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.     

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.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/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.     

Indirect estimation of stomach volume of rainbow trout Oncorhynchus mykiss (Walbaum)

The main hypothesis of this study was that if stomach volume is correlated with food intake it can be estimated without laborious and destructive direct measurement. Rainbow trout, Oncorhynchus mykiss (Walbaum), ca. 500–1300 g, were starved for 1, 4, 8 or 16 days at 15°C after which they were fed in excess with dry pellets containing known amounts of X‐ray‐dense markers. Immediately after feeding the fish were killed, X rayed and weighed. Then the stomach was dissected, its contents removed and weighed, and stomach volume was measured. X‐ray plates were developed and feed intake was estimated based on the amount of marker. All measured variables correlated positively with stomach volume. The best fit for linear regression models was obtained for fish starved for 4 days, where stomach content (dry mass) explained 94%, food intake (based on X‐ray measurement) 77% and fish mass 62% of the variation in stomach volume. However, as stomach content measurement can be a lethal, or at least very stressful, event for the fish, the accuracy of food intake measurement (X‐ray) could be increased using multiple regression. In multiple linear regressions, R²‐values varied between 0.79 (16‐day starvation) and 0.91 (1‐day starvation) with food intake and fish mass as explanatory variables for stomach volume. These results indicate that the stomach volume in rainbow trout can be estimated satisfactorily using indirect methods, which are not detrimental to the fish, although feeding history may affect the accuracy of the estimates.     

Effects of different artificial photoperiods and temperatures on haematological parameters of rainbow trout (Oncorhynchus mykiss)

This study assessed the effects of two artificial photoperiods (LD 24:0 and LD 12:12) and three temperature regimes (9, 11, and 18°C) for 30 days on haematological parameters of trout (Oncorhynchus mykiss) kept in freshwater. Samples were taken at days 7, 14, and 30 during exposure to treatments. A higher mortality (22%) and lower oxygen concentration (<8.0 mg/l) were associated with the combination of photoperiod LD 24:0 and 18°C. The LD 24:0 photoperiod (independently of temperature) increased the haematocrit and the number of erythrocytes at days 7, 14, and 30 (P < 0.01). A temperature of 18°C (independently of photoperiod regimes) diminished the number of total leucocytes, lymphocytes and thrombocytes (P < 0.01). The LD 24:0 photoperiod (also independently of temperature) lowered the number of lymphocytes only after 14 days of experimentation (P < 0.01). Interaction between artificial photoperiod and temperature was only observed at day 14 for polychromatophils (P < 0.01). These results resemble the effects of stress caused by elevated temperatures and the application of continuous light photoperiods, indicating that survival risks may develop in trout farming when this combination is met.     

Lipid-stimulated somatostatin secretion in rainbow trout,Oncorhynchus mykiss

Previous work has shown that somatostatins (SS) affect teleost lipid metabolism indirectly by inhibition of insulin (INS) and directly by stimulation of hepatic lipolysis. In the present study, rainbow trout (Oncorhynchus mykiss) were used to characterize further the lipid-SS relationship by evaluating how lipid, contributes to SS secretion bothin vivo andin vitro. In vivo hyperlipidemia was induced for up to 3 h by short-term (2 min) infusion of a triacylglycerol (TG)-rich lipid emulsion (20% Intralipid^®). Plasma total lipid concentration increased 118 and 155% over control levels 1 h and 3 h, respectively, after infusion; much of this increase was due to elevated plasma fatty acids (FA), which increased 39 and 520%, respectively, over the same time-frame. The hyperlipidemic pattern was attended by a significant increase in the plasma concentration of SS. The specific effects of fatty acids were evaluated on isolated Brockmann bodies. Palmitic acid and oleic acid stimulated SS release 378 and 82%, respectively, over baseline levels. These results indicate that lipids, and in particular fatty acids, modulate SS secretion in rainbow trout.     

Effect of esterification on the absorption of astaxanthin in rainbow trout, Oncorhynchus mykiss (Walbaum)

Gastrointestinal and serum absorption of astaxanthin was studied in rainbow trout, Oncorhynchus mykiss (Walbaum) (217 ± 2 g) fed diets supplemented with either esterified astaxanthin (from Haematococcus pluvialis) or free astaxanthin (synthetic, as 8% w/w beadlets) at similar levels (50 mg kg^?1). After 56 days of feeding, there was a significant difference (P = 0.0582) between steady‐state serum astaxanthin concentrations for fish fed free (2.0 ± 0.3 μg mL^?1) or esterified astaxanthin (1.3 ± 0.1 μg mL^?1) at the 90% confidence level. However, following ingestion of a single meal supplemented with free or esterified astaxanthin, the rates of astaxanthin absorption into serum were not significantly different (P > 0.1) (0.8 ± 0.2 µg mL^?1 h^?1 and 1.0 ± 0.4 µg mL^?1 h^?1 respectively). In fish fed both free or esterified astaxanthin, higher absorption (P < 0.05) of astaxanthin by the ileal (0.8 ± 0.14 μg g^?1 and 0.9 ± 0.15 μg g^?1 respectively) compared with the posterior (0.2 ± 0.01 μg g^?1 and 0.3 ± 0.14 μg g^?1 respectively) intestine was recorded. This confirmed the role of the anterior intestine in carotenoid absorption. Non‐detectable levels of esters in digesta taken from the hind intestine suggest the anterior intestine is also the primary region for ester hydrolysis.     

Relationships between growth and disease resistance in rainbow trout,Oncorhynchus mykiss (Walbaum)

Rainbow trout from 23 families were evaluated for growth and resistance to the bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum and infectious haematopoietic necrosis (IHN) caused by IHN virus. Average family weights were between 161 and 263 g with an average of 225 g at 213 days post‐fertilization with specific growth rates ranging from 2.37 to 2.88. Per cent survival of fish challenged with F. psychrophilum was between 18% and 100%, while for those challenged with IHNV, the range was between 12% and 93%. Significant positive correlations were found for end body weight and resistance to IHN (P < 0.05) and for early body weight and resistance to BCWD (P < 0.1). However, no significant correlations were detected between resistance to both pathogens or disease resistance and overall genetic diversity or diversity within the major histocompatibility locus.     

Initiation of tetraploid breeding line development in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. For initiating the development of tetraploid breeding lines in rainbow trout, Oncorhynchus mykiss (Walbaum), using different populations as a genetic basis, four populations out of a total of 11 were selected according to gene marker differences. Egg batches resulting from single-pair and group matings and representing purebred and crossbred progenies of these populations were exposed to a late pressure shock for inhibition of first mitosis. In six out of 26 treated egg batches tetraploid embryos were identified by chromosome examinations. In the case of successful ploidization single-pair matings yielded exclusively tetraploid embryos, while group matings contained some diploids. Successful tetraploidization was dominated by mating partners from one population. In total 413 putative tetraploid fry were available for further rearing. Besides diploids, triploids obtained by thermally induced retention of the second polar body were kept as controls mainly for methods to identify genome manipulated trout. Survival rates of tetraploids were low and loss frequencies did not stabilize till the age of 9 months. Thirty-three tetraploid fish, individually checked by measurements of DNA content, nuclear cell size and number of nucleoli per cell, were found at the age of 17 months. Body weights of survivors were similar for tetraploids and diploids throughout the whole testing procedure.     

Red blood cell indices of rainbow trout Oncorhynchus mykiss (Walbaum) in aquaculture

A direct non‐parametric method was used to calculate reference (physiological) haematology values for farmed 10–12‐month rainbow trout of the Kamloops strain (mean weight: 330±131 g) with respect to red blood cell counts (RBCc), haematocrit values (Hct), haemoglobin concentrations (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentrations (MCHC). The fish in the selected reference group (n=798) were given dry pelleted diets that contained 37–47% crude protein, 7–18% crude fat and 108–300‐mg vitamin E, 1.08–5‐mg folic acid, 0.018–0.05‐mg vitamin B₁₂, 48–64.5‐mg iron, 4.5–8.4‐mg copper and 0.18–0.24‐mg selenium supplied per kg of diet. Ethoxyquin and butylhydroxytoluol were used to protect the fat component against oxidation. The fish were kept at a stocking density of 50kg per cubic metre in tanks provided with running freshwater (dissolved oxygen 8.4–11.5 mg L^?1, with O₂ saturation of 77–98%) at an ambient temperature of 0.2–16°C. Blood was sampled between September and November at a photoperiod of 9–13 h:11–15 h (light:dark). Reference ranges for the preceding haematological indices were as follows in immature females (males): RBCc, 0.77–1.42T L^?1 (T – tera, 10¹²) (0.98–1.55T L^?1); Hct, 0.304–0.502 (0.34–0.546); Hb, 54–93 g L^?1 (59–97 g L^?1); MCV, 282–469 fL (279–434 fL); MCH, 51–86 pg (47–78 pg); MCHC, 0.15–0.22 (0.15–0.2). In males, values for RBCc, Hct and Hb were significantly higher (P=0.01 and 0.0000 respectively) and those for MCV, MCH and MCHC were significantly lower (P=0.01 and 0.0002 respectively) than in immature females. Nutritional and environmental factors affecting erythropoiesis in trout and some correlations between haematological (RBCc, Hb, Hct) and biochemical indices of the blood plasma (total protein, cholesterol) are discussed.     

Attachment of Vibrio pathogens to cells of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. The attachment of Vibrio pathogens to cells of rainbow trout, Oncorhynchus mykiss (Walbaum), was studied by use of species-specific monoclonal antibodies and indirect FITC- immunofluorescence microscopy. Vibrio anguillarum, V. ordalii and V. parahaemolyticus attached to cultured cells of rainbow trout gonads, various tissues of cryostat sections of whole fish, and smears of gills, intestine, buccal mucosa and skin. The attachment was inhibited by prior incubation of bacteria with monoclonal antibodies at titres of 1:32, or less. Other Vibrio pathogens used in this study did not attach to any trout cells. The research provides approaches to study the mechanisms of bacterial attachment in the onset of Vibrio infections.     

Effects of temperature on disease progression and swimming stamina in Ichthyophonus-infected rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss , were infected with Ichthyophonus sp. and held at 10 °C, 15 °C and 20 °C for 28 days to monitor mortality and disease progression. Infected fish demonstrated more rapid onset of disease, higher parasite load, more severe host tissue reaction and reduced mean-day-to-death at higher temperature. In a second experiment, Ichthyophonus -infected fish were reared at 15 °C for 16 weeks then subjected to forced swimming at 10 °C, 15 °C and 20 °C. Stamina improved significantly with increased temperature in uninfected fish; however, this was not observed for infected fish. The difference in performance between infected and uninfected fish became significant at 15 °C ( P  =   0.02) and highly significant at 20 °C ( P  =   0.005). These results have implications for changes in the ecology of fish diseases in the face of global warming and demonstrate the effects of higher temperature on the progression and severity of ichthyophoniasis as well as on swimming stamina, a critical fitness trait of salmonids. This study helps explain field observations showing the recent emergence of clinical ichthyophoniasis in Yukon River Chinook salmon later in their spawning migration when water temperatures were high, as well as the apparent failure of a substantial percentage of infected fish to successfully reach their natal spawning areas.     

Electrophoretic characterization of three hatchery-reared strains of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Significant differences were found in allelic frequency distribution among three strains of rainbow trout, Oncorhynchus mykiss (Walbaum). The isocitrate dehydrogenase ( IDH-3, 4 * 1.1.1.42) locus was fixed in two of the strains while the superoxide dismutase ( SOD-I * 1.15.1.1) locus was absent in one of the strains.
Similarity indices ranged from 0·973 to 0·988 on the scale of 1·000 among pairwise comparisons of the strains while Nei's genetic distance was 0·012 between Mount Lassen and Manx, 0·025 between Mount Lassen and Tagwerker and 0·027 between Manx and Tagwerker. Gene diversity was highest in the Mount Lassen strain, followed by Manx, with Tagwerker having the least. These differences in gene diversity were attributed to breeding and management practices used at the hatchery. The need for characterization of strains of rainbow trout, especially in crossbreeding programmes that aim to increase genetic variation, was emphasized by these results.