Flavio Pironi

Immunocytochemical localization of piscidin in mast cells of infected seabass gill.

Annual losses of approximately 5-10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worms attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.

HISTOPATHOLOGY AND ULTRASTRUCTURE OF PLATICHTHYS FLESUS NATURALLY INFECTED WITH ANISAKIS SIMPLEX S.L. LARVAE (NEMATODA: ANISAKIDAE)

The histopathology, ultrastructure, and immunohistochemistry of the alimentary canal of flounder Platichthys flesus (L.), naturally infected with the nematode Anisakis simplex s.l. (Rudolphi 1809) from the River Forth (Scotland), were investigated and described. Eight of the 16 flounders were infected with A. simplex s.l. larvae (L3); parasites were encapsulated by serosa on the external surface of the hosts digestive tract (intensity of infection 1–8 parasites per host), although nematode larvae were found encysted under the peritoneal visceral serosa of the host spleen and liver and, occasionally, in the liver parenchyma (intensity of infection 3–10 parasites per host). In all sites, different structural elements were recognized within the capsule surrounding larvae. Among the epithelial cells of the intestine of 5 flounders with larvae encysted on external surface of the gut, the presence of several rodlet cells (RCs) was observed. Furthermore, often the occurrence of macrophage aggregates (MAs) was noticed in infected liver and spleen, mainly around the parasite larvae. Eight neuropeptide antisera were tested with immunohistochemistry methods on gut sections of 4 P. flesus infected with extraintestinal nematodes. Sections from the gut of 5 uninfected flounder were used for comparative purposes. In the tunica mucosa of parasitized P. flesus, several endocrine epithelial cells were immunoreactive to anti-CCK-39 (cholecystokinin 39) and -NPY (neuropeptide Y) sera; furthermore, in the myenteric plexus, a high number of neurons were immunoreactive to antibombesin, -galanin, and several to -NPY and -PHI (peptide histidine isoleucine) sera.

Cell types and structures involved in tench, Tinca tinca (L.), defence mechanisms against a systemic digenean infection

Histopathological and ultrastructural investigations were conducted on 36 tench, Tinca tinca (L.), from Lake Trasimeno (Italy). The gills, intestine, liver, spleen, kidney and heart of 21 individuals were found to harbour an extensive infection of larvae of an unidentified digenean trematode. The eyes, gonads, swim bladder and muscles were uninfected. The parasites in each tissue type were embedded in a granulomatous proliferation of tissue, forming a reactive fibroconnective capsule around each larva. Most of the encysted larvae were metacercariae, in a degenerative state, but on occasion some cercariae were found. Many of the granulomas were either necrotic or had a calcified core. Within the granuloma of each, the occurrence of granulocytes, macrophages, rodlet cells and pigment-bearing macrophage aggregates was observed. Hearts bore the highest parasitic infection. Whilst the presence of metacercariae within the intestine was found positioned between the submucosa and muscle layers, metacercariae in the liver were commonly found encysted on its surface where the hepatocytes in close contact with the granuloma were observed to have electron-lucent vesicles within their cytoplasm. Metacercariae encysting adjacent to the cartilaginous rods of gill filaments were seen to elicit a proliferation of the cartilage from the perichondrium. Rodlet cells, neutrophils and mast cells were frequently observed in close proximity to, and within, infected gill capillaries. Given the degenerated state of most granulomas, a morphology-based identification of the enclosed digeneans was not possible.

The response of intestinal mucous cells to the presence of enteric helminths: their distribution, histochemistry and fine structure.

Histochemical and ultrastructural investigations were conducted on the mucous cells of the intestine of brown trout, Salmo trutta L., naturally infected with the cestode Cyathocephalus truncatus (Pallas, 1781) and the acanthocephalan Echinorhynchus truttae Shrank, 1788. A subpopulation of 45 S. trutta were examined of which 15 specimens harboured E. truttae, 15 of which were infected with C. truncatus and 15 fish, the control group, were uninfected. In histological sections, hyperplasia and hypertrophy of the mucous cells were evident at the site of parasite infection. Enhanced mucus secretion was also recorded in infected fish. The number of mucous cells close to the site of parasite attachment within the intestine was significantly higher than the number detected in uninfected individuals and in infected individuals at sites 1 cm or greater from the point of parasite attachment. There were no significant differences between the number of mucous cells found at the latter two sites. Alcian blue and periodic acid-Schiffs staining of representative histological sections revealed a significant increase in the number of mucous cells staining positively for acid glycoconjugates compared to the number of cells found in the intestines of uninfected S. trutta. In transmission electron microscopy sections, each mucous cell typically possessed an elongated, basally positioned nucleus. The cytoplasm was observed to possess numerous electron dense and lucent vesicles, in addition to well-developed rough endoplasmic reticulum, Golgi apparatus and a few round mitochondria.

Piscidins in the intestine of European perch, Perca fluviatilis, naturally infected with an enteric worm

This study set out to determine how an enteric parasite, the thorny-headed worm Acanthocephalus lucii, affected the expression of antimicrobial peptides (piscidins) in its host population, the European perch (Perca fluviatilis) collected from Lake Piediluco in Central Italy. A total of 87 perch were examined; 44 (50.5%) were infected with A. lucii (1-18 worms fish(-1)). Pathological changes and immune response were assessed using histological, ultrastructural and immunohistochemical techniques. The acanthocephalans only penetrated the surficial zone of the intestinal wall and induced only slight inflammation. The main damage was destruction of the mucosal epithelium covering the villi adjacent to the parasites attachment site, and included necrosis and degeneration. Infected intestine had numerous mast cells (MCs), often in close proximity to, and within, the capillaries, and were associated with fibroblasts of the submucosal layer. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bounded granules. Immunostaining of intestine with antibodies against the antimicrobial peptides piscidin 3 and piscidin 4 showed subpopulations of MCs that were positive. Piscidin-positive MCs were mainly observed among the epithelial cells of the intestine, but also within the submucosa. In both uninfected and parasite-infected perch, the number of MCs positive for piscidin 4 was higher than those immunoreactive with piscidin 3 (p < 0.05). For both piscidins, there was no significant difference in the number of positive MCs between parasite-infected and uninfected intestine (p > 0.05). However, uninfected fish showed higher immunostaining intensity for piscidin 3 than infected conspecifics (p < 0.05).

The origin and function of cement gland secretion in Pomphorhynchus laevis (Acanthocephala).

Cement gland protein in male and inseminated female individuals of an acanthocephalan parasite of fish, Pomphorhynchus laevis (Müller, 1776), was localized by immunohistochemistry using an antibody specific for cement protein. Male P. laevis possess 3 pairs of round to oval cement glands ranging from 0.5 to 0.9 mm in length and from 0.3 to 0.7 mm in width. Each gland has an outer portion containing nuclear fragments and other cellular organelles surrounding a space for storage of gland products. Very little work has been carried out on the nature of the cement gland secretions. We have previously reported that the major component of cement is a protein with molecular weight of 23 kDa; in fresh glands it is white in colour. Immunohistochemical studies herein reported were carried out using a polyclonal antibody raised against purified P. laevis p23 cement protein (anti-p23PL). Localization of p23 cement protein at the light microscope level, by means of the anti-p23PL antibody, shows that p23 is present within the cytoplasmic layer of the gland as well as in the gland duct lumen. Interestingly, the p23 cement protein was also identifiable at the posterior ends of females retaining the cap. Positivity to anti-p23PL antibody was obtained not only in the external part of the copulatory cap, but also within the vaginal tract and at the base of the uterine duct. Thus, we report herein the first photographic evidence that the copulatory cap is not a simple gonopore lid but it is really an intravaginal plug.

Liver of the fish Gymnotus inaequilabiatus and nematode larvae infection: Histochemical features and expression of proliferative cell nuclear antigen

Histopathological lesions due to third-larval stage of nematode Brevimulticaecum sp. within the liver of a subpopulation of 31 Gymnotus inaequilabiatus from the Pantanal Region (Brazil) were studied with histochemical and immunohistochemical methods. In 93.5% of fish, livers harboured nematode larvae and the intensity of infection ranged from 8 to 293. In livers with highest number of larvae, the hepatic tissue was occupied primarily by the nematodes. Each larva was encircled by focal inflammatory granulomatous reaction. Within the thickness of the granuloma, three concentric layers were recognized: an inner layer of densely packed epithelioid cells, a middle layer of mast cells (MCs) entrapped in a thin fibroblast-connective mesh and an outer layer of fibrous connective tissue with fibroblasts. Epithelioid cells and fibroblasts within the thickness of the granuloma wall were positive for proliferative cell nuclear antigen (PCNA). Moreover, several hepatocytes in infected liver were immunoreactive to PCNA. Occurrence of rodlet cells and MCs in parenchyma, in close proximity to the encysted nematode larvae and near the blood vessel of infected liver, was observed. Macrophage aggregates (MAs) were numerous within the granulomas and scattered in parenchyma of the infected liver. High quantity of haemosiderin was encountered in MAs and hepatocytes of infected liver.