Systemic infection with Uronema-like ciliates in farmed turbot, Scophthalmus maximus (L.)

Histophagous ciliates caused high mortality among turbot in a commercial fish farm in southern Norway. The ciliates spread systemically in fry (< 0.3 g). In the early stages of infection the ciliates were found in connective tissue in skin and fins, as well as in nervous tissue. In terminal stages the whole organism was infected. In large turbot (500–1000 g), the ciliates were predominantly found in the central nervous system, causing liquefaction of the nervous tissue. The ciliates were not identified to species but resembled species in the genus Uronema (Scuticociliatida).     

Glucose tolerance in turbot, Scophthalmus maximus (L.)

Eighty turbot, Scophthalmus maximus (L.), (average weight 61 g) were injected intraperitoneally with exactly 1 g glucose per kg body weight. There was a peak in plasma glucose 3 h post injection. Thereafter a gradual decrease to basal levels was seen within 24 h. Plasma triacylglycerol concentrations showed a rapid decline during the first 24 h, and thereafter stable values. Blood haematocrit values decreased from 20% before injection to 16% 72 h after injection. Liver glycogen concentrations showed an initial decrease from 8 to 5 g 100 g^?1 (w.w) during the first 12 h, and thereafter stable values, while muscle glycogen concentrations increased during the first 12 h, and thereafter showed a gradual decline until 72 h. This response was most probably caused by secondary changes upon handling in combination with the direct response to a glucose load. Thus turbot was able to restore alterations in carbohydrate metabolism efficiently within 24 h.     

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.)

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.     

Observations on the pathogenicity of Haemogregarina sachai Kirmse, 1978, in farmed turbot Scophthalmus maximus (L.)

Abstract. Cultured turbot Scophthalmus maximus (L.) from three farms at Hunterston, Scotland were found to be infected with Haemogregarina sachai . Up to 6% of some populations were affected with gross tumorous lesions of the musculature and viscera. The lesions consisted of necrotic tissue with a caseous centre. Histologically, there was an accumulation of parasitized reticulo-endothelial cells, cell debris and pyknotic nuclei. Parasitaemias of up to 36% of all blood cell types were observed. The majority of infected cells were neutrophils and monocytes. Infected leucocytes were hypertrophied. There was an erythrocytopenia of infected fish with increases in erythroblasts, monocytes and neutrophils but a decrease in thrombocytes and lymphocytes. Transmission experiments between fish were unsuccessful.     

Pathology of Edwardsiella tarda infection in turbot, Scophthalmus maximus (L.)

Macroscopic and histopathological changes in cultured turbot, Scophthalmus maximus (L.), in Spain caused by infection with Edwardsiella tarda are described. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. Histopathological lesions were found in the kidney, spleen and liver. In the kidney and spleen these were characterized by a severe apostematous inflammatory reaction, with a large number of abscesses. The liver was affected to a lesser degree and only some phagocytes loaded with bacteria were observed. Ultrastructural observations indicated that macrophages were the main cell type implicated in the inflammatory response. Most of the bacteria observed within the phagocyte cytoplasm showed no degenerative changes and some were dividing. Degenerative changes observed in macrophages indicate their failure in preventing the infection.     

Metabolizable energy from dietary carbohydrates in turbot, Scophthalmus maximus (L.)

Abstract. In a feeding experiment, the metabolizability of dietary carbohydrates in diets for turbot, Scophthalmus maximus (L.), was estimated and expressed as percentage of the metabolizability of dietary lipids. Four isoenergetic and isonitrogenous dietary treatments were applied with varying percentages of non-protein energy presented as starch. In order to obtain results allowing quantitative interpretation, an experimental design was used in which effects are reflected in varying body fat depositions only. Two methods were used simultaneously to overcome practical problems associated with differences in energy density between carbohydrates and lipids. The conventional method was based on equal feeding rates combined with the inclusion of inert filler in the diets, whereas the alternative method applied different dietary nutrient percentages in combination with different feeding rates. Although similar results were obtained, the alternative method was more convenient to use than the conventional method. Because of the experimental strategy applied, no significant effects on growth and protein utilization were obtained, but lipid deposition rates were significantly reduced at increased percentages of non-protein energy presented as starch. The metabolizable fraction of the gross dietary carbohydrate energy was estimated as 67% of that of dietary lipids. This reduction does not indicate that turbot have any systematic deficiency in metabolizing dietary carbohydrates.     

Pathology of Enterococcus sp. infection in farmed turbot, Scophthalmus maximus L.

Abstract The lesions of an enterococcal septicaemia occurring in turbot, Scophthalmus maximus (L.), cultured in several farms in northwest Spain are described. The agent of this septicaemia was identified previously as an Enterococcus -like bacterium. Two main patterns of lesions were observed in the diseased turbot: a focal form characterized by exophthalmia, muscular haemorrhages, acute branchitis, and suppurative inflammation of periorbital tissues, eyeball, meninges and brain; and another generalized form with similar lesions, but showing more extensive haemorrhages, ulceration and purulent inflammation of the skin, desquamative enteritis, and necrosis of spleen and kidney. Fatty changes of the liver were observed in all turbot examined (diseased and controls), which indicates that these alterations were not related to the infection. Experimentally inoculated turbot developed lesions corresponding to the generalized form of the natural disease. Although the Enterococcus strain was recovered in pure culture from all the organs, Gram-positive bacteria could not be visualized histologically in the lesions of muscle and brain.     

Dietary protein requirement for young turbot (Scophthalmus maximus L.)

This study was conducted to determine the optimum dietary protein level for young (an initial weight of 89 g) turbot, Scophthalmus maximus L. Duplicate groups of the fish were fed the five isoenergetic diets containing the various protein levels ranging from 290 to 570 g kg^?1 diet for 45 days. Survival was not affected by dietary protein level. Weight gain and feed efficiency were improved with dietary protein level up to 490 g kg^?1 diet. Dietary protein requirement of young turbot using the broken‐line model was estimated to be 494 g kg^?1 diet based on weight gain response. Protein efficiency ratio was not influenced by dietary protein level. The highest protein retention was obtained from the fish fed the 490 g protein kg^?1 diet. Proximate composition of the fish was not significantly affected by dietary protein level. In considering these results, it was concluded that the 494 g protein kg^?1 diet with 100 g lipid kg^?1 diet (15 MJ kg^?1 diet) provided optimal growth of young turbot under these experimental conditions.     

Ulcerative skin and fin lesions in reared turbot, Scophthalmus maximus (L.)

Abstract. In this report, the authors describe the evolution of an ulcerative disease that occurred during July and August 1986 in cultured juvenile turbot, Scophthalmus maximus (L.), in northwestern Spain. The daily fish mortality ranged from less than 1 to 7·5%, and the total of cumulative losses were 23·2%. The start of the epizootic was closely associated with a sudden increase of water temperature to values higher than 20·C. Peaks of mortality were observed during the development of the disease. Affected turbot showed erratic swimming, respiratory distress and inappetence. The lesions began as white discoloured foci on the skin, operculum, and base of dorsal and caudal fins which could suffer a total destruction of the interradial membrane. In an advanced stage of this condition, the epithelial surface of the centre of the lesions sloughed off and shallow and haemorrhagic ulcers were produced. Three distinct areas were observed in the ulcerative lesions with a clear predominance of different microorganisms: (1) a peripheric pale zone invaded by a ciliate protozoan resembling Cryptocaryon spp.; (2) an intermediate greyish-dark area containing large numbers of fastidious myxobacteria which failed to grow in culture media; and (3) a central haemorrhagic zone where we have isolated two halophilic, urease positive Vibrio strains (closely related to V. carchariae ). No microbial involvement was detected in the internal organs of affected fish. The virulence assays conducted with the Vibrio strains failed to reproduce the signs of the disease, either by intraperitoneal inoculation or by water challenge after skin abrasion. The use of alternate baths of formalin/malachite green and nitrofurazone resulted in an effective therapy to control the mortalities.     

Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen.     

An amoeba associated with gill disease in turbot,Scophthalmus maximus (L.)

An amoeba associated with a severe gill disease affecting farmed turbot, characterized by pronounced hyperplasia of the branchial epithelia and accumulation of mucus between the secondary lamellae, is described for the first time. The amoeba was isolated, cloned and cultured on Bactoagar. Light microscopy showed that trophozoites had a mean length of 12 μm (range 8–20 μm), with a nucleus 3–5 μm in diameter containing a large central nucleolus 1–3 μm in diameter. Transmission electron microscopy showed that the mitochondria, Golgi bodies, rough endoplasmic reticulum, microfilaments, cytoplasmic microtubules and glycocalyx were similar to those previously reported for the genus Platyamoeba. These and other findings strongly suggest that the amoeba isolated is of the genus Platyamoeba, while its morphometric characteristics suggest that it may represent a new species. This is the first report of a species of the genus Platyamoeba in association with gill disease in turbot.     

大菱鲆种质资源研究与开发

综合介绍了大菱鲆种质资源的分类、自然分布和生物学特性。从生化和分子生物学方面阐述了大菱鲆自然群体遗传结构及国内外研究现状。详述了大菱鲆野生资源的利用和养殖开发现状．特别是我国对引进的大菱鲆种质资源的养殖开发。指明了我国目前在引种及养殖方面存在的不足．提出了建立全国性大菱鲆良种引育中心,以保证我国大菱鲆养殖业的持续、稳定、健康发展的建议。     

Pathology associated with a viral erythrocytic infection in turbot, Scophthalmus maximus (L.)

Abstract. Since 1992, frequent erythrocytic viral infections have occurred in cultured turbot, Scophthalmus maximus (L.), on several fish farms in Galicia, Spain, causing important mortalities. Affected fish showed haemorrhages in the head, mouth and fins, exophthalmia and abdominal distension. The disease was also characterized by the presence of pale blue inclusions in affected erythrocytes. The examination of these erythrocytes by electron microscopy showed virus particles of 50–160 nm in size in the cytoplasm, budding av the cell membrane and extracellularly. The histopathological study revealed occasional necrosis of kidney haematopoietic tissue, an increase in the size and number of melanomacrophage centres of the spleen and kidney, and oedematous areas with hepatocytes containing eosinophilc bodies in the liver. Degeneration of muscle fibres was accompanied by an intense inflammatory oedema. Dermatitis as well as uveitis and corneal oedema were consistent lesions.     

Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn²⁺ and Cu²⁺ and inactivated by Fe³⁺. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.     

Cytotoxic activity against prelabelled RTG-2 cells in the turbot, Scophthalmus maximus (L.)

Leucocytes derived from head kidney, blood and spleen of turbot, Scophthalmus maximus (L.), exhibited cytotoxic activities against an established cell line (RTG-2) derived from rainbow trout, Oncorhynchus mykiss (Walbaum). The optimal effector:target ratio, temperature and incubation period for the activity was determined. Cytotoxicity was not caused by released factors, as effector:target contact was needed. Visualization of the cultures under the transmission electron microscope corroborated the contact between leucocytes and target cells and suggested the implication of monocyte-like cells in this cell-mediated cytotoxicity.     

Myxosporean infection causing intestinal disease in farmed turbot, Scophthalmus maximus (L.), (Teleostei: Scophthalmidae)

The present report describes an intestinal disease which causes important losses in farmed turbot. Mortality rates were higher in summer and reached 100% in all tanks where the disease was confirmed. Affected fish showed external signs consisting of anorexia, sunken eyes and a typical prominent bony ridge on the skull. These signs can be considered the pathognomonic signs of the disease, together with the gut lesions seen in the histological study. Pallor of the internal organs, intestinal haemorrhages and the presence of liquid in the intestine were also observed, with ascites in heavily infected fish. Histopathological damage was evident in the gut, with severe enteritis, detachment of epithelium, haemorrhages and inflammation of the subepithelial connective tissue. The myxosporean aetiology was demonstrated in all the fish showing the characteristic signs of disease. Myxosporean stages, including scarce spores, were found in the affected epithelium or free in the intestinal lumen together with epithelial debris. The present study demonstrates the importance of this myxosporean disease which represents a serious threat for turbot culture. This is also the first record of a member of the Myxosporea in turbot.     

Ocular bacterial signatures of exophthalmic disease in farmed turbot (Scophthalmus maximus)

As is the case at other sites in the body (e.g. the gut, skin and mouth), the ocular microbiota plays a crucial role in their host, as disturbances of the composition and function of the ocular microbiota are known to be associated with ocular disorders. Exophthalmic disease (ED) is a significant cause of high mortality in fish species, including farmed turbot (Scophthalmus maximus). However, the relationship between alterations in the ocular microbiota and ED in turbot is unclear. In this work, we collected turbot samples from farmed ponds with ED and healthy samples to understand changes in the ocular microbiota of turbot suffering from ED. We compared the structural and metabolic differences of ocular bacterial communities from farmed turbot with exophthalmic disease and those of healthy controls. Besides less microbial diversity found in turbot with ED regarding the control group, we also found that Aeromonas was the dominant bacteria both in controls and ED samples, but the abundance of Aeromonas was significantly greater in ED individuals. Moreover, the results of correlation test further suggest that Aeromonas overgrowth was correlated with the progress of the disease and shifts in ocular microbiota functional pathways in turbot. These findings emphasize that an increased abundance of Aeromonas serves as an ocular bacterial signature associated with ED in turbot, which provide basic information useful for diagnoses, prevention and treatment of ocular diseases occurring in cultured fish.     

大菱鲆仔稚鱼发育早期肠道菌群结构形成的影响因素

为研究鱼类发育早期肠道菌群结构的演变过程及影响因素,运用高通量测序技术,分析了处于不同发育阶段的大菱鲆(Scophthalmus maximus)仔稚鱼肠道、受精卵、不同类型的饵料和水源中的菌群结构,以及它们之间的相关性。结果显示,以不同的OTU(Operational Taxonomic Units,可操作分类单元)作为分类依据,发现大菱鲆仔稚鱼的肠道菌群结构在开口摄食后不久已趋于稳定,其优势菌与受精卵所携带的细菌关联较大。并且在大菱鲆仔稚鱼不同的发育时期,这一菌群的结构非常稳固,几乎不受水和饵料中优势细菌的影响而发生改变。乳球菌属的Lactococcus piscium菌株一直是大菱鲆仔稚鱼肠道中的优势菌种,在不同发育时期的优势度高达45%~65%。本研究还发现,大菱鲆仔稚鱼肠道可能对定植的菌种具有选择性,一些水环境和饵料中的非优势菌,如Streptococcus sp.,Pseudomonas sp.,Carnobacterium sp.等细菌也会定植于肠道,成为大菱鲆肠道中的次优势菌。