B04009: Evaluation and validation of alternative indicators of viral contamination in bivalve molluscan shellfish

Study Duration: December 2000 to November 2002

Contractor: Centre for Environment, Fisheries and Aquaculture Sciences (CEFAS)

Shellfish transmitted diseases tend to be of viral origin and usually cause outbreaks of gastroenteritis or infectious hepatitis. The main reason for this is that many bivalve shellfish species, such as oysters and mussels, are produced in shallow, inshore estuaries that are often contaminated with human sewage. Bivalve shellfish are filter feeders. This means that, in the process of feeding, they can concentrate and retain human pathogens derived from sewage contamination. The risk to the consumer from the accumulation of human enteric viruses, such as Norovirus (NV) and hepatitis A (HAV) in shellfish, is increased by the fact that many species are eaten raw or lightly cooked, and also because the whole animal is consumed.

Monitoring of the virological quality of shellfish and/or the waters in which they grow would be both desirable and beneficial. However the main viruses of concern, NV and HAV, are non-culturable or very difficult to culture in the laboratory. Their detection relies upon use of complex molecular methods, which are technically difficult, time consuming, costly, poorly standardised and restricted to a few specialist laboratories. It is for these reasons that commensal enteric bacteria, such as faecal coliforms and E. coli, have been adopted as surrogate indicator organisms to assess the quality of shellfish flesh and to predict the risk of exposure to enteric pathogenic viruses.

Across the EU, the criteria laying down the microbiological standards for bivalve molluscs are set out in European Directive 91/492/EEC. These regulations make use of E. coli to assess the faecal contamination of shellfish, to instigate appropriate control measures (e.g. depuration or relaying in clean waters) and to set end product standards. However, the continuing occurrence of NV and HAV associated diseases linked to the consumption of shellfish that are fully compliant with end product standards suggests that reliance on the traditional faecal indicators does not adequately protect the shellfish consumer.

The project aimed to evaluate and validate the use of viral indicator organisms as alternatives to the recommended microbial contamination indicator, E. coli, in sewage contaminated shellfish and shellfish sold for direct human consumption.

A comprehensive review of existing sources of information was undertaken. This included scrutiny of published scientific/technical journals and analysis of existing in-house data. This approach allowed the most promising candidate viral indicators to be identified.

The second phase of the research was to optimise methods to detect these viral indicators in the laboratory to permit their application to the complex matrix that constitutes shellfish tissue. This was followed by the evaluation and validation stage, where selected alternative indicators were tested on 'real' samples during intensive series of field trials.

All samples were analysed for pathogenic viruses (NV and HAV) and E. coli. This allowed the performance of the alternative indicators to be compared both with the current microbiological indicator (E. coli) and the actual pathogens of interest in shellfish harvesting areas and as an endproduct test in shellfish available for human consumption.

This research was designed to identify viral indicators that may be used to predict the safety of shellfish more effectively than E. coli. Regular monthly monitoring of commercial shellfish harvesting areas in the UK for oysters (Crassostrea gigas) and mussels (Mytilus edulis) were carried out and samples were analysed for Norovirus (NV), hepatitis A virus (HAV), E. coli and the selected 'alternative' indicators (enterovirus, human adenovirus, FRNA bacteriophage, Bacteroides fragilis bacteriophage and somatic coliphages).

The correlation between the occurrence of viral pathogens and selected indicators was examined and revealed a statistically significant relationship between the presence of human pathogenic viruses and all prospective indicators in those areas designated as being heavily contaminated. It was concluded that in areas significantly affected by human sewage pollution, E. coli is an appropriate and adequate indicator for the risk of viral contamination. In cleaner shellfish areas the correlation between all faecal pollution indicators and presence of human enteric viruses was less robust. In such areas, by definition, numbers of E. coli are low or absent; and human enteric viruses may still be isolated. In these areas B. fragilis bacteriophage was generally found in insufficient quantities to be useful as an indicator.

Numbers of somatic bacteriophage were highly variable and did not correlate well with the occurrence of enteric viral pathogens. Adenovirus was the most commonly isolated human enteric virus and could be applied as a molecular index of viral contamination from human origin in shellfish. Absence of human adenoviruses was a parameter that may improve levels of consumer protection against contamination with human enteric viruses. However, the methodology for adenovirus detection depends on PCR (and other molecular techniques) which are not currently sufficiently well standardised and controlled for adoption in routine laboratories.

Among the faecal pollution indicators studied, FRNA bacteriophage showed the most promise as an alternative 'viral' indicator organism for areas where E. coli levels are low or absent. In areas classified using EU microbiological standards as category A and category B, FRNA bacteriophage demonstrated the best predictive capability. Distribution of FRNA bacteriophage was also shown to be seasonally dependent, with higher numbers recorded during the winter months. Higher numbers of NV are also observed, but not of other enteric viruses in winter. Following verification, a distinct relationship between FRNA bacteriophage levels and NV positives were observed.

The results from this study are encouraging with respect to the possible future use of FRNA bacteriophage as an indicator of viral contamination in molluscan shellfish. The methodology is well standardised and accredited, and can be easily adopted in more routine food control laboratories.

Final report is available from the FSA Library and Information centre. To obtain a copy, please contact the Enquiry Desk, Dr. Elsie Widdowson Library and Information Services, Food Standards Agency ( Tel: +44 (0) 20 7276 8181/8182 or by e-mail to: library&info@foodstandards.gsi.gov.uk ).

Rangdale, R. E. (2001) Microbiological Indicators of Faecal Contamination in Shellfish Harvesting Areas. Oral presentation Environment Agency Workshop, CEFAS Weymouth Dorset. UK. (3rd July 2001).

Lees, D. N. (2001) Norwalk-like Virus Contamination of Shellfish and the Development of Better Regulatory Controls. Oral presentation: Recents Avenços En La Transmissio De Virus A Traves Del Mediambibient. University of Barcelona, Spain. (22nd October 2001).

Lees, D. N. (2002) Viruses and Bivalve Shellfish: Where do we go from here. Keynote oral presentation: 4th International Conference on Molluscan Shellfish Safety, June 4-8 2002, Santiago de Compostela, Galicia, Spain.

Contact: Email: science@foodstandards.gsi.gov.uk