In-vivo induced antigen technology of Aeromonas salmonicida

Furunculosis is a bacterial disease of fish that is responsible for a significant impact on the fish aquaculture industry. The causative agent is Aeromonas salmonicida, which is found worldwide and can infect many species of cold- and warm-water fishes. The disease has acute, sub-acute and chronic forms and may have local or systemic effects. Fish that recover from furunculosis can remain asymptomatic carriers. During infection, pathogens express genes that enable them to adapt to different environments. Although a number of Aeromonas salmonicida virulence factors and genes have been identified under standard in vitro culturing conditions, there is no information about genes that are up-regulated within the fish host during Aeromonas salmonicida infection. The aim of this project is to identify bacterial genes expressed within fishes during infection with Aeromonas salmonicida subspecies salmonicida. The work will be undertaken by means of the in vivo induced antigen technology (IVIAT), an immunoscreening technique that identifies bacterial antigens expressed during infection. Screening for genes induced in vivo by Aeromonas salmonicida subspecies salmonicida represents a strategy to identify novel virulence factors and to define global expression patterns relevant to pathogenesis. Novel genes identified during the project will be appropriate for consideration for therapeutic, vaccine or diagnostic applications. The results of the project will provide new insights into the mechanism of infection of Aeromonas salmonicida.

Aeromonas salmonicida is an important bacterial fish pathogen. Several sub-species have been described, although the best known sub-species is A. salmonicida subsp. salmonicida; mostly known as a pathogen of salmonids. The disease has acute, sub-acute and chronic forms and Fish that recover from furunculosis can remain asymptomatic carriers. Aeromonas salmonicida infections had a particularly significant impact until the mid-nineties when an efficient vaccine was introduced in the salmonid industry. However, this vaccine can have severe side effect on the fish. Moreover, the introduction of new species to be cultivated, such as cod or halibut, is an area of concerns as conflicting reports exist regarding the efficacy of current vaccines to protect these new species.Bacterial pathogens often regulate the expression of their virulence gene, only expressing them during the infection process. While this often complicates the investigation of the virulence factors of these bacteria it also unable to highlight mechanisms that have been selected to be over-expressed during the invasion process. This is the basis of the method called in vivo induced antigen technology (IVIAT) that was applied for the present study: antibodies were harvested from fish exposed to the pathogen of interest. These antibodies were then adsorbed against an in vitro culture of the bacterium of interest, removing the ones that react with proteins expressed in vitro so that only the antibodies targeting antigens specifically expressed during the infection process remained. These antibodies were then used to screen a genomic library composed of 12074 random sections from the genome of A. salmonicida subsp. salmonicida. Positive sections were sequenced and identified based on homology. This approach yield 8 genes for which the corresponding protein appeared to be more highly expressed in vivo: UDP-3-O-acyl-N-acetylglucosamine deacetylase (involved in cell wall synthesis), RNA polymerase sigma factor RpoD (a regulator of gene expression); TonB (that provides energy for transport across the cell membrane) alongside the effector of the type 3 secretion system AopO; Lactoylglutathione lyase (involved to resistance to pH and oxidative stress); a LamB-like maltoporin as well as two hypothetical conserved protein. Afterwards, reverse transcription quantitative PCR (RT-qPCR) was then performed on each of these genes and confirmed that they were significantly more highly transcribed during the infectious process.This project allowed us to improve our understanding of the virulence process among aeromonaceae and their interaction with their fish host.