Communications: Tolerance of Rainbow Trout to Dissolved Oxygen Supplementation and a Yersinia ruckeri Challenge

Abstract

Dissolved oxygen accounted for a significant proportion of the variation in mortality rate of rainbow trout Oncorhynchus mykiss acclimated for 10 weeks to a range of dissolved oxygen levels and challenged with Yersinia ruckeri. Moderate levels of oxygen supersaturation (150%) resulted in greater cumulative mortality (17.9%) among fish exposed to a 0.5-h static bath challenge with Y. ruckeri (1 × 10⁷ colony-forming units/mL). In contrast, hypoxic (70%) and normoxic (100%) levels of dissolved oxygen resulted in 12.8% and 10% cumulative mortality. Higher oxygen levels provided no additional “protection” against infection by Y. ruckeri in this study.     

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.     

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.     

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     

Differing Resistance of Atlantic Salmon Strains and Rainbow Trout to Gyrodactylus salaris Infection

Abstract

Laboratory studies were conducted on the susceptibility of different strains of Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss to infection with the monogenean Gyrodactylus salaris. This parasite, probably originating from the Baltic Sea region, is known to minimally affect Neva River (Baltic Sea) Atlantic salmon. However, following its introduction into Norway, G. salaris has caused severe mortality and morbidity among Norwegian Atlantic salmon, which are considered a highly susceptible strain. The cohabitation experiment included one stock of rainbow trout and four different strains of Atlantic salmon from the Baltic Sea region (Mörrum River, Sweden), Europe (Skjern River, Denmark; Conon River, Scotland), and North America (Bristol Cove River, Canada). Fish were exposed to a Norwegian strain of G. salaris, and parasite population development and distribution were monitored for 7 weeks. Rainbow trout exhibited low susceptibility to G. salaris infection, whereas Conon River and Skjern River Atlantic salmon were highly susceptible and exhibited high mortality rates. Mörrum River Atlantic salmon exhibited intermediate susceptibility and low mortality. Bristol Cove River Atlantic salmon harbored relatively low parasite numbers, but fish mortality was high. Our experiment showed that the Danish Skjern River strain of Atlantic salmon is highly susceptible to G. salaris infection, further supporting the hypothesis that Atlantic Ocean strains are more susceptible to G. salaris infection than are Baltic strains.     

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.     

牛磺酸对虹鳟仔鱼生长、体成分和免疫指标的影响

本试验研究了牛磺酸对虹鳟仔鱼生长、体成分和免疫指标的影响。试验共分4个处理,分别添加0、500、1 000和1 500 mg/kg牛磺酸,试验共进行12周。结果表明:与未添加牛磺酸组相比,12周时添加1 000 mg/kg组体重、体长和平均日增重显著下降(P<0.0 5),饵料系数极显著提高(P<0.0 1)。添加1 5 0 0 mg/kg组体脂肪显著下降(P<0.0 5)。4周时,添加1 000和1 500 mg/kg组提高超氧化物岐化酶(superoxide dismutase,SOD)和总抗氧化能力(P<0.05),但酸性磷酸酶却下降(P<0.05)。添加500(P<0.05)和1 500 mg/kg组(P<0.01)丙二醛也升高。添加牛磺酸各组碱性磷酸酶活性显著提高(P<0.05)。12周龄时,添加1 500 mg/kg组显著降低SOD水平(P<0.05),添加1 000和1 500 mg/kg组总抗氧化能力也显著下降(P<0.05),添加牛磺酸各组丙二醛、碱性磷酸酶和酸性磷酸酶显著下降(P<0.05)。本试验得出,添加500~1 500 mg/kg牛磺酸不能改善虹鳟仔鱼生长性能,但可提高4周时免疫力,但长时间添加(16周)却降低免疫力。     

Effects of Hyperoxic Rearing Water on Blood Hemoglobin and Hematocrit Levels of Rainbow Trout

Abstract

Rainbow trout Oncorhynchus mykiss cultured under hatchery conditions were exposed to hyperoxia by injection of oxygen into the rearing water. In two separate studies of 67 and 73 d, mean hemoglobin of experimental fish reared at oxygen levels averaging 147–220% saturation was 7.8–8.7% lower than that of control fish reared at mean oxygen saturations of 66–74%. Mean hematocrit of experimental fish was also 3.1–5.7% lower than that of control fish. Fish reared in hyperoxic conditions did not differ in growth, survival, or behavior from control fish. Fish exposed to hyperoxia rapidly adjusted hemoglobin and hematocrit to levels similar to those of control fish after oxygen injection was stopped.     

Outer membrane protein profiles of Yersinia ruckeri

The outer membrane protein (OMP) profiles of 135 isolates of Yersinia ruckeri, obtained from nine European countries (100 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), and including four reference strains, were examined by SDS-PAGE. Outer membranes were isolated by selective solubilisation of the cytoplasmic membrane with 0.5% (w/v) sodium N-lauroyl sarcosinate (Sarkosyl). Outer membrane proteins were stable after in vitro passage and there was no variation in OMP profiles due to colony selection. With the exception of a 39.5 kDa peptidoglycan-associated protein there was also no variation at different stages of the growth cycle. The 39.5 kDa protein was not produced during logarithmic growth phase but increased in abundance as the stationary phase progressed. Interstrain variation occurred in the possession of a 36.5 or 38 kDa heat-modifiable protein and in the possession of peptidoglycan-associated proteins in the molecular weight range 36.5 to 40.5 kDa. Based on variation of these proteins five OMP-types, designated OMP-types 1-5, were identified among the 135 isolates examined. Outer membrane protein analysis was demonstrated to be useful in epidemiological studies of Y. ruckeri.     

Early Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV) Infection in Rainbow Trout

Abstract

A series of experiments was carried out with infectious hematopoietic necrosis virus (IHNV; 193-110 isolate) in rainbow trout Oncorhynchus mykiss (weight, ～1.2 g) to determine the duration of the patent period and the timing of onset of the infectious periods. We first attempted to transmit IHNV to recipient fish from infected rainbow trout 2–3 d after they had been exposed. No infection transfer occurred despite high titers (10^4.79 to 10^4.91 plaque-forming units 5–8 d postexposure (dpe). To determine the number of secondary cases produced by one infectious individual, we exposed approximately 50 rainbow trout (weight, ～1.5 g) in each of seven replicate tanks to a donor fish that had been infected with virus by bath exposure 3 d earlier. The prevalence of infection in recipient fish rose from 0.84% at 2 dpe to 7.9% at 6 dpe. Maximum incidence (22 cases) occurred between 2 and 4 dpe. No disease-specific mortalities occurred in recipient fish during the experiment. The titer of virus in both recipient and donor fish increased from 2 to 4 dpe. There was a positive correlation between the level of infection among donors and prevalence values among recipient fish (r ² = 0.60). The level of challenge by one infectious fish under the conditions provided was enough for infection transfer from sick cohabitant to susceptible fish but was not enough for initiation of a full-scale epizootic among recipients.     

A Comparative Investigation of Arcobacter cryaerophilus Infection among Albino Crosses and High- and Low-Body-Weight Rainbow Trout

Abstract

Arcobacter cryaerophilus was isolated from naturally infected rainbow trout Oncorhynchus mykiss, and its pathogenicity was tested by intramuscular injection into healthy 1-year-old high-body-weight (HBW) and low-body-weight (LBW) normally pigmented rainbow trout and albino crosses. Experimental infections caused deaths with gross clinical abnormalities such as exophthalmia, liver damage, bloody hemorrhagic kidney and heart, and swollen intestines. No significant differences in deaths were observed among the three infected fish groups. Hematocrit levels in blood of the experimentally infected HBW rainbow trout were significantly less than in healthy fish. No significant decreases were observed in the serum total protein of both the experimentally infected albino crosses and the high weight groups. Albumin and creatinine concentrations in serum were not significantly different among the three treatments.     

Effects of Acute Stress on Blood Clotting and Yeast Killing by Phagocytes of Rainbow Trout

Abstract

The effects of different levels of acute stress on blood parameters in rainbow trout Oncorhynchus mykiss were examined. Experimental fish were exposed to a combination of stressors, including handling, confinement, and thermal shock, and were bled 30 s to 30 min later. Control fish were sampled quickly without intentional stress. Two activities of blood were investigated: (1) whole-blood clotting (coagulation) times and (2) the ability of heparinized plasma to influence the survival of yeast soaked in this plasma and subsequently exposed to trout phagocytes. The speed of blood clotting increased with increasing levels of stress, whereas blood from many resting fish failed to clot. Yeast incubated in plasma from stressed fish before exposure to pronephric phagocytes experienced a greater reduction in colony-forming units (cfus) than yeast incubated in plasma from resting fish. Different levels of stress did not induce significant differences in reduction of yeast cfus. These data support the idea that acute stress may actually stimulate primary defense mechanisms rather than suppress them. Furthermore, whole-blood clotting times seemed to be reliable indicators of the severity of stressors to which fish were exposed. We postulate that factors appearing in blood very quickly, such as catecholamines and specific plasma proteins, play important roles in both the killing activities of phagocytes and the blood-clotting process.     

Effects of a Prophylactic Chloramine-T Treatment on Growth Performance and Condition Indices of Rainbow Trout

Abstract

Chloramine-T has been commonly used as a therapeutic agent for the treatment of bacterial gill disease and related phenomena in the salmonid aquaculture industry, and it is now commonly used as a prophylactic agent to prevent diseases in commercial salmonid hatcheries. Rainbow trout Oncorhynchus mykiss (average weight 98 g) were exposed to chloramine-T at 10 mg/L for 1 h twice weekly (a commonly used regime in the aquaculture industry) for 11 weeks. Fish were fed ad libitum without feed wastage throughout the 11 -week trial. Body weight, specific growth rate, feed conversion index, and appetite were assessed at the end of weeks 3, 6, 9, and 11. Chloramine-T treatment was not associated with either clinical disease or mortality. However, by the end of the trial, growth (based on body weight) of treated fish was significantly suppressed (δ = 7.3%) compared with control fish. Growth suppression was attributed to a significant reduction (δ = 9.3%) of feed conversion efficiency in treated fish. Based on specific growth rates, chloramine-T had an early negative effect on growth. The effect was diminished in later weeks (although not completely lost), which suggests some degree of compensation by the fish to the chemical agent.     

Regulatory Effects of Water Temperature on Loma salmonae (Microspora) Development in Rainbow Trout

Abstract

Water temperature, a pivotal factor influencing interactions between teleosts and pathogens, was examined to determine its effects on the kinetics of xenoma formation and dissolution subsequent to experimental exposure of rainbow trout Oncorhynchus mykiss to the microsporidian gill pathogen Loma salmonae. The permissive water temperature range in which xenomas developed was between 9° and 20°C. Parasite development was arrested at temperatures outside this range, as indicated by the absence of visible xenomas among exposed fish. In addition, when these trout were subsequently moved to temperatures within the permissive range, xenomas failed to develop. Water temperature, within the permissive range, had no significant effect on either the number of xenomas that formed or the proportion of fish that developed xenomas following gastric intubation with a standard dose of spores. The relationship between water temperature and xenoma onset-time was best described (R ² = 88.3%) by polynomial regression analysis: onset = 320 ? 33.4T + 0.9547T ², where T is temperature (°C). Xenoma onset rate was also described through a modified degree-days model, yielding a predictive equation appropriate for use under conditions of fluctuating temperature. The thermal units, expressed as days × (°C above 7°C) necessary for xenoma onset were 298.6 on average. Xenoma dissolution rates, from the time of onset, also appeared to have a trend; more rapid dissolution occurred as temperatures increased. However, this trend correlated minimally with regression models.     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Whole-Body Assay and Immunohistochemistry

Abstract

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.     

O-serotyping of Yersinia ruckeri with special emphasis on European isolates.

Yersinia ruckeri is the aetiological agent of enteric redmouth disease (ERM), an acute to chronic bacteraemic infection of salmonid fish. The O-serotypes of 127 isolates of Y. ruckeri obtained from Europe (96 isolates), North America (23 isolates), Australia (six isolates) and South Africa (two isolates), as well as four reference strains, were determined by slide agglutination test and microplate agglutination assay. A serotyping scheme is proposed based on heat-stable O-antigens; the serotypes were designated O1, O2, O5, O6 and O7. The proposed scheme is compared to serotyping schemes described by other authors. All five O-serotypes were present in both Europe and North America, whereas only serotype O1 isolates were identified in Australia and South Africa. These findings suggest that European and North American populations of Y. ruckeri are interrelated, thus supporting previous evidence which suggests that the organism was introduced into Europe from North America by the importation of asymptomatic infected carrier fish. Conversely, the results suggest that Australia and South Africa are more isolated from the dissemination of Y. ruckeri. Serotypes O5, O6 and O7 have not previously been recognized in Europe and these findings will have important implications on the diagnosis of ERM and on the vaccination of fish against this disease. It is suggested that the Australian isolate previously described as serotype III is a rough-type mutant and that other isolates described in the literature as serotype III have been incorrectly serotyped and are, in fact, serotype O1. To avoid further confusion it is suggested that the scheme described here be adopted for serological studies of Y. ruckeri.     

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     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Organ Assay and Electron Microscopy

Abstract

Sequential spread of infectious hematopoietic necrosis virus (IHNV) to tissues of rainbow trout Oncorhynchus mykiss was examined following immersion infection with two different isolates of IHNV, a pathogenic strain and a nonpathogenic strain from rainbow trout. Virus strain 193–110 was highly pathogenic to 1-month-old rainbow trout and caused 100% mortality within 13 d, whereas strain RB-76 was much less virulent, causing 50% mortality by the 19th day. Virus titers of 1-month-old fingerling fish dying soon after infection were significantly higher than titers of those dying later. Assays of dissected tissues showed that gills of infected 2-month-old fingerlings contained virus as early as 16 and 20 h postinfection, with definite replication occurring at 48 h. The early presence of the virus in the gills followed shortly by appearance of the virus in the kidneys and spleen indicated that the virus spreads rapidly to the target organs. Virus was detected in many other organs at lower levels on the third day and increased to higher levels during the following days. Heart tissue had high titers later in the infection. When 4-month-old rainbow trout were infected with strain 193–110, the mortality was reduced and delayed, whereas those infected with strain RB-76 produced no mortality. Assays on the day of death of these older fingerlings infected with strain 193–110 revealed that fish dying soon after infection also had higher titers than those dying later. Electron microscopic examination offish organs showed the presence of typical IHNV particles budding off from various tissue cells of affected organs, including gill tissue. The destructive effect of the virus was particularly noticeable in the disarrangement of heart muscle organelles.