Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua).

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12 °C or 17 °C. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by elevated temperature and infection, except for the number of mucus cells in the 12 °C group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17 °C. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.

The skin mucosal barrier of lumpfish (Cyclopterus lumpus L.) is weakened by exposure to potential aquaculture production-related stressors.

Various cleaner fish species, such as the lumpfish (Cyclopterus lumpus L.), are used in the sea cage production of Atlantic salmon (Salmo salar L.) as a control measure against the ectoparasitic salmon louse (Lepeophtheirus salmonis). Nonetheless, during severe lice infestation, alternative treatments are required to control parasitic burden. The aim of this study was to gain insight into how lumpfish skin responds to different chemicals used to treat parasites. The authors collected skin from lumpfish from both research facilities (tank-reared fish) and commercial production (cage-reared fish) and used operational welfare indicators, in vitro models, histology and transcriptomics to study how the skin responded to two anti-parasitic oxidative chemicals, hydrogen peroxide (H2 O2 ) and peracetic acid. Lumpfish sampled from the farm were classified as clinically healthy or weak based on their morbidity status, and fish from each category were used to gain insight into how the therapeutics affect the skin barrier. Differences between healthy and weakened (moribund) fish, and between treated fish from each of the two groups, were observed. Histological examination showed an overall reduced skin quality in fish characterized as moribund, including different grades of exposed bony plates. In vitro oxidant-treated lumpfish skin had reduced the migration capacity of keratocytes, a weakened epidermal barrier, and altered gene transcription, changes that are known predisposing factors to secondary infections. Skin from non-treated, healthy fish sampled from commercial farms exhibited similar features and attributes to oxidant-exposed tank-reared fish from a research facility, suggesting that apparently healthy cage-held lumpfish exhibited stress responses in the epidermal barrier. The results of the study outline the risks and consequences lumpfish can face if accidentally subjected to potential anti-parasitic oxidant treatments aimed at Atlantic salmon. It also strengthens the evidence behind the requirement that lumpfish should be removed from the cages before being potentially exposed to this type of treatment and outlines the potential risks of differing husbandry practices upon lumpfish health, welfare and resilience.

Intermittent administration of peracetic acid is a mild environmental stressor that elicits mucosal and systemic adaptive responses from Atlantic salmon post-smolts.

BACKGROUND: Fish encounter oxidative stress several times during their lifetime, and it has a pervasive influence on their health and welfare. One of the triggers of oxidative stress in fish farming is the use of oxidative disinfectants to improve rearing conditions, especially in production systems employing recirculation technology. Here we report the physiological and morphological adaptive responses of Atlantic salmon (Salmo salar L.) post-smolts to intermittent exposure to a potent oxidative agent peracetic acid (PAA). Fish reared in semi-commercial scale brackish water recirculating aquaculture system (RAS) were exposed to 1 ppm PAA every 3 days over 6 weeks. Mucosal and systemic responses were profiled before exposure, 22 and 45 days during the intermittent PAA administration. RESULTS: Oxidative stress was likely triggered as plasma antioxidant capacity increased significantly during the exposure period. Adaptive stress response to the periodic oxidant challenge was likewise demonstrated in the changes in plasma glucose and lactate levels. PAA-induced alterations in the transcription of antioxidants, cytokines, heat shock proteins and mucin genes showed a tissue-specific pattern: downregulation was observed in the gills and olfactory rosette, upregulation occurred in the skin, and no substantial changes in the liver. Further, PAA exposure resulted in histological changes in key mucosal organs (i.e. olfactory rosette, skin and gills); pathological alterations were predominant in the gills where cases of epithelial lifting, hypertrophy and clubbing were prevalent. In addition, intermittent PAA administration resulted in an apparent overproduction of mucus in the nasal mucosa. Lastly, PAA did not dramatically alter the ability of salmon to mount a physiological stress response in the presence of a secondary stressor, though some subtle interference was documented in the kinetics and magnitude of plasma cortisol and glucose response post-stress. CONCLUSIONS: The present study collectively demonstrated that intermittent oxidant exposure was a mild environmental stressor that salmon could mount strong adaptive responses at systemic and mucosal levels. The results will be valuable in optimising the rearing conditions of post-smolts in RAS, especially in adopting water treatment strategies that do not considerably interfere with fish health and welfare.

Transcriptome Responses of Atlantic Salmon (Salmo salar L.) to Viral and Bacterial Pathogens, Inflammation, and Stress.

Transcriptomics provides valuable data for functional annotations of genes, the discovery of biomarkers, and quantitative assessment of responses to challenges. Meta-analysis of Nofima's Atlantic salmon microarray database was performed for the selection of genes that have shown strong and reproducible expression changes. Using data from 127 experiments including 6440 microarrays, four transcription modules (TM) were identified with a total of 902 annotated genes: 161 virus responsive genes - VRG (activated with five viruses and poly I:C), genes that responded to three pathogenic bacteria (523 up and 33 down-regulated genes), inflammation not caused by infections - wounds, melanized foci in skeletal muscle and exposure to PAMP (180 up and 72 down-regulated genes), and stress by exercise, crowding and cortisol implants (33 genes). To assist the selection of gene markers, genes in each TM were ranked according to the scale of expression changes. In terms of functional annotations, association with diseases and stress was unknown or not reflected in public databases for a large part of genes, including several genes with the highest ranks. A set of multifunctional genes was discovered. Cholesterol 25-hydroxylase was present in all TM and 22 genes, including most differentially expressed matrix metalloproteinases 9 and 13 were assigned to three TMs. The meta-analysis has improved understanding of the defense strategies in Atlantic salmon. VRG have demonstrated equal or similar responses to RNA (SAV, IPNV, PRV, and ISAV), and DNA (gill pox) viruses, injection of bacterial DNA (plasmid) and exposure of cells to PAMP (CpG and gardiquimod) and relatively low sensitivity to inflammation and bacteria. Genes of the highest rank show preferential expression in erythrocytes. This group includes multigene families (gig and several trim families) and many paralogs. Of pathogen recognition receptors, only RNA helicases have shown strong expression changes. Most VRG (82%) are effectors with a preponderance of ubiquitin-related genes, GTPases, and genes of nucleotide metabolism. Many VRG have unknown roles. The identification of TMs makes possible quantification of responses and assessment of their interactions. Based on this, we are able to separate pathogen-specific responses from general inflammation and stress.

Consequences of continuous ozonation on the health and welfare of Atlantic salmon post-smolts in a brackish water recirculating aquaculture system.

This study investigated the biological consequences of 45-day continuous ozonation on Atlantic salmon (Salmo salar) post-smolts in a brackish water recirculating aquaculture system (RAS). There was no significant difference in survival, operational welfare indicators, and average weight at termination between the ozone-treated and control groups. Plasma biochemical analyses revealed that the creatinine level was significantly higher in the ozone-treated group than in the control at termination. Histological evaluation of skin health showed no significant difference between the two groups. On the other hand, quantitative histopathology disclosed that the ozone group exhibited a better gill health status than did the control group, particularly at the end of the trial. Mucosal transcriptomics revealed a distinct response profile between the gills and skin. At day 45, there were no differentially expressed genes (DEG) identified in the skin, in contrast to 242 ozone-induced DEGs in the gills. Assessment of the transcriptomic profiles over time revealed that temporal effects were of greater impact in skin compared to gills, regardless of the treatment. The treatment did not result in metabolomic dysregulation and the overall profile lent support to the transcriptomics data that temporal effects had a greater influence on the changes observed. Exposure to handling-confinement stress revealed that ozone treatment did not alter the ability of post-smolts to respond to a secondary stressor. In summary, the suite of health and welfare indicators collectively indicated that continuous ozonation resulted in minimal physiological perturbations in salmon post-smolts. The results are expected to contribute to optimising the rearing conditions for post-smolts in RAS.

Impact of a candidate vaccine on the dynamics of salmon lice (Lepeophtheirus salmonis) infestation and immune response in Atlantic salmon (Salmo salar L.).

Infection with parasitic copepod salmon louse Lepeophtheirus salmonis, represents one of the most important limitations to sustainable Atlantic salmon (Salmo salar L.) farming today in the North Atlantic region. The parasite exerts negative impact on health, growth and welfare of farmed fish as well as impact on wild salmonid populations. It is therefore central to ensure continuous low level of salmon lice with the least possible handling of the salmon and drug use. To address this, vaccination is a cost-effective and environmentally friendly control approach. In this study, efficacy of a vaccine candidate, containing a peptide derived from ribosomal protein P0, was validated post infestation with L. salmonis, at the lab-scale. The sampling results showed good potential of the vaccine candidate when administered intraperitoneally in the host, in reducing the ectoparasite load, through reduction of adult female lice counts and fecundity and with greater presumptive effect in F1 lice generation. The sampling results correlated well with the differential modulation of pro-inflammatory, Th1, Th2 and T regulatory mediators at the transcript level at different lice stages. Overall, the results supports approximately 56% efficacy when administered by intraperitoneal injection. However, additional validation is necessary under large-scale laboratory trial for further application under field conditions.

Influence of Dietary Supplementation of Probiotic Pediococcus acidilactici MA18/5M During the Transition From Freshwater to Seawater on Intestinal Health and Microbiota of Atlantic Salmon (Salmo salar L.).

The aim of this study was to assess the effect of the transfer from freshwater to seawater on the distal intestinal bacterial communities of Atlantic salmon (Salmo salar L.) and to evaluate the effect of dietary inclusion of Pediococcus acidilactici MA18/5M (at 1.19 × 106 CFU/g). In this context, fish health and antiviral response were also investigated. A 12-week feeding trial was conducted in a flow-through rearing system involving 6 weeks in freshwater and 6 weeks in seawater. Fish received a control and probiotic diet. The composition of the salmon gut bacterial communities was determined by high-throughput sequencing of digesta and mucosa samples from both the freshwater and seawater stage. The main phyla detected during both freshwater and seawater stages were Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria. Significant differences were observed between the intestinal microbiota in the digesta and the mucosa. Both probiotic supplementation and the seawater transfer (SWT) had a substantial impact on the microbial communities, with most pronounced changes detected in the mucosal communities after SWT. This last finding together with a significantly higher antiviral response (mx-1 and tlr3 gene expression) in the distal intestine of fish fed the probiotic diet suggest a causal link between the microbiota modulation and activation of antiviral response. Feeding probiotics during the freshwater stage did not significantly increase survival after infectious pancreatic necrosis virus (IPNV) challenge after SWT, although higher survival was observed in one out of two replicate challenge tanks. In conclusion, this study demonstrated that both dietary probiotic supplementation and transfer from freshwater to seawater have an important role in modulating the bacterial communities in the distal intestine of Atlantic salmon. Furthermore, supplementation of the diet with P. acidilactici MA18/5M can modulate antiviral response.

Atlantic salmon (Salmo salar) mounts systemic and mucosal stress responses to peracetic acid.

Peracetic acid (PAA), a strong organic peroxide, is considered a relatively sustainable disinfectant in aquaculture because of its broad effectivity against many pathogens at low concentrations and because it degrades spontaneously to harmless residues. The impacts of PAA on fish health must be determined before its use as either a routine disinfectant or chemotherapeutant. Here we investigated the systemic and mucosal stress responses of Atlantic salmon (Salmo salar) to PAA. In experiment 1, salmon were exposed to different nominal concentrations (0, 0.6, and 2.4 ppm) of PAA for 5 min, followed by a re-exposure to the same concentrations for 30 min 2 weeks later. Sampling was performed before exposure to PAA and at 2 h, 48 h, and 2 w after exposures. In experiment 2, fish were subjected to crowding stress prior to PAA exposure at 4.8 ppm for 30 min. The fish were sampled before exposure and 1 h, 4 h, and 2 w after. The two trials were performed in a recirculation system. Both systemic (i.e., plasma cortisol, glucose, lactate, total antioxidant capacity) and mucosal (i.e., expression of antioxidant coding genes in the skin and gills) stress indicators were affected by the treatments at varying levels, and it was apparent that the fish were able to mount a robust response to the physiological demands of PAA exposure. The cortisol levels increased in the early hours after exposure and returned to basal level afterwards. Prior exposure history to PAA did not markedly affect the levels of plasma lactate and glucose when fish were re-exposed to PAA. Crowding stress before PAA treatment, however, did alter some of the stress indicators (i.e., lactate, glucose and expression of antioxidant genes in the gills), suggesting that stress history serves as both a confounding and compounding factor on how stress responses to PAA are mobilised. Nonetheless, the changes were not substantial. Gene expression profile analyses revealed that the antioxidant system was more responsive to PAA in the gills than in the skin. The increased antioxidant capacity in the plasma, particularly at 2.4 ppm and higher, indicates that antioxidants were produced to neutralise the internal redox imbalance resulting from PAA exposure. In conclusion, the results show that salmon were able to mount a robust adaptive response to different PAA doses and exposure times, and a combined exposure to stress and PAA. These results underscore the potential of PAA as a chemotherapeutant for salmon at PAA concentrations commonly applied to control parasitic infestations.

Differences in gene expression in Atlantic salmon parr and smolt after challenge with Piscine orthoreovirus (PRV).

Heart and skeletal muscle inflammation (HSMI) are a disease of farmed Atlantic salmon (Salmo salar) associated with Piscine orthoreovirus (PRV). The disease appears mainly during the marine production phase. This study examined if smoltification and transfer to seawater could compromise immune responses to PRV. Parr and smolts of the same origin were challenged by cohabitation with intraperitoneally injected salmon. Peak levels of PRV in spleen of cohabitants were reached after 8 weeks, but at a lower level in parr compared to smolts. Thereafter the virus levels declined, but remained significantly lower in parr than in smolts. Both groups developed typical HSMI histopathological heart lesions, which were most prominent after 10 weeks. Microarray and qPCR analyses revealed slightly lower expression of immune genes in spleen and head kidney of smolts before challenge. Infected parr showed earlier induction of genes involved in innate antiviral immunity, as well as for genes related to B and T cell responses. Gene expression profiles also indicated stimulation of heme and iron metabolism and erythropoiesis in smolts, which may indicate replacement of PRV-infected erythrocytes.

Comparison of transcriptomic responses to pancreas disease (PD) and heart and skeletal muscle inflammation (HSMI) in heart of Atlantic salmon (Salmo salar L).

Pancreas disease (PD) and heart and skeletal muscle inflammation (HSMI) are viral diseases associated with SAV (salmonid alphavirus) and PRV (piscine reovirus), which induce systemic infections and pathologies in cardiac and skeletal muscle tissue of farmed Atlantic salmon (Salmo salar L), resulting in severe morbidity and mortality. While general features of the clinical symptoms and pathogenesis of salmonid viral diseases are relatively well studied, much less is known about molecular mechanisms associated with immunity and disease-specific changes. In this study, transcriptomic analyses of heart tissue from PD and HSMI challenged Atlantic salmon were done, focusing on the mature phases of both diseases at respectively 28-35 and 42-77 days post infection. A large number of immune genes was activated in both trials with prevalence of genes associated with early innate antiviral responses, their expression levels being slightly higher in PD challenged fish. Activation of the IFN axis was in parallel with inflammatory changes that involved diverse humoral and cellular factors. Adaptive immune response genes were more pronounced in fish with HSMI, as suggested by increased expression of a large number of genes associated with differentiation and maturation of B lymphocytes and cytotoxic T cells. A similar down-regulation of non-immune genes such as myofiber and mitochondrial proteins between diseases was most likely reflecting myocardial pathology. A suite of genes important for cardiac function including B-type natriuretic peptide and four neuropeptides displayed differential expression between PD and HSMI. Comparison of results revealed common and distinct features and added to the understanding of both diseases at their mature phases with typical clinical pictures. A number of genes that showed disease-specific changes can be of interest for diagnostics.

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages.

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1ß and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.

CpG DNA induces protective antiviral immune responses in Atlantic salmon (Salmo salar L.).

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN show promise as vaccine adjuvants and immunoprotective agents in animal models. Here we report that pretreatment with CpG ODN in animals induces nonspecific protection against viral infection. A panel of different synthetic CpG ODN was tested for the in vitro effects in Atlantic salmon (Salmo salar L.) leukocytes. The ODN were tested for their capacity to stimulate proliferation of peripheral blood leukocytes and to induce production of interferon-like factors in head kidney leukocytes. These studies revealed that the sequence and number of the CpG motifs as well as the lengths of the ODN contribute to their stimulatory activity. ODN with the 6-mer CpG motif (5'-GTCGTT-3') showed the highest stimulatory activity and were shown to induce protection against infectious pancreatic necrosis virus when injected in Atlantic salmon. Expression of the Mx transcript, as an indicator of alpha/beta interferon induction, was induced in the CpG-injected fish. These results suggest that CpG DNA in fish induces early, nonspecific antiviral protection.