Progress 09/01/19 to 08/31/24
Outputs
Target Audience:The audience for this grant project includes a diverse group of stakeholders, primarily within the seafood industry, such as shellfish producers and oyster farmers who are directly impacted by contamination risks in oysters. These industry stakeholders are integral to the implementation of innovative depuration methods, which aim to reduce pathogens and improve food safety in seafood products. Additionally, the project engages with broader scientific and regulatory communities focused on foodborne illness prevention, public health, and sustainable aquaculture practices. The project also reaches academic communities, particularly those training future professionals in food safety and aquaculture, while contributing to the development of new, cost-effective, and environmentally sustainable methods for ensuring safer seafood production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A postdoc and later PhD student worked on this project. The latter is to defend his dissertation on March 25th 2025 and should also submit this work to the journal Aquaculture soon. How have the results been disseminated to communities of interest?As in previous reports noted, we have extensively spoken at national conferences about this project and the publication is pending. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The project initially aimed to explore probiotic depuration as a method for improving food safety in oysters, with a focus on industry collaboration and real-world applicability. Key stakeholders were identified as partners to facilitate the integration of probiotic depuration into commercial shellfish processing. The research was designed to address Vibrio spp. contamination in oysters, a critical issue for public health and seafood industry sustainability. As the project progressed, the research placed greater emphasis on evaluating the efficacy of depuration and identifying the most effective conditions for its application in a commercial facility to reduce Vibrio parahaemolyticus in oysters. This shift was driven by significant gaps in both existing research and federal depuration guidelines that needed to be addressed. Establishing a foundational understanding of depuration processes was essential before assessing the potential role of probiotics. Our findings contribute to filling these critical knowledge gaps, and our study is anticipated to be published in Aquaculture later this year. In essence, we accomplised to compare thedepuration efficacy across three oyster species--Crassostrea gigas, C. sikamea, and C. virginica--and tested the suitability of non-pathogenic V. parahaemolyticus as a surrogate for commercial validation studies. Using a pilot-scale recirculating depuration system, the research successfully demonstrated that depuration at 11°C achieved a significant (>3.0-log CFU/mL) reduction of V. parahaemolyticus in C. gigas and C. sikamea within five days. Additionally, the findings supported the use of non-pathogenic V. parahaemolyticus strains for future industry-scale validations. This phase of the project provided critical data on depuration efficiency under controlled conditions, offering insights into species-specific differences in bacterial clearance rates. The results confirm that depuration is an effective method for reducing V. parahaemolyticus in oysters, though adjustments to temperature and duration may be necessary to meet food safety targets across different oyster species. These findings serve as a foundation for further research into probiotic applications and other enhancements to depuration methods.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lunda S, Burroughs S, Hesser J, Dewey B, DePaola A, Waite-Cusic J, Schubiger C. Pilot-scale depuration of three species of oysters demonstrates varying rates of reduction of Vibrio parahaemolyticus. 72nd Annual Pacific Fisheries Technologists Conference, February 20-23, 2022, poster. (Awarded best student poster presentation).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lunda S, Burroughs S, Hesser J, Waite-Cusic J, Schubiger C. Pilot-scale depuration demonstrates the suitability of non-pathogenic Vibrio parahaemolyticus as a surrogate for commercial-scale validation studies. World Aquaculture Society 2022 Triennial Meeting, February 28 March 4, 2022, oral presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Pilot-scale depuration demonstrates the suitability of non-pathogenic Vibrio parahaemolyticus as a surrogate for commercial-scale validation studies. Pacific Coast Shellfish Growers Association 76th Annual Conference, September 20-22, 2022, oral presentation.
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2025
Citation:
Tentative Title: Optimizing oyster production: Improving food safety, disease resistance, and investigating the microbiome. Will be submitted and defended by March 25, 2025.
- Type:
Other Journal Articles
Status:
Submitted
Year Published:
2025
Citation:
Spencer L. Lunda, Samantha Kilgore, Jennifer M. Hesser, Joy G. Waite-Cusic, Carla B. Schubiger: Pilot-scale depuration demonstrates the suitability of non-pathogenic Vibrio parahaemolyticus as a surrogate for commercial-scale validation studies. (Submitted to biorxiv March 6th, 2025; assigned number BIORXIV/2025/641953; this is subsequently routed for transfer for publication in the peer-reviewed journal PLOS ONE with the same title and author list).
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
No work was conducted during this project report as the project has concluded. No money was spent.
Publications
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Target audiences are oyster producers as we are establishing effective and efficient post-harvest processing technology to help make oysters safer for raw consumption and ultimately reduce seafood-borne illesses and product recalls. In addition, this work might inform FDA and programs of the Interstate Shellfish Sanitation Conference (ISSC). Particularly the National Shellfish Sanitation Program NSSP which is a federal and state cooperative program seeking to improve the sanitation of shellfish destined for human consumption and to provide the standards for raw or post-harvest processed shellfish. During this project period, we have interacted with industry (oyster farms and depuration facilites) and FDA. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Publication is ready to be submitted
Impacts
What was accomplished under these goals?
Dose dependency was tested in oysters using a variety of probiotic candidates that have shown effects in inhibiting or killing Vibrio parahaemolyticus on agar plates. Adding probiotics did not significantly increase depuration ratios. However, due to the fact that inocculation concentrations need to be very high to ultimately identify a 3.52 log reduction, the difference of probiotic to V. parahemolyticus was ultimately not high enough to see clear effects. For oysters sold in the United States, standards mandate a reduction of at least 3.52 log in V. parahaemolytius levels and a final concentration in treated oysters of < 30 MPN/g. Additionally, post-harvest processing (PhP) can allow for the sale of oysters harvested from sites currently closed due to the occurrence of V. parahaemolyticus-associated illness; a >3.0 log reduction is required to meet this standard for Pacific Coast oysters. A validation study plan approved by the ISSC is required to demonstrate the efficacy of the proposed PHP method. However, many assumptions in the FDA/ISSC regulations have not been tested and could render shellfish-depuration facilities validations problematic. For example, our investigations have revealed that to establish the mandated 3.52 log reduction, oysters have to be infected with articifically high concentrations of Vibrio parahaemolyticus in the order of up to 7log CFU per ml. In addition, to achieve final V. parahaemolyticus concentrations of less than 30 MPN/g is problematic as the pathogen seems to have migrated into mollusk tissues rendering continuous depuration without effects. Even up to seven days of constant depuration did not reveal a continous purge of Vibrio, instead depuration was most effective in the first 48 hours but then tapered off. This could be related to Vibrio only loosely associated with the internal surfaces of the oysters to be purged early and easily (oysters were rinsed in ethanol to eliminate Vibrio contamination from the shell), but a high amount of Vibrio might remain within tissues making them more resistant to depuration However, ultimately the methods established in this project period were successful to achieve a concentration of at least 5 log CFU/ml of V. parahemolyticus in three different species of oysters. However, it was noted that activity of oysters is of importance to accumulate pathogen by active filter feeding. This activity could be manipulated by changing water temperature, salinity, disturbances of the oysters, availability of food in the water (microalgae) etc. More work will be conducted in the next project period to assess this hypothesis.
Publications
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Our target audience are oyster producers as we are establishing effective and efficient post-harvest processing technologies that help make oysters safer for raw consumption and ultimately reduce seafood-borne illnesses. Changes/Problems:The pandemic keeps impeding our research due to us not being able to purchase necessary research items (or having them back-ordered) and use our research spaces. Some of our research spaces are in NOAA-owned buildings and these buildings only opened in early summer '21 and now at the beginning of fall '21, these spaces are closing again due to an uptick of covid -19 infections. What opportunities for training and professional development has the project provided?We had trained a postdoctoral scholar on this project for 12 months, however, due to the pandemic this time included some Covid related closures and quarantines,and she left at the end of Feb 2021. In the meantime, a graduate student has continued some of the work. In addition, we trained an undergraduate research intern during the summer. How have the results been disseminated to communities of interest?Since January 21, we have met regularly with an oyster producer (Taylor Shellfish) to discuss depuration. Also, we are currently preparing a manuscript for publication. What do you plan to do during the next reporting period to accomplish the goals?We need to isolate, screen, and test more probiotics to achieve the objectives. Also, we plan to publish 1-2 manuscripts during the next 12 months.
Impacts
What was accomplished under these goals?
In our last report, we explained that we changed course from the original probiotics (presented in preliminary data in the proposal). These probiotics,while providingsuccessful preliminary data, unfortunately, revealed to be from the genus Vibrio after sequencing.Due to the potential for horizontal gene transfer of virulent factors, we decided to instead test a probiotic cocktail that we developed in our lab for a different project with oyster larvae. Ourpostdoctoral scholar tested single and combination treatments of our original probiotics but wasn't able to either get sufficient levels of Vibrio parahaemolyticus into the oysters or did not observe a significant difference in depuration efficiency. We discovered that she had some technical mistakes in her work, so we repeated some of the postdoc's work, including trials with higher probiotic concentrations to avoid dose-dependency issues. While we were able to establish a new protocol that can be easily replicated, unfortunately, our cocktail that works generally well on oyster larvae and seems to increase their general vigor (possibly the immune response, this investigation is pending on another project), failed to produce significant results. However, we also noticed that probiotic applications are leading to somewhat variable results. It also seems that Vibrio parahemoyticus seems to integrate itself to a higher degree into the microbiome of the oyster gut, something that should be investigated in the future. In addition, we are still trying to pinpoint the factors that lead to the variability of our results. These factors possibly include subtle differences in the water quality (pH, temperature, oxygenation, salinity, etc.), bacterial population coming to oysters and bioparticles, and phages. Also, possible variabilities might develop from repeated freeze-thaw of freezer stocks. To avoid that, we will be trying to freeze-dry bacteria to have a more stable population upon reconstitution though we also acknowledge that freeze-drying itself can lead to problems. However, freeze-drying would be the preferred large-scale application form that would be advantageous because of the long shelf-life and easy application by the producers (no training or special equipment needed). We are currently trying to isolate probiotics that perform similarly to the original probiotics, which unfortunately turned out to be Vibrio species, which we think should not be used as probiotics, due to their potential to take up virulence genes via horizontal gene transfer (discussed in last years report). While probiotic isolation is underway, we established baseline procedures necessary for the large-scale application of depuration facilities. These facilities will need to be validated for their efficiency and safety. Several overarching idea concepts need to be evaluated. We were able to establish a protocol that leads to a consistent inoculation level in oysters and also works in the three most important US farmed oyster species,Pacific oyster (Crassostrea gigas), Kumomoto oysters (C. sikamea), and the Eastern oyster (C. virginica). We compared these species on Vibrio uptake and depuration under identical conditions. For the depuration we worked with an oyster farm to build a small-scale prototype of a depuration unit: The results were that: Gigas and Kumamotos take up and depurate Vibrios at a similar rate. Kumamotosmay depurate at a slightly higher rate than Gigas, but this may also be due to the generally smaller size of Kumos compared to Gigas (slightly higher beginning concentrations of Vibrio, so the overall depuration effect is slightly higher at the end of sampling). Virginica consistently took up less Vibrio, and also purged Vibrio at a slower rate than the other two species at the depuration temperatures tested. This could be due to the warmertemperatures preferred by Virginica - they appear to simply be less active, both in taking up Vibrio and in purging it. Further, in order for a facility to validate their depuration units, working with a non-human pathogenic V. parahaemolyticus cocktail as a surrogate would be essential due to health risks (aerosols, contaminated water, etc) and the difficulty to effectively sterilize large scale depuration tanks. Therefore, we also evaluated whether pathogenic and non-pathogenic V. parahaemolyticus cocktails get accumulated and depurated the same way. The non-pathogenic cocktail seems to depurate at either the same rate, or slightly slower, so it should serve as a conservative surrogate for pathogenic Vibrio strains. Since the non-pathogenic cocktail appears to be more conservative in depuration rate, this should mean that successful depuration of nonpathogenic strains would also indicate successful depuration of pathogenic strains. We also established whether oysters need to be individually infected or whether a batch infection protocol would work. The idea was that batch infected oysters would have highly variable concentrations of V. parahaemolyticus due to different filtering activities of some oysters or some oysters delayed filtering start. The hypothesis is also that the concentration of the pathogen in the surrounding water equals the concentration of the pathogen in the oyster. With batch vs. individual, we didn't see significant differences in either mean CFU/mL, or standard deviation for initial Vibrio uptake (which did not turn out to be true). However, it seems that batch inoculation works for inoculating the oysters - we did see some variable results later in the course of depuration (around day 3), but we also saw those with individual inoculation, so we are thinking that the oysters themselves are more variable in uptake of Vibrio, likely based on their microbiome. Also, it seems that some of the Vibrios might be added to the microbiome and are therefore very difficult to get below the required thresholds for depuration. We will spend more time evaluating this. In conclusion, despite the challenges and Covid roadblocks, we are very proud to have developed data that will be very applicable and important tothe oyster farming industry. And we will focus in the remaining time of this grant, on finding probiotics to increase the depuration efficiency. The data presented in this reportwill be published later this year.
Publications
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:The PD met with Oregon Oyster Farms (a potential stakeholder) early in this reporting period to talk about this project, the objectives, and their collaboration. Oregon Oyster Farms is a producer of oysters located upriver in the Yaquina Bay. Their location is at a traditional, historic location for oyster farming in the Pacific NW.Oregon Oyster Farms is providing us with market-ready oysters. We are picking them up at the oyster docks within a few hours of harvest. Due to COVID-19 outbreaks in the seafood community in Newport, oysters were unavailable for a few weeks. We have also been in contact with Taylor shellfish as a consultant on their depurationfacility project that was in progress of being builtthis spring. The COVID-19 pandemic seems to have halted their progress. Changes/Problems:Timeline changes: We searched and found a postdoctoral scholar suitable for this project during the summer 2019, unfortunately, the candidate decided early August to take another position. Our second candidate was not able to start until January 1 2020, due to graduation in December. Family reasons pushed her start date back to March 1 2020. In hindsight, I should have probably notified the program director about this delay, but at the time I was convinced we would make up for lost time. I like to point out that no funds were spent from this award until March 1st 2020, when the postdoc actually started. Unfortunately, two weeks after the start of the postdoctoral scholar, the COVID-19 shut down happened. The start of research has been a slow process because we had to find a new lab space (we previously worked in a room in an NOAA building, but NOAA as of End of August has not reopened). We now have a new lab, with a new biosafety hood and are happy with the postdoctoral scholar. We are excitedthat we are slowly getting back to somewhat normal research. And we havespent the time composing a review paper on depuration methodologies that we expect to submit in the next couple of weeks. Also while our oyster supplier was shut down due to COVID-19 outbreaks in the seafood community, we started engineering a GFP-tagged V. parahaemolyticus and V. vulnificus that will be very valuable to anyone studying these seafood borne pathogens. What opportunities for training and professional development has the project provided?We are training a postdoctoral scholar who is funded on this project. We have also had an undergraduate student involved that was helping composing a review paper on depuration technologies while we all worked remotely this summer. How have the results been disseminated to communities of interest?Mostly by personal interactions with two stakeholders so far. What do you plan to do during the next reporting period to accomplish the goals?First, we need to troubleshoot why our pathogen retention is half of what other study authors are seeing and then finish the optimization of V. parahemolyticus depuration (objective b). We are also currently engineering a Vibrio vulnificus and V. parahaemolyticus with a GFP chromosomal insert. Such tagged strains could advance and streamline further research done in our and other labs in the US.
Impacts
What was accomplished under these goals?
Genomic sequencing led to the revelation that the probiotics used in the preliminary data were non-pathogenic Vibrio. While there are labs that use Vibrio as probiotics in the growth of oyster larvae, recent publications and discussions made us pivot from those and decide to find non-vibrio probiotics for this project. The rationale is that 50% of all Vibrio species are pathogenic and that vibrios can readily acquire virulence factors via horizontal gene transfer. We decided that this is too risky and we are confident there are non-vibrio probiotics in the ocean that have modes of action against vibrios, such as competitive exclusion or direct inhibition. So our first goal was to screen our probiotic stocks that we are currently testing in another project for oyster larvae survival, growth, and settlement. This could be a two-fer if probiotics fed to oyster larvae early in their development, could remain in the oysters and prohibit or actively reduce human pathogenic Vibrio accumulation in adult oysters. When the water treatment control was compared against probiotic treatment, small improvements in Vp reduction were seen with most of the 8 non-vibrio probiotics tested. Our goal is to achieve the mandatory reduction of Vibrio parahaemolyticus of below 99.9698% (3.52 log) within 24 hours. D16 and B11 were both able to accomplish that after 24h in 30% and 25% of independent trials, respectively. A 48 h continuation of those trials did not perform better than when oysters were immersed in clean seawater. However, those results are encouraging as the probiotic concentration was relatively low and within -/+ log of the Vp infection dose (which was 10^7 CFU/ml infection pressure in the water). A bit puzzling is that our tested water sources can strongly reduce Vp concentration by immersion alone, this outperforms published data, such as Aagesen et al., 2018 who used a recirculation depuration system with UV sterilization and saw a pathogen retention of 56.4%+/-14.8 after 24h and 43.9%+/-13.7. Meanwhile, our pathogen retention is 90.52%+/- 15.9. This average is also lower than our previous preliminary data that was collected with urban distilled water vs the water that we are now sourced from the Oregon State University Hatfield Marine Science Center at the rural Oregon coast. This led us to compare different water sources as standalone immersion depuration. Granted, this is a very limited trial with 2 replicates; all different water sources led to a reduction of around the legal limit of approximately 99.97% with at least one replicate. The various water sources are filtered seawater from the Yaquina Bay (OSU SW), autoclaved OSU SW, artificial seawater made with distilled water from the Hatfield Marine Science Center, and instant ocean salts (artificial SW), and artificial seawater (SW) that was autoclaved.
Publications
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