Most recently, several human cases severely affected by an infection with “Ca. Neoehrlichia mikurensis” have been described in Europe (5, 12, 23, 24). As are all other members of the family Anaplasmataceae, this pathogen is an obligate intracellular bacterium. It appears to display an endothelial cell tropism (8), and symptoms observed in some of these patients, such as aneurysm, thromboembolic complications, subcutaneous hemorrhages, and erythematous rashes (5, 12, 23, 24), may result from this affinity. Of the six patients infected by “Ca. Neoehrlichia mikurensis” reported hitherto, five were undergoing immunosuppressive therapy (12, 23, 24). In addition, a dog affected by “Ca. Neoehrlichia mikurensis” postoperatively suffered from chronic neutropenia (4). The contact to a tick infected with “Ca. Neoehrlichia mikurensis” could have occurred during immunosuppressive treatment or latent bacteria acquired from a previous tick contact could have been activated by immunosuppressive treatment or postoperative trauma.

To detect DNA of Borrelia sp., “Ca. Neoehrlichia mikurensis” and A. phagocytophilum in a random subsample of these questing ticks, as well as in ticks removed from patients, we amplified a fragment of the 16S rRNA gene for each of the first two pathogens by nested PCR and a fragment of the p44 gene for A. phagocytophilum by conventional PCR (for primers and references see Table 1). DNA was obtained by using a QIAamp DNA minikit (Qiagen, Hilden, Germany) and stored at −30°C. For the amplification of DNA of “Ca. Neoehrlichia mikurensis” and A. phagocytophilum, aliquots of DNA suspensions (1 μl) were diluted to 25 μl by using 200 μM concentrations of each deoxynucleoside triphosphate, 1 U of Taq polymerase (Qiagen), and 5 pmol of the outer primer pair EC9 and EC12A for “Ca. Neoehrlichia mikurensis” or 10 pmol of the primer pair MSP3F and MSP3R for A. phagocytophilum and Coral-PCR-buffer supplied with the Taq polymerase. The mixture was placed in a thermocycler (PTC 200; MJ Research Biozym, Diagnostic, Hess. Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 40 cycles of 20 s denaturation at 94°C, 1 min for the annealing reaction at 55°C with a 20-s extension at 72°C, and a final extension for 2 min at 72°C. For “Ca. Neoehrlichia mikurensis,” 0.2 μl of the amplification product was transferred to a fresh tube containing 24.8 μl of the reaction mixture described above, except that 5 pmol of the inner primer pair IS58-62f and IS58-594r was used. This mixture was subjected to the same amplification conditions as described above. PCR products were detected by electrophoresis in a 1.5% agarose gel stained with ethidium bromide. Any PCR products were sequenced. To detect and identify spirochetal genospecies, we amplified spirochetal DNA as described previously (15). Each PCR amplification product was purified by using a QIAquick-Spin PCR column (Qiagen) according to the manufacturer's instructions. Amplified DNA fragments were directly sequenced in both directions using the inner primers by the dideoxynucleotide chain termination method with a Licor DNA4200 sequencer (Licor Biosciences, Bad Homburg, Germany). Each resulting sequence was compared to sequences of the same gene fragment from various spirochete genospecies. The following sequences (identified by the accession numbers under which they were deposited) were used for comparison: X85196 and X85203 for B. burgdorferi sensu stricto; X85190, X85192, and X85194 for B. afzelii; X85193, X85199, and M64311 for B. garinii; X98228 and X98229 for B. lusitaniae; X98232 and X98233 for B. valaisiana; AY147008 for B. spielmanii; and AY253149 for B. miyamotoi. A complete match (no more than two nucleotide changes) was required for identification. A nested PCR amplifying a fragment of the ospA gene served to differentiate B. bavariensis (cand. sp. nov.) from B. garinii (16). To examine variability, a fragment of the groESL gene was amplified in any sample harboring “Ca. Neoehrlichia mikurensis” DNA and sequenced (20). The DNA was extracted, reaction vials were prepared for amplification, templates were added, and products were electrophoresed in separate rooms. As an additional precaution, the reaction mixtures were prepared in a designated PCR workstation (Labcaire Systems, North Somerset, United Kingdom), and templates were added to the mixtures in a second PCR workstation. Benches and equipment were wiped down with a DNA decontamination solution (DNAerase; MP Biomedicals, Eschwege, Germany) after each use. In each sixth reaction mix, water was added instead of extracted DNA to serve as negative control.

We examined whether any of 111 nymphal or adult I. ricinus ticks removed from 35 persons harbored “Ca. Neoehrlichia mikurensis,” A. phagocytophilum, or spirochetes. About a fifth of patient-associated nymphs (19.1%) and a third of adults (31.8%) were infected by spirochetes (Table 3). Patient-derived nymphs appeared to be twice as likely to be infected by “Ca. Neoehrlichia mikurensis” as adults (9% versus 4.5%). No such tick was infected by A. phagocytophilum. B. afzelii was the most frequently detected genospecies, infecting more than half (54.2%) of all spirochete-infected ticks. B. burgdorferi sensu stricto and B. garinii were detected in 4.5 and 3.6% of the ticks, respectively, whereas the nonpathogenic spirochetes B. valaisiana, B. lusitaniae, and B. miyamotoi infected fewer than 1% of ticks removed from people. Three nymphs harbored B. afzelii and “Ca. Neoehrlichia mikurensis” simultaneously. None of the people from whom these ticks were removed displayed symptoms typical for any of these pathogens.

On average, every twelfth encounter with an I. ricinus tick may bear the risk of acquiring “Ca. Neoehrlichia mikurensis” (8.1% infection rate). In ticks removed from people, this pathogen appeared to be somewhat more frequent than in questing ticks. The difference may result from a bias, because three quarters of the patient-derived ticks were acquired in a German region where we determined that 18% of adult ticks harbored “Ca. Neoehrlichia mikurensis,” or it may result from a higher infection rate in nymphs, constituting most of the patient-removed ticks, than in adults. In comparison, <3% of ticks removed from asymptomatic patients in Northern Italy were infected with “Ca. Ehrlichia walkeri” (2). The 16S rRNA fragment amplified in our patient-derived ticks differed only in one base pair from that of “Ca. Ehrlichia walkeri” and may indicate closely related, if not identical, pathogens. None of the seven different persons to whom at least one tick infected with “Ca. Neoehrlichia mikurensis” had attached reported symptoms related to an infection with this pathogen. We assume that its transmission by the feeding tick is not immediate. Similarly, Lyme disease spirochetes, as well as A. phagocytophilum, appear to not be transmitted before the second day of feeding (6, 13). Alternatively, symptoms related to “Ca. Neoehrlichia mikurensis” infection may only become apparent in immunocompromised patients (12, 23, 24).