Search

It is well known that healthy gut microbiota is essential to promote host health and well-being. The intestinal microbiota of endothermic animals as well as fish are classified as autochthonous or indigenous, when they are able to colonize the host’s epithelial surface or are associated with the microvilli, or as allochthonous or transient (associated with digesta or are present in the lumen). Furthermore, the gut microbiota of aquatic animals is more fluidic than that of terrestrial vertebrates and is highly sensitive to dietary changes. In fish, it is demonstrated that [a] dietary form (live feeds or pelleted diets), [b] dietary lipid (lipid levels, lipid sources and polyunsaturated fatty acids), [c] protein sources (soybean meal, krill meal and other meal products), [d] functional glycomic ingredients (chitin and cellulose), [e] nutraceuticals (probiotics, prebiotics, synbiotics and immunostimulants), [f] antibiotics, [g] dietary iron and [h] chromic oxide affect the gut microbiota. Furthermore, some information is available on bacterial colonization of the gut enterocyte surface as a result of dietary manipulation which indicates that changes in indigenous microbial populations may have repercussion on secondary host–microbe interactions. The effect of dietary components on the gut microbiota is important to investigate, as the gastrointestinal tract has been suggested as one of the major routes of infection in fish. Possible interactions between dietary components and the protective microbiota colonizing the digestive tract are discussed.

This study builds upon previous work studying antimicrobial peptide (AMP) gene expression in rainbow trout ( Oncorhynchus mykiss ) fed a peptidoglycan (PG) enriched diet. The aims here were 1) to evaluate how long AMP expression is elevated in skin with continuous feeding of fish with the PG enriched diet for 21 or 28 days, and 2) to assess the impact of stopping PG feeding at day 14 when sampled at day 21 or 28. The rainbow trout were divided into 6 groups, with two fed a control commercial diet for the duration of the experiment and the other four given the same diet enriched with 10 mg PG/Kg for 14 days (PG 1–14) or continuously (PG continuous), the former reverting back to the commercial diet at day 14. No mortalities occurred during the study and there were no significant differences in growth among the fish in the different diet groups. The expression of six AMP genes was studied by real-time PCR in the skin, since these genes were shown to be induced in response to the PG enriched diets in a previous experiment. We show that continuous PG treatment for 21 or 28 days maintained high levels of AMP expression, although in general the levels decreased with time on the diets. Withdrawal of the PG diets at day 14 resulted in a fall in expression level especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated levels (x10) in comparison to fish given a control diet. These results confirm that orally administered PG clearly enhances the trout innate immune system and could be used as a means to boost fish defences. Future studies should be conducted to verify the impact on survival after pathogen challenge in trout fed PG enriched diets under these regimes.

Suppressor of cytokine signalling (SOCS) family members are crucial in the control and attenuation of cytokine induced responses via activation of the JAK/STAT, TLR and NF-kB signalling pathways. SOCS proteins orchestrate the termination of many types of immune responses and are often the targets of microbial pathogens exploiting SOCS mechanisms to evade the host's immune response. Through whole and lineage specific genome duplication events, the teleost cytokine/SOCS network is complex. Not only are the orthologues of all mammalian SOCS members present, namely cytokine inducible Src homology 2 (SH2)-containing protein (CISH) and SOCS-1 to -7, but multiple gene copies exist that may potentially become functionally divergent. In this paper we focus on the CISH genes in rainbow trout (Oncorhynchus mykiss), and have cloned two further paralogues, CISHa2 and CISHb2, additional to the known CISHa1 and CISHb1 genes. We present for the first time a comparative expression analysis of these four paralogues, to establish whether subfunctionalisation is apparent. In vivo examination of gene expression revealed a higher constitutive expression level of CISHa paralogues compared to CISHb expression in adult trout tissues. All CISHs were relatively highly abundant in immune tissues but CISHa2 and CISHb2 had highest expression in the heart and muscle. An inverse picture of CISH abundance during trout ontogeny was seen, and further hints at differential roles of the four genes in immune regulation and development. Stimulation of head kidney (HK) leukocytes with trout recombinant interleukin (rIL)-15 and rIL-21 had a major effect on CISHa2 and to a lesser extent CISHa1 expression. In HK macrophages rIL-1β, phytohemagglutinin, and phorbol 12-myristate 13-acetate also had a strong impact on CISHa2 expression. Yersinia ruckeri infection caused a temporally and spatially differential onset of CISH expression that may be viewed in the context of pathogen evasion strategies. These data, against the backdrop of fish specific whole genome duplication events and functional divergence, provide the first evidence for differential roles of the four trout CISH genes in immune control and development.

Heart and Skeletal Muscle Inflammation (HSMI) is an emerging viral disease caused by a novel Atlantic salmon reovirus (ASRV) affecting farmed fish. Primary symptoms associated with HSMI include myocardial and skeletal muscle necrosis indicating a severe inflammatory process. Recently, we applied the concept of clinical nutrition to moderate the long-term inflammatory process associated with HSMI in salmon subjected to experimental ASRV challenge. The use of functional feeds with lower lipid (hence energy) content reduced the inflammatory response to ASRV infection and the severity of associated heart lesions. The aim of the present study was to elucidate possible mechanisms underpinning the observed effects of the functional feeds, focussing on eicosanoid and fatty acid metabolism in liver and head kidney. Here we show that liver was also a site for histopathological lesions in HSMI showing steatosis reflecting impaired lipid metabolism. This study is also the first to evaluate the expression of a suite of key genes involved in pathways relating diet and membrane phospholipid fatty acid compositions, and the inflammatory response after ASRV infection. The expression of hepatic Δ6 and Δ5 desaturases was higher in fish fed the functional feeds, potentially increasing their capacity for endogenous production and availability of anti-inflammatory EPA. Effects on mobilization of lipids and changes in the LC-PUFA composition of membrane phospholipids, along with significant changes in the expression of the genes related to eicosanoid pathways, showed the important role of the head kidney in inflammatory diseases caused by viral infections. The results from the present study suggest that clinical nutrition through functional feeding could be an effective complementary therapy for emerging salmon viral diseases associated with long-term inflammation.

The aim of this study was to investigate the effect of feeding rainbow trout (Oncorhynchus mykiss) peptidoglycan (PG) enriched diets on antimicrobial peptide (AMP) gene expression. Fish were divided into 5 groups and fed diets containing 0, 5, 10, 50 and 100 mg PG/Kg, and sampled 1, 7 and 14 days later. The expression of eight AMP genes (four defensins, two cathelicidins and two liver expressed AMPs) was determined in skin, gill, gut and liver, tissues important for first lines of defence or production of acute phase proteins. Up-regulation of many AMPs was found after feeding the PG enriched diets, with sequential expression seen over the time course studied, where defensins were typically expressed early and cathelicidins and LEAPs later on. A number of clear differences in AMP responsiveness between the tissues examined were also apparent. Of the four PG concentrations used, 5 mg PG/Kg did not always elicit AMP gene induction or to the same degree as seen with the other diets. The three higher dose groups generally showed similar trends although differences in fold change were more pronounced in the 50 and 100 mg PG/Kg groups. Curiously several AMPs were down-regulated after 14 days of feeding in gills, gut and liver. Nevertheless, overall the PG enriched diets had a positive effect on AMP expression. Further investigations now need to be undertaken to confirm whether this higher AMP gene expression correlates with protection against common bacterial diseases and if PG enriched diets have value as a means to temporarily boost the piscine immune system.

Transforming growth factor-β (TGF-β) binding to the TGF-β type I (TGFBR1) and type II (TGFBR2) receptors delivers a plethora of cell-type specific effects. Moreover, the responses to TGF-β are tuned by regulatory mechanisms at the receptor level itself. To further elucidate TGF-β family signal transduction in teleosts, we therefore cloned the first complete set of a putative TGF-β receptor complex in salmonids. Rainbow trout TGFBR1 and TGFBR2 are transmembrane proteins with a serine/threonine kinase domain and are highly conserved within vertebrates. High expression levels in muscle and brain indicate regulation of the TGF-β system in muscular and nervous systems. Lipopolysaccharide (LPS) induced expression of both receptor chains in RTgill cells while bacterial and viral mimics modulated the two receptors inversely in head kidney (HK) macrophages. In addition, T cell mitogens lowered receptor levels in HK leukocytes. These data provide the first insights into TGF-β type I and II receptor modulation during immune responses in teleost fish.

An initial bioinformatics investigation followed by cloning and sequencing analysis, has led to the identification of three novel members (omDB-2, omDB-3, omBD-4) of the β-defensin family in rainbow trout ( Oncorhynchus mykiss ). The contiguous sequences could be translated to give predicted peptides of 62 (omDB-2), 63 (omDB-3) and 68 (omDB-4) amino acids (aa) in length, with mature peptides of 43 (omDB-2), 39 (omDB-3) and 42 (omDB-4) aa, with no obvious proregion present. Analysis of the gene organization found that all three new genes contained three exons divided by two introns, as seen in defensin genes of other fish species. Constitutive expression of all the trout defensins was detected by RT-PCR in a wide range of mucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout β-defensin genes were induced in head kidney primary leucocyte cultures at 4 h post-stimulation, with omDB-1 and omDB-3 particularly highly expressed. These data suggest that β-defensins are likely an important component of the innate defences of fish, and reveal an added level of antimicrobial peptide complexity in fish to that known previously.

The present study addressed the adherence of Carnobacterium divergens and Aeromonas salmonicida subsp. salmonicida to the intestinal lining of Atlantic salmon (Salmo salar L.) using an ex vivo method–intestinal sacfollowing feeding with pea protein concentrate, extracted sunflower or feather meal at 200 g/kg inclusion level. Control diet was a 450 g/kg fishmeal diet. The experimental feeds were fed to two groups each for seven weeks at EWOS Innovations’ facilities in Lonningdal, Norway. Ex vivo intestinal challenge was carried out at Institute for Marine Research, Bergen. Excised intestines of salmon from all feeding groups were exposed to a probiotic, C. divergens or a pathogen, A. salmonicida either alone or in combination and control samples were exposed to sterile saline solution. Exposure to A. salmonicida caused severe damage to the intestinal ultrastructure of the mid intestine, but after exposure to C. divergens, sterile saline solution or any of the combination treatments, morphology remained mostly unaltered indicating an alleviating effect of the probiotic. Feather meal intensified the damaging effect of exposure to A. salmonicida and there were otherwise no effect of diet on the morphology. qPCR analysis of adhered C. divergens and A. salmonicida showed that although the pathogen has a higher adherence efficiency, C. divergens was more efficient at displacing the pathogen if allowed to adhere to the mucosal lining first indicating that the probiotic should be present prior to the pathogen for optimal effect. There were no dietary effects on bacterial adherence. The present study shows that use of some commercially available alternative feed ingredients may not affect the probiotic abilities of C. divergens or make the fish more susceptible to disease through intestinal invasion.

Background Cardiomyopathy syndrome (CMS) is a severe cardiac disease of Atlantic salmon (Salmo salar) recently associated with a double-stranded RNA virus, Piscine Myocarditis Virus (PMCV). The disease has been diagnosed in 75-85 farms in Norway each year over the last decade resulting in annual economic losses estimated at up to €9 million. Recently, we demonstrated that functional feeds led to a milder inflammatory response and reduced severity of heart lesions in salmon experimentally infected with Atlantic salmon reovirus, the causal agent of heart and skeletal muscle inflammation (HSMI). In the present study we employed a similar strategy to investigate the effects of functional feeds, with reduced lipid content and increased eicosapentaenoic acid levels, in controlling CMS in salmon after experimental infection with PMCV. Results Hepatic steatosis associated with CMS was significantly reduced over the time course of the infection in fish fed the functional feeds. Significant differences in immune and inflammatory responses and pathology in heart tissue were found in fish fed the different dietary treatments over the course of the infection. Specifically, fish fed the functional feeds showed a milder and delayed inflammatory response and, consequently, less severity of heart lesions at earlier and later stages after infection with PMCV. Decreasing levels of phosphatidylinositol in cell membranes combined with the increased expression of genes related with T-cell signalling pathways revealed new interactions between dietary lipid composition and the immune response in fish during viral infection. Dietary histidine supplementation did not significantly affect immune responses or levels of heart lesions. Conclusions Combined with the previous findings on HSMI, the results of the present study highlight the potential role of clinical nutrition in controlling inflammatory diseases in Atlantic salmon. In particular, dietary lipid content and fatty acid composition may have important immune-modulatory effects in Atlantic salmon that could be potentially beneficial in fish balancing the immune and tissue responses to viral infections. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-462) contains supplementary material, which is available to authorized users.

The present study investigated the effect of various alternative diet ingredients partially replacing fishmeal (FM) on digestive and metabolic parameters in Atlantic salmon (Salmo salar L.) post-smolts (initial body mass 305 ± 69 g) following 12 weeks of feeding. Experimental diets containing 20 % extracted sunflower (ESF), pea protein concentrate (PPC), soy protein concentrate (SPC), feather meal (FeM) and poultry by-product (PBY) were compared to a reference diet containing FM as the main protein source. For the different intestinal compartments trypsin, lipase, bile salts, dry matter and chyme-associated leucine aminopeptidase (LAP) were measured from the content and LAP was measured in the tissue. Selected metabolites were measured in plasma samples. In general, use of plant proteins resulted in low C-LAP activity, low plasma cholesterol and high plasma magnesium. The plasma levels of cholesterol and Mg reflecting were most likely reflections of the composition of the diet, while the LAP activity in chyme may indicate lower epithelial cell turnover. Other responses varied depending on the plant protein source. Results from the animal protein substitution also varied both between diets and compartments; however, both materials increased lipase activity in DI. FeM resulted in a significant increase in both total and specific LAP activities suggesting an attempt to increase the digestive capacity in response to low digestibility of the diet while PBY showed very little difference from the FM-fed control fish. The present trial indicates that 20 % PPC, SPC and PBY can partially replace FM in diets for Atlantic salmon. The qualities of ESF and FeM used in this trial show little promise as FM replacement at 20 % inclusion level.

The relationship between lipid and digestible energy content of the feed and growth performance has been exploited with great effect in Atlantic salmon (Salmo salar). The precise metabolic consequences of so-called "high-energy" feeds have not been fully defined, but increased and altered tissue lipid deposition patterns impacting on carcass and product quality have been reported. Recent studies on global gene expression have shown that dietary lipid and digestible energy content can have significant effects on gene expression in salmonids. In addition, we recently showed that functional feeds with reduced digestible energy significantly improved outcomes in response to inflammatory disease in salmon. The present study aimed to elucidate and clarify the effects of dietary digestible energy content (22, 20 and 18 MJ/kg; HE, ME and LE diets, respectively) on lipid and fatty acid metabolism in salmon fed diets containing graded amounts of lipid. Specifically the effects on liver lipid and fatty acid compositions, and on the hepatic expression of genes of lipid and fatty acid metabolism were determined. Final weight and weight gain were significantly higher, and FCR lower, in fish fed the HE diet. Crude lipid content was significantly lower in fish fed the LE diet compared to fish fed the two higher energy contents. Significantly lower total lipid and triacylglycerol levels were recorded in liver of fish fed the LE diet compared to fish fed the higher energy diets. Liver lipids in salmon fed the LE diet had generally significantly higher proportions of saturated fatty acids and long-chain polyunsaturated fatty acids (LC-PUFA), and lower monounsaturated fatty acids, C18 and n - 6 PUFA. Consistent with this, salmon fed the LE diet showed increased liver expression of both Δ6 and Δ5 fatty acyl desaturases in comparison to fish fed the diets with higher energy levels. Fatty acid synthase expression showed a clear upward trend as dietary energy decreased, and sterol regulatory element binding protein 2 and liver X receptor showed reciprocal trends that were consistent with the level of dietary cholesterol that reflects digestible energy content. Although not statistically significant, these trends were biologically logical, significant and relevant. Expression of genes of fatty acid oxidation was less consistent. Overall, reduced dietary digestible energy/lipid content alone, without major changes in dietary fatty acid composition, altered the expression of key genes of lipid and fatty acid metabolism resulting in general up-regulation of biosynthetic pathways.

13 pages, 9 figures, 2 tables, The rainbow trout (Oncorhynchus mykiss) TGF-β1 sequence was one of the first fish cytokines described. Studies of its expression suggest it is constitutively expressed but displays refractory inducibility. Here we describe a second TGF-β1 (TGF-β1b) gene that is novel in several respects. TGF-β1b possesses typical TGF-β features, including a CXC motif and an integrin binding site, a tetrabasic cut site and a mature peptide of 112 amino acids (aa) containing nine conserved cysteine residues. The mature peptide is 83% identical to the first TGF-β1 sequence described in rainbow trout, that we designate TGF-β1a, and relative to TGF-β1a shows higher homology to Atlantic salmon TGF-β1b, zebrafish TGF-β1a, and sea bass and seabream TGF-β1. The gene organisation of salmonid TGF-β1b genes, as inferred from Atlantic salmon whole genome shotgun contigs, is a 6 exon/5 intron structure with exons 3 and 4 of salmonid TGF-β1a genes apparently fused together. The two trout TGF-β1 genes have a wide distribution in vivo, with highest expression found in immune tissues for both isoforms indicating that TGF-β1 has a predominant role in immunity of fish. Expression of both genes was also seen during the ontogeny of trout, with TGF-β1a relatively constant in expression level but TGF-β1b increasing over time. Immune responses in head kidney (HK) macrophages induced by pathogen associated molecular patterns (PAMPs), pro-inflammatory cytokines, mitogens and pathway activators highly elevated the expression level of TGF-β1b but not that of TGF-β1a. TGF-β1b expression was also increased by polyinosinic:polycytidylic acid (poly(I:C)) and/or lipopolysaccharide (LPS) stimulation in three different trout cell lines studied. Finally we show that TGF-β1b is potentially involved in defense against infection with viral haemorrhagic septicemia virus (VHSV), which had no effect on TGF-β1a expression. Thus, it is likely the TGF-β1b gene represents a copy which fulfils the major immune orchestrating functions of TGF-β1 as seen in other vertebrates., TW received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland).

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.

The intracellular suppressors of cytokine signaling (SOCS) family members, including CISH and SOCS1 to 7 in mammals, are important regulators of cytokine signaling pathways. So far, the orthologues of all the eight mammalian SOCS members have been identified in fish, with several of them having multiple copies. Whilst fish CISH, SOCS3, and SOCS5 paralogues are possibly the result of the fish-specific whole genome duplication event, gene duplication or lineage-specific genome duplication may also contribute to some paralogues, as with the three trout SOCS2s and three zebrafish SOCS5s. Fish SOCS genes are broadly expressed and also show species-specific expression patterns. They can be upregulated by cytokines, such as IFN-γ, TNF-α, IL-1β, IL-6, and IL-21, by immune stimulants such as LPS, poly I:C, and PMA, as well as by viral, bacterial, and parasitic infections in member- and species-dependent manners. Initial functional studies demonstrate conserved mechanisms of fish SOCS action via JAK/STAT pathways.

Currently there are numerous gaps in existing knowledge about exogenous nucleotide application to fish including various aspects of digestion, absorption, metabolism, and influences on various physiological responses, especially expression of immunogenes and modulation of immunoglobulin production. Additional information is also needed in regard to age/size-related responses and appropriate doses and timing of administration. Thus further research in these areas should be pursued. Immunostimulants Abstract It is well established that proper nutrition is essential for maintenance of normal growth and health of all animals including various aquatic species. During the last two decades increased attention has focus on immunostimulants and nucleotides to reduce susceptibility to various stressors and diseases, as well as enhance the overall health of fish. The immune response can be modulated by β-glucans and high-M-alginate. β-glucans are glucose polymers that are major structural components of the cell wall of yeast, fungi, and bacteria, but also of cereals like oat and barley. There is much structural variation in the β-glucans from these different sources, which may influence their physiological functions. Alginate is a polysaccharide composed of β-1,4-D-mannuronic acid (M) and α-L-glucuronic acid (G). In vitro as well as in vivo studies in fish show that especially β-glucans derived from fungi and yeast and alginate have immune modulating properties. Most frequently evaluated are effects on macrophage activation and on lysozyme, respiratory burst and leukocyte activity, which have been suggested to contribute to the increased resistance against infections, after immunostimulant exposure. Although more fish studies are needed, it is tempting to suggest that dietary β-glucans and alginate may be useful tools to prime the host immune system and increase resistance against invading pathogens. As no knowledge is available regarding short versus long-term effects and efficiency, more knowledge is needed on this topic.

In the present study the impact on gut microbiology and indigenous gut histology of Atlantic salmon ( Salmo salar L.) was investigated following feeding of a control and a prebiotic (EWOS prebiosal ® ) diet and ex vivo exposure to saline or the probiotic bacterium Carnobacterium divergen s. The results showed that ex vivo exposure of C. divergens at 10 8 CFU ml -1 did not cause cell damage to the intestine tract of Atlantic salmon. Furthermore, prior provision of dietary prebiotic elevated the ability of C. divergens to adhere to the epithelium or mucus layer in the proximal intestine, where culturable heterotrophic bacterial levels (which were identified as C. divergens ) were elevated by 234% compared to the control. This effect was not apparent in the distal intestine. The ability of isolated carnobacteria from the ex vivo experiment to inhibit growth of two fish pathogenic bacteria ( Yersinia ruckeri and Aeromonas salmonicida ssp. salmonicida ) was assessed. Extracellular products from all 11 of the isolated carnobacteria strains, plus the type strain Carnobacterium inhibens CCUG 31728, inhibited the in vitro growth of Y. ruckeri . However, only extracellular products from C. divergens isolate 57 inhibited the growth of A. salmonicida ssp. salmonicida .