Rickettsia spp.

in Ixodes ricinus Ticks

in Bavaria, Germany
R. WÖLFEL,a R. TERZIOGLU,a J. KIESSLING,b S. WILHELM,b,c
S. ESSBAUER,a M. PFEFFER,a AND G. DOBLERa
a Department of Virology and Rickettsiology, Bundeswehr Institute of
Microbiology, Neuherbergstr. 11, 80937 Munich, Germany
b Institute
of Medical Microbiology, Epidemic and Infectious Diseases,
LMU Munich, Germany
c Institute
of Animal Hygiene and Veterinary Public Health,
University of Leipzig, Leipzig, Germany

ABSTRACT: This study aims to provide information on the occurrence of

spotted fever rickettsiae in Ixodes ricinus ticks in southern Germany. A
total of 2,141 I. ricinus ticks was collected in Bavaria. Pools of 5-10 ticks
were studied by a PCR targeting the rickettsial citrate synthase gene gltA.
The average prevalence rate was 12% (257 of 2,141). Sequencing data
exclusively identified Rickettsia helvetica DNA. Results and other data
demonstrate the possible role of R. helvetica in I. ricinus as a source of
human infections in southern Germany.

KEYWORDS: Ixodes ricinus; Rickettsia helvetica; Bavaria; Germany

INTRODUCTION

The most prevalent and widely distributed tick species in Germany is the
sheep tick, Ixodes (I.) ricinus. In many European countries these ticks were
found to be infected with spotted fever group (SFG) rickettsiae.1 In Germany,
Rickettsia (R.) helvetica has thus far only been identified in ticks from Baden-
Wuerttemberg.2 This study was set up to provide information on the occurrence
of SFG rickettsiae in I. ricinus ticks in Bavaria, southern Germany.

MATERIALS AND METHODS

A total of 2,141 I. ricinus ticks (151 female, 174 male, 1204 nymph, 612 lar-
vae) were collected in six different areas of Bavaria (administrative districts of
Address for correspondence: Roman Wölfel, Department of Virology and Rickettsiology, Bun-
deswehr Institute of Microbiology, Neuherbergstrasse 11, 80937 Munich, Germany. Fax: +49-89-3168-
3292.
e-mail: RomanWoelfel@Bundeswehr.org

Ann. N.Y. Acad. Sci. 1078: 509–511 (2006). 

C 2006 New York Academy of Sciences.

doi: 10.1196/annals.1374.133

509
510 ANNALS NEW YORK ACADEMY OF SCIENCES

Erlangen, Freyung-Grafenau, Hengersberg, Muehldorf, Munich, Traunstein)

by both flagging and from trapped rodents (Clethrionomys glareolus, Apode-
mus spp.). For nucleic acid (NA) extraction all larvae and nymphs were pooled
together to a pool size of 10 and a few of the adults were pooled to a pool
size of 4 (see TABLE 1). All samples were stored at –80◦ C until PCR was per-
formed. Ticks were homogenized in 1 mL Eagle’s minimal essential medium
using a Fast Prep device as described by the supplier (Qbiogene, Heidelberg,
Germany). NAs were extracted using QIAamp Viral RNA Mini Kit (Qiagen
GmbH, Hilden, Germany) according to the manufacturer’s protocol. Samples
were screened for the presence of rickettsia DNA by PCR amplification of a
fragment of the citrate synthase-encoding gene (gltA).3 A plasmid containing a
gltA-fragment of R. rickettsii was used as positive control in each test. Purified
amplicons were submitted to a commercial subcontractor for automated dye-
terminator cycle sequencing. Sequences were analyzed by BLAST sequencing
analysis in the GenBank database.

RESULTS

All 2,141 ticks (254 pooled, 57 single samples) were examined for Rick-
ettsia spp. Using the gltA primers, PCR products of rickettsial DNA of 340-bp
size were detected in 147 of the 311 studied samples. The positive samples
were not clustered to any distinct geographical area of the six regions investi-
gated. Sequencing of the positive gltA PCR products identified in all of these
R. helvetica showed 100% identity. The distribution of positive samples is
summarized in TABLE 1.

DISCUSSION

I. ricinus ticks play an important role as the vector of pathogens of medical

and veterinary importance. Our results support the occurrence of R. helvetica

TABLE 1. Distribution of ticks, tick samples, and pooled samples and distribution of var-
ious developing stages in different samples
Parameters Adult males Adult females Nymphs Larvae Total
No. of ticks 174 151 1,204 612 2,141
Pools∗ single Pools∗ single Pools∗ Pools∗
No. of samples 35 34 32 23 125 62 311
No. of positive samples 18 4 25 3 63 34 147
No. of ticks in 72 4 100 3 630 340 1,149
positive samples
% Positive ticks 12.6–43.7 18.5–68.2 5.2–52.3 5.5–55.5 6.9–53.6
∗ Pool sizes: for adult ticks = 4; for nymphs and larvae = 10.
WÖLFEL et al.: RICKETTSIA IN I. RICINUS TICKS 511

in I. ricinus in Germany2 by expanding its geographical range to Bavaria.

With the assumption that only one tick in each pooled sample was positive for
rickettsial DNA, the minimum infection rate would be 6.9% (147 of 2,141). If,
on the other hand, all ticks in the pooled sample were positive, the maximum
prevalence would be 53.6% (1,149 of 2,141). We assume that the frequency
of positive ticks in the pooled samples can be expected to reflect that of the
entire selection of the tick material. Therefore, it is probable to assume that the
average prevalence rate corresponds to 12% (257 of 2,141). Data from other
parts of Germany and Switzerland show similar infection rates.2,4 These results
imply that transmission of R. helvetica to humans may occur quite frequently.
The presence of R. helvetica in ticks in Bavaria has a diagnostic importance
because this rickettsia species has been recognized as a human pathogen.1
However, to date, no clinical cases of human R. helvetica infections have been
reported in Germany. Nevertheless, further studies are needed since contact
with I. ricinus ticks during outdoor activities is common. Thus more attention
should be paid to the incidence of tick-borne rickettsioses.

ACKNOWLEDGMENTS

We thank Kathrin Hartelt (Baden-Wuerttemberg State Health Office) for

providing the DNA of Rickettsia spp. We acknowledge the skillful assistance of
Heike Prabel and Mirko Köhler in field collection of ticks. The views expressed
in this article are those of the authors and do not reflect the official policy or
position of the German Department of Defense, or the German government.

REFERENCES

1. PAROLA, P. 2004. Tick-borne rickettsial diseases: emerging risks in Europe. Comp.

Immunol. Microbiol. Infect. Dis. 27: 297–304.
2. HARTELT, K., R. OEHME, H. FRANK, et al. 2004. Pathogens and symbionts in ticks:
prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rick-
ettsia sp., and Babesia sp. in Southern Germany. Int. J. Med. Microbiol. 293 (Suppl
37): 86–92.
3. ROUX, V., E. RYDKINA, M. EREMEEVA & D. RAOULT. 1997. Citrate synthase gene
comparison, a new tool for phylogenetic analysis, and its application for the rick-
ettsiae. Int. J. Syst. Bacteriol. 47: 252–261.
4. BURGDORFER, W., A. AESCHLIMANN, O. PETER, et al. 1979. Ixodes ricinus: vector of
a hitherto undescribed spotted fever group agent in Switzerland. Acta Trop. 36:
357–367.