Background

The hard tick Ixodes ricinus Linné 1758, known as vector for different pathogenic agents, serves as the main vector for spirochetes of the Borrelia burgdorferi sensu lato (s.l.) complex in Central Europe [1]. This genospecies complex includes the causative agents for Lyme borreliosis, the most frequent arthropod-borne human disease in the temperate northern hemisphere [2]. To date, 19 named spirochetes belong to the B. burgdorferi s.l. complex worldwide, however, several genospecies still remain unnamed [3–5]. In Europe, eleven genospecies of the Borrelia burgdorferi s.l. complex could be detected: B. burgdorferi sensu stricto (s.s.), B. afzelii, B. bavariensis, B. bissettii, B. garinii, B. lusitaniae, B. spielmanii, B. valaisiana, B. kurtenbachii (formerly included in the B. bissettii species), B. finlandensis and B. carolinensis[6–17]. All named species, with the exception of B. finlandensis and B. carolinensis, are implicated in different manifestations of Lyme borreliosis as, for example, erythema migrans, acrodermatitis chronica atrophicans, Lyme arthritis and neuroborreliosis [3, 5, 18].

In Europe, several reservoir hosts for Lyme disease-associated Borrelia spp. are known as, for example, small rodents, hedgehogs, squirrels, lizards and various bird species [16, 19–23]. Transmission of B. burgdorferi s.l. from ticks to hosts may occur through different developmental stages of I. ricinus, as transstadial transmission is very efficient. Even though larvae are occasionally affected, transovarial transmission in ticks seems to be inefficient [24]. Prevalence rates of B. burgdorferi s.l. in I. ricinus range from 6.1% in France to 22.5% in Western Switzerland [14, 25–28]. In Germany, prevalences of Borrelia-positive questing I. ricinus ticks range from 3.1% in Northern Germany to 27.0% in Thuringia or locally up to 37% in Bavaria [13, 29–33]. The present study was conducted to determine the Borrelia infection rate in Hanoverian ticks in 2010 and at the same time to serve as fifth-year-follow-up study to monitor changes or stagnation of tick infection rates when compared to data from 2005 [9]. For this, 2,100 ticks were collected in different recreational areas in 2010 in the northern German city of Hanover and subsequently analysed for B. burgdorferi s.l. infections.

Methods

Tick material

Questing ticks were collected each month from April to October 2010 in ten different recreation areas in the city of Hanover, the capital of the Northern German federal state Lower Saxony [34]. The city of Hanover was elected as the “German Capital of Biodiversity” in 2011 and is nicknamed “The green metropolis” as it houses numerous parks and the largest continuous urban woodland in Europe. At each sampling site, 30 ticks were collected per month resulting in 210 ticks per defined location over the sampling period and a total of 2,100 ticks overall. Tick species and developmental stages were determined microscopically based on morphological parameters [35].

DNA isolation and detection of Borrelia spp

Genomic DNA isolation was carried out as described previously [34, 36]. Genomic DNA was eluted twice with 70 μl and 60 μl double-distilled water, respectively, to obtain a final volume of 100 μl genomic DNA. Detection of B. burgdorferi s.l. was achieved by minor groove binder probe-based qPCR targeting the 5S-23S intergenic spacer (IGS) region described by Strube et al. [37] with the modification that Absolute Blue QPCR low ROX mix (Thermo Fisher; containing Thermo-Start™ DNA Polymerase) was used and tick DNA template was increased to 10 μl. All qPCR reactions were performed as duplicates and each run included a negative template control as well as plasmid standard positive controls containing 100 - 106 copies of the Borrelia 5S-23S IGS and Ixodes ITS2 region, respectively [37]. Samples were resubjected to qPCR analysis, if only one well was determined positive and species identification was unsuccessful with the Reverse Line Blot (RLB). If no amplification could be detected in the second qPCR run, samples were considered as questionable positives.

Identification of B. burgdorferi s.l. genospecies by Reverse Line Blot (RLB) and Sanger sequencing

Species identification of Borrelia-positive tick samples was achieved by RLB. Sanger sequencing was additionally applied to samples that were determined B. garinii-positive in RLB to further differentiate between B. garinii and B. bavariensis.

RLB was preceded by amplification of the B. burgdorferi s.l. 5S-23S IGS region using biotin linked forward primer 5SCB and reverse primer 23SN2 (10 pmol each) as described by Rijpkema et al. [38]. For the 25 μl reaction set up, 12.5 μl Thermo-Start™ PCR Master Mix (Thermo Scientific, Surrey, England), 1 μl of each primer and 5 μl tick DNA template were added to the corresponding amount of H2O. Cycling conditions were based on the protocol by Burri et al. [39] with addition of a polymerase activation step (15 min, 94°C). Each run included positive controls using template DNA of the following Borrelia genospecies (isolates): B. afzelii (PGau), B. bavariensis (PBi), B. bissettii (DN127), B. burgdorferi s.s. (Pka2), B. garinii (TN), B. lusitaniae (PotiB2), B. spielmanii (PHap) and B. valaisiana (VS116). Cross reactivity and specificity of the RLB were determined by using B. duttonii, B. recurrentis and Treponema phagedenis isolates as negative controls in addition to a non-template control.

RLB technique was performed as described by Rijpkema et al. [38] with few modifications: PCR products were hybridized to 7 different oligonucleotide probes: B. afzelii (AF; 10 μM), B. garinii (GA; 10 μM), B. burgdorferi s.s. (SS; 20 μM) [38], B. bissettii (BISNE2; 10 μM), B. lusitaniae (LUSINE2; 10 μM), B. spielmanii (SpiNE3; 800 μM) [40] and B. valaisiana (VSNE; 20 μM) [41]. Moreover, a modified probe for B. burgdorferi s.l. (SL2; 5’-[AmC6T]-CCATATTTTTATCTTCCATCTCTA-3’; 500 μM) was added as positive control for successful hybridization procedure. Furthermore, a probe for relapsing fever-like spirochetes (RFLNE; 250 μM) [40] was added to the RLB. All reactions were performed as duplicates.

Following sample hybridization as described by Rijpkema et al. [38], the membrane was washed with 2× SSPE-0.5% SDS at 45°C for 15 min, then incubated with streptavidin-peroxidase for 30 min at 42°C, and finally washed with 2× SSPE-0.5% SDS for 10 min at 42°C. Chemiluminescent hybridization signals were achieved by use of ECL detection reagent (GE Healthcare) and detected with Bio Imaging System MF-ChemiBIS 3.2 (Biostep, Jahnsdorf, Germany) during 2 to 10 min exposition (depending on signal strength). Reactions were performed as duplicates and all samples were tested at least twice.

To further differentiate between B. garinii and B. bavariensis, GA positive samples were reamplified and products showing visible gel bands were custom sequenced. Identification of those two species was based on four SNPs occurring in the 5S-23S IGS region of B. bavariensis and B. garinii (Figure 1).

Figure 1
figure 1

SNPs in the partial 5S-23S intergenic spacer region of B. garinii and B. bavariensis . SNPs used for genospecies differentiation are indicated by numbers. The displayed alignment is based on GenBank accession numbers FJ546505 (Bgar: B. garinii, strain IPT157), FJ546507 (Bgar: B. garinii, strain IPT165) and FJ546495 (Bbav: B. bavariensis, strain PBi).

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Data was statistically analysed by application of chi-square test followed by Yates correction with SigmaStat® software (version 3.11) with subsequent Bonferroni-Holm correction. For analysis, α was defined with 0.05 and H0 was rejected if P ≤ 0.05. Ticks classified as questionable were included as positives in statistical analysis.

Comparison of B. burgdorferi s.l. tick infection rates in 2010 vs. 2005

To compare results concerning infection rates of different tick stages obtained in 2010 to those obtained in 2005, questionable results were added to positive tested ticks in 2005 [9]. As a result, modified 2005 data was used for analysis as follows: 153 positive adults divided into 75 male and 78 female ticks (75 positive and 3 questionable), 96 nymphs (92 positive and 4 questionable) and 2 larvae (1 positive and 1 questionable). Data of adult and nymphal stages were statistically analysed as described above. Final significances were based on corrected α-values. To compare larval stages collected in 2005 with those collected in 2010, Fisher’s exact test was used due to low sample size. Final significances were based on corrected α-values.

Coinfections of ticks with B. burgdorferi s.l. and Rickettsiales

To analyze the tick coinfection rates with Borrelia and Rickettsiales, obtained data on B. burgdorferi s.l. infections were compared with data on infections with Rickettsiales published previously [34]. Statistical analysis of present coinfections with Rickettsia spp. or A. phagocytophilum and comparison with data from 2005 was conducted as described above. Comparison of coinfections with all three pathogens between years was carried out by using Fisher’s exact Test (SigmaStat® software version 3.11).

Results

Collection of 2,100 questing ticks resulted in 372 adult ticks (196 males and 176 females), 1,697 nymphs and 31 larvae all identified as I. ricinus. A total of 22.7% (476/2,100) of collected ticks was found to be infected with B. burgdorferi s.l.. Male adults showed an infection rate of 29.6% (58/196) whereas 37.5% (66/176) of female adults were determined positive resulting in an overall infection rate of 33.3% (124/372) for adult ticks. Nymphs showed an infection rate of 20.3% (344/1,697) and larval infection rates were determined as 25.8% (8/31). Statistically significant differences concerning developmental stages were observed between adults and nymphs (P < 0.001; αloc = 0.017). On level below, adult males (P < 0.001; αloc = 0.0083) as well as adult females (P = 0.003; αloc = 0.01) were significantly more often infected than nymphs. A detailed overview of infection rates of different stages during the collection period is shown in Table 1.

Table 1 B. burgdorferi s.l.-infected Hanoverian ticks (positive/total ticks) in 2010
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Over the collection period, two main infection peaks of ticks could be observed with a first increase in June (25.3%; 76/300) and July (28%; 83/300) as well as a second peak in October (32.7%; 98/300), the latter is simultaneously the highest seasonal prevalence. The lowest Borrelia-prevalence was determined in May (17.7%; 53/300). Statistically significant differences were observed between October and the months of April, May and August. P-values and adjusted α-values are shown in Figure 2.

Figure 2
figure 2

Seasonal variations of B. burgdorferi s.l.-infected Hanoverian ticks in 2010. Connection lines indicate significant differences between prevalence rates.

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Concerning the distribution of infected ticks at different sampling locations, several statistical significances were observed. The highest infection rate was found at “Misburger Wald” consisting of 32.9% (69/210) infected ticks, followed by “Mecklenheide” (30.0%; 63/210). The locations with lowest infection rates were “Bornumer Holz” and “Maschpark” (both 16.2%; 34/210) followed by “Annateiche” (16.7%; 35/210). Statistically significant differences were determined between “Misburger Wald” vs. “Bornumer Holz”, “Maschpark” and “Annateiche” as well as between “Mecklenheide” vs. “Bornumer Holz” and “Maschpark”. An overview of infection rates of tick stages at all sampling locations is provided in Table 2. Adjusted α-values and corresponding P-values are shown in Figure 3. Borrelia spp. infection rates in ticks at the different sampling locations are visualized in Figure 4.

Table 2 Distribution of B. burgdorferi s.l.-infected ticks (positives/total ticks) at different collection sites in the city of Hanover in 2010
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Figure 3
figure 3

Local distribution of B. burgdorferi s.l.-infected Hanoverian ticks in 2010. Connection lines indicate significant differences between total prevalence rates.

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Figure 4
figure 4

B. burgdorferi s.l.-infected Hanoverian ticks in 2010 at the different sampling locations. Green dots: <20% infected ticks; blue dots: ≥20 < 30% infected ticks; pink dots: ≥30% infected ticks. 1: Mecklenheide; 2: Große Heide; 3: Misburger Wald; 4: Annateiche; 5: Seelhorster Wald; 6: Ricklinger Teiche; 7: Bornumer Holz; 8: Georgen Garten; 9: Eilenriede; 10: Maschpark. The white dashed line represents the city border (Map source: Google Earth).

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Identification and distribution of B. burgdorferi s.l. genospecies

In 65.5% (312/476) of Borrelia qPCR-positive ticks, RLB confirmed B. burgdorferi s.l. infection. Data was composed of 39 male adults, 40 female adults, 232 nymphs and 1 larva. Samples containing ≥104 5S-23S IGS copies showed a detection rate of 77.8% (7/9), ≥103 copies 95.7% (44/46), ≥102 88.9% (96/108) and ≥101 copies resulted in 74.2% (66/89) detection rate. Ticks containing ≤10 5S-23S IGS copies showed a detection rate of 44.2% (99/224). B. burgdorferi s.l. genospecies identification by RLB was successful in 282 of the 476 qPCR-positive ticks (59.2%). A detailed overview of detected genospecies is given in Table 3. Overall, B. spielmanii, B. bissettii and B. lusitaniae were mainly associated with multiple-infections.

Table 3 Distribution of different B. burgdorferi s.l. genospecies in positive Hanoverian ticks in 2010
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Out of 54 tick samples with a positive signal using the B. garinii including B. bavariensis RLB probe, 44 samples were sequenced via Sanger sequencing and revealed 18.2% (8/44) B. bavariensis, 45.5% (20/44) B. garinii and 15.9% (7/44) non-identifiable samples. The remaining 20.5% (9/44) samples were assigned to other genospecies (B. afzelii, B. burgdorferi s.s., B. spielmanii and B. valaisiana) corresponding to the genospecies coinfections identified by RLB.

Coinfections of ticks with B. burgdorferi s.l. and Rickettsiales

The total coinfection rate of the 2100 analysed samples with Rickettsiales was 7.8% (153/2,100). Coinfections with Rickettsia spp. [R. helvetica in all typable samples (34)] were found in 7.3% (153/2,100) of the examined ticks and coinfections with A. phagocytophilum were detected in 0.3% (7/2,100) of ticks. Infection with all three pathogens was found in 0.1% (3/2100) of samples. Adult stages (43/372; 11.6%) were statistically significant (P = 0.005; αloc = 0.01) more often infected with Rickettsiales than nymphs (120/1697; 7.1%). Concerning Rickettsia spp. coinfections, statistically significant differences (P = 0.002; αloc = 0.01) were observed between adults (42/372; 11.3%) and nymphs (111/1,697; 6.5%). No coinfections were found in examined larvae. A detailed overview about infection rates and tick stages is provided in Table 4.

Table 4 Coinfections with B. burgdorferi s.l. and Rickettsiales in Hanoverian ticks in 2010
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Comparison of tick infections with B. burgdorferi s.l. and coinfections with Rickettsiales between 2010 and 2005

Comparing infection rates of Hanoverian I. ricinus ticks with Borrelia spp. 5 years apart, the infection rate of adult stages and nymphs remained mainly unchanged with 35.5% (153/433) and 18.9% (96/507) infected individuals in 2005 [9] compared to 33.3% (124/372) and 20.3% (344/1,976) infected individuals in 2010. However, a statistically significant increase (P = 0.002) was observed for larval stages with 3.3% (2/60) infected larvae in 2005 whereas 25.8% (8/31) Borrelia-positive larvae were found in 2010. Concerning detection of different B. burgdorferi s.l. genospecies, it should be noted that in 2005, B. garinii, including B. bavariensis, was the most frequently detected genospecies, followed by B. afzelii and B. spielmanii. In 2010, B. afzelii was the most frequent detected species followed by B. garinii including B. bavariensis and B. spielmanii. Comparison of Borrelia spp. and Rickettsiales coinfection rates revealed no statistically significant difference between both years: Collected ticks were infected with B. burgdorferi s.l. and Rickettsia spp. at a rate of 9.1% (99/1,098) in 2005 [42] and 7.3% (153/2,100) in 2010. Coinfection rates with B. burgdorferi s.l. and A. phagocytophilum were 0.9% (15/1,646) in 2005 [43] and 0.3% (7/2,100) in 2010. Coinfections with all three pathogens were detected in 1.3% (5/391) of ticks in 2005 [42] and 0.1% (3/2,100) in 2010.

Discussion

The hard tick I. ricinus serves as main vector for spirochetes of the B. burgdorferi s.l. complex in Central Europe. Parts of this complex are responsible for Lyme disease, a sickness that has increased rapidly during the past 20 years in the northern hemisphere [5, 44]. In several studies, infection rates of ticks with B. burgdorferi s.l. in Germany were investigated to assess the potential infection risk for humans resulting in a broad range of infection rates ranging from 3.1% in Northern Germany to 27.0% in Thuringia and 36.2% in Bavaria [29–33]. Besides data collection in different geographical regions, it is of importance to monitor tick infection rates over time to assess whether human infection risk increases or decreases. Thus, the present study served not only as a status survey for the Northern German state capital Hanover but also as fifth-year-follow-up survey of Borrelia infections in I. ricinus ticks.

The total infection rate of B. burgdorferi s.l.-infected ticks in 2010 was 22.7% with a significant difference between adults and nymphs (33.3% and 20.3% infected individuals, respectively), but not between adults and larvae (25.8% infected individuals). The expected highest burden in adult ticks is in accordance with different previous studies [6, 9, 31] and is most likely related to the combination of transstadial Borrelia transmission and the number of blood meals for development, which is connected to a higher probability to acquire bacteria from infected hosts. Comparison of B. burgdorferi s.l. infection rates in I. ricinus ticks of 2010 and 2005 resulted in approximately the same percentage of infected adults (33.3% in 2010 and 35.5% in 2005) and nymphs (20.3% in 2010 and 18.9% in 2005) whereas tick larvae showed a significantly different B. burgdorferi s.l. infection rate (P = 0.002) between 2005 (3.3%) and 2010 (25.8%). Due to low numbers of collected larvae (60 in 2005; 31 in 2010), the significant increase has to be verified in further studies and should be interpreted with caution. The rather high prevalence of Borrelia-infected larvae in 2010 might be explained by an interrupted blood meal with failed further development as well as by transovarial transmission. Even though transovarial transmission of B. burgdorferi s.l. was found to be rather inefficient [24, 45], it is the most plausible explanation as it seems unlikely that a quarter of collected larvae were removed during feeding from a Borrelia-infected host. However, solely in one of eight Borrelia- positive tested larvae, B. burgdorferi s.l. was confirmed by RLB. Overall, RLB confirmed 312 (65.5%) out of 476 Borrelia qPCR-positive ticks and identified 282 (59.2%) successfully concerning their genospecies. Reason for the generally lower RLB-positive rate is most likely a higher sensitivity of qPCR. The 30 tick samples in which RLB resulted in detection of B. burgdorferi s.l., but genospecies identification failed, might be explained by the higher sensitivity of the SL2 probe compared to genospecies-specific probes. Furthermore, not all in Europe occurring B. burgdorferi s.l. genospecies were detected by RLB – probes are not available for B. kurtenbachii, B. carolinensis and B. finlandensis. Moreover, ticks might have been infected with Borrelia miyamotoi, a Borrelia species associated with the relapsing fever group. B. miyamotoi, originally detected in Japan in I. persulcatus[46], was also previously found in I. ricinus ticks in Europe [47, 48]. In contrast to B. burgdorferi s.l., transovarial transmission could be demonstrated [49, 50]. However, the primer-probe-combination [9] used in the present study does not detect B. miyamotoi, but it is specific for the B. burgdorferi s.l. complex.

Regarding seasonal distribution of B. burgdorferi s.l. infection in Hanoverian I. ricinus ticks, the two peak course of infected ticks in June/July and October may depend on biological and climatic conditions like host disposability, temperature and humidity as these factors can affect pathogen transmission from hosts to ticks [51–54]. Concerning different sampling sites in the city of Hanover, tick infection rates range from 16.2% at the location “Maschpark” up to 32.9% at “Misburger Wald”. These differences in numbers of Borrelia-infected ticks might be explained by different incidences of Borrelia-infected hosts like small rodents or birds at single sampling locations.

B. burgdorferi s.l. genospecies identification resulted in findings comparable to results from previous studies, where B. afzelii was the predominant detected species in ticks [33, 55, 56], but also B. garinii including B. bavariensis and B. spielmanii were frequent findings in Borrelia-infected I. ricinus ticks [6, 12, 55]. Comparing mentioned genospecies distribution in 2010 with data from Hanoverian ticks collected in 2005, B. afzelii, B. garinii including B. bavariensis and B. spielmanii were the most frequently detected species in both studies. B. afzelii was the most mono-infecting species, B. spielmanii was found more often in multiple-infections than as mono-infecting species and frequent findings of double-infections contained a combination of B. afzelii and B. spielmanii[9]. Double- or multiple-infections (cf. Table 3) may result from blood meals on different hosts each infected with one genospecies, from one host having a multiple infection causing ticks to take up a number of genospecies, or from co-feeding of infected ticks. Several combinations including B. afzelii or B. garinii as detected in the present study were also observed by Pichon et al.[57]. The detected combination of B. afzelii and B. burgdorferi s.s. was also described amongst others in red squirrels [23].

Concerning coinfections of Borrelia-positive ticks with Rickettsiales in 2010, statistically significant differences were observed between different tick stages. This is in accordance with data from 2005, where adult ticks also showed higher infection rates than nymphs [42, 43]. No statistically significant differences in coinfection rates were found between years.

Conclusion

In summary, comparing B. burgdorferi s.l. infections in Hanoverian I. ricinus ticks in 2010 with data from 2005, a significant increase of infected larvae was determined whereas nymphs and adult ticks did not show significantly changed infection rates. Whether this development of infected larvae is an actual fact through rather effective transovarial transmission under field conditions due to unknown factors, or an isolated event should be evaluated by further studies. The Hanoverian tick infection rate and distribution of B. afzelii, B. garinii and B. spielmanii as most abundant genospecies will be further monitored in the next fifth-year-follow-up study starting in April 2015.