Effects of sanitation, freezing and frozen storage on enteric viruses in berries and herbs

International Journal of Food Microbiology 126 (2008) 30–35

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Effects of sanitation, freezing and frozen storage on enteric viruses in berries and herbs S. Butot, T. Putallaz, G. Sánchez ⁎ Quality & Safety Assurance Department, Nestlé Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Lausanne, Switzerland

A R T I C L E

I N F O

Article history: Received 31 October 2007 Received in revised form 29 April 2008 Accepted 29 April 2008 Keywords: Enteric viruses Berries Vegetables Survival Sanitation

A B S T R A C T Norovirus (NV) and hepatitis A virus (HAV) are foodborne enteric viruses associated with outbreaks of disease following consumption of fresh or frozen produce. Model experiments were performed to determine the effectiveness of certain commercial processes for the removal of enteric viruses that might be present in berries and herbs. The survival and persistence of HAV, NV, rotavirus (RV) and feline calicivirus (FCV), a surrogate for NV, in frozen produce over time were determined. Survival and inactivation of HAV, RV and FCV were assessed by viral culture and quantitative reverse transcription-PCR (RT-PCR), whereas NV persistence was determined by quantitative RT-PCR only. Freezing did not significantly reduce the viability of any of the viruses except the infectivity of FCV in strawberries. Frozen storage for 3 months had limited effects on HAV and RV survival in all tested food products, whereas in frozen raspberries and strawberries FCV infectivity showed the highest decay rate due to acid pH. To simulate postharvesting conditions, fresh berries and herbs were rinsed with tap, warm or chlorinated water or with a chlorine dioxide (ClO2) solution. Available chlorine at a concentration of 200 ppm and ClO2 at 10 ppm reduced measurable enteric viruses in raspberry and parsley samples by less than 2 log10 units. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Recent epidemiological evidence indicates that enteric viruses, in particular norovirus (NV), which cause acute gastroenteritis, but also hepatitis A virus (HAV) and rotavirus (RV), are the leading cause of foodborne illness in developed countries (Fleet et al., 2000; Koopmans and Duizer, 2004; Widdowson et al., 2005). While consumption of raw or improperly cooked shellfish remains the main cause of outbreaks of disease caused by foodborne viruses, various berries are increasingly being recognized as vehicles for enteric viruses (Gaulin et al., 1999; Ponka et al., 1999; Hedlund et al., 2000; Le Guyader et al., 2004; Hjertqvist et al., 2006; Fell et al., 2007) or hepatitis A virus (Reid and Robinson, 1987; Ramsay and Upton, 1989; Niu et al., 1992; Hutin et al., 1999; Calder et al., 2003). Recently, six norovirus outbreaks that occurred in Europe and involved up to 1100 people were associated with the consumption of frozen berries imported from Poland (Cotterelle et al., 2005; Falkenhorst et al., 2005; Korsager et al., 2005). Vegetables, including various types of salads and green onions have also been associated with outbreaks of viral hepatitis and gastroenteritis

⁎ Corresponding author. Tel.: +41 21 785 8692; fax: +41 21 785 8553. E-mail address: [email protected] (G. Sánchez). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.04.033

(Rosenblum et al., 1990; Warner et al., 1991; Dentinger et al., 2001; Long et al., 2002; Grotto et al., 2004). Recently, a hepatitis A outbreak caused by the ingestion of contaminated green onions resulted in three deaths among a total of 601 cases (Wheeler et al., 2005). Produce may be contaminated during cultivation before harvest by contact with inadequately treated sewage or sewage polluted water. Contamination may also occur during processing, storage, distribution or final preparation. This could happen because food is contacted by infected people, contaminated water, or fomites. However, except for shellfish, foods are seldom tested for viruses. Frequently, foodborne outbreaks are suspected of being caused by viruses but, because of the lack of sensitive and reliable methods, this suspicion can rarely be confirmed by isolation of the virus from the implicated food. Hence the safety of food products cannot be assured by testing for viruses, but can be by the prevention of contamination and the implementation of manufacturing processes that inactivate or eliminate them. Various studies have addressed enteric virus survival of commercial processes (Table 1). It is difficult to draw general conclusions from these studies, because of differences in the experimental conditions and methods that were used. However, most of the studies found that viruses remained viable for periods exceeding the shelf lives of products. Information is still lacking on the survival of viruses on frozen produce and the efficiency of current commercial processes for

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Table 1 Survival of enteric viruses in berries and vegetables (adapted from Koopmans and Duizer, 2004) Food product

Likelihood of virus presence in raw materialsa

Process

Virus inactivation (log10)

Risk for the consumer if viruses present before processingb

Carrot (Badawy et al., 1985; Croci et al., 2002)

Possible

Storage at 4 °C for 4 days

Variable

Fennel (Croci et al., 2002) Green onions (Kurdziel et al., 2001) Lettuce (Badawy et al., 1985; Kurdziel et al., 2001; Croci et al., 2002)

Possible Likely Likely

Storage at 4 °C for 7 days Storage at 4 °C for 15 days Storage at 4 °C for 9 days

Lettuce (Croci et al., 2002) Lettuce and strawberries (Bidawid et al., 2000) Raspberries (Kurdziel et al., 2001) Strawberries (Kurdziel et al., 2001) Strawberries (Mariam and Cliver, 2000) Strawberries (Lukasik et al., 2003) Strawberry purée and sliced green onions (Kingsley et al., 2005) Strawberry mashes (Deboosere et al., 2004)

Likely Likely Likely Likely Likely Likely Likely

Washing (5 min potable water) Gamma irradiation (3 KGy) Storage at 4 °C for 9 days Freezing Washing with 2 ppm ClO2 Washing with 200 ppm chlorine High hydrostatic pressure (375 MPa, 5 min) 85 °C, 5 min

HAV N 4 RV b 1 HAV N 4 PV b 1 HAV b 2 PV b 2 RV b 1 HAV b 1 HAV = 1 PV b 1 PV b 2 HAV b 1 PV b 2 HAV N 4

High Medium High Medium High Medium Negligible

HAV b 2

Medium

Likely

Negligible High Medium

a

Possible: sporadic contamination with NV, HAV, RV, or PV has been reported on these food items. Likely: contamination with NV, HAV, RV, or PV is reported frequently on these food items. b Suggested risk based on published literature concerning reported outbreaks and the effectiveness of various processing methods to inactivate enteric viruses. Negligible risk: product highly unlikely to contain infectious viruses; treatment results in at least 4 log10 inactivation of common foodborne viruses. Medium risk: product may contain infectious viruses in numbers that may cause disease; treatment results in approximately 2 log10 inactivation of common foodborne viruses. High risk: products in which the level of viruses is likely to be high enough to cause disease in healthy individuals; treatment results in less than 1 log10 inactivation of common foodborne viruses. Variable risk: treatment results in significant differences in inactivation of several common foodborne viruses.

their removal or inactivation. Therefore, studies were undertaken to provide information on enteric virus survival in frozen produce and the fate of viruses in processed produce. 2. Materials and methods 2.1. Viruses, cells, and infections Clinical stool samples positive for NV genogroup I (Valetta strain; kindly provided by the RIVM, Bilthoven, The Netherlands) and genogroup II (Lordsdale; kindly provided by Dr. Buesa, University of Valencia, Spain) were used as NV reference material. The cytopathogenic HM-175 strain of HAV (ATCC VR-1402), the human rotavirus (RV) strain Wa (ATCC VR-2018) and the F9 strain of feline calicivirus (FCV; ATCC VR-782) were propagated and assayed in FRhK-4, MA-104 and CRFK cell monolayers, respectively. Semi purified stocks were subsequently produced from the same cells by centrifugation of infected cell lysates at 660 g for 30 min. Infectious viruses were enumerated by determining the 50% tissue culture infective dose (TCID50) with eight wells per dilution and 20 μl of inoculum per well. 2.2. Viral genome quantification A LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) was used for real-time RT-PCR reactions throughout the study. Norovirus GI and GII were quantified with a Platinum® Quantitative RT-PCR ThermoScript™ One-Step System (Invitrogen AG, Basel, Switzerland) as described previously (da Silva et al., 2007). Standard curves for NV GI and GII were generated by amplifying 10-fold dilutions of the stool extract by real-time RT-PCR. The crossing points obtained from the assay of each dilution were used to plot a standard curve by assigning a value of 1 RT-PCR unit (PCRU) to the highest dilution showing a positive crossing point value and progressively ten-fold higher values to the lower dilutions. Amplification efficiencies calculated from the slopes given by the LightCycler were 2.077 and 2.021 for NV GI and NV GII, respectively. A real-time RT-PCR for FCV was developed using primers and probe targeting the p30 region (Dr. Lowther, CEFAS, personal

communication). Briefly, real-time RT-PCR was performed using a QuantiTect®Probe RT-PCR kit (QIAGEN GmbH, Hilden, Germany) consisting of 10 µl QuantiTect Probe, 0.2 µl QuantiTect RT Mix, 1 µM of FCVFWD (5V-GCCAATCAGCATGTGGTAACC-3V) and FCVREV (5V-GCACATCATATGCGGCTCTG-3V) primers, 0.2 µM of the TaqMan probe (5V FAM-CCCAGGCCAAATCAAACACCGAATTAA-3V TAMRA) and 8 U of RNase inhibitor. Five µl of RNA was transferred to a LightCycler capillary tube containing 15 µl of master mix. RT was performed at 50 °C for 30 min. Amplification was performed for 1 cycle of 95 °C for 15 min and 45 cycles of 95 °C for 0 s and 60 °C for 1 min. A standard curve for FCV was generated by performing real-time RT-PCR on 10-fold dilutions of FCV-extracted RNA. The crossing points obtained from the assay of each dilution were used to plot a standard curve by assigning the corresponding TCID50 values. Amplification efficiency calculated from the slope given by the LightCycler was 2.161. HAV RNA was quantified with a LightCycler hepatitis A virus quantification kit (Roche Diagnostics) as previously described (Sanchez et al., 2006). The LightCycler hepatitis A virus quantification kit contains HAV RNA standards that allow the number of RNA copies per sample to be estimated. Amplification efficiency calculated from the slope given by the LightCycler was 2.047. The kit also includes an internal control to prevent misidentification of false negatives. A real-time RT-PCR for RV was based on a previously published method (Pang et al., 2004), which was adapted for the LightCycler (Butot et al., 2007). An RV standard curve was generated by performing real-time RT-PCR on 10-fold dilutions of Wa-extracted RNA. The crossing points obtained from the assay of each dilution were used to plot a standard curve by assigning the corresponding TCID50 values. Amplification efficiency calculated from the slope given by the LightCycler was 1.683. 2.3. Virus recovery from produce Viruses were released from the food surface as previously described by Butot et al. (2007). Briefly, food samples were gently shaken for 15 min at room temperature with 60 ml of an elution buffer containing 50 mM Glycine, 100 mM Tris and 1% (wt/vol) beef extract pH 9.5. Then the food sample and diluent were transferred to a filter

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stomacher bag (Tempo Sacs, bioMérieux, Marcy l'Etoile, France). The recovered elution buffer was adjusted to pH 7.0 ± 0.2 and centrifuged at 3500 g for 15 min. Raspberry and strawberry samples were treated with pectinase by adding 300 µl of pectinase (Pectinex; Sigma, Buchs, Switzerland) to the elution buffer and gently shook for 30 min at room temperature before centrifugation. The supernatant was concentrated in a volume of 500 µl using a Centricon Plus-70 centrifugal filter device (100 K NMWL, Millipore, Molsheim, France). Before testing on cell cultures, concentrated samples were decontaminated by sequential filtering through 0.45 and then 0.22 μm Spin Centrifuge Tube Filters (Corning, NY, USA) pretreated with 300 μl of PBS containing 10% fetal calf serum. Filtered samples were divided in two subsamples, one of 350 µl for the cell culture assays and other of 150 µl for the RNA extraction. RNA extraction was performed using a QIAamp® Viral RNA Mini Kit (QIAGEN) according to the manufacturer's instructions. Concentrated samples of raspberries were pretreated with 300 µl of Plant RNA Isolation Aid (Ambion, Cambridgeshire, United Kingdom) before RNA extraction. A negative control was included with each group of RNA extracts. RNA extracts were either immediately analyzed by real-time RT-PCR or stored at −80 °C until use. Nucleic acid suspensions were analyzed twice by the specific real-time RT-PCR method, which also allowed us to estimate the number of recovered viruses or PCRU. Positive controls, with the same concentration of viruses as the suspension used to inoculate berries and herbs, were analyzed in parallel by real-time RT-PCR, to determine the fraction of particles recovered by the concentration procedure. 2.4. Titration of viruses To determine infectivity titers, filtered concentrated samples were titrated by inoculating 20 μl of serial 5-fold dilutions of FCV, HAV or RV into each well containing CRFK, FRhk-4 or MA-104 cells, respectively, grown in 96-well plates. Eight wells of negative controls were included in each plate. After incubation at 37 °C in a CO2 incubator for 3, 14 or 7 days, for plates inoculated with FCV, HAV or RV respectively, the cells were observed for cytopathic effects. The infectivity titers were determined as TCID50 per ml. The decay of viruses was calculated as log10 (Nx/N0) where N0 is the infectious virus titer for untreated produce and Nx is the infectious virus titer for treated produce. 2.5. Stability studies in frozen berries and herbs Locally purchased strawberries, raspberries, blueberries, parsley and basil were used in the study. Dilutions of viruses in phosphatebuffered saline (PBS) were inoculated onto the surfaces of each 15 g portion of fresh produce by distributing 50 µl over 10 spots. Fifteengram portions of fresh berries and herbs were inoculated with about 1.6 × 106 TCID50 of HAV, 1.2 × 105 PCRU of NV GI, 2 × 106 PCRU of NV GII, 1.6 × 106 TCID50 of FCV and 4 × 104 TCID50 of RV. Inoculated samples were air-dried in a laminar flow hood for 60 min. Batches of three samples were analyzed on day 0 and the remaining samples were stored at −20 °C. After each of 2, 30 and 90 days of storage, batches of

three samples were thawed and processed as described above. Virus survival was calculated as log10 (Nt/N0), where N0 is the virus titer recovered from fresh berries and Nt is the virus titer recovered at various time intervals after freezing. 2.6. Washing experiments Triplicate 15 g portions of produce inoculated as previously described were washed by stirring for 30 s in 200 ml of tap water at 18 °C with 0 ppm free chlorine (FC); warm water at 43 °C with 0 ppm FC; or chlorinated water at 18 °C with 200 ppm FC. After washing, all samples were rinsed in 200 ml of tap water, as is recommended for fruits and vegetables washed with chlorinated solutions (FDA, 2006). All containers used in the experiments were soaked in 12.5% nitric acid, rinsed with distilled water, and autoclaved before use. A solution containing 200 ppm FC was prepared from a stock solution of sodium hypochlorite containing 6 to 14% active chlorine (Sigma). FC was measured using the Chlor-Test kit (Merck, Darmstadt, Germany) according to the manufacturer's instructions. After rinsing, samples were processed as described above. The inactivation of viruses was calculated as before. 2.7. Generation of chlorine dioxide solutions A ClO2 solution was obtained by adding 2 l of deionized water to a pouch containing a ClO2 system (Selective Micro Clean 2 L50A, Selective Micro Technologies, Beverly, USA). The 50 ppm solution was diluted to 5, 10 and 25 ppm with deionized water. The concentrations of ClO2 solutions were tested immediately before their application to produce, with low and wide range test strips (Selective Micro Technologies), according to the manufacturer's instructions. 2.8. Chlorine dioxide wash Fifteen-gram portions of raspberries or parsley inoculated as previously described were immersed in 200 ml of a ClO2 solution at ambient temperature for 1 or 10 min, with gentle stirring. Each treatment was carried out in triplicate. The inactivation of viruses was calculated as before. 2.9. Statistical analysis The significance of differences among the mean numbers of viruses determined after the various treatments were decided by the Student's t test with a significance level of P b 0.05 (Microsoft Office Excel; Microsoft, Redmond, WA, USA). 3. Results 3.1. Virus survival on frozen produce Freezing reduced the numbers of most viruses on all produce by less than one log10 unit, as determined by real-time RT-PCR or TCID50. The exceptions were FCV on strawberries and raspberries, the TCID50

Table 2 Survival of norovirus (NV), feline calicivirus (FCV), hepatitis A virus (HAV) and rotavirus (RV) in produce after freezing as determined by real-time RT-PCR (PCRU or RNA copies) and viral culture (TCID50); tabulated values are mean values for log10 (N2/N0) ± standard error where N0 is the virus titer before and N2 is the titer after frozen storage for 2 days Product

NV GI PCRU

NV GII PCRU

Blueberries Raspberries Strawberries Basil Parsley

−0.9 ± 0.07 0.1 ± 0.48 −0.1 ± 0.05 0.3 ± 0.08 −0.3 ± 0.14

−0.9 ± 0.09 0.0 ± 0.19 −0.4 ± 0.20 0.1 ± 0.10 −0.7 ± 0.12

FCV

HAV

PCRU

TCID50

− 0.6 ± 0.09 − 0.6 ± 0.18 − 0.2 ± 0.43 0.2 ± 0.03 − 0.8 ± 0.24

0.3 ± 0.15 −1.1 ± 0.39 −2.7 ± 0.10 −0.4 ± 0.10 −0.6 ± 0.34

RNA copies 0.0 ± 0.18 0.2 ± 0.30 0.7 ± 0.07 0.2 ± 0.10 0.0 ± 0.31

RV TCID50

PCRU

TCID50

−0.4 ± 0.19 0.0 ± 0.27 0.0 ± 0.18 − 0.1 ± 0.14 − 0.1 ± 0.25

−0.8 ± 0.15 0.2 ± 0.06 0.7 ± 0.16 0.1 ± 0.02 −0.5 ± 0.10

−1.2 ± 0.13 0.9 ± 0.18 − 0.1 ± 0.17 −0.2 ± 0.15 − 0.1 ± 0.04

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Table 3 Removal of norovirus (NV), feline calicivirus (FCV), hepatitis A virus (HAV) and rotavirus (RV) in produce after washing with tap water as determined by real-time RT-PCR (PCRU or RNA copies) and viral culture (TCID50); tabulated values are mean values for log10 (Nx/N0) ± standard error where N0 is the virus titer before and Nx is the titer after washing with cold water Product

NV GI PCRU

NV GII PCRU

Blueberries Raspberries Strawberries Basil Parsley

− 0.8 ± 0.81 − 0.1 ± 0.17 − 0.6 ± 0.17 N −3.4 − 0.5 ± 0.07

−1.5 ± 0.18 −0.2 ± 0.36 −0.8 ± 0.30 − 1.1 ± 0.22 −0.9 ± 0.32

FCV

HAV

RV

PCRU

TCID50

RNA copies

TCID50

PCRU

TCID50

− 1.8 ± 0.27 −2.7 ± 0.16 − 1.3 ± 0.43 − 1.4 ± 0.30 −0.8 ± 0.07

− 2.6 ± 0.58 −1.7 ± 0.14 −1.9 ± 0.29 − 2.5 ± 0.58 −1.3 ± 0.27

−1.3 ± 0.30 −0.4 ± 0.21 −0.9 ± 0.37 −1.2 ± 0.15 −1.0 ± 0.18

−0.9 ± 0.23 −0.6 ± 0.15 −0.8 ± 0.21 −1.1 ± 0.23 −0.5 ± 0.04

−1.5 ± 0.27 −1.4 ± 0.09 −2.6 ± 1.1 −1.0 ± 0.22 −0.8 ± 0.29

−2.2 ± 0.16 0.0 ± 0.44 − 1.5 ± 0.17 −0.8 ± 0.44 −0.9 ± 0.33

Table 4 Removal of norovirus (NV), feline calicivirus (FCV), hepatitis A virus (HAV) and rotavirus (RV) in produce after washing with warm water as determined by real-time RT-PCR (PCRU or RNA copies) and viral culture (TCID50); tabulated values are mean values for log10 (Nx/N0) ± standard error where N0 is the virus titer before and Nx is the titer after washing with warm water Product

NV GI PCRU

NV GII PCRU

Blueberries Raspberries Strawberries Basil Parsley

−0.9 ± 0.26 0.0 ± 0.11 −0.8 ± 0.09 N− 3.4 −0.7 ± 0.10

−2.1 ± 0.39 −0.2 ± 0.30 −1.1 ± 0.11 −1.1 ± 0.08 −1.7 ± 0.26

FCV

HAV

RV

PCRU

TCID50

RNA copies

TCID50

PCRU

TCID50

−2.3 ± 0.13 − 2.1 ± 0.34 − 2.1 ± 0.40 −1.5 ± 0.09 −1.3 ± 0.04

−3.3 ± 0.14 −1.3 ± 0.26 N −3.5 −2.9 ± 0.07 −1.8 ± 0.08

−1.6 ± 0.47 −0.5 ± 0.16 −0.9 ± 0.27 −1.3 ± 0.06 −0.6 ± 0.07

−0.5 ± 0.22 −0.6 ± 0.35 −0.6 ± 0.19 −1.1 ± 0.11 −0.03 ± 0.0

−1.2 ± 0.14 −1.7 ± 0.31 −3.0 ± 0.57 −1.2 ± 0.35 −1.6 ± 0.34

−1.9 ± 0.72 − 0.5 ± 1.08 −1.4 ± 0.18 −1.1 ± 0.35 −1.2 ± 0.29

values of which were reduced by more than 1 log10 unit although the corresponding PCRU values were reduced by less than one log10, and RV on blueberries (Table 2). Generally, HAV and RV numbers remained the same in all the tested frozen products during 90 days of storage, as determined by real-time RT-PCR or TCID50. The exceptions were RV on blueberries and basil, the values of which were reduced by 1 log10 unit after 2 days of storage. The numbers of FCV recovered from raspberries progressively declined during the frozen storage. TCID50 values were reduced by more than 2 log10 units although PCRU values were reduced by only about 1 log10 unit. In strawberries there was a rapid initial fall in numbers followed by tailing because of a diminishing inactivation rate as determined by TCID50. The corresponding PCRU values were reduced by less than 0.5 log10 units. NV GII was less resistant than NV GI under the tested conditions. Blueberries had the greatest degree of NV GII inactivation, with the virus titer being reduced by 2.3 log10 after 90 days of storage. However there was no more than 1 log difference in the reductions found for the two norovirus genogroups. 3.2. Effects of washing The reductions of HAV, NV GI and NV GII on most products obtained by washing with cold water were less than 1.5 log10 units, except with basil, while NV GI was not recovered after washing with tap water (Tables 3) or with warm or chlorinated water. Generally, the effects of warm water washes in HAV, NV GI and GII titers were not significantly different (P N 0.05) from the effects of washing with cold water (Table 4).

When comparing only real-time RT-PCR detection after the different washing treatments, HAV, NV GI and GII show similar behavior except for NV GI in basil which was completely removed after washing. Washing produce with chlorinated water was significantly (P b 0.05) more effective for removing FCV than washing with cold or warm water (Table 5). Washing with chlorinated water resulted in significant reductions of NV GI, NV GII and HAV in blueberries, strawberries and basil. There was appreciably less reduction of NV GI, NV GII and HAV titers in raspberries and parsley (Table 5). HAV was more resistant than other virus to all washing treatments. 3.3. Effect of chlorine dioxide One-minute washing with ClO2 at 5, 10, 25 or 50 ppm had limited effects on HAV and NV titers (data not shown). To improve efficacy of ClO2 treatment, the treatment time was extended to 10 min, but with no better effects (Table 6). FCV was only used with parsley as in raspberries it is inactivated by the low pH. Inactivation of HAV by washing parsley with ClO2 was significantly (P b 0.05) greater as determined by TCID50 than by RNA copies. Even so, the reduction in the infectious titer was b2 log10 units after washing with 10 ppm of ClO2 for 10 min. 4. Discussion Treatments of the sorts commonly applied to produce are apparently unable to wholly remove or inactivate enteric viruses. The occurrence of outbreaks of viral disease due to the consumption of

Table 5 Inactivation of norovirus (NV), feline calicivirus (FCV), hepatitis A virus (HAV) and rotavirus (RV) in produce after washing with chlorinated water as determined by real-time RT-PCR (PCRU or RNA copies) and viral culture (TCID50); tabulated values are mean values for log10 (Nx/N0) ± standard error where N0 is the virus titer before and Nx is the titer after washing with water containing 200 ppm free chlorine Product

Blueberries Raspberries Strawberries Basil Parsley

NV GI PCRU

−3.4 ± 0.8 0.0 ± 0.34 N −3.1 N −3.4 −0.9 ± 0.03

NV GII PCRU

− 3.0 ± 0.62 − 0.9 ± 0.15 −1.4 ± 0.23 −1.6 ± 0.12 −1.8 ± 0.10

FCV

HAV

RV

PCRU

TCID50

RNA copies

TCID50

PCRU

TCID50

− 4.0 ± 0.66 N −3.0 − 3.4 ± 0.66 − 2.5 ± 0.28 − 2.0 ± 0.15

N−3.5 N−3.5 N−3.5 N−3.5 N−2.7

−2.2 ± 0.23 −0.7 ± 0.07 −1.9 ± 0.25 −1.4 ± 0.05 −1.1 ± 0.07

−2.4 ± 0.34 −0.6 ± 0.21 −1.8 ± 0.26 −2.4 ± 0.21 −1.4 ± 0.04

−2.2 ± 0.15 −1.8 ± 0.15 −4.1 ± 0.35 −1.6 ± 0.21 −0.4 ± 0.03

N −3.0 −1.2 ± 0.33 N −3.0 −1.3 ± 0.17 −1.0 ± 0.16

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Table 6 Inactivation of norovirus (NV), feline calicivirus (FCV), hepatitis A virus (HAV) and rotavirus (RV) in produce after washing with chlorine dioxide (ClO2) as determined by real-time RTPCR (PCRU or RNA copies) and viral culture (TCID50); tabulated values are mean values for log10 (Nx/N0) ± standard error where N0 is the virus titer before and Nx is the titer after washing with water containing 5 or 10 ppm chlorine dioxide for 10 min Product

ClO2 treatment

NV GI PCRU

NV GII PCRU

Raspberries

5 ppm 10 ppm 5 ppm 10 ppm

−0.58 ± 0.07 −0.50 ± 0.12 −0.40 ± 0.06 −0.62 ± 0.17

− 0.71 ± 0.07 −0.98 ± 0.07 −0.64 ± 0.04 −1.19 ± 0.12

Parsley

produce that had been frozen several months (Hjertqvist et al., 2006; Fell et al., 2007) indicates that if produce is contaminated before freezing substantial fractions of the viruses may remain infectious during frozen storage. So far, no data on that matter has been available, except for a reported reduction of b2 log10 units of Poliovirus (PV) in frozen strawberries after 15 days of storage (Kurdziel et al., 2001). Considering that PV is known to be less resistant to environmental conditions than other enteric viruses (Abad et al., 1994), it is not surprising that all the human viruses used in this study were little affected by freezing. The simple practice of washing raw fruits and vegetables using cold or warm water has been shown to remove some of the bacteria on produce, but studies showing the efficacy of these treatments on enteric viruses are few. Rinsing vegetables with cold water has been reported to reduce HAV titers by b1 log10 unit (Croci et al., 2002), but PV was not removed from strawberries by rinsing with warm water (Lukasik et al., 2003). Our results show that rinsing with cold or warm water had limited effects in removing enteric viruses from produce. In contrast, washing produce with cold water reduced FCV titers by an average of 2 log10 units, in line with results reported by Gulati et al. (2001). Generally, results for RV were very inconsistent since in some instances reductions determined by real-time RT-PCR were significantly higher than those determined by TCID50 values. These unexpected results may be due to the low real-time RT-PCR amplification efficiency. Chlorine is the most widely used sanitizing agent for produce, at concentrations of 50 to 200 ppm FC with exposure times of 1 to 2 min. Virus inactivation by washing produce with chlorinated water containing 200 ppm FC varied with both the type of virus and the type of product. Chlorinated water had a limited effect on enteric virus titers when used to decontaminate raspberries and parsley, probably because of their different surface. For instance, raspberries have crevices and hairlike projections which may shield the viruses against environmental modifications. Kniel et al. (2002) found that raspberries retained more Toxoplasma gondii oocysts than blueberries, and this was attributed to the fine hairlike projections covering raspberries. Unlike chlorine and hypochlorite the efficacy of chlorine dioxide is little affected by pH and organic matter, and it does not react with ammonia to form chloramines. Despite that, chlorine dioxide was not very effective for inactivation of viruses in raspberries and parsley, even at three times the recommended concentration of 3 ppm (FDA, 2006). Because of the large number of outbreaks of viral disease associated with consumption of frozen raspberries, and the limited effects of chlorinated water and chlorine dioxide, alternative means of sanitizing raspberries are evidently required. NV GI and GII showed completely different behaviors on basil. NV GI was completely removed from basil surface by washing, suggesting weak attachment of this NV strain to basil surfaces. These results are in agreement with reported results for butterhead lettuce, with which attachment varied between virus strains (Vega et al., 2005); and the fact that different NV strains bind to different receptors (Tan and Jiang, 2005). Research with human NV has been hampered by the lack of suitable laboratory animals and the inability to propagate the virus in

FCV

HAV

PCRU

TCID50

RNA copies

TCID50

– – −0.43 ± 0.19 −0.81 ± 0.23

– – −0.73 ± 0.35 −1.31 ± 0.07

−0.70 ± 0.06 −0.72 ± 0.09 −0.40 ± 0.08 −0.30 ± 0.09

− 0.97 ± 0.21 −0.79 ± 0.07 −1.05 ± 0.14 −1.75 ± 0.19

vitro. An in vitro cell culture assay for human noroviruses has recently been developed, but this assay is not yet suitable for routine analyses (Straub et al., 2007). The F9 strain of FCV is the most generally used surrogate for human NV. Inactivation of FCV increased with decreasing pH of the produce. This is not entirely surprising since the F9 strain of FCV has been reported to be an inappropriate surrogate for NV in acid conditions (Slomka and Appleton, 1998; Hewitt and Greening, 2004; Cannon et al., 2006). It has recently been reported that FCV was not recovered from strawberries after 2 days storage at room temperature whereas it was recovered from other food matrices that were not of low pH for much longer periods (Mattison et al., 2007). FCV was completely inactivated by chlorinated water with chlorine at 200 ppm, which is the maximum concentration allowed by the FDA (FDA, 2006), contrary to the results of Gulati et al. (2001). The difference may be due by the concentrations of chlorine reported by Gulati et al. (2001) being different to those in the waters used with the food. Since they did not check the final concentration of FC, nor did they treat glassware to make it chlorine demand free. FCV is evidently not a suitable surrogate for noroviruses. The murine norovirus (MNV-1), might be a good alternative since it is genetically similar to human norovirus and is capable of surviving extreme acid conditions (Cannon et al., 2006). In some instances, infectious virus titers were reduced although the corresponding PCRU values were not affected. These findings indicate that some combinations of treatment and food may alter the viral protein capsid and cause a loss of infectivity even though the viral genome is still protected. Similar findings have been reported for other inactivation treatments (Hewitt and Greening, 2004; Lamhoujeb et al., in press). Most of the current methods for virus detection in food are based on RT-PCR or real-time RT-PCR. The detection of viral genomes by these methods is evidently insufficient to confirm the presence of infectious viruses, so PCR results must be interpreted with caution when estimating the risks from viruses in foods. Koopmans and Duizer (2004) summarized the risks of consuming products that may have become contaminated with enteric viruses before processing. The risks were categorized as negligible, low, medium, and high depending on whether the subsequent process gave reductions in infectivity for common foodborne viruses of at least 4, 3, 2, or 1-log units, respectively. Product that is frozen or washed with water was classed as being of high risk if viruses are present in it before processing. Our results support the high risk classification for freezing treatment and for fresh produce that is washed with cold water, whereas fresh produce washed with chlorinated water would be classed as being of variable risk, except for raspberries and parsley with which there is a high likelihood of viruses being present if they were contaminated before processing. Consequently, the emphasis should be on prevention of contamination before or during processing by implementation of good agricultural, hygienic and manufacturing practices, and hazard analysis critical control points (HACCP) systems. Acknowledgment We would like to thank Dr. Lowther (CEFAS) for designing the probe and primer sequences for FCV.

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