Genomic Analysis of Pasteurella atlantica Provides Insight on Its Virulence Factors and Phylogeny and Highlights the Potential of Reverse Vaccinology in Aquaculture.

Pasteurellosis in farmed lumpsuckers, Cyclopterus lumpus, has emerged as a serious disease in Norwegian aquaculture in recent years. Genomic characterization of the causative agent is essential in understanding the biology of the bacteria involved and in devising an efficient preventive strategy. The genomes of two clinical Pasteurella atlantica isolates were sequenced (≈2.3 Mbp), and phylogenetic analysis confirmed their position as a novel species within the Pasteurellaceae. In silico analyses revealed 11 genomic islands and 5 prophages, highlighting the potential of mobile elements as driving forces in the evolution of this species. The previously documented pathogenicity of P. atlantica is strongly supported by the current study, and 17 target genes were recognized as putative primary drivers of pathogenicity. The expression level of a predicted vaccine target, an uncharacterized adhesin protein, was significantly increased in both broth culture and following the exposure of P. atlantica to lumpsucker head kidney leucocytes. Based on in silico and functional analyses, the strongest gene target candidates will be prioritized in future vaccine development efforts to prevent future pasteurellosis outbreaks.

Microbial Communities in a Flow-Through Fish Farm for Lumpfish (Cyclopterus lumpus L.) During Healthy Rearing Conditions.

Lumpfish can efficiently remove sea lice from Atlantic salmon in net-pens, and production of lumpfish in closed fish farms is a new, fast developing industry in Norway. However, periodic outbreaks of bacterial diseases in the fish farms represent a large problem, both economically and ethically. Therefore it is important to obtain a better understanding of how microbial communities develop in these production facilities. Knowledge on the characteristics of microbial communities associated with healthy fish could also enable detection of changes associated with disease outbreaks at an early stage. In this study we have monitored microbial communities in a fish farm for lumpfish during normal operational conditions with no disease outbreak by using 16S rRNA gene amplicon sequencing. The study involved weekly samplings of water and biofilms from fish tanks, and fish. The results revealed that the microbial communities in fish tank water were different from the intake water. The water and biofilm in fish tanks were highly similar in regards to microbial community members, but with large differences in relative abundances for some taxa. The sampled fish were associated with mostly the same taxa as in tank water and biofilm, but more variation in relative abundances of different taxonomic groups occurred. The microbial communities in the fish farm seemed stable over time, and were dominated by marine bacteria and archaea within Alphaproteobacteria, Epsilonproteobacteria, Flavobacteria, Gammaproteobacteria, Thaumarchaeota, Planctomycetes, Sphingobacteriia, and Verrucomicrobiae (>10% relative abundance). Bacterial genera known to include fish-pathogenic strains were detected in all types of sample materials, but with low relative abundances (<5%). Exceptions were some samples of fish, biofilm and water with high relative abundance of Tenacibaculum (<85.8%) and Moritella (<82%). In addition, some of the eggs had a high relative abundance of Tenacibaculum (<89.5%). Overall, this study shows that a stable microbial community dominated by various genera of non-pathogenic bacteria is associated with a healthy environment for rearing lumpfish. Taxa with pathogenic members were also part of the microbial communities during healthy conditions, but the stable non-pathogenic bacteria may limit their growth and thereby prevent disease outbreaks.

Exploring the Effect of Phage Therapy in Preventing Vibrio anguillarum Infections in Cod and Turbot Larvae.

The aquaculture industry is suffering from losses associated with bacterial infections by opportunistic pathogens. Vibrio anguillarum is one of the most important pathogens, causing vibriosis in fish and shellfish cultures leading to high mortalities and economic losses. Bacterial resistance to antibiotics and inefficient vaccination at the larval stage of fish emphasizes the need for novel approaches, and phage therapy for controlling Vibrio pathogens has gained interest in the past few years. In this study, we examined the potential of the broad-host-range phage KVP40 to control four different V. anguillarum strains in Atlantic cod (Gadus morhua L.) and turbot (Scophthalmus maximus L.) larvae. We examined larval mortality and abundance of bacteria and phages. Phage KVP40 was able to reduce and/or delay the mortality of the cod and turbot larvae challenged with V. anguillarum. However, growth of other pathogenic bacteria naturally occurring on the fish eggs prior to our experiment caused mortality of the larvae in the unchallenged control groups. Interestingly, the broad-spectrum phage KVP40 was able to reduce mortality in these groups, compared to the nonchallenge control groups not treated with phage KVP40, demonstrating that the phage could also reduce mortality imposed by the background population of pathogens. Overall, phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge assays with V. anguillarum pathogens suggested that application of broad-host-range phages can reduce Vibrio-induced mortality in turbot and cod larvae, emphasizing that phage therapy is a promising alternative to traditional treatment of vibriosis in marine aquaculture.

Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.).

The expression levels of three commonly used housekeeping genes, EF1-alpha, RPS20 and Beta-Actin, were examined in seven different tissues and leucocytes from non-stimulated Atlantic salmon (Salmo salar L.). The tissues analysed by quantitative real-time PCR were gill, liver, intestine, muscle, spleen, head kidney leucocytes (HKL) and peripheral blood leucocytes (PBL). The experiments were performed to investigate the transcriptional stability within and between tissues and leucocytes and between individuals. For all tissues and leucocytes, an appropriate reference gene was identified except for muscle tissue. HKL were used as a calibrator and the expression of EF1-alpha varied maximally 2.5-fold in five out of the six tissues and leucocytes investigated relative to the expression of 18S rRNA. The RPS20 gene was more intermediate and varied at least by a factor of two and maximally by a 20-fold factor. Beta-Actin was generally the most regulated gene showing high variations for gill (5.8x) and spleen tissue (10.3x) relative to the calibrator. A suitable reference gene for muscle tissue was not found since the expression varied between 8.3- and 25-fold for the three genes compared to the calibrator. By comparing the expression results of the non-stimulated tissues and leucocytes using the Normfinder programme, it was further shown that EF1-alpha was the most stably expressed gene both between individuals and the different tissues/leucocytes. Stimulation with lipopolysaccharide (LPS) of TO cells and HKL from Atlantic salmon was additionally performed to reveal whether an immune stimulating agent would change the expression level of EF1-alpha, RPS20 and Beta-Actin. LPS stimulation of cells revealed that RPS20 and EF1-alpha were least regulated by the LPS treatment in the TO cells relative to 18S rRNA, but in HKL, Beta-Actin was the most appropriate gene. However, the variations were overall maximally two-fold in LPS-stimulated TO cells and HKL, which make all three genes suitable as reference genes in this case. A further experiment showed that no RT- and/or PCR inhibitors were present in the non-stimulated tissues and cells, indicating true transcriptional differences.