Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins | 5010 - Food (not readily associated with specific plant and animal products) | 1101 - Virology | 40% |
| 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins | 5010 - Food (not readily associated with specific plant and animal products) | 2020 - Engineering | 40% |
| 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins | 5010 - Food (not readily associated with specific plant and animal products) | 1020 - Physiology | 20% |
Viruses have been identified as the source of a number of disease outbreaks associated with the consumption of fresh produce. Knowledge of the physical and chemical interactions of the virus with the morphology and chemistry of the fresh vegetable leaf and tomato is required for effective prevention of viral contamination and effective produce sanitation. This knowledge is, however, lacking. We hypothesize that the attachment/detachment mechanisms of viruses to/from fresh vegetable leaves and tomatoes depend on topography and chemical characteristics of the cuticular surfaces. We further hypothesize that because the topography and chemical characteristics (cuticular wax deposition) of the vegetable leaves and tomato fruits vary among vegetable species and over the period of leaf and tomato development and expansion, the attachment/detachment of the virus will also depend on plant species and leaf and fruit age. The knowledge gained from testing these hypotheses will allow the selection of sanitation methods based on the combined effect of ultrasonication and sanitizer to enhance the inactivation and removal of attached viruses from the produce. Two diarrhea causing culturable viruses, murine norovirus and rotavirus, will be selected as model organisms. Leafy salad greens and tomatoes will be selected as model produce. Synthetic polydimethylsiloxane (PDMS) surfaces with topography and hydrophobicity similar to the selected produce will be used as model reproducible produce surfaces. The experimental plan will be designed for the following specific objectives: 1) Determine the attachment/detachment mechanisms of murine norovirus and rotavirus on selected leafy vegetable plants and tomatoes grown in a greenhouse; 2) Develop an effective virus decontamination protocol for these produce; and 3) Determine the damage to the viral capsid and genome associated with combinations of ultrasonication and/or new sanitizer compositional treatments.
Four tasks are planned: 1) A number of vegetables and tomatoes will be grown in a greenhouse and harvested to develop a correlation between cuticular characteristics and virus attachment or virus sanitation. A selected number of vegetables will be grown and harvested at different ages. 2) The leaf and the tomato cuticle will be characterized. Chemical composition will be determined using selective solvent extraction followed by chromatography. The surface roughness will be measured using a laser confocal scanning micrograph method developed by Feng's lab. Surface contact angle and images by atomic force and scanning electron microscopy will be determined as a guide to create analogous topographies on PDMS surfaces using a soft lithography technique. The PDMS surface will be further modified with layer-by-layer surface coating techniques using the compound mixtures extracted from fresh leaves. 3) Interaction force between norovirus and rotavirus with freshly harvested leaf surfaces, tomato cuticular surfaces, and with the PDMS surface with similar topography and hydrophobicity to the produce surface will be determined in solutions with systematically varied composition. Atomic force microscopy and attachment assays will be applied for this task. 4) Genomic and protein analysis will be used to determine the mechanisms of viral damage associated with the combined effects of ultrasonication and/or a new sanitizer composition.
Target Audience
Members of the target audience included fresh produce growers, consumers, food processors, researchers, and regulators.
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided training for two graduate students and one post-doctoral associate.
Dissemination Streams
The results have been presented inpeer-reviewed journals and in conference presentations.
Next Reporting Steps
Nothing Reported
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.
Training & Professional Development
The project provided training for two graduate students.
Dissemination Streams
Results have been disseminated throughpeer-reviewed publication.
Next Reporting Steps
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. <br><br>
<br>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. <br><br><b>Publications</b><br>
Target Audience
The information is useful forvegetable growers, consumers, and researchers.
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided training for two graduate students and one post-doctoral associate.
Dissemination Streams
We published a peer-reviewed article. We also presented the results at two conferences [see Products section].
Next Reporting Steps
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. <br><br>
<br>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. <br><br><b>Publications</b><br>
Target Audience
Our project intends to reach scientists, policy makers, andthe produce safety industry.
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided training for two graduate students and one post-doctoral associate.
Dissemination Streams
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.
Next Reporting Steps
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. <br><br>
<br>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. <br><br><b>Publications</b><br>