Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / George C. Stewart / Fusobacterium necrophorum, a gram negative anaerobe and an opportunistic pathogen, causes necrotic infections in humans and animals. Two subspecies of F. necrophorum, subsp. necrophorum and subsp. funduliforme are described. Leukotoxin (Lkt), a secreted protein encoded by a tricistronic operon (lktBAC), is the major virulence factor of F. necrophorum. The concentration of Lkt produced by subsp. necrophorum is higher than that of subsp. funduliforme. Quantitative-PCR was used to determine the relative expression of lktA by the two subspecies of bovine origin. The mRNA transcript of lktA was detectable in early-log phase of growth in subsp. necrophorum, whereas in subsp. funduliforme, the lktA transcript was detected only in the mid-log phase. Q-PCR analysis revealed that subsp. necrophorum had 20-fold more lktA transcript than subsp. funduliforme. The amount of lktA transcript declined by late-log phase in both subspecies; but lktA mRNA levels in subsp. necrophorum was 8-fold higher than in subsp. funduliforme. Leukotoxin protein stability assays showed the Lkt to be stable in both subspecies despite the decrease in the concentration of the protein during late-log phase. The subspecies identity of human F. necrophorum strains and whether they possess lktA and leukotoxin activity are not known. Human clinical isolates (n = 4) of F. necrophorum were identified as subsp. funduliforme based on 16S rRNA sequence and absence of hemagglutinin gene. Four human strains had the lkt promoter, lktB, and lktC similar to that of subsp. funduliforme. One strain had full length lktA, while other three strains exhibited considerable heterogeneity. All four strains secreted Lkt that was toxic to human leukocytes. Fusobacterium equinum, formerly F. necrophorum, is a newly recognized species. It is associated with infections of the respiratory tract in horses. Little is known about the virulence factors of F. equinum. Southern hybridization revealed that F. equinum strains had lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The toxicity of culture supernatants of isolates to equine leukocytes was variable. Our data indicate that F. equinum isolates possess lktA gene and exhibit leukotoxin activity. The importance of leukotoxin as a virulence factor in human and equine fusobacterial infections needs to be investigated.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, an emerging tick-borne pathogen, causes human monocytic ehrlichiosis (HME). The relationship between E. chaffeensis and its target cells in ticks and vertebrates is critical as the organism must persist in them. We hypothesize that E. chaffeensis alters gene expression in support of adapting to dual hosts. In support of testing this hypothesis, we developed an ORF-based microarray and performed global transcriptional analysis on the pathogen grown in macrophage and tick cells. The analysis revealed the expression of about 30% of all the predicted E. chaffeensis genes, in macrophages or tick cell. Two-thirds of the transcribed genes are common for both host cell backgrounds. About 20% of the commonly expressed genes also varied in expression levels which ranged from two to five fold. Microarray data was verified by RT-PCR for a subset of randomly selected genes. Together, this is the first report describing the global host cell-specific gene expression patterns in E. chaffeensis. Differential gene expression may be an important adaptive mechanism used by E. chaffeensis for its continued survival in dual hosts. To test this hypothesis, we established many basic protocols and tools needed for performing mutational analysis in E. chaffeensis. Four antibiotic selection markers; gentamicin, chloramphenicol, spectinomycin and rifampin, and two promoters constitutively expressed in E. chaffeensis, genes rpsL and tr, were identified. Two regions of the genome were also identified for performing initial mutational analysis. Several plasmid constructs were also made. The optimal conditions for introducing these plasmids into host cell-free viable E. chaffeensis organisms were also established. The molecular evaluation of several E. chaffeensis transformants using these plasmids suggested that the plasmids gained entry, but failed to get integrated into the genome or remain in the bacteria for longer periods of time. In summary, we demonstrated global host cell-specific differential gene expression in E. chaffeensis by employing microarray analysis. Numerous host-specific expressed genes will be important for studies leading to effective methods of control. We also established several basic protocols and tools needed for performing mutational analysis useful in evaluating the impact of the loss of expression of uniquely expressed genes.

Master of Science / Food Science Institute, Animal Science and Industry / Daniel Y.C. Fung / Within the United States, it has been estimated that 60 deaths and 73,000 illnesses are caused by Escherichia coli O157:H7 infection annually (Gavin et al., 2004). Multiple effects have been known to occur with the onset of infection from E. coli O157:H7 in which some of these can become life-threatening. Escherichia coli O157:H7 is defined as a Shiga-toxin-producing E. coli strain (STEC). This microbial pathogen is a gram-negative bacillus organism that is motile, non-sorbitol fermenting, and β-glucuronidase negative. The infectious dose of E. coli O157:H7 can be as low as ten cells (Food and Drug Administration, 2009). Consumption of contaminated food, mainly undercooked ground beef and non or incorrectly pasteurized milk, are the primary sources of E. coli O157:H7 infection in human. Cattle, in particular, are considered chief asymptomatic reservoirs for this pathogen. Carried in their gut, feces, and milk, cattle carry this Shiga toxin-producing E. coli in ranges from 10[superscript]2 to 10[superscript]5 CFU/g. Although colonized with E. coli O157:H7, cattle and other ruminants show no adverse side effects from the pathogenic bacteria. There is also a difference in the prevalence of this pathogen between human and cattle. There has been a low incidence of illness caused by E. coli O157:H7 in humans when compared to the high prevalence of E. coli 057:H7 found in cattle and their environment. It has been discovered, through population genetic analysis, that E. coli O157:H7 and other O157:H- isolates make up a clone complex. In spite of the clonal nature of E. coli O157:H7 and other O157:H[superscript]- isolates, there are significant characteristics showing variability between the clone complex. These variability aspects can possibly account for the rapid divergence of E. coli strains including the recently discovered divergence of E. coli O157:H7 in to two separate lineages. Other possible reasons for a non-linear relationship between cattle prevalence and human infection include diversity of the Shiga Toxin-Encoding bacteriophage and receptors in cattle verses human, and finally the difference between the production of Locus of Enterocyte Effacement (LEE) in both human and cattle lineages.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / With introduction of the 2009 pandemic H1N1 virus (pH1N1) into swine herds, reassortment between the pH1N1 and endemic swine influenza viruses (SIVs) has been reported worldwide. Recently, reassortant H3N2 and H1N2 variant SIVs that contain the M gene from pH1N1 virus and the remaining seven genes from North American triple-reassortant (TR) SIVs have emerged. These variant viruses have caused more than 300 cases of human infections and one death in the USA, creating a major public health concern. To date, the pathogenicity and transmissibility of H1N2 variant viruses in pigs has not been investigated. Through passive surveillance, we have isolated two genotypes of reassortant H1N2 viruses with pH1N1 genes from diseased pigs in Kansas. Full genome sequence and phylogenetic analysis showed that one is a swine H1N2 variant virus (swH1N2v) with the M gene from pH1N1; the other is a reassortant H1N2 virus (2+6 rH1N2) with six internal genes from pH1N1 and the two surface genes from endemic North American TR H1N2 SIVs. Furthermore, we determined the pathogenicity and transmissibility of the swH1N2v, a human H1N2 variant (huH1N2v), and the 2+6 rH1N2 in pigs using an endemic TR H1N2 SIV (eH1N2) isolated in 2011 as a control. All four viruses were able to infect pigs and replicate in the lungs. Both H1N2 variant viruses caused more severe lung lesions in infected pigs when compared to the eH1N2 and 2+6 rH1N2 viruses. Although all four viruses are transmissible in pigs and were detected in the lungs of contact animals, the swH1N2v shed more efficiently than the other three viruses in the respective sentinel animals. The huH1N2v displayed delayed and inefficient nasal shedding in sentinel animals. Taken together, the human and swine H1N2 variant viruses are more pathogenic and the swH1N2v more transmissible in pigs and could pose a threat to public and animal health.

Doctor of Philosophy / Department of Human Nutrition / Tonatiuh Melgarejo / Background and Objective: Crohn’s disease (CD) patients express low levels of host defense peptides (HDPs) especially β-defensins, which may compromise intestinal barrier function. Antibiotic treatment for bacterial infections in CD is limited and rarely curative, making it necessary to find alternative therapeutic approaches. We therefore investigated to what extent an essential amino acid; L-isoleucine (L-ILE) might induce the expression of human β-defensins (HBDs) in colonic epithelial cells as an alternative approach to help patients with CD. Antimicrobial activity of HBD2 was also assessed against four bacterial isolates which can cause secondary infections in CD. Methods: HTB-37 Caco-2 cells were stimulated with L-ILE at a concentration of 0 - 500µg/ml for 6 hours. Total RNA was extracted using RNeasy Micro Kit (QIAGEN). Reverse transcription was carried out with Superscript ®III First-Strand Synthesis System. The cDNA was amplified using specific primers for HBD1-3. Antimicrobial activity of HBD2 was determined using the broth dilution assay. Results: HBD1 was constitutively expressed under all conditions. HBD2 was expressed in HTB-37 cells after stimulation with L-ILE. Below 25µg/ml L- ILE stimulation, no expression of HBD2 was observed. HBD2 exhibited antimicrobial activity against bacterial isolates tested, with a MIC of 32, 64 and 128 µg/ml for both strains of E. coli, S. aureus and P. aeruginosa respectively. Conclusions: Our results indicate that L-ILE stimulation of HTB-37 Caco-2 cells can induce HBD2 expression. Data collected from our in vitro studies might have major implications for modifying the intestinal microbiota towards a healthier state in CD patients. Promoting the expression of HBD2 by colonic cells may lead to a lower rate of infection in these patients. Future in vivo studies are warranted to determine the potential clinical use of intra colonic administration of L-ILE in CD patients. The observed antimicrobial activity of HBD2 against bacterial isolates provides evidence that it is a crucial component of mucosal epithelial defense against infections which can complicate disease symptoms in CD.

A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.

A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.

Doctor of Philosophy / Food Science Program / Daniel Y.C. Fung / Curtis L. Kastner / This dissertation was designed to evaluate antimicrobial, quality, and shelf life effects of controlled phase carbon dioxide (CPCO2) on beef trimmings destined for ground beef. Critical parameters included pressure, temperature, exposure times, modified atmosphere conditions, and days of simulated retail display. 1500 psi CPCO2 for 15 min achieved 0.83, 0.96, 1.00, and 1.06 log reductions for Total Plate Count (TPC), Generic E. coli (GEC), E. coli O157:H7 (O157), and Salmonella spp. (SS), respectively. Bacterial reductions in ground beef and beef trimmings were similar (P≥0.05). CIE L*, a*, and b* values in raw patties showed no differences (P≥0.05) immediately after CPCO2 application on beef trimmings. Nevertheless, significant (P<0.05) interactions were found in pressure by packaging for L*, in pressure by packaging by days of simulated retail display for a*, and in packaging by days of simulated retail display for b* scores. Nevertheless, after 5 days of simulated retail display, L*, a*, and reflectance (630/580nm) ratios were similar for all treatments (P≥0.05), and b* scores were most acceptable with 1500 CPCO2 (P≥0.05), regardless of the packaging conditions. After 5 days of display, cooked patties showed similar (P≥0.05) values for crude protein (%CP) and crude fat (%CF), the extent of lipid oxidation (TBARS), was higher (P0.05) in aerobic trays than flushed packages with 100% CO2. Ground beef patties manufactured from beef trimmings treated with CPCO2scored higher values for tenderness (P0.05) than other treatments. In addition, no differences (P0.05) for juiciness, beef flavor intensity, or off flavor intensity were found between non-treated and the 1500 psi CPCO2 treated patties. Microbial control of spoilage organisms and foodborne pathogens in ground beef patties with CPCO2 application in beef trimmings was effective (0.6 to 1.2 logs). Lethality levels are comparable to other intervention strategies. Discoloration of beef trimmings after CPCO2 application may not be a concern for grinding purposes. Further packaging with 100% CO2 is viable for controlling spoilage and pathogenic microorganisms after packaging and during refrigerated storage, although discoloration of raw ground beef patties packaged with 100% CO2 may be a concern for product marketing.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Sanjeevkumar Narayanan / Antimicrobials are included in finishing cattle diets for growth promotion, feed efficiency, and protection against liver abscesses. The inclusion of in-feed antimicrobials at or below therapeutic concentrations may provide a selective pressure for antimicrobial resistant microorganisms. Additionally, heavy metals such as copper and zinc may be included in cattle diets because of growth-promoting effects. Heavy metal resistance genes are on transferable plasmids that also contain antimicrobial resistance genes. The objectives of this research were to 1) determine the prevalence of food-borne pathogens, Salmonella and E. coli O157, in cattle fed diets with or without monensin and tylosin and 0 or 25% wet corn distiller's grains (WDGS), 2) determine the prevalence of food-borne pathogens in cattle fed elevated concentrations of copper and zinc 3) evaluate the effect of antimicrobials on antimicrobial susceptibility of food-borne pathogens and commensal fecal bacteria, and 4) determine a possible association between in-feed antimicrobials and the concentration of antimicrobial resistance genes in the feces of cattle. Inclusion of 25% WDGS was associated with a higher prevalence of E. coli O157 on one of two sample collection days; however, there was no association between the use of monensin and tylosin, or copper and zinc on the prevalence of food-borne pathogens. Including monensin and tylosin in cattle diets was associated with an increased resistance of enterococci to macrolides, but was not related to concentration of the common macrolide resistance gene, ermB. In cattle fed diets with copper and/or zinc, no differences were observed in antimicrobial susceptibility or the concentration of antimicrobial resistance genes. In conclusion, results indicate that including growth-promoting antimicrobials in cattle diets at below therapeutic concentrations only limitedly impacted antimicrobial susceptibility and concentration of fecal antimicrobial resistance genes; however, this research encompassed only a select number of microorganisms. The positive association between WDGS and E. coli O157 prevalence in cattle has important implications for food safety, and warrants further investigation.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Escherichia coli O157 is a major foodborne pathogen. The bovine gut is the primary reservoir and the organism is shed in the feces, which serves as the major source of contamination. The objectives of our research on E. coli O157 were to 1) determine prevalence and characterize presence in the rectoanal mucosa of cattle, 2) rationalize preferential prevalence in the hindgut, 3) evaluate fecal prevalence and concentration in relation to carcass contamination 4) determine the efficacy of preharvest intervention strategies to reduce fecal prevalence and/or concentration in cattle. We determined that E. coli O157 isolated from the rectoanal mucosa were similar to fecal isolates. We evaluated methods of enumeration in cattle feces to identify fecal samples with > 10[superscript3] and 10[superscript4] CFU of E. coli O157/g. We observed that prevalence of E. coli O157 on carcasses was correlated with high-shedders. We identified gluconic acid as a substrate which stimulates growth of E. coli O157 in fermentations with ruminal microbial or fecal microbial inocula from cattle. This may explain the preference to persist or colonize the hindgut because gluconic acid is a component of colonic mucin. Additionally, we evaluated the effects of cattle diets (two grain types and two grain processing methods), which present different amounts of fermentable starch to the hindgut, on prevalence of E. coli O157. Dry-rolled grain-based diets reduced prevalence of E. coli O157, possibly because of increased flow of starch to the hindgut. Competitive exclusion cultures of E. coli in in vitro fermentations reduced E. coli O157 in ruminal microbial inoculum, but in fecal microbial inoculum cultures were only efficacious when gluconic acid also was added. Lastly, we evaluated a vaccine which targeted the siderophore receptor/porin proteins of E. coli O157. The vaccine reduced prevalence, the total number of days cattle tested positive, and the total number of days cattle were identified as high-shedders of E. coli O157. In summary, our research adds further knowledge to the literature about E. coli O157 in the hindgut, provides methods to identify high-shedding animals, demonstrates the importance of high-shedding animals, and offers information about potential preharvest interventions.