To compare the optimum recoveries of an inoculation of <i>Salmonella enterica</i> serotype Typhimurium, fluorescent microspheres (1.0 μm diameter, carboxylate-modified, crimson FluoSpheres®, Molecular Probes, Eugene, OR), or a combination of both from stainless steel, three recovery methods, including a standard rinse, a one-ply composite tissue (Kimwipe®) or a sonicating brush were used. Findings were used to assess the effectiveness of fluorescent microspheres as surrogates for <i>S.</i> Typhimurium. For each method, ten coupons (304 grade, 2.5 x 8 cm) were inoculated with either 100 μl of a <i>S.</i> Typhimurium culture, or a solution of fluorescent microspheres, or both, at approximate concentrations of 10⁶. After drying for one hour, coupons were sampled using either a rinse of 100 ml of phosphate buffered saline solution (PBS) for one min, a Kimwipe® tissue method, or submerged in PBS and subjected to a sonicating brush for one min. After treatments, PBS solutions were analyzed using duplicate plate counting (<i>Salmonella</i>) or hemacytometry (microspheres). For microspheres and <i>Salmonella</i>, recovery by sonicating brush > rinse > Kimwipe® method. Additionally, the retention of microspheres on the steel ranged from 16 to 25% (mean from five coupons each recovery method). Microspheres yielded a significantly higher recovery rate (11 – 60%) than <i>Salmonella</i> (~1%) for each recovery method, therefore the microspheres used in this study, are not appropriate surrogates for <i>S.</i> Typhimurium for future recovery studies on stainless steel. However, due to their low standard deviations for their mean percent recovery, they hold the opportunity to provide better accuracy and reproducibility. / Master of Science

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.

M.S.

Tesis por compendio / [ES] Salmonella es una de las principales causas de toxiinfecciones alimentarias en el mundo. En Europa, en el último año tuvo lugar un total de 94.203 casos de salmonelosis, de los cuales 8.730 fueron en España. La principal fuente de la infección son los productos de origen animal, principalmente los huevos y la carne de pollo. Los Programas Nacionales de Control de Salmonella, han logrado reducir la prevalencia de la bacteria a nivel de campo. Sin embargo, cada año siguen surgiendo nuevos casos de salmonelosis en la especie humana, y se sigue detectando la presencia de la bacteria en las explotaciones avícolas. Por ello, se continúan buscando nuevas alternativas en la lucha contra la bacteria, como puede ser el empleo de bacteriófagos. Los bacteriófagos o fagos son virus ubicuos en el ambiente que están ampliamente distribuidos en la naturaleza. Son microorganismos que atacan específicamente a bacterias, alterándolas hasta destruirlas. Estas características hacen de los fagos una herramienta muy prometedora para la eliminación de Salmonella en explotaciones avícolas como medida complementaria a la limpieza y desinfección, y como una herramienta más de control para incluir en las buenas prácticas de higiene a nivel de campo. En este contexto, en esta tesis doctoral hemos centrado nuestros estudios en conocer la diversidad fágica en las explotaciones avícolas y estudiar la aplicación de la terapia fágica para el control de Salmonella como medida complementaria a la limpieza y desinfección. Para ello, se realizaron tres experimentos durante el periodo de enero de 2017 y diciembre de 2019, en los que se aislaron y multiplicaron fagos de muestras procedentes de granjas avícolas. El objetivo del primer experimento fue evaluar la prevalencia de fagos frente a Salmonella en granjas comerciales de gallinas ponedoras y broilers. Los resultados de este estudio indican que las granjas avícolas podrían representar una importante fuente de fagos de Salmonella. Además, se ha observado una estrecha relación entre la prevalencia de fagos frente a Salmonella y la prevalencia de serotipos de Salmonella en las granjas avícolas, lo que sugiere que los fagos coexisten con su serotipo. su concentración y aplicarlos frente a las mismas granjas avícolas positivas a Salmonella. El objetivo del segundo experimento fue evaluar el efecto de los fagos contra Salmonella Infantis y Salmonella Enteritidis en las superficies de las granjas, y evaluar la aplicación del procedimiento de los fagos como desinfectante contra Salmonella en las condiciones de campo. Se observó una reducción de la concentración de S. Infantis y S. Enteritidis en las superficies de la nave en 4,55 log10 UFC/mL y 3,85 log10 UFC/mL, respectivamente. La mayor reducción para ambos serotipos se obtuvo el 5º día después de la primera aplicación. Estos resultados ponen de manifiesto que los fagos podrían ser una herramienta prometedora para utilizar en combinación con los procedimientos de limpieza y desinfección. Por último, el objetivo del tercer experimento fue evaluar la aplicación experimental de los autofagos para el control de S. Enteritidis en una granja de gallinas ponedoras contaminada de forma natural. Este estudio pone en evidencia que los autofagos se podrían emplear, no solo como una medida para reducir la excreción de Salmonella por parte de los animales infectados, sino como medida complementaria en la limpieza y desinfección de las instalaciones con una gran eficacia. Además, el hecho de que el autofago eliminara la Salmonella del medio ambiente, aún con los animales en el interior de la nave, podría evitar la re-contaminación horizontal entre animales infectados y no infectados. Los resultados más relevantes que se han obtenido de esta tesis doctoral es que el sector avícola dispone de una herramienta prometedora, económica y ecológica capaz de reducir significativamente la prevalencia de Salmonella cuando las medida / [EN] Millions of human salmonellosis cases are reported worldwide every year, and the disease results in thousands of deaths. In 2018, a total of 94,203 confirmed cases in humans were reported in Europe of which 8,730 were in Spain. The main source of infection is poultry products as eggs and chicken meat. National Salmonella Control Programmes have succeeded in reducing the prevalence of the bacteria at the field level. However, new cases of salmonellosis in humans continue to emerge every year and the presence of the bacterium continues to be detected in poultry farms. Therefore, new alternatives continue to be sought in the fight against the bacteria, such as the use of bacteriophages. Bacteriophages or phages are ubiquitous viruses in the environment and are widely distributed in nature. They are microorganisms that specifically attack bacteria, altering them until they are destroyed. These characteristics make phages a very promising tool for the elimination of Salmonella in poultry farms as a complementary tool for cleaning and disinfection, and as another control tool to be included in good hygiene practices at field level. Thus, in this doctoral thesis, we have focused our studies on determining the phage diversity in poultry farms and studying the application of phage therapy for the control of Salmonella as a complementary measure to cleaning and disinfection. To this end, three experiments were carried out during the period from January 2017 to December 2019, where phages were isolated, purified, and multiplied from different samples taken from poultry farms and their epidemiology was studied. The objective of the first experiment was to assess Salmonella-phage prevalence in commercial poultry farms in terms of the production livestock type: layers or broilers. The results of this study indicated that poultry farms could represent an important source of Salmonella phages. Moreover, we have shown a close relationship between Salmonella phage prevalence and Salmonella serovar prevalence in poultry farms, suggesting that phages co-exist within their serovar. The purpose of the second experiment was to assess the effect of phages against Salmonella Infantis and Salmonella Enteritidis on farm surfaces and evaluate phage application procedure as sanitiser against Salmonella in field conditions. Results showed S. Infantis and S. Enteritidis decreased by 4.55 log10 CFU/mL and 3.85 log10 CFU/mL, respectively; the maximum reduction in Salmonella was on the 5th day, after 108 PFU/mL and 103 PFU/mL phage application. These results highlight phages as a promising tool together with cleansing and disinfection. However, more studies are needed to demonstrate their efficacy as sanitisers in poultry farms. Finally, the aim of the third experiment was to assess the application of autophages for the control of S. Enteritidis in a naturally contaminated laying hen farm. This study shows that autophages could be used not only as a measure to reduce the excretion of Salmonella by infected animals but also as a complementary measure in the cleaning and disinfection of the facilities. In addition, the fact that autophages eliminated the Salmonella from the environment, even with the animals inside the facility, could avoid horizontal transmission of Salmonella among infected and non-infected animals. The most relevant results obtained from this doctoral thesis are that the poultry sector has a promising, economic and ecological tool, as phage therapy, able to significantly reduce the prevalence of Salmonella when current measures cannot eliminate it from poultry farms. / [CA] Salmonella és una de les principals causes de toxiinfeccions alimentàries en el món. En A Europa, en l'últim any es van reportar 94.203 casos, dels quals 8.730 es van produir a Espanya. La principal font d'infecció són els productes d'origen animal, principalment els ous i la carn de pollastre. Els Programes Nacionals de Control de Salmonella han aconseguit reduir la prevalença a nivell de camp. No obstant, cada any continuen sorgint nous casos de salmonel·losi, i es continua detectant la presència de la bacteria en algunes explotacions avícoles. Per això, es continua buscant noves alternatives en la lluita contra la bacteria, com pot ser l'ocupació d'additius en el pinso, un bon maneig a nivell de camp o l'ús de bacteriòfags. Els bacteriòfags o fags són virus ubics en l'ambient i es troben àmpliament distribuïts en la naturalesa. Són microorganismes que ataquen específicament a les bacteries, alterant-les fins a destruir-les. Estes característiques fan dels bacteriòfags una ferramenta molt prometedora per a l'eliminació de Salmonella en explotacions avícoles com a mesura complementària a la neteja i desinfecció i com una ferramenta més de control per a incloure en les bones pràctiques d'higiene. En este context, en esta tesi doctoral hem centrat els nostres estudis a conèixer la diversitat fàgica en les explotacions avícoles i estudiar l'aplicació de la teràpia fágica per al control de Salmonella com a mesura complementària a la neteja i desinfecció. Per a això, es van realitzar tres experiments durant el període de gener de 2017 i desembre de 2019, en els que es van aïllar y multiplicar bacteriòfags de diferents mostres procedents de granges avícoles. L'objectiu del primer experiment va ser avaluar la prevalença de bacteriòfags enfront de Salmonella en granges comercials de gallines ponedores i pollastres. Els resultants de este estudi indiquen que les granges avícoles podrien representar una important font enfront de bacteriòfags de Salmonella. A més, s'ha observat una estreta relació entre la prevalença de bacteriòfags enfront de Salmonella i la prevalença de serotips de Salmonella en les granges avícoles, la qual cosa suggereix que els bacteriòfags coexisteixen amb el seu serotip. L'objectiu del segon experiment va ser avaluar l'efecte dels bacteriòfags contra Salmonella Infantis i Salmonella Enteritidis en les superfícies de les granges, i avaluar l'aplicació del procediment dels bacteriòfags com a desinfectant contra Salmonella en les condicions de camp. La concentració de Salmonella Infantis i Salmonella Enteritidis va disminuir 4,55 log10 UFC/mL i 3,85 log10 UFC/mL, respectivament, després de dos aplicacions consecutives de bacteriòfags. La major reducció de S. Infantis i S. Enteritidis es va obtindre el 5º dia després de la primera aplicació. Estos resultats manifesten que els bacteriòfags són una ferramenta prometedora per a utilitzar en combinació amb els procediments de neteja i desinfecció. Finalment, l'objectiu del tercer experiment va ser avaluar l'aplicació experimental dels autòfags per al control de S. Enteritidis en una granja de gallines ponedores. Este estudi posa en evidència que els autofagos es podrien emprar com a mesura complementària en la neteja i desinfecció de les instal·lacions amb una gran eficàcia i per a reduir els recomptes de Salmonella en animals infectats. A més, el fet de que l'autòfag eliminarà la Salmonella del medi ambient, encara amb els animals en l'interior de la nau, podria evitar la recontaminació horitzontal entre animals infectats i no infectats. Els resultats més rellevants que s'han obtingut d'esta tesi doctoral és que el sector avícola disposa d'una ferramenta prometedora, econòmica i ecològica capaç de reduir significativament la prevalença de Salmonella quan les mesures actuals no són capaços d'eliminar-la de les granges avícoles. / Agradecer al Centro de Calidad Avícola y Alimentación Animal de la Comunidad Valenciana (CECAV) por su apoyo para realizar este proyecto. En especial, al comité técnico de Asociación Avícola Valenciana (ASAV) por la iniciativa de este proyecto y a los miembros de la Junta Directiva de CECAV por la financiación. / Sevilla Navarro, S. (2020). Selection and assessment of bacteriophages against salmonella spp. in the poultry industry [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/149561 / Compendio

Salmonella and Campylobacter are the leading causes of bacterial foodborne illness in the United States. Commonly found in the gastrointestinal tract of poultry, Salmonella and Campylobacter may show little to no signs of infection in birds. The objective of this dissertation was to evaluate the influence on mRNA abundance of nutrient transporters and host defense peptides (HDPs) during a Salmonella or a Campylobacter challenge in young commercial broilers. Comparisons were made between non-challenged and challenged (106, 107, or 108 colony forming units of Salmonella or Campylobacter) broilers on expression of nutrient transporters and host defense peptides in the duodenum, jejunum, ileum and cecum at various days after inoculation. During a Salmonella challenge, changes in mRNA abundance of nutrient transporters and avian beta-defensins (AvBD) vary by day, tissue and challenge dose. ZnT1 may play an important role during a Salmonella challenge as mRNA abundance of ZnT1 significantly increased (P<0.05) by day 7 in the 108 group compared to the control. Early changes in LEAP2 mRNA abundance were observed in the 106 group than the 107 and 108 groups. However, at a later time point post challenge, a lower abundance of almost all AvBD mRNA (P<0.05) was observed in the lower gastrointestinal tract especially in the 107 and 108 groups compared to the control group, indicating that the pathogen may be influencing intestinal expression of AvBD mRNA. In Campylobacter, analyses revealed that expression of zinc transporter 1 (ZnT1) increased (P<0.05) in the duodenum, ileum and ceca in the 106 group on day 7. An increase (P<0.05) in the expression of avian beta-defensins were observed on day 14 in the ileum and ceca in the 106 group compared to the control group. Pathogens like Salmonella and Campylobacter may have an influence on the mRNA abundance of nutrient transporters and HDPs. Manipulation of these genes may ensure the survivability of these pathogens. Through sequestration of nutrients, the pathogen would have the ability to colonize the host and replicate. However, it must evade the host immune system as well. The processing of infected poultry with these pathogens may lead to foodborne illness in humans. Further research is needed to investigate possible methods to counter the influence these pathogens have on host immunity genes. / Ph. D. / Foodborne pathogens such as Salmonella and Campylobacter are commonly found in the gastrointestinal tract of poultry, causing little to no disease symptoms in poultry. Consumption of uncooked or mishandled meat and eggs from infected poultry could result in foodborne illness in humans. Little is known, however, about the influence of Salmonella and Campylobacter on the intestinal expression of nutrient transporters and immune genes such as host defense peptides in broiler chickens. Nutrient transporters are responsible for the transport of a variety of nutrients across the intestinal lumen to the blood. Host defense peptide are small peptides, which can be effective against invading bacteria, viruses and fungi. Therefore, the effects of Salmonella and Campylobacter at low, medium, and high challenge doses were determined in broiler chickens. In chickens challenged with Salmonella, changes in the expression of nutrient transporters and host defense peptides were dependent on day, intestinal segment and challenge dose. The expression of the zinc transporter increased in chickens challenged with the highest Salmonella dose. In chickens challenged with Campylobacter, changes in expression of nutrient transporters and host defense peptides were also observed. Expression of the zinc transporter increased in chickens challenged with the lowest Campylobacter dose. Expression of host defense peptides increased in chickens challenged with the lowest Campylobacter challenge dose. These results indicate that cellular zinc levels as well as host defense peptides may play an important role in modulating a Salmonella and Campylobacter infection.

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.

The effect of pressure on the log reduction of six strains of E. coli O157:H7 and five serovars of Salmonella enterica were investigated in tryptic soy broth, sterile distilled water and commercially sterile orange and apple juice. Samples were subjected to high hydrostatic pressure (HHP) at 300 and 550 MPa for 2 minutes at 6°C, and then held for 24 hours at 4°C following treatment. E. coli O157:H7 strain E009 was the most pressure resistant, having a decrease of only 0.77 log10 CFU/ml directly after pressurization in TSB. S. Agona was the most pressure resistant Salmonella serovar tested with a decrease of 3.79 log¬10 CFU/ml in TSB at 550 MPa. The two most pressure resistant cultures were then used in a subsequent study using HHP in conjunction with antimicrobials (dimethyl dicarbonate [DMDC] at 62.5 and 125 ppm, hydrogen peroxide at 150 and 300 ppm, cinnamic acid, potassium salt at 125 and 250 ppm, potassium sorbate [KS] at 500 and 1000 ppm and sodium benzoate [NaB] at 500 and 1000). For both E. coli O157:H7 and Salmonella, the most effective antimicrobial was DMDC having a 5.79 and 5.96 log10 CFU/ml decrease directly following pressurization, respectively. Other treatments that were significantly different from the samples treated with no antimicrobial were hydrogen peroxide, and NaB at 500 ppm for E. coli O157:H7 and a treatment of NaB at 1000 ppm for S. Agona. After 24 hours at 4°C, S. Agona samples with added antimicrobials had a close to or above 5-log10 CFU/ml reduction. DMDC should be further investigated as an antimicrobial agent that can work in conjunction with HHP. / Master of Science

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.

Seed sanitization is crucial for microgreen production to prevent pathogens that compromise quality and safety. This study examined the effects of various sanitization methods on germination, mold suppression, microbial load, and Salmonella spp. survival on leek (Allium porrum L.) seeds. Seeds were treated with Hydrogen Peroxide (HP) at 0.27%, Hot Water (HW) at 85 ± 2°C, Tsunami® 100 (TS) at 0.04%, Vinegar (VN) at 1.1%, and Deionized Water (DI). Germination and mold percentages were recorded over 5 days. HW and HP treatments resulted in significantly higher germination rates (72% and 71.4%) compared to other treatments, with HW effectively preventing mold growth. Microbiological analysis showed that HW treatment significantly reduced aerobic plate count (APC) to 4.6 log CFU/g compared to 5.6 log CFU/g in control seeds. HW also had the lowest coliform count at 1 log CFU/g and significantly reduced Enterobacteriaceae, yeast, and mold counts. Both HW and TS treatments effectively reduced Salmonella, with HW showing the most significant reductions. These findings demonstrate that HW treatment enhances germination, inhibits mold growth, and reduces bacterial load, including Salmonella, on leek seeds. Implementing appropriate sanitization methods ensures high-quality and safe microgreens, improving product quality, consumer health, and overall food safety standards.

Orientador: Roberto de Oliveira Roça / Resumo: O objetivo deste estudo foi determinar a excreção de patógenos e inocuidade da carcaça de bovinos alimentados com diferentes níveis de silagem inoculada de grãos úmidos de destilaria desengordurados (WDG). Um total de 100 bovinos machos não castrados, 50% Angus e 50% Nelore, foram divididos aleatoriamente entre quatro dietas (N = 25) compostas por diferentes níveis de silagem de WDG (0, 15, 30 e 45% da matéria seca dietética). Amostras de fezes foram colhidas por meio de suabe da junção reto anal, 15 dias antes do abate, para determinar as ocorrências e quantificação de Escherichia coli produtora de toxina Shiga (STEC), E. coli enteropatogênica (EPEC) e Salmonella spp. por meio da técnica qPCR. Também foram colhidas, 52 dias antes do abate, amostras de fezes de cada animal do piso do curral, logo após defecação, as quais foram submetidas a análises físico-químicas. Logo após o abate, a ocorrência e a contagem de indicadores higiênicos e sanitários, E. coli não patogênica, coliformes totais e bactérias aeróbias mesófilas, assim como, a ocorrência de STEC, EPEC e Salmonella spp., foram determinados a partir de amostras colhidas por meio de esponja da superfície das carcaças das regiões do coxão, flanco, peito e pescoço. Os resultados quantitativos foram submetidos a análises de variância e os dados binários foram submetidos a análises logísticas com razão de chances. Todas as análises estatísticas foram realizadas no software estatístico SAS 9.4 considerando um nível de signifi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to determine the pathogen excretion and carcass safety of cattle fed different levels of inoculated silage from degreased wet distillery grains (WDG). A total of 100 male, 50% Angus and 50% Nelore male bulls were randomly divided into four diets (N = 25) composed of different levels of WDG silage (0, 15, 30 and 45% of dietary dry matter) . Stool specimens were collected by rectal anal junction swab 15 days prior to slaughter to determine the occurrences and quantification of Shiga toxin-producing Escherichia coli (STEC), E. coli enteropathogenic (EPEC) and Salmonella spp. by the qPCR technique. Samples of faeces from each animal on the corral floor were also collected, 52 days before slaughter, immediately after defecation, which were submitted to physical-chemical analysis. Immediately after slaughtering, the occurrence and counting of hygienic and sanitary indicators, non-pathogenic E. coli, total coliforms, and mesophilic aerobic bacteria, as well as the occurrence of STEC, EPEC and Salmonella spp., were determined from samples collected by medium of the surface of the carcasses of the regions of the tail, flank, chest and neck. The quantitative results were submitted to analysis of variance and the binary data were submitted to logistic analyzes with odds ratio. All statistical analyzes were performed in SAS 9.4 statistical software considering a significance level of 5%. The results showed that there was no difference between treatments fo... (Complete abstract click electronic access below) / Doutor