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Listen to this siteFriday 30 April 2004
This research study aims to provide information on the survival and behaviour of Noroviruses and potential viral indicators in sewage treatment and marine environments.
Study Duration : September 1999 to October 2002
Contractor : Centre of Environment, Fisheries and Aquaculture Sciences (CEFAS)
In the UK the main food safety risk associated with the consumption of bivalve molluscs is gastroenteritis caused by Noroviruses (NV). The protection of vulnerable shellfisheries is heavily dependent on adequate sewage treatment and inactivation of any residual pathogen load through effective dilution/dispersion of effluent in the sea following discharge. NV are non-culturable and molecular methods for their detection have only recently been developed. As a result, despite their acknowledged importance as agents of viral illness, virtually nothing is known about their inactivation/removal during sewage treatment or survival in the environment. Monitoring data from conventional bacterial faecal pollution indicators (such as E. coli ) is used to inform decisions on sewage treatment, location of discharges and priority given to intermittent discharges (such as combined storm overflows). It is however not known whether such indicators adequately reflect the behaviour and survival of NV.
The main objectives of this project will be to provide information on: the occurrence of NV in sewage effluents; the inactivation/removal of NV during passage through conventional biological sewage treatment; the survival characteristics of NV in the sea following effluent discharge; the linkage between NV discharge in effluents and their uptake in shellfish; and the relationship of NV during the above studies to other more commonly monitored enteric viruses (enteroviruses), conventional faecal pollution indicators ( E. coli ) and a potential viral indicator (FRNA bacteriophage).
Initial work will focus on the development of a quantitative Polymerase Chain Reaction (PCR) assay to detect NV strains in sewage effluent using a real time fluorogenic 5' nuclease assay (TaqMan). The NV occurrence in sewage effluent and inactivation/removal during treatment will be examined at three sewage treatment works, all sampled fortnightly for crude sewage, primary and final effluents over a one year period. In addition, at two of the sewage treatment works, virus presence in discharged effluent will be compared with virus presence in shellfish laid at an adjacent field site. NV occurrence will also be compared with enterovirus and FRNA bacteriophage by TaqMan PCR and with the indicator organisms E. coli and FRNA bacteriophage by viability assay.
The survival of NV in marine water will be investigated by seeding virus into seawater in the laboratory and measuring the degradation of NV over time using the developed TaqMan assay. NV inactivation will be compared with poliovirus, FRNA bacteriophage and E. coli . The inactivation kinetics of these organisms will also be investigated under simulated winter (low light and temperature) and summer (high light and temperature) conditions. Sunlight will be simulated using a solar simulator with the correct balance of UVA and UVB found in natural sunlight in England.
Little is known about whether conventional faecal indicators reflect the behaviour of viruses or survival characteristics during sewage treatment, in the environment following discharge or in contaminated foods, e.g. shellfish.
This project aimed to address these points and to investigate the relationship between Noroviruses and enteric viruses, faecal pollution indicators and a potential viral indicator.
The project developed a method for the detection and enumeration of Noroviruses (NV) in sewage effluents, a quantitative PCR assay using a real time fIuorogenic 5' nuclease assay (TaqMan). TaqMan assays were also developed to detect human enteroviruses and FRNA bacteriophage - a candidate viral indicator organism.
NV'were detected in 96% of crude sewage samples and 75% of final sewage effluents. NV concentrations were high compared with other organisms - crude sewage, for example, contained an average of 4.5 log 10 virus per ml. Although NV were present year round, significantly higher levels were found during the winter months. The occurrence of peak viral contamination loads in effluent during the winter months may contribute towards the noted seasonality of shellfish associated disease outbreaks.
E. coli levels were reduced by 3 logs during sewage treatment whereas NV (and other viruses) were reduced by only 1 log. These results indicate that sewage treatment is considerably more effective for removal of indicator bacteria than for enteric viruses. A broad correlation was found between NV levels in discharged effluent and in shellfish impacted by those effluents.
Laboratory investigations showed that although sunlight has an impact on virus survival, it is much less significant for bacterial indicators. By contrast water temperature seemed to have only a marginal effect on survival times. Thus in the winter period when the UV intensity of natural sunlight is Iow viruses can survive for long periods in the sea. The data suggests that the use of bacterial indicator data may significantly underestimate the potential risk from viral contamination. Survival of NV in the sea during the winter may play a part in the documented winter incidence of shellfish associated NV gastroenteritis outbreaks.
This data demonstrates that NV are a very common constituent of sewage effluents and underscores the potential for such discharges to cause viral contamination in shellfisheries.
The study also showed that conventional faecal pollution indicators do not adequately reflect the characteristics of viruses in such situations. The findings should help inform discussions on the most effective combination of control measures to protect vulnerable shellfisheries from enteric virus contamination.
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 ).
Henshilwood, K., Cross, L., Lees, D. N. (2002) The Survival of Norwalk-like Virus and Potential Viral Indicators in Sewage Treatment Processes and in the Marine Environment. Oral presentation: IWA 3rd World Water Congress, Melbourne, Australia (7-12 April 2002).
Cross, L., Henshilwood, K., Lees, D. N. (2001) The Development of Quantitative Molecular Assays for Determination of Enteric Viral Load within Environmental Samples. Poster presentation Winchester University: Conference on Real-Time PCR (September 2001).
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