Comparison of chlorine and peroxyacetic-based disinfectant to inactivate Feline calicivirus, Murine norovirus and Hepatitis A virus on lettuce

In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission to humans of enteric viruses, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur during all steps of food processing, primary production is a critical stage on which prevention measures must be focused to minimize the risk of infection to consumers. Postharvest sanitation may be a valid technological solution for decreasing the bacterial load on fresh raw material, but there is a lack of data concerning the effectiveness of this process on enteric viruses. In this study, we compared the survival of two human norovirus surrogates, the feline calicivirus (FCV), and the murine norovirus (MNV-1), and of HAV on lettuce after water washing with bubbles and with or without ultrasound, and washing with bubbles in the presence of active chlorine (15 ppm) or peroxyacetic acid-based disinfectant (100 ppm). Cell culture and quantitative RT-PCR assays were used to detect and quantify the viruses on the surface of the lettuce after the sanitizing treatments. Levels of viral inactivation on the lettuce leaves were not significantly different between washing with bubbles and washing with bubbles plus ultrasound and were not dependant on the quantification method. A simple washing without disinfectant resulted in a decrease of approximately 0.7 log units in the quantity of virus detected for HAV and FCV and of 1.0 log unit for MNV-1.In the experimental set-up including a washing step (with or without ultrasound) followed by washing for 2 min in the presence of disinfectants, 15 ppm of active chlorine was found more effective for inactivating FCV (2.9 log units) than HAV and MNV-1 (1.9 log units and 1.4 log units, respectively) whereas 100 ppm of peroxyacetic-based biocide was found effective for inactivating FCV (3.2 log units) and MNV-1 (2.3 log units), but not HAV (0.7 log units). Quantitative RT-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious viruses on disinfected lettuce, except for MNV-1 processed with chlorine (15 ppm). In comparison with water washing, a substantial additional decrease of genomic FCV titer (1.1 log units) but no significant reduction of the genomic titers of HAV and MNV-1 were found on lettuce treated with chlorine (15 ppm). No significant effect of the disinfection step of lettuce with peroxyacetic-based biocide (100 ppm peracetic acid) was found by qRT-PCR on all genomic viral titers tested. This study illustrates the necessity of determining the effectiveness of technological processes against enteric viruses, using a relevant reference such as HAV, in order to reduce the risk of hepatitis and gastroenteritis by exposure to vegetables.     

The survival of hepatitis A virus in fresh produce

Fresh produce has been repeatedly implicated as the source of human viral infections, including infection with hepatitis A virus (HAV). The objective of the present study was to evaluate the HAV adsorption capacity of the surface of various fresh vegetables that are generally eaten raw and the persistence of the HAV. To this end, the authors experimentally contaminated samples of lettuce, fennel, and carrot by immersing them in sterile distilled water supplemented with an HAV suspension until reaching a concentration of 5 log tissue culture infectious dose (TCID50)/ml. After contamination, the samples were stored at 4 degrees C and analysed at 0, 2, 4, 7, and 9 days. To detect the HAV, RT-nested-PCR was used; positive samples were subjected to the quantitative determination using cell cultures. The three vegetables differed in terms of their adsorption capacity. The highest quantity of virus was consistently detected for lettuce, for which only a slight decrease was observed over time (HAV titre = 4.44 +/- 0.22 log TCID50/ml at day 0 vs. 2.46 +/- 0.17 log TCID50/ml at day 9, before washing). The virus remained vital through the last day of storage. For the other two vegetables, a greater decrease was observed, and complete inactivation had occurred at day 4 for carrot and at day 7 for fennel. For all three vegetables, washing does not guarantee a substantial reduction in the viral contamination.     

Adhesion of human pathogenic enteric viruses and surrogate viruses to inert and vegetal food surfaces

Enteric viruses, particularly human Noroviruses (NoV) and hepatitis A virus (HAV), are key food-borne pathogens. The attachment of these pathogens to foodstuff and food-contact surfaces is an important mechanism in the human contamination process. Studies were done to investigate the nature of the physicochemical forces, such as hydrophobic and electrostatic ones, involved in the interaction virus/matrix but, at this day, only few data are available concerning surface properties of viruses and prediction of the adhesion capacity of one specific virus onto matrices is still very difficult. The purpose of this study was to propose a reference system, including a representative virus surrogate, able to predict as close as possible behaviour of pathogenic viruses in term of adhesion on inert (stainless steel and polypropylene) and food surfaces (lettuce leaves, strawberries and raspberries). The adhesion of human pathogenic enteric viruses, cultivable strain of HAV and non-cultivable strains of human NoV (genogroups I and II), have been quantified and compared to these of human enteric viruses surrogates, included the MNV-1 and three F-specific RNA bacteriophages (MS2, GA and Qβ). A standardized approach was developed to assess and quantify viral adhesion on tested matrices after a contact time with each virus using real-time RT-PCR. Methods used for virus recovery were in accordance with the CEN recommendations, including a bovine Enterovirus type 1 as control to monitor the efficiency of the extraction process and amplification procedure from directly extracted or eluted samples. The adhesion of human pathogenic viruses, ranging from 0.1 to 2%, could be comparable for all matrices studied, except for NoV GII on soft fruits. Adhesion percentages obtained for the studied surrogate virus and phages were shown to be comparable to those of HAV and NoV on inert and lettuce surfaces. The MNV-1 appeared as the best candidate to simulate adhesion phenomena of all human pathogenic enteric viruses on all studied surfaces, while MS2 and GA bacteriophages could be a good alternative as model of viral adhesion on inert and lettuce surfaces. These results will be usable to design relevant experimental systems integrating adhesion behaviour of enteric viruses in the assessment of the efficiency of a technological or hygienic industrial process.     

Analytical Methods for Virus Detection in Water and Food

Potential ways to address the issues that relate to the techniques for analyzing food and environmental samples for the presence of enteric viruses are discussed. It is not the authors?? remit to produce or recommend standard or reference methods but to address specific issues in the analytical procedures. Foods of primary importance are bivalve molluscs, particularly, oysters, clams, and mussels; salad crops such as lettuce, green onions and other greens; and soft fruits such as raspberries and strawberries. All types of water, not only drinking water but also recreational water (fresh, marine, and swimming pool), river water (irrigation water), raw and treated sewage are potential vehicles for virus transmission. Well over 100 different enteric viruses could be food or water contaminants; however, with few exceptions, most well-characterized foodborne or waterborne viral outbreaks are restricted to hepatitis A virus (HAV) and calicivirus, essentially norovirus (NoV). Target viruses for analytical methods include, in addition to NoV and HAV, hepatitis E virus (HEV), enteroviruses (e.g., poliovirus), adenovirus, rotavirus, astrovirus, and any other relevant virus likely to be transmitted by food or water. A survey of the currently available methods for detection of viruses in food and environmental matrices was conducted, gathering information on protocols for extraction of viruses from various matrices and on the various specific detection techniques for each virus type.     

UV light inactivation of hepatitis A virus, Aichi virus, and feline calicivirus on strawberries, green onions, and lettuce

A majority of illnesses caused by foodborne viruses are associated with fresh produce. Fruits and vegetables may be considered high-risk foods, as they are often consumed raw without a specific inactivation step. Therefore, there is a need to evaluate nonthermal treatments for the inactivation of foodborne pathogens. This study investigates the UV inactivation of three viruses: feline calicivirus (a surrogate for norovirus), and two picornaviruses, hepatitis A virus and Aichi virus. Three produce types were selected for their different surface topographies and association with outbreaks. Green onions, lettuce, and strawberries were individually spot inoculated with 10(7) to 10(9) 50% tissue culture infective doses (TCID50) of each virus per ml and exposed to UV light at various doses (< or = 240 mW s/cm2), and viruses were eluted using an optimized recovery strategy. Virus infection was quantified by TCID50 in mammalian cell culture and compared with untreated recovered virus. UV light applied to contaminated lettuce resulted in inactivation of 4.5 to 4.6 log TCID50/ml; for contaminated green onions, inactivation ranged from 2.5 to 5.6 log TCID50/ml; and for contaminated strawberries, inactivation ranged from 1.9 to 2.6 log TCID50/ml for the three viruses tested. UV light inactivation on the surface of lettuce is more effective than inactivation on the other two produce items. Consistently, the lowest results were observed in the inactivation of viruses on strawberries. No significant differences (P > 0.05) for virus inactivation were observed among the three doses applied (40, 120, and 240 mW s/cm2) on the produce, with the exception of hepatitis A virus and Aichi virus inactivation on green onions, where inactivation continued at 120 mW s/cm2 (P < 0.05).     

Chlorine disinfection of produce to inactivate hepatitis A virus and coliphage MS2

Disinfection of produce is principally used to inactivate spoilage microbes and may also reduce the risk of consumer exposure to enteric pathogens. However, the rate and extent of enteric virus inactivation by free chlorine on produce has not been adequately characterized. Experiments were performed to determine the kinetics of free chlorine inactivation of hepatitis A virus (HAV) and the indicator virus coliphage MS2 on strawberries (SBs), cherry tomatoes (CTs), and head lettuce (HL). The oxidant demand of these produce items also was determined. When produce items were exposed to approximately 20 parts per million (ppm) solution of free chlorine for 5-10 min, HAV and MS2 were inactivated by 90-99% and in some cases virus inactivation was >/=99%. Exposure of strawberries to approximately 200 ppm free chlorine resulted in more rapid and extensive inactivation of both viruses. The produce items tested in this study exhibited a demand for chlorine which varied by produce type, and chlorine residuals declined over time. These results demonstrate the potential for chlorine to reduce the levels of infectious viruses on different produce types, but adequate contact time and chlorine residual are required to achieve maximum virus inactivation. The difference in chlorine demand between SBs, CTs, and HL suggests that varying disinfection practices are needed for the wide variety of processed fruits and vegetables. The inactivation kinetics of MS2 and HAV were similar, suggesting that MS2 and perhaps other similar bacterial viruses may be used as process indicators and surrogates for determining the disinfection efficacy of produce in the laboratory or in actual practice.     

Inactivation of hepatitis A virus and a calicivirus by high hydrostatic pressure

Potential application of high hydrostatic pressure processing (HPP) as a method for virus inactivation was evaluated. A 7-log10 PFU/ml hepatitis A virus (HAV) stock, in tissue culture medium, was reduced to nondetectable levels after exposure to more than 450 MPa of pressure for 5 min. Titers of HAV were reduced in a time- and pressure-dependent manner between 300 and 450 MPa. In contrast, poliovirus titer was unaffected by a 5-min treatment at 600 MPa. Dilution of HAV in seawater increased the pressure resistance of HAV, suggesting a protective effect of salts on virus inactivation. RNase protection experiments indicated that viral capsids may remain intact during pressure treatment, suggesting that inactivation was due to subtle alterations of viral capsid proteins. A 7-log10 tissue culture infectious dose for 50% of the cultures per ml of feline calicivirus, a Norwalk virus surrogate, was completely inactivated after 5-min treatments with 275 MPa or more. These data show that HAV and a Norwalk virus surrogate can be inactivated by HPP and suggest that HPP may be capable of rendering potentially contaminated raw shellfish free of infectious viruses.     

A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables

Fresh-cut vegetables are prone to be contaminated with foodborne pathogens during growth, harvest, transport and further processing and handling. As most of these products are generally eaten raw or mildly treated, there is an increase in the number of outbreaks caused by viruses and bacteria associated with fresh vegetables. Foodborne pathogens are usually present at very low levels and have to be concentrated (i.e. viruses) or enriched (i.e. bacteria) to enhance their detection. With this aim, a rapid concentration method has been developed for the simultaneous recovery of hepatitis A virus (HAV), norovirus (NV), murine norovirus (MNV) as a surrogate for NV, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica. Initial experiments focused on evaluating the elution conditions suitable for virus release from vegetables. Finally, elution with buffered peptone water (BPW), using a Pulsifier, and concentration by polyethylene glycol (PEG) precipitation were the methods selected for the elution and concentration of both, enteric viruses and bacteria, from three different types of fresh-cut vegetables by quantitative PCR (qPCR) using specific primers. The average recoveries from inoculated parsley, spinach and salad, were ca. 9.2%, 43.5%, and 20.7% for NV, MNV, and HAV, respectively. Detection limits were 132 RT-PCR units (PCRU), 1.5 50% tissue culture infectious dose (TCID₅₀), and 6.6 TCID₅₀ for NV, MNV, and HAV, respectively. This protocol resulted in average recoveries of 57.4%, 64.5% and 64.6% in three vegetables for E. coli O157:H7, L. monocytogenes and Salmonella with corresponding detection limits of 10³, 10² and 10³ CFU/g, respectively.Based on these results, it can be concluded that the procedure herein is suitable to recover, detect and quantify enteric viruses and foodborne pathogenic bacteria within 5 h and can be applied for the simultaneous detection of both types of foodborne pathogens in fresh-cut vegetables.     

Inactivation of feline calicivirus as a surrogate for norovirus on lettuce by electron beam irradiation

Caliciviridae, including norovirus, are considered important sources of human gastroenteritis. As leafy green vegetables are commonly consumed without additional processing, it is important to evaluate interventions to reduce the presence of human pathogens in these products. Feline calicivirus was used as a model for small round structured viruses on lettuce. The lettuce was inoculated by immersion to simulate contamination from irrigation or wash water. The inoculated lettuce was then exposed to electron beam irradiation at various dose levels to determine survival. The D??-value of the calicivirus on lettuce was determined to be 2.95 kGy. Irradiation to reduce bacterial pathogens on cut lettuce could also reduce the risk associated with small round structured viruses on lettuce.     

Inactivation of MS2 F(+) coliphage on lettuce by a combination of UV light and hydrogen peroxide

The efficacy of a produce decontamination method based on a combination of UV light (254 nm) and hydrogen peroxide (H2O2) to inactivate the MS2 F(+) coliphage inoculated onto iceberg lettuce was evaluated. Lettuce inoculated with 6.57 log PFU of MS2 was reduced by 0.5 to 1.0 log unit when illuminated with UV light alone for 20 to 60 s (12.64 to 18.96 mJ/cm2). In contrast, a 3-log reduction in MS2 was achieved with 2% (vol/vol) H2O2 spray delivered at 50 degrees C. No significant increase in log count reduction (LCR) was observed when H2O2 and UV light were applied simultaneously. However, H2O2 sprayed onto lettuce samples for 10 s, followed by a further 20-s UV illumination, resulted in an LCR of 4.12 that compares with the 1.67 obtained with 200 ppm of calcium hypochlorite wash. No further increase in MS2 inactivation was achieved by the use of either longer H2O2 spray or UV illumination times. The extent of MS2 reduction was significantly (P < 0.05) decreased when the H2O2 spray was delivered at 10 or 25 degrees C compared with 50 degrees C. In the course of aerobic storage at 4 degrees C, lettuce treated with UV light and H2O2 (10 or 25 degrees C) developed discoloration (polyphenol accumulation) within 6 days. In contrast, lettuce treated with UV light and H2O2 at 50 degrees C developed less discoloration within this time period and was comparable to untreated controls. This study demonstrated that the combination of UV light and H2O2 represents an alternative to hypochlorite-based washes to reduce the carriage of viruses on fresh produce.     

Inactivation of hepatitis A virus and norovirus surrogate in suspension and on food-contact surfaces using pulsed UV light (pulsed light inactivation of food-borne viruses)

This study was conducted to evaluate the inactivation of murine norovirus (MNV-1) and hepatitis A virus (HAV) by pulsed ultraviolet (UV) light. MNV-1 was used as a model for human norovirus. Viral suspensions of about 10⁶ PFU/ml were exposed to pulses of UV light for different times and at different distances in a Xenon Steripulse device (model RS-3000C). Inactivation studies were also carried out on 1-cm² stainless steel and polyvinyl chloride disks with 10⁵ PFU/ml. Inactivation of MNV-1 and HAV at 10.5 cm from the UV source was greater on inert surfaces than in suspension. The presence of organic matter (fetal bovine serum) reduced the effectiveness of pulsed light both in suspension and on surfaces. However, 2-s treatment in the absence of FBS completely inactivated (5 log reduction) the viral load at different distances tested, whether in suspension (MNV-1) or on disks (MNV-1 and HAV). The same treatment in the presence of fetal bovine serum (5%) allowed a reduction of about 3 log. This study showed that short duration pulses represent an excellent alternative for inactivation of food-borne viruses. This technology could be used to inactivate viruses in drinking water or on food-handling surfaces.     

Seed decontamination as an intervention step for eliminating Escherichia coli on salad vegetables and herbs

The efficacy of seed decontamination to enhance the safety of salad vegetables and herbs was evaluated. Seeds (celery, coriander, lettuce, spinach and watercress) were inoculated (at a level of 3–5 log cfu g⁻¹) with either Escherichia coli P36 or Listeria monocytogenes NCTC 7973 and decontaminated with ozone gas, acidified sodium chlorite (ASC) or quaternary ammonium salt preparation (QAS). None of the treatments applied were initially effective at inactivating E coli on lettuce or spinach seeds as the bacterium could be recovered on the subsequent seedlings. However, as the cultivation period progressed, E coli numbers on plants derived from decontaminated seeds declined to below the level of detection. Interestingly, E coli persisted on the surface of lettuce and spinach leaves from inoculated non‐treated seeds throughout the 42‐day cultivation period. E coli also persisted on coriander derived from inoculated non‐treated seeds although it was isolated sporadically on plants derived from QAS‐decontaminated seed. E coli numbers progressively declined on celery and watercress regardless of the seed decontamination treatment being applied. No L monocytogenes was recovered from any of the seedlings, possibly because of the growth‐suppressing effect of endogenous microflora. The results suggest that effective on‐farm controls, such as seed decontamination, should be considered in order to reduce the risk of pathogens associated with salad vegetables and herbs. Copyright © 2005 Society of Chemical Industry     

Survival of poliovirus on soft fruit and salad vegetables

A series of studies were performed, using poliovirus, to ascertain the potential for enteric pathogenic viruses to survive on various foodstuffs. The extraction protocols, which could be performed in just a few hours, were developed for use with quantities of food that would normally constitute a portion for consumption. The protocols were based on elution of viruses from food surfaces, followed by differential centrifugation to remove food debris and concentrate viruses. The studies were mostly performed using fresh produce stored at refrigeration temperature for 2 weeks or so, which was considered to represent the maximum time elapsing between purchase and consumption. Each food sample was inoculated with a viral suspension, and samples were analyzed immediately and at intervals throughout the experiment. Statistical analyses were performed on the results, and the decimal reduction times (D-values), or number of days after which the initial virus numbers had declined by 90%, were calculated. In summary, the resulting D-values were as follows: lettuce, 11.6 days; green onion, no decline; white cabbage, 14.2 days; fresh raspberries, no decline; and frozen strawberries, 8.4 days. The results showed that enteric viruses may persist on fresh fruit and vegetables for several days under conditions commonly used for storage in households. Therefore, if contamination has occurred before purchase, there will always be a risk of infection from consumption of the food.