The effect of glycyrrhizin on rainbow trout, Oncorhynchus mykiss (Walbaum), leucocyte responses

Abstract. Treatment of rainbow trout macrophages with glycyrrhizin (GL), an aqueous extract of licorice, Glycyrrhiza glabra , enhanced their respiratory burst activity. Maximal effects were seen using concentrations of 10–100 μml⁻¹. GL also modulated trout lymphocytes, increasing proliferation responses to the mitogen phytohaemagglutinin some two-fold over a range of GL concentrations. In addition, GL elicited the release of a macrophage activating factor (MAF) from head kidney leucocytes, as assessed by the ability of generated supernatants to increase respiratory burst activity of target macrophages. MAF activity was most apparent using 100 μg ml⁻¹ GL to induce MAF release and a 48-h incubation period with the target macrophages. Finally, GL was shown to enhance the release of MAF in response to the mitogen concanavalin A. The possible use of GL as a stimulant of fish innate defences is discussed.     

The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum)

The anaesthetic effects of clove-oil-derived eugenol were studied in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Acute lethality and the effects of multiple exposures to eugenol were measured. The estimated 8-96 h LC₅₀ for eugenol was found to be approximately 9 p.p.m. Times to induction and recovery from anaesthesia were measured and compared with MS-222 under similar conditions. Eugenol generally induced anaesthesia faster and at lower concentrations than MS-222. The recovery times for fish exposed to eugenol were six to 10 times longer than in those exposed to similar concentrations of MS-222. Clove oil eugenol was determined to be an acceptable anaesthetic with potential for use in aquaculture and aquatic research. Doses of 40-60 p.p.m. eugenol were found to induce rapid anaesthesia with a relatively short time for recovery in juvenile trout.     

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum , A. hydrophila , A. hydrophila subsp. dhakensis , A. salmonicida subsp. salmonicida , A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10–25 bands, of which major bands were seen in the region of 32.5–47.5 and 62–83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83–173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Surveillance of health status on eight marine rainbow trout, Oncorhynchus mykiss (Walbaum), farms in Denmark in 2006

The health status of eight marine rainbow trout farms was followed from mid-June to mid-September 2006 by sampling both dead and healthy fish approximately every 2 weeks for bacteriological and virological investigation. No fish pathogenic viruses were detected, but all farms experienced disease and mortality as a result of various bacterial infections. Yersinia ruckeri was found on four and Renibacterium salmoninarum on five of the farms, but only during the first part of the surveillance period. This indicates that the fish carried the infection from fresh water, and cleared the infection in salt water. Aeromonas salmonicida subsp. salmonicida caused mortality on five farms, but persisted throughout the sampling period. Although A. salmonicida was probably carried from fresh water, the fish were not able to clear the infection in the sea. Vibrio anguillarum caused mortality on six of the farms throughout the sampling period, O1 being the dominant serovar, and Photobacterium damselae subsp. damselae was found on seven farms as a cause of disease. During the period of highest water temperatures Vibrio parahaemolyticus and Vibrio vulnificus were detected in dead fish in five and two farms, respectively, although their significance as causative pathogens is questionable. Vibrio vulnificus has not previously been found in rainbow trout in Denmark. Both mortality and number of antimicrobial treatments during the period were considerably higher in unvaccinated compared with vaccinated fish. Resistance to commonly used antimicrobials was low or absent.     

Photobacterium damselae subsp. damselae, an emerging pathogen in Danish rainbow trout, Oncorhynchus mykiss (Walbaum), mariculture

A selection of 16 field isolates of Photobacterium damselae from marine rainbow trout farms in Denmark was subjected to phenotypic and genotypic characterization and pathogenicity to fish. All isolates belonged to the subspecies damselae , being positive for haemolysis, motility and urease. There were considerable differences in haemolytic properties, some isolates presenting a broad zone of haemolysis and others only a narrow zone. Pulsed-field gel electrophoresis revealed a high diversity indicating that P. damselae subsp. damselae is an opportunistic, not clonal pathogen in Danish marine rainbow trout. Virulence of the strains to rainbow trout was highly variable with LD₅₀ values ranging from 3.9 × 10³ to 1.5 × 10⁸ cfu at 20 °C. The virulence was significantly higher at 20 °C than at 13 °C. The strains with the strongest haemolytic properties were the most virulent suggesting a strong involvement of haemolysin in the pathogenesis. The pathological changes were consistent with a bacterial septicaemia and the haemorrhages were more pronounced than for most other bacterial infections.     

Oxygen-induced gas bubble disease in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. An Aquatector oxygen injection unit was used to supersaturate a hatchery water supply to 200% oxygen saturation (18–20mg/l) and increase the total gas pressure to 120% of saturation. Nitrogen saturation was reduced to near 100%. Rainbow trout, Oncorhynchus mykiss (Walbaum), held in the treated water developed signs of gas bubble disease in 4 days, and 50% died within 20 days. We demonstrated that supersaturated total gas pressure due to excessive oxygen saturation causes gas bubble disease in the absence of supersaturated nitrogen gas. It is recommended that users of oxygen injection systems closely adjust the amount of oxygen added to the water to keep the total gas pressure near saturation.     

Immunogenicity of amoebic antigens in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Rainbow trout developed a humoral immune response against numerous antigens of sonicated amoebae which were emulsified with Freund's complete adjuvant and injected into the peritoneum. The amoebae were cultured from the gills of Atlantic salmon, Salmo salar L., affected by amoebic gill disease. Antibodies in fish sera were detected by both enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Non-spedfie reactivity in fish serum against Escherichia coli, the bacterium used in co-cultivation of amoebae in vitro, was removed by immunoadsorption. Results obtained using ELISA and immunoblotting were comparable and indicated no significant difference in response to immunization with 10, 50 or 250 μg of sonieatcd amoebic protein. Amoebae contained immunogenie components of > 100, 100, 89, 49, 37 and 34kDa.     

Seasonal production of rainbow trout, Oncorhynchus mykiss (Walbaum), in ponds using different feeding practices

Abstract. In temperate regions of North America, the culture of rainbow trout, Oncorhynchus mykiss (Walbaum), in ponds is constrained to the period mid-November to early April by water temperature. Feeding practice may influence production time. The effects of three feeding practices on growth and body composition of rainbow trout raised in ponds during a 142-day winter growth period were investigated. Feeding practices were (1) hand-feeding to satiation, (2) feeding according to a fish size/water temperature chart, and (3) feeding by demand feeder.
Fish fed to satiation consumed 66% more diet than fish fed according to a feeding chart and 163% more than fish fed by demand feeders. Fish fed to satiation had significantly higher (P < 0·05) weight gain, harvest weight, specific growth rate, and protein gain with no significant increase (P > 0·05) in percentage body fat or feed conversion ratio (FCR) compared to the other two treatments. Survival percentage did not differ significantly (P > 0·05) between treatments. These data indicate that by using satiation feeding, marketable size rainbow trout can be produced in temperate region ponds without increases in percentage body fat or FCR.     

Blood electrolyte and prolactin regulation in rainbow trout, Oncorhynchus mykiss (Walbaum), exposed to nitrite

Abstract. Rainbow trout, Oncorhynchus mykiss (Walbaum), exposed to environmental nitrite for 3h showed significantly reduced chloride concentrations from 120·9 ± 0·5 to 113·8 ± 2-4 mmol/1, the result of nitrite uptake by the gills with a compensatory loss of blood chloride. This change could be prevented by injection of 10 mg/kg sulpiride, which stimulates prolactin secretion through antagonism of dopamine D₂ receptors, thus increasing circulating prolactin levels. Prolactin is known to decrease membrane permeability and the increased levels of the hormone probably account for the relatively unchanged (compared to controls) plasma chloride and potassium levels in nitrite-exposed, sulpiride-treated fish.     

A comparative tissue distribution study of oxytetracycline in rainbow trout, Oncorhynchus mykiss (Walbaum), and chinook salmon, Oncorhynchus tshawytscha (Walbaum)

Oxytetracycline (OTC), a broad-spectrum antibiotic, is used widely to treat bacterial diseases in farmed fish. In the present study, the time course of OTC concentrations in freshwater rainbow trout, Oncorhynchus mykiss (Walbaum), and seawater chinook salmon, Oncorhynchus tshawytscha (Walbaum), were compared, tissue by tissue, after receiving a bolus dose of the antibiotic (5 mg kg^–1 or 50 mg kg^–1) intra-arterially (i.a.). The OTC concentration–time profiles of rainbow trout tissues were found to be very similar to those of the corresponding tissues in chinook salmon. Therefore, neither water salinity nor fish species seemed to play an important role in the disposition and elimination of OTC in these salmonids. In a separate experiment, rainbow trout were implanted surgically with a urinary cannula and received a single dose of OTC (50 mg kg^–1) i.a. Urine was collected from the cannula daily for 13 days. The amount of OTC excreted into the bile was found to be larger than that eliminated by the urine. These results show the similarity of OTC pharmacokinetics in freshwater rainbow trout and seawater chinook salmon and render support in using a single fish species to study the pharmacokinetics of a drug for other species in the same taxon.