Projects

Sea Grant Aquaculture Research Program 2010: Eliminating human-pathogenic Vibrio vulnificus from Gul

Objectives

The overall objective of this project is to test the efficacy of depuration under high salinity conditions in eliminating the human pathogen Vibrio vulnificus. We proposed:

  1. To construct a salinity-control recirculating aquaculture system (RAS).
  2. To test the efficacy of high salinity depuration using hatchery reared oysters.
  3. To test the efficacy of high salinity depuration using commercial oysters.

Methodology

Objective 1: to construct a high salinity depuration system. A salinity controlled recirculating depuration unit will be constructed consisting of a 380 L depuration tank with particulate cartridge filters, ammonia removal media filter and UV light sterilization. Flow rate into the tank will not exceed 40 L/min to maximize UV efficiency. As water exits the tank it will flow through a 5 µm and 1 µm cartridge filter and then through an ammonia removal media filter before passing through a UV light sterilizer. This system will ensure elimination on V. vulnificus in water. Three of these depuration units will be constructed to identify optimal depuration salinity.

Objective 2: to test the efficacy of high salinity depuration using hatchery reared oysters. Experimental depuration trials will investigate three salinities: 15 ppt (1/2 full strength seawater), 25 ppt, and 35 ppt (near full strength seawater). Filtered seawater (1µm) will be salinity adjusted for each treatment with the addition of Instant Ocean salt. Temperature will be maintained at a consistent 25ºC. One year-old, hatchery reared oysters at the Auburn University Shellfish Laboratory (AUSL) (100 individuals) will be placed into a 380 L raceway tank on mesh trays suspended 13 cm above the tank bottom. At day 0, 3, 6, 10 and 14, oysters (12 individuals) from each tank will be randomly selected and analyzed for the presence of V. vulnificus. The Most Probable Number (MPN) method followed by hybridization with a species-specific oligonucleotide probe (vvhA) as recommended by FDA will be used to enumerate V. vulnificus counts (FDA, 2002). All bacterial samples will be analyzed in triplicate. Experimental trials will be replicated three times in a Randomized Complete Block Design. The standard error will be calculated for all of the treatment replications. To determine if difference between data set existed, the Randomized Complete Block Design test will be performed using one-way ANOVA analysis procedure in the statistical analysis system (SAS 9.1.3 for Windows; SAS Institute, Cary, NC, USA). Treatments will also be compared to oysters maintained in an ambient water flow though system as a control. After three runs we will reassess what salinity works best and refine treatments to identify optimal or best range of salinity. After identifying optimal salinity, in year two, we could hold salinity constant at the optimal and run replicates that vary temperature to identify optimal temperature.

Objective 3: to test the efficacy of high salinity depuration using commercial oysters. Commercially harvested oysters supplied by BonSecour Fisheries Inc (Bon Secour, AL) will be collected at the processing plant and transport immediately to the AUSL. Upon arrival, oysters will be scrubbed clean of any fouling organisms and rinsed with freshwater to remove any exogenous material. One hundred oysters will be placed into a 380 L raceway tank on mesh trays suspended 13 cm above the tank bottom and depurated as described above using the identified optimal salinity. Vibrio vulnificus detection and enumeration will be done following FDA protocols.

Rationale

Reportedly 54% of American consumers will eat more oysters if those oysters were depurated to increase food safety. Although the Federal Drug Administration (FDA)-approved post-harvest technologies do produce safe oysters all those methods transform delicious, live seafood into a processed food that typically does not meet consumer expectations. Depuration could help the oyster industry to meet market demands for fresh and safe oysters.

However, little information is available regarding the efficacy of varied methods for oyster depuration. The overriding objective of the present study is to test the efficacy of using post-harvest depuration through an inshore high-salinity recirculation system so that raw oysters can be delivered to the consumer and eaten safely without further processing.

Since 2007 our group has been working on assessing the efficacy of a flow-through system to eliminate the human pathogen V. vulnificus from Gulf Coast oysters. Similar to what has been reported by other groups, we found that elimination of artificially-seeded oysters with a laboratory grown strain of V. vulnificus can be accomplished. However, depuration of wild strains of V. vulnificus from oysters yielded highly variable results. Out of 11 depuration flow-through trials performed in 2008-2009 we achieved effective reduction in only six. We were able to show a strong positive correlation between low V. vulnificus numbers post-depuration and high salinity. Our previous results with flow-through depuration suggest that salinity is the key parameter in reducing V. vulnificus counts from oyster meat. However, a flow-though system with a salinity control unit (by means of mixing a brine solution for example) is unpractical since they require too much salt. We plan to depurate oysters using a recirculating aquaculture system (RAS) at constant high salinity throughout the depuration process.

Although our preliminary data showed variable success in reducing this pathogenic bacterium from oysters, we believe we have identified the main source of variation: salinity. This conclusion is also supported by previous studies showing success with offshore relaying of oysters. We intend to perform an exhaustive series of experiments under control conditions that will prove if depuration is indeed a valuable alternative to post-harvest methods.

Positive results could allow Gulf Coast oysters to be depurated in the summer months and sold as a live, non-processed product, which will satisfy consumer demands while increasing the safety of the Gulf Coast oysters.

The planned project falls under the focus area ‘Safe and sustainable seafood supply’. It has been recognized by Sea Grant that a healthy seafood industry requires development of new technologies and marketing tools to enhance quality assurance and profitability. Specifically, the Mississippi-Alabama Sea Grant Consortium (MASGC) has recognized the many challenges the seafood industry faces these days and has identified as one its goals ‘A healthy domestic seafood industry that harvests, produces, processes, and markets seafood responsible and efficiently (http://www.masgc.org)’. The development of a new high-salinity depuration system to eliminate the human pathogen V. vulnificus from oysters will aid the local Mississippi-Alabama oyster industry to produce a safe, naturally processed product. Critically, farmed oysters are an essential component of the Gulf of Mexico oyster industry and there is regional interest in developing intensive oyster aquaculture; this new industry would benefit tremendously from the proposed research. Additionally, this new technology will have a positive impact on the seafood processing industry, restaurants and retailers and the public. It has the potential to decrease the number of fatalities associated to the consumption of Gulf Coast oysters and to improve the image and marketing of oysters grown and harvested in the region.