Progress 09/01/13 to 08/31/17
Outputs
Target Audience:Members of the target audience included fresh produce growers, consumers, food processors, researchers, and regulators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training for two graduate students and one post-doctoral associate. How have the results been disseminated to communities of interest?The results have been presented inpeer-reviewed journals and in conference presentations. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
All three objectives have been accomplished as documented below: Objective 1. Determine the attachment/detachment mechanisms of rotavirus on selected leafy vegetable plants and tomatoes grown in a greenhouse. We have determined the number of rotaviruses adsorbed on produce surfaces using group A porcine rotaviruses and 24 cultivars of leafy vegetables and tomato fruits. We have also characterized the physicochemical properties of each produce's outermost surface layer, known as the epicuticle. The number of rotaviruses found on produce surfaces varied among cultivars. Three-dimensional crystalline wax structures on the epicuticular surfaces were found to significantly contribute to the inhibition of viral adsorption to the produce surfaces (p = 0.01). We found significant negative correlations between the number of rotaviruses adsorbed on the epicuticular surfaces and the concentrations of alkanes, fatty acids, and total waxes on the epicuticular surfaces. Partial least square model fitting results suggest that alkanes, ketones, fatty acids, alcohols, contact angle and surface roughness together can explain 60% of the variation in viral adsorption. The results suggest that various fresh produce surface properties need to be collectively considered for efficient sanitation treatments. Up to 10.8% of the originally applied rotaviruses were found on the produce surfaces after three washing treatments, suggesting a potential public health concern regarding rotavirus contamination. In summary, vegetables such as endive without wax crystals on the surface are prone to viral contamination and more attention from both growers and consumers should be given to these vegetables to avoid contamination and to sanitize them. Objective 2. Develop an effective virus decontamination protocol forproduce. Part A. Sanitation efficacy for rotavirus attached to real leaves We have determined the disinfection efficacies of an oxidant-based sanitizer and a new surfactant-based sanitizer for porcine rotavirus (PRV) strain OSU. 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-log10 PRV disinfection on endive surfaces (low wax content) and 3-log10 disinfection of the cultivars with higher wax contents. In contrast, the surfactant-based sanitizer showed similar PRV disinfection efficacies (up to 3-log10) that were independent of leaf wax content. For vegetables with naturally occurring wax crystals, 2 minutes ofwashing with either surfactant-based (0.25% MA with 0.025%TDS) or oxidant-based (50 ppm) sanitizer are recommended. For vegetables without naturally occurring wax crystals, 2 minutes of washing with surfactant-based sanitizer is recommended. Oxidant-based sanitizer will requires longer timer or higher dose. We also estimated the infection risks caused by consumption of fresh produce ('Totem' Belgian endive (Cichorium intybus), containing low epicuticular wax concentration on its surface and 'Starbor' kale (Brassica oleracea), containing high epicuticular wax concentration on its surface) disinfected with either oxidant- or surfactant-based sanitizer. Both cultivar vegetables were assumed to be contaminated with irrigation water containing rotavirus. Surface area and weight of cultivar vegetables measured in this study, previously obtained disinfection data, and literature-based infection parameters were combined in a quantitative microbial risk assessment model to estimate risk of rotavirus infection from consuming fresh produce. Risks were estimated to be sanitizer- and produce-dependent, as the disinfection efficacy of rotavirus adhering to the surface of fresh produce varied. We concluded that properties of produce surface and vegetable sanitizer affected rotavirus infection risks via consumption of these vegetables. Part B. Sanitation efficacy for rotavirus attached to artificial leaves We have determined the efficacy of using an artificial surface that mimics the physico-chemical characteristics of Romaine lettuce and Carmel spinach as a model to study the attachment of porcine rotavirus (PRV), strain OSU, and human rotavirus (HRV), strain ST3. PRV and HRV were spot inoculated on the adaxial surface of fresh produce surfaces of Romaine lettuce and Carmel spinach and to their homolog artificial surfaces. PRV and HRV inoculated on the surfaces were allowed to incubate for two hours at room temperature to promote attachment and were quantified using the plaque forming using assay (PFU). PFU results indicated that no significant differences in attachment were observed for PRV that was inoculated in fresh produce surfaces of Romaine lettuce and Carmel spinach or their homologs artificial surfaces (attachment Romaine lettuce fresh produce and artificial surface = 6.0 ± 0.3 log10 PFU, 5.3 ± 0.7 log10 PFU, 5.4 ± 0.7 log10 PFU; attachment Carmel spinach = 6.1 ± 0.2 log10 PFU, 5.4 ± 0.4 log10 PFU, 5.9 ± 0.2 log10 PFU) and no significant differences were observed for HRV that was inoculated in fresh produce surfaces of Romaine lettuce and Carmel spinach or their homologs artificial surfaces (attachment Romaine lettuce fresh produce and artificial surface = 5.0 ± 0.6 log10 PFU, 4.4 ± 0.2 log10 PFU, 4.4 ± 0.3 log10 PFU; attachment Carmel spinach = 5.4 ± 0.6 log10 PFU, 4.7 ± 0.5 log10 PFU, 4.8 ± 0.8 log10 PFU). In addition, porcine rotavirus (PRV), strain OSU, and human rotavirus (HRV), strain ST3 attached to fresh produce surfaces of Romaine lettuce and Carmel spinach and artificial surfaces of same cultivars were exposed to a one minute sanitization treatment. The washing system included using oxidant-base sanitizers and surfactant-base sanitizer with and without the addition of ultrasound (25 kHz). Our results indicate that the combination of the oxidant-base sanitizer sodium hypochlorite + ultrasound was efficacious in removing PRV attached to fresh produce surfaces of Romaine lettuce and Carmel spinach compared to attachment to the artificial surfaces tested (up to 1.9 log10 PFU increment in viral reduction for fresh produce). Also, our results indicate a synergistic effect resulting from the combination of the surfactant-base sanitizer 0.5% malic acid + 0.05% TDS + ultrasound; an increment in reduction of viral counts of up to 1.5 log10 PFU for HRV and 1.2 log10 PFU for PRV attached to fresh produce and artificial surfaces was observed when ultrasound was added to the surfactant-base sanitizer washing system. Objective 3. Determine the damage to the viral capsid and genome associated with new sanitizer compositional treatments. We found a statistical difference in 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-based sanitizer-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. Thus, for rotavirus strains that depend on sialic acid receptor for infection, oxidant-based sanitizer needs to damage the VP4 to prevent the attachment step in the virus life cycle. 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.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Palma, S., Nguyen. T.H. and Feng, H. 2017. Use of silicon-based artificial surfaces of Romaine lettuce and spinach leaves for attachment and removal evaluation of Porcine rotavirus strain OSU and Human rotavirus strain ST3. Institute of Food Technologists Annual Meeting, Las Vegas, Nevada.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Fuzawa, M., Smith, R.L., Ku, K.M., Feng, H., Juvik, J.A. and Nguyen, T.H. 2017. Roles of vegetable surface properties and sanitizer type on rotavirus infection risks by consumption of contaminated fresh produce. AEESP, June 22nd, University of Michigan at Ann Arbor.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Fuzawa, M., Ku, K.M., Smith, R.L. and Nguyen, T.H. 2017. Impact of vegetable surface properties and sanitizer type on rotavirus infection risks from consumption of fresh produce: Quantitative microbial risk assessment. IWA Water Microbiology, University of North Carolina at Chapel Hill. May 2017.
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:Members of the target audience include leafy green growers and consumers. Changes/Problems:During the 2017 Spring semester (January through May) the PI for this project will be on sabatical. During this sabatical the PIwill be fully active on the project and no delays in the project are anticipated. What opportunities for training and professional development has the project provided?The project provided training for two graduate students. How have the results been disseminated to communities of interest?Results have been disseminated throughpeer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?We will determine efficacy of chlorine with respect to viral contamination,the mechanisms of viral disinfection by chlorine,and the attachment of rotavirus on artificial leaf surfaces and sanitation efficacy for the rotavirus attached to the artificial leaf surface. In addition, twomore manuscripts will be submitted.
Impacts
What was accomplished under these goals?
Task 3a. Application of chemicals that mimic epicuticular wax composition on the PDMS artificial surface. Our 2015publication (Influence of Epicuticular Physicochemical Properties on Porcine Rotavirus Adsorption to 24 Leafy Green Vegetables and Tomatoes) concluded that chemical composition (alkanes, fatty acids, total waxes) has a negative correlation with rotavirus adsorption to plant surfaces and indicatesthat epicuticular wax on plants plays an important role in allowing human pathogenic bacteria and viral attachment on fresh produce. The objective of this task was to investigate if epicuticular wax can be replicated on the artificial surface using laboratory-grade chemicals. Using a chemical composition profile obtained earlier on this project; several chemical solutions were prepared and added to the surface that mimics spinach and romaine lettuce. A mixture of fatty acids, fatty alcohols and alkanes were selected from the profile; for spinach the 26-carbon fatty alcohol "1-Hexacosanol" was selected, the 14-carbon fatty acid "Myristic acid" was selected and 21-carbon alkane "Heneicosane" was selected. For romaine lettuce the 28-carbon fatty alcohol "Octacosanol" was selected, the 14-carbon fatty acid "Myristic acid" was selected and 18-carbon alkane "Octadecenol" was selected. Elemental composition (surface chemistry) of PDMS surfaces treated with chemical solutions was analyzed using X-Ray Photoelectron Spectroscopy (XPS). According to the XPS output spinach artificial surface elemental composition is O2 % is 26.5 ± 0.3, C % is 52.3 ± 0.4 and Si % is 21.3 ± 0.4; the romaine lettuce artificial composition is O2 % is 24.05 ± 0.22, C % is 57.7 ± 0.3 and Si % is 18.3 ± 0.3. Task 3b. Effect of Leaf Surface Chemical Properties on the Efficacy of Sanitizer for Rotavirus Inactivation. The use of sanitizers is essential for produce safety. However, little is known about how the sanitizer efficacy varies with respect to the chemical surface properties of produce. To answer this question, the disinfection efficacy of an oxidant-based sanitizer and a new surfactant-based sanitizer for porcine rotavirus strain OSU (PRV) was examined. PRV was attached to the leaf surfaces of two kale cultivars with high epicuticular wax content, and one cultivar of endive with low epicuticular wax content and then treated with each sanitizer. The efficacy of the oxidant-based sanitizer correlated with leaf wax content as evidenced in 1-log10 PRV disinfection on endive surfaces (low wax content) and 3-log10 disinfection with the cultivars with higher wax content. In contrast, the surfactant-based sanitizer showed similar PRV disinfection efficacies (up to 3-log10) independent of the leaf's wax content. A statistical difference was observed with disinfection efficacies of the oxidant-based sanitizer for suspended and attached PRV, while the surfactant-based sanitizer showed similar PRV disinfection efficacies. A significant reduction of entry and replication of the PRV was observed after treatment with either disinfectant. Moreover, the oxidant-based sanitizer-treated PRV reduced sialic-acid specific binding to the host cells, whereas the surfactant-based sanitizer increased non-specific 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 different surface characteristics of fresh produce. Task 3c: Rotavirus attachment on PDMS artificial surfaces (ongoing project). The objective is to investigate the survival rate and attachment on the artificial plant surface of two rotavirus strains. Group A, porcine rotavirus (OSU strain), initial stock concentration 7.5 PFU/ml; and tissue culture-adapted human rotavirus strain "ST3" (serotype 4), initial stock concentration 7.6 PFU/ml. 100 μl of either rotavirus strain was spot inoculated on the epicuticular side of the PDMS surfaces that mimic spinach and romaine lettuce, samples were incubated at room temperature for 2 hours in a BSL2 cabinet to allow further viral attachment. Plaque forming unit (PFU) assay was performed to determine rotavirus attachment and survival on an artificial surface. Porcine rotavirus "OSU" attachment on lettuce surface was 3.7 ± 0.2 PFU/ml and on the spinach surface was 3.7 ± 0.1 PFU/ml. Human rotavirus "ST3" attachment on lettuce surface was 3.6 ± 0.1 PFU/ml and on the spinach surface 3.3 ± 0.4 PFU/ml.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
M. Fuzawa, K.-M. Ku, S.P. Palma-Salgado, K. Nagasaka, H. Feng, J.A. Juvik, D. Sano, J.L. Shisler and T.H. Nguyen. 2016. Effect of Leaf Surface Chemical Properties on the Efficacy of Sanitizer for Rotavirus Inactivation. Applied and Environmental Microbiology doi: 10.1128/AEM. 01778-16.
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:The information is useful forvegetable growers, consumers, and researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training for two graduate students and one post-doctoral associate. How have the results been disseminated to communities of interest?We published a peer-reviewed article. We also presented the results at two conferences [see Products section]. What do you plan to do during the next reporting period to accomplish the goals?1. We will determine effectiveness of sanitizers with respect to viral contamination. 2. We will determine the mechanisms of viral disinfection by sanitizers. 3. We will determine the attachment of pathogens on artificial leaf surfaces and sanitation efficacy for the pathogen attached to the artificial leaf surface. 4. Two more manuscripts will be submitted.
Impacts
What was accomplished under these goals?
Task 1. Development of a reproducible artificial surface with topography and hydrophobicity similar tospinach leaves and leafy green salad vegetables using soft lithography and rapid replication methods. A reproducible artificial surface with topography and hydrophobicity similar to spinach leaves was developed using soft lithography replication. Polydimethylsiloxane (PDMS) was mixed at different ratios (0%, 1.5%, 4%, and 7%) with surfactant Caprol PGE-860, poured onto a silicon wafer mold with features resembling the surface of spinach leaves, and baked at 100oC for 3 minutes. The artificial spinach surfaces had contact angles of 118°, 89°, 70°, and 64°, respectively. Produce from five leafy green salad vegetables with varied epicuticular physiochemical properties were used. To prepare the PDMS molds, plants were collected at commercial harvest maturity and rinsed with distilled water to remove soil and debris from the surface. The plants were taped to the bottom of a 2 inch aluminum dish; degassed PDMS mixture was poured onto the aluminum dishes and allowed solidification for 16 hours under refrigeration followed by a 4 hours baking under controlled temperature (35°C) and relative humidity (60%). The obtained inverted PDMS master molds were treated with a solution of 1% Hydroxypollymethylcellulose and stored at room temperature. A second inversion was reproduced by pouring degassed PDMS mixture onto PDMS molds and dried at 100 °C for 3 minutes. Hardened PDMS surface was removed from PDMS master and stored at room temperature for further use. In addition, two components (a 28 carbons fatty alcohol (-Octacosanol) and 28 carbons fatty acid (Octacosanoic acid)) found in epicuticular wax composition were added to artificial surfaces to mimic chemical characteristics. 'Totem' Belgian endive and 'Two Star' lettuce were the only plants suitable for replication from all five leafy greens selected; the contact angle measurements of Belgian endive fresh sample was 58.3° ± 3.5 and the contact angle measurement of artificial surface were 58.0 °± 3.6. The contact angle measurement of Belgian endive spin coated with Octacosanol and Octacosanoic acid were 60.0 °± 7.5 and 69.1°± 8.7 respectively. Task 2. Examination of the effect of bacterial surface hydrophobicity and epicuticular wax composition on attachment and removal of E. coli K-12. The surface hydrophobicity of E. coli K-12 was determined with sessile drop method and microbial adhesion to hydrocarbons (MATH) test. Both the sessile drop and MATH methods indicated that E. coli K-12 surfaces are hydrophilic (contact angle 17.6° ± 2.3°, and adhesive to hydrocarbons 22.1% ± 8.7%). A lower E. coli attachment (6.3 ± 0.7 to 6.6 ± 0.4 Log CFU/cm2) was observed on hydrophobic leaves (contact angle > 100°), whereas the highest attachment (6.8 ± 0.3 Log CFU/cm2) was observed on hydrophilic leave surfaces with a contact angle of 49o. The highest E. coli removal (2.8 ± 0.01 Log CFU/cm2) was observed on produce treated with malic acid + TDS. Produce surfaces with higher wax concentrations (alkane, ketone, or total wax) had significantly higher bacteria removal. Task3. Examination of attachment of E. coli O157:H7 strain 87-23 to artificial spinach surface and its removal using a selected sanitizer and a surfactant. Attenuated E. coli O157:H7 cells were spot-inoculated onto the PDMS surfaces, and were dried in a laminar-flow fume hood for 2 hours at 22oC. Dried samples were washed in a beaker containing either distilled water, chlorinated water (20 ppm free chlorine), or chlorinated water with a surfactant (0.1%) having one of three HLB values (11, 6.5, 3.9). The E. coli cells surviving on washed samples were enumerated via spread plating with a selective medium. For the same proportions of surfactant in the samples, those washed with distilled water achieved 1.8, 1.4, 1.6 and 2.2 Log CFU/g, reductions in E. coli O157:H7 counts, respectively. Samples washed with chlorine or with chlorine in combination with 0.1% surfactant (for any HLB value tested) had no detectable surviving bacteria. Task 4. Sanitation efficacy of rotavirus attached to leaf surface. 'Red Russian' kale, 'Starbor' kale and 'Totem' Belgian endive were selected as model produce and harvested at market maturity. Porcine rotavirus was used as model viral pathogen. Free chlorine (sodium hypochlorite solution, pH = 7) was used at 50 ppm. The infectious rotaviruses in elution buffer were quantified through integrated cell culture and qPCR assay (ICC-qPCR). 'Totem' Belgian endive (log10 N/No = -3.37 ± 0.5 at 0.5 min) showed the highest rate of rotavirus removal within the first 30 s, followed by 'Red Russian' kale (log10 N/No = -2.34 ± 0.67) and 'Starbor' kale (log10 N/No = -1.80 ± 1.2). These results implied that the rotavirus removal from leaf surfaces might be related to the wax concentration of the epicuticular layers of each species. The major virus reduction in this experiment was observed within 1 minute. When exposed longer to free chlorine, the rotavirus removal did not go higher but stayedconstant. This was because very few infectious viruses were left after one minute ofsanitization. The consumption of chlorine by the organic compounds on vegetable surfaces was not likely responsible for the "steady phase" of virus removal, since maximum 50 ppm of free chlorine was found to be consumed by organic compounds present on 'Red Russian' kale surfaces.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
L. Lu, K.-M. Ku, S.P. Palma-Salgado, A.P. Storm, H. Feng, J.A. Juvik and T.H. Nguyen. Influence of epicuticular physicochemical properties on porcine rotavirus adsorption to 24 leafy green vegetables and tomatoes. PLoS ONE, 2015, 10, e0132841.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Palma Salgado, S., Kang, K.M., Juvik, J.A. and Feng, H. Effects of bacterial surface hydrophobicity and epicuticular wax composition of produce leaves on attachment and removal of E. coli K-12. Institute of Food Technologists Annual Meeting, Chicago, IL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Palma Salgado, S., Liu, Y., Nguyen, T.H., Pearlstein, A.J., Luo, Y. and Feng, H. Polydimethylsiloxane-based artificial plant surface for studying bacteria-plant surface interactions. Institute of Food Technologists Annual Meeting, New Orleans, LA.
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience: Our project intends to reach scientists, policy makers, andthe produce safety industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided training for two graduate students and one post-doctoral associate. How have the results been disseminated to communities of interest? Results were disseminated through a publication in the Journal ofChemical Physics and at theFourth International Conference on Emerging Contaminants and theInstitute of Food Technologists annual meeting. What do you plan to do during the next reporting period to accomplish the goals? 1. We will investigate the influence of sanitizers on chemical composition of the leafy green produce; 2. We will determine the effectiveness of sanitizers with respect to viral contamination; and 3. We will determine the mechanisms of viral disinfection by sanitizers.
Impacts
What was accomplished under these goals?
We conducted viral attachment assays with OSU porcine rotaviruses on 24 genotypes of leafy vegetables and tomato fruits, and characterized the physiochemical properties of each produce’s surface, known as the epicuticular wax layer. Based on our results, significant negative correlations exist between viral attachments and the contents of alkanes, fatty acids, ketones and total wax in the epicuticular layers. For the first time, 3-D wax crystals on the epicuticular layer were found to significantly contribute to inhibition of viral adhesion to produce surfaces. The six major epicuticular factors including alkanes, fatty acids, alcohols, ketones, contact angle, and surface roughness were used for partial least square (PLS) prediction model. The PLS model explained 60.2% of viral attachment using comprehensive physiochemical data. The alkanes showed the highest variable importance for projection (VIP) value, followed by fatty acids, contact angle, ketones, alcohols, and surface roughness. The VIP score is a measure of a variable’s importance relative to the modeling prediction based on multivariate regression analysis. In summary, our results indicated that even after washingthree timeswith phosphate buffer saline, up to 107 of porcine rotaviruses could be found to still attach to a surface area of 1 cm2 of a wide range of different genotypes of produce. These results suggest a potential public health concern regarding the rotavirus contamination during food processing.In addition to leafy green, we have also worked on creating artificial leaf using PDMS. However, we did not find a significant difference between viral attachment to PDMS with a narrow range of contact angles and roughness.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Phan, A.D, Tracy, D.A., Nguyen, T. L. H., Viet,N. A., Phan, T.-L. and Nguyen, T.H. Electric potential profile of a spherical soft particle with a charged core. Journal of Chemical Physics, 139 (24), pp. 244908- 244913, 2013.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Lu, L., Ku, K-M, Palma-Salgado, S., Storm, A., Feng, H., Juvik, J. and Nguyen, T.H. Correlation between properties of epicuticular layer and porcine rotavirus attachment from 24 salad vegetables. The Fourth International Conference on Emerging Contaminants in the Environment. Iowa City.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Palma Salgado, S., Liu, Y., Nguyen, T.H., Pearlstein, A.J., Feng, H. and Luo, Y. Polydimethylsiloxane-based artificial plant surface for studying bacteria-plant surface Interactions. Institute of Food Technologists annual meeting, New Orleans, LA.
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