Branchial Ionic Flux Responses in Rainbow Trout to Chloramine-T after Acclimation to Different Levels of Water Hardness

Abstract

Whole-body unidirectional and net Na⁺ and Cl^– fluxes were determined in rainbow trout Oncorhynchus mykiss exposed to chloramine-T (9 mg/L) alone or in combination with 17 mmol NaCl/L (0.1 % weight per volume, w/v) after acclimation to either city of Ottawa tap water (TW: Na⁺ 0.121 ± 0.004 mmol/L; K⁺ 0.015 ± <0.001 mmol/L; Ca²⁺ 0.351 ± 0.004 mmol/L; Cl^– 0.141 ± 0.002 mmol/L) or artificial soft water (ASW: Na⁺ 0.043 ± 0.003 mmol/L; K⁺ 0.005 ± <0.001 mmol/L; Ca²⁺ 0.063 ± 0.002 mmol/L; Cl^– 0.026 ± 0.002 mmol/L). Preexposure fluxes revealed that ASW-acclimated fish had reduced rates of Na⁺ uptake and efflux compared with TW-acclimated fish. However, rates of Cl^– uptake among ASW-acclimated fish were comparable with those of TW-acclimated fish. Exposure to chloramine-T in TW resulted in significant increases in the Cl^– efflux, giving rise to a significant and negative net flux. Exposure of ASW-acclimated fish to chloramine-T caused a significant negative increase in the Na⁺ efflux and net flux but only a negative increase in the Cl^– net flux. The addition of 17 mmol NaCl/L (0.1% w/v) eliminated the negative net ionic fluxes, resulting in significant increases in influx. The combined use of chloramine-T with NaCl is a potential method of remediating ionic disturbances that may be caused by treating trout with chloramine-T.     

Gill Ectoparasites of Juvenile Rainbow Trout and Brown Trout in the Upper Colorado River

Abstract

During 1996 and 1997, 112 rainbow trout Oncorhynchus mykiss and 204 brown trout Salmo trutta, all young of the year, were sampled from a 40-km study area of the upper Colorado River and were examined for gill parasites. Ambiphrya, Chilodonella, Ichthyobodo, Apiosoma, Trichodina, Trichodinella, Tripartiella, Epistylis, and an unidentified cochliopodid amoeba were the representative protozoan genera observed on fish examined. Significant month–year–species interactions (P = 0.0295) were revealed, reflecting the changes in infestation prevalence among months, years, and species of salmonid. Greater ectoparasite richness was observed in downstream sections of the study area, most notably near Hot Sulphur Springs, Colorado. Peaks of infestation intensity and ectoparasite richness occurred in August and September of both years, presumably because of high mean water temperatures and low flows during that time.     

虹鳟酪氨酸酶基因的生物信息学分析及其在不同发育阶段和组织中的表达分析

酪氨酸酶（tyrosinase,Tyr）基因在调控动物色素合成的过程中发挥关键作用。为了解tyr基因与虹鳟（Oncorhynchus mykiss）体色变异的关系,本研究对虹鳟tyr-1和tyr-2基因进行了蛋白序列分析,并利用实时荧光定量PCR检测两种基因在野生型虹鳟（虹鳟）和黄色突变型虹鳟（金鳟）体色发生的19个不同时期和成鱼13种不同组织中的表达差异。蛋白序列分析结果表明,Tyr-1和Tyr-2都是亲水性蛋白,且蛋白结构主要是无规则卷曲和α-螺旋;实时荧光定量PCR结果显示,tyr-1基因在胚胎各时期均有表达,tyr-1基因表达量在虹鳟受精期最高,桑椹期次之,且显著高于其他时期（P<0.05）;tyr-1基因表达量在金鳟16-细胞期最高,且显著高于其他时期（P<0.05）;tyr-2基因分别从虹鳟囊胚期和金鳟原肠期开始表达,表达量均在心跳期达到最高且显著高于其他时期（P<0.05）。出膜后,tyr-1、tyr-2基因分别在虹鳟5日龄和金鳟3月龄表达量最高。在胚胎各时期中,tyr-1、tyr-2基因在虹鳟中表达量普遍高于金鳟,出膜后普遍低于金鳟。在各组织中,tyr-1基因在皮肤、眼睛、肾脏、肝脏等组织中有较高表达,tyr-2基因主要在皮肤、眼睛中有较高表达,在其他组织中表达量较低。以上结果表明,tyr-1和tyr-2基因与虹鳟体色变异具有一定的相关性,为深入阐明虹鳟体色变异机制和体色遗传改良奠定了理论基础。     

Effects of Salinity on Yersinia ruckeri Infection of Rainbow Trout and Brown Trout

Abstract

Juvenile rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta acclimated to freshwater or salinities of 9.0‰ or less were exposed to Yersinia ruckeri, the bacterial pathogen that causes enteric redmouth disease (ERM). Both species of fish were kept in the same recirculating systems after bacterial exposure. Rainbow trout mortality was significantly (P < 0.05) different in each salinity: 96.5% in freshwater, 89.5% in water of 1.1‰ salinity, 81.3% in 3.0‰ salinity, and 75.0% in 9.0‰ salinity (model SE = 1.0). All deaths occurred between 3 and 12 d after exposure to Y. ruckeri. Only 2.3% of brown trout in all salinities died, and differences among treatments were not significant. For both fish species, Y. ruckeri was isolated from liver, spleen, and trunk kidney of fish dying during this experiment, and lesions of rainbow trout were consistent with ERM. Yersinia ruckeri was not isolated from brown trout surviving for 21 d after bacterial exposure but was isolated from 3 of 24 surviving rainbow trout; a polymerase chain reaction assay detected the DNA of Y. ruckeri in 3 additional rainbow trout survivors. Neither the lesions of fish with ERM nor the percentage of surviving fish subclinically infected with Y. ruckeri was affected by salinity. Bacterial growth in vitro was not affected by low (≤9.0‰) salinity; however, bacterial adhesion to polystyrene was significantly reduced as salinity increased. Although mortality caused by Y. ruckeri was significantly lower for rainbow trout in water with slightly increased salinity, none of the salinities tested was effective in preventing serious losses caused by this pathogen in recirculating systems.     

Detection of Myxobolus cerebralis in Rainbow Trout and Oligochaete Tissues by Using a Nonradioactive in Situ Hybridization (ISH) Protocol

Abstract

A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebralis, the causative organism of whirling disease, in its primary host, rainbow trout Oncorhynchus mykiss, and in its alternate oligochaete host, Tubifex tubifex. A cocktail of three oligonucleotide primers (derived from the small subunit ribosomal DNA sequence) directed at target sequences of the parasite DNA was tailed at the 3′ end with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP). Labeled probes were hybridized to parasite DNA present in deparaffinized tissue sections from infected trout and oligochaetes. The bound probes were visualized after modifications of existing ISH protocols. By using the new ISH procedure, the parasite was found in target tissues of subclinically and clinically infected fish and tubificid oligochaetes after exposures of these hosts to triactinomyxons and mature spores, respectively. The probe did not bind with salmonid tissues infected with two other myxosporean parasites, Ceratomyxa shasta or the PKX organism, or to a Myxobolus sp. infecting the cartilage of plain sculpin Myoxocephalus jaok. These initial results indicate that ISH is an effective and specific test for detecting Myxobolus cerebralis in its fish and oligochaete hosts.     

Experimental Infection of Rainbow Trout with Saprolegnia parasitica

Abstract

A method was developed to experimentally induce saprolegniasis in rainbow trout Oncorhynchus mykiss. The development of a reliable method to produce infected fish is essential to efforts to determine the efficacy of various antifungal treatments. Three methods for inducing saprolegniasis were evaluated in waters containing known concentrations of Saprolegnia parasitica zoospores. These methods included application of the following stressors to fish: (1) abrasion and dewatering, (2) water temperature increase, and (3) a combination of abrasion, dewatering, and temperature increase. Neither physical abrasion nor temperature increase stress alone was effective for inducing saprolegniasis. Only 25.9% of fish stressed by abrasion and dewatering alone became infected. Application of both abrasion and temperature stress, however, induced saprolegniasis in 77.8% of fish tested. Most of these fish became infected after 5 d of stress treatments. No fish became infected or died in the positive control group (not stressed but exposed to S. parasitica zoospores) or the negative control group (not stressed or challenged). This method should enable researchers to induce saprolegniasis in rainbow trout to study its pathogenesis or to test the efficacy of antifungal treatments. In conducting efficacy studies, it is important that therapeutic treatments begin promptly after the first signs of saprolegniasis are observed because the disease can progress very quickly and often results in mortality.     

Purification and Partial Characterization of a Rainbow Trout Egg Lectin

Abstract

A lectin that agglutinates rabbit red blood cells (RBCs) and human type B RBCs was isolated from ova of rainbow trout Oncorhynchus mykiss. Hemagglutination of rabbit RBCs was inhibited completely by 10 mM L-rhamnose but not by certain other sugars, 100 mM EDTA, or 100 mM 2-mercaptoethanol. Partial purification of this hemagglutinating material was achieved by affinity chromatography of an H₂O-dialyzed yolk homogenate on rhamnose-linked Sepharose. A polyacrylamide gel electrophoresis (SDS-PAGE) performed on this sample revealed two polypeptides with approximate molecular masses of 19 kilodaltons (kDa) and 30 kDa. By fast-phase liquid chromatography, proteins with a molecular mass of less than 20 kDa were separated from other elements of the affinity-purified hemagglutinating material. These proteins were found to lack hemagglutinating activity. When a western blot with rabbit antilectin antiserum was performed against yolk extract, rainbow trout serum, or yolk from larvae, a 30-kDa polypeptide was detected within all three samples. If the rainbow trout serum and egg lectins are the same molecule, then the biological function of the rainbow trout egg lectin may include host defense or perhaps a basic, homeostatic mechanism such as glycoprotein transport     

Assessment of Detoxifying Markers for Florfenicol in Rainbow Trout Liver

Florfenicol (FF) is employed in fish farms to contest or prevent bacterial infections. However, this pharmaceutical may produce reactive oxygen species that may cause biochemical changes in antibiotic-treated fish. The aim of this study was to evaluate the effect of FF on Rainbow Trout Oncorhynchus mykiss treated for 10 d with 7.5 and 15 mg/kg FF followed by a withdrawal period of 5 d. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyoxalase I and glyoxalase II, total glutathione, lactic dehydrogenase, and alkaline phosphatase were investigated in the livers of treated and untreated fish. A general impairment of antioxidant enzymes and metabolic indicators was measured in FF-treated Rainbow Trout. Onset of oxidative damage may have occurred during the antibiotic treatment as a consequence of the effect of FF toxicity at mainly the highest dose. Nevertheless, the rise in levels of total glutathione and glutathione S-transferase even after the withdrawal period may shield the antibiotic-mediated oxidative processes.

Received December 22, 2015; accepted May 26, 2016 Published online October 28, 2016     

Dietary Ascorbyl Monophosphate Depresses Lipid Peroxidation in Rainbow Trout Spermatozoa

Abstract

This study was designed to analyze lipid peroxidation in spermatozoa of rainbow trout Oncorhynchus mykiss by an optimized thiobarbituric acid (TBA) method, and to evaluate the effect of graded levels of dietary antioxidant (ascorbic acid in the form of ascorbyl monophosphate, AP) on TBA values of spermatozoa. Sperm from rainbow trout fed diets supplemented with AP at 0, 110, or 870 mg/kg were sampled during the reproductive season. The group given the unsupplemented diet had the lowest ascorbic acid concentration in seminal plasma during most of the spermiation season compared with groups fed diets with AP While the ascorbic acid concentration in seminal plasma decreased toward the end of the reproductive season, spermatozoa malondialdehyde production, indicative of increasing peroxidation, tended to increase. At the end of the season, significant differences (P < 0.05) were found in peroxidation levels in spermatozoa from fish fed different levels of ascorbic acid. The most abundant polyunsaturated fatty acid in sperm lipids, docosahexaenoic acid (C22:6), was significantly lower in lipids from spermatozoa of the AP-devoid (control) group than in the 870-mg/kg supplement group. By the end of the reproductive season, spermatozoa from the control group had significantly higher peroxidation levels than did spermatozoa from fish given AP (110 and 870 mg/kg). Thus, it is the first evidence in fish that dietary AP supplements can increase seminal plasma ascorbic acid concentrations, and it suggests that peroxidative damage to spermatozoa can be reduced during the reproductive season, which in turn may affect sperm fertilizing ability.     

Case Report: Strawberry Disease in Farmed Chilean Rainbow Trout

Strawberry disease is a chronic, nonlethal skin condition that affects Rainbow Trout Oncorhynchus mykiss in the United States and several European countries, where it is also known as red-mark syndrome. We provide the first identification and characterization of three strawberry disease outbreaks occurring at two aquaculture farms in southern Chile. Clinically affected fish weighing an average of 400 g presented multiple bright-red, usually raised, skin lesions on the flank, ventral surface, and dorsal surface. A PCR using Rickettsia-like-organism (RLO)-specific primers was performed on nine affected fish, and all skin samples were positive for the RLO 16S ribosomal RNA sequence. All PCR results for Flavobacterium psychrophilum and other bacterial and viral pathogens were negative. Histopathological examination of the skin lesions revealed extensive dermatitis, with severe lymphocytic infiltration in advanced cases. This report is the first to describe strawberry disease in farmed Chilean Rainbow Trout. Additional studies are needed to evaluate the risk for Rainbow Trout culture; fish challenge experiments should be performed to fulfill Koch’s postulates and to demonstrate that RLO is the cause of this disease.

Received December 27, 2014; accepted October 23, 2015.     

Toxicity of Copper Sulfate to Flavobacterium psychrophilum and Rainbow Trout Eggs

Abstract

Tests were conducted to determine the concentrations of copper sulfate needed to kill Flavobacterium psychrophilum, the cause of bacterial coldwater disease, either in vitro or on Rainbow Trout Oncorhynchus mykiss eggs. For the in vitro test, a plastic strip dipped in a solution of F. psychrophilum was exposed for 15 min to copper sulfate solutions of 0, 1, 5, 10, 20, 35, 50, 75, or 100 mg/L. Bacteria were “too numerous to count” at concentrations ≤10 mg/L CuSO₄; significant reductions in prevalence relative to untreated controls were noted for concentrations ≥35 mg/L. However, CFUs were still observed at 50 and 75 mg/L (20% of plates with tryptone yeast extract salts media). No yellow-pigmented CFUs typical of F. psychrophilum were observed at 100 mg/L CuSO₄. For the in vivo test, eggs were exposed for 15 min to 100, 300, 500, and 700 mg/L CuSO₄ or 100 mg/L iodine (control). Survival to hatch was significantly lower at 500 (44.3 ± 15.2%, mean ± SD) or 700 mg/L CuSO₄ (1.7 ± 0.8%) than for controls treated with 100 mg/L iodine (93.6 ± 0.9%) or at copper sulfate concentrations ≤300 mg/L. The 15-min LD₅₀ and LD₁₀ for copper sulfate were 461 mg/L (95% confidence interval: 457–466 mg/L) and 259 mg/L (251–266 mg/L). The prevalence of yellow CFUs at 100 mg/L CuSO₄ (40.0%) was significantly higher than in untreated controls. Significant reductions in yellow CFUs were achieved using 300, 500, or 700 mg/L CuSO₄ (7.5, 2.5, or 0.0% of plates with CFUs, respectively) or 100 mg/L iodine (2.5%), relative to untreated control eggs. Overall, since the concentrations of copper sulfate required to eliminate F. psychrophilum were toxic to the eggs, copper sulfate is not recommended for coldwater disease control in Rainbow Trout eggs based on conditions and parameters in this study.

Received July 7, 2011; accepted March 17, 2013     

Gill Infection Model for Columnaris Disease in Common Carp and Rainbow Trout

Challenge models generating gill lesions typical for columnaris disease were developed for the fry of both Common Carp Cyprinus carpio and Rainbow Trout Oncorhynchus mykiss by means of an immersion challenge and Flavobacterium columnare field isolates were characterized regarding virulence. Carp inoculated with highly virulent isolates revealed diffuse, whitish discoloration of the gills affecting all arches, while in trout mostly unilateral focal lesions, which were restricted to the first two gill arches, occurred. Light microscopic examination of the gills of carp exposed to highly virulent isolates revealed a diffuse loss of branchial structures and desquamation and necrosis of gill epithelium with fusion of filaments and lamellae. In severe cases, large parts of the filaments were replaced with necrotic debris entangled with massive clusters of F. columnare bacterial cells enwrapped in an eosinophilic matrix. In trout, histopathologic lesions were similar but less extensive and much more focal, and well delineated from apparently healthy tissue. Scanning and transmission electron microscopic observations of the affected gills showed long, slender bacterial cells contained in an extracellular matrix and in close contact with the destructed gill tissue.

This is the first study to reveal gill lesions typical for columnaris disease at macroscopic, light microscopic, and ultrastructural levels in both Common Carp and Rainbow Trout following a challenge with F. columnare. The results provide a basis for research opportunities to examine pathogen–gill interactions.

Received January 10, 2014; accepted July 27, 2014     

Temperature Related Absorption and Excretion of Sulphadimidine in Rainbow Trout,Salmo Gairdneri

The main purpose of the present study was to see if a kinetic model and a computer program for characterizing the rate of uptake and clearance of chemicals in aquatic organisms (Biofac, OECD’s Chemicals Testing Programme) was applicable to pharmacokinetic studies in fish. The program seems suitable for such studies.The effect of temperature upon rate of absorption of sulphadimidine from medicated water and elimination of the same drug was investigated in rainbow trout. A rise in the temperature from 7° C to 14° C more than doubled the calculated rate constants of absorption and elimination. The biological half life (t_½ β) and the time to reach 90 % of steady state (t_{90% ss}) at 14° C were approximately half the values at 7° C.     

Blood Parameters and Immune Status of Rainbow Trout with Proliferative Kidney Disease

Abstract

Blood parameters, disease resistance, and the immune response were sequentially evaluated in rainbow trout Oncorhynchus mykiss with proliferative kidney disease (PKD). The fish were maintained under laboratory conditions, and the study group went through a full cycle of the disease. Hematological and serological changes occurred primarily in those fish with severe kidney lesions. Fish infected with the parasite that causes PKD demonstrated a greater resistance to bacterial challenge, and their immune responses were heightened when compared with those of uninfected fish. These data suggest that PKD alone is not a predisposing factor for secondary infections if the fish does not incur severe renal lesions.     

Treatment of Ichthyophthiriasis in Rainbow Trout and Common Carp with Common and Alternative Therapeutics

Abstract

The goal of this laboratory study was to provide better knowledge about the treatment of ichthyophthiriasis (causative agent: Ichthyophthirius multifiliis, a ciliate bacteria) in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio. The following questions were investigated: (1) the effectiveness of different chemicals (formalin, sodium chloride, hydrogen peroxide, Perotan, Virkon, Aquahumin, Baycox, and Ivomec) and at different concentrations and durations of application, (2) the number of treatments and the time intervals between treatments that were necessary to remove the parasite, and (3) how treatment effectiveness differed between the two species. The most effective treatment was a 37% stock solution of formalin at 110 μL/L of bath water for 1 h in rainbow trout and for 2 h in common carp. Aquahumin (150 μL/L for 2 h) was effective in slightly or moderately infected rainbow trout and at low water temperatures, but it was not effective for common carp. All other tested chemicals were ineffective. With formalin and Aquahumin, five treatments were necessary to remove I. multifiliis infestation. At 10 ± 1°C, the parasites were eradicated when the treatment was performed at 48-h intervals. At 18 ± 1°C the infestation was eliminated when treatment was performed at 24-h intervals but not at 48-h intervals. At 25 ± 1°C, treatment at 24-h intervals was ineffective; however, shorter intervals between treatments might improve treatment efficacy at this temperature. In contrast, the number of treatment repetitions played a minor role, and parasites were eliminated with five treatments in all experiments when the type of chemical and treatment interval were optimal.     

Studies on the Motility and Cryopreservation of Rainbow Trout (Salmo Gairdneri) Spermatozoa

The very short duration of vigorous movement (1 1/2 to 7 min) in fresh water and physiological solutions make trout spermatozoa difficult subjects for cryopreservation studies. Solutions consisting of 250 to 280 mmol sucrose and 5 to 12% dimethyl sulphoxide (DMSO) (4 parts) did not activate trout spermatozoa (1 part), but after dilution with fresh water vigorous motility could be fully restored. These sucrose-DMSO solutions were employed in cryopreservation studies. Using straws and a fast freezing — fast thawing procedure, post-thaw dilution with fresh water resulted in 25%-60% of spermatozoa becoming motile, all with vigorous forward progression. Some existing methods for the cryopreservation of other freshwater fish spermatozoa were repeated on trout without success.     

Physiological and Immunological Effects of Adjuvanted Aeromonas salmonicida Vaccines on Juvenile Rainbow Trout

Abstract

The effects of injectable vaccines against Aeromonas salmonicida on oxygen consumption, growth, kidney lysozyme activity, and anti-A. salmonicida plasma antibody titers of juvenile rainbow trout Oncorhynchus mykiss were examined. The vaccines were A. salmonicida bacterin only, bacterin adjuvanted with levamisole, bacterin in emulsified oil, microencapsulated bacterin, microencapsulated bacterin with muramyl dipeptide, microencapsulated bacterin with β-1,3-glucan, and microencapsulated bacterin with Vibrio anguillarum lipopolysaccharide (LPS). The greatest and broadest ranges of responses were caused by the microencapsulated bacterin with V. anguillarum LPS. Oxygen consumption rates and specific growth rates were significantly higher over the course of 1 month among fish treated with the LPS vaccine. These fish also maintained a higher anti-A. salmonicida plasma antibody titer and kidney lysozyme activity for a substantially longer period than fish receiving the other treatments.     

养殖虹鳟与大西洋鲑肉质和营养成分比较研究

试验选取10尾贵州都匀养殖场的虹鳟(1562±32)g和10尾市场出售的挪威大西洋鲑(1856±45 g),采集其背部肌肉,分别进行质构特性及营养成分分析。结果表明:质构特性上,虹鳟肌肉的失水率显著低于大西洋鲑(P<0.05),剪切力显著高于大西洋鲑(P<0.05);常规成分中,虹鳟肌肉的粗脂肪含量显著低于大西洋鲑(P<0.05),二者的粗蛋白含量无显著差异(P>0.05);脂肪酸组成上,虹鳟肌肉的n-6多不饱和脂肪酸含量显著低于大西洋鲑(P<0.05),但基于脂肪酸组成获得的饱和度(S/P)、动脉粥样硬化指数(AI)、凝血指数(TI)和降胆固醇/致高血脂比率(HH)等指标无显著性差异(P>0.05);氨基酸组成方面,虹鳟肌肉内的人体必需氨基酸比例显著低于大西洋鲑(P<0.05),鲜味氨基酸含量显著高于大西洋鲑(P<0.05)。结果表明,养殖虹鳟在肉质质构上相比大西洋鲑较“干硬”,且虹鳟肌肉多不饱和脂肪酸和必需氨基酸含量均低于大西洋鲑。     

Myxobolus neurotropus Infecting Rainbow Trout in Alaska,a New Geographic Record

Abstract

The Alaska Department of Fish and Game fish pathology laboratory received a rainbow trout Oncorhynchus mykiss from the Alaska Peninsula that was suspected of having whirling disease based on the display of aberrant swimming behavior and a deformed spine. We tested for Myxobolus cerebralis using standard pepsin–trypsin digest and molecular procedures, which yielded negative results. However, many oval shaped myxospores were observed in brain smears and were confirmed to be those of the morphologically similar M. neurotropus based on a diagnostic assay using PCR. The known geographic distribution of this parasite includes Idaho, Washington, Utah, Oregon, California, and now, Alaska. Whether this species is an emerging parasite is not known because it was only described a few years ago. Given the severe infection found in this rainbow trout, perhaps the considerable displacement of neurological tissues and subsequent pressure on peripheral nerves could have contributed to the spinal curvature and accompanied abnormal swimming. Conversely, the M. neurotropus infection may have been incidental and the spinal deformity may have actually been due to one of several nonspecific developmental or congenital causes. Further studies on geographic distribution and impact on host fitness will probably determine the importance of this species to fish health.

Received March 1, 2012; accepted June 15, 2012