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Evaluation of Norovirus Reduction in Environmentally Contaminated Pacific Oysters During Laboratory Controlled and Commercial Depuration

Evaluation of Norovirus Reduction in Environmentally Contaminated Pacific Oysters During... Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT- PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17–21 °C and after 4 days at 11–15 °C, compared to 44% reduction at 7–9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram −1 (gcg ; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant dif- ference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the −1 norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg . These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry. Keywords Depuration · Human norovirus · Risk management · Oysters · RT-qPCR · ISO 15216-1 Introduction throughout the world to manage their production. In Europe, regulatory controls predominantly centre around the sanitary Norovirus infections are the most common cause of non- classification of harvesting areas into three categories, A, B bacterial gastroenteritis worldwide (Marshall et al. 2003; or C, based on increasing Escherichia coli concentrations Marshall and Bruggink 2011). Filter-feeding bivalve mollus- (Anonymous 2004, 2019). Each classification category calls can shellfish such as mussels, clams and oysters can become for die ff ring degrees of post-harvest treatment: from no addi - contaminated with human norovirus when grown in areas tional treatment for shellfish harvested from class A waters, impacted by human sewage discharges. Such shellfish pre- to relay and depuration for shellfish grown in class C areas. sent a recognised public health risk when consumed raw One of the most widely practiced post-harvest treatments or lightly cooked (Bellou et al. 2013) and regulations exist is depuration, whereby bivalve shellfish undergo self-puri- fication in land-based tanks of clean seawater. The process was originally designed in the beginning of the twentieth * Sinéad Keaveney century to prevent bacterial illness associated with shellfish Sinead.Keaveney@marine.ie consumption. In order to comply with EU regulations shellfish har - Marine Institute, Rinville, Oranmore, Ireland 2 vested from class B areas, accounting for approximately 60% Centre for Food Safety, University College Dublin, Dublin, of overall oyster production across the EU (EFSA 2019) Ireland Vol.:(0123456789) 1 3 230 Food and Environmental Virology (2021) 13:229–240 must be depurated prior to going to market. Despite these 90–94% reduction) and based on that evidence the process regulatory obligations virtually eliminating bacterial illness was deemed ineffective (McLeod et al. 2017). However, associated with bivalve shellfish, there are many reports of the initial viral loads in such conditioned animals would such product causing outbreaks of viral illness (Morse et al. be considered to be much higher than those seen in oysters 1986; Chalmers and McMillan 1995; Lee et al. 1999; King- from classified production areas across Europe as confirmed sley et al. 2002; Baker et al. 2010; LeBlanc et al. 2016). by the EFSA baseline survey conducted between November In particular, such outbreaks have been associated with the 2016 and October 2018 (EFSA 2019). Much less is therefore consumption of oysters (Le Guyader et al. 2010; Westrell known about the efficacy of norovirus reduction of environ- et al. 2010; Alfano-Sobsey et al. 2011; Rajko-Nenow et al. mentally contaminated oysters from classified production 2013). Oysters harvested from A classified areas, where areas under controlled laboratory conditions or in commer- post-harvest treatment is not mandatory, have also been cial operations. Evidence for viral reduction during modified associated with outbreaks of illness (Doré et al. 2010). Oys- depuration procedures was reported by (Doré et al. 1998) ters present additional risks due to a number of factors such using FRNA bacteriophage where a significant reduction in as being grown in intertidal areas often impacted by sew- FRNA bacteriophage was observed when water temperature age and being consumed raw or only lightly cooked. This was increased to 18 °C. The introduction of quantitative real- has resulted in an increasing number of commercial oys- time PCR for the detection and quantification of norovirus in ter producers in Ireland and elsewhere that harvest from A bivalve shellfish, particularly the method described in ISO classified production areas to include depuration as an extra 15216-1:2017 (Anonymous 2017), has been an important step in their HACCP (Hazard Analysis and Critical Control step in the area of risk management of norovirus. This tool Point) or risk management procedures (Rupnik et al. 2018). has allowed better understanding of the relative differences Minimum time and water temperature used in commercial in norovirus concentrations when applying risk management depuration are not stipulated in EU regulation. In Ireland, it procedures (Rupnik et al. 2018), as well as understanding the is recommended that depuration should be carried out for a concentrations of norovirus found in classified production minimum of 42 h with a water temperature of no less than areas across the EU (EFSA 2019). 8 °C (Anonymous 2018). This treatment has been shown As far as the authors are aware, this is the first study using to consistently reduce E. coli concentrations to below the the ISO 15216-1 standard method based on real-time RT- regulatory limit of 230 MPN/100 g, but not to reduce noro- PCR to measure norovirus RNA concentrations in oysters virus to the same extent (Schwab et al. 1998; McLeod et al. contaminated in their natural environment during controlled 2009). However, some studies have indicated that depura- laboratory depuration studies and enhanced commercial dep- tion time and seawater temperature are both factors that may uration procedures. Our aim was to provide evidence to sup- influence virus reduction during bivalve shellfish depuration port the hypothesis that appropriate depuration conditions (Lees et al. 2010). The potential for norovirus reduction in can be successfully used in the commercial setting as part of oysters during enhanced depuration procedures was identi- risk management measures to reduce the risk to consumer fied previously by this group (Rupnik et al. 2018) prompting by decreasing the norovirus concentrations in market-ready this study to investigate norovirus reduction in oysters under shellfish. controlled laboratory conditions. Artificial contamination of shellfish with viral pathogens is a common approach to studying depuration processes and Materials and Methods has been described previously (Muniain-Mujika et al. 2002; Choi and Jiang 2005; Nappier et al. 2008; McLeod et al. Oysters 2009; Polo et al. 2014b). As shellfish are known to efficiently bioaccumulate viruses in as little as several hours (Flannery Triploid Pacific oysters ( Crassostrea gigas) contaminated et  al. 2012; Souza et al. 2013, 2018; Pilotto et al. 2019), with norovirus in their growing areas were used in all labora- this approach allows for a rapid generation of animals con- tory depuration trials. Oysters from a commercial classified taminated with one or multiple viral strains. Concentrations production area were monitored for norovirus GI and GII 11 −1 as high as 10 genome copies per gram (gcg ) of murine contamination on a weekly basis and results of the moni- norovirus (MNV1) were achieved during 24h bioaccumula- toring were used to schedule the depuration experiments. tion in C. gigas by Pilotto et al. (2019), whereas both GI Once the norovirus concentrations increased to, or above, −1 and GII human norovirus genogroups, simultaneously bio-300 gcg a batch of oysters were harvested, washed and 6 −1 accumulated in C. gigas oysters to 10 gcg (Maalouf et al. transported to the laboratory within 27 h under temperature- 2011). Maximum reductions of the norovirus concentrations controlled conditions (< 15 °C). On receipt in the labora- in such artificially contaminated shellfish were previously tory, oysters were stored in dry, cool conditions and used in reported in the region of 1–1.2 log unit (equivalent to depuration trials within 24 h. 1 3 Food and Environmental Virology (2021) 13:229–240 231 cycles). Producer B performs depurations typically for Laboratory Depuration Trials 72  h at an accurately controlled stable  18  °C. Twenty- four and 22 depuration cycles carried out by Producer A Two 0.6m tanks (Depur Systems, UK) were filled with 400–450 L potable mains water and 15 kg of Seamix Artifi- and B, respectively, were examined. Pre-depuration oyster samples were collected on the day depuration commenced cial Sea Water salt mix (NaCl 66.1%; MgSO 16.3%; MgCl 4 2 12.7%; CaCl 3.3% and KCl 1.6%; Peacock Salt, UK) was and were followed by a second, paired sample taken after the completion of the depuration process. Each sample added to each and stirred until fully dissolved. The amount of salt added increased water salinity to between 28 and consisted of 12 live animals that were transported into the laboratory under chilled conditions (< 15 °C) within 27 h 32ppt mimicking that of the estuary. This artificial seawa- ter was circulated in the tank overnight to fully equilibrate (Producer A) or 5 h (Producer B). Upon arrival, oysters were stored at 4 °C for up to 24 h before processing. When before depuration experiments commenced. Water tem- perature was maintained within 1 °C of target temperature evaluating the commercial depuration processes, both nor- ovirus genogroups were quantified in the pre-depuration with a combination of 300  W aquarium heaters placed directly in the tank and an externally located 750 W water samples. Samples in which the virus concentration was −1 greater than 150 gcg for at least one genotype were chiller (D&D, DC750). Bar sprinklers located above one side wall of the tank were used for aeration and water flow selected and subsequently followed with a sample taken after completion of depuration. This allowed for concur- was maintained using an external pump at 2000–2200 L/h. One 36 W UV-C lamp fitted inline on each tank was used rent analysis of the effect of depuration on both norovirus genotypes in oysters. to sterilise the circulating water. Dissolved oxygen levels were measured once daily using a hand-held OxyGuard device. Oysters were placed loosely in shallow plastic trays, approximately 70–80 animals per tray. In total, four trays Norovirus Detection in Oysters per tank (between 280 and 320 oysters) were placed in each depuration tank and subjected to depuration. Triplicate sam- Preparation of Oyster Samples for Norovirus Analysis ples of 12 oysters were collected at commencement of the experiment (Day 0) and at 24-h intervals thereafter (Days Oysters were analysed for the presence of norovirus GI and GII in accordance with ISO 15216-1:2017 (Anony- 1–7). Oysters were stored at 4 °C for up to 24 h before fur- ther processing. A full depuration cycle was run for 7 days mous 2017). Briefly, oysters were cleaned under running potable water. Ten oysters per sample were shucked and (approx.168 h) after which the tanks were drained, cleaned, and filled with freshly prepared artificial sea water before the digestive tissues (DT) dissected out. The dissected DT was finely chopped using a sterile razor blade and mixed the next experiment. well. Two grams of DT were then spiked with 10 µl of the internal process control (IPC) virus (Mengo virus strain Shelf Life Test MC ) for evaluation of virus extraction efficiency similar to that described by (Costafreda et al. 2006) and treated Forty-five oysters remaining in the depuration tanks after −1 completion of the laboratory trials at 8 °C and 18 °C were with 2 ml Proteinase K (100 µg  ml ). Samples were incu- bated at 37 °C for 60 min with shaking at 150 rpm fol- placed on shallow trays and refrigerated (5 °C) for 4 weeks. Once a week, oysters were inspected and any gaping or unre- lowed by 15 min at 60 °C. Finally, after centrifugation at 3000×g for 5 min, supernatants were retained for RNA sponsive to percussion animals were counted and disposed of. extraction. Commercial Depuration Viral RNA Extraction Paired samples, pre- and post-depuration, were collected RNA was extracted from 500  µl of the DT supernatants from two separate oyster producers between January 2018 and December 2019 (Producer A) and February 2018 to using NucliSENS® magnetic extraction reagents (bioMé- rieux) and the NucliSENS® MiniMAG® extraction platform April 2019 (Producer B). Producer A operates in a class A area, whilst Producer B operates in an area with a seasonal and eluted into 100 µl of elution buffer. RNA extracts were stored at − 80 °C until the RT-qPCR analysis was conducted. A classification during December through to March and class B for the rest of the year. Depuration is routinely RNA was also extracted from 10 µl of the IPC sample for evaluation of extraction efficiency. A single negative extrac- used in both facilities with Producer A typically depurat- ing oysters for 7 days with an average water temperature tion control (water only) was processed alongside the oyster samples. of 14 °C (ranging from 12 to 16 °C between the depuration 1 3 232 Food and Environmental Virology (2021) 13:229–240 Determination of the Norovirus Concentration Using controls (blank sample carried through the RNA extraction One‑Step RT‑qPCR step) were included in each RT-PCR analysis to control for cross-contamination. Oysters were analysed for the norovirus concentrations using standardised quantitative real-time reverse transcription PCR Analysis of Results (RT-qPCR) (Anonymous 2017). RT-qPCR analysis was car- ried out using the Applied Biosystems AB7500 instrument LOD and LOQ (Applied Biosystems, Foster City, CA) and the RNA Ultra- sense one-step RT-qPCR system (Invitrogen). The reaction The limits of detection (LOD) and quantification (LOQ) was prepared by combining 5 µl of the extracted RNA sam- for Norovirus GI and GII were established based on Euro- ple and 20 µl of the reaction mix containing 500 nM forward pean Union Reference Laboratory (EURL) for monitor- primer, 900 nM reverse primer, 250 nM sequence specific ing the bacteriological and viral contamination of bivalve probe,1 × ROX reference dye and 1.25 µl of enzyme mix. molluscs guidance using dilution series of oyster digestive Previously described primers QNIF4 (da Silva et al. 2007), gland material contaminated with both, norovirus GI and NV1LCR (Svraka et al. 2007) and TM9 probe (Hoehne and GII (CEFAS 2016). The LOD and LOQ for norovirus GI and −1 Schreier 2006) were used for the detection of norovirus GI, GII was determined as 20 and 100 gcg , respectively. For and QNIF2 (Loisy et al. 2005), COG2R (Kageyama et al. statistical analysis, and in order to facilitate geometric mean 2003) and QNIFS probe (Loisy et al. 2005) were used for calculation, samples in which norovirus was not detected −1 the detection of norovirus GII. The Mengo110, Mengo209 were assigned a value of 10 gcg (half of the LOD) and −1 primers and Mengo147 probe were used in IPC assay (Pintó those that tested positive below 100 gcg (< LOQ) were −1 et al. 2009). The 96-well optical reaction plate was incu- assigned a value of 50 gcg (half of LOQ). bated at 55 °C for 60 min, 95 °C for 5 min, and then 45 cycles of PCR were performed, with 1 cycle consisting of Relative Concentrations 95 °C for 15 s, 60 °C for 1 min, and 65 °C for 1 min. All samples were analysed for norovirus GI and GII in dupli- Due to variances in starting norovirus concentration, and cate. All control materials used in the RT-qPCR assays were to allow for comparison between experiments and statis- prepared as described by Flannery et al. (2012). To enable tical analysis, all concentrations were converted to values quantification of norovirus RNA in copies per µl, a log relative to Day 0 (initial load), as follows: C = C /C , 10 Ri i 0 dilution series of the norovirus GI and GII DNA plasmids where C – Relative concentration of sample i; C –noro- Ri i 1 5 −1 (ranging from 1 × 10 to 1 × 10 copies/µl) were included virus concentration gcg obtained for sample i using RT- in duplicate on each RT-qPCR run. The number of RNA qPCR and C –geometric mean of initial load concentra- copies in norovirus-positive samples was determined by tion in a given experiment. For example, 2023, 1781 and −1 comparing the C value to the standard curves. The final 1459 norovirus GII gcg were detected in samples A, B concentration was then adjusted to reflect the volume of and C collected at Day 0 giving a geometric mean of 1739 −1 sample analysed and expressed as the number of detectable norovirus GII gcg . Therefore, the relative result for sam- −1 −1 virus genome copies per gram of DT. The presence of inhibi- ple A = 2023 gcg /1739 gcg = 1.163; sample B = 1781 −1 −1 −1 tors was checked by spiking an additional 5 µl of each sam- gcg /1739 gcg = 1.024 and sample C = 1459 gcg /1739 −1 ple RNA with 1 µl of either norovirus GI or norovirus GII gcg = 0.839. This principle was applied to all samples. external control RNA (ECRNA; 10 RNA transcripts/µl). The threshold cycle (C ) value obtained for samples spiked Assigning of Data for Laboratory Depuration Trials with the ECRNA was compared to the results obtained in the absence of the sample (5 µl of water used instead) and Norovirus test results were assigned into 3 separate groups used to estimate RT-PCR inhibition expressed as a percent- based on the water temperature used in a given depuration age. In accordance with ISO 15216-1:2017. Oyster samples trial: Low—depuration experiments carried out at 8 ± 1 °C with RT-PCR inhibition below the 75% were accepted for (n = 5); Medium—depuration experiments carried out at 12 inclusion in this study. Extraction efficiency was assessed or 14 °C (± 1 °C; n = 3) and High—depuration experiments by comparing the C value of the sample spiked with IPC carried out at 18 or 20 °C (± 1 °C; n = 7). virus to a standard curve obtained by preparing log dilu- tions of the RNA extracted from 10 µl Mengo virus and was Statistical Analysis subsequently expressed as percentage extraction efficiency. Samples with the extraction efficiency greater than 1% were R (version 3.6.0) using the libraries ‘car’ and ‘PMCMR’ was accepted for inclusion in this study (Anonymous 2017). used for statistical analysis. The normality of the data was No template controls (water only) and negative extraction tested by performing the Shapiro–Wilk test and the Levene 1 3 Food and Environmental Virology (2021) 13:229–240 233 test was used to evaluate the homogeneity of variances. To achieved by day 3 in the high temperature trials with rela- evaluate the differences between three temperatures condi- tive virus concentration reduced to 0.26 ± 0.11 (reduction tions (low, medium and high) during the depuration study a of 74% or 0.59 log units). A similar degree of reduction Kruskal–Wallis rank ANOVA with the pairwise test for mul- was observed in the medium temperature trials on day 4 tiple comparison of mean rank sums (Dunn’s test) as posthoc with relative concentration reduced to 0.27 ± 0.32 (reduc- tests was used. To test the difference of norovirus concentra- tion of 73%, 0.57 log units). By contrast, norovirus GII tions during commercial depuration the Mann–Whitney test concentrations were not reduced in oysters depurated under was applied. The significance level for all statistical analysis low temperature for the first 2 days of depuration. In fact, was set at p ≤ 0.05. an increase in norovirus GII concentration was observed in these oysters at day 1 (relative concentration 1.35 ± 0.33) and the first signs of virus concentration reduction were only Results observed at day 3. The maximum reduction of the initial viral load to 0.65 ± 0.29 (reduction of 44% or 0.25 log Laboratory Depuration Trials units) was observed under low temperature conditions by day 4. Non-parametric Kruskal–Wallis ANOVA showed that Stable conditions in the depuration tanks were maintained there were significant differences in the norovirus GII con- throughout each experiment with dissolved oxygen (DO ) centrations observed between the low and high temperature levels in excess of 80% in all trials. Norovirus GI concen- (p < 0.001) and between low and medium temperature condi- trations detected in C. gigas before depuration were either tions (p < 0.001), but not between medium and high water below or close to the limit of quantification (LOQ) of the temperatures (p = 0.14). −1 test (100 gcg ) and therefore were excluded from further Norovirus GII reductions during all depuration trials con- analysis in this study. Before depuration, norovirus GII con- ducted at medium and high water temperatures displayed centrations in the environmentally contaminated oysters a two-phase virus reduction kinetic. After the initial rapid −1 ranged between 178 and 16,426 norovirus GII gcg with a reduction of norovirus GII, the rate of depuration decreased −1 geometric mean of 906 norovirus GII gcg (Table 1). and no further significant reduction was observed between Norovirus GII reduction in oysters during laboratory days 3 and 7 or days 4 and 7 for high and medium tempera- experiments are shown in Fig. 1. First indications of noro- tures, respectively (Fig. 1). The two-phase virus reduction virus GII reduction in the medium and high temperature kinetic was not evident in the low temperature trials, with a conditions were visible during the initial 24  h (Fig.  1). delayed and shortened viral reduction stage. Virus reduction continued until day 3 at the high temper- No oyster mortalities were observed during the first ature conditions and until day 4 at the medium tempera- 2  weeks of shelf life test conducted upon completion of ture conditions. Maximum reduction of norovirus GII was the depuration experiments with all oysters responding to Table 1 Norovirus GII −1 Experiment Water Time point [days]/norovirus GII concentration [gcg ] concentrations measured in temperature Pacific oysters during laboratory 0 1 2 3 4 5 6 7 [°C] depuration trials 1 8 16,426 15,792 7803 7463 10,742 5443 11,210 7558 2 8 290 310 284 142 < LOQ 249 147 225 3 8 5573 14,505 7428 5981 2965 3334 3548 2293 4 8 1739 1970 2309 1601 2122 4798 2087 3020 5 8 850 1247 956 467 662 888 512 606 6 12 452 143 231 202 < LOQ 219 181 < LOQ 7 12 405 234 193 < LOQ < LOQ < LOQ < LOQ < LOQ 8 14 1739 2465 1451 1442 1599 1595 1813 1248 9 20 452 129 183 110 < LOQ < LOQ < LOQ < LOQ 10 18 405 236 147 < LOQ < LOQ < LOQ < LOQ < LOQ 11 18 178 102 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 12 20 178 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 13 20 290 168 < LOQ < LOQ < LOQ < LOQ 224 233 14 20 5573 4670 5896 2296 2244 1769 1513 2054 15 18 850 845 389 365 522 474 301 310 1 3 234 Food and Environmental Virology (2021) 13:229–240 Fig. 1 Log reduction (lines) 10 Low Medium High of norovirus GII at 3 water temperature regimes studied and norovirus GII concentra- 1.8 -0.2 tions (bars) detected in oysters during laboratory depuration 1.6 -0.1 trials. Results shown as relative 1.4 0 to norovirus GII concentration at day 0, error bars ± geometric 1.2 0.1 standard deviation 1 0.2 0.8 0.3 0.6 0.4 0.4 0.5 0.2 0.6 0 0.7 0123456 7 Depuraon duraon [days] percussion. Mortality rates were comparable for the two oys- The geometric mean concentrations in the pre-depuration −1 −1 ter populations tested past week two, despite the different samples were 251 gcg and 281 gcg and maximum con- −1 water temperature regimes used in depuration trials (paired centrations detected were 761 and 1658 gcg for norovirus t test p = 0.3724). GI and GII, respectively (Table 2). Following depuration, the geometric mean virus concentrations were < LOQ for both Evaluation of Commercial Depuration genogroups with the maximum concentrations of norovi- −1 rus GI and GII detected in any sample 468 and 300 gcg , Producer A Commercial Depuration Results respectively. The difference in norovirus concentrations in oysters before and after depuration were found to be sta- A total of 13 paired samples (pre and post depuration) con- tistically significant for both genogroups (Mann–Whitney taminated with norovirus GI, and 20 paired samples con- non-parametric t-test, p < 0.01 and p < 0.001, for norovi- taminated with norovirus GII, prior to depuration were rus GI and GII, respectively). A higher level of reduction identified from Producer A and included in this evaluation. was observed on average for norovirus GII compared to GI Table 2 Norovirus GI and Producer A B GII concentrations detected in oysters pre- and post-depuration Depuration conditions 12–16 °C, 7 days 18 °C, 3 days collected from Producer A and Norovirus genogroup GI GII GI GII GII n = 13 20 20 13 12 −1 Pre-depuration [gcg ]  Average 285 344 242 844 806  Geometric mean 251 281 229 526 488  Min–Max 150–761 155–1658 151–409 167–2802 167–2802 −1 Post-depuration [gcg ]  Average 121 65 129 317 158  Geometric mean 82 45 91 66 49  Min–Max < LOD-468 < LOD-300 < LOD-366 < LOD-2232 < LOD-780 Geometric mean reduction 67.58% 83.95% 60.41% 87.47% 89.92% Log reduction 0.49 0.79 0.40 0.90 1.00 −1 −1 < LOD—results below the limit of detection (< 20 gcg norovirus GI, < 20 gcg norovirus GII) Analysis results on an adjusted sample population, with one outlier pair, failing to reduce norovirus GII concentration in oysters during depuration, excluded from norovirus GII results 1 3 Relave norovirus GII concentraon Log reducon Food and Environmental Virology (2021) 13:229–240 235 −1 (Table 2) where norovirus GII was reduced by 83.95% (0.79 contaminated with < 1000 norovirus GII gcg were reduced log units) compared to 67.58% (0.49 log units) reduction to < LOQ (Table 3). Samples contaminated with norovirus 10 10 −1 of norovirus GI. GII at concentrations > 1000 gcg prior to depuration were reduced to concentrations < LOQ in 40% of the cases. None Producer B Commercial Depuration Results of the samples examined in the commercial setting had con- −1 centrations of norovirus GI greater than 1000 gcg . A total of 20 paired samples (pre- and post- depuration) contaminated with norovirus GI and 13 paired samples contaminated with norovirus GII were identified from Discussion Producer B and included in this evaluation. The geomet- ric mean concentration in the pre-depuration samples were Norovirus contamination in oyster production areas is an −1 −1 229 gcg and 526 gcg , for norovirus GI and GII, respec- ongoing issue for food safety regulators and oyster produc- tively (Table 2) with the maximum concentrations of 409 ers alike. The primary objective for both is the production −1 −1 gcg norovirus GI and 2802 gcg norovirus GII. Following of food that is safe for human consumption. Data published depuration, the geometric mean virus concentrations were in a recent EFSA baseline survey highlights the high preva- significantly reduced to < LOQ for both norovirus GI and lence of norovirus contamination in oysters from classified −1 GII with maximum concentrations detected at 366 gcg production areas during the winter months (EFSA 2019). −1 norovirus GI and 2232 gcg norovirus GII. The differ - Peak norovirus concentration and prevalence was observed ence in norovirus concentrations in oysters before and after in the months of January and February when almost 65% depuration were found to be statistically significant for both of samples tested were positive for norovirus with a mean −1 genogroups (Mann–Whitney non-parametric t-test, p < 0.001 concentration of 661 gcg (EFSA 2019). This important and p < 0.05 for norovirus GI and GII, respectively). In one data demonstrates the need for effective mitigation meas- of the 13 depuration runs analysed post depuration the con- ures to reduce norovirus concentrations before reaching the −1 centration for norovirus GII (2232 gcg ) did not decrease consumer. Whilst there are currently no legislative criteria from the pre-depuration concentration of 1304 norovirus GII for norovirus in oysters intended for consumption, there is −1 gcg . On removal of this outlier pair the overall level of recognition from international food safety regulators for reduction demonstrated for norovirus GII was almost 90% or the need to introduce such a criterion to protect consum- 1 log units (p < 0.01). As observed for Producer A, a higher ers. Therefore, oyster producers need effective virus reduc- level of reduction was achieved for oysters contaminated tion measures in order to meet any such microbiological with norovirus GII (87.47–89.92% or 0.90–1.00 log reduc- criterion for norovirus in the future. Previously, we dem- tion) compared to norovirus GI (60.41% or 0.40 log reduc- onstrated the potential of risk management procedures to tion), despite the different depuration conditions employed. reduce the risk of norovirus contamination in a production Overall in the commercial settings, there was a signifi- area as well as the reduction of norovirus concentrations cant difference in the level of reduction between norovirus in end-product by depuration (Rupnik et al. 2018). In that GI and GII (p < 0.001) where norovirus GI was reduced on particular study extended depuration periods of up to 9 days average by 63.4% (0.43 log units) compared to an on aver- were applied during the high-risk winter season. In addi- age reduction of 85.4% (0.84 log units) for norovirus GII tion, during that study minimum depuration temperatures (Table 3). The extent of reduction to < LOQ also differed were generally above the recommended minimum tempera- between genogroups when the pre-depuration concentra- ture of 8 °C (Anonymous 2018) with the mean temperature −1 tions were < 1000 gcg (p < 0.05). In this instance 51.5% of over all depuration cycles of 13.3 °C during the winter. This −1 samples contaminated with < 1000 norovirus GI gcg were prompted further investigation of the ability of enhanced reduced to < LOQ post-depuration, whilst 78.6% of samples depuration conditions to reduce norovirus concentrations Table 3 Overall efficacy of commercial norovirus GI and GII depuration in Pacific oysters based on all paired results from Producer A and B, combined Norovirus genogroup GI GII Geometric mean reduction 63.40% 85.40% Log reduction 0.43 0.84 Pre-depuration concentration % Samples depurated to < LOQ −1 < 1000 gcg 51.5 78.6 −1 > 1000 gcg n/a 40.0 1 3 236 Food and Environmental Virology (2021) 13:229–240 −1 in environmentally contaminated oysters destined for con- higher virus concentrations (> 1000 gcg ). This study has sumption. To our knowledge this is the first report of the demonstrated the impact of the initial viral load prior to application of a standardised test method (Anonymous 2017) commencing depuration trials on the variability in concen- to evaluate norovirus reduction during both, laboratory and trations observed especially in the early stages (day 1–3). commercial depuration processes to environmentally con- Nevertheless, the concentrations detected after day 3 in the taminated oysters. medium and high temperature trials in many of the samples The results of the laboratory depuration trials indicate were at or below the LOQ for norovirus GII. This empha- that norovirus, whilst not completely removed, was signifi- sises the importance of having knowledge of the starting cantly reduced when the water temperature used for depu- concentration of norovirus in oysters prior to depuration and ration was increased to > 11 °C, but not at lower tempera- should be a key consideration for operators who wish to tures. Other studies have previously reported similar findings reduce norovirus concentrations to levels that may be below where norovirus was reduced, but not eliminated from con- any future microbiological criterion. taminated shellfish, including clams, mussels and oysters A two-phase reduction of norovirus in oysters was (Ueki et al. 2007; Neish 2013; Polo et al. 2014a; Pilotto et al. observed during the laboratory depuration trials, similar to 2019) using a variety of time and temperature combinations. that reported in clams and mussels (Polo et al. 2014c). In this The initial temporary increase in norovirus concentrations study once the maximum reduction was achieved after day 3 observed here at Day 1, and by others at Day 1–2 (McLeod and day 4 no further significant decline in virus concentra- et al. 2009; Polo et al. 2014c), could be explained by tran- tions took place. A slight fluctuation of norovirus concentra- sient capture and subsequent release of norovirus particles tions were detected during the second reduction phase (i.e. from animal tissue outside of DT, such as gills and mantle post day 3/4) in some experiments particularly on day 5 and (Maalouf et al. 2010). In addition, shellfish are known to 6. It is not clear what the exact cause is for these increases slow down their metabolism and respiration rate at lower in virus concentration however it could be a reflection of the water temperatures (Dittman 1997; Li et al. 2017) and in limitations of the RT-PCR method itself, especially when such conditions relocation of virus particles from other tis- working with target concentrations close to, or below, the sues to DT and out as faeces or pseudofeces is less efficient LOQ, a natural variability between samples or a potential and requires longer time. re-contamination event due to sampling procedures. The The use of environmentally contaminated oysters in the optimal combination of time and temperature providing depuration trials presented here, allowing for best represen- the most rapid reduction in norovirus was three days at the tation of virus concentrations found in commercially har- high temperature range with 74% (0.59 log units) reduc- vested oysters, relied on long-term presence of the virus in tion observed. However, a similar final reduction occurred environment in order to complete all planned experiments. in the medium water temperature range albeit one day later Premature drop of norovirus GII concentration to levels (73%, 0.57 lo g units). More limited norovirus reduction −1 below the critical lower limit (of 300 gcg ) experienced was achieved during depuration at the low temperature with during this study resulted in lower number of depuration maximum reduction of 44% (0.25 log units) observed trials completed at medium water temperatures, compared after day 4. The rate of reduction observed in the increased to low or high water temperatures. While such an ade- temperature trials (medium and high) were significantly dif- quately balanced data set could have provided improved, ferent from that observed from the low temperature trials. more robust statistics, the data set obtained during this study This indicates that by setting depuration water temperature provides statistically significant differences between the dif- to > 11 °C and by increasing the time allowed for depura- ferent temperature regimes studied. Additional depuration tion to 3–4 days, but not beyond, provides optimal reduction studies, focusing on the medium water temperatures of 11 of norovirus from contaminated oysters whilst maintaining to 15 °C and using environmentally contaminated oysters, product shelf life. would be welcomed in the future to supplement the data To investigate norovirus reduction during depuration in presented here. the commercial setting we evaluated commercial practices Initial viral load has been thought to have an impact undertaken by two separate oyster producers. Producer A on the depuration outcome whereby the higher the virus employs longer depuration times (7 days) but lower water concentration prior to depuration, the higher the remain- temperature (approximately 14 °C), while Producer B mini- ing virus load, if all other parameters were kept consistent mises the depuration time (3 days) but maximises the tem- (McLeod et al. 2017). The same finding was also observed perature (18 °C). In both scenarios, similar levels of viral in the study described here where oysters contaminated reduction for norovirus GI and GII were achieved despite the −1 with < 1000 norovirus GII gc g , were reduced to < 100 difference in time and temperature combinations (Table  3). −1 gcg in 1–4 days. However, the same post-depuration con- As with the laboratory trials the concentration of norovi- centrations were not achieved in oysters contaminated with rus in the pre-depuration samples was an important feature 1 3 Food and Environmental Virology (2021) 13:229–240 237 of the overall success of commercial norovirus reduction A greater reduction of norovirus GII was demonstrated as judged by a target concentration of < LOQ post-depura- in the commercial setting than in the laboratory trials with tion. Almost four in five of all commercial oyster samples 84–87% (0.80–0.94 log ) reduction achieved by both com- −1 tested prior to depuration with a concentration < 1000 gcg mercial depuration processes compared with 74% (0.59 norovirus GII contained concentrations < LOQ post-depu- log ) in laboratory trials. The improved reduction observed ration (Table 3). On the other hand, only 40% of samples in the commercial setting could be caused by a number of −1 with > 1000 gcg prior to depuration, were found to have factors particularly those that affect the condition of the oys- concentrations < LOQ for norovirus GII post-depuration. ters themselves. Depuration is a complex process despite Despite a small number of commercial samples (n = 5) con- the simplicity of the infrastructure required. Individual −1 taining in excess of the 1000 gcg norovirus GII included in animals can respond differently to stress factors, such as this evaluation, combined with findings from the laboratory sudden changes in temperature, salinity, lack of food and designed trials, these results suggest that whilst enhanced physical disturbance (shaking, transport or rough handling) depuration conditions can reduce norovirus concentrations (Lacoste et al. 2001; Marigómez et al. 2017; Peteiro et al. in oysters, this only becomes an effective consumer health 2018; Seuront et al. 2019). Distressed animals may have intervention strategy when the contamination in the oys- trouble resuming normal filtration and therefore may fail to ter harvesting area is also managed. Applying enhanced purge impurities. Indeed, in all the laboratory trials visual depuration conditions to oysters that are contaminated to inspection confirmed that oysters resumed filtration within −1 levels > 1000 gcg , is unlikely to reduce norovirus concen- an hour from submerging in water, suggesting minimal trations to an acceptable level and will have limited public disruption. However, it is possible that a few animals were health benefit. under some degree of distress during harvest, transport, All but one laboratory depuration trial conducted at upon submerging into heated water or while collecting the either medium or high water temperatures where the initial daily samples. In contrast to the laboratory setting, where a −1 viral load in oysters was below 1000 gcg yielded con- portion of oysters were removed from each depuration tank centrations < LOQ after 4 days of depuration. In addition, daily potentially unsettling the oyster population, the com- one commercial depuration run (Producer B) also failed to mercial depuration tanks were left undisturbed for the full reduce norovirus GII where the pre- and post-depuration duration of depuration (either 3 or 7 days). The tanks were concentration were similar. It is not clear what caused the drained once the process was completed and only then the poor depuration, or lack thereof, in these two instances but oysters were removed, and the final post-depuration sample it does indicate that not all depuration runs will yield suf- taken for analysis. This suggests the need for minimal han- ficient or similar reductions, despite consistent time and dling and disturbance of the oysters during the depuration temperature conditions. This underlines the importance of process to ensure greatest reduction. monitoring the operation of commercial depuration systems Many studies investigating norovirus removal or reduc- to ensure reliable reduction of norovirus. tion from bivalves have involved contaminating various In both commercial depuration scenarios studied here the shellfish species through bioaccumulation. Such laboratory overall reduction rates were higher for norovirus GII than contamination can result in very high starting concentra- GI. Different behaviour between the viral strains during bio- tions which may not be reflective of concentrations naturally accumulation and subsequent depuration have been reported found in bivalves, especially oysters. Here, we used environ- previously (Nappier et al. 2008; Maalouf et al. 2011; Polo mentally contaminated oysters from approved production et  al. 2014c) and could explain the differences observed areas to study norovirus reduction. Therefore, this repre- here. Norovirus GI has been shown to bind to histo-blood sented concentrations of norovirus found in oysters prior to like ligands in oyster digestive tissue, exhibiting prolonged post-harvest treatment and destined for human consumption. persistence in oysters over GII norovirus indicating that its The concentration prior to depuration in both the labora- removal from oyster tissue may prove a greater challenge (Le tory and commercial settings was significantly higher than Guyader et al. 2012). Laboratory trials as conducted for nor- the concentrations measured after depuration once the water ovirus GII in this study are required for GI to fully elucidate temperature was > 11 °C. Of course, the question remains the depuration kinetics for this genogroup. Unfortunately for surrounding the public health significance of these remain- the laboratory trials in this study, the production area from ing norovirus concentrations due to the lack of a routine which the oysters were obtained does not have a history of norovirus infectivity assay despite promising developments regular norovirus GI contamination at concentrations suit- with the human enteroid system (Ettayebi et al. 2016; Cos- able for such studies. Nonetheless, the results obtained from tantini 2018). The illness outcome for any given norovirus the commercial settings indicate the potential of enhanced concentration in contaminated oysters will depend on the depuration conditions to reduce norovirus GI concentrations norovirus genotype, which could be single or multiple, the to below the LOQ. immune and genetic susceptibility of a host (Noda et al. 1 3 238 Food and Environmental Virology (2021) 13:229–240 2008), as well as the size and number of oysters ingested method for detection of norovirus in shellfish may provide by the consumer. However, a link between increasing num- an important risk management tool for the oyster industry ber of virus genome copies detected and risk of infection in light of potential forthcoming legislation. has been reported previously (Lowther et al. 2012). A low likelihood of outbreaks was shown to be associated with Funding This study was funded by Department of Agriculture, Food oysters containing norovirus in concentrations below 100 and the Marine (Grant No. 14/SF/852). −1 gcg , levels readily achievable in this study in the depura- tion trials using increased water temperature and times. Hunt Open Access This article is licensed under a Creative Commons Attri- et al. (2020) demonstrated that norovirus GII was found to bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long be log-normally distributed across individual animals within as you give appropriate credit to the original author(s) and the source, a population and that for mean concentrations below 100 provide a link to the Creative Commons licence, and indicate if changes −1 gcg it was likely that some of the oysters in the tested pop- were made. The images or other third party material in this article are ulation contain no or very little amount of virus (< LOD). In included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in contrast, all oysters in the tested population were found to be the article’s Creative Commons licence and your intended use is not contaminated when the mean concentration tested above 300 permitted by statutory regulation or exceeds the permitted use, you will −1 gcg (Hunt 2019). Additionally, two recent studies inves- need to obtain permission directly from the copyright holder. To view a tigated the FRNA bacteriophage type II (norovirus surro- copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. gate) removal from oysters during depuration using genomic and infectivity methods (Leduc et al. 2020; Younger et al. 2020). These studies indicated that viruses may either be References inactivated in oysters during depuration (Leduc et al. 2020) Alfano-Sobsey, E., Sweat, D., Hall, A., Breedlove, F., Rodriguez, R., or, as suggested by Younger et al. (2020), destroyed and Greene, S., et  al. (2011). Norovirus outbreak associated with removed. Despite the differences in the concluded viral undercooked oysters and secondary household transmission. reduction mechanisms these findings could indicate that the Epidemiology and Infection, 28, 1–7. virus genome copies detected using RT-PCR methods are, Anonymous. (2004). Regulation (EC) 854/2004 of the European Par- liament and of the Council of 29 April 2004 laying down specific at least in part, only footprints left by the infectious virus rules for the organisation of official controls on products of animal particles and they no longer have the capability of causing origin intended for human consumption. infection especially at low concentrations (i.e. ≤ LOQ). Anonymous. (2017). ISO 15216-1:2017 Microbiology of food and A recently published EFSA baseline survey provides com- animal feed—horizontal method for determination of hepatitis A virus and norovirus in food using realtime RT-PCR—Part 1: prehensive scientific data for norovirus prevalence in Euro- method for quantification. International Organization for Stand- pean oyster harvesting areas and will form the basis for dis- ardization, Geneva, pp 15216–1 cussion amongst EU authorities attempting to place a safety Anonymous. (2018). SFPA Guidance Document for Inspecting LBM limit on norovirus concentration in oysters. Should a regula- Purification Centres. Ref: Regulation (EC) 853/2004 Annex III Section VII tory microbiological criterion for norovirus arise, reduction Anonymous. (2019). 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(2013). Norovirus genotypes implicated in two oyster-related jurisdictional claims in published maps and institutional affiliations. illness outbreaks in Ireland. Epidemiology and Infection, 142(10), 2096–2104. https ://doi.org/10.1017/S0950 26881 30030 14. Rupnik, A., Keaveney, S., Devilly, L., Butler, F., & Doré, W. (2018). The impact of winter relocation and depuration on norovirus 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food and Environmental Virology Springer Journals

Evaluation of Norovirus Reduction in Environmentally Contaminated Pacific Oysters During Laboratory Controlled and Commercial Depuration

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Springer Journals
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Copyright © The Author(s) 2021
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1867-0334
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1867-0342
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10.1007/s12560-021-09464-2
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Abstract

Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT- PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17–21 °C and after 4 days at 11–15 °C, compared to 44% reduction at 7–9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram −1 (gcg ; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant dif- ference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the −1 norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg . These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry. Keywords Depuration · Human norovirus · Risk management · Oysters · RT-qPCR · ISO 15216-1 Introduction throughout the world to manage their production. In Europe, regulatory controls predominantly centre around the sanitary Norovirus infections are the most common cause of non- classification of harvesting areas into three categories, A, B bacterial gastroenteritis worldwide (Marshall et al. 2003; or C, based on increasing Escherichia coli concentrations Marshall and Bruggink 2011). Filter-feeding bivalve mollus- (Anonymous 2004, 2019). Each classification category calls can shellfish such as mussels, clams and oysters can become for die ff ring degrees of post-harvest treatment: from no addi - contaminated with human norovirus when grown in areas tional treatment for shellfish harvested from class A waters, impacted by human sewage discharges. Such shellfish pre- to relay and depuration for shellfish grown in class C areas. sent a recognised public health risk when consumed raw One of the most widely practiced post-harvest treatments or lightly cooked (Bellou et al. 2013) and regulations exist is depuration, whereby bivalve shellfish undergo self-puri- fication in land-based tanks of clean seawater. The process was originally designed in the beginning of the twentieth * Sinéad Keaveney century to prevent bacterial illness associated with shellfish Sinead.Keaveney@marine.ie consumption. In order to comply with EU regulations shellfish har - Marine Institute, Rinville, Oranmore, Ireland 2 vested from class B areas, accounting for approximately 60% Centre for Food Safety, University College Dublin, Dublin, of overall oyster production across the EU (EFSA 2019) Ireland Vol.:(0123456789) 1 3 230 Food and Environmental Virology (2021) 13:229–240 must be depurated prior to going to market. Despite these 90–94% reduction) and based on that evidence the process regulatory obligations virtually eliminating bacterial illness was deemed ineffective (McLeod et al. 2017). However, associated with bivalve shellfish, there are many reports of the initial viral loads in such conditioned animals would such product causing outbreaks of viral illness (Morse et al. be considered to be much higher than those seen in oysters 1986; Chalmers and McMillan 1995; Lee et al. 1999; King- from classified production areas across Europe as confirmed sley et al. 2002; Baker et al. 2010; LeBlanc et al. 2016). by the EFSA baseline survey conducted between November In particular, such outbreaks have been associated with the 2016 and October 2018 (EFSA 2019). Much less is therefore consumption of oysters (Le Guyader et al. 2010; Westrell known about the efficacy of norovirus reduction of environ- et al. 2010; Alfano-Sobsey et al. 2011; Rajko-Nenow et al. mentally contaminated oysters from classified production 2013). Oysters harvested from A classified areas, where areas under controlled laboratory conditions or in commer- post-harvest treatment is not mandatory, have also been cial operations. Evidence for viral reduction during modified associated with outbreaks of illness (Doré et al. 2010). Oys- depuration procedures was reported by (Doré et al. 1998) ters present additional risks due to a number of factors such using FRNA bacteriophage where a significant reduction in as being grown in intertidal areas often impacted by sew- FRNA bacteriophage was observed when water temperature age and being consumed raw or only lightly cooked. This was increased to 18 °C. The introduction of quantitative real- has resulted in an increasing number of commercial oys- time PCR for the detection and quantification of norovirus in ter producers in Ireland and elsewhere that harvest from A bivalve shellfish, particularly the method described in ISO classified production areas to include depuration as an extra 15216-1:2017 (Anonymous 2017), has been an important step in their HACCP (Hazard Analysis and Critical Control step in the area of risk management of norovirus. This tool Point) or risk management procedures (Rupnik et al. 2018). has allowed better understanding of the relative differences Minimum time and water temperature used in commercial in norovirus concentrations when applying risk management depuration are not stipulated in EU regulation. In Ireland, it procedures (Rupnik et al. 2018), as well as understanding the is recommended that depuration should be carried out for a concentrations of norovirus found in classified production minimum of 42 h with a water temperature of no less than areas across the EU (EFSA 2019). 8 °C (Anonymous 2018). This treatment has been shown As far as the authors are aware, this is the first study using to consistently reduce E. coli concentrations to below the the ISO 15216-1 standard method based on real-time RT- regulatory limit of 230 MPN/100 g, but not to reduce noro- PCR to measure norovirus RNA concentrations in oysters virus to the same extent (Schwab et al. 1998; McLeod et al. contaminated in their natural environment during controlled 2009). However, some studies have indicated that depura- laboratory depuration studies and enhanced commercial dep- tion time and seawater temperature are both factors that may uration procedures. Our aim was to provide evidence to sup- influence virus reduction during bivalve shellfish depuration port the hypothesis that appropriate depuration conditions (Lees et al. 2010). The potential for norovirus reduction in can be successfully used in the commercial setting as part of oysters during enhanced depuration procedures was identi- risk management measures to reduce the risk to consumer fied previously by this group (Rupnik et al. 2018) prompting by decreasing the norovirus concentrations in market-ready this study to investigate norovirus reduction in oysters under shellfish. controlled laboratory conditions. Artificial contamination of shellfish with viral pathogens is a common approach to studying depuration processes and Materials and Methods has been described previously (Muniain-Mujika et al. 2002; Choi and Jiang 2005; Nappier et al. 2008; McLeod et al. Oysters 2009; Polo et al. 2014b). As shellfish are known to efficiently bioaccumulate viruses in as little as several hours (Flannery Triploid Pacific oysters ( Crassostrea gigas) contaminated et  al. 2012; Souza et al. 2013, 2018; Pilotto et al. 2019), with norovirus in their growing areas were used in all labora- this approach allows for a rapid generation of animals con- tory depuration trials. Oysters from a commercial classified taminated with one or multiple viral strains. Concentrations production area were monitored for norovirus GI and GII 11 −1 as high as 10 genome copies per gram (gcg ) of murine contamination on a weekly basis and results of the moni- norovirus (MNV1) were achieved during 24h bioaccumula- toring were used to schedule the depuration experiments. tion in C. gigas by Pilotto et al. (2019), whereas both GI Once the norovirus concentrations increased to, or above, −1 and GII human norovirus genogroups, simultaneously bio-300 gcg a batch of oysters were harvested, washed and 6 −1 accumulated in C. gigas oysters to 10 gcg (Maalouf et al. transported to the laboratory within 27 h under temperature- 2011). Maximum reductions of the norovirus concentrations controlled conditions (< 15 °C). On receipt in the labora- in such artificially contaminated shellfish were previously tory, oysters were stored in dry, cool conditions and used in reported in the region of 1–1.2 log unit (equivalent to depuration trials within 24 h. 1 3 Food and Environmental Virology (2021) 13:229–240 231 cycles). Producer B performs depurations typically for Laboratory Depuration Trials 72  h at an accurately controlled stable  18  °C. Twenty- four and 22 depuration cycles carried out by Producer A Two 0.6m tanks (Depur Systems, UK) were filled with 400–450 L potable mains water and 15 kg of Seamix Artifi- and B, respectively, were examined. Pre-depuration oyster samples were collected on the day depuration commenced cial Sea Water salt mix (NaCl 66.1%; MgSO 16.3%; MgCl 4 2 12.7%; CaCl 3.3% and KCl 1.6%; Peacock Salt, UK) was and were followed by a second, paired sample taken after the completion of the depuration process. Each sample added to each and stirred until fully dissolved. The amount of salt added increased water salinity to between 28 and consisted of 12 live animals that were transported into the laboratory under chilled conditions (< 15 °C) within 27 h 32ppt mimicking that of the estuary. This artificial seawa- ter was circulated in the tank overnight to fully equilibrate (Producer A) or 5 h (Producer B). Upon arrival, oysters were stored at 4 °C for up to 24 h before processing. When before depuration experiments commenced. Water tem- perature was maintained within 1 °C of target temperature evaluating the commercial depuration processes, both nor- ovirus genogroups were quantified in the pre-depuration with a combination of 300  W aquarium heaters placed directly in the tank and an externally located 750 W water samples. Samples in which the virus concentration was −1 greater than 150 gcg for at least one genotype were chiller (D&D, DC750). Bar sprinklers located above one side wall of the tank were used for aeration and water flow selected and subsequently followed with a sample taken after completion of depuration. This allowed for concur- was maintained using an external pump at 2000–2200 L/h. One 36 W UV-C lamp fitted inline on each tank was used rent analysis of the effect of depuration on both norovirus genotypes in oysters. to sterilise the circulating water. Dissolved oxygen levels were measured once daily using a hand-held OxyGuard device. Oysters were placed loosely in shallow plastic trays, approximately 70–80 animals per tray. In total, four trays Norovirus Detection in Oysters per tank (between 280 and 320 oysters) were placed in each depuration tank and subjected to depuration. Triplicate sam- Preparation of Oyster Samples for Norovirus Analysis ples of 12 oysters were collected at commencement of the experiment (Day 0) and at 24-h intervals thereafter (Days Oysters were analysed for the presence of norovirus GI and GII in accordance with ISO 15216-1:2017 (Anony- 1–7). Oysters were stored at 4 °C for up to 24 h before fur- ther processing. A full depuration cycle was run for 7 days mous 2017). Briefly, oysters were cleaned under running potable water. Ten oysters per sample were shucked and (approx.168 h) after which the tanks were drained, cleaned, and filled with freshly prepared artificial sea water before the digestive tissues (DT) dissected out. The dissected DT was finely chopped using a sterile razor blade and mixed the next experiment. well. Two grams of DT were then spiked with 10 µl of the internal process control (IPC) virus (Mengo virus strain Shelf Life Test MC ) for evaluation of virus extraction efficiency similar to that described by (Costafreda et al. 2006) and treated Forty-five oysters remaining in the depuration tanks after −1 completion of the laboratory trials at 8 °C and 18 °C were with 2 ml Proteinase K (100 µg  ml ). Samples were incu- bated at 37 °C for 60 min with shaking at 150 rpm fol- placed on shallow trays and refrigerated (5 °C) for 4 weeks. Once a week, oysters were inspected and any gaping or unre- lowed by 15 min at 60 °C. Finally, after centrifugation at 3000×g for 5 min, supernatants were retained for RNA sponsive to percussion animals were counted and disposed of. extraction. Commercial Depuration Viral RNA Extraction Paired samples, pre- and post-depuration, were collected RNA was extracted from 500  µl of the DT supernatants from two separate oyster producers between January 2018 and December 2019 (Producer A) and February 2018 to using NucliSENS® magnetic extraction reagents (bioMé- rieux) and the NucliSENS® MiniMAG® extraction platform April 2019 (Producer B). Producer A operates in a class A area, whilst Producer B operates in an area with a seasonal and eluted into 100 µl of elution buffer. RNA extracts were stored at − 80 °C until the RT-qPCR analysis was conducted. A classification during December through to March and class B for the rest of the year. Depuration is routinely RNA was also extracted from 10 µl of the IPC sample for evaluation of extraction efficiency. A single negative extrac- used in both facilities with Producer A typically depurat- ing oysters for 7 days with an average water temperature tion control (water only) was processed alongside the oyster samples. of 14 °C (ranging from 12 to 16 °C between the depuration 1 3 232 Food and Environmental Virology (2021) 13:229–240 Determination of the Norovirus Concentration Using controls (blank sample carried through the RNA extraction One‑Step RT‑qPCR step) were included in each RT-PCR analysis to control for cross-contamination. Oysters were analysed for the norovirus concentrations using standardised quantitative real-time reverse transcription PCR Analysis of Results (RT-qPCR) (Anonymous 2017). RT-qPCR analysis was car- ried out using the Applied Biosystems AB7500 instrument LOD and LOQ (Applied Biosystems, Foster City, CA) and the RNA Ultra- sense one-step RT-qPCR system (Invitrogen). The reaction The limits of detection (LOD) and quantification (LOQ) was prepared by combining 5 µl of the extracted RNA sam- for Norovirus GI and GII were established based on Euro- ple and 20 µl of the reaction mix containing 500 nM forward pean Union Reference Laboratory (EURL) for monitor- primer, 900 nM reverse primer, 250 nM sequence specific ing the bacteriological and viral contamination of bivalve probe,1 × ROX reference dye and 1.25 µl of enzyme mix. molluscs guidance using dilution series of oyster digestive Previously described primers QNIF4 (da Silva et al. 2007), gland material contaminated with both, norovirus GI and NV1LCR (Svraka et al. 2007) and TM9 probe (Hoehne and GII (CEFAS 2016). The LOD and LOQ for norovirus GI and −1 Schreier 2006) were used for the detection of norovirus GI, GII was determined as 20 and 100 gcg , respectively. For and QNIF2 (Loisy et al. 2005), COG2R (Kageyama et al. statistical analysis, and in order to facilitate geometric mean 2003) and QNIFS probe (Loisy et al. 2005) were used for calculation, samples in which norovirus was not detected −1 the detection of norovirus GII. The Mengo110, Mengo209 were assigned a value of 10 gcg (half of the LOD) and −1 primers and Mengo147 probe were used in IPC assay (Pintó those that tested positive below 100 gcg (< LOQ) were −1 et al. 2009). The 96-well optical reaction plate was incu- assigned a value of 50 gcg (half of LOQ). bated at 55 °C for 60 min, 95 °C for 5 min, and then 45 cycles of PCR were performed, with 1 cycle consisting of Relative Concentrations 95 °C for 15 s, 60 °C for 1 min, and 65 °C for 1 min. All samples were analysed for norovirus GI and GII in dupli- Due to variances in starting norovirus concentration, and cate. All control materials used in the RT-qPCR assays were to allow for comparison between experiments and statis- prepared as described by Flannery et al. (2012). To enable tical analysis, all concentrations were converted to values quantification of norovirus RNA in copies per µl, a log relative to Day 0 (initial load), as follows: C = C /C , 10 Ri i 0 dilution series of the norovirus GI and GII DNA plasmids where C – Relative concentration of sample i; C –noro- Ri i 1 5 −1 (ranging from 1 × 10 to 1 × 10 copies/µl) were included virus concentration gcg obtained for sample i using RT- in duplicate on each RT-qPCR run. The number of RNA qPCR and C –geometric mean of initial load concentra- copies in norovirus-positive samples was determined by tion in a given experiment. For example, 2023, 1781 and −1 comparing the C value to the standard curves. The final 1459 norovirus GII gcg were detected in samples A, B concentration was then adjusted to reflect the volume of and C collected at Day 0 giving a geometric mean of 1739 −1 sample analysed and expressed as the number of detectable norovirus GII gcg . Therefore, the relative result for sam- −1 −1 virus genome copies per gram of DT. The presence of inhibi- ple A = 2023 gcg /1739 gcg = 1.163; sample B = 1781 −1 −1 −1 tors was checked by spiking an additional 5 µl of each sam- gcg /1739 gcg = 1.024 and sample C = 1459 gcg /1739 −1 ple RNA with 1 µl of either norovirus GI or norovirus GII gcg = 0.839. This principle was applied to all samples. external control RNA (ECRNA; 10 RNA transcripts/µl). The threshold cycle (C ) value obtained for samples spiked Assigning of Data for Laboratory Depuration Trials with the ECRNA was compared to the results obtained in the absence of the sample (5 µl of water used instead) and Norovirus test results were assigned into 3 separate groups used to estimate RT-PCR inhibition expressed as a percent- based on the water temperature used in a given depuration age. In accordance with ISO 15216-1:2017. Oyster samples trial: Low—depuration experiments carried out at 8 ± 1 °C with RT-PCR inhibition below the 75% were accepted for (n = 5); Medium—depuration experiments carried out at 12 inclusion in this study. Extraction efficiency was assessed or 14 °C (± 1 °C; n = 3) and High—depuration experiments by comparing the C value of the sample spiked with IPC carried out at 18 or 20 °C (± 1 °C; n = 7). virus to a standard curve obtained by preparing log dilu- tions of the RNA extracted from 10 µl Mengo virus and was Statistical Analysis subsequently expressed as percentage extraction efficiency. Samples with the extraction efficiency greater than 1% were R (version 3.6.0) using the libraries ‘car’ and ‘PMCMR’ was accepted for inclusion in this study (Anonymous 2017). used for statistical analysis. The normality of the data was No template controls (water only) and negative extraction tested by performing the Shapiro–Wilk test and the Levene 1 3 Food and Environmental Virology (2021) 13:229–240 233 test was used to evaluate the homogeneity of variances. To achieved by day 3 in the high temperature trials with rela- evaluate the differences between three temperatures condi- tive virus concentration reduced to 0.26 ± 0.11 (reduction tions (low, medium and high) during the depuration study a of 74% or 0.59 log units). A similar degree of reduction Kruskal–Wallis rank ANOVA with the pairwise test for mul- was observed in the medium temperature trials on day 4 tiple comparison of mean rank sums (Dunn’s test) as posthoc with relative concentration reduced to 0.27 ± 0.32 (reduc- tests was used. To test the difference of norovirus concentra- tion of 73%, 0.57 log units). By contrast, norovirus GII tions during commercial depuration the Mann–Whitney test concentrations were not reduced in oysters depurated under was applied. The significance level for all statistical analysis low temperature for the first 2 days of depuration. In fact, was set at p ≤ 0.05. an increase in norovirus GII concentration was observed in these oysters at day 1 (relative concentration 1.35 ± 0.33) and the first signs of virus concentration reduction were only Results observed at day 3. The maximum reduction of the initial viral load to 0.65 ± 0.29 (reduction of 44% or 0.25 log Laboratory Depuration Trials units) was observed under low temperature conditions by day 4. Non-parametric Kruskal–Wallis ANOVA showed that Stable conditions in the depuration tanks were maintained there were significant differences in the norovirus GII con- throughout each experiment with dissolved oxygen (DO ) centrations observed between the low and high temperature levels in excess of 80% in all trials. Norovirus GI concen- (p < 0.001) and between low and medium temperature condi- trations detected in C. gigas before depuration were either tions (p < 0.001), but not between medium and high water below or close to the limit of quantification (LOQ) of the temperatures (p = 0.14). −1 test (100 gcg ) and therefore were excluded from further Norovirus GII reductions during all depuration trials con- analysis in this study. Before depuration, norovirus GII con- ducted at medium and high water temperatures displayed centrations in the environmentally contaminated oysters a two-phase virus reduction kinetic. After the initial rapid −1 ranged between 178 and 16,426 norovirus GII gcg with a reduction of norovirus GII, the rate of depuration decreased −1 geometric mean of 906 norovirus GII gcg (Table 1). and no further significant reduction was observed between Norovirus GII reduction in oysters during laboratory days 3 and 7 or days 4 and 7 for high and medium tempera- experiments are shown in Fig. 1. First indications of noro- tures, respectively (Fig. 1). The two-phase virus reduction virus GII reduction in the medium and high temperature kinetic was not evident in the low temperature trials, with a conditions were visible during the initial 24  h (Fig.  1). delayed and shortened viral reduction stage. Virus reduction continued until day 3 at the high temper- No oyster mortalities were observed during the first ature conditions and until day 4 at the medium tempera- 2  weeks of shelf life test conducted upon completion of ture conditions. Maximum reduction of norovirus GII was the depuration experiments with all oysters responding to Table 1 Norovirus GII −1 Experiment Water Time point [days]/norovirus GII concentration [gcg ] concentrations measured in temperature Pacific oysters during laboratory 0 1 2 3 4 5 6 7 [°C] depuration trials 1 8 16,426 15,792 7803 7463 10,742 5443 11,210 7558 2 8 290 310 284 142 < LOQ 249 147 225 3 8 5573 14,505 7428 5981 2965 3334 3548 2293 4 8 1739 1970 2309 1601 2122 4798 2087 3020 5 8 850 1247 956 467 662 888 512 606 6 12 452 143 231 202 < LOQ 219 181 < LOQ 7 12 405 234 193 < LOQ < LOQ < LOQ < LOQ < LOQ 8 14 1739 2465 1451 1442 1599 1595 1813 1248 9 20 452 129 183 110 < LOQ < LOQ < LOQ < LOQ 10 18 405 236 147 < LOQ < LOQ < LOQ < LOQ < LOQ 11 18 178 102 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 12 20 178 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 13 20 290 168 < LOQ < LOQ < LOQ < LOQ 224 233 14 20 5573 4670 5896 2296 2244 1769 1513 2054 15 18 850 845 389 365 522 474 301 310 1 3 234 Food and Environmental Virology (2021) 13:229–240 Fig. 1 Log reduction (lines) 10 Low Medium High of norovirus GII at 3 water temperature regimes studied and norovirus GII concentra- 1.8 -0.2 tions (bars) detected in oysters during laboratory depuration 1.6 -0.1 trials. Results shown as relative 1.4 0 to norovirus GII concentration at day 0, error bars ± geometric 1.2 0.1 standard deviation 1 0.2 0.8 0.3 0.6 0.4 0.4 0.5 0.2 0.6 0 0.7 0123456 7 Depuraon duraon [days] percussion. Mortality rates were comparable for the two oys- The geometric mean concentrations in the pre-depuration −1 −1 ter populations tested past week two, despite the different samples were 251 gcg and 281 gcg and maximum con- −1 water temperature regimes used in depuration trials (paired centrations detected were 761 and 1658 gcg for norovirus t test p = 0.3724). GI and GII, respectively (Table 2). Following depuration, the geometric mean virus concentrations were < LOQ for both Evaluation of Commercial Depuration genogroups with the maximum concentrations of norovi- −1 rus GI and GII detected in any sample 468 and 300 gcg , Producer A Commercial Depuration Results respectively. The difference in norovirus concentrations in oysters before and after depuration were found to be sta- A total of 13 paired samples (pre and post depuration) con- tistically significant for both genogroups (Mann–Whitney taminated with norovirus GI, and 20 paired samples con- non-parametric t-test, p < 0.01 and p < 0.001, for norovi- taminated with norovirus GII, prior to depuration were rus GI and GII, respectively). A higher level of reduction identified from Producer A and included in this evaluation. was observed on average for norovirus GII compared to GI Table 2 Norovirus GI and Producer A B GII concentrations detected in oysters pre- and post-depuration Depuration conditions 12–16 °C, 7 days 18 °C, 3 days collected from Producer A and Norovirus genogroup GI GII GI GII GII n = 13 20 20 13 12 −1 Pre-depuration [gcg ]  Average 285 344 242 844 806  Geometric mean 251 281 229 526 488  Min–Max 150–761 155–1658 151–409 167–2802 167–2802 −1 Post-depuration [gcg ]  Average 121 65 129 317 158  Geometric mean 82 45 91 66 49  Min–Max < LOD-468 < LOD-300 < LOD-366 < LOD-2232 < LOD-780 Geometric mean reduction 67.58% 83.95% 60.41% 87.47% 89.92% Log reduction 0.49 0.79 0.40 0.90 1.00 −1 −1 < LOD—results below the limit of detection (< 20 gcg norovirus GI, < 20 gcg norovirus GII) Analysis results on an adjusted sample population, with one outlier pair, failing to reduce norovirus GII concentration in oysters during depuration, excluded from norovirus GII results 1 3 Relave norovirus GII concentraon Log reducon Food and Environmental Virology (2021) 13:229–240 235 −1 (Table 2) where norovirus GII was reduced by 83.95% (0.79 contaminated with < 1000 norovirus GII gcg were reduced log units) compared to 67.58% (0.49 log units) reduction to < LOQ (Table 3). Samples contaminated with norovirus 10 10 −1 of norovirus GI. GII at concentrations > 1000 gcg prior to depuration were reduced to concentrations < LOQ in 40% of the cases. None Producer B Commercial Depuration Results of the samples examined in the commercial setting had con- −1 centrations of norovirus GI greater than 1000 gcg . A total of 20 paired samples (pre- and post- depuration) contaminated with norovirus GI and 13 paired samples contaminated with norovirus GII were identified from Discussion Producer B and included in this evaluation. The geomet- ric mean concentration in the pre-depuration samples were Norovirus contamination in oyster production areas is an −1 −1 229 gcg and 526 gcg , for norovirus GI and GII, respec- ongoing issue for food safety regulators and oyster produc- tively (Table 2) with the maximum concentrations of 409 ers alike. The primary objective for both is the production −1 −1 gcg norovirus GI and 2802 gcg norovirus GII. Following of food that is safe for human consumption. Data published depuration, the geometric mean virus concentrations were in a recent EFSA baseline survey highlights the high preva- significantly reduced to < LOQ for both norovirus GI and lence of norovirus contamination in oysters from classified −1 GII with maximum concentrations detected at 366 gcg production areas during the winter months (EFSA 2019). −1 norovirus GI and 2232 gcg norovirus GII. The differ - Peak norovirus concentration and prevalence was observed ence in norovirus concentrations in oysters before and after in the months of January and February when almost 65% depuration were found to be statistically significant for both of samples tested were positive for norovirus with a mean −1 genogroups (Mann–Whitney non-parametric t-test, p < 0.001 concentration of 661 gcg (EFSA 2019). This important and p < 0.05 for norovirus GI and GII, respectively). In one data demonstrates the need for effective mitigation meas- of the 13 depuration runs analysed post depuration the con- ures to reduce norovirus concentrations before reaching the −1 centration for norovirus GII (2232 gcg ) did not decrease consumer. Whilst there are currently no legislative criteria from the pre-depuration concentration of 1304 norovirus GII for norovirus in oysters intended for consumption, there is −1 gcg . On removal of this outlier pair the overall level of recognition from international food safety regulators for reduction demonstrated for norovirus GII was almost 90% or the need to introduce such a criterion to protect consum- 1 log units (p < 0.01). As observed for Producer A, a higher ers. Therefore, oyster producers need effective virus reduc- level of reduction was achieved for oysters contaminated tion measures in order to meet any such microbiological with norovirus GII (87.47–89.92% or 0.90–1.00 log reduc- criterion for norovirus in the future. Previously, we dem- tion) compared to norovirus GI (60.41% or 0.40 log reduc- onstrated the potential of risk management procedures to tion), despite the different depuration conditions employed. reduce the risk of norovirus contamination in a production Overall in the commercial settings, there was a signifi- area as well as the reduction of norovirus concentrations cant difference in the level of reduction between norovirus in end-product by depuration (Rupnik et al. 2018). In that GI and GII (p < 0.001) where norovirus GI was reduced on particular study extended depuration periods of up to 9 days average by 63.4% (0.43 log units) compared to an on aver- were applied during the high-risk winter season. In addi- age reduction of 85.4% (0.84 log units) for norovirus GII tion, during that study minimum depuration temperatures (Table 3). The extent of reduction to < LOQ also differed were generally above the recommended minimum tempera- between genogroups when the pre-depuration concentra- ture of 8 °C (Anonymous 2018) with the mean temperature −1 tions were < 1000 gcg (p < 0.05). In this instance 51.5% of over all depuration cycles of 13.3 °C during the winter. This −1 samples contaminated with < 1000 norovirus GI gcg were prompted further investigation of the ability of enhanced reduced to < LOQ post-depuration, whilst 78.6% of samples depuration conditions to reduce norovirus concentrations Table 3 Overall efficacy of commercial norovirus GI and GII depuration in Pacific oysters based on all paired results from Producer A and B, combined Norovirus genogroup GI GII Geometric mean reduction 63.40% 85.40% Log reduction 0.43 0.84 Pre-depuration concentration % Samples depurated to < LOQ −1 < 1000 gcg 51.5 78.6 −1 > 1000 gcg n/a 40.0 1 3 236 Food and Environmental Virology (2021) 13:229–240 −1 in environmentally contaminated oysters destined for con- higher virus concentrations (> 1000 gcg ). This study has sumption. To our knowledge this is the first report of the demonstrated the impact of the initial viral load prior to application of a standardised test method (Anonymous 2017) commencing depuration trials on the variability in concen- to evaluate norovirus reduction during both, laboratory and trations observed especially in the early stages (day 1–3). commercial depuration processes to environmentally con- Nevertheless, the concentrations detected after day 3 in the taminated oysters. medium and high temperature trials in many of the samples The results of the laboratory depuration trials indicate were at or below the LOQ for norovirus GII. This empha- that norovirus, whilst not completely removed, was signifi- sises the importance of having knowledge of the starting cantly reduced when the water temperature used for depu- concentration of norovirus in oysters prior to depuration and ration was increased to > 11 °C, but not at lower tempera- should be a key consideration for operators who wish to tures. Other studies have previously reported similar findings reduce norovirus concentrations to levels that may be below where norovirus was reduced, but not eliminated from con- any future microbiological criterion. taminated shellfish, including clams, mussels and oysters A two-phase reduction of norovirus in oysters was (Ueki et al. 2007; Neish 2013; Polo et al. 2014a; Pilotto et al. observed during the laboratory depuration trials, similar to 2019) using a variety of time and temperature combinations. that reported in clams and mussels (Polo et al. 2014c). In this The initial temporary increase in norovirus concentrations study once the maximum reduction was achieved after day 3 observed here at Day 1, and by others at Day 1–2 (McLeod and day 4 no further significant decline in virus concentra- et al. 2009; Polo et al. 2014c), could be explained by tran- tions took place. A slight fluctuation of norovirus concentra- sient capture and subsequent release of norovirus particles tions were detected during the second reduction phase (i.e. from animal tissue outside of DT, such as gills and mantle post day 3/4) in some experiments particularly on day 5 and (Maalouf et al. 2010). In addition, shellfish are known to 6. It is not clear what the exact cause is for these increases slow down their metabolism and respiration rate at lower in virus concentration however it could be a reflection of the water temperatures (Dittman 1997; Li et al. 2017) and in limitations of the RT-PCR method itself, especially when such conditions relocation of virus particles from other tis- working with target concentrations close to, or below, the sues to DT and out as faeces or pseudofeces is less efficient LOQ, a natural variability between samples or a potential and requires longer time. re-contamination event due to sampling procedures. The The use of environmentally contaminated oysters in the optimal combination of time and temperature providing depuration trials presented here, allowing for best represen- the most rapid reduction in norovirus was three days at the tation of virus concentrations found in commercially har- high temperature range with 74% (0.59 log units) reduc- vested oysters, relied on long-term presence of the virus in tion observed. However, a similar final reduction occurred environment in order to complete all planned experiments. in the medium water temperature range albeit one day later Premature drop of norovirus GII concentration to levels (73%, 0.57 lo g units). More limited norovirus reduction −1 below the critical lower limit (of 300 gcg ) experienced was achieved during depuration at the low temperature with during this study resulted in lower number of depuration maximum reduction of 44% (0.25 log units) observed trials completed at medium water temperatures, compared after day 4. The rate of reduction observed in the increased to low or high water temperatures. While such an ade- temperature trials (medium and high) were significantly dif- quately balanced data set could have provided improved, ferent from that observed from the low temperature trials. more robust statistics, the data set obtained during this study This indicates that by setting depuration water temperature provides statistically significant differences between the dif- to > 11 °C and by increasing the time allowed for depura- ferent temperature regimes studied. Additional depuration tion to 3–4 days, but not beyond, provides optimal reduction studies, focusing on the medium water temperatures of 11 of norovirus from contaminated oysters whilst maintaining to 15 °C and using environmentally contaminated oysters, product shelf life. would be welcomed in the future to supplement the data To investigate norovirus reduction during depuration in presented here. the commercial setting we evaluated commercial practices Initial viral load has been thought to have an impact undertaken by two separate oyster producers. Producer A on the depuration outcome whereby the higher the virus employs longer depuration times (7 days) but lower water concentration prior to depuration, the higher the remain- temperature (approximately 14 °C), while Producer B mini- ing virus load, if all other parameters were kept consistent mises the depuration time (3 days) but maximises the tem- (McLeod et al. 2017). The same finding was also observed perature (18 °C). In both scenarios, similar levels of viral in the study described here where oysters contaminated reduction for norovirus GI and GII were achieved despite the −1 with < 1000 norovirus GII gc g , were reduced to < 100 difference in time and temperature combinations (Table  3). −1 gcg in 1–4 days. However, the same post-depuration con- As with the laboratory trials the concentration of norovi- centrations were not achieved in oysters contaminated with rus in the pre-depuration samples was an important feature 1 3 Food and Environmental Virology (2021) 13:229–240 237 of the overall success of commercial norovirus reduction A greater reduction of norovirus GII was demonstrated as judged by a target concentration of < LOQ post-depura- in the commercial setting than in the laboratory trials with tion. Almost four in five of all commercial oyster samples 84–87% (0.80–0.94 log ) reduction achieved by both com- −1 tested prior to depuration with a concentration < 1000 gcg mercial depuration processes compared with 74% (0.59 norovirus GII contained concentrations < LOQ post-depu- log ) in laboratory trials. The improved reduction observed ration (Table 3). On the other hand, only 40% of samples in the commercial setting could be caused by a number of −1 with > 1000 gcg prior to depuration, were found to have factors particularly those that affect the condition of the oys- concentrations < LOQ for norovirus GII post-depuration. ters themselves. Depuration is a complex process despite Despite a small number of commercial samples (n = 5) con- the simplicity of the infrastructure required. Individual −1 taining in excess of the 1000 gcg norovirus GII included in animals can respond differently to stress factors, such as this evaluation, combined with findings from the laboratory sudden changes in temperature, salinity, lack of food and designed trials, these results suggest that whilst enhanced physical disturbance (shaking, transport or rough handling) depuration conditions can reduce norovirus concentrations (Lacoste et al. 2001; Marigómez et al. 2017; Peteiro et al. in oysters, this only becomes an effective consumer health 2018; Seuront et al. 2019). Distressed animals may have intervention strategy when the contamination in the oys- trouble resuming normal filtration and therefore may fail to ter harvesting area is also managed. Applying enhanced purge impurities. Indeed, in all the laboratory trials visual depuration conditions to oysters that are contaminated to inspection confirmed that oysters resumed filtration within −1 levels > 1000 gcg , is unlikely to reduce norovirus concen- an hour from submerging in water, suggesting minimal trations to an acceptable level and will have limited public disruption. However, it is possible that a few animals were health benefit. under some degree of distress during harvest, transport, All but one laboratory depuration trial conducted at upon submerging into heated water or while collecting the either medium or high water temperatures where the initial daily samples. In contrast to the laboratory setting, where a −1 viral load in oysters was below 1000 gcg yielded con- portion of oysters were removed from each depuration tank centrations < LOQ after 4 days of depuration. In addition, daily potentially unsettling the oyster population, the com- one commercial depuration run (Producer B) also failed to mercial depuration tanks were left undisturbed for the full reduce norovirus GII where the pre- and post-depuration duration of depuration (either 3 or 7 days). The tanks were concentration were similar. It is not clear what caused the drained once the process was completed and only then the poor depuration, or lack thereof, in these two instances but oysters were removed, and the final post-depuration sample it does indicate that not all depuration runs will yield suf- taken for analysis. This suggests the need for minimal han- ficient or similar reductions, despite consistent time and dling and disturbance of the oysters during the depuration temperature conditions. This underlines the importance of process to ensure greatest reduction. monitoring the operation of commercial depuration systems Many studies investigating norovirus removal or reduc- to ensure reliable reduction of norovirus. tion from bivalves have involved contaminating various In both commercial depuration scenarios studied here the shellfish species through bioaccumulation. Such laboratory overall reduction rates were higher for norovirus GII than contamination can result in very high starting concentra- GI. Different behaviour between the viral strains during bio- tions which may not be reflective of concentrations naturally accumulation and subsequent depuration have been reported found in bivalves, especially oysters. Here, we used environ- previously (Nappier et al. 2008; Maalouf et al. 2011; Polo mentally contaminated oysters from approved production et  al. 2014c) and could explain the differences observed areas to study norovirus reduction. Therefore, this repre- here. Norovirus GI has been shown to bind to histo-blood sented concentrations of norovirus found in oysters prior to like ligands in oyster digestive tissue, exhibiting prolonged post-harvest treatment and destined for human consumption. persistence in oysters over GII norovirus indicating that its The concentration prior to depuration in both the labora- removal from oyster tissue may prove a greater challenge (Le tory and commercial settings was significantly higher than Guyader et al. 2012). Laboratory trials as conducted for nor- the concentrations measured after depuration once the water ovirus GII in this study are required for GI to fully elucidate temperature was > 11 °C. Of course, the question remains the depuration kinetics for this genogroup. Unfortunately for surrounding the public health significance of these remain- the laboratory trials in this study, the production area from ing norovirus concentrations due to the lack of a routine which the oysters were obtained does not have a history of norovirus infectivity assay despite promising developments regular norovirus GI contamination at concentrations suit- with the human enteroid system (Ettayebi et al. 2016; Cos- able for such studies. Nonetheless, the results obtained from tantini 2018). The illness outcome for any given norovirus the commercial settings indicate the potential of enhanced concentration in contaminated oysters will depend on the depuration conditions to reduce norovirus GI concentrations norovirus genotype, which could be single or multiple, the to below the LOQ. immune and genetic susceptibility of a host (Noda et al. 1 3 238 Food and Environmental Virology (2021) 13:229–240 2008), as well as the size and number of oysters ingested method for detection of norovirus in shellfish may provide by the consumer. However, a link between increasing num- an important risk management tool for the oyster industry ber of virus genome copies detected and risk of infection in light of potential forthcoming legislation. has been reported previously (Lowther et al. 2012). A low likelihood of outbreaks was shown to be associated with Funding This study was funded by Department of Agriculture, Food oysters containing norovirus in concentrations below 100 and the Marine (Grant No. 14/SF/852). −1 gcg , levels readily achievable in this study in the depura- tion trials using increased water temperature and times. Hunt Open Access This article is licensed under a Creative Commons Attri- et al. (2020) demonstrated that norovirus GII was found to bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long be log-normally distributed across individual animals within as you give appropriate credit to the original author(s) and the source, a population and that for mean concentrations below 100 provide a link to the Creative Commons licence, and indicate if changes −1 gcg it was likely that some of the oysters in the tested pop- were made. The images or other third party material in this article are ulation contain no or very little amount of virus (< LOD). In included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in contrast, all oysters in the tested population were found to be the article’s Creative Commons licence and your intended use is not contaminated when the mean concentration tested above 300 permitted by statutory regulation or exceeds the permitted use, you will −1 gcg (Hunt 2019). Additionally, two recent studies inves- need to obtain permission directly from the copyright holder. To view a tigated the FRNA bacteriophage type II (norovirus surro- copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. gate) removal from oysters during depuration using genomic and infectivity methods (Leduc et al. 2020; Younger et al. 2020). These studies indicated that viruses may either be References inactivated in oysters during depuration (Leduc et al. 2020) Alfano-Sobsey, E., Sweat, D., Hall, A., Breedlove, F., Rodriguez, R., or, as suggested by Younger et al. (2020), destroyed and Greene, S., et  al. (2011). Norovirus outbreak associated with removed. Despite the differences in the concluded viral undercooked oysters and secondary household transmission. reduction mechanisms these findings could indicate that the Epidemiology and Infection, 28, 1–7. virus genome copies detected using RT-PCR methods are, Anonymous. (2004). Regulation (EC) 854/2004 of the European Par- liament and of the Council of 29 April 2004 laying down specific at least in part, only footprints left by the infectious virus rules for the organisation of official controls on products of animal particles and they no longer have the capability of causing origin intended for human consumption. infection especially at low concentrations (i.e. ≤ LOQ). Anonymous. (2017). ISO 15216-1:2017 Microbiology of food and A recently published EFSA baseline survey provides com- animal feed—horizontal method for determination of hepatitis A virus and norovirus in food using realtime RT-PCR—Part 1: prehensive scientific data for norovirus prevalence in Euro- method for quantification. International Organization for Stand- pean oyster harvesting areas and will form the basis for dis- ardization, Geneva, pp 15216–1 cussion amongst EU authorities attempting to place a safety Anonymous. (2018). SFPA Guidance Document for Inspecting LBM limit on norovirus concentration in oysters. Should a regula- Purification Centres. Ref: Regulation (EC) 853/2004 Annex III Section VII tory microbiological criterion for norovirus arise, reduction Anonymous. (2019). Commission implementing regulation (EU) 2019/ strategies such as enhanced depuration as described in this 627–of 15 March 2019—laying down uniform practical arrange- study may become an important intervention measure for ments for the performance of official controls on products of ani- food business operators to successfully comply with such mal origin intended for human consumption in accordance with Regulation (EU) 2017/ 625 of the European Parliament and of the a criterion. The application of enhanced depuration condi- Council and amending Commission Regulation (EC) No 2074 / tions, taking into consideration time and temperature, to 2005 as regards official controls environmentally contaminated oysters provides a practical Baker, K., Morris, J., McCarthy, N., Saldana, L., Lowther, J., Collin- tool for oyster producers enabling the reduction of norovirus son, A., & Young, M. (2010). An outbreak of norovirus infection linked to oyster consumption at a UK restaurant. 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