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.     

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

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

寄生于两种湖泊淡水鱼的伊格诺图斯胃瘤线虫IV期幼虫PCR分析、形态结构和组织病理

为探明寄生于翘嘴鲌（Culter alburnus Basilewsky）和花（鱼骨）（Hemibarbus maculatus Bleeker）腹腔内线虫的分类地位、形态特征及其寄生部位的组织损伤作用,进行了18S rDNA和ITS rDNA序列的PCR分析以及虫体和组织病理的显微观察。结果表明该线虫为伊格诺图斯胃瘤线虫（Eustrongylides ignotus）的IV期幼虫,明确了其ITS rDNA的基因序列。虫体除头部具有6个棘状突起和6个乳头状突起以及尾部具有尾斑突外,还发现其具有凹纹的体侧斑突等一些新的形态结构特征,补充了一些主要形态结构数据。根据体肌肌细胞数量和其一端伸入体腔的特点判定线虫体肌属于多肌型。食道由背侧腺和下腹腺组成,食道腔横切面呈三放射形。依据组成肠道的单层柱状上皮细胞数量,判定该线虫肠道属于多胞型。卵巢、输卵管、精巢和输精管外层均由以纤维细胞为主要成分的结缔组织构成。病理观察显示部分线虫以包囊形式寄生于肝脏、肠系膜和脾脏的腹腔组织器官,形成由纤维细胞和血管组成的包囊和大量嗜酸性白细胞浸润的主要炎症反应,包囊经历了形成、增大、壁增厚和后期包囊壁中细胞坏死而破裂的过程,严重感染的个体出现肝脏充血、细胞坏死和含铁血黄素沉着,脾脏缺血和含铁血黄素沉着以及肠系膜中的胰腺细胞分泌颗粒增多等病理变化。结果表明,该IV期幼虫对寄主内脏组织器官具有不同程度的组织病理损伤作用。     

Microbial origin of the abdominal swelling affecting farmed larvae of gilt-head seabream, Sparus aurata L.

The aetiological agents of the abdominal swelling affecting farmed larvae of gilt-head seabream, Sparus aurata L., were studied. Four Vibrio strains were isolated from larvae of S. aurata affected by this disease, and all strains reproduced the disease in healthy larvae under controlled infection experiments, producing a significant increase of the mortality rates compared to the control (non-inoculated larvae). Several enzymatic properties, which can act as .virulence factors, were demonstrated both in the extracellular products (ECPs) and In live cells of the strains tested. Histopathological examinations of the infected fish larvae revealed important changes of the anterior intestine and liver characterized by a marked hyperthrophy of the intestinal epithelium and hepatocytes, and by a separation of the mucosal and submucosal layers in the digestive tube. These histological alterations were associated with the constant presence of cocobacillar bacteria in the anterior intestine and in the liver. However, the precise pathogenic mechanisms of the strains tested have not been completely elucidated yet.     

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.     

Histopathology of abnormal livers and other organs of starry flounder Platichthys stellatus (Pallas) from the estuary of the Duwamish River, Seattle, Washington, U.S.A.

Abstract. Starry flounder Platichthys stellatus (Pallas) from the Duwamish River estuary, which has received large volumes of pollutants in the past, and starry flounder from two relatively unpolluted reference areas were examined for pathological conditions. Gross and microscopic examination of starry flounders from the Duwamish River estuary revealed one or more of the following unusual liver characteristics in 92% (monthly range 85–100%) of the fish: liver discolouration, liver enlargement, severe fatty vacuolation of the hepatocytes, centrolobular cellular degeneration and necrosis of hepatocytes, megalocytic hepatocytes, proliferating perivascular fibroblasts displacing adjacent liver parenchyma, hepatic structural disarray and increased basophilia of hepatocytes in localized areas. Livers of starry flounders, usually spent spawners, from one of the reference areas also had one or more of these characteristics, although at a much lower frequency (<13%), and these included liver discolouration, increased fatty vacuolation of the hepatocytes and hepatic structural disarray.     

Mast cells in the gills and intestines of naturally infected fish: evidence of migration and degranulation

Immunopathological and ultrastructural studies were carried out on the gills of bream, Abramis brama, naturally infected with the copepod Ergasilus sieboldi and on the intestine of brown trout, Salmo trutta, infected with the acanthocephalan Echinorhynchus truttae. Infected gills showed extensive tissue damage due to copepod attachment, including hyperplasia, as well as proliferation of mast cells, rodlet cells and mucous cells. In parasitized gills of bream, mast cells were more abundant than in uninfected gills. They were free within the lacunae, as well as outside and inside the blood vessels of the primary lamellae, and made intimate contact with vascular endothelial cells and with neutrophils. In some infected gills, degranulation of mast cells was common. Infected intestines of brown trout had more mast cells than uninfected intestines, and these cells were often in close proximity to, and inside, the capillaries and lying close to fibroblasts of the muscularis layer and stratum granulosum. Intense degranulation of mast cells was encountered in all intestinal layers, especially near the E. truttae body.     

Morphologic study of the liver of lambari (Astyanax altiparanae) with emphasis on the distribution of cytokeratin

Studies on the morphology of the liver of teleosts reflect some controversy in the interpretation of the data, but also provide confirmation of variations in the structure of the organ in several species. Thus, we intend to understand the specific structural organization of the liver of Astyanax altiparanae. Specimens were collected in the city of Andirá, Paraná, Brazil. The livers were processed according to histological routine for inclusion in Paraplast, and the sections were stained with HE and Mallory’s trichrome or followed the protocol for fluorescence immunohistochemistry, anti-cytokeratin. The liver of A. altiparanae was covered by a capsule of connective tissue, without delimiting lobes. The hepatocytes had an arrangement in cords around sinusoids. Melanomacrophage centers were observed. The vascular components and intrahepatic pancreatic acini were distributed between hepatocytes. Presence of cytokeratin was detected in tissues that lined the liver and endothelial cells of sinusoids. The comparison of the liver of A. altiparanae to other characids corroborates with the fact that there is variation in the morphology of the liver even between closely related species. Moreover, it appears that in this species, endothelial cells of sinusoids can synthesize the cytokeratin filaments required for the regulation of blood flow in capillaries in adults.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

Melanotic visceral fibrosis associated with larval infections of Posthodiplostomum minimum and Proteocephalus sp. in bluegill, Lepomis macrochirus Rafinesque, in central Iowa, U.S.A.

Abstract. Severe melanotic liver and visceral fibroses in bluegill associated with infections of plerocercoids of Proteocephalus sp. and metacercariae of the digenean Posthodiplostomum minimum , were studied by light and transmission electron microscopy. Fibrosis was most evident in bluegill carrying heavy worm burdens. Tubular and multilocular melanotic and non-melanotic cysts were formed of concentric layers of non-cellular eosinophilic connective tissue surrounding central spaces containing basophilic material. Moribund and deteriorating plerocercoids and metacercariae were surrounded by, and sometimes invested with, fibrous tissue. Living plerocercoids in the liver were surrounded by necrotic and compressed hepatocyte zones. Fibro-blasts, epithelioid cells and eosinophilic granulocytes were common in compressed cell zones. Intrahepatic fibrotic cysts were surrounded by granulomatous zones. Parenchyma in fibrotic livers showed increased numbers of macrophages, melano-macrophage centres and engorged blood vessels with thickened walls. Histochemical tests for lipofuscin and haemosiderin were strongly positive in melanotic liver tissue. Ultrastructural changes in hepatocytes adjacent to fibrotic cysts and epithelioid zones included intranuclear lipid and glycogen inclusions, chromatin alterations and mitochrondrial degeneration. Bile canaliculi, sinusoids and perisinusoidal spaces showed reduced micro villarsurfacesand a decrease in luminar diameter. Degenerating hepatocytes contained phagolysosomes and myelin bodies.     

The histopathology of larval anisakid nematode infections in the liver of whiting, Merlangius merlangus (L.), with some observations on blood leucocytes of the fish

Abstract. The cellular response of whiting, Merlangius merlangus (L.), to Contracaecum and Anisakis larvae parasitic in the liver was characterized by the presence of neutrophils, macrophages and proliferating fibroblasts. A capsule formed around the larvae after the removal of all necrotic debris by phagocytic cells. Melanin granules were deposited around the capsule. Four types of blood leucocytes were identified; thrombocytes, lymphocytes, monocytes and neutrophils. Phagocytic activity of the neutrophils and macrophages was suggested by the presence of dark stained bodies within them.     

鲫圆形碘泡虫病组织病理学研究

采用常规石蜡切片、苏木精-伊红染色的方法,研究了鲫（ Carassius auratus auratus ）自然感染圆形碘泡虫（Myxobolus rotundus）后的组织病理变化。结果表明,感染圆形碘泡虫后,在鲫的头部、鳃和体表能形成大小不一的圆形或椭圆形乳白色孢囊,其孢囊为典型的3层结构：外层的结缔组织、双层质膜的外质以及各个发育阶段的孢子或营养体组成的内质。病理切片可见鱼体各组织器官均有不同程度的病变,主要表现为鳃丝粘连、肿胀增生;肝脏发生以肝血窦为中心的透明、空泡变性;肾小管内皮脱落,肾小球萎缩;肠道粘膜层脱落,肌层细胞肿胀病变;脾脏的含铁血黄素增多等异常现象。     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

Massive Hepatic Necrosis and Nodular Regeneration in Largemouth Bass Fed Diets High in Available Carbohydrate

Largemouth bass Micropterus salmoides are piscivorous fish raised on farms then sold live in Asian fish markets on the east and west coasts of the United States. In the winter of 1998, a major producer of feed‐trained bass suffered a significant increase in fish mortality both during shipping and while the fish were still in ponds. No bacterial, viral, or significant parasitic pathogens were found at necropsy. Livers of affected fish were pale and translucent with 3–10 mm pink nodules on their surface and deeper in the parenchyma. Histological examination of these livers showed that the translucent regions of the liver contained few hepatocytes and were composed of tissue consistent with a chronic inflammatory response. Also present were eosinophils, islands of pancreatic and biliary cells, and granulomas that did not stain positively for mycobacteria. The pink nodules were areas of multifocal regeneration of normal hepatocytes. Sequential studies of bass in ponds revealed that the bass were progressively accumulating glycogen in their hepatocytes to an extent sufficient to explain the massive necrosis of that organ. In order to determine the effect of diets varying in available carbohydrate on fish growth, survival, and liver glycogen content, a 12‐wk feeding trial was conducted in aquaria with juvenile largemouth bass. Nitrogen‐free extract values indicated that an extruded trout diet, a steelhead trout diet, and a diet designed to contain 45% protein and 25% fat, contained 35, 27, and 21% carbohydrate, respectively. Weight gain was lowest in fish fed the extruded trout diet, while liver glycogen was significantly higher in fish fed diets with >27% carbohydrate than in fish fed the diet with 21% carbohydrate. The farmer switched to a diet similar to the 45–25 diet used in our trial. Subsequently, 16‐mo‐old fish examined in October 2000 had no hepatic nodules or necrosis, were significantly larger and less variable in size than previous crops, and the farmer experienced no significant losses in ponds or during shipping.     

Experimental infection of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae with infectious pancreatic necrosis virus: detection of virus by immunohistochemistry and in situ hybridization

Three different concentrations (10⁷, 10⁵ and 10³ TCID₅₀ ml^-1) of infectious pancreatic necrosis virus (IPNV) serotype Sp isolated from Atlantic halibut, Hippoglossus hippoglossus L., were used to bath-challenge Atlantic halibut yolk-sac larvae. The larvae challenged with 10⁷ TCID₅₀ ml^-1 suffered significantly higher cumulative mortality than the other challenged groups and the control group, and affected individuals displayed necrosis of the intestine, liver and kidney. In larvae from the groups challenged with 10⁷ and 10⁵ TCID₅₀ ml^-1, IPNV was detected by immunohistochemistry and in situ RNA/DNA hybridization in the intestine, liver and kidney. In addition, some individuals stained IPNV-positive in the heart and eye/brain region. Detection by in situ hybridization did not appear to be more sensitive than immunohistochemistry. However, background staining was virtually absent in comparison with immunohistochemistry, and the staining seemed to be more distinctly localized to the cytoplasm of infected cells. The results show that farmed halibut yolk-sac larvae can be infected by IPNV immediately after hatching, with resulting high mortality. As the larvae are not immunologically mature at this stage of development, vaccination is not recommended.     

Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics

Several studies have been demonstrated that phosphotransfer network, through the adenylate kinase (AK) and pyruvate kinase (PK) activities, allows for new perspectives leading to understanding of disease conditions associated with disturbances in energy metabolism, metabolic monitoring and signalling. In this sense, the aim of this study was to evaluate whether experimental infection by Aeromonas caviae alters hepatic AK and PK activities of silver catfish Rhamdia quelen. Hepatic AK and PK activities decreased in infected animals compared to uninfected animals, as well as the hepatic adenosine triphosphate (ATP) levels. Also, a severe hepatic damage was observed in the infected animals due to the presence of dilation and congestion of vessels, degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure. Therefore, we have demonstrated, for the first time, that experimental infection by A. caviae inhibits key enzymes linked to the communication between sites of ATP generation and ATP utilization. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes contributes directly to hepatic damage and for a severe energetic imbalance, which may contribute to disease pathophysiology.     

Experimental infection of Atlantic halibut,Hippoglossus hippoglossus L., yolk-sac larvae with infectious pancreatic necrosis virus: detection of virus by immunohistochemistry and in situ hybridization*

Three different concentrations (10⁷, 10⁵ and 10³ TCID₅₀ ml^?1) of infectious pancreatic necrosis virus (IPNV) serotype Sp isolated from Atlantic halibut, Hippoglossus hippoglossus L., were used to bath-challenge Atlantic halibut yolk-sac larvae. The larvae challenged with 10⁷ TCID₅₀ ml^?1 suffered significantly higher cumulative mortality than the other challenged groups and the control group, and affected individuals displayed necrosis of the intestine, liver and kidney. In larvae from the groups challenged with 10⁷ and 10⁵ TCID₅₀ ml^?1, IPNV was detected by immunohistochemistry and in situ RNA/DNA hybridization in the intestine, liver and kidney. In addition, some individuals stained IPNV-positive in the heart and eye/ brain region. Detection by in situ hybridization did not appear to be more sensitive than immunohistochemistry. However, background staining was virtually absent in comparison with immunohistochemistry, and the staining seemed to be more distinctly localized to the cytoplasm of infected cells. The results show that farmed halibut yolk-sac larvae can be infected by IPNV immediately after hatching, with resulting high mortality. As the larvae are not immunologically mature at this stage of development, vaccination is not recommended.     

Ultrastructural effects of the coccidium, Eimeria funduli Duszynski, Solangi and Over street, 1979 on the liver of killifishes

Abstract. Ultrastructural studies were made on the inflammatory response in livers of two killifishes, Fundulus grandis and F. similis , experimentally infected with the coccidium Eimeria funduli. Coinciding with the formation and development of gamonts of E. funduli , changes took place in the liver and leucocytes appeared in that organ. Near areas of developing macrogamonts, many hepatocytes degenerated whereas others contained residual bodies which indicated sublethal damage. The damage was probably associated with merozoite formation and release. Filament-rich perisinusoidal cells hypertrophied during the development of E. funduli and contributed to walling off aggregates of oocysts from uninfected liver tissue. Leucocytes involved in the host response included lymphocytes, monocytes, heterophils, eosinophils and an unidentified third type of granulocyte. The heterophil was the first granulocyte to respond to the parasite and sometimes appeared to penetrate the parasitophorous vacuole of degenerating macrogamonts. Degranulating eosinophils were often observed near damaged tissues. Both the monocyte and eosinophil were phagocytically active. The monocyte was more phagocytically active and contained both host cells and parasite debris, whereas the eosinophil contained only debris from degenerating macrogamonts. The presence of immature granulocytes in the liver suggests that at least some granulocyte formation occurred in the liver.