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Title:   Author  item
                                            Richards, Gary
  Submitted to: Meeting Abstract
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 6/10/2005
                                    Publication  Date: 8/10/2005
                                    Citation: Richards, G.P. 2005.  The colony overlay procedure for peptidases to detect and enumerate total Vibrionaceae in molluscan shellfish and their growing waters. First Interstate Shellfish Safety Conference/International Conference on Molluscan Shellfish Safety Joint Workshop and Integration of Science into Policy:  Special Emphasis on Vibrios and Viruses Abstract p.33.Interpretive Summary:
                                Technical Abstract: Since 1925, molluscan shellfish harvesting has been regulated in the United States based on sanitary surveys of shellfish growing waters.  Surveys have relied on the use of coliform standards which have effectively eliminated outbreaks of typhoid fever and other bacterial illnesses.  Only the Vibrionaceae have not been controlled by the implementation of coliform or fecal coliform standards.  The Vibrionaceae are indigenous to marine waters, and subsequently, can not be predicted based on the levels of fecal coliform contamination.  Vibrionaceae contain members of the genera Vibrio, Aeromonas, Plesiomonas, and Photobacterium.  We developed an enzyme assay known as the colony overlay procedure for peptidases (COPP) that detects and quantifies Vibrionaceae family members in seawater and shellfish based on the presence of the enzyme phosphoglucose isomerase and its lysyl aminopeptidase activity.  This procedure is relatively simple, rapid, and inexpensive compared to other Vibrio testing methods.  Dilutions of oyster homogenates or seawater are spread on plates of tryptic soy agar plus 0.5% added NaCl and incubated at 37°C overnight.  Countable plates are overlaid for 10 min with a cellulose acetate membrane containing a commercially available synthetic substrate, L-Lys-7-amino-4-trifluoromethylcoumarin.   The membrane is then observed under UV light.  Fluorescent foci corresponding with bacterial colonies indicate the presence of Vibrionaceae family members.  Total Vibrionaceae counts may be determined by multiplying the total number of fluorescent foci detected by the dilution factor.  High levels of total Vibrionaceae may serve as an indicator for the possible presence of pathogenic Vibrio species and signal a point when shellfish beds should be closed to harvesting or when additional, more sophisticated testing for specific pathogens is warranted.  In a monthly survey of oysters and seawater between May of 2004 and May of 2005 involving approximately 150 samples recovered from the Delaware Bay, we detected levels of Vibrionaceae exceeding 10(6) per gram of oyster and 10(3) per ml of seawater during the summer months.  Vibrio vulnificus was particularly prevalent in oysters, with counts exceeding 10(5) per gram from May – August, while Vibrio parahaemolyticus levels exceeded 104 per gram in May and July.  The COPP assay may find utility in identifying peak periods when Vibrionaceae are at their highest levels in East, West, and Gulf Coast oysters and growing waters and allow the correlation of total counts with the incidence of Vibrio outbreaks in those areas.  The COPP assay is equally applicable in the United States and the European Union where shellfish are currently regulated based on coliform levels in seawater and shellfish meats, respectively.


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