In vitro evaluation of gut contractile response to histamine in rainbow trout (Oncorhynchus mykiss Walbaum, 1792)

The contractile response of intestinal strips in rainbow trout (Oncorhynchus mykiss Walbaum, 1792) to the administration of histamine was assessed by means of the organ bath technique. Intestinal strips were isolated from 16 clinically healthy fish and mounted in organ baths. Histamine was compared with the full agonist serotonin, to evaluate their contractile efficacy and potency. Serotonin elicited a concentration-related contraction in all examined intestinal strips, whereas histamine induced the contraction only in 14 exemplars. Of these, seven exhibited a concentration-related response. A sigmoidal curve was fitted from data (R(2) = 0.55) and its best fit values were compared with those of serotonin. Interestingly, histamine exhibited the same efficacy (E(max)) as serotonin (F-test, p > 0.05), but showed lower potency (by an order of magnitude) (F-test, p < 0.01). Moreover, the effect of the H1 antagonist, pyrilamine, has been tested to exclude aspecific contraction due to other agonists eventually released in situ following histamine administration. Pyrilamine showed a marked concentration-related antagonist action on the contractility induced by histamine with complete contractility antagonism at 10(-4)M. The authors suggest that the responses to histamine measured in the present study reflect a less sensitive response to an exogenous source of histamine, possibly due to bacterial metabolism.     

Morphological and molecular characterisation of Gyrodactylus salmonis (Platyhelminthes, Monogenea) isolates collected in Mexico from rainbow trout (Oncorhynchus mykiss Walbaum)

Gyrodactylus salmonis (Yin et Sproston, 1948) isolates collected from feral rainbow trout, Oncorhynchus mykiss (Walbaum) in Veracruz, southeastern Mexico are described. Morphological and molecular variation of these isolates to G. salmonis collected in Canada and the U.S.A. is characterised. Morphologically, the marginal hook sickles of Mexican isolates of G. salmonis closely resemble those of Canadian specimens - their shaft and hook regions align closely with one another; only features of the sickle base and a prominent bridge to the toe permit their separation. The 18S sequence determined from the Mexican specimens was identical to two variable regions of SSU rDNA obtained from a Canadian population of G. salmonis. Internal transcribed spacer (ITS) regions (spanning ITS1, 5.8S and ITS2) of Mexican isolates of G. salmonis are identical to ITS sequences of an American population of G. salmonis and to Gyrodactylus salvelini Kuusela, Zi?tara et Lumme, 2008 from Finland. Analyses of the ribosomal RNA gene of Mexican isolates of G. salmonis show 98-99% similarity to those of Gyrodactylus gobiensis Gl?ser, 1974, Gyrodactylus salaris Malmberg, 1957, and Gyrodactylus rutilensis Gl?ser, 1974. Mexican and American isolates of G. salmonis are 98% identical, as assessed by sequencing the mitochondrial cox1 gene. Oncorhynchus mykiss is one of the most widely-dispersed fish species in the world and has been shown to be an important vector for parasite/disease transmission. Considering that Mexican isolates of G. salmonis were collected well outside the native distribution range of all salmonid fish, we discuss the possibility that the parasites were translocated with their host through the aquacultural trade. In addition, this study includes a morphological review of Gyrodactylus species collected from rainbow trout and from other salmonid fish of the genus Oncorhynchus which occur throughout North America.     

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.     

Induction, characterisation and pathogenicity in rainbow trout Oncorhynchus mykiss (Walbaum) of Lactococcus garvieae L-forms.

Difficulties with induction and cultivation of L-forms, particularly those derived from Gram positive parent cells, have constrained to some degree the ability to evaluate the pathogenicity of these morphotypes. Induction of L-forms of Lactococcus garvieae was undertaken using either charcoal or inactivated horse serum media supplemented with ampicillin, benzylpenicillin or erythromycin, the drug of choice for treatment of infections in rainbow trout, Oncorhynchus mykiss, (Walbaum), and NaCl as an osmotic stabiliser. Lysozyme treated cells could be cultured in a cell wall deficient state using media consisting of charcoal, NaCl and either ampicillin or benzylpenicillin. The influence of some amino acids for induction of L-forms was assessed by disc diffusion and combined interaction. Analysis of variance of colony counts indicated that the amino acids glycine, DL-methionine, L-threonine and L-serine (P<0.03), and the presence of charcoal were beneficial and that inactivated horse serum was detrimental to L-form development. Electron microscopy revealed that the cell wall of L-forms was missing and this cell had a greatly expanded volume compared to parent cells. Electrophoresis of whole cell proteins showed some variation of electropherotype between parent and L-form cells. L-forms expressed greater quantities of proteins with molecular mass of 36 and 66 kDa and parent cells contained greater quantities of proteins of molecular mass 29, 43 and 60 kDa. Additional proteins of molecular mass 32, 44 and 53 kDa were present in L-form extracts, and in parent cells of 34, 38, 40, 42, 85 and 123 kDa which may represent cell wall associated proteins or alterations in expression due to different growth rates. Intraperitoneal challenge of rainbow trout with L-forms failed to produce overt infection even in immune-suppressed fish, but L-forms were shown by indirect fluorescent antibody test to remain inkidney tissue. Fish were susceptible to infection when challenged with parent cells of L. garvieae.     

Comparative protection of two different commercial vaccines against Yersinia ruckeri serotype O1 and biotype 2 in rainbow trout (Oncorhynchus mykiss)

Differentially extended specific protection by two commercial vaccines against Yersinia ruckeri serotype O1 biotype 2 was studied following 30 s immersion exposure. Rainbow trout were challenged intra-peritoneally (i.p.) with Y. ruckeri serotype O1, biotype 2 (≈10⁶ to 10⁷ CFU/fish) at 4, 6 and 8 months after vaccination with vaccines containing either biotype 1 (AquaVac^® ERM) or both biotypes 1 and 2 (AquaVac^® RELERA?). The specific pattern of vaccine-mediated protection was evaluated by relative percentage survival (RPS) analysis at 4 and 6 months post-vaccination and by obtaining gross pathological observations at 4 and 8 months respectively. We determined specific significant and superior protection in terms of increased survivability in AquaVac^® RELERA? vaccinated fish and observed correspondingly fewer pathological changes. The challenge trials indicated a longer protection for at least 6 months without any booster vaccination. A specific and adaptive response induced by AquaVac^® RELERA? vaccine against Y. ruckeri biotype 2 was clearly indicated. In addition, some degree of cross protection rendered by AquaVac^® ERM containing biotype 1 during infection with Y. ruckeri biotype 2 was also noted.     

Proteomic analysis of rainbow trout (Oncorhynchus mykiss, Walbaum) serum after administration of probiotics in diets

The response of rainbow trout (Oncorhynchus mykiss, Walbaum) towards probiotics present in the feed was investigated by examining the proteome of serum as a measure of the acute phase response (APR). Proteomic analysis by two-dimensional electrophoresis (2D) concurrently with mass spectrometry was used to detect APR related proteins in rainbow trout serum following feeding with probiotics Aeromonas sobria GC2 and Bacillus sp. JB-1. Three candidate proteins increased following use of GC2, and were putatively identified as NADH dehydrogenase, dystrophin and mKIAA0350. Conversely, one of the proteins, which were induced following use of JB-1 was identified as transferrin.     

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.     

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.     

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.     

Comparative single‐dose pharmacokinetics of orally administered florfenicol in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792) at health and experimental infection with Streptococcus iniae or Lactococcus garvieae

This study evaluates changes in the pharmacokinetic behavior of a single oral dose of florfenicol in rainbow trouts experimentally infected with Lactococcus garvieae or Streptococcus iniae. One hundred and fifty fish were randomly divided into three equal groups: 1—healthy fish, 2—fish inoculated with S. iniae (2.87 × 10⁷ CFU/ml, i.p.), and 3—fish inoculated with L. garvieae (6.8 × 10⁵ CFU/ml, i.p.). Florfenicol was administered to all groups at 15 mg/kg by oral gavage. Blood sampling was performed at 0, 2, 3, 6, 8, 12, 24, 48, 72, and 120 hr after drug administration to each group, and plasma concentration of florfenicol was assayed by HPLC method. The MICs of florfenicol were 1.2 μg/ml and 5 μg/ml against L. garviae and S. iniae, respectively. Healthy fish showed higher values for most of the PK/PD parameters as compared to fish infected with L. garvieae which was reversed in fish infected with S. iniae. Fish infected with L. garvieae showed decreased relative bioavailability accompanied by increased volume of distribution at steady‐state (Vd_ss) and total body clearance (Cl_B)_. Infection with S. iniae increased the peak concentration of drug after administration (Cmax) and decreased elimination half‐life (T_{1/2 β})_, central compartment volume (V_c), and Vd_ss. In conclusion, infection with these bacteria can affect the pharmacokinetic behavior of florfenicol in rainbow trouts as shown by decreased bioavailability and increased total body clearance and volume of distribution in L. garvieae infection and decreased volume of distribution accompanied by increased Cmax in S. iniae‐infected fish.     

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     

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.     

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.     

Molecular detection of an Ehrlichia-like agent in rainbow trout (Oncorhynchus mykiss) from Northern California

Ehrlichia DNA was identified by nested PCR in rainbow trout (Oncorhynchus mykiss) collected from a creek in northern California where Potomac horse fever is endemic. Ehrlichia DNA was found in tissues from several organs including the gills, heart, spleen, liver, kidneys and intestine of trout and from three different adult digenetic trematodes (Deropegus sp., Crepidostomum sp., Creptotrema sp.) parasitizing the gallbladder and/or the intestine of the trout. Sequencing of PCR-amplified DNA from the 16S rRNA gene indicated that the source organism was most closely related to the sequences of E. risticii (level of sequence similarity 96.0%), the SF agent (95.9%), E. sennetsu (95.8%), and Neorickettsia helminthoeca (95.3%). The data suggest that trout and parasitic trematodes may be involved in the epidemiology of an Ehrlichia-like agent belonging to the E. sennetsu genogroup. Whether the fish agent infects horses, dogs, or human beings, and whether it causes disease, remain to be determined.     

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%.     

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.