The condition is commonly referred to as “SBM-induced enteritis” (SBMIE). Recent studies revealed that a bacterial meal (BM) containing mainly Methylococcus capsulatus grown on natural gas prevents the development of SBMIE in Atlantic salmon (Romarheim et al., 2011; Romarheim et al., 2013). Protection is dose-dependent and requires somewhere in between 15 and 30% of BM in the diet. Our most recent large scale gene expression profiling of inflamed and healthy DI protected by 30% of BM outlined
candidate biomarkers of BM-mediated protection (Škugor et al., 2013). Findings from this study and published literature until now suggest that protection by BM involves immunomodulation but also improvement of the status of non-immune epithelial cells.
The aim of this study, presented at Aquaculture Europe 2013, by F. E. Reveco et al, Norwegian University of Life Sciences, was to perform in-depth investigation of the protective effects of diets with increasing amount of BM against SBMIE in Atlantic salmon.
Materials and methods
In this experiment, Atlantic salmon (273g) fed with eight experimental diets (n=50 fish/tank; 2 tanks/treatment) for 7-wk. The diets consisted of a FM based diet as a negative control diet and seven experimental diets containing 200g/kg SBM with increasing levels of BM: 25, 50, 100, 150, 200 or 300g/kg as positive control diets. The tanks were supplied with seawater (32.5g NaCl/l). Three fish per tank were sampled for studying transcriptome changes in the DI by qPCR. We have selected 25 genes as most promising biomarkers based on our latest microarray study (Škugor et al., 2013) and published studies that described their roles as relevant in SBMIE by the magnitude of their expression and roles in immunity, metabolism, and maintenance of epithelial barrier (Marjara et al., 2012).
A number of genes responded to low dietary level of BM; however, most pronounced changes were observed at 15% and higher level of BM inclusion. Of particular interest were the immunity related biomarkers.
The expression of interleukin (IL) 17D linearly increased similar to the expression pattern of properdin factor P 2 (PFC2) while non-specific cytotoxic cell receptor protein-1 (NCCRP-1) decreased with increasing levels of BM. High levels of BM induced the expression of two representatives of antimicrobial proteins, lysozyme (LYZ) and nattectin (NATTE), which were strongly down-regulated in the SBM group. The expression of mannosidase, alpha, class 2B, member 1 (MAN2B1) followed this expression pattern. MAN2B1 is a promising indicator of lysosomal function, an organelle crucially involved in antigen presentation (AP).
Alteration of AP pathways was addressed by profiling the expression of MHC class I gene; noticeable difference was observed only between the SBM (down-regulated) and 30% BM group (up-regulated).
BM changed the direction of expression of genes linked to epithelial/cell integrity and proliferation (proliferating cell nuclear antigen, arginase, calcium-activated chloride channel-2 and anti-apoptotic transmembrane protein 85).
The expression of nonspecific 5-aminolevulinate synthase, which is involved in the first step of heme biosynthesis, linearly decreased as BM increased in the diet. E3 ubiquitin-protein ligase LINCR, a gene responsive to bacterial LPS, was strongly induced by SBM but suppressed by BM. A number of genes that function in different metabolic pathways were also analyzed: gammaglutamyl hydrolase, an important player in the cellular homeostasis of folate; epidermal retinal dehydrogenase 2 (EPHD-2), a vitamin A-metabolizing enzyme; and cysteine dioxygenase type 1, a regulator of cellular cysteine concentrations. These genes were positively correlated to the BM increase in diets.
Discussions and conclusion
The histology of fish analysed in this study improved gradually with increasing levels of BM in the diet, with SBMIE no longer being observed at 200g BM/kg and higher (Romarheim et al., 2013). This change is also reflected at the transcriptomic level. A large proportion of genes that have been previously described as relevant in SBMIE showed strong correlation with either BM or SBM exposure. Marjara et al. (2012) reported strong induction of IL17A during SBMIE, which indicated involvement of T helper 17 cells in the pathogenesis. On the other side, our findings revealed highest expression of IL17D in BM and FM-fed fish, implying an important role for IL17D in the normal intestinal physiology. The expression of PFC2 further supported the notion that a certain level of physiological inflammation should be present in healthy intestine that is constantly bombarded with potentially damaging dietary antigens and pathogens. The immunomodulatory properties of BM are best illustrated by the expression pattern of LYZ and NATTE. These secreted antimicrobial proteins are probably involved in the regulation of gut microbial homeostasis while at the same time posing little threat to own tissues.
On the other side, BM effectively reduced the expression of NCCRP-1, indicative of reduced infiltration of cytotoxic cells that probably contribute to the tissue damage. Furthermore, counteracting effects of BM against SBM-induced damage were supported by the expression of several genes with putative beneficial roles in the intestinal tissue.
In conclusion, this study confirmed complex modes of BM-mediated protection against SBMIE in Atlantic salmon. We outlined several biomarkers of protection conferred by BM enriched diets. Suppression of potentially damaging immune responses was accompanied with the induction of antimicrobial effectors while at the same time, BM contributed to improved amino acid and vitamin metabolism and the integrity of epithelial barrier.