Immunoreactivity of red mark syndrome (RMS) trout skin to TLR5

RMS is an inflammatory skin disorder of farmed rainbow trout (Oncorhynchus mykiss) occurring in several European countries and which aetiology has been widely discussed. Recently specific investigations identified a Rickettsia-like-organisms (RLO)-related DNA or a Midichloria-like organisms (MLO)-related DNA in tissues and detected intracytoplasmic microorganisms morphologically consistent with bacteria belonging to Rickettsiales order. The aim of this study was to evaluate the tissue immunoreactivity of Toll-like receptor 5 (TLR5), a receptor for bacterial flagellin that plays a critical role in early innate immunity. Methodology: Skin tissues from 17 trout with RMS and 5 unaffected trout were analysed by immunohistochemistry to determine the labelling expression of TLR5 using a rabbit polyclonal anti-TLR5 antibody (Sigma-Aldrich) with an avidin–biotin–peroxidase-complex (ABC) technique. A blocking peptide-based protocol for TLR5 was used as negative control. Labelling was evaluated by one author semi-quantitatively based on the percentage of immunopositive cells and staining intensity to obtain an immunoreactivity score. A total of 10 HPF fields were assessed for each sample. Scores from 1 to 4 related to a low expression, scores from 5 to 8 as intermediate whereas scores greater than 8 represented high expression. Results: Trout with RMS showed a diffuse and intense immunoreactivity of TLR5 both in the epidermis and in the layers involved by inflammatory infiltrates. Especially macrophages were strongly immunolabeled. A significant difference in TLR5 expression between RMS and normal skin was observed since the expression score in trout with RMS had a mean value of 9,9 whereas in normal skin it was 3,6. Conclusions: In the veterinary literature several biomolecular studies on fish response to bacterial diseases demonstrated an overexpression of TLR5. Also our research confirms an up-regulation of TLR5 in the affected tissues compared to the controls and suggests that TLR5 could have an important role for the trout innate immune response versus the RMS etiological agent. A recent study demonstrated the presence of a flagellar apparatus in ‘Candidatus Midichloria mitochondrii’, a member of the order Rickettsiales, thus the evidence in trout with RMS of bacteria belonging to Midichloraceae might be associated to TLR5 activation.