Histopathology and the inflammatory response of European perch, Perca fluviatilis muscle infected with Eustrongylides sp. (Nematoda)

Abstract

Background: The European perch, Perca fluviatilis L. is a common paratenic host of dioctophymatid nematodes belonging to the genus Eustrongylides. In this host, once infected oligochaetes, which serve as the first intermediate host, are ingested, Eustrongylides migrates through the intestine and is frequently encountered within the musculature, free within the body cavity, or encapsulated on the viscera. The current study details the first Italian record of Eustrongylides sp. with larvae reported in the muscle of P. fluviatilis.

Methods: Uninfected and nematode-infected muscle tissues of perch were fixed and prepared for histological evaluation and electron microscopy. Some sections were subjected to an indirect immunohistochemical method using anti-PCNA, anti-piscidin 3 and anti-piscidin 4 antibodies.

Results: A total of 510 P. fluviatilis (TL range 15-25 cm) from Lake Trasimeno, Perugia were post-mortemed; 31 individuals had encysted nematode larvae within their musculature (1-2 worms fish(-1)). Histologically, larvae were surrounded by a capsule with an evident acute inflammatory reaction. Muscle degeneration and necrosis extending throughout the sarcoplasm, sarcolemmal basal lamina, endomysial connective tissue cells and capillaries was frequently observed. Within the encapsulating reaction, macrophage aggregates (MAs) were seen. Immunohistochemical staining with the proliferating cell nuclear antigen (PCNA) revealed numerous PCNA-positive cells within the thickness of the capsule and in the immediate vicinity surrounding Eustrongylides sp. larvae (i.e. fibroblasts and satellite cells), suggesting a host response had been initiated to repair the nematode-damaged muscle. Mast cells (MCs) staining positively for piscidin 3, were demonstrated for the first time in response to a muscle-infecting nematode. The piscidin 3 positive MC's were seen principally in the periphery of the capsule surrounding the Eustrongylides sp. larva.

Conclusions: A host tissue response to Eustrongylides sp. larvae infecting the musculature of P. fluviatilis was observed. Numerous fibroblasts, MAs and MCs were seen throughout the thick fibroconnectival layer of the capsule enclosing larvae. PCNA positive cells within the capsule suggest that host repair of nematode damaged muscle does occur, while the presence of the antimicrobial peptide piscidin 3 is shown for the first time. This is first report of Eustrongylides sp. in an Italian population of P. fluviatilis.

Figure 1

An encysted Eustrongylides sp. larva (arrow) within the epiaxial muscle of Perca fluviatilis L.

Figure 2

Alcian blue and periodic acid Schiff (PAS) stained histological sections through the skeletal muscle of Perca fluviatilis L. (a) Larval specimens of Eustrongylides sp. (arrow heads) within the muscle of P. fluviatilis where the nematode has replaced large portions of the skeletal muscle resulting in compression (arrow); scale bar = 200 μm. (b) PAS-positive macrophage aggregates (arrows) and multifocal fibro-epithelioid granulomata (arrow heads) are evident within the thickness of the capsule enclosing the Eustrongylides sp. larva. Note the PAS-positive necrotic material (asterisk); scale bar = 100 μm. (c) Histological section through nematode-infected muscle that has been stained with a PCNA-antibody. Positive fibroblasts (arrows) are scattered among the collagen fibres. Part of the nematode (asterisk) can be seen compressing the muscle tissues; scale bar = 20 μm. (d) PCNA-positive satellite cells (arrows) in the outer part of the myotubes (asterisk) can be seen; scale bar = 10 μm.

Figure 3

Transmission electron micrographs of Eustrongylides-infected Perca fluviatilis L. muscle. (a) Low magnification of the macrophages aggregates (MAs) in association with the Eustrongylides sp. larvae. The MAs appeared as group of large oval to round shaped cells (arrows) with a vacuolated and foamy appearance to their cytoplasm; scale bar = 3.3 μm. (b) Micrograph shows a macrophage aggregate with an eccentric polar nucleus (arrow) with marginal accumulations of chromatin. There are also cytoplasmic inclusions of various differing electron-densities, scale bar = 0.7 μm. (c) Two fibroblasts within the thickness of the capsule enclosing the larvae of the Eustrongylides sp., which are characterised by possessing elongated nuclei (arrows) and narrow cytoplasm, scale bar = 2.2 μm. (d) High magnification of a satellite cell beneath the basal lamina of the myofibres (asterisk) which possesses an elongated nucleus (arrow) and scarce-narrow cytoplasm; scale bar = 0.9 μm.