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.Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that occurs naturally in estuarine environments worldwide (21). Its densities in the environment and seafoods vary greatly by season, location, sample type, fecal pollution, and analytical methodology (5, 8-10, 14, 21, 22, 36). Pathogenic V. parahaemolyticus generally produces a thermostable direct hemolysin (TDH), the product of the tdh gene (20). More than 90% of clinical V. parahaemolyticus isolates but fewer than 1% of food or environmental strains produce TDH or possess tdh (9,10,14,22,26,31,35). The frequency of TDH or tdh detection in environmental samples and seafoods ranges from 0 to 6% (5,14,22,26,31,34). Quantitative data for tdh ϩ V. parahaemolyticus have been reported in only one study and are based on one or two isolates from each of four oyster samples (10).A series of oyster-associated outbreaks of V. parahaemolyticus (3, 4, 6, 7) in 1997 (Washington State) and 1998 (Texas, New York and Connecticut, and Washington State) prompted the development of nonradioactive DNA probes (28, 29) and direct-plating methods for rapid and efficient determination of the abundance of total and pathogenic V. parahaemolyticus in oysters. The Food and Drug Administration (FDA) initiated a surveillance program using these new methods in Alabama in March 1999. The objective of this study was to correlate quantitative data on the abundance of total and pathogenic V. parahaemolyticus with each other and with environmental parameters such as water temperature and salini...

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a speciesspecific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10 4 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh ؉ and trh ؉ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.Vibrio parahaemolyticus is found worldwide in estuarine waters and is the leading cause of gastroenteritis from seafood in the United States, with most infections resulting from the consumption of raw or mishandled seafood (2, 29). The tlh (thermolabile hemolysin) gene is a species-specific marker for V. parahaemolyticus (42,26), while the tdh (thermostable direct hemolysin) (33, 34) and trh (thermostable-related hemolysin) (35) genes are pathogenicity markers for V. parahaemolyticus. Pathogenic V. parahaemolyticus bacteria in the environment and food samples typically comprise 0.3 to 3% of the total V. parahaemolyticus population (4,8,20,22,43). DNA-based assays targeting these genes were developed for the detection and enumeration of V. parahaemolyticus bacteria (7,10,21,26,27,30,38,44). However, the ability of the PCR assays to detect low levels of pathogenic V. parahaemolyticus bacteria in the presence of a high background of nonpathogenic V. parahaemolyticus bacteria was not reported by the authors (10,30,44). The preferential amplification of nonpathogenic members of the total V. parahaemolyticus population due to the muchhigher copy number of the target could preclude the detection of pathogenic strains pres...

Vibrio parahaemolyticus is indigenous to coastal environments and a frequent cause of seafood-borne gastroenteritis in the United States, primarily due to raw-oyster consumption. Previous seasonal-cycle studies of V. parahaemolyticus have identified water temperature as the strongest environmental predictor. Salinity has also been identified, although it is evident that its effect on annual variation is not as pronounced. The effects of other environmental factors, both with respect to the seasonal cycle and intraseasonal variation, are uncertain. This study investigated intraseasonal variations of densities of total and pathogenic V. parahaemolyticus organisms in oysters and overlying waters during the summer of 2004 at two sites in the northern Gulf of Mexico. Regression analyses indicated significant associations (P < 0.001) between total V. parahaemolyticus densities and salinity, as well as turbidity in water and in oysters at the Mississippi site but not at the Alabama site. Pathogenic V. parahaemolyticus organisms in Mississippi oyster and water samples were detected in 56% (9 out of 16) and 78% (43 out of 55) of samples, respectively. In contrast, 44% (7 out of 16) of oyster samples and 30% (14 out of 47) of water samples from Alabama were positive. At both sites, there was greater sample-to-sample variability in pathogenic V. parahaemolyticus densities than in total V. parahaemolyticus densities. These data suggest that, although total V. parahaemolyticus densities may be very informative, there is greater uncertainty when total V. parahaemolyticus densities are used to predict the risk of infection by pathogenic V. parahaemolyticus than previously recognized.Vibrio parahaemolyticus is the leading cause of Vibrio-associated gastroenteritis in the United States (15,19,20,32) and has been isolated from oysters, blue crabs, finfish, and planktonic copepods (6,7,18,22). Vibrio infections are most common in individuals living in states bordering the Gulf of Mexico (23) and are usually associated with the consumption of raw shellfish, primarily oysters (9, 20). Recent outbreaks (4,5,10,14) raised the awareness of public health officials concerning shellfish throughout coastal states and prompted the Interstate Shellfish Sanitation Conference to develop an Interim Control Plan for regulating shellfish harvest areas based on V. parahaemolyticus densities in shellfish (31). The Interim Control Plan employs a colony lift technique using DNA probes that target the species-specific thermolabile hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene associated with pathogenic V. parahaemolyticus strains (32). A similar DNA probe colony hybridization method has been developed to target the tdh-related hemolysin gene (trh), which is also associated with pathogenic strains of V. parahaemolyticus (27). While these colony lift techniques make it possible to investigate the distribution of pathogenic V. parahaemolyticus strains directly, they have a relatively high limit of detection (LOD). Thus, the colony...

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.Vibrio parahaemolyticus is the leading cause of seafood-associated bacterial gastroenteritis in the United States (29) and is a major cause of food-borne illness in the world (21, 41). However, the relationship between strains isolated from estuarine environments, those isolated from seafood, and human clinical isolates is poorly understood. The presence of thermostable direct hemolysin (TDH) is a proven virulence factor (31), and TDH occurs in over 90% of clinical