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Given the abundance, heterogeneity and ubiquity of parasitic organisms, understanding how they influence biodiversity, evolution, health and ecosystem functionality is crucial, especially currently when anthropogenic pressures are altering host–parasite balances. This review describes the features, roles and impacts of metazoan parasites of fish occurring in transitional waters (TW). These aquatic ecosystems are highly productive and widespread around the globe and represent most favourable theatres for parasitism given the availability of hosts (invertebrates, fishes and birds) and an increased probability of parasite transmission, especially of those having complex life cycles. Fascinating examples of how parasitism can influence different hierarchical levels of biological systems, from host individuals and populations to entire aquatic communities, through effects on food webs come from this kind of ecosystem. Edible fish of commercial value found in TW can harbour some parasite species, significantly reducing host health, marketability and food safety, with possible economic and public health consequences. Many TW are historically exploited by humans as sources of relevant ecosystem services, including fisheries and aquaculture, and they are highly vulnerable ecosystems. Alteration of TW can be revealed through the study of parasite communities, contributing, as bioindicators, for assessing environmental changes, health and restoration. Fish parasites can provide much information about TW, but this potential appears to be not fully exploited. More studies are necessary to quantify the ecological, economic and medical impacts fish parasites can have on these important ecosystems.

Defence mechanisms of fish can be divided into specific and non-specific that act in concert and are often interdependent. Most fish in both wild and cultured populations are vulnerable to metazoan parasites. Endoparasitic helminths include several species of digeneans, cestodes, nematodes, and acanthocephalans. Although they may occur in large numbers, helminth infections rarely result in fish mortality. Conversely, some ectoparasites cause mass mortality in farmed fish. Given the importance of fish innate immunity, this review addresses non-specific defence mechanisms of fish against metazoan parasites, with emphasis on granulocyte responses involving mast cells, neutrophils, macrophages, rodlet cells, and mucous cells. Metazoan parasites are important disease agents that affect wild and farmed fish and can induce high economic loss and, as pathogen organisms, deserve considerable attention. The paper will provide our light and transmission electron microscopy data on metazoan parasites-fish innate immune and neuroendocrine systems. Insights about the structure and functions of the cell types listed above and a brief account of the effects and harms of each metazoan taxon to specific fish apparati/organs will be presented., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The Northern pike, Esox lucius, is a large, long-lived, top-predator fish species and occupies a broad range of aquatic environments. This species is on its way to becoming an important model organism and has the potential to contribute new knowledge and a better understanding of ecology and evolutionary biology. Very few studies have been done on the intestinal pathology of pike infected with helminths. The present study details the first Italian record of adult Acanthocephalus lucii reported in the intestine of E. lucius., Results: A total of 22 pike from Lake Piediluco (Central Italy) were examined, of which 16 (72.7%) were infected with A. lucii. The most affected areas of gastrointestinal tract were the medium and distal intestine. The intensity of infection ranged from 1 to 18 parasites per host. Acanthocephalus lucii penetrated mucosal and submucosal layers which had a high number of mast cells (MCs) with an intense degranulation. The cellular elements involved in the immune response within the intestine of pike were assessed by ultrastructural techniques and immunohistochemistry using antibodies against met-enkephalin, immunoglobulin E (IgE)-like receptor (FCεRIγ), histamine, interleukin-6, interleukin-1β, substance P, lysozyme, serotonin, inducible-nitric oxide synthase (i-NOS), tumor necrosis factor-α (TNF-α) and the antimicrobial peptide piscidin 3 (P3). In intestines of the pike, several MCs were immunopositive to 9 out of the 11 aforementioned antibodies and infected fish had a higher number of positive MCs when compared to uninfected fish., Conclusions: Pike intestinal tissue response to A. lucii was documented. Numerous MCs were seen throughout the mucosa and submucosal layers. In infected and uninfected intestines of pike, MCs were the dominant immune cell type encountered; they are the most common granulocyte type involved in several fish-helminth systems. Immunopositivity of MCs to 9 out of 11 antibodies is of great interest and these cells could play an important key role in the host response to an enteric helminth. This is the first report of A. lucii in an Italian population of E. lucius and the first account on positivity of MCs to piscidin 3 and histamine in a non-perciform fish.

The histopathology and ultrastructure of the intestine of mullets, Liza ramada and Liza saliens, from Comacchio lagoons (northern Italy) naturally infected with myxozoans and helminths were investigated and described. Sixty-two (80.5%) of 77 mullets harboured one or more of the following parasites species: Myxobolus mugchelo (Myxozoa), Neoechinorhynchus agilis (Acanthocephala), Haplosplanchnus pachysomus and Dicrogaster contractus (Digenea). Co-occurrence of helminths with myxozoans was common. The main damage caused by digeneans was destruction of the mucosal epithelium of the villi, necrosis and degeneration of intestinal epithelial cells. More severe intestinal damage was caused by acanthocephalans which reach the submucosa layer with their proboscis. At the site of helminths infection, several mast cells (MCs), rodlet cells (RCs), mucous cells and few neutrophils and macrophages were observed in the epithelium. RCs and mucous cells exhibited discharge activity in close vicinity to the worm's tegument. M. mugchelo conspicuous plasmodia were encysted mainly in muscle and submucosa layers of the intestine. Indeed, spores of M. mugchelo were documented within the epithelial cells of host intestine and in proximity to MCs. Degranulation of the MCs near the myxozoans was very frequent., (© 2017 John Wiley & Sons Ltd.)

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., (© 2017 John Wiley & Sons Ltd.)

Histopathological, immunofluorescence and ultrastructural studies were conducted on the intestines of four fish species infected with different taxa of enteric helminths. Brown trout (Salmo trutta trutta), eel (Anguilla anguilla) and tench (Tinca tinca) obtained from Lake Piediluco (central Italy) were examined. Brown trout and eel were infected with two species of acanthocephalans, and tench was parasitized with a tapeworm species. In addition to the above site, specimens of chub (Squalius cephalus) and brown trout infected with an acanthocephalan were examined from the River Brenta (north Italy). Moreover, eels were examined from a brackish water, Comacchio lagoons (north Italy), where one digenean species was the predominant enteric worm. All the helminths species induced a similar response, the hyperplasia of the intestinal mucous cells, particularly of those secreting acid mucins. Local endocrine signals seemed to affect the production and secretion of mucus in the parasitized fish, as worms often were surrounded by an adherent mucus layer or blanket. This is the first quantitative report of enteric worm effects on the density of various mucous cell types and on the mucus composition in intestine of infected/uninfected conspecifics. We provide a global comparison between the several fish-helminth systems examined., (© 2016 John Wiley & Sons Ltd.)

This paper provides histological and ultrastructural data on Myxobolus mugchelo (Myxozoa), a parasite of the intestinal wall of thinlip mullet Liza ramada from the Comacchio lagoons (Northern Adriatic Sea, Italy). About 80% of the examined mullets were infected with this myxozoan. In histological sections, the plasmodia appeared rounded to ovoid and spindle shaped, measured 181.61 ± 97 × 122.79 ± 53.16 μm (mean ± standard deviation, SD) in size. Early sporogenic plasmodia were located primarily in the muscle layer where no pronounced inflammatory response was detected. Plasmodia containing mature spores were situated closer to mucosal folds and were encircled by numerous immune cells, especially mast cells. Mature spores were generally oval and 6.06 ± 0.40 × 3.48 ± 0.92 μm in size. The spores showed symmetrical and smooth valves. The anterior end of the spores contained two equal pyriform polar capsules measuring 2.19 ± 0.47 × 1.59 ± 0.29 μm. Inside the polar capsules, an isofilar polar filament displayed 5-6 coils perpendicular or slightly oblique to the longitudinal axis. The wall of the polar capsule was filled with a hyaline substance contrasting with the very dense internal matrix. The mucus layer surrounded the spore surface. A thorough comparison with the Myxobolus species infecting intestine of fish is provided. This is the first report on occurrence of this myxozoan species in L. ramada population of the North Adriatic Sea.

Immunohistochemical, immunofluorescence and ultrastructural studies were conducted on a sub-population of 20 wels catfish Silurus glanis from a tributary of the River Po (Northern Italy). Fish were examined for the presence of ecto- and endo-parasites; in the intestine of 5 fish, 11 specimens of cestode Glanitaenia osculata were noted and was the only helminth species encountered. The architecture of intestine and its cellular features were nearly identical in either the uninfected S. glanis or in those harboring G. osculata. Near the site of worm's attachment, mucous cells, several mast cells (MCs), few neutrophils and some endocrine cells (ECs) were found to co-occur within the intestinal epithelium. MCs and neutrophils were abundant also in the submucosa. Immunohistochemical staining revealed that enteric ECs were immunoreactive to met-enkephalin, galanin and serotonin anti-bodies. The numbers of ECs, mucous cells and MCs were significantly higher in infected wels catfish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the biotinylated lectin Sambucus nigra Agglutinin and the rabbit polyclonal anti-met-enkephalin or anti-serotonin, with parallel transmission electron microscopy, showed that ECs often made intimate contact with the mucous cells and epithelial MCs. The presence of numerous MCs in intestinal epithelium shows S. glanis to be an interesting model fish to study processes underlying intestinal inflammation elicited by an enteric worm. Immune cells, ECs and mucous cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions together will be discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The European eel, Anguilla anguilla, is a major warm-water fish species cultured in North and South Europe. Seventy-one A. anguilla collected between 2010 and 2015 from the Comacchio lagoons were examined. Fish were infected and damaged by larvae (L3) of the nematode Contracaecum rudolphii A, which were encapsulated within the thickness of the intestinal wall and within the external visceral peritoneum (serosa). Conspicuous granulomas, visible at sites of infection, were arranged in a trilayer, formed by a series of concentric whorls. The cells involved in the immune response and their distribution in the granuloma layers were assessed by immunohistochemical, immunofluorescence, and ultrastructural techniques. The outer part of the granuloma contained macrophages, macrophage aggregates, and mast cells (MCs) scattered among fibroblasts. This layer was vascularized, with degranulation of MCs occurring in close proximity to the capillaries. The middle layer was rich in MCs and fibroblasts. The inner layer, closest to the parasite larva, consisted mainly of dark epithelioid cells, some of which were necrotic. Non-necrotic epithelioid cells formed desmosomes between themselves or with fibroblasts. Within the granulomas, numerous cells of different types were positive to proliferative cell nuclear antigen antibody, indicating a high degree of cellular proliferation around the larvae., (© 2016 John Wiley & Sons Ltd.)

Background: A survey on endoparasitic helminths from freshwater fishes in the Pantanal Region (Mato Grosso do Sul, Brazil) revealed the occurrence of third-larval stage of the nematode Brevimulticaecum sp. (Heterocheilidae) in most organs of Gymnotus inaequilabiatus (Gymnotidae) also known by the local name tuvira. The aim of the present study was to examine Brevimulticaecum sp.-infected tuvira liver at the ultrastructural level and clarify the nature of granulomas and the cellular elements involved in the immune response to nematode larvae., Methods: Thirty-eight adult specimens of tuvira from Porto Morrinho, were acquired in January and March 2016. Infected and uninfected liver tissues were fixed and prepared for histological and ultrastructure investigations., Results: The prevalence of infection of tuvira liver by the nematode larvae was 95 %, with an intensity of infection ranging from 4 to 343 larvae (mean ± SD: 55.31 ± 73.94 larvae per liver). In livers with high numbers of nematode larvae, almost entire hepatic tissue was occupied by the parasites. Hepatocytes showed slight to mild degenerative changes and accumulation of pigments. Parasite larvae were surrounded by round to oval granulomas, the result of focal host tissue response to the infection. Each granuloma was typically formed by three concentric layers: an outer layer of fibrous connective tissue with thin elongated fibroblasts; a middle layer of mast cells entrapped in a thin fibroblast-connective mesh; and an inner layer of densely packed epithelioid cells, displaying numerous desmosomes between each other. Numerous macrophage aggregates occurred in the granulomas and in the parenchyma., Conclusions: Our results in tuvira showed that the larvae were efficiently sequestered within the granulomas, most of the inflammatory components were confined within the thickness of the granuloma, and the parenchyma was relatively free of immune cells and without fibrosis. Presumably this focal encapsulation of the parasites permits uninfected portions of liver to maintain its functions and allows the survival of the host.

Most individual fish in farmed and wild populations are infected with parasites. Upon dissection of fish, helminths from gut are often easily visible. Enteric helminths include several species of digeneans, cestodes, acanthocephalans and nematodes. Some insights into biology, morphology and histopathological effects of the main fish enteric helminths taxa will be described here. The immune system of fish, as that of other vertebrates, can be subdivided into specific and aspecific types, which in vivo act in concert with each other and indeed are interdependent in many ways. Beyond the small number of well-described models that exist, research focusing on innate immunity in fish against parasitic infections is lacking. Enteric helminths frequently cause inflammation of the digestive tract, resulting in a series of chemical and morphological changes in the affected tissues and inducing leukocyte migration to the site of infection. This review provides an overview on the aspecific defence mechanisms of fish intestine against helminths. Emphasis will be placed on the immune cellular response involving mast cells, neutrophils, macrophages, rodlet cells and mucous cells against enteric helminths. Given the relative importance of innate immunity in fish, and the magnitude of economic loss in aquaculture as a consequence of disease, this area deserves considerable attention and support., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A sub-population of 34 specimens of chub, Squalius cephalus, was sampled from the River Brenta (Northern Italy) and examined for ecto- and endo-parasites. Pomphorhynchus laevis (Acanthocephala) was the only enteric helminth encountered. Immunofluorescence and ultrastructural studies were conducted on the intestines of chub. Near the site of parasite's attachment, mucous cells, mast cells (MCs), neutrophils and rodlet cells (RCs) were found to co-occur within the intestinal epithelium. The numbers of mucous cells, MCs and neutrophils were significantly higher in infected fish (Mann-Whitney U test, p < 0.05). Dual immunofluorescence staining with the lectin Dolichos Biflorus Agglutinin (DBA) and the macrophage-specific MAC387 monoclonal antibody, with parallel transmission electron microscopy, revealed that epithelial MCs often made intimate contact with the mucous cells. Degranulation of a large number of MCs around the site of the acanthocephalan's attachment and in proximity to mucous cells was also documented. MCs and neutrophils were abundant in the submucosa. Immune cells of the intestinal epithelium have been described at the ultrastructural level and their possible functions and interactions are discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: In vertebrates, the presence of enteric worms can induce structural changes to the alimentary canal impacting on the neuroendocrine system, altering the proper functioning of the gastrointestinal tract and affecting the occurrence and relative density of endocrine cells (ECs). This account represents the first immunohistochemistry and ultrastructure-based study which documents the intimate relationship between the intestinal mucous cells and ECs in a fish-helminth system, investigating the potential effects of enteric neuromodulators on gut mucus secretion/discharge., Methods: A modified dual immunohisto- and histochemical staining technique was applied on intestinal sections from both infected and uninfected fish. Sections were incubated in antisera to a range of neuromodulators (i.e. leu-enkephalin, met-enkephalin, galanin and serotonin) and the glycoconjugate histochemistry of the mucous cells was determined using a subsequent alcian blue - periodic acid Schiff staining step. Dual fluorescent staining on sections prepared for confocal laser scanning microscopy and transmission electron microscopy were also used to document the relationship between ECs and mucous cells., Results: From a total of 26 specimens of Squalius cephalus sampled from the River Paglia, 16 (i.e. 62 %) specimens were found to harbour an infection of the acanthocephalan Pomphorhynchus laevis (average intensity of infection 9.2 ± 0.8 parasites host(-1), mean ± standard error). When acanthocephalans were present, the numbers of mucous cells (most notably those containing acidic or mixed glycoconjugates) and ECs secreting leu-enkephalin, met-enkephalin, galanin, serotonin were significantly higher than those seen on sections from uninfected fish. The relationship between met-enkephalin-like or serotonin-like ECs and lectin DBA positive mucous cells was demonstrated through a dual fluorescent staining. The presence of tight connections and desmosomes between mucous and ECs in transmission electron micrographs provides further evidence of this intimate relationship., Conclusions: The presence of P. laevis induces an increase in the number of enteric ECs that are immunoreactive to leu- and met-enkephalin, galanin, and serotonin anti-sera. The mucous cells hyperplasia and enhanced mucus secretion in the helminth-infected intestines could be elicited by the increase in the number of ECs which release these regulatory substances.

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.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Granulomas caused by migration of larvae of a helminth parasite, Triaenophorus nodulosus, within the liver of perch (Perca fluviatilis) from Rimov Dam Lake (Czech Republic) were assessed by transmission electron microscopy. Lesions were found in the liver of 29 out of 34 perch examined (85.2%) and there were between 1 and 15 T. nodulosus larvae identified per host. Pathological changes were more severe in livers containing more granulomas. Within the granulomas, there were three concentric regions: an outer layer of fibrous connective tissue, a middle clear epithelioid layer and a central dark spindle cell layer. The outer layer contained mast cells, fibroblasts, thick collagen bundles and epithelioid cells. The granulomas contained few lymphocytes and macrophages. Hepatocytes adjacent to the granulomas showed pronounced degeneration (ranging from vacuolar degeneration to acute cellular swelling)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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 parasite's 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)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Immunohistochemical and ultrastructural studies were conducted on the gills of gilthead seabream, Sparus aurata L., naturally infected with the copepod ectoparasite Ergasilus lizae (Krøyer, 1863) in order to assess pathology and the host immune cell response. Gills of 56 gilthead seabream were screened for ectoparasites; 36 specimens (64.3%) harbored E. lizae. Intensity of infection was 32.7 ± 8.7 (mean ± SE). Pathological alterations to the gills of the host were more pronounced in close proximity to the copepod site of attachment. The parasite attached to the gills by means of its modified second antennae, occluded the arteries, provoked epithelial hyperplasia and hemorrhages and most often caused lamellar disruption. Numerous granular cells were encountered near the site of E. lizae attachment. In both infected and uninfected gills, the granular cells lay within the filaments and frequently occurred within the connective tissue inside and outside the blood vessels of the filaments. The type of granular cell was identified by immunohistochemical staining by using the monoclonal antibody G7 (mAb G7), which specifically recognizes acidophilic granulocytes (AGs) of S. aurata and with an anti-histamine antibody (as a marker for mast cells, MCs) on sections from 13 uninfected gills and 21 parasitized gills. The use of mAb G7 revealed that, in gills harboring copepods, the number of G7-positive cells (i.e., AGs; 32.9 ± 3.9, mean number of cells per 45,000 μm2 ± SE) was significantly higher than the density of the same cells in uninfected gills (15.3 ± 3.8; ANOVA, P < 0.05). Few histamine-positive granular cells (i.e., MCs) were found in the uninfected and parasitized gills. Here, we show, for the first time in S. aurata infected gills, that AGs rather than MCs are recruited and involved in the response to E. lizae infection in seabream.

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., (© 2012 Blackwell Publishing Ltd.)

Background: Changes 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., Methods: A 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., Results: The 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)., Conclusions: An 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.

Background: Among 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., Results: Immunopathological 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 parasite's 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., Conclusions: The 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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Light and ultrastructure studies were carried out on gill of sea bream, Sparus aurata L., naturally infected with Ergasilus sieboldi (Copepoda) to assess pathology and host cell responses. Thirty S. aurata were examined, and 23 (74%) were infected, the intensity of infection ranging from 3 to 50 parasites per host. The copepod encircled gill lamellae with its second antennae, occluded arteries, compressed the epithelium, provoked hyperplasia and haemorrhage, and often caused tissue disruption. Adjacent to the site of attachment, rodlet cells (RCs), mast cells (MCs) and mucous cells were observed. In parasitized fish, mucous cells were more abundant in infected gills than in uninfected (t-test, P<0.01), while no significant differences were encountered in the numbers of RCs and MCs between gill of infected and uninfected fish (t-test, P>0.01). In both infected and uninfected gill, the RCs were within the primary lamella and also sometimes occurred in secondary lamella. In healthy and infected gill, 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. Infected and uninfected gill mucous cells stained positively for neutral muco-substances (PAS positive). In all sea bream, gill mucous cells presented a central or eccentric electron-dense core within the mucus granules., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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-Schiff's 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.

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 worm's 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., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Immunopathological and ultrastructural studies were carried out on the gut of 30 specimens of powan Coregonus lavaretus (L.) from Lake Piediluco, Italy. The digestive tracts of 10 (33.3%) of the powan were found to harbour an acanthocephalan Dentitruncus truttae (Sinzar 1955). The numerous trunk spines of D. truttae reduced the number of mucosal folds near the parasite site of infection. The acanthocephalan induced hyperplasia and hypertrophy of the intestinal mucous cells and many worms were surrounded with an adherent mucous gel. Near the site of acanthocephalan attachment, the number of mucous cells was significantly higher (P<0.01) in comparison to those found in uninfected intestines. Rodlet cells (RCs) were present in the epithelial layer in both infected and uninfected fish, with no significant difference in the numbers observed (P>0.05). In infected intestine, mast cells were more abundant than in uninfected gut (P<0.01). Migration of the mast cells and their intense degranulation at the site of infection were suggested. Immunohistochemical tests applied to sections of intestinal tissue of both infected and uninfected powan revealed that the parasitized C. lavaretus had a larger number of mast cells positive for met-enkephalin and serotonin antisera.

Fish serve as a good model for studying vertebrate immune systems because they have a relatively simple system. Descriptions of histopathological effects of helminth parasites on fish are few and far between with limited observations made on the identification of the inflammatory cells involved in the host reaction. Recently, two cell types found within teleosts received a great deal of attention, namely mast cells and rodlet cells. Fish most cells also known as eosinophilic granule cells, are morphologically and functionally similar to their mammalian counterparts. Acute tissue damage causes mast cells degranulation and the release of mediators of inflammation, whereas, an increase in the number of these cells is usually found in chronically inflamed tissues. Rodlet cells, however, are exclusive to fish and are characterized by a distinct cell cortex and conspicuous inclusions, called rodlets, which accounts for their name. Piscidin has also been encountered within rodlet cells. As important cell types within the immune system of fish, both mast cells and rodlet cells have been seen to increase in number in infected fish, notably at the sites of pathogen attachment or infection. The present survey will provide data from studies with the light microscope on the response of mast cells and rodlet cells in a range of fish species (Anguilla anguilla, Oncorhynchus mykiss, Salmo trutta, Coregonus lavoretus) infected with a range of different parasite genera including representatives from the Digenea, Cestoda, Nematoda and Acanthocephala.

Brown trout, Salmo trutta L., were infected with the acanthocephalan Dentitruncus truttae with the most affected areas being the anterior (near the pyloric caeca) and middle intestine. The parasite attached with a proboscis which usually penetrated the mucosa, lamina propria, stratum compactum, stratum granulosum and, sometimes, the muscularis layer. Around the parasite's body was an area of inflammatory tissue. At the point of attachment the lamina propria was thickened and the stratum compactum, stratum granulosum and muscularis layer were disrupted by proboscis penetration. Rodlet cells were more numerous in infected fish (P<0.01), and were found in the epithelial layer away from the worm. Infected intestines had larger numbers of mast cells (P<0.01), often in close proximity to, and inside, the blood capillaries and associated with fibroblasts of the muscularis layer and the stratum granulosum. Their migration toward the site of infection was suggested. Intense degranulation of mast cells was encountered in all intestinal layers especially near the parasite's body. Immunohistochemical tests were conducted on sections of intestinal tissue of uninfected and infected fish revealing the presence of met-enkephalin and serotonin (5-HT) in immuno-related cells of the intestine wall. Infected trout had larger numbers of elements positive to met-enkephalin and serotonin antisera. These data provided evidence for the role of the immune system of brown trout in the modulation of the inflammatory response to D. truttae. Results are discussed with respect to host immune response to an intestinal helminth.

Scanning electron microscope (SEM) investigations on the holdfast elements, proboscis hooks, and trunk spines of Dentitruncus truttae (Acanthocephala, Palaeacanthocephala), an endoparasite of Salmo trutta (brown trout), provide more data about the surface of these taxonomic relevant structures. In both acanthocephalan sexes, the fully everted cylindrical proboscis possessed 18 longitudinal rows of hooks with 18 hooks per row (rarely 19-20). Hook length varied according to position on the proboscis; apical hooks were 40-52 microm long, middle hooks were 31.7-36.6 microm, and basal hooks were 38.1-40 microm. Starting from the anterior end of the metasoma, numerous cuticular spines (26.7-30 microm in length) were visible and their number progressively decreased posteriorly. SEM observations of D. truttae hooks and spines revealed the presence of many surface striations on each proboscis hook. These surface striations were absent from trunk spines. From the base of the hook, the striations ran parallel toward the point of convergence. Additionally, survey of longitudinal and transversal sections of the hook using transmission electron microscope confirmed that the hook surface was not smooth. SEM comparison with the hooks of several palaeacanthocephalan species, as well as with the hooks of species belonging to Eoacanthocephala and Polyacanthocephala, indicated that the striations are currently exclusive to D. truttae proboscis hooks., ((c) 2007 Wiley-Liss, Inc.)

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 host's 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.

European minnows, Phoxinus phoxinus L., are commonly infected with Diplostomum phoxini Faust, 1918 metacercariae. A sub-sample of 34 minnows collected from the River Endrick, Stirlingshire, Scotland revealed that 50% of the population were infected (n = 17), with the cerebellum, medulla oblongata and the optic lobe regions of the minnow brain bearing the heaviest infections (13.7 +/- 2.6 mean +/- S.E.; 1-38 range). Serial histological sections through the brains of both uninfected and infected minnows revealed the presence of rodlet cells in the latter, which were occasionally observed in close proximity to the tegument of a metacercaria. Rodlet cells were the only type of host inflammatory cells found in this study and their role in the host's immune response to parasitic infection is commented upon.

The pathological changes induced by an infection of Diphyllobothrium dendriticum (Nitzsch, 1824) plerocercoids in powan, Coregonus lavaretus (L.), from Loch Lomond, Scotland, were assessed using immunohistochemical and ultrastructural techniques. In a sample of 26 powan, the occurrence of encysted plerocercoids of D. dendriticum on the outer surface of the stomach was 38.5% (n = 10) with the number of cysts ranging from 4 to 15 and measuring 4.2 +/- 1.0 mm x 3.4 +/- 0.9 mm (mean +/- SD). Histological examination of intestinal samples also revealed plerocercoids (2-21) encapsulated within a proliferation of mesenteric fibrous tissues of the gastric wall and, occasionally, by the gut lamina propria-submucosa and lamina muscularis. In section, cysts were tri-layered and were formed from a series of concentric whorls of fibroblast and collagen fibre-based connective elements. The extent of necrosis within each muscle layer and the serosa of the stomach differed, notably within the latter that was marked by a chronic inflammatory reaction and fibrosis. Within the cyst and around it, a large number of degranulating mast cell/eosinophilic granule cells were seen, in addition to melano-macrophage centres. Immunohistochemical staining of sections of infected stomach revealed a high density of elements, in close proximity to plerocercoids, staining positive for serotonin, bombesin, substance P and galanin. Uninfected material did not present the same levels of activity. Sections through both infected and uninfected tissue were also tested for elements containing vasoactive intestinal peptide, met-enkephalin, calcitonin gene-related peptide, neuropeptide Y and nitric oxide synthase, but these were absent.

The spatial distribution and histopathological changes induced by metacercariae of the digenean trematode Diplostomum phoxini (Faust, 1918) in the brains of European minnows Phoxinus phoxinus (L.) from the River Endrick, Scotland, were studied by light and electron microscopy. Post-mortem examination of a sample of 34 minnows revealed that 50% (n = 17) of the population was infected with 13.7 +/- 2.6 (mean +/- SE; range 1 to 38) metacercariae per infected host. Serial histological sections of the infected minnow brains revealed that the metacercariae were unevenly distributed throughout the brain, with aggregations occurring in the cerebellum, the medulla oblongata and the optic lobes. In fish with highest intensities of infection, over 40% of the cerebellar area and about 30% of the medulla oblongata area were occupied by larvae. Metacercariae disrupt the integrity of brain tissue, with individuals being found in small pockets surrounded by cellular debris. Metacercariae were rarely encountered on the surface of the brain. Electron microscopic examination of infection sites revealed that the granular layer surrounding metacercariae was necrotic, exhibited nuclear degradation and was marked by vacuolation of the cytoplasm. Rodlet cells, the only inflammatory cell types recorded in this study, were found only in parasitized brains and in close proximity to the teguments of metacercariae. It is hypothesised that secretions released from the teguments of metacercariae are a counter response to protect the metacercariae from the fish brain's cellular defence mechanisms.

The mortality of juvenile European sea bass, Dicentrarchus labrax (L.), in the spring of the last 5 years in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans (Wagener 1857) Diesing 1858. The histopathological examination of 38 sets of gills from hosts measuring 16.46 +/- 0.26 cm in total length (mean+/-S.E.) and weighing 45.98 +/- 2.37 g (mean+/-S.E.) were conducted using light and transmission electron microscopy. Twenty-eight (73.6%) D. labrax specimens were infected (34.61 +/- 4.42, mean intensity+/-S.E.; 5-100, range) with the majority of D. aequans attaching to the median and apical portions of the primary gill filaments. The sites of attachment were marked by the common presence of haemorrhages and a white mucoid exudate. In histological sections, the opisthaptors of the parasites were observed to penetrate deeply, lying in close proximity to the basal membrane of primary lamella where they induced a hyperplastic response. Disruption and fusion of the secondary lamellae were common in all infected specimens with several individuals also exhibiting a marked erosion and inflammation of the epithelium of the primary and secondary lamellae. In infected fish, cellular changes in the epithelium underlying the bodies of worms were noted typified by an elevation in the number of mucous and rodlet cells and a reduction in the number of chloride cells.

A histopathological and immunohistochemical study on the intestines of 45 specimens of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), from Loch Awe, Scotland, revealed a number of cellular deviations in individuals naturally infected with the pseudophyllidean cestode Eubothrium crassum (Bloch, 1779). Twenty-five individuals (55.5%) were infected with an average worm burden of 18.84 +/- 4.06 (mean +/- SE) cestodes per host (range, 2-80 worms; total 471 worms). The cestodes, measuring an average 8.23 +/- 1.10 cm (mean +/- SE; range, 5.3-13.0 cm) in length, were found attached by their scolices to the mucosal lining of the distal portion of the pyloric caeca. Within the caeca, the strobila evoked a mild catarrhal enteritis, namely an enhanced mucus production with epithelial cellular desquamation, a leucocytic infiltration of the lamina propria-submucosa and vacuolization of the intestinal epithelial cells. Eosinophilic granular cells of the stratum granulosum exhibited granular depletion, while within the catarrh, the presence of a high number of rodlet cells was noticed. Immunohistochemically, the occurrence of E. crassum caused a significant reduction in the number of bombesin-, gastrin-releasing peptide and glucagon-like immunoreactive endocrine cells, but an increase in the relative densities of endocrine cells containing cholecystokinin-8- and gastrin-like substances. There were, however, no significant differences in the number of endocrine cells that were immunoreactive to secretin, neuropeptide Y and peptide histidine-isoleucine antisera in the digestive tracts of either the infected or non-infected O. mykiss.

Histopathological, ultrastructural and immunohistochemical investigations were conducted on 26 specimens of powan Coregonus lavaretus (L.) from Loch Lomond (Scotland). The hearts of all 26 powan (15 females and 11 males) investigated harboured metacercariae of the digenean trematode Ichthyocotylurus erraticus (Rudolphi, 1809). The vast majority of metacercariae were located either singly or as an aggregation of white cysts on the surface of the bulbus arteriosus. The intensity of infection ranged from 2 to 200 larvae heart(-1), although the number of metacercariae found on male powan did not exceed 13. Histochemically, the parasite cyst wall gave a strong positive reaction with periodic acid schiff (PAS) and a faint positive signal with Azan-Mallory stain. All the metacercariae cysts were embedded in a granulomatous proliferation of heart epicardium tissue, forming a reactive fibroconnective capsule around the parasite. The capsule enclosing the parasite (produced by the host's reaction to the parasite) measured 13.57 to 90.20 microm (37.43 +/- 3.56) in thickness. Within the capsule wall, eosinophilic granular cells (EGCs), granulocytes, melanocytes and, in some instances, partially degenerated or vacuolated epithelioid cells were observed in close proximity to the cyst wall. Pigment-bearing macrophages were scattered throughout the granulomatous host-tissue reaction and as macrophage aggregates (MAs) within the capsules surrounding parasites. Immunohistochemical tests were applied to infected heart sections using 12 different antisera. Nerve fibres immunoreactive to bombesin, substance P (SP), and atrial natriuretic peptide (ANP) antisera were observed in close proximity to the parasite larvae. The presence of a serotonin-like substance was also observed within host immune-cells surrounding trematode cysts. Large cells of the epicardium were found to be immunoreactive to met-enkephalin and vasoactive intestinal peptide (VIP) antisera but not immunoreactive to anti-protein gene-product 9.5 (PGP9.5) sera.

Immunohistochemical and histopathological studies were conducted on a population of 3-spined sticklebacks Gasterosteus aculeatus (L.) from Loch Airthrey (Stirling, Scotland) naturally infected with the microsporean Glugea anomala (Moniez 1887). Of the 55 host specimens that were examined, 16 (29.09%) were infected, the intensity of infection ranging from 1 to 4 xenomas per fish, which were principally located within the central portion of the body lateral flank musculature. All 32 G. anomala xenomas examined were mature, their diameter ranging from 936 to 2232 Pum, and their walls of presented a laminar structure. Subcutaneously situated xenomas protruded from the fish body surface, whilst xenomas encountered within the intestine were seen to cause distortion. Light and electron microscopical observations confirmed a host cellular reaction around the xenoma, seen by the presence of eosinophile granule cells (EGCs), and some neutrophils. The occurrences of rodlet cells among the intestinal epithelial cells, and in close proximity to the xenoma wall, were observed in certain specimens. Outside the xenoma wall, macrophage aggregates (MAs) were commonly encountered. Within the xenoma wall, the presence of eosinophile granular cells immunoreactive to the anti-serotonin serum was also recorded. Further immunohistochemical tests revealed that a high number of nerve fibres running along the white lateral muscle fibres were immunoreactive to bombesin-, galanin-, and leu-enkephalin-antisera. Nerve fibres containing bombesin- and leu-enkephalin-like substances were also observed in the connective inflammatory tissue around the protozoan cyst, while neurons in the spinal ganglia were immunoreactive to met-enkephalin, and serotonin antisera. The control for the specificity of immunohistochemical reactions was performed using preabsorption tests of each antiserum with the corresponding antigen, and no immunoreactivity was noticed. The data presented are discussed in relation to the occurrence of G. anomala, which alters the pattern of nerve fibres present in the host. Specifically, the protozoan induces a response in the stickleback nervous system, the reaction of which is revealed through the application of immunohistochemical techniques.

Intraspecific and interspecific effects on the growth and body size of helminths are rarely studied in natural situations, yet knowing what determines helminth sizes and thus fecundity is crucial to our understanding of helminth ecology and epidemiology. The determinants of average individual worm mass were investigated in four common species of helminths parasitic in trout, Salmo trutta. In the acanthocephalan Echinorhynchus truttae, there was a negative relationship between the intensity of infection by conspecifics and average individual worm size. However, in the acanthocephalans Pomphorhynchus laevis and Acanthocephalus anguillae and in the cestode Cyathocephalus truncatus, the relationship was positive: individual worms were larger on average when co-occurring with many conspecifics than when co-occurring with very few. In addition, the average mass of individual C. truncatus in a host decreased as the total mass of other helminth species in the same host increased. This interspecific effect involves the whole helminth community, as the combined effect of all other helminth species is a better predictor of reduced mass in C. truncatus than the mass of any other species taken on its own. These results illustrate the importance of considering helminth interactions and helminth growth in a natural setting.

The presence of intestinal helminths in the alimentary canal of brown trout, Salmo trutta L., can alter the number of cells that synthesize modulatory peptides. A total of 167 brown trout were collected from tributaries of the River Brenta (northern Italy), of which 119 (71.3%) specimens were infected with enteric helminths, 28 with the acanthocephalan Pomphorhynchus laevis Müller, 1776 with intensity of infection ranging from 1 to 162 (18.57 +/- 30.79) worms per host and 67 fish with the cestode Cyathocephalus truncatus Pallas, 1781. Intensity of infection with C. truncatus ranged from 1 to 85 (6.87 +/- 12.59) per fish. In 24 fish there were concurrent infections of both species of helminths. The caecal and middle regions of the intestine were the most heavily parasitized. Immunohistochemical tests showed a decrease in endocrine cells (ECs) of the diffuse endocrine system (DES) positive to gastrin, cholecystokinin-8, bombesin and secretin antisera in the intestine of the infected trout. The number of ECs immunoreactive to anti-glucagon serum did not show differences in the digestive tract of uninfected brown trout and in conspecifics parasitized with P. laevis. The density of cells containing glucagon-like material was low in the fish parasitized with C. truncatus. The results suggest that endoparasitic helminths induce alterations in the DES of infected S. trutta.

Immunohistochemical, ultrastructural and pathological studies were carried out on the gills of bream Abramis brama (L.) from Lake Mondsee, Austria, that were naturally infected with Ergasilus sieboldi Nordmann, 1832. Of a total of 14 specimens of bream examined, the gills of 7 (50%) were parasitized with this copepod and the intensity of infection ranged from 1 to 23 crustaceans per host. Histopathological investigations of infected gill showed extensive tissue damage due to attachment and feeding of the crustacean. Parasites attached close to the base of filaments near the gill arch. Pressure exerted by the ectoparasite attached to the lateral margin of the gill filaments induced atrophy of the secondary lamellae. Tissue reactions included hyperplasia and mucous cell proliferation of the respiratory epithelium. Mucous cells displayed an intense immunohistochemical reactivity with the anti-nitric oxide synthase antibody. In parasitized primary and secondary lamellae, a high number of eosinophilic granular cells and rodlet cells were noticed. Rodlet cells represent an inflammatory cell type closely linked to other piscine inflammatory cells. Presence of a high number of inflammatory cells at the site of E. sieboldi attachment is related to intense host cellular reaction.

Immunohistochemical and pathological studies were carried out on the digestive tract of parasitized and uninfected specimens of Salmo trutta (L.). A total of 124 brown trout were collected on several occasions from 3 tributaries of the Brenta River, northern Italy. Twenty-eight individuals of S. trutta (22.6%) were parasitized with Pomphorhynchus laevis (Miller, 1776). The occurrence of P. laevis in the trout gut significantly increased the number of endocrine cells immunoreactive to calcitonin gene-related peptide (CGRP), beta-endorphin, met-enkephalin, neuropeptide Y (NPY) and Substance P (SP) antisera. Moreover, bombesin-, cholecistokinin-8- (CCK-8), leu-enkephalin- and serotonin- (5-HT)-like immunoreactive cells were less numerous in the intestine of the parasitized brown trout. A strong positive immunoreactivity was observed in nerve fibres and neurones of the myenteric plexus of the parasitized fish; the antisera involved in this positive reactivity were bombesin, met-enkephalin, SP and vasoactive intestinal peptide (VIP). More neurones immunoreactive to anti-CGRP and anti-5-HT sera were noted in the myenteric plexus and in the inner layer of the tunica muscularis of the infected fish. Most of the above-mentioned neuromodulators are known to control gut motility, digestive/absorptive processes, as well as the immune response. The changes induced by parasites in the neuroendocrine system of the brown trout are discussed.

Species co-occurrences and interspecific associations between intensity of infection were studied in helminth communities of three populations of brown trout, Salmo trutta, from northern Italy. Of the eight helminth species, only four were common enough to be included in the analyses: Pomphorhynchus laevis, Acanthocephalus anguillae, Echinorhynchus truttae and Cyathocephalus truncatus. The observed frequencies of co-occurrences of the different species, based on presence/absence data, did not differ from those predicted by a null model derived from prevalence data. However, the intensity of infection (number of individuals per fish) of different helminth species were generally, but not always significantly, negatively correlated in tests of pairwise associations. Variation in fish sizes and its effect on infection levels, and whether or not two helminth species used the same or different intermediate hosts, had no influence on these findings. Of the few significant negative associations found between pairs of helminth species, none was found in more than one fish population. This suggests that interspecific associations may be condition-dependent: even in apparently similar localities, the same combinations of helminth species show different associations. Without evidence of replicability, it is almost impossible to conclude to the consistent role of competition between any pair of helminth species in the field.