Evaluation of Norovirus Reduction in Environmentally Contaminated Pacific Oysters During Laboratory Controlled and Commercial Depuration.

Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT-PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17-21 °C and after 4 days at 11-15 °C, compared to 44% reduction at 7-9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram (gcg-1; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant difference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg-1. These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry.

The Impact of Winter Relocation and Depuration on Norovirus Concentrations in Pacific Oysters Harvested from a Commercial Production Site.

Oysters contaminated with norovirus present a significant public health risk when consumed raw. In this study, norovirus genome copy concentrations were determined in Pacific oysters (Magallana gigas) harvested from a sewage-impacted production site and then subjected to site-specific management procedures. These procedures consisted of relocation of oysters to an alternative production area during the norovirus high-risk winter periods (November to March) followed by an extended depuration (self-purification) under controlled temperature conditions. Significant differences in norovirus RNA concentrations were demonstrated at each point in the management process. Thirty-one percent of oyster samples from the main harvest area (Site 1) contained norovirus concentrations > 500 genome copies/g and 29% contained norovirus concentrations < 100 genome copies/g. By contrast, no oyster sample from the alternative harvest area (Site 2) or following depuration contained norovirus concentrations > 500 genome copies/g. In addition, 60 and 88% of oysters samples contained norovirus concentrations < 100 genome copies/g in oysters sampled from Site 2 and following depuration, respectively. These data demonstrate that site-specific management processes, supported by norovirus monitoring, can be an effective strategy to reduce, but not eliminate, consumer exposure to norovirus genome copies.

Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters.

Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml(-1), respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml(-1) compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml(-1). A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g(-1) and 1050 PFU 100 g(-1) respectively.

Norovirus genotypes present in oysters and in effluent from a wastewater treatment plant during the seasonal peak of infections in Ireland in 2010.

We determined norovirus (NoV) concentrations in effluent from a wastewater treatment plant and in oysters during the peak period of laboratory-confirmed cases of NoV infection in Ireland in 2010 (January to March). Weekly samples of influent, secondary treated effluent, and oysters were analyzed using real-time quantitative reverse transcription-PCR for NoV genogroup I (GI) and genogroup II (GII). The mean concentration of NoV GII (5.87 × 10(4) genome copies 100 ml(-1)) in influent wastewater was significantly higher than the mean concentration of NoV GI (1.40 × 10(4) genome copies 100 ml(-1)). The highest concentration of NoV GII (2.20 × 10(5) genome copies 100 ml(-1)) was detected in influent wastewater during week 6. Over the study period, a total of 931 laboratory-confirmed cases of NoV GII infection were recorded, with the peak (n = 171) occurring in week 7. In comparison, 16 cases of NoV GI-associated illness were reported during the study period. In addition, the NoV capsid N/S domain was molecularly characterized for selected samples. Multiple genotypes of NoV GI (GI.1, GI.4, GI.5, GI.6, and GI.7) and GII (GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, and GII.17), as well as 4 putative recombinant strains, were detected in the environmental samples. The NoV GII.4 variant 2010 was detected in wastewater and oyster samples and was the dominant strain detected in NoV outbreaks at that time. This study demonstrates the diversity of NoV genotypes present in wastewater during a period of high rates of NoV infection in the community and highlights the potential for the environmental spread of multiple NoV genotypes.

Characterisation of norovirus contamination in an Irish shellfishery using real-time RT-qPCR and sequencing analysis.

Norovirus (NoV) is the single most important agent of foodborne viral gastroenteritis worldwide. Bivalve shellfish, such as oysters, grown in areas contaminated with human faecal waste may become contaminated with human pathogens including NoV. A study was undertaken to investigate NoV contamination in oysters (Crassostrea gigas) from a shellfishery over a 24month period from October 2007 to September 2009. Oyster samples were collected monthly from a commercial shellfish harvest area classified as category B under EU regulations, but that had had been closed for commercial harvesting due to its previous association with NoV outbreaks. Real-time reverse transcription quantitative PCR (RT-qPCR) was used to determine the concentration of human NoV genogroups I and II (GI and GII) in monthly samples. Total NoV (GI and GII) concentrations in NoV positive oysters ranged from 97 to 20,080genome copiesg(-1) of digestive tissue and displayed a strong seasonal trend with greater concentrations occurring during the winter months. While NoV GII concentrations detected in oysters during both years were similar, NoV GI concentrations were significantly greater in oysters during the winter of 2008/09 than during the winter of 2007/08. To examine the NoV genotypes present in oyster samples, sequence analysis of nested RT-PCR products was undertaken. Although NoV GII.4 is responsible for the vast majority of reports of outbreaks in the community, multiple NoV genotypes were identified in oysters during this study: GI.4, GI.3, GI.2, GII.4, GII.b, GII.2, GII.12, and GII.e. NoV GI.4 was the most frequently detected genotype throughout the study period and was detected in 88.9% of positive samples, this was followed by GII.4 (43.7%) and GII.b (37.5%). This data demonstrates the diversity of NoV genotypes that can be present in sewage contaminated shellfish and that a disproportionate number of non-NoV GII.4 genotypes can be found in environmental samples compared to the number of recorded human infections associated with non-NoV GII.4 genotypes.

Concentration of norovirus during wastewater treatment and its impact on oyster contamination.

The concentrations of Escherichia coli, F-specific RNA bacteriophage (FRNA bacteriophage), and norovirus genogroup I (NoV GI) and norovirus genogroup II (NoV GII) in wastewater were monitored weekly over a 1-year period at a wastewater treatment plant (WWTP) providing secondary wastewater treatment. A total of 49 samples of influent wastewater and wastewater that had been treated by primary and secondary wastewater treatment processes (primary and secondary treated wastewater) were analyzed. Using a real-time reverse transcription-quantitative PCR (RT-qPCR), the mean NoV GI and NoV GII concentrations detected in effluent wastewater were 2.53 and 2.63 log(10) virus genome copies 100 ml(-1), respectively. The mean NoV concentrations in wastewater during the winter period (January to March) (n = 12) were 0.82 (NoV GI) and 1.41 (NoV GII) log units greater than the mean concentrations for the rest of the year (n = 37). The mean reductions of NoV GI and GII during treatment were 0.80 and 0.92 log units, respectively, with no significant difference detected in the extent of NoV reductions due to season. No seasonal trend was detected in the concentrations of E. coli or FRNA bacteriophage in wastewater influent and showed mean reductions of 1.49 and 2.13 log units, respectively. Mean concentrations of 3.56 and 3.72 log(10) virus genome copies 100 ml(-1) for NoV GI and GII, respectively, were detected in oysters sampled adjacent to the WWTP discharge. A strong seasonal trend was observed, and the concentrations of NoV GI and GII detected in oysters were correlated with concentrations detected in the wastewater effluent. No seasonal difference was detected in concentrations of E. coli or FRNA bacteriophage detected in oysters.

Use of FRNA bacteriophages to indicate the risk of norovirus contamination in Irish oysters.

Male-specific (F) RNA bacteriophages have been proposed as indicators for human enteric viruses in shellfish. This study compared the use of Escherichia coli and FRNA bacteriophages to indicate the presence and level of noroviruses in Crassostrea gigas. A total of 167 samples from category A and B shellfish harvesting areas were analyzed for E. coli and FRNA bacteriophages by standard methods and for noroviruses (NoV) by using a previously described real-time PCR assay. FRNA bacteriophage and NoV levels in shellfish showed a seasonal trend and were elevated during the winter period (October through March). Conversely, E. coli levels did not reflect this seasonal trend. Categorizing samples on the basis of E. coli levels according to European Union regulatory limits failed to indicate the occurrence or level of NoV in shellfish. However, by grouping shellfish samples on the basis of FRNA bacteriophage levels a clear correlation was observed with NoV levels. The use of FRNA bacteriophages to predict the occurrence of NoV in shellfish could provide improved public health protection and should be considered when developing risk management procedures for shellfisheries.