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Silaghi, Cornelia (2008): Prevalence and genetic analysis of Anaplasma phagocytophilum and Spotted Fever Group rickettsiae in the tick Ixodes ricinus in urban and periurban sites in southern Germany. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

In recent years, Anaplasma phagocytophilum and Rickettsia spp. have been detected in Ixodes ricinus in Germany and a focal distribution has been suggested for A. phagocytophilum. In the present study the prevalence of A. phagocytophilum and spotted fever group (SFG) rickettsiae was investigated in I. ricinus. DNA-extracts were taken from 2,862 unfed I. ricinus ticks (adults and nymphs) from eight sites in Munich, sampled over a five-month period. Single samples from three comparative sites outside of Munich were also included. A real-time PCR targeting the msp2 gene of A. phagocytophilum was used for screening and a nested PCR targeting the 16S rRNA gene for sequencing of 30% of positives. Screening for Rickettsia spp. was performed with a PCR targeting the citrate synthase gene (gltA), followed by PCRs detecting the ompA gene for all gltA positives, and the ompB and 16S rRNA genes for clarifying results of some samples. The overall prevalence was 2.90% (95% CI 2.27 to 3.48%) for A. phagocytophilum and 5.28% (95% CI 4.31 to 6.17%) for SFG rickettsiae. Only 0.31% of the ticks investigated were coinfected. Statistical analysis revealed that prevalence of A. phagocytophilum in ticks from city parks in Munich was significantly higher than in ticks from natural forest, whereas the prevalence of Rickettsia spp. was the opposite. For both, the prevalence in adults was significantly higher than in nymphs. Although wide ranges of prevalence were observed monthly, the variations were not significant along the observational period. Sequence analysis of 16S rRNA PCR products (n=31) revealed 100% homology to Ehrlichia sp. “Frankonia 2”, only one differed in one nucleotide position. All differed in one nucleotide position from the HGA agent detected in human patients. All rickettsial PCR products were also sequenced. All gltA sequences of R. helvetica (n=138) were 100% identical to each other and differed in one nucleotide position from the prototype sequence. Two different R. monacensis strains (n=13) were detected, which differed in up to 4 nucleotide positions in gltA, ompA and ompB. Further rickettsial strains (n=3) most probably belonging to rickettsial endosymbionts were also found. These results show, by molecular methods, a wide distribution of A. phagocytophilum and SFG rickettsiae in I. ricinus ticks in Southern Germany. SFG rickettsiae which are thought to be involved in human disease (R. helvetica and R. monacensis) had a significantly higher prevalence in natural forest areas. Prevalence of A. phagocytophilum was significantly higher in city parks; the genetic strain has not yet been associated with human infection.