Virulence and required genes in the fish pathogen Vibrio anguillarum

• Main Author: McMillan, Stuart
• Other Authors: Desbois, Andrew, Centre for environment, fisheries and aquaculture science (CEFAS)
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: University of Stirling 2016
• Subjects: Vibrio anguillarum; Galleria mellonella; transposon mutagenesis; alternative hosts; Fishes Diseases; Vibrionaceae; Bacterial diseases; Greater wax moth; Atlantic salmon
• Online Access: http://hdl.handle.net/1893/25417; http://dspace.stir.ac.uk/bitstream/1893/25417/1/McMillan_PhD.2016.pdf

Vibrio 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. ...