, Pfeffer M. "Candidatus Neoehrlichia mikurensis" in rodents in an area with sympatric existence of the hard ticks Ixodes ricinus and Dermacentor reticulatus, Germany. Parasit Vectors. 2012;5:285.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Vayssier-Taussat M, Le Rhun D, Buffet JP, Maaoui N, Galan M, Guivier E, et al. "Candidatus Neoehrlichia mikurensis" in bank voles, France. Emerg Infect Dis. 2012;18(12):2063–5.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Svitálkova ZH, Haruštiaková D, Mahríková L, Mojšová M, Berthová L, Slovak M, et al. "Candidatus Neoehrlichia mikurensis" in ticks and rodents from urban and natural habitats of south-western Slovakia. Parasit Vectors. 2016;9:2.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Fehr JS, Bloemberg GV, Ritter C, Hombach M, Luscher TF, Weber R, Keller PM. Septicemia caused by tick-borne bacterial pathogen "Candidatus Neoehrlichia mikurensis". Emerg Infect Dis. 2010;16(7):1127–9.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Li H, Jiang JF, Liu W, Zheng YC, Huo QB, Tang K, et al. Human infection with "Candidatus Neoehrlichia mikurensis", China. Emerg Infect Dis. 2012;18(10):1636–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. von Loewenich FD, Geissdorfer W, Disque C, Matten J, Schett G, Sakka SG, Bogdan C. Detection of "Candidatus Neoehrlichia mikurensis" in two patients with severe febrile illnesses: evidence for a European sequence variant. J Clin Microbiol. 2010;48(7):2630–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Welinder-Olsson C, Kjellin E, Vaht K, Jacobsson S, Wenneras C. First case of human "Candidatus Neoehrlichia mikurensis" infection in a febrile patient with chronic lymphocytic leukemia. J Clin Microbiol. 2010;48(5):1956–9.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Hunfeld KP, Hildebrandt A, Gray JS. Babesiosis: recent insights into an ancient disease. Int J Parasitol. 2008;38(11):1219–37.

    Article  CAS  PubMed  Google Scholar 

  59. Martinot M, Zadeh MM, Hansmann Y, Grawey I, Christmann D, Aguillon S, et al. Babesiosis in immunocompetent patients, Europe. Emerg Infect Dis. 2011;17(1):114–6.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Gigandet L, Stauffer E, Douet V, Rais O, Moret J, Gern L. Prevalence of three zoonotic Babesia species in Ixodes ricinus (Linné, 1758) nymphs in a suburban forest in Switzerland. Vector Borne Zoonotic Dis. 2011;11(4):363–6.

    Article  PubMed  Google Scholar 

  61. Hartelt K, Oehme R, Frank H, Brockmann SO, Hassler D, Kimmig P. Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in southern Germany. Int J Med Microbiol. 2004;37:86–92.

    Google Scholar 

  62. Skotarczak B, Cichocka A. Isolation and amplification by polymerase chain reaction DNA of Babesia microti and Babesia divergens in ticks in Poland. Ann Agric Environ Med. 2001;8(2):187–9.

    CAS  PubMed  Google Scholar 

  63. Silaghi C, Woll D, Hamel D, Pfister K, Mahling M, Pfeffer M. Babesia spp. and Anaplasma phagocytophilum in questing ticks, ticks parasitizing rodents and the parasitized rodents - analyzing the host-pathogen-vector interface in a metropolitan area. Parasit Vectors. 2012;5:191.

    Article  PubMed  PubMed Central  Google Scholar 

  64. Hamšiková Z, Kazimírová M, Haruštiaková D, Mahríková L, Slovák M, Berthová L, et al. Babesia spp. in ticks and wildlife in different habitat types of Slovakia. Parasit Vectors. 2016;9:291.

    Article  Google Scholar 

  65. Stanczak J, Cieniuch S, Lass A, Biernat B, Racewicz M. Detection and quantification of Anaplasma phagocytophilum and Babesia spp. in Ixodes ricinus ticks from urban and rural environment, northern Poland, by real-time polymerase chain reaction. Exp Appl Acarol. 2015;66(1):63–81.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Chauvin A, Moreau E, Bonnet S, Plantard O, Malandrin L. Babesia and its hosts: adaptation to long-lasting interactions as a way to achieve efficient transmission. Vet Res. 2009;40(2):21.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Gray J, von Stedingk LV, Gurtelschmid M, Granstrom M. Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils. J Clin Microbiol. 2002;40(4):1259–63.

    Article  PubMed  PubMed Central  Google Scholar 

  68. Casati S, Sager H, Gern L, Piffaretti JC. Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland. Ann Agric Environ Med. 2006;13(1):65–70.

    CAS  PubMed  Google Scholar 

  69. Rieille N, Bressanelli S, Freire CC, Arcioni S, Gern L, Péter O, Voordouw MJ. Prevalence and phylogenetic analysis of tick-borne encephalitis virus (TBEV) in field-collected ticks (Ixodes ricinus) in southern Switzerland. Parasit Vectors. 2014;7:443.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Lommano E, Burri C, Maeder G, Guerne M, Bastic V, Patalas E, Gern L. Prevalence and genotyping of tick-borne encephalitis virus in questing Ixodes ricinus ticks in a new endemic area in western Switzerland. J Med Entomol. 2012;49(1):156–64.

    Article  CAS  PubMed  Google Scholar 

  71. Burri C, Moran Cadenas F, Douet V, Moret J, Gern L. Ixodes ricinus density and infection prevalence of Borrelia burgdorferi (sensu lato) along a North-facing altitudinal gradient in the Rhone Valley (Switzerland). Vector Borne Zoonotic Dis. 2007;7(1):50-58.

  72. Jouda F, Perret JL, Gern L. Ixodes ricinus density, and distribution and prevalence of Borrelia burgdorferi (sensu lato) infection along an altitudinal gradient. J Med Entomol. 2004;41(2):162–9.

    Article  PubMed  Google Scholar 

  73. Pilloux L, Aeby S, Gaumann R, Burri C, Beuret C, Greub G. The high prevalence and diversity of Chlamydiales DNA within Ixodes ricinus ticks suggest a role for ticks as reservoirs and vectors of Chlamydia-related bacteria. Appl Environ Microbiol. 2015;81(23):8177–82.

    Article  PubMed  PubMed Central  Google Scholar 

  74. Cathomas F. Vorkommen von Ixodes ricinus und Borrelia burgdorferi (sensu lato) in städtischen Naherholungsgebieten am Beispiel von Basel (Schweiz). Mitt Naturforsch Ges Basel. 2005;8:63–79.

    Google Scholar 

  75. University of Bristol Online Photographic Guide to Ticks, Bristol University tick ID [http://www.bristoluniversitytickid.uk//]. Accessed July 2016.

  76. Hillyard P. Ticks of North-West Europe Synopses of the British Fauna, No. 52. London: The Linnean Society of London; 1996.

    Google Scholar 

  77. Phylogeny.fr webpage. [http://phylogeny.lirmm.fr/phylo_cgi/index.cgi]. Accessed Oct 2016.

  78. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, Gapped BLAST. PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. NCBI Nucleotide database [https://www.ncbi.nlm.nih.gov/nucleotide/]. Accessed 02. Dec 2016.

  80. Alekseev AN, Dubinina HV, Van De Pol I, Schouls LM. Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J Clin Microbiol. 2001;39(6):2237–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Poupon M-A, Lommano E, Humair PF, Douet V, Rais O, Schaad M, et al. Prevalence of Borrelia burgdorferi (sensu lato) in ticks collected from migratory birds in Switzerland. Appl Environ Microbiol. 2006;72(1):976–9.

    Article  CAS  PubMed Central  Google Scholar 

  82. Rijpkema SG, Molkenboer MJ, Schouls LM, Jongejan F, Schellekens JF. Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi (sensu lato) in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes. J Clin Microbiol. 1995;33(12):3091–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Gern L, Douet V, Lopez Z, Rais O, Cadenas FM. Diversity of Borrelia genospecies in Ixodes ricinus ticks in a Lyme borreliosis endemic area in Switzerland identified by using new probes for reverse line blotting. Ticks Tick Borne Dis. 2010;1(1):23–9.

    Article  PubMed  Google Scholar 

  84. R Development Core Team R: A language and environment for statistical computing. Vienna, Austria, 2008.

  85. Sassera D, Beninati T, Bandi C, Bouman EA, Sacchi L, Fabbi M, Lo N. Candidatus Midichloria mitochondrii', an endosymbiont of the tick Ixodes ricinus with a unique intramitochondrial lifestyle. Int J Syst Evol Microbiol. 2006;56(Pt 11):2535–40.

    Article  CAS  PubMed  Google Scholar 

  86. Pfaffle M, Littwin N, Muders SV, Petney TN. The ecology of tick-borne diseases. Int J Parasitol. 2013;43(12-13):1059–77.

    Article  PubMed  Google Scholar 

  87. Perez D, Kneubuhler Y, Rais O, Gern L. Seasonality of Ixodes ricinus ticks on vegetation and on rodents and Borrelia burgdorferi (sensu lato) genospecies diversity in two Lyme borreliosis-endemic areas in Switzerland. Vector Borne Zoonotic Dis. 2012;12(8):633–44.

    Article  PubMed  PubMed Central  Google Scholar 

  88. Drelich A, Andreassen A, Vainio K, Kruszynski P, Wasik TJ. Prevalence of tick-borne encephalitis virus in a highly urbanized and low risk area in southern Poland. Ticks Tick Borne Dis. 2014;5(6):663–7.

    Article  PubMed  Google Scholar 

  89. Mehlhorn H, Mehlhorn T, Muller M, Vogt M, Rissland J. Tick survey for prevalent pathogens in peri-urban recreation sites in Saarland and Rhineland-Palatinate (Germany). Parasitol Res. 2016;115(3):1167–72.

    Article  PubMed  Google Scholar 

  90. Corrain R, Drigo M, Fenati M, Menandro ML, Mondin A, Pasotto D, Martini M. Study on ticks and tick-borne zoonoses in public parks in Italy. Zoonoses Public Health. 2012;59(7):468–76.

    Article  CAS  PubMed  Google Scholar 

  91. Kahl O, Schmidt K, Schonberg A, Laukamm-Josten U, Knulle W, Bienzle U. Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in Berlin (West). Zentralbl Bakteriol Mikrobiol Hyg A. 1989;270(3):434–40.

    CAS  PubMed  Google Scholar 

  92. Maetzel D, Maier WA, Kampen H. Borrelia burgdorferi infection prevalences in questing Ixodes ricinus ticks (Acari: Ixodidae) in urban and suburban Bonn, western Germany. Parasitol Res. 2005;95(1):5–12.

    Article  PubMed  Google Scholar 

  93. Burri C, Dupasquier C, Bastic V, Gern L. Pathogens of emerging tick-borne diseases, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp., in Ixodes ticks collected from rodents at four sites in Switzerland (Canton of Bern). Vector Borne Zoonotic Dis. 2011;11(7):939–44.

    Article  PubMed  Google Scholar 

  94. Leutenegger CM, Pusterla N, Mislin CN, Weber R, Lutz H. Molecular evidence of coinfection of ticks with Borrelia burgdorferi (sensu lato) and the human granulocytic ehrlichiosis agent in Switzerland. J Clin Microbiol. 1999;37(10):3390–1.

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Liz JS, Anderes L, Sumner JW, Massung RF, Gern L, Rutti B, Brossard MPCR. detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small mammals in western Switzerland. J Clin Microbiol. 2000;38(3):1002–7.

  96. Pusterla N, Leutenegger CM, Huder JB, Weber R, Braun U, Lutz H. Evidence of the human granulocytic ehrlichiosis agent in Ixodes ricinus ticks in Switzerland. J Clin Microbiol. 1999;37(5):1332–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Wicki R, Sauter P, Mettler C, Natsch A, Enzler T, Pusterla N, et al. Swiss Army Survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi (sensu lato), and tick-borne encephalitis virus in ticks. Eur J Clin Microbiol Infect Dis. 2000;19(6):427–32.

    Article  CAS  PubMed  Google Scholar 

  98. Brouqui P, Dumler JS, Lienhard R, Brossard M, Raoult D. Human granulocytic ehrlichiosis in Europe. Lancet. 1995;346(8977):782–3.

    Article  CAS  PubMed  Google Scholar 

  99. Maurer FP, Keller PM, Beuret C, Joha C, Achermann Y, Gubler J, et al. Close geographic association of human neoehrlichiosis and tick populations carrying "Candidatus Neoehrlichia mikurensis" in eastern Switzerland. J Clin Microbiol. 2013;51(1):169–76.

    Article  PubMed  PubMed Central  Google Scholar 

  100. Hildebrandt A, Gray JS, Hunfeld KP. Human babesiosis in Europe: what clinicians need to know. Infection. 2013;41(6):1057–72.

    Article  CAS  PubMed  Google Scholar 

  101. Moutailler S, Valiente Moro C, Vaumourin E, Michelet L, Tran FH, Devillers E, et al. Co-infection of ticks: The rule rather than the exception. PLoS Negl Trop Dis. 2016;10(3)

  102. Diuk-Wasser MA, Vannier E, Krause PJ. Coinfection by Ixodes tick-borne pathogens: Ecological, epidemiological, and clinical consequences. Trends Parasitol. 2016;32(1):30–42.

    Article  PubMed  Google Scholar 

  103. Tijsse-Klasen E, Sprong H, Pandak N. Co-infection of Borrelia burgdorferi (sensu lato) and Rickettsia species in ticks and in an erythema migrans patient. Parasit Vectors. 2013;6:347.

    Article  PubMed  PubMed Central  Google Scholar 

  104. Horowitz HW, Aguero-Rosenfeld ME, Holmgren D, McKenna D, Schwartz I, Cox ME, Wormser GP. Lyme disease and human granulocytic anaplasmosis coinfection: impact of case definition on coinfection rates and illness severity. Clin Infect Dis. 2013;56(1):93–9.

    Article  PubMed  Google Scholar 

  105. Andersson M, Raberg L. Wild rodents and novel human pathogen "Candidatus Neoehrlichia mikurensis", southern Sweden. Emerg Infect Dis. 2011;17(9):1716–8.

    Article  PubMed  PubMed Central  Google Scholar 

  106. Gern L, Lienhard R, Peter O. Diseases and pathogenic agents transmitted by ticks in Switzerland. Rev Med Suisse. 2010;6(266):1906–9. (In French)

    CAS  PubMed  Google Scholar 

  107. Lo N, Beninati T, Sassera D, Bouman EA, Santagati S, Gern L, et al. Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus. Environ Microbiol. 2006;8(7):1280–7.

    Article  CAS  PubMed  Google Scholar 

  108. Courtney JW, Kostelnik LM, Zeidner NS, Massung RF. Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol. 2004;42(7):3164–8.

  109. Teal AE, Habura A, Ennis J, Keithly JS, Madison-Antenucci SA. New real-time PCR assay for improved detection of the parasite Babesia microti. J Clin Microbiol. 2012;50(3):903–8.

  110. Ornstein K, Barbour AGA. reverse transcriptase-polymerase chain reaction assay of Borrelia burgdorferi 16S rRNA for highly sensitive quantification of pathogen load in a vector. Vector Borne Zoonotic Dis. 2006;6(1):103–12.

    Article  PubMed  Google Scholar 

  111. Coipan EC, Fonville M, Tijsse-Klasen E, van der Giessen JW, Takken W, Sprong H, Takumi K. Geodemographic analysis of Borrelia burgdorferi (sensu lato) using the 5S-23S rDNA spacer region. Infect Genet Evol. 2013;17:216–22.

    Article  CAS  PubMed  Google Scholar 

  112. Najm NA, Meyer-Kayser E, Hoffmann L, Herb I, Fensterer V, Pfister K, Silaghi CA. molecular survey of Babesia spp. and Theileria spp. in red foxes (Vulpes vulpes) and their ticks from Thuringia, Germany. Ticks Tick Borne Dis. 2014;5(4):386–91.

    Article  PubMed  Google Scholar 

  113. Jado I, Escudero R, Gil H, Jimenez-Alonso MI, Sousa R, Garcia-Perez AL, et al. Molecular method for identification of Rickettsia species in clinical and environmental samples. J Clin Microbiol. 2006;44(12):4572–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Rothen J, Githaka N, Kanduma EG, Olds C, Pfluger V, Mwaura S, et al. Matrix-assisted laser desorption/ionization time of flight mass spectrometry for comprehensive indexing of East African ixodid tick species. Parasit Vectors. 2016;9:151.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Tick collection was supported by the Swiss Army. We particularly thank Roger Herger for sustaining this study, and all people involved in tick collection. We thank Cornelia Silaghi and Alexander Mathis (Institute of Parasitology, University of Zürich, Zürich, Switzerland) for providing us with DNA from various Babesia spp. as well as a key to morphological tick identification. Many thanks go to Alexis Dumoulin (Institute Central des Hôpitaux du Valais, Sion, Switzerland) for providing us with R. helvetica DNA and Reto Lienhard (ADMED Microbiologie, La Chaux-de-Fonds, Switzerland) for providing us with A. phagocytophilum DNA. We thank Christoph Grünig and Patrick Pfistner (Microsynth, Switzerland) for performing Sanger sequencing and sequence data analyses. Finally, many thanks go to Matthias Wittwer, Nicole Liechti, Marc Strasser and Olivier Engler for valuable discussions.

Funding

The project was funded with in-kind contributions.

Availability of data and materials

The authors declare that the data underlying analyses on pathogen prevalence and carriage of multiple pathogens are available within the article. Sequences generated using Sanger (capillary electrophoresis) sequencing have been deposited in the GenBank database under the accession numbers MF121944–MF121977. Information on NGS analyses are available within supplementary information files. NGS sequencing data are available from the corresponding author upon reasonable request.

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Authors and Affiliations

  1. Spiez Laboratory, Federal Office for Civil Protection, Austrasse, Spiez, Switzerland

    Corinne P. Oechslin, Daniel Heutschi, Nicole Lenz, Christian M. Beuret, Stephen L. Leib & Rahel Ackermann-Gäumann

  2. Institute for Infectious Diseases, University of Bern, Friedbühlstrasse, Bern, Switzerland

    Corinne P. Oechslin, Nicole Lenz & Stephen L. Leib

  3. Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland

    Corinne P. Oechslin

  4. ZHAW Life Science and Facility Management, Grüental, Wädenswil, Switzerland

    Werner Tischhauser

  5. retired, Infectious Diseases, Central Institute of Valais Hospitals, Sion, Switzerland

    Olivier Péter

  6. Laboratory of Ecology and Evolution of Parasites, Institute of Biology, University of Neuchâtel, Emile Argand, Neuchâtel, Switzerland

    Olivier Rais

  7. Medical Services Directorate, Swiss Armed Forces, Ittigen, Switzerland

    Sergei Bankoul

  8. Swiss National Reference Centre for tick-transmitted diseases, Spiez, Switzerland

    Rahel Ackermann-Gäumann

Authors
  1. Corinne P. Oechslin
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Contributions

SB contributed to organizational support in the collection of ticks by military personnel and discussed the study progress with DH on a monthly base. DH organized and assisted in the tick collection done by the Swiss Army and performed the real-time PCR screening of tick samples. CB technically supported the real-time PCR screening. OP and WT collected ticks at different collection sites and different time points, in addition to the ticks collected by the Swiss Army. OR analyzed samples with suspected mixed Borrelia infections using reverse line blot. CO analyzed selected samples using NGS, including all laboratory and data analysis work and contributed in writing the manuscript. NL did the statistical analyses and contributed in writing the manuscript, too. Together with SLL, RAG initiated the project. RAG coordinated the study, morphologically identified all collected ticks and purified nucleic acids from all samples. Also, RAG coordinated the Sanger sequencing with Microsynth, evaluated the data and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Rahel Ackermann-Gäumann.

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Additional file

Additional file 1:

NGS protocol, bioinformatics pipeline, detailed NGS results and discussion. (DOCX 22 kb)

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Oechslin, C.P., Heutschi, D., Lenz, N. et al. Prevalence of tick-borne pathogens in questing Ixodes ricinus ticks in urban and suburban areas of Switzerland. Parasites Vectors 10, 558 (2017). https://doi.org/10.1186/s13071-017-2500-2

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Parasites & Vectors

ISSN: 1756-3305

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