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United States Department of Agriculture

Agricultural Research Service

2008 Annual Report

1a.Objectives (from AD-416)
Research will focus on four main objectives designed to enhance the safety of aquaculture products, to: a) continue to develop rapid, enzyme-based assays to detect bacterial pathogens in aquaculture products; b) identify RT-PCR inhibitors and develop real-time molecular methods to detect and quantify viral pathogens in shellfish tissues; c) investigate physical and chemical parameters influencing the efficiency of high hydrostatic pressure inactivation of hepatitis A virus, norovirus, and surrogate viruses; and d) investigate the mechanisms of enteric virus persistence within live shellfish.

Develop more effective means for decontaminating fresh and minimally processed fruits and vegetables containing human pathogens to ensure food safety and security by assessing the efficacy of new and/or improved intervention technologies. This maintains the flexibility to expand research efforts on produced when and where necessary.

1b.Approach (from AD-416)
We propose to use a wide variety of protein chemistry, biochemistry, microbiology, virology, molecular biology, and food technology principles and techniques to: a) develop molecular biological and enzyme-based assays to detect specific pathogens in shellfish tissues as well as processing interventions to inactivate enteric viruses that contaminate shellfish; b) screen for, identify and characterize novel enzyme activities associated with bacterial pathogens to develop rapid, enzyme-based assays for their decteion; c) develop improved virus extraction procedures for shellfish with the intent to characterize and eliminate potential inhibitors of real-time RT-PCR methods; d)explore the mechanism by which high pressure processing inactivates hepatitis A virus and noroviruses to determine the physical and chemical parameters that influence processing effectiveness, e) participate in a human volunteer study to determine the effectiveness of high pressure processing to inactivate noroviruses in oysters; and f) evaluate the mechanism by which enteric viruses persist within shellfish with the goal of developing improved shellfish disinfection and detection methods. We will accomplish these tasks in collaboration with Federal, State, and industry partners and distribute new methods and information to our stakeholders, especially the aquaculture industry and regulatory agencies. Together, these studies will enchance seafood safety and quality for all Americans.

3.Progress Report
A new, rapid, simple, and inexpensive enzyme-based assay for fecal coliforms (including E. coli) in seawater and shellfish was developed. This method can streamline existing assays for fecal coliforms, currently mandated by the FDA, for monitoring shellfish safety and for the regulation of harvesting. Weaknesses were identified in the standard method for detecting fecal coliforms in shellfish harvesting waters. Improved monitoring tools will reduce the incidence of shellfish-borne illness.

A study was completed to field-test an enzyme-based assay for vibrios using ribbed mussels obtained from Assateague Island National Seashore, MD. The assay successfully detected and quantified total Vibrionaceae levels within the shellfish, thus allowing vibrios in mussels to be monitored as potential indicators of water quality and shellfish safety.

A study was completed to evaluate the effects of temperature and pH on high pressure processing (HPP) of hepatitis A virus. Warmer temperatures and acidic pH’s were found to enhance hepatitis A virus inactivation.

Laser light scatter patterns and an automated computer recognition system were used to identify different species of vibrios growing on agar plates. The patterns were found useful in differentiating a wide variety of vibrios including known pathogenic and non-pathogenic species. For oysters, the method identified vibrios down to the species level.

2006-2010 Action Plan National Program Component 108, Problem Statement: 1.2.1, Detection [of pathogens].

1. Novel enzyme assay for fecal coliform detection. Fecal coliform bacteria, including E. coli, are frequently monitored as indicators of food safety, but monitoring is complicated, labor intensive, and costly. The Colony Overlay Procedure for Peptidases (COPP assay for short) is an enzyme-based test that we developed to detect members of the Vibrionaceae family of bacteria in shellfish and seawater. Vibrionaceae, especially Vibrio parahaemolyticus and Vibrio vulnificus, are attributable to outbreaks of illness and the closure of shellfish harvesting areas around the world. This year, the COPP assay was modified to detect not only Vibrionaceae family members, but to simultaneously detect fecal coliforms as well. This method is more rapid, simple, and cost effective than other analytical procedures. It can be used as a monitoring tool for fecal coliforms and Vibrionaceae in shellfish and marine waters, and is practical enough for potential use in regulating shellfish harvesting. 2006-2010 Action Plan National Program Component 108, Problem Statement: 1.2.1, Detection [of pathogens]

2. Field testing the COPP assay. Environmental bacteria in the Vibrionaceae family pose a threat to shellfish and human health and have been difficult to evaluate due to the lack of testing methods. Working with our partners at Delaware State University, we evaluated our recently developed, enzyme-based Colony Overlay Procedure for Peptidases (COPP assay) for detecting total Vibrionaceae levels in ribbed mussels from six sites along Assateague Island National Seashore, Maryland. Mussels in that area contained high levels of Vibrionaceae family members (over 7 million/gram of tissue) during the summer months and may serve as sentinels of environmentally-derived Vibrio contamination. Monitoring for total Vibrionaceae levels may provide a better indicator of water and shellfish safety than the current method, which relies on identifying specific pathogenic strains. Our field testing of the COPP assay showed that it was rapid, simple, and cost effective for monitoring Vibrionaceae in mussels and that it may have applications in monitoring aquaculture farms and native grown shellfish in an effort to ensure seafood safety. 2006-2010 Action Plan National Program Component 108, Problem Statement: 1.2.1, Detection [of pathogens]

3. Evaluation of Virus Inactivation by High Hydrostatic Pressure with the University of Delaware (see separate annual report for further details). Hepatitis A virus (HAV) in shellfish and produce is a significant concern in food safety. A study was completed to evaluate the effects of temperature and pH on high pressure processing (HPP) of hepatitis A virus. Results indicate that colder processing temperatures retard the inactivation of HAV; however, for inactivation of HAV within oysters, this temperature effect is less pronounced. For pH experiments, HAV in acidic environments is more sensitive to HPP than at higher more neutral pH’s. This study provides oyster and other processors with specific information on HPP conditions useful for HAV inactivation. 2006-2010 Action Plan National Program Component 108, Problem Statement: 1.2.4, Processing Intervention Strategies

4. Diffraction of laser light can identify Vibrio colonies. The identification of specific Vibrio species from seafoods is complicated and time-consuming. We participated in a study hosted by Purdue University to evaluate laser light diffraction patterns from Vibrio colonies as a potentially more rapid screening technique for Vibrio pathogens. Diffraction patterns were different among the Vibrio species. Prototypes for the laser system, including computer recognition software, have been engineered and are being deployed for evaluation at other laboratories. This equipment should be useful in screening shellfish, other foods, and environmental samples for specific pathogenic Vibrio species. 2006-2010 Action Plan National Program Component 108, Problem Statements: 1.2.1, Detection [of pathogens]

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings6
Number of Newspaper Articles and Other Presentations for Non-Science Audiences1

Review Publications
Richards, G.P., Watson, M.A., Crane, E.J., Bushek, D., Burt, I. 2008. Shewanella and photobacterium in oysters and seawater from the delaware bay. Applied and Environmental Microbiology. 74(11):3323-3327.

Last Modified: 10/13/2015
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