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The Impact of Winter Relocation and Depuration on Norovirus Concentrations in Pacific Oysters Harvested from a Commercial Production Site

The Impact of Winter Relocation and Depuration on Norovirus Concentrations in Pacific Oysters... Oysters contaminated with norovirus present a significant public health risk when consumed raw. In this study, norovirus genome copy concentrations were determined in Pacific oysters (Magallana gigas) harvested from a sewage-impacted produc- tion site and then subjected to site-specific management procedures. These procedures consisted of relocation of oysters to an alternative production area during the norovirus high-risk winter periods (November to March) followed by an extended depuration (self-purification) under controlled temperature conditions. Significant differences in norovirus RNA concentra- tions were demonstrated at each point in the management process. Thirty-one percent of oyster samples from the main harvest area (Site 1) contained norovirus concentrations > 500 genome copies/g and 29% contained norovirus concentrations < 100 genome copies/g. By contrast, no oyster sample from the alternative harvest area (Site 2) or following depuration contained norovirus concentrations > 500 genome copies/g. In addition, 60 and 88% of oysters samples contained norovirus concentra- tions < 100 genome copies/g in oysters sampled from Site 2 and following depuration, respectively. These data demonstrate that site-specific management processes, supported by norovirus monitoring, can be an effective strategy to reduce, but not eliminate, consumer exposure to norovirus genome copies. Keywords Human norovirus · RT-qPCR · Oysters · Depuration · Monitoring Introduction The public health risks associated with bivalve molluscan shellfish are clearly recognised and regulations exist through- Norovirus infections are the most common cause of non- out the world to manage their production. In Europe, regu- bacterial gastroenteritis worldwide. In Europe, the peak of latory controls primarily centre around the sanitary classifi- illness cases occurs in the winter months, with most patients cation of harvesting areas into three categories based on E. experiencing relatively mild symptoms. During these epi- coli concentrations (Anonymous 2004). Each classification demic winter periods, the norovirus distribution in the category requires harvested shellfish to be treated to differing human population, and therefore in sewage, is ubiquitous degrees depending on the level of harvest area contamination. (Flannery et al. 2012; Kitajima et al. 2012). Filter-feeding In addition, market-ready oysters must comply with an E. coli bivalve molluscan shellfish such as mussels, clams and oys- standard of < 230 MPN/100 g shellfish flesh. Acceptable post- ters can become contaminated with human norovirus when harvest treatments available to ensure oysters meet the E. coli grown in areas impacted by sewage discharges. Such shell- standard include self-purification in land-based tanks of clean fish present an identified public health risk when consumed seawater by a process called depuration, relaying bivalve shell- raw or lightly cooked (Bellou et al. 2013). fish in clean seawater areas for an extended period and heat treatment by approved processes. Despite controls, virtually eliminating outbreaks of bacterial illness following shellfish * William Doré consumption, numerous outbreaks of norovirus gastroenteri- bill.dore@marine.ie tis associated with molluscan shellfish consumption continue to occur. In particular, such outbreaks have been associated Marine Institute, Rinville, Oranmore, Ireland 2 with the consumption of oysters (Ang 1998; Baker et al. 2010; Centre for Food Safety, University College Dublin, Dublin, Chalmers and McMillan 1995; Le Guyader et al. 2008). This Ireland Vol:.(1234567890) 1 3 Food and Environmental Virology (2018) 10:288–296 289 is principally because (1) oysters are most often consumed contamination. The most significant of them is a secondary raw, (2) norovirus has been demonstrated to specifically bind wastewater treatment plant (WWTP), serving a popula- to oyster tissues and (3) oysters are grown in intertidal areas tion equivalent of 1200 with the discharge outlet located often impacted by sewage discharges (McLeod et al. 2017). approximately 1.5 km away from Site 1. Further significant Norovirus-related gastroenteritis outbreaks have occurred even contamination sources (such as septic tanks or other waste- when oysters have been demonstrated to be fully compliant water treatment plants) may also impact on the produc- with regulatory end-product standards (Baker et al. 2010; tion site but these are more distant. Throughout the study Chalmers and McMillan 1995; Doré et al. 2010). Therefore, period, this production area was classified as a category A the combination of harvest area controls and post-harvest treat- site, based on E. coli monitoring under European regula- ments as currently practiced are not considered to fully protect tions (Anonymous 2004). oyster consumers from the risk associated with norovirus con- During the production, Pacific oysters (Magallana tamination (EFSA 2012). gigas) were generally harvested from Site 1 during the Controls based on acceptable limits for norovirus are nec- months of April to September inclusive. Over the win- essary to further protect consumers. Although recognised as ter months (October to March, inclusive), production being required (EFSA 2012), controls based on virus stand- switched to Site 2, an alternative category A site situated ards in bivalve molluscan shellfish have not been forthcoming. at the estuary mouth and approximately 4.5 km away from To some extent, this has been due to the lack of standardised the WWTP discharge outlet (Fig. 1). In September 2014 and reliable procedures for the detection and quantification of and again in September 2015, large consignments of oys- norovirus in oysters. Such a tool has become available more ters were transferred from Site 1 to Site 2 prior to any recently with an introduction of a standardised quantitative significant norovirus contamination occurring in Site (1). real-time reverse transcription PCR (RT-qPCR) method able On occasion, throughout both winter periods, a number of to monitor norovirus genome copy concentrations (“ISO further consignments of oysters were transferred to Site (2) 15216-1:2017” 2017). It has already been used by us and other Oysters moved during the winter months were relocated laboratories to establish the prevalence and concentration of in Site 2 for at least 2 months before harvesting and were norovirus in oyster harvest areas (Lowther et al. 2012a) and in therefore considered to have equilibrated to the norovirus outbreak investigations (Baker et al. 2010; Doré et al. 2010). concentrations associated with Site 2 before harvest took In this study, we monitored norovirus RNA concentrations place. in oysters at a production site known to be impacted by sew- Following harvest from sites 1 (summer), and 2 (win- age, contaminated with norovirus, and previously associated ter), all oysters were depurated before placing on the mar- with a large-scale illness outbreak (Doré et al. 2010). Risk ket. During the summer, oysters were depurated for 2–3 management procedures were introduced by the producer dur- days at ambient temperatures. During the winter months, ing commercial production in response to this outbreak. A oysters were depurated for between 2 and 9 days at ele- two-stage management approach was followed at the site. First, vated temperatures. during the high-risk winter period, oysters were only harvested from an alternative local site which had previously been identi- fied as being subject to less norovirus contamination than the main site. Second, following harvest, oysters were subjected to depuration for an extended period of up to 9 days under controlled temperature conditions. During a 14-month period, we used the standardised RT-qPCR procedure to determine norovirus genome copy concentrations in oysters in the main production site, the alternative harvest area, and following dep- uration to determine the impact of the management procedures on potential consumer exposure to norovirus genome copies. Materials and Methods Sampling Sites and Management Controls Procedures During Commercial Production Fig. 1 Schematic (not to scale) representation of sampling locations. (1) Main production area, (2) alternative winter harvest site, (3) depu- The main harvest site (Site 1) is located in an estu- ration tanks and (4) WWTP discharge point. Approximate distance ary that has a number of potential sources of sewage from (1) to (4) is 1.5 km and (2) to (4) is 4.5 km 1 3 290 Food and Environmental Virology (2018) 10:288–296 Winter Sampling Schedule Depuration Procedures Between January and March 2015, a sample was collected Depuration undertaken by the shellfish producer was rou- tinely performed in recirculating tanks of seawater using from the unused Site 1 and duplicate samples were collected from Site 2 each week. In total, five samples were collected ultraviolet (UV) disinfection. In total, seven depuration tanks were used during the study period. Tank dimensions weekly from the depuration tanks during the winter months. This consisted of triplicate samples of oysters purified for were approximately 6 by 0.7 by 1.3 m. The maximum vol- ume of each tank was 6000 L of seawater pumped from a 2–3 days (short-term depuration) and duplicate samples of oysters depurated for 7–9 days (long-term depuration). nearby pit on the estuary shore that flooded during high water. This seawater was filtered and UV treated prior to Between November 2015 and March 2016, duplicate samples were collected weekly from Site 1 and 2 as well as entering the depuration system. The seawater flow rate was approximately 10 m /h providing effective aeration following short-term and long-term depuration. In total, 55 samples were taken from Site 1 and 65 sam- and adequate dissolved oxygen levels in the tanks to allow active functioning of the oysters. Each tank was fitted ples from Site 2 during the winter months. In the same period, 74 and 50 samples were collected following short- with 4 UV sterilisation lamps (55 W, UVc 200–280 nm) and a set of three 300 W aquarium water heaters, which term (2–3 days; mean 2.42 days) and long-term (7–9 days; mean 7.24 days) depuration, respectively. were used when the seawater temperature dropped below 12 °C. During the study, the average seawater temperature Summer Sampling Schedule measured during depuration in winter (October to March) was 13.3 °C (min = 8.1 °C and max = 19.7 °C). Prior to Sampling frequency was reduced during the summer months depuration, oysters were washed briefly and placed in shallow, open mesh plastic trays (8–10 kg of oysters per in line with the expected reduction in norovirus detection rates. Between April and October 2015, a single sample was tray). Trays were stacked up to four layers high, with a maximum of 800 kg of oysters placed in each of the tanks collected weekly from the unused Site 2, duplicate sam- ples were harvested from Site 1 and following short-term for between 2 and 9 days. depuration. In total, 50 samples were collected from Site 1 and 36 Oyster Sampling samples from Site 2 during the summer months. In the same period, 89 samples were collected following short-term Between January 2015 and April 2016, samples consist- depuration. ing of 10 live oysters were collected at the three produc- tion points on a weekly basis. Sampling schedules varied Preparation of Oyster Samples for Norovirus Analysis throughout the study period depending on season and are outlined in Table 1. Oysters were prepared in accordance with ISO 15216- All oyster samples collected during the study period were transported to the laboratory under chilled condi- 1:2017 ( 2017). Briefly, oysters were cleaned under running potable tions (< 15 °C) and received within 48 h of harvesting. Analysis began within 24 h of receipt to the laboratory. water. Ten oysters per sample were shucked and the diges- tive tissues (DT) dissected out. The dissected DT was finely Table 1 The study sampling Winter 2014/2015 Summer 2015 Winter 2015/2016 schedule January to March 2015 April to October 2015 November 2015 to March 2016 Site 1 1 (13) 2 (50) 2 (42) Site 2 2 (26) 1 (36) 2 (39) Depurations (total) of which 5 (62) 2 (89) 4 (62)  Short term (2–3 days) 3 (36) 2 (89) 2 (38)  Long term (7–9 days) 2 (26) – 2 (24) Oysters were sampled weekly from the three designated sampling points: Site 1, Site 2 and following depu- ration (short term and long term) The number of samples collected weekly from each site is indicated. The total number of samples collected from each site is shown in brackets. All samples consisted of 10 live oysters 1 3 Food and Environmental Virology (2018) 10:288–296 291 chopped using a sterile razor blade and mixed well. Two The number of RNA copies in norovirus-positive samples grams of DT was then spiked with 10 µl of the internal pro- was determined by comparing the C value to the standard cess control (IPC) virus (Mengo virus strain MC ) for evalu- curves. The final concentration was then adjusted to reflect ation of virus extraction efficiency similar to that described the volume of sample analysed and expressed as the number by Costafreda et al. (2006) and treated with 2 ml Protein- of detectable virus genome copies per gram of DT. −1 ase K (100  µg  ml ). Samples were incubated at 37  °C The presence of inhibitors was checked by spiking an for 60 min with shaking at 150 rpm followed by 15 min additional 5 µl of each sample RNA with 1 µl of either noro- at 60 °C. Finally, after centrifugation at 3000×g for 5 min, virus GI or norovirus GII external control RNA (ECRNA; supernatants were retained for RNA extraction. 10 RNA transcripts/µl). The threshold cycle (C ) value obtained for samples spiked with the ECRNA was compared Viral RNA Extraction to the results obtained in the absence of the sample (5 µl of water used instead) and used to estimate RT-PCR inhibition RNA was extracted from 500  µl of the DT supernatants expressed as a percentage. In accordance with ISO 15216- using NucliSENS® magnetic extraction reagents (bioMé- 1:2017, oyster samples with RT-PCR inhibition below the rieux) and the NucliSENS® MiniMAG® extraction platform 75% were accepted for inclusion in this study. and eluted into 100 µl of elution buffer. RNA extracts were Extraction efficiency was assessed by comparing the stored at − 80 °C until the RT-qPCR analysis was conducted. C value of the sample spiked with IPC virus to a stand- RNA was also extracted from 10 µl of the IPC sample for ard curve obtained by preparing log dilutions of the RNA evaluation of extraction efficiency. A single negative extrac- extracted from 10 µl Mengo virus, and was subsequently tion control (water only) was processed alongside the oyster expressed as percentage extraction efficiency. Samples with samples. the extraction efficiency greater than 1% were accepted for inclusion in this study (“ISO 15216-1:2017” 2017). Determination of the Norovirus Concentration No template controls (water only) and negative extraction Using One‑Step qRT‑PCR controls (blank sample carried through the RNA extraction step) were included in each RT-PCR analysis in order to Oysters were analysed for the norovirus concentrations using control for cross-contamination. standardised quantitative real-time reverse transcription PCR (RT-qPCR) (“ISO 15216-1:2017” 2017). Data Analysis RT-qPCR analysis was carried out using the Applied Bio- systems AB7500 instrument (Applied Biosystems, Foster The limit of quantification (LOQ) for both, norovirus GI City, CA) and the RNA Ultrasense one-step qRT-PCR sys- and norovirus GII assays was 100 genome copies/g. Results tem (Invitrogen). The reaction was prepared by combining were presented as total norovirus concentration calculated 5 µl of the extracted RNA sample and 20 µl of the reaction as a sum of GI and GII results. To facilitate the statistical mix containing 500 nM forward primer, 900 nM reverse analysis of the results, any result demonstrating a norovirus primer, 250  nM sequence specific probe, 1x ROX refer - concentration < LOQ was assigned a value of half of the ence dye and 1.25 µl of enzyme mix. Previously described LOQ value (50 genome copies/g) for each genogroup. primers QNIF4 (da Silva et al. 2007), NV1LCR (Svraka An unpaired, two-tailed t test was performed in Micro- et  al. 2007) and TM9 probe (Hoehne and Schreier 2006) soft Excel to compare the norovirus concentration results were used for the detection of norovirus GI, and QNIF2 obtained from the three sampling points. Results with values (Loisy et al. 2005), COG2R (Kageyama et al. 2003) and p < 0.05 were deemed statistically significant. QNIFS probe (Loisy et al. 2005) were used for the detection of norovirus GII. The Mengo110, Mengo209 primers and Mengo147 probe were used in IPC assay (Pinto et al. 1999). Results The 96-well optical reaction plate was incubated at 55 °C for 60 min, 95 °C for 5 min, and then 45 cycles of PCR were Norovirus concentrations in oysters followed a clear sea- performed, with 1 cycle consisting of 95 °C for 15 s, 60 °C sonal trend at both Sites 1 and 2 (Fig. 2). Peak norovirus for 1 min and 65 °C for 1 min. All samples were analysed RNA concentrations were detected in oysters in the win- for norovirus GI and GII in duplicate. All control materials ter during the period between November and March, while used in the RT-qPCR assays were prepared as described by norovirus was absent, or present at concentrations below 100 Flannery et al. (2012). To enable quantification of norovirus genome copies/g, in all but one oyster sample in the remain- RNA in copies per µl, a log dilution series of the norovirus der of the year. This seasonal trend was also present in oys- GI and GII DNA plasmids (ranging from 1 × 10° to 1 × 10 ters following depuration. Norovirus RNA concentrations copies/µl) were included in duplicate on each RT-qPCR run. detected in oyster samples from Site 1 and 2 were similar 1 3 292 Food and Environmental Virology (2018) 10:288–296 1400 during both the winter periods covered by this study. The peak concentrations of norovirus in oysters from Site 1 were 1124 genome copies/g in January 2015 and 1191 genome copies/g in December 2015. In oysters from Site 2, the peak concentrations of norovirus were 361 genome copies/g in February 2015 and 467 genome copies/g in January 2016. During the winter periods, norovirus detection rate in oysters at Site 1 was at 91% compared to 89% at Site 2, and 81% in marketed oysters following depuration (Table 2). However, there was a significant gradation in the norovi- rus RNA concentrations detected in oysters sampled at Site 1 and 2, and following depuration during the same period (p < 0.05). The maximum and mean norovirus RNA con- centrations detected in oysters from Site 1 were 1191 and 353 genome copies/g, respectively. This is compared with maximum of 467 and mean of 117 genome copies/g at Site 2. Following depuration, these figures were reduced to 341 1400 c 1200 and 58 gc/g (< LOQ), respectively. 1000 To determine the theoretical compliance of oyster batches with potential future acceptable limits for norovirus in oys- ters, the percentage of oyster samples conforming to arbi- trarily selected norovirus concentration intervals was deter- mined for each point in the production process (Fig.  3). Norovirus RNA concentrations in excess of 500 genome copies/g were detected in 31% of oyster samples taken at Site 1 during the winter, whereas no oysters from Site 2 or following depuration contained norovirus RNA concentra- tions above that value. In the same period, a total of 60% of oyster samples contained norovirus RNA concentrations Fig. 2 Total norovirus (genogroup I and II) concentrations in genome copies/g detected in oysters from a Site 1—main production area, below 100 genome copies/g at Site 2 compared with 29% of b Site 2—alternative winter harvest site and c post depuration (2–9 samples from Site 1. Following both short- and long-term days). Samples were collected weekly from the three production depuration, the frequency of samples containing less than points between January 2015 and April 2016. Samples in which 100 norovirus genome copies/g increased to 88% overall. norovirus was not detected are represented by a triangle, samples containing < 100 genome copies/g (< LOQ) are represented by an X The percentage of samples containing norovirus concen- and diamonds indicate samples with results greater than 100 genome tration below 100 genome copies/g in oysters subjected to copies/g Table 2 Norovirus detection rates and mean total norovirus RNA concentrations in oyster samples Production area Marketed oysters Depuration period b b Site 1 (n = 55) Site 2 (n = 65) n = 124 Short (n = 74) Long (n = 50) Percent positive 91 89 81 84 78 c c c Mean NoV conc. gc/g 353 117 58 65 47 Percent of positive samples < LOQ 20 49 69 68 70 Min–max NoV conc. gc/g n.d.–1191 n.d.–467 n.d.–341 n.d.–341 n.d.–204 Total norovirus RNA concentrations in each sample, calculated as a sum of GI and GII genogroup results were used to determine the mean con- centration. Samples were collected from Site 1, Site 2 and post-depuration during winter periods of January to March 2015 and October 2015 to March 2016 n.d not detected Oysters following depuration for either short- or long-term periods Short and long depuration periods were 2–3 days (mean 2.4 days) and 7–9 days (mean 7.2 days), respectively Mean norovirus concentration in genome copies/g determined by assigning a value of 50 to all < LOQ results 1 3 total NoV gc g-1 total NoV gc g-1 total NoV gc g-1 12/26/2014 3/11/2015 5/25/2015 8/8/2015 10/22/2015 1/5/2016 3/20/2016 Food and Environmental Virology (2018) 10:288–296 293 n.d. <LOQ 101-200 201-500 > 500 [gc g-1] Following the outbreak associated with oysters harvested 100% from Site 1, the producer introduced additional management 90% 80% controls which included the use of an alternative harvest site 70% (Site 2) during the winter (November to March) and depu- 60% 50% ration for extended periods under controlled temperature 40% conditions. Nevertheless, the additional controls resulted in 30% 20% a limited reduction in the frequency of norovirus detection 10% in marketed oysters during winter with norovirus detected 0% Site 1Site 2short term long term in 91, 89 and 81% of oysters from the main production depuration depuration site (Site 1), alternative harvest site (Site 2) and following depuration, respectively. However, a reduction in norovirus Fig. 3 Impact of winter management interventions on norovirus concentration was observed in the oysters following imple- concentrations in oysters. Total norovirus concentrations detected mentation of the management procedures. It was notable in oysters at Site 1 (n = 55), Site 2 (n = 62) and following short-term (n = 74) and long-term depuration (n = 50) were assigned to arbitrary that the majority (84%) of oyster samples following depu- concentration intervals; bottom to top: n.d.; <LOQ; 101–200; 201– ration contained norovirus RNA concentrations below the 500 and > 500 genome copies/g. The percentage numbers of each LOQ. Therefore, while management procedures employed sample falling within an assigned category are given for each site. during this study did not significantly lower the frequency n.d. not detected of norovirus detection in marketed oysters, they reduced the concentration of norovirus copies, to which consumers were depuration for 2–3 days was 84% compared with 92% of exposed. oysters depurated for 7–9 days. The overall impact on potential illness cases of imple- menting the management procedures employed during this study cannot be determined and remains uncertain. Noro- virus has a low infective dose with one study based on a Discussion human exposure trial reporting the 50% human infectious dose (HID ) to be between 18 and 1015 genome equivalents During this study, norovirus detection in oysters followed a for norovirus GI.I (Teunis et al. 2008). In addition, illness clear seasonal trend with both a higher frequency of detec- outbreaks following the consumption of oysters with noro- tion and increased norovirus RNA concentrations detected virus RNA concentrations < 100 genome copies/g have been in the winter season (November to March). This confirms reported (Thebault et al. 2013). Therefore, the low concen- finding in other studies conducted in Europe (EFSA 2012; trations of norovirus as detected in market ready oysters in Flannery et al. 2009; Lowther et al. 2012b) and highlights a this study could still have the potential to make consumers need for additional targeted risk management interventions ill. However, by its nature, the RT-qPCR method detects at this time of year. It is significant that high concentrations genome copies only and does not distinguish between infec- (> 1000 genome copies/g) were detected in oysters sampled tious and non-infectious virus copies. This further increases from the main harvest area (Site 1) during the high-risk the uncertainty of the illness outcome for a given concen- winter months. Similar norovirus RNA concentrations have tration of norovirus genome copies. In addition, a second previously been detected in oysters associated with illness study has reported a higher HID for norovirus GI.I at 1320 outbreaks (Doré et al. 2010; Lowther et al. 2012a; Rajko- (95% CI 440–3760) genome equivalents for susceptible indi- Nenow et al. 2013). Based on E. coli monitoring data and viduals (Atmar et al. 2014). It is clear that there remains despite the high concentrations of norovirus detected, Site 1 significant uncertainty regarding the dose response models was classified as ‘Class A’ harvesting area under EU regula- developed for norovirus thus far. This makes it extremely tions. Shellfish harvested from a Class A area can be placed difficult to determine the likely illness outcomes associ- directly on the market even during the high-risk winter ated with a given concentration of norovirus genome copies period. In 2010, oysters produced at Site 1 were associated detected in oysters. In reality, the illness outcome for any with a major illness outbreak (Doré et al. 2010). At that time, given norovirus concentration in oysters will vary depending oysters were marketed directly from Site 1 without addi- on a range of factors including differences between norovi- tional post-harvest treatment or following minimal depura- rus genotype and host susceptibility based on immunity, and tion periods (< 48 h) and without temperature control. Given genetic susceptibility (Noda et al. 2008). A very significant the norovirus RNA concentrations detected in this study, it is factor may also be the type of contamination event impact- likely that if oysters from Site 1 had continued to be placed ing on the oysters. For example, oysters contaminated with on the market directly, with or without minimal treatment, nearby untreated sewer overflows will likely contain a higher further illness outbreaks would have occurred. ratio of infectious norovirus than oysters contaminated with 1 3 294 Food and Environmental Virology (2018) 10:288–296 disinfected sewage from a distant location for any given Depuration is one of the most widely practiced post- norovirus genome copy concentration. However, this will harvest treatments during the production of raw oysters depend on the efficacy of the type of wastewater treatment (Lees et al. 2010). The process was originally designed in applied, for example, recent evidence suggests that chlo- the beginning of the twentieth century to prevent bacterial rine-based sewage treatment may not substantially inactivate illness associated with shellfish consumption. It has been norovirus (Kingsley et al. 2017). Despite these uncertain- documented on numerous occasions that depuration is una- ties, a clear relationship between increasing genome copy ble to achieve complete elimination of viruses (reviewed by concentrations in oysters and illness outcome has been McLeod et al. 2017). We confirm this finding here with the reported (Lowther et al. 2012a). It is apparent that the risk majority of oysters still containing norovirus following dep- of an illness event rises with increasing number of genome uration during the high-risk winter period. However, depura- copies present, even if this increased risk is not quantifiable. tion as practiced in this study did have an overall impact on Conversely, the decrease in norovirus concentration due to the concentrations of norovirus in oysters. Previous stud- the procedures reported here must therefore be considered ies have indicated that the time and seawater temperature likely to reduce the risk of illness associated with oyster are both factors that may influence virus reduction during consumption. Further, the fact that there were no reported bivalve shellfish depuration (Lees et al. 2010). Nevertheless, incidences of illness associated with consumption of more the minimum depuration times and temperatures are not stip- than 3 million oysters sold from this production site over ulated in EU regulation. In Ireland, in common with many the study period would appear to suggest that the health risk other countries in Europe, it is recommended that depuration was indeed reduced. However, it is also the case that the vast should be carried out for a minimum of 42 h at temperatures majority of norovirus-related gastroenteritis is unreported not less than 8 °C. This minimal time temperature regime and one large-scale study indicates that approximately only has been shown to effectively reduce E. coli but achieves a 1 in 300 cases of norovirus gastroenteritis occurring in the minimal reduction of viruses. During this study, extended community may be recorded in the national statistics (Tam depuration periods of up to 9 days were applied during the et al. 2012). Therefore, the lack of documented incidences winter season. In addition, minimum depuration tempera- during this study does not, in itself, indicate a lack of risk. tures were generally significantly above the recommended It is possible that sporadic unreported illness occurred fol- minimum with the mean temperature over all depuration lowing the consumption of oysters from the site particularly cycles of 13.3 °C during the winter. It is notable that these during the high-risk winter period. Clearly, further charac- depuration conditions were routinely applied by the producer terisation of the relationship between norovirus genome on a commercial basis without any deterioration of shellfish copy concentrations determined in oysters by RT-qPCR quality and were considered economically viable. and illness outcome is required to be able to fully assess the Interestingly, even under these enhanced conditions (ele- impact of management procedures adopted by producers. vated temperature and extended time), only a slight drop In this study, harvesting was switched to a less contami- in the number of oyster samples containing norovirus was nated site during the high-risk winter period. Monitoring of observed. However, there was a notable drop in the aver- both the main harvest and the alternative site for norovirus age concentration of norovirus in oysters before and after confirmed that on average oysters in the alternative harvest depuration with most (90%) of oysters following depura- site contained lower concentrations of norovirus. This was tion for 7–9 days containing < 100 genome copies/g. This despite the fact that there was no difference in the num- is compared with just 55% of oysters containing norovirus ber of oyster samples positive for norovirus. It is therefore RNA concentrations < 100 genome copies/g prior to depura- worth noting that, in this context, the standardised RT-qPCR tion. A possible explanation for the reduction in norovirus method provided a robust and reliable tool to allow charac- RNA concentrations observed in this study is that norovirus terisation of the two harvest areas in relation to the extent of was sequestered into tissues outside of the digestive tissue. norovirus contamination. Initial virus concentration has been Given that the ISO standard method used in this study exam- demonstrated to have an impact on the outcome of virus ines the digestive tissue only, we cannot determine from this depuration i.e. the higher the initial virus concentration, the study whether this is the case but we can find no evidence higher the final virus concentration if all other parameters in the literature of such sequestration into alternative tis- are equal (McLeod et al. 2017). By using the alternative site, sues. On average oysters purified for 7–9 days contained norovirus RNA concentrations in oysters sent for depuration slightly lower concentrations of norovirus than oysters depu- were reduced compared to oysters from the main harvest rated for 2–3 days indicating that depuration for extended area. This would undoubtedly contribute to the fact that the periods may further reduce norovirus RNA concentrations. majority of norovirus-positive oyster samples contained However, the additional reduction in norovirus RNA con- norovirus RNA concentrations below 100 genome copies/g centrations in oysters depurated beyond the 3 days was not following depuration. significantly different (p > 0.05) and its value as an added 1 3 Food and Environmental Virology (2018) 10:288–296 295 Bellou, M., Kokkinos, P., & Vantarakis, A. (2013). Shellfish-borne viral public health control is questionable. The limited value of outbreaks: A systematic review. Food and Environmental Virol- depuration periods extended beyond the 3 days as observed ogy, 5(1), 13–23. https ://doi.org/10.1007/s1256 0-012-9097-6. here is consistent with laboratory-based studies reported Chalmers, J. W., & McMillan, J. H. (1995). An outbreak of viral gastro- elsewhere for other shellfish species (Polo et al. 2015) and enteritis associated with adequately prepared oysters. Epidemiol- ogy and Infection, 115(1), 163–167. oysters (McLeod et al. 2017). Interestingly, laboratory-based Costafreda, M. I., Bosch, A., & Pintó, R. M. (2006). Development, depuration studies have demonstrated that virus reduction is evaluation, and standardization of a real-time taqman reverse a two-phase process (Polo et al. 2014). The first phase is a transcription-PCR assay for quantification of Hepatitis A virus relatively rapid process related directly to shellfish filtration in clinical and shellfish samples. Applied and Environmental Microbiology, 72(6), 3846–3855. rate. However, subsequent low-level norovirus persistence is Doré, B., Keaveney, S., Flannery, J., & Rajko-Nenow, P. (2010). associated with a second phase demonstrating a significantly Management of health risk associated with oysters harvested slower reduction rate where viruses appear to be refractory from a norovirus contaminated area, Ireland, February–March to the initial depuration process, possibly because they are 2010. EuroSurveillance, 15(19), 1–5. EFSA. (2012). Scientific opinion on norovirus (NoV) in oysters: intrinsically bound to specific norovirus receptors in the Methods, limits and control options. EFSA Journal 10(1), 2500. oyster tissue. Flannery, J., Keaveney, S., & Dore, W. (2009). Use of FRNA bac- In summary, in the absence of current regulatory stand- teriophage to indicate the risk of norovirus contamination in ards, we believe that a site-specific management approach, Irish Oysters. Journal of Food Protection, 72(11), 2358–2362. Flannery, J., Keaveney, S., Rajko-Nenow, P., O’Flaherty, V., & Doré, such as described here and supported by norovirus moni- W. (2012). Concentration of norovirus during wastewater treat- toring, can reduce consumer exposure to norovirus genome ment and its impact on oyster contamination. Applied and Envi- copies. This may provide additional, if not complete, con- romental Microbiology, 78(9), 3400–3406. sumer protection. However, despite the anecdotal evidence Hoehne, M., Schreier, E. (2006). Detection of norovirus genogroup I and II by multiplex real-time RT-PCR using a 3′-minor groove presented here, i.e. lack of illness reports, it is not possible to binder-DNA probe. BMC Infectious Diseases. h tt ps : // do i . determine the public health benefits of this approach. There- org/10.1186/1471-2334-6-69. fore, there remains a clear requirement for further work to ISO 15216-1:2017. (2017). Microbiology of the food chain—Hori- better characterise the relationship between norovirus RNA zontal method for determination of hepatitis a virus and norovi- rus using real-time RT-PCR—Part 1: Method for quantification. concentrations in oysters as judged by RT-qPCR and illness https ://www.iso.org/stand ard/65681 .html. outcomes. Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S., Hoshino, F. B., Takeda, N., & Katayama, K. (2003). Broadly Acknowledgements This work was supported by the Department of reactive and highly sensitive assay for norwalk-like viruses Agriculture Food and Marine research programme Food Institutional based on real-time quantitative reverse transcription-PCR. Jour- Research Measure (FIRM) Grant 14/SF/852. nal of Clinical Microbiology, 41(4), 1548–1557. Kingsley, D. H., Johnna, P., Fay, K., Calci, R., Pouillot, J., Woods, Open Access This article is distributed under the terms of the Crea- H., Chen, B. A., Niemira, & Van Doren, J. M. (2017). Evalua- tive Commons Attribution 4.0 International License (http://creat iveco tion of chlorine treatment levels for inactivation of human noro- mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- virus and MS2 bacteriophage during sewage treatment. Applied tion, and reproduction in any medium, provided you give appropriate and Environmental Microbiology 83(23), e01270-17. https :// credit to the original author(s) and the source, provide a link to the doi.org/10.1128/AEM.01270 -17. Creative Commons license, and indicate if changes were made. Kitajima, M., Haramoto, E., Phanuwan, C., Katayama, H., & Furu- mai, H. (2012). Molecular detection and genotyping of human noroviruses in influent and effluent water at a wastewater treat- ment plant in Japan. Journal of Applied Microbiology, 112(3), 605–613. References Le Guyader, F. S., Le Saux, J. C., Ambert-Balay, K., Krol, J., Serais, O., Parnaudeau, S., Giraudon, H., et al. (2008). Aichi virus, Ang, L. H. (1998). An outbreak of viral gastroenteritis associated with norovirus, astrovirus, enterovirus, and rotavirus involved in eating raw oysters. Communicable Disease and Public Health, clinical cases from a french oyster-related gastroenteritis out- 1(1), 38–40. break. Journal of Clinical Microbiology, 46(12), 4011–4017. Anonymous. (2004). Regulation (EC) 854/2004 of the European Parlia- Lees, D., Younger, A., & Dore´, B. (2010). Depuration and relaying. ment and of the Council of 29 April 2004 Laying down Specific In G. Rees, K. Pond, D. Kay, J. Bartram, & J. Santo Domingo Rules for the Organisation of Official Controls on Products of (Eds.), Safe management of shellfish and harvest waters. Lon- Animal Origin Intended for Human Consumption. Vol. L226. don: International Water Association. Atmar, R. L., Antone, R., Opekun, M. A., Gilger, Mary, K., Estes, Loisy, F., Atmar, R. L., Guillon, P., Cann, P. Le, Pommepuy, M., Sue, E., Crawford, Frederick, H., Neill, Graham, D. Y., Ramani, & Le Guyager, F. S. (2005). Real-time RT-PCR for norovirus S., Hill, H., Ferreira, J. (2014). Determination of the 50% human screening in shellfish. Journal of Virological Methods, 123, 1–7. infectious dose for norwalk virus. The Journal of Infectious Dis- Lowther, J. A., Gustar, N. E., Hartnell, R. E., & Lees, D. N. (2012a). eases, 209(7), 1016–1022. https ://doi.org/10.1093/infdi s/jit62 0. Comparison of norovirus RNA levels in outbreak-related oysters Baker, K., Morris, J., McCarthy, N., Saldana, L., Lowther, J., Collin- with background environmental levels. Journal of Food Protec- son, A., & Young, M. (2010). An outbreak of norovirus infection tion, 75(2), 389–393. linked to oyster consumption at a UK Restaurant, February 2010. Lowther, J. A., Gustar, N. E., Powell, A. L., Hartnell, R. E., & Lees, Journal of Public Health, 33(2), 205–211. D. N. (2012b). Two-year systematic study to assess norovirus 1 3 296 Food and Environmental Virology (2018) 10:288–296 contamination in oysters from Commercial harvesting areas in illness outbreaks in Ireland. Epidemiology and Infection, 142(10), the United Kingdom. Applied and Environmental Microbiology, 2096–2104. https ://doi.org/10.1017/S0950 26881 30030 14. 78(16), 5812–5817. https ://doi.org/10.1128/AEM.01046 -12. Silva, A. K., da, J. C., Le Saux, S., Parnaudeau, M., Pommepuy, M., McLeod, C., Polo, D., Saux, J.-C. L., & Guyader, F. S. L. (2017). Elimelech, & Le Guyader, F. S. (2007). Evaluation of removal of Depuration and relaying: A review on potential removal of noro- noroviruses during wastewater treatment, using real-time reverse virus from oysters. Comprehensive Reviews in Food Science and transcription-PCR: Different behaviours of genogroups I and II. Food Safety. https ://doi.org/10.1111/1541-4337.12271 . Applied and Environmental Microbiology, 73(24), 7891–7897. Noda, M., Fukuda, S., & Nishio, O. (2008). Statistical analysis of attack Svraka, S., Duizer, E., Vennema, H., de Bruin, E., van de Veer, B., rate in norovirus foodbourne outbreaks. International Journal of Dorresteijn, B., & Koopmans, M. (2007). Etiological role of Food Microbiology, 122, 216–220. viruses in outbreaks of acute gastroenteritis in The Netherlands Pinto, B., Pierotti, R., Canale, G., & Reali, D. (1999). Characteriza- from 1994 through 2005. Journal of Clinical Microbiology, 45(5), tion of ‘faecal streptococci’ as indicators of faecal pollution and 1389–1394. distribution in the environment. Letters in Applied Microbiology, Tam, C., Viviana, L., Adak, B., Bolton, E., Dodds, J., Cowden, J., 29, 258–263. Evans, M., et al. (2012). The second study of infectious intestinal Polo, D., Alvarez, C., Diez, J., Darriba, S., Longa, A., & Romalde, disease in the community (IID2 study). Project Number: B18021. J. L. (2014). Viral elimination during commercial depuration of London: UK Food Standards Agency. shellfish. Food Control, 43(September), 206–212. h t t p s : / / d o i . Teunis, P. F. M., Moe, C. L., Liu, P., Miller, S. E., Lindesmith, L., org/10.1016/j.foodc ont.2014.03.022. Baric, R. S., Le Pendu, J., & Calderon, R. L. (2008). Norwalk Polo, D., Feal, X., & Romalde, J. L. (2015). Mathematical model for virus: How infectious is it? Journal of Medical Virology, 80, viral depuration kinetics in shellfish: An useful tool to estimate the 1468–1476. risk for the consumers. Food Microbiology, 49(August), 220–225. Thebault, A., Teunis, P. F. M., Pendu, J. L., Le Guyader, F. S., Denis, https ://doi.org/10.1016/j.fm.2015.02.015. J-B (2013). Infectivity of GI and GII noroviruses established from Rajko-Nenow, P., Keaveney, S., Flannery, J., McIntyre, A., & Dore, oyster related outbreaks. Epidemics, 5(2), 98–110. https ://doi. W. (2013). Norovirus genotypes implicated in two oyster-related org/10.1016/j.epide m.2012.12.004. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food and Environmental Virology Springer Journals

The Impact of Winter Relocation and Depuration on Norovirus Concentrations in Pacific Oysters Harvested from a Commercial Production Site

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Springer Journals
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Copyright © 2018 by The Author(s)
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Biomedicine; Virology; Food Science; Chemistry/Food Science, general
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1867-0334
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1867-0342
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10.1007/s12560-018-9345-5
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Abstract

Oysters contaminated with norovirus present a significant public health risk when consumed raw. In this study, norovirus genome copy concentrations were determined in Pacific oysters (Magallana gigas) harvested from a sewage-impacted produc- tion site and then subjected to site-specific management procedures. These procedures consisted of relocation of oysters to an alternative production area during the norovirus high-risk winter periods (November to March) followed by an extended depuration (self-purification) under controlled temperature conditions. Significant differences in norovirus RNA concentra- tions were demonstrated at each point in the management process. Thirty-one percent of oyster samples from the main harvest area (Site 1) contained norovirus concentrations > 500 genome copies/g and 29% contained norovirus concentrations < 100 genome copies/g. By contrast, no oyster sample from the alternative harvest area (Site 2) or following depuration contained norovirus concentrations > 500 genome copies/g. In addition, 60 and 88% of oysters samples contained norovirus concentra- tions < 100 genome copies/g in oysters sampled from Site 2 and following depuration, respectively. These data demonstrate that site-specific management processes, supported by norovirus monitoring, can be an effective strategy to reduce, but not eliminate, consumer exposure to norovirus genome copies. Keywords Human norovirus · RT-qPCR · Oysters · Depuration · Monitoring Introduction The public health risks associated with bivalve molluscan shellfish are clearly recognised and regulations exist through- Norovirus infections are the most common cause of non- out the world to manage their production. In Europe, regu- bacterial gastroenteritis worldwide. In Europe, the peak of latory controls primarily centre around the sanitary classifi- illness cases occurs in the winter months, with most patients cation of harvesting areas into three categories based on E. experiencing relatively mild symptoms. During these epi- coli concentrations (Anonymous 2004). Each classification demic winter periods, the norovirus distribution in the category requires harvested shellfish to be treated to differing human population, and therefore in sewage, is ubiquitous degrees depending on the level of harvest area contamination. (Flannery et al. 2012; Kitajima et al. 2012). Filter-feeding In addition, market-ready oysters must comply with an E. coli bivalve molluscan shellfish such as mussels, clams and oys- standard of < 230 MPN/100 g shellfish flesh. Acceptable post- ters can become contaminated with human norovirus when harvest treatments available to ensure oysters meet the E. coli grown in areas impacted by sewage discharges. Such shell- standard include self-purification in land-based tanks of clean fish present an identified public health risk when consumed seawater by a process called depuration, relaying bivalve shell- raw or lightly cooked (Bellou et al. 2013). fish in clean seawater areas for an extended period and heat treatment by approved processes. Despite controls, virtually eliminating outbreaks of bacterial illness following shellfish * William Doré consumption, numerous outbreaks of norovirus gastroenteri- bill.dore@marine.ie tis associated with molluscan shellfish consumption continue to occur. In particular, such outbreaks have been associated Marine Institute, Rinville, Oranmore, Ireland 2 with the consumption of oysters (Ang 1998; Baker et al. 2010; Centre for Food Safety, University College Dublin, Dublin, Chalmers and McMillan 1995; Le Guyader et al. 2008). This Ireland Vol:.(1234567890) 1 3 Food and Environmental Virology (2018) 10:288–296 289 is principally because (1) oysters are most often consumed contamination. The most significant of them is a secondary raw, (2) norovirus has been demonstrated to specifically bind wastewater treatment plant (WWTP), serving a popula- to oyster tissues and (3) oysters are grown in intertidal areas tion equivalent of 1200 with the discharge outlet located often impacted by sewage discharges (McLeod et al. 2017). approximately 1.5 km away from Site 1. Further significant Norovirus-related gastroenteritis outbreaks have occurred even contamination sources (such as septic tanks or other waste- when oysters have been demonstrated to be fully compliant water treatment plants) may also impact on the produc- with regulatory end-product standards (Baker et al. 2010; tion site but these are more distant. Throughout the study Chalmers and McMillan 1995; Doré et al. 2010). Therefore, period, this production area was classified as a category A the combination of harvest area controls and post-harvest treat- site, based on E. coli monitoring under European regula- ments as currently practiced are not considered to fully protect tions (Anonymous 2004). oyster consumers from the risk associated with norovirus con- During the production, Pacific oysters (Magallana tamination (EFSA 2012). gigas) were generally harvested from Site 1 during the Controls based on acceptable limits for norovirus are nec- months of April to September inclusive. Over the win- essary to further protect consumers. Although recognised as ter months (October to March, inclusive), production being required (EFSA 2012), controls based on virus stand- switched to Site 2, an alternative category A site situated ards in bivalve molluscan shellfish have not been forthcoming. at the estuary mouth and approximately 4.5 km away from To some extent, this has been due to the lack of standardised the WWTP discharge outlet (Fig. 1). In September 2014 and reliable procedures for the detection and quantification of and again in September 2015, large consignments of oys- norovirus in oysters. Such a tool has become available more ters were transferred from Site 1 to Site 2 prior to any recently with an introduction of a standardised quantitative significant norovirus contamination occurring in Site (1). real-time reverse transcription PCR (RT-qPCR) method able On occasion, throughout both winter periods, a number of to monitor norovirus genome copy concentrations (“ISO further consignments of oysters were transferred to Site (2) 15216-1:2017” 2017). It has already been used by us and other Oysters moved during the winter months were relocated laboratories to establish the prevalence and concentration of in Site 2 for at least 2 months before harvesting and were norovirus in oyster harvest areas (Lowther et al. 2012a) and in therefore considered to have equilibrated to the norovirus outbreak investigations (Baker et al. 2010; Doré et al. 2010). concentrations associated with Site 2 before harvest took In this study, we monitored norovirus RNA concentrations place. in oysters at a production site known to be impacted by sew- Following harvest from sites 1 (summer), and 2 (win- age, contaminated with norovirus, and previously associated ter), all oysters were depurated before placing on the mar- with a large-scale illness outbreak (Doré et al. 2010). Risk ket. During the summer, oysters were depurated for 2–3 management procedures were introduced by the producer dur- days at ambient temperatures. During the winter months, ing commercial production in response to this outbreak. A oysters were depurated for between 2 and 9 days at ele- two-stage management approach was followed at the site. First, vated temperatures. during the high-risk winter period, oysters were only harvested from an alternative local site which had previously been identi- fied as being subject to less norovirus contamination than the main site. Second, following harvest, oysters were subjected to depuration for an extended period of up to 9 days under controlled temperature conditions. During a 14-month period, we used the standardised RT-qPCR procedure to determine norovirus genome copy concentrations in oysters in the main production site, the alternative harvest area, and following dep- uration to determine the impact of the management procedures on potential consumer exposure to norovirus genome copies. Materials and Methods Sampling Sites and Management Controls Procedures During Commercial Production Fig. 1 Schematic (not to scale) representation of sampling locations. (1) Main production area, (2) alternative winter harvest site, (3) depu- The main harvest site (Site 1) is located in an estu- ration tanks and (4) WWTP discharge point. Approximate distance ary that has a number of potential sources of sewage from (1) to (4) is 1.5 km and (2) to (4) is 4.5 km 1 3 290 Food and Environmental Virology (2018) 10:288–296 Winter Sampling Schedule Depuration Procedures Between January and March 2015, a sample was collected Depuration undertaken by the shellfish producer was rou- tinely performed in recirculating tanks of seawater using from the unused Site 1 and duplicate samples were collected from Site 2 each week. In total, five samples were collected ultraviolet (UV) disinfection. In total, seven depuration tanks were used during the study period. Tank dimensions weekly from the depuration tanks during the winter months. This consisted of triplicate samples of oysters purified for were approximately 6 by 0.7 by 1.3 m. The maximum vol- ume of each tank was 6000 L of seawater pumped from a 2–3 days (short-term depuration) and duplicate samples of oysters depurated for 7–9 days (long-term depuration). nearby pit on the estuary shore that flooded during high water. This seawater was filtered and UV treated prior to Between November 2015 and March 2016, duplicate samples were collected weekly from Site 1 and 2 as well as entering the depuration system. The seawater flow rate was approximately 10 m /h providing effective aeration following short-term and long-term depuration. In total, 55 samples were taken from Site 1 and 65 sam- and adequate dissolved oxygen levels in the tanks to allow active functioning of the oysters. Each tank was fitted ples from Site 2 during the winter months. In the same period, 74 and 50 samples were collected following short- with 4 UV sterilisation lamps (55 W, UVc 200–280 nm) and a set of three 300 W aquarium water heaters, which term (2–3 days; mean 2.42 days) and long-term (7–9 days; mean 7.24 days) depuration, respectively. were used when the seawater temperature dropped below 12 °C. During the study, the average seawater temperature Summer Sampling Schedule measured during depuration in winter (October to March) was 13.3 °C (min = 8.1 °C and max = 19.7 °C). Prior to Sampling frequency was reduced during the summer months depuration, oysters were washed briefly and placed in shallow, open mesh plastic trays (8–10 kg of oysters per in line with the expected reduction in norovirus detection rates. Between April and October 2015, a single sample was tray). Trays were stacked up to four layers high, with a maximum of 800 kg of oysters placed in each of the tanks collected weekly from the unused Site 2, duplicate sam- ples were harvested from Site 1 and following short-term for between 2 and 9 days. depuration. In total, 50 samples were collected from Site 1 and 36 Oyster Sampling samples from Site 2 during the summer months. In the same period, 89 samples were collected following short-term Between January 2015 and April 2016, samples consist- depuration. ing of 10 live oysters were collected at the three produc- tion points on a weekly basis. Sampling schedules varied Preparation of Oyster Samples for Norovirus Analysis throughout the study period depending on season and are outlined in Table 1. Oysters were prepared in accordance with ISO 15216- All oyster samples collected during the study period were transported to the laboratory under chilled condi- 1:2017 ( 2017). Briefly, oysters were cleaned under running potable tions (< 15 °C) and received within 48 h of harvesting. Analysis began within 24 h of receipt to the laboratory. water. Ten oysters per sample were shucked and the diges- tive tissues (DT) dissected out. The dissected DT was finely Table 1 The study sampling Winter 2014/2015 Summer 2015 Winter 2015/2016 schedule January to March 2015 April to October 2015 November 2015 to March 2016 Site 1 1 (13) 2 (50) 2 (42) Site 2 2 (26) 1 (36) 2 (39) Depurations (total) of which 5 (62) 2 (89) 4 (62)  Short term (2–3 days) 3 (36) 2 (89) 2 (38)  Long term (7–9 days) 2 (26) – 2 (24) Oysters were sampled weekly from the three designated sampling points: Site 1, Site 2 and following depu- ration (short term and long term) The number of samples collected weekly from each site is indicated. The total number of samples collected from each site is shown in brackets. All samples consisted of 10 live oysters 1 3 Food and Environmental Virology (2018) 10:288–296 291 chopped using a sterile razor blade and mixed well. Two The number of RNA copies in norovirus-positive samples grams of DT was then spiked with 10 µl of the internal pro- was determined by comparing the C value to the standard cess control (IPC) virus (Mengo virus strain MC ) for evalu- curves. The final concentration was then adjusted to reflect ation of virus extraction efficiency similar to that described the volume of sample analysed and expressed as the number by Costafreda et al. (2006) and treated with 2 ml Protein- of detectable virus genome copies per gram of DT. −1 ase K (100  µg  ml ). Samples were incubated at 37  °C The presence of inhibitors was checked by spiking an for 60 min with shaking at 150 rpm followed by 15 min additional 5 µl of each sample RNA with 1 µl of either noro- at 60 °C. Finally, after centrifugation at 3000×g for 5 min, virus GI or norovirus GII external control RNA (ECRNA; supernatants were retained for RNA extraction. 10 RNA transcripts/µl). The threshold cycle (C ) value obtained for samples spiked with the ECRNA was compared Viral RNA Extraction to the results obtained in the absence of the sample (5 µl of water used instead) and used to estimate RT-PCR inhibition RNA was extracted from 500  µl of the DT supernatants expressed as a percentage. In accordance with ISO 15216- using NucliSENS® magnetic extraction reagents (bioMé- 1:2017, oyster samples with RT-PCR inhibition below the rieux) and the NucliSENS® MiniMAG® extraction platform 75% were accepted for inclusion in this study. and eluted into 100 µl of elution buffer. RNA extracts were Extraction efficiency was assessed by comparing the stored at − 80 °C until the RT-qPCR analysis was conducted. C value of the sample spiked with IPC virus to a stand- RNA was also extracted from 10 µl of the IPC sample for ard curve obtained by preparing log dilutions of the RNA evaluation of extraction efficiency. A single negative extrac- extracted from 10 µl Mengo virus, and was subsequently tion control (water only) was processed alongside the oyster expressed as percentage extraction efficiency. Samples with samples. the extraction efficiency greater than 1% were accepted for inclusion in this study (“ISO 15216-1:2017” 2017). Determination of the Norovirus Concentration No template controls (water only) and negative extraction Using One‑Step qRT‑PCR controls (blank sample carried through the RNA extraction step) were included in each RT-PCR analysis in order to Oysters were analysed for the norovirus concentrations using control for cross-contamination. standardised quantitative real-time reverse transcription PCR (RT-qPCR) (“ISO 15216-1:2017” 2017). Data Analysis RT-qPCR analysis was carried out using the Applied Bio- systems AB7500 instrument (Applied Biosystems, Foster The limit of quantification (LOQ) for both, norovirus GI City, CA) and the RNA Ultrasense one-step qRT-PCR sys- and norovirus GII assays was 100 genome copies/g. Results tem (Invitrogen). The reaction was prepared by combining were presented as total norovirus concentration calculated 5 µl of the extracted RNA sample and 20 µl of the reaction as a sum of GI and GII results. To facilitate the statistical mix containing 500 nM forward primer, 900 nM reverse analysis of the results, any result demonstrating a norovirus primer, 250  nM sequence specific probe, 1x ROX refer - concentration < LOQ was assigned a value of half of the ence dye and 1.25 µl of enzyme mix. Previously described LOQ value (50 genome copies/g) for each genogroup. primers QNIF4 (da Silva et al. 2007), NV1LCR (Svraka An unpaired, two-tailed t test was performed in Micro- et  al. 2007) and TM9 probe (Hoehne and Schreier 2006) soft Excel to compare the norovirus concentration results were used for the detection of norovirus GI, and QNIF2 obtained from the three sampling points. Results with values (Loisy et al. 2005), COG2R (Kageyama et al. 2003) and p < 0.05 were deemed statistically significant. QNIFS probe (Loisy et al. 2005) were used for the detection of norovirus GII. The Mengo110, Mengo209 primers and Mengo147 probe were used in IPC assay (Pinto et al. 1999). Results The 96-well optical reaction plate was incubated at 55 °C for 60 min, 95 °C for 5 min, and then 45 cycles of PCR were Norovirus concentrations in oysters followed a clear sea- performed, with 1 cycle consisting of 95 °C for 15 s, 60 °C sonal trend at both Sites 1 and 2 (Fig. 2). Peak norovirus for 1 min and 65 °C for 1 min. All samples were analysed RNA concentrations were detected in oysters in the win- for norovirus GI and GII in duplicate. All control materials ter during the period between November and March, while used in the RT-qPCR assays were prepared as described by norovirus was absent, or present at concentrations below 100 Flannery et al. (2012). To enable quantification of norovirus genome copies/g, in all but one oyster sample in the remain- RNA in copies per µl, a log dilution series of the norovirus der of the year. This seasonal trend was also present in oys- GI and GII DNA plasmids (ranging from 1 × 10° to 1 × 10 ters following depuration. Norovirus RNA concentrations copies/µl) were included in duplicate on each RT-qPCR run. detected in oyster samples from Site 1 and 2 were similar 1 3 292 Food and Environmental Virology (2018) 10:288–296 1400 during both the winter periods covered by this study. The peak concentrations of norovirus in oysters from Site 1 were 1124 genome copies/g in January 2015 and 1191 genome copies/g in December 2015. In oysters from Site 2, the peak concentrations of norovirus were 361 genome copies/g in February 2015 and 467 genome copies/g in January 2016. During the winter periods, norovirus detection rate in oysters at Site 1 was at 91% compared to 89% at Site 2, and 81% in marketed oysters following depuration (Table 2). However, there was a significant gradation in the norovi- rus RNA concentrations detected in oysters sampled at Site 1 and 2, and following depuration during the same period (p < 0.05). The maximum and mean norovirus RNA con- centrations detected in oysters from Site 1 were 1191 and 353 genome copies/g, respectively. This is compared with maximum of 467 and mean of 117 genome copies/g at Site 2. Following depuration, these figures were reduced to 341 1400 c 1200 and 58 gc/g (< LOQ), respectively. 1000 To determine the theoretical compliance of oyster batches with potential future acceptable limits for norovirus in oys- ters, the percentage of oyster samples conforming to arbi- trarily selected norovirus concentration intervals was deter- mined for each point in the production process (Fig.  3). Norovirus RNA concentrations in excess of 500 genome copies/g were detected in 31% of oyster samples taken at Site 1 during the winter, whereas no oysters from Site 2 or following depuration contained norovirus RNA concentra- tions above that value. In the same period, a total of 60% of oyster samples contained norovirus RNA concentrations Fig. 2 Total norovirus (genogroup I and II) concentrations in genome copies/g detected in oysters from a Site 1—main production area, below 100 genome copies/g at Site 2 compared with 29% of b Site 2—alternative winter harvest site and c post depuration (2–9 samples from Site 1. Following both short- and long-term days). Samples were collected weekly from the three production depuration, the frequency of samples containing less than points between January 2015 and April 2016. Samples in which 100 norovirus genome copies/g increased to 88% overall. norovirus was not detected are represented by a triangle, samples containing < 100 genome copies/g (< LOQ) are represented by an X The percentage of samples containing norovirus concen- and diamonds indicate samples with results greater than 100 genome tration below 100 genome copies/g in oysters subjected to copies/g Table 2 Norovirus detection rates and mean total norovirus RNA concentrations in oyster samples Production area Marketed oysters Depuration period b b Site 1 (n = 55) Site 2 (n = 65) n = 124 Short (n = 74) Long (n = 50) Percent positive 91 89 81 84 78 c c c Mean NoV conc. gc/g 353 117 58 65 47 Percent of positive samples < LOQ 20 49 69 68 70 Min–max NoV conc. gc/g n.d.–1191 n.d.–467 n.d.–341 n.d.–341 n.d.–204 Total norovirus RNA concentrations in each sample, calculated as a sum of GI and GII genogroup results were used to determine the mean con- centration. Samples were collected from Site 1, Site 2 and post-depuration during winter periods of January to March 2015 and October 2015 to March 2016 n.d not detected Oysters following depuration for either short- or long-term periods Short and long depuration periods were 2–3 days (mean 2.4 days) and 7–9 days (mean 7.2 days), respectively Mean norovirus concentration in genome copies/g determined by assigning a value of 50 to all < LOQ results 1 3 total NoV gc g-1 total NoV gc g-1 total NoV gc g-1 12/26/2014 3/11/2015 5/25/2015 8/8/2015 10/22/2015 1/5/2016 3/20/2016 Food and Environmental Virology (2018) 10:288–296 293 n.d. <LOQ 101-200 201-500 > 500 [gc g-1] Following the outbreak associated with oysters harvested 100% from Site 1, the producer introduced additional management 90% 80% controls which included the use of an alternative harvest site 70% (Site 2) during the winter (November to March) and depu- 60% 50% ration for extended periods under controlled temperature 40% conditions. Nevertheless, the additional controls resulted in 30% 20% a limited reduction in the frequency of norovirus detection 10% in marketed oysters during winter with norovirus detected 0% Site 1Site 2short term long term in 91, 89 and 81% of oysters from the main production depuration depuration site (Site 1), alternative harvest site (Site 2) and following depuration, respectively. However, a reduction in norovirus Fig. 3 Impact of winter management interventions on norovirus concentration was observed in the oysters following imple- concentrations in oysters. Total norovirus concentrations detected mentation of the management procedures. It was notable in oysters at Site 1 (n = 55), Site 2 (n = 62) and following short-term (n = 74) and long-term depuration (n = 50) were assigned to arbitrary that the majority (84%) of oyster samples following depu- concentration intervals; bottom to top: n.d.; <LOQ; 101–200; 201– ration contained norovirus RNA concentrations below the 500 and > 500 genome copies/g. The percentage numbers of each LOQ. Therefore, while management procedures employed sample falling within an assigned category are given for each site. during this study did not significantly lower the frequency n.d. not detected of norovirus detection in marketed oysters, they reduced the concentration of norovirus copies, to which consumers were depuration for 2–3 days was 84% compared with 92% of exposed. oysters depurated for 7–9 days. The overall impact on potential illness cases of imple- menting the management procedures employed during this study cannot be determined and remains uncertain. Noro- virus has a low infective dose with one study based on a Discussion human exposure trial reporting the 50% human infectious dose (HID ) to be between 18 and 1015 genome equivalents During this study, norovirus detection in oysters followed a for norovirus GI.I (Teunis et al. 2008). In addition, illness clear seasonal trend with both a higher frequency of detec- outbreaks following the consumption of oysters with noro- tion and increased norovirus RNA concentrations detected virus RNA concentrations < 100 genome copies/g have been in the winter season (November to March). This confirms reported (Thebault et al. 2013). Therefore, the low concen- finding in other studies conducted in Europe (EFSA 2012; trations of norovirus as detected in market ready oysters in Flannery et al. 2009; Lowther et al. 2012b) and highlights a this study could still have the potential to make consumers need for additional targeted risk management interventions ill. However, by its nature, the RT-qPCR method detects at this time of year. It is significant that high concentrations genome copies only and does not distinguish between infec- (> 1000 genome copies/g) were detected in oysters sampled tious and non-infectious virus copies. This further increases from the main harvest area (Site 1) during the high-risk the uncertainty of the illness outcome for a given concen- winter months. Similar norovirus RNA concentrations have tration of norovirus genome copies. In addition, a second previously been detected in oysters associated with illness study has reported a higher HID for norovirus GI.I at 1320 outbreaks (Doré et al. 2010; Lowther et al. 2012a; Rajko- (95% CI 440–3760) genome equivalents for susceptible indi- Nenow et al. 2013). Based on E. coli monitoring data and viduals (Atmar et al. 2014). It is clear that there remains despite the high concentrations of norovirus detected, Site 1 significant uncertainty regarding the dose response models was classified as ‘Class A’ harvesting area under EU regula- developed for norovirus thus far. This makes it extremely tions. Shellfish harvested from a Class A area can be placed difficult to determine the likely illness outcomes associ- directly on the market even during the high-risk winter ated with a given concentration of norovirus genome copies period. In 2010, oysters produced at Site 1 were associated detected in oysters. In reality, the illness outcome for any with a major illness outbreak (Doré et al. 2010). At that time, given norovirus concentration in oysters will vary depending oysters were marketed directly from Site 1 without addi- on a range of factors including differences between norovi- tional post-harvest treatment or following minimal depura- rus genotype and host susceptibility based on immunity, and tion periods (< 48 h) and without temperature control. Given genetic susceptibility (Noda et al. 2008). A very significant the norovirus RNA concentrations detected in this study, it is factor may also be the type of contamination event impact- likely that if oysters from Site 1 had continued to be placed ing on the oysters. For example, oysters contaminated with on the market directly, with or without minimal treatment, nearby untreated sewer overflows will likely contain a higher further illness outbreaks would have occurred. ratio of infectious norovirus than oysters contaminated with 1 3 294 Food and Environmental Virology (2018) 10:288–296 disinfected sewage from a distant location for any given Depuration is one of the most widely practiced post- norovirus genome copy concentration. However, this will harvest treatments during the production of raw oysters depend on the efficacy of the type of wastewater treatment (Lees et al. 2010). The process was originally designed in applied, for example, recent evidence suggests that chlo- the beginning of the twentieth century to prevent bacterial rine-based sewage treatment may not substantially inactivate illness associated with shellfish consumption. It has been norovirus (Kingsley et al. 2017). Despite these uncertain- documented on numerous occasions that depuration is una- ties, a clear relationship between increasing genome copy ble to achieve complete elimination of viruses (reviewed by concentrations in oysters and illness outcome has been McLeod et al. 2017). We confirm this finding here with the reported (Lowther et al. 2012a). It is apparent that the risk majority of oysters still containing norovirus following dep- of an illness event rises with increasing number of genome uration during the high-risk winter period. However, depura- copies present, even if this increased risk is not quantifiable. tion as practiced in this study did have an overall impact on Conversely, the decrease in norovirus concentration due to the concentrations of norovirus in oysters. Previous stud- the procedures reported here must therefore be considered ies have indicated that the time and seawater temperature likely to reduce the risk of illness associated with oyster are both factors that may influence virus reduction during consumption. Further, the fact that there were no reported bivalve shellfish depuration (Lees et al. 2010). Nevertheless, incidences of illness associated with consumption of more the minimum depuration times and temperatures are not stip- than 3 million oysters sold from this production site over ulated in EU regulation. In Ireland, in common with many the study period would appear to suggest that the health risk other countries in Europe, it is recommended that depuration was indeed reduced. However, it is also the case that the vast should be carried out for a minimum of 42 h at temperatures majority of norovirus-related gastroenteritis is unreported not less than 8 °C. This minimal time temperature regime and one large-scale study indicates that approximately only has been shown to effectively reduce E. coli but achieves a 1 in 300 cases of norovirus gastroenteritis occurring in the minimal reduction of viruses. During this study, extended community may be recorded in the national statistics (Tam depuration periods of up to 9 days were applied during the et al. 2012). Therefore, the lack of documented incidences winter season. In addition, minimum depuration tempera- during this study does not, in itself, indicate a lack of risk. tures were generally significantly above the recommended It is possible that sporadic unreported illness occurred fol- minimum with the mean temperature over all depuration lowing the consumption of oysters from the site particularly cycles of 13.3 °C during the winter. It is notable that these during the high-risk winter period. Clearly, further charac- depuration conditions were routinely applied by the producer terisation of the relationship between norovirus genome on a commercial basis without any deterioration of shellfish copy concentrations determined in oysters by RT-qPCR quality and were considered economically viable. and illness outcome is required to be able to fully assess the Interestingly, even under these enhanced conditions (ele- impact of management procedures adopted by producers. vated temperature and extended time), only a slight drop In this study, harvesting was switched to a less contami- in the number of oyster samples containing norovirus was nated site during the high-risk winter period. Monitoring of observed. However, there was a notable drop in the aver- both the main harvest and the alternative site for norovirus age concentration of norovirus in oysters before and after confirmed that on average oysters in the alternative harvest depuration with most (90%) of oysters following depura- site contained lower concentrations of norovirus. This was tion for 7–9 days containing < 100 genome copies/g. This despite the fact that there was no difference in the num- is compared with just 55% of oysters containing norovirus ber of oyster samples positive for norovirus. It is therefore RNA concentrations < 100 genome copies/g prior to depura- worth noting that, in this context, the standardised RT-qPCR tion. A possible explanation for the reduction in norovirus method provided a robust and reliable tool to allow charac- RNA concentrations observed in this study is that norovirus terisation of the two harvest areas in relation to the extent of was sequestered into tissues outside of the digestive tissue. norovirus contamination. Initial virus concentration has been Given that the ISO standard method used in this study exam- demonstrated to have an impact on the outcome of virus ines the digestive tissue only, we cannot determine from this depuration i.e. the higher the initial virus concentration, the study whether this is the case but we can find no evidence higher the final virus concentration if all other parameters in the literature of such sequestration into alternative tis- are equal (McLeod et al. 2017). By using the alternative site, sues. On average oysters purified for 7–9 days contained norovirus RNA concentrations in oysters sent for depuration slightly lower concentrations of norovirus than oysters depu- were reduced compared to oysters from the main harvest rated for 2–3 days indicating that depuration for extended area. This would undoubtedly contribute to the fact that the periods may further reduce norovirus RNA concentrations. majority of norovirus-positive oyster samples contained However, the additional reduction in norovirus RNA con- norovirus RNA concentrations below 100 genome copies/g centrations in oysters depurated beyond the 3 days was not following depuration. significantly different (p > 0.05) and its value as an added 1 3 Food and Environmental Virology (2018) 10:288–296 295 Bellou, M., Kokkinos, P., & Vantarakis, A. (2013). Shellfish-borne viral public health control is questionable. The limited value of outbreaks: A systematic review. Food and Environmental Virol- depuration periods extended beyond the 3 days as observed ogy, 5(1), 13–23. https ://doi.org/10.1007/s1256 0-012-9097-6. here is consistent with laboratory-based studies reported Chalmers, J. W., & McMillan, J. H. (1995). An outbreak of viral gastro- elsewhere for other shellfish species (Polo et al. 2015) and enteritis associated with adequately prepared oysters. Epidemiol- ogy and Infection, 115(1), 163–167. oysters (McLeod et al. 2017). Interestingly, laboratory-based Costafreda, M. I., Bosch, A., & Pintó, R. M. (2006). Development, depuration studies have demonstrated that virus reduction is evaluation, and standardization of a real-time taqman reverse a two-phase process (Polo et al. 2014). The first phase is a transcription-PCR assay for quantification of Hepatitis A virus relatively rapid process related directly to shellfish filtration in clinical and shellfish samples. Applied and Environmental Microbiology, 72(6), 3846–3855. rate. However, subsequent low-level norovirus persistence is Doré, B., Keaveney, S., Flannery, J., & Rajko-Nenow, P. (2010). associated with a second phase demonstrating a significantly Management of health risk associated with oysters harvested slower reduction rate where viruses appear to be refractory from a norovirus contaminated area, Ireland, February–March to the initial depuration process, possibly because they are 2010. EuroSurveillance, 15(19), 1–5. EFSA. (2012). Scientific opinion on norovirus (NoV) in oysters: intrinsically bound to specific norovirus receptors in the Methods, limits and control options. EFSA Journal 10(1), 2500. oyster tissue. Flannery, J., Keaveney, S., & Dore, W. (2009). Use of FRNA bac- In summary, in the absence of current regulatory stand- teriophage to indicate the risk of norovirus contamination in ards, we believe that a site-specific management approach, Irish Oysters. Journal of Food Protection, 72(11), 2358–2362. Flannery, J., Keaveney, S., Rajko-Nenow, P., O’Flaherty, V., & Doré, such as described here and supported by norovirus moni- W. (2012). Concentration of norovirus during wastewater treat- toring, can reduce consumer exposure to norovirus genome ment and its impact on oyster contamination. Applied and Envi- copies. This may provide additional, if not complete, con- romental Microbiology, 78(9), 3400–3406. sumer protection. However, despite the anecdotal evidence Hoehne, M., Schreier, E. (2006). Detection of norovirus genogroup I and II by multiplex real-time RT-PCR using a 3′-minor groove presented here, i.e. lack of illness reports, it is not possible to binder-DNA probe. BMC Infectious Diseases. h tt ps : // do i . determine the public health benefits of this approach. There- org/10.1186/1471-2334-6-69. fore, there remains a clear requirement for further work to ISO 15216-1:2017. (2017). Microbiology of the food chain—Hori- better characterise the relationship between norovirus RNA zontal method for determination of hepatitis a virus and norovi- rus using real-time RT-PCR—Part 1: Method for quantification. concentrations in oysters as judged by RT-qPCR and illness https ://www.iso.org/stand ard/65681 .html. outcomes. Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S., Hoshino, F. B., Takeda, N., & Katayama, K. (2003). Broadly Acknowledgements This work was supported by the Department of reactive and highly sensitive assay for norwalk-like viruses Agriculture Food and Marine research programme Food Institutional based on real-time quantitative reverse transcription-PCR. Jour- Research Measure (FIRM) Grant 14/SF/852. nal of Clinical Microbiology, 41(4), 1548–1557. Kingsley, D. H., Johnna, P., Fay, K., Calci, R., Pouillot, J., Woods, Open Access This article is distributed under the terms of the Crea- H., Chen, B. A., Niemira, & Van Doren, J. M. (2017). Evalua- tive Commons Attribution 4.0 International License (http://creat iveco tion of chlorine treatment levels for inactivation of human noro- mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- virus and MS2 bacteriophage during sewage treatment. Applied tion, and reproduction in any medium, provided you give appropriate and Environmental Microbiology 83(23), e01270-17. https :// credit to the original author(s) and the source, provide a link to the doi.org/10.1128/AEM.01270 -17. Creative Commons license, and indicate if changes were made. Kitajima, M., Haramoto, E., Phanuwan, C., Katayama, H., & Furu- mai, H. (2012). Molecular detection and genotyping of human noroviruses in influent and effluent water at a wastewater treat- ment plant in Japan. Journal of Applied Microbiology, 112(3), 605–613. References Le Guyader, F. S., Le Saux, J. C., Ambert-Balay, K., Krol, J., Serais, O., Parnaudeau, S., Giraudon, H., et al. (2008). Aichi virus, Ang, L. H. (1998). An outbreak of viral gastroenteritis associated with norovirus, astrovirus, enterovirus, and rotavirus involved in eating raw oysters. Communicable Disease and Public Health, clinical cases from a french oyster-related gastroenteritis out- 1(1), 38–40. break. Journal of Clinical Microbiology, 46(12), 4011–4017. Anonymous. (2004). Regulation (EC) 854/2004 of the European Parlia- Lees, D., Younger, A., & Dore´, B. (2010). Depuration and relaying. ment and of the Council of 29 April 2004 Laying down Specific In G. Rees, K. Pond, D. Kay, J. Bartram, & J. Santo Domingo Rules for the Organisation of Official Controls on Products of (Eds.), Safe management of shellfish and harvest waters. Lon- Animal Origin Intended for Human Consumption. Vol. L226. don: International Water Association. Atmar, R. L., Antone, R., Opekun, M. A., Gilger, Mary, K., Estes, Loisy, F., Atmar, R. L., Guillon, P., Cann, P. Le, Pommepuy, M., Sue, E., Crawford, Frederick, H., Neill, Graham, D. Y., Ramani, & Le Guyager, F. S. (2005). Real-time RT-PCR for norovirus S., Hill, H., Ferreira, J. (2014). Determination of the 50% human screening in shellfish. Journal of Virological Methods, 123, 1–7. infectious dose for norwalk virus. The Journal of Infectious Dis- Lowther, J. A., Gustar, N. E., Hartnell, R. E., & Lees, D. N. (2012a). eases, 209(7), 1016–1022. https ://doi.org/10.1093/infdi s/jit62 0. Comparison of norovirus RNA levels in outbreak-related oysters Baker, K., Morris, J., McCarthy, N., Saldana, L., Lowther, J., Collin- with background environmental levels. Journal of Food Protec- son, A., & Young, M. (2010). An outbreak of norovirus infection tion, 75(2), 389–393. linked to oyster consumption at a UK Restaurant, February 2010. Lowther, J. A., Gustar, N. E., Powell, A. L., Hartnell, R. E., & Lees, Journal of Public Health, 33(2), 205–211. D. N. (2012b). Two-year systematic study to assess norovirus 1 3 296 Food and Environmental Virology (2018) 10:288–296 contamination in oysters from Commercial harvesting areas in illness outbreaks in Ireland. Epidemiology and Infection, 142(10), the United Kingdom. Applied and Environmental Microbiology, 2096–2104. https ://doi.org/10.1017/S0950 26881 30030 14. 78(16), 5812–5817. https ://doi.org/10.1128/AEM.01046 -12. Silva, A. K., da, J. C., Le Saux, S., Parnaudeau, M., Pommepuy, M., McLeod, C., Polo, D., Saux, J.-C. L., & Guyader, F. S. L. (2017). Elimelech, & Le Guyader, F. S. (2007). Evaluation of removal of Depuration and relaying: A review on potential removal of noro- noroviruses during wastewater treatment, using real-time reverse virus from oysters. Comprehensive Reviews in Food Science and transcription-PCR: Different behaviours of genogroups I and II. Food Safety. https ://doi.org/10.1111/1541-4337.12271 . Applied and Environmental Microbiology, 73(24), 7891–7897. Noda, M., Fukuda, S., & Nishio, O. (2008). Statistical analysis of attack Svraka, S., Duizer, E., Vennema, H., de Bruin, E., van de Veer, B., rate in norovirus foodbourne outbreaks. International Journal of Dorresteijn, B., & Koopmans, M. (2007). Etiological role of Food Microbiology, 122, 216–220. viruses in outbreaks of acute gastroenteritis in The Netherlands Pinto, B., Pierotti, R., Canale, G., & Reali, D. (1999). Characteriza- from 1994 through 2005. Journal of Clinical Microbiology, 45(5), tion of ‘faecal streptococci’ as indicators of faecal pollution and 1389–1394. distribution in the environment. Letters in Applied Microbiology, Tam, C., Viviana, L., Adak, B., Bolton, E., Dodds, J., Cowden, J., 29, 258–263. Evans, M., et al. (2012). The second study of infectious intestinal Polo, D., Alvarez, C., Diez, J., Darriba, S., Longa, A., & Romalde, disease in the community (IID2 study). Project Number: B18021. J. L. (2014). Viral elimination during commercial depuration of London: UK Food Standards Agency. shellfish. Food Control, 43(September), 206–212. h t t p s : / / d o i . Teunis, P. F. M., Moe, C. L., Liu, P., Miller, S. E., Lindesmith, L., org/10.1016/j.foodc ont.2014.03.022. Baric, R. S., Le Pendu, J., & Calderon, R. L. (2008). Norwalk Polo, D., Feal, X., & Romalde, J. L. (2015). Mathematical model for virus: How infectious is it? Journal of Medical Virology, 80, viral depuration kinetics in shellfish: An useful tool to estimate the 1468–1476. risk for the consumers. Food Microbiology, 49(August), 220–225. Thebault, A., Teunis, P. F. M., Pendu, J. L., Le Guyader, F. S., Denis, https ://doi.org/10.1016/j.fm.2015.02.015. J-B (2013). Infectivity of GI and GII noroviruses established from Rajko-Nenow, P., Keaveney, S., Flannery, J., McIntyre, A., & Dore, oyster related outbreaks. Epidemics, 5(2), 98–110. https ://doi. W. (2013). Norovirus genotypes implicated in two oyster-related org/10.1016/j.epide m.2012.12.004. 1 3

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Published: May 3, 2018

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