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.     

Edwardsiellosis in farmed rainbow trout (Oncorhynchus mykiss)

Edwardsiella tarda was isolated from naturally infected rainbow trout (Oncorhynchus mykiss) in raceway culture on a commercial fish farm where the fish were kept at a density of 110 kg m^?3 and at a water temperature of 14°C and with a photoperiod of 13 h light:11 h dark. The clinical signs of diseased fish (150 ± 20 mm standard length) were anorexia and lethargy. The most striking lesions in the fish were in the liver. There were hyperaemia and haemorrhages; on histopathological examination, the liver displayed inflammatory infiltrate in portal area, focal necrosis, dilatation of blood sinus and activation of sinusoidal cells. Infection experiments, performed 2 years after isolation of the original culture of E. tarda, were carried out under laboratory conditions at water temperatures of 15, 18 or 24°C. All experimental fish (common carp, Prussian carp, tench), intraperitoneally injected with 8 × 10⁶ cells, demonstrated a total resistance to E. tarda.     

Transfer of toxaphene and chlordane into farmed rainbow trout, Oncorhynchus mykiss (Walbaum) via feed

The study was carried out to quantitate the transfer of toxaphene and chlordane compounds from commercial fish feed into the edible part of rainbow trout, Oncorhynchus mykiss (Walbaum) under normal rearing conditions. Trout were fed with unspiked high energy feed for salmon (fat content 26–30%) over a period of 19 months. The average weight of trout increased from 10 g to more than 2092 g, reaching sizes of 51 cm length. Considerable amounts of toxaphene and chlordane residues were transferred from fish feed into trout muscle. Toxaphene concentrations increased up to 8.6 µg (Σ toxaphene indicator compounds 1–3) kg^?1 wet weight (w.w.) and chlordane reached 5.3 µg Σoxy‐, trans‐, cis‐chlordane + t‐nonachlor kg^?1 w.w. Results are also discussed on the contaminant levels based on the fat content and the effect of sexual maturation is considered in this study. The data allow the establishment of transfer rates for toxaphene and chlordane congeners from high energy diet into the edible part of farmed rainbow trout.     

Quantitative expression (Walbaum) of immunological factors in rainbow trout, Oncorhynchus mykiss (Walbaum), after infection with either Flavobacterium psychrophilum, Aeromonas salmonicida, or infectious haematopoietic necrosis virus

To further enhance our understanding of immunological gene expression in rainbow trout, Oncorhynchus mykiss, after infection with naturally occurring pathogens, a series of probes and primers were developed for the quantification of immune factors. Separate groups of specific-pathogen-free rainbow trout were infected with either Flavobacterium psychrophilum, Aeromonas salmonicida or infectious haematopoietic necrosis virus (IHNV). Three different concentrations of each pathogen were used and samples from infected and mock-infected fish were taken at either 1 or 5 days after infection. Ten fish were sampled at each time point for individual sections of liver, spleen and head kidney. Organ specimens from five of the fish were used to re-isolate and quantify the pathogen at the time the samples were taken. Total RNA was extracted from the organs of the remaining five animals. Using real-time polymerase chain reaction with fluorescent-labelled probes, the RNA from these organs was examined for the level of expression of the following immunological factors; an interferon related protein (MX-1), interleukin-8 (IL-8), the cytotoxic T-cell marker CD-8 and complement factor C3 (C3). They were also measured for the level of beta-actin, which was used as a standardization control for cellular RNA expression. Infection with IHNV produced the greatest change in expression level for all the immunological related factors examined in this study. IHNV elicited the best dose response profile, which was typically seen at 5-days post-infection for MX-1, C3, IL-8 and CD-8. Infection with A. salmonicida and F. psychrophilum showed elevated, but variable expression levels for several of the genes tested.     

Association between MHC II beta chain gene polymorphisms and resistance to infectious haematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792)

Infectious haematopoietic necrosis virus (IHNV) is a serious pathogenic threat to rainbow trout (Oncorhynchus mykiss) and other salmonids. Major histocompatibility complex (MHC) polymorphisms have been closely linked to disease susceptibility. In this study, polymerase chain reaction‐single‐strand conformation polymorphism, cloning and sequencing were used to examine MHC class II β chain gene (DAB) polymorphisms and analyse their association with IHNV in rainbow trout. The DAB exon 2 was amplified from 104 resistant and 91 susceptible fish. Forty‐five different alleles were identified, 30 of which were novel. Individuals possessed between one and four alleles, indicating that the genome harbours at least two closely related DAB exon 2 loci. The percentages of amino acid mutations in the peptide binding region (PBR) and non‐PBR were 66.67% and 45.28% respectively. The rate of non‐synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions, suggesting the existence of balancing selection for maintenance of polymorphisms at the DAB exon 2 locus. The alleles Onmy‐DAB*0201 (P < 0.001), Onmy‐DAB*0904 (P < 0.001) and Onmy‐DAB*1102 (P < 0.01) were associated with susceptibility to IHNV, while Onmy‐DAB*0103, Onmy‐DAB*0601 and Onmy‐DAB*1402 were associated with resistance (P < 0.001). These alleles could be used as molecular markers for IHNV resistance breeding in rainbow trout.     

Oxygen-induced gas bubble disease in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. An Aquatector oxygen injection unit was used to supersaturate a hatchery water supply to 200% oxygen saturation (18–20mg/l) and increase the total gas pressure to 120% of saturation. Nitrogen saturation was reduced to near 100%. Rainbow trout, Oncorhynchus mykiss (Walbaum), held in the treated water developed signs of gas bubble disease in 4 days, and 50% died within 20 days. We demonstrated that supersaturated total gas pressure due to excessive oxygen saturation causes gas bubble disease in the absence of supersaturated nitrogen gas. It is recommended that users of oxygen injection systems closely adjust the amount of oxygen added to the water to keep the total gas pressure near saturation.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum)

The influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum), was studied experimentally in the laboratory and at a fish farm. In experiment I, the effect of parasitic infection on columnaris disease was studied using F. columnare carrier fish. The fish were exposed to Diplostomum spathaceum cercariae and a set of other stressors in order to induce clinical columnaris infection. Parasitic infection and other stressors failed to induce the disease. Disease occurred when the fish were challenged with F. columnare, but D. spathaceum infection did not enhance the severity of the infection. In experiment II, the influence of rearing density and water temperature was studied. Overall mortality was highest in fish at normal rearing density with high temperature (+23 degrees C). At low temperature (+18 degrees C) mortality was not affected by rearing density, but the transmission of columnaris disease was faster at normal rearing density at both temperatures. This supports the view that reduction of fish density could be used in prevention of columnaris disease especially if water temperature is high. Because the lower rearing density can also decrease the transmission of ectoparasites and penetrating endoparasites, it could be an efficient tool in ecological disease management.     

Immunogenicity of amoebic antigens in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Rainbow trout developed a humoral immune response against numerous antigens of sonicated amoebae which were emulsified with Freund's complete adjuvant and injected into the peritoneum. The amoebae were cultured from the gills of Atlantic salmon, Salmo salar L., affected by amoebic gill disease. Antibodies in fish sera were detected by both enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Non-spedfie reactivity in fish serum against Escherichia coli, the bacterium used in co-cultivation of amoebae in vitro, was removed by immunoadsorption. Results obtained using ELISA and immunoblotting were comparable and indicated no significant difference in response to immunization with 10, 50 or 250 μg of sonieatcd amoebic protein. Amoebae contained immunogenie components of > 100, 100, 89, 49, 37 and 34kDa.     

Cellular components of probiotics control Yersinia ruckeri infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss , conferred protection against a new biogroup of Yersinia ruckeri . Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80–100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with ' Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.     

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.