Contribution on nflammation/cell proliferation markers to the study of RMS pathogenesis

Red mark syndrome (RMS) is a chronic non-lethal skin disease affecting rainbow trout (O. mykiss). It was first noticed in Scotland in 2003, spread rapidly throughout the U.K. and has also been reported in Austria, Germany, Italy, Serbia and U.S.A. Single/multiple skin lesions are macroscopically detectable on the flanks and ventral/dorsal body surfaces, ranging from small pink spots to large bright red areas. Histology shows a lymphocytic/macrophage infiltration in the dermis, enlargement of scale pockets and necrosis of the surrounding connective tissue. Advanced stages are characterised by osteoclastic resorption of scales, and lymphocytes infiltration into the subcutaneous adipose tissue reaching the underling muscle bundles. In the present study, samples of skin and internal organs from naturally infected trouts farmed in northern Italy were evaluated macroscopically and histologically; an immunohistochemical approach was also carried out aiming to study inflammatory cells recruitment and proliferation rate. The evaluation was performed considering the severity of the skin lesion according to Galeotti et al., 2011. The following markers were performed on 4 µm-thick sections from formalin/Bouin’s fixed paraffin embedded specimens: rabbit anti human CD3 (A-0452, Dako); rabbit anti rainbow trout IgT (Prof. O. Sunyer); rabbit anti salmonid HSP70 (AS05061A, Agrisera); rabbit anti human GM-CSFRα (sc-690, Santa Cruz Biotech.); rabbit anti histamine (H7403, Sigma-Aldrich); rabbit anti-IL-1 (Santa Cruz Biotech.); mouse anti PCNA (2586, Cell Signaling Technology); mouse anti AE1/AE3 Cytokeratin (M3515, Dako); mouse anti E Cadherin (M3612, Dako). Anti CD3 and IL-1 did not cross-react. Anti trout IgT marked a limited number of scattered cells in the dermis, and numerous cells at the base of the intestinal mucosae. HSP70 positive cells were detectable within scale pockets involved in inflammatory changes. Numerous GM-CSFRα positive macrophage like-cells and some positive fibroblasts were scattered in the spongiosum derma, especially surrounding the scales. Anti-Cytokeratin and E Cadherin marked the epithelial cells. PCNA positive cells have been semiquantitatively scored (0=absence of staining; 1=up to 25% or 2=25-50% or 3=50-75% or 4=>75% positive cells) separately in epidermis, dermis and hypodermis. In the two latter mesenchymal areas PCNA positive cells were apart scored in infiltrating lymphocytes, vascular endothelium and stromal fibroblasts. Data were compared by the nonparametric Spearman correlation test among lesions macroscopically graded as initial, intermediate and severe. Any difference in proliferation was found in epidermis and in the 3 dermal components (3 compared groups). In hypodermis any reaction was apparent in endothelium and in fibroblasts but only lymphocytes were PCNA immuno-reactive. Statistic revealed, only for these latter, an increasing percentage of proliferating lymphocytes from initials to severe lesions.