Molecular and immunological analysis of New Zealand isolates of Mycoplasma ovipneumoniae : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand
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Date
2021
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Massey University
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Abstract
Mycoplasma ovipneumoniae is a pathogen of caprinae expected to infect the majority of domestic sheep in early life. M. ovipneumoniae is suspected to be the primary cause of chronic non-progressive pneumoniae (CNP) in lambs. Currently there is no effective vaccine against M. ovipneumoniae, and many commonly used antibiotics have limited effect. Diagnosis of M. ovipneumoniae infections can be difficult, due in part to the limited overt symptoms seen in cases of CNP. Despite the significance of M. ovipneumoniae infections there is limited information on the molecular and immunological data of M. ovipneumoniae. Thus, the major focus of this thesis was to identify potential immunogenic proteins for a vaccine or diagnostic purposes and construct genomes from New Zealand isolates of M. ovipneumoniae.
Draft genomes were produced for three New Zealand isolates of M. ovipneumoniae, (isolates 16, 90, and 103), and annotated. Using antisera produced against these isolates, immunogenic proteins were identified from hydrophobic membrane protein fractions of M. ovipneumoniae using Western blotting. Those protein bands were subjected to mass spectrometry analysis, and by comparing mass spectrometry data to the genomes, six novel proteins, GAPDH, P146, MATE, hypothetical protein 1, hypothetical protein 2, hypothetical protein 3, and two previously described immunogenic proteins, EF-Tu and HSP70, were identified. Five of the proteins were produced as recombinant proteins for further study.
The nucleotide sequence of EF-Tu was edited using an overlap polymerase chain reaction (PCR) to convert the sequence to Escherichia coli codon preference. This sequence was then cloned into the pET22b (+) vector for expression of histidine-tagged fusion protein. Additionally, EF-Tu, GAPDH, P146, hypothetical protein 3 and an epitope of hypothetical protein 1 were produced as recombinant proteins by GenScript. Antisera were produced in sheep against each of the recombinant proteins for further studies. Enzyme-linked immunosorbent assay (ELISA) confirmed that each protein was immunogenic and elicited antibody responses in sheep. Immunofluorescence microscopy confirmed the localisation of native EF-Tu and GAPDH proteins on the surface of M. ovipneumoniae cells.
Antisera produced against whole cell antigens from M. ovipneumoniae cross-reacted with P146 and hypothetical protein 3 suggesting a potential role for these proteins to be used to detect animals infected with M. ovipneumoniae. Sheep vaccinated with M. ovipneumoniae produced weak IFN-γ responses but stronger IL-17 responses in whole blood cultures stimulated with whole cell antigens. Of the five proteins tested as recall antigens in the IL-17A assay, recombinant GAPDH produced a strong IL-17A response in sheep vaccinated with M. ovipneumoniae suggesting a potential role for GAPDH in a diagnostic assay for measuring IL-17A responses in M. ovipneumoniae infected sheep.
Mycoplasmas are known to adhere to host epithelial cells as part of the pathogenesis process. A bovine endometrial epithelial cell line was established as a model to study adherence of M. ovipneumoniae to epithelial cells and determine if antibodies against the surface proteins could interfere with adherence. M. ovipneumoniae cells adhered to the endometrial epithelial cells. Adherence was inhibited by antibodies directed against whole cells of M. ovipneumoniae but not against the recombinant proteins.
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Keywords
Mycoplasma diseases in animals, New Zealand, Genetic aspects, Immunological aspects, Sheep, Diseases