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:
Bacteriophages (phages) are bacterial viruses that are the most prevalent life form on Earth. They are found in high numbers in the oceans of the world, in fresh and terrestrial environments and in (and on) animals including humans. The isolation of phages against fish pathogens has led to their use in various aquaculture applications to treat diseases in fish and shellfish in order to reduce mortalities. ARS has been instrumental over the past few years in isolating and characterizing phages against larval shellfish pathogens, principally Vibrio coralliilyticus, and has developed a treatment with potential commercial applications. Under the milestone “Complete sequencing of phages against fish vibrios and perform bioinformatic analyses”, ARS has completed DNA sequencing and bioinformatics analysis of phages against the shellfish pathogens Vibrio coralliilyticus and Vibrio tubiashii, another larval oyster pathogen. This research was performed under a Cooperative Research and Development Agreement (CRADA). ARS studies included the complete sequencing and assembly of phage DNAs followed by bioinformatics analyses to determine if there were deleterious genes within the genomes – genes that could preclude the phages’ use in commercial applications. A mixture (cocktail) of these phages was found to reduce larval oyster mortalities by nearly 100% in some applications. We determined that two additional V. coralliilyticus strains (known as OCN008 and OCN014), which are potent coral pathogens, were also highly pathogenic toward larval oysters especially at slightly elevated seawater temperatures. ARS submitted and published a manuscript on that this year. In order to develop a cocktail that would encompass the elimination of these two additional vibrios along with six other V. coralliilyticus that are maintained by ARS, additional phages with broader host specificity were sought from Hawaiian seawater. In addition, East Coast oysters were screened monthly for 6 months for phages against V. coralliilyticus. Only one phage with broad host specificity was identified and that was from the Hawaiian seawater. Sequencing of that phage genome relied first on DNA sequencing of the host V. coralliilyticus strain (RE22) in which the phage was propagated so that the phage DNA sequence could be distinguished from the V. coralliilyticus RE22 sequence. Consequently, the complete genome sequence for V. coralliilyticus RE22 was determined and that led to the successful assembly and bioinformatics analysis of the new phage. Bioinformatics analyses were performed using on-line search engines (BLAST and PHASTER searches) on all the phage candidates. Results showed that one of the three phages in the treatment cocktail contained unacceptable genetic traits, necessitating its removal, while the new phage displayed broad specificity toward V. coralliilyticus strains and did not have deleterious genes. Consequently, it was included in the new cocktail. Host specificity testing revealed that this latest generation cocktail contained phages that could combat all eight V. coralliilyticus strains in the ARS culture collection and a V. tubiashii strain, making this cocktail highly desirable for commercialization. An invention disclosure entitled “Bacteriophages cocktail to reduce larval shellfish mortalities caused by vibrios in hatchery settings” was submitted on this work. Another group of phages, termed male-specific coliphages, like the MS-2 RNA phage and the M13 DNA phage, are commonly associated with the intestinal tract of warm-blooded animals including humans, so their presence in freshwater, seawater, and foods can serve as an indicator of sewage pollution. These phages are similar in basic composition to human enteric viruses, such as norovirus and hepatitis A virus, and may persist in the environment where they bioconcentrate within shellfish in a manner analogous to enteric viruses. There is interest by researchers and shellfish regulators in using these common phages as potential indicators for the presence of pathogenic enteric viruses in water and shellfish as well as in other foods. Currently under the National Shellfish Sanitation Program (NSSP), there is a mandatory 3 week harvest closure after a flood event or a sewage release. However current NSSP regulations stipulate that if male-specific coliphage is demonstrated to be low enough within shellfish meats that this mandatory closure may be lifted by individual state authorities at their own discretion. It is generally understood that human enteric viruses and phages do not readily depurate (purge) from oysters when placed in clean seawater. Reductions in virus levels are known to be substantially affected by water temperatures. Under the milestone “Complete evaluation of MS-2 phage as a surrogate for human norovirus in live oysters”, we characterized human norovirus, MS-2 and M13 coliphage reductions in oysters at cool, moderate and warm seawater temperatures to provide regulatory authorities with more insight into how enteric virus levels in shellfish may potentially correlate with male specific coliphage. Human norovirus and MS-2 work has been completed and published. Results indicate that MS-2 does behave substantially like human norovirus although norovirus appears to be slightly more persistent within oyster meats than MS-2. An M13 phage study is nearing completion with publication submission anticipated in the fall of 2018.
1. Identified cocktail of phages to prevent larval oyster mortalities. Shellfish hatcheries have experienced dramatic losses in larval oysters due to certain Vibrio bacteria. Losses reduce the availability of seed oysters needed by the commercial shellfish industry. ARS researchers at Dover, Delaware, have identified a host of vibrios that kill larval oysters and corals and identified bacterial viruses (phages) which broadly infect and kill these vibrios. A mixture (cocktail) of these phages effectively knocked out the vibrios and greatly enhanced the survival of larval oysters in a hatchery setting. Commercialization of the cocktail is anticipated soon.
2. Potential interventions for virus-contaminated berries. Enteric virus contamination is a serious concern for the fruit industry. ARS researchers at Dover, Delaware, have shown that gaseous chlorine dioxide inactivates viruses on blueberries with limited impact on berry color. This may offer a viable means of making berry fruits safer. Evaluation of several other berry types in large scale is now ongoing.
Kehlet-Delgado, H., Richards, G.P., Hase, C., Mueller, R.S. 2017. Three draft genomes of Vibrio coralliilyticus strains isolated from bivalve hatcheries. Genome Announcements. Volume 5: Issue 41 e01162-17.
Ushijima, B., Richards, G.P., Watson, M.A., Schubiger, C.B., Hase, C.C. 2018. Factors affecting infection of corals and larval oysters by vibrio coralliilyticus. PLoS One. https://doi.org/10.1371/journal.pone.0199475.
Kingsley, D.H., Perez, R., Niemira, B.A., Fan, X. 2018. Evaluation of gaseous chlorine dioxide for the inactivation of tulane virus on blueberries. International Journal of Food Microbiology. 273:23-32.
Kingsley, D.H., Perez, R.E., Boyd, G., Sites, J.E., Niemira, B.A. 2018. Evaluation of 405 nm monochromatic light for inactivation of tulane virus on blueberry surfaces. Journal of Applied Microbiology. 124:1017-1022.
Kingsley, D.H., Chen, H., Meade, G.K. 2017. Persistence of MS-2 bacteriophage within Eastern Oysters. Food and Environmental Virology. 10:83-88.
Kingsley, D.H., Fay, J., Calci, K., Pouillot, R., Woods, J., Chen, H., Niemira, B.A., Van Doren, J.M. 2017. Evaluation of chlorine treatment levels on inactivation of human norovirus and MS2 bacteriophage during sewage treatment. Applied and Environmental Microbiology. 83:e01270-17.
Kingsley, D.H. 2017. High pressure processing's potential to inactivate norovirus and other fooodborne viruses. In: Nova Science Publishers. Noroviruses: Outbreaks Contral and Prevention Strategies. Jesus Romalde. p. 173-221.