Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in Dutch Ixodes ricinus ticks

A sensitive and specific PCR hybridization assay was developed for the simu ltaneous detection and identification of Ehrlichia and Borrelia burgdorferi sensu late. In separate assays the 16S rRNA gene of Ehrlichia species and the 23S-5S rRNA spacer region of B, burgdorferi sensu late were amplified a nd labeled by PCR, These PCR products were used in a reverse line blot hybr idization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. Hybridization of the samples with the oligonucl eotide probes on this membrane enabled the simultaneous detection and ident ification of Ehrlichia, B. burgdorferi, and Bartonella species in 40 differ ent samples. The application of the assay to DNA extracts from 121 Ixodes r icinus ticks collected from roe deer demonstrated that 45% of these ticks c arried Ehrlichia DNA. More than half of these positive ticks carried specie s with 16S rRNA gene sequences closely related to those of E. phagocytophil a and the human granulocytic ehrlichiosis agent. The majority of the other positive ticks were infected with a newly identified Ehrlichia-like species . In addition, 13% of the ticks were infected with one or more B. burgdorfe ri genospecies. In more than 70% of the ticks 16S rRNA gene sequences for B artonella species or other species closely related to Bartonella were found . In five of the ticks both Ehrlichia and B. burgdorferi species were detec ted.