Interaction of human norovirus and its surrogates with fresh produce

Date
2016
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Publisher
University of Delaware
Abstract
Produce has been identified as the most common source of foodborne outbreaks in the United States. Among those outbreaks, human norovirus (huNoV) is the leading cause. HuNoV can contaminate produce at any point from farm to table. In order to reduce contamination events, the Food Safety Modernization Act (FSMA) has mandated the implementation of good agricultural practices. However, due to the variety of growing conditions, commodity and cultivar types, as well as pre- and postharvest practices, it is still a great challenge to provide best practices to ensure produce safety. Sprouted seeds have been involved in numerous foodborne outbreaks in the United States and across the world. Additionally, microgreens are gaining in popularity, but there is a lack of information pertaining to the microbiological safety of microgreens, particularly of those grown hydroponically. The potential risks associated with virus contamination of crops within a hydroponic system have not been studied to date. In order to better prevent foodborne outbreaks and protect public health, it is urgent to investigate the interaction between foodborne pathogens and fresh produce. Many studies have focused on bacterial pathogens, but little knowledge exits on the interaction between huNoV and fresh produce. Furthermore, measuring norovirus infectivity is still a challenge due to a lack of appropriate cell lines and limitations associated with volunteer studies. Using surrogates to predict the behavior of huNoV is considered as a promising method to characterize its survivability in different environmental conditions. The objectives of this project were to 1) investigate the survival and transfer of enteric viruses during seed storage and germination; 2) address how those viruses can be inactivated by intervention strategies before germination, including traditional chlorine washes as well as the novel non-thermal processing technology, application of aqueous ozone; 3) evaluate viruses and bacteria for their ability to become internalized from root to edible tissues of microgreens and secondly evaluate virus survival in re-circulated water without adequate disinfection; 4) increase the titers of Tulane virus (TV), and characterize the interaction between TV and Caco-2 cells, to better understand the mechanism of huNoV infection. For seeds and sprouted seeds, viruses including murine norovirus (MNV), TV, and hepatitis A virus (HAV) were persistent and remained infectious for a prolonged period of time during seed storage with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID50 /g after 50 days, respectively. Additionally, contaminated alfalfa seeds were allowed to germinate, virus was transferred from seeds to sprouts and was located in all tissues with low titers (~1-3 log PFU/g for MNV and TV, or ~2.5- 3.5 log TCID50/g for HAV) as well as spent water (~1-3 log PFU/ml for MNV and TV, or ~2-3.5 log TCID50/ml for HAV) during germination. These findings highlight the importance of sanitation and prevention procedures before germination. Further, traditional calcium hypochlorite treatment as well as a non-thermal technology of aqueous ozone were applied on inoculated seeds to determine their inactivation effectiveness. Data showed that both calcium hypochlorite and aqueous ozone resulted in significant reductions of viruses (and bacteria) inoculated on seeds. Calcium hypochlorite at 20,000 ppm was more effective than 2,000 ppm for all the organisms tested. The reductions of 20,000 ppm calcium hypochlorite were 3.75 ± 0.42 log PFU/g and 2.29 ± 0.16 log PFU/g for MNV and TV in alfalfa seeds, respectively; whereas huNoV GII was reduced in seeds by 1.65 ± 0.40 log genomic copies/g. The effectiveness on viral inactivation decreased as the organic load increased. For calcium hypochlorite treatment, it is likely that both MNV and TV behave similarly at lower levels of hypochlorite; however, MNV is more sensitive to chlorine than is TV at relatively high levels (20,000 ppm) of calcium hypochlorite with ~ 1 log PFU/ml more reduction than that of TV. For aqueous ozone treatment, TV (reductions range from 1.66 to 3.83 log PFU/g) in alfalfa seeds was significantly more resistant compared to MNV (reductions range from 4.04 to 5.60 log PFU/g) in terms of infectivity. Interestingly, viral genomes were relatively resistant in seeds; reduction of TV genomic copies present in seeds was similar to that of huNoV with Dvalues (genomic copies) of 27.04 s and 27.73 s, respectively; whereas MNV had significantly greater reductions in genomic copies with D-value of 24.37 s. TV was determined to be more environmental robust than MNV with less reduction in infectivity observed both on seeds treated by calcium hypochlorite and aqueous ozone. Therefore, with greater retention of infectivity and more robust to disinfectant inactivation than MNV, TV makes it as a promising worst-case model for estimating huNoV. For microgreens, both viruses and bacteria were detected in kale and mustard microgreen roots and were translocated to edible tissues via contaminated irrigation water. The levels of viral and bacterial uptake in edible portions and roots were relatively persistent during harvest (~1-2 log PFU/sample for viruses, and ~ 2-3 log CFU/sample for bacteria, respectively). Cross-contamination occurred easily. Even after an initial contamination event is removed, viruses can still be present and recirculated in water, taken up through the roots of microgreens, and transferred to edible tissues. These findings reinforce the need for adequate and diligent sanitation. The information on the transfer and internalization of viruses and bacteria in microgreens via contaminated water, as well as previously determined pre-harvest inactivation rates of pathogens present in fresh produce will be useful to conduct quantitative microbial risk assessment in the future, and the effectiveness of appropriate sanitation can be determined. Results showed that good agriculture practice as well as diligent sanitation are necessary to prevent foodborne outbreaks, which is the goal of produce guidelines and regulations. INDEX WORDS: Human Norovirus, Murine Norovirus, Tulane Virus, Produce, Decontamination, Cross-contamination, Pre-harvest, Transfer, Survival
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