Genetic manipulation of type D Pasteurella multocida for vaccine development

Progressive Atrophic Rhinitis (PAR) is a serious complex disease of young swine characterized by sneezing, atrophy of the nasal turbinates, shortening and twisting of the snout and reduction in weight gain. Although the aetiology of the disease is complex, infection with the bacterium toxigenic Pasteurella multocida, is considered essential. A dermonecrotic toxin (DNT) produced by toxigenic strains of type D P. multocida is central to the resorption of the nasal bone structures characteristic of the infection. The P. multocida DNT gene toxA has been previously cloned, sequenced and genetically manipulated in order to develop a vaccine for PAR. These earlier studies demonstrated that DNT-specific antibodies produced in pigs by vaccination with the purified genetically inactivated DNT derivative (toxoid) resulted in the protection of the animals against experimentally induced PAR. An alternative approach to using a subunit vaccine for PAR is to express the toxoided gene from P. multocida either from the chromosome or a plasmid thus providing a live vaccine that could present to the porcine immune system a full spectrum of bacterial antigens in addition to the DNT.;The first requirement for the development of live vaccine candidates against PAR based on P. multocida was an efficient gene transfer system that allowed the stable maintenance and expression of the toxoided toxA gene in P. multocida. Studies are presented which describe the isolation, nucleotide sequence and analysis of a naturally occurring 5.36 kb streptomycin and sulphonomide resistance plasmid isolated from P. multocida, designated PiGI.;A second strategy for the development of potential live recombinant vaccines for PAR required the construction of P. multocida toxA mutants using site-specific chromosomal mutagenesis. Various different methods were attempted before it was shown that targeted inactivation of the P. multocida chromosomal toxA gene could be achieved with the use of a conjugative suicide vector based on the plasmid pGP704.;Finally, mutation of the P. multocida gene was shown to attenuate the organism in a murine septicemia model. The ability of the P. multocida recombinant strains to function as live vaccines for PAR was also modelled in the murine infection system. (Abstract shortened by UMI.).;The overall aim of this project was to develop an experimental PAR vaccine based on an attenuated P. multocida expressing toxoided DNT. This was achieved by two methods, expression of DNT from a multicopy plasmid in a non-toxigenic strain of P. multocida and expression from a chromosomal locus through manipulation of the P. multocida chromosomal DNT gene by site-directed mutagenesis.