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Various control methods used in the meat and poultry processing environment to mitigate listeriosis were evaluated using a dynamic in-plant Monte Carlo model. These control methods included food contact surface testing, sanitation, post-processing lethality treatment, and product formulation with microbial growth inhibitors. The dynamic in-plant model served as an input into the risk assessment model developed by the FDA and FSIS in 2003 which predicts the number of deaths and illnesses resulting from the use of each control method. The use of growth inhibitors combined with a post-processing lethality step was estimated to save over 200 more lives than the FSIS proposed minimum sampling standard. An analysis of data collected by the National Alliance for Food Safety and Security (NAFSS) found that retail-sliced deli meats have a greater prevalence and concentration of L. monocytogenes than prepackaged deli meats. Cross contamination at the retail level is suspected due to clustering of sample positives by store and the influence of sampling time of day on the prevalence of L. monocytogenes. The comparative risk of Listeria monocytogenes in retail sliced versus prepackaged deli meats was evaluated using a modified version of the 2003 FDA-FSIS risk assessment model which considered slicing location and the use of growth inhibitors. The comparative risk ratio for the number of deaths from retail-sliced versus prepackaged deli meats was found to be 9.1 and retail-sliced product with a growth inhibitor was found to be at greater risk for listeriosis than prepackaged product without growth inhibitor. / Master of Science

Log phase cells of Listeria monocytogenes Scott A were heat shocked in Trypticase Soy + 0.6% Yeast Extract broth at 40, 44, and 45°C for 3, 10 and 20 min at each temperature, followed by heating at 55â C for 50 minutes in order to determine an optimum heat shock response. Most heat shock temperatures significantly increased thermal resistance (p < 0.05). Increasing heat shock temperature and time allowed the organism to survive much longer at 50 to 65°C than nonheat shocked cells. The optimal heat shock condition was 45°C for 20 min where D-values at 55°C increased 2.3 fold in non-selective agar and 1.6 fold in selective agar in comparison to non-heat shocked cells. However, cells heat shocked at 48°C for 10 min gave more consistent results; these cells were heated at 50, 55, 60, and 65°C to determine a z-value. Although D-values notably increased due to heat shocking, z-values remained constant regardless of the plating medium. When aerobically heat shocked cells (45°C for 10 min) were plated on a non-selective or a selective medium, a 1.4x increase in D-value was observed when enumerated under strictly anaerobic conditions. Aerobically heat shocked cells (48°C for 10 min) added to shrimp samples retained the increased heat resistance at 55°C when enumerated on a nonselective medium compared to the non-heat shocked cells. Heat shocking conditions may be created in pasteurization or minimal thermal processing of foods allowing increased heat resistance of pathogenic and spoilage microorganisms. / Master of Science

This study examines the extent to which the equivalency provision presented in the SPS agreement is able to foster trade negotiations between countries adopting different food safety measures. The study examines the role of scientific evidence as well as the political, economic, and cultural factors in impacting the national regulatory process and the international trade negotiations. It focuses on the limitations of science in allowing countries to reach consensus in contentious trade-related debates laden with risk uncertainty and missing data. The study consists of comparing the key components of the U.S. and French regulatory systems to identify the cultural basis for the differences in the perception of listeria risk and in preferences to control it. The stringent standards adopted in the U.S. and the preference for pasteurization are attributed to the complete separation of the regulatory functions form those of food production, the open style of decision-making which allows private citizens to review and comment on administrative actions, the unwillingness of U.S. regulators to expose vulnerable individuals to deadly pathogens, and the reliance on quantitative data to validate the effectiveness of pasteurization. The more flexible standards impacting listeria regulation in France are attributed to the the integration of regulatory functions with those of food production, the consumer preference for natural products, the public's trust in the government's regulatory decisions, and the belief that the determination of appropriate safety measures should be left up to the producers. / Master of Science

Survival of Listeria monocytogenes in apple, orange, red grape, and white grape juice was evaluated. A six-strain cocktail of L. monocytogenes was used to inoculate (approx. 7 log cfu/ml) fruit juices, which were stored at 4, 10 and 24°C for up to 61 days. Inoculated red grape juice was stored for up to 5 hours only. Samples were withdrawn at appropriate intervals, neutralized with 1.0 N NaOH, serially diluted in 0.1% peptone water, and surface plated onto Tryptic Soy Agar + 0.6% Yeast Extract (TSAYE) and Modified Oxford Agar (MOX), followed by incubation at 32°C for 48 hours. When L. monocytogenes was no longer detected by direct plating, samples were enriched for L. monocytogenes using Listeria Enrichment Broth (LEB), followed by isolation on MOX. L. monocytogenes remained viable in white grape, apple, and orange juices for up to 12, 24 and 61 days, respectively. Over time, recovery of Listeria on TSAYE versus MOX was not significantly different (P>0.05), indicating that limited acid-injury developed during storage. The results of this study demonstrate the ability of L. monocytogenes to survive in apple, orange, and white grape juices during refrigerated and abusive storage conditions. Therefore, measures to prevent or eliminate L. monocytogenes in the fruit juice-processing environment are necessary to ensure the safety of juice products for public consumption. / Master of Science

Batches of fromage blanc, a soft white cheese were prepared from whole pasteurized cow's milk. Processing and formulation methods were used in cheese making to reduce Listeria monocytogenes in artificially contaminated cheese. Treatments implemented included use of additional starter culture in formulation (25% more starter culture than original formulation), use of a higher temperature draining process (at 45oC instead of 22oC), addition of the anti-listerial bacteriocin nisin (Danisco Nisaplin) in formulation at different levels (125 ppm, 250 ppm, 400 ppm), and combinations of these treatments. Characteristics including pH, fat content, protein content, and color were evaluated for each treatment cheese. Statistically significant differences (p<0.0001) were found between the population (log CFU/g) values of L. monocytogenes in the different treatment cheeses and control cheese. Treatments using additional starter culture or higher temperature draining alone were not successful in significantly reducing numbers of L. monocytogenes, but when combined, a 1 log reduction resulted. Of the different concentrations of nisin used in cheese formulation, the level of 250 ppm nisin was used in combination treatments. The treatments using 250 ppm nisin were able to reduce numbers of L. monocytogenes by 2 log 24h after addition. Combination treatments with 250 ppm nisin and additional starter culture in formulation reduced the level of L. monocytogenes by only 1 log, while combination treatments coupling 250 ppm nisin with a higher temperature draining and treatments with 250 ppm nisin, additional starter culture, and a higher temperature draining were able to reduce the pathogen by 2 log. There were statistically significant (p<0.0001) differences found between cheese treatments for values of pH, fat content, and protein content. This soft cheese could be standardized for each of these parameters by the processor before packaging and sale of cheese. There were no statistically significant (p>0.05) differences found between colorimetric values for different cheese treatments. / Master of Science

Tree fruit growers need information on pathogen dynamics following harvest bags contamination to determine effective sanitation interventions for decontaminating these surfaces. Therefore, the objectives of this research were (i) to determine the survival of generic E. coli, Salmonella, and L. monocytogenes on different harvest bag materials (ii) to quantify the transfer of generic E. coli, Salmonella, and L. monocytogenes from different harvest bag materials to fresh unwaxed apples and (iii) to determine the efficacy of different sanitizers for decontaminating different harvest bag materials. For Obj. 1, harvest bag materials were inoculated with rifampicin-resistant (80ppm; R) E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail. All surfaces were air-dried and held at 22 °C and either 30 or 80% relative humidity for 90 d (E. coli), or at 22 °C and 55% relative humidity (RH) for 21 d (L. monocytogenes and Salmonella). For Obj. 2, harvest bag materials were inoculated with E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail and air dried as previously mentioned. For E. coli trials, bacterial transfer to unwaxed 'Red Delicious' apples was assessed for 2 inoculum dry times (1 or 4 h), 2 contact times (5 or 25 minutes), and 2 pressure scenarios (0.0 or 0.1kg/cm2). For Salmonella or L. monocytogenes trials, transfer was assessed for 1 inoculum dry time (1 h), and 1 contact time (5 minutes). For Obj. 3, coupons were inoculated with L. monocytogenes or Salmonella cocktails and were air-dried. Following inoculation, coupons were exposed to different sanitizer treatments: chlorine, peroxyacetic acid (PAA), isopropyl alcohol with quaternary ammonium compounds (IPAQuats), steam, and water. Regression models were fitted, and Tukey's post hoc test was performed at P<0.05. E. coli exhibited survival for extended durations at 30 % than at 80% RH. In addition, E. coli survived at higher concentrations on canvas surfaces than on cordura and nylon surfaces. Generally, E. coli survived for more than 21 d across all surfaces and exhibited a triphasic die-off pattern. Similarly, L. monocytogenes and Salmonella exhibited die-off in phases with an initial rapid die-off followed by more gradual die-off rates up to 21 d. Canvas materials also promoted better L. monocytogenes and Salmonella survival than cordura surfaces. Contact time did not significantly impact the transfer of E. coli from harvest bag surfaces to apples (P=0.55). However, pressure, inoculum dry time and material type significantly impacted the transfer of E. coli to 'Red Delicious' apples (P≤0.03). The transfer rates of Salmonella did not differ between canvas and cordura surfaces (P=0.46). However, cordura transferred L. monocytogenes at significantly higher rates than canvas surfaces (P<0.001). Of the sanitizer treatments that were used on L. monocytogenes or Salmonella inoculated surfaces, IPAQuats was the most effective achieving over 4.5 log CFU/coupon reduction on both canvas and cordura surfaces. Our studies demonstrated that bacteria could survive for over 21 d under different conditions and could transfer from contaminated harvest bag surfaces to apples underlining the importance of cleaning and sanitizing harvest bags with sanitizers like IPAQuats. / Doctor of Philosophy / Tree fruit growers need information on pathogen behavior on harvest bag surfaces to determine effective sanitation interventions for decontaminating these surfaces. Therefore, the objectives of this research were (i) to determine the survival of generic E.coli, Salmonella, and L. monocytogenes on different harvest bag materials (ii) to quantify the transfer of generic E. coli, Salmonella, and L. monocytogenes from different harvest bag materials to fresh unwaxed apples and (iii) to determine the efficacy of different sanitizers for decontaminating different harvest bag materials. For Obj. 1, harvest bag materials were inoculated with E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail. All surfaces were air-dried and held for 90 d (E. coli), or 21 d (L. monocytogenes and Salmonella). For Obj. 2, harvest bag materials were inoculated with E. coli (TVS353) or Salmonella strain cocktail or L. monocytogenes strain cocktail and air dried as previously mentioned. For E. coli trials, bacterial transfer to unwaxed 'Red Delicious' apples was assessed for 2 drying conditions (wet or visibly dry), 2 contact times (5 and 25 minutes), and 2 pressure scenarios (0.0 and 0.1kg/cm2). For Salmonella or L. monocytogenes trials, transfer was assessed for wet surfaces only and apples sat on surfaces for 5 minutes. For Obj. 3, harvest bags were inoculated with L. monocytogenes or Salmonella cocktails and exposed to different sanitizer treatments including chlorine, peroxyacetic acid (PAA), isopropyl alcohol with quaternary ammonium compounds (IPAQuats), steam, and water. Regression models were fitted, and Tukey's post hoc test was performed at P<0.05. Canvas surfaces promoted better E. coli survival compared to cordura and nylon surfaces. Similarly, canvas materials also supported better L. monocytogenes and Salmonella survival compared to cordura surfaces. Contact time did not significantly impact the transfer of E. coli from harvest bag surfaces to apples (P=0.55). However, pressure, inoculum dry time and material type significantly impacted the transfer of E. coli to 'Red Delicious' apples (P≤0.03). The transfer rates of Salmonella did not differ between canvas and cordura surfaces (P=0.46). However, cordura transferred L. monocytogenes at significantly higher rates than canvas surfaces (P<0.001). Of the sanitizer treatments that were used on L. monocytogenes or Salmonella inoculated surfaces, IPAQuats was the most effective achieving over 4.5 log CFU/coupon reduction on both canvas and cordura surfaces. Our studies demonstrated that bacteria could survive for over 21 d under different conditions and could transfer from contaminated harvest bag surfaces to apples underlining the importance of cleaning and sanitizing harvest bags with sanitizers like IPAQuats.

Prior research reveals foodborne pathogens, as well as enteric bacteria, can thrive in agricultural soils. Understanding how macro- and micronutrients, as well as meteorological factors and observational factors, impact pathogen prevalence may promote a better understanding of how pathogens persist in agricultural soils. This study aimed to (i), characterize associations between soil properties (e.g., macro- and micro-nutrient levels) and microbial targets (e.g., S. enterica and L. monocytogenes prevalence, fecal indicator bacteria concentration). Three produce farms in Virginia were selected from different regions (i.e., Blue Ridge Highlands, Piedmont, Coastal Plains). Farms were sampled four times to capture seasonal differences. Five soil samples were collected from 20 plots (25m2) and pooled in equal quantities to form one sample per plot. A total of 240 samples were collected. Listeria and S. enterica samples (25g) were processed using a modified FDA BAM method, while generic Escherichia coli (gEC) and total coliform (TC) samples (5g) were enumerated using Petrifilm. Presumptive Listeria and S. enterica positive samples were confirmed by PCR using a single gene. Bayesian mixed models were used to evaluate associations with each foodborne pathogen and indicator organism with factors of interest. S. enterica prevalence was 4.2% (10/240) in soil samples. Of the ten S. enterica positive samples, nine samples (90%) were from one farm in eastern VA. Listeria spp. prevalence was 10% (24/240) with L.monocytogenes prevalence being 2.5% (6/240). The average gEC and TC concentrations in soil samples were 1.53 (range 0.95-4.01) and 4.21 (range 1.23-7.12) log CFU/g, respectively. Bayesian mixed models revealed that pH impacted prevalence of L. monocytogenes and gEC (MAP=5.48, 95% CI=0.75,345.39, PD=0.98, ROPE=0.01), and (MAP=4.87, 95% CI=2.31,12.22, PD=1.00, ROPE=0.00). There was no evidence of an association between S. enterica prevalence and factors of interest. S. enterica was 11.55 times more likely to be detected on Farm C (where prevalence was highest) compared to other farms (95 % CI= 1.36, 1155.27, PD=0.98, ROPE=0.00). Findings show that while soil nutrient trends differ across all farms, it is difficult to determine the strength of these trends due to strong regional distinctions. / Master of Science in Life Sciences / Fresh produce is essential to our food supply but is often a source of foodborne outbreaks since they are often consumed raw and have absence of a "kill step". Importantly, numerous produce outbreaks are often traced back to the production environment with water and soil and common methods of contamination. Additionally, growers are required to identify potential sources of contamination to minimize public health risks. For example, the FDA Produce Safety Rule mandates that growers identify measures that can be taken to prevent contamination from sources including soil and water. Many growers will often test their soils to determine nutrient levels so they can determine appropriate fertilizer amounts to apply to their crops. By understanding how the level of nutrients as well as weather patterns and management factors impact microbial detection, scientists and growers can gain a better understanding of how pathogens persist in agricultural soils. This study looked at sampling soil from three farms in three different regions of Virginia (i.e., Blue Ridge Highlands, Piedmont, and Coastal Plains). Sampling occurred four times across two seasons (fall and summer). Five soil samples were pooled to form one composite sample per plot which totals twenty samples per farm. A total of 240 samples were collected overall for this study. Samples were processed for Listeria and Salmonella (foodborne pathogens) using a modified method from the FDA Bacteriological Analytical Manual (BAM), while generic Escherichia coli and total coliforms (indicator organisms) were computed using Petrifilm. Polymerase Chain Reaction (PCR) was used to confirm the presence of Listeria and Salmonella. Key results from this study revealed that different regions had a significant impact on the presence or absence of Listeria and Salmonella in Virginia agricultural soils. For example, Salmonella was more likely to be detected in the Coastal Plains region, where interestingly, the prevalence was highest (90%; 9/10). The prevalence of Listeria spp. (63%; 15/24) and L. monocytogenes (83%; 5/6 ) were highest in the Piedmont region. Additionally, there was no association between the occurrence of Salmonella and factors of interest (e.g., soil properties, weather factors, observational factors). Additionally, the study found L. monocytogenes was more likely to be detected when soil pH increased. These findings reveal that while soil nutrient, weather, and observational trends differ across all farms, sampling region and time of year create challenges in determining trends due to clear regional differences. This study offers insights into how growers can potentially utilize soil testing (a practice they are already doing) to identify how pathogens may be present in their agricultural soils.

Master of Science / Food Science Institute / Daniel Y.C. Fung / James L. Marsden / Listeria monocytogenes is known to be an environmental contaminant in food processing facilities. Floor drains in processing environments harbor Listeria spp. due to continuous presence of humidity and organic substrates. The cleaning and washing activities undertaken may translocate the bacterial cells from the drain to the surrounding environment, thus contaminating food products being produced. This study validates the effectiveness of Johnson Diversey ‘Eliminex’ Foaming Drain Cleaner and Johnson Diversey ‘Final Step’ 512 sanitizer for inhibition of Listeria monocytogenes in drain surfaces and evaluates the potential for translocation of L. monocytogenes from drains to food contact surfaces in the surrounding environment using Listeria innocua as a surrogate. A 7x 7 x 8 feet flexi glass chamber was built in which a 10 inch diameter drain mounted on an aluminum cabinet was placed. The drain was inoculated with the surrogate organism, L. innocua, at specific time intervals and then treated with the given chemicals. Sponge samples were taken and bacterial populations were recovered on Tryptic Soy Agar (TSA), Modified Oxford Medium (MOX) and Thin Agar Layer MOX (TALMOX). Stainless steel coupons (6.4 x 1.9 x 0.1 cm) were hung at 3 different heights 1, 3 and 5 feet inside the chamber and cell translocation from the drain on to the stainless steel coupons was studied. Reductions up to 4 Log CFU/area or ml were seen at the drain surface, drain crate, drain pipe and wash water for both free cells and cells entrapped in biofilms Treatment had a significant effect (p<0.05) on the reduction of bacterial cells. The wash water showed the greatest reduction from 8 Log CFU/ml to est. 0.23 Log CFU/ml. The given cleaner and sanitizer were found to be effective for reducing Listeria spp. on drain surfaces. Results for the second part indicated translocation at all three heights with percentage translocation ranging between 2-17%. Significantly higher translocation (p<0.05) was seen at 1 foot, followed by 3 feet and 5 feet indicating the closer the height to the drain, the greater the number of bacterial cells that are able to transfer from the drain to the surrounding environment.

Memoria para optar al título de Ingeniero en Alimentos / El chorizo español es un producto cárnico listo para el consumo, altamente apetecido por los consumidores de embutidos y carnes. Su intenso sabor y sus distintas formas de consumirlo lo sitúan en una posición privilegiada, sin embargo, es importante tener en cuenta los riesgos potenciales que podría tener el producto por una mala elaboración o por contaminación durante o posterior al proceso de fabricación, especialmente de tipo microbiológica, donde uno de los microorganismos más importantes tanto por su resistencia como por su nocividad es Listeria monocytogenes. El objetivo de este estudio fue determinar el efecto de la radiación sobre L. innocua como marcador de L. monocytogenes y como afecta las propiedades sensoriales en el producto. Fueron determinadas las características fisicoquímicas y microbiológicas del producto, midiendo pH, actividad de agua y recuento de contaminación natural del chorizo español con L. monocytogenes. Se determinó la sobrevida de L. innocua como marcador de L. monocytogenes en el producto inoculado durante un periodo de 11 d, almacenado en condiciones de refrigeración (4±1°C). Se realizó la dosimetría específica para el producto a través de ensayos realizados en el Irradiador experimental de 60Co, obteniendo los tiempos utilizados para cada dosis, las cuales fueron de 0,5; 1,0; 2,0 y 3,0 kGy. Se determinó el valor D10 y posteriormente se efectuó la irradiación del producto con dosis de 4D10 y 5D10 en el producto inoculado con L. innocua para ver el efecto sobre las características organolépticas del producto irradiado. El efecto en las propiedades sensoriales con las dosis utilizadas fue evaluado por un panel de 15 jueces entrenados, a través de un test triangular a los 0 y 7 d. Los valores obtenidos para pH y aw fueron 4,5 y 0,8432 respectivamente. En cuanto a la contaminación inicial de L. monocytogenes en el producto, el recuento arrojó un resultado de <10 ufc/g en todos los ensayos realizados. Para el estudio de la sobrevida de L. innocua en el producto inoculado, se obtuvo como carga inicial en el día 0 una concentración de 1,5 x 102 ufc/g, y para el día 11 una concentración de 5 x 101 ufc/g. A partir del estudio dosimétrico se obtuvieron las posiciones de máxima y mínima radiación, siendo las posiciones 2 y 5 respectivamente. También se establecieron los tiempos de irradiación para las distintas dosis utilizadas en este estudio, los cuales oscilaron entre 40 a 242 minutos aproximadamente. El valor D10 calculado fue de 0,65 kGy para el producto inoculado con L. innocua como marcador de L. monocytogenes. Los resultados del test triangular arrojaron diferencias significativas al día 0 para la muestra con mayor dosis de irradiación con respecto a la muestra no irradiada, mientras que la con menor dosis no mostró diferencias significativas. Los resultados del día 7 fueron iguales para las 2 muestras analizadas, los cuales no mostraron diferencias significativas entre las muestras, independiente de la dosis de irradiación. De acuerdo a los resultados del estudio, la radiación ionizante es un buen tratamiento para productos con alta composición grasa, pero manteniendo una dosis de irradiación menor a 5 kGy, con la cual es posible obtener una reducción de microorganismos contaminantes sin afectar la calidad sensorial / Spanish sausage is a ready-to-eat meat product highly desired by consumers of sausages and meats. Its intense flavor and its different ways of consuming it place it in a privileged position, however, it is important to take into account the potentials risks that could have the product due to poor processing or contamination during and after the manufacturing process, especially microbiological, where one of the most important microorganisms due to its resistance and harmfulness is Listeria monocytogenes. The objective of this study was to determine the effect of radiation on L. innocua as a surrogate of L. monocytogenes and how it affects the sensory properties in the product. The physicochemical and microbiological characteristics of the product were determined by measuring pH, water activity and natural contamination of the Spanish chorizo with L. monocytogenes. The survival of L. innocua as a surrogate of L. monocytogenes in the inoculated product was determined for a period of 11 days, stored under refrigeration conditions (4 ± 1° C). The specific dosimetry for the product was carried out through tests performed in a 60Co experimental irradiator, obtaining the required times for each dose, which were 0,5; 1,0; 2,0 and 3,0 kGy. The D10 value was determined and the irradiation of the product, inoculated with L. innocua, with doses of 4D10 and 5D10 was carried out in order to evaluate the effect on the organoleptic characteristics of the irradiated product. A panel of 15 trained judges, through a triangular test at 0 and 7 days, performed the sensory evaluation. The values obtained for pH and aw were 4,5 and 0,8432 respectively. As for the initial contamination of L. monocytogenes in the product, the count had a result of <10 CFU/g in all tests performed. For the study of survival of L. innocua in the inoculated product, a concentration of 1,5 x 102 CFU/g was obtained as initial loading on day 0 and for day 11 the contamination was reduced to 5 x 101 CFU/g. From the dosimetric study, the positions of maximum and minimum radiation were obtained, with positions 2 and 5 respectively. The irradiation times were also established for the different doses used in this study, which ranged from approximately 40 to 242 minutes. The calculated D10 value was 0,65 kGy for the product inoculated with L. innocua as a surrogate of L. monocytogenes. The results of the triangular test revealed significant differences at day 0 for the sample with a higher irradiation dose compared to the non-irradiated sample, while the one with the lowest dose did not show significant differences. The results of day 7 were the same for the 2 sets analyzed, which did not show significant differences between the samples, independent of irradiation dose. According to the results of the study, ionizing irradiation is a good treatment for products with high fat composition. By using an irradiation dose of less than 5 kGy, is possible to obtain a reduction of the microbiological contamination without affecting the sensory quality