Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / Escherichia coli O157:H7 is responsible for over 70,000 cases of human illness every year in the United States. Most cases occur in children under the age of five, the elderly, or other immune-compromised people. A small percentage of these cases will develop a life threatening complication, hemolytic uremic syndrome. Cattle are the reservoir host for E. coli O157:H7 and serve as the main source of contamination of meat products and other food sources. The beef cattle industry is diverse with producers caring for as few as one to as many as thousands of cattle. The first objective of this research was to examine three major production systems (conventional, organic, and natural) in the U.S. and the published performance effects of the various technologies used in each system. The second objective was to determine if a newly licensed E. coli O157:H7 SRP® (SRP) vaccine administered to cows pre-partum could achieve successful passive transfer in their offspring. The third objective was to determine if colostrum obtained from SRP vaccinated heifers could protect against an oral challenge with an E. coli K99+ strain. The fourth objective was to examine the shedding characteristics, health, and performance effects of calves born to SRP-vaccinated cows that also receive SRP vaccination themselves. The technologies used in conventional beef cattle production resulted in significant improvements in health and performance of beef cattle. Vaccinating cows pre-partum with SRP resulted in passive transfer in calves consuming their colostrum. Calves that achieved successful passive transfer shed less E. coli K99+ and had improved fecal consistency compared to placebo. When calves were vaccinated with SRP at branding, weaning, and arrival to the feedyard there was no difference in fecal E. coli O157:H7 shedding on arrival to the feedyard or at harvest. Vaccinating calves with SRP had no effects on performance or health outcomes. Vaccinating cattle with SRP may provide protection against other pathogenic E. coli strains and warrants further investigation. The timing of vaccination appears to be an important consideration in order to ensure maximum vaccine efficacy.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / Halogenated organic compounds have had widespread and massive applications in industry, agriculture, and private households, for example, as degreasing solvents, flame retardants and in polymer production. They are released to the environment through both anthropogenic and natural sources. The most common chlorinated solvents present as contaminants include tetrachloroethylene (PCE, perchloroethene), trichloroethene (TCE), trichloroethane (TCA), and carbon tetrachloride (CT). These chlorinated solvents are problematic because of their health hazards and persistence in the environment, threatening human and environmental health. This contribution provides insight on PCE degradation at laboratory and field scale at a former dry cleaning site in Manhattan, KS. Biostimulation experiments included combinations and concentrations of the following nutrients: soy oil methyl esters (SOME), yeast extract (YE), glucose, lactate, methanol and cheese whey. Bioaugmentation studies used KB-1 bacterial consortium (commercially available culture containing Dehalococcoides). This culture is known to complete the degradation of PCE to a safe end product, ethene. Concentrations of PCE and its degradation intermediates were monitored in the gas phase of the microcosm vials. Biostimulation of the natural ground water and soil microflora did not completely degrade PCE as cis-DCE (c-DCE) accumulated in the sample. Bioaugmented microcosms containing YE and SOME created reducing conditions for KB-1 culture, resulting in ~ 90% dechlorination of PCE to methane and c-DCE. Cheese whey microcosms containing 0.05% cheese whey inhibited the KB-1 culture. This inhibition was due to a drop of pH that inhibited the culture activity. Lower concentrations of cheese whey (e.g. 0.01% to 0.025%) reduced PCE and generated methane in KB-1 augmented microcosms. Based on microcosm results, a pilot bioremediation field study was conducted for a dry cleaning site contaminated with PCE. Ground water flow threatened public water wells located 1.5 miles from the source. Concentrations of PCE in the aquifer was 15 mg/L above the maximum contaminant level of 5 µg/L. Tracer studies with potassium bromide (KBr) were conducted before, during and after the bioremediation study. Nutrient solutions prepared with YE, SOME, lactate and glucose were used for biostimulation and preconditioning of ground water prior to KB-1 injection. Nutrients were provided twice during the pilot study to supplement microbial growth and cheese whey was used. During biostimulation no degradation beyond DCE was evident. The addition of KB-1 reduced PCE and DCE concentrations in the monitoring wells of the pilot study area. Total chlorinated ethene concentrations did not reach background levels 2 years after the last nutrient addition. Tracer results showed that microbial growth decreased ground water velocity during the study, but returned to normal conditions 1 year after the last nutrient addition. In this study we were able to show that native microbial population was not able to degrade PCE to final end products. Therefore, it was necessary to introduce KB-1 culture a long with nutrients to support complete reductive dechlorination of PCE.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is a Gram negative, rickettsial organism responsible for human monocytic ehrlichiosis, an emerging disease in people. E. chaffeensis infection to a vertebrate host occurs when the pathogen is inoculated by an infected tick, Amblyomma americanum. White-tailed deer is a reservoir host for this pathogen. The strategies employed by E. chaffeensis in support of its dual host adaptation and persistence are not clear. One of the possible mechanisms by which the pathogen adapts and persists, is by altering its gene expression in response to its host cell environments. Recently, we reported that E. chaffeensis protein expression including that from a 28 kDa outer membrane protein multigene locus (p28-Omp), is influenced by macrophage and tick cell environments. E. chaffeensis expresses p28-Omp gene 14 product predominantly when it is grown in tick cells and p28-Omp gene 19 protein in macrophages. We hypothesize that E. chaffeensis achieves its host-specific gene expression by employing transcriptional regulation by sensing the host cell signals. In support of this hypothesis, transcriptional analysis of 14 and 19 genes was performed utilizing several RNA analysis methods. The results supported our hypothesis that the gene regulation occurs at mRNA level in a host cell-specific manner. This analysis also identified transcription start sites and located putative promoters for the p28-Omp genes 14 and 19. Promoter regions of genes 14 and 19 were mapped to identify gene-specific differences, RNA polymerase binding sequences and the putative regulatory elements that may influence the promoter activities. Electrophoretic mobility shift assays revealed interaction of E. chaffeensis proteins with gene 14 and 19 promoters. Several E. chaffeensis putative regulatory proteins were expressed as recombinants and their effects on a p28-Omp gene promoter activity were evaluated. In summary, we demonstrated that the differences in the E. chaffeensis p28-Omp genes 14 and 19 are the result of their regulation at transcriptional level in response to the host cell environment. We also identified RNA polymerase binding regions and several DNA sequences that influenced promoter activity. This is the first description of a transcriptional machinery of E. chaffeensis. The data from these studies provide important insights about molecular mechanisms of gene regulation in E. chaffeensis.

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / The enterococci are gaining much notoriety as common nosocomial pathogens. One aspect of their pathogenesis, especially characteristic to infectious endocarditis and urinary tract infections, involves their ability to transition from the sessile state of existence to surface adherent structured communities called biofilms. Existence as biofilms, affords enterococci protection against a number of growth limiting challenges including antibiotic therapy and host immunity. In the current study a mechanistic role for two Fsr quorum-regulated extracellular proteases- gelatinase (GelE) and its cotranscribed serine protease (SprE), were explored in biofilm development of E. faecalis V583. Confocal imaging of biofilms suggested that GelE[superscript]– mutants were significantly reduced in biofilm biomass compared to V583, whereas the absence of SprE appeared to accelerate the progression of biofilm development. Culture supernatant and biofilm analysis confirmed that decreased biofilms observed in GelE[superscript]– mutants resulted from their inability to undergo autolysis and release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas SprE[superscript]– mutants produced significantly more eDNA as components of the biofilm matrix. The governing principle behind GelE mediated autolysis and eDNA release in E. faecalis V583 was demonstrated to be fratricide. GFP reporter assays of V583 populations confirmed that GBAP (gelatinase biosynthesis-activating pheromone encoded by fsrD) quorum non-responders (GelE[superscript]–SprE[superscript]–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE[superscript]+SprE[superscript]+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, comparison of virulence effects of isogenic extracellular protease mutants (∆gelE, ∆sprE and ∆gelEsprE) relative to parental strain (V583) in a rabbit model of enterococcal endocarditis confirmed a critical role for GelE in the infection process. In conclusion, the data presented in this thesis are consistent with significant roles for GelE and SprE in biofilm mediated pathogenesis of enterococcal infections.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / John C. Reese / Keeping in view the important roles of bacteria in almost every aspect of insect’s life, the current study is the first systemic and intensive work on microbes associated with Hessian fly, a serious pest of wheat crop. A whole body analysis of Hessian fly larvae, pupae, or adults suggested that a remarkable diversity of bacteria is associated with different stages of the insect life cycle. The overriding detection of genera Acinetobacter and Enterobacter throughout the life cycle of Hessian fly suggested a stable and intimate relationship with the insect host. Adult Hessian flies have the most dissimilar bacterial composition from other stages with Bacillus as the most dominant genus. Analysis of 5778 high quality sequence reads obtained from larval gut estimated 187, 142, and 262 operational taxonomic units at 3% distance level from the 1st, 2nd, and 3rd instar respectively. Pseudomonas was the most dominant genus found in the gut of all three instars. The 3rd instar larval gut had the most diverse bacterial composition including genera Stenotrophomonas, Pantoea, Enterobacter, Ensifer, and Achromobacter. The transovarial transmission of major bacterial groups provided evidence of their intimate relationship with the Hessian fly. The Hessian fly is known to manipulate wheat plants to its own advantage. This study demonstrated that the combination of a decrease in carbon compounds and an increase in nitrogen compounds in the feeding tissues of Hessian fly-infested plants results in a C/N ratio of 17:1, nearly 2.5 times less than the C/N ratio (42:1) observed in control plants. We propose that bacteria associated with Hessian fly perform nitrogen fixation in the infested wheat, which was responsible for shifting the C/N ratio. The following findings made in the current study i.e. the presence of bacteria encoding nitrogenase (nifH) genes both in Hessian fly and infested wheat, exclusive expression of nifH in infested wheat, presence of diverse bacteria (including the nitrogen fixing genera) in the Hessian fly larvae, presence of similar bacterial microbiota in Hessian fly larvae and at the feeding site tissues in the infested wheat, and reduction in survival of Hessian fly larvae due to loss of bacteria are consistent with this hypothesis. The reduction in Hessian fly longevity after the loss of Alphaproteobacteria in first instar larvae, highest proportion of Alphaproteobacteria in insects surviving after the antibiotic treatments and the nitrogen fixation ability of associated Alphaproteobacteria strongly implies that Alphaproteobacteria are critical for the survival of Hessian fly larvae. This study provides a foundation for future studies to elucidate the role of associated microbes on Hessian fly virulence and biology. A better understanding of Hessian fly-microbe interactions may lead to new strategies to control this pest.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Copper, as copper sulfate, is often supplemented at elevated concentrations in swine diets, particularly in piglets, to promote growth. Growth promotional effects of copper are believed to be similar to that of antibiotics in that gut microbial flora is altered to reduce loss of nutrients and suppress pathogens. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and E. faecalis, resistance is conferred by a plasmid-borne transferable copper resistance (tcrB) gene. The plasmid also carries macrolide [erm(B)] and glycopeptide (vanA) antibiotics resistance genes. The objectives of the research were to 1) determine the prevalence of tcrB gene in fecal enterococci of piglets in relation to normal (16.5 ppm) and elevated level (125 ppm) of copper supplementation, 2) determine the relationship of tcrB gene and susceptibilities to copper, erythromycin, and vancomycin, and 3) determine the transferability of tcrB gene in enterococci by conjugation. Weaned piglets, housed in pens, fed normal (16.5 ppm; control) or elevated level of copper (125 ppm) were used. Fecal samples were collected weekly for isolation of enterococci. Isolates were speciated by multiplex PCR and sodA gene sequence analysis. The prevalence of tcrB-positive enterococcal isolates was higher (P < 0.05) in the copper supplemented group than the control group. The prevalence of tcrB was affected by sampling days (P < 0.05) with a significant treatment and sampling time interaction (P < 0.05). The tcrB positive isolates were either E. faecium or E. faecalis, and majority of isolates was E. faecium. The mean MIC of copper for tcrB-positive isolates (21.1 mM) was higher (P < 0.001) compared to tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin, tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains suggests the possibility that copper supplementation may exert selection pressure for antibiotic resistance. The positive association between copper supplementation and prevalence of tcrB gene has important implications for antimicrobial resistance and food safety, which warrants further investigation.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen that causes infection in people and several vertebrate animals. One of the striking features of E. chaffeensis infection is the prolonged persistence in its vertebrate and tick hosts. The mechanism of persistent infection and the reasons for the host immune system failure to clear the infection are not well understood. One hypothesis is that differential gene expression serves as an important adaptive mechanism used by E. chaffeensis in support of its continued survival in both tick and vertebrate hosts. One way to test this hypothesis is by performing mutational analysis. However, the methods for introducing mutations in this pathogen have not yet been documented and are challenging, possibly due to its obligate, intraphagosomal growth requirement. Recently, a novel gene mutation method called ‘TargeTron Gene Knockout System’ that is based on the modified group II intron insertion strategy has been developed. This method appears to be effective in creating mutations in a wide range of gram positive and gram negative bacterial organisms. The group II intron can be programmed for insertion into virtually any desired DNA target with possibly high frequency and specificity. In this study, I focus on creating mutations in E. chaffeensis using the TargeTron gene knockout system. I prepared modified group II intron constructs retargeting for insertion into three E. chaffeensis genes: Ech_0126 (a transcriptionally silent gene), macrophage-specific expressed gene (p28-Omp 19, Ech_1143) and tick cell-specific expressed gene (p28-Omp 14, Ech_1136). In support of driving the expression of the modified group II introns in E. chaffeensis, the pathogen- specific high-expressing gene promoter (tuf) was inserted upstream to the transcription start site. In addition, a chloramphenicol acetyltransferase gene with E. chaffeensis rpsl promoter was introduced for use as a selection marker. The constructs were then evaluated by transforming into E. chaffeensis. Transformants with mutations, introduced in two of the three genes (Ech_0126 and Ech_1143), were identified by PCR and Southern blot methods. Although the mutants are detectable for up to 48 hours, establishment of stable transformants remains to be challenging. The outcomes of this project will have important implications in defining the pathogenesis of E. chaffeensis, particularly to assess the differences in the organism in tick and vertebrate hosts.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Escherichia coli O157 is a major foodborne pathogen that causes enteritis in humans ranging in severity from mild to bloody diarrhea to hemolytic uremic syndrome and even death. Cattle are asymptomatic carriers and fecal shedding of the organism is the major source of contamination of food and water for human infections. Distiller’s grains (DG) are ethanol fermentation co-products that are valuable feed ingredients for use in cattle diets. Previous research suggests an association between feeding DG and an increased fecal shedding of Escherichia coli O157:H7. The objectives of the research were to evaluate fecal E. coli O157:H7 prevalence and concentration in cattle fed diets with and without DG, determine if the association was dependent on inclusion level or form (wet or dried), evaluate the association in populations of cattle at harvest, and evaluate a potential intervention strategy. Our results indicated that cattle fed DG had a higher prevalence and shed a higher concentration of E. coli O157 than cattle fed diets without DG. The relationship was not dependent on the DG form, however, it was affected by the inclusion level of DG in the diet. Cattle that were fed 40% DG had a higher E. coli O157:H7 prevalence than cattle fed control or 20% DG diets and cattle fed 20% DG had a prevalence that was not statistically different from control cattle. The same response was observed in a subpopulation of cattle, termed super-shedders, which shed E. coli O157:H7 at higher concentrations than the general population. At harvest, we did not find differences in E. coli O157:H7 or super-shedder prevalence between cattle fed diets with or without DG, however, study design limitations affected the power of the study. Finally, previous work had shown that cattle fed dry-rolled grains had a decreased prevalence of E. coli O157:H7 when compared to cattle fed steam-flaked grains. We evaluated the effect of feeding DG and dry-rolled corn (DRC), alone or in combination, and observed no difference in E. coli O157 prevalence between cattle fed either DG or DRC diets. In conclusion, DG supplementation increased the prevalence and concentration of E. coli O157:H7 in cattle.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Gary A. Anderson / Brucellosis is one of the major endemic zoonotic diseases worldwide, and it has history dating back to 1937 in Iraq when it was first isolated by an Iraqi physician. In order to establish a solution for the continuous devastating impacts of the disease in humans and livestock, the Brucellosis Control Program was established in 1995. The main responsibilities of this program were setting and implementing the appropriate strategies for controlling the disease. After the war in 2003, the United Nation organization for Food and Agriculture (FAO) developed a strategic plan to control the disease. The main goal of the project was to improve productivity in the livestock sector and reduce the prevalence of disease in small ruminants (sheep and goats) to less than 2%, and less than 0.2% in cattle and buffalo. Achieving such goals ultimately would reduce the disease incidence among the human population from more than 27.2 cases/100,000 persons in 2002, to less than 4 cases/100,000 people within 15 years. A serological surveillance was conducted and revealed the apparent prevalence of the disease in sheep and goats, cattle, buffalo, and camels was 6.51%, 1%, 1.48%, and 0.02%, respectively in Iraqi governorates except the three northern governorates of Kurdistan province . Based on surveillance results, a vaccination policy was the only appropriate strategy that could be chosen to control the disease. Four vaccination campaigns were implemented in 2006, 2007, 2008, and 2009, with a total number of vaccinated animals each year at 10099972, 4698482, 753153, and 1833482 head, respectively. The primary satisfactory outcome of the program was the apparent decline in livestock abortions leading to obvious increases in productivity. Regarding the incidence of brucellosis among the human population, the apparent decline in the middle and south of Iraq began with the vaccination phase of the control program in 2006. The results represented a significant decrease in human cases after only four vaccination campaigns of a program that was intended to continue for 15 years.

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.