Levels of Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas) From Washington State Following Ambient Exposure and Chilling.

Vibrio parahaemolyticus illnesses, often associated with the consumption of raw or undercooked oysters, are most common in summer months when higher temperatures increase V. parahaemolyticus levels in the environment. In Washington, post-harvest controls focus on the time from harvest to temperature-controlled storage to minimize V. parahaemolyticus illness risk. This study examined the effect of post-harvest ambient storage on V. parahaemolyticus levels in Pacific oysters. Additionally, the effects of cooling method, icing and/or refrigeration, on V. parahaemolyticus levels in oysters were evaluated. Five independent trials were conducted during July and August of 2015. For each trial, oysters were harvested from Totten Inlet and exposed to ambient conditions for 0 h (immediately cooled), 1 h, 5 h, or 9 h, and then either iced or refrigerated. Total and pathogenic (tdh+/trh+) V. parahaemolyticus levels were determined via MPN real-time PCR. Data from each trial were analyzed independently due to differences in initial V. parahaemolyticus levels. Total V. parahaemolyticus levels in oysters increased relative to control (0 h I) levels after the 1 h ambient exposure in only one trial, but pathogenic V. parahaemolyticus levels did not significantly increase after the 1 h exposure. Total and pathogenic V. parahaemolyticus levels increased by 0.8-1.9 log MPN/g in 5 h exposed oysters and by 1.0-2.9 log MPN/g in 9 h exposed oysters compared to levels in 0 h I samples. Mean maximum temperature of 5 h and 9 h exposed samples increased to ≈29°C compared to ≈21°C in 0 h and 1 h exposures, which likely contributed to observed increases in V. parahaemolyticus levels. Total and pathogenic V. parahaemolyticus levels increased more often in oysters cooled by refrigeration than by ice; this was most notable for the longer ambient exposure samples. Overall, these data support shorter post-harvest ambient exposure as well as rapid cooling of oysters to minimize risk of V. parahaemolyticus illness.

A systematic review of post-harvest interventions for Vibrio parahaemolyticus in raw oysters.

BACKGROUND: Non-cholera Vibrio bacteria are a major cause of foodborne illness in the United States. Raw oysters are commonly implicated in gastroenteritis caused by pathogenic Vibrio parahaemolyticus. In response to outbreaks in 1997-1998, the US Food and Drug Administration developed a nation-wide quantitative microbial risk assessment (QMRA) of V. parahaemolyticus in raw oysters in 2005. The QMRA identified information gaps that new research may address. Incidence of sporadic V. parahaemolyticus illness has recently increased and, as oyster consumption increases and sea temperatures rise, V. parahaemolyticus outbreaks may become more frequent, posing health concerns. Updated and region-specific QMRAs will improve the accuracy and precision of risk of infection estimates. OBJECTIVES: We identify research to support an updated QMRA of V. parahaemolyticus from oysters harvested in Chesapeake Bay and Puget Sound, focusing on observational and experimental research on post-harvest practices (PHPs) published from 2004 to 2019. METHODS: A predefined search strategy was applied to PubMed, Embase, Scopus, Science.gov, NAL Agricola, and Google Scholar. Study eligibility criteria were defined using a population, intervention, comparator, and outcome statement. Reviewers independently coded abstracts for inclusion/exclusion using predefined criteria. Data were extracted and study quality and relevance evaluated based on published guidance for food safety risk assessments. Findings were synthesized using a weight of evidence approach. RESULTS: Of 12,174 articles retrieved, 93 were included for full-text review. Twenty-seven studies were found to be high quality and high relevance, including studies on cold storage, high hydrostatic pressure, depuration, and disinfectant, and other PHPs. High hydrostatic pressure consistently emerged as the most effective PHP in reducing abundance of V. parahaemolyticus. DISCUSSION: Limitations of the knowledge base and review approach involve the type and quantity of data reported. Future research should focus on PHPs for which few or no high quality and high relevance studies exist, such as irradiation and relaying.

Associations of Environmental Conditions and Vibrio parahaemolyticus Genetic Markers in Washington State Pacific Oysters.

Vibrio parahaemolyticus is a naturally occurring bacterium in estuarine waters and is a major cause of seafood-borne illness. The bacterium has been consistently identified in Pacific Northwest waters and elevated illness rates of vibriosis in Washington State have raised concerns among growers, risk managers, and consumers of Pacific oysters (Crassostrea gigas). In order to better understand pre-harvest variation of V. parahaemolyticus in the region, abundance of total and potentially pathogenic strains of the bacterium in a large number of Washington State Pacific oyster samples were compared with environmental conditions at the time of sampling. The Washington Department of Health regularly sampled oysters between June and September at over 21 locations from 2014 to 2018, resulting in over 946 samples. V. parahaemolyticus strains carrying three genetic markers, tlh, trh, and tdh, were enumerated in oyster tissue using a most probable number-PCR analysis. Tobit regressions and seemingly unrelated estimations were used to formally assess relationships between environmental measures and genetic markers. All genetic markers were found to be positively associated with temperature, independent of the abundance of other genetic markers. Surface water temperature displayed a non-linear relationship, with no association observed between any genetic marker in the warmest waters. There were also stark differences between surface and shore water temperature models. Salinity was not found to be substantially associated with any of the genetic variables. The relative abundance of tdh+ strains given total V. parahaemolyticus abundance (pathogenic ratio tdh:tlh) was negatively associated with water temperature in colder waters and decreased exponentially as total V. parahaemolyticus abundance increased. Strains carrying the trh gene had a pronounced positive association with strains carrying the tdh gene but was also negatively associated with the tdh:tlh pathogenic ratio. These results suggest that there are ecological relationships of competition, growth, and survival for V. parahaemolyticus strains in the oyster tissue matrix. This work also improves the overall understanding of environmental associations with V. parahaemolyticus in Washington State Pacific oysters, laying the groundwork for future risk mitigation efforts in the region.

Effects of Dry Storage and Resubmersion of Oysters on Total Vibrio vulnificus and Total and Pathogenic (tdh+/trh+) Vibrio parahaemolyticus Levels.

Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are the two leading causes of bacterial illnesses associated with raw shellfish consumption. Levels of these pathogens in oysters can increase during routine antifouling aquaculture practices involving dry storage in ambient air conditions. After storage, common practice is to resubmerge these stored oysters to reduce elevated Vv and Vp levels, but evidence proving the effectiveness of this practice is lacking. This study examined the changes in Vv and in total and pathogenic (thermostable direct hemolysin gene and the tdh-related hemolysin gene, tdh+ and trh+) Vp levels in oysters after 5 or 24 h of dry storage (28 to 32°C), followed by resubmersion (27 to 32°C) for 14 days. For each trial, replicate oyster samples were collected at initial harvest, after dry storage, after 7 days, and after 14 days of resubmersion. Oysters not subjected to dry storage were collected and analyzed to determine natural undisturbed vibrio levels (background control). Vibrio levels were measured using a most-probable-number enrichment followed by real-time PCR. After storage, vibrio levels (excluding tdh+ and trh+ Vp during 5-h storage) increased significantly (P < 0.001) from initial levels. After 7 days of resubmersion, Vv and total Vp levels (excluding total Vp in oysters stored for 5 h) were not significantly different (P < 0.1) from levels in background oysters. Vv and total and pathogenic Vp levels were not significantly different (P > 0.1) from levels in background oysters after 14 days of resubmersion, regardless of dry storage time. These data demonstrate that oyster resubmersion after dry storage at elevated ambient temperatures allows vibrio levels to return to those of background control samples. These results can be used to help minimize the risk of Vv and Vp illnesses and to inform the oyster industry on the effectiveness of routine storing and resubmerging of aquaculture oysters.

Prevalence and Characterization of High Histamine-Producing Bacteria in Gulf of Mexico Fish Species.

Recent developments in detection and enumeration of histamine-producing bacteria (HPB) have created powerful molecular-based tools to better understand the presence of spoilage bacteria and conditions, resulting in increased risk of scombrotoxin fish poisoning. We examined 235 scombrotoxin-forming fish from the Gulf of Mexico for the presence of high HPB. Photobacterium damselae subsp. damselae was the most prevalent HPB (49%), followed by Morganella morganii (14%), Enterobacter aerogenes (4%), and Raoultella planticola (3%). The growth characteristics and histamine production capabilities of the two most prevalent HPB were further examined. M. morganii and P. damselae had optimum growth at 35°C and 30 to 35°C and 0 to 2% and 1 to 3% NaCl, respectively. P. damselae produced significantly (P < 0.001) higher histamine than M. morganii in inoculated mahimahi and Spanish mackerel incubated at 30°C for 24 h, but histamine production was not significantly different between the two HPB in inoculated tuna, possibly due to differences in muscle composition and salt content. Results in this study showed that P. damselae was the most prevalent high HPB in Gulf of Mexico fish. In addition, previously reported results using the traditional Niven's method may underreport the prevalence of P. damselae. Molecular-based methods should be used in addition to culture-based methods to enhance detection and enumeration of HPB.

Abundance of Vibrio cholerae, V. vulnificus, and V. parahaemolyticus in oysters (Crassostrea virginica) and clams (Mercenaria mercenaria) from Long Island sound.

Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)-real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh(+) and/or trh(+)) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and -0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.

Comparison of plating media for recovery of total and virulent genotypes of Vibrio vulnificus in U.S. market oysters.

Vibrio vulnificus is the leading cause of seafood associated mortality in the United States and is generally associated with consumption of raw oysters. Two genetic markers have emerged as indicators of strain virulence, 16S rDNA type B (rrnB) and virulence correlated gene type C (vcgC). While much is known about the distribution of V. vulnificus in oysters, a limited number of studies have addressed the more virulent subtypes. Therefore, the goals of this study were to (1) determine the suitability of media for recovery of total and virulent genotypes of V. vulnificus and (2) evaluate the geographical and seasonal distribution of these genotypes. Market oysters from across the United States and the strains isolated from them during a year-long study in 2007 were used. For media evaluation, VVA and CPC+ were compared using direct plating of oyster tissues while mCPC and CPC+ were compared for isolation following MPN enrichment. Representative isolates from each media/method were tested for rrn and vcg types to determine their seasonal and geographical distribution. No statistically significant difference was observed between VVA/CPC+ or mCPC/CPC+ for isolation of total or virulent (rrnB/vcgC) genotypes of V. vulnificus. Overall, 32% of recovered isolates possessed the virulent genotype. The prevalence of these genotypes was highest in oysters from the Gulf Coast during Oct-Dec, and demonstrated a statistically significant geographical and seasonal pattern. This is the first report on the distribution of virulent V. vulnificus genotypes across the United States, which provides novel insight into the occurrence of this pathogen.

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the coastal and estuarine waters of Louisiana, Maryland, Mississippi, and Washington (United States).

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.

Biochemical, serological, and virulence characterization of clinical and oyster Vibrio parahaemolyticus isolates.

In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2α or T3SS2ß genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2α genes, and all isolates negative for tdh and positive for trh possessed all T3SS2ß genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2α, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2ß genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.

Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus.

Pathogenic vibrios are a global concern for seafood safety and many molecular methods have been developed for their detection. This study compares several molecular methods for detection of total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus, in MPN enrichments from oysters and fish intestine samples. This study employed the DuPont Qualicon BAX® System Real-Time PCR assay for detection of V. parahaemolyticus and V. vulnificus. Multiplex real-time PCR detection of total (tlh+), tdh+, and trh+V. parahaemolyticus was conducted on the Cepheid SmartCycler II. Total (rpoD) and tdh+V. parahaemolyticus were also detected using LAMP. V. vulnificus detection was performed using real-time PCR methods developed for the SmartCycler and the AB 7500 Fast. Recommended template preparations were compared to BAX® lysis samples for suitability. There was no significant difference in detection of V. parahaemolyticus and V. vulnificus using the BAX® or SmartCycler assays. The AB assay showed no difference from other methods in detection of V. vulnificus unless boiled templates were utilized. There was a significant difference in detection of tdh+V. parahaemolyticus between SmartCycler and LAMP assays unless the total (tlh+) V. parahaemolyticus gene target was omitted from the SmartCycler assay; a similar trend was observed for trh+V. parahaemolyticus.

Bacterial and viral pathogens in live oysters: 2007 United States market survey.

Two samples of market oysters, primarily from retail establishments, were collected twice each month in each of nine states during 2007. Samples were shipped refrigerated overnight to five U.S. Food and Drug Administration laboratories on a rotating basis and analyzed by most probable number (MPN) for total and pathogenic Vibrio parahaemolyticus and V. vulnificus numbers and for the presence of toxigenic V. cholerae, Salmonella spp., norovirus (NoV), and hepatitis A virus (HAV). Levels of indicator organisms, including fecal coliforms (MPN), Escherichia coli (MPN), male-specific bacteriophage, and aerobic plate counts, were also determined. V. parahaemolyticus and V. vulnificus levels were distributed seasonally and geographically by harvest region and were similar to levels observed in a previous study conducted in 1998-1999. Levels of pathogenic V. parahaemolyticus were typically several logs lower than total V. parahaemolyticus levels regardless of season or region. Pathogenic V. parahaemolyticus levels in the Gulf and Mid-Atlantic regions were about two logs greater than the levels observed in the Pacific and North Atlantic regions. Pathogens generally associated with fecal pollution were detected sporadically or not at all (toxigenic V. cholerae, 0%; Salmonella, 1.5%; NoV, 3.9%; HAV, 4.4%). While seasonal prevalences of NoV and HAV were generally greater in oysters harvested from December to March, the low detection frequency obscured any apparent seasonal effects. Overall, there was no relationship between the levels of indicator microorganisms and the presence of enteric viruses. These data provide a baseline that can be used to further validate risk assessment predictions, determine the effectiveness of new control measures, and compare the level of protection provided by the U.S. shellfish sanitation system to those in other countries.

Survey of postharvest-processed oysters in the United States for levels of Vibrio vulnificus and Vibrio parahaemolyticus.

From June through October 2004, the U.S. Food and Drug Administration collected oysters (61 samples) that had been subjected to postharvest processing (PHP) methods, including mild heat treatment, freezing, and high hydrostatic pressure, from processors and retail markets in various states to determine Vibrio vulnificus and V. parahaemolyticus levels. Presence in a 25-g sample and most probable number (MPN) using standard enrichment and selective isolation procedures were utilized. Suspect colonies were isolated and identified using DNA probe colony hybridization. Neither species of vibrio was detected in 25-g portions of most samples regardless of the PHP. The lowest frequency of isolation of either pathogen (<10%) was observed with the mild heat process. Few (12 to 13%) frozen samples collected at the processor but not at retail contained >30 MPN/g of either pathogen. The mean levels of either organism in PHP oysters observed in the present study were 5 to 6 log less than in unprocessed raw Gulf Coast oysters. Of the 70 V. vulnificus isolates examined, only 5 possessed the putative virulence marker, type B 16S rRNA. Neither the thermostable direct hemolysin (tdh) nor the tdh-related hemolysin (trh) virulence gene was detected in any of the 40 V. parahaemolyticus isolates examined in the present study. These data suggest that if there is any selective advantage to pathogenic strains of V. vulnificus and V. parahaemolyticus, these differences are minimal. These results indicate that all PHP treatments greatly reduce exposure of V. vulnificus and V. parahaemolyticus to raw-oyster consumers. Consequently, these PHP oysters pose a much lower risk of illness to consumers due to these pathogens.

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.

Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters.

Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g(-1). Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh(+) V. parahaemolyticus than previously reported.

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.

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.