P3-214 Development of an In Vitro Assay for the Determination of Pathogenicity of Vibrio vulnificus

Wednesday, July 12, 2017
Exhibit Hall (Tampa Convention Center)
Joey Marchant-Tambone , FDA Gulf Coast Seafood Laboratory , DAUPHIN ISLAND , AL
Jessica Jones , FDA Gulf Coast Seafood Laboratory , Dauphin Island , AL
Paul Gulig , University of Florida, College of Medicine , Gainesville , FL
Introduction: Vibrio vulnificus (Vv) is the leading cause of mortality in United States shellfish consumers. However, Vv is ubiquitous in the environment and not all Vv isolated from the environment are pathogenic. As no reliable markers of pathogenicity have been identified, there is no rapid method to differentiate pathogenic strains.

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.