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The rickettsial plaque. Evidence for direct cytopathic effect of Rickettsia rickettsii
Abstract:
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 sever 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.
Insights
This study developed a plaque assay to observe Rickettsia rickettsii infection in chick embryo cells. The assay revealed distinct zones within plaques, correlating rickettsial load with cell damage and release mechanisms.
Area of Science:
- Microbiology
- Cell Biology
- Pathology
Background:
- Rickettsia rickettsii causes Rocky Mountain spotted fever, a severe tick-borne illness.
- Understanding rickettsial infection mechanisms at the cellular level is crucial for developing treatments.
Purpose of the Study:
- To establish a quantitative plaque assay for studying Rickettsia rickettsii infection in cell monolayers.
- To investigate the temporospatial relationship between rickettsial infection and cellular pathology.
Main Methods:
- Primary chick embryo cell monolayers were infected with Rickettsia rickettsii.
- A plaque assay system with agarose overlay was employed.
- Cells were analyzed using supravital staining, immunofluorescence, and electron microscopy.
Main Results:
- Plaques exhibited four distinct zones: peripheral, marginal, necrotic, and central.
- Increased Rickettsia rickettsii quantity correlated with greater cytopathology, including endoplasmic reticulum dilation and necrosis.
- Rickettsiae were predominantly found in the cytosol, with evidence of cell lysis for release.
Conclusions:
- The developed plaque assay provides a predictable, quantitative model for studying Rickettsia rickettsii pathogenesis.
- This model facilitates experimental investigation into the mechanisms of rickettsial-induced cell injury.
- Findings elucidate rickettsial distribution and release mechanisms within host cells.