Internalization of Murine Norovirus 1 by Lactuca sativa during Irrigation

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Hydroponically grown microgreens are gaining in popularity, but there is a lack of information pertaining to their microbiological safety. The potential risks associated with virus contamination of crops within a hydroponic system have not been studied to date. Here a human norovirus (huNoV) surrogate (murine norovirus [MNV]) was evaluated for its ability to become internalized from roots to edible tissues of microgreens. Subsequently, virus survival in recirculated water without adequate disinfection was assessed. Kale and mustard seeds were grown on hydroponic pads (for 7 days with harvest at days 8 to 12), edible tissues (10 g) were cut 1 cm above the pads, and corresponding pieces (4 cm by 4 cm) of pads containing only roots were collected separately. Samples were collected from a newly contaminated system (recirculated water inoculated with ϳ3 log PFU/ml MNV on day 8) and from a previously contaminated system. (A contaminated system without adequate disinfection or further inoculation was used for production of another set of microgreens.) Viral titers and RNA copies were quantified by plaque assay and real-time reverse transcription (RT)-PCR. The behaviors of MNV in kale and mustard microgreens were similar (P > 0.05). MNV was detected in edible tissues and roots after 2 h postinoculation, and the levels were generally stable during the first 12 h. Relatively low levels (ϳ2.5 to ϳ1.5 log PFU/sample of both edible tissues and roots) of infectious viruses were found with a decreasing trend over time from harvest days 8 to 12. However, the levels of viral RNA present were higher and consistently stable (ϳ4.0 to ϳ5.5 log copies/sample). Recirculated water maintained relatively high levels of infectious MNV over the period of harvest, from 3.54 to 2.73 log PFU/ml. Importantly, cross-contamination occurred easily; MNV remained infectious in previously contaminated hydroponic systems for up to 12 days (2.26 to 1.00 PFU/ml), and MNV was detected in both edible tissues and roots. Here we see that viruses can be recirculated in water, even after an initial contamination event is removed, taken up through the roots of microgreens, and transferred to edible tissues. The ease of product contamination shown here reinforces the need for proper sanitation. Hydroponics is gaining in popularity, with consistent growth across the United States (1). Specifically growing microgreens hydroponically is a new trend in the food industry (1). Microgreens have been defined as salad crop shoots harvested for consumption within 10 to 20 days of seedling emergence (2). Microgreens are considered a gourmet food to add taste, color, and texture to dishes; they mainly appear in fine and upscale restaurants and have been gaining attention and popularity during the past few years due to the fresh appearance and health benefits (3, 4). Both microgreens and sprouts are usually grown within indoor facilities under controlled environmental conditions to minimize potential contamination of foodborne pathogens. However, microgreens are different ...

Section: Discussionmentioning

confidence: 77%

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confidence: 99%

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confidence: 99%

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Survival and Transfer of Murine Norovirus within a Hydroponic System during Kale and Mustard Microgreen Harvesting

Wang

Kniel

2016

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“…A number of studies reported that HuNoV and its surrogates can internalize inside lettuce and spinach plants under normal conditions (28,43,44) and in lettuce under certain biotic and extreme abiotic stress a Numbers in bold indicate a significant effect (P Ͻ 0.05) for that factor. Statistical analyses were performed by two-way ANOVA.…”

Section: Discussionmentioning

confidence: 99%

Abiotic Stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and Its Surrogates on Preharvest Leafy Greens

Esseili

Gao

Tegtmeier

et al. 2016

Foodborne outbreaks of human noroviruses (HuNoVs) are frequently associated with leafy greens. Because there is no effective method to eliminate HuNoV from postharvest leafy greens, understanding virus survival under preharvest conditions is crucial. The objective of this study was to evaluate the survival of HuNoV and its surrogate viruses, murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV), on preharvest lettuce and spinach that were subjected to abiotic stress (physical damage, heat, or flood). We also examined the bacteria culturable from the phyllosphere in response to abiotic stress and in relation to viral persistence. Mature plants were subjected to stressors 2 days prior to inoculation of the viruses on leaves. We quantified the viral RNA, determined the infectivity of the surrogates, and performed bacterial counts on postinoculation days (PIDs) 0, 1, 7, and 14. For both plant types, time exerted significant effects on HuNoV, MNV, SaV, and TV RNA titers, with greater effects being seen for the surrogates. Infectious surrogate viruses were undetectable on PID 14. Only physical damage on PID 14 significantly enhanced HuNoV RNA persistence on lettuce, while the three stressors differentially enhanced the persistence of MNV and TV RNA. Bacterial counts were significantly affected by time and plant type but not by the stressors. However, bacterial counts correlated significantly with HuNoV RNA titers on spinach and with the presence of surrogate viruses on both plant types under various conditions. In conclusion, abiotic stressors and phyllosphere bacterial density may differentially influence the survival of HuNoV and its surrogates on lettuce and spinach, emphasizing the need for the use of preventive measures at the preharvest stage.H uman noroviruses (HuNoVs) are the leading cause of acute gastroenteritis and foodborne illnesses in the United States, causing 19 million to 21 million cases, 570 to 800 deaths, and $777 million in health care-associated costs annually (1). These singlestranded RNA viruses belong to the Caliciviridae family and are 28 to 35 nm in diameter and nonenveloped. Although these viruses cause self-limiting gastroenteritis, more serious infections can develop in high-risk groups, such as the elderly and immunocompromised populations (2). The virus is mainly transmitted to humans via the fecal-oral route either by contact with an infected person or fomites or by ingestion of contaminated food and water (2). Leafy greens are frequently associated with HuNoV outbreaks (1) and are globally recognized to be a high priority in terms of the microbial safety of fresh produce (3). Contamination of leafy greens with HuNoVs can occur at any stage along the farm-to-fork chain (3) through a number of sources, including fecally contaminated water used for irrigation, improperly treated sewage sludge used for fertilization, and food harvesters or food handlers who use improper hygiene practices (4, 5). Because HuNoVs have a low infectious dose (ϳ18 to 1,000 viral particles) (6) ...

“…Viruses, including norovirus and rotavirus, cause foodborne outbreaks, especially due to their persistence in the environment and their low infectious doses (10 to 100 particles) (7,8). Murine norovirus, Tulane virus, and sapovirus have been found to attach to the surface of or within the tissues of romaine lettuce and strawberries (9)(10)(11)(12)(13), which suggests that preharvest viral contamination is a public health concern. To reduce the incidence of viral foodborne illnesses caused by contaminated produce, it is essential to understand the environmental and agricultural factors that control the stability and, therefore, the infectivity of foodborne viruses.…”

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confidence: 99%

Effect of Leaf Surface Chemical Properties on Efficacy of Sanitizer for Rotavirus Inactivation

Fuzawa

Palma-Salgado

et al. 2016

The use of sanitizers is essential for produce safety. However, little is known about how sanitizer efficacy varies with respect to the chemical surface properties of produce. To answer this question, the disinfection efficacies of an oxidant-based sanitizer and a new surfactant-based sanitizer for porcine rotavirus (PRV) strain OSU were examined. PRV was attached to the leaf surfaces of two kale cultivars with high epicuticular wax contents and one cultivar of endive with a low epicuticular wax content and then treated with each sanitizer. The efficacy of the oxidant-based sanitizer correlated with leaf wax content as evidenced by the 1-log 10 PRV disinfection on endive surfaces (low wax content) and 3-log 10 disinfection of the cultivars with higher wax contents. In contrast, the surfactant-based sanitizer showed similar PRV disinfection efficacies (up to 3 log 10 ) that were independent of leaf wax content. A statistical difference was observed with the disinfection efficacies of the oxidant-based sanitizer for suspended and attached PRV, while the surfactant-based sanitizer showed similar PRV disinfection efficacies. Significant reductions in the entry and replication of PRV were observed after treatment with either disinfectant. Moreover, the oxidant-basedsanitizer-treated PRV showed sialic acid-specific binding to the host cells, whereas the surfactant-based sanitizer increased the nonspecific binding of PRV to the host cells. These findings suggest that the surface properties of fresh produce may affect the efficacy of virus disinfection, implying that food sanitizers should be carefully selected for the different surface characteristics of fresh produce. IMPORTANCEFood sanitizer efficacies are affected by the surface properties of vegetables. This study evaluated the disinfection efficacies of two food sanitizers, an oxidant-based sanitizer and a surfactant-based sanitizer, on porcine rotavirus strain OSU adhering to the leaf epicuticular surfaces of high-and low-wax-content cultivars. The disinfection efficacy of the oxidant-based sanitizer was affected by the surface properties of the vegetables, while the surfactant-based sanitizer was effective for both high-and low-wax leafy vegetable cultivars. This study suggests that the surface properties of vegetables may be an important factor that interacts with disinfection with food sanitizers of rotaviruses adhering to fresh produce. There are 48 million estimated annual incidents of foodborne illness in the United States (1-3). Of these incidents, 128,000 cases require hospitalization and 3,000 result in death (1-3). The annual cost associated with foodborne illness, including human morbidity and mortality, is estimated to range between $14.1 and $152 billion (4-6). Viruses, including norovirus and rotavirus, cause foodborne outbreaks, especially due to their persistence in the environment and their low infectious doses (10 to 100 particles) (7,8). Murine norovirus, Tulane virus, and sapovirus have been found to attach to the surface of or within ...