Figure

Figure. Tick sampling sites in Germany during 2008 and 2009 (green shading): Saarland, sites A–C, along the border with France (sampled March–September 2008). Bavaria, site D (sampled April and September 2009). Saxony,...

Questing ticks were collected from vegetation by flagging in 3 regions in Germany (9 sites total) in March–September 2008 and April–October 2009 (Figure). Three sites, including renatured gravel pits and walking areas near villages and cities (A–C), were located in the federal state of Saarland. One site in southern Germany was in a natural alluvial forest north of Munich, popular for hiking and dog walking (D), and in East Germany (Saxony), 3 sites were former brown coal surface-mining areas near the city of Leipzig (E–G). Ongoing renaturation and flooding of the pit holes during past decades created a highly valuable recreational area with artificial lakes and surrounding meadows and forest (www.leipzigerneuseenland.de). Here, sampling was carried out around Lake Cospuden (436 ha, fully flooded for the past 10 years, 51.5 m deep; Figure). Further sampling sites were located on a renatured former waste disposal area (H), now a popular urban recreation area, and in an alluvial forest near a popular game park (I), both within Leipzig.

Ticks were stored either in 70% ethanol or kept at 4°C, identified to species level, and homogenized (ticks in ethanol: 2 mL Eppendorf tubes [Eppendorf, Hamburg, Germany] with one 5-mm steel bead, 80 µL phosphate-buffered saline, and in Tissue Lyser [QIAGEN, Hilden, Germany] for 5 min at 30.000U/min; live ticks: 2.0 mp PRECELLYS Kit Mk28, 320 µL minimum essential medium cell culture medium [Sigma-Aldrich, Taufkirchen, Germany], in a PRECELLYS 24 dual [Peqlab, Erlangen, Germany] for 2 × 5,000 rpm for 30 s, with a 15-s break). DNA was extracted from each tick separately (females, males, nymphs) or in pools of 5 individuals (nymphs only) with the QIAGEN DNA Mini Kit (QIAGEN) by using the protocol for animal tissue. Quality and quantity of extracted DNA were tested with a NANO DROP ND-1000 spectrophotometer (Peqlab).

Ticks were screened for rickettsial DNA by using a PCR amplifying part of the gltA gene as described (8). Some tick samples positive for Rickettsia spp. were selected to amplify and determine the sequence of the outer membrane protein–coding genes ompA and ompB (9). New primers were developed for the first part of the ompA gene: OmpA-MMX1-for 5′-ACAAGCTGGAGGAAGCCTAGC-3′; OmpA-MMX1-rev 5′-TCTCCCGCTCCTTTGAAAACTAT-3′.

Of the 3,076 ticks collected, 1,359 were identified as D. reticulatus ticks (542 males, 817 females, from all sites except site I), 15 as D. marginatus ticks (8 males, 7 females, only at site B), and 1,702 as I. ricinus ticks (714 males, 658 females, 330 nymphs, from all sites). DNA was extracted from most D. reticulatus ticks and almost half of the I. ricinus ticks (Table). In the gltA PCR, 849 bands of the correct size were obtained in the investigated samples (Table; 749/1,322 (56.7%) of D. reticulatus ticks; 2/15 (13.3 %) of D. marginatus ticks; 98/730 (13.4%–17.4%) of I. ricinus ticks (772 nymphs in pools of up to 5). The rate of infection differed by tick species, sex of the tick, and collection site; for example, the infection rate was 20.5%–76.4% in female D. reticulatus ticks (Table). Sequencing of 24.1% (n = 205) of the gltA PCR-products verified the specific amplification of rickettsial DNA in 192 cases (93.6%); sequencing for the remaining 13 amplicons could not be determined because the sequencing result was of poor quality. In 1 I. ricinus tick from site B, 87%–88% identity to R. asiatica, R. canadensis, and R. helvetica was identified, but amplification of ompA and ompB failed to further verify the species.

From each site, 1–6 positive tick samples were analyzed for the ompA and ompB genes. The ompB genes of R. raoultii (n = 27), R. helvetica (n = 8), and R. slovaca (n = 2), and ompA genes of R. raoultii (n = 25) and R. slovaca (n = 2) were 100% identical in the amplified part of the respective species, regardless of geographic origin. R. raoultii from our study showed 100% identity (ompA) and 99% identity (ompB) to R. raoultii strain Marne (GenBank accession nos. DQ365800 and DQ365797); for R. helvetica (ompB), 100% identity to GenBank entry AF123725; and for R. slovaca, 100% identity to GenBank entry AF123723 (ompB), and U83454 (ompA). R. raoultii, R. slovaca, and R. helvetica were found only in D. reticulatus, D. marginatus, and I. ricinus ticks, respectively. Sequences (ompA and ompB) from this study were deposited in GenBank (accession nos. HQ232215–HQ232278).

In Germany, the most common tick is I. ricinus; D. reticulatus ticks have a focal distribution, and D. marginatus ticks have been described on only a few occasions because the latter require warm and dry habitats (2,10,11). Climate change and structural landscape changes have been discussed as reasons for the creation of new tick habitats (12). Brown coal surface-mining sites of the former German Democratic Republic undergo extensive renaturation, thus providing new biotopes for many plant and animal species, including ticks.

In Germany, R. slovaca was first described in 1977 (2) and again recently (11). Even though the sample size in the present study was small, comparable prevalence rates were detected (13%). R. raoultii was first detected in Russia and has recently been described as a new species (13). The average infection rate of R. raoultii in this study was 56.7% and, in the renatured brown coal surface-mining sites, ≈80.1%. The latter rate is high in comparison with results of previous studies (11,12). R. helvetica prevalence in I. ricinus ticks was similar to results of other studies in Germany (8).

Our results confirm the presence of these rickettsial pathogens in Germany. In addition, we identified previously unknown areas where Rickettsia spp. are endemic. This finding is of major concern to public health: both R. slovaca and R. raoultii can cause TIBOLA, even though R. slovaca is considered to be more pathogenic (7,14). A case of TIBOLA caused by R. slovaca was identified in a human patient in an R. slovaca–endemic area in western Germany (11). The pathogenicity of R. helvetica has not been fully clarified, but serologic evidence shows human infections in France (3), and DNA of R. helvetica was recently identified in a patient in Sweden who had meningitis (15).

Renaturation of industrial sites specifically provides new areas for human recreation and, simultaneously, new habitats for many plant and animal species. Previously nonexistent opportunities for intensive contact between vector ticks and humans are now available. Thus, human-made habitats may lead to increased emerging diseases, especially in tick-borne rickettsioses, because renaturation areas may form favorable biotopes for enhanced human–vector interactions.