Rainbow trout (Oncorhynchus mykiss) urea cycle and polyamine synthesis gene families show dynamic... more Rainbow trout (Oncorhynchus mykiss) urea cycle and polyamine synthesis gene families show dynamic expression responses to inflammation, Fish and Shellfish Immunology (2019), doi:
Supplementing the diet with functional ingredients is a key strategy to improve fish performance ... more Supplementing the diet with functional ingredients is a key strategy to improve fish performance and health in aquaculture. The amino acids of the urea and nitric oxide (NO) cycles-arginine, ornithine and citrulline-perform crucial roles in the immune response through the generation of NO and the synthesis of polyamine used for tissue repair. We previously found that citrulline supplementation improves and maintains circulating free arginine levels in rainbow trout more effectively than arginine supplementation. Here, to test whether supplementation of urea cycle amino acids modulates the immune response in rainbow trout (Oncorhynchus mykiss), we supplemented a commercial diet with high levels (2% of total diet) of either arginine, ornithine or citrulline during a 7-week feeding trial, before challenging fish with the bacterium Aeromonas salmonicida. We carried out two separate experiments to investigate fish survival and 24h post-infection to investigate the immediate response of free amino acid levels, and transcriptional changes in genes encoding urea cycle, NO cycle and polyamine synthesis enzymes. There were no differences in percentage fish mortality between diets, however there were numerous highly significant changes in free amino acid levels and gene expression to both dietary supplementation and infection. Out of 26 amino acids detected in blood plasma, 8 were significantly changed by infection and 9 by dietary supplementation of either arginine, ornithine or citrulline. Taurine, glycine and aspartic acid displayed the largest decreases in circulating levels in infected fish, while ornithine and isoleucine were the only amino acids that increased in concentration. We investigated transcriptional responses of the enzymes involved in arginine metabolism in liver and head kidney; transcripts for polyamine synthesis enzymes showed highly significant increases in both tissues across all diets following infection. The paralogous arginase-encoding genes, Arg1a, Arg1b, Arg2a and Arg2b, displayed complex responses across tissues and also due to diet and infection. Overall, these findings improve our understanding of amino acid metabolism following infection and suggests new potential amino acid targets for improving the immune response in salmonids.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2020
Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, gro... more Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein deposition and the immune response. In mammals, supplementation of arginine's precursor amino acid, citrulline, is known to increase circulating arginine to levels beyond direct arginine supplementation, however, citrulline supplementation is poorly studied in fish. To address this knowledge gap, we supplemented the diet of rainbow trout with arginine and its precursor amino acids, ornithine and citrulline, at 3 levels (0.5%, 1% and 2% of the total diet) during a 14-week experiment. We sampled fish at 3h and 24h post-feeding to investigate immediate and steady-state effects, respectively. There were no differences in fish growth for any of the diets across a range of indicators. In blood plasma, out of 26 amino acids detected, 11 and 6 displayed significant changes 24h and 3h post-prandial, respectively. Arginine, ornithine and citrulline levels were all significantly increased by the citrulline supplemented diets. In muscle, 8 amino acids were significantly altered by supplemented diets, while there were no significant changes in liver. Arginine was increased by 2% citrulline supplementation in muscle tissue. We also investigated the transcriptional responses of urea cycle, nitric oxide cycle and rate-limiting polyamine synthesis enzymes, related to arginine's metabolism, in liver. At both time points, only 2 enzymes were significantly altered by the supplemented diets, however several significant changes were observed comparing 3h and 24h post-prandial expression levels. Of these, the paralogous polyamine synthesis enzyme encoding genes ODC1 and ODC2 displayed the largest increases in 3h post-prandial fish. These findings demonstrate that endogenous synthesis of arginine is possible from a citrulline supplemented diet and improve our understanding of arginine metabolism in fish.
Diseases which cause skeletal muscle myopathy are some of the most economically damaging diseases... more Diseases which cause skeletal muscle myopathy are some of the most economically damaging diseases in Atlantic salmon, Salmo salar L., aquaculture. Despite this, there are limited means of assessing fish health non-destructively. Previous investigation of the serum proteome of Atlantic salmon, Salmo salar L., during pancreas disease (PD) has identified proteins in serum that have potential as biomarkers of the disease. Amongst these proteins, the enzyme enolase was selected as the most viable for use as a biomarker of muscle myopathy associated with PD. Western blot and immunoassay (ELISA) validated enolase as a biomarker for PD, whilst immunohistochemistry identified white muscle as the source of enolase. Enolase was shown to be a specific marker for white muscle myopathy in salmon, rising in serum concentration significantly correlating with pathological damage to the tissue.
The efficacy of cellular components of probiotics Kocuria SM1 and Rhodococcus SM2 to protect rain... more 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 × 10 5 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; OD 550nm) from 0.039 to 0.043-0.045, peroxidase (OD 550nm) 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.
Salmonid alphavirus is the aetological agent of pancreas disease (PD) in marine Atlantic salmon, ... more 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 significance Salmonid 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
Whether through direct supplementation of bacteria or by prebiotic supplementation thought to fav... more Whether through direct supplementation of bacteria or by prebiotic supplementation thought to favour subsets of bacteria, modulation of gut microbiota constitutes an important and promising alternative to the use of prophylactic and growth promoting antibiotics in worldwide aquaculture. We fed a commercial base feed, alone or supplemented with either proprietary β-glucan, β-glucan and organic acids, citrus flavonoid or yeast cell wall supplements, to rainbow trout over a period of four weeks. Fish from each feed group were then subjected to experimental, waterborne infection withYersinia ruckeriO1 biotype 2. Following experimental feeding, the β-glucan and organic acids supplemented group showed significantly improved feed conversion and lipid efficiency ratios. Furthermore, the β-glucan, β-glucan and organic acids and citrus flavonoid supplements proved to significantly reduce the risk of mortality in rainbow trout during experimental infection as shown by hazard ratio analysis. Re...
The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces the availab... more The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces the availability of n-3 long-chain PUFA (LC-PUFA) to marine fish such as gilthead seabream. The aim of this study was to examine compositional and physiological responses to a dietary gradient of n-3 LC-PUFA. Six iso-energetic and iso-nitrogenous diets (D1-D6) were fed to seabream, with the added oil being a blend of FO and VO to achieve a dietary gradient of n-3 LC-PUFA. Fish were sampled after 4 months feeding, to determine biochemical composition, tissue fatty acid concentrations and lipid metabolic gene expression. The results indicated a disturbance to lipid metabolism, with fat in the liver increased and fat deposits in the viscera reduced. Tissue fatty acid profiles were altered towards the fatty acid compositions of the diets. There was evidence of endogenous modification of dietary PUFA in the liver which correlated with the expression of fatty acid desaturase 2 (fads2). Expression of ster...
The present study aimed to investigate whether alternative dietary protein sources modulate the m... more 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. Reg...
In Atlantic salmon (Salmo salar L.), and also in other fish species, certain plant protein ingred... more 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.
Recently outbreaks of enteric redmouth disease in previously vaccinated salmonids was attributed ... more Recently outbreaks of enteric redmouth disease in previously vaccinated salmonids was attributed to biotype 2 (non-motile, lipase negative) isolates of Yersinia ruckeri. Currently, isolates of biotype 2 are the most widely encountered form in Europe and North America. Biochemical analysis of over 90 clinical isolates revealed that there are potentially eight novel phenotypes associated with disease. Cell surface characteristics of biotype 2 isolates displayed a unique O antigen structure. A multilocus sequence typing scheme was developed using 4 "housekeeping" gene loci in order to understand the phylogeny and population structure of Y. ruckeri. The scheme highlighted that although there is low species diversity, this is characteristic of a small population size which is not limited to fish species or geographical region. The study suggests that biotype 2 isolates have arisen due to changes in the population structure in the natural environment not as a direct mutation caused by vaccination. Analysis of extracellular products revealed that Y. ruckeri is a poor producer of excreted compounds. However, it was observed that these compounds are homogenous to all serotypes and biotypes. Antigenic characterisation highlighted that the O antigen is the serospecific antigen between biotypes although there was marked cross reaction with whole cell proteins. In this context, vaccination studies revealed that the O antigen is the dominant immunogenic molecule involved in protection against the disease. Virulence studies demonstrated that all serogroups and biotypes were virulent to rainbow trout. Y. ruckeri was able to stimulate the innate immune response in order to survive and proliferate in vaccinated rainbow trout.
Atrogin-1 is a conserved ubiquitin E3 ligase that is central to the early stages of skeletal and ... more Atrogin-1 is a conserved ubiquitin E3 ligase that is central to the early stages of skeletal and cardiac muscle wasting and degradation following starvation and inflammatory diseases. The control of protein turnover is different between endothermic and ectothermic animals reflecting the body energy requirements. Here we have characterised the promoter of the atrogin-1 gene in a phylogenetically diverse group of vertebrates and show conserved FOXO elements are present in all species examined. We have examined the gene expression responses in primary muscle cell culture to key immune modulators (IL-1β, interferon type 1 and interferon γ) and to the anabolic hormone insulin like growth factor (IGF-1). We show that the IL-1β and interferon type 1 increased atrogin-1 mRNA expression whereas IGF-1 suppressed atrogin-1 expression. The proximal promoter of salmon atrogin-1 was used to transfect primary muscle cell cultures and we found all three cytokines increased promoter activity whereas...
In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, t... more In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin...
While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancrea... more While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on-farm qualitative test and an in-laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (D 340). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the D 340 in infected fish rose from undetectable to a maximum at 6 weeks post-infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.
Rainbow trout (Oncorhynchus mykiss) urea cycle and polyamine synthesis gene families show dynamic... more Rainbow trout (Oncorhynchus mykiss) urea cycle and polyamine synthesis gene families show dynamic expression responses to inflammation, Fish and Shellfish Immunology (2019), doi:
Supplementing the diet with functional ingredients is a key strategy to improve fish performance ... more Supplementing the diet with functional ingredients is a key strategy to improve fish performance and health in aquaculture. The amino acids of the urea and nitric oxide (NO) cycles-arginine, ornithine and citrulline-perform crucial roles in the immune response through the generation of NO and the synthesis of polyamine used for tissue repair. We previously found that citrulline supplementation improves and maintains circulating free arginine levels in rainbow trout more effectively than arginine supplementation. Here, to test whether supplementation of urea cycle amino acids modulates the immune response in rainbow trout (Oncorhynchus mykiss), we supplemented a commercial diet with high levels (2% of total diet) of either arginine, ornithine or citrulline during a 7-week feeding trial, before challenging fish with the bacterium Aeromonas salmonicida. We carried out two separate experiments to investigate fish survival and 24h post-infection to investigate the immediate response of free amino acid levels, and transcriptional changes in genes encoding urea cycle, NO cycle and polyamine synthesis enzymes. There were no differences in percentage fish mortality between diets, however there were numerous highly significant changes in free amino acid levels and gene expression to both dietary supplementation and infection. Out of 26 amino acids detected in blood plasma, 8 were significantly changed by infection and 9 by dietary supplementation of either arginine, ornithine or citrulline. Taurine, glycine and aspartic acid displayed the largest decreases in circulating levels in infected fish, while ornithine and isoleucine were the only amino acids that increased in concentration. We investigated transcriptional responses of the enzymes involved in arginine metabolism in liver and head kidney; transcripts for polyamine synthesis enzymes showed highly significant increases in both tissues across all diets following infection. The paralogous arginase-encoding genes, Arg1a, Arg1b, Arg2a and Arg2b, displayed complex responses across tissues and also due to diet and infection. Overall, these findings improve our understanding of amino acid metabolism following infection and suggests new potential amino acid targets for improving the immune response in salmonids.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2020
Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, gro... more Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein deposition and the immune response. In mammals, supplementation of arginine's precursor amino acid, citrulline, is known to increase circulating arginine to levels beyond direct arginine supplementation, however, citrulline supplementation is poorly studied in fish. To address this knowledge gap, we supplemented the diet of rainbow trout with arginine and its precursor amino acids, ornithine and citrulline, at 3 levels (0.5%, 1% and 2% of the total diet) during a 14-week experiment. We sampled fish at 3h and 24h post-feeding to investigate immediate and steady-state effects, respectively. There were no differences in fish growth for any of the diets across a range of indicators. In blood plasma, out of 26 amino acids detected, 11 and 6 displayed significant changes 24h and 3h post-prandial, respectively. Arginine, ornithine and citrulline levels were all significantly increased by the citrulline supplemented diets. In muscle, 8 amino acids were significantly altered by supplemented diets, while there were no significant changes in liver. Arginine was increased by 2% citrulline supplementation in muscle tissue. We also investigated the transcriptional responses of urea cycle, nitric oxide cycle and rate-limiting polyamine synthesis enzymes, related to arginine's metabolism, in liver. At both time points, only 2 enzymes were significantly altered by the supplemented diets, however several significant changes were observed comparing 3h and 24h post-prandial expression levels. Of these, the paralogous polyamine synthesis enzyme encoding genes ODC1 and ODC2 displayed the largest increases in 3h post-prandial fish. These findings demonstrate that endogenous synthesis of arginine is possible from a citrulline supplemented diet and improve our understanding of arginine metabolism in fish.
Diseases which cause skeletal muscle myopathy are some of the most economically damaging diseases... more Diseases which cause skeletal muscle myopathy are some of the most economically damaging diseases in Atlantic salmon, Salmo salar L., aquaculture. Despite this, there are limited means of assessing fish health non-destructively. Previous investigation of the serum proteome of Atlantic salmon, Salmo salar L., during pancreas disease (PD) has identified proteins in serum that have potential as biomarkers of the disease. Amongst these proteins, the enzyme enolase was selected as the most viable for use as a biomarker of muscle myopathy associated with PD. Western blot and immunoassay (ELISA) validated enolase as a biomarker for PD, whilst immunohistochemistry identified white muscle as the source of enolase. Enolase was shown to be a specific marker for white muscle myopathy in salmon, rising in serum concentration significantly correlating with pathological damage to the tissue.
The efficacy of cellular components of probiotics Kocuria SM1 and Rhodococcus SM2 to protect rain... more 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 × 10 5 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; OD 550nm) from 0.039 to 0.043-0.045, peroxidase (OD 550nm) 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.
Salmonid alphavirus is the aetological agent of pancreas disease (PD) in marine Atlantic salmon, ... more 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 significance Salmonid 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
Whether through direct supplementation of bacteria or by prebiotic supplementation thought to fav... more Whether through direct supplementation of bacteria or by prebiotic supplementation thought to favour subsets of bacteria, modulation of gut microbiota constitutes an important and promising alternative to the use of prophylactic and growth promoting antibiotics in worldwide aquaculture. We fed a commercial base feed, alone or supplemented with either proprietary β-glucan, β-glucan and organic acids, citrus flavonoid or yeast cell wall supplements, to rainbow trout over a period of four weeks. Fish from each feed group were then subjected to experimental, waterborne infection withYersinia ruckeriO1 biotype 2. Following experimental feeding, the β-glucan and organic acids supplemented group showed significantly improved feed conversion and lipid efficiency ratios. Furthermore, the β-glucan, β-glucan and organic acids and citrus flavonoid supplements proved to significantly reduce the risk of mortality in rainbow trout during experimental infection as shown by hazard ratio analysis. Re...
The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces the availab... more The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces the availability of n-3 long-chain PUFA (LC-PUFA) to marine fish such as gilthead seabream. The aim of this study was to examine compositional and physiological responses to a dietary gradient of n-3 LC-PUFA. Six iso-energetic and iso-nitrogenous diets (D1-D6) were fed to seabream, with the added oil being a blend of FO and VO to achieve a dietary gradient of n-3 LC-PUFA. Fish were sampled after 4 months feeding, to determine biochemical composition, tissue fatty acid concentrations and lipid metabolic gene expression. The results indicated a disturbance to lipid metabolism, with fat in the liver increased and fat deposits in the viscera reduced. Tissue fatty acid profiles were altered towards the fatty acid compositions of the diets. There was evidence of endogenous modification of dietary PUFA in the liver which correlated with the expression of fatty acid desaturase 2 (fads2). Expression of ster...
The present study aimed to investigate whether alternative dietary protein sources modulate the m... more 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. Reg...
In Atlantic salmon (Salmo salar L.), and also in other fish species, certain plant protein ingred... more 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.
Recently outbreaks of enteric redmouth disease in previously vaccinated salmonids was attributed ... more Recently outbreaks of enteric redmouth disease in previously vaccinated salmonids was attributed to biotype 2 (non-motile, lipase negative) isolates of Yersinia ruckeri. Currently, isolates of biotype 2 are the most widely encountered form in Europe and North America. Biochemical analysis of over 90 clinical isolates revealed that there are potentially eight novel phenotypes associated with disease. Cell surface characteristics of biotype 2 isolates displayed a unique O antigen structure. A multilocus sequence typing scheme was developed using 4 "housekeeping" gene loci in order to understand the phylogeny and population structure of Y. ruckeri. The scheme highlighted that although there is low species diversity, this is characteristic of a small population size which is not limited to fish species or geographical region. The study suggests that biotype 2 isolates have arisen due to changes in the population structure in the natural environment not as a direct mutation caused by vaccination. Analysis of extracellular products revealed that Y. ruckeri is a poor producer of excreted compounds. However, it was observed that these compounds are homogenous to all serotypes and biotypes. Antigenic characterisation highlighted that the O antigen is the serospecific antigen between biotypes although there was marked cross reaction with whole cell proteins. In this context, vaccination studies revealed that the O antigen is the dominant immunogenic molecule involved in protection against the disease. Virulence studies demonstrated that all serogroups and biotypes were virulent to rainbow trout. Y. ruckeri was able to stimulate the innate immune response in order to survive and proliferate in vaccinated rainbow trout.
Atrogin-1 is a conserved ubiquitin E3 ligase that is central to the early stages of skeletal and ... more Atrogin-1 is a conserved ubiquitin E3 ligase that is central to the early stages of skeletal and cardiac muscle wasting and degradation following starvation and inflammatory diseases. The control of protein turnover is different between endothermic and ectothermic animals reflecting the body energy requirements. Here we have characterised the promoter of the atrogin-1 gene in a phylogenetically diverse group of vertebrates and show conserved FOXO elements are present in all species examined. We have examined the gene expression responses in primary muscle cell culture to key immune modulators (IL-1β, interferon type 1 and interferon γ) and to the anabolic hormone insulin like growth factor (IGF-1). We show that the IL-1β and interferon type 1 increased atrogin-1 mRNA expression whereas IGF-1 suppressed atrogin-1 expression. The proximal promoter of salmon atrogin-1 was used to transfect primary muscle cell cultures and we found all three cytokines increased promoter activity whereas...
In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, t... more In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin...
While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancrea... more While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on-farm qualitative test and an in-laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (D 340). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the D 340 in infected fish rose from undetectable to a maximum at 6 weeks post-infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.
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