A Proof-of-Concept Study to Develop a Peptide-Based Vaccine against Salmon Lice Infestation in Atlantic Salmon (Salmo salar L.).

Proteins present in blood samples from Atlantic salmon (Salmo salar) infected with salmon lice (Lepeophtheirus salmonis) were analyzed using liquid chromatography-high-resolution mass spectrometry. Bioinformatic analyses revealed 1820 proteins, of which 58 were assigned to lice. Among these, peroxiredoxin-2, an antioxidant protein, was found relevant with respect to blood feeding of the parasite. The three-dimensional structure analysis of the protein revealed a surface amino acid sequence of interest. A 13-amino-acid peptide was selected as a potential antigen due to its predicted solubility, antigenicity, probable non-allergenic, and non-toxic nature. This peroxiredoxin-2-derived peptide was synthesized, combined with a commercially available adjuvant, and used for vaccination. The test vaccine demonstrated a 60-70% protection rate against early-stage Lepeophtheirus salmonis infection in a challenge trial in Norway. Additionally, the vaccine was tested against salmon lice (Caligus rogercresseyi) in Chile, where a remarkable 92% reduction in the number of adult lice was observed. Thus, in combination with the selected adjuvant, the peptide showed antigenic potential, making it a suitable candidate for future vaccine development. The approach described holds promise for the development of peptide vaccines against various ectoparasites feeding on blood or skin secretions of their hosts.

Chemical Profiling and Biological Activity of Extracts from Nine Norwegian Medicinal and Aromatic Plants.

There is an increased interest in identifying beneficial compounds of plant origin that can be added to animal diets to improve animal performance and have a health-promoting effect. In the present study, nine herb species of the Norwegian wild flora or which can be cultivated in Norway were selected for phytogenic evaluation (hops, maral root, mint, oregano, purslane, rosemary, roseroot, sweet wormwood, yarrow). Dried herbs were sequentially extracted with dichloromethane (DCM), ethanol (EtOH) and finally water (H2O) by ultrasound-assisted extraction (UAE). The UAE protocol was found to be more rational than conventional Soxhlet with respect to DCM extraction. Total extraction yield was found to be highest for oregano (Origanum vulgare) with 34.4 g 100-1 g dry matter (DM). H2O-extracts gave the highest yields of the three solvents, with up to 25 g 100-1 g DM for purslane (Portulaca oleracea ssp. sativa) and mint (Mentha piperita). EtOH- and H2O-extracts were the most efficient extracts with respect to free radical scavenging capacity (ABTS (=2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and oregano, mint, hops (Humulus lupulus) and maral root-leaves (Leuzea carthamoides) were found to be the most efficient antioxidant sources. Hops (EtOH-extract) contained α- and ß-acids, xanthohumols, chlorogenic acid and the hitherto unreported 3-O-glucosides of kaempferol and quercetin. Maral root-leaves contained among other compounds hexosides of the 6-hydroxy- and 6-methoxy-kaempferol and -quercetin, whereas roseroot (Rosea rhodiola) revealed contents of rosavin, rhodiosin and rhodionin. Sweet wormwood (Artemisia annua) contained chlorogenic acid and several derivatives thereof, scopoletin and poly-methylated flavones (eupatin, casticin, chrysoplenetin). Antimicrobial potential of different plant extracts was demonstrated against Gram-positive and Gram-negative bacteria using the indicator organisms Staphylococcus aureus, and Escherichia coli, and the Atlantic salmon bacterial pathogens Moritella viscosa, Tenacibaculum finnmarkense and Aliivibrio wodanis. DCM extracts possessed the highest activities. Data demonstrate the potential ability of herb extracts as natural antimicrobials. However, future safety studies should be performed to elucidate any compromising effect on fish health.

Beyond standard PSE testing: An exploratory study of bioimpedance as a marker for ham defects.

During post-mortem conversion from muscle to meat, diverse quality anomalies can emerge. Recent pork defects are often accompanied by deteriorating fibre structure. Here we investigate how bioimpedance response, an indicator of structural disintegration, can help in detecting quality defects. We, first, measured the relationship between standard meat quality variables (pHu, CIELAB, drip loss) and bioimpedance (BI) response. To screen for defect-biomarkers that are linked to aberrant bioimpedance and physicochemical indicators of quality decline, we performed LC-MS/MS proteomic analysis on samples, classified with a multivariate-based separation into good versus poor quality. We found that BI correlated significantly with, e.g., colour and drip loss. Proteomics revealed eleven proteins to be unique for either, good or poor ham quality groups, and maybe linked to structural degradation. In all, our data supports a wider integration of BI testing in pork quality testing to assess structural disintegration, which can render ham unsuitable for, e.g., costly curing.

Plant-Based Diets Induce Transcriptomic Changes in Muscle of Zebrafish and Atlantic Salmon.

With the expansion of the aquaculture industry in the last two decades, there has been a large increase in the use of plant ingredients in aquafeeds, which has created new challenges in fish growth, health and welfare. Fish muscle growth is an important trait that is strongly affected by diet, but our knowledge on the effect of plant protein-based diets on global gene expression in muscle is still scant. The present study evaluated nutrigenomic effects of the inclusion of proteins from pea, soy and wheat into aquafeeds, compared to a control diet with fishmeal as the main protein source using the zebrafish model by RNA-seq; these results were extended to an important aquaculture species by analyzing selected differentially expressed genes identified in the zebrafish model on on-growing Atlantic salmon fed with equivalent plant protein-based diets. Expression of selected Atlantic salmon paralogues of the zebrafish homologs was analyzed using paralogue-specific qPCR assays. Global gene expression changes in muscle of zebrafish fed with plant-based diets were moderate, with the highest changes observed in the soy diet-fed fish, and no change for the pea diet-fed fish compared to the control diet. Among the differentially expressed genes were mylpfb, hsp90aa1.1, col2a1a, and odc1, which are important in regulating muscle growth, maintaining muscle structure and function, and muscle tissue homeostasis. Furthermore, those genes and their paralogues were differentially expressed in Atlantic salmon fed with the equivalent percentage of soy or wheat protein containing diets. Some of these genes were similarly regulated in both species while others showed species-specific regulation. The present study expands our understanding on the molecular effects of plant ingredients in fish muscle. Ultimately, the knowledge gained would be of importance for the improved formulation of sustainable plant-based diets for the aquaculture industry.

Sensitivity to Dietary Wheat Gluten in Atlantic Salmon Indicated by Gene Expression Changes in Liver and Intestine.

Feed safety is a necessity for animal health and welfare as well as prerequisite for food safety and human health. Wheat gluten (WG) is considered as a valuable protein source in fish feed due to its suitability as a feed binder, high digestibility, good amino acid profile, energy density and most importantly, due to its relatively low level of anti-nutritional factors (ANFs). The main aim of this study was to identify the impact of dietary WG on salmon health by analysing growth, feed efficiency and the hepatic and intestinal transcriptomes. The fish were fed either control diet with fishmeal (FM) as the only source of protein or diets, where 15% or 30% of the FM were replaced by WG. The fish had a mean initial weight of 223 g and approximately doubled their weight during the 9-week experiment. Salmon fed on 30% WG showed reduced feed intake compared to the 15% and FM fed groups. The liver was the less affected organ but fat content and activities of the liver health markers in plasma increased with the inclusion level of WG in the diet. Gene expression analysis showed significant changes in both, intestine and liver of fish fed with 30% WG. Especially noticeable were changes in the lipid metabolism, in particular in relation to the intestinal lipoprotein transport and sterol metabolism. Moreover, the intestinal transcriptome of WG-fed fish showed shifts in the expression of a large number of genes responsible for immunity and tissue structure and integrity. These observations implied that the fish receiving WG-containing diet were undergoing nutritional stress. Overall, the study provided evidence that a high dietary level of WG can have a negative impact on the intestinal and liver health of salmon with symptoms similar to gluten sensitivity in humans.

Biotransformation of phytoestrogens from soy in enzymatically characterized liver microsomes and primary hepatocytes of Atlantic salmon.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Development and Validation of a Liquid Chromatography High-Resolution Mass Spectrometry Method for the Simultaneous Determination of Mycotoxins and Phytoestrogens in Plant-Based Fish Feed and Exposed Fish.

New protein sources in fish feed require the assessment of the carry-over potential of contaminants and anti-nutrients from feed ingredients into the fish, and the assessment of possible health risks for consumers. Presently, plant materials including wheat and legumes make up the largest part of aquafeeds, so evaluation of the transfer capabilities of typical toxic metabolites from plant-infesting fungi and of vegetable phytoestrogens into fish products is of great importance. With the aim of facilitating surveillance of relevant mycotoxins and isoflavones, we have developed and validated a multi-analyte LC-HRMS/MS method that can be used to ensure compliance to set maximum levels in feed and fish. The method performance characteristics were determined, showing high specificity for all 25 targeted analytes, which included 19 mycotoxins and three isoflavones and their corresponding aglycons with sufficient to excellent sensitivities and uniform analytical linearity in different matrices. Depending on the availability of matching stable isotope-labelled derivates or similar-structure homologues, calibration curves were generated either by using internal standards or by matrix-matched external standards. Precision and recovery data were in the accepted range, although they varied between the different analytes. This new method was considered as fit-for-purpose and applied for the analysis of customised fish feed containing wheat gluten, soy, or pea protein concentrate as well as salmon and zebrafish fed on diets with these ingredients for a period of up to eight weeks. Only mycotoxin enniatin B, at a level near the limit of detection, and low levels of isoflavones were detected in the feed, demonstrating the effectiveness of maximum level recommendations and modern feed processing technologies in the Norwegian aquaculture industry. Consequently, carry-over into fish muscle was not observed, confirming that fillets from plant-fed salmon were safe for human consumption.