Characterization of serum and mucosal antibody responses and relative per cent survival in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and challenge with Flavobacterium psychrophilum

Serum and mucosal antibody responses of juvenile rainbow trout, Oncorhynchus mykiss, were characterized by enzyme‐linked immunosorbent assay (ELISA) following immunization with various preparations of formalin‐killed Flavobacterium psychrophilum cells. The protective nature of these preparations was then determined by immunizing rainbow trout fry and challenging with the bacterium. Juvenile rainbow trout immunized intraperitoneally (i.p.) with formalin‐killed F. psychrophilum emulsified with Freund's complete adjuvant (FCA), and i.p. with formalin‐killed F. psychrophilum either with or without culture supernatant generated significant serum antibody responses by 6 and 9 weeks, respectively. Significant mucosal antibody responses were detected by 9 weeks only in fish immunized i.p. with killed F. psychrophilum/FCA. Following immunization and bacterial challenge of rainbow trout fry, protective immunity was conferred in F. psychrophilum/FCA and saline/FCA groups with relative per cent survival values of up to 83 and 51, respectively. Significant protection was not observed in treatment groups immunized by immersion or i.p. without adjuvant at the challenge doses tested. Results suggest that stimulation of non‐specific immune factors enhances the ability of fish to mount a protective immune response, but specific antibody appears necessary to provide near complete protection. In this study, an ELISA was developed to monitor anti‐F. psychrophilum antibody production in trout. The relationship of such responses to protective immunity suggests that future vaccination strategies against coldwater disease may require stimulation of both the innate and adaptive arms of the immune response.     

Effects of Oral Administration of Specific Antibodies to Rainbow Trout Oncorhynchus mykiss

A new product called oralized fish serum concentrate (OFSC) was evaluated for a possible effect against various bacterial pathogens in rainbow trout. The OFSC produced from immune trout sera was found to contain fully functional antibodies and complement component C3. The antibodies detected in the serum concentrate were specific to Vibrio anguillarum (O1 and O2) and Aeromonas salmonicida , which had been used for vaccination of the fish prior to serum collection. The functionality of the specific antibodies in OFSC was not reduced after 6 wk storage at -20 C, 5 C, and 20 C. The serum was mixed with commercial trout feed and used for feeding rainbow trout fry (first feed period). After oral delivery of OFSC to rainbow trout for 1 mo, samples of gut content and gut tissue contained functional antibodies. In gutted fish no functional antibodies were found. This suggests that antibodies from OFSC are unable to be transferred across the gut wall in a functional state. Oral administration of OFSC did not increase survival of rainbow trout in an immersion challenge with Vibrio anguillarum .     

Antibody response in sea bass (Dicentrarchus labrax L.) in relation to water temperature and oxygenation

The influence of water temperature and water oxygenation on the specific antibody response was evaluated by indirect ELISA in sea bass (Dicentrarchus labrax L.) immunized against human‐γ‐ globulins emulsified in Freund's complete adjuvant. A higher antibody response was observed in fish reared at 24 and 30 °C, than at 12 and 18 °C. For the fish group reared at 24 °C, the immunological observation was carried out until 300 days after immunization. At the end of this period, fish kept at 24 °C still showed immunological competence, although the antibody response began to decrease significantly from 120 days after the immunization. Fish reared under mild hyperoxia and normoxia conditions had higher antibody responses than fish reared under mild hypoxia conditions. Moreover, at the end of the experimental period of 56 days the antibody response of fish reared under hyperoxia conditions was significantly higher than the immune response detected in fish reared under normoxia and hypoxia conditions.     

Vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against cryptobiosis: efficacy of the vaccine in fresh and sea water

Adult rainbow trout, Oncorhynchus mykiss (Walbaum), maintained in either fresh or sea water were vaccinated with a live Cryptobia salmositica vaccine. All vaccinated fish were protected 4 weeks later against the cryptobiosis, while unvaccinated rainbow trout developed the disease (e.g. high parasitaemia and severe anaemia) after challenge with virulent C. salmositica . There was also no disease in vaccinated fish when they were transferred from fresh to sea water immediately after vaccination. Complement fixing antibodies (CFAbs) were detected in vaccinated fish and the CFAbs lysed parasites under in vitro conditions. The antibody titres increased rapidly at one week post-challenge in vaccinated fish in fresh water and vaccinated fish transferred from fresh water to sea water after vaccination. However, the production of CFAbs was delayed by one week in vaccinated fish in sea water and the antibody titre was significantly lower than that in fish maintained in fresh water.     

Challenge studies of European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), with epizootic haematopoietic necrosis virus

A challenge model for comparison of the virulence of epizootic haematopoietic necrosis virus (EHNV) to European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), was tested. The model investigated intraperitoneal (IP), bath and cohabitation routes at 10, 15 and 20 °C for 5–6 g fish and 15 °C for 20 g perch. In the IP challenges of perch, significant mortality occurred at 15 °C and 20 °C. In challenge trials for rainbow trout, significant mortalities were observed in IP and bath challenges at 20 °C. The mortality observed in IP challenged 20 g perch was not significantly different from that recorded for 6 g fish challenged IP. No significant mortality was observed in any other treatment groups. Re-isolation of ranavirus was confirmed by IFAT and was consistently associated with dead or moribund fish in the trial groups challenged with EHNV. The findings indicate that EHNV does not pose a high risk for wild perch and trout populations in Europe by natural exposure. Mortality appears to be primarily a function of environmental factors, with temperature playing an important role, and not just the presence of the virus in the fish.     

Characterization of differentially expressed genes in liver in response to the rearing temperature of rainbow trout Oncorhynchus mykiss and their heritable differences

To characterize thermal-responsive genes in fish, firstly, juvenile rainbow trout were reared in four different temperature conditions (average temperatures were 10, 14, 18, and 22 °C, respectively) and differentially expressed genes were identified. Gene expression in the liver was analyzed by the differential display method, followed by validation using real-time PCR. Subsequently, to examine whether the identified genes show heritable differences, the gene expression levels were compared among juveniles of three genetically distinct lines of rainbow trout (a strain and two closed colonies) by rearing at two different temperature conditions (average 14 and 22 °C). By rearing at 22 °C, growth retardation was observed compared with fish reared at 14 and 18 °C, and six genes were identified as differentially expressed genes in response to the rearing temperature in the gene expression analyses. With the increase in rearing temperature, gene expressions of a complement C1q and two ribosomal proteins were significantly up-regulated. On the other hand, three metabolic genes (betaine homocysteine methyltransferase, triosephosphate isomerase, and glucose-6-phosphatase) were down-regulated, indicating a metabolic depression due to high temperature. In the subsequent analyses, in response to the rearing temperature (14 and 22 °C), there was a trend that the complement C1q and glucose-6-phosphatase genes showed different expression patterns among the three rainbow trout lines, suggesting heritable differences in these genes. Our study provides information on thermal-responsive genes in fish, and we anticipate it will facilitate further investigation in the thermal biology of fish.     

Immunohistochemical evaluation of experimental Vagococcus salmoninarum infection in rainbow trout (Oncorhynchus mykiss,Walbaum 1792)

The aim of this study was to investigate the pathogenesis and histopathological and immunohistochemical findings in rainbow trout (Oncorhynchus mykiss) following experimental vagococcosis. For this purpose, 60 rainbow trout were used. The experimental study used the pathogen Vagococcus salmoninarum. The fish were intraperitoneally (IP) administered with an inoculate containing 0.1 mL of the bacteria, resulting in a dose of 1.2 × 10⁹ cfu mL^?1 per fish. For histopathological observations, tissue samples were taken from fish that died during the experiment and fish that survived until the end of the trial (60th day). All the tissue samples were immunohistochemically stained by the avidin–biotin–peroxidase complex and immunofluorescence methods using polyclonal antibody to detect V. salmoninarum antigens. In immunoperoxidase staining, positive reactions to bacterial antigens were most commonly seen in the kidney, heart and liver. In the immunofluorescence analysis, the distribution of antigens in the tissue and organs was similar to that observed with the immunoperoxidase staining. The results reveal an important correlation between histochemical and immunohistochemical staining in demonstrating the distribution of V. salmoninarum antigens in the affected tissues.     

Cellular immune response in rainbow trout Salmo gairdneri Richardson to Yersinia ruckeri O-antigen monitored by the passive haemolytic plaque assay test

Abstract. The specificity and kinetics of the immune response of rainbow trout ( Salmo gairdneri ) to single injections of an O-antigen extracted from the bacterial pathogen Yersinia ruckeri , which causes enteric redmouth in fish, were investigated by the passive haemolytic plaque assay and serum antibody quantitation. Doses ranging from 5 ng to 500 mg in 10-fold increments were injected intraperitoneally into groups of trout held at 17 × 1°5°C. The occurrence of plaque forming cells (PFC) and humoral antibody was followed for 35 days after injection. Trout gave an immune response to doses of 500 ng and above. Seven days after injection no humoral antibody was detected, but PFC were found in the spleen. The maximum PFC numbers occurred 11 days after injection. On day 21, few PFC were found, whereas serum antibody titres were highest. The antibody from immunized trout showed little or no cross-reactions with sheep red blood cells passively labelled With antigens from other fish pathogens.     

Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) skin and gill

Antimicrobial polypeptides (AMPPs) are increasingly recognized as a critical component of innate host defense. Among the AMPPs, polypeptides related to histones have been identified from many animals. Using peptide mapping, we further confirm the identity of two histone-like proteins from fish as members of the H2B (sunshine bass) and H1 (rainbow trout) histone groups. We optimized the conditions for measuring rainbow trout HLP-1/H2B via sandwich ELISA. We used two antibodies, one to the amino terminus and one to the carboxyl terminus, of trout histone H2B, as the capture antibodies, and we used peroxidase-labeled antibody raised to calf histone H2B as the secondary antibody. Specificity of the detecting antibody was confirmed by specific reactivity with histone H2B in tissue extracts via western blotting. The test was reproducible and capable of detecting as little as 5 ng of histone H2B (0.05 μg/ml). Histone H2B levels expressed in gill tissue of juvenile, healthy rainbow trout were well within concentrations that are lethal to important fish pathogens. However, there was a significant, age (size)-dependent decline in histone H2B concentrations as fish matured, until levels became virtually undetectable in market-size fish. In contrast, levels in skin appeared to remain high and unchanged in small versus large fish. Antibacterial activity in skin and gill tissues was closely correlated with histone H2B concentration measured via ELISA, which supports our previous finding that histones are the major AMPPs in rainbow trout skin and gill.     

Humoral immune response of turbot, Scophthalmus maximus (L.), to antigens from Tetramicra brevifilum Matthews & Matthews, 1980 (Microspora)

Abstract. The humoral immune response of turbot, Scophthalmus maximus (L.), to antigens from the microsporean parasite Tetramicra brevifilum Matthews & Matthews, 1980, was studied. Thirty days after intraperitoneal immunization with whole T. brevifilum spores in Freund's complete adjuvant, double indirect ELISA indicated that initial production of antibodies to parasite surface antigens was considerably higher than production of antibodies to the antigens contained in a crude extract (CE) of spores. Following re-immunization without adjuvant on day 30, levels of antibodies to surface antigens gradually declined, whilst levels of antibodies to CE antigens increased. The antibody response of intraperitoneally immunized fish was characterized by Western blotting of total soluble antigens obtained by heating and reduction of T. brevifilum spores at 95–100°C in Tris-HCl buffer containing SDS and dithiothreitol: a series of bands with molecular weights between 20 and 53 kDa was recognized by immunized turbot sera. Four additional bands (with molecular weights between 15 and 18kdA) were recognized by serum from re-immunized fish. ELISA studies of sera from naturally infected fish revealed a surprisingly low incidence of strong T. brevifilum seropositivity (61% individuals); antibodies to surface antigens predominated in seropositive individuals. The low background response levels and high sensitivity of the ELISA used in this study indicate that the assay is of value for the monitoring of serum antibody levels in turbot. However, given the relatively low seropositivities observed in naturally infected turbot, particularly to CE antigens, the use of anti- T. brevifilum serum antibody levels for the diagnosis of infection by this parasite may lead to false negative results.     

Feeding increasing levels of corn gluten meal induces suboptimal muscle pigmentation of rainbow trout (Oncorhynchus mykiss)

A 24‐week growth trial was conducted to evaluate the effects of feeding levels of corn gluten meal (CGM) on growth performance and pigment deposition in the muscle of rainbow trout (Oncorhynchus mykiss). Three isonitrogenous and isoenergetic (digestible energy basis) experimental diets were formulated to contain increasing levels of CGM (0%, 9% and 18%) and 50 mg kg^?1 of astaxanthin. Each diet was fed in triplicate to groups of 75 fish (initial average body weight = 549 g fish^?1) reared at 8.5°C. The inclusion of CGM did not significantly (P > 0.05) affect final body weight, thermal growth efficiency (TGC) or feed efficiency. Carotenoid concentration determined by liquid chromatography showed a significant (P < 0.05) linear reduction in the concentration of one astaxanthin isomer, all‐trans astaxanthin and all‐trans lutein in the muscle of fish in response to increasing levels of CGM. Tristimulus colour analysis of the muscle showed a significant (P < 0.05) linear reduction in a* (redness) and C*ab (chroma). Salmofan^? score showed a significant (P < 0.05) linear and quadratic reduction in response to increasing levels of CGM. In conclusion, the inclusion of CGM up to 18% does not significantly impact growth performance of rainbow trout. However, the concentration of all‐trans astaxanthin as well as the expression of important colour attributes of the muscle can be negatively affected at levels exceeding 9% of CGM in the diet. More research on this topic is needed to discern the mechanism(s) behind the negative effects of dietary CGM and/or its intrinsic yellow pigments on muscle pigmentation of rainbow trout.     

Spatial patterns in fish biomass and relative trophic level abundance in a wastewater enriched river

Abstract –  It is generally accepted that nutrient enrichment of aquatic systems will lead to increased production at the top trophic level (fish). We found that in the wastewater enriched Bow River, Alberta rainbow trout ( Oncorhynchus mykiss ) biomass increased over 25-fold, and brown trout ( Salmo trutta ) biomass increased 5-fold, however total sportfish biomass did not increase below the nutrient input point source. This was due to a dramatic downstream decrease in mountain whitefish ( Prosopium williamsoni ) biomass to 2% of the average biomass upstream of the municipal effluent source. The spatial pattern over a 177-km river section encompassing the city of Calgary, showed that the increase in trout abundance approximately tracked the expected nutrient concentrations in the river, but with a downstream lag of 20–30 km. Mountain whitefish biomass over the 177 km was inversely related to the dominant trout species, rainbow trout. Invertebrate abundance, macrophyte biomass and phytoplankton biomass all increased below the wastewater treatment plant outfalls. However, periphyton data were highly variable and showed no response. We propose several hypotheses as regards the factors that may have led to the decrease in mountain whitefish, based on the data from all trophic levels and the spatial pattern for fish biomass. Proposed factors influencing the mountain whitefish decline were; altered competitive ability because of macrophyte abundance, ammonia toxicity and barriers to movement (weirs).     

Comparison of brown trout (Salmo trutta) reared in fresh water and sea water to freshwater rainbow trout (Oncorhynchus mykiss): II. Phosphorus balance

Brown trout and rainbow trout (average weight 100 g) were reared in fresh water at 12 °C under the same conditions before transferring brown trout to sea water, in order to compare phosphorus utilisation in both species. Apparent phosphorus availability, orthophosphate excretion and phosphorus accretion in the fish were directly determined. Thus, actual phosphorus mass balance was built. Rainbow trout raised in fresh water had a higher phosphorus retention coefficient (maximum 50 %) than brown trout reared in fresh water (maximum 45 %). Transferring brown trout to sea water induced a reduction in phosphorus retention (maximum 39 %). Orthophosphate excretion, ranging 7–20 mg phosphorus per kg wet weight per day, represented 10–20 % of ingested phosphorus. Phosphorus availability was lower in brown trout raised in sea water (65 %) than brown trout raised in fresh water (76 %). Phosphorus balance measurements showed that 90 to 98 % of phosphorus flow could be accounted for.     

Susceptibility of selected freshwater fish species to a UK Lactococcus garvieae isolate

Gram-positive cocci recovered from diseased rainbow trout from a farm in England were characterized by different methods, including pulsed field gel electrophoresis, as virulent Lactococcus garvieae serogroup 2 (pulsotype A1). Groups of rainbow trout were kept at a range of temperatures and injected intraperitoneally (i.p.) with one of the UK isolates, L. garvieae 00021. The 18 °C and 16 °C groups showed 67% and 28% mortality, respectively, by day 27 post-injection. Fish kept at 14 °C or lower were less susceptible (≤3% mortality). Raising the temperature of all groups to 18 °C at day 27 post-injection did not result in recurrence of the disease, even though viable bacteria were recovered from all groups 42 days later. Grayling were highly susceptible, with 65% mortalities when challenged with 200 colony forming unit fish⁻¹ by i.p. injection and 37% mortalities when exposed to effluent water from tanks containing affected rainbow trout. Other fish species tested, Atlantic salmon, brown trout and seven cyprinid species, were less susceptible. Viable L. garvieae was isolated from the internal organs of all species tested at the end of the trials, suggesting that they may pose a threat as possible carriers to susceptible farmed and wild fish.     

Subcellular components of Vibrio harveyi and probiotics induce immune responses in rainbow trout, Oncorhynchus mykiss (Walbaum), against V. harveyi

Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (∼93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi . Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of ∼93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens.     

Detection of Renibacterium salmoninarum in salmonid kidney samples: a comparison of results using double-sandwich ELISA and isolation on selective medium

Abstract A total of 1239 kidney samples from four species of salmonid fish, Atlantic salmon, Salmo salar L., brown trout, Salmo trutta L., Arctic charr, Salvelinus alpinus (L.), and rainbow trout, Oncorhynchus mykiss (Walbaum), were screened for Renibacterium salmoninarum using double-sandwich ELISA and bacterial isolation. For bacterial isolation, samples were homogenized, washed, plated onto S-KDM and incubated for 12 weeks. Samples for ELISA were kept frozen until tested. After thawing, 25% homogenates in PBS were heated at 100°C for 15 min in the presence (2.5% v/v) of HemoDe solvent (terpene and butylated hydroxyanisole) and then centrifuged. The supernatant was tested with polyclonal antibodies against whole bacterium in a double-sandwich ELISA. In seven out of 12 groups tested, all samples were negative in both tests. Positive ELISA results occurred in five groups. Renibacterium salmoninarum was isolated on SKDM from samples in four out of these five groups. The ELISA test gave significantly higher numbers of positive samples in three out of the four groups showing positive results in both tests.     

Soybean meal-induced enteritis in Atlantic salmon (Salmo salar L.) at different temperatures

This study evaluates the effect of temperature on the development of intestinal disorders when Atlantic salmon are fed soybean meal (SBM). In this study 20% of the dietary fishmeal was replaced by solvent-extracted Hipro SBM. Atlantic salmon reared at two different water temperatures (8 °C and 12 °C), were fed a control diet and an experimental diet for 20 days. Samples were taken at days 7 and 20. The extent of the morphological changes was assessed using a semi-quantitative scoring system developed for this purpose. The study demonstrates that enteritis is affected by temperature. The intestinal disorders were more severe in fish reared at 12 °C compared with those reared at 8 °C. It can be concluded from this study that temperature changes the speed but not the type of SBM-induced enteritis expressed as a delay on the response when Atlantic salmon are kept at lower temperatures.     

Designing an in‐house ELISA to detect antibody against viral haemorrhagic septicaemia virus using recombinant N protein in Iranian farmed rainbow trout (Oncorhynchus mykiss)

Viral haemorrhagic septicaemia (VHS) is one of the most important viral diseases in rainbow trout that has caused great losses to Iranian rainbow trout aquaculture industry in the last 3 years. Therefore, rapid and reliable diagnosis of VHS virus infections is of great importance. An enzyme linked immunosorbent assay (ELISA) method was performed to study serum antibodies against viral haemorrhagic septicaemia virus (VHSV) using recombinant fragments of their N protein. For this purpose, the virus was first isolated from an infected farm. A part of the nucleocapsid (1–505 bp) gene was amplified by RT‐PCR using specific primers. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of Escherichia coli. Then, recombinant plasmids were tested for protein expression in E. coli Rosetta strain. SDS‐PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 61 KDa. Analysis of trout serum samples from seven previously infected farms and two VHS free farms showed that the designed ELISA method was effective in diagnosing the infected fish. The results revealed that the developed serological assay using designed ELISA based on recombinant protein (N) has the potential to be used in monitoring studies and to determine the prevalence of VHS in rainbow trout farms. The present data allow evaluating the levels of nonneutralizing antibodies without crude virus preparations.     

Normal and aberrant tissue distribution of Loma salmonae (Microspora) within rainbow trout, Oncorhynchus mykiss (Walbaum), following experimental infection at water temperatures within and outside of the xenoma-expression temperature boundaries

Temperatures above 20 °C or below 9 °C interrupt the life cycle of the gill intracellular microsporidian parasite Loma salmonae (Microspora) prior to sporogony, inhibiting the production of xenomas. This study intended to characterize this life-cycle failure. Juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), were experimentally infected with L. salmonae spores, and the effect of water temperature on the progress of infection, as determined by polymerase chain reaction, was compared for fish held at water temperatures of 5, 15 and 21 °C. At 15 °C, parasite DNA was first detected in the heart (3 days post-exposure [PE]), and then in the gills and spleen (2 weeks PE). Branchial xenomas developed by week 4 PE. In contrast, at 5 °C, the arrival of the parasite in the heart was delayed until 7 days PE. However, even though parasite DNA was detected in the gills at 7 days PE, xenomas failed to form in the gill, and by week 4 PE, parasite DNA was no longer detected. In fish held at 21 °C, parasite DNA was detected in the heart, gills and spleen by 3 days post-infection, and similar results were observed at 7 days PE. Xenomas also failed to form in these fish and parasite DNA was no longer detected by week 2 PE. Within the range of temperatures tested in this study, spore germination and delivery of their DNA into the host through the intestinal wall was not blocked by temperature. At 5 or 21 °C, migration to the heart and gills occurred, but at aberrant periods of time. The normal life cycle of L. salmonae may depend on the completion of relatively lengthy, but yet unknown, stages of development within the heart, prior to reaching the gill. This development may be adversely affected by temperature, and explain the temperature limits of this parasite.