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United States Department of Agriculture

Agricultural Research Service


item Richards, Gary
item Bushek, David

Submitted to: Meeting Abstract
Publication Type: Other
Publication Acceptance Date: 2/9/2005
Publication Date: 8/14/2005
Citation: Richards, G.P., Bushek, D. 2005. A novel, enzyme-based assay for the rapid and simple detection of vibrios in shellfish and seawater. International Association for Food Protection. Abstract P4-27. p. 130.

Interpretive Summary:

Technical Abstract: We discovered an enzyme present in members of the Vibrionaceae family, but not present in non-Vibrio pathogens. This enzyme is a lysyl aminopeptidase associated with phosphoglucose isomerase of all Vibrio species tested to date. A colony overlay procedure for peptidases (COPP) was developed for the rapid detection of bacteria containing this enzyme activity. For this procedure, cellulose acetate membranes containing a synthetic fluorogenic substrate were overlayed onto bacterial colonies on trypticase soy agar plates containing 1% NaCl to detect and quantify vibrios in seawater and shellfish. Within 10 min, Vibrionaceae family members produced fluorescent spots on the membranes where the substrate was cleaved by the enzyme. We used this COPP technique in a monthly survey of oysters and seawater from moderate and low salinity areas of the Delaware Bay from May through December, 2004. Six sets of oysters and 6 seawater samples were collected for analysis each month. Results indicated the presence of greater than 10e4 Vibrionaceae per gram of oyster during the summer months. Biochemical testing of isolates showed the presence of Vibrionaceae family members including species of Vibrio, Aeromonas, Plesiomonas, Photobacterium, and Shewanella. During June and July, Vibrio vulnificus was most frequently isolated and was at levels greater than 10e4/g of oyster and greater than 10e2/ml of seawater from the Delaware Bay. The COPP technique is rapid, simple, and inexpensive. It was useful for monitoring rising levels of Vibrionaceae family members in shellfish and seawater as water temperatures increased. Subsequent correlation of pathogenic species and shellfish-associated disease with total Vibrio levels could lead to the application of the COPP assay in the establishment of regulatory guidelines for total Vibrio levels in shellfish.

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