Veterinary Biosciences - Theses
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ItemThe effect of tonB deletion on the expression of the genes encoding Shiga toxin, TonB-dependent receptors and fimbriae in the 16001 oedema disease strain of E. coli( 2018)Oedema disease is caused by Shiga toxin producing Escherichia coli (STEC) and generally affects piglets at one to two weeks after weaning. The stress related to dietary change and loss of lactogenic immunity after weaning are believed to contribute to the onset of the disease. Affected piglets typically have oedematous lesions in multiple organs, including the stomach, colon and eyelids, and also neurological signs, such as ataxia and paralysis. Occasionally, the disease can result in sudden death without any apparent clinical signs. The Shiga toxin and fimbriae are the two key virulence factors and they play an essential role in the pathogenesis of the disease. Multiple attempts have been made to develop active, passive and maternal approaches to immunization. These studies have mainly focussed on attenuating the organism by manipulating these virulence factors at the molecular level. Although the disease is endemic and has a considerable economic impact, there is no commercial vaccine available in Australia. Control measures mainly focus on antibiotic treatment but this has been challenged by the emergence of drug resistance in the strain. Iron is one of the most important micronutrients for the growth of bacteria and its availability to pathogens is restricted both inside and outside the host. Pathogenic bacteria respond by elaboration of siderophores in order to transport iron across the outer membrane, and the TonB protein supplies the necessary energy for this transport. The aims of this research project were to develop tonB mutants of an Australian STEC strain (16001) isolated from pigs with oedema disease by deleting the tonB gene using the lambda Red recombination system and to investigate whether the transcription of the virulence factors Shiga toxin 2e (stx2e) and the F18 fimbriae and TonB-dependent transporter genes was influenced by the tonB deletion. The lambda Red recombination system was used to replace the target gene with an antibiotic resistance cassette, and successful mutagenesis was confirmed by molecular and phenotypic characterisation of the mutant. Sequencing studies showed that the tonB gene was replaced with a kanamycin resistance gene. The mutant had a slower growth rate and produced greater concentrations of siderophores on chrome azurol S (CAS) agar. In general, phenotypic characterization of the tonB mutant indicated that it had reduced intracellular iron levels as a result of the tonB deletion. The aim of deleting tonB was to reduce intracellular iron levels, which was anticipated to attenuate the organism and also upregulate the iron regulon to increase the production of protective antigens. During iron shortage, siderophore synthesis and expression of siderophore transporters is increased, thus eliciting an enhanced immune response against these antigens. Upregulation of the genes for some outer membrane receptors, as well as those for the key virulence factors Stx2e and F18 fimbriae, was demonstrated in this study. In conclusion, the deletion mutant generated in this study may be able to be used as the basis for development of a vaccine candidate for control of this important disease of pigs.