Master of Science / Department of Clinical Sciences / David E. Anderson / Drug delivery systems for time release of recombinant human bone morphogenetic protein-2 (rhBMP-2) and antibiotics in orthopedic surgeries continue to be developed. Recently, a biodegradable novel polymeric matrix has been developed for this purpose. We hypothesized that impregnation of the matrix with rhBMP-2 would enhance bone healing. The objectives of the study were to characterize elution of rhBMP-2 and two antimicrobials (tigecycline, tobramycin) from the matrix, and bone response to the matrix in the presence or absence of rhBMP-2 and antimicrobials. In vitro elution of tigecycline, tobramycin, and rhBMP-2 from the matrix was investigated. Drug concentration in media were measured on days 1-6, 8, 10, 13, 15, 17, 21, 25, 28, and 30 using high pressure liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS; antimicrobials) and ELISA (rhBMP-2). In vivo testing was done using a unicortical defect created into each tibia of twenty adult goats. Animals were randomly assigned to one of 5 groups: 1) control (untreated defect); 2) matrix; 3) matrix+ antimicrobials (tigecycline+tobramycin); 4) matrix+rhBMP-2; and 5) matrix+antimicrobials+rhBMP-2. Plasma concentration of tigecycline and tobramycin and serum concentration of rhBMP-2 were measured by the above techniques on days 1-7, 9, 11, 13, 15, 17, 22, 26, and 30. Bone response was assessed on days 0, 14, and 30 using radiographic scoring and dual energy X-ray absorptiometry (bone mineral density [BMD]). After euthanasia on day 30, histomorphologic analyses of the bone defects were done. Categorical variables were analyzed using a generalized linear model, and continuous variables using an ANOVA with P < 0.05 considered significant. In vitro elution was characterized by a rapid release on day 1 followed by a slow release until day 30 for both antimicrobials and rhBMP-2. Plasma antimicrobial concentrations showed continued release throughout the study period. Serum rhBMP-2 concentration, radiographic scores and BMD were not significantly different between groups. Periosteal and endosteal reaction surface areas were significantly greater surrounding the defects in group 4 (matrix+rhBMP-2). There was no significant difference between the groups for the percent of bone filling the defect. The matrix served as an appropriate antimicrobial and rhBMP-2 delivery system and successfully stimulated bone production when rhBMP-2 was present.

Doctor of Philosophy / Department of Anatomy and Physiology / Chris R. Ross / The immediate antiviral defense residing in the innate immune system of multicellular organisms critically determines the outcome of viral infection. This dissertation presents a study of the "effectors" and "receptors" of porcine innate immunity in infection caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is the most devastating pathogen impacting the swine industry. In the first investigation, eleven novel porcine host defense peptides (HDPs), [Beta]-defensins (pBDs), were identified and characterized. All of these peptides have a consensus [Beta]-defensin motif and phylogenetically are similar to orthologs from other species. A differential expression pattern for these 11 newly identified genes was found. For example, pBD-2 and pBD-3 were expressed in bone marrow, lung, skin and other lymphoid tissues. pBD-2 and pBD-3 were further characterized for their gene structure, and antimicrobial activity of synthetic peptides. The second study was conducted to evaluate PRRSV-induced differential expression of porcine HDPs and direct antiviral activity of selected HDPs against PRRSV. In vitro incubation of PRRSV with synthetic pBD-3 or protegrin-4 (PG-4) significantly inhibited viral infectivity. Using nine protegrin-derived peptides, it was determined that cyclization of PG-4 increased anti-PRRSV activity and mutation of some residues in PG-4 diminished some of the activity. These findings suggest the potential role of porcine HDPs as a group of innate antiviral effectors. In the third and fourth investigations, porcine Toll-like receptor (TLR) 3 and TLR7 were identified and functionally expressed. Increased expression of TLR3 was observed in PRRSV-infected porcine lungs. Stimulation of porcine alovelar macrophages with poly (I:C), a synthetic TLR3 ligand, increased expression of interferon-[Beta] and suppressed PRRSV infectivity. Activation of porcine TLR3 overexpressed in a PRRSV-sensitive cell line, elicited antiviral responses to PRRSV infection. Partial silencing of TLR3 in PAMs resulted in increased PRRSV infection. In summary, these data provide molecular information on porcine TLR3 and TLR7, and their involvement in PRRSV pathogenesis, which may elicit new strategies to prevent this costly swine disease.

Master of Science / Department of Anatomy and Physiology / Bruce D. Schultz / Epithelial cells lining the vas deferens modify the luminal contents to which sperm are exposed in response to neuroendocrine, autocrine and lumicrine transmitters. The role and identity of vas deferens epithelial potassium channels that provide the correct luminal environment for sperm maturation and delivery have not yet been determined. Cultures of vas deferens epithelial cells isolated from adult pigs were employed to investigate contributions of selected ion channels to net flux. A two-pore potassium channel, TASK-2, was identified on the apical membrane of cultured primary porcine vas deferens epithelial cells (1°PVD). Bupivacaine, a known TASK-2 inhibitor, when added to the apical bathing solution, inhibited forskolin- stimulated short circuit current, Isc, in a concentration dependent manner with a maximum inhibition of 72 ± 6% and an IC50 of 7.4 ± 2.2 µM. Apical exposure of 1°PVD cells to quinidine, lidocaine, and clofilium (other known TASK-2 blockers) inhibited forskolin-stimulated Isc in a concentration dependent manner. Fitting a modified Michalis-Menten function to the data revealed IC50 values of 274 µM, 531 µM, and 925 µM, respectively. Riluzole, a two-pore potassium channel activator, stimulated bupivacaine-sensitive Isc, further confirming the contribution of TASK-2 to net ion flux. Western blotting demonstrated the presence of TASK-2 immunoreactivity in 1°PVD cell lysates, while immunocytochemistry demonstrated apical localization of the targeted epitope in virtually all cells lining native porcine vas deferens. These results suggest that TASK-2 likely plays a role in vas deferens epithelial ion transport that may account for the reportedly high concentration of potassium in the male reproductive duct lumen. TASK-2 likely contributes to male fertility as an integral member of the regulated transport processes that account for the luminal environment to which sperm are exposed.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Joel M. DeRouchey / Steven S. Dritz / A total of 9,979 pigs were used in 11 experiments to quantify production responses under field conditions in growing pigs to PCV2 vaccination, biofuel co-products and dietary supplemental enzymes. Experiments 1 and 2 were conducted to determine the efficacy of a commercial 2-dose Porcine Circovirus Type 2 (PCV2) vaccine. Growth performance and mortality (P < 0.05) of vaccinated pigs improved compared to non-vaccinated pigs in both experiments with the vaccine causing a greater increase in ADG in vaccinated barrows than vaccinated gilts in Exp. 2. Experiment 3 compared the efficacy of 1-dose and 2-dose commercial PCV2 vaccines, where vaccinated pigs had greater ADG (P < 0.05) than vaccinated pigs regardless of vaccine type. The 2-dose group was heavier (P < 0.05) than the control group while the 1-dose group was intermediate. Therefore, PCV2 vaccines were efficacious under field conditions. Experiments 4, 5, and 6 were conducted to evaluate de-oiled corn dried distillers grains with solubles (dDGS) in grow-finish pigs. In Exp. 4, analyzed CP and AA content were higher, but lysine digestibility and energy content were lower in dDGS than traditional dried distillers grains with solubles (DDGS). In Exp. 5, 0 to 30% dDGS in nursery diets did not affect growth performance (P > 0.52). In Exp. 6, 0 to 30% dDGS reduced (linear; P < 0.01) ADG and ADFI, tended to improve (linear; P > 0.07) G:F, decreased (linear; P < 0.01) carcass yield, and increased (linear; P < 0.01) fat iodine values. Experiment 7 was conducted to determine the AA digestibility and energy concentration of novel high-CP distillers co-products from corn (HPC-DDG) and sorghum (HPS-DDGS). Digestibility of AA was higher for HPC-DDG but lower in HPS-DDGS than traditional DDGS. Both co-products had lower energy than traditional DDGS. Finally, Exp. 8, 9, 10, and 11 were used in a meta-analysis to evaluate supplementary dietary enzymes in pigs. Supplemental enzymes, alone or in combination, did not improve grow-finish pig performance (P > 0.58) regardless of dietary DDGS level. In conclusion, these experiments provide important empirical data to quantify production responses of various interventions and dietary ingredients under actual field conditions.

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 Anatomy and Physiology / Mark L. Weiss / When mouse embryonic stem cells (ESCs) were grown on mitotically inactivated rat umbilical cord-derived stromal cells (RUCs) in the absence of leukemia inhibitory factor (LIF) and 2-mercaptoethanol (2-MCE), the ESCs showed alkaline phosphatase (AP) staining. ESCs cultured on RUCs maintain expression of the following pluripotency genes, Nanog, Sox2 and Oct4 and grow at a slower rate when compared with ESCs grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). Differences in gene expression for the markers of pluripotency Oct4, Sox2 and Nanog, AP staining and ESC growth rate were also observed after LIF and 2-MCE were removed from the co-cultures. Reverse transcriptase polymerase chain reaction (RT-PCR) suggested differences in Sox2 and Nanog mRNA expression, with both genes being expressed at higher levels in the ESCs cultured on RUCs in the absence of LIF/2-MCE as compared to ESCs cultured on MEFs. Semi-quantitative RT-PCR indicated that Nanog expression was higher when ESCs were grown on RUCs in the absence of LIF and 2-MCE as compared to MEFs in the same treatment conditions. Bisulfite-mediated methylation analysis of the Nanog proximal promoter suggested that the maintenance of Nanog gene expression found in ESCs grown on RUCs after culture for 96 hours in the absence of LIF/2-MCE may be due to prevention of methylation of the CpG dinucleotides in the Nanog proximal promoter as compared to ESCs grown on MEFs. Thus, RUCs may release factors into the medium that maintain the pluripotent state of mouse ESCs in the absence of LIF and 2-MCE.

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.

Master of Science / Department of Clinical Sciences / Daniel U. Thomson / Bovine respiratory disease complex (BRDC) is the most common, and costly, disease in feed yard cattle. A review of the literature shows a correlation between the diagnosis of BRDC ante-mortem and respiratory lesions at slaughter. The objectives of the studies reported here were to: 1) validate a thoracic auscultation scoring system by correlating ante-mortem lung sounds with post-mortem lung lesions and 2) evaluate thoracic auscultation and rectal temperature as diagnostic tools to predict case outcome in the feeder cattle treated for BRDC. First, a prospective cohort study involving thirty four head of cattle that had been realized from commercial cattle feeding operations were used to validate the use of a lung auscultation scoring system to identify cattle suffering from BRDC. Ante-mortem auscultation scores were compared to post-mortem lung lesions evaluated using a previously described scoring system. There was a positive correlation (P < .0001) between ante-mortem lung auscultation scores and post-mortem lung lesion scores in the population of feeder cattle that were tested. Subsequently, a retrospective cohort study was conducted using data obtained from three commercial feed yards. Cattle enrolled in the study (n = 4,341 head) were treated for BRDC between January 2007 to October 2007 by trained feed yard personnel. Data recorded included animal identification, rectal temperature, lung score, and antibiotic therapy at first treatment. Treatment outcome data were recorded by feed yard personnel utilizing an animal health computer. The outcome data tracked for this study included subsequent BRDC treatment or death of the animal. Our findings indicated that as lung auscultation score (P < .0001) or rectal temperature (P < .0001) increased there was an increased risk for cattle to require a second BRDC treatment. Also, we observed an increased risk for death loss in cattle with higher lung auscultation scores (P < .0001) or higher rectal temperature (P < .0001) at the time of treatment for BRDC. We have demonstrated that lung auscultation score and rectal temperature can be used as tools to predict treatment outcome in cattle treated for BRDC. Future research with these tools could be used to develop more precise therapeutic protocols for BRDC in feeder cattle.