Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain.

Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have been classified as free of infection. Clinical signs of nodavirus infection were reproduced in fish infected following bath exposure or intramuscular injection, demonstrating horizontal transmission of the disease. Nodavirus was always detected in the brain of diseased fish but also in many recovered fish. The new assay enables us to confirm the presence of the virus at an early phase in the production cycle and may represent a useful tool to prevent or slow down the spread of nodavirus to new locations.

Tissue tropism of salmonid alphaviruses (subtypes SAV1 and SAV3) in experimentally challenged Atlantic salmon (Salmo salar L.).

Diagnosis of SAV infections has traditionally been based upon clinical observations together with a set of histopathological findings in exocrine pancreas, heart and skeletal muscle, but recently, real-time RT-PCR assays have been developed as a supplement for the detection of SAV. The aim of this study was to determine tissue tropism of SAV1 and SAV3 in Atlantic salmon Salmo salar L. in order to identify the most suitable tissues for real-time RT-PCR diagnostic assays. The results indicated that the pseudobranch and the heart (ventricle) are the most useful tissues for such assays, regardless of disease status. The pyloric caecae with associated pancreatic tissue is unsuitable for diagnosis using this method. The use of real-time RT-PCR enabled viral RNA detection at all stages of the disease, including in surviving fish six months after infection. Considering the short production cycle of farmed salmonids, this suggests that surviving Atlantic salmon may become life-long asymptomatic carriers of SAV after an infection.

Genetic stability within the Norwegian subtype of salmonid alphavirus (family Togaviridae).

Salmonid alphavirus (SAV) (family Togaviridae) causes mortality in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss W.) in Norway, France, UK, and Ireland. At least three subtypes of SAV exist: SPDV in UK/Ireland, SDV in France/UK, and the recently reported Norwegian salmonid alphavirus (NSAV) in western Norway. During 2003 and 2004, disease caused by NSAV was reported for the first time in northern Norway, more than 800 km away from the enzootic area in western Norway. The present study has investigated the phylogenetic relationships among 20 NSAV isolates, based on a 1221-nt-long segment covering part of the capsid gene, E3, and part of the E2 gene, collected over a period of eight years. The results revealed genetic homogeneity among NSAV isolates, including those from northern Norway. The SDV or SPDV subtypes were not found in diseased Norwegian fish. A substitution rate of 1.70 (+/-1.03) x 10(-4) nt subst/site/year was obtained for the NSAV subtype by maximum likelihood analysis. The second aim of this study was to clarify whether NSAV changes genotypically in cell culture by culturing a NSAV isolate through 20 passages in CHSE-214 cells. Sequencing of almost the entire genome (11530 nt) after 20 passages revealed four nucleotide substitutions, all resulting in amino acid substitutions. One of these substitutions, serine to proline in E2 position 206, was also found to have occurred in field isolates.

New subtype of salmonid alphavirus (SAV), Togaviridae, from Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss in Norway.

In Europe, 2 closely related alphaviruses (Togaviridae) are regarded as the causative agents of sleeping disease (SD) and salmon pancreas disease (SPD): SD virus (SDV) has been isolated from rainbow trout Oncorhynchus mykiss in France and the UK, while SPD virus (SPDV) has been isolated from salmon Salmo salar in Ireland and the UK. Farmed salmonids in western Norway also suffer from a disease called pancreas disease (PD), and this disease is also believed to be caused by an alphavirus. However, this virus has not yet been characterised at the molecular level. We have cultured a Norwegian salmonid alphavirus from moribund fishes diagnosed with cardiac myopathy syndrome (CMS) and fishes diagnosed with PD. The virus has also been found in salmon suffering from haemorrhagic smolt syndrome in the fresh water phase. The genomic organisation of the Norwegian salmonid alphavirus is identical to that in SPDV and SDV, and the nucleotide sequence similarity to the other 2 alphaviruses is 91.6 and 92.9%, respectively. Based on the pathological changes, host species and the nucleotide sequence, we suggest naming this virus Norwegian salmonid alphavirus (NSAV). Together with SPDV and SDV it constitutes a third subtype of salmonid alphavirus (SAV) species within the genus Alphavirus, family Togaviridae.

Haemorrhagic smolt syndrome (HSS) in Norway: pathology and associated virus-like particles.

Atlantic salmon Salmo salar pre-smolt, smolt and post-smolt, with clinical signs of haemorrhagic smolt syndrome (HSS) have been found in several locations along the Norwegian coast (Rogaland to Troms). Affected fish had pale gills and bleeding at the fin bases, but seemed to be in good physical condition with no obvious weight loss. The internal organs and body cavity showed distinct bleedings. Petechiae were found on the gastrointestinal tract, swim bladder and peritoneum, visceral adipose tissue, heart and somatic musculature. The liver was bright yellow and sometimes mottled with petechiae and ecchymoses. Acitic fluid was found in the visceral cavity and fluid was also present in the pericardial cavity. Histological examination revealed haemorrhage in most organs. The glomeruli were degenerated and the renal tubules were filled with erythrocytes. The aims of this study were to describe the pathology and discover, if possible, the aetiology of the HSS. Tissues were collected for light and transmission electron microscopy (TEM), immunofluorescence (IFAT), reverse transcription (RT)-PCR diagnostics (screening for infectious salmon anaemia virus [ISAV], viral haemorrhagic septicaemia virus [VHSV], salmon pancreas disease virus [SPDV], sleeping disease virus [SDV] and infectious haematopoetic necrosis virus [IHNV]), and tissue homogenates (heart, liver, kidney and spleen) were sterile-filtered and inoculated into cell cultures. Homogenates made from several tissues were also injected intraperitoneally into salmon and rainbow trout Oncorhynchus mykiss. The diagnostic tests revealed no consistent findings of any pathogens, with the exception of TEM which showed 2 types of virus-like particles: Type I was 50 to 60 nm in diameter and Type II about 50 nm in diameter. These virus-like particles were found in salmon from all farms affected by HSS and screened by TEM. Several different cells, blood vessel endothelial cells, endocardial cells, heart myofibres, and leukocytes were associated with the 2 virus-like particles. The Type I particle seems to be an infectious pancreatic necrosis (IPN)-like virus, while (based on the number of target cells, particle morphology, budding and uptake into target cells) Type II particle could be a togavirus.

Experimental infection of Atlantic salmon (Salmo salar) with marine Eubothrium sp. (Cestoda: Pseudophyllidea): observations on the life cycle, aspects of development and growth of the parasite.

The life cycle of marine Eubothrium sp. (Cestoda: Pseudophyllidea), from Atlantic salmon (Salmo salar L.) was experimentally completed in one year and included only one intermediate host (Acartia tonsa Dana) (Copepoda: Calanoida). Adult cestodes were collected from farmed salmon, and ripe eggs released by the cestodes were fed to Acartia tonsa. Ingested eggs hatched in the gut and the larvae developed in the haemocoel of the copepod for 15 days at 16 degrees C. A total of 170 seawater-reared salmon were exposed to infected copepods and the total prevalence of Eubothrium sp. in the salmon after infection was 95.3%, with a mean intensity of 15.0 (range 1-87). The infected salmon were kept in the laboratory where the growth of the cestodes was studied for eleven months. Mean length of the cestodes increased with time, but a large variation among the cestodes was observed. Growth and maturation of the cestodes were dependent on host size and the number of worms present in the intestine. No evidence of mortality of Eubothrium sp. was observed during the experimental period.

On the occurrence and morphology of Gyrodactylus pterygialis from saithe Pollachius virens in a Norwegian fjord.

A total of 308 Pollachius virens from a fjord near Bergen, western Norway, were examined for Gyrodactylus pterygialis over a 14-mo period; G. pterygialis showed the highest prevalence and density in winter/spring (November, January, March, and April pooled) but was absent in some months (August, September, and November). The morphology of the opisthaptoral hard parts of G. pterygialis were studied throughout the survey, and their variation is discussed with respect to host species, host locality, and different temperatures. On the basis of the present and previous descriptions, it is suggested that G. pterygialis is a parasite infecting gadine fishes of the genera Pollachius and Gadus, and that records from Pacific Eleginus and White Sea herring are distinct from G. pterygialis sensu Bychowsky and Poljansky, 1953.