Duplicate tanks of c.280 g Atlantic salmon (Salmo salar L.) were fed for 60 days on diets in which fishmeal was substituted with graded levels of extracted soybean meal (SBM) comprising 0%, 10%, 15%, 20%, 25% or 35% of total protein. The effects on feed intake, growth, feed conversion, apparent digestibility and utilization of macronutrients and energy, pathohistological response of the distal intestine (DI), activities of digestive enzymes in the mid and distal intestinal mucosa, and faecal trypsin and plasma insulin concentrations were studied. A negative, dose-dependent effect of SBM was observed on nearly all performance parameters with a notable exception of feed intake. The lowest SBM inclusion level of 10% resulted in moderate pathohistological changes in the DI. Each subsequent increase in SBM level increased the number of fish displaying severe changes. In contrast to the mid-intestine (MI), all enzyme activities in the distal intestinal mucosa decreased dose-dependently with increasing SBM inclusion. Faecal trypsin increased up to an SBM inclusion level of 20% and then levelled off. Plasma insulin increased from 0% to 15% SBM inclusion and then decreased. The results suggest that caution should be exercised in the use of even low levels of extracted SBM in salmon feeds. KEY WORDS

Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.

Extracted soybean meal (SBM) in the diet for Atlantic salmon, Salmo salar L., causes an inflammatory response in the distal intestine. The morphological changes of the epithelial cells and a characterization of the inflammatory cell infiltrate of the distal intestinal mucosa were studied using a panel of enzyme and immunohistochemical markers. The salmon (average body weight 927 g) used in the study were fed either a fishmeal‐based diet (control diet) or a diet in which 30% of the fishmeal protein was replaced with SBM protein (SBM diet). In salmon fed SBM, there were markedly reduced enzyme reactivities in the distal intestinal epithelial cells, both in the brush border [5′‐nucleotidase (5′N), Mg2+‐ATPase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP)] and in the intracellular structures [alkaline and acid phosphatase, non‐specific esterase (NSE) and alanine aminopeptidase (AAP)]. There appeared to be an increased presence of cells of monocytic lineage, including macrophages, as well as neutrophilic granulocytes and immunoglobulin (Ig) M in the lamina propria of the SBM‐fed fish. The mid intestine showed little response to the diet. The results suggest that toxic/antigenic component(s) of SBM affect the differentiation of the distal intestinal epithelial cells and may help explain the reduced nutrient digestibilities previously reported in salmonids fed extracted SBM.

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This study was conducted to investigate the long-term effects of feeding plant products from both traditional breeding and from biotechnology on intestinal somatic indices, histology and cell proliferation in first-feeding Atlantic salmon, Salmo salar L. (initial weight 0.21 +/- 0.02 g). A standard fishmeal diet (standard fishmeal) was formulated to contain fishmeal as the sole protein source and suprex maize as the main starch source. Six experimental diets were then developed: two in which some of the fishmeal was replaced with commercially available, genetically modified Roundup Ready full-fat soybean meal (GM-soy) or commercially available, non-GM full-fat soybean meal (nGM-soy) at a level of 12.5% of the total diet, and four diets in which the suprex maize was replaced with two lines of GM-maize (Dekalb 1; D1 and Pioneer 1; P1), both products of event MON810, and their half-sibling non-GM counterparts (Dekalb 2; D2 and Pioneer 2; P2), at a level of 12.1% of total diet. Each diet was fed to fish in triplicate tanks and the experiment lasted for 8 months, during which the fish reached a final weight of 101-116 g. There was no significant effect of diet on the intestinal indices, nor were histological changes observed in the pyloric caeca or mid intestine. In the distal intestine, one of nine sampled fish fed nGM-soy showed moderate changes, two of nine sampled fish fed GM-soy showed changes, one with moderate and one with severe changes, and two of nine fish fed nGM-maize D2 had moderate changes. Using a monoclonal antibody against proliferating cell nuclear antigen (PCNA), cell proliferative responses to the experimental diets were assessed. In fish fed both soy diets, a significantly higher (P < 0.05) cell proliferation response was observed in the distal intestine concomitant with an increased localization of PCNA positive cells along the whole distal intestinal folds. The PCNA response among the nGM-soy group was significantly higher compared with all the other diet groups. In contrast, for fish exposed to dietary maize (type D) compared with fish fed the standard fishmeal, the soy-diets (GM-soy and nGM-soy) and maize (type P), a significantly lower (P < 0.05) cell proliferation response was observed in the distal intestine. Results indicated that the GM plant products investigated in this study, at about 12% inclusion level, were as safe as commercially available non-GM products, at least in terms of their effect on indices and histological parameters of the Atlantic salmon intestinal tract.

T-cell-mediated hypersensitivity could be central in soybean meal (SBM)-induced intestinal changes in salmon. However, tools for immunohistochemical detection of T cells have been lacking in teleosts, including Atlantic salmon. Application of a specific histochemical protocol allowed demonstration of T-cell-like reactivities in formalin-fixed, paraffin-embedded tissues using an antibody reacting to a conserved region of human CD3epsilon (Dako A0452). Characteristic staining was observed in cells of the thymus as well as distal intestine, skin, gills and spleen. These cells were negative for immunoglobulin M (IgM). Intestinal intraepithelial leucocytes were CD3epsilon positive. During the SBM-induced enteropathy, the mixed inflammatory infiltrate in the lamina propria of the distal intestine included many lymphocytes with a T-cell-like reactivity. Real-time polymerase chain reaction revealed significantly increased expression of a complex polypeptide (CD3pp), CD4 and CD8beta (P < 0.05) in the distal intestine of SBM-fed fish compared to fish meal-fed reference fish. Increased reactivity for extracellular IgM in the lamina propria and a positive material between the epithelial cells at the tips of the folds was observed, possibly due to leakage of IgM through an abrogated epithelial barrier. In conclusion, a T-cell-like response appears to be involved in this example of a food-sensitive enteropathy.

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