Investigation of F-RNA Bacteriophage as a Tool in Re-Opening Australian Oyster Growing Areas Following Sewage Spills

Abstract

Oysters contaminated with human enteric viruses from sewage are implicated in foodborne outbreaks globally. Bacteriophages have been identified as potential indicators for these viruses, but have not been used in shellfish management outside of the USA. This study aimed to determine the background levels of F-RNA phage in five Australian oyster growing areas with a history of sewage spills and closures, over an 18-month period. In addition, oysters from five growing areas impacted by adverse sewage events were investigated for F-RNA phage, Escherichia coli, norovirus (NoV) and hepatitis A virus (HAV). F-RNA phage ≤ 60 pfu/100 gm shellfish flesh were found to represent a conservative background level in the surveyed areas. Following two of the five sewage spills, elevated phage levels were observed in most sample sites less than 4 days post spill. By 7 days, most sites from all events had phage < 30 pfu/100 gm. NoV was detected in day 1 and day 6 samples from one event when all phage were ≤ 30 pfu/100 gm. NoV was also detected in a day 3 sample from another event with < 30 phage pfu/100 gm, however, multiple replicate samples had elevated phage levels. The results of this study add evidence on the potential use of F-RNA phage as a tool in early re-opening of oyster harvest areas post sewage spills. However, it also highlights the need to better understand situations where phage testing may be ineffectual, and the importance of sampling at multiple sites and over multiple time points, to effectively capture evidence of contamination.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. ABS (2019). ABS 3218.0 – Regional Population Growth Australia, 2017–18 https://www.abs.gov.au/AUSSTATS/abs@.nsf/DetailsPage/3218.02017-18, accessed 15th October 2020.

  2. ASQAAC (2016). Australian Shellfish Quality Assurance Program, Operations Manual. http://safefish.com.au/technical-program.

  3. Bellou, M., Kokkinos, P., & Vantarakis, A. (2013). Shellfish-borne viral outbreaks: a systematic review. Food and Environmental Virology, 5(1), 13–23.

    CAS  Article  Google Scholar 

  4. Blatchley, E. R. I., Gong, W. L., Alleman, J. E., Rose, J. B., Huffman, D. E., Otaki, M., et al. (2007). Effects of wastewater disinfection on waterborne bacteria and viruses. Water Environment Research, 79(1), 81–92.

    CAS  Article  Google Scholar 

  5. Brake, F., Kiermeier, A., Ross, T., Holds, G., Landinez, L., & McLeod, C. (2018). Spatial and temporal distribution of Norovirus and E. coli in Sydney Rock oysters following a sewage overflow into an estuary. Food and Environmental Virology, 10(1), 7–15.

    Article  Google Scholar 

  6. CEFAS. (2007). Enumeration of male-specific RNA bacteriophages in bivalve molluscan shellfish. Weymouth, UK: CEFAS Standard Operating Procedure.

    Google Scholar 

  7. Dore, W. J., Henshilwood, K., & Lees, D. N. (2000). Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish. Applied and Environmental Microbiology, 66(4), 1280–1285.

    CAS  Article  Google Scholar 

  8. Dore, W. J., & Lees, D. N. (1995). Behaviour of Escherichia coli and male-specific bacteriophage in environmentally contaminated bivalve molluscs before and after depuration. Applied and Environmental Microbiology, 61(8), 2830–2834.

    CAS  Article  Google Scholar 

  9. EC (2004). Annex II of Commission Regulation (EC) No 854/2004 on laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32004R0854&from=en.

  10. EFSA. (2019). Scientific report on analysis of the European baseline survey of norovirus in oysters. EFSA Journal, 17(7), 5762–5848. https://doi.org/10.2903/j.efsa.2019.5762.

    Article  Google Scholar 

  11. FDA (2009). National Shellfish Sanitation Program (NSSP) Guide for the control of molluscan shellfish: 2009 Revision http://www.fda.gov/downloads/Food/GuidanceRegulation/FederalStateFoodPrograms/UCM350004.pdf.

  12. FDA (2015). National Shellfish Sanitation Program (NSSP) guide for the control of molluscan shellfish: 2015 revision. http://www.fda.gov/Food/GuidanceRegulation/FederalStateFoodPrograms/ucm2006754.htm.

  13. Flannery, J., Keaveney, S., & Dore, W. (2009). Use of FRNA bacteriophages to indicate the risk of norovirus contamination in Irish oysters. Journal of Food Protection, 72(11), 2358–2362.

    CAS  Article  Google Scholar 

  14. Havelaar, A. H., Furuse, K., & Hogeboom, W. M. (1986). Bacteriophages and indicator bacteria in human and animal faeces. Journal of Applied Bacteriology, 60(3), 255–262.

    CAS  Article  Google Scholar 

  15. Hewitt, J., Rivera-Aban, M., & Greening, G. E. (2009). Evaluation of murine norovirus as a surrogate for human norovirus and hepatitis A virus in heat inactivation studies. Journal of Applied Microbiology, 107(1), 65–71. https://doi.org/10.1128/JVI.02346-05.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Hodgson, K. R., Torok, V. A., & Turnbull, A. R. (2017). Bacteriophage as enteric viral indicators in bivalve mollusc management. Food Microbiology, 65, 284–293.

    CAS  Article  Google Scholar 

  17. ISO/CEN (2013). Microbiology of food and animal feed - Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR - Part 1: Method for quantification ISO/TS 15216–1:2013, Corrected Version 2013–05–01. CEN/TC 275 - Food analysis - Horizontal methods.

  18. Lodo, K. L., Veitch, M. G. K., & Green, M. L. (2014). An outbreak of norovirus linked to oysters in Tasmania. Communicable Diseases Intelligence, 38(1), E16–E19.

    PubMed  Google Scholar 

  19. Long, S. C., El-Khoury, S. S., Oudejans, S. J. G., Sobsey, M. D., & Vinjé, J. (2005). Assessment of sources and diversity of male-specific coliphages for source tracking. Environmental Engineering Science, 22(3), 367–377.

    CAS  Article  Google Scholar 

  20. Lowther, J. A., Cross, L., Stapleton, T., Gustar, N. E., Walker, D. I., Sills, M., et al. (2019). Use of F-specific RNA bacteriophage to estimate infectious Norovirus levels in oysters. Food and Environmental Virology, 11, 247–258.

    CAS  Article  Google Scholar 

  21. Lowther, J. A., Gustar, N. E., Powell, A. L., O’Brien, S., & Lee, D. N. (2018). A one-year survey of Norovirus in UK oysters collected at the point of sale. Food and Environmental Virology, 10, 278–287.

    CAS  Article  Google Scholar 

  22. MPI (2013). Enumeration of Escherichia coli in Bivalve Molluscan Shellfish MPI Method v9. https://www.mpi.govt.nz/dmsdocument/25886-enumeration-of-e-coli-in-bms-mpi-method.

  23. Scholes, P., Greening, G., Campbell, D., Sim, J., Gibbons-Davies, J., Dohnt, G., et al. (2009). Microbiological quality of shellfish in estuarine areas. http://www.toiteorapublichealth.govt.nz/vdb/document/57.

  24. Stewart, J. R., Gast, R. J., Fujioka, R. S., Solo-Gabriele, H. M., Meschke, J. S., Amaral-Zettler, L. A., et al. (2008). The coastal environment and human health: Microbial indicators, pathogens, sentinels and reservoirs, Proceedings from Centers for Oceans and Human Health Investigators Meeting, Woods Hole, MA, USA. A Global Access Science Source Environmental Health. https://doi.org/10.1186/1476-069X-7-S2-S3.

    Article  Google Scholar 

  25. Torok, V., Hodgson, K., McLeod, C., Tan, J., Malhi, N., & Turnbull, A. (2018). National survey of foodborne viruses in Australian oysters at production. Food Microbiology, 69, 196–203.

    CAS  Article  Google Scholar 

  26. USFDA (2010). Laboratory manuals (food). BAM appendix 2: Most Probable Number from serial dilutions. https://www.fda.gov/food/laboratory-methods-food/bam-appendix-2-most-probable-number-serial-dilutions.

  27. WHO. (2001). Indicators of microbial water quality (Vol. Chapter 13, Water Quality: Guidelines, Standards and Health). London, UK: IWA Publishing.

    Google Scholar 

Download references

Acknowledgements

We acknowledge that this project (FRDC 2015/037) was supported by funding from the Fisheries Research and Development Corporation on behalf of the Australian Government. We also acknowledge the financial support of TasWater, Central Coast Council, Port Macquarie-Hastings Council, Shoalhaven Council and New South Wales Food Authority. We would like to acknowledge and thank the steering committee members for their contributions and input into the project: Anthony Zammit and Phil Baker, New South Wales Food Authority; Maree Smith, Port Macquarie-Hastings Council; Wayne Hutchinson, Oysters Australia; Joanna Waugh and Stephen Shinners, Central Coast Council; Andrew McVey, Shoalhaven City Council; David Holmes, TasWater: Kate Wilson, Tasmanian Shellfish Quality Assurance Program; Justin Goc, Barilla Bay Oysters; and Brandon Armstrong, Armstrong Oysters. We also acknowledge contributions from New South Wales Shellfish Program, Oysters Australia and Tasmanian Shellfish Quality Assurance Program and the NSW and Tasmanian oyster growers and samplers for their assistance in collecting and transporting oyster samples, in particular Brandon Armstrong, Wayne Moxham, Anthony Munn, Justin Goc, Josh Poke, James Calvert, Peter Kosmeyer and Tony Byrne. We acknowledge the technical assistance of Linda Friedrich and Navreet Malhi, SARDI Food Sciences. We also acknowledge Phil Baker, NSW Food Authority, and Ruari Colquhoun, University of Tasmania, for generation of maps.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Valeria A. Torok.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Torok, V.A., Hodgson, K.R., Jolley, J. et al. Investigation of F-RNA Bacteriophage as a Tool in Re-Opening Australian Oyster Growing Areas Following Sewage Spills. Food Environ Virol (2021). https://doi.org/10.1007/s12560-021-09462-4

Download citation

Keywords

  • Shellfish management
  • Food safety
  • Foodborne viruses
  • Norovirus
  • Hepatitis A virus
  • Escherichia coli bacteriophage MS2