HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDIES OF PARASITE ASSOCIATED GRANULOMA DEVELOPMENT IN VISCERAL ORGANS OF GREY MULLETS (OSTEICHTHYES: MUGILIDAE) FROM SARDINIAN LAGOONS

Grey mullets (Osteichytes: Mugilidae) are cosmopolitan fish that inhabit estuaries and lagoons and are particularly important in the fisheries and economy in particular Sardinian areas. Parasitic diseases have been found to be the main health problem in these populations (1). Fish immune response against these parasites is mainly represented by chronic granuloma development (2). The aim of this work was to describe the structure of parasitic granulomas and their temporal progression in visceral organs of grey mullets by histological and immunohistochemical techniques. A total of 239 grey mullets were collected from four different Sardinian lagoons (western Mediterranean Sea) in two seasonal samplings. Fish were euthanized (Tricaine Methanesulfonate) and a complete necropsy was performed. Samples of visceral organs, where macroscopic granulomas were detected, were processed for histological examination, stained with Hematoxylin-eosin (HE), Masson's Trichrome (MT) and investigated by immunohistochemical techniques using anti-cytokeratin AE1/AE3 and anti-Vimentin antibodies. Quantitative assessment of epithelioid cells, fibroblasts and collagen component of granulomas was performed with a semiquantitative grading score system, whereas rodlet cells (RCs) and eosinophilic granular cells (EGCs) were quantified with an image analysis software (Rasband, W.S., ImageJ). Microscopical features of the lesions were analysed using Stata 11.2 software (StataCorp LP). Histopathological examination revealed two groups of different granuloma categories according to the aetiological agent (digenean trematodes or Myxosporea). Granulomas associated to metacercariae of digenean parasites revealed a higher number of EGCs (ρ=0.5197, P<0.05), whereas granulomas due to spore of Myxobolus sp. were significantly associated with a higher number of RCs (ρ=0.4296 , P<0.05). Three developmental stages were identified during the evolution of the granulomas on the basis of common histopathologic features in both parasitic groups. Early stage granulomas were characterised by an intact parasite and minimal inflammatory response. In the intermediate stage granulomas, epithelioid cells (CK AE1/AE3 positive) were evident and represented the most characteristic cells. In late stage, fibroblasts (Vimentin positive) were noticed in large numbers in the outer portion (capsule) of granulomas. At this stage, collagen fiber development showed a significant correlation with the presence of EGCs (ρ=0.4707, P<0.05). The immunitary response of Mugilidae to different classes of metazoan parasites seems to display a low specificity but, even if characterised by a common encapsulation mechanism, some differences were identified in the cell composition and associated inflammation development.