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

      Rajko-Nenow, Paulina; Keaveney, Sinéad; Flannery, John; O'Flaherty, Vincent; Doré, William (Elsevier, 2012)
      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 24 month 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 20,080 genome copies g− 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

      Flannery, John; Keaveney, Sinéad; Rajko-Nenow, Paulina; O’Flaherty, Vincent; Doré, William (American Society for Microbiology, 2012)
      Concentrations of E. coli, FRNA bacteriophage, norovirus genogroup I (NoV GI) and II (NoV GII) in wastewater were monitored weekly over a one-year period at a wastewater treatment plant (WWTP) providing secondary treatment. A total of 49 samples of influent, primary and secondary-treated wastewater were analyzed. Using a real-time RT-qPCR, mean NoV GI and NoV GII concentrations detected in effluent wastewater were 2.53 and 2.63 log10 virus genome copies 100 ml-1 respectively. Mean NoV concentrations in wastewater during the winter period (January to March inclusive) (n=12) were 0.82 (NoV GI) and 1.41 (NoV GII) log units greater than mean concentrations for the rest of the year (n=37). The mean reduction of NoV GI and GII during treatment was 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 log10 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 concentrations of NoV GI and GII detected in oyster 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.