Objective 1. Explore the use of a cocktail containing phages and predatory bacteria to kill Vibrio parahaemolyticus in market oysters. Objective 2. Compare and contrast Halobacteriovorax and phage levels in oysters, seawater, and sediment as a prerequisite to the development of future prediction and forecast models for pathogenic vibrios in market oysters. Objective 3. Probe the biology, host range, and infectivity of predatory bacteria to optimize their potential use as treatment against shellfish-borne vibrios. Objective 4. Develop novel and comprehensive methods for virus detection in shellfish that may also have potential for other foods. Objective 5: Identify novel in vitro propagation methods for human norovirus and hepatitis E virus. Objective 6: Evaluate inactivation technologies for virus-contaminated shellfish and other foods.
Under objective 1, a cocktail of phages and predatory bacteria will be formulated from isolates collected during surveys of Delaware Bay oysters. Cocktail effectiveness in eliminating V. parahaemolyticus (VP) from seawater will be tested followed by efficacy testing of the cocktail against VP in naturally-contaminated, market-size oysters. Under objective 2, a quantitative Halobacteriovorax (HBX) assay will be developed using a most probable number (MPN) based approach to quantify HBX in seawater, oysters and marine sediments. Positive tubes will be determined by plaque assay. Alternative, enzyme-based assays will also be explored. In the second phase of this objective, information will be collected on HBX and total and pathogenic VP abundances in oysters, seawater and sediments for the development of future prediction and forecast models for pathogenic vibrios in oysters. Phage abundances will also be monitored by plaque assay. The goal of objective 3 is to further our understanding of factors that affect the biology, host range and infectivity of predatory bacteria under various environmental conditions and how HBX impact pathogenic VP levels in oysters and their environment. Among questions to be answered are: whether HBX replicates within oyster gut or gill tissues; what is the generation time for HBX in VP; do HBX persist or die in the absence of host vibrios, do environmental conditions (temperature, salinity, or pH) affect HBX infection and replication within host cells, and what is the host range of HBX isolates. Under objective 4, metagenomics will be used to detect viruses in shellfish. The principal challenges and limitations will be sample preparation and sensitivity, so several virus extraction procedures will be investigated. All methods will be evaluated for purity and yield of virus RNA using shellfish samples seeded with surrogate viruses. Laboratory-spiked shellfish and wild shellfish impacted by sewage outfalls or from other areas prone to contamination will be evaluated. The goal of objective 5 is to identify novel in vitro propagation methods for human norovirus (HuNoV) and hepatitis E virus (HEV). Two established embryonic cell lines from zebrafish will be investigated for HuNoV replication. After incubation for up to 2 weeks, virus yields will be determined by RT-qPCR. The feasibility of a surrogate trout HEV assay will also be investigated as a potential model system for HEV inactivation. Trout HEV will also be evaluated in nonthermal virus inactivation studies. Other potential HEV cultivation techniques will be investigated to assess the infectivity and inactivation of genotype 3 zoonotic HEV including a 3-dimensional, microgravity culture system. Under objective 6, inactivation technologies for virus-contaminated shellfish will be evaluated including: high pressure processing (HPP) of frozen oysters to reduce or eliminate HuNoV; the use of X-rays with and without singlet oxygen enhancers to inactivate surrogates for HuNoV, hepatitis A virus and HEV; and targeted heating with infrared or radiofrequency to eliminate viruses and bacteria in specific shellfish tissues.
This is the first report for project 8072-42000-090-000D, which began on January 31, 2021, entitled “Innovative Detection and Intervention Technologies Mitigating Shellfish-borne Pathogens”. Bacterial and viral contamination of molluscan shellfish, like oysters, clams, and mussels, constitute a serious health threat to the consumer of raw or lightly cooked products. Among the pathogens of significance are Vibrio parahaemolyticus (VP) bacteria and human norovirus. VP is recognized as the principal bacterial cause of illness associated with the consumption of shellfish. Vibrios are naturally occurring marine pathogens which proliferate to high levels in the U.S. and other coastal waters, particularly during the warm summer months. Levels fluctuate dramatically, thus making an assessment of their prevalence difficult. One potential cause for these fluctuations appears to be the presence of bacterial predators against VP and other Vibrio species. One of the major predatory bacteria is in the genus Halobacteriovorax (HBX). Under Objective 1, we made significant progress to understand the role of HBX in attacking different strains of VP and reducing their levels in seawater. Strains of HBX against vibrios were previously isolated by project scientists from seawater collected from Hawaii, Alabama, and the Delaware Bay. Under this year’s milestone, “to complete host specificity study with HBX”, ARS obtained twenty well characterized strains of clinically derived VP from the U.S. Food and Drug Administration (FDA) laboratory in Dauphin Island, Alabama under a material transfer agreement. These strains were obtained primarily from states on the Atlantic, Gulf, and Pacific coasts of the United States and from Hawaii. They represented different sequence types and serotypes of VP. All the strains were isolated from patients with VP infections. Our aim was to determine the susceptibility of the twenty VP to five different predatory HBX to examine whether some HBX were more effective in killing the VP than others. It has been widely speculated that HBX prefer to prey on bacteria that are from the same general habitat (location), so this study also included an evaluation of whether there was any difference in susceptibly of VP to infection by HBX isolated from similar and different locations around the country. Results showed that the HBX were broadly invasive toward nearly all the VP isolates; however, there were some interesting exceptions. For instance, one VP strain isolated from a patient in Hawaii was only preyed upon by the Hawaiian HBX isolate. Conversely, a VP isolated from a patient in Texas was preyed upon by all four HBX isolates from the continental U.S., but not by the Hawaiian strain. We also compared whether the VP sequence type or serotype influenced HBX infectivity. They do not appear to. Overall, the HBX seem quite capable of infecting a wide range of clinically important VP isolates and may be the reason for the rapid swings observed in VP levels in environmental samples over the summer months. Of special note is the fact that HBX only persist at high levels in seawater when their Vibrio host is present. When HBX kills off most of the VP in laboratory cultures, the HBX levels rapidly diminish. Consequently, it appears that there is a cycling phenomenon occurring in coastal waters and likely in the shellfish themselves where VPs increase first, which prompts the HBX to increase, and then as the VP levels decline to low levels due to predation from HBX, the HBX start to decline. This cycling phenomenon likely protects both VP and HBX from extinction so that this cycle can continue repeatedly throughout the summer months. Results from this study will allow us to formulate a cocktail of HBX strains that are most effective against pathogenic VP strains. Future work under this project will evaluate the use of cocktails containing HBX and bacteriophages for the development of new processing technologies to kill VP in market oysters. Human norovirus is the most common source of food-borne illness in the United States. Previously, we showed that high pressure processing inactivates noroviruses in oysters and that initial pressurization at refrigeration temperatures enhance the inactivation of noroviruses. Under Objective 6, we evaluated whether noroviruses would be inactivated by high pressure processing at an ultralow temperature (-40 degrees Celsius). Results showed that norovirus inactivation was substantially greater at an initial pressurization temperature of -40 degrees Celsius compared to pressurization at refrigeration temperatures slightly above freezing. This was observed for both murine norovirus and Tulane virus, which serve as surrogates for human norovirus. These findings suggest that high pressure processing may provide a means to inactivate human noroviruses within frozen shellfish and other foods. Demonstration of inactivation of these viruses directly within frozen oyster meats will be the subject of future research under this project.
1. Naturally occurring marine predatory bacteria kills major human pathogen (Vibrio Parahaemolyticus). Vibrio parahaemolyticus (VP) is the most common source of seafood-associated bacterial illness in the United States, causing closures of shellfish harvesting areas, recalls of products, and countless sick consumers. ARS scientists in Dover, Delaware, identified naturally occurring marine predatory bacteria known as Halobacterioxorax (HBX), which tunnel into VP and readily kill them. ARS obtained from the U.S. FDA twenty strains of known pathogenic VP isolated from sick individuals from widely varying geographic locations and determined whether five strains of HBX, which ARS isolated from Mid-Atlantic, Gulf Coast and Hawaiian seawater, could infect and kill these VP. Most of the VP strains were readily infected and killed by the HBX regardless of where the VP and HBX originated from, thus refuting previous suspicions that HBX from one locale prefer prey (VP) from the same locale. This broad specificity of HBX toward clinically relevant VP strains further supports ARS’s plan to develop new disinfection technologies to enhance shellfish safety by using HBX to kill pathogenic VP in market oysters.
Madison, D., Richards, G.P., Sulakvelidze, A., Langdon, C. 2022. Bacteriophages improve survival and metamorphosis of larval Pacific oysters (Crassostrea gigas) exposed to Vibrio coralliilyticus (strain RE98). Aquaculture. 555(2022)738242. https://doi.org/10.1016/j.aquaculture.2022.738242.