engleski

Rickettsia felis – like bacteria from Haemaphysalis sulcata ticks in the mosquito C6/36 cell line

Objectives: A Rickettsia felis-like bacterium was recently described in Haemaphysalis sulcata ticks collected from sheep in southern Croatia. However, the description of the particular Rickettsia was based only on the sequence analysis of the complete gltA gene and portions of 17 kDa and ompB genes. The aim of this study was therefore to isolate the bacterium and establish its growth in cell culture. The presence of Rickettsia in ticks was additionally confirmed by transmission electron microscopy (TEM). Methods: Four H. sulcata ticks, collected from one sheep in southern Croatia, were selected for the study. They were repeatedly immersed in 5% bleach and 70% ethanol and washed with sterile water. DNA was extracted from one tick, for which infection with rickettsiae was previously confirmed by molecular analysis. The remaining three adult ticks were used for the inoculation of mosquito C6/36 cells. Ticks were homogenized in Leibovitz– 15 medium with 5% FCS. Two shell vials, containing a monolayer of approximately 40, 000 C6/36 cells, were inoculated with 300 &micro ; l of the homogenate each. After centrifugation, 1 ml of 5% Leibovitz– 15 medium with or without antibiotics was added to the respective shell vials. They were incubated at 26°C. The rest of the homogenate was used for DNA extraction. The infection of cells was assessed every third day by Diff-Quik staining and by PCR and sequencing of portion of 17 kDa gene. When 80% of C6/36 cells were infected, cells were harvested and inoculated into two 25-cm2 flasks containing a monolayer of fresh C6/36 cells. TEM was performed on infected C6/36 cells as well as on internal organs of H. sulcata ticks infected with R. felis-like bacteria. Results: After 32 days, 80% of C6/36 cells in shell vial culture were infected. The presence of R. felis-like bacteria in infected cells was confirmed by PCR and sequencing. The isolate was passaged for four times in 25-cm2 flasks and within 15 days, 90 % of cells were always infected. TEM demonstrated multiple rickettsiae with typical morphology in infected C6/36 cells and in tick salivary glands and midgut epithelial cells. Conclusions: The successful isolation and establishment of R. felis-like bacteria from H. sulcata ticks in the mosquito C6/36 cell line and TEM evidence of Rickettsia in internal organs of infected ticks are reported herein. These results in combination with molecular analysis of gltA, 17kDa, ompA and ompB genes will warrant naming this novel organism as Rickettsia croatica, strain Kastelanii.

Rickettsia felis bacteria; Haemaphysalissulcata; mosquito cell line

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