questing ticks were collected from vegetation by flagging in 3 regions in germany ( 9 sites total ) in march 
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 .
tick sampling sites in germany during 2008 and 2009 ( green shading ) : saarland , sites a 
g , in lake cospuden renatured brown coal surface - mining area ( blue shading ) ; site h , renatured waste disposal site ( sampled april and september / october 2009 ) ; and site i , game park ( sampled in april 2009 ) .
ticks were stored either in 70% ethanol or kept at 4c , identified to species level , and homogenized ( ticks in ethanol : 2 ml eppendorf tubes [ eppendorf , hamburg , germany ] with one 5-mm steel bead , 80 l 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 l 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 . * na , not available . 
in the regions saarland and saxony , some of the nymphs were placed in pools of up to 5 specimens ; range of positive ticks indicates the possible minimum and maximum number of ticks positive for rickettsia spp . in a pool .
from each site , 16 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 .
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) .
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 
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 .
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 .
 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 . 
The Study
Conclusions