In vivo and in vitro cell-mediated immune responses of rainbow trout, Oncorhynchus mykiss (Walbaum), against Cryptobia salmositica Katz, 1951 (Sarcomastigophora: Kinetoplastida)

Abstract. Delayed-type hypersensitivity (DTH) reaction (an in vivo manifestation of cell-mediated immunity) was detected in Oncorhynchus mykiss maintained on a pantothenic-acid-supplemented diet 2 weeks after infection with Cryptobia salmositica. The reaction was similar to that in mammals with mononuclear cell infiltration into the dermis and muscle layers and the presence of oedema. DTH reaction was also displayed by fish on a pantothcnic-acid-supplemented diet that had recovered from the infection and were protected against further infection. The reaction was less marked in infected or protected fish on a pantothenie-acid-deficient diet. Inhibition of macrophage migration (an in vitro expression of cell-mediated immunity) was observed when head kidney cell suspensions from protected fish maintained on either pantothenic acid supplemented or deficient diets were incubated with Cryptobia antigen. No inhibition of migration was evident when head kidney cell suspensions from the above fish were incubated without antigen, nor was it evident when cells from uninfected fish were used. The occurrence of a typical DTH reaction in rainbow trout and the feasibility of assessing it by measuring the thickness of the induration provides a simple and practical method for assessing cell-mediated immunity in large scale vaccination programmes against pathogens.     

Dietary modulation of humoral immune response and anaemia in rainbow trout, Oncorhynchus mykiss (Walbaum), infected with Cryptobia salmositica Katz, 1951

Abstract. The humoral immune response in Cryptobia-salmositica -infected rainbow trout on pantothenic-acid-deficient/low protein (19%) diets was depressed. There was no significant difference in parasitaemia between fish on the pantothenic-acid-deficient diet and those on the supplemented diet; however, the parasitaemia in fish on 19% protein diet was significantly lower than that in fish on 38% protein diet. The low humoral responders, in contrast to the high responders (on 38 and 29% protein diets), had less severe anaemia and the packed cell volume at the end of the experiment was not significantly different from that at 2 weeks post-infection. Red cells from the low responders were Coombs' negative while those from the high responders were Coombs' positive from 4 to 6 weeks post-infection. The authors suggest that, in the low responders, red cell destruction is essentially due to the'lytic component' of the parasite antigen in contrast to the involvement of both the ^llytic' and the 'immune complex-forming' components in the high responders. The present study indicates that certain deficient diets are useful in modulating the parasitaemia and also in decreasing red cell destruction. Therefore, dietary modification may help in altering the course of such infections and the progression of the disease.     

Impact of a pathogenic haemoflagellate, Cryptobia salmositica Katz, on the metabolism and swimming performance of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Oxygen consumption of juvenile rainbow trout (5 g at 13°C) at moderate swimming speeds did not change significantly when infected with Cryptobia salmositica. However, significant reductions of as much as 44% of the maximum aerobic scope for activity and 24% of the critical swimming speed were observed when the parasitaemia reached a maximum of 57.6 × 10⁶ ml⁻¹ fish blood at 3 weeks post- infection. Blood haematocrit was significantly reduced from the initial 34.1 to 19.7% at 4 weeks post- infection, probably as a result of haemolysis by the parasite. The destruction of red blood cells clearly led to lower oxygen carrying capacity, and reduced respiratory and swimming performance.     

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.     

Comparative in vitro studies on virulent and avirulent strains of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida)

Abstract. The optimum temperature for in vitro multiplication of Cryptobia salmositica was 10°C. The avirulent strain multiplied more rapidly than the virulent strain. The haemolytic components, lytic component (LC) and immune complex-forming component (ICC), were secreted by the two strains into the culture medium and were detectable from one week post-inoculation. The haemolytic activity in the supernatant increased with increasing parasite numbers in both strains. Although cultures of the avirulent strain had higher parasite numbers than those of the virulent strain, the haemolytic activity was significantly lower than that of the virulent strain. Antiserum against ICC was produced in rabbit by immunization with ICC-coated rainbow trout red blood cells.     

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.     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

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.     

Siderophores produced by Vibrio anguillarum in vitro and in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Ten strains of Vibrio anguillarum produced three different types of iron-binding compounds when cultured under different conditions. These were (1) a common phenolate siderophore produced by all strains. (2) a hydroxamate siderophore produced by three strains and (3) a second phenolate siderophore, tentatively identified as anguibactin, produced by V. anguillarum strain 775 and two other strains, all of which contained a plasmid of 45–50 Md. The relative affinities of these siderophores, determined by competition for ⁵⁵Fe was: anguibactin < hydroxamate siderophore < common phenolate siderophore. However, under these conditions, none removed iron from purified aerobactin. Experimental infection of rainbow trout. Oncorhynchus mykiss (Walbaum), showed that only the common phenolate siderophore was detected in the kidney and spleen of fish infected with strains 91079 and NCIMB6. The hydroxamate siderophore produced in vitro by strain NCIMB6 was not detected in vivo. However, in the kidney of fish infected with strain 775, both the common phenolate siderophore and anguibactin were detected, showing that a second uptake system is required by strain 775 in vivo and that the iron-uptake system based on the common phenolate siderophore is defective.     

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.     

Use of probiotics to control furunculosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aerobic heterotrophic bacteria were isolated from the intestinal contents of Atlantic salmon, Salmo salar , rainbow trout, Oncorhynchus mykiss , and turbot, Scophthalmus maximus , on tryptone soya agar and De Man Rogosa and Sharpe agar, of which 11 of 177 (6% of the total) of the isolates were antagonistic to Aeromonas salmonicida . Four of these cultures, which were identified tentatively as A. hydrophila , Vibrio fluvialis , Carnobacterium sp. and an unidentified Gram-positive coccus, were beneficial to fish when fed singly or as an equi-mixture. Feed supplemented with the putative probiotics indicated survival of the organisms in the gastrointestinal tract for 7 days. Feeding with the probiotics for 7 and 14 days led to better survival following challenge with A. salmonicida . There was no indication of serum or mucus antibodies to A. salmonicida , but there was an increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity in the 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.