Microbial Food Safety Research Unit. Microbial Food Safety Research Unit Collaborators. Microbial Safety of Aquaculture Products Center of Excellence

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1 The Colony Overlay Procedure for Peptidases: Applications for Monitoring Seafood Safety Gary P. Richards, Ph.D. U.S. Department of Agriculture Agricultural Research Service Microbial Food Safety Research Unit Delaware State University Dover, Delaware Microbial Food Safety Research Unit Research Directions Predictive Microbiology Stress/Sensory Response Interventions/Omics Aquaculture Safety Microbial Food Safety Research Unit Collaborators Hatfield Quality Meats University of Wisconsin University of Georgia University of Bologna Michigan State University Delaware State University Drexel University Rutgers University University of Delaware Purdue University Univ. of MD Eastern Shore Virginia Polytech. Inst. Teagasc - Ireland J.L. Foods Pilgrim s Pride (Wampler) Institute of Food Research UK FSIS, FDA, APHIS, CDC C.I.A.D. - Mexico American Meat Institute The Institute for Genomics Res. Qualicon/Dupont Kraft/Oscar Mayer University of Nebraska National Cattleman s Beef Assoc. Embrapa/Labex - Brazil Microbial Safety of Aquaculture Products Center of Excellence Research on enteric virus assay methods Enzyme-based assays for vibrios Virus processing interventions (HPP) Vibrionaceae Family Vibrio cholerae V. parahaemolyticus V. vulnificus V. alginolyticus V. anguillarum V. fluvialis V. harveyi Aeromonas Plesiomonas Photobacterium t 1

2 Shellfish Safety Aquaculture is an Important Source of Fresh Fish and Shellfish Since 1925, shellfish sanitation in the US has relied on coliform standards which have effectively eliminated outbreaks of typhoid fever US standards are based on sanitary surveys of water quality based on fecal coliform levels Testing for pathogenic vibrios is relatively recent and the assays are impractical for routine use Major Vibrionaceae Pathogens in Aquaculture Vibrio anguillarum, salt water Aeromonas salmonicida, salt water Aeromonas hydrophila, fresh and brackish water Photobacterium damselae, fresh and salt water Research Needs Improved methods to monitor for vibrios in shellfish harvesting areas and in landbased aquaculture systems Vibrio Research Characterized new enzyme New Assay Concept: Colony Overlay Procedure for Peptidases (COPP Assay) Developed enzyme-based assay for vibrios l It s so simple a Caveman could do it! Isolated new enzyme in Vibrio family members 2

3 Homogenize and Dilute Spread Plate on Tryptic Soy Agar Overlay Membrane onto Colonies on the Plate Incubate 10 Minutes View Membrane Under UV Oyster Soil Bacterial Plates Overlays 3

4 Delaware Bay New Jersey Oyster Seed Beds Collected oysters and seawater May 2004 thru June 2006 Cohansey seed bed low salinity site New Beds seed bed high salinity site Samples were plated on tryptic soy agar (TSA), supplemented with NaCl (1% final conc.) Applied COPP assay to picked colonies: fluorescent colonies scored as positive non-fluorescent colonies scored as negative Estimated total bacteria (cfu) from 10-fold serial dilution Picked colonies for COPP assay Biochemically characterized 1421 colonies with API 20E test kits Results - Overview 276 samples collected and processed 6,416 colonies picked for COPP assay 3,108 COPP+ (48.4%) 3,084 COPP - (48.1%) 224 COPP+/- ( 3.5%) 1,421 API s run (Aug 04 to June 06) Human and fish pathogenic species detected 4

5 Seasonal occurrence of the most commonly encountered Vibrio spp. in Delaware Bay Summertime levels in oysters CFU's CFU's 100,000 10,000 1,000 1, V. vulnificus Vibrio vulnificus Vibrio parahaemolyticus V. parahaemolyticus Aug-04 Oct-04 Dec-04 Feb-05 Apr-05 Jun-05 Aug-05 Oct-05 Dec-05 Feb-06 Apr-06 Jun-06 HSO HSW LSO LSW 10 V. vulnificus: >10,000/gram 1 V. parahaemolyticus: >1,000/g Vibrio V. hollisae: >10,000/gram V. hollisae 10,000 1, Aug-04 Sep-04 Oct-04 Nov-04 Dec-04 Jan-05 Feb-05 Mar-05 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 HSO HSW LSO LSW Colony Overlay Procedure for Peptidases Detected all Vibrio family members Rapid, simple, and quantitative Inexpensive Applicable for the detection of total vibrios in seawater, fish, and shellfish Based on the presence of an enzyme found only in Vibrio family members 10 1 Aug-04 Sep-04 Oct-04 Nov-04 Dec-04 Jan-05 Feb-05 Mar-05 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 HSO HSW LSO LSW COPP Assay for Regulatory Use Determine normal, seasonal levels of total vibrios Monitor for spikes in levels Compare levels and spikes with conventional testing methods and with incidence of illness Identify correlations Determine if total Vibrio levels (COPP assay) may be used to regulate shellfish harvesting COPP assay in aquaculture and in monitoring shellfish safety Concept based on the premise that as total Vibrionaceae levels increase in seawater, so too will the pathogenic strains Similar to the way we currently regulate shellfish harvesting based on total levels of fecal coliforms Monitoring for total Vibrionaceae levels in market samples COPP Assay in a Recirculating Aquaculture System Determine normal levels of vibrios by COPP testing of water, fish, or shellfish Monitor for spikes in levels Compare levels with overall fish health during spike periods Determine if spikes serve as an index of disease Implement remedial actions when spikes occur COPP Assay for Market Surveys Evaluate fish and shellfish in the marketplace for total Vibrio levels using COPP assay Monitor for V. parahaemolyticus and V. vulnificus levels using cultural and PCR-based assays Correlate total Vibrio levels with specific pathogens Identify pros and cons of the COPP assay vs. molecular procedures 5

6 Potential Collaborations Identify COPP-type assays for other enzymes/ foodborne pathogens Perform market surveys to validate use of COPP vs. molecular methods for vibrios Evaluate the use of COPP as an indicator of fish health/safety in aquaculture settings Contact Me for More Information