INTRODUCTION

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Microorganisms of the tribe Ehrlichieae are obligate intracellular bacteria that reside within cytoplasmic vacuoles (phagosomes) of monocytes, granulocytes, or platelets of various mammalian species. Presently, the genus Ehrlichia can be divided into three distinct clusters based on the nucleotide sequence homology of the 16S rRNA genes (1, 11). These genogroups carry the name of the group member that was first characterized: Ehrlichia canis group (E. canis, E. chaffeensis, E. muris, and E. ewingii), Ehrlichia phagocytophila group (E. phagocytophila, E. equi, human granulocytic ehrlichiosis [HGE] agent, and E. platys), and Ehrlichia sennetsu group (E. sennetsu and E. risticii). E. canis was originally described as a pathogen in 1935, when an outbreak was observed among experimental Algerian dogs (10). In 1953, E. sennetsu was the first ehrlichial agent shown to be pathogenic for humans (20). To date, however, the mononucleosis-like Sennetsu fever rarely occurs outside Japan. More recently, two new ehrlichial organisms, which elicit illnesses with fever, leukopenia, and thrombocytopenia in humans, were found in the United States. E. chaffeensis, the cause of human monocytic ehrlichiosis, was discovered in 1986 (1, 18), and the HGE agent was first reported in 1994 (7).

Both monocytic and granulocytic ehrlichiosis appear to be transmitted by ticks. Recent serological and PCR studies suggest that granulocytic ehrlichiosis and HGE infection also exist outside the United States in some European countries where Ixodes ricinus ticks, Lyme borreliosis, and tick-borne encephalitis are endemic (3, 6, 8, 12, 14, 23, 24, 26, 27, 29-32). To date, however, only four cases of HGE have been diagnosed in Europe, all of which occurred in patients from Slovenia (17, 25). In order to provide a firm basis for future estimates of the likelihood of HGE infections in central Europe, we analyzed the rate of Ehrlichia infections in I. ricinus ticks from southern Germany. Our data not only demonstrate a significant prevalence of granulocytic ehrlichiae in the German tick population but also provide evidence for a further heterogeneity of the E. phagocytophila 16S rRNA genogroup.

	MATERIALS AND METHODS

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Tick collection. A total of 287 morphologically adult ticks of the species I. ricinus were collected in two regions of southern Germany, Frankonia and southwest Baden-Württemberg, during a 5-month period in the spring and summer of 1998. Of these, 12 ticks were removed from two deer. The other ticks were collected from the fur or skin of four domestic dogs (two each living in Frankonia and southwest Baden-Württemberg), because this method is much more efficient than flagging and also selects for adult ticks that were actually attracted to mammals. The observed prevalences of granulocytic ehrlichiae and Borrelia burgdorferi infection in the collected tick population were not due to an inapparent ehrlichiosis or borreliosis of any of the dogs, because the vast majority of nonfed and fed ticks removed from the same dogs before or after obtaining the positive ticks were clearly negative in the same nested PCRs.

DNA purification. The ticks were examined and classified by morphology and then frozen at 70°C until further processing was done. The DNA was extracted with a QIAamp tissue kit (Qiagen, Hilden, Germany) with some modifications. Each individual tick was placed in a 1.5-ml microcentrifuge tube and mechanically homogenized with a micropestle. After addition of 180 µl of ATL lysis buffer and 20 µl of proteinase K stock solution (20 mg/ml), the samples were incubated overnight at 55°C. If the tick samples were larger than 100 mg, the amounts of ATL buffer and proteinase K stock solution were doubled. After addition of AL buffer and ethanol according to the manufacturer's instructions, the samples of heavily engorged ticks were centrifuged to pellet the residual insoluble material, and the supernatant was applied to the QIAamp spin column. The QIAamp tissue extraction protocol was then followed as described by the manufacturer except that the DNA was eluted twice with 100 µl of AE buffer. Purified DNA was stored at 20°C until used for PCR analysis.

PCR amplification of tick mitochondrial 16S rDNA. The quality of the prepared DNA was first assessed with primers (16S+1 and 16S2) specific for tick mitochondrial 16S ribosomal DNA (rDNA) in a single-round PCR which yields a 325-bp product (5). A 1.5-µl portion of extracted genomic tick DNA was amplified in a 50-µl reaction mixture containing 1× PCR buffer (Pharmacia Biotech, Freiburg, Germany) (50 mM KCl, 1.5 mM MgCl₂, 10 mM Tris-HCl [pH 9.0], with the addition of 0.1% Triton X-100), a 0.2 mM concentration of each deoxyribonucleoside triphosphate (dNTP), a 1 µM concentration of each primer, and 0.2 µl of Taq polymerase (5,000 U/ml; Pharmacia Biotech). Amplification was performed in a Perkin-Elmer 480 thermal cycler with a three-step program as follows: 3 min of denaturation at 94°C, 1 min of annealing at 54°C, and 1 min of extension at 72°C; followed by 36 cycles of 1 min at 92°C, 1 min at 54°C, and 1 min at 72°C; and a final extension at 72°C for 7 min. The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light. Mitochondrial 16S rDNA was successfully amplified for all investigated tick samples (data not shown).

Borrelia- and ehrlichia-specific nested PCRs. Primary PCR amplification was performed with a pair of universal (degenerate) primers (POmod and PC3mod) which recognize conserved sequences of the 5' and 3' ends, respectively, of the 16S rRNA genes of all eubacteria, and produce a 756-bp (E. risticii) to 762-bp (HGE) fragment upon amplification of ehrlichial templates (7, 33). The primary PCR mixtures contained 1 µl of purified template DNA, 1× PCR buffer (Perkin-Elmer, Weiterstadt, Germany) (50 mM KCl, 10 mM Tris-HCl [pH 8.3], with the addition of 1% Tween 20), 1.5 mM MgCl₂, a 0.2 mM concentration of each dNTP, a 0.4 µM concentration of each primer, and 0.2 µl (= 1 U) of Taq polymerase (5,000 U/ml; Pharmacia). The reaction mixtures were overlaid with mineral oil and incubated for 4 min at 95°C; thermally cycled 32 times at 95°C for 1 min, 42°C for 1 min, and 72°C for 2 min; and then incubated at 72°C for 7 min to allow complete strand extension. Reaction products were kept at 4 or 20°C for later use in nested PCRs.

Positive and negative controls. For the detection of granulocytic ehrlichiae in (engorged) ticks, horse blood infected with the Rosa isolate of the HGE agent (kindly provided by Eva Olsson, National Veterinary Institute, Stockholm, Sweden) (24) or tick cell cultures infected with a canine HGE isolate (kindly provided by Uli Munderloh, University of Minnesota, St. Paul) (21) was used as a positive control for the preparation of DNA and for the subsequent nested PCR. DNA from E. canis, E. equi, E. chaffeensis, or the HGE agent (kindly provided by Stephen Dumler, John Hopkins University, Baltimore, Md.) was used as a positive control for species-specific PCR.

Cloning of PCR products and DNA sequencing. The products of positive nested PCR runs were ligated into the plasmid vector pGEM-T by using the pGEM-T Easy Vector System kit (Promega, Mannheim, Germany). The ligation products were transformed into Escherichia coli ElectroMAX DH10B cells (Gibco BRL Life Technologies GmbH, Karlsruhe, Germany). Transformants containing inserted PCR products were selected by blue-white color screening on Luria-Bertani agarose with IPTG (isopropyl--D-thiogalactopyranoside), X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside), and ampicillin by standard protocols. Plasmid DNA was purified from overnight cultures by using the Qiagen plasmid kit (Qiagen GmbH). The vector inserts were sequenced by using fluorescence-labeled dideoxynucleotide technology with an Li-COR model 4200 automated DNA sequencer (MWG-Biotech, Ebersberg, Germany). All reported sequences are based on sequencing of both DNA strands and were confirmed with at least one additional independently obtained primary PCR product, which was sequenced either directly or after cloning into pGEM-T Easy as described above.

Nucleotide sequence accession numbers. The DNA sequences reported in this study have been submitted to GenBank, where they received the following accession numbers: AF136712 for E. phagocytophila genogroup strain Frankonia II, AF136713 for E. phagocytophila genogroup strain Frankonia I, and AF136714 for E. phagocytophila genogroup strain Baden.

	RESULTS

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Prevalence of ehrlichia-positive ticks. A total of 287 adult I. ricinus ticks from southern Germany were examined for the presence of DNAs of the E. phagocytophila genogroup, E. chaffeensis, E. canis, and B. burgdorferi by nested PCR. The distribution of 275 ticks collected from dogs, according to origin, sex, engorgement, and status of infection with B. burgdorferi and/or the E. phagocytophila genogroup, is given in Table 1. Six (2.2%) and 65 (21.8%) of the 275 ticks were positive for the E. phagocytophila genogroup and B. burgdorferi, respectively. Of 83 ticks from southwest Baden-Württemberg, one (1.2%) and 13 (15.7%) were positive for the E. phagocytophila genogroup and B. burgdorferi, respectively. Among the 192 ticks from Frankonia, 5 (2.6%) and 52 (27.1%) were found to carry DNA of the E. phagocytophila genogroup or B. burgdorferi, respectively. Two ticks, an unfed female and a male tick from Frankonia, were coinfected with both bacteria. Of 12 engorged female ticks collected from two deer, 8 (66.7%) and 1 (8.3%) were positive for the E. phagocytophila genogroup and B. burgdorferi, respectively. 16S rDNA specific for E. canis or E. chaffeensis was not detected in the 287 ticks analyzed by nested PCR.

Sequence analysis. The 546-bp nucleotide sequences obtained from the Ehrlichia PCR amplicons were all identified as part of the 16S rRNA gene of the E. phagocytophila genogroup and were highly homologous (99.8 to 99.9%), but not identical, to the published HGE sequence (7). Three E. phagocytophila 16S rRNA genogroup variants were detected (Table 2): variant 1 (Baden) was found in the only positive tick from dogs in Baden-Württemberg (with a G instead of an A at position 76), variant 2 (Frankonia I) was found in the eight positive ticks from deer in Frankonia (with a G instead of an A at position 76 and an A instead of a G at position 84), and variant 3 (Frankonia II) was found in the five positive ticks from dogs in Frankonia (with an A instead of a G at position 376).

	DISCUSSION

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This study was undertaken to provide data on the prevalence of Ehrlichia infections in I. ricinus ticks in two areas of southern Germany where B. burgdorferi and the tick-borne encephalitis virus are known to be endemic. The reported prevalences of infection of I. ricinus ticks with bacteria of the E. phagocytophila genogroup in Europe were 0.8% in Switzerland (E. phagocytophila in free-living, adult I. ricinus ticks) (28), 3.2% in Slovenia (HGE in free-living, adult I. ricinus ticks) (26), 3.1 and 9.2% at the east and west coasts of Sweden, respectively (HGE in I. ricinus nymphs) (32), and 24.4% in a region of Italy (E. phagocytophila in I. ricinus nymphs) (8). In this study the overall prevalence of the E. phagocytophila genogroup in ticks (2.2%) was found to be about 10 times lower than the prevalence of B. burgdorferi (21.8%); this value, however, is still 10-fold higher than the rate of infection of I. ricinus with the tick-borne encephalitis virus in southern Germany (Baden-Württemberg) (ca. 0.2%) (13). These numbers imply that transmission of granulocytic ehrlichiae by ticks to mammals, including humans, may occur quite frequently in southern Germany. This hypothesis is supported by the results of Fingerle et al., who found that 14% of serum samples from forestry workers in southern Germany reacted positively in an immunofluorescence assay with HGE-infected HL-60 cells (14). Of the total of 14 ehrlichia-positive ticks (from dogs or deer) in our study, two harbored both granulocytic ehrlichiae and B. burgdorferi. Therefore, humans could become coinfected through the bite of a single tick. Simultaneous infection of humans with both pathogens has already been reported (22) and may lead to unusual clinical manifestations.

The high prevalence (66.7%) of granulocytic ehrlichiae in the female deer ticks might result from an infection of one or both of the two hosts with this pathogen. Unfortunately, deer-derived blood was not available to analyze this possibility. On the other hand, three of the four ehrlichia-negative ticks were engorged with amounts of blood similar to those for the ehrlichia-positive ticks (tick weight, 18 to 38 mg), and four of the eight ehrlichia-positive ticks showed no macroscopic evidence of blood feeding (tick weight, <14 mg), although they were already attached to deer skin. Thus, there was no direct correlation between the engorgement status of the 12 ticks from deer and the presence of E. phagocytophila genogroup 16S rDNA. Similar observations have been made in the past with a tick population collected from cattle with ehrlichiosis (28).

In this study, we detected three sequence variants (Frankonia I, Frankonia II, and Baden) of the E. phagocytophila 16S rRNA genogroup (Table 2). The nucleotide sequence of the 546-bp portion of the 16S rRNA gene of the variant Baden was described earlier for Ehrlichia type II in Swedish ticks (32). Likewise, the sequence of the variant Frankonia I from ticks of Frankonian deer was identical to the sequence of a granulocytic Ehrlichia variant previously found in the blood from Maryland white-tailed deer (19), in Wisconsin white-tailed deer (4), in Rhode Island ticks (19), and in Swedish ticks (Swedish Ehrlichia type III) (32) but was clearly distinct (96% homology) from that of another ehrlichia-like agent isolated from deer in Oklahoma and Georgia (9). In contrast, to our knowledge the sequence variant Frankonia II, found in ticks from dogs living in Frankonia, has not been reported before. Together, these data suggest that the granulocytic Ehrlichia variant of white-tailed deer originally discovered in Maryland and Wisconsin might also exist in Europe and that within different regions of southern Germany at least three different E. phagocytophila 16S rRNA genogroup variants are prevalent. Further sequencing of both highly and less conserved genes will be required to determine whether these and the previously reported 16S rRNA sequence variations are just clones differing at a single hot spot, reflect strains of the same species, or represent separate granulocytic Ehrlichia species. Furthermore, we do not yet know whether any of these E. phagocytophila 16S rRNA genogroup variants can infect humans.