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

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.