Theses & dissertations

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    Coccidiosis and Necrotic Enteritis (NE): Testing Molecular Tools for Quantifying Clostridium and Eimeria Simultaneously in Experimentally Challenged Broiler Chickens
    (University of Guelph, ) Heibein, Will; Barta, John
    Various species of Eimeria infect the intestinal tract of poultry, with Eimeria maxima commonly associated with the development of necrotic enteritis (NE). Restrictions to prophylactic use of Category 2 antibiotics in Canadian poultry make outbreaks of NE caused by Clostridium perfringens more likely. Infections with Eimeria species, combined with poor diet and sanitation, are key drivers of C. perfringens infection and NE. This study quantified C. perfringens and E. maxima DNA from within the intestinal tract of experimentally infected broilers using a newly developed, duplexed, probe-based qPCR assay. C. perfringens showed little change in quantification but expressed lesion development in birds infected with E. maxima, particularly from 5 days-post-infection. Inoculation of day-of-age chicks with C. perfringens permitted colonization and persistence throughout the intestinal tract for several weeks. Birds inoculated with C. perfringens prior to E. maxima, and vice versa, were more likely to exhibit lesions consistent with NE.
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    Extending the Longevity of AAV-mediated Lung Gene Therapy
    (University of Guelph, ) Thomas, Sylvia; Wootton, Sarah
    AAV-mediated gene therapy has progressed rapidly in the past three decades, permitting the use of this vector for the treatment of several diseases. Although the first administration of an AAV vector into a human patient occurred in 1995 to correct cystic fibrosis in the lungs, this organ remains a challenging treatment site and elusive target for gene therapy. Limitations are attributed to the requirement for lung tropic vectors, the defensive barriers of the lungs, immune system considerations, the obstacles in the journey to reach the target cell and be granted entry, and cell turnover. In monogenic disorders such as surfactant protein B deficiency, additional hurdles appear in achieving long term expression due to the developing lung. This disease affects neonates, causing respiratory distress leading to death within a year of birth. Novel vectors such as capsid engineered AAV6.2FF may be able to deliver the gene of interest, but the gene addition delivery of AAV will be maintained in an episomal fashion and eventually wane in expression. Therefore, persistent expression will need to be achieved through either readministration or permanent gene correction. The following chapters feed into each other in progressing this vector forward and extending its persistence in the lungs. In this thesis, we assess the vector performance of AAV6.2FF, analyzing its biodistribution and stability, along with investigating mechanisms to extend expression in the lungs. Here, we administered 1e11 vector genome (vg) of AAV6.2FF with an alkaline phosphatase reporter gene by five routes of administration into C57BL/6 mice and viewed robust and localized expression in the lungs through intranasal instillation. Separately, we examined the thermal stability of this vector and determined that an excipient formulation change to include the non-ionic surfactant chemical PF-68 improved stability under different exposure conditions. In addition, we investigated mechanisms to extend the persistence of AAV6.2FF gene expression in lungs by mitigating the immune response with a stealth design change to the genome that permitted readministration along with demonstrating that permanent gene correction with AAV6.2FF is possible, providing optimistic conclusions that both readministration and gene editing can extend survival in surfactant protein B deficiency.
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    Oxidative Stress Renders T Cells Susceptible To Oncolytic Virus Infection
    (University of Guelph, ) Vanderkamp, Sierra; Bridle, Byram
    Oncolytic viruses selectively infect and kill cancer cells and induce tumour-specific immune responses. However, success has been limited partially due to the ability of some oncolytic viruses, like vesicular stomatitis virus (VSV) to cause off-target infection of leukocytes, including activated T cells, thereby reducing therapeutic potential. This could be problematic for multi-dosing protocols in which the first dose of VSV activates T cells that can then be infected with subsequent doses of the virus. The aim of the research presented in this thesis was to investigate potential mechanisms behind off-target infection of T cells by VSV, as well as ways to abrogate this infection, both in vitro and in vivo, to improve VSV-based cancer immunotherapies. All research was summarized in manuscript format to facilitate publication.
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    AAV-Mediated Monoclonal Antibody Expression for the Prevention of Pseudomonas Aeruginosa Infections
    (University of Guelph, ) Lopes, Jordyn; Wootton, Sarah
    In vivo expression of pathogen specific monoclonal antibody (mAb) genes using adeno-associated virus (AAV) vectors leads to robust and sustained accumulation of mAbs in the blood and at mucosal surfaces following intramuscular administration. Here we evaluated whether AAV-mediated mAb expression could protect against lethal challenge with Pseudomonas aeruginosa (PA). We show that our AAV6.2FF platform mediates long-term serum and mucosal expression of highly protective mAbs targeting the exopolysaccharide Psl and the PcrV component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. Intramuscular administration of AAV6.2FF-αPcrV, AAV6.2FF-αPsl and AAV6.2FF-MEDI3902 provided significant protection from lethal pneumonia in mice challenged with PA strains PAO1 and PA14 and dramatically reduced bacterial burden in the lung and dissemination to other organs. These results support further investigation into the use of AAV vectored immunoprophylaxis for the prevention and treatment of PA infections and other bacterial pathogens for which current treatment strategies are limited.
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    The role of inflammatory factors in the pathogenesis of Mycoplasma bovis pneumonia
    (University of Guelph, ) Vulikh, Ksenia; Caswell, Jeff
    Bovine respiratory disease (BRD) is a major cause of morbidity and mortality in cattle production worldwide. Mycoplasma bovis is one of the most important pathogens involved in BRD, and it is associated with severe caseonecrotic bronchopneumonia characterized by the presence of necrotic foci with ghost-like cells in the lungs. The pathogenesis of this disease is poorly understood, and the role of inflammation in its development is unclear. This thesis investigates the hypothesis that M. bovis alone does not cause severe disease, but local inflammation at the site of infection exacerbates the development of macrophage cell death and caseonecrotic lesions. The research consisted of two parts: an in vivo experiment using partially colostrum-deprived calves raised in an isolation unit and exposed to various pro-inflammatory factors followed by infection with M. bovis, and an in vitro experiment using bovine monocyte-derived macrophages activated with Mannheimia haemolytica lysate and infected with different M. bovis strains. The in vivo experiment aimed to develop an improved animal model for the study of M. bovis pathogenesis and to evaluate the effects of different inflammatory stimuli on the development and severity of caseonecrotic lesions. The in vitro experiment aimed to determine the type of macrophage cell death initiated by M. bovis, and to compare the cytotoxicity and cytokine production of different M. bovis strains. The results showed that M. bovis infection did not significantly harm macrophages or cause caseonecrotic lesions in the absence of prior inflammation, but it did so when inflammation was induced by live M. haemolytica or bacterial lysates. The results also revealed that M. bovis strains varied in their ability to kill macrophages and induce cytokine expression, and that strains isolated from sick or dead animals tended to be more cytotoxic than those from healthy animals. The results indicate that inflammation is a key factor in the pathogenesis of M. bovis-induced caseonecrotic bronchopneumonia, suggesting that control of harmful inflammatory responses could minimize the severity of this disease. The thesis also suggested directions for future research to elucidate the mechanisms of M. bovis pathogenicity and the immune response to this infection.