Genetic characterization of salmonid alphavirus in Norway.

Pancreas disease (PD), caused by salmonid alphavirus subtype 3 (SAV3), emerged in Norwegian aquaculture in the 1980s and is now endemic along the south-western coast. In 2011, the first cases of PD caused by marine salmonid alphavirus subtype 2 (SAV2) were reported. This subtype has spread rapidly among the fish farms outside the PD-endemic zone and is responsible for disease outbreaks at an increasing numbers of sites. To describe the geographical distribution of salmonid alphavirus (SAV), and to assess the time and site of introduction of marine SAV2 to Norway, an extensive genetic characterization including more than 200 SAV-positive samples from 157 Norwegian marine production sites collected from May 2007 to December 2012 was executed. The first samples positive for marine SAV2 originated from Romsdal, in June 2010. Sequence analysis of the E2 gene revealed that all marine SAV2 included in this study were nearly identical, suggesting a single introduction into Norwegian aquaculture. Further, this study provides evidence of a separate geographical distribution of two subtypes in Norway. SAV3 is present in south-western Norway, and marine SAV2 circulates in north-western and Mid-Norway, a geographical area which since 2010 constitutes the endemic zone for marine SAV2.

Mortality and weight loss of Atlantic salmon, Salmon salar L., experimentally infected with salmonid alphavirus subtype 2 and subtype 3 isolates from Norway.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) has a significant negative economic impact in the salmonid fish farming industry in northern Europe. Until recently, only SAV subtype 3 was present in Norwegian fish farms. However, in 2011, a marine SAV 2 subtype was detected in a fish farm outside the PD-endemic zone. This subtype has spread rapidly among fish farms in mid-Norway. The PD mortality in several farms has been lower than expected, although high mortality has also been reported. In this situation, the industry and the authorities needed scientific-based information about the virulence of the marine SAV 2 strain in Norway to decide how to handle this new situation. Atlantic salmon post-smolts were experimentally infected with SAV 2 and SAV 3 strains from six different PD cases in Norway. SAV 3-infected fish showed higher mortality than SAV 2-infected fish. Among the SAV 3 isolates, two isolates gave higher mortality than the third one. At the end of the experiment, fish in all SAV-infected groups had significantly lower weight than the uninfected control fish. This is the first published paper on PD to document that waterborne infection produced significantly higher mortality than intraperitoneal injection.

Use of molecular epidemiology to trace transmission pathways for infectious salmon anaemia virus (ISAV) in Norwegian salmon farming.

BACKGROUND: Infectious Salmon Anaemia (ISA) is a disease affecting farmed Atlantic salmon, and most salmon producing countries have experienced ISA outbreaks. The aim of the present study was to use epidemiological and viral sequence information to trace transmission pathways for ISA virus (ISAV) in Norwegian salmon farming. METHODS: The study covers a period from January 2007 to July 2009 with a relatively high rate of ISA outbreaks, including a large cluster of outbreaks that emerged in Northern Norway (the North-cluster). Farms with ISA outbreaks and neighbouring salmon farms (At-risk-sites) were tested for the presence of ISAV, and epidemiological information was collected. ISAV hemagglutinin-esterase (HE) and fusion (F) protein genes were sequenced and phylogenetic analyses were performed. Associations between sequence similarities and salmon population data were analysed to substantiate possible transmission pathways. RESULTS: There was a high degree of genetic similarity between ISAV isolates within the North-cluster. ISAV was detected in 12 of 28 At-risk-sites, and a high proportion of the viruses were identified as putative low virulent genotypes harbouring the full length highly polymorphic region (HPR); HPR0 of the HE protein and the amino acid glutamine (Q) in the F protein at position 266. The sequences from HPR0/F (Q(266)) genotypes revealed larger genetic variation, lower viral loads and lower prevalence of infection than HPR-deleted genotypes. Seaway distance between salmon farms was the only robust explanatory variable to explain genetic similarity between ISAV isolates. DISCUSSION: We suggest that a single HPR-deleted genotype of ISAV has spread between salmon farms in the North-cluster. Furthermore, we find that HPR0/F (Q(266)) genotypes are frequently present in farmed populations of Atlantic salmon. From this, we anticipate a population dynamics of ISAV portrayed by low virulent genotypes occasionally transitioning into virulent genotypes, causing solitary outbreaks or local epidemics through local transmission.

Immunohistochemical and Taqman real-time PCR detection of mycobacterial infections in fish.

Real-time PCR and immunohistochemistry (IHC) assays were developed to detect fish mycobacterial infections at the genus level, based on the RNA polymerase ß subunit (rpoB) gene and polyclonal anti-Mycobacterium rabbit serum, respectively. The PCR assay positively identified a number of pathogenic mycobacteria including Mycobacterium abscessus, M. avium ssp. avium, M. bohemicum, M. chelonae ssp. chelonae, M. farcinogenes, M. flavescens, M. fortuitum ssp. fortuitum, M. gastri, M. gordonae, M. immunogenicum, M. malmoense, M. marinum, M. montefiorense, M. phlei, M. phocaicum, M. pseudoshottsii, M. salmoniphilum, M. senegalense, M. shottsii, M. smegmatis, M. szulgi and M. wolinskyi. A detection limit equivalent to 10(2) cfu g(-1) was registered for M. salmoniphilum-infected fish tissue. The IHC precisely localized both free and intracellular mycobacteria in tissues and detected mycobacterial infections down to 10(2) cfu g(-1) tissue. Both assays were found to be more sensitive than Ziehl-Neelsen (ZN) staining, where the detection limit was below 8 × 10(3) cfu g(-1) tissue. Although specificity testing of the real-time PCR against a panel of non-Mycobacterium spp. revealed a degree of cross-reaction against pure DNA extracted from Nocardia seriolae and Rhodococcus erythropolis, no cross-reactions were identified (by either real-time PCR or IHC) on testing of formalin-fixed paraffin-embedded (FFPE) tissues confirmed to be infected with these bacteria. The broad applicability of both assays was confirmed by analysis of FFPE tissues from a range of fish species infected with diverse Mycobacterium spp. The results indicate that both assays, alone or in combination, constitute sensitive tools for initial, rapid diagnosis of mycobacteriosis in fish. This should in turn allow rapid application of more specific studies, i.e. culture based, to identify the specific Mycobacterium sp. involved.

Epidemiological investigation of infectious salmon anaemia (ISA) outbreaks in Norway 2003-2005.

Epidemiological information was summarized from 32 outbreaks of infectious salmon anaemia (ISA) on salmon farming sites in Norway in 2003-2005. Virus isolates from the outbreak sites were genotyped, and the genotyping was used to assess possible associations between outbreak sites due to adjacent location, sharing fish farming authorisation, sharing smolt suppliers or sharing broodfish origin of the fish. The ISA outbreaks were distributed along most of the Norwegian coast and showed a variable clinical picture. The virus genotypes clustered into three genogroups. Pairs of outbreak sites matched for adjacent location or registered under the same authorisation, all shared genogroup, which was a significantly higher number of corresponding genogroups than expected by chance. For outbreak sites sharing smolt suppliers, corresponding genogroups appeared in 7 out of 12 matched pairs, which was not significant. An evaluation of broodfish origin associated with genogroups did not support transmission linked to broodfish origin. In conclusion, genotyping of virus isolates from ISA outbreaks supports associations between adjacent outbreaks. This is consistent with horizontal transmission. The present study failed to find evidence for vertical transmission (patterns of genogroups related to smolt suppliers or broodfish companies were not identified).

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.

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.

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.).

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.