Use of small extracellular vesicles for diagnosis of Mycoplasma bovis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Genetics at Massey University, Manawatū, New Zealand

Figure 1 is re-used with the publisher's permission.Background: Mycoplasma bovis (M. bovis) is a pathogenic bacterium responsible for causing numerous production and welfare issues in cattle herds. Eradication efforts worldwide are limited by ineffective antibiotics, intracellular infection of host cells, immune evasion, and insufficient diagnostic tools. Current diagnostic tests are inadequate to assess M. bovis infection as they rely upon direct detection of M. bovis or indirect detection via serology (M. bovis–specific antibodies). Therefore, with aim to improve diagnostics of M. bovis infection, small extracellular vesicles (sEVs) were utilised. These nanoparticles act as intercellular messengers and contain material representative of their cell of origin. Their use as diagnostic and therapeutic tools has enabled a variety of diseases and infections to be assessed and/or treated. The aim of this project was to develop an upscaled in vitro model of M. bovis infection within a bioreactor flask to test the hypothesis that the protein cargo of host cell sEVs were altered in response to M. bovis infection. Methods: A control culture of a bovine endometrial epithelial cell line (bEEL cells) and a co–culture of bEEL cells and M. bovis were established within bioreactor flasks. Using Size Exclusion Chromatography columns, sEVs were isolated from the harvests of the bioreactor flasks. Liquid Chromatography Tandem Mass Spectrometry assessed the sEV proteome to compare differences created by M. bovis infection. Results: Infection was indicated by a continued presence of M. bovis within the co–culture. Changes in the regulation of various proteins, such as inhibition of host cell endopeptidases, was demonstrated in co–culture sEVs as a response to M. bovis infection. Adherence of M. bovis to bEEL cells was certain, but intracellular infection remained inconclusive. Conclusion: Data from this study implies that a co–culture can be successfully established within a bioreactor flask, and that the proteome of sEVs is altered in response to infection by M. bovis