Edwardsiellosis in farmed rainbow trout (Oncorhynchus mykiss)

Edwardsiella tarda was isolated from naturally infected rainbow trout (Oncorhynchus mykiss) in raceway culture on a commercial fish farm where the fish were kept at a density of 110 kg m^?3 and at a water temperature of 14°C and with a photoperiod of 13 h light:11 h dark. The clinical signs of diseased fish (150 ± 20 mm standard length) were anorexia and lethargy. The most striking lesions in the fish were in the liver. There were hyperaemia and haemorrhages; on histopathological examination, the liver displayed inflammatory infiltrate in portal area, focal necrosis, dilatation of blood sinus and activation of sinusoidal cells. Infection experiments, performed 2 years after isolation of the original culture of E. tarda, were carried out under laboratory conditions at water temperatures of 15, 18 or 24°C. All experimental fish (common carp, Prussian carp, tench), intraperitoneally injected with 8 × 10⁶ cells, demonstrated a total resistance to E. tarda.     

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.     

Seasonal variation of muscle metabolic organization in rainbow trout (Oncorhynchus mykiss)

This study examined how muscle metabolic organization varied during an annual cycle in which rainbow trout (Oncorhynchus mykiss) were held in outdoor holding ponds in which they were exposed to natural changes in temperature (range 0.2 to 15.6°C) and photoperiod. We examined the activities of glycolytic and mitochondrial enzymes in red and white muscle to evaluate whether trout enhance their capacity for lipid and carbohydrate oxidation during cold-acclimization. When assayed at habitat temperature, the enzyme activities generally increased in spring to reach a maximum in summer followed by a decrease in the fall. This led to significantly higher activities at warm than cold periods for all enzymes measured in red muscle and all but one in white muscle. The activities at 10°C provided little evidence for compensatory adjustments of aerobic capacity. Particularly in red muscle, enzyme levels at 10°C were generally lower during cold than warm periods. The variation of enzyme activities throughout the cycle was not due to changes in protein concentration, as the same responses were observed when activities were expressed per g wet mass or per mg protein. Although the aerobic capacity did not increase with cold-acclimatization, the relative capacity for lipid oxidation was higher in winter than in summer trout. In contrast, the relative capacity for aerobic glycolysis was higher in summer than in winter trout. Thus, the metabolic capacities of trout muscle undergo seasonal reorganization.     

Development of a protein binding assay for teleost insulin-like growth factor (IGF)-like: relationships between growth hormone (GH) and IGF-like in the blood of rainbow trout (Oncorhynchus mykiss)

Using rainbow trout plasma protein (IGF-BP) which specifically binds human insulin-like growth factor (IGF) (Niu and Le Bail 1993), we have developed an assay to measure plasma IGF-like levels in different teleost species. Before the assay and to prevent interference by IGF-BP, IGF-like was extracted from all samples, using SP Sephadex C-25 in acidic conditions. After this treatment, contamination of the IGF fraction by IGF-BP which was estimated by binding assay, was approximately 5%, and was not detectable by western ligand blot. Human IGF-I was used as standard and labelled hormone. Sensitivity of the assay was 0.15–0.40 ng/ml (ED90) and ED50 was 1–3 ng/ml. hIGF-II crossreaction was partial and no significant displacement was observed with Insulin from different species or with other hormones. Inhibition curves were obtained with plasma IGF fractions (but not with tissue extracts) from teleost and mammals and are parallel to the standard curve. These results suggest that the protein binding assay can quantify an IGF-like factor in the plasma of teleost and that the binding sites of IGF are well conserved during vertebrate evolution. Using this IGF binding assay, IGF-like was measured in parallel with growth hormone (GH) in plasma from young rainbow trout killed every 1.5h throughout one day. The daily profiles for both hormones, which appear pulsatile, are similar. A significant correlation was observed between GH levels and IGF-like levels with a 1.5h delay. Analogous observations were obtained in individual catheterized adult rainbow trout. Although plasma GH levels differ greatly between fish, less variability is found with IGF-like. In a third experiment, rainbow trout were starved or submitted to bovine GH treatment for four weeks. Starved fish, in which plasma GH levels increased, had plasma IGF-like level significantly lower than in fed fish. In bGH injected fish, plasma IGF-like level was significantly higher than in non-injected fish. These results suggest that, as in mammals, IGF-like secretion depends on plasma GH level and could be modulated by the nutritional status of fish.     

Hemoglobin and subunit multiplicity in the rainbow trout (Oncorhynchus mykiss) hemoglobin system

Hemoglobin (Hb) multiplicity is a commonly used index of phylogenetic differentiation and molecular adaptation in fish. Despite improvements in technologies for characterizing protein structure, our knowledge of the component composition of the Hb system in rainbow trout, Oncorhychus mykiss, has remained almost unchanged through three decades. The Hb is considered to consist of four components, of which two (HbI and HbIV) have been extensively characterised with regard to structure and function. Using a variety of molecular techniques we demonstrate the presence of at least nine different Hb fractions composed of nine (five α and four β globin) chains, and that HbI and HbIV consist of two and five individual components, respectively. These findings indicate that the published data on trout HbI and HbIV refer to mixtures of isoHbs and need to be reappraised. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

An in vitro method to determine phosphorus digestibility of rainbow trout Oncorhynchus mykiss (Walbaum) feed ingredients

An in vitro method was developed to assess the digestibility of phosphorus in 12 plant and animal feed ingredients for rainbow trout Oncorhynchus mykiss (Walbaum). The method simulates the gastrointestinal tract of the rainbow trout with regard to pH and gastrointestinal enzymes. Phosphorus solubility was measured after acid digestion (pH 3) with and without gastric enzymes, after alkaline digestion (pH 9) with and without intestinal enzymes, and after a two-step process involving acid and alkaline digestion. Commercially available digestive enzymes from mammals were compared with digestive enzymes from rainbow trout. Correlating in vitro digestibility with in vivo digestibility showed that acid digestion with both commercial enzymes ( r ²=0.98, P  < 0.05) and trout enzymes ( r ²=0.94, P  < 0.05) predicted the in vivo digestibility of animal feed ingredients. Alkaline digestion with both enzyme systems (commercial r ²=0.79; trout r ²=0.74, P  < 0.05) or without ( r ²=0.82, P  < 0.05) enzymes predicted the in vivo digestibility of ingredients from animal byproducts but not those from plant products. The in vitro digestibility with two enzyme steps (acid and alkaline) predicted in vivo digestibility of plant and animal ingredients ( r ²=0.79 for commercial enzymes and r ²=0.74 for trout enzymes) better than did one-step acid or alkaline digestion.     

Epidermal growth factor receptor-protein kinase interactions in hepatic membranes of rainbow trout (Oncorhynchus mykiss)

The association between the presence of EGF binding and its associated tyrosine kinase activity was investigated in the hepatic membranes of rainbow trout (Oncorhynchus mykiss). Time course experiments indicted that the binding of EGF to hepatic membranes was temperature dependent and resulted in increased phosphorylation of membrane components. The phosphorylation reaction was dependent on the presence of divalent ions (Mg²⁺ and Mn²⁺) but not monovalent ions (Na⁺ and K⁺) or calcium. Western blot analysis of phosphorylated trout membranes indicated protein bands at 150 and 180 kDa which is similar to that observed in mammals. Use of specific substrates and protein kinase inhibitors also indicated that the increase in EGF stimulated phosphorylation was related to an increase in tyrosine kinase activity. These data support the hypothesis that trout have an EGF-like receptor that has binding and biological activities similar to that observed in mammalian species.     

Relationship between vitamin C and plasma concentrations of testosterone in female rainbow trout,Oncorhynchus mykiss

Two-year old rainbow trout females were fed diets containing 0, 30, 110, 220, 440 and 870 mg kg^-1 ascorbyl-2-monophosphate Mg⁺ salt (groups 1, 2, 3, 4, 5, and 6, respectively) from August until March. At the time of spawning (February–March) blood was sampled and the ovulating females were hand stripped. Estradiol (E₂) and testosterone (T) concentrations in plasma, and ascorbic acid (AA) concentrations in plasma and eggs were determined. The mean plasma concentrations of T were higher in group 4, 5, or 6 than in group 2 or 3 (p < 0.05). Moreover, the average plasma concentration of T in fish fed the diets with AA level below National Research Council (NRC) recommendations (groups 1, 2 and 3) was significantly lower (p<0.01) than the average plasma concentration in fish fed diets with AA level above NRC recommendations (groups 4, 5, and 6). These data are consistent with the hypothesis that AA can influence production of steroids in female rainbow trout.Corresponding author     

Biochemical characterization of growth hormone receptor in rainbow trout (Oncorhynchus mykiss) before and after purification

The aim of this work was to verify if, compared to mammals, the lower molecular weight of GH-R previously reported in salmonid is real or due to the experimental process. For this purpose, we compared the apparent molecular weight of GH-R, obtained by SDS-PAGE after cross-linking with ⁵⁰ I-rtGH, obtained from rainbow trout crude liver membrane preparation, incubated in different buffers with those obtained after purification with affinity chromatography.Using crude liver membrane preparation, two specific bands of ⁵⁰ I-rtGH-protein complex were observed: the major one corresponds to a MW of 70 kDa and the minor one to 45 kDa. However, the pattern of electrophoresis varied according to the different incubation buffers tested. Digestion of the cross-linked complex with -galactosidase and phospholipase did not significantly modify the position of bands, whilst N-glycosidase F induced a large smear including 4 more pronounced bands (50, 65, 97 and > 130 kDa), the heavier band corresponding to the most intensive signal.GH receptors were purified using solubilisation and affinity chromatography. The yield of the liver GH-R from crude liver membrane preparation by the solubilization technique was optimized (48%) using Triton 1% for 1 h (12°C ). Specific binding sites in the solubilized membrane proteins were saturable when incubated with increasing ⁵⁰ I-rtGH concentrations, and revealed a high affinity constant (Ka=0.7×10⁹ M^–1). After affinity chromatography, specific binding activity was increased 64,000 fold. However, the purity of the preparation was partial and the purification yield was very low (about 0.3%). This enriched fraction, analysed by SDS-PAGE after cross-linking, showed a very intense band (about 63 kDa) which disappeared with an excess of cold rtGH.These results suggest that the lower molecular weight observed in salmonid (41 kDa), compared to mamals, is not due to the experimental process. The significance of GH-R size difference between salmonids and mammals is discussed.     

In vivo adherence of Flavobacterium psychrophilum to mucosal external surfaces of rainbow trout (Oncorhynchus mykiss) fry

The adherence of Flavobacterium psychrophilum to surfaces of epithelial tissues has been inconclusively suggested as a mechanism, which enables the bacterium to invade the host. Hence, the present study aimed to examine the adherence of the cells of two colony phenotypes, smooth and rough, of F. psychrophilum to mucosal tissues of rainbow trout fry and to test the skin mucus as a nutrient for the growth of F. psychrophilum. Fish were immersed in water containing 10⁶ CFU mL^?1 F. psychrophilum for each colony phenotype. Mucosal tissue samples from fins, gills, skin and eyes, and swab samples from spleen and kidney were taken and inoculated onto TYES agar plates. Colony phenotypes of F. psychrophilum were identified and number of colonies counted. The results showed that cells of both phenotypes initially (0 h) adhered to all mucosal surfaces, but only the rough cells were still present on tissues 1 h post‐immersion. Both phenotypes showed a tissue tropism with the fin tissue being the most adhered. Furthermore, skin mucus promoted the growth of both colony phenotypes. We suggest that the growth of F. psychrophilum cells in skin mucus apparently facilitates the bacterial adherence to mucosal surfaces, and the subsequent invasion into the host.     

Effects of ascorbic acid enrichment by immersion of rainbow trout (Oncorhynchus mykiss, Walbaum 1792) eggs and embryos

This study was conducted to examine the effects of different forms and concentrations of ascorbic acid (vitamin C), and different enrichment times (24 and 48 h post ovulation) on egg, embryo and alevin ascorbate concentrations and survival of rainbow trout (enrichment was at the ova stage). In experiments 1 and 2, fertilized eggs were immersed in water containing ascorbate at 0 (control), 100, 1000 mg L^?1 l ‐ascorbic acid (AA) and 2000 mg L^?1 l ‐ascorbyl monophosphate (AP). In experiment 3, 0 (control), 500 and 1000 mg L^?1 AA neutralized (N) with NaOH, 1000 mg L^?1 AA non‐neutralized (NN), 1000 and 2000 mg L^?1 AP immersions were used. The mean total ascorbic acid (TAA) and dehydroascorbic acid (DHA) concentrations were measured before fertilization, at 3 and 24 h after fertilization, at the eyed stage, and in hatched alevins. We observed significant differences in TAA concentration at different immersion levels at 3 and 24 h after fertilization. Survival decreased significantly depending on the level of vitamin C, pH of the solutions and immersion time. We suggest that when broodstock rainbow trout do not have enough vitamin C in their ovaries, immersion of eggs in 1000 mg L^?1 of neutralized AA may be useful.     

Metabolism of exogenous histamine in rainbow trout (Oncorhynchus mykiss)

Information on the metabolism of exogenous histamine in fish is of much concern regarding the effect of dietary histamine on fish. Histamine catabolic enzymes, diamine oxidase and histamine N-methyl transferase were measured in the tissues of rainbow trout. Diamine oxidase was detected in the stomach, pylorus caeca and intestine. Histamine N-methyl transferase was detected only in the liver.A change in the contents of histamine and its metabolites was observed in the tissues of rainbow trout after oral administration of histamine. A large amount of imidazole acetic acid was observed in the serum, kidney, liver and muscle. On the other hand, 1-methyl histamine was detected only in the liver. Histamine and its metabolites, imidazole acetic acid and 1-methyl histamine were metabolized and diminished within 48 hr in all tissues.These results showed that histamine was metabolized by two metabolic routes in rainbow trout. One is the main pathway producing imidazole acetic acid by intestinal diamine oxidase and the other is the complementary pathway producing 1-methyl histamine by liver N-methyl-transferase.     

Mineral availability from barley low phytic acid grains in rainbow trout (Oncorhynchus mykiss) diets

Phosphorus storage within plant seeds occurs mainly as phytic acid, which has profound implications on the use of seeds as food material. Phytic acid phosphorus is unavailable to non‐ruminants, leaches ionic minerals during digestion, and is excreted at elevated levels as a waste product. This presents a problem in nonruminant livestock production including current efforts to develop renewable grain and legume products for use in fish feeds. The development of lines of several cereal grain species that have reduced seed phytic acid concentration provides a novel approach to dietary and environmental problems associated with seed phytic acid. Utilizing four isogenic strains of barley (one normal for seed phytic acid, and three low phytic acid lines that produce seeds with approximately 50, 70 and 95% reductions in phytic acid) the apparent digestibility of nutrients in formulated diets containing these barleys at a level of 30% was measured using rainbow trout (Oncorhynchus mykiss). Also examined was the apparent availability of several minerals including phytate phosphorus and total phosphorus. Results from these studies corresponded well with the results of other animal studies that evaluated low phytic acid cereal grains. With increasing reductions in seed phytic acid, seed available phosphorus increased and faecal phosphorus was reduced by up to 50%. Calcium availability increased, copper and sulphur decreased, and the other tested minerals demonstrated either increased or decreased availability in a manner uncorrelated to grain phytic acid concentration.     

Cortisol and testosterone accumulation in a low pH recirculating aquaculture system for rainbow trout (Oncorhynchus mykiss)

Steroids accumulate in recirculating aquaculture system (RAS), although explanatory factors for such accumulation are still poorly explored. This study investigated the effect of water exchange rate and pH in six replicated RAS on the concentration of the stress hormone cortisol in rainbow trout blood plasma and in the holding water and of the sex steroids testosterone, 11‐ketotestosterone (11‐KT) and 17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) over a 70‐day experimental period. Three combinations of water exchange rate and pH were used each treatment, with two replications: (i) high water exchange rate (±1700 L kg^?1 feed) and neutral pH (±7.3), (ii) low water exchange rate (±500 L kg^?1 feed) and neutral pH (±7.3) and (iii) low water exchange rate (±500 L kg^?1 feed) and low pH (±5.8). Plasma cortisol concentrations at day 70 were higher (24.4 ± 9.5 ng mL^?1) for fish kept at low pH when compared to fish kept at neutral pH (12.0 ± 0.1 and 8.7 ± 0.2 ng mL^?1). Water cortisol and testosterone concentrations at day 35 were higher at low pH than at neutral pH, whereas water 11‐KT and 17,20β‐P did not differ among treatments. At day 70, there were no significant differences between low and high pH. These results demonstrate that low pH contributes to increased plasma cortisol concentrations and to its accumulation in water, possibly indicating a stress response to low pH. The higher concentration of testosterone but not of the other sex hormones point to unspecified reproductive effects that need further investigation.     

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.     

Diel changes in plasma arginine vasotocin, isotocin, and melatonin in rainbow trout (Oncorhynchus mykiss)

The diel changes in plasma AVT, IT and Mel in rainbow trout (Oncorhynchus mykiss) were studied to assess potential relationships. Blood was sampled at 05:00, 11:00, 16:00, 22:30 and 05:00 in freshwater-adapted fish and at 22:30 in brackish water-adapted fish maintained under natural photoperiod. A few of the FW-acclimated fish were assigned to one of two experimental groups and adapted to DD or LL lighting regimes. Blood samples were taken at 11:00 and 22:30. Hormones were extracted from plasma by solid phase extraction and determined by high-performance liquid chromatography. Marked diel variations in AVT and Mel were detected in fish maintained under natural photoperiod. Plasma AVT (fmol ml^–1) increased during the light to reach the maximal level at the end of that phase (261.7±23.1). Thereafter, AVT concentration decreased and became minimal at 05:00 (68.9±11.5) 3 h before the sunrise. Plasma Mel (pmol ml^–1) increased between 16:00 and 22:30 when a peak value was reached (1204.0±55.5). Thereafter, Mel levels decreased and were minimal after the onset of the light phase (242.8±37.0). IT levels displayed no significant diel changes. Linear regression analysis indicated the negative correlation between plasma Mel and AVT for five collecting times of the daily 24 h cycle in freshwater fish and at 22:30 in brackish water fish. A similar correlation occurred at 11:00 in the DD group and at 22:30 in the LL group. To elucidate the character of the Mel-AVT relationships further studies are required.