Animal Welfare Issues in Capture-Based Aquaculture.

Capture-based aquaculture (CBA) represents a type of intensive aquaculture production system for some economically valuable fish species, such as bluefin tuna (Thunnus thynnus), eel (Anguilla spp.) and Atlantic cod (Gadus morhua). In CBA, fish are captured from the wild in certain periods of the year, and following a recovery phase, they are kept in rearing facilities for a period of time, until they reach the market size. In this case, the fish are wild and have not gone through domestication like other fish species that are reproduced and farmed under the established farming systems. Therefore, these fish are not genetically adapted to live under the intensive farming conditions, and thus their welfare may be compromised in different manners compared to their domesticated counterparts. This review presents an overview of the current situation of CBA, while focusing on the assessment of fish welfare in CBA. The most commonly used fish welfare indicators will be discussed in relation to the different stages of CBA.

A Truncated Galectin-3 Isolated from Skin Mucus of Atlantic Salmon Salmo salar Binds to and Modulates the Proteome of the Gram-Negative Bacteria Moritella viscosa.

The mucus of fish skin plays a vital role in innate immune defense. Some mucus proteins have the potential to incapacitate pathogens and/or inhibit their passage through the skin. In this study the aim was to isolate and characterize galectin(s), ß-galactosides binding proteins, present in skin mucus. A novel short form of galectin-3 was isolated from Atlantic salmon skin mucus by α-lactose agarose based affinity chromatography followed by Sephadex G-15 gel filtration. Mass spectrometric analysis showed that the isolated protein was the C-terminal half of galectin-3 (galectin-3C). Galectin-3C showed calcium independent and lactose inhabitable hemagglutination, and agglutinated the Gram-negative pathogenic bacteria Moritella viscosa. Galectin-3 mRNA was highly expressed in skin and gill, followed by muscle, hindgut, spleen, stomach, foregut, head kidney, and liver. Moritella viscosa incubated with galectin-3C had a modified proteome. Proteins with changed abundance included multidrug transporter and three ribosomal proteins L7/12, S2, and S13. Overall, this study shows the isolation and characterization of a novel galectin-3 short form involved in pathogen recognition and modulation, and hence in immune defense of Atlantic salmon.

Nutrient Digestibility, Growth, Mucosal Barrier Status, and Activity of Leucocytes From Head Kidney of Atlantic Salmon Fed Marine- or Plant-Derived Protein and Lipid Sources.

Nutrient digestibility, growth, and mucosal barrier status of fish skin, gills, and distal intestine were studied in Atlantic salmon fed feeds based on marine or plant-derived ingredients. The barrier status was assessed by considering the expression of four mucin genes, five genes that encode antimicrobial proteins, distal intestine micromorphology, and design-based stereology of the midgut epithelium. In addition, the head kidney leukocytes were examined using flow cytometry; to understand the differences in their counts and function. Five experimental feeds containing the main components i) fishmeal and fish oil (BG1), ii) soybean meal (BG2; to induce enteritis), iii) fishmeal as the main protein source and rapeseed oil as the main lipid source (BG3), iv) a mix of plant protein concentrates as the protein sources and fish oil as the lipid source (BG4), and v) plant and marine ingredients in the ratio 70:30 (BG5) were produced for the study. Atlantic salmon with initial weight 72.7 ± 1.2 g was offered the experimental feeds for 65 days. The results revealed that the weights of all fish groups doubled, except for fish fed BG2. Fish fed the BG2 diet had lower blood cholesterol concentration, developed enteritis, had lower expression of muc2 in the distal intestine, and had a compromised barrier status in the intestine. Expression of both the mucin genes and genes that encode antimicrobial peptides were tissue-specific and some were significantly affected by diet. The fish fed BG1 and BG3 had more head kidney lymphocyte-like cells compared to BG5-fed fish, and the phagocytic activity of macrophage-like cells from the head kidney was the highest in fish fed BG1. The intestinal micromorphology and the mucosal mapping suggest two different ways by which plant-based diets can alter the gut barrier status; by either reducing the mucous cell sizes, volumetric densities and barrier status (as noted for BG2) or increasing volumetric density of mucous cells (as observed for BG4 and BG5). The results of the compromised intestinal barrier in fish fed plant ingredients should be further confirmed through transcriptomic and immunohistochemical studies to refine ingredient composition for sustainable and acceptable healthy diets.

High nervous necrosis virus (NNV) diversity in wild wrasse (Labridae) in Norway and Sweden.

Wild goldsinny wrasse Ctenolabrus rupestris, corkwing wrasse Symphodus melops and ballan wrasse Labrus bergylta were collected at 8 sampling sites in Sweden and Norway during summer 2014. Brain tissue from 466 wrasses were analyzed for nervous necrosis virus (NNV) infections by real-time RT-PCR, and positive samples were subjected to sequencing and phylogenetic analysis of partial segments of the RNA2 and RNA1 genes. This study shows that NNV is present in wild ballan, corkwing and goldsinny wrasse along the coastline of Sweden and Norway. The overall prevalence in the sampled labrids was 6.7%. Prevalence was 6.4% in goldsinny, 6.3% in corkwing and 18% in ballan wrasse. The wrasse RNA2 NNV sequences revealed high genetic variability and were divided into 3 clusters within the cold water barfin flounder NNV (BFNNV) and warm water cluster red-spotted grouper NNV (RGNNV) genogroups. Within the BFNNV genogroup, wrasse NNVs clustered in 2 sub-genogroups, with grey mullet NNV (GMNNV) and with Atlantic halibut NNV (AHNNV). These groups were previously dominated by virus originating from Atlantic cod Gadus morhua and Atlantic halibut Hippoglossus hippoglossus from the northeast Atlantic. The presence of NNV in wild wrasse and the surprising high genetic variability observed in this study should be considered before moving wild-caught wrasse between geographically distant sites. The results show that use of wild-caught wrasse as brood fish in wrasse farming represents a risk of introducing NNV into aquaculture.

Transcriptional regulation of cytokines in the intestine of Atlantic cod fed yeast derived mannan oligosaccharide or ß-glucan and challenged with Vibrio anguillarum.

Immunomodulatory feed additives are expected to exert their primary influence at the intestinal level through the expression of cytokines, which in turn affect the immune responses in fish. In two separate experiments a yeast-derived mannan oligosaccharide product (YM) or a purified ß-glucan (BG) product were fed to Atlantic cod (Gadus morhua L.) for 5 weeks, after which they were bath-challenged with a bacterial pathogen--Vibrio anguillarum. The transcription of selected cytokines (proinflammatory--il1b, il8, ifng; anti-inflammatory--il10) in different intestinal segments was analysed using qPCR. In the case of YM study, the effect of the compound was observed in both the posterior intestine and rectum of Atlantic cod, upon challenge with the pathogen. iIl1b expression in the posterior intestine and rectum of post-challenge fish was significantly higher than that of pre-challenge fish. In the case of il8 the difference was confined to rectum. The expression of ifng was altered only in the anterior intestine upon YM feeding. In the BG trial, the additive had a differential effect on the expression of the cytokine genes. In anterior intestine and rectum, the purified ß-glucan additive significantly elevated the expression of il1b when challenged with V. anguillarum. An effect of BG on the anti-inflammatory cytokine il10 was visible in the rectum after the pathogen challenge. The differential responses of cytokines in the intestine of fish upon exposure to V. anguillarum suggest that both mannan oligosaccharides and ß-glucans impact the ability of Atlantic cod to respond to the pathogen.

Horizontal transmission of nervous necrosis virus between turbot Scophthalmus maximus and Atlantic cod Gadus morhua using cohabitation challenge.

Experimental horizontal transmission of nervous necrosis virus (NNV) originating from halibut Hippoglossus hippoglossus was studied through cohabitation of intraperitoneally (i.p.) injected fish with uninfected fish for 125 d. The experimental groups consisted of i.p. injected turbot Scophthalmus maximus or i.p. injected Atlantic salmon Salmo salar with turbot, salmon or Atlantic cod Gadus morhua cohabitants. The initial weights were cod 10 g, salmon 40 g and turbot 3 g. NNV was detected in brain, eye and spleen by real-time reverse transcriptase PCR (qRT-PCR) in cod cohabitated with i.p. injected turbot after 90 and 125 d, suggesting NNV infection was transmitted horizontally from the turbot to cod. NNV was not detected in salmon that were cohabitated with i.p. challenged turbot or salmon. This study shows that NNV strains belonging to the Barfin Flounder Nervous Necrosis Virus (BFNNV) clade may be transmitted from halibut to cod via water. Hence there is a potential risk of horizontal transmission of the virus from farmed halibut to farmed and wild cod. The lack of detection of NNV in cohabitant salmon suggests that this fish species is less susceptible than cod, or not susceptible, to horizontal NNV transmission. This result might be influenced by the size of salmon, viral load in i.p. injected cohabitants or insufficient duration of the experiment.

Molecular diagnosis of francisellosis, a systemic granulomatous inflammatory disease in Atlantic cod, Gadus morhua L.

A PCR-based assay for the detection of Francisella noatunensis causing francisellosis in Atlantic cod, Gadus morhua has been developed. Seven sets of primers targeting the flanking regions of the genes (rpoA, sdhA, atpA, rpoB, pgm, groEL and 16S rRNA) of the pathogen were designed. Among the primers, groEL was found to be the most suitable gene candidate for detecting the pathogen, due to its high sensitivity at various annealing temperatures and specificity in detection. The detection limit of the assay was 100 pg of bacterial DNA per milliliter or 100 fg bacterial DNA (approximately 50 genome equivalents) per PCR reaction, however, the sensitivity of the reaction decreased by 1 log dilution in the presence of 1 mg mL(-1) of serum and mucus samples as inhibitors. Nevertheless, the assay can potentially be used as a direct and non-lethal method to detect the pathogen in fish. Thus this PCR assay is a specific and sensitive molecular method to diagnose francisellosis in Atlantic cod, and will be helpful for controlling the infection through prompt detection of the disease in farms.

Changes in the intestinal microbiota of wild Atlantic cod Gadus morhua L. upon captive rearing.

The commensal microbiota plays an important role in the well-being of the host organism, and it would be worthwhile to know the tenacious communities among them. Therefore, a study was undertaken to examine the changes in constitution of the intestinal microbiota of wild fish consequential to captivity. At first, the composition of intestinal microorganisms of Atlantic cod caught from the coastal area off Bodø, Norway, was examined. Thereafter, the changes in the bacterial community of the captive fish after offering them artificial feed or subjecting them to starvation were studied. The microbiota from the intestinal contents and wall segments were analyzed quantitatively by spread plate technique and DAPI staining and qualitatively by denaturing gradient gel electrophoresis. The study revealed that the counts of intestinal microbes in wild-caught Atlantic cod were not affected by captive rearing for 6 weeks, either when fed or when starved. However, the diversity of intestinal bacterial community was reduced in response to artificial feeding, whereas the change was restricted upon starvation.

Detection of Francisella piscicida in Atlantic cod (Gadus morhua L) by the loop-mediated isothermal amplification (LAMP) reaction.

The loop-mediated isothermal amplification (LAMP) reaction was evaluated for its speed and sensitivity in detecting the presence of Francisella piscicida, the causative agent of francisellosis in Atlantic cod (Gadus morhua). Four primer sets, consisting of two outer and two inner, were designed from the groEL gene of the pathogen. The LAMP reaction was optimised at 63 degrees C for 1h using bacterial genomic DNA as the template and the products were visualised under ultra-violet light and analysed by agarose gel electrophoresis. A ladder-like pattern of bands, specific for F. piscicida, was amplified from positive samples. The method was highly specific for the detection of F. piscicida and was 100 times more sensitive than conventional PCR. In addition, the LAMP assay was able to detect the pathogen in kidney and splenic samples of naturally-infected Atlantic cod.

Vibrio tapetis-like strain isolated from introduced Manila clams Ruditapes philippinarum showing symptoms of brown ring disease in Norway.

The Manila clam Ruditapes philippinarum was introduced to Norway in 1987 and was produced in 2 hatcheries until 1991. Clam seed was planted at 6 sites. Two sites were on the Island of Tysnes, south of Bergen. Surviving adult Manila clams were recovered in 1995 and 1996. In the present study, Manila clams from the original seeding that displayed morphological signs of brown ring disease (BRD) were recovered in June 2003 (n=7) and in June 2004 (n=17). Samples from extrapallial fluid, tissues and haemolymph were inoculated on marine agar. Replicate subcultures on selective media were used to select potential Vibrio tapetis strains, and in total, 190 bacterial strains were isolated. One of these strains clustered within the V tapetis clade and was named NRP 45. DNA:DNA hybridisation with the type strain CECT4600 showed 52.7 and 57.3% DNA:DNA similarity. Hybridisation of NRP 45 and the V tapetis LP2 strain, isolated from corkwing wrasse Symphodus melops, produced 46.6 and 44.4% re-association. Partial gene segments encoding 16S rRNA, gyrase B protein (GyrB) and chaperonin 60 protein (Cpn60) were characterised and compared to CECT 4600. NRP 45 showed 5 differences in the 1416 nucleotides (nt) of the 16S rRNA encoding gene (99.6% similarity), while the GyrB encoding gene had 62 substitutions of 1181 nt compared (94.8% similarity) and the Cpn60 encoding gene had 22 substitutions out of 548 nt compared (96% similarity). This is the first finding of BRD and the first isolation of a V. tapetis-like bacterial strain from a bivalve in Norway.

Nodavirus in farmed Atlantic cod Gadus morhua in Norway.

Viral encephalopathy and retinopathy (VER) was diagnosed in 5 to 24 g sized farmed Atlantic cod Gadus morhua kept in sea cages at Parisvatn, Hordaland county, on the west coast of Norway. Moderate mortality (10 to 15%) was observed, along with anorexia and abnormal swimming behaviour, such as looping or spiral swimming and reduced coordination. Nodavirus was detected by 2 different real-time RT-PCR assays, and this was later confirmed by immunohistochemistry. This is the first report of an outbreak of VER in farmed cod in Norway, and the first report that VER affect cod exceeding 5 g in size.

Viral encephalopathy and retinopathy (VER) in Atlantic salmon Salmo salar after intraperitoneal challenge with a nodavirus from Atlantic halibut Hippoglossus hippoglossus.

Homogenate of tissue from juveniles of Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) was used to challenge smolt of Atlantic salmon Salmo salar with an initial average weight of 110 g. The nodavirus was administered in the form of an intraperitoneal injection, and the fish were kept for 134 d post challenge. Genotype characterisation of the nodavirus was performed by sequencing the RNA1 and RNA2 segments, and a quantitative real-time PCR (Q-PCR) assay was developed. Tissues from different organs were stained by immunohistochemistry (IHC). Samples were collected at random on Days 7, 25, 45, 69, 125 and 134 after challenge. Mortality, clinical signs and pathology of VER were observed only in the challenged group. The Q-PCR detected positive fish only in the challenged group, all of which were positive on all days of sampling. An increase in relative virus concentrations was observed from Day 7 to Day 25 post challenge. The increased level of virus concentration was maintained in the medulla oblongata throughout the experiment, suggesting persistence or slow elimination of the virus over time. The IHC detected positive cells on Days 34, 70 and 74. These results suggest that the nodavirus is transported to the medulla oblongata from the intraperitoneal injection site and is able to replicate in salmon. When injected, this nodavirus isolate caused mortality and established a persistent infection in the challenged salmon throughout the experiment. This susceptibility suggests that co-location of salmon and marine species should be avoided until further studies of possible transmission have been carried out.