The present study aimed to investigate whether alternative dietary protein sources modulate the microbial communities in the distal intestine (DI) of Atlantic salmon, and whether alterations in microbiota profiles are reflected in modifications in host intestinal function and health status. A 48-day feeding trial was conducted, in which groups of fish received one of five diets: a reference diet in which fishmeal (diet FM) was the only protein source and four experimental diets with commercially relevant compositions containing alternative ingredients as partial replacements of fishmeal, i.e., poultry meal (diet PM), a mix of soybean meal and wheat gluten (diet SBMWG), a mix of soy protein concentrate and poultry meal (diet SPCPM), and guar meal and wheat gluten (diet GMWG). Samples were taken of DI digesta and mucosa for microbial profiling using high-throughput sequencing and from DI whole tissue for immunohistochemistry and expression profiling of marker genes for gut health. Regardless of diet, there were significant differences between the microbial populations in the digesta and the mucosa in the salmon DI. Microbial richness was higher in the digesta than the mucosa. The digesta-associated bacterial communities were more affected by the diet than the mucosa-associated microbiota. Interestingly, both legume-based diets (SBMWG and GMWG) presented high relative abundance of lactic acid bacteria in addition to alteration in the expression of a salmon gene related to cell proliferation (pcna). It was, however, not possible to ascertain the cause-effect relationship between changes in bacterial communities and the host's intestinal responses to the diets.IMPORTANCE The intestine of cultivated Atlantic salmon shows symptoms of compromised function, which are most likely caused by imbalances related to the use of new feed ingredients. Intestinal microbiota profiling may become in the future a valuable endpoint measurement in order to assess fish intestinal health status and effects of diet. The present study aimed to gain information about whether alternative dietary protein sources modulate the microbial communities in the Atlantic salmon intestine and whether alterations in microbiota profiles are reflected in alterations in host intestinal function and health status. We demonstrate here that there are substantial differences between the intestinal digesta and mucosa in the presence and abundance of bacteria. The digesta-associated microbiota showed clear dependence on the diet composition, whereas mucosa-associated microbiota appeared to be less affected by diet composition. Most important, the study identified bacterial groups associated with diet-induced gut dysfunction that may be utilized as microbial markers of gut health status in fish.

The efficacy of cellular components of probiotics Kocuria SM1 and Rhodococcus SM2 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against vibriosis was assessed. Groups of fish (average weight = 10–15 g) were immunized intraperitoneally (i.p.) with 0.1 ml of subcellular materials, i.e. 0.2 ± 0.05 mg protein per fish, comprising extracellular proteins (ECPs), cell wall proteins (CWPs) and whole cell proteins (WCPs) of SM1 and SM2, respectively, or with 0.1 ml of phosphate-buffered saline (PBS) to serve as the control. Seven days after administration, fish from each group were challenged i.p. with 0.1 ml of a suspension in PBS of 3 × 105 cells ml−1 per fish of Vibrio anguillarum. Use of CWPs and WCPs demonstrated significantly (P < 0.05) better protection against V. anguillarum insofar as mortalities were reduced to 11–17% [relative percent survival (RPS) = 80–87%], although ECPs fared less well (mortalities = 33–38%; RPS = 56–62%; P > 0.05), compared to 86% mortalities of the controls. The mode of action reflected activation of innate immune factors by CWPs and WCPs, demonstrating significantly (P < 0.05) increased expression of respiratory burst (optical density; OD550nm) from 0.039 to 0.043–0.045, peroxidase (OD550nm) from 0.26 to 0.37–0.55, and bacterial killing activities (i.e. percentage of surviving bacteria reduced from 79% to 56–57% for SM2). Moreover, an elevation of leucocyte number (from 1.93% to 1.98–2.93%; P > 0.05) and immunoglubolin level (from 27 mg ml−1 to 28.5–33 mg ml−1; P > 0.05) were observed with the experimental groups. These results indicate that cell components of the probiotics stimulate an immune response

In Atlantic salmon (Salmo salar L.), and also in other fish species, certain plant protein ingredients can increase fecal water content creating a diarrhea-like condition which may impair gut function and reduce fish growth. The present study aimed to strengthen understanding of the underlying mechanisms by observing effects of various alternative plant protein sources when replacing fish meal on expression of genes encoding proteins playing key roles in regulation of water transport across the mucosa of the distal intestine (DI). A 48-day feeding trial was conducted with five diets: A reference diet (FM) in which fish meal (72%) was the only protein source; Diet SBMWG with a mix of soybean meal (30%) and wheat gluten (22%); Diet SPCPM with a mix of soy protein concentrate (30%) and poultry meal (6%); Diet GMWG with guar meal (30%) and wheat gluten (14.5%); Diet PM with 58% poultry meal. Compared to fish fed the FM reference diet, fish fed the soybean meal containing diet (SBMWG) showed signs of enteritis in the DI, increased fecal water content of DI chyme and higher plasma osmolality. Altered DI expression of a battery of genes encoding aquaporins, ion transporters, tight junction and adherens junction proteins suggested reduced transcellular transport of water as well as a tightening of the junction barrier in fish fed the SBMWG diet, which may explain the observed higher fecal water content and plasma osmolality. DI structure was not altered for fish fed the other experimental diets but alterations in target gene expression and fecal water content were observed, indicating that alterations in water transport components may take place without clear effects on intestinal structure.

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.

Salmonid alphavirus is the aetological agent of pancreas disease (PD) in marine Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, with most outbreaks in Norway caused by SAV subtype 3 (SAV3). This atypical alphavirus is transmitted horizontally causing a significant economic impact on the aquaculture industry. This histopathological and proteomic study, using an established cohabitational experimental model, investigated the correlation between tissue damage during PD and a number of serum proteins associated with these pathologies in Atlantic salmon. The proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting. A number of humoral components of immunity which may act as biomarkers of the disease were also identified. For example, creatine kinase, enolase and malate dehydrogenase serum concentrations were shown to correlate with pathology during PD. In contrast, hemopexin, transferrin, and apolipoprotein, amongst others, altered during later stages of the disease and did not correlate with tissue pathologies. This approach has given new insight into not only PD but also fish disease as a whole, by characterisation of the protein response to infection, through pathological processes to tissue recovery.Biological significanceSalmonid alphavirus causes pancreas disease (PD) in Atlantic salmon, Salmo salar, and has a major economic impact on the aquaculture industry. A proteomic investigation of the change to the serum proteome during PD has been made with an established experimental model of the disease. Serum proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting with 72 protein spots being shown to alter significantly over the 12 week period of the infection. The concentrations of certain proteins in serum such as creatine kinase, enolase and malate dehydrogenase were shown to correlate with tissue pathology while other proteins such as hemopexin, transferrin, and apolipoprotein, altered in concentration during later stages of the disease and did not correlate with tissue pathologies. The protein response to infection may be used to monitor disease progression and enhance understanding of the pathology of PD.

The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces 24 the availability of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) to marine fish 25 such as gilthead seabream. The aim of this study was to examine compositional and 26 physiological responses to a dietary gradient of n-3 LC-PUFA. Six isoenergetic and 27 isonitrogenous diets (D1-D6) were fed to seabream, with the added oil being a blend of 28 FO and VO to achieve a dietary gradient of n-3 LC-PUFA. Fish were sampled after four 29 months feeding, to determine biochemical composition, tissue fatty acid concentrations 30 and lipid metabolic gene expression. The results indicated a disturbance to lipid 31 metabolism, with fat in the liver increased and fat deposits in the viscera reduced. 32Tissue fatty acid profiles were altered towards the fatty acid compositions of the diets. 33There was evidence of endogenous modification of dietary PUFA in the liver which 34 correlated with the expression of fatty acid desaturase 2 (fads2). Expression of sterol 35 regulatory element-binding protein 1 (srebp1), fads2 and fatty acid synthase increased 36 in the liver, while peroxisome proliferator-activated receptor alpha 1 pathways appeared 37 to be supressed by dietary VO in a concentration-dependent manner. The effects in 38 lipogenic genes appear to become measurable in D1-D3, which agrees with the weight 39 gain data suggesting that disturbances to energy metabolism and lipogenesis may be 40 related to performance differences. These findings suggested that suppression of beta-41 oxidation and stimulation of srebp1-mediated lipogenesis may play a role in 42 contributing toward steatosis in fish fed n-3 LC-PUFA deficient diets. 43 44 3 Introduction 45Sustainable expansion of aquaculture requires reduction in the use of fishmeal (FM) and 46 fish oil (FO) in aquafeed formulations (1)(2)(3)(4) . Both raw materials, particularly FO, are rich 47 in the two key n-3 (or omega-3) long-chain (≥ C 20 ) polyunsaturated fatty acids (LC-48 PUFA), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 3), recognised as essential fatty acids (EFA) for the majority of marine fish species (5) . 50 DHA is an essential component of neural and retinal membranes (6) and both EPA and 51 DHA are precursors for an extensive range of autocrine signalling molecules (e.g. 52 eicosanoids, resolvins, protectins, etc.) (7) . Dietary deficiency of n-3 LC-PUFA has 53 impacts on the health (8) , metabolism (9, 10) , composition (11, 12) and growth (13) of marine 54 fish. 55Typically, an aquafeed for a given marine fish species contains a combination of 56 FO to supply essential n-3 LC-PUFA and vegetable oils (VO) that, while devoid of LC-57 PUFA, supply dietary energy (14, 15) . Marine fish lack sufficient activity of the LC-PUFA 58 biosynthesis pathway to satisfy requirements (5) . In terms of fatty acid composition, the 59 key effects of high inclusion levels of VO are an increase in C 18 unsaturated fatty acids 60 (α-linolenic acid, linoleic...

Aims:  The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. Methods and Results:  The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole‐cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron‐regulated outer membrane proteins (IROMP) of c. 90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. Conclusions:  The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. Significance and Impact of the Study:  There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.

The potential benefits of a commercial preparation of heptanoate (NOREL, HEPTON®) were evaluated in an 11‐week gilthead sea bream feeding trial (May–August), using a factorial design with four isoproteic and isoenergetic diets. Fish meal (FM) was added at 200 g/kg in D1–D2 diets and at 50 g/kg in D3–D4 diets, which also contained fish peptones and plant proteins as source of proteins. Heptanoate was added at 3 g/kg in D2 and D4 diets. All fish grew from 13–14 g to 81–84 g with an overall feed efficiency (FE) of 0.91–0.94. An early impairment of FE (weeks 1–4) was found with the standard FM‐based diet (D1), but this detrimental condition was reversed by heptanoate, increasing FE from 0.88 in D1 fish to 0.99 in D2 fish. Further improvements were progressively diluted over time, remaining D2 and D3–D4 fed fish almost undistinguishable through all the trial. Heptanoate supplementation produced higher hepatic glycogen depots, but no signs of histopathological damage were found in liver or intestine. Other lasting heptanoate effects included changes in plasma antioxidant capacity, plasma cortisol and growth hormone levels, and measures of respirometry in swimming performance tests. Altogether, it supports the potential use of heptanoate to speed up adaptive and healthy metabolic states of farmed fish to cope with challenging culture conditions.