PCR survey for Paramoeba perurans in fauna, environmental samples and fish associated with marine farming sites for Atlantic salmon (Salmo salar L.).

Amoebic gill disease (AGD) caused by the amoeba Paramoeba perurans is an increasing problem in Atlantic salmon aquaculture. In the present PCR survey, the focus was to identify reservoir species or environmental samples where P. perurans could be present throughout the year, regardless of the infection status in farmed Atlantic salmon. A total of 1200 samples were collected at or in the proximity to farming sites with AGD, or with history of AGD, and analysed for the presence of P. perurans. No results supported biofouling organisms, salmon lice, biofilm or sediment to maintain P. perurans. However, during clinical AGD in Atlantic salmon, the amoeba were detected in several samples, including water, biofilm, plankton, several filter feeders and wild fish. It is likely that some of these samples were positive as a result of the continuous exposure through water. Positive wild fish may contribute to the spread of P. perurans. Cleaner fish tested positive for P. perurans when salmon tested negative, indicating that they may withhold the amoeba longer than salmon. The results demonstrate the high infection pressure produced from an AGD-afflicted Atlantic salmon population and thus the importance of early intervention to reduce infection pressure and horizontal spread of P. perurans within farms.

Liquid fat, a potential abiotic vector for horizontal transmission of salmonid alphavirus?

Viral diseases represent serious challenge in marine farming of Atlantic salmon (Salmo salar L). Pancreas disease (PD) caused by a salmonid alphavirus (SAV) is by far the most serious in northern Europe. To control PD, it is necessary to identify virus transmission routes. One aspect to consider is whether the virus is transported as free particles or associated with potential vectors. Farmed salmonids have high lipid content in their tissue which may be released into the environment from decomposing dead fish. At the seawater surface, the effects of wind and ocean currents are most prominent. The aim of this study was primarily to identify whether the lipid fraction leaking from dead infected salmon contains SAV. Adipose tissue from dead SAV-infected fish from three farming sites was submerged in beakers with sea water in the laboratory and stored at different temperature and time conditions. SAV was identified by real-time RT-PCR in the lipid fractions accumulating at the water surface in the beakers. SAV-RNA was also present in the sea water. Lipid fractions were transferred to cell culture, and viable SAV was identified. Due to its hydrophobic nature, fat with infective pathogenic virus at the surface may contribute to long-distance transmission of SAV.

Lack of evidence for vertical transmission of SAV 3 using gametes of Atlantic salmon, Salmo salar L., exposed by natural and experimental routes.

Pancreas disease (PD) is an important cause of losses in farmed salmonids in Norway, the United Kingdom and Ireland. As the spread of salmonid alphavirus (SAV), the causal agent, to naïve populations is of major concern to the farming industry, it is important to uncover the transmission routes of the virus. This study was conducted to investigate the potential for vertical transmission of SAV subtype 3. Progeny of broodstock with signs of late-stage PD and persistent RT-PCR signals for SAV were followed from fertilization to smoltification in an experimental facility. Fertilized ova were either not disinfected or taken through one of three different disinfection regimes. Also, ova and milt from uninfected broodfish from a different population were exposed to a cell-cultured strain of SAV 3 immediately before fertilization to simulate a viraemic phase in parent fish. A group of uninfected controls were also included in the study. Fertilized ova from bath exposed and negative control groups were double disinfected. Following fertilization, experimental fish went through a normal freshwater phase. However, fry were stressed at first feeding to enhance replication of possibly latent virus. Smoltification was induced by an artificial light regime, and experimental fish were followed to the late smoltification phase. Selected samples were investigated by real-time RT-PCR for SAV, by histology for evidence of PD and by serology for neutralising antibodies against SAV. All analysed samples of progeny were negative. This result shows that SAV 3 is not readily transmitted vertically from parents to offspring. Additional negative PCR results from salmon sampled in commercial hatcheries support these findings. Also, recent studies have shown that risk factors for the horizontal transmission route explain the vast majority of PD outbreaks in Norway. It is concluded that if it happens at all, vertical transmission is of minor importance in the spread of SAV 3.

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.

Isolation and characterisation of segment 1 of the infectious salmon anaemia virus genome.

The isolation and characterisation of the largest genomic segment of infectious salmon anaemia virus (ISAV) is reported. Following identification of ISAV-specific clones from a cDNA library, a rapid amplification of cDNA ends-PCR strategy was designed to obtain the sequence of the full length mRNA transcript. The full length open reading frame (ORF) of this gene was shown to be 2169 nucleotides in length, encoding a putative protein of 722 aa. This sequence was demonstrated by RT-PCR to be specific to ISAV-infected cell cultures. The start codon of this ORF was preceded by the ISAV consensus sequence 5' GCTAAGA 3' indicating the full 5' end of the gene to have been obtained. Based on protein size and amino acid composition, this protein was shown to be similar to the PB2 protein of other orthomyxoviruses. Furthermore, a bipartite nuclear localisation signal was identified in the C-terminus of the protein as is found on all of the influenza virus P proteins. Expression of the putative PB2 as a green fluorescent marker protein-fusion protein confirmed that this protein exhibited nuclear localisation in a fish cell line. Sequences of the ISAV segment 1 gene were obtained from Scottish, Norwegian and Canadian ISAV isolates. Analyses confirmed the close genetic relationship between Norwegian and Scottish ISAV and indicated that this segment was among the most conserved of the ISAV genes identified to date. Thus, this evidence strongly suggests that the genomic segment 1 of ISAV encodes a polymerase protein which is thought to be analagous in function to the PB2 protein of influenza viruses.

Strain variation, based on the hemagglutinin gene, in Norwegian ISA virus isolates collected from 1987 to 2001: indications of recombination.

Infectious salmon anemia (ISA) is caused by a virus that probably belongs to the Orthomyxoviridae and was first recorded in Norway in 1984. The disease has since spread along the Norwegian coast and has later been found in Canada, Scotland, the Faroe Islands, Chile, and the USA. This study presents sequence variation of the hemagglutinin gene from 37 ISA virus isolates, viz. one isolate from Scotland, one from Canada and 35 from Norway. The hemagglutinin gene contains a highly polymorphic region (HPR), which together with the rest of the gene sequence provides a good tool for studies of epizootics. The gene shows temporal and geographical sequence variation, where certain areas are dominated by distinct groups of isolates. Evidence of transmission of ISA virus isolates within and between regions is given. It is suggested that the hemagglutinin gene from different isolates may recombine. Possible recombination sites are found within the HPR and in the 5'-end flanking region close to the HPR.

Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection.

The emergence of infectious salmon anaemia virus (ISAV) in Canada and Scotland and frequent new outbreaks of the disease in Norway strongly suggest that there are natural reservoirs for the virus. The main host for the ISA virus is probably a fish occurring in the coastal area, most likely a salmonid fish. Since sea trout is an abundant species along the Norwegian coast, common in areas where ISA outbreaks occur, and possibly a life-long carrier of the ISA virus, a study was initiated to evaluate reverse transcriptase polymerase chain reaction (RT-PCR) for diagnosis of the virus in experimentally infected trout. Several tissues (kidney, spleen, heart and skin) were collected from the trout during a 135 d period. The following diagnostic methods for detection of the ISA virus were compared: cell culture (Atlantic Salmon Kidney, ASK cells), challenge of disease-free salmon with blood from the infected trout, and RT-PCR. The RT-PCR was the most sensitive of these methods. With the help of this technique it was possible to pick out positive individuals throughout the experimental period of 135 d. Challenge of disease-free salmon were more sensitive than cell culture (ASK cells). These 2 latter methods require use of the immunofluorescent antibody test (IFAT) or RT-PCR for verification of presence of ISA virus.