PFAS levels in fish species in the Po River (Italy): New generation PFAS, fish ecological traits and parasitism in the foreground.

Per- and polyfluoroalkyl substances (PFAS) are resistant to breakdown and are now considered ubiquitous and concerning contaminants. Although scientific and legislative interest in these compounds has greatly increased in recent decades, our knowledge about their environmental fate and their effects on organisms is still incomplete, especially those of the new generation PFAS. In this study, we analysed the level of PFAS contamination in the fish fauna of the Po River, the most important waterway in Italy, to evaluate the influence of different factors (such as fish ecological traits and parasitism) on the accumulation of 17 PFAS. After solvent extraction and purification, hepatic or intestinal tissues from forty specimens of bleak, channel catfish, and barbel were analysed by liquid chromatography coupled with mass spectrometry (LOQ = 2.5 ng/g w.w.). The prevalent PFAS were perfluorooctane sulfonate (PFOS), present in all samples at the highest concentration (reaching a maximum of 126.4 ng/g and 114.4 ng/g in bleak and channel catfish, respectively), and long-chain perfluoroalkyl carboxylic acids (PFDA and PFUnDA). Perfluorooctanoic acid and new generation PFAS (Gen X and C6O4) were not detected. Comparison of the hepatic contamination between the benthic channel catfish and the pelagic bleak showed similar concentrations of PFOS (p > 0.05) but significantly higher concentrations of other individual PFAS and of the sum of all measured PFAS (p < 0.05) in bleak. No correlation was found between the hepatic level of PFAS and fish size in channel catfish. For the first time, PFAS partitioning in a parasite-fish system was studied: intestinal acanthocephalans accumulated PFOS at lower levels than the intestinal tissue of their host (barbel), in contrast to what has been reported for other pollutants (e.g., metals). The infection state did not significantly alter the level of PFAS accumulation in fish, and acanthocephalans do not appear to be a good bioindicator of PFAS pollution.

Intestinal granular cells of a cartilaginous fish, thornback ray Raja clavata: Morphological characterization and expression of different molecules.

This investigation aims to fill gaps in our understanding of the intestinal immune cells of elasmobranchs. Whole digestive tracts of fifteen thornback ray Raja clavata were provided by a trawl fleet from the Gulf of Asinara (Sardinia, western Mediterranean Sea). Histochemical, immunohistochemical and ultrastructural observations were conducted on the spiral intestine. Three types of granular cells were identified; type I in epithelium, types II and III in lamina propria-submucosa, with each of them containing cytoplasmic granules with different ultrastructural characteristics. Data on size and density of each granular cell type are provided. Immunostaining of intestinal sections showed the reactivity of the granular cells: type I cells were positive for lysozyme, mast cell tryptase and tumor necrosis factor-ɑ based on antibody staining; type III cells were immune-reactive to anti-interleukin 6 antibody, whilst type II cells were negative to all the antibodies used. Comparison of each granular cell type with immune cells of teleosts or mammals and an hypothesis on their nature and function are reported. A potential role for granular cells in intestinal cellular immunity is also discussed with respect to type I and type III cells having similarities to Paneth cells and neutrophils, respectively.

Histochemical and immunohistochemical characterization of rodlet cells in the intestine of two teleosts, Anguilla anguilla and Cyprinus carpio.

Rodlet cells (RC) are characterized by a distinctive cell cortex and conspicuous inclusions named "rodlets." These cells are particularly abundant and large in size in intestine of eels. Histochemical, immunohistochemical and ultrastructural investigations were carried out on European eel Anguilla anguilla and Common carp Cyprinus carpio from Northern Italy. Eight biotinylated lectins were used to probe for specific carbohydrate residues in deparaffinized, hydrated intestinal sections of eel and carp. Five antibodies were tested on intestinal sections of both fish species: inducible nitric oxide synthase (i-NOS), leu-enkephalin, lysozyme, serotonin and tumour necrosis factor-α. Lectin histochemistry revealed rodlet cells (RCs) of the eel intestine to react with two of the eight lectins tested, specifically Concanavalin A (ConA) and Sambucus Nigra Agglutinin (SNA). This contrasted to lectin staining of RCs in the intestine of common carp, where four of the eight lectins showed a positive reaction; Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), SNA and ConA. RCs in eel and carp intestine were immunoreactive with antibodies to lysozyme and i-NOS. The occurrence of the inflammatory peptides lysozyme and i-NOS in RCs of the eel and common carp poses in favour that these cells are involved in the mechanism of defence against pathogens.

Histopathological and ultrastructural assessment of two mugilid species infected with myxozoans and helminths.

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.

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.

A fish model for the study of the relationship between neuroendocrine and immune cells in the intestinal epithelium: Silurus glanis infected with a tapeworm.

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.

Protective responses of intestinal mucous cells in a range of fish-helminth systems.

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.

Fish innate immunity against intestinal helminths.

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.

Anguilla anguilla intestinal immune response to natural infection with Contracaecum rudolphii A larvae.

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.

European sea bass gill pathology after exposure to cadmium and terbuthylazine: expert versus fractal analysis.

The objective of this study was to compare expert versus fractal analysis as new methods to evaluate branchial lamellar pathology in European sea bass Dicentrarchus labrax (Linnaeus, 1758) experimentally exposed to cadmium and to terbuthylazine. In particular, guided expert quantitative and fractal analysis were performed on selected images from semithin sections to test possible differences according to exposure class (unexposed, cadmium exposed, or terbuthylazine exposed) and the discrimination power of the two methods. With respect to guided expert quantitative analysis, the following elementary pathological features were assessed according to pre-determined cover classes: 'epithelial lifting', 'epithelial shrinkage', 'epithelial swelling', 'pillar cells coarctation', 'pillar cells detachment', 'channels fusion', 'chloride cells swelling' and 'chloride cells invasion'. Considering fractal analysis, DB (box dimension), DM (mass dimension), Dx (mean fractal dimension) as fractal dimensions and lacunarity from DM and Dx scan types were calculated both from the outlined and skeletonized (one pixel wide lines) images. Despite significant differences among experimental classes, only expert analysis provided good discrimination with correct classification of 91.7 % of the original cases, and of 87.5 % of the cross-validated cases, with a sensitivity of 95.45 % and 91.3 %, respectively, and a specificity of 75 % in both cases. Guided expert quantitative analysis appears to be a reliable method to objectively characterize fish gill pathology and may represent a powerful tool in environmental biomonitoring to ensure proper standardization and reproducibility. Though fractal analysis did not equal the discrimination power of the expert method, it certainly warrants further study to evaluate local variations in complexity or possible multiple scaling rules.

Ultrastructural assessment of granulomas in the liver of perch (Perca fluviatilis) Infected by tapeworm.

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).

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.

The use of fractal dimension and lacunarity in the characterization of mast cell degranulation in rainbow trout (Onchorhynchus mykiss).

Fractal analysis is a reliable method for describing, summarizing object complexity and heterogeneity and has been widely used in biology and medicine to deal with scale, size and shape management problems. The aim of present survey was to use fractal analysis as a complexity measure to characterize mast cells (MCs) degranulation in a rainbow trout ex vivo model (isolated organ bath). Compound 48/80, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, was adopted as MCs degranulation agent in trout intestinal strips. Fractal dimension (D), as a measure of complexity, 'roughness' and lacunarity (λ), as a measure of rotational and translational invariance, heterogeneity, in other words, of the texture, were compared in MCs images taken from intestinal strips before and after compound 48/80 addition to evaluate if and how they were affected by degranulation. Such measures were also adopted to evaluate their discrimination efficacy between compound 48/80 degranulated group and not degranulated group and the results were compared with previously reported data obtained with conventional texture analysis (image histogram, run-length matrix, co-occurrence matrix, autoregressive model, wavelet transform) on the same experimental material. Outlines, skeletons and original greyscale images were fractal analysed to evaluate possible significant differences in the measures values according to the analysed feature. In particular, and considering outline and skeleton as analysed features, fractal dimensions from compound 48/80 treated intestinal strips were significantly higher than the corresponding untreated ones (paired t and Wilcoxon test, p < 0.05), whereas corresponding lacunarity values were significantly lower (paired Wilcoxon test, p < 0.05) but only for outline as analysed feature. Outlines roughness increase is consistent with an increased granular mediators interface, favourable for their biological action; while lacunarity (image heterogeneity) reduction is consistent with the biological informative content decrease, due to granule content depletion. In spite of the significant differences in fractal dimension and lacunarity values registered according to the analysed feature (greyscale obtained values were, on average, lower than those obtained from outlines and skeletons; General Linear Model, p < 0.01), the discrimination power between not degranulated and degranulated MCs was, on average, the same and fully comparable with previously performed texture analysis on the same experimental material (outline and skeleton misclassification error, 20% [two false negative cases]; greyscale misclassification error, 30% [two false negative cases and one false positive case]). Fractal analysis proved to be a reliable and objective method for the characterization of MCs degranulation.

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.

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.

Infiltration and activation of acidophilic granulocytes in skin lesions of gilthead seabream, Sparus aurata, naturally infected with lymphocystis disease virus.

Light, ultrastructural and immunocytochemical investigations were carried out on the skin of gilthead seabream, Sparus aurata L., naturally infected with lymphocystis iridovirus, to assess pathology and host cellular responses. Of 220,000 young seabream examined, 32,400 (14.7%) had clinical signs of lymphocystis and within 6 months of disease appearance, 45% of clinically affected fish had died. A subsample of 20 S. aurata (80.0 ± 12.5mm total length, mean ± S.D.), including 10 with lymphocystis on the skin and 10 clinically normal, were examined via immunohistochemistry. Affected skin displayed macroscopic, wart-like clusters of hypertrophic fibroblasts which arose from the dermis and were covered by the epithelium. Clusters were encountered on the head, trunk and fins, but there was no evidence of visceral lymphocystis. The lymphocysts were surrounded by numerous granular cells that were positive for the antimicrobial peptide (AMP) piscidin 3 and underwent intense degranulation. To identify the type of granular cells involved in this viral disease, a double immunohistochemical staining with the monoclonal antibody G7 (mAb G7), which is specific for seabream acidophilic granulocytes (AGs), and with anti-histamine (as a marker for mast cells, MCs) was applied to the skin sections of the 10 clinically normal fish and 10 fish with lymphocystis. In infected skin, the number of G7-positive cells (i.e., AGs) (18.5 ± 10.5, mean number of cells per 20,000 µm(2) ± S.D.) was significantly higher compared to their density in uninfected skin (1.4 ± 2.2) (t test, p<0.01). Notably, the AGs that infiltrated the skin lesions of infected animals were found to be degranulated and to produce the pro-inflammatory cytokine interleukin-1ß. No histamine-positive granular cells (i.e., MCs) were encountered in the lymphocystis lesions. The present study shows the response of skin to lymphocystis disease virus (LCDV) and provides evidence that AGs, but not MCs, are recruited and activated in response to this skin infection.

The impact of an oil spill on organs of bream Abramis brama in the Po River.

An oil spill into the River Lambro occurred on 23 February 2010 and reached the Po River the following day. Breams captured here on 1 March 2010, along with a sample from a control site, were examined by light and electron microscopy. The main affected organs were skin and gill with slight or no damage to liver, kidney, and intestine. The gills exhibited lamellar aneurisms, fusion of secondary lamellae, edema with epithelial lifting, mucous cell hypertrophy, and mucus hypersecretion. Significantly higher mucous cell density was observed in the skin of exposed fish. Histochemical staining revealed that acid glycoconjugates were prevalent in epidermal mucous cells in the exposed Abramis brama, whereas neutral and mixed glycoconjugates were dominant in the control fish. Rodlet cells were significantly more abundant in the kidney of exposed fish and showed ultrastructural differences compared to controls. These histopathologic effects were indicators of chemical stress due to exposure to oil. The present study is one of the first which explores the acute effects of this incident and makes part of a few reports focused on freshwater oil spill.

Degranulation of mast cells due to compound 48/80 induces concentration-dependent intestinal contraction in rainbow trout (Oncorhynchus mykiss Walbaum) ex vivo.

Rainbow trout (Oncorhynchus mykiss) intestinal strips (n = 10) were mounted in an isolated organ bath and the effect of incremental doses of compound 48/80 was recorded. Compound 48/80 induced concentration-related contraction in all the examined strips following a sigmoidal dose-response curve fit. Values for maximal contraction (E(max) , g cm(-2)), negative logarithm of the EC(50) (pD(2)), and hill slope were, respectively (mean±standard error), 12.88 ± 0.51, 1.88 ± 0.05, 1.49 ± 0.27. The histological modification induced on mast cells (MCs) due to compound 48/80 was characterized by mean of gray-levels and texture analysis. Significant differences were observed between gray-levels values (Linear mixed model, P<0.01), contrast, and entropy (Linear mixed model, P<0.05) of MCs from compound 48/80-treated strips compared with MCs from untreated strips. Moreover, maximal intestinal contraction (due to compound 48/80) correlates positively and significantly (Pearson and Spearman correlations, P<0.05) with degranulation intensity determined by means of gray-levels analysis. Four antisera were tested on intestinal sections and no MCs positive to serotonin, substance P, met-enkephalin, and bombesin were found. This study demonstrates that compound 48/80 induces the degranulation of trout intestinal MCs ex vivo, and that the aforementioned degranulation promotes a concentration-dependent intestinal contraction.

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.

Cellular response in semi-intensively cultured sea bream gills to Ergasilus sieboldi (Copepoda) with emphasis on the distribution, histochemistry and fine structure of mucous cells.

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.