Ce diaporama a bien été signalé.
Nous utilisons votre profil LinkedIn et vos données d’activité pour vous proposer des publicités personnalisées et pertinentes. Vous pouvez changer vos préférences de publicités à tout moment.
Assessing the impact of wastewater treatment plant     effluent on noroviruscontamination in shellfisheries  EPA STRIVE pr...
Norovirus and Gastroenteritis   The most common cause of infectious intestinal disease in the    community     – US: 23 m...
Size of the NoV problem Infectious intestinal disease studies show large underreporting for norovirus compared with other ...
Role of shellfish in spread of NoV?                                                   person-to-person                    ...
Role of shellfish in spread of NoV?                                                   person-to-person                    ...
Virus removal during wastewater treatment?   Limited information on NoV removal during wastewater    treatment and surviv...
Assessing the impact of WWTP plant effluenton norovirus contamination in shellfisheries      EPA STRIVE project: 2008-EH-M...
Microbiological parameters and methodology used  Indicator organism /       Matrix                         Methodology  Pa...
WWTP plants and sampling   Wastewater treatment plants    – WWTP1        • Secondary treatment (activated sludge process)...
WWTP1 - 12 month data                                    NoV GII concentration in effluent ( ) and                       ...
Mean log10 concentrations of NoV in effluent    wastewater and oysters by season (WWTP1)                               Mea...
Mean log10 concentrations of NoV in effluent    wastewater and oysters by season (WWTP1)                               Mea...
Mean log10 concentrations of E. coli, FRNA   bacteriophage and NoV GI and GII at wastewater             treatment stages (...
Mean log10 concentrations of E. coli, FRNA   bacteriophage and NoV GI and GII at wastewater             treatment stages (...
Mean log10 concentrations of E. coli, FRNA   bacteriophage and NoV GI and GII at wastewater             treatment stages (...
Interpretation of PCR results?    PCR capable of detecting infectious and non-infectious     virus     – Virus genome or ...
Genus               Genogroup          Source  Levivirus               MS2            Mammals other than humans           ...
Detection of NoV GII (Real-time PCR) and FRNA GAbacteriophage (Infectivity assay and Real-time PCR)                       ...
Detection of NoV GII (Real-time PCR) and FRNA GAbacteriophage (Infectivity assay and Real-time PCR)                       ...
Log10 reductions of FRNA bacteriophage                and NoV GII                       Treatment                   Activa...
Impact of CSO discharges and NoV GII                               concentrations in oysters                           4  ...
Concentration of infectious and total FRNA GAbacteriophage in final effluent and CSO discharges                           ...
Concentration of infectious and total FRNA GAbacteriophage in final effluent and CSO discharges    Mean difference Log10 G...
Indicator and Index roles                                FRNA bacteriophage         NoV                                 (i...
Key conclusions & recommendations (1/3)   Real-time RT qPCR is an inappropriate method to determine    NoV (and other vir...
Key conclusions & recommendations (2/3)   FRNA bacteriophage provide a good indication of infectious    virus removal dur...
Key conclusions & recommendations (3/3)   CSO discharges contain a greater concentration of infectious    virus (as judge...
Future Work   Methods to distinguish between infectious and non-    infectious NoV    – Project lead NUIG partner with MI...
Acknowledgements   Steering committee    – Sandra Kavanagh (EPA), Tadhg O’Connor (DEHLG), Noel      O’Keeffe (Cork County...
Shellfish Microbiology teamMarine Environment and Food SafetyServices, Marine Institute  Bill Doré  Sinéad Keaveney  John ...
Prochain SlideShare
Chargement dans…5
×

Assessing the impact of wastewater treatment plant effulent on norovirus contamination in shellfisheries - Sinead Keaveney

1 184 vues

Publié le

Publié dans : Technologie
  • Soyez le premier à commenter

Assessing the impact of wastewater treatment plant effulent on norovirus contamination in shellfisheries - Sinead Keaveney

  1. 1. Assessing the impact of wastewater treatment plant effluent on noroviruscontamination in shellfisheries EPA STRIVE project: 2008-EH-MS-7-53 EPA STRIVE RESEARCH CONFERENCE June 28th 2012, Trinity College, Dublin
  2. 2. Norovirus and Gastroenteritis The most common cause of infectious intestinal disease in the community – US: 23 million cases annually (Mead et al., 1999) “Relatively mild” gastroenteritis including nausea, diarrhoea, vomiting, fever and abdominal pain – Infectious period: 1-4 days, illness duration of about 2-4 days – Excess deaths in epidemic years (Harris et al., 2008) Seasonal distribution – “Winter vomiting disease” Person to person spread major route of infection – Hospitals, cruise ships, care settings – Strain diversity (Human genogroups; GI and GII)
  3. 3. Size of the NoV problem Infectious intestinal disease studies show large underreporting for norovirus compared with other pathogens Communicable disease surveillance 1 centre (CDSC) 1 248 GP 2.3 1,562 Community 3.2 Norovirus Salmonella
  4. 4. Role of shellfish in spread of NoV? person-to-person spread recombination Virus shedding in feces: 104-1010 viral particles/g new variant Waste water treatment: critical control point Discharge to the environmentcontaminated Global tradewith multiple strains Shellfish (oysters)
  5. 5. Role of shellfish in spread of NoV? person-to-person spread recombination Virus shedding in feces: 104-1010 viral particles/g new variant No virus standards EPA license requirements Discharge to the environmentcontaminated Global tradewith multiple strains Shellfish (oysters)
  6. 6. Virus removal during wastewater treatment? Limited information on NoV removal during wastewater treatment and survival in the environment NoV is difficult to detect and quantify in environmental sample – No culture system – Low target numbers – Environmental inhibitors to molecular detection – Real time PCR (RT-qPCR) method available which allows NoV quantification
  7. 7. Assessing the impact of WWTP plant effluenton norovirus contamination in shellfisheries EPA STRIVE project: 2008-EH-MS-7-53Overall project objectives Quantify NoVs in sewage influent, intermediate stages and effluent in a secondary WWTP and identify the extent of NoV removal Determine the extent of the reduction of NoV levels using UV disinfection Determine the relative contribution of CSO discharges and continuous inputs of NoVs in shellfisheries Establish (T90 values) for norovirus in seawater under typical winter and summer conditions
  8. 8. Microbiological parameters and methodology used Indicator organism / Matrix Methodology Pathogen Virus extraction using proteinase K Shellfish followed by RT-qPCR (CEN, 2010 Food Environ. Virol. 2:146-155) Norovirus Virus concentration (adapted from Katayama Wastewater et al., 2002, Appl. Environ. Microbiol. 68; 1033-1039) followed by RT-qPCR (CEN/ISO) ISO 10705-1, Part 1 Shellfish (plus probe hybridisation assay for GA) FRNA andbacteriophage RT-qPCR for GA (adapted from Wolf et al., wastewater 2007,J. Virol. Methods. 149;123-128) Shellfish and ISO/TS 16649-3: Most Probable Number E. coli (MPN) method wastewater
  9. 9. WWTP plants and sampling Wastewater treatment plants – WWTP1 • Secondary treatment (activated sludge process) – WWTP 2 • UV treatment • CSO discharges Oysters – Close proximity to the outfall of both plants Water Research Facility (Tuam WWTP, Co. Galway) – NUI Galway – UV treatment
  10. 10. WWTP1 - 12 month data NoV GII concentration in effluent ( ) and oysters ( ) 6Log10 genome copies /g or /100 ml 5 4 3 2 1 r=0.68 (p=<0.05) 0 Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May
  11. 11. Mean log10 concentrations of NoV in effluent wastewater and oysters by season (WWTP1) Mean concentration ± SDNoV Effluent Oystersgenogroup Season (n) (copies/ 100ml) (copies/g) GI All data (49) 2.53 ± 0.57 3.53 ± 0.87 April-Dec (37) 2.32 ± 0.68 3.12 ± 0.68 Jan-Mar (12) 3.06 ± 0.55 4.43 ± 0.50 GII All data (49) 2.63 ± 0.71 3.73 ± 0.55 April-Dec (37) 2.27 ± 0.39 3.21 ± 0.56 Jan-Mar (12) 3.53 ± 0.65 4.86 ± 0.54
  12. 12. Mean log10 concentrations of NoV in effluent wastewater and oysters by season (WWTP1) Mean concentration ± SDNoV Effluent Oystersgenogroup Season (n) (copies/ 100ml) (copies/g) GI All data (49) 2.53 ± 0.57 3.53 ± 0.87 April-Dec (37) 2.32 ± 0.68 3.12 ± 0.68 Jan-Mar (12) 3.06 ± 0.55 4.43 ± 0.50 GII All data (49) 2.63 ± 0.71 3.73 ± 0.55 April-Dec (37) 2.27 ± 0.39 3.21 ± 0.56 Jan-Mar (12) 3.53 ± 0.65 4.86 ± 0.54
  13. 13. Mean log10 concentrations of E. coli, FRNA bacteriophage and NoV GI and GII at wastewater treatment stages (WWTP1) Wastewater treatment stage Influent Final effluentn = 49 Concn. Concn. Log (range) (range) reductionE. coli 6.54 ± 0.59 5.06 ± 0.58 1.49 ± 0.63MPN 100 ml-1 (3.73-7.54) (3.54-6.20)FRNA 5.54 ± 0.51 3.41 ± 0.77 2.13 ± 0.76bacteriophage (3.87-6.82) (2.00-5.84)pfu 100 ml-1NoV GI 3.32 ± 0.64 2.53 ± 0.57 0.79 ± 0.49copies 100 ml-1 (2.05-4.76) (1.26-4.06)NoV GII 3.55 ± 0.89 2.63 ± 0.71 0.92 ± 0.76copies 100 ml-1 (1.81-5.34) (1.51-4.08)
  14. 14. Mean log10 concentrations of E. coli, FRNA bacteriophage and NoV GI and GII at wastewater treatment stages (WWTP1) Wastewater treatment stage Influent Final effluentn = 49 Concn. Concn. Log (range) (range) reductionE. coli 6.54 ± 0.59 5.06 ± 0.58 1.49 ± 0.63MPN 100 ml-1 (3.73-7.54) (3.54-6.20)FRNA 5.54 ± 0.51 3.41 ± 0.77 2.13 ± 0.76bacteriophage (3.87-6.82) (2.00-5.84)pfu 100 ml-1NoV GI 3.32 ± 0.64 2.53 ± 0.57 0.79 ± 0.49copies 100 ml-1 (2.05-4.76) (1.26-4.06)NoV GII 3.55 ± 0.89 2.63 ± 0.71 0.92 ± 0.76copies 100 ml-1 (1.81-5.34) (1.51-4.08)
  15. 15. Mean log10 concentrations of E. coli, FRNA bacteriophage and NoV GI and GII at wastewater treatment stages (WWTP1) Wastewater treatment stage Influent Final effluentn = 49 Concn. Concn. Log (range) (range) reductionE. coli 6.54 ± 0.59 5.06 ± 0.58 1.49 ± 0.63MPN 100 ml-1 (3.73-7.54) (3.54-6.20)FRNA 5.54 ± 0.51 3.41 ± 0.77 2.13 ± 0.76bacteriophage (3.87-6.82) (2.00-5.84)pfu 100 ml-1NoV GI 3.32 ± 0.64 2.53 ± 0.57 0.79 ± 0.49copies 100 ml-1 (2.05-4.76) (1.26-4.06)NoV GII 3.55 ± 0.89 2.63 ± 0.71 0.92 ± 0.76copies 100 ml-1 (1.81-5.34) (1.51-4.08)
  16. 16. Interpretation of PCR results? PCR capable of detecting infectious and non-infectious virus – Virus genome or virus capsid may be damaged preventing infection – Problem in environmental samples – Damaged virus may still be detected by RT-qPCR – No infectivity assay for NoV Therefore… – Use FRNA bacteriophage to compare virus infectivity results against PCR results • Infectious virus V “Total” virus
  17. 17. Genus Genogroup Source Levivirus MS2 Mammals other than humans GA High frequency in human Allolevivirus Qβ Low frequency in humans SP A range of non-human hosts FRNA bacteriophage GAGA Infectivity assay (pfu/100 ml) GA Real-time PCR assay (GA genome copies/100 ml) Total GA bacteriophage bacteriophage detected by probe (classical test) hybridisation Adapted qualitative real-time PCR assay for GA (Wolf et al., 2008) and developed assay to allow for quantification using GA DNA standards
  18. 18. Detection of NoV GII (Real-time PCR) and FRNA GAbacteriophage (Infectivity assay and Real-time PCR) ct ct ct ct fe fe fe fe In In In In influent secondary UV oysters
  19. 19. Detection of NoV GII (Real-time PCR) and FRNA GAbacteriophage (Infectivity assay and Real-time PCR) ct ct ct ct fe fe fe fe In In In In influent influent secondary secondary UV UV oysters oysters
  20. 20. Log10 reductions of FRNA bacteriophage and NoV GII Treatment Activated Sludge UV Total (WWTP1) (Tuam WRF) reductionInfectious GA 2.13 1.8 3.93(pfu 100ml-1)NoV GII 0.92 0.52 1.44(copies 100ml-1)
  21. 21. Impact of CSO discharges and NoV GII concentrations in oysters 4 12 3 413 m 3.5 10Log10 genome copies g-1 3 Log10 NoV discharged 8 2.5 2 6 1.5 LOD 4 1 2 0.5 0 0 0 12 24 36 48 60 72 84 96 Time (hours) CSO event () NoV GII in CSO discharge () NoV GII in oysters ()
  22. 22. Concentration of infectious and total FRNA GAbacteriophage in final effluent and CSO discharges LOD Infectivity assay () PCR assay () CSO events
  23. 23. Concentration of infectious and total FRNA GAbacteriophage in final effluent and CSO discharges Mean difference Log10 GA: CSO = 0.1 Mean difference Log10 GA: UV treated = 2.51
  24. 24. Indicator and Index roles FRNA bacteriophage NoV (infectivity assay) (real-time RT-qPCR)Indicator of virus reduction during wastewater treatment – desirablecriteriaUbiquitous in wastewater Yes No (absent in summer in some WWTP)Detects infectious virus only Yes NoIndex of virus risk in bivalve shellfish - desirable criteriaConcentration elevated in No/Yes (size of impact, Yes (extent of humanlocations of higher risk animal sources) impact sources)Concentration elevated at Yes (concentrationstimes of higher risk No (constant year round) related to current infections in population)Concentration directly No Yes (EFSA, 2012)related to risk of infectionGeneral criteriaCheap and easy to analyse Yes (approx. €20 – 30) No (approx. €200)ISO methods available Yes No
  25. 25. Key conclusions & recommendations (1/3) Real-time RT qPCR is an inappropriate method to determine NoV (and other viruses) removal during WWT – There is a requirement to establish methods that distinguish between infectious and non-infectious NoV Real-time RT qPCR can be used to determine the concentration of NoV in oysters and provides a relative index of the potential risk to consumers – Risk management plans can be developed with NoV monitoring of harvest areas forming a useful element in those plans
  26. 26. Key conclusions & recommendations (2/3) FRNA bacteriophage provide a good indication of infectious virus removal during WWT – Consideration should be given to introducing criteria for virus reduction using FRNA bacteriophage as an indicator – Consideration should be given to introducing a programme of before and after monitoring for FRNA bacteriophage to assess ongoing compliance with any such criteria UV disinfection can provide and additional ~2 log10 reduction in infectious virus (FRNA bacteriophage) – The introduction of UV disinfection should be considered at WWTPs that are demonstrated to impact shellfish growing areas
  27. 27. Key conclusions & recommendations (3/3) CSO discharges contain a greater concentration of infectious virus (as judged by FRNA bacteriophage) than fully treated wastewater effluent – Appropriate guidelines that limit the impact of CSO discharges in shellfish production areas, as far a reasonably practical, should be developed. Further research is required to fully establish the relative impact of CSO discharges on shellfish production areas. – Other potential contamination sources during storm events
  28. 28. Future Work Methods to distinguish between infectious and non- infectious NoV – Project lead NUIG partner with MI – 3 year Ph.D. Alternative wastewater treatment processes – Project lead NUIG partner with MI – Barrier methods (membrane filters) – Additional UV disinfection
  29. 29. Acknowledgements Steering committee – Sandra Kavanagh (EPA), Tadhg O’Connor (DEHLG), Noel O’Keeffe (Cork County Council), Vincent O’Flaherty (NUIG), Terry McMahon (MI) Galway City Council – Ray Brennan Cork County Council – Noel O’Keeffe Wastewater Research Facility (WRF) Tuam, Co. Galway – Eoghan Clifford, NUIG EPA
  30. 30. Shellfish Microbiology teamMarine Environment and Food SafetyServices, Marine Institute Bill Doré Sinéad Keaveney John Flannery Paulina Rajko-Nenowwww.marine.ie

×