Histological, digestive, metabolic, hormonal and some immune factor responses in Atlantic salmon, Salmo salar L., fed genetically modified soybeans

The paper reports the second and final part of an experiment aiming to study physiological and health-related effects of genetically modified (GM) soybean meal (SBM) type Roundup Ready soybean (RRS) in diets for post-smolt Atlantic salmon. For 3 months salmon were fed diets containing 172 g kg(-1) full-fat SBM from RRS (GM-soy) or an unmodified, non-isogenic line (nGM-soy), or a reference diet with fishmeal as the sole protein source (FM). Slight differences in anti-nutrient levels were observed between the GM and nGM-soy. Histological changes were observed only in the distal intestine of the soy-fed fish. The incidence of moderate inflammation was higher in the GM-soy group (9 of 10 sampled fish) compared with the nGM-soy group (7 of 10). However, no differences in the concomitant decreases in activities of digestive enzymes located in the brush border (leucine aminopeptidase and maltase) and apical cytoplasm (acid phosphatase) of enterocytes or in the number of major histocompatibility complex class II+ cells, lysozyme activity, or total IgM of the distal intestine were observed. GM compared with nGM-soy fed fish had higher head kidney lysozyme (11,856 vs. 10,456 units g(-1) tissue) and a tendency towards higher acid phosphatase (0.45 vs. 0.39 micromol h(-1) kg(-1) body mass in whole tissue) activities, respectively. Plasma insulin and thyroxin levels, and hepatic fructose-1,6-bisphosphatase and ethoxyresorufin-O-deethylase activities were not significantly affected. It is not possible, however, to conclude whether the differences in responses to GM-soy were due to the genetic modification or to differences in soy cultivars in the soy-containing diets. Results from studies using non-modified, parental line soybeans as the control group are necessary to evaluate whether genetic modification of soybeans in diets poses any risk to farmed Atlantic salmon.     

Vaccine-associated granulomatous inflammation and melanin accumulation in Atlantic salmon, Salmo salar L., white muscle

The purpose of this study was to investigate the nature of variably sized pigmented foci encountered in fillets of farmed Atlantic salmon, Salmo salar L. The material was sampled on the fillet production line and on salmon farms from fish with an average size of 3 kg from various producers. The fish had been routinely vaccinated by injection. Gross pathology, histology, immunohistochemistry using antisera against major histocompatibility complex (MHC) class II beta chain and transmission electron microscopy (TEM) were used to characterize the changes. Macroscopically, melanized foci were seen penetrating from the peritoneum deep into the abdominal wall, sometimes right through to the skin, and also embedded in the caudal musculature. Histological investigation revealed muscle degeneration and necrosis, fibrosis and granulomatous inflammation containing varying numbers of melano-macrophages. Vacuoles, either empty or containing heterogeneous material, were frequently seen. The presence of abundant MHC class II+ cells indicated an active inflammatory condition. TEM showed large extracellular vacuoles and leucocytes containing homogeneous material of lipid-like appearance. The results showed that the melanized foci in Atlantic salmon fillet resulted from an inflammatory condition probably induced by vaccination. The described condition is not known in wild salmon and in farmed salmon where injection vaccination is not applied.     

Time after seawater transfer influences immune cell abundance and responses to SAV3 infection in Atlantic salmon

Pancreas disease (PD) caused by salmonid alphavirus (SAV) severely affects salmonid aquaculture during the seawater phase. To characterize immune cells in target tissues for SAV infection, heart, pancreas and pyloric caeca were analysed from two groups of fish adapted to seawater for 2 and 9 weeks. The sections were scored for the relative abundance of cells expressing MHC class II, IgM, CD3, CD8 or neutrophil/granulocyte markers using immuno‐histochemical techniques. In general, necrosis of tissue was more severe in fish infected at 2 weeks post‐seawater transfer (wpt) compared with those infected at 9 wpt. At 9 wpt, there were higher numbers of MHC II⁺ cells in heart, pancreas and pyloric caeca, IgM⁺ cells in heart and pancreas, and CD3⁺ cells in pancreas compared to those infected at 2 wpt. The majority of the immune cells infiltrating PD‐affected tissues were MHC II⁺ and CD3⁺ cells suggesting that antigen‐presenting cells and T lymphocytes are the main types of immune cells responding to SAV infection. All the investigated cell types were also observed in pyloric caeca of infected fish, suggesting that this tissue may play a role in the immune response to SAV.     

Vaccination and triploidy increase relative heart weight in farmed Atlantic salmon,Salmo salar L.

Heart morphology is particularly plastic in teleosts and differs between farmed and wild Atlantic salmon. However, little is known about how different culture practices and sex affect heart morphology. This study investigated how vaccination, triploidy and sex affected heart size and heart morphology (ventricle shape, angle of the bulbus arteriosus) in farmed Atlantic salmon for 18 months following vaccination (from c. 50–3000 g body weight). In addition, hearts were examined histologically after 7 months in sea water. All fish sampled were sexually immature. Vaccinated fish had significantly heavier hearts relative to body weight and a more triangular ventricle than unvaccinated fish, suggesting a greater cardiac workload. Irrespective of time, triploids had significantly heavier hearts relative to body weight, a more acute angle of the bulbus arteriosus and less fat deposition in the epicardium than diploids. The ventricle was also more triangular in triploids than diploids at seawater transfer. Sex had transient effects on the angle of the bulbus arteriosus, but no effect on relative heart weight or ventricle shape. From a morphological perspective, the results indicate that vaccination and triploidy increase cardiac workload in farmed Atlantic salmon.     

MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II β chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II β chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II⁺ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II⁺ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II⁺ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II⁺ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.     

Variations in mucous cell numbers in gills of Atlantic salmon (Salmo salar) presmolt in commercial freshwater farms in Norway

Fish gills are heavily exposed to the external milieu and may react against irritants with different cellular responses. We describe variations in mucous cell counts in gills from healthy Atlantic salmon (Salmo salar) presmolts in five recirculating aquaculture system (RAS) farms and one flow‐through farm. Based on certain criteria, mucous cells were histologically quantified in a defined lamellar region of the gills and the counts were analysed. Immunohistochemistry (IHC) was used to investigate epithelial responses. The median number of total mucous cells in the defined region was 59 per fish. Between the farms, the medians varied from 31 to 101 with the lowest in the flow‐through farm. A regression model was fitted with “total mucous cells” as the dependent variable and with “fish length” and “fish farm” as independent variables. The proportion of variation in mucous cell counts explained by the model was twice as high when “fish farm” was included compared to only “fish length.” IHC revealed proliferative responses in coherence with high mucous cell numbers. Conclusively, the variation in mucous cell counts depends on combined farm‐related factors. Establishing a baseline for mucous cell counts is fundamental in the development of high‐throughput monitoring programmes of gill health in farmed fish.     

Laser Micro-dissection and Quantitative RT-PCR as a Tool for Cell Identification

The mechanisms of embryo-maternal communication during the first days of embryonic life are largely unknown. Using the bovine as a model, the aims of our study were to morphologically characterize the interaction between the pre-implantation embryo and the epithelium of the maternal ampulla, isthmus and uterotubal junction by light and scanning electron microscopy. For this purpose, oviducts were removed from cows revealing a functional corpus luteum on day 3 after insemination. These were compared to oviducts removed on day 3 (metestrus) of the estrous cycle. Three days after insemination, the majority of the epithelial cells in the ampulla were secretory cells distinctly protruding into the oviductal lumen. Contrary the ampulla of cows on day 3 of the cycle predominantly revealed ciliated cells in the oviductal epithelium. As shown by Periodic Acid Schiff reaction (PAS) with and without amylase digestion, the secretory cells of the ampulla synthesized merely glycoproteins during metestrus, but large amounts of glycogen during pregnancy. In the isthmus no morphological differences were seen between pregnant and cyclic cows. The most conspicuous finding during pregnancy was seen in the uterotubal junction: Vital cumulus cells embedded in between epithelial cells had developed short cytoplasmic processes intensely contacting the epithelial uterine cells. The embryos obtained ex vivo were regularly covered with a thick layer of homogenous extracellular matrix. Contrary embryos produced in vitro – both with and without coculture with oviductal cells –revealed a clearly visible zona pellucida with spongy appearance and numerous pores. Our results imply that already during the first days of life there is intense interaction of the pre-implantation embryo and the maternal genital tract part of which may be mediated by cumulus cells.     

Expression of insulin-like growth factor-I in the gastrointestinal tract of Atlantic salmon (Salmo salar L.)

The expression of insulin-like growth factor (IGF-I) in the gut of Atlantic salmon (Salmo salar L.) smolt was investigated at the cellular level using in situ hybridization. A hybridization protocol was adapted for use on formalin fixed, paraffin embedded tissues and was based on a digoxigenin-labelled oligoprobe that recognised a conserved region of IGF-I mRNA. The specificity of the anti-sense IGF-I probe and the performance of the hybridization protocol were evaluated using a sense IGF-I probe, an Ep stein-Barr virus probe and an insulin probe cocktail on serial sections of fish gut, mouse thyroid and human lymph node tissue. In the gut of Atlantic salmon smolt, IGF-I was found to be expressed in single epithelial cells or small clusters of epithelial cells in the pyloric ampulla and in the pyloric caeca. Expression was not detected in the lamina propria of the gut or in epithelial cells of the stomach, midgut or hindgut. The restriction of IGF-I expression to the pyloric ampulla and pyloric caeca is consistent with the role of these gut segments in osmoregulation and the scattered epithelial expression supports the putative autocrine/paracrine mechanism of action of IGF-I in osmoregulation.