Comparison between quantitative real-time reverse transcription PCR results for norovirus in oysters and self-reported gastroenteric illness in restaurant customers.

Norovirus is the principal agent of bivalve shellfish-associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This study attempts to compare norovirus RNA detection in Pacific oysters (Crassostrea gigas) by quantitative real-time reverse transcription PCR (RT-PCR) and human health risk. Self-reported customer complaints of illness in a restaurant setting (screened for credible norovirus symptoms) were compared with presence and levels of norovirus as determined by real-time RT-PCR for the batch of oysters consumed. No illness was reported for batches consistently negative for norovirus by real-time RT-PCR. However, norovirus was detected in some batches for which no illness was reported. Overall presence or absence of norovirus showed a significant association with illness complaints. In addition, the batch with the highest norovirus RNA levels also resulted in the highest rate of reported illness, suggesting a linkage between virus RNA levels and health risks. This study suggests that detection of high levels of norovirus RNA in oysters is indicative of a significantly elevated health risk. However, illness may not necessarily be reported after detection of norovirus RNA at low levels.

Application of mitochondrial DNA analysis for microbial source tracking purposes in shellfish harvesting waters.

We present a method for the reliable detection and source characterisation of faecal pollution in water and shellfish matrices, utilising real-time PCR analysis of mitochondrial DNA targets. In this study we designed real-time PCR (TaqMan) probes to target human, bovine, ovine and swine mtDNA. PCR amplification using species-specific TaqMan probes on faecal matter and mixed effluent slurries revealed no cross-reactions between species of interest and other vertebrate faecal matter. Performed as a single blind experiment we were able to correctly identify faecal material in 17/20 effluents (85% correct). mtDNA degrades relatively quickly in faecally-spiked water samples (approximately 2 weeks), a similar timeframe of environmental persistence to several bacterial faecal indictors, highlighting its applicability. The procedure described here is specific, rapid (<5 hours) and sensitive. These results confirm the suitability of using species-specific mtDNA as an indicator in source tracking studies in surface waters, shellfish harvesting areas and shellfish matrices.

Environmental occurrence and clinical impact of Vibrio vulnificus and Vibrio parahaemolyticus: a European perspective.

Vibrio vulnificus and Vibrio parahaemolyticus are ubiquitous Gram-negative bacterial pathogens found naturally in marine and estuarine waters, and are a leading cause of seafood-associated bacterial illness. These pathogens are commonly reported in the USA and in many Asian countries, including China, Japan and Taiwan; however, there is growing concern that V. vulnificus and V. parahaemolyticus may represent an important and increasing clinical problem in Europe. Several factors underlie the need for a greater understanding of these non-cholera vibrios within a European context. First, there is a growing body of evidence to suggest that V. vulnificus and V. parahaemolyticus infections are increasing, and tend to follow regional climatic trends, with outbreaks typically following episodes of unusually warm weather. Such findings are especially alarming given current predictions regarding warming of marine waters as a result of global climatic change. Second, a myriad of epidemiological factors may greatly increase the incidence as well as clinical burden of these pathogens - including increasing global consumption and trade of seafood produce coupled to an increase in the number of susceptible individuals consuming seafood produce. Finally, there is currently a lack of detailed surveillance information regarding non-cholerae Vibrio infections in Europe, as these pathogens are not notifiable in many countries, which probably masks the true clinical burden of many human infections. This review will present a pertinent overview of both the environmental occurrence and clinical impact of V. vulnificus and V. parahaemolyticus in Europe.

Rapid in situ detection of virulent Vibrio vulnificus strains in raw oyster matrices using real-time PCR.

Vibrio vulnificus is a Gram-negative bacterial pathogen responsible for the vast majority of bacterially mediated fatalities from the consumption of raw or undercooked seafood in the USA. Vibrio vulnificus-associated septicaemia can occur rapidly (< 24 h); however, methods for the isolation and confirmation of V. vulnificus from seafood samples typically require several days. A real-time PCR assay was developed for V. vulnificus biotype 1 that provides a rapid means of identifying a gene fragment (vcgC) previously indicated as a strong predictor of potential virulence. PCR probe specificity was confirmed by amplification of 17 clinical V. vulnificus strains and by the lack of amplification with seven non-pathogenic V. vulnificus isolates and a wide range of closely related bacteria. Oyster and seawater samples were amended with a range of environmentally realistic concentrations of C-genotype V. vulnificus cells, which were quantitatively and unambiguously identified according to biotype. Of some significance, we utilized a sample processing and nucleic acid extraction procedure that allowed identification of pathogenic strains of V. vulnificus from oyster matrices without prior enrichment or culturing of strains. This outlined approach allowed the detection of as little as 50 cfu of V. vulnificus in less than 5 h, which compares favourably with culture-based approaches. The results indicate the applicability of this approach for monitoring purposes or as a potential diagnostic tool in clinical settings.

Detection and identification of tdh- and trh-positive Vibrio parahaemolyticus strains from four species of cultured bivalve molluscs on the Spanish Mediterranean Coast.

Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.

Prevalence and potential pathogenicity of Vibrio parahaemolyticus in Chinese mitten crabs (Eriocheir sinensis) harvested from the River Thames estuary, England.

Chinese mitten crabs (Eriocheir sinensis) have been described as an alien invasive species in the River Thames, United Kingdom, and elsewhere in Europe. The crabs can cause considerable physical damage to the riverbeds and threaten native ecosystems. Trapping has been considered an option, but such attempts to control mitten crab populations in Germany in the 1930s failed. In the United Kingdom, it has been suggested that commercial exploitation of the species could be employed as a control option. This study was conducted as part of a larger program to assess the suitability of a commercial Chinese mitten crab fishery in the River Thames. Crabs and water samples from the River Thames between 2003 and 2006 were examined for the human pathogenic bacterium Vibrio parahaemolyticus. All samples throughout this testing period were positive for V. parahaemolyticus. The putative pathogenicity markers, thermostable direct hemolysin and thermostable direct-related hemolysin, were detected in one sample, indicating that the crabs possessed the potential to cause V. parahaemolyticus-associated illness if consumed without further processing. Levels of V. parahaemolyticus were higher during the summer than in the winter. This is the first study of V. parahaemolyticus prevalence in European-adapted Chinese mitten crabs.

Comparison of V. parahaemolyticus isolated from seafoods and cases of gastrointestinal disease in the UK.

In this study the prevalence of Vibrio parahaemolyticus in shellfish and estuarine waters from the UK was examined using cultural and nucleic acid hybridisation approaches. Forty-nine isolates derived from environmental sources were characterised using serotyping, PCR, nucleic acid hybridisation and pulsed field gel electrophoresis (PFGE). The serotypic and molecular profiles of these isolates were compared to 20 clinical isolates, including representatives of the pandemic O3:K6 clone. Thirty percent of environmental samples were positive for V. parahaemolyticus. The tdh gene was identified in 12% of samples tested. Environmentally derived tdh+ strains were highly heterogeneous with neither association between isolates from similar origins nor seafood type. Previously uncharacterised clinical strains from UK patients with travel related V. parahaemolyticus associated gastroenteritis, were unrelated to tdh+ or tdh- environmental isolates but 2 were clonally indistinguishable from the pandemic O3:K6 strain responsible for outbreaks in Spain, Korea, Japan and Laos.

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.