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.