Red Mark Syndrome/Cold Water Strawberry Disease: emergence in Italy and histopathological investigations.

RMS/CWSD is a severe dermatitis affecting rainbow trout (O. mykiss), firstly described in Scotland in 2003. In the present paper we document for the first time some outbreaks occurring in Italy, by providing an accurate histological description of the lesions. The disease was signalled and surveyed in two farms located in north Italy, during spring period 2011. The water temperature 9\ub0C. The percentage of affected fish ranged between 10-15%, and their size was 450 g. Fish did not present other apparent concomitant signs of diseases, and mortality was absent. The necropsy findings allowed to classify based on the severity, the skin lesions as follows: PATTERN 1, flat and small lesions, characterised by slight desquamation and rare haemorrhages; PATTERN 2, larger than the previous, further sub-divided in 2a, round, slightly protruding, with serous/fibrinous exudates, various intensity of reddening, centrally ulcerated, and 2b, irregular shape, flat or poorly protruding, scarce presence of exudates, marked reddening, centrally ulcerated; PATTERN 3, large, markedly haemorrhagic, swollen, with presence of marginal exudates and deeply ulcerated. Skin tissue samples representative of each pattern were collected, as well as internal organs (heart, liver, kidney and spleen). The specimens were fixed with 4% buffered formalin or Bouin solution, then addressed to histology. Sections were stained with H.E., Toluidine blue, Masson\u2019s Trichrome, Giemsa. Moreover immunohistochemistry (IHC) was performed using antibodies specific for: pan-cytokeratin, AE1-AE2 cytokeratins, cadherin, myeloperoxidase, lysozyme, CD3, CD68. The histological description of patterns is the following. PATTERN 1, slight loss of epidermis, with moderate necrosis, severe monocyte-lymphocyte infiltration in the dermal layer and dermal-epidermal junction, severe lymphocyte infiltration in the subcutaneous layer, involving also the muscle. PATTERN 2a, loss of epidermis in the centre of the lesion, presence of lymphocyte infiltrate and necrosis, in the dermal-epidermal junction presence of necrosis with detachment of the layers. In the dermis spongiosum presence of congestion, haemorrhages, massive infiltration of macrophages and lymphocytes, multinucleated cells, high mitotic index and necro-fibrinuous exudate. In the subcutaneous tissue presence of a monomorphic histiocyte-like infiltrate, high mitotic index. The infiltrate involves the adipose tissue displaying granulomatous aspects (panniculitis-like), and involves also the muscle. Often the infiltrate is neurotrophic. PATTERN 2b, in the epidermal layer the pattern is similar to the one observed in PATTERN 2a. In the stratum spongiosum, presence of lymphocyte infiltrate in proximity to scales and within the scale pockets, dermal necrosis, congestion and haemorrhages, presence of histiocyte-like cells. In the stratum compactum presence of a mononuclear infiltrate. In the sub-cutaneous layer evidence of a strong lymphocyte-histiocyte infiltrate, being the latter more abundant. The architecture of the skin and muscular layers is deeply modified. PATTERN 3, the lesions are similar to the previous, but are more severe in terms of ulcerative-necrotic processes affecting skin. The antibody markers did not reveal any positivity, except for cytokeratins and E cadherin at epidermis. The histopathological patterns are dominated by a severe cellular infiltrate, initially composed by lymphocytes and subsequently partially replaced by histiocytes, located in the subcutis. Histiocytosis seems to be the prevalent feature. The dynamic of the lesion proceeds from subcutus, towards the derma to the epidermis (that can be considered an epiphenomenon). It is however interesting the flogistic lesion localized in the dermal-epidermal junction, assimilable to lichenoid-dermatitis features already described in mammals