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We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

Human granulocytic Ehrlichiosis (HGE) is a newly emerging acute febrile illness which is likely transmitted by ticks of the Ixodes ricinus/I. persulcatus complex. First seroepidemiological surveys on the prevalence of HGE antibodies, detection of DNA of granulocytotropic Ehrlichiae in I. ricinus and one case of HGE from Slovenia confirmed by serology and PCR (polymerase chain reaction) suggest that HGE might exist all over Europe. The purpose of the present study was a) to determine the prevalence of antibodies against the HGE agent in sera collected from persons at high risk for exposure to I. ricinus with that of a control population and b) to determine the prevalence of granulocytic Ehrlichiae in I. ricinus ticks from Southern Germany. We studied sera from 150 forestry workers and 105 patients with an established diagnosis of Lyme disease as tick-exposed populations. Sera from 103 healthy blood donors without a history of known tick bites served as controls. A significantly higher prevalence of HGE antibodies (P < or = 0.01) was present among patients with Lyme borreliosis (12 of 105 were positive; 11.4%) and forestry workers (21 of 150 were positive; 14%) compared to blood donors (2 of 103 were positive; 1.9%). Furthermore, 510 adult and nymphal I. ricinus were investigated by PCR for the presence of granulocytic Ehrlichiae with primers specific for the E. phagocytophila group. In eight (1.6%) of the investigated ticks the expected amplification product was detectable, indicating a low prevalence of infected ticks especially when compared with B. burgdorferi. The presented data strongly suggests that the HGE agent or a closely related organism exists in Southern Germany and therefore HGE should be considered in the differential diagnosis of febrile illnesses. However, final evidence can be provided only after isolation of the organism from patients.

To date, human granulocytic ehrlichiosis (HGE), the causative agent of which is likely transmitted by ticks in the Ixodes ricinus-Ixodes persulcatus complex, has not been diagnosed with certainty in patients outside the United States. The presence of a closely related vector tick, I. ricinus, as well as the occurrence of similar Ehrlichia spp. of veterinary importance, suggests that this disease is likely to be present in Europe. The aim of the present study was to compare the prevalence of antibodies against the HGE agent in sera collected from patients in groups at high risk for exposure to I. ricinus with that of a control population. Risk groups consisted of 150 forestry workers and 105 patients with an established diagnosis of Lyme disease. The control group was 103 healthy blood donors without a history of tick bites. We used a patient isolate of the HGE agent from Minnesota (J. L. Goodman, C. Nelson, B. Vitale, J. E. Madigan, J. S. Dumler, T. J. Kurtti, and U. G. Munderloh, N. Engl. J. Med. 334:209-215, 1996) propagated in HL60 cells as the source of antigen for a specific immunofluorescence assay (IFA). Elevated IFA titers (> or = 1:80) were present in 21 of 150 (14%) serum samples from forestry workers and in 12 of 105 (11.4%) serum samples from Lyme disease patients, but in only 2 of 103 (1.9%) serum samples from blood donors (P < or = 0.01 for either of the at-risk groups versus blood donors). The results of this study suggest that the HGE agent or a closely related organism exists in southern Germany and that seroconversion to it is common among groups exposed to Ixodes ticks. Final proof that HGE occurs in Germany will require the isolation of the causative agent from patients. HGE should be considered in the differential diagnosis of febrile illnesses in individuals exposed to Ixodes ticks in Europe as well as in North America.