| Summary: | Filter-feeding bivalve molluscs bio-accumulate pathogens and are the principal vectors of human seafood-related illness. Whilst the adoption of bacterial indicators for shellfish monitoring has reduced the threat of bacterial infections associated with their consumption, enteric viral pathogens, particularly Norovirus, continue to pose a significant threat to human health. Approaches that may further help to reduce the incidence of human viral infections associated with the consumption of bivalve molluscs include the adoption of viral indicators (bacteriophages), direct pathogen detection and microbial source tracking (MST) in shellfish and their overlying waters. Bacteriophages infecting the human-specific Bacteroides fragilis strain GB-124 have recently been used successfully as a low-cost MST tool, and they may represent a potential surrogate for the presence of human Norovirus in bivalve molluscan shellfisheries. However, little is known about their presence in shellfish overlying waters and shellfish matrices, or how their presence correlates with the presence of human Norovirus. A two-year field-based research study was therefore undertaken to assess the occurrence of bacterial (E. coli, faecal coliforms and intestinal enterococci) and viral indicators (somatic coliphages, F-RNA and GB-124 phages) in mussel (Mytilus edulis) sample matrices (overlying waters, flesh and intravalvular liquid and digestive gland) in a river estuary in Southeast England. Furthermore, the occurrence of human Norovirus in mussel digestive gland was analysed using a real-time quantitative PCR. Based on the E. coli levels recorded in mussels, the site would be classified as a class ‘B’ harvesting area. There was a marked seasonality in the occurrence of all indicators and Norovirus. Autumn and winter months accounted for the highest levels. Somatic coliphages were the most abundant phage (and were always detected), followed by F-RNA and GB-124 phages, which were undetected in 7.1% and 40% of samples respectively, mainly during the summer and spring months. The mussel digestive gland was the most ‘sensitive’ matrix, recording the highest percentage of positive samples for all indicators. All bacteriophages correlated better with the occurrence of Norovirus in all mussel matrices than did E. coli. Somatic coliphages demonstrated the highest Spearman’s correlation coefficients in mussel overlying waters (rho=0.859), and mussel flesh and intravalvular liquid (rho=0.761), while F-RNA phages demonstrated the highest coefficients in mussel digestive gland (rho=0.879). GB-124 phages were more consistently detected during autumn and winter months, and demonstrated the highest correlation coefficient with Norovirus (rho=0.840) in the mussel digestive gland matrix, when these seasons were analysed separately. Importantly, the highest level of GB-124 phages recorded coincided with the highest concentrations of Norovirus (even exceeding somatic coliphages concentrations), indicating a strong human faecal contamination. Therefore, enumeration of bacteriophages may offer a practical tool to indicate contamination of shellfish and their overlying waters by enteric viral pathogens (either during routine monitoring or as part of a risk assessment of proposed harvesting areas), potentially representing a highly valuable contribution to human health protection.
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