Changes of Non-Specific Immune Parameters in Rainbow Trout,Oncorhynchus mykiss,After Exposure to Antimicrobial Agents Used in Aquaculture

Changes of plasma lysozyme and serum bactericidal activity, ceruloplasmin, total plasma protein, serum glucose, hematocrit and leucocrit levels in rainbow trout (Oncorhynchus mykiss) were studied after exposure to formalin and chloramine-T, which are used in aquaculture as anti-ectoparasitic and antibacterial agents. Fish were exposed to formalin at a concentration of 250 ppm for 60 min and exposed to chloramine-T for 5 ppm for 3 h. The parameters were analyzed for rainbow trout exposed to formalin and chloramine-T soon after treatments and rainbow trout exposed to freshwater after 24 h (return) following the treatments. Plasma lysozyme activity decreased after exposure to formalin; however, it was not affected by chloramine-T exposure. Serum bactericidal activity reduced after both formalin and chloramine-T exposure and did not change after returning to freshwater. Ceruloplasmin and total plasma protein were not affected by both treatments. Serum glucose levels of exposed fish elevated and remained at higher levels during recovery period when compared to control values. Hematocrit values of fish exposed to formalin increased; however, no significant change was observed in fish exposed to chloramine-T. Leucocrit levels of fish treated with both formalin and chloramine-T were higher than that of controls.     

Experimental validation of geosmin uptake in rainbow trout,Oncorhynchus mykiss (Waldbaum) suggests biotransformation

The bioconcentration of waterborne geosmin in rainbow trout, Oncorhynchus mykiss (Waldbaum) was assessed. Fifty rainbow trout with a mean (SD) weight of 226.6 (29.0) g and lipid content of 6.2 (0.6) % (w/w) were exposed to geosmin in static water for 0, 2, 4, 6, 8, 12, 24, 36, 48 and 120 hr, with one tank containing five fish for each exposure period. Geosmin concentrations were measured in fish tissue and water samples collected over time. With time the geosmin concentration in the fish increased and decreased in the water. However, the total absolute amount of geosmin in the system declined over time which could be explained by induction of biotransformation. This is in accordance with the decreasing lipid normalized geosmin levels in the liver compared with the liver‐free carcass. Geosmin distribution within rainbow trout clearly is not exclusively governed by the lipid content of tissues. In vivo geosmin bioconcentration in rainbow trout is slower and the body burden reached is lower than the generally accepted theoretical model predicts.     

Effects of dietary yeast inclusion and acute stress on postprandial plasma free amino acid profiles of dorsal aorta‐cannulated rainbow trout

In a 4‐week experiment, 15 cannulated rainbow trout were fed three diets based on fish meal (FM), Saccharomyces cerevisiae yeast (SC) and Wickerhamomyces anomalus and S. cerevisiae yeast mix (WA). Fish were fed daily, and blood samples were collected on day 7 of each week at 0, 3, 6, 12 and 24 hr after feeding. In the final week, fish were exposed to a 1‐min netting stressor. All essential and non‐essential plasma amino acid levels except methionine were similar between fish fed diets FM, SC and WA. Plasma methionine and sarcosine were significantly higher in fish fed diets SC and WA, possibly due to the crystalline methionine level, form or feeding regime. Hydroxy‐proline and 3‐methyl‐histidine were higher in fish fed diet FM, which can be explained by the higher levels present in fish meal compared with yeast. In stressed fish, there were no dietary effects on plasma amino acid levels, but significant increases in taurine and cystathionine were found in stressed compared with unstressed fish. These results demonstrate that yeast‐based diets produce similar plasma amino acid profiles to fish meal and suggest that yeast may be a suitable fish meal replacement in diets for rainbow trout.     

Susceptibility of Chinook salmon,Oncorhynchus tshawytscha (Walbaum), and rainbow trout,Salmo gairdneri Richardson,to infection with Vibrio anguillarum following sublethal copper exposure*

Abstract. Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and rainbow trout, Salmo gairdneri Richardson, were used to determine if sublethal copper exposure would increase their susceptibility to Vibrio anguillarum infection. Fish were pretreated with copper at fractional levels of the 96 h copper LC₅₀ before exposure to the pathogen. Mortality by vibriosis was greater among fish exposed to 9% of the copper LC₅₀ for 96 h than unexposed fish. Peak susceptibility to vibriosis depended in part on the interaction of exposure time and copper concentration. The higher copper concentrations produced peak susceptibility to infection in shorter time periods. After the peak of susceptibility, sensitivity to infection declined to near control levels in those fish where exposure was continued. Rainbow trout stressed by copper required about 50% fewer pathogens to induce a fatal infection than non-copper exposed fish.     

Timing and duration of constant light affects rainbow trout (Oncorhynchus mykiss) growth during autumn–spring grow-out in freshwater

Photoperiod enhancement of growth is becoming an area of increasing interest as a means of enhancing rainbow trout production efficiency in commercial practice. This paper examines the possible implications of shortening periods of constant light (LL) exposure on rainbow trout growth during autumn–spring grow-out under ambient water temperatures in freshwater to portion size. Triplicate groups of juvenile all-female rainbow trout were permanently exposed to LL in October, November, December or January. Growth was monitored and compared with those maintained under a simulated natural photoperiod (SNP) until the following May. Permanent exposure to LL (all treatments) resulted in significantly greater weight gain of rainbow trout than those under SNP. Furthermore, greatest growth was achieved when fish were left permanently exposed to LL from October. These findings suggest there may be implications for fish farmers if the period of photoperiod exposure is reduced, or timing of application is not considered with regards to ambient water temperatures.     

Effect of Dietary Vitamin C on Weight Gain,Tissue Ascorbate Concentration,Stress Response,and Disease Resistance of Channel Catfish Ictalurus punctatus1

Juvenile channel catfish Ictalurus punctatus (average initial weight, 6.5 g/fish) were fed twice daily to apparent satiation with practical-type diets containing 0, 50, 150, or 250 mg supplemental vitamin C/kg from L-ascorbyl-2-polyphosphate for 10 wk under laboratory conditions. At the end of the feeding period, one half of the fish were stressed for 2 h by confinement and both stressed and nonstressed fish were exposed to a virulent strain of Edwardsiella ictaluri. Weight gain and feed conversion efficiency were lower for fish fed the basal diet than those fed diets containing supplemental vitamin C. No differences were observed in weight gain and feed conversion among fish fed diets containing supplemental vitamin C. There were no differences in feed consumption and survival (prior to experimental infection) among treatments. No vitamin C deficiency signs except reduced weight gain were observed in fish fed the basal diet. Serum cortisol concentrations were higher in stressed fish than in non-stressed fish. Dietary vitamin C level had no effect on serum cortisol concentration. As dietary vitamin C increased, ascorbate concentration in serum and liver increased. Confinement stress had no effect on serum and liver ascorbate concentrations. Cumulative mortality of channel catfish 21 d subsequent to experimental infection with E. ictaluri was higher for stressed fish than for nonstressed fish. Regardless of stress or nonstress, overall mortality for fish fed the basal diet was lower than the fish fed diets containing supplemental vitamin C. There were no differences in post-infection antibody levels among treatments or between stressed and nonstressed fish. Results from this study indicate that channel catfish require no more than 50 mg/kg dietary vitamin C for normal growth, stress response, and disease resistance.     

Effects of slaughter methods on physical,biochemical and microbiological quality of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> and mirror carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> filleted in pre-, in- or post-rigor periods

Rainbow trout Oncorhynchus mykiss and mirror carp Cyprinus carpio were slaughtered by either percussion or asphyxiation. For determination of the physical (texture value) and biochemical quality attributes [pH, lactic acid, total volatile basic nitrogen (TVB-N), and malondialdehyde (MDA) values] of trout and carp, the fish were filleted immediately (within 2 h), 12 h after harvest (pre-rigor), 24 and 36 h after harvest (in-rigor), and finally 48 and 60 h after harvest (post-rigor). For microbiological quality attribute (mesophilic and psychrophilic bacteria count) analyses, the fish were filleted immediately (within 2 h) after harvest (pre-rigor), 24 h after harvest (in-rigor) and 48 and 72 h after harvest (post-rigor). By percussion slaughtering followed by pre-rigor filleting, it was possible to process rainbow trout and mirror carp without inflicting excessive handling stress. Percussion slaughter delayed onset of rigor and the percussion-slaughtered fish exhibited a long pre-rigor period (≈24 h), giving plenty time for pre-rigor processing. Percussion slaughtering and pre-rigor filleting of fish was considered to be superior to the traditional asphyxiation slaughtering with respect to texture, TVB-N and MDA attributes.     

Effect of Oyster Mushroom,Pleurotus ostreatus,Extract on Hemato‐Immunological Parameters of Rainbow Trout,Oncorhynchus mykiss

The current study investigated the effects of oyster mushroom Pleurotus ostreatus, extract on immunological and hematological parameters of rainbow trout, Oncorhynchus mykiss. The fish were fed diets containing 0 (control), 1, and 2% supplementation of the oyster mushroom extract for 6 wk. Blood samples were collected weekly for the first 6 weeks. After 6 wk of feeding, the fish were challenged with Lactococcus garvieae and mortality was recorded. The results of this study showed that feeding rainbow trout an oyster mushroom extract–supplemented diet stimulated phagocytic activity of phagocytic cells, lysozyme activity, and myeloperoxidase activity in serum, but it did not significantly affect the total serum immunoglobulin level. At the same time, a significant increase was found in the number of neutrophils, monocytes, and total white blood cells. Fish fed the diet supplemented with oyster mushroom extract showed reduced mortalities following L. garvieae infection compared with controls. These results show that supplementation of fish diets with oyster mushroom extract at 1 and 2% concentrations significantly imrpoves hematological parameters and modulates the immune response against L. garvieae in rainbow trout.     

Stress-induced changes in the affinity and abundance of cytosolic cortisol-binding sites in the liver of rainbow trout,Oncorhynchus mykiss (Walbaum), are not accompanied by changes in measurable nuclear binding

Plasma cortisol levels and the number (N_max) and affinity (K_d) of specific hepatic cortisol-binding sites were determined in rainbow trout subjected to chronic confinement stress for 14 days. Confinement significantly elevated plasma cortisol levels to 47.3 ± 13.5 ng ml^–1 within 24h and although levels declined to 8.0 ± 3.0 ng ml^–1 after 14 days, they were significantly higher throughout than levels in unstressed control fish (< 2.0 ng ml^–1). There was a 60% reduction in cytosolic N_max in stressed fish during the first 24h of confinement (35.8 ± 7.9 cf. 129.0 ± 15.2 fmol mg^–1 protein), a decline which was sustained at 7 days after the onset of stress but, although numbers of binding sites in the liver of stressed fish were still lower than in unstressed fish, the difference was no longer significant after 14 days of confinement. There was an accompanying significant rise in the K_d of cortisol binding in stressed fish during confinement, from 4.0 ± 0.6 nM at time 0 to 8.4 ± 0.8 nM after 24 h confinement. This increment in K_d was sustained at a level significantly higher than in control fish throughout the 14 day confinement period, despite marked reductions in cortisol levels and increases in N_max in stressed fish. Throughout the study, specific binding of cortisol could not be consistently detected in high-salt nuclear extracts from stressed or unstressed fish, suggesting either that high-affinity binding sites for cortisol were absent from these preparations, that receptors were present but unable to interact with ligand because they were occupied, or that receptors were present but not being extracted. These possibilities were investigated using a range of extraction procedures, by varying the temperature of incubation, by employing dexamethasone as ligand and by examining binding in purified, intact, nuclei. Estradiol was employed as a methodological control throughout and substantial amounts of specific estradiol binding were detected in all compartments and preparations. Specific cortisol-binding sites were detected in intact nuclei of both stressed and unstressed fish, at levels an order of magnitude lower than estradiol binding in the same preparations. These data demonstrate that activation of the pituitary-interrenal axis leads to significant changes in the nature of target-tissue binding of cortisol in rainbow trout, and reveal a clear difference in the subcellular distribution of binding-sites for estradiol and cortisol, which reflects the situation in mammalian tissues.     

Physiological Responses of Rainbow Trout Oncorhynchus mykiss to Crowding and Anesthesia with AQUI-S™

Abstract.— Following exposure to the anesthetic AQUI-S™, plasma cortisol concentration in immature rainbow trout was measured as (mean) 293 ± 48 ng/ mL, which was significantly ( P > 0.05) higher than the mean concentration in resting fish. Cortisol concentrations remained significantly ( P > 0.05) elevated for at least 24 h after treatment. This was accompanied by a significant increase and decrease in hematocrit and plasma potassium, respectively. These perturbations continued for at least 48 h following recovery from anesthesia. Plasma concentrations of total protein and sodium remained unchanged following anesthesia with AQUI-S™. Crowding stress is commonly encountered by fish during manipulation in aquaculture situations. Anesthetising fish prior to, and during, manipulation may reduce the associated stress. Changes in cortisol values resulting from crowding (30 min; 0.1 kg/L) during anesthesia with AQUI-S™ were not appreciably different from those in fish crowded without anesthesia. Thus, anesthesia with AQUI-S™ at the recommended dose of 17 mg/L did not appear to be effective for alleviating the stress of crowding under the conditions of our experiments.     

Changes in selected stress and immune‐related genes in Atlantic cod,Gadus morhua,following overcrowding

Using Atlantic cod, Gadus morhua, as a model for the stress response in gadoid fish, the changes in the expression of some stress and immune genes as well as the profiles of plasma cortisol were examined. Adult fish were kept at a density of ca. 100 kg m^?3 by lowering the water level in the rearing tank for 1 h and this short‐term crowding stress was repeated thrice over a 12‐h interval period. Blood samples were collected before exposure and at 2, 24 and 72 h post crowding. Plasma cortisol level significantly increased at 2 h post crowding but returned to pre‐crowding levels 24 h after exposure. The relative expression of the stress response genes, glucose transporter‐3 and a putative heat shock protein 70 significantly increased at 2 and 24 h post crowding respectively. Significant up‐regulation of the pro‐inflammatory cytokines, interleukin (IL)‐1β and IL‐8, as well as anti‐bacterial genes, g‐type lysozyme and bactericidal permeability‐increasing protein/lipopolysaccharide‐binding protein (BPI/LBP) was also observed at 2 h and the levels were maintained until 72 h post exposure, except for BPI/LBP which had maximum up‐regulation at 24 h. The present observations have implications with respect to fish welfare and assessment of the health status of the farmed fish.     

The response of coral trout (Plectropomus leopardus) to capture, handling and transport and shallow water stress

The stress response of coral trout (Plectropomus leopardus ) to wild capture or controlled shallow water stressors was investigated. Stress associated with capture from the wild, handling and transport and shallow water evoked significant changes in circulating levels of cortisol, glucose, lactate, haemoglobin (Hb) and haematocrit (Hct) in coral trout. Plasma glucose, Hb and Hct increased over 30 to 60 min in response to the controlled stressor and then returned toward unstressed levels, even if the stressor persisted. Cortisol increased to maximum levels over the 60 min following the onset of the stressor and remained elevated for 4 h in the case of a single 30 min shallow water event or 3 d in response to the capture, handling and transport. The difference between the responses of blood glucose and cortisol suggests that cortisol does not maintain blood glucose during stress in coral trout. The concentrations of circulating thrombocytes, lymphocytes and granulocytes were also significantly affected by shallow water stress. As lymphocytes and granulocytes are important immune components, reduced concentrations of these cells may explain the increases in disease susceptibility, commonly observed in stressed fish during live transport.     

Altered stress responses in rainbow trout following a dietary administration of cortisol and β-napthoflavone

Previous studies have demonstrated that fish inhabiting polluted waterways often have an impaired stress response at the organismal level. Given the possible link between the organismal (i.e. cortisol) and cellular (i.e. heat shock proteins; hsp) stress responses, we conducted this study to examine the ability of rainbow trout to respond to a 2 h, +14 °C heat stress (HS) challenge following a 28 d, sub-chronic exposure to increased concentrations of cortisol (5 mg kg⁻¹ b.w.), β-napthoflavone (bnf; 50 mg kg⁻¹ b.w.), and a combination of both (mixture), through the diet (1.5% b.w. every 48 h). While control fish responded to the HS by significantly increasing components of their organismal (cortisol, glucose, and lactate) and cellular (hepatic hsp70 protein) stress responses 6 and 24 h post HS, cortisol-, bnf-, and mixture-fed fish had impaired stress responses at both levels of organization. Additionally, hepatic hsp70 levels were significantly reduced 6 h post HS in cortisol-fed fish. While bnf-fed fish had significantly higher EROD activity, cortisol potentiated EROD activity in the mixture-fed fish. Similarly, plasma cortisol concentrations in the mixture-fed fish were significantly lower relative to cortisol-fed fish. These data are the first to indicate that sub-chronically stressed fish can have impaired stress responses at both the organismal and cellular levels. These findings raise questions regarding: (a) the universal and simple applicability of biological indicators of stress in fish; (b) the possible functional relationship between these two levels of stress responses; and (c) the importance of hsps in the generalized stress response of the whole organism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antioxidant and immune defences of rainbow trout (Oncorhynchus mykiss) offered plant oils differing in fatty acid profiles from early stages

The antioxidant defence and immune response of rainbow trout (Oncorhynchus mykiss) that had received plant oils, rich in either n‐3 polyunsaturated fatty acids (PUFA; linseed oil) or n‐6 PUFA (safflower oil) was evaluated upon antigen exposure. The fish employed in this study had been offered the diets for 18 months from the first feeding. Rainbow trout from each group were injected intraperitoneally with formalin‐killed bacteria (Aeromonas hydrophila) or were sham‐injected and observations were made 24 h later. Though the fish fed safflower oil seemed to be under relatively greater oxidative stress, the antioxidant defences (superoxide dismutase, catalase, glutathione peroxidase) were as effective as in those fed linseed oil. The humoral (alternate complement activity and lysozyme activity) and cellular (phagocytic activity and lymphocyte proliferation) immune responses were not significantly affected by the oil offered. With the exception of reactive oxygen production that was significantly greater in the linseed oil fed fish, both groups did not differ greatly in their immune responses after antigen exposure. Thus, fish fed safflower oil that was deficient in n‐3 PUFA was able to sustain most of the critical responses similar to those fed linseed oil suggesting that plant oils of both fatty acid categories were effective for this fresh water fish.     

The patterns of plasma free amino acids after force-feeding in rainbow trout Oncorhynchus mykiss (Walbaum) with and without dorsal aorta cannulation

Two experiments were conducted to compare the patterns of plasma free amino acid concentrations after force-feeding in rainbow trout Oncorhynchus mykiss (Walbaum) with and without dorsal aorta cannulation. In the first experiment, 35 rainbow trout averaging 504 ± 7.8 g (mean ± SD) were divided into seven groups of five fish each. After 48 h starvation, a group of fish was anaesthetized and blood samples were taken at one of the following time periods: 0, 4, 8, 12, 24, 36 and 48 h after feeding. In the second experiment, five dorsal aorta cannulated rainbow trout averaging 511 ± 6.2 g (mean ± SD) were kept in a cage. After 48 h starvation, the fish were anaesthetized and blood samples were taken from the same fish at 0, 4, 8, 12, 24, 36 and 48 h after feeding. In the first experiment, the concentration of all plasma free amino acids except histidine and glycine peaked at 4 h and returned to the basal level 24 h after feeding. In the second experiment, the concentration of all plasma free amino acids except isoleucine, leucine, phenylalanine and tryptophan also peaked at 4 h and returned to the basal level 24 h after feeding. These results showed that the pattern of plasma free amino acid concentrations from fish with and without dorsal aorta cannulation were similar.     

Inhibition of the attachment of Flavobacterium branchiophilum to the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Preliminary experiments are described which aimed to identify compounds that could inhibit the attachment of Flavobacterium branchiophilum strains LAB4a and ATCC 35035 to the gills of rainbow trout. Total inhibition was never achieved, regardless of the compound tested. Formalin-killed or acetone-killed F. branchiophilum cells retained at least some of their adherent nature, relative to untreated (live) cells. Adherence was reduced by 22–33% following immersion of fish in one litre of water containing 0.21 mg of a homologous crude fimbrial extract. When fish were immersed in water containing hyperimmune rainbow trout antiLAB4a serum, a dose-dependent decrease in attachment (a reduction of 15% to 63%) of LAB4a to the gills was observed. Rainbow trout anti-LAB4a serum also reduced the attachment of ATCC 35035 to the gills, but this reduction was not significant. Adherence of LAB4a was not inhibited following exposure of fish to group 1 carbohydrates (arabinose, mannose and xylose), group 2 carbohydrates (dextrose, galactose and lactose), group 3 carbohydrates (galactosamine, glucosamine and fucose) or group 4 carbohydrates (N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid and the globoceramide glycolipid from human erythrocytes). In contrast, when rainbow trout erythrocytes were incubated with the bacteria prior to bath challenge, this resulted in an 87% and 53% reduction in gill-associated LAB4a and ATCC 35035 antigen, respectively, following immersion of rainbow trout in this suspension.     

The ability of clove oil and MS-222 to minimize handling stress in rainbow trout (Oncorhynchus mykiss Walbaum)

The effects of 60‐mg L^?1 clove oil and 60‐mg L^?1 tricaine methanesulphonate (MS‐222) on the blood chemistry of rainbow trout were compared after exposure to handling stress via caudal puncture blood sampling. Fish sampled by caudal puncture and subsequently exposed to anaesthetics showed a typical handling stress response over a 48‐h period. There were no significant differences between the responses of fish exposed to equal concentrations of clove oil and MS‐222, with the following exceptions: the blood glucose at full anaesthesia, and lactate at full recovery increased significantly in the clove oil‐exposed fish. In a subsequent experiment, the stress response observed in fish sampled by caudal puncture and exposed to clove oil and MS‐222 was compared with a non‐anaesthetized control group. The increases in plasma cortisol levels were significantly lower at recovery in fish treated with either anaesthetic compared with the control fish. Fish exposed to MS‐222 had significantly higher cortisol levels at 1 h. These findings show that few differences exist between the anaesthetic effects of clove oil and MS‐222 on the physiological response of fish to stress. However, clove oil is more effective at reducing the short‐term stress response induced by handling and blood sampling, and is recommended as an alternative fish anaesthetic.     

Influence of water quality on the outbreak of proliferative kidney disease – field studies and exposure experiments

This paper presents data from field studies and exposure experiments and the possible association of limno- and physicochemical parameters with outbreaks of proliferative kidney disease (PKD) in rainbow and brown trout. The investigations were carried out at Singold Brook in southern Germany. Exposure experiments and sampling of wild fish were performed in Singold at least twice a year in 1992, 1994, 1996 and 2000. Both wild fish collected from the specific sites and experimentally exposed rainbow trout were investigated histologically for the occurrence of PKD. At the time of sampling, various water parameters at the respective river sites were measured. The results indicate strongly the existence of a correlation between organic pollution of water, the presence of Bryozoa and the outbreak of PKD.     

Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Freshwater fish are able to mount a protective immune response against the parasite Ichthyophthirius multifiliis (Ich) following a non‐lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in protection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of immune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naïve rainbow trout. This study indicates that antibodies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction.