Outbreak of viral haemorrhagic septicaemia (VHS) in seawater-farmed rainbow trout in Norway caused by VHS virus Genotype III.

We describe the finding of a novel viral haemorrhagic septicaemia virus (VHSV) Genotype III strain that caused disease of both a neurological and septicaemic nature in seawater-farmed rainbow trout Oncorhynchus mykiss in Storfjorden, Norway. In November 2007, an outbreak of VHS associated with slightly elevated mortality was confirmed at a seawater site rearing rainbow trout (90 to 440 g). Within 3 to 4 mo, the disease was recognised in 3 neighbouring sea sites with ongrowing rainbow trout. The clinical, gross pathological and histopathological findings were in accordance with VHS, and the diagnosis was confirmed by the detection of VHSV in brain and internal tissues by immunohistochemistry, cell culture and reverse transcriptase PCR (RT-PCR). Sequence analysis of the G-gene revealed that the isolated virus clustered with VHSV Genotype III and that the Norwegian isolate represents a unique strain of VHSV. The pathogenicity of the virus strain to rainbow trout and Atlantic salmon Salmo salar was examined using infection experiments. In immersion trials, the Norwegian isolate produced a cumulative mortality of 70% in rainbow trout, while nearly 100% mortality was obtained after intraperitoneal injection of the virus. For Atlantic salmon, no mortality was observed in immersion trials, whereas 52% mortality was observed after intraperitoneal injection. The Norwegian isolate thus represents the first VHSV of Genotype III pathogenic to rainbow trout.

Evidence for in vivo hepatic uptake of a galactose-terminated glycoprotein in fish (Salmo alpinus L.).

125I-labelled asialofetuin injected intravenously into chars (Salmo alpinus L.) was cleared from the blood with a half-time of approx. 60 min. The injected asialofetuin was found to be accumulating in the liver when different organs were dissected out at various time points after the injection. 125I-labelled fetuin was not concentrated in the liver, and the uptake of 125I-labelled asialofetuin was almost completely inhibited by simultaneously injecting an excess unlabelled asialofetuin, indicating a specific uptake mechanism for the galactose-terminated glycoprotein in char liver. Denatured human serum albumin was not taken up by the liver, but was recovered in the kidneys, suggesting that the liver in char is devoid of macrophages. It therefore seems reasonable to assume that the injected 125I-labelled asialofetuin is taken up by the parenchymal cells of the char liver.

Isolation of pronephros cells which endocytose chemically modified proteins in the rainbow trout.

Modified serum albumin is cleared from the blood by kidney cells in salmonid fishes. The present study deals with isolation of cells from pronephros which endocytose formaldehyde-treated human serum albumin (fHSA). Radioactively labelled fHSA or dinitrophenyl-conjugated albumin (DNP-HSA) were injected intravenously into rainbow trouts. Pronephros cells, containing the endocytosed protein, were isolated and further separated by centrifugal elutriation and density-gradient centrifugation. Most of the radioactive protein was elutriated together with small cells. After centrifuging the cells through a Percoll density gradient, radioactive protein was located in cells recovered in the upper part of the gradient. In mammals, fHSA and other modified proteins are mainly taken up by sinusoidal endothelial cells in the liver via a "scavenger receptor"0. Our results suggest that a comparable function in salmonids is located in a subpopulation of relatively small cells in kidney tissue, possibly sinusoidal lining cells. The separation techniques used seemed to be suitable for isolation of different populations of pronephros cells.

In vitro degradation of endocytosed protein in pronephros cells of the char (Salmo alpinus L.). The effects of temperature and inhibitors.

In vitro degradation of 125I-formaldehyde treated human serum albumin (fHSA) in char (Salmo alpinus L.) pronephros cells was studied. The labelled protein was injected intravenously and after various intervals of time pronephros cells were isolated and degradation of internalized protein was measured. No degradation could be observed in cells isolated 30 min after injection. The degradation was very effective in cells isolated at later time points (60-90 min); as much as 65% of the initial cell associated labelled protein was degraded during 90 min incubation at 15 degrees C. The effect of temperature on degradation showed a linear course in the temperature range 0-20 degrees C when plotted in an Arrhenius plot. Monensin and ammonium ions inhibited degradation while colchicine had no effect when pronephros cells were isolated 75 min after the injection.

Endocytosis of a mannose-terminated glycoprotein and formaldehyde-treated human serum albumin in liver and kidney cells from fish (Salmo alpinus L.).

The uptake and degradation of a mannose-terminated glycoprotein, yeast invertase, in char (Salmo alpinus L.) tissue was studied after intravenously injection of the 125I-labelled protein. 125I-labelled formaldehyde-treated human serum albumin (fHSA) and native HSA was also injected for comparison. Labelled invertase was rapidly cleared from blood and at about the same rate as labelled fHSA (at 8 degrees C). Approximately 50% of the initial concentration remained in blood 15 min after the injection of the ligands. Acid soluble degradation products appeared in the circulation about 60 min after the injection of the proteins. 125I-labelled invertase was recovered in the liver, pronephros and kidney. The clearance of labelled invertase from blood and the uptake in the organs were inhibited by co-injection of excess unlabelled invertase. fHSA was taken up in the pronephros and kidney tissue, while HSA was not taken up in any organs. In vitro degradation of the labelled ligands was studied in isolated pronephros cells, which had taken up the proteins in vivo. The degradation of invertase in isolated cells was partly inhibited by ammonium chloride. Ammonium chloride and chloroquine inhibited degradation of fHSA, but not leupeptin. These results together suggest that invertase and fHSA were taken up in the organs described by the receptor-mediated endocytosis. The degradation was partly or wholly lysosomal.

Spleen and kidney of Atlantic salmon (Salmo salar L.) show histochemical changes early in the course of experimentally induced infectious salmon anaemia (ISA).

Infectious salmon anaemia (ISA) is a disease of farmed Atlantic salmon (Salmo salar L.) in Norway that affects both erythrocytic and leucocytic cells. Both cell types are possible target cells for the aetiological ISA agent, which is probably a virus. In the present study the distribution and phenotype of leucocyte populations in the spleen and head kidney of Atlantic salmon that were developing ISA have been examined. Frozen tissues were collected from fish at various times after inoculation with ISA-infective material. Immune and enzyme histochemical techniques were used to characterise the response of leucocyte populations. Acid phosphatase positive macrophages predominantly in the red pulp of the spleen appeared to have engulfed erythrocytes at day 4 after infection. Evidence of degradation products of phagocytosed erythrocytes was present in macrophages in red pulp of the spleen at day 7 after infection, in addition to the usual site of erythrophagocytosis in melanomacrophage accumulations. Signs of erythrophagocytosis were not found in the head or body portions of the kidney. The activation of macrophages in the spleen at day 7 was suggested by decreased reactivity for the enzyme 5' nucleotidase. From day 7, clusters of immunoglobulin positive (Ig +) cells were present in the head kidney, while from day 11, the ellipsoids of the spleen showed reactivity for Ig and complement factor C3. These observations are discussed in relation to early immunoglobulin production and possible immune complex trapping. The present results suggest that the leucocyte populations in Atlantic salmon respond to ISA infection through macrophage activation and the initiation of an immune response.

Desaturation and chain elongation of essential fatty acids in isolated liver cells from rat and rainbow trout.

Isolated hepatocytes from rainbow trout and rat were incubated with 14C-labeled linoleic acid, linolenic acid, dihomogammalinolenic acid or eicosapentaenoic acid. The most striking difference in the desaturase activity was the lower level of delta 5 desaturase in trout than in rat. No delta 4 desaturation of 22:4(n-6) to 22:5(n-6) was observed in either of the two species, while the conversion of 22:5(n-3) to 22:6(n-3) was significant in both groups and highest in rainbow trout. The chain-elongating activity was remarkably similar in the two species, except for the "dead-end" elongation which was distinctly more important in fish.

Characterization and applications of a monoclonal antibody against infectious salmon anaemia virus.

The preparation of the first monoclonal antibody (MAb) against the orthomyxovirus-like infectious salmon anaemia (ISA) virus is described. Characterization of the MAb included isotyping, enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining of virus infected cell cultures (SHK-1 cells), immunoelectron microscopy (IEM) of negatively stained virus preparations, virus neutralization assay and haemagglutination inhibition assay. The MAb reacted with ISA virus preparations both with immunofluorescent staining and in ELISA. No reactions were observed in cell cultures infected with other viruses infecting salmonids including infectious pancreatic necrosis (IPN) virus, viral haemorrhagic septicaemia (VHS) virus and infectious haematopoietic necrosis (IHN) virus. The MAb was also shown to neutralize ISA virus infection in cell cultures and to inhibit the haemagglutination reaction. IEM demonstrated binding to the surface of negatively stained ISA virions. Thus, it is concluded that the MAb binds to the haemagglutinin on the virion surface. Furthermore, using immunofluorescent staining of virus infected cell cultures, reactivity against all the 13 ISA virus strains currently available was demonstrated. Using the MAb, a simple, rapid direct immunofluorescent assay for ISA virus detection and titration in 96-well tissue culture plates was developed. Infectivity titrations by this method correlated well with titration by cytopathic effects. The reliability of the assay was demonstrated by close agreement in virus infectivity titres among different assays for the same virus that were performed on the same day and on different days. A method for detection of viral antigen in cryosections from ISA diseased fish is also reported that may prove useful for the diagnosis and control of ISA.

Acute and persistent experimental nodavirus infection in spotted wolffish Anarhichas minor.

Spotted wolffish Anarhichas minor (approx. 0.7 g) were found to be susceptible to infection with a nodavirus isolated from Atlantic halibut (AHNV) by bath-challenge. During the acute stage of infection, 4 to 8 wk post-challenge, viral encephalopathy and retinopathy (VER) were diagnosed by histopathology, immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR). Accumulated mortality was 52% in the challenged group. The surviving fish were sampled 16 wk post-challenge, by which time they had grown to approximately 17 g. No clinical signs of VER were observed in these fish. RT-PCR examination revealed the presence of nodavirus in several organs of the survivors, but no immunopositive cells were detected by IHC. Nodavirus was reisolated from fish at the last sampling in SSN-1 cells, showing that nodavirus retains virulence in persistently infected wolffish for at least 16 wk post-bath-challenge.

Isolation in cell culture of nodavirus from farmed Atlantic halibut Hippoglossus hippoglossus in Norway.

Isolation in cell culture of nodavirus from Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) is described. The cell line SSN-1 was inoculated with tissue material from affected juveniles (60 d after first feeding). Extensive cytopathic effects (CPE) developed approximately 5 d after inoculation, and were also observed after several passages in the same cell line. Cells from infected cultures showed reactivity with an antiserum against sea bass Dicentrarchus labrax nodavirus in an indirect immunofluorescence test. Analysis of infected cells with reverse transcriptase-polymerase chain reaction (RT-PCR) resulted in a product of the predicted size using primers specific for striped jack Pseudocaranx dentex nodavirus. Electron micrographs of infected SSN-1 cells demonstrated virus particles that were approximately less than 30 nm. Challenge of Atlantic halibut larvae (4 d post-hatching) with supernatants from infected SSN-1 cells resulted in development of VER as verified by immunohistochemistry performed on larvae sampled from Day 9 after challenge. The present results show that a nodavirus from Atlantic halibut has been isolated using the SSN-1 cell line and that virus propagated in cell culture retained virulence.

Experimental infection of Atlantic halibut Hippoglossus hippoglossus with nodavirus: tissue distribution and immune response.

Atlantic halibut Hippoglossus hippoglossus, age 8 mo and weighing 20 g, were challenged by either intraperitoneal injection (i.p.) or by bath exposure using nodavirus isolated from Atlantic halibut. Fish were sampled at intervals over a 41 d period, starting on Day 5 post-challenge. Although no clinical disease or mortality was recorded, the data show that nodavirus did successfully propagate in i.p.-challenged fish. Using conventional end-point reverse transcription (RT)-PCR, nodavirus was detected in the kidney of all examined i.p.-challenged fish, and further in the head, heart, liver and posterior intestine of most of these individuals. Quantitative real-time RT-PCR revealed that the amount of virus in head samples from the i.p.-challenged group increased during the experiment. The presence of nodavirus in nervous tissue of i.p.-challenged fish was detected by immunohistochemistry from Day 13 post-challenge. In the retina, virus positive cells were found adjacent to the circumferential germinal zone at the ciliary margin towards the iris. In the brain, a few positive cells were detected in the tectum opticum. An ELISA was developed to detect anti-nodavirus activity in plasma. The method included an optimized coating procedure, which allowed the use of non-purified nodavirus as the coating antigen in a simple indirect ELISA. An anti-nodavirus antibody response was detected from Day 19 post-challenge in i.p.-challenged fish, while a response was not detected in the bath-challenged or control fish. This experiment demonstrates a subclinical nodavirus infection in Atlantic halibut at a post-juvenile stage induced by i.p. injection of virus.

First identification of infectious salmon anaemia virus in North America with haemorrhagic kidney syndrome.

Haemorrhagic kidney syndrome (HKS), a serious disease affecting Atlantic salmon on the east coast of Canada, was determined to be caused by infectious salmon anaemia virus (ISAV) through the isolation of the pathogen on the SHK-1 (salmon head kidney) cell line and confirmation by ISAV-specific immunofluorescent antibody test (IFAT) and reverse transcriptase polymerase chain reaction (RT-PCR). In addition, the defining histopathology of HKS could be reproduced following the injection of material that rendered challenged fish ISAV-positive by cell culture in the absence of any other detectable pathogen. Preliminary nucleotide sequence comparison does not suggest any direct epidemiological connection between the Canadian and Norwegian isolates.

Infectious salmon anaemia virus (ISAV) in experimentally challenged Atlantic cod (Gadus morhua).

Juvenile Atlantic cod, Gadus morhua, (6 g) were challenged with infectious salmon anaemia virus (ISAV) either by intraperitoneal (i.p.) injection or by cohabitation with ISA-diseased Atlantic salmon (Salmo salar). Samplings of cod were performed over a period of 45 days and various tissue samples were collected. The presence of ISAV RNA (segment 8) in samples was assessed by both conventional RT-PCR and a competitive quantitative real-time RT-PCR. In the i.p.-challenged group, ISAV RNA was detected in fish from all samplings, i.e. at days 7, 15, 21, 30 and 45 post-challenge. At day 7 post-challenge, all individual fish were positive, and so were the vast majority of individual tissue samples. At later samplings, the fraction of positive brain samples remained high (approximately 75%). In contrast, the positive fraction of other tissues/organs declined during the experiment. Analysis of positive brain samples by a quantitative real-time RT-PCR analysis showed that the level of ISAV RNA increased significantly (approximately 20 times) between days 7 and 30 post-challenge and remained high at day 45, indicating that a replication of ISAV had taken place. ISAV RNA was not detected in any control or cohabitation-challenged fish. No abnormal behaviour, clinical disease or, most notably, mortality was observed in any of the challenge or control groups.

Pancreas disease in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in Norway.

The present paper describes, for the first time, clinical signs and pathological findings of pancreas disease (PD) in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in sea water in Norway. Similarities and differences with reports of PD from Ireland and Scotland are discussed. Samples of 68 rainbow trout from disease outbreaks on 14 farms and from 155 Atlantic salmon from outbreaks on 20 farms collected from 1996 to 2004 were included in the present study. The histopathological findings of PD in Atlantic salmon and rainbow trout in sea water were similar. Acute PD, characterized by acute necrosis of exocrine pancreatic tissues, was detected in nine Atlantic salmon and three rainbow trout. Salmonid alphavirus (SAV) was identified in acute pancreatic necroses by immunohistochemistry. Most fish showed severe loss of exocrine pancreatic tissue combined with chronic myositis. Myocarditis was often but not consistently found. Kidneys from 40% and 64% of the rainbow trout and Atlantic salmon, respectively, had cells along the sinusoids that were packed with cytoplasmic eosinophilic granules. These cells resembled hypertrophied endothelial cells or elongated mast cell analogues. Histochemical staining properties and electron microscopy of these cells are presented. SAV was identified by RT-PCR and neutralizing antibodies against SAV were detected in blood samples.

Susceptibility and immune responses following experimental infection of MHC compatible Atlantic salmon (Salmo salar L.) with different infectious salmon anaemia virus isolates.

Infectious salmon anaemia virus (ISAV) is an aquatic orthomyxovirus causing a multisystemic disease in farmed Atlantic salmon (Salmo salar) where disease development, clinical signs, and histopathology vary to a large extent. Here, an experimental trial was designed to determine the effect of variation in viral genes on virus-host interactions, as measured by disease susceptibility and immune responses. The fish were infected using cohabitant transmission, representing a natural route of infection. Variation caused by host factors was minimized using MHC compatible A. salmon half-siblings as experimental fish. Virus isolates were selected according to HE genotype, as European ISAV isolates can be genotyped according to deletion patterns in their hemagglutinin-esterase (HE) surface glycoprotein, and the course of disease they typically induce, classified as acute versus protracted. The different ISAV isolates induced large variations in death prevalence, ranging from 0-47% in the test-group and 3-75% in the cohabitant fish. The use of MHC compatible experimental fish made it possible to determine the relative contribution of humoral versus cellular response in protection against ISA. Ability to induce a strong proliferative response correlated with survival and virus clearance, while induction of a humoral response was less protective.

Demonstration of infectious salmon anaemia (ISA) viral antigens in cell cultures and tissue sections.

Rabbit polyclonal antibodies and mouse monoclonal antibodies (MAb) directed against infectious salmon anaemia virus (ISAV) were produced using a virus prepared in a newly established cell line culture developed from Atlantic salmon head kidney (SHK-1) cells. These antibodies were used to establish an indirect fluorescent antibody test for the detection of viral antigens in cell cultures and tissue cryosections. Specific fluorescence was detected in ISAV-infected cell cultures using rabbit polyclonal and MAb. One selected MAb produced specific staining for ISAV in the tissue sections from all the examined organs of ISAV-infected Atlantic salmon 20 d post infection. Fluorescence was mainly found in the endothelial cells and in single cells scattered throughout the parenchyma of the organs.

Effects of fasting on plasma lipids and cholesterol esterification in plasma, liver and intestinal mucosa in the char (Salmo alpinus L.).

1. CoA-dependent cholesterol esterification, measured as esterification of 3H-cholesterol, was demonstrated in homogenates of liver and intestinal mucosa of the char (Salmo alpinus L.). 2. Plasma concentration of triacylglycerols, unesterified and total cholesterol were significantly reduced to 43, 58 and 72% of the control values, respectively, after 6 weeks fasting. 3. The rate of cholesterol esterification in plasma and liver homogenate was significantly lower in the fasted fish compared to the controls, but the esterification activity in the homogenate of intestinal mucosa increased twofold in the fish fasted for 6 weeks.

Esterification of cholesterol in fish plasma: studies on the cholesterol esterifying enzyme in plasma of char (Salmo alpinus L.).

1. Plasma of sea char (Salmo alpinus L.) has the ability to esterify cholesterol in vitro. Heat inactivation of the plasma totally inhibited the esterification. 2. Arrhenius plot of the esterification of [3H]cholesterol was linear in the temperature range 5-35 degrees C. 3. Cholesterol esterification in sea char plasma could not be reversibly inhibited by sulphydryl-blocking agents. Total equilibration of the added [3H]cholesterol and the endogenous cholesterol could therefore not be obtained. 4. Comparison of esterification of endogenous and labelled cholesterol revealed that the isotopic method was valid only in qualitative measurements.

Endocytosis of galactose-terminated glycoproteins by isolated liver cells of the rainbow trout (Salmo gairdneri).

Intravenously injected 125I-labeled galactose-terminated glycoproteins were mainly recovered in the liver of the rainbow trout. After injection of [14C]sucrose-labeled asialofetuin, the liver cells were isolated and separated by differential centrifugation. The radioactivity was located in the parenchymal cells. Uptake of asialoglycoproteins in liver cells was inhibited by EGTA, lactose and excess unlabeled ligand. Degradation was inhibited by ammonium chloride, suggesting a lysosomal process. Internalization of 125I-asialoglycoproteins was demonstrated by removing receptor-bound ligand with EGTA at different time points during the incubation. The cellular uptake occurred even at 0 degree C.

Cholesterol esterification and lipids in blood plasma of the char (Salmo alpinus L.) during sexual maturation.

1. The initial rate of cholesterol esterification and the concentration of triacylglycerols in plasma were generally lower in prespawning than in immature chars. No typical pattern of variation of plasma cholesterol could be observed. 2. The observed variations in plasma cholesterol esterification and lipids may be caused by reduced feeding in the prespawning period. 3. The initial rate of cholesterol esterification in plasma was positively correlated with the concentrations of triacylglycerols and unesterified cholesterol in plasma, respectively.