Spotted Fever Group Rickettsiae in Ticks, Germany

To explore increased risk for human Rickettsia spp. infection in Germany, we investigated recreational areas and renatured brown coal surface-mining sites (also used for recreation) for the presence of spotted fever group rickettsiae in ticks. R. raoultii (56.7%), R. slovaca (13.3%), and R. helvetica (>13.4%) were detected in the respective tick species.

To explore increased risk for human Rickettsia spp. infection in Germany, we investigated recreational areas and renatured brown coal surface-mining sites (also used for recreation) for the presence of spotted fever group rickettsiae in ticks. R. raoultii (56.7%), R. slovaca (13.3%), and R. helvetica (>13.4%) were detected in the respective tick species.
R ickettsia species of the spotted fever group are causing emerging infectious diseases (1). Since 1977, Rickettsia slovaca, found in Dermacentor marginatus ticks, was the only known Rickettsia sp. in Germany until 2002, when the following were identifi ed: R. monacensis and R. helvetica in Ixodes ricinus ticks, Rickettsia sp. RpA4 (now R. raoultii) in D. reticulatus ticks, R. felis in Ctenocephalides felis cat fl eas, and R. massiliae in I. ricinus ticks (1,2). All of these species cause tick-borne rickettsioses in humans, including tick-borne lymphadenopathy (TIBOLA) (3)(4)(5)(6)(7). The aim of this study was to explore the interface between the vector tick and humans by investigating the presence of Rickettsia spp. in ticks at highly frequented recreational areas and renatured brown coal surface-mining sites that also are used for leisure.

The Study
Questing ticks were collected from vegetation by fl agging 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 fl ooding of the pit holes during past decades created a highly valuable recreational area with artifi cial lakes and surrounding meadows and forest (www.leipzigerneuseenland.de). Here, sampling was carried out around Lake Cospuden (436 ha, fully fl ooded 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, identifi ed to species level, and homogenized (ticks in ethanol: 2 mL Eppendorf tubes [Eppendorf, Hamburg, Germany] with one 5-mm steel bead, 80 μL phosphatebuffered 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 proteincoding genes ompA and ompB (9). New primers were developed for the fi rst part of the ompA gene: OmpA-MMX1for 5′-ACAAGCTGGAGGAAGCCTAGC-3′; OmpA-MMX1-rev 5′-TCTCCCGCTCCTTTGAAAACTAT-3′.

Conclusions
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 fi rst 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 fi rst 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 confi rm the presence of these rickettsial pathogens in Germany. In addition, we identifi ed previously unknown areas where Rickettsia spp. are endemic. This fi nding 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 identifi ed in a human patient in an R. slovaca-endemic area in western Germany (11). The pathogenicity of R. helvetica has not been fully clarifi ed, but serologic evidence shows human infections in  France (3), and DNA of R. helvetica was recently identifi ed in a patient in Sweden who had meningitis (15).
Renaturation of industrial sites specifi cally 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, humanmade habitats may lead to increased emerging diseases, especially in tick-borne rickettsioses, because renaturation areas may form favorable biotopes for enhanced humanvector interactions.