Vibrio parahaemolyticus (Vp) is a bacterium that is naturally found in estuarine and marine waters around the world. This organism can accumulate in filter-feeding organisms, such as oysters, especially during summer months. Human pathogenic strains in shellfish caused dozens of cases of food borne illness in 1997-98 and 2006, and continue to be responsible for sporadic outbreaks. While existing procedures to control Vp contamination in shellfish are generally effective in reducing the number of outbreaks, they are inefficient methods for oyster culturing in Washington and elsewhere, where oysters are predominantly cultured on the bottom on cultch or as single oysters. The demand for raw oysters during the summer months is also increasing rapidly. Therefore, cost-effective techniques to depurate Vp would greatly assist the industry.
The overall goal was to test Pacific oysters (Crassostrea gigas) under varying water temperatures and immersion times within a laboratory-scale recirculating depuration system equipped with a generator of electrolyzed oxidizing water and measure the effects of these treatments on Vp loading in the oysters. Generation of bactericidal oxidizing molecules is a technology now available that is similar to but an improvement over conventional ozone generators. The following objectives were tested to address Vp depuration. 1) To test a depuration system which uses the generation of oxidation products for bactericidal effects effective at reducing concentrations of pathogenic Vp bacteria; 2) To evaluate time and temperature conditions for depuration under controlled tank conditions to facilitate a significant reduction of Vp by addition of bactericidal oxidants in a closed system; and 3) To determine the specific locations of Vp bioaccumulation in oyster tissues after direct injection and water bath incubation using a fluorescently labeled antibody. This summary applies only to the results of objectives 1 and 2.
Bacterial cultures preparation: Five clinical strains of Vp [10290 (O4:K12), 10292 (O6:K18), 10293 (O1:K56) 1997 Washington outbreak strain, BE98-2029 (O3:K6) 1998 Texas outbreak strain, and 027-1C1 (O5:K15) 1997 Oregon outbreak strain] were individually enriched and pelletized. Cell pellets were resuspended in 50 ml of NaCl solution (1%) to produce a culture cocktail.
EO water production: EO water containing chlorine contents of approximately 30 ppm (pH 2.8-2.9) was produced with an electrolyzed water generator (Model V-500, Electric Aquagenics Unlimited, Inc., Lindon, UT) according to manufacturer's instructions. Salt (NaCl) was added to the EO water to reach a concentration of 3% NaCl and the water was used within 20 min after production.
Oyster Preparation: Pacific oysters were obtained from an oyster farm in Willapa Bay, Washington. The oysters were placed in artificial seawater (ASW) at room temperature for 3-4 h before being inoculated with Vp. Naturally contaminated Pacific oysters were obtained from Hood Canal of Washington. These oysters were left at ambient temperature for about 18 h before treatments to allow growth of Vp in oysters.,
Bacterial inoculation: Oysters obtained from Willapa Bay were transferred from ASW to a tank of fresh ASW containing Vp. The inoculation was conducted overnight with water being circulated at a flow rate of about 10L/h. Oysters and ASW were analyzed for Vp before the inoculation process.
Oyster depuration: Oyster depurations were conducted using two identical laboratory-scale recirculating systems each equipped with a 15W Gamma UV sterilizer (Aquatic Eco-Systems) at four temperatures (22, 15, 10, and 5C). For each temperature process, 40 laboratory-inoculated oysters were depurated in 40 liters of recirculating ASW or EO water (30 ppm chlorine) containing 3% NaCl at circulating flow rate of 25 L/min. Populations of Vp in oysters were analyzed at 0, 3, 6, 9, 12, 24, 36, and 48 h during the process. Both ASW and EO water were replaced with freshly prepared solutions at each sampling time. Naturally contaminated oysters were depurated in ASW at 5C and analyzed for Vp every 12 h for up to 96 h.
Microbiological tests: At each sampling time, 5 oysters from each process were picked for analyzing Vp with a 3-tube most probable number (MPN) method. The oysters were shucked with a sterile shucking knife in a sterile stainless tray. Each shucked oyster meat was placed in a sterile blender jar and blended with 9 volumes of sterile alkaline peptone water (APW) at high speed for 1 min to prepare a 1:10 sample suspension. Additional ten-fold dilutions of each oyster homogenate were prepared with sterile APW. All sample dilutions were individually inoculated into test tubes containing alkaline peptone salt broth (APS). The inoculated APS tubes were incubated at 35-37°C for 16-18 h. One loopful (3 mm) of the enriched APS from the top 1 cm of a turbid tube was streaked onto individual thiosulfate-citrate-bile salts-sucrose agar (TCBS, Difco Laboratories, Sparks, MD) plates. The plates were incubated at 35-37°C for 18-24 h. Formation of colonies that are round (2-3 mm diameter) and green or bluish on the plates was considered positive for Vp. Total populations of Vp in oysters were determined by converting numbers of APS tubes that were positive for Vp to MPN/g using a MPN table. Results were reported as means of triplicate determinations (excluding the highest and lowest results).
Statistical analysis: Results of microbiological tests were transformed into log10 values for statistical analysis. Bacterial populations at different times of each treatment were analyzed with two-sample t test (S-Plus, Insightful Corp., Seattle, WA) with significant differences established at P=0.05.
Contamination of Vp in oysters could be reduced by about 90% after 6 h of EO water depuration at room temperature (22C). Decreasing water temperatures did not enhance the efficacy of EO water in reducing Vp in oysters. However, depuration with ASW at temperatures below 15C greatly increased the efficacy in decontamination. Depuration with ASW at 5C for 48 h resulted in 3.65 log MPN/g of reduction of Vp in laboratory-inoculated oysters and 2.89 log MPN/g of Vp in naturally contaminated oysters. While more experiments are needed to determine the efficacy of low temperature depuration in reducing Vp in oysters in a commercial operating system, refrigerated live holding at a central processing facility may be an attractive alternative if the alternative is costly fleets of refrigerated boats and trucks required to ship live oysters in the summer months.
Publications and presentations of project results are available. The materials with links below may be viewed in PDF format (file sizes are indicated).