Incorporating Phage Therapy into WPI Dip Coatings for Applications on Fresh Whole and Cut Fruit and Vegetable Surfaces.

There is a significant unmet need to develop antimicrobial solutions to reduce the risk of contamination in fresh produce. Bacteriophages have been proposed as a potential approach for controlling foodborne pathogens. This study evaluated the combination of edible dip coatings with T7 bacteriophages on whole and cut produce. The evaluation includes an assessment of phage loading, phage storage stability, antimicrobial activity, and phage stability during simulated gastric digestion on sliced cucumbers, sliced apples, and whole cherry tomatoes. In this evaluation, phages coated on fresh produce using edible whey protein isolate (WPI) were compared with phages coated from an aqueous suspension (control coating). The results demonstrated that WPI coatings load more phages than the control and enhanced phage stability during cold storage (4 °C) for cut apples and whole cherry tomatoes. Phage stability decreased by 1 to 3 log(PFU) in a simulated gastric environment. Phage antimicrobial activity against Escherichia coli BL21 decreased 2 to 4 log(CFU) of bacteria on cut apples and whole cherry tomatoes, while no significant bacterial reduction was observed for sliced cucumbers. Overall, the results show that WPI dip coating provides phage loading, stability, and antimicrobial activity to produce surfaces compared to the control coating, and thus may be considered an effective approach for extending phage therapy on fresh produce. PRACTICAL APPLICATION: The practical application is to prevent bacterial cross contamination of fresh produce by using a combination of edible coating with bacteriophages. The results demonstrate enhanced loading and stability of phages on fresh produce when used in combination with an edible coating.

Efficacy of Combination Treatment with Sodium Metasilicate and Sodium Hypochlorite for Inactivation of Norovirus on Fresh Vegetables.

In recent years, fresh vegetables have frequently been associated with the foodborne transmission of enteric viruses, such as human norovirus (NoV). Therefore, several studies have focused on developing methods to inactivate foodborne viruses for preventing outbreaks of foodborne illnesses. Sodium hypochlorite (NaOCl) is commonly used as a disinfectant, but results in undesirable effects on the appearance and taste of foods and can generate toxic byproducts when it exceeds the allowable concentration. Here, we evaluated the efficacy of a range of NaOCl concentrations (50-1000 ppm) for reducing the amounts of human NoV (NoV GII.4) on lettuce (Lactuca sativa), celery (Apium graveolens L.), and white cabbage (Brassica oleracea ssp. capitata). In addition, the combination treatment of NaOCl and sodium metasilicate (SMS, 0.1-0.5%) pentahydrate was evaluated for its ability to decrease the populations of NoV GII.4 in the three food samples. An immunomagnetic separation procedure combined with reverse transcription quantitative polymerase chain reaction was used for virus detection. For lettuce, celery, and cabbage, the NoV GII.4 recovery rates were 57.3% ± 6.5%, 52.5% ± 1.7%, and 60.3% ± 3.9%, respectively, using a glycine/NaCl elution buffer (0.25 M glycine/0.14 M NaCl, pH 9.5). The reductions of NoV GII.4 were 3.17, 3.06, and 3.27 log10 genomic copies/µL for lettuce, celery, and cabbage, respectively, at 1000 ppm NaOCl, while a reduction of ∼3 log10 genomic copies/µL was obtained when the samples were treated with a combination of 100 ppm NaOCl and 0.4% SMS pentahydrate. Taken together, these results demonstrated that combined treatment with NaOCl and SMS pentahydrate was an efficient strategy to reduce the concentration of NaOCl for control of NoV GII.4 contamination in fresh vegetables.

Internalization and dissemination of human norovirus and Tulane virus in fresh produce is plant dependent.

Human norovirus (NoV) is a leading cause of fresh produce associated outbreaks. Previous research indicates that the roots of growing leafy greens and berries internalize human NoV. However the effect of plant type and inoculum level on internalization rates has not been directly compared. In this study we compared the internalization and dissemination rates of human NoV and its surrogate, Tulane virus (TV) in green onion, radishes, and Romaine lettuce. We also evaluated the effect inoculum level and plant growth matrix on the rate of viral internalization. In the hydroponic growth system, we detected internalization and dissemination of human NoV RNA in green onions. In hydroponically growing green onions inoculated with high titer TV, we found higher rates of internalization and dissemination compared to green onions inoculated with low titer TV. In soil growth systems, no infectious TV was detected in either green onion or radishes. However, in Romaine lettuce plants grown in soil approximately 4 log10 PFU/g was recovered from all tissues on day 14 p.i. Overall, we found that the type of plant, growth matrix, and the inoculum level influences the internalization and dissemination of human NoV and TV.

High hydrostatic pressure inactivation of murine norovirus and human noroviruses on green onions and in salsa.

In this study, high hydrostatic pressure (HHP) was evaluated as an intervention for human noroviruses (HuNoVs) in green onions and salsa. To determine the effect of water during HHP treatment on virus inactivation, a HuNoV surrogate, murine norovirus 1 (MNV-1), was inoculated onto green onions and then HHP-treated at 350MPa with or without water at 4 or 20°C. The presence of water enhanced HHP inactivation of MNV-1 on green onions at 4°C but not at 20°C. To test the temperature effect on HHP inactivation of MNV-1, inoculated green onions were HHP-treated at 300MPa at 1, 4 and 10°C. As the temperature decreased, MNV-1 became more sensitive to HHP treatment. HHP inactivation curves of MNV-1 on green onions and salsa were obtained at 300 or 350MPa for 0.5-3min at 1°C. All three inactivation curves showed a linear relationship between log reduction of MNV-1 and time. D values of HHP inactivation of MNV-1 on green onions were 1.10 and 0.61min at 300 and 350MPa, respectively. The D value of HHP inactivation of MNV-1 in salsa at 300MPa was 0.63min. HHP inactivation of HuNoV GI.1 and GII.4 on green onions and salsa was also conducted. To achieve >3 log reduction of HuNoV GI.1, HHP treatments for 2min at 1°C should be conducted at 600MPa and 500MPa for green onions and salsa, respectively. To achieve >3 log reduction of HuNoV GII.4, HHP treatments for 2min at 1°C should be conducted at 500MPa and 300MPa for green onions and salsa, respectively.

Histochemical visualization of ROS and antioxidant response to viral infections of vegetable crops grown in Azerbaijan.

Extremes of environmental conditions, such as biotic stresses, strongly affect plant growth and development and may adversely affect photosynthetic process. Virus infection is especially problematic in crops, because unlike other diseases, its impact cannot be reduced by phytosanitary treatments. The vegetable crops (Solanum lycopеrsicum L, Cucurbita melo L., Cucumis sativus L., Piper longum L., Solánum melongéna L., Vicia faba L.) showing virus-like symptoms were collected from fields located in the main crop production provinces of Azerbaijan. Infection of the plants were confirmed by Enzyme-linked immunosorbent assay using commercial kits for the following viruses: Tomato yellow leaf curl virus, Tomato mosaic virus, Tomato chlorosis virus, Melon necrotic spot virus and Cucumber mosaic virus, Bean common mosaic virus and Bean yellow mosaic virus. Generation sites of superoxide and hydrogen peroxide radicals and activities of enzymes involved in the detoxification of reactive oxygen species (catalase, glutathione reductase, ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase) were examined in uninfected leaves and in leaves infected with viruses. High accumulation of superoxide and hydrogen peroxide radicals was visualized in infected leaves as a purple discoloration of nitro blue tetrazolium and 3,3'-diaminobenzidine tetrahydrochloride. It was found that the activities of APX and CAT significantly increased in all infected samples compared with non-infected ones. Dynamics of GR and Cu/Zn-SOD activities differed from those of CAT and APX, and slightly increased in stressed samples. Electrophoretic mobility profiling of APX, GPX and CAT isoenzymes was also studied.

Comparison of RNA extraction methods for the detection of a norovirus surrogate in ready-to-eat foods.

Four nucleic acid extraction methods were evaluated for the purpose of quantifying a norovirus surrogate (murine norovirus [MNV-1]) concentrated from different food samples. Simple (strawberries and lettuce) and complex (sliced turkey breast, soft-shell clams, and potato salad) food matrices were inoculated with a viral suspension containing high (4×10(5) PFU) or low (4×10(3) PFU) numbers of viral particles. MNV-1 was eluted using either the Pulsifier™ or repetitive pipetting. The four methods were based on using magnetic silica (MiniMAG), non-magnetic silica (bioMérieux Basic kit), silica membrane (Qiagen kit), and phenol (TriReagent) for RNA extraction. The greatest recovery of viral RNA from simple matrices was obtained using magnetic silica for both inoculation levels. For strawberries, the addition of pectinase during the elution step improved RNA recovery when the Pulsifier was used with silica membrane extraction and when repetitive pipetting was used with magnetic silica extraction. In the case of complex matrices, the extraction of high or low numbers of MNV-1 was highest overall using magnetic silica. The exception was soft-shell clams with a high viral load, in which the greatest recovery was obtained with the phenol-based method. In general, magnetic silica was the most effective for extracting both high and low numbers of MNV-1 particles from a wide range of foods.

Importance and genetic diversity of vegetable-infecting tospoviruses in India.

A survey for Peanut bud necrosis virus (PBNV), Watermelon bud necrosis virus (WBNV), Capsicum chlorosis virus (CaCV), and Iris yellow spot virus (IYSV) was conducted between 2002 and 2009 in the major vegetable-growing areas in India. PBNV was documented widely in tomato and chili peppers in 14 states representing southern, north-western, north-eastern, and central regions and WBNV was predominantly detected in watermelons and cucurbits in all except north-eastern regions. In addition, the expanded host range of PBNV to watermelons and other cucurbits and WBNV to tomato and chili peppers was observed leading to natural mixed infection of the two viruses. IYSV was found in onion in southern, central, and north-eastern regions and CaCV in tomato and chili peppers in northern and southern regions, respectively. Phylogenetic analysis of the nucleocapsid gene revealed segregation of field isolates of PBNV and WBNV into two distinct subclades, whereas isolates of CaCV and IYSV each clustered into a single clade. A proposal for establishing WBNV as a distinct tospovirus species is made based on the molecular characterization of small- (S) and medium- (M) RNA segments.

Comparative recovery of foodborne viruses from fresh produce.

A large percentage of foodborne outbreaks are caused by viruses, and outbreaks associated with fresh produce have increased over the past decade within the United States. Virus recovery from food is of the utmost importance in determining the cause of viral outbreaks. While there are many experimental studies investigating viruses on fruits and vegetables, there is a lack of standard techniques concerning the initial inoculation and recovery of viruses. This study investigates the efficiency of methodology in the recovery of three viruses, hepatitis A virus (HAV), Aichi virus, and feline calicivirus, on three different produce surfaces (lettuce, green onions, and strawberries). To do so, three common times of virus inoculation were examined (0.5, 4, and 12 h) along with two routes of inoculation (immersion and spot inoculation), and then two recovery methods were compared (physical removal and chemical extraction/blending) utilizing three different recovery eluents (2% media, beef extract, and phosphate-buffered saline). Results suggested that incubation time did not significantly affect the survival of the viruses on green onions and strawberries, while a significant decrease (p 0.05); however, the percent recovery was greater by extraction/blending methodology.

Procedure for rapid concentration and detection of enteric viruses from berries and vegetables.

Several hepatitis A virus (HAV) and norovirus (NV) outbreaks due to consumption of berries and vegetables have been reported during recent years. To facilitate the detection of enteric viruses that may be present on different fresh and frozen products, we developed a rapid and sensitive detection method for HAV, NV, and rotavirus (RV). Initial experiments focused on optimizing the composition of the elution buffer, improving the viral concentration method, and evaluating the performance of various extraction kits. Viruses were extracted from the food surface by a direct elution method in a glycine-Tris (pH 9.5) buffer containing 1% beef extract and concentrated by ultrafiltration. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. The average detection limits were 1 50% tissue culture infective dose (TCID(50)), 54 RT-PCR units, and 0.02 TCID(50) per 15 g of food for HAV, NV, and RV, respectively. Based on our results, it is concluded that this procedure is suitable to detect and quantify enteric viruses within 6 h and can be applied for surveillance of enteric viruses in fresh and frozen products.