Master of Science / Food Science Institute / Elizabeth Boyle / Intrinsic factors impact Listeria monocytogenes growth in ready-to-eat poultry products. Sliced deli turkey was formulated with in-going concentrations of 1.5% NaCl or 0.75% NaCl/0.75% KCl, 0 ppm or 200 ppm NaNO[subscript]2, and using 10% or 45% pump for a total of 8 treatments. Turkey roasts were sliced and inoculated with a 5-strain L. monocytogenes cocktail or peptone water (control), vacuum packaged, and stored at 4[degree]C. Treatments were sampled on days 0, 7, 14, 21, 28, 42, 63, and 91 of storage to determine L. monocytogenes mean log growth and aerobic plate count (APC). The pH, water activity, residual nitrite concentration, and percent fat, moisture, protein, and sodium were measured using control treatments on each sampling day. There was a nitrite by day and a percent pump by day interaction (P<0.05) for L. monocytogenes and APC populations. Listeria monocytogenes populations in treatments containing 200 ppm NaNO[subscript]2 were 0.70 to 2.39 log CFU/cm[superscript]2 lower compared with products formulated with 0 ppm NaNO[subscript]2. Using 10% pump reduced L. monocytogenes populations by 0.62 to 1.50 log CFU/cm[superscript]2 on days 7 to 28 and at day 63 compared with 45% pump treatments. Incorporating 1.5% NaCl or 0.75% NaCl/0.75% KCl into formulations did not affect (P>0.05) L. monocytogenes populations during storage. On days 7 through 91, APC populations were 0.76 to 2.96 log CFU/cm[superscript]2 lower with inclusion of 200 ppm NaNO[subscript]2 compared to 0 ppm NaNO[subscript]2. There was a treatment by day interaction (P<0.05) for L. monocytogenes populations and APC. The initial inoculum level of L. monocytogenes averaged 2.21 log CFU/cm[superscript]2 and was similar (P>0.05) for all treatments on day 0. Listeria monocytogenes populations increased (P<0.05) from day 0 to 14 by 1.30 to 5.04 log CFU/cm[superscript]2. Overall, L. monocytogenes populations increased during storage and by day 91 L. monocytogenes populations were similar regardless of NaNO[subscript]2 level used except for treatments formulated with 0.75% NaCl/0.75% KCl and 10% pump. Listeria monocytogenes and APC populations were influenced by nitrite concentration and percent pump, while inclusion of NaCl or NaCl/KCl did not affect L. monocytogenes growth during refrigerated storage in vacuum packed sliced deli turkey.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / Antimicrobial resistance (AMR) has arisen as one of the most important public health concerns in the last 60 years. AMR results from pathogenic strains of bacteria adapting to antimicrobial-containing environments through mutations or through horizontal gene transfer (HGT) of genetic material containing resistance genes. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. This dissertation explores the factors, specifically catecholamines and antimicrobials that influence the conjugation frequencies of enteric bacteria including Salmonella, E. coli and Enterococcus. We found that the catecholamine norepinephrine (NE) at physiological concentrations enhanced conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an E. coli recipient in vitro. Additional experiments determined the influence of the antimicrobial concentrations above, equal to and below the minimum inhibitory concentration (MIC) under in vitro conditions on conjugation efficiencies using an Enterococcus to Enterococcus mating pair in addition to the Salmonella to E. coli mating pair. Conjugation occurred in all concentrations, but efficiencies of transfer were consistently low in 0 MIC and 1 MIC, with increased activity both above and below 1 MIC. These data were fit to a previously described mathematical model and the rate constant E that relates the rate of gene transfer to drug concentration was determined. The data showed highly similar patterns of conjugation efficiencies when compared to the rate constant E. A final study we measured conjugation frequencies when donor Salmonella Typhimurium and the E. coli recipient were exposed to both variable concentrations of oxytetracycline and NE. Conjugation was increased pre- and post- MIC, but conjugation frequencies were not enhanced further by the combination of the oxytetracycline and the NE. This dissertation defines the role of outside factors in conjugative gene transfer, and may provide future insight into better control of AMR.

Master of Agribusiness / Department of Agricultural Economics / Michael Woolverton / To meet the demand of the 2007 Energy Bill will require a new approach to ethanol production in the United States. The question persists: how can the ethanol industry in the United States produce 21 billion gallons of ethanol from cellulosic sources? This challenge will require changes in the facilities currently manufacturing ethanol, the collection and storage methods to which the Midwestern farmer is accustomed, and a drastic change in farm production practices. Several different methods of cellulosic ethanol production are being examined. One such method is to change the focus from starch based ethanol to ethanol produced by harvest, collection, and manufacture from corn cobs. Research has included surveys, development of economic models, and focus group meetings to determine the feasibility of corn cobs as a viable raw material source for cellulosic ethanol. Findings indicate that: corn cob collection is feasible for the Midwestern farmer. According to the economic models presented in this thesis, Midwestern farmers can benefit economically from the collection of corn cobs. Further, the collection of corn cobs allows for current ethanol plants to be upgraded with new technology without major change in the manufacturing processes. The focus of this research was to determine which method of corn cob collection was preferable for Midwestern corn producers.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / H. Morgan Scott / Copper, as copper sulfate, is often used as an alternative to in-feed antibiotics for growth promotion in both swine and cattle diets. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a plasmid-borne transferable copper resistance gene (tcrB). The plasmid also carries tetracycline [tet(M)] and macrolide [erm(B)] resistance genes. Because of the genetic link between acquired copper (tcrB) and antibiotic resistance in Enterococcus spp., we hypothesized that copper supplementation may exert selection pressure for enterococci to become resistant to macrolides and tetracyclines, and possibly to other antibiotics. We conducted studies in cattle and swine to investigate the relationship between copper supplementation and the fecal prevalence of tcrB-positive enterococci, as well as its potential co-selection for macrolide and tetracycline resistance. The prevalence was higher in animals fed diets supplemented with elevated level of copper compared to normal level (P < 0.05). The tcrB-positive isolates belonged to either E. faecium or E. faecalis; the majority was E. faecium. All tcrB-positive isolates also contained both erm(B) and tet(M) genes; however, none of them harbored the vanA gene. Median minimum inhibitory concentrations (MIC) of copper for tcrB-positive and tcrB-negative enterococci were 22 mM and 4 mM, respectively (P < 0.0001). The overall prevalence of erm(B) and tet(M) genes among enterococcal isolates of cattle were 46.8 % and 57.5%, respectively; in contrast,100% of the swine isolates were positive for both erm(B) and tet(M) genes. The transferability of the tcrB gene was demonstrated by filter mating assay. Multi-locus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci are potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut. The occurrence of vancomycin resistant enterococci in swine in the US is very rare. Strains of E. faecium positive for vanA, that confers resistance to vancomycin, were isolated and characterized from swine feces. The swine strains belonged to clonal complex 17, a well-adapted hospital clone throughout the world.

Doctor of Philosophy / Food Science - Animal Sciences & Industry / Kelly J. K. Getty / Produce such as tomatoes, lettuce, and cantaloupes have been associated repeatedly with food outbreaks connected to various Salmonella serovars, Listeria monocytogenes, and Escherichia coli O157:H7. The aim of this research was to validate washing solutions and techniques in reducing pathogens on produce surfaces. Lettuce (25 ± 0.3g) and tomatoes were inoculated with E. coli O157:H7 and Salmonella spp., respectively. Samples were treated with tap water (TW) or a chemical wash treatment (CWT; containing citric acid) for 30, 60, or 120 s. Reduction of E. coli O157:H7 and Salmonella spp. populations on the surface of leaf lettuce and tomatoes, respectively, were greater (P<0.05) for CWT (ca. 3.0 logs) than for TW (ca. 2.3- 2.5 logs). Cantaloupes were washed with TW, 9% vinegar solution, or a commercial antimicrobial for fruit and vegetables treatment (CAFVT; containing lactic acid) for 2 min using a washing system. Cantaloupes were cut into wedges or cubes and stored at 4ºC for aerobic plate counts (APC) on days 0, 1, 3, and 6. APC populations of cubed and wedged cantaloupes were different over time (P=0.00052); cantaloupes washed with 9% vinegar solution showed the lowest APC populations after day 1 and 3 of storage. Salmonella spp. or L. monocytogenes inoculated cantaloupes were washed with CPW for 30, 60 or 120 s. Washing cantaloupes for 120 s with CPW showed greater (P<0.05) reductions of Salmonella spp. and L. monocytogenes populations (1.26 and 1.12 log₁₀ CFU/cm²) than TW (ca. 0.63 log₁₀ CFU/cm²) on cantaloupe surface. Lettuce leaves were inoculated with rifampicin-resistant E. coli surrogates and then washed with CAFVT, 5% vinegar solution or TW for 2 min with agitation (washing system) or without. Log reductions of CAFVT (2.25 log₁₀ CFU/g) were greater (P=0.0145) than those by tap water (1.34 log₁₀ CFU/g), but similar to 5% vinegar solution (2.09 log₁₀ CFU/g). Washing lettuce with continuous agitation achieved higher (P=0.0072) E. coli reductions (2.26 log₁₀ CFU/g) than without agitation (1.53 log₁₀ CFU/g). Overall, incorporation of wash solutions or agitation (washing system) in the washing process compared to TW alone reduced greater (P<0.05) APC, pathogens, or surrogates populations from lettuce, tomato, and cantaloupe surfaces.

Master of Science / Department of Biochemistry / Rachel Zufferey / Glycerolipids are the most abundant lipids and are important constituents of various virulence factors in the protozoan parasite Leishmania. The dihydroxyacetone phosphate acyltransferase LmDAT catalyzes the first step of the ether, and possibly ester glycerolipid biosynthetic pathway. A L. major null mutant of LmDAT grew slowly, died rapidly during the stationary phase of growth, and more importantly, was attenuated in virulence in mice. The goal of this study was to determine the molecular basis responsible for the attenuated virulence. Western blot analysis revealed that the ∆lmdat/∆lmdat null mutant synthesized altered versions of the virulence factor lipophosphoglycans that were not released in the media, suggesting that its lipid anchor structure was altered. The ∆lmdat/∆lmdat strain differentiated into virulent metacyclics, but with lower efficiency compared to the wild type. Using the autophagosomal marker ATG8-GFP, the ∆lmdat/∆lmdat line produced twice as many autophagosomes as the wild type, suggesting that it is either defective in degradation of autophagosomes or that autophagy is simply induced. In conclusion, the attenuated virulence of ∆lmdat/∆lmdat may be explained by i) its inability to synthesize and release normal forms of lipophosphoglycan, ii) its inability to fully differentiate into virulent metacyclics, and iii) altered autophagy.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / Escherichia coli O157:H7 is a human food-borne pathogen and cattle feces are a major source of contamination. Immunization against E. coli O157 may be a practical pre-harvest intervention strategy. A siderophore receptor/porin proteins (SRP) based vaccine has been developed to decrease the prevalence of E. coli O157 in cattle. Two studies were conducted to determine the efficacy of the SRP vaccine. In the first study, thirty calves were randomly assigned to one of two groups: control or SRP vaccine. Two weeks after the second vaccination, calves were orally inoculated with nalidixic acid-resistant (Nal[superscriptR]) E. coli O157. Fecal samples were collected for five weeks. Calves were necropsied on day 35 to collect gut contents and tissue swabs to determine Nal[superscriptR] E. coli O157:H7. The number of calves that were culture positive for E. coli O157 were lower (P= 0.07) in vaccinated group compared to the control. In the second study, cattle in two feedlots were randomized to SRP vaccine or control. Cattle were vaccinated on days 0 and 21. Rectal fecal samples were collected on day 0, and pen floor samples were collected on days 21, 35, and 70. Rectal fecal samples, RAMS, and hide swab samples were collected on d 85. Cattle were weighed on days 0, 21, and 85. Vaccination significantly reduced (P = 0.04) fecal E. coli O157 prevalence. There was also a decrease (P < 0.05) in E. coli O157 prevalence on hides and in fecal samples on day 85 in vaccinated cattle compared to the control.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / Enterococci are a group of commensal bacteria that are important nosocomial pathogens. They are abundant in human sewage and wastewater treatment facilities (WWTF). This study focused on the role of house flies, Musca domestica, in the ecology of enterococci at WWTF in both field and laboratory experiments. The first study objective focused on sampling and characterizing enterococci from house flies and wastewater sludge from four WWTF in northeastern Kansas. Enterococci were quantified, identified, and screened for antibiotic resistance and virulence traits, and genotyped. The profiles of enterococci (spp. diversity, antibiotic resistance and virulence) from WWTF sludge and the house flies were similar, indicating that the flies successfully acquired the bacteria from the WWTF substrate. Enterococci with the greatest amount of antibiotic resistant and virulence traits originated from the WWTF that processed meat waste from a commercial sausage plant. Genotyping of E. faecalis revealed clonal matches from sludge and house flies. The second study objective involved tracking the fate of E. faecalis in the digestive tract of house flies in laboratory assays. Colony forming unit (CFU) counts were highest in the midgut at 1 h and declined during the first 24 h. In the labellum, foregut and hindgut, E. faecalis concentrations were more variable but were overall higher after 24 h. Observations from CFU counts and visualizations under a dissecting microscope revealed that E. faecalis peaked in the crop after 48 h suggesting active proliferation in this region. The third objective of the study involved tracking the emergence of calyptrate muscoid flies from stockpiled biosolid cake at one of the four WWTF. Traps were employed at the site for a total of 47 weeks, totaling 386 trap-weeks. A total of 11,349 calyptrate muscoid flies were identified with the two most common species being stable flies (Stomoxys calcitrans) (9,016, 80.2%) and house flies (2022, 18.0%). Numbers of stable flies and house flies peaked around mid-July each year and a second, smaller peak was observed for stable flies 5-8 weeks later. Estimated annual emergence of stable flies was 551,404 and for house flies 109,188.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / As the world population continues to grow and the uncertainty of petroleum and food availability transpires, alternative resources will be needed to meet our demands. Single cell oil (SCO) from oleaginous yeast is a renewable noncrop-based resource that can be used for the production of petroleum counterparts. Currently, commercial production is limited, mainly due to high production costs and competition from cheaper alternatives. As a result, improved fermentation techniques, utilization of low-valued feedstocks and efficient downstream processing would be highly valuable. The major objectives of this study were to: 1) optimize fermentation conditions for the development of a novel fed-batch fermentation to enhance oil production using Lipomyces starkeyi, 2) determine the major lipids produced by L. starkeyi, 3) utilize low-valued hemicellulose-rich feedstocks for oil production, and 4) demonstrate the use of 2-methyltetrahydrofuran (2-MeTHF) and cyclopentyl methyl ether (CPME) as greener solvents for oil extraction. Under optimized fermentation conditions, the oil yield increased from 78 to 157 mg oil/g sugar when supplying xylose rather than glucose as the major carbon source. A novel repeated fed-batch fermentation supplying glucose for growth and xylose for lipid accumulation generated the highest oil yield of 171 mg oil/g sugar, oil content of 60% (dry mass basis) and oil productivity of 143 mg oil/L/hr. Oleic acid accounted for 70% of the total fatty acid profile indicating that oil from L. starkeyi is a naturally high source of oleic acid; an added benefit for the biofuel, cosmetic, food, and oleochemical industries. Hemicellulose-rich corn bran and wheat bran were successfully used to produce oil; oil yields of 125 and 71 mg oil/g sugar were reported for whole and de-starched bran hydrolysates, respectively. Compared to traditional methods, biphasic oil extraction systems of 2-MeTHF and CPME had an 80 and 53% extraction efficiency and 64 and 49% selectivity, respectively. The information from this study will be useful for the development of an integrated approach to improve the viability of SCO biochemical platforms for the production of advanced biofuels and renewable chemicals.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Harvey Morgan Scott / A randomized controlled field trial was designed to evaluate the effects of two treatment strategies on ceftiofur and tetracycline resistances in feedlot cattle. The two strategies consisted of administering ceftiofur crystalline-free acid administration (CCFA) at either one or else all of the steers within a pen, and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. Both strategies were hypothesized to reduce ceftiofur resistance. The effects of treatment strategies were evaluated via metagenome-based and culture-based assays. In this 26-day study, 176 steers were allocated to 16 pens of 11 steers each. The two strategies were randomly assigned to the pens in a two-way full-factorial manner resulting in four treatment groups. The blaCMY-2, blaCTX-M, tet(A), tet(B), and 16S rRNA gene copies/g feces were quantified using qRT-PCR from fecal community DNA. Antimicrobial susceptibility profiles were determined using microbroth dilution technique from the non-type-specific (NTS) E. coli isolates (n=1,050). The NTS E. coli DNA was screened for the presence of blaCMY-2, tet(A), and tet(B) genes. Pens in which all the steers received CCFA treatment showed an increase in blaCMY-2 and blaCTX-M log10 gene copies/g feces and in the proportion of ceftiofur-resistant and blaCMY-2 positive NTS E. coli. This was in contrast to the pens where only one animal received CCFA treatment. There was a significant decrease in quantities of tetracycline genes in community DNA in pens where all animals received CCFA treatment. In contrast to metagenome-based assay results, culture-based assays indicated an increase in the proportion of tetracycline resistant NTS E. coli upon CCFA treatment. Thereafter, chlortetracycline administration led to rapid expansion both of ceftiofur (blaCMY-2, blaCTX-M) and tetracycline [tet(A) and tet(B)] log10 gene copies/g feces. Chlortetracycline treatment delayed the return of the ceftiofur resistance prevalence to baseline among NTS E. coli and thus did not lead to the hypothesized decrease in ceftiofur resistance. Our data suggest that chlortetracycline use is contraindicated when attempting to avoid expansion of resistance to critically important 3rd generation cephalosporins in feedlot cattle. Further studies are required to better establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together at varying ratios on the levels of antimicrobial resistance.