Pasteurella atlantica in Norwegian lumpsuckers : Characterisation, pathogenicity, vaccine development and immune responses

Abstract

Lumpsuckers (Cyclopterus lumpus L.) have gained popularity as a cleaner fish to biologically control salmon lice infestations in farmed Atlantic salmon in Norway. Starting in 2012, most producers in Norway have been raising lumpsuckers in captivity from milt and eggs collected from wild-caught fish. Since then, this industry has grown exponentially to meet the demands of Atlantic salmon producers. However, this growth was not without its issues. A number of bacterial diseases have been recorded from farmed lumpsuckers, with Pasteurella atlantica being one of the most concerning to farmers, given it is an emerging pathogen first diagnosed in Norwegian lumpsuckers in 2012.

The main objectives of this project were to characterise P. atlantica, investigate the pathogenicity and the resulting immune responses in lumpsuckers, develop and test vaccines, and identify vaccine targets for future vaccine development.

The first milestone was the establishment of P. atlantica culture in liquid medium. Through this work, bacterial growth was achieved in tryptic soy broth supplemented with foetal calf serum. This meant that bacteria could be produced in large volumes as required for experiments and vaccine development.

Subsequently, a variety of challenge models were tested to study pathogenesis of P. atlantica in lumpsuckers. The bath challenge model was identified as the most reliable model, which could be further used during vaccine testing. Through these experiments, it was found that an asymptomatic carrier status occurs following exposure to the bacteria, highlighting the importance of fish health screening programs.

To that end, whole cell inactivated vaccines were developed against P. atlantica to investigate their protection potential. However, only limited protection was conferred following experimental challenge of vaccinated lumpsuckers despite serology following vaccination showing high specific antibody levels. Additionally, in vitro experiments to investigate interactions between P. atlantica and lumpsucker head kidney leucocytes demonstrated potential intracellular behaviour, as well as auto-aggregation and adherence of the bacteria to host cells.

Whole genome sequencing was then used to investigate the genome of P. atlantica to shed light on virulence factors that may be involved in disease and may be suitable as vaccine targets, paving the way for further work in reverse vaccinology. Furthermore, as the pathogen was still unclassified taxonomically at this point, phylogenetic analysis was additionally carried out which suggested the taxonomic position of P. atlantica within the Family Pasteurellaceae.

In silico analysis subsequently identified a putative uncharacterised adhesin protein as being a major virulence factor and potential vaccine target and possessed similarities to the adhesins YadA and Hia found in Yersinia spp. and Haemophilus influenzae, respectively.

Gene expression analysis was then used to assess the regulation of this adhesin in the presence and absence of lumpsucker head kidney leucocytes. It was observed that the gene encoding the putative adhesin is upregulated significantly, both during bacterial culture as well as when bacterial cells are exposed to lumpsucker head kidney leucocytes in vitro, complementing the in silico analysis.

The knowledge gained from this work has significant implications for vaccine development. Through reverse vaccinology, a potential vaccine target has been identified that, through further research, could be used to develop subunit vaccines such as recombinant or DNA/mRNA vaccines. These promising results provide grounds for further research against a serious emerging disease in aquaculture with the aim of improving lumpsucker health and welfare.