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Knowledge of construction duration is pertinent to a number of project planning functions prior to detailed design development. Funding, financing, and resource allocation decisions take place early in project design development and are significantly influenced by the construction duration. Currently, there is not an understanding of the project factors having a statistically significant relationship with highway construction duration. Other industry sectors have successfully used statistical regression analysis to identify and model the project parameters related to construction duration. While the need is seen for such work in highway construction, there are very few studies which attempt to identify duration-influential parameters and their relationship with the highway construction duration. This research identifies the project factors, known early in design development, which influence highway construction duration. The factors identified are specific to their respective project types and are those factors which demonstrate a statistically-significant relationship with construction duration. This work also quantifies the relationship between the duration-influential factors and highway construction duration. The quantity, magnitude, and sign of the factor coefficient yields evidence regarding the importance of the project factor to highway construction duration. Finally, the research incorporates the duration-influential project factors and their relationship with highway construction duration into mathematical models which assist in the prediction of construction duration. Full and condensed models are presented for Full-Depth Section and Highway Improvement project types. This research uses statistical regression analysis to identify, quantify, and model these early-known, duration-influential project factors. The results of this research contribute to the body of knowledge of the sponsoring organization (Virginia Department of Transportation), the highway construction industry, and the general construction industry at large. Ph. D.

Farmers markets (FM) have become increasingly popular almost tripling over the past two decades due to the rising interest in local and/or organic foods. Within this same time period, notable farmers market foodborne illness outbreaks; such as E. coli O157:H7 and strawberries and Salmonella and peas; have occurred, and emphasize the importance of food safety practices at farmers markets. Some farmers may be encouraged to follow Good Agricultural Practices (GAPs) to limit the contamination of fresh produce; however, GAPs programs are complex and also very driven by wholesale buyers. Furthermore, many FM growers are unfamiliar with GAPs or do not believe it is applicable to them. The Food Safety Modernization Act (FSMA) was passed in 2011, and shifted the food safety paradigm from a reactive to proactive approach introducing seven different rules including the Preventive Controls for Human Foods (PCHFR) and the Produce Safety Rule (PSR). Each FSMA rule does contain exemptions for smaller-scale operations (e.g., farms, facilities) that allows them to be excluded from the rule, or excluded from certain aspects of the rule. Generally, most FM vendors may satisfy exemptions from the FSMA regulations (e.g., be exempt from the FSMA Produce Safety Rule because of commodities); however, to our knowledge, no studies have assessed the regulatory compliance requirements of FM vendors to the FSMA regulations. This dissertation explored the regulatory requirements of FM vendors, and the accessibility of FSMA materials for these audiences. Additionally, due to the sudden and unprecedented global COVID-19 pandemic, an objective was added that explored how COVID-19 influenced behavior changes among this audience. Interviews were conducted and found that all vendors (100%) were exempt from the PCHFR and that most vendors (67%) were exempt from the PSR, some were qualified exempt (28%), and a few (5%) were covered. This study also found that farmers market vendors received information mainly from University Cooperative Extension sources. The COVID-19 pandemic greatly impacted hygiene and health practices at FMs. Market personnel (managers and vendors) implemented many different hygiene and health practices at their markets. The major source of COVID-19 food safety information for market personnel was local and federal government; as well as University Cooperative Extension. Accessibility analyses showed that many FM or COVID-19 resources were not accessible due to populations that rely on produce safety resources generated by Cooperative Extension due to (i) navigation or web accessibility errors, (ii) high literacy level requirements, and (iii) lack of keep (i.e., resources were not up-to-date, or continuously managed or monitored). These findings will inform the development of FM targeted resources, that are also, accessible to a more diverse and inclusive audience. One example is a produce safety resource on the updated agricultural water requirements that is developed to an 8th grade reading level, with no broken links or additional navigations errors, and if a PDF version is available, proper headers and titles. Doctor of Philosophy Farmers markets (FMs) have increased in popularity over the two decades with because of interest by consumers to support local agriculture, buy organic products, or because they believe these foods to be safer from bacteria and viruses. Shopping at a FMs does not necessarily make the products any safer compared to buying it from a grocery store. It is important that farmers markets follow proper food safety practices during the growing, harvesting, and preparation of produce for sale. One example is the prevention of animal feces from getting on the produce or in a water source that would be used. Another would be to follow proper hygiene practices such as handwashing after using the bathroom. These two examples and others are detailed in a law passed by President Obama in 2011 called the Food Safety Modernization Act (FSMA), which allows the Food and Drug Administration to begin its proactive instead of reactive food safety approach. FSMA does not have to be followed by all food entities. Depending on how much a farm makes in revenue (

Solar radiation influences virtually all biological process on earth. Yeasts, the microbial driver of ethanol fermentation, evolved on the surface of vegetation and had to adapt to survive photonic assault. Past research has demonstrated that white light affects yeast metabolism along with the ability to entrain circadian rhythms, although no known genetic mechanism accounts for this. High intensity narrow wavelength light-emitting diodes were employed to illuminate synthetic cultures under fermentation. Multiple colors along the visible spectrum were used, corresponding to the peak absorbance wavelengths of Saccharomyces sp. yeast. Impacts in primary metabolite evolution were found, dependent on wavelength. Longer wavelengths produced higher amounts of acetic acid and glycerol; shorter wavelengths produced more ethanol. Because past research showed light timing had pronounced effects, illumination schemes on the scale of milliseconds to hours were tested for ethanol production. Light schemes on the scale of enzymatic reactions, yeast generation times, and circadian rhythms produced the most ethanol. Discrete blocks and duration of illumination were used to elucidate where light had the most influence over yeast metabolism and fermentation. Late lag phase and mid log phase illumination impacted ethanol fermentation more than any other period of time. Light effects were tested on apple juice to see if they extended from synthetic media to natural products. Significant impacts on ethanol production were discovered and flavor/aroma impacts were noted. Light, color, intensity, and timing have all been shown to control and affect fermentation with both positive and negative effects established. Doctor of Philosophy Sun light influences virtually all biological process on earth. Yeasts, the microbial drivers of ethanol fermentation, evolved on the surface of vegetation and had to adapt to survive destructive effects of the sun. Past research has demonstrated that white light affects yeast metabolism along with the ability to develop growth cycles similar to day / night patterns, although it is currently not believed this possible due to the biology of yeast. High intensity single color light-emitting diodes were employed to illuminate laboratory formulated cultures under fermentation. Multiple colors along the visible spectrum were used, corresponding to the peak absorbance wavelengths of Saccharomyces sp. yeast. Green/yellow/red wavelengths produced higher amounts of acetic acid (vinegar) and glycerol; blue and ultraviolet wavelengths produced more ethanol. Because past research showed light timing could change how yeast grow and consumed carbohydrates, light timing on the scale of milliseconds to hours were tested for ethanol production. Light timing on the scale of milliseconds, hours, and daylight cycles produces the most ethanol. Discrete blocks and duration of illumination were used to find where during fermentation light had the most impact. It was found that from immediately after the beginning of fermentation to the middle of fermentation is where yeast responded the most strongly. Light effects were tested on apple juice to see if they extended laboratory cultures to natural products. Significant changes in the amount of ethanol produced were discovered and changes in the taste and smell of fermented apple juice were noted. Light, color, intensity, and timing have all been shown to control and affect fermentation with both positive and negative effects established.

Public health costs of foodborne campylobacteriosis are estimated to cost more than a billion dollars in the United States annually. This pathogen has been primarily associated with chicken production and processing which is a ~$33 billion industry. To further identify practices that could reduce Campylobacter presence, concentration, and persistence in chicken prior to consumption a quantitative microbial risk assessment (QMRA) was conducted for boneless, skinless chicken breast meals prepared and consumed domestically in the contiguous United States. This QMRA model was developed with @RISK software (Palisade Corp., Ithaca, NY) and data inputs can be easily modified and updated. QMRA is a powerful analytic method that can be utilized to model the dynamics between the food pathogen, food commodity, and ingestion. It provides insight into the impacts of the process in its interaction and its surrounding environment. The baseline model determined that consumption of this product resulted in annual means of infections: 328,257, illnesses: 108,174, hospitalizations: 27,754, deaths: 37, cases of Campylobacter-associated Guillain-Barré Syndrome (GBS): 1,373, and cases of Campylobacter-associated Irritable Bowel Syndrome (IBS): 9,501. The associated annual economic burdens were ~$192 million for acute campylobacteriosis, ~$77 million for GBS, and ~$96 million for IBS. The effects of targeting and modifying the baseline model's inputs within the farm-to-fork process were studied as follows: Post grow-out (1) prevalence and (2) concentration of Campylobacter on chickens at the farm prior to transport, (3) transport crate cleaning frequency prior to loading, (4) temperature storage conditions at post processing/ pre-retail, (5) the frequency of handwashing in the preparation and handling of a chicken meal, and (6) the combination Campylobacter mitigation models using the inputs from (2) and (4). Mean yearly illnesses are estimated to decrease by approximately half if on-farm Campylobacter prevalence was lowered to 35-50% from the baseline level of 76%. An additional ~50,000 illnesses can be expected if the proportion of home preparers who do not wash their hands increases from 8.3% (baseline) to 20%. The combined effects of reducing on-farm Campylobacter concentrations and increasing the proportion of product frozen in-plant have greater impacts on reducing yearly campylobacteriosis and associated costs than either intervention alone. Doctor of Philosophy Campylobacter is a major foodborne bacterial pathogen that is not well known by the general public, although public health costs are estimated to cost more than a billion dollars in the United States annually. Chicken production is a ~$33 billion industry that is affected by the contamination of this pathogen. In this study, a quantitative microbial risk assessment (QMRA) was performed to study the impact of Campylobacter on chicken meals prepared in the home. Societal and economic burdens were evaluated as well. The models developed provide comprehensive simulations that describe the spread, persistence, and the concentration of Campylobacter throughout the farm to fork process in the annual consumption of boneless, skinless chicken breast meals prepared and consumed domestically in the contiguous United States. Additional simulation models were created to compare methods for reducing Campylobacter along the food chain that could lead to fewer cases of campylobacteriosis in the United States, or what could happen if there was a breakdown in a hygiene step in the preparation of the chicken meal.

The Food Safety Modernization Act (FSMA) Produce Safety Rule (PSR) establishes science-based minimum standards for the safe growing, harvesting, packing, and holding of produce on farms. Operations covered under the FSMA PSR must meet standards for worker training, health and hygiene. Produce operators (e.g., growers, packers) subject to the PSR will need to comply with the new worker health, hygiene and training requirements. The need for a training curriculum to assist produce operators who are covered under the FSMA PSR is needed for compliance. This training curriculum includes all required standards outlined in subpart D including visual representations of personal health and hygiene; as well as, content to confidently train farm and packinghouse workers. This training curriculum was presented to >20 Virginia Cooperative Extension (VCE) agents and specialists in a train-the trainer model, who also evaluated the curriculum. As a result of the train-the trainer sessions, the curriculum has been refined by incorporation of VCE agent and specialist feedback, and is ready for use by stakeholders and extension educators nationwide. The training is currently available in English and Spanish, and is best taught using technology (e.g., Microsoft PowerPoint), but can be taught without technology (e.g., using printed aids). Overall, this curriculum will assist stakeholders in obtaining FSMA PSR compliance specific to subpart D in approximately

Low water activity foods (LWAF), specifically nuts and dried fruits, have been generally considered safe because they do not support the growth of foodborne pathogens. However, many pathogens have been noted to survive in LWAF for considerable periods of time, and a number of recent outbreaks and recalls have implicated various types of nuts and dried fruits. The Food Safety Modernization Act requires food processors to develop preventive control plans that make ready-to-eat LWAF safer for consumers. The presented research was designed to investigate several aspects of LWAF safety by evaluating a steam process as a strategy to remove pathogen contamination from LWAF, modeling the inactivation of such treatments, and studying the thermal resistances of two E. coli strains in low-water activity solutions. Low-temperature, vacuum-assisted steam (vacuum-steam) was evaluated as a potential intervention and preventive control to remove pathogens from the surface of LWAF without using high-heat treatments that could damage product quality. The presented work examined the efficacy of vacuum-steam (4 log CFU/g from the surfaces of the foods. Additionally, Pediococcus acidilactici, proved to be a surrogate organism for these pathogens and could be used to challenge and validate similar treatments within processing plants. The data were fit to models, which showed that food type significantly impacted the fit, with the Weibull model best describing bacterial inactivation kinetics on raisins and macadamia nuts, and the Gompertz model best describing reductions on the apricot halves. The models were challenged for validation of their abilities to predict times required for 3-log reductions using internal and external datasets, determining the usefulness to industry members who wish to design similar thermal treatments for LWAF. Comparing predicted values from internally constructed models to observed values generated from external data, models were shown to be limited in scope and application and could only be applied to pathogen inactivation on different LWAF or thermal processes in certain circumstances. First-order and Weibull model predictions of bacterial reductions on dried apricots had varied success in predicting times for 3-log reductions on other thermally treated LWAF. However, the models of bacterial reductions on thermally treated macadamia nuts frequently overestimated the times required for 3-log bacterial reductions for other LWAF. In an effort to understand the effect that reduced water activity has specifically on STEC, two strains were investigated for induced thermal resistance due to osmotic stress. Thermal resistance of STEC strains (O121:H19 and O157:H7) were evaluated on the basis of strain variation, culture preparation, and water activity (D- and z-values). At the lowest treatment temperature (56°C), O121 displayed greater heat resistance than O157, and the broth-grown samples exhibited greater heat resistance than the lawn-grown cells, but significant differences were not observed at higher temperatures. Samples in reduced-water activity solutions displayed reduced thermal resistance at 56°C, but the z-values were 29-43% higher than those of high-water activity samples. While water activity has been shown to impact thermal resistance of pathogens, comparisons of STEC thermal resistance according to the D- and z-values revealed that other factors also play roles in pathogen thermal resistance on LWAF. Results from the collection of experiments conclude that efficacy of thermal treatments is impacted by the physiological state of the cells, stress experienced in the food matrix, and characteristics of the food, including water activity and composition. Doctor of Philosophy Consumers expect foods they purchase to be safe to consume by themselves and family members, particularly those that are ready-to-eat with no additional cooking requirements. Many of these foods are low-water activity foods (LWAF), like nuts and dried fruits, with very little water content that could be used by bacteria. These foods may be preferred snack foods due to their affordability, long shelf lives, and health benefits over other types of snack foods. Until recently, LWAF were generally considered safe because they do not support the growth of foodborne pathogens due to the lack of moisture or water within the food. However, a number of recent outbreaks related to various types of nuts and dried fruits have proven that many pathogens can survive in dried foods, even if not actively growing, for considerable amounts of time. Designed to address these types of food safety issues, the Food Safety Modernization Act recognizes risks associated with foods and responded with regulations requiring food processors to take steps to make ready-to-eat LWAF, like nuts and dried fruits, safer for consumers. A popular strategy is to treat foods with heat to destroy pathogens, however the quality attributes of some nuts and dried fruits could be damaged by high-heat treatments like roasting. An alternative process uses a vacuum to form steam at lower temperatures, allowing for efficient heat transfer through water droplets to the surface of the foods, thus causing less damage to the foods without introducing too much moisture. This research evaluated how this process could be used by food processors to remove harmful bacteria from the surfaces of whole macadamia nuts, dried apricot halves, and raisins. Results indicated that the low-temperature steam treatments successfully reduced Salmonella, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) by >4 log CFU/g (>99.99%) from the surfaces of the foods. Additionally, a nonpathogenic lactic acid bacterium, Pediococcus acidilactici, exhibited similar or greater heat tolerance, which would allow food processors to use it as a substitute, or surrogate, for in-plant studies without introducing harmful bacteria into the food processing environment. Mathematical models were used to describe the trends of bacterial death due to the steam treatments, and the results indicated that the type of food significantly impacted the reduction of bacteria. The models were tested using additional data collected within our own laboratory, as well as others. Results indicated that some of the models could be used as predictors of bacterial death for similar LWAF but can only be applied with caution and consideration for the type of food and process. Additionally, two different E. coli strains associated with outbreaks (O121:H19 and O157:H7) were investigated to understand impacts of strain variation, growth method, and water activity on thermal resistance. Some differences in heat resistance were observed between the strains and between the growth methods. Additionally, the reduced water activity seemed to decrease the bacteria's ability to withstand some heat treatments. Overall, thermal resistance studies indicated that several factors, in addition to water activity, impact pathogens' development of resistance to heat treatments. The experiments' results show that there are complex relationships between bacteria and the food they inhabit. Food processors must consider these relationships in order to design the best thermal processes to make LWAF safe for consumers.

Solanaceous crops such as peppers and more commonly tomatoes have been linked to Salmonella outbreaks that have occurred in the United States. The source of contamination for some of these outbreaks was traced back to the preharvest environment. Sources of contamination at the preharvest level of production often include irrigation water, soil, and the use of biological soil amendments of animal origin (BSAAO). This dissertation aims to (i) determine how factors such as the use of BSAAO's, Salmonella strain, soil type, and irrigation influence the survival of Salmonella in agricultural soils and (ii) determine the risk of Salmonella internalization in to transplanted solanaceous crops (tomatoes and bell peppers) and identify factors that influence internalization through the roots such as mode of contamination, soil contamination level, and root damage. Sandy loam (SL) and clay loam (CL) soils both unamended and amended with poultry litter (PL), irrigated either daily or weekly were inoculated with one of twelve strains of Salmonella enterica and monitored for growth and survival until no longer detected. Tomato plants were transplanted into one of three contamination treatments: contaminated soil (A) or irrigation water administered either once (B, single exposure event) or for 7 d following transplantation (C, repeated exposure event). Plants were sampled 1, 2, 3, 7 and 90 d post-transplantation for Salmonella internalization. Pepper plant roots were left intact, damaged, or removed and placed in inoculated water (6.2 log CFU/ml). Pepper plants in the soil contamination study, were transplanted into soil inoculated with Salmonella at different contamination levels (High, Mid, and Low). Samples were collected 1, 2, 3, and 7d post-transplant. Plants were surface sanitized and enumerated for Salmonella internalization by plant section. Strains in soils treated with PL survived significantly (P

The Food Safety Modernization Act was a broadly impactful piece of legislation signed into law in 2011. This legislation is composed of seven core rules that together made sweeping changes to the regulations surrounding how foods are grown, harvested, and processed. These regulations affected many different industries, including the brewing industry and its supporting counterparts. Due to the expansive nature of the regulations, there have been issues with interpretations during their rollout and direct guidance from the FDA was not always immediately available. This meant that during initial years following passage of the legislation, non-government groups were left to gather information and disseminate answers to questions on maintaining compliance. A review of available literature and resources has found that even though there has now been clarification on how many of the provisions affect various industries directly from the FDA, resources on how to achieve compliance in specific areas are not always available and can sometimes be hidden behind a paid membership. Due to this, there exists a state where some resources may be unavailable to potential industry participants. There is value in providing free resources to potential participants, future research on sustainable practices with industry byproducts, and in identifying shortcomings in the legislative rollout for the benefit of future regulatory endeavors. MALS

Water used in fresh produce washing is a potential vehicle of foodborne pathogen contamination. This work focused on assessing the sanitizing efficacy of ethanol mist to reduce Salmonella populations on the surfaces of tomatoes and cantaloupes. Ethanol (70%) mist was applied to whole tomatoes and cantaloupe rind plugs using a Biomist sanitation system, which uses CO2 as a carrier gas to spray vapors through a fine droplet spray nozzle. Fresh red tomatoes (Solanum lycopersicum) and cantaloupe (Cucumis melo) plugs were inoculated with Salmonella enterica Newport to a concentration of log 7 CFU/tomato and log 7 CFU/cm2 respectively. Application time (5, 10, and 15 sec), dry vs. wet surface, and stem scar contamination were evaluated on tomatoes, while only application time was evaluated on cantaloupe. Application of ethanol mist for 10 seconds was the most effective treatment time, reducing Salmonella by 3.3 log CFU/tomato. Application of ethanol mist was more effective on dry opposed to wet tomato surfaces by approximately 0.7 log CFU/tomato. Ethanol mist application to inoculated tomato stem scars reduced Salmonella 1.2 log CFU/tomato. Additionally, Salmonella decreased by 1.3 log CFU/cm2 on cantaloupe rind plugs at 10 seconds, again the most effective treatment time. Ethanol mist resulted in sufficient reductions of Salmonella populations on dry tomato surfaces, but was limited in effectiveness if the surface was wet, or if Salmonella adhered to the stem scar. Furthermore, this technique was overall not an efficient method to sanitize cantaloupe surfaces. Master of Science in Life Sciences Water is often used in washing and moving fresh produce during harvesting. However, this water is often found to be a source of contamination that can cause the fruits or vegetables to become unsafe to eat. In order to resolve this problem, a mist-type sanitizing system is being explored to wash fresh produce in packinghouses. In this experiment, a mist of ethanol (70%) was applied to whole tomato and cantaloupe plugs using a Biomist sanitation system, which turns liquids into a mist. Whole fresh tomatoes and cantaloupe plugs were infected with Salmonella enterica bacteria. Tomatoes and cantaloupe plugs were treated with ethanol mist for various times of 5, 10, or 15 seconds. Additionally, tomatoes were treated with ethanol to compare a wet surfaced tomato, as well as the tomatoes stem scar (where the vine of the tomato is attached during growing). Ethanol mist application was most effective at 10 seconds, but there was very little difference in bacterial elimination when all the times were compared to the untreated tomatoes. In addition, the mists effectiveness decreased if the surface of the tomato was wet compared to dry, and was even less effective if the bacteria were located where the vine attaches. Also, the ethanol mist has very little effect if bacterial contamination is present on a cantaloupes surface. If a harvested tomato remains dry post harvest, ethanol mist may make for a good washing system. However, it is probably not the best for rough surfaced produce like cantaloupes.

Listeria monocytogenes has emerged as a food safety concern for a number of produce commodities. While L. monocytogenes contamination can occur throughout the supply chain, contamination from the packinghouse environment represents a particular challenge and has been linked to recalls. This study aimed to investigate the prevalence, persistence, and diversity of Listeria monocytogenes (LM) and other Listeria species (LS) in produce packinghouses. A longitudinal study was performed in 11 packinghouses (commodities included micro-green, peach, apple, tomato, broccoli, cauliflower, and cucumber) in three US states. In each packinghouse, 34 to 46 sites representing zones 2-4 were selected and swabbed. Packinghouses were visited 4 times and samples were processed for Listeria by US Food and Drug Administration's Bacteriological Analytical Manual methods. Presumptive Listeria-positive isolates were confirmed by PCR. Species and allelic type (AT) were identified by sigB sequencing. Among the 1,584 samples tested, 3.2%, 2.7%, and 0.6% of the samples were positive for LM, LS, and both LM and LS, respectively. Five different species of Listeria were identified with L. monocytogenes being the most prevalent species. A high AT diversity (0.95 Simpson's Diversity Index) was observed amongst Listeria isolates. There were 15 instances of Listeria repeated isolation (site testing positive ≥2 times). Upon analysis of subtype data, only 3 sites tested positive for the same Listeria AT >2 times. Data showed in this longitudinal study that Listeria prevalence and persistence in packinghouses was low (e.g.

Encouraging procurement of food via Farm to School programs has become an emphasis of many schools throughout our nation and state. Farm to school produce/products benefits children and communities in many different ways. Contact between farmers and school management needs to be fostered and nurtured to be successful and provide the students with the freshest and safest product possible. School divisions often need a starting point to begin the process of procurement between the local farmer and the school nutrition department. Development of a checklist and other tools for farmers and school division nutrition managers/cafeteria managers to use when gaining access to fresh local produce may be beneficial for school nutrition departments trying to start a farm to school program. This project developed a checklist and fact sheets to lead and guide school nutrition directors and producers seeking to increase purchases and consumption throughout the state of Virginia. Farm to school is a vital program and allows farmers to reach markets that may have been untouched in the past. We are excited to share our resources and information with educators, farmers, and nutrition directors as well as our colleagues in extension. MALS

The topic of genetically modified organisms (GMOs), and its many equivalent terms, has been magnified in recent years, and with it, the uncertainty of the meaning of GMOs. Consumers are challenged with conflicting information on the safety of GMOs and whether or not consuming products sourced from genetically modified (GM) sources will negatively affect their health. The scientific community seems to be at odds with consumer advocacy groups. While scientists back the safety and risk of genetically modified foods, claiming equivalency to conventional food alternatives, consumer advocacy groups’ positions disagree with this opinion and are seeking point-of-purchase labeling differentiating foods made with genetic modification from those without. This paper will discuss these relevant topics of GMOs, the sourcing and availability of genetically modified foodstuffs, and the labeling and pending U.S. government regulation on genetically modified or “bioengineered” foods. Scientists assert genetic modification has been occurring in one form or another in nature for centuries. In attempt to demystify the bioengineered food spectrum, a panorama of various forms of genetic modification will be outlined, and the climate of consumers’ beliefs, attitudes and concerns over genetically modified foods will be examined. MALS

Consumers desire tender, juicy, and flavorful cuts of beef. Mechanical tenderization (MT) and enhancement methods applied to lower valued beef cuts can improve tenderness, flavor or juiciness, increasing desirability for the consumer. However, these processes can introduce pathogens that may be present on the exterior of the meat into the sterile interior. This process renders an ‘intact’ product ‘non-intact’ and requires altered cooking methods to ensure safety. The primary pathogens of concern for beef products are Shiga-toxin producing Escherichia coli (STEC). STEC are associated with approximately 265,000 illnesses and 3,600 hospitalizations annually. Since 2006, there have been 6 STEC outbreaks in the United States and 18 cases in Canada attributed to MT beef (MTB). The pathogen has also been implicated in 136 non-intact beef-related recalls. Due to the potential food safety hazards associated with MTB, mandatory labeling of these products was mandated in 2015 to inform consumers on how to safely handle the product. While this is a good step to inform consumers, it is unclear how familiar they are with the terms associated with these processes. Consumer’s knowledge, beliefs, attitudes, awareness, and behaviors related to MTBs is quite limited. This study uses an exploratory sequential mixed-methods design, to assess consumer knowledge of MTB. Qualitative focus groups conducted throughout urban and rural North Carolina and Virginia found that although participants purchased MTBs, they were unaware of the process, did not prepare MTBs properly, wanted to know more about the process, and wanted applicable risk messages. A nationwide survey developed from the focus group findings found that demographic differences were associated with knowledge of; and how participants interact with MTBs. How demographics influence consumer’s beef safety knowledge, practices, and risky behaviors was further explored. Demographic characteristics were highly correlated with consumers’ behaviors surrounding beef storage, refrigerator temperature knowledge, defrosting behaviors, meat washing, and meat preparation behaviors. Collectively, the mixed methods research design provided insight into specific demographic characteristics related to consumer attitudes, beliefs, and behaviors surrounding beef safety. This data will help inform the development of well-crafted, culturally, and socially relevant risk messaging that may promote safe handling behaviors. Ph. D. In the United States, every year, the Center for Disease Control estimates that 48M people are sickened, 128,000 people are hospitalized, and 3,000 people die from foodborne illnesses. The most common illnesses arise from Salmonella, Norovirus, Campylobacter, Escherichia coli [E. coli], Listeria, and Clostridium perfringens; these bacteria and viruses have been on the news as being associated with flour recalls, cantaloupe, eggs, cheeses, berries, and even at restaurants like Chipotle! The bacteria of concern in this study is E. coli. Most E. coli is not pathogenic, and extremely common in the environment; living in mammalian (e.g. humans, cows, sheep, insects, etc.) gut and within the environment (e.g. in the soil). However, there are some pathogenic variants, like Shiga-toxin producing E. coli [STEC] that have been associated with 265,000 annual illnesses and deaths. The main reservoirs of many pathogenic E. coli are within the intestines of ruminant mammals, including cattle. If mishandled, feces can contaminate and cause human illness as a result of improper handling and preparation. The contamination can occur through meat, water, and fecal-oral routes; often, improperly cooking and handling beef products can lead to illness. In the U.S., there have been 27 multi-state STEC associated food outbreaks. While the U.S. beef industry is the largest in the world, five multi-state outbreaks were related to E. coli O157:H7 contamination in ground beef products. Additionally, between 2005 – 2018, 136 of 171 recalls were due to STEC-contaminated beef products. Non-intact beef products (e.g. ground beef) are the most commonly recalled types of beef products. Consumers purchase beef products on extrinsic (i.e. price, weight, cut) and intrinsic (i.e. color, fat, safety) factors with a desire for tenderness, juiciness, and flavor. Tenderization processes (e.g. mechanical tenderization) or other enhancement processes (e.g. marinades) can increase tenderness on lower-value cuts but may introduce pathogens from the exterior to the sterile interior. To prevent illness, it is necessary to prepare beef products to the recommended USDA-specified temperature using a thermometer to check. Up until this point, consumer knowledge of and behaviors towards mechanically tenderized beef products [MTB]s and other enhanced beef products had not been characterized. This study uses an exploratory sequential mixed-methods study design (qualitative study guides quantitative study) to assess consumer knowledge of MTBs, enhanced beef products, and food safety / beef safety handling behaviors. Focus groups conducted throughout urban and rural North Carolina and Virginia found that although participants purchased MTBs, they were unaware of the process, did not prepare them properly, wanted to know more about MTBs, and requested different ways of obtaining information. A nationwide survey developed from the focus group findings found that demographic differences were associated with knowledge of and interaction with MTBs and enhanced beef products. However, people were still generally unaware of MTBs, despite a recent 2016 labeling mandate for all MTBs. The Theory of Planned Behavior was used to further explore reported consumer’s food safety knowledge, practices, and risky behaviors in the kitchen. Demographic characteristics were highly correlated with consumers’ behaviors of beef storage, refrigerator temperature knowledge, defrosting behaviors, meat washing, and meat preparation behaviors. Collectively, the mixed methods research design provided insight into specific demographic characteristics related to consumer attitudes, beliefs, and behaviors surrounding beef safety. Additional research, alternative non-didactic strategies, and collaborations within health and public services is necessary to accommodate for specific demographics, cultures, and social groups. Well-crafted, culturally, and socially relevant targeted risk messaging must be developed to increase awareness and promote ease-of-access.

Listeria monocytogenes (L. mono.) is a top pathogen of concern in the cheese industry. The organism is prevalent in the environment and has been known to inhabit dairy manufacturing facilities, including long-term survival in brine tanks. Along with good hygiene and sanitation practices, validated L. mono. control methods must be employed to ensure extended-use brine tanks do not become reservoirs for Listeria monocytogenes, potentially leading to serious health issues in cheese consumers. Control methods considerations include L. mono. cells in suspension, cells attached to protective biofilms, and effectiveness in the presence of organic matter. This literature review investigated the availability of scientific research on effective control methods for Listeria monocytogenes in cheese brines. Research on L. mono. control in other applicable areas was considered where information related specifically to cheese brine environments was not found. Potential effective control methods were identified, including membrane filtration and oxidizing agents such as peroxyacetic acid and hydrogen peroxide. Further research is needed to fill knowledge gaps related to successful implementation of these controls in cheese brines. MALS

Several produce-borne outbreaks have been associated with the use of contaminated water during pre-harvest applications. Salmonella has been implicated in a number of these outbreaks. The purpose of this study was to evaluate the microbial quality of agricultural surface water used in pre-harvest production on the Eastern Shore of Virginia in accordance to the Food Safety Modernization Act’s Produce Safety Rule water standards. The study also examined the prevalence, concentration, and diversity of Salmonella in those water sources. Water samples (1 L) from 20 agricultural ponds were collected during the 2015 and 2016 growing seasons (n=400). Total aerobic bacteria, total coliforms, and generic E. coli were enumerated for each sample. Population levels of each microorganism were calculated per 100 mL sample and log transformed, when necessary. Samples (250 mL) were also enriched for Salmonella. Presumptive Salmonella isolates were confirmed by PCR of the invA gene and were serotyped. In 2016, the concentration of Salmonella in each sample was also estimated by MPN. Indicator bacteria, and environmental and meteorological factors were analyzed for their association with the detection of a Salmonella-positive water sample using logistic regression analysis. Seventeen of the 20 ponds met the FSMA PSR standards for production agricultural water. Three ponds did not meet the standards because the statistical threshold value exceeded the limit. Salmonella was detected in 19% of water samples in each year (38/200; 2015 and 38/200; 2016). Of the 118 Salmonella isolates serotyped, 14 serotypes were identified with the most prevalent being S. Newport. Generic Escherichia coli concentration, total aerobic bacteria concentration, and farm were significantly associated with the likelihood of detecting a Salmonella-positive sample. The average concentration of Salmonella in all samples was 4.44 MPN/100 mL, with the limit of detection being 3 MPN/100 mL. The highest concentration of Salmonella was 93 MPN/100 mL. These data will assist in understanding the risks of production water poses to produce contamination events. MALS

E.coli 0157:H7 is one of the major bacterial diseases transmitted through drinking water and is a major contaminant on leafy greens. Results from current traditional plating methods including P-A analysis is time consuming and this slow process is an hinderance as a preventive control for food-borne outbreaks. In this experiment a fluorescence immonoassay outlined in fig. 7, is proposed for detecting E.coli 0157:H7 in water with a moderate amount of preparatory steps and less than 5 minutes for the detection of this pathogen. In examining the relationship between E.coli 0157: H7 CFU/0.1mL to fluorescence intensity, our model data evaluated with Minitab 16, revealed a P(0.001)= 0, and F=271.56 demonstrating a stong relationship of E.coli 0157:H7 detection with this immunoassay. E.aerogenes the negative contol for the immunoassay revealed a P(0.001)=0.677 and F=0.31, validating the specificity of the model. E.coli 0157:H7 was detected at 1 CFU/0.1mL with the proposed model and least three sigma (3σ) levels above the baseline for the control blank, thereby showing ≥ 99.7% probability of locating E.coli 0157:H7 when compared to the experiment control. MALS

Aquacultured products assist the human demands for seafood so that foodfish supplies can remain sustainable and consistent. Although the fish-farming industry has seen dramatic growth and intensification in recent years, the latter has led to an increase in bacterial diseases and fish health management problems, resulting in major economic losses around the world. In addition to the lack of understanding of fish physiology, these complications are exacerbated by the inappropriate and controversial use of antibiotics. This work addressed these issues in striped catfish (Pangasius hypophthalmus) and Nile tilapia (Oreochromis niloticus), two economically important foodfish, by investigating alternative, more cost-effective options to promote fish health. The first two studies established reference intervals for immunology, hematology and plasma chemistry analytes in striped catfish in a recirculating aquaculture system (RAS). In a third study, the immunomodulatory effects after directly feeding probiotic strains of Bacillus subtilis NZ86 and O14VRQ in Nile tilapia were ascertained. This last study revealed that supplementation with both of the probiotic strains for 51 days stimulated several local and systemic innate immune responses of tilapia. When these transient probiotic bacteria were present in the gut, a pro-inflammatory environment was developed as evidenced by the localized higher expression of the cytokines tumor necrosis factor (TNF) – α and interleukin (IL) – 1β. Significant increases (p < 0.05) were noted differentially by both probiotic strains throughout the trial in plasma lysozyme content, alternative complement activity, and in the peripheral blood leukocyte profiles. Additionally, there were trends for increased levels of phagocytosis and respiratory burst in leukocytes of the anterior kidney and spleen at the end of the trial, suggesting the potential use of these probiotic strains for improved immune-competence. These findings help to understand and clarify the potential mechanism of action associated with the increased disease resistance recorded in preliminary studies with the same probiotic strains. Implementation of the tools established and validated in this work could be useful in evaluating fish welfare situations involving striped catfish grown in RAS conditions, and also show promise for a healthier foodfish supply where antibiotic applications practices could be minimized. Ph. D.

There are over 200 farmers markets in the state of Virginia. They are regulated by local and state governments. They use the 2013 U.S. Food Code and Cottage Food Laws and regulations that work for farmers markets. How do you adapt these for mobile farmers markets? With more than a dozen mobile farmer markets operating in the state of Virginia, a standard should be applied when it comes to them handling raw agricultural products. How do we go about this when they are not considered the same as a brick and mortar farmers market, but rather regarded as a delivery service? Could we use the same regulations and practices used in governing brick and mortar markets to satisfy this niche? Due to them becoming wildly popular, it’s imperative that we find an applicable method to avoid any slipping through the cracks for this newer market. Ensuring, they fall under the same regulations as a brick and mortar market could also serve as a double-edged sword. Although, they have many similarities they also have vast differences. A conflict that comes into play are the transient properties of the mobile market which affect the temperature, amount of handling, and the possible introduction of foodborne illness. In this thesis I would like to explain why the need for mobile farmers markets arose and how coming up with best management practices can sustain it for years to come. MALS

Airborne pathogenic bacteria can present a significant public health risk. Pathogenic Listeria monocytogenes can colonize numerous surfaces as well, through direct and indirect cross contamination. The physical environment can also affect the transmission and viability of Listeria (distance from the source, temperature, humidity, air flow). The purpose of this work was to explore the ability of Listeria innocua (a surrogate for L. monocytogenes) to contaminate a surface after it has become aerosolized in a bioaerosol chamber and a walk-in cooler. L. innocua was nebulized into a 154 L biosafety chamber (~5 log CFU in 1 mL) at two relative humidity (RH) levels (83% and 65%). Oxford Listeria agar plates, stainless steel coupons and polyethylene (HDPE) coupons in the chamber were exposed to the aerosolized bacteria for 5, 10, 20 or 40 minutes. Also, at these times, air samples (100 L) were collected on to gelatin filters which were transferred to Oxford agar plates. In the second part of the research, L. innocua was nebulized into an 11 m3 walk-in cooler where RH ranged from ~29-37%. Aerosolized bacteria were collected on to Oxford agar plates for 10 min intervals and with 50 or 100 L air samples. Recovery of L. innocua from steel, plastic and agar was significantly higher at 83% RH (2.7 cells/cm2) compared to 65% RH (0.45 cells/cm2). Mean cell recovery from air samples (gelatin filters) was significantly higher (p

In this study, potential of high hydrostatic pressure (HHP) and essential oils (EOs) as natural antimicrobials was evaluated to produce E. coli safe and quality beef product. First, the individual and combined effects of antimicrobial activity (minimum inhibitory concentration) of basil, black cumin, cilantro, cumin, fenugreek, ginger, oregano, black pepper, rosemary, thyme, turmeric oil emulsions on E. coli ATCC 25922 with and without HHP treatment were evaluated. Cumin, oregano and thyme EOs showed highest antimicrobial activity against E. coli ATCC 25922. The synergy of selected EOs against E. coli ATCC 25922 was determined using the checkerboard method to obtain fractional inhibitory concentration index. Although their combinations did not show synergy, they expressed synergy when combined with HHP (400 MPa, 10 min, 20 °C) and the best combination was cumin and oregano EOs with HHP. Effects of HHP and EO combinations on inactivation of E. coli ATCC 25922 in beef were investigated using response surface methodology (RSM). Statistical analysis showed the model was significant for predicting log reduction with high accuracy. The significant model terms were pressure and time. Compared to control, HHP/EO treated samples showed no-post growth when stored up to 120 days at 4°C. Presented results suggests that the combination of HHP and antimicrobials has not only improved the process parameters (lowered pressure, time, and EO concentration) but also prevented recovery of E. coli ATCC 25922 during storage. RSM was employed to analyze the synergistic effects of HHP and EOs on beef quality (color, texture and lipid oxidation). Color indices were significantly affected by pressure, time and their interactions. Above 400 MPa the discoloration was similar to cooked beef and EO addition did not help color improvement. However, EOs showed significant antioxidant activity on both treated and untreated samples during storage. In conclusion, there is a great potential of HHP and EO combinations to enhance pathogen inactivation while keeping the quality of beef. Moreover, presence of EOs can prolong the shelf life of pressure treated beef. Therefore, the combination of HHP and EO is very promising for meat industry. Ph. D.

Salmonella enterica has been associated with several outbreaks due to consumption of low water activity foods including spices. Consequently, to improve microbiological quality, spices are commonly treated before ultimately reaching consumers. These processes may result in sub-lethal injury to cells, which can lead to an underestimation of microbial populations when plating on selective media. Sub-lethally injured cells are difficult to enumerate due to reduced ability to grow on selective microbiological media. Poor recovery of sub-lethally-injured cells may obscure process validation results and lead to overestimation of process effect. Therefore, this work was performed to determine the influence of agar overlay and media supplementation methods on the recovery of Salmonella from steam and ethylene oxide treated peppercorns and cumin seeds. Traditional agar overlay allowed recovery of significantly more Salmonella (p < 0.05) from inoculated peppercorns treated with steam (65.5C, 15 sec.) than selective media (XLT4) or solid agar overlay. Additional supplementation of the TSA base to contain 3,3'-thiodipropionic acid further improved Salmonella recovery from steam treated peppercorns (p < 0.05). For peppercorns and cumin seeds subjected to ethylene oxide, neither sodium pyruvate + yeast extract, 3,3'-thiodipropionic acid, glycerophosphate, ATP, nor magnesium enhanced recovery compared to overlay alone but both methods recovered significantly higher numbers than XLT-4 alone (p < 0.05) for both cumin seeds and peppercorns. Master of Science in Life Sciences

Current methods to reduce the native microbiota and potential pathogens on spices include steam treatments and ethylene oxide (EtO) fumigation. The objectives of this research were to identify the effectiveness of a lab-scale steam apparatus and a commercial EtO process on the inactivation of Salmonella enterica or Enterococcus faecium NRRL B-2354 inoculated whole black peppercorns and cumin seeds. Peppercorns and cumin seeds were inoculated with Salmonella or Enterococcus and processed in a lab-scale steam apparatus at 16.9 PSIA and two references temperatures (165°F and 180°F) and in a commercial ethylene oxide fumigation chamber using a standard commercial EtO fumigation process. Cells were enumerated by serial dilution and plating onto TSA with a thin overlay of selective media. Inoculation preparation influenced inactivation of Salmonella on peppercorns with greater reductions reported for TSA-grown cells compared to within a biofilm. To achieve an assured 5-log reduction of TSA-inoculated Salmonella on peppercorns exposure for 125s and 100s at 165°F and 180°F, respectively is required. For cumin seeds temperatures of 165°F for 110s were needed or 65s at 180°F to assure 5 log reduction. EtO fumigation significantly reduced both microorganisms on both spices (p

Approximately 1600 illnesses and 260 deaths in the United States are caused by food contaminated with Listeria monocytogenes (CDC, 2014; Scallan, et al, 2011). Listeria monocytogenes is a Gram-positive bacterium (Vaquez-Boland, Kuhn et al, 2011) that causes the infection listeriosis within the body. L. monocytogenes can be found in ready to eat (RTE) foods. Unlike other foodborne pathogens, one of the organisms unique characteristics is that it is a psychrotroph, meaning it can grow in refrigeration temperatures; therefore, refrigeration is not the most effective way to control the pathogens growth (figure 1) (Frazer, 1998), even when temperatures almost reach freezing. MALS

Salmonella contamination of spices and other low water activity foods is a growing concern for the food industry due to increased frequency of salmonellosis outbreaks and detection-based product recalls. The impact of inoculation preparation on the survival of a Salmonella enterica and its proposed surrogate, Enterococcus faecium NRRL B-2385, on the whole black peppercorns and cumin seeds was examined. Three liquid inoculation methods (biofilm-inclusion, agar-grown, broth-grown) for Salmonella enterica and surrogate Enterococcus faecium and one dry transfer method for Salmonella enterica were developed then applied to whole peppercorn and cumin seeds. Spices were returned to original water activity (aw 0.3) and stored for 28 days with periodic sampling (0, 1, 7, 14, 21, 28 days) and surviving bacteria enumerated. Average log reductions (LR) over time were statistically analyzed to determine differences in stability during storage. Inoculation preparation was associated with significant differences in recovered Salmonella and Enterococcus from both peppercorn and cumin over the storage period. At 28 days, the most stable inoculations of Salmonella resulted from the biofilm-inclusion (-0.04 CFU/g LR) and agar grown (-0.75 CFU/g LR) methods on peppercorn and the biofilm inclusion method (-0.28 CFU/g LR) on cumin. Log reductions of Enterococcus faecium (-0.02 CFU/g LR biofilm-inclusion-peppercorn, -0.19 CFU/g LR agar-grown-peppercorn, -0.61 CFU/g LR biofilm-inclusion-cumin) were comparable to Salmonella after 28d desiccated storage. These results will guide the inoculation strategies for validating inactivation processes for reducing Salmonella on whole spices, and for comparisons of inactivation of Salmonella and its proposed surrogate Enterococcus faecium. Master of Science in Life Sciences

Salmonella embedded biofilms were formed on whole black peppercorns and treated with ethanol vapor under atmospheric pressure conditions and under vacuum assisted pressure conditions. The effect of ethanol vapor, heat and vacuum pressure on the survival of multiple Salmonella enterica serovars within a biofilm contained on low moisture food products (Aw 0.30-0.40) was observed in this study. Samples were exposed to zero, one, five and ten minute ethanol vapor treatments at atmospheric boiling temperature (Atmospheric Pressure Boiling Method, AB), and at reduced temperature and pressure conditions, -20 inHg (Vacuum-Assisted Boiling Method, VB). The AB treatments showed 4.0 log CFU/g reductions on nonselective media that included native microbiota, and 6.0 log CFU/g reductions on Salmonella selective media. The VB treatments showed 2.69 log reductions on Tryptic Soy Agar and 4.55 log reductions on Xylose Lysine Tergitol-4 agar. Ethanol vapor treatments should be further investigated as an alternative to ethylene oxide or ionizing radiation processes to treat dry spices to control Salmonella. Master of Science in Life Sciences

Risk analysis is a powerful science-based tool that can be used to control and mitigate microbial food safety hazards. Codex recommends conducting preliminary risk management activities (PRMAs) to initiate risk analysis and to plan the risk assessment process. The information learned from these PRMAs should be utilized to construct a quantitative microbial risk assessment (QMRA) model. Then, risk management activities can utilize the QMRA model to identify and select microbial risk management (MRM) options. In this project, Codex recommendations for conducting risk analysis were followed to analyze the risk of acquiring salmonellosis from whole broiler (meat chickens) consumption within the United States. At the first stage, the risk of Salmonella on whole broilers was quantitatively estimated by attributing reported annual salmonellosis to whole broilers. A quantitative microbial risk assessment (QMRA) model was constructed to build an informative risk analysis model based on performance criteria, while minimizing associated modeling complications. The QMRA model was constructed in Excel® (Microsoft Corporation, Redmond, WA, USA) with the @RISK® Add-ins software (Palisade Corp., Ithaca, NY, USA). @RISK® software was used to perform Monte Carlo simulations that account for attendant uncertainties. After the model was tested and calibrated, it estimated the annual salmonellosis cases from whole broilers as 216,408 case/year that corresponds to the number of salmonellosis reported by Center for Disease and Control Prevention (CDC). Furthermore, sensitivity analysis was performed where 16 sensitive inputs (potential places for food safety interventions) and 10 data gaps (inputs that significantly affect the overall uncertainty) were reported. Some QMRA model results were transformed to MRM metrics. These MRM metrics, including ALOPs (Appropriate Level of Protection), FSOs (Food Safety Objectives), POs (Performance Objectives), and PC (Performance Criteria), were calculated along with a sampling plan for a food safety control system. The MRM metrics were utilized to identify and plan food control interventions such as risk communication, auditing, inspection, and monitoring. Furthermore, the QMRA model was utilized to identify and to quantitatively evaluate food safety interventions that affect Salmonella prevalence and/or concentration. Ph. D.

Fresh market tomatoes have been associated with 15 multistate Salmonella outbreaks between 1973 and 2010. While, S. enterica survival has been studied in tomato plants, field studies have been limited. To understand pathogen growth and survival, in crop fields, surrogate or attenuated organisms must be developed and validated. The purpose of this study was to compare the growth and survival of seven attenuated S. enterica Typhimurium and Newport strains against virulent strains S. Typhimurium ATCC14028 and S. Newport J1892 in optimum (TSB and TSB+kan) and minimal M9 growth media, and in commercial, red ripe tomatoes. Bacterial growth in media was assessed via BioScreen. Tomatoes were separately inoculated with 7 Log CFU/g of each isolate via vacuum infiltration, surface spot inoculation, or diced inoculation. Populations of each strain were determined on Days 0, 1, 3, and 5. In media, there were few differences in overall growth and growth rates between mutant isolates and wild-type (P0.05); however, Salmonella strains in diced tomatoes had a higher growth rate than that in spot treated tomatoes (P>0.05). The growths of all the isolates in tomatoes indicated that under the tested conditions, isolates acted similarly to their WT counterparts. Thus, these strains may be able to be used as surrogate organisms in field studies. Master of Science in Life Sciences