Interaction of human norovirus and its surrogates with fresh produce
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
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