1. Assessing the impact of
wastewater treatment plant
effluent on norovirus
contamination in shellfisheries
EPA STRIVE project: 2008-EH-MS-7-53
EPA STRIVE RESEARCH CONFERENCE
June 28th 2012, Trinity College, Dublin

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. Size of the NoV problem
Infectious intestinal disease studies show large under
reporting 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. 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 environment
contaminated Global trade
with multiple
strains
Shellfish (oysters)

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 environment
contaminated Global trade
with multiple
strains
Shellfish (oysters)

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. Assessing the impact of WWTP plant effluent
on norovirus contamination in shellfisheries
EPA STRIVE project: 2008-EH-MS-7-53
Overall 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. 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
and
bacteriophage 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. 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. WWTP1 - 12 month data
NoV GII concentration in effluent ( ) and
oysters ( )
6
Log10 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. Mean log10 concentrations of NoV in effluent
wastewater and oysters by season (WWTP1)
Mean concentration ± SD
NoV Effluent Oysters
genogroup 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. Mean log10 concentrations of NoV in effluent
wastewater and oysters by season (WWTP1)
Mean concentration ± SD
NoV Effluent Oysters
genogroup 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. Mean log10 concentrations of E. coli, FRNA
bacteriophage and NoV GI and GII at wastewater
treatment stages (WWTP1)
Wastewater treatment stage
Influent Final effluent
n = 49 Concn. Concn. Log
(range) (range) reduction
E. coli 6.54 ± 0.59 5.06 ± 0.58 1.49 ± 0.63
MPN 100 ml-1 (3.73-7.54) (3.54-6.20)
FRNA 5.54 ± 0.51 3.41 ± 0.77 2.13 ± 0.76
bacteriophage (3.87-6.82) (2.00-5.84)
pfu 100 ml-1
NoV GI 3.32 ± 0.64 2.53 ± 0.57 0.79 ± 0.49
copies 100 ml-1 (2.05-4.76) (1.26-4.06)
NoV GII 3.55 ± 0.89 2.63 ± 0.71 0.92 ± 0.76
copies 100 ml-1 (1.81-5.34) (1.51-4.08)

14. Mean log10 concentrations of E. coli, FRNA
bacteriophage and NoV GI and GII at wastewater
treatment stages (WWTP1)
Wastewater treatment stage
Influent Final effluent
n = 49 Concn. Concn. Log
(range) (range) reduction
E. coli 6.54 ± 0.59 5.06 ± 0.58 1.49 ± 0.63
MPN 100 ml-1 (3.73-7.54) (3.54-6.20)
FRNA 5.54 ± 0.51 3.41 ± 0.77 2.13 ± 0.76
bacteriophage (3.87-6.82) (2.00-5.84)
pfu 100 ml-1
NoV GI 3.32 ± 0.64 2.53 ± 0.57 0.79 ± 0.49
copies 100 ml-1 (2.05-4.76) (1.26-4.06)
NoV GII 3.55 ± 0.89 2.63 ± 0.71 0.92 ± 0.76
copies 100 ml-1 (1.81-5.34) (1.51-4.08)

15. Mean log10 concentrations of E. coli, FRNA
bacteriophage and NoV GI and GII at wastewater
treatment stages (WWTP1)
Wastewater treatment stage
Influent Final effluent
n = 49 Concn. Concn. Log
(range) (range) reduction
E. coli 6.54 ± 0.59 5.06 ± 0.58 1.49 ± 0.63
MPN 100 ml-1 (3.73-7.54) (3.54-6.20)
FRNA 5.54 ± 0.51 3.41 ± 0.77 2.13 ± 0.76
bacteriophage (3.87-6.82) (2.00-5.84)
pfu 100 ml-1
NoV GI 3.32 ± 0.64 2.53 ± 0.57 0.79 ± 0.49
copies 100 ml-1 (2.05-4.76) (1.26-4.06)
NoV GII 3.55 ± 0.89 2.63 ± 0.71 0.92 ± 0.76
copies 100 ml-1 (1.81-5.34) (1.51-4.08)

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. 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 GA
GA 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. Detection of NoV GII (Real-time PCR) and FRNA GA
bacteriophage (Infectivity assay and Real-time PCR)
ct
ct
ct
ct
fe
fe
fe
fe
In
In
In
In
influent secondary UV oysters

19. Detection of NoV GII (Real-time PCR) and FRNA GA
bacteriophage (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. Log10 reductions of FRNA bacteriophage
and NoV GII
Treatment
Activated
Sludge UV Total
(WWTP1) (Tuam WRF) reduction
Infectious GA
2.13 1.8 3.93
(pfu 100ml-1)
NoV GII
0.92 0.52 1.44
(copies 100ml-1)

21. Impact of CSO discharges and NoV GII
concentrations in oysters
4 12
3
413 m
3.5
10
Log10 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. Concentration of infectious and total FRNA GA
bacteriophage in final effluent and CSO discharges
LOD
Infectivity assay () PCR assay ()
CSO events

23. Concentration of infectious and total FRNA GA
bacteriophage in final effluent and CSO discharges
Mean difference Log10 GA: CSO = 0.1
Mean difference Log10 GA: UV treated = 2.51

24. Indicator and Index roles
FRNA bacteriophage NoV
(infectivity assay) (real-time RT-qPCR)
Indicator of virus reduction during wastewater treatment – desirable
criteria
Ubiquitous in wastewater Yes No (absent in summer in
some WWTP)
Detects infectious virus only Yes No
Index of virus risk in bivalve shellfish - desirable criteria
Concentration elevated in No/Yes (size of impact, Yes (extent of human
locations of higher risk animal sources) impact sources)
Concentration elevated at Yes (concentrations
times of higher risk No (constant year round) related to current infections
in population)
Concentration directly
No Yes (EFSA, 2012)
related to risk of infection
General criteria
Cheap and easy to analyse Yes (approx. €20 – 30) No (approx. €200)
ISO methods available Yes No

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. 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. 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. 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. 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. Shellfish Microbiology team
Marine Environment and Food Safety
Services, Marine Institute
Bill Doré
Sinéad Keaveney
John Flannery
Paulina Rajko-Nenow
www.marine.ie