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Food Control 43 (2014) 206e212

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Food Control

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Viral elimination during commercial depuration of shellfish

David Polo a, Cristina Álvarez b, Jorge Díez c, Susana Darriba b, Ángeles Longa c,Jesús L. Romalde a,*

aDepartamento de Microbiología y Parasitología, CIBUS-Facultad de Biología, Universidad de Santiago de Compostela, 15782, Santiago de Compostela,Spainb Instituto Tecnolóxico para o Control do Medio Mariño de Galicia (INTECMAR), 36611 Vilagarcía de Arousa, SpaincConsello Regulador da Denominación de Orixe Protexida do Mexillón de Galicia, 36600 Vilagarcía de Arousa, Spain

a r t i c l e i n f o

Article history:Received 7 November 2013Received in revised form6 March 2014Accepted 18 March 2014Available online 27 March 2014

Keywords:DepurationShellfishFþRNA bacteriophagesEnteric virusesHepatitis A virusNorovirus

* Corresponding author. Tel.: þ34 881816908; fax:E-mail address: jesus.romalde@usc.es (J.L. Romald

http://dx.doi.org/10.1016/j.foodcont.2014.03.0220956-7135/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The effectiveness of depuration for the removal of hepatitis A virus (HAV), norovirus (NoV) genogroupsI (GI) and II (GII), and FþRNA bacteriophage (FþRNA) was evaluated for pullet carpet shell clams(Venerupis pullastra) and Mediterranean mussels (Mytilus galloprovincialis). The objective was to comparethe behaviour of the different pathogens under commercial depuration conditions during 7 days in anauthorized plant. Standard double agar overlay method (ISO 10705-1) was employed for FþRNA quan-tification. Recently developed ISO/TS 15216:2013 standard method, based on RT-real time PCR, wereemployed for the quantification of HAV and NoV. The reduction of FþRNA showed a two-phase depu-ration kinetic. The average reduction rates were 1-log units for clams and 2-log units for mussels, withresidual levels after the process of 6.3 � 103 and 8.3 � 101 FþRNA/100 g, respectively. HAV, NoV GI andGII were detected intermittently throughout the entire process, ranging mostly from 103 to 105 RNAcopies/g digestive tissue (DT). NoV GI showed the higher viral levels followed by NoV GII and HAV. All ofthem were detected in clams after seven days of depuration, however, in mussels only NoV GI wasdetected after the process. Generally, clams showed slower depuration rates and higher contaminationlevels for all viruses analysed.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Shellfish are one of the most commonly transmission vector ofdifferent human enteric pathogens. Among them, the enteric vi-ruses are the most frequently involved in foodborne outbreaks, andNorovirus (NoV) and hepatitis A virus (HAV) the leading etiologicalagents of food-borne illness (including shellfish), attending to thenumber of cases and to the illness severity, respectively (Koopmans& Duizer, 2004; Lees, 2000).

NoV, member of the family Caliciviridae, is the most importanthuman pathogen of diarrhoea worldwide, being the main cause offood-borne outbreaks of acute gastroenteritis and sporadic infec-tious gastroenteritis (Atmar & Estes, 2006). HAV, member of thefamily Picornaviridae, displays a high degree of genetic conserva-tion throughout the genome and is less common in countries with ahigh standard of hygiene. However, it may cause infection later in

þ34 881816966.e).

life, with the risk of a more severe disease outcome (Hollinger &Emerson, 2001).

The inability of sewage treatments to completely remove orinactivate viruses is well known (Da Silva et al., 2007; Iwai et al.2009). According to their prevalence in the community, entericviruses are discharged from sewage outfalls into fresh, marine andestuarine waters, and may subsequently be concentrated by shell-fish, due to their filter-feeding activity. Globalization of food pro-duction and shellfish imports favours the transmission anddissemination of viruses around the world (Polo, Vilariño, Manso, &Romalde, 2010). Furthermore, the traditional way of consumingshellfish, raw or slightly cooked, and whole, including digestivetissues (where viruses are mainly bio-accumulated), increases theinfection risk and makes the bivalves a high-risk food group(Romalde et al., 1994).

Shellfish depuration is a commercial compulsory processrequired in many countries for the fresh shellfish commercializa-tion. The natural pumping activity of the bivalves is used in theprocess to purge the contaminants and reduce the likelihood ofhuman pathogen transmission to consumers. Nevertheless, for

D. Polo et al. / Food Control 43 (2014) 206e212 207

decades the study of food-borne diseases linked to contaminatedmolluscs has mainly focused on bacterial pathogens. Consequently,controlling these pathogens has been the main objective of theshellfish sanitary controls and depuration systems and practices.The bacterial indicators are indeed the unique parameter used incurrent standards and depuration controls (Anon, 2004).

The compliance with the end product bacterial standards (�230Escherichia coli/100 g shellfish flesh in EU) is frequently seen as ev-idence of satisfactory design and operation of purification plants.However, virus removal is known to be less effective than bacterialremoval and the compliance with standards cannot guarantee theviral absence (Loisy, Atmar, LeSaux, et al., 2005; Richards, McLeod, &LeGuyader, 2010; Romalde et al., 2002; Schwab, Neill, Estes,Metcalf,& Atmar, 1998; Ueki et al., 2007), a fact evidenced by the periodicoutbreaks of Hepatitis A and gastroenteritis following the con-sumption of depurated shellfish (Chalmers &McMillan,1995; Helleret al., 1986; Le Guyader et al., 2003, 2006).

From a virological point of view, shellfish safety continues to bea sanitary challenge and the severe impact of the viral entericdiseases on human populations has brought awareness by Euro-pean authorities. Recently, it has been developed a standardmethod based on real time RT-PCR (RT-qPCR) for NoV and HAVquantification in foodstuffs, to be incorporated into EU legislationas a reference method (ISO/TS 15216).

This study focuses on the evaluation of the effectiveness anddepuration kinetic of HAV, NoV (genogroups I and II) and FþRNAbacteriophage (a potential indicator for enteric viruses) from clamand mussels. The objective was to compare the behaviour of theseviruses under commercial depuration in an authorized plant,extending the 48 h purification period applied commercially for alonger period of 7 days.

2. Material and methods

2.1. Samples

A total of 13 and 9 depuration trials were carried out with pulletcarpet shell clams (Venerupis pullastra) and Mediterranean mussels(Mytilus galloprovincialis) respectively. Molluscs were naturallycontaminated during 15 days in an intertidal zone near of a sewagedischarge zone. After this period, the initial level of contamination

Fig. 1. Commercial depuration system. IP: Intake point; SS: Seawater system supply; PT: PrimPU: Production unit of Cl2; AC: Aerator column; AT: Aerator tank; MF: Micro filter; V: VeDischsrge point; *: Intake and discharge points are separated in time and space to avoid cr

(t0) for FþRNA, HAV and NoV was determined and then molluscswere collected and transferred to the depuration plant to undergopurification during 7 days. Each trial was carried out with 200 kg ofmollusc. Bivalves were randomly sampled before (t0) and duringthe purification process (t1et7) every 24 h. All samples were keptat 4 �C during shipment, washed and scrubbed thoroughly inwater,shucked aseptically and analysed within 24 h.

2.2. Depuration system

Depuration process was carried out in an authorized commer-cial depuration plant in a separate area to avoid interferences withthe normal operations in the plant. The depuration system con-sisted on a vertical system with two or three bins per column(100 m3 each), with open water flow circuit and water treatmentby chlorination (Fig. 1). Initial concentration of chlorine in thedisinfection tank (Fig. 1: DT) was around 2 ppm. Due to the toxicnature of chlorine, the water is dechlorinated through a cascadesystem to provide vigorous aeration before contact with theshellfish (Fig. 1: AC and AT). This avoids the presence of chlorine inthe depuration bins. Physico-chemical parameters of the water(conductivity, salinity, dissolved oxygen, pH, Temperature, andchlorine) were exhaustively controlled over the complete depu-ration period.

2.3. FþRNA bacteriophages detection and quantification

For the FþRNA bacteriophages analysis, shellfish flesh washomogenised in a blender for 2 min with peptone water (0.1%pH ¼ 7.4; 1:2 w:v). Diluted homogenates were then centrifuged at1000� g for 5 min at room temperature. The supernatant wasdecanted and a 1:10 and 1:100 dilutions with peptone water wereprepared. Ten millilitres of the undiluted supernatant and ten-folddilutions were then assayed by using 1-ml portions in 90 mm-petridishes.

Quantification was performed according to the standard doubleagar overlay method ISO 10705-1 with Salmonella typhimuriumWG49 as bacterial host. Briefly, replicate 1-ml portions of undi-luted, 1:10 and 1:100 shellfish homogenates and 1-ml portions ofa WG49 host culture were added to 2.5-ml portions of molten 1%tryptoneeyeast extract agar at 45 �C. The melted agar and sample

ary tank for filtration and settlement; DT Cl2: Disinfection tank with chlorine (Cl2); Cl2nturi Device; � : Water flow regulator; DB: Depuration bins; DS: Drain system; DP:oss contamination.

D. Polo et al. / Food Control 43 (2014) 206e212208

were mixed by inversion and poured onto previously prepared 2%tryptoneeyeast extracteglucose agar base in a 90-mm petri dish.The overlays were inverted and incubated at 37 �1 �C overnight,resulting in the formation of a confluent lawn of host cells. Themethod includes procedures for production and quality control of S.typhimurium WG49 host cells and FRNA bacteriophage (MS2) pos-itive control material. The results were expressed in plaque formingunits of FþRNA bacteriophages/100 g of shellfish flesh (FþRNA/100g) and the detection limit was 30 FþRNA/100 g.

2.4. HAV and NoV detection and quantification

Viral recovery from shellfish was carried out according to theISO/TS 15216-1:2013 with minor modifications. Briefly, clams andmussels (20 and 10 individuals respectively) were shucked and thedigestive tissues (DT) were removed by dissection and pooled to geta final weight between 2 and 3 g. Known amounts of mengovirusclone (vMC0) (10 ml of mengovirus stock) were spiked to eachsample pool as an independent nucleic acid extraction efficiencycontrol (Costafreda, Bosch, & Pintó, 2006) and then homogenizedwith one volume (1:1 w/v) in peptone water (0.1%; pH 7.4). Ho-mogenates were strongly shaken for 1 h and centrifuged at 1000� gfor 5 min, recovering the supernatant. Viral RNA was extracted induplicate from each homogenate using Nucleospin RNA Virus Kit(MachereyeNagel; Dürem, Germany), using a sample volume of150 ml according to themanufacturer’s protocol. The RNAwas eluted

Table 1Viral detection and quantification for trials carried out with clams (Venerupis pullastra). Remollusk flesh for FþRNA bacteriophages.

Virus Trial t0 t1 t2 t3

NoV GI 1 þ þ 4.12 þ345 þ67 5.69 � 103

89 7.17 � 104 3.1

10111213

NoV GII 123 3.34567 9.58 � 103

89 8.36 � 104 5.1

1011 8.94 � 103 2.54 � 103 3.03 � 104 2.712 1.74 � 105

13 4.01 � 104 3.37 � 104

FþRNA 1 7.6 � 104 1.6 � 104 1.3 � 104 12 8.6 � 104 5.1 � 104 5.0 � 104 13 1.4 � 105 2.1 � 105 1.5 � 105 84 5.1 � 104 5.9 � 104 3.7 � 104 15 5.2 � 104 1.5 � 105 3.1 � 104 16 2.4 � 105 1.8 � 105 1.3 � 105 37 7.3 � 104 7.9 � 104 6.2 � 104 38 1.9 � 104 1.5 � 104 6.4 � 103

9 5.6 � 103 4.7 � 104 3.0 � 104 110 2.3 � 104 4.6 � 104 2.6 � 104 211 7.6 � 103 1.3 � 104 7.0 � 103 212 4.0 � 103 1.1 � 104 9.1 � 103 113 1.3 � 104 9.2 � 103 7.8 � 103 4

þ, Positive sample but outside of the quantification range.

in RNAsa-free sterile water, quantified in the same day and storedat �80 �C.

The RT-qPCR for HAV and NoV (GI and GII) were performed onan Mx3005p QPCR System (Stratagene, USA) thermocycler. Plat-inum Quantitative RT-PCR Thermoscript One-step System kit(Invitrogen, Saint Aubin, France) was used for a 25 ml total volume,using 5 ml of extracted RNA. Quantification was also carried outfollowing the principles outlined in the CEN/ISO technical specifi-cation (ISO/TS 15216-2:2013).

To test for the extraction efficiency, values were determined bycomparing the Cycle threshold (Ct) values for the Mengovirus-positive amplification control with the Ct value for the tested vi-rus, and was classified as valid (>5%) or invalid (<5%). To test thepresence of RT-PCR inhibitors and calculate the RT-qPCR efficiency,external controls were included for each reaction. The Ct value of asample extracted RNA (2.5 ml) mixed with external control (2.5 ml)was compared to the Ct value of the external control mixed onlywith RNA-free sterile water, and then the efficiency was classifiedas valid (>25%) or invalid (<25%). Following the CEN/ISO method,samples with a <5% extraction efficiency or a <25% RT-qPCR effi-ciency were re-extracted and tested again.

Primer sets and probes used were: HAV240(�) (50-GGA-GAGCCCTGGAAGAAAG-30), HAV68(þ) (50-TCACCGCCGTTTGCCTAG-30) and HAV150 (6-FAM-CCTGAACCTGCAGGAATTAA-MGB) for HAV(Costafreda et al., 2006). NV1LCR (�) (CCT-TAG-ACG-CCA-TCA-TCA-TTY-AC), QNIF4 (þ) (CGY-TGG-ATG-CGN-TTY-CAT-GA) and NV1Lpr

sults are expressed as no. of RNA copies/g DT for NoV GI and GII, and as FþRNA/100 g

t4 t5 t6 t7

1 � 106

4.68 � 106

5 � 104 7.48 � 103

1.63 � 106

2.91 � 104

þ6 � 103 1.19 � 105

3.60 � 103

2 � 104 9.58 � 103

2.54 � 103

3 � 104 2.93 � 104

1.46 � 104

.7 � 104 1.5 � 104

.8 � 104 2.4 � 104 1.6 � 104 1.0 � 104

.2 � 104 4.3 � 104 2.2 � 104 2.5 � 104

.6 � 104 1.4 � 104 6.6 � 103 1.6 � 103

.1 � 104 1.0 � 104 3.3 � 104 1.7 � 104

.7 � 104 3.2 � 104 2.1 � 104 1.1 � 104

.1 � 104 1.7 � 104 1.0 � 104 5.8 � 103

5.2 � 103 1.0 � 103 8.0 � 102

.1 � 104 6.4 � 103 3.1 � 103 8.4 � 102

.3 � 104 4.1 � 103 2.4 � 103 1.6 � 103

.0 � 103 3.5 � 103 4.8 � 102 6.0 � 101

.2 � 104 1.1 � 103 2.4 � 102 1.2 � 102

.0 � 103 7.2 � 102 1.4 � 103 7.2 � 102

D. Polo et al. / Food Control 43 (2014) 206e212 209

(6-FAM-TGG-ACA-GGA-GAY-CGC-RAT-CT-BHQ1) for NoV GI (DaSilva et al., 2007; Svraka et al., 2007). COG2R (�) (TCG-ACG-CCA-TCT-TCA-TTC-ACA), QNIF2d (þ) (ATG-TTC-AGR-TGG-ATG-AGR-TTC-TCW-GA) and QNIFS (6-FAM-AGC-ACG-TGG-GAG-GGC-GAT-CG-BHQ1) for NoV GII (Kageyama et al., 2003; Loisy, Atmar, Guillon,et al., 2005).

Negative controls containing no nucleic acid as well as positivecontrols were introduced in each run. A sample displaying a Ct� 39,with no evidence of amplification in the negative controls, wasconsidered as positive and quantified. A sample displaying a39 � Ct � 41, was considered as positive but outside of the quan-tification range. Quantification was estimated by standard curvesconstructed with serial dilutions of RNA in the case of HAV and RNAtranscripts for NoV GI and GII, plotting the number of genomecopies against the Ct. Results were expressed as number of RNAviral genome copies per gram of digestive tissue (RNAc/g DT). Thelimit of detection was 1 �102 RNAc/g DT.

2.5. Statistical analysis

ANOVA analysis was performed to compare the number RNAc/gDT obtained among the different depuration times (t0-t7). Chi-square test was performed between viral levels and frequenciesof detection, at a confidence level of 95%. A Pearson correlationwasrun to determine the relationship between t0 and t7 viral levels. Allstatistical analyses were performed using the statistical packageIBM SPSS v20.0.0 software.

3. Results

Physico-chemical parameters of did not showed importantvariations both between trial as within the same trial. Averageconductivity ranged from 49.9 to 51.5 mS/cm. Average dissolvedoxygen varied from 7.0 to 8.0 ppm, and never descended below5 ppm. Water pH ranged from 7.4 to 8.7 and no residual chlorinewas detected in the depuration bins. The depuration plant is located

Table 2Viral detection and quantification for trials carried out with mussels (Mytilus galloprovinFþRNA/100 g mollusk flesh for FþRNA bacteriophages.

Virus Trial t0 t1 t2 t3

NoV GI 12 þ3456 2.1 � 105

7 1.2 � 105 1.8 � 104 2.4 � 105 68 8.9 � 103 1.1 � 104 2.4 � 104 29 1.5 � 104 1.1 � 104 þ

NoV GII 1234 1567 2.1 � 104 7.3 � 103 4.4 � 103

8 2.5 � 104 1.0 � 104 1.2 � 104 29 8.1 � 104 4.8 � 104 3.9 � 104 3

FþRNA 1 3.1 � 104 2.5 � 104 1.9 � 104 12 2.3 � 104 5.5 � 103 1.8 � 102 13 1.0 � 104 6.7 � 103 1.8 � 104 14 2.7 � 103 2.7 � 103 1.1 � 103 05 4.0 � 104 2.0 � 104 8.4 � 102 16 3.6 � 103 2.3 � 103 8.0 � 102 07 3.1 � 103 1.6 � 103 3.6 � 102 18 1.8 � 103 1.1 � 103 6.6 � 102 19 6.6 � 102 3.8 � 103 1.3 � 103 3

þ, Positive sample but outside of the quantification range.

inside the estuary, taking the water from the environment. Thisimplies a variable temperature and salinity along the year. Tem-perature ranged from 10.2 to 18 �C and from 12.2 to 24 �C in clamsand mussels, respectively; however, in the same trial the variationwas never greater than 3 �C. The average salinity ranged from 31.8to 33.4 and 33.7 to 34.7 in clams and mussels, respectively.

FþRNA bacteriophages initial levels ranged from 4.0 � 103 to1.4 � 105 FþRNA/100 g in clams, and 6.6 � 102 to 4.0 � 104 FþRNA/100 g in mussels (Tables 1 and 2). In clams, the average concen-tration before and after the seven days of the purification processwas 6.1 � 104 and 6.3 � 103 FþRNA/100 g, respectively, with anaverage reduction rate of 1 log unit (90% of removal). In mussels,the average concentration before and after depuration processwere 1.3 � 104 and 8.3 � 101 FþRNA/100 g respectively, whichrepresents a removal rate of >2 log units (>99%) (Fig. 2). All trialsshowed statistically significant (p < 0.05) reduction rates in FþRNAcounts at the end of the depuration. Pearson’s correlation analysisshowed a positively lineal correlation between the initial and thefinal load of bacteriophages in clams (r ¼ 0.65, p ¼ 0.02). However,in mussels this correlation was not observed (r ¼ �0.06, p ¼ 0.89).

Concerning to the FþRNA removal kinetics, all trials showed atwo-phase depuration kinetic, i.e. a logarithmic reduction trendwith a more pronounced decrease in the first days and a subse-quent stabilization in the removal rate in the remaining days. Sta-tistical analysis showed the t4 stage in clams and t5 in mussels asthe point from which the average reduction of the FþRNA loadwere no longer statistically significant (Fig. 2).

With regard to HAV and NoV quantification, 38.5% of the clamtrials (5 out of 13) and 55% of the mussel trials (5 out of 9) showedinitial contamination (t0) with at least one of the virus analysed.However, there were other trials that showed viral contaminationat intermediate depuration stages (t1et7) but not at t0. Taking thisinto account, all the trials, except two (trial 4 and 6) in clams andone (trial 1) in mussels, were positive for at least one of the virusesduring the depuration process. This represents 84.6% in clams and88.9% in mussels of viral presence.

cialis). Results are expressed as no. of RNA copies/g DT for NoV GI and GII, and as

t4 t5 t6 t7

4.3 � 103

5.0 � 105

.5 � 104 1.2 � 104 6.1 � 103

.4 � 104 6.1 � 103 þ

.9 � 105 3.6 � 103

.9 � 104 1.1 � 104

.4 � 103 1.2 � 103

.7 � 103 4.2 � 103 4.2 � 102

.2 � 102 3.0 � 102 1.8 � 102

.1 � 104 1.4 � 103 1.2 � 102 1.8 � 102

.0 � 100 3.0 � 102 1.2 � 102

.2 � 102 5.4 � 102 1.2 � 102 0.0 � 100

.0 � 100 3.0 � 102 6.0 � 101 0.0 � 100

.4 � 103 1.5 � 103 0.0 � 100 0.0 � 100

.8 � 103 4.8 � 102 1.8 � 102 1.2 � 102

.3 � 103 1.2 � 102 6.0 � 101 6.0 � 101

Fig. 2. Average concentration of FþRNA bacteriophages (expressed as no. of FþRNA/100 g)in clams and mussels along the depuration period and their correspondent trendline(expressed in % of FþRNA/100 g) and R2. t0, sampling at transfer of mollusks from envi-ronment to depuration system; t1et7, day 1e7 samplings during purification process.

D. Polo et al. / Food Control 43 (2014) 206e212210

In clams NoV GII was the most prevalent virus followed by NoVGI and HAV. In mussels NoV GI was the most prevalent virus fol-lowed by NoV GII and HAV. However, both in clams and musselsNoVGI showed the higher viral levels followed byNoV GII and HAV.Overall quantification levels ranged from 103 to 106 RNAc/g DT inclams and from 103 to 105 RNAc/g DT in mussels. However, most ofthe samples varied from 103 to 105 RNAc/g DT, and only threesamples reached values higher than 105 RNAc/g DT, all of them inclam trials (Tables 1 and 2). HAV was only quantified in two clamsamples (w103 RNAc/g DT) at t4 and t7 stage (trial 2 and 12,respectively), and in one mussel sample (w104 RNAc/g DT) at t4stage (trial 2) (data not shown).

At the end of depuration, 5 out of 11 contaminated trials (inclams) and 3 out of 8 (in mussels) showed viral contamination. Inclams, NoVGI, GII and HAVwere detected at t7. Inmussels only NoVGI was detected at t7. No statistical correlation could be establishedby chi-square test between FþRNA and HAV or NoV at a confidencelevel of 95%.

4. Discussion

The studies about viral depuration in shellfish using RT-qPCR arerelatively scarce and most of them are focused on oysters and/orcarried out at laboratory scale. Little is available about thecomparative abilities of naturally contaminated mussels and clamsto depurate human viruses and bacteriophages in commercialconditions. This study attempts to provide an overview on the viralelimination under an extended commercial depuration process of 7days in an authorized plant. The large size of samples used, theprofessional equipment of the depuration plant and the standardmethods used for viral quantification confer high robustness andreliability to the analysed data.

Most common water disinfection methods used in commercialdepuration are chlorine, ultraviolet light (UV) and ozone. Chlorinecan affect the shellfishpumping activity, cause organoleptic changesin shellfish meat and give rise a chlorinated by-products, like tri-halomethanes, identified as possible carcinogenic agents (Lee,Lovatelli, & Ababouch, 2008). Depuration plants overcome thesedrawbacks removing residual chlorine by degassing with thio-sulphate and/or a vigorous aeration before the water is introducedinto the depuration tanks (Lees, Younger, & Dore, 2010). UV andozone disinfection have gained popularity in recent years but their

high initial and/or running costs difficult their implementation inbigger facilities. Efficiency of UV is directly related to depth andturbidityand its efficacy inhighflow rates, at least for now, is limited(Jackson & Ogburn, 1999). Ozone can be influenced by temperature,pH, dissolved compounds and also can form by-products (bro-mates), potentiallycarcinogenic. As chlorine, residual ozonemust beremoved by degassing (Lees et al., 2010). Chlorine, employed here, ismore commonly used in big open flow depuration facilities withgreater quantity of water than closed systems.

FþRNA bacteriophage was significantly reduced in 7 days. Inaddition, they showed differences in the reduction rate betweenclams andmussels, showing a faster depuration rate inmussels. Thelesser sensitivity to environmental stress and the higher pumpingcapacity ofmussels with respect to other bivalves like clams (Bayne,Thompson, & Widdows, 1976) could favour its adaptability to thedepuration process and could increase their removal rates.

Other studies reported that successful elimination of bacterio-phages from oysters is dependent on initial contaminant level andthat heavily contaminated shellfish failed to clear bacteriophagewithin 7 days (Doré, Henshilwood, & Lees, 1998; Lees et al., 2010).Here, higher levels of bacteriophages were detected for clams, and apositive correlation between initial and final bacteriophages levelswas observed in clams, but not inmussels. In addition, clams showedlower removal rate for bacteriophages and higher final levels of NoVthan mussels. These facts could reflect again the higher depurationcapacity of mussels respect to clams or a preferential viral bio-accumulation due to biological properties of their tissues. Thesefindings could also be a reflection of the results of previous studiesthat report a higher percentage of outbreaks due to the consumptionof clams than mussels (Bellou, Kokkinos, & Vantarakis, 2012).

Is important to take into account that the quantification tech-niques used for bacteriophages (based on infectivity) and forenteric viruses (based on genome copies) differs substantially, socomparisons must be made with caution. Although, non-infectiousparticlesmay be also detected, in the absence of an infectivity assay,the RT-qPCR is the best method applicable. In addition, Dancer,Rangdale, Lowther, and Lees (2010) reported that norovirus RNAmay persist in seawater but does not bioaccumulate efficiently inPacific oysters. This could have important implications for depu-ration studies since NoV RNA in water does not significantlycontribute to positive RT-qPCR results.

Recent studies indicate that molluscs can selectively accumulatenorovirus strains based on viral carbohydrate ligands of histo-bloodgroup like antigens present in gastrointestinal cells of oysters, clamsand mussels (Le Guyader, Atmar, & Le Pendu, 2012; Le Guyaderet al., 2006; Tian, Engelbrektson, Jiang, Zhong, & Mandrell, 2007).In addition, bivalve haemocytes have been recently proposed as asite of viral persistence (Provost, Ozbay, Anderson, Richards, &Kingsley, 2011). Differences in depuration rates between viraland/or bivalve species (Maalouf et al., 2011; Nappier, Graczyk, &Schwab, 2008) can be the consequence of this specific attachmentof virus to the molluscs tissues and presence, amount and/or dis-tribution of these specific ligands and/or the specific resistance ofcertains viruses to intracellular processes such the acidic digestionwithin haemocytes. This also could explain the higher prevalence ofNoV, principally NoV GI strains, in shellfish-related outbreaks (LeGuyader et al., 2012; Verhoef et al., 2010).

With regard to the depuration kinetics FþRNA bacteriophageshowed two-phase depuration kinetic. For NoV the absence ofdetection in the initial time of many trials and/or in consecutivetimes of the depuration process, possibly due to the heterogeneityof the viral distribution and the large sample size used in each trial,precluded the establishment of a kinetic pattern. These aspectsmust be present in the future in order to define and establish pro-tocols for the control of the viral depuration process effectiveness.

D. Polo et al. / Food Control 43 (2014) 206e212 211

Despite of this fact, previous studies in our laboratory have shownsimilar two-phase depuration kinetics for HAV and MNV-1 in Ma-nila clams (Venerupis philippinarum) and Mediterranean mussel(Polo, Álvarez, Vilariño, Longa, & Romalde, 2014; unpublished data).Love, Lovelace, and Sobsey (2010) reported similar results in oysters(Crassostrea virginica) and hard shell clams (Mercenariamercenaria).

Bacteriophage elimination kinetics under depuration appears toreflect those of enteric viruses in a more representative way thanE. coli, and could therefore complement the use of bacterial in-dicators for the sanitary quality of shellfish (Doré & Lees, 1995;Power & Collins, 1989). However, FþRNA bacteriophages are nothuman specific and animal faeces from land runoff also causeFþRNA bacteriophage contamination but without health risk(Havelaar et a., 1986). On the other hand, viruses like HAV and NoVcould be evolved to exploit molluscs as vector of human infectionand therefore use mechanism like specific ligands or their higherresistance to acidic conditions that other viruses that are morerapidly depurated like FþRNA bacteriophages, poliovirus or felinecalicivirus do not posses (McLeod, Hay, Grant, Greening, & Day,2009; Provost et al., 2011; Ueki et al., 2007).

It has often been suggested that process temperature increasethe depuration rate (Doré et al., 1998; Jaykus, Hemard, & Sobsey,1994; Pommepuy et al., 2003; Power & Collins, 1990). Here, theapplication of an open circuit depuration implied a seasonal vari-ation of the water temperature, from 13 to 15 �C in the cold monthsand from 15 to 18 �C inwarmmonths. No differences were found inNoV depuration with regard to the water temperature. However inFþRNA a higher reduction during the warm months was observed,specially in mussels (data not shown). The use of higher watertemperatures could favour the removal rate due to the increase inthe pumping rate and enzymatic activity. However, there arespecies-specific physiological limits in the optimal depurationtemperature and the impact of using warmer water temperature indifferent bivalve and viral species, geographical areas or even sea-son of the year remains uncertainly and further studies are required(Lees et al., 2010; Love et al., 2010; Pommepuy et al., 2003).

5. Conclusions

The results seem to indicate that commercial depuration canreduce at certain extent the risk of viral infection, but not enough toensure the viral absence in marketable shellfish. An improvedprotocol in depuration plants is necessary with a better under-standing of differences in contamination levels between in-dividuals and/or within batches to developing more appropriateand representative sampling strategies. In addition, informationabout differences on the behaviour of different mollusc and/or viralspecies under the same depuration conditions is crucial for theimprovement of the process and the developing of new legislationthat encompasses these pathogens.

Acknowledgements

This work was supported in part by Grant 10MMA200010PRfrom Xunta de Galicia (Spain).

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