Call number: LD2668 .T4 1963 K57 / Master of Science

Call number: LD2668 .T4 1965 A31 / Master of Science

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Previous research evaluated a laboratory strain of Bacillus licheniformis (BL) in a model swine epithelium and found it exerted anti-inflammatory effects on Salmonella enterica serovar Typhimurium (S)-induced secretion of interleukin-8 (IL-8). The current investigation evaluated the anti-inflammatory actions of Bacillus bacteria available commercially as feed additives for the swine industry. Three isolates were obtained from the product, two Bacillus subtilis (BS1 and BS3) and one Bacillus licheniformis (BL2). Swine jejunal epithelial IPEC-J2 cells were seeded into wells on permeable membrane supports and allowed to form confluent monolayers. Treatments included apical pretreatment with BL, BS1, BL2, or BS3 for 17 h without S, and the same Bacillus treatments but with 10[superscript]8 CFU S added in the final 1 h of Bacillus incubation. Two additional treatments included negative control wells receiving no bacteria (C) and positive control wells receiving only S. Following bacterial incubation, wells were washed and fresh media containing gentamicin was added. Cells were incubated for an additional 5 h, after which apical and basolateral media were recovered for quantitation of IL-8 and bacitracin. In addition, inserts with epithelial cells that had received S were lysed and lysates cultured to determine treatment effects on S invasion. Exposure to S alone provoked an increase in IL-8 secretion from IPEC-J2 cells compared to C wells (P < 0.001 for both the apical and basolateral directions). Pre-treatment with each Bacillus isolate followed by challenge with S reduced S-induced IL-8 secretion in both apical and basolateral compartments compared to the wells receiving only S (P < 0.001; except for BS3 apical, P < 0.01). Secretion of bacitracin could only be detected in BL2 and BL2+S. Fewer S colonies could be cultured from lysates of BL2+S than S, BS1+S, and BS3+S treatments (P < 0.001). Results suggest that Bacillus subtilis and Bacillus licheniformis have the ability to intervene in secretion of the neutrophil chemoattractant IL-8 from swine intestinal epithelial cells. This effect on chemokine secretion by gastrointestinal epithelial cells in vitro could not be explained solely by production of bacitracin or reduced invasion of epithelial cells by S.

Salmonella is one of the most important pathogens of mankind and animals alike, causing several billion pounds worth of damage worldwide each year. We have sequenced, annotated and published 4 genomes of Salmonella of well-defined virulence in farm animals. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals. Specifically, we have examined pathway detrition and mutagenesis and linked this to host specificity of the serovars. With the advent of next generation sequencing there has been a boom in genomic sequence submission, and an onslaught of -omics data has ensued. Integrating these different data types is complex and there is little available to visualise this data in the context of its genome. We present GeneBook, a web-based tool that synchronously integrates disparate datasets, displaying a fully annotated genome, enriched with publicly available data and the user's private experiments. It is accessed through a user-friendly interface that allows scientists to interrogate genomic features across multiple, heterogeneous, experiments.

Master of Science / Department of Grain Science and Industry / Cassandra Jones / Salmonella contamination in animal food production facilities is a growing concern. The bacteria has been the cause of 40% of pet food recalls in the past 5 years, and there are potential human health implications because pet food is a direct human contact food. A potential method to reduce Salmonella contamination in pet food is through the use of acidifiers and desiccants to destroy and inhibit growth of bacteria. The objective of this thesis was to quantify Salmonella contamination in livestock feed and pet food manufacturing facilities, and propose mitigation measures to mitigate the presence of pathogens in animal food. Therefore, the objective of Experiment 1 was to investigate sources of Salmonella contamination throughout livestock feed (n = 2) and pet food (n = 2) manufacturing facilities on a specific sampling day. Salmonella was present in all four facilities. However, one of the livestock feed manufacturing facilities had more than double the Salmonella-positive locations than all other facilities. This experiment demonstrated that surface type and location should be taken into consideration when controlling Salmonella contamination. In Experiments 2 and 3, the use of a commercial powdered dry acidulant, sodium bisulfate, was studied as a coating of dog kibble to reduce and prevent Salmonella growth over time. The coating reduced Salmonella concentration, and its efficacy was not impacted by altering the bulk density or surface area of the kibble. Experiment 4 was conducted to determine the efficacy of sodium bisulfate added to poultry mash to reduce or prevent Salmonella growth over time. The inclusion of the dry acidulant did not reduce Salmonella concentration; however, storage time reduced Salmonella contamination in poultry feed. In summary, Salmonella contamination exists in manufacturing facilities, but the location and magnitude of contamination differs. Furthermore, sodium bisulfate effectively reduces Salmonella contamination when applied as a pet food coating, but not in poultry feed.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds remains problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. I have constructed several mutagenicity tester strains that co-express combinations of full-length human cytochrome P450 2E1, rat cytochrome P450 reductase, and human cytochrome b5 in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt-; and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. Coexpressing human cytochrome b5 with cytochrome P450 2E1 and cytochrome P450 reductase potentiates the mutagenicity observed with dialkylnitrosamines. These strains were sensitive to nitrosamines with varying alkyl side chains, including dimethylnitrosamine, diethylnitrosamine, dipropylnitrosamine, and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples, and may serve as useful tools in investigating the molecular properties of proteins in the cytochrome P450 monooxygenase system.

Salmonellosis is a foodborne disease, poultry and poultry products are the main source of the disease. Many countries including Iraq still use antibiotics to control Salmonella. The increase in antibiotic resistant bacteria putatively caused by the overuse of antibiotics in agriculture has provided the incentive to look for alterna-tives to antibiotic to control diseases in livestock. Lactic acid bacteria with probiotic characteristics have the potential to beneficially affect the gut microflora of chick-ens and help reduce the incidence of Salmonella infection. In this study, Lactobacillus plantarum NCIMB 41607 was examined in vitro for in-hibitory effects against Salmonella and in vivo for its effects on the gut microflora and on the incidence of Salmonella infection. In separate studies Lb. plantarum was administered to chicks as fermented moist feed (containing 175 mmol/L lactic acid and 1x109 CFU/g of Lb. plantarum), in drinking water or applied to dry feed. Rifampicin resistance was used as a biomarker for the Lb. plantarum NCIMB 41607. Traditional and molecular microbiology including DGGE, RISA, and FISH were used with electron microscopy to assess the effect of Lb. plantarum on bac-terial population in the digestive system of chicks. Lactobacillus plantarum (NCIMB 41607) was found to reduce the growth of Salmonella Typhimurium and Salmonella Enteritidis by 4 Log CFU/g in a chicken simulated digestive system in vitro. The presence of rifampicine resistant Lactobacillus plantarum in the gut was con-firmed by replica plating and fluorescent in situ hybridisation with a strain specific probe. In general, the bacterial population in the guts of chicks fed Lactobacillus plantarum was more diverse in the guts of chicks fed control feed. There was no significant effect on Salmonella infection in Ross chicks compared with control. However, with specific pathogen free chicks the Salmonella infection was reduced. The intestinal villi lengths in FMF group were increased. These studies demonstrate that probiotics can affect the microflora in the chicken gut. Lactobacillus plantarum NCIMB 41607 may have the potential to control of Salmonella Typhimurium infection in chicken and continued research is advised.

The EspF protein is translocated into host cells by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF sequences differ between EPEC and EHEC serotypes in terms of the number of SH3-binding polyproline rich repeats and specific residues in these regions as well as residues in the amino domain involved in cellular localization. In this study we have compared the capacity of different espF alleles to inhibit: (i) bacterial phagocytosis by macrophages; (ii) translocation through an M-cell co-culture system; (iii) uptake by and translocation through cultured bovine epithelial cells. The espFO157 allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast, espFO157 was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonisation site of EHEC O157 in cattle and a site containing M-like cells. As functional differences could not be simply assigned to variation in established interactions of EspF with Sorting Nexin 9 and N-WASP, yeast-2-hybrid screening was used to identify additional host proteins that may interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was identified from this screen. Mad2L2 was then demonstrated to interact with EspF variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays. While Mad2L2 has been shown to be targeted by the non homologous Shigella effector protein IpaB to limit epithelial cell turnover, we presume that EspF interactions with this protein may indicate a similar function to promote EPEC and EHEC colonization. The final section of work addressed whether bacterial interactions can actually induce M-cell differentiation on follicle-associated epithelium. The work focused on bovine rectal primary cell cultures interacting with Salmonella enterica serovar Typhimurium. The type III secreted protein, SopB, was required for Salmonella to: III (i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii) transform a subset of epithelial cells to a cell type that phenotypically and functionally resembles specialized antigen sampling M cells; (iii) induce RANKL and downstream RelB dependent NFkB signaling. The work suggests that Salmonella may induce this cellular transformation to promote its invasion and colonization of intestinal mucosa.

Memoria para optar al Título Profesional de Médico Veterinario / En los últimos años se han utilizado biotecnologías, como los bacteriófagos, para controlar patógenos bacterianos asociados a enfermedades transmitidas por alimentos (ETA), tales como Campylobacter spp., E. coli 0157H7 y Salmonella enterica. Si bien en un inicio se realizaron fagoterapias directamente en los animales de abasto, hace poco más de una década se inició el biocontrol de estos agentes biológicos directamente en los alimentos, tanto a temperatura de refrigeración como ambiental. Los resultados internacionales indican que la aplicación directa de ellos logra reducir en rangos variables los recuentos bacterianos, sin producir cambios organolépticos. El objetivo de este estudio fue establecer la efectividad de una mezcla de fagos líticos nativos en la reducción de los recuentos de Salmonella Enteritidis (SE), en dos matrices alimentarias como son las cecinas cocidas, particularmente el jamón de pavo y la vienesa pollo. Para esto se trabajó con dos grupos de 25 muestras cada uno: el grupo experimental se contaminó con SE y se le aplicó la mezcla de fagos (MOI 105), en tanto que el grupo control sólo se contaminó con la cepa desafío. La dosis de contaminación varió según la temperatura de incubación de las muestras. Una vez contaminada y aplicada la mezcla de fagos, las muestras se incubaron por 10 días a temperatura ambiente (18 ºC) y a temperatura de refrigeración (4 ºC) para luego realizarles recuento bacteriano. Pasados los 10 días se observó que la aplicación de la mezcla de bacteriófagos redujo significativamente (p < 0,0001) los recuentos de SE en jamón de pavo mantenidos a temperatura ambiente, logrando una leve reducción de 0,48 unidades logarítmicas de SE/g mientras que en las muestras que permanecieron a temperatura de refrigeración se obtuvieron mayores reducciones del orden de 1,72 unidades logarítmicas de SE/g. Para vienesa pollo, la mezcla de fagos redujo los recuentos en 1,13 unidades logarítmicas de SE (p < 0,05) para el grupo que permaneció a temperatura ambiente mientras que en el grupo a temperatura de refrigeración se logró obtener reducciones significativas de 0,48 unidades logarítmicas (p < 0,05) de SE. Los resultados obtenidos indican que la efectividad de esta mezcla de fagos líticos depende de la matriz alimentaria y que podría ser una alternativa para el biocontrol de SE en jamón de pavo y vienesa de pollo a temperatura ambiente y de refrigeración por 10 días. / In recent years, biotechnological tools, as bacteriophages, have been used to control bacterial pathogens associated with food-borne diseases, such as Campylobacter spp., E. coli 0157:H7 and Salmonella enterica. While initially performed direct phagetherapies were performed in livestock, just over a decade ago the biocontrol direct in Food began, both cooling and room temperature. The international data have shown that the bacteriophage direct application reduce, in variable ranges, the bacterial counts, with no organoleptic changes. The aim of this study was to establish the effectiveness of a native lytic bacteriophage cocktail in reducing Salmonella Enteritidis (SE) counts, in two processed food matrices, as turkey breast ham and chicken sausage. Thus, two groups of 25 samples each one were differentiated: the experimental group was inoculated with SE and receive the phage cocktail (MOI 105), in so far as the control group was only inoculated with the bacterial strain. The inoculation dose varied according to the storage temperature of the samples. Once contaminated and added with the phage cocktail, the samples were incubated for 10 days at room (18 ºC) and cooling (4 ºC) temperature, to next perform the bacterial count. After 10 days a significant bacterial count reduction (p < 0.0001) was observed due to the application of the phage cocktail in turkey breast ham stored at room temperature, achieving a slight reduction of 0.48 log CFU/g, while in the samples stored at cooling temperature higher reductions were obtained, around 1.72 log UFC/g. In chicken sausage, the phage cocktail reduced the bacterial counts in 1.13 log UFC/g (p < 0.05) in the samples stored at room temperature, while in the samples stored at cooling temperature the reduction were of 0.48 log CFU/g (p < 0.05). The present results indicates that the effectiveness of this lytic bacteriophage cocktail depends strongly in the type of food matrix, and that could be an alternative tool for the biocontrol of SE in turkey ham and chicken sausages at room and cooling temperatures for 10 days. / Financiamiento: Proyecto FONDECYT 1110038.