Determination of Norovirus Contamination in Oysters from Two Commercial Harvesting Areas over an Extended Period, Using Semiquantitative Real-Time Reverse Transcription PCR

Abstract

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses, which cause gastroenteritis, are the principal agents of shellfish-related illness. Fecal-indicator quality standards based on Escherichia coli are well established in Europe and elsewhere. However, norovirus outbreaks after consumption of shellfish meeting these standards still occur, and the need to improve consumer health protection is well recognized. Alternative approaches proposed include direct monitoring of viral pathogens and the use of alternative indicator organisms capable of providing a better indication of virus risk. This study applies a recently developed TaqMan PCR assay to assess norovirus contamination in shellfish. Comparison was made with E. coli as the existing sanitary standard and a male-specific RNA bacteriophage as a possible alternative. Two commercial pacific oyster (Crassostrea gigas) harvesting areas were monitored over a 31-month period. The results show peaks of norovirus contamination in both areas during winter months, with average levels approximately 17 times higher in oysters sampled October to March than during the remainder of the year, consistent with epidemiological data for the United Kingdom showing oyster-associated illness is confined to winter months. While there was no apparent association with E. coli, an association between levels of norovirus contamination and the male-specific RNA bacteriophage was noted, with average norovirus levels over 40 times higher in samples with male-specific RNA bacteriophage counts of >1,000 PFU/100 g than in samples with <100 PFU/100 g. Overall, these results suggest that norovirus monitoring in shellfish production areas could be an effective strategy for reduction of virus risk.

Two-Year Systematic Study To Assess Norovirus Contamination in Oysters from Commercial Harvesting Areas in the United Kingdom

“…In common with previous studies in Northern Europe (14,15,23,29), a very marked seasonality was observed, with the highest prevalences and levels being found during the winter periods. Possible contributors to this seasonal pattern of occurrence were previously suggested to include the greater persistence of viral particles under typical Northern European winter environmental conditions of low solar irradiation, low temperature, and higher turbidity (1, 7); more effective viral clearance in oysters during summer months due to the higher rate of shellfish metabolism in warmer seawaters (12,17); and the higher prevalence of noroviruses in the human population, and thus seeded into the marine environment, during the winter (6,26,30).…”

“…Currently, there is little systematic monitoring data available to inform risk managers on the possible applications, levels, and impacts of a norovirus standard; only relatively few long-term surveys using real-time PCR technology have been carried out in European Union member states. These surveys have often focused on limited geographical areas (35, 39), although a survey of Irish oysters from 21 sites using a semiquantitative method found norovirus genogroup II (GII) in 37% of samples and demonstrated a seasonal pattern of contamination, with higher prevalences and levels during the winter months from October to March (14).Similarly, previous studies in the United Kingdom have shown the presence of viral RNA in a significant proportion of oyster samples and indicated a distinct winter seasonality (27,29). However, those studies generally targeted problematical production areas (e.g., due to gastroenteritis outbreaks), and therefore, the data may not be representative.…”

“…Similarly, previous studies in the United Kingdom have shown the presence of viral RNA in a significant proportion of oyster samples and indicated a distinct winter seasonality (27,29). However, those studies generally targeted problematical production areas (e.g., due to gastroenteritis outbreaks), and therefore, the data may not be representative.…”

Environmental Transmission of Human Noroviruses in Shellfish Waters

“…The observed winter peak of viral prevalence in shellfish is consistent with the higher prevalence of NoV seen in sewage and with the consequential winter seasonality of shellfish-related gastroenteritis outbreaks in temperate climates (106). In the United Kingdom, average levels of NoV in Pacific oysters (Crassostrea gigas) during winter months (October to March) may be as much as 17 times higher than those during the remainder of the year (110). This seasonal pattern has been observed to mirror variations in water temperature (104) and in salinity and rainfall (103).…”

“…Many studies have detected NoV, sometimes at elevated levels, in oysters harvested from areas compliant with health status classification A (Ͻ230 most probable number [MPN] of E. coli/100 g) and classification B (Ͻ4,600 MPN of E. coli/100 g in 90% of samples) under the European system (32,33,110). Such contaminated shellfish may require extended relaying before they can be safely introduced into the market (32,119).…”