Massimo Lorenzoni

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.

Intestinal immune response of Silurus glanis and Barbus barbus naturally infected with Pomphorhynchus laevis (Acanthocephala).

Immunopathological and ultrastructural studies were conducted on the intestine of barbel Barbus barbus and sheatfish Silurus glanis that were naturally infected with the acanthocephalan Pomphorhynchus laevis. Enteric helminths often cause inflammation of the digestive tract, inducing the recruitment of different types of immune cells at the site of infection. The results of our study clearly demonstrated that mast cells (MC) were the dominant immune cells which occur at the site of inflammation in both hosts. MC were associated with fibroblasts and were found in close proximity to, and inside, the capillaries of the intestine, thus, migration of mast cells via the bloodstream was suggested. Significant degranulation of MC was present. Immunohistochemical staining revealed met‐enkephalin and serotonin (5‐HT) in intestinal MC of both uninfected and infected barbel and the absence of the antimicrobial peptides piscidin 3 and piscidin 4 in both species. Data are discussed with respect to host immune response to an intestinal helminth and compared with other host‐parasite systems.

Proliferative cell nuclear antigen (PCNA) expression in the intestine of Salmo trutta trutta naturally infected with an acanthocephalan

BackgroundChanges in the production of proliferating cell nuclear antigen (PCNA), a 36 kd protein involved in protein synthesis, within intestinal epithelia can provide an early indication of deviations to normal functioning. Inhibition or stimulation of cell proliferation and PCNA can be determined through immunohistochemical staining of intestinal tissue. Changes in the expression of PCNA act as an early warning system of changes to the gut and this application has not been applied to the fields of aquatic parasitology and fish health. The current study set out to determine whether a population of wild brown trout, Salmo trutta trutta (L.) harbouring an infection of the acanthocephalan Dentitruncus truttae Sinzar, 1955 collected from Lake Piediluco in Central Italy also effected changes in the expression of PCNA.MethodsA total of 29 brown trout were investigated, 19 of which (i.e. 65.5%) were found to harbour acanthocephalans (5–320 worms fish-1). Histological sections of both uninfected and infected intestinal material were immunostained for PCNA.ResultsThe expression of PCNA was observed in the epithelial cells in the intestinal crypts and within the mast cells and fibroblasts in the submucosa layer which is consistent with its role in cell proliferation and DNA synthesis. The number of PCNA-positive cells in both the intestinal epithelium and the submucosa layer in regions close to the point of parasite attachment were significantly higher than the number observed in uninfected individuals and in infected individuals in zones at least 0.7 cm from the point of parasite attachment (ANOVA, p < 0.05).ConclusionsAn infection of the acanthocephalan D. truttae within the intestinal tract of S. t. trutta effected a significant increase in the number of PCNA positive cells (mast cells and fibroblasts) at the site of parasite attachment when compared to the number of positive cells found in uninfected conspecifics and in tissue zones away from the point of parasite attachment.

Histological damage and inflammatory response elicited by Monobothrium wageneri (Cestoda) in the intestine of Tinca tinca (Cyprinidae)

BackgroundAmong the European cyprinids, tench, Tinca tinca (L.), and the pathological effects their cestodes may effect, have received very little or no attention. Most literature relating to Monobothrium wageneri Nybelin, 1922, a common intestinal cestode of tench, for example, has focused on aspects of its morphology rather than on aspects of the host-parasite interaction.ResultsImmunopathological and ultrastructural studies were conducted on the intestines of 28 tench, collected from Lake Piediluco, of which 16 specimens harboured tight clusters of numerous M. wageneri attached to the intestinal wall. The infection was associated with the degeneration of the mucosal layer and the formation of raised inflammatory swelling surrounding the worms. At the site of infection, the number of granulocytes in the intestine of T. tinca was significantly higher than the number determined 1 cm away from the site of infection or the number found in uninfected fish. Using transmission electron microscopy, mast cells and neutrophils were frequently observed in close proximity to, and inside, the intestinal capillaries; often these cells were in contact with the cestode tegument. At the host-parasite interface, no secretion from the parasites tegument was observed. Intense degranulation of the mast cells was seen within the submucosa and lamina muscularis, most noticeably at sites close to the tegument of the scolex. In some instances, rodlet cells were encountered in the submucosa. In histological sections, hyperplasia of the mucous cells, notably those giving an alcian blue positive reaction, were evident in the intestinal tissues close to the swelling surrounding the worms. Enhanced mucus secretion was recorded in the intestines of infected tench.ConclusionsThe pathological changes and the inflammatory cellular response induced by the caryophyllidean monozoic tapeworm M. wageneri within the intestinal tract of an Italian population of wild tench is reported for the first time.

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.

Growth and reproduction of the goldfish Carassius auratus: a case study from Italy

The goldfish Carassius auratus (Linnaeus) is a scaly, high-bodied, laterally compressed fish; its mouth is small and terminal, without barbels. The dorsal fin is long with a slightly serrated third spine. This species is very similar to the crucian carp Carassius carassius (Linneaus), but is more elongated and has a slightly concave dorsal fin and slightly larger scales (Lelek 1987). There has been considerable confusion concerning the taxonomic status of C. auratus. Many authors have recognized two subspecies in its native range: C. a. auratus (goldfish, Chinese goldfish, or Asian goldfish) from Asia, and Carassius auratus gibelio Bloch (Prussian carp, gibele carp, or European goldfish) from Eastern Europe (Hanfling et al. 2005). Howells (1992, in Nico and Schofield 2006) reported that goldfish typically observed in the US waters are crucian carp goldfish hybrids. Goldfish commonly hybridises with the carp Cyprinus carpio Linnaeus, giving rise to individuals that are intermediate in morphology between the two parent species. Recent studies have indicated Gherardi: Biological Invaders in Inland Waters ch13 Page Proof page 259 13.4.2007 3:58pm Compositor Name: PDjeapradaban

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).

Perch liver reaction to Triaenophorus nodulosus plerocercoids with an emphasis on piscidins 3, 4 and proliferative cell nuclear antigen (PCNA) expression

Histopathological lesions caused by plerocercoids of Triaenophorus nodulosus within the liver of perch, Perca fluviatilis, from Lake Trasimeno were studied. Livers harbored 1-3 parasite larvae and pathological alterations were more marked in those with 3 plerocercoids. In the liver, larvae were encysted, surrounded by a capsule of host tissue; two of 14 plerocercoids were necrotic. In infected livers, some hepatocytes showed degenerative changes, i.e. swelling and hydropic degeneration, notably those in close proximity to larvae. By comparison, hepatocytes in uninfected livers or in regions away from the point of infection appeared normal. The occurrence of macrophage aggregates (MAs) distributed among the mast cells (MCs) was observed around the encysted larvae. The cellular elements involved in the immune response within liver were assessed by immunohistochemical techniques and by the use of antibodies against the antimicrobial peptides piscidins 3 and 4, which revealed a sub-population of positive MCs. In infected livers, numerous MCs that were immunopositive to P4 and a few that were positive to P3 were found around T. nodulosus larvae. Histological sections of both uninfected and infected liver were immunostained with proliferative cell nuclear antigen (PCNA) antibody. Within the capsule and in close proximity to the parasite larvae, various cell types (i.e., MCs, fibroblasts and epithelioid cells) and a significantly higher number of PCNA-positive hepatocytes that were immunoreactive to PCNA were found compared to uninfected livers (ANOVA, P<0.05). No parasites of any type were found in gill, spleen, kidney or gonad of P. fluviatilis and the intestine of 3 perch were infected with few specimens of Acanthocephalus lucii.

Fine structure and cellular responses at the host–parasite interface in a range of fish–helminth systems

A series of ultrastructural-based studies were conducted on the interface region in different fish-helminth systems: (a) an intestinal infection of the cestode Monobothrium wageneri in tench, Tinca tinca; (b) an extensive intestinal submucosa and mucosal infection in tench by metacercariae of an unidentified digenean trematode; (c) an intestinal infection in brown trout, Salmo trutta, by the acanthocephalan Dentitruncus truttae; (d) an extraintestinal infection by larvae of the acanthocephalan, Pomphorhynchus laevis in three-spined sticklebacks, Gasterosteus aculeatus; and (e) an infection in the livers of Eurasian minnow, Phoxinus phoxinus, by larvae of the nematode Raphidascaris acus. Endoparasitic helminths frequently cause inflammation of the digestive tract and associated organs, inducing the recruitment of various immune cells to the site of infection. In each of the fish-helminth systems that were studied, a massive hyperplastic granulocyte response involving mast cells (MCs) and neutrophils in close proximity to the helminths was documented. The current study presents data on the interface region in each fish-helminth system and documents the penetration of mast cells granules within the tegument of P. laevis larvae. No extracellular vesicles containing tegumental secretions from any of the four different taxa of endoparasitic helminths species at the host-parasite interface region were seen.

Innate immune defence mechanisms of tench, Tinca tinca (L.), naturally infected with the tapeworm Monobothrium wageneri

A histochemical and ultrastructural investigation of the cellular inflammatory response within the intestines of tench Tinca tinca L. naturally infected with the caryophyllidean cestode Monobothrium wageneri was conducted and the data obtained compared to those in uninfected counterparts. Cestode infections within the intestines were evident through the appearance of raised inflammatory swellings induced by the deep penetration of their scolices into the intestinal wall. Cestodes typically attached in tight clusters, inducing a massive hyperplastic granulocyte response of mast cells and neutrophils, which were significantly more numerous (P < 0·01) in the intestines of infected (n = 14) than of uninfected (n = 9) tench. Neutrophils were more abundant than mast cells (P < 0·01) in host tissues in close proximity to the parasite tegument. In transmission electron microscopy sections, mast cells and neutrophils were frequently observed in contact with or inside capillaries, and in close proximity to the cestode. Degranulation of both cell types was seen in the submucosa and lamina muscularis, notably in the immediate tissues surrounding the scolex of M. wageneri. No tegumental secretions were seen at the host–parasite interface. Occasional rodlet cells were encountered in the submucosa of infected fish.