Nonisotopic detection of Loma salmonae (microspora) in rainbow trout (Oncorhynchus mykiss) gills by in situ hybridization

Loma salmonae, a microsporidian parasite of salmonids of the genus Oncorhynchus, is a significant cause of economic loss in pen-reared chinook salmon (O. tschawytscha). Final stages of L. salmonae infections are easily recognized by the xenomas that form in the gills during sporogony. However, early prexenoma stages of infection (3 weeks or less after infection) are difficult to detect on histologic slides. An L. salmonae-specific single-stranded DNA probe labeled with digoxigenin was used to detect these prexenoma stages of L salmonae by in situ hybridization in experimentally infected rainbow trout. This method allows detection of the parasite in the gills only 2 weeks after infection, providing a sensitive and specific way of detecting L. salmonae during the early stages of infection.     

Pharmacokinetics of enrofloxacin in fingerling rainbow trout (Oncorhynchus mykiss)

The pharmacokinetics of intravenously and orally administered enrofloxacin was determined in fingerling rainbow trout (Oncorhynchus mykiss). Doses of 5 or 10 mg enrofloxacin/kg body weight were administered intravenously to 26 fish for each dose and blood was sampled over a 60-h period at 15 degrees C. Two groups of fish were treated orally with 5, 10, or 50 mg/kg (80 fish/dose at each temperature) and held at 15 degrees C or 10 degrees C during the 60-h sampling period. Following intravenous administration, the serum concentration-time data of enrofloxacin in rainbow trout were best described by a two-compartment open model for both doses of 5 and 10 mg enrofloxacin/kg. The hybrid rate constants alpha and beta did not differ between doses. The distributional phase was rapid with a half-life of 6-7 min for both doses. Overall half-lives of elimination were 24.4 h (95% CI = 20.2-30.8) and 30.4 h (24.2-41.0), respectively, for the 5- and 10-mg/kg doses. A large Vd(area) was observed following dosing of either 5 or 10 mg enrofloxacin/kg,: 3.22 and 2.56 l/kg, respectively. Whole body clearance for 5 mg/kg was 92 ml/h.kg and 58 ml/h.kg at the 10-mg/kg dose. Following oral administration, the serum concentration-time data for enrofloxacin were best described as a one-compartment open model with first-order absorption and elimination. Apparent Ka over all doses at 10 degrees C averaged 62% less than apparent Ka at 15 degrees C. Estimates of the apparent t(1/2)e over both temperatures ranged from 29.5 h (18.4-73.4) to 56.3 h (38.3-106.6). Bioavailability averaged 42% over all doses at 15 degrees C and was decreased to an average of 25% at 10 degrees C. Peak serum concentrations appeared between 6 and 8 h following dosing. A dose of 5 mg/kg/day was estimated to provide average steady-state serum concentrations at 10 degrees C that are approximately 4.5 times the highest reported MIC values for Streptococcus spp., the fish pathogen least sensitive to enrofloxacin. Owing to the long apparent half-life of elimination of enrofloxacin in fingerling trout, it would take approximately 5 to 9 days to achieve these predicted steady-state serum concentrations; this estimate is important when considering the duration of therapy in clinical trials.     

Residues of clenbuterol in tissues of the rainbow trout (Oncorhynchus mykiss)

There have been many studies on the efficacy of ₂-adrenergic drugs as feed additives but no data are available at present on the use of clenbuterol in fish production. To evaluate the residues of clenbuterol in tissues of fish, 50 trout (Oncorhynchus mykiss) were fed for 21 days on a fish feed containing 5 ppm of the drug. The livers, muscles and skins of sample groups of fish were analysed by HPLC with visible spectrophotometric detection on days 15 and 21 of treatment and at intervals during a 30-day withdrawal time. Clenbuterol reached its highest levels in the liver (mean 440 ppb; SD=±159;n=5) on day 15 of treatment, with a slow depletion curve; 24±3 ppb was still present at the end of the withdrawal period. At this time, residues were still present in the edible tissues, i.e. muscle (5±1 ppb) and skin (7±3 ppb). Side-effects were noted during the first week of treatment.Abbreviations ppb parts per billion - BHT butylated hydroxy toluene - CV coefficient of variation - DAD diode array detector - HPLC high-performance liquid chromatography     

Accumulation and depletion kinetics of erythromycin in rainbow trout (Oncorhynchus mykiss)

Erythromycin is an antimicrobial agent recommended for the control and treatment of diseases caused by gram-positive bacteria. Few studies, however, have determined the metabolic and pharmacokinetic aspects of this antimicrobial agent in fish. The aim of the present study, therefore, was to determine the accumulation and depletion time of erythromycin after administration of medicated feed containing 52 mg kg(-1) body weight day(-1) for 8 days in rainbow trout (Oncorhynchus mykiss). Results were analyzed following the European Agency for Evaluation of Medicinal Products guidelines. We measured a withdrawal time of 187°C-day (°C-day=water temperature×days), lower than the value (500°C-day) recommended by Council Directive 2004/28/EC for veterinary medicinal products. Our results provide data to establish therapeutic regimens for the use of erythromycin in aquaculture.     

Immunostimulation in the rainbow trout (Oncorhynchus mykiss) following intraperitoneal administration of Ergosan

The present work provides information concerning the immunostimulatory activity of Ergosan, an algal based product, injected intraperitoneally in the rainbow trout (Oncorhynchus mykiss). Ergosan is composed of 0.002% unspecified plant extract, 1% alginic acid from Laminaria digitata, and 98.998% algal based carrier. Migration of leucocytes into the peritoneal cavity was stimulated at doses > or =1 mg ml(-1). A single dose of 1mg significantly augmented the proportion of neutrophils, degree of phagocytosis, respiratory burst activity and expression of interleukin-1beta (IL-1beta), interleukin-8 (IL-8) and one of the two known isoforms of trout tumour necrosis factor-alpha (TNF2) in peritoneal leucocytes at 1 day post-injection. Humoral immune parameters were less responsive to intraperitoneal Ergosan administration, with complement stimulation only evident in the 1mg treated group at 2 days post-injection. Antiprotease and lysozyme activity were unaffected by Ergosan over a 7-day time period at the doses examined.     

Effect of florfenicol on the immune response of rainbow trout (Oncorhynchus mykiss)

Florfenicol, a drug effective against several bacterial diseases of fish, was tested for possible immunomodulatory effects. The aim of the study was to follow the kinetics of the immune response after vaccination with simultaneous oral antibiotic treatment. The fish were immunised with a commercial oil-based divalent (furunculosis/vibriosis) vaccine and were simultaneously given oral antibiotic treatment. The specific immune response was monitored by analysing the levels of specific antibodies with ELISA. As an indicator of the non-specific immune response the phagocytic activity of circulating leucocytes was measured by a chemiluminescence assay. Total circulating leucocyte counts and differentials were also monitored. The disease resistance was evaluated by challenge tests at the end of the experiment. The results showed that florfenicol did not have any significant effect on antibody production and circulating leucocyte levels but caused a suppression in chemiluminescence response/phagocytic cell 5-6 weeks after vaccination. The survival after challenge was slightly suppressed by the florfenicol treatment. The RPS-value for the vaccinated group was 98% and for the florfenicol-treated group was 88%.     

Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis by probiotic bacteria

We analysed the effect of probiotic supplementation on the control of lactococcosis in rainbow trout. Probiotic strains Leuconostoc mesenteroides CLFP 196 and Lactobacillus plantarum CLFP 238 were administered orally to fish for 30 days at 10(7) CFU g(-1) feed. Thirty days after the start of the probiotic feeding, fish were challenged with Lactococcus garvieae. Probiotic supplementation reduced fish mortality significantly, from 78% in the control group to 46-54% in the probiotic groups.     

Vitamin E-responsive myopathy in rainbow trout fry (Oncorhynchus mykiss).

Unacceptably high mortalities in rainbow trout fry (Oncorhynchus mykiss) six to 10 weeks after they started to feed were recorded in two spring water trout hatcheries in Northern Ireland in May 1989. Muscle degeneration and necrosis were consistent with histopathological findings in both outbreaks, and this myopathy was similar to that previously described in salmonids and other species associated with vitamin E and selenium deficiency. A feed trial was designed to investigate the hypothesis that the vitamin E requirement of rainbow trout fry on these farms was higher than the current minimum recommendations. Three groups of fry were fed diets containing 147, 239 and 532 iu/kg alpha-tocopherol. The mortality in the groups was inversely related to the dietary alpha-tocopherol concentration, and there was severe myopathy in fry fed the diet containing 147 iu/kg alpha-tocopherol, mild myopathy in fry fed 239 iu/kg alpha-tocopherol but no myopathy in fry fed 532 iu/kg alpha-tocopherol.     

Pharmacokinetics of morphine and its metabolites in freshwater rainbow trout (Oncorhynchus mykiss)

In this study, we injected morphine sulfate IP into rainbow trout and measured the concentration of morphine and all potential metabolites in plasma using LC-MS/MS at a series of times after the injection. The pharmacokinetics of morphine were similar to those previously reported for seawater-acclimated rainbow trout, i.e. they were about one order of magnitude slower than in similarly sized mammals. The only metabolite of morphine present in the plasma was morphine-3-β- d -glucuronide (M3G); morphine-6-β- d -glucuronide (M6G) was not detected. M3G gradually increased after the morphine injection, peaked about 2 days later, then gradually decreased. In mammals, M3G plasma levels exceed morphine levels extremely rapidly, i.e. in less than an hour, regardless of dose, route of administration, or species. In trout, it took 2 days for M3G levels to exceed morphine levels. This is the first study of the metabolites of morphine in any ectotherm. We conclude that trout can metabolize morphine, but at a rate much slower than in mammals.     

Pathological effects of Arcobacter cryaerophilus infection in rainbow trout (Oncorhynchus mykiss Walbaum)

Arcobacter cryaerophilus was isolated from naturally infected rainbow trout (Oncorhynchus mykiss Walbaum), and its pathogenicity was tested by intramuscular injection into 40 healthy 1-year-old rainbow trout at 16 degrees C. The lethal dosage of 50% end point (LD50) for A. cryaerophilus was calculated 2.25 x 10(4) viable cells. Experimental infection caused deaths with gross clinical abnormalities such as degenerated opercula and gills, liver damage, haemorrhagic kidney and serous fluid in swollen intestines. The counts of A. cryaerophilus in kidney, liver and gills of experimentally infected fish ranged from 1.59 x 10(10) colony forming units (cfu)/g to 7.41 x 10(12) cfu/g. The means of erythrocyte (RBC) count, haematocrit level, serum cholesterol, triglyceride, albumin and total protein concentrations in the blood of the experimentally infected rainbow trout group were significantly lower than in the healthy fish. Leukocyte (WBC) counts of the experimentally infected rainbow trout were significantly higher than those of healthy fish. The present work shows that the selected blood characteristics may be good indicators of response to infections in rainbow trout.     

Immunosuppressive effect of infectious pancreatic necrosis virus on rainbow trout (Oncorhynchus mykiss)

The infectivity of infectious pancreatic necrosis virus (IPNV) for rainbow trout (Oncorhynchus mykiss) mononuclear leukocyte subpopulations was investigated to determine the mechanisms of immunosuppression caused by the virus. IPNV was recovered from nylon wool-adherent, surface immunoglobulin (Ig)-positive leukocytes of head kidney, spleen and peripheral blood collected from virus-inoculated fish with higher titers than non-adherent, Ig-negative cells. Non-adherent cell population showed mitogenic response to phytohemagglutinin and concanavalin A but not to lipopolysaccharide. Conversely, the responses of adherent cells to these mitogens were weak. Mitogenic response and non-specific cytotoxicity of head kidney leukocytes significantly decreased by the inoculation of fish with the virus. These results suggest that the suppression of immune responses is involved in the establishment of carrier state in fish after infection with IPNV.     

Influence of zearalenone on selected biochemical parameters in juvenile rainbow trout (Oncorhynchus mykiss)

Zearalenone (ZEA) is a mycoestrogen frequently found in food and animal feed materials all over the world. Despite its hydrophobic character, ZEA is also found in surface and ground waters which suggests an environmental risk for aquatic animals. Knowledge concerning mycotoxin-related mechanisms of toxicity is still incomplete, e.g. little is known about the influence of ZEA exposure on fish. The aim of this study was to investigate the effect of ZEA on selected biochemical parameters in juvenile rainbow trout after 24, 72, and 168 h of intraperitoneal exposure (10 mg/kg of body weight). The analysis showed a slight tendency towards prolonged blood clotting time and significant iron deficiency in the liver and ovary of exposed animals. However, no differences in aminotransferase (AlaAT, AspAT) activity or glucose levels in fish plasma was observed. The results of this study suggest that although trout exposed to ZEA did not exhibit any distinct symptoms of liver damage, the mycotoxin tested was able interfere with blood coagulation and iron-storage processes.     

Physiological and endocrinological responses during prolonged exercise in hatchery-reared rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) were subjected to vigorous exercise (1.5 body length s-1), low exercise (0.5 body length s-1) or still-water (0.0 body length s-1). Hematocrit, glucose, growth hormone (GH), cortisol and triiodo-L-thyronine (T3) were monitored at the start of exercise, after 24 h, and after 2, 4, 8, 16 and 32 days of continuous swimming. Morphological indices and food intake were also monitored. At the end of the experiment, trout subjected to low exercise had gained significantly (p < 0.05) more weight than both the control (still-water) and vigorously exercised fish. This low exercised group of fish also consumed more food than the 2 other groups. Hematocrit increased significantly in both exercised groups at the onset of swimming but returned to pre-exercise levels within 8 hrs. Plasma glucose appeared to be generally unaffected by exercise. Likewise, plasma concentrations of both GH and T3 were not influenced by exercise. Plasma cortisol levels increased in an exercise dependent fashion at the onset of swimming, but returned to pre-swimming levels within 24 h and there was no apparent effect of sustained swimming. The results suggest: (i) the onset of exercise elicits transient changes in plasma components, (ii) the observed weight gain in low exercising salmonids occur without increases in circulating levels of GH or T3.     

Occurrence of enteric redmouth disease in rainbow trout (Oncorhynchus mykiss) on farms in Croatia

During the spring of 1996 and autumn of 1997 unusual mortality outbreaks among rainbow trout fry and yearlings occurred at two different trout farms, resulting in mortality of 20 and 10 per cent, respectively. Generally, the affected fish, swimming at the water surface, were reluctant to eat and were dark pigmented with visible haemorrhages around and within the oral cavity. Bacterial isolates from moribund fish from both cases were identified as Yersinia ruckeri by standard biochemical tests and API 20E. The isolated strains were found to be sensitive to tetracycline, chloramphenicol, co-trimoxazole, nalidixic acid, flumequine, enrofloxacin, carbenicillin and gentamicin. Microplate agglutination assay confirmed that both isolates belonged to serotype O1. The pathogenicity of the isolated bacteria was confirmed by challenge experiment. Titres of specific antibodies were determined in the sera of survivors. The titre was highest on the 21st day postchallenge and was detectable until the 81st day.     

Effect of sulphadiazine and trimethoprim on the immune response of rainbow trout (Oncorhynchus mykiss)

A combination of sulphadiazine and trimethoprim at a ratio of 5:1 (SDZ/TMP) was tested for possible immunomodulatory effects. The aim of the study was to follow the immune response after vaccination with simultaneous drug treatment. The fish were immunised with a commercial oil-based divalent (furunculosis/vibriosis) vaccine and were simultaneously given oral drug treatment. The specific immune response was monitored by analysing the levels of specific antibodies with ELISA. As indicators of the nonspecific immune response, the lysozyme activity of serum was measured and the phagocytic activity of circulating leucocytes was monitored by a chemiluminescence assay. Total circulating leucocyte counts and differentials were also monitored. The disease resistance was evaluated by challenge tests at the end of the experiment.The results indicate that SDZ/TMP at a ratio of 5:1 does not interfere negatively with the immune response in rainbow trout after vaccination. A slight stimulation in the antibody response as well as in the chemiluminescence response of circulating granulocytes was recorded in fish treated with the drugs in connection with vaccination. The drugs did not significantly affect the survival after challenge.