Doctor of Philosophy
School of Biological Sciences - Faculty of Science
Matic, Jake, The development of non antibiotic resistant recombinant vaccines against mycoplasma hyopneumoniae, PhD thesis, School of Biological Sciences, , University of Wollongong, 2008. http://ro.uow.edu.au/theses/441
Mycoplasma hyopneumoniae is a respiratory pathogen of pigs that has substantial global economic impact and currently there is no vaccine available that prevents colonisation. This study focuses on the characterisation of novel M. hyopneumoniae vaccine candidates and the development of a live, attenuated vaccine expressing heterologous M. hyopneumoniae antigens. The putative M. hyopneumoniae vaccine antigen, the lipoyl binding domain of the dihydrolipoyl dehydrogenase subunit (PdhD) of the pyruvate dehydrogenase complex, was strongly recognised by porcine hyperimmune sera. Western blot analysis with PdhD antisera detected the protein in geographically diverse M. hyopneumoniae isolates. However PdhD was only weakly recognised by convalescent swine sera indicating it is not likely to contribute significantly to the protective convalescent response. Therefore it was not used in further vaccination experiments. Live vaccine delivery systems expressing two antigens from M. hyopneumoniae, adhesin (Adh) and ribonucleotide reductase (NrdF), were constructed using either plasmid-based expression (PBE) or chromosomally-based expression (CBE) systems. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter trapping technique whereby the promoterless operon was stably integrated into the chromosome of Salmonella enterica serovar Typhimurium aroA (STM-1) and the expression of antigens assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained using the in vivo induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems with PBE producing much higher quantities of both antigens. The stability of antigen expression was higher in the CBE system with 60-100% of individual cells still expressing antigen after 60 generations without selection. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice and significant systemic IgM and IgG responses against both antigens amongst all CBE groups were detected. No significant immune response against either antigen was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally-located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.
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