Purpose: A cytotoxicity assay may be able to provide insight into the potential pathogenicity of Vv isolated from the environment. A mouse model has been developed and is the standard used to evaluate pathogenicity of Vv strains. However, this is time-consuming and expensive and not feasible to use as a screening method. This goal of this study was to develop a cell-based method to evaluate pathogenicity and aid in the identification of genetic markers for virulence potential without having to use a live animal model.
Methods: Environmental and clinical Vv isolates were tested in a cytotoxicity assay using RAW 264.9 (RAW) cells and compared to results from the standard mouse bio-assay using the Pearson Correlation. Briefly, using an LDH-releasing assay per the manufacturer’s protocol, a set of 27 Vv isolates from environmental and clinical sources were tested for their cytotoxic effect on the RAW cells.
Results: Mouse mortality ranged from 0% to 90% and cytotoxicity in RAW cells ranged from 18% to 257%. Although some background interference was observed with the cell assay, there was a statistically significant correlation between mouse mortality and RAW cytotoxicity (r=0.811, p=8.96 x 10-7).
Significance: The results indicated the potential for the RAW cell model to reflect mouse (and, presumably, human) virulence. Development of a reliable cell-based model to identify pathogenic Vv will aid in identification of virulence genes, which can be used to develop rapid, molecular screening methods.