Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters.

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 species-specific 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.

Evaluation of an alkaline phosphatase-labeled oligonucleotide probe for the detection and enumeration of the thermostable-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase-labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.

Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters.

BACKGROUND: Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis in the United States, typically is associated with the consumption of raw oysters gathered from warm-water estuaries. We describe a recognized outbreak of V. parahaemolyticus infection associated with the consumption of seafood from Alaska. METHODS: After we received reports of the occurrence of gastroenteritis on a cruise ship, we conducted a retrospective cohort study among passengers, as well as active surveillance throughout Alaska to identify additional cases, and an environmental study to identify sources of V. parahaemolyticus and contributors to the outbreak. RESULTS: Of 189 passengers, 132 (70 percent) were interviewed; 22 of the interviewees (17 percent) met our case definition of gastroenteritis. In our multiple logistic-regression analysis, consumption of raw oysters was the only significant predictor of illness; the attack rate among people who consumed oysters was 29 percent. Active surveillance identified a total of 62 patients with gastroenteritis. V. parahaemolyticus serotype O6:K18 was isolated from the majority of patients tested and from environmental samples of oysters. Patterns on pulsed-field gel electrophoresis were highly related across clinical and oyster isolates. All oysters associated with the outbreak were harvested when mean daily water temperatures exceeded 15.0 degrees C (the theorized threshold for the risk of V. parahaemolyticus illness from the consumption of raw oysters). Since 1997, mean water temperatures in July and August at the implicated oyster farm increased 0.21 degrees C per year (P<0.001 by linear regression); 2004 was the only year during which mean daily temperatures in July and August at the shellfish farm did not drop below 15.0 degrees C. CONCLUSIONS: This investigation extends by 1000 km the northernmost documented source of oysters that caused illness due to V. parahaemolyticus. Rising temperatures of ocean water seem to have contributed to one of the largest known outbreaks of V. parahaemolyticus in the United States.