Actin assessment in addition to specific immuno-fluorescence staining to demonstrate rickettsial growth in cell culture.

Rickettsiae are able to spread within infected cell mono-layers by modifying intra-cellular actin formations. The study analyzes whether a visualization of actin modifications in addition to specific immuno-fluorescence staining of rickettsiae might facilitate the proof of rickettsial growth in cell culture. Cell mono-layers of Vero E6 und BGM cells were infected with Rickettsia honei. Intra-cellular actin was fluorescence stained with TRITC-(tetra-methyl-5,6-isothiocyanate)-labeled phalloidin in addition to specific immuno-fluorescence staining of rickettsiae with FITC-(fluorescein-isothiocyanate)-labeled antibodies. DNA of bacteria and cells was counter-stained with DAPI (4´,6-diamino-2-phenyl-indole). Cell cultures infected with Vaccinia virus were used as positive controls, cell cultures infected with Coxiella burnetii as negative controls. High concentrations of R. honei are necessary to demonstrate characteristic modifications of the intra-cellular actin. This effect is more pronounced in Vero E6 cells than in BGM cells. Actin staining with phalloidin is not suited for an early proof of rickettsial growth in cell culture but may confirm unclear findings in specific immuno-fluorescence staining in case of sufficient bacterial density.