Location: Food Safety and Intervention Technologies Research
2021 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
This is the final report for project 8072-42000-081-00D, entitled “Development of Detection and Intervention Technologies for Bacterial and Viral Pathogens affecting Shellfish,” which ended January 30, 2021. A new NP108 OSQR-approved project 8072-42000-090-00D, entitled “Innovative Detection and Intervention Technologies Mitigating Shellfish-borne Pathogens,” has been established effective January 31, 2021. Among the most consequential shellfish-borne pathogens are bacteria of the genus Vibrio, which constitute both human and shellfish pathogens, and enteric viruses, particularly human norovirus and hepatitis A virus. Vibrios have been problematic in oyster hatcheries, which supply the commercial industry with seed oysters needed for commercial operations. Oyster hatcheries have been crippled by high larval mortalities caused by Vibrio coralliilyticus (Vcor) and Vibrio tubiashii (Vt). Under Objective 1, “to 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,” bacteriophages (phages) were evaluated for potential therapeutic applications to reduct Vibrio-associated mortalities in larval oysters. Phages are bacterial viruses that can infect and kill bacteria. We characterized 16 phages that we isolated from Hawaiian seawater and tested mixtures (cocktails) of these phages as potential treatments against Vcor and Vt infection. One cocktail, consisting of three different phages, successfully reduced mortalities by up to 91%, which was well above our stated goal of reducing mortalities by 50%. Phages in the cocktail were active against eight strains of Vcor that are problematic in hatcheries. We also demonstrated that the phage cocktail is effective in reducing mortalities from Vcor in commercially valuable Eastern and Pacific oyster species. Phages in this cocktail were also DNA (genomic) sequenced, and two were unlike any phages listed in the GenBank database. The third phage was similar to a phage isolated from the Great Barrier Reef, Australia. This proof-of-principle testing demonstrated that phage therapy can reduce losses in oyster hatcheries to prevent shortages of seed oysters needed by the commercial oyster industry. This work was recently published and featured on the cover of Applied and Environmental Microbiology (May 2021, Vol. 87, issue 10). Working with university partners, we also showed that four strains of Vcor were more virulent at slightly elevated seawater temperatures. Significantly increased mortalities of larval oysters were observed at 27 degrees C compared to 25 degrees C. The genes responsible for this increased virulence were also identified. Recommendations were provided to shellfish hatcheries to maintain seawater around 25 degrees C to reduce mortalities caused by Vcor. At the request of the Smithsonian Marine Station at Fort Pierce, Florida, we screened our phages against 5 of their Vcor isolates that were taken from diseased corals in Florida and the Caribbean. The Smithsonian is assessing the effects of Vcor on the dieoff of coral reefs. One of our phages effectively targeted 4 of the 5 strains of Vcor that they provided. Under an MTA, we provided this phage to the Smithsonian for further study. We discovered a new strain of Vcor designated AIC-7 in a New Jersey oyster hatchery. Under a collaboration, AIC-7 was provided to Oregon State University for genomic sequencing and the sequence was published in GenBank. This isolate was also provided under an MTA to a shellfish hatchery to serve as a positive control in their Vcor testing procedures.
Objective 1 was also intended to evaluate the use of phages against the human pathogenic vibrios V. parahaemolyticus (Vpara) and V. vulnificus (Vv) in commercial shellfish processing. Vpara is the primary bacterial cause of seafood-associated illnesses in the U.S., while Vv is the main bacterial cause of shellfish-related deaths in the U.S., most of which are caused by the consumption of raw or undercooked oysters. Due to the pandemic, work on these pathogens was only partially met. A survey for phages was performed using market oysters from the Delaware Bay. Phages were readily detected against three strains of Vpara of which two were notable pandemic strains. No phages were detected against Vv. Phages against Vpara had very narrow host specificity, supporting the notion that pandemic strains of Vpara are likely present in the Bay and may be emerging public health risks associated with the consumption of raw or undercooked oysters. Nine of these phages were further characterized by electron microscopy. Since some Vpara strains are also pathogenic toward shrimp, we provided eight phage isolates under an MTA to a phage-based biotechnology company to evaluate their use in phage therapy to reduce Vpara-associated mortalities in shrimp aquaculture.
Under Objective 2, “to evaluate a modified depuration process with marine Bdellovibrio and related bacteria to eliminate Vibrio in market oysters,” we characterized predatory bacteria (Bdellovibrios such as Halobacteriovorax [HBX]) that kill a wide variety of human pathogens. In the process, we also isolated and identified through genomic sequencing, another bacterium (Pseudoalteromonas piscicida) and showed for the first time that it is also predatory. We demonstrated through electron microscopy that P. piscicida transfers enzyme-containing vesicles from its surface to the surface of Vpara. Enzymes in the vesicles then digest holes in the cell wall of the vibrios, effectively killing them and releasing nutrients that P. piscicida can use as food, making them true predators. Our sequencing of three P. piscicida genomes revealed a host of genes for enzymes that could potentially control vibrios and other pathogens in various processing scenarios. Publication of the DNA sequences in GenBank also led to much interest in these isolates and the opportunity to transfer our isolates to others. Cultures of P. piscicida were provided under MTAs to researchers in Zurich, Switzerland for the identification of novel enzymes; to the University of Texas in Austin to evaluate whether enzymes in P. piscicida could decompose chitin in the shells of crabs, shrimp, and lobsters to facilitate more economical, sustainable, and environmentally friendly ways to dispose of shell waste by shellfish processors; and to the Lawrence Berkeley National Laboratory in California, where they are exploring whether different prey bacteria develop resistance to P. piscicida. In addition, we trained a visiting government scientist from Italy on our techniques for HBX culturing and quantification, and we provided cultures and instructions to them under an MTA. They have since redirected their laboratory’s efforts toward HBX research by identifying HBX in the Aegean Sea and successfully using HBX to reduce Vpara in mussels.
Under Objective 3, ”to 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,“ we used an assay we developed, known as the porcine gastric mucin-conjugated magnetic beads assay (or PGM-MBs assay for short) to demonstrate that traditional chlorine sewage treatment does not inactivate significant amounts of human norovirus, indicating that large amounts of norovirus are released into streams and estuaries throughout the US and elsewhere. The persistence of human norovirus and MS-2 bacteriophage, which is used as an indicator of sewage exposure, has been characterized. We showed that human norovirus persists slightly better than MS-2 within oysters but overall persistence of NoV and MS-2 has a reasonable correlation and therefore MS-2 is a suitable indicator for the potential presence of norovirus within shellfish. We successfully propagated human norovirus in vitro using the published method of a group at Baylor College of Medicine, but unfortunately the replication method will require substantial improvement before it will be practical for determining human norovirus in shellfish and other foods.
Under Objective 4, “to 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,” we evaluated treatments to enhance the effectiveness of high pressure processing (HPP) and/or extend the shelf-life of HPP-treated oysters were evaluated. Efforts included the use of ozonated water in the HPP package and modified atmosphere (carbon dioxide) treatments after HPP. We showed that neither treatment was effective in reducing human norovirus in oysters. We also determined that treatment with copper ions was ineffective for inactivating viruses within live shellfish. Fortunately, several other interventions to inactivate viruses have been more successful. Laser inactivation experiments showed that singlet oxygen produced by visible laser light likely causes virus inactivation. The use of singlet oxygen enhancers in combination with 405 nm blue light emitted by an LED was able to substantially reduce the norovirus surrogate, Tulane virus, on the surface of blueberries. Gaseous chlorine dioxide has been successfully evaluated for inactivation of hepatitis A virus and norovirus surrogates on berries. Results suggest that chlorine dioxide gas could be an effective mitigation technology for virus-contaminated produce.
Accomplishments
1. Bacteriophage therapy. Bacteriophage therapy reduces larval oyster mortalities. Shellfish hatcheries have experienced intermittent outbreaks of larval oyster mortalities causing shortages of seed oysters needed by the commercial oyster industry. Many outbreaks have been associated with the naturally-occurring marine bacteria Vibrio coralliilyticus and Vibrio tubiashii. ARS scientists in Dover, Delaware, identified and characterized 16 bacterial viruses (bacteriophages) that can kill these vibrios, and developed an effective mixture (cocktail) of three bacteriophages to reduce larval mortalities by up to 91% after exposure to ordinarily lethal doses of these vibrios. Together, this cocktail was effective against eight strains of V. coralliilyticus commonly found in U.S. waters and a strain of V. tubiashii. This new technology provides proof of principle that phage therapy can significantly reduce larval oyster mortalities, thus supporting the needs of shellfish hatcheries and the commercial oyster industry.
2. Norovirus. Norovirus successfully propagated. Inability to reproduce human norovirus has been a roadblock to food safety research. ARS scientists at Dover, Delaware, evaluated a method developed by a group from Baylor College of Medicine using human organoid cultures to propagate human norovirus. Human organoid cells were successfully cultured and norovirus was successfully propagated, albeit limited, as determined by molecular (reverse-transcription-PCR) testing. Although propagation was achieved, this method is too complex and time-consuming for routine norovirus assays or for the determination of human norovirus inactivation without substantial improvements.
Review Publications
Annous, B.A., Buckley, D., Kingsley, D.H. 2021. Efficacy of chlorine dioxide gas against hepatitis A virus on blueberries, blackberries, raspberries, and strawberries. Food and Environmental Virology. 13,241-247. https://doi.org/10.1007/s12560-021-09465-1.
Kingsley, D.H., Annous, B.A. 2021. Evaluation of SDS and GRAS liquid disinfectants for mitigation of potential HAV contamination of berries. Journal of Applied Microbiology. 15123. https://doi.org/10.1111/jam.15123.
Richards, G.P., Watson, M.A., Madison, D., Soffer, N., Needleman, D.S., Soroka, D.S., Uknalis, J., Baranzoni, G., Church, K.M., Poison, S.W., Elston, R., Langdon, C., Sulakvelictze, A. 2021. Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, characterization and remediation of larval oyster mortalities. Applied and Environmental Microbiology. 87. https://doi.org/10.1128/AEM.00008-21.
Parveen, S., Jacobs, J., Ozbay, G., Chintapenta, L., Almuhaideb, E., Meredith, J., Ossai, S., Abbott, A., Grant, A., Brohawn, K., Chigbu, P., Richards, G.P. 2020. Seasonal and geographical differences in total and pathogenic vibrio parahaemolyticus and vibrio vulninficus levels in seawater and oysters from the Delaware and Chesapeake Bays using several methods. Applied and Environmental Microbiology. 86(23). https://doi.org/10.1128/AEM.01581-20.
