Location: Food Safety and Intervention Technologies Research
2020 Annual Report
Objectives
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.
Approach
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.
Progress Report
Molluscan shellfish, like oysters and clams, are commonly contaminated with bacteria and viruses that are present in the marine environment. Among the more consequential bacteria are members of the genus Vibrio, which are naturally-occurring marine organisms. Some vibrios, like Vibrio coralliilyticus and Vibrio tubiashii, are pathogens of larval oysters and contribute to major mortality events in commercial shellfish hatcheries, while other vibrios are serious human pathogens. Vibrio parahaemolyticus and Vibrio vulnificus are among the human pathogenic vibrios associated with the consumption of contaminated shellfish, particularly raw or lightly cooked oysters. Vibrio parahaemolyticus is attributed to over 30,000 cases of illness in the United States annually and is considered the most prevalent cause of seafood-associated bacterial illness. Some strains of V. parahaemolyticus are also significant pathogens in shrimp aquaculture. Illnesses from V. vulnificus are less common; however, the mortality rate is exceedingly high among those infected, estimated at 35% in the United States and higher in other parts of the world. Marine waters and oysters contain a plethora of bacterial viruses, known as bacteriophages (phages for short), which attack and kill a wide variety of vibrio species. Phages are one of nature’s ways to maintain bacterial balance within the environment. Under the milestone for Objective 1, “to complete characterization of phage growth characteristics, ARS identified phages against pandemic strains of V. parahaemolyticus in Delaware Bay oysters and completed their characterization based on morphology, host specificity and ability to form plaques (discrete areas of vibrio death caused by the phages) in agar cultures. The consistent formation of plaques is an indicator of the likely presence of lytic phages which generally kill their host cells quickly, making lytic phages desirable for application in phage therapy. The other type of phages, known as lysogenic phages, incorporate their DNA into the host cell’s genome and are undesirable for use in phage therapy. They can make the host bacterium more virulent. Tests showed that the phages we isolated are likely lytic phages, suitable for use in phage therapy. Characterization of the phages demonstrated that they had relatively narrow host ranges, where they could infect and kill only some V. parahaemolyticus strains, but not others. The use of these phages in a commercial processing intervention to reduce human pathogenic strains of V. parahaemolyticus or V. vulnificus in wild-caught oysters may be inadequate because of their narrow host range. Consequently, these phages will be combined with ARS-isolated Vibrio predatory bacteria to formulate a treatment against the multitude of V. parahaemolyticus and V. vulnificus strains likely to be present in commercially harvested oysters. ARS provided phages against V. parahaemolyticus under a Material Transfer Agreement to Intralytix, Inc., a phage-based biotechnology company that seeks to develop treatments to reduce disease caused by V. parahaemolyticus in shrimp aquaculture. Objective 1 also involves the formulation of a mixture (cocktail) of phages to be used as a treatment to reduce larval oyster mortalities caused by V. coralliilyticus and V. tubiashii in oyster hatcheries. ARS has entered these phage DNA sequences into GenBank and has deposited frozen phage cultures into the American Type Culture Collection Patent Depository, as prerequisites for submission of a patent application over the coming months. An ARS-isolated Hawaiian phage against V. coralliilyticus was provided under a Material Transfer Agreement to the Smithsonian Marine Station at Fort Pierce, Florida for their evaluation of the use of these phages to reduce stoney coral disease and associated coral mortalities caused by V. coralliilyticus. Preliminary studies showed that one of 17 phages in ARS’s collection of Hawaiian phages did kill 4 out of 5 strains of V. coralliilyticus associated with diseased coral reefs in Florida and the Caribbean. there were no milestones under Objectives 2 or 3 this year; however, the Objective 4 milestone was “to evaluate ozone and copper in depuration water and /or with high pressure processing”. Copper (Cu+0 and Cu+1) has been reported to inactivate norovirus surrogates and human norovirus. Toward this milestone, ARS evaluated the application of Cu+1 ions in water where murine norovirus-contaminated oysters were held to determine if adding copper could inactivate the virus within live oyster tissues. Little if any virus inactivation was observed as compared to nontreated controls. In addition, copper caused a persistent blue green tint in the treated oysters suggesting that this method might not be commercially feasible. A second hypothesis was tested regarding high pressure processing (HPP) with ozone. Previous work indicated that 4000-5000 atmospheres of pressure was needed to inactivate noroviruses and hepatitis A virus in oysters. Processors would prefer to treat shellfish at 2700-3000 atm pressure, since the higher-pressure machines are expensive, primarily because of the engineering tolerances required for elevated pressures. It is known that at about 2000 atm the oyster tissue separates from the shell, which is desirable and prevents the need for labor intensive shucking. However, 2000 atm is inadequate to eliminate hepatitis A and norovirus from oysters. If a machine only needed to process oysters at 2000 atm to inactivate the viruses and at the same time could shuck the oysters, HPP would be financially more feasible. During HPP, oyster tissues take up water or any other liquid that is within the pressurization vessel. It was hypothesized that oyster tissues could potentially take up ozonated water at the lower pressure resulting in a virus-free, ozone-treated, shucked oyster. Using a cheaper HPP machine would further promote the use of HPP technology as a more practical and cost-effective process to eliminate enteric viruses from potentially contaminated oyster. The potential of driving ozonated water into oyster tissues while shucking the oyster at 2000 atm was evaluated. Results showed that the HPP effectively shucked the oysters as expected; however, no beneficial effects were observed on the reduction of the human norovirus surrogate murine norovirus in oysters subjected to 2000 atm of pressure and 5-8 ppm of ozonated water.
Accomplishments
1. Bacterial viruses as indicators of norovirus in shellfish. Noroviruses are a common cause of oyster-borne illness, jeopardizing the well-being of the commercial oyster industry and shellfish consumer. Male-specific coliphages (MSC) are bacterial viruses monitored by regulators as indicators of sewage and potential norovirus contamination. ARS investigators at Dover, Delaware, evaluated the persistence of MSCs in oysters as a function of water temperature and compared their persistence with that of human norovirus in live oysters. Results indicated that persistence was comparable in different type of MSCs, and was similar to human norovirus, suggesting that MSCs may be used as a marker for potential human norovirus contamination in oysters. This information provides the shellfish industry and shellfish safety regulators with additional options by which to assess virus contamination of market oysters.
