1a. Objectives (from AD-416):
The safety of aquaculture products, particularly molluscan shellfish, is jeopardized by Vibrio and enteric virus contamination and the lack of effective processing interventions. Among the foods of greatest concern are raw or lightly-cooked oysters and clams, which result in substantial health risks to consumers. The objectives of this project are designed to: identify practical intervention methods to eliminate vibrios in shellfish using bacteriophages and Bdellovibrio-and like-organisms (Vibrio predatory bacteria) and to develop and validate methods for enteric virus detection and elimination from shellfish. 1: Develop and evaluate intervention and control strategies for Vibrio species, with specific emphasis on the identification, characterization and application of bacteriophage to remediate shellfish mortalities in hatchery settings, and for use in commercial shellfish processing. 2: Evaluate a modified depuration process with marine Bdellovibrio and related bacteria to eliminate Vibrio in market oysters. 3: Develop and validate technologies to improve current virus detection and testing methods, including distinguishing infectious versus non-infectious virus; technologies for virus replication, for example, development of a cell culture propagation method for human norovirus; virus surrogates; and long-term virus persistence. 4: Develop and validate emerging technologies for inactivation of enteric virus-contaminated shellfish and other foods using novel applications of high pressure and laser-induced resonance energy.
1b. Approach (from AD-416):
Under objective 1, we will seek to reduce Vibrio-associated mortalities in larval oyster hatcheries by 50% using a mixture of bacteriophages (phages), and to reduce human pathogenic vibrios in market oysters using a second mixture of phages. Under a CRADA with Intralytix, Inc. and Oregon State University, ARS will continue efforts to commercialize this phage treatment against the larval shellfish pathogens V. tubiashii and V. coralliilyticus for use in shellfish hatcheries. Phages that we already isolated and identified will be further characterized genetically, morphologically, and mechanistically as potential candidates for commercialization. At the completion of these studies, efforts will shift to an evaluation of phages against the human pathogens V. parahaemolyticus and V. vulnificus in market-sized oysters. Oysters will be challenged with streptomycin-resistant strains of V. parahaemolyticus and V. vulnificus, allowed to bioaccumulate these vibrios in tanks of seawater and then treated with phages to determine the Vibrio reduction rates in the shellfish. Under objective 2, we will evaluate a modified depuration process with predatory bacteria known as Bacteriovorax species (recently renamed Halobacteriovorax) to reduce or eliminate Vibrio parahaemolyticus and Vibrio vulnificus in market-sized oysters. Work will be performed using Halobacteriovorax strains that we isolated and partially characterized from the U.S. Atlantic, Gulf, and Hawaiian coasts. Concurrent with the above research will be studies to better characterize Halobacteriovorax and related bacteria that inhibit pathogenic vibrios and other bacteria. Under objective 3, we will develop and validate our porcine gastric mucin-magnetic beads (PGM-MBs) assay to distinguish infectious from non-infectious human noroviruses (NoV), determine if a correlation exists between long-term persistence of NoV within oysters and MS-2 phages at different water temperatures, and attempt to develop an in vitro replication system for NoV. The degree to which MS-2 phages mimic NoV in their ability to remain viable within shellfish and survive chlorination levels found in sewage treatment will be determined. The persistence of NoV in shellfish, oyster hemocytes, and in sewage effluent will be evaluated along with potential interventions to eliminate viral contamination. Under objective 4, we will seek to overcome barriers to the widespread commercial use of high pressure processing (HPP) for oysters and identify substances, like ozone or copper ions, that may inactivate NoV during HPP treatment. We will also evaluate the use of modified atmosphere packaging for shellfish using “oxygen scavenger” technology to enhance freshness of HPP-treated oysters during transit. We will seek to understand how laser induced resonance energy can destroy small icosahedral viruses, like NoV. This work will be performed in collaboration with researchers at Delaware State University and the University of Maryland.
3. Progress Report:
Progress was made on all objectives, all of which fall under National Program 108 – Food Safety, Component I Foodborne Contaminants. Progress on this project focuses on Problem Statement 3, Microbial Contaminants: Technologies for Detection and Characterization; and Problem Statement 5, Intervention and Control Strategies including Mycotoxins. Oysters, clams and mussels are popular shellfish consumed throughout the United States; however, they are subject to contamination by naturally occurring bacteria and sewage-borne bacteria and viruses which lead to foodborne illness in unsuspecting consumers. Two, naturally-occurring bacteria (known as Vibrio vulnificus and Vibrio parahaemolyticus) cause most of the shellfish-related human illnesses and deaths in the United States. These bacteria are present in seawater and shellfish, especially during the warm summer months. Other vibrio species have caused major losses to larval shellfish in hatcheries which produce seed oysters and clams needed to maintain the commercial shellfish industry. In addition to these naturally-occurring vibrios, sewage pollution of our oceans is responsible for the introduction of viruses, like the human norovirus (dubbed the cruise-ship virus by some) into productive shellfish harvesting areas. Norovirus causes severe gastroenteritis (diarrhea and vomiting) and can be transmitted by not only shellfish, but any food which is contaminated by sewage or the hands of a sick individual, thus norovirus is the principle cause of foodborne illness in the United States with an estimated 5.5 million cases annually. Our work is dedicated toward resolving these problems. Under Objective 1, we developed and evaluated intervention and control strategies for Vibrio species, with specific emphasis on the identification, characterization and application of bacteriophage to remediate shellfish mortalities in hatchery settings and for potential use to eliminate human pathogenic vibrios from commercial shellfish. Bacteriophage are naturally-occurring bacterial viruses capable of infecting and killing vibrios and other bacteria. This year, ARS isolated and characterized additional phages from Hawaiian seawater, developed improved methods to extract and sequence the DNA from five phage strains, and is evaluating the sequences for undesirable genes that might preclude these phages from being used in commercial hatcheries. A three-way Cooperative Research and Development Agreement (CRADA) was established between ARS, a university, and a phage-based technology company. Phage production has been up-scaled by the biotech company and large-scale hatchery trials are underway at a university as a prelude to commercialization. Research is underway on objective 2, to evaluate a modified depuration process with marine Bdellovibrio and related bacteria (Pseudoalteromonas species) to eliminate vibrios in market oysters in an effort to reduce human illnesses and deaths. Commercial depuration is a well-known process where shellfish are placed in tanks of recirculating seawater and allowed to purge bacterial contaminants into the water over a 3-day period. The water is continually recirculated and disinfected, usually via ultraviolet light in the U.S. and ozone in Europe. Conventional depuration effectively removes many sewage-borne bacteria from the shellfish, but is not very effective in removing naturally-occurring vibrios. Our plan is to add a step before the shellfish are depurated by adding vibrio predatory bacteria and/or phages to the tanks before ultraviolet disinfection is applied. This year, ARS identified, characterized, and published a paper on a new type of predatory bacteria known as Pseudoalteromonas piscicida. It appears to attack vibrios in two ways. The first is by direct transfer of digestive vesicles from its surface to the surface of vibrios, followed by the digestion of holes in the vibrios. The second method is by the secretion of vibrio-inhibitory substances, including a host of digestive enzymes which we characterized. We also DNA sequenced the genomes of three Pseudoalteromonas piscicida strains that we isolated from Delaware coastal seawater. Our characterization of these strains is underway and DNA sequence data has been submitted to GenBank. Presently, we are evaluating the use of Pseudoalteromonas piscicida and previously identified Halobacteriovorax species to determine their ability to reduce vibrios in market oysters. ARS also collaborated with two Historically Black Colleges and Universities in a study to validate a rapid, simple, and inexpensive method developed by ARS to detect total pathogenic vibrios in shellfish. Current methods for vibrio detection are time-consuming, complicated, and relatively expensive, so a faster method is needed. The first year of the study has been completed and the final year is underway. Preliminary work suggests the usefulness of the ARS method to detect pathogenic vibrios in shellfish. Research is underway under objective 3, to develop and validate technologies to improve current virus detection and testing methods, including distinguishing infectious versus non-infectious viruses; technologies for virus replication, for example, development of a cell culture propagation method for human norovirus; virus surrogates; and long-term virus persistence. Using the PGM-MB assay for norovirus (previously developed by ARS) evaluation of the efficiency of chlorine-based sewage treatment against human norovirus was performed. Results indicate that chlorine–based sewage treatment does not inactivate norovirus. This revelation will be of significance to regulatory agencies with respect to shellfish safety. A manuscript is in preparation. Characterization of the persistence of human norovirus and MS-2 bacteriophage (norovirus indicator) within live oysters has been evaluated with respect to water temperature. Results indicate that colder water dramatically enhances persistence of both human norovirus and MS-2 within live oysters. The manuscript on human noroviruses has been published and the MS-2 manuscript is currently under revision. Evaluation of the PGM-MB assay for viability within live shellfish has been performed. Results have been inconsistent, suggesting that this method may not be ideal for routine evaluation of human norovirus viability within shellfish. Evaluation of a DNA male specific coliphage as an indicator of sewage-contaminated shellfish is in the planning stages. Data on coliphage is needed because it is proposed as a hygienic indicator for shellfish. Also under objective 3, a limited replication system for human norovirus was recently published by a university and involves human organoid cells. We have obtained these cells and training under a Material Transfer Agreement and are evaluating this replication system. Evaluation of its feasibility to assess the viability of human norovirus samples is underway. Under objective 4, we showed previously that femtopulse visible light lasers can inactivate murine norovirus, a surrogate for human norovirus. While a resonance vibrational mechanism was thought to be the mechanism for virus inactivation, recent work has demonstrated that the actual mechanism is most likely the formation of reactive oxygen molecules, which serve as a disinfectant. The finding is based on demonstrated inactivation by constant wave length laser light, reduced inactivation when an oxygen scavenger (sodium bisulfite) or a singlet oxygen quencher (B-carotene) is present. We also initiated an evaluation of the role of copper ions as a means to depurate live oysters to reduce norovirus using the Tulane virus, another norovirus surrogate. Preliminary results are promising. Evaluation of the potential of blue laser light (405 nm light) and chlorine dioxide to inactivate Tulane virus on the surface of blueberries has been substantially completed. Blue light inactivation was limited without addition of singlet enhancer molecules such as riboflavin (vitamin B2) and rose bengal (red food dye #105); however, the presence of these molecules did substantially enhance inactivation by blue light. Results with Tulane virus indicates that chlorine dioxide can be used to inactivate viruses on the surface of blueberries at concentrations that do not cause bleaching of the fruit.
1. Identified new Vibrio predatory bacteria. Vibrio bacteria, particularly Vibrio vulnificus and Vibrio parahaemolyticus, cause illnesses and deaths to consumers of raw or partially cooked shellfish. Processing interventions are needed to kill vibrios in shellfish to enhance seafood safety. ARS researchers at Dover, Delaware, identified three novel marine bacterial strains (Pseudoalteromonas piscicida) as predators of human pathogenic vibrios. These bacteria effectively reduce vibrios and other human pathogens in the marine environment and may provide the key to the development of processing interventions to reduce shellfish-associated illness.
2. Method validation to enhance human norovirus detection. Most shellfish contamination by human noroviruses is from faulty sewage treatment processes and the release of large quantities of norovirus into the coastal environment. A method developed by ARS researchers at Dover, Delaware known as the PGM-MB binding assay, detects viable human noroviruses and has been applied to evaluate the efficacy of sewage treatment. Results demonstrate that human norovirus is not inactivated by chlorine-based sewage treatment. This data has been provided to the U.S. Food and Drug Administration (FDA) as part of their ongoing shellfish risk assessment project and may affect the way the FDA regulates the harvesting of shellfish.
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