Novel Rickettsia raoultii strain isolated and propagated from Austrian Dermacentor reticulatus ticks.

BACKGROUND: Continuous culture of tick cell lines has proven a valuable asset in isolating and propagating several different vector-borne pathogens, making it possible to study these microorganisms under laboratory conditions and develop serological tests to benefit public health. We describe a method for effective, cost- and labor-efficient isolation and propagation of Rickettsia raoultii using generally available laboratory equipment and Rhipicephalus microplus cells, further demonstrating the usefulness of continuous tick cell lines. R. raoultii is one of the causative agents of tick-borne lymphadenopathy (TIBOLA) and is, together with its vector Dermacentor reticulatus, emerging in novel regions of Europe, giving rise to an increased threat to general public health. METHODS: Dermacentor reticulatus ticks were collected in the Donau-Auen (Lobau) national park in Vienna, Austria. The hemolymph of ten collected ticks was screened by PCR-reverse line blot for the presence of rickettsial DNA. A single tick tested positive for R. raoultii DNA and was used to infect Rhipicephalus microplus BME/CTVM2 cells. RESULTS: Sixty-five days after infection of the tick-cell line with an extract from a R. raoultii-infected tick, we observed intracellular bacteria in the cultured cells. On the basis of microscopy we suspected that the intracellular bacteria were a species of Rickettsia; this was confirmed by several PCRs targeting different genes. Subsequent sequencing showed 99-100 % identity with R. raoultii. Cryopreservation and resuscitation of R. raoultii was successful. After 28 days identical intracellular bacteria were microscopically observed. CONCLUSIONS: R. raoultii was successfully isolated and propagated from D. reticulatus ticks using R. microplus BME/CTVM2 cells. The isolated strain shows significant molecular variation compared to currently known sequences. Furthermore we show for the first time the successful cryopreservation and resuscitation of R. raoultii.