Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evaluation of cross protection induced by a furunculosis vaccine

Turbot was shown to be sensitive to injection challenges by Aeromonas salmonicida subsp. achromogenes (Asa). A systemic disease was induced and the bacterium was isolated from various internal organs. Histopathological changes involved haemorrhages, necrosis and degeneration in skin and muscle, haemorrhages and necrosis in kidney, degeneration in the heart muscle, and fusion of the secondary gill lamellae. A polyvalent commercial salmon vaccine, containing A. salmonicida subsp. salmonicida as one of five antigens, did not confer protection in turbot against an experimental Asa infection 13 weeks post-vaccination. Vaccination induced a significant antibody response against Asa cells but not against extracellular products of the bacterium. The results of the study indicate that Asa may be a potential threat to turbot farming and that the development of new turbot vaccines is needed.     

Protection against atypical furunculosis in Atlantic halibut,Hippoglossus hippoglossus (L.); comparison of a commercial furunculosis vaccine and an autogenous vaccine

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.     

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.     

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.     

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.     

Haemorrhagic areas in the mouth of farmed turbot, Scophthalmus maximus (L.)

Abstract. An epizootic in juvenile turbol reared on a farm located in The Ria de Vigo, Galicia, Northwest Spain, is described. The diseased turbot did not display unusual swimming behavior; the external signs of the disease were abdominal distension and haemorrbagic areas in the mouth. Internal examination of the fist showed an accumulation of reddish fluid in the peritoneal cavity. Microbiological analysis of the diseased fish revealed the presence, in pure culture in all the organs and lesions examined, of a bacterium which was characterized biochemically as Vibrio splendidus biotype I. The virulence tests showed that the V. splendidus biotype I isolate was pathogenic for rainbow trout (LD₅₀: 2.2 × 10⁴) and also for turbot (LD₅₀: 1.2 × 10⁴). The treatment of the fish using flumequine incorporated into the feed was effective in offsetting the mortality rate.     

Cryopreservation of turbot Scophthalmus maximus (L.) sperm: fertilization and hatching rates

The present paper assesses the fertilization and hatching rates of an artificial fertilization series (n=1153) using fresh and cryopreserved sperm from 49 specimens of turbot Scophthalmus maximus (L.), carried out to confirm the results of a previous study, with the ultimate aim of transferring these cryo‐preservation techniques to commercial hatcheries. No significant differences were found between the fertilization rates of the two groups (fresh and cryopreserved sperm) when their respective fertility rates were >69.2%, which was the case in 75% of all fertilizations. Likewise, no significant differences in hatching rates were found. In order to use the cryopreserved sperm more efficiently, a key concern for commercial use of this technique, we also experimented with a lower sperm:diluent ratio (1:1) than used previously at our centre. We also compared the traditional 0.5‐mL straws with 2‐mL cryotubes able to contain a higher volume of sperm, finding no significant differences in the resulting fertilization and hatching rates. In conclusion, the use of cryopreservation for turbot sperm presents major advantages for broodstock management in commercial hatcheries.     

Effects of oral vaccination against vibriosis in turbot, Scophthalmus maximus (L.), and sea bass, Dicentrarchus labrax (L.)

Abstract. Turbot, Scophthalmus maximus (L.), and sea bass, Dicentrarchus labrax (L.), are susceptible to vibriosis, a septicaemia caused in France by Vibrio anguillarum 408. A bacterin produced with this strain by Rhône–Merieux has been tested orally in both species and, as a comparison, by intraperitoneal injection. During a challenge carried out 4 weeks later by intraperitoneal inoculation, the protection observed in orally vaccinated fishes was significant (72 % in sea bass and 70 % in turbot). Eleven weeks after vaccination, this protection is still significant. The mean titre of serum agglutinins was weakly increased in orally vaccinated fishes. Bacteriostatic antibodies have also been searched for in serum, but compared to agglutination, the applied technique leads to lower titres and it has not been possible to prove the presence of such antibodies after oral vaccination. Passive immunizations using serum from orally vaccinated fishes (2 months after vaccination) confers some protection against a challenge carried out by inoculation of virulent vibrio. Therefore, the production of serum factors following oral vaccination of sea bass and turbot may be involved in generalized protection of fish.     

Experimental infection of turbot, Scophthalmus maximus (L.), by Moritella viscosa, vaccination effort and vaccine-induced side-effects

Moritella viscosa is the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic. The bacterium has also been isolated from various marine fish species. Bacterial diseases have been a limiting factor in farming of turbot, but M. viscosa has not so far been isolated. In this study, turbot was shown to be sensitive to M. viscosa infection in experimental challenges. Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon. A multivalent commercial salmon vaccine, containing M. viscosa as one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination. Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination. Vaccination did not induce a specific anti-M. viscosa response, while elevated anti-M. viscosa antibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge. The vaccine did, however, induce an antibody response against Aeromonas salmonicida, another vaccine component. Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant. The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups.     

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.     

Larval growth of turbot, Scophthalmus maximus (L.) produced with fresh and cryopreserved sperm

The present paper assesses the fertilization and hatching rates, as well as the growth, of larvae obtained from four artificial fertilizations (AF) using fresh and cryopreserved sperm of the turbot Scophthalmus maximus (L.). Larvae growth in both sperm groups, measured in terms of length and weight at culture days 0, 7, 14 and 31, are compared, as well as their growth rates. The two groups' fertilization and hatching rates were not significantly different. Likewise, no significant differences in length and wet weight of 7‐ and 14‐day‐old larvae were found using fresh and cryopreserved sperm; however, significant differences were found in 31‐day‐old larvae, which were more attributable to the variability inherent in larval turbot culture, and to variability in the reproductive specimens used in our study, than to the type of sperm employed. These results indicate that the type of sperm used in artificial fertilization, i.e. fresh or cryopreserved, is not a determining factor, either for fertilization and hatching, or for subsequent larval development. Our results also confirm once again the high quality of cryopreserved turbot sperm, and its usefulness in commercial hatcheries.     

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.     

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron‐like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1‐TRBIV‐MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT‐PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT‐PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post‐vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon‐induced antiviral molecules were observed in the vaccinated fish 60 h post‐vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.     

Growth and survival of young turbot (Scophthalmus maximus L.) produced with cryopreserved sperm

This study assessed the growth and survival over a year of two groups of 4‐month‐old turbot Scophthalmus maximus (L.) derived from artificial fertilization with fresh (FG) and cryopreserved sperm (CG). Growth in both groups, measured monthly in terms of length and weight, were compared. Survival was also recorded. No significant differences were found when we compared weight and length data in both groups. Growth rates were similar between FG and CG young turbot during 1 year. Likewise, the same survival rate (92.2%) was found in both groups. Our results show the good survival and growth of young turbot obtained from cryopreserved sperm, and confirm the cryopreservation technique as a useful tool for raising turbot for commercial purposes.     

Atypical Aeromonas salmonicida infection in naturally and experimentally infected cod, Gadus morhua L.

Cod, Gadus morhua L., of wild origin, were reared at different temperatures for 12 months. During this period, moribund and newly dead fish were examined and samples collected for bacteriology and histopathology. Atypical Aeromonas salmonicida was isolated from 10 individuals reared at or above 7 °C. The isolates were homogeneous with respect to biochemical and antibiogram characters and similar to the ssp. achromogenes National Collection of Industrial and Marine Bacteria, UK, type strain 1110 and reference strains that have been isolated from salmonids and haddock in Iceland. Histopathological analysis of the naturally infected cod showed typical ulceration associated with atypical A. salmonicida infection and also widespread granulomatous formations. One‐year‐old cod of farmed origin, kept at 9 °C, received intraperitoneal or intramuscular injection with different doses of atypical A. salmonicida, isolated from the above wild cod. Mortalities were monitored for 28 days and the LD₅₀ calculated. The route of bacterial injection influenced the mortality rate and LD₅₀ value and affected, to some extent, the pathological changes observed and humoral immune parameters. Pathological changes, including haemorrhage, early stages of granuloma formation and necrotic changes, were seen in several organs. Infection appeared to induce non‐specific antibody activity against trinitrophenyl (TNP)‐haptenated protein and may have activated the complement system. Specific antibody response against atypical A. salmonicida was not detected.     

Effects of silymarin on growth performance,antioxidant capacity and immune response in turbot (Scophthalmus maximus L.)

A 9‐week feeding trial was conducted using triplicate groups of turbot (6.50 ± 0.01 g) to explore the potential effects of silymarin. Three concentrations of silymarin (100, 200, and 400 mg/kg) were added to the plant protein‐based diet. Fish were randomly distributed into fiberglass tanks (30 fish per tank). The results showed that adding 100 mg/kg silymarin significantly improved the growth performance, with no effects on feed utilization. The antioxidant capacity in the liver was significantly improved in the 100 mg/kg and 200 mg/kg silymarin groups by not only inducing the activities of superoxide dismutase (SOD) and catalase but also increasing the messager RNA (mRNA) expression levels of SOD, glutathione peroxidase, and peroxiredoxin 6. Meanwhile, supplying 100 and 200 mg/kg of silymarin enhanced the heights of villi and enterocytes. Silymarin supplementation reduced the mRNA expression of interleukin‐8 and tumor necrosis factor‐α but induced the expression of transforming growth factor‐β (TGF‐β) in the intestine. These results indicated that silymarin was a potential nutraceutical that could enhance the growth performance and health status of turbot fed in a high plant protein diet. Adding 100 mg/kg silymarin to the plant protein diet achieved optimal performance in turbot.     

Physiological and morphological effects of severe hypoxia,hypoxia and hyperoxia in juvenile turbot (Scophthalmus maximus L.)

We studied the physiological status of juvenile turbot (Scophthalmus maximus L.) under severe hypoxia (1 and 2 mg/L dissolved oxygen, DO), hypoxia (3 and 5 mg/L DO), hyperoxia (11 and 14 mg/L DO) and normoxia (7 mg/L DO, control) conditions. The respiratory rates, haematology parameters, acid–base balance status and gill structure were analysed to find the effects of different DO concentration on turbot. Fish mortality was only observed under severe hypoxia conditions. Severe hypoxia caused an increase in respiratory rates and red blood cell counts, as well as an increase in haemoglobin and haematocrit levels in the fish. In fish exposed to hypoxia conditions, the respiratory rate increased overall as the DO concentration decreased. Lower pCO₂ and HCO₃^? levels led to a high blood pH, while the pO₂ remained stable. In hyperoxia groups, respiratory rate decreased as the DO concentration increased. The levels of pCO₂ and HCO₃^? significantly increased (P < 0.05), while the pO₂ level and blood pH did not change obviously. The gill structure was damaged after prolonged exposure to hyperoxia, but no obvious damage was found in hypoxia groups. The fish that survived the hypoxia or hyperoxia treatment were able to restore the structural integrity of the gills after 14 days' recovery. The results suggest that juvenile turbots can tolerate a wide range of DO concentrations. However, even mild hyperoxia condition (11 mg/L DO), which is widely used in fish culture, has adverse effects on juvenile turbot physiology.