Soy as an alternative protein source in salmonid aqua-feeds: an example of a cell-mediated hypersensitivity reaction in the gut?

The increasing use of alternative protein feed ingredients in formulated feeds for carnivorous fish species requires vigilance regarding effects on intestinal health and hence, general fish health and welfare. Soybean meal (SBM) causes an inflammatory response in the distal intestine of salmonids. Aetiological component(s) of full-fat and extracted SBM have not been identified. The enteropathy severely limits SBM's use as an alternative protein source to fishmeal. The pathogenic mechanisms were investigated in Atlantic salmon, Salmo salar L., post-smolts (mean initial body weight 172 g) in seawater fed extracted SBM (25% inclusion level). These were compared to fish fed a control diet containing fishmeal (FM) as the sole protein source. The trial lasted 3 weeks and both diets were with and without the inclusion of a broad-spectrum antibiotic, oxytetracycline (OTC), to evaluate the involvement of gut microflora in the development of the enteropathy. Immunohistochemical reactivity of markers for putative T cells (anti-cluster of differentiation 3-epsilon [CD3?] of the T-cell receptor [TCR] complex) and immunoglobulin (anti-salmonid IgM) as well as programmed cell death (apoptosis; anti-caspase 3), protein degenerative/regenerative processes (anti-heat shock protein 70; HSP70), and proliferation (anti-proliferative cell nuclear antigen; PCNA), were investigated in cryosections or formalin-fixed, paraffin-embedded tissue sections from the distal intestine. Real-time (rt) PCR with primers for the T cell markers CD3??, CD4, CD8?, and CD8? was also carried out on RNA extracted from thymus and distal intestine homogenates. The Dako A0452 anti-human CD3? appeared to react to putative T cells in salmon since characteristic staining was observed in cells of the thymus as well as tissues with immunosurveillance function: distal intestine, skin, gills, and spleen. These cells were IgM negative. A dramatic increase in the number of CD3?-positive cells was observed in the SBM-fed fish. Real-time PCR confirmed the RNA expression of CD3??, CD4, CD8?, and CD8? in thymus and distal intestine as well as up-regulation in the distal intestine of SBM-fed fish. Intestinal intraepithelial lymphocytes (IEL) at the base of the epithelium were CD3? positive. Increased numbers of epithelial cells reactive to anti-caspase-3, HSP70, and PCNA were observed in salmon with SBM-induced enteropathy. OTC inclusion did not influence the development of the enteropathy in this study. The results indicate that SBM-induced enteropathy in the distal intestine of Atlantic salmon is a T lymphocyte mediated (i.e. Type IV) hypersensitivity reaction. Following three weeks of feeding, the mucosa was apparently infiltrated with a mixed population of T cells: CD4-positive T helper (TH) cells and CD8-positive cytotoxic cells; assuming that these markers are specific for these differentiated T cells in salmon as they are for mammals. The enteropathy is accompanied by increased numbers of epithelial cells undergoing apoptosis, proliferation, and cellular repair processes at the apex, base, and sides of the villous folds, respectively.