[Isolation of Rickettsia slovaca by shell-vial cell culture method from Dermacentor marginatus ticks]

Rickettsia species are gram negative, small pleomorphic coccobacilli, obligate intracellular bacteria. The majority of these bacteria are transported by the ticks. Rickettsia slovaca and Rickettsia raoultii are among the Rickettsia species in the spotted fever group and carried by Dermacentor species ticks, that cause tick-borne lymphadenopathy (Tickborne Lymphadenopathy-TIBOLA or Dermacentor-borne necrotic erythema and lymphadenopathy- DEBONEL). Rickettsia species are obligate intracellular bacteria and they can be cultivated in cell cultures. The chance of isolation of Rickettsia species from clinical and tick specimens has been increased by the shell-vial centrifugation cell culture method. The aim of this study was to isolate R.slovaca from two Dermacentor marginatus species ticks which were detected in humans by the use of shell-vial centrifugation cell culture method using VERO cell. Before proceeding to the culture stage, an algorithm including description, disinfection, dissection of ticks and methods of hemolymph, homogenization, DNA extraction and real-time PCR was performed. Iodine-alcohol disinfection was performed following identification of the ticks with the use of a stereo microscope. Ticks hemolymph were obtained with extremity dissection of ticks and indirect fluorescence antibody (IFA) test was performed with Rickettsia positive sera. Ticks identified as containing Rickettsia like bacteria in hemolymph were homogenized and DNA extraction was performed from homogenate of ticks. By real-time PCR, using Rickettsia genus-specific PanR8 primers and probes, PCR positive Rickettsia spp. were determined among the ticks. Vero cells that have formed monolayer in vials were used for Rickettsia culture. Homogenized tick samples which were positive for Rickettsia in hemolymph and real-time PCR methods were inoculated to cell culture vials. Suspended bacteria in the inoculum were allowed to approach the cells by the shell-vial centrifugation method. Cells were scraped after five days of incubation in 5% CO2 at 36°C. An aliquot of the determined cell suspension was fixed to the slides and then IFA was performed using Rickettsia positive sera. In fluorescence microscopy examination, adhered and proliferated bacteria were observed on Vero cells. This cell suspension was again inoculated into the Vero cell cultures to increase bacterial replication and taken for 5-7 days of incubation. DNA was extracted from the suspension of the cultivated bacteria. Conventional PCR of citrate synthase (gltA) and outer membrane protein A (ompA) gene regions were used to identify Rickettsia species. DNA sequence analysis of PCR amplification products were determined. DNA sequence results were compared to Genbank data and found that the gltA sequence was 100%, similar to R.slovaca with accession number AY129301.1 and the ompA sequence was 100%, similar to R.slovaca with accession number KF791242.1.  In addition, the two strains isolated in phylogenetic analysis were found to be R.slovaca. As a result, R.slovaca isolation from of D.marginatus type ticks has been reported for the first time in our country by the cell culture method.