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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Microbial and Chemical Food Safety » Research » Research Project #439581

Research Project: Innovative Detection and Intervention Technologies Mitigating Shellfish-borne Pathogens

Location: Microbial and Chemical Food Safety

2023 Annual Report

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.

Progress Report
This is the second-year report for project 8072-42000-090-000D entitled “Innovative Detection and Intervention Technologies Mitigating Shellfish-borne Pathogens”. The most common bacterial and viral causes of shellfish-associated illnesses in the United States are Vibrio parahaemolyticus (Vp) and human norovirus, which are most frequently associated with the consumption of raw or lightly cooked oysters. A multitude of different Vibrio species are naturally present in oysters and seawater, but only a few are known human pathogens with Vp at the top of the list. One of the goals of this project is to identify naturally occurring predators of Vp that might be useful in the development of a new processing technology to reduce Vp in market oysters. Toward this goal, we evaluated the effectiveness of four known predatory bacteria known as Halobacteriovorax (HBX) and five bacterial viruses known as bacteriophages to infect and kill 23 human pathogenic strains of Vp. Under Objective 1, a mixture (cocktail) of two HBX that ARS isolated from seawater from the Gulf Coast of Alabama and from Hawaii was formulated. This cocktail effectively killed all 23 strains of clinically relevant Vp. Our initial plan was to include one or more bacteriophages in the cocktail, but current research showed that bacteriophages isolated from the Delaware Bay had very narrow specificity toward different Vp strains. Their lack of broad specificity and the ability of the HBX to broadly infect Vp strains led to the decision to include only the HBX in the cocktail. A simple two-component cocktail will also be more economical to produce over more complex cocktails if it is adopted for use in future oyster processing operations. Research will continue over the next two summers to determine the extent to which the cocktail reduces Vp in naturally contaminated oysters. Under Objective 2, we evaluated potential methods to quantify HBX in oysters, seawater, and sediment. Past efforts at quantification have relied on filtration of seawater and sediment as well as oyster homogenates to remove solids and larger bacteria, while allowing the relatively small HBX to pass through the filters. We showed that this method also filters out most of the HBX, even in seawater, as the filters become blocked. A multi-tube, semi-quantitative assay method known as a most probable number (MPN) assay was evaluated to determine whether tubes containing sample dilutions (potentially containing HBX) plus added host VP could be used to detect viable HBX strains in environmental samples. Samples were inoculated into seawater followed by incubation for 3 days. Any HBX present would infect the added Vp, and then a small amount of culture was dotted or streaked onto agar plates containing Vp cells in the medium. Plates were incubated and then examined for clear spots (plaques) where live HBX are replicating. This is confirmation of viable HBX presence. Disadvantages of this test are that it is very labor intensive, slow (incubation takes 10-14 days), and only provides approximate counts. Another possible method was explored in the quest for better quantification of HBX. It involved an enzyme (serine hydrolase) produced by HBX that is reportedly capable of digesting a biopolymer, called medium chain length polyhydroxyalconate (mcl-PHA), which is produced within some bacteria. A novel method was evaluated where a small amount of mcl-PHA was dissolved in acetone and a drop was placed along the inside of a glass test tube and allowed to drip down the inside of the tube. Over several minutes, the acetone evaporated, leaving a translucent white streak down the inside of the tube. Tubes were sterilized and then sterile seawater was placed in the tube to a level where the bottom half of the plastic was covered, but the top half was not. Vibrio and predatory HBX bacteria were inoculated into the seawater and the mcl-PHA streak was monitored daily for potential degradation as HBX levels increased. A positive test would have been some visual degradation or possible break in the plastic. Unfortunately, the mcl-PHA showed no evidence of digestion over a week-long incubation period, so this effort was discontinued. The enzyme was perhaps too dilute to digest the plastic within the timeframe allowed. An additional method was evaluated involving the use of a deep blue dye known as resazurin which reportedly is converted by HBX to a pink byproduct. In liquid and solid (agar) cultures of HBX and VP, no visible color change was apparent. Consequently, to date, no truly quantitative method has yet been developed for viable HBX in oysters or their environment. Under Objective 3, we conducted studies on the host range of HBX isolates toward Vp. Data from that study was instrumental in formulating the cocktail mentioned in Objective 1, above. Other research under Objective 3 involved studies to determine whether HBX accumulates and replicates within specific Vibrio-infected oyster tissues. Delaware Bay oysters were obtained and challenged in aquaria with both Vp and HBx and monitored for tissue contamination. Results showed that oyster gills and digestive tissues, readily accumulated the vibrios. HBX added to the water was detected in low levels in both the gills and digestive tissues, but the lack of methods to accurately quantify the levels in tissues was problematic. Low levels of bacteriophages naturally present in the oysters also replicated within and killed some Vp, further complicating an evaluation of the effects of HBX on the Vp levels. Under Objective 6, the potential to enhance the effectiveness of X-ray and E-beam treatments against viruses was investigated. Although irradiation is very high energy, viruses, being small targets, are resistant and require doses much higher than what is needed for most bacteria. Experimental evidence indicates that the foodborne viruses are susceptible to singlet oxygen, a short-lived form of molecular oxygen that is highly reactive. This form of oxygen is generated in response to lower energy visible and ultraviolet (UV) irradiation particularly when edible singlet oxygen enhancers, such as riboflavin (vitamin B2), are present. Furthermore, ionizing irradiation tends to deplete riboflavin in foods suggesting that riboflavin may absorb energy from radicals generated during irradiation. To test the hypothesis that high energy irradiation might convert molecular oxygen to reactive oxygen species in the presence of singlet oxygen enhancers, virus stocks of murine norovirus and Tulane virus, two research surrogate viruses for human norovirus were treated by E-beam and X-ray. Unfortunately, there was no discernable difference observed between virus-treated stocks in the presence or absence of riboflavin.


Review Publications
Marcano Olaizola, A., Kuis, R., Johnson, A., Kingsley, D.H. 2022. Stimulated raman generation of aqueous singlet oxygen without photosensitizers. Photochemistry and Photobiology. 235.
Kingsley, D.H., Chang, S., Annous, B.A., Pillai, S.D. 2022. Evaluation of riboflavin as an enhancer for X-Ray and EBeam irradiation treatment of Tulane virus. Radiation Physics and Chemistry.