Plaque assay of rickettsiae in a mammalian cell line

Abstract

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.