Growth and survival of Vibrio parahaemolyticus in postharvest American oysters

Oysters at the retail stage of distribution generally contain greater densities of Vibrio parahaemolyticus than do oysters at harvest. The objective of this study was to determine the effects of postharvest storage at 26 and 3 degrees C on the growth and survival of naturally occurring V. parahaemolyticus in shellstock American oysters (Crassostrea virginica). Oysters were collected monthly from May 1998 through April 1999 from Mobile Bay, Alabama, and their V. parahaemolyticus densities were determined after 0, 5, 10, and 24 h of postharvest storage at 26 degrees C. After 24 h of storage at 26 degrees C, oysters were transferred to a refrigerator at 3 degrees C and analyzed 14 to 17 days later. V. parahaemolyticus numbers were determined by a direct plating method involving an alkaline-phosphatase-labeled DNA probe that targets the species-specific thermolabile hemolysin gene (tlh-AP) to identify suspect isolates. From April to December, when water temperatures at harvest were >20 degrees C, the geometric mean harvest density of V. parahaemolyticus was 130 CFU/g. When water temperatures were <20 degrees C, the geometric mean harvest density was 15 CFU/g. After harvest, V. parahaemolyticus multiplied rapidly in live oysters held at 26 degrees C, showing a 50-fold increase (1.7 log CFU/g) at 10 h and a 790-fold increase (2.9 log CFU/g) at 24 h (April through December). Average V. parahaemolyticus numbers showed a sixfold decrease (0.8 log CFU/g) after approximately 14 days of refrigeration. These results indicate that V. parahaemolyticus can grow rapidly in unrefrigerated oysters.     

Oyster-to-oyster variability in levels of Vibrio parahaemolyticus

This study examined the variability in the levels of total and pathogenic Vibrio parahaemolyticus in individual oysters. Twenty oysters were collected on three occasions (in June, July, and September 2001) from a site near Mobile Bay, Ala. Ten of these oysters were tested immediately, and 10 were tested after 24 h of storage at 26 degrees C. Levels of total and pathogenic V. parahaemolyticus were determined by alkaline phosphatase-labeled DNA probe procedures targeting the thermolabile hemolysin and thermostable direct hemolysin genes, respectively. Similar V. parahaemolyticus levels (200 to 2,000 CFU/g) were found in nearly 90% of the oysters (for all sampling occasions) prior to storage. The log-transformed densities (means +/- standard deviations) of V. parahaemolyticus in oysters immediately after harvest were 2.90 +/- 0.91, 2.88 +/- 0.36, and 2.47 +/- 0.26 log10 CFU/g for June, July, and September, respectively. After storage for 24 h at 26 degrees C, the mean V. parahaemolyticus densities increased approximately 13- to 26-fold. Before storage, pathogenic V. parahaemolyticus was detected in 40% (10 to 20 CFU/g) of the oysters collected in June and July but was not detected in any oysters collected in September. After storage, pathogenic V. parahaemolyticus was detected in some oysters at levels of > 100 CFU/g. These data should aid in the development of sampling protocols for oyster monitoring programs and in the determination of exposure distributions associated with raw oyster consumption.     

温暖月份零售带壳牡蛎中副溶血性弧菌的定量研究

为了解温暖月份零售带壳牡蛎中副溶血性弧菌 (VP)的污染情况 ,2 0 0 3年 4～ 8月在福建省福州和厦门两地共收集带壳牡蛎 113份 ,样品分别来自水产品批发市场 (18% ) ,零售市场 (4 6 % )和饭店 (36 % )。采用Vitek鉴定系统和最可能数法进行VP的定量分析。结果显示 ,带壳牡蛎中VP密度的几何均数为 6 0MPN 10 0g ,4 1 6 %的样品VP密度低于 30MPN 10 0g的最低检出限 ,仅厦门2个样品菌量超过 2 4 0 0 0MPN 10 0g。两个地区、不同采样点和不同月份之间样品VP密度的几何均数差别均有统计学意义 (P <0 0 1)。厦门样品污染菌量高于福州 ;批发市场样品菌量最高 ;5月份样品菌量最高 ,为 14 9MPN 10 0g ,而 6～ 8月样品菌量约为 4 0MPN 10 0g。零售环节带壳牡蛎VP的检出率较高。未来应加强对生食海产品中VP污染状况的监测。     

福建省带壳牡蛎中副溶血性弧菌的市场调查

为了解零售带壳牡蛎中副溶血性弧菌(VP)的污染情况，2003年4月～2004年3月每月在福建省福州和厦门两地收集带壳牡蛎，样品共252份，分别来自水产品批发市场(11％)、零售市场(50％)和饭店(39％)。采用Vitek鉴定系统和最可能数(MPN)法进行VP的定性和定量分析。结果显示，带壳牡蛎VP几何平均密度为46MPN，100g，46％的试样VP密度低于30MPN/100g的最低检出限，仅厦门2个试样菌量超过104MPIN/100g。两个地区、不同采样点和不同季节之间试样vP平均密度差别均有显性。厦门试样菌量高于福州；批发市场试样菌量最高；春季试样菌量(93MPN/100g)高于其它季节(约为40MPN/100g)。研究结果可以用于估计生食牡蛎人群VP的暴露量。     

Vibrio vulnificus and Vibrio parahaemolyticus in U.S. retail shell oysters: a national survey from June 1998 to July 1999.

From June 1998 to July 1999, 370 lots of oysters in the shell were sampled at 275 different establishments (71%, restaurants or oyster bars; 27%, retail seafood markets: and 2%, wholesale seafood markets) in coastal and inland markets throughout the United States. The oysters were harvested from the Gulf (49%). Pacific (14%), Mid-Atlantic (18%), and North Atlantic (11%) Coasts of the United States and from Canada (8%). Densities of Vibrio vulnificus and Vibrio parahaemolyticus were determined using a modification of the most probable number (MPN) techniques described in the Food and Drug Administration's Bacteriological Analytical Manual. DNA probes and enzyme immunoassay were used to identify suspect isolates and to determine the presence of the thermostable direct hemolysin gene associated with pathogenicity of V. parahaemolyticus. Densities of both V. vulnifcus and V. parahaemolyticus in market oysters from all harvest regions followed a seasonal distribution, with highest densities in the summer. Highest densities of both organisms were observed in oysters harvested from the Gulf Coast, where densities often exceeded 10,000 MPN/g. The majority (78%) of lots harvested in the North Atlantic, Pacific, and Canadian Coasts had V. vulnificus densities below the detectable level of 0.2 MPN/g; none exceeded 100 MPN/g. V. parahaemolyticus densities were greater than those of V. vulnificus in lots from these same areas, with some lots exceeding 1,000 MPN/g for V. parahaemolyticus. Some lots from the Mid-Atlantic states exceeded 10,000 MPN/g for both V. vulnificus and V. parahaemolyicus. Overall, there was a significant correlation between V. vulificus and V. parahaemolyticus densities (r = 0.72, n = 202, P < 0.0001), but neither density correlated with salinity. Storage time significantly affected the V. vulnificus (10% decrease per day) and V. parahaemolyticus (7% decrease per day) densities in market oysters. The thermostable direct hemolysin gene associated with V parahaemolyticus virulence was detected in 9 of 3,429 (0.3%) V. parahaemolyticus cultures and in 8 of 198 (4.0%) lots of oysters. These data can be used to estimate the exposure of raw oyster consumers to V. vulnificus and V. parahaemolyticus.     

Occurrence of Vibrio parahaemolyticus in Two Oregon Oyster-growing Bays

ABSTRACT: Occurrence of Vibrio parahaemolyticus in 2 Oregon oyster-growing areas (Yaquina andTillamook Bays) was studied from November 2002 to October 2003. Vibrio parahaemolyticus was detected in 15.0% of oyster, 20.0% of seawater, and 47.5% of sediment samples with very low levels of pathogenic strains being detected in oysters (≤3.6 most probable number [MPN] /g). The densities of total and pathogenic V. parahaemolyticus were higher in sediment (≤1100 and ≤43 MPN/g) than in seawater (≤15 and ≤3.6 MPN/100 mL) or oyster (≤43 and ≤3.6 MPN/ g). Densities of V. parahaemolyticus in both bays were positively correlated to water temperatures ( P < 0.01), with higher densities in samples being detected in summer, especially July and August. There was no correlation between the densities of V. parahaemolyticus and water salinity or the densities of V. parahaemolyticus and bacterial populations in seawater. Freshly harvested oysters should be kept at refrigeration temperatures to prevent rapid growth of pathogenic V. parahaemolyticus in contaminated oysters.     

Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters

The objectives of this study were to investigate the seasonal distribution of total and pathogenic Vibrio parahaemolyticus in the Chesapeake Bay oysters and waters, and to determine the degree of association between V. parahaemolyticus densities and selected environmental parameters. Oyster and water samples were collected monthly from three sites in Chesapeake Bay, Maryland from November 2004 through October 2005. During collection of samples, water temperature, salinity, turbidity, dissolved oxygen, pH, chlorophyll a, and fecal coliform levels in oysters were also determined. V. parahaemolyticus levels were enumerated by a quantitative direct-plating method followed by DNA colony hybridization; presence/absence was further determined by overnight broth enrichment followed by either standard colony isolation or real-time PCR. The thermolabile hemolysin (tlh) gene and thermostable direct hemolysin (tdh) gene were targeted for detection of total and pathogenic V. parahaemolyticus, respectively, for both direct plating and enrichment. The thermostable related hemolysin (trh) gene, which is a presumptive pathogenicity marker, was targeted only for the enrichment approach. By direct plating, colonies producing tlh signals were detected in 79% of oyster samples at densities ranging from 1.5x10(1) to 6.0x10(2) CFU/g. Pathogenic V. parahaemolyticus (tdh+) was detected in 3% (level was 10 CFU/g) of oyster samples while no V. parahaemolyticus was detected in water samples. By the enrichment approach with standard colony isolation, 67% of oyster and 55% of water samples (n=33) were positive for total V. parahaemolyticus, and all samples were negative for pathogenic V. parahaemolyticus. In contrast, enrichment followed by real-time PCR detected tlh, tdh and trh in 100%, 20% and 40% of oyster and 100%, 13% and 40% of water enrichments collected from June to October 2005, respectively. V. parahaemolyticus densities in oysters varied seasonally and were found to be positively correlated with water temperature, turbidity, and dissolved oxygen.     

Bactericidal effects of wine on Vibrio parahaemolyticus in oysters

The bactericidal effects of wines on Vibrio parahaemolyticus in oysters were studied to evaluate potential inactivation of V. parahaemolyticus in contaminated oysters by wine consumption. Shucked whole oyster and oyster meat homogenate were inoculated with V. parahaemolyticus and mixed with red or white wine. Survivals of V. parahaemolyticus in inoculated oysters were determined at 7 and 25 degrees C. Populations of V. parahaemolyticus in inoculated whole oysters (5.52 log most probable number [MPN] per g) decreased slightly to 4.90 log MPN/g (a 0.62-log reduction) after 24 h at 7 degrees C but increased to 7.37 log MPN/g over the same period at 25 degrees C. However, the populations in wine-treated whole oysters decreased by >1.7 and >1.9 log MPN/g after 24 h at 7 and 25 degrees C, respectively. Both red and white wines were more effective in inactivating V. parahaemolyticus in oyster meat homogenate than in whole oyster. Populations of V. parahaemolyticus in oyster meat homogenate (7.8 x 10(3) MPN/g) decreased rapidly to nondetectable levels (< 3 MPN/g) after 30 min of mixing with wine at 25 degrees C (a 3.89-log MPN/g reduction). These results suggest that chewing oysters before swallowing when eating raw oysters may result in greater inactivation of V. parahaemolyticus if wine is consumed. More studies are needed to determine the bactericidal effects of wine on V. parahaemolyticus in the complicated stomach environment.     

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.     

Response of Vibrio parahaemolyticus 03:K6 to a hot water/cold shock pasteurization process.

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments world wide. Pathogenic strains of these bacteria are often transmitted to humans through consumption of raw oysters, which flourish in the same estuaries. Previous studies reported the effective use of hot water pasteurization followed by cold shock to eliminate from raw oysters naturally and artificially incurred environmental strains of V. vulnificus and V. parahaemolyticus common to the Gulf of Mexico. The present study focused on the use of the same pasteurization method to reduce a highly process resistant Vibrio strain, V. parahaemolyticus O3:K6 to non-detectable levels. Oysters were artificially contaminated with 10(4) and 10(6) V. parahaemolyticus 03:K6 cfu g(-1) oyster meat. Contaminated oysters were pasteurized between 50 and 52 degrees C for up to 22 min. Samples of processed oysters were enumerated for V. parahaemolyticus O3:K6 at 2-min intervals beginning after the 'come-up time' to achieve an oyster internal temperature of at least 50 degrees C. The D value (D(52)deg C) was 1.3-1.6 min. V. parahaemolyticus O3:K6 proved more process resistant than non-pathogenic environmental strains found in Gulf of Mexico waters. A total processing time of at least 22 min at 52 degrees C was recommended to reduce this bacterium to non-detectable levels (< 3 g(-1) oyster meat).     

High salinity relay as a postharvest processing strategy to reduce vibrio vulnificus levels in Chesapeake Bay oysters (Crassostrea virginica)

In 2009 the U.S. Food and Drug Administration (FDA) announced its intention to implement postharvest processing (PHP) methods to eliminate Vibrio vulnificus from oysters intended for the raw, half-shell market that are harvested from the Gulf of Mexico during warmer months. FDA-approved PHP methods can be expensive and may be associated with unfavorable responses from some consumers. A relatively unexplored PHP method that uses relaying to high salinity waters could be an alternative strategy, considering that high salinities appear to negatively affect the survival of V. vulnificus. During relay, however, oysters may be exposed to rapid and large salinity increases that could cause increased mortality. In this study, the effectiveness of high salinity relay to reduce V. vulnificus to <30 most probable number (MPN) per g and the impact on oyster mortality were assessed in the lower Chesapeake Bay. Two relay experiments were performed during the summer and fall of 2010. Oysters collected from three grow-out sites, a low salinity site (14 to 15 practical salinity units [psu]) and two moderate salinity sites (22 to 25 psu), were relayed directly to a high salinity site (≥30 psu) on Virginia's Eastern Shore. Oysters were assayed for V. vulnificus and Vibrio parahaemolyticus (another Vibrio species of concern) densities at time 0 prior to relay and after 7 and 14 days of relay, using the FDA MPN enrichment method combined with detection by real-time PCR. After 14 days, both V. vulnificus and V. parahaemolyticus densities were ≤0.8 MPN/g, and decreases of 2 to 3 log in V. vulnificus densities were observed. Oyster mortalities were low (≤4%) even for oysters from the low salinity harvest site, which experienced a salinity increase of approximately 15 psu. Results, although preliminary and requiring formal validation and economic analysis, suggest that high salinity relay could be an effective PHP method.     

An evaluation of the use of remotely sensed parameters for prediction of incidence and risk associated with Vibrio parahaemolyticus in Gulf Coast oysters (Crassostrea virginica)

The U.S. Food and Drug Administration recently published a Vibrio parahaemolyticus risk assessment for consumption of raw oysters that predicts V. parahaemolyticus densities at harvest based on water temperature. We retrospectively compared archived remotely sensed measurements (sea surface temperature, chlorophyll, and turbidity) with previously published data from an environmental study of V. parahaemolyticus in Alabama oysters to assess the utility of the former data for predicting V. parahaemolyticus densities in oysters. Remotely sensed sea surface temperature correlated well with previous in situ measurements (R(2) = 0.86) of bottom water temperature, supporting the notion that remotely sensed sea surface temperature data are a sufficiently accurate substitute for direct measurement. Turbidity and chlorophyll levels were not determined in the previous study, but in comparison with the V. parahaemolyticus data, remotely sensed values for these parameters may explain some of the variation in V. parahaemolyticus levels. More accurate determination of these effects and the temporal and spatial variability of these parameters may further improve the accuracy of prediction models. To illustrate the utility of remotely sensed data as a basis for risk management, predictions based on the U.S. Food and Drug Administration V. parahaemolyticus risk assessment model were integrated with remotely sensed sea surface temperature data to display graphically variations in V. parahaemolyticus density in oysters associated with spatial variations in water temperature. We believe images such as these could be posted in near real time, and that the availability of such information in a user-friendly format could be the basis for timely and informed risk management decisions.     

Effects of electrolyzed oxidizing water treatment on reducing Vibrio parahaemolyticus and Vibrio vulnificus in raw oysters

Contamination of Vibrio parahaemolyticus and Vibrio vulnificus in oysters is a food safety concern. This study investigated effects of electrolyzed oxidizing (EO) water treatment on reducing V. parahaemolyticus and V. vulnificus in laboratory-contaminated oysters. EO water exhibited strong antibacterial activity against V. parahaemolyticus and V. vulnificus in pure cultures. Populations of V. parahaemolyticus (8.74 x 10(7) CFU/ml) and V. vulnificus (8.69 x 10(7) CFU/ml) decreased quickly in EO water containing 0.5% NaCl to nondetectable levels (> 6.6 log reductions) within 15 s. Freshly harvested Pacific oysters were inoculated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus at levels of 10(4) and 10(6) most probable number (MPN)/g and treated with EO water (chlorine, 30 ppm; pH 2.82; oxidation-reduction potential, 1131 mV) containing 1% NaCl at room temperature. Reductions of V. parahaemolyticus and V. vulnificus in oysters were determined at 0 (before treatment), 2, 4, 6, and 8 h of treatment. Holding oysters inoculated with V. parahaemolyticus or V. vulnificus in the EO water containing 1% NaCl for 4 to 6 h resulted in significant (P < 0.05) reductions of V. parahaemolyticus and V. vulnificus by 1.13 and 1.05 log MPN/g, respectively. Extended exposure (> 12 h) of oysters in EO water containing high levels of chlorine (> 30 ppm) was found to be detrimental to oysters. EO water could be used as a postharvest treatment to reduce Vibrio contamination in oysters. However, treatment should be limited to 4 to 6 h to avoid death of oysters. Further studies are needed to determine effects of EO water treatment on sensory characteristics of oysters.     

Temperature Effects on the Depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American Oyster (Crassostrea virginica)

ABSTRACT:  This study investigated temperature effects on depuration for reducing Vibrio parahaemolyticus and Vibrio vulnificus in American oyster ( Crassostrea virginica ). Raw oysters were inoculated with 5-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10⁴ to 10⁵ MPN (most probable number)/g and depurated in artificial seawater (ASW) at 22, 15, 10, and 5 °C. Depuration of oysters at 22 °C had limited effects on reducing V. parahaemolyticus or V. vulnificus in the oysters. Populations of V. parahaemolyticus and V. vulnificus were reduced by 1.2 and 2.0 log MPN/g, respectively, after 48 h of depuration at 22 °C. Decreasing water temperature to 15 °C increased the efficacy of depuration in reducing V. parahaemolyticus and V. vulnificus in oysters. Reductions of V. parahaemolyticus and V. vulnificus in oysters increased to 2.1 and 2.9 log MPN/g, respectively, after 48 h of depuration at 15 °C. However, depurations at 10 and 5 °C were less effective than at 15 °C in reducing the Vibrio spp. in oysters. Extended depuration at 15 °C for 96 h increased reductions of V. parahaemolyticus and V. vulnificus in oysters to 2.6 and 3.3 log MPN/g, respectively.     

Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments and may be transmitted to humans by ingestion of raw oysters. This study focused on the use of low temperature pasteurization, to reduce these Vibrio spp. to nondetectable levels, thus reducing the risk of infection associated with raw oyster consumption. Artificially inoculated V. vulnificus and V. parahaemolyticus and naturally-contaminated V. vulnificus in live oysters were pasteurized at 50%deg;C for up to 15min. Samples of processed and unprocessed oysters were enumerated for V. vulnificus, V. parahaemolyticus, and aerobic spoilage bacteria for 0-14 days. Low temperature pasteurization was effective in reducing these pathogens from > 100000 to non-detectable levels in less than 10min of processing. Spoilage bacteria were reduced by 2-3 logs, thus increasing the shelf-life for up to 7 days beyond live unprocessed oysters. Vibrio vulnificus in control oysters was reduced by 102 during ice storage alone. Following pasteurization and during a temperature storage abuse study (24h at 22°C), V. vulnificus was not recovered. During this storage period spoilage bacteria exceeded 1 million/g oyster meat.     

Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest

The densities of total and pathogenic Vibrio parahaemolyticus in 671 samples of molluscan shellfish harvested in 1999 and 2000 from 14 sites in seven Gulf and Atlantic coast states were determined at 2-week intervals over a period of 12 to 16 months in each state. Changes in V. parahaemolyticus densities in shellfish between harvest and sample analysis were minimized with time and temperature controls. Densities were measured by direct plating techniques, and gene probes were used for identification. Total and pathogenic V. parahaemolyticus organisms were identified with probes for the thermolabile direct hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene, respectively. An enrichment procedure involving 25 g of shellfish was also used for the recovery of pathogenic V. parahaemolyticus. The densities of V. parahaemolyticus in shellfish from all harvest sites were positively correlated with water temperature. Shellfish from the Gulf Coast typically had higher densities of V. parahaemolyticus than did shellfish harvested from the North Atlantic or mid-Atlantic coast. Vibrio parahaemolyticus counts exceeded 1,000 CFU/g for only 5% of all samples. Pathogenic (tdh+) V. parahaemolyticus was detected in approximately 6% of all samples by both procedures, and 61.5% of populations in the positive samples from the direct plating procedure were at the lower limit of detection (10 CFU/g). The frequency of detection of pathogenic V. parahaemolyticus was significantly related to water temperature and to the density of total V. parahaemolyticus. The failure to detect pathogenic V. parahaemolyticus in shellfish more frequently was attributed to the low numbers and uneven distribution of the organism.     

Depuration of Oysters (Crassostrea gigas) Contaminated with Vibrio parahaemolyticus and Vibrio vulnificus with UV Light and Chlorinated Seawater

The efficacy of depuration using UV light and chlorinated seawater for decontaminating Vibrio parahaemolyticus and Vibrio vulnificus from oysters was investigated. Oysters were contaminated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10(4) to 10(5) CFU ml(-1) for bioaccumulation. The depuration was conducted in a closed system in which 350 liters of seawater was recirculated at a rate of 7 liters/min for 48 h at room temperature. Counts of V. parahaemolyticus or V. vulnificus were determined at 0, 6, 18, 24, and 48 h. Three treatments were conducted: T1, control treatment; T2, UV treatment; and T3, UV plus chlorine treatment. After 48 h of depuration of V. parahaemolyticus, T3 reduced the count by 3.1 log most probable number (MPN) g(-1) and T2 reduced the count by 2.4 log MPN g(-1), while T1 reduced the count by only 2.0 log MPN g(-1). After 48 h of depuration of V. vulnificus, T2 and T3 were efficient, reducing the counts by 2.5 and 2.4 log MPN g(-1), respectively, while T1 reduced the count by only 1.4 log MPN g(-1). The UV light plus chlorine treatment was more efficient for controlling V. parahaemolyticus in oysters. Both UV light and UV light plus chlorine were efficient for V. vulnificus. The present study is the first report showing the efficacy of depuration systems for decontaminating V. parahaemolyticus and V. vulnificus in oysters cultivated on the Brazilian coast. This study provides information on processes that can contribute to controlling and preventing such microorganisms in oysters and could be used for effective postharvest treatment by restaurants and small producers of oysters on the coast of Brazil.     

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.     

Inactivation of vibrio parahaemolyticus and vibrio vulnificus in phosphate-buffered saline and in inoculated whole oysters by high-pressure processing

Inactivation studies for Vibrio parahaemolyticus TX-2103 (serotype O3:K6) and Vibrio vulnificus MO-624 (clinical isolate) were conducted in phosphate-buffered saline (PBS) and in inoculated oysters under high-pressure processing conditions. V. parahaemolyticus was more resistant than V. vulnificus in PBS at all pressures and times. A 6-log reduction of V. parahaemolyticus and V. vulnificus in PBS at 241 MPa required 11 and 5 min, respectively, which included a 3-min pressure come-up time. A 4.5-log reduction of V. parahaemolyticus in oysters at 345 MPa required 7.7 min, which included a 6.7-min pressure come-up time. More than a 5.4-log reduction of V. vulnificus in oysters at 345 MPa occurred during the 6-min pressure come-up time. Both V. parahaemolyticus and V. vulnificus in PBS and in oysters were reduced to nondetectable numbers at 586 MPa during the 8- and 7-min pressure come-up times, respectively.     

Effect of intertidal exposure on Vibrio parahaemolyticus levels in Pacific Northwest oysters

Interest in Vibrio parahaemolyticus (Vp) increased in the United States following Vp-associated gastroenteritis outbreaks in 1997 and 1998 involving the West Coast and other areas. The present study evaluated multiple aspects of Vp ecology in the Pacific Northwest with three objectives: (i) to determine the effect of low-tide exposure on Vp levels in oysters, (ii) to determine the relationship between total and pathogenic Vp, and (iii) to examine sediments and aquatic fauna as reservoirs for pathogenic Vp. Samples were collected from intertidal reefs along Hood Canal, Wash., in August 2001. Fecal matter from marine mammals and aquatic birds as well as intestinal contents from bottom-dwelling fish were tested. Total and pathogenic Vp levels in all the samples were enumerated with colony hybridization procedures using DNA probes that targeted the thermolabile direct hemolysin (tlh) and thermostable direct hemolysin (tdh) genes, respectively. The mean Vp densities in oysters were four to eight times greater at maximum exposure than at the corresponding first exposure. While tdh-positive Vp counts were generally < or = 10 CFU/g at first exposure, counts as high as 160 CFU/g were found at maximum exposure. Vp concentrations in sediments were not significantly different from those in oysters at maximum exposure. Pathogenic (tdh positive) Vp was detected in 9 of 42 (21%) oyster samples at maximum exposure, in 5 of 19 (26%) sediment samples, but in 0 of 9 excreta samples. These results demonstrate that summer conditions permit the multiplication of Vp in oysters exposed by a receding tide.