Theses and Dissertations at Montana State University (MSU)
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Item Experimental infection of specific pathogen-free domestic lambs with Mycoplasma ovipneumoniae(Montana State University - Bozeman, College of Agriculture, 2021) Johnson, Thea Haviland; Chairperson, Graduate Committee: Diane Bimczok; Diane Bimczok, Kerri Jones, Cassie Mosdal, Steven Jones, SK, CB, AS, and B. Tegner Jacobson were co-authors of the article, 'Immunoglobulin transfer, survival, and growth in motherless lambs fed a bovine serum-based colostrum replacer' which is contained within this thesis.; Kerri Jones, B. Tegner Jacobson, Julia Schearer, Noah Adams, Isaak Thornton, Cassie Mosdal, Steven Jones, Mark Jutila, Agnieszka Rynda-Apple, Thomas Besser and Diane Bimczok were co-authors of the article, 'Experimental infection of specific-pathogen-free domestic lambs with Mycoplasma ovipneumoniae causes asymptomatic colonization of the upper airways that is resistant to antibiotic treatment' in the journal 'Veterinary microbiology' which is contained within this thesis.Mycoplasma ovipneumoniae (M. ovi) is a respiratory pathogen commonly found in sheep and goats. It is associated with mild to moderate respiratory disease in domestic lambs, but severe pneumonia outbreaks in wild ruminants, specifically bighorn sheep. The goal of our study was to better understand the role of M. ovi as a respiratory pathogen in domestic sheep and to explore potential antibiotic treatment approaches. We first established a flock of specific pathogen-free (SPF) lambs through supervised lambing and motherless rearing in a Large Animal BSL-2 facility. Lambs were fed a colostrum replacer that yielded low mortality, steady weight gain and serum IgG and protein concentrations comparable to those of lambs raised on ewe colostrum. We inoculated the SPF lambs with field isolates of M. ovi and monitored the lambs for eight weeks for colonization with the bacteria, M. ovi-specific antibodies, clinical symptoms, and cellular and molecular correlates of lung inflammation. After eight weeks, lambs were treated with the macrolide antibiotic gamithromycin and observed for an additional four weeks. Stable colonization of the upper respiratory tract with M. ovi was established in all four M. ovi-inoculated, but in none of the four mock-infected lambs. All M. ovi-infected lambs developed a robust antibody response to M. ovi within 2 weeks. However, we did not observe significant clinical symptoms, evidence of lung damage or inflammation in any of the infected lambs. Interestingly, treatment with gamithromycin failed to reduce M. ovi colonization. These observations indicate that, in the absence of co-factors, M. ovi causes asymptomatic colonization of the upper respiratory tract of that is resistant to clearance by the host immune response as well as by gamithromycin treatment in domestic lambs.Item Developing and implementing genetic tools designed to understand host takeover by Chlamydia trachomatis.(Montana State University - Bozeman, College of Letters & Science, 2019) Kessy, Enock Joel; Chairperson, Graduate Committee: Blake WiedenheftChlamydia are gram negative obligate intracellular parasites that are responsible for millions of new infections in humans and animals every year. C. trachomatis is the number one cause of bacterial sexually transmitted infections in the United States, the number one cause of infectious blindness worldwide. Since 2001, there has been a steady increase in the number of new cases of C. trachomatis infections each year. Despite the prevalence and medical importance of C. trachomatis, we still know relatively little about the lifecycle of this parasite and the host factors that are essential for the lifecycle of C. trachomatis. To address this critical gap in our knowledge, my thesis work aimed to develop and implement genetic tools to understand host takeover by C. trachomatis. In this thesis I present results suggesting that I have transformed C. trachomatis with a plasmid carrying the Cas9 gene from Campylobacter jejuni. Additional experiments are necessary to determine if the CjCas9 is expressed, nuclease active, and functional for programable editing in C. trachomatis. In addition to my work aimed at developing a CRISPR-Cas9-based genetic engineering system in C. trachomatis, I also participated in a genome wide knockout screen aimed at identifying human genes necessary for completion of the C. trachomatis lifecycle. The CRISPR-Cas9 genome wide knockout screen identified 103 genes as critical factors for C. trachomatis. To validate results for the screen I have been involved in creating clonal cell lines with deletions in three of the genes that form the Adaptor Protein (AP) Complex (i.e., AP3S2, AP1B2 and AP1G2). The genes have been deleted and future experiments are aimed at measuring the impact of these genes on the C. trachomatis lifecycle.