The rickettsial plaque. Evidence for direct cytopathic effect of Rickettsia rickettsii

Monolayers of primary chick embryo cells were infected with Rickettsia rickettsii in a plaque assay system with 0.5 per cent agarose overlay. Plaques appeared 5 days after inoculation and were examined on day 6 by supravital staining, immunofluorescence for R. rickettsii, and electron microscopy. In all studies maintenance of the topographic integrity of the monolayer and the plaque allowed assessment of temporospatial relationships of rickettsial infection and cytopathologic changes. Plaques comprised four zones: peripheral, marginal, necrotic and central. The cells of the peripheral zone were viable, mildly infected, and ultrastructurally normal. Cells of the marginal, necrotic, and central zones exhibited a correlation between quantity of rickettsiae and presence of cytopathology. Pathologic changes included severe dilation of rough endoplasmic reticulum and necrosis. Quantitative assessment of the distribution of rickettsiae ultrastructurally showed the overwhelming predominance of cytosol location and suggested the ultrastructural sequence of events for rickettsial release from cells via lysis of cell membrane at the end of filopodia. These studies of the plaque technic provide a predictable, quantitative model for experimental investigations into mechanisms of cell injury by rickettsiae.