DETECTION AND PRODUCTION OF VEROTOXIN-1 OF ESCHERICHIA-COLI O157-H7 IN FOOD

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng@ml) was detected in samples that were incubated at 37-degrees-C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30-degrees-C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37-degrees-C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30-degrees-C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk. Beef that was contaminated with E. coli O157:H7 and incubated at temperatures near 37-degrees-C could contain substantial levels of VT1; however, production of VT1 (> 0.5 to 1.0 ng/g) is not likely to occur in milk or beef under normal handling and storage conditions.