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dc.contributor.advisorDesbois, Andrew-
dc.contributor.authorMcMillan, Stuart-
dc.identifier.citationMcMillan, S., D. Verner-Jeffreys, J. Weeks, B. Austin, and A. P. Desbois, 2015, Larva of the greater wax moth, Galleria mellonella, is a suitable alternative host for studying virulence of fish pathogenic Vibrio anguillarum: Bmc Microbiology, v. 15, p. 10.en_GB
dc.description.abstractVibrio anguillarum infects many fish species in aquaculture, reducing farm productivity and negatively impacting fish welfare. Deeper understanding of the biology of V. anguillarum, particularly during infections in vivo, will help to improve disease prevention and control. Thus, the aim of this thesis was to provide further insight into the infection biology of V. anguillarum with a view to identifying better ways to reduce the impact of this pathogen in aquaculture. Conventional studies on virulence, particularly those aiming to identify novel virulence factors, often employ transposon mutagenesis where the functions of individual genes in the bacterium are disrupted. These mutant libraries are screened to identify those with attenuated virulence, allowing subsequent identification of the gene responsible. Usually the native fish host would be used but such studies are increasingly difficult to perform due to regulations on vertebrate experiments and ethical concerns. As a result, alternative invertebrate hosts are now an important means to studying microbial infections, but few models have been assessed for bacterial pathogens of fish. In this thesis, larvae of the greater wax moth Galleria mellonella were evaluated as an alternative host to investigate V. anguillarum virulence. Wild-type V. anguillarum isolates killed larvae in a dose-dependent manner, replicated in the haemolymph, and larvae infected with a lethal dose of bacteria could be rescued by antibiotic therapy, thus indicating that V. anguillarum established an infection in G. mellonella. Crucially, virulence of 11 wild-type V. anguillarum isolates correlated significantly between larva and Atlantic salmon infection models, and studies with isogenic mutants knocked out for various virulence determinants revealed conserved roles for some in larva and fish infections, including the pJM1 virulence plasmid and rtxA toxin. Thereafter, 350 strains from a V. anguillarum random transposon insertion library were screened for attenuated virulence in G. mellonella. In total, 12 strains had reduced virulence and in these mutants the transposon had inserted into genes encoding several recognised and putative virulence factors, including a haemolytic toxin (vah1) and proteins involved in iron sequestration (angB/G and angN). Importantly, the transposon in one strain had inserted into an uncharacterised hypothetical protein. Preliminary investigations found this putative novel virulence factor to contain a GlyGly-CTERM sorting domain motif, with sequence similarity to VesB of Vibrio cholerae which is involved in post-translational processing of cholera toxin. Finally, three transposon insertion libraries were mass sequenced on a MiSeq platform to identify V. anguillarum genes lacking transposon insertions. These genes were assumed to be ‘required’ for viability in the conditions under which the mutants were selected, in this case tryptone soya agar. In total, 248 genes lacked a transposon insertion and were the putative ‘required’ genes, and these may be important chemotherapeutic targets for new approaches to combat V. anguillarum infections. This thesis has furthered our understanding of the biology of the important fish pathogen V. anguillarum using an ethically acceptable approach, and the findings may assist with new ways to reduce the burden of this bacterium in aquaculture.en_GB
dc.publisherUniversity of Stirlingen_GB
dc.subjectVibrio anguillarumen_GB
dc.subjectGalleria mellonellaen_GB
dc.subjecttransposon mutagenesisen_GB
dc.subjectalternative hostsen_GB
dc.subject.lcshFishes Diseasesen_GB
dc.subject.lcshBacterial diseasesen_GB
dc.subject.lcshGreater wax mothen_GB
dc.titleVirulence and required genes in the fish pathogen Vibrio anguillarumen_GB
dc.typeThesis or Dissertationen_GB
dc.type.qualificationnameDoctor of Philosophyen_GB
dc.rights.embargoreasonI require time to complete further work and then write articles for publication which will include work within this thesis.en_GB
dc.contributor.funderCentre for environment, fisheries and aquaculture science (CEFAS)en_GB
Appears in Collections:Aquaculture eTheses

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