An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 x 10(1) TCID(50) mL(-1) indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 x 10(-2) TCID(50) mL(-1)kg(-1)) and rose to levels above the minimum infective dose (4.2 x 10(1) TCID(50) mL(-1)kg(-1)) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 x 10(1) TCID(50) mL(-1)kg(-1) 15 days post-infection. These data provide important information relevant to the management of ISA.

Estimation of infectious dose and viral shedding rates for infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts.

Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post-smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 10(2) to 10(-4) 50% end-point tissue culture infectious dose (TCID(50)) mL(-1) sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post-smolts was <10(-1) TCID(50) mL(-1) by bath immersion (4 h at 10 degrees C). The peak shedding rate for IPNV following intraperitoneal challenge using post-smolts was estimated to be 6.8 x 10(3) TCID(50) h(-1) kg(-1) and occurred 11 days post-challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.

Distribution of infectious pancreatic necrosis virus (IPNV) in wild marine fish from Scottish waters with respect to clinically infected aquaculture sites producing Atlantic salmon, Salmo salar L.

This study represents the first large-scale investigation of IPNV in Scottish wild marine fish. Kidney samples were taken from 30 627 fish comprising 37 species and 45 isolations were made from nine different species, illustrating these as reservoirs of IPNV in Scottish waters. The estimated prevalence of IPNV in the Scottish marine environment was low at 0.15% (90% confidence intervals, (CI) of 0.11-0.19%). This was significantly greater in fish caught less than 5.0 km from IPN-positive fish farms in Shetland, at 0.58% (90% CI of 0.45-0.77%). This prevalence persisted and did not significantly decrease over the 16-month period of study. The estimated prevalence of IPNV for each positive species was less than 1% with the statistically non-significant exceptions of flounder, Platichthys flesus (L.), at 12.5% (90% CI of 0.64-47.06%) and saithe, Pollachius virens (L.), at 1.11% (90% CI of 0.49-2.19%). The 45 isolates were titrated and all but two were below the detection limit of the test (<55 PFU g(-1)). Titres of 3.8 x 10(2) PFU g(-1) and 2.8 x 10(1) PFU g(-1) were calculated from common dab, Limanda limanda (L.), and saithe, respectively. This study provides evidence that clinical outbreaks of IPN in farmed Atlantic salmon may cause a localized small increase in the prevalence of IPNV in wild marine fish.

Genetic analysis of infectious pancreatic necrosis virus from Scotland.

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish, and is one of the most serious economic diseases in aquaculture. In Scotland, an increase in IPN virus (IPNV) outbreaks in seawater Atlantic salmon, Salmo salar, has been reported in recent years. The aim of this study was to analyse the VP2 gene from recent IPNV isolates from Scotland, to determine whether there are epidemiological links between IPNV isolates from farms (13), wild fish (17) and the environment (6) in order to investigate potential wild and farmed fish interactions. Comparison of the nucleotide sequence of the VP2 gene revealed that 34 of 36 isolates were 97.1-100% similar and the deduced amino acid sequences showed 97-100% identity. Two isolates from wild fish exhibited the most divergence at 85-87.3% similarity to the other isolates at the nucleotide level and 88.2-90.8% identity at the deduced amino acid level. Phylogenetic analyses revealed that 34 of 36 of the isolates from Scotland were genetically closely related to the A2 (Sp) serotype of IPNV. The two wild isolates from seatrout, Salmo trutta, and flounder, Platichthys flesus, were most closely related to the European A5 (Te) serotype. This study represents a comprehensive IPNV phylogenetic study that indicates that there are closely related or identical isolates in circulation in the marine environment, which adds evidence that disease interactions between wild and farmed fish may occur. This type of analysis is a useful tool in the management and control of fish diseases because it can assist in the identification of epidemiological links and highlight potential risks to aquaculture.

Detection of infectious pancreatic necrosis virus (IPNV) from the environment in the vicinity of IPNV-infected Atlantic salmon farms in Scotland.

Infectious pancreatic necrosis virus (IPNV) has been isolated from mussels, sediment and surface water in the vicinity of clinically infected salmon farms, at shore bases supplying the farms and for several hundred metres distance from farms in the direction of current flow. There was evidence of decreasing prevalence of IPNV in mussels from Shetland once IPN outbreaks subsided, indicating they are an unlikely source of re-infection on farms. There was little evidence of persistence in the environment, although conclusions were complicated by the presence of IPNV on neighbouring farms 1 year after the outbreak.

Modelling for disease preparedness and response.

Modelling is a useful tool for the understanding and hence, potentially, control of disease. In an emergency situation, obtaining data to validate detailed models may be difficult. However, modelling can be useful in aiding the management of disease, if the objectives are not too ambitious. For example, import risk analysis can use models to minimise the risk of outbreaks. Modelling can also be used to maximise the power of surveillance data to ensure that any outbreak is detected as rapidly as possible. Methods of modelling spread of disease, and measures to prevent this, including SIR-style epidemiological models, models of disease control zones and contact network models can be used to identify and minimise the potential scale of an epidemic. Finally, economic and ecological models can be used to assess the impact of outbreaks. A range of simple models is likely to be of more use than a single comprehensive model.

Genotyping of marine viral haemorrhagic septicaemia virus isolated from the Flemish Cap by nucleotide sequence analysis and restriction fragment length polymorphism patterns.

A total of 14 viral haemorrhagic septicaemia virus (VHSV) isolates obtained from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap, a fishing ground in the North Atlantic Ocean near Newfoundland, were characterised using restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis. RFLP analysis was performed on a 1259 bp fragment of the glycoprotein (G) gene, and a 305 nucleotide region within the nucleoprotein (N) gene was used for sequence analysis. Representative strains of the 4 established genotypes were employed for comparative purposes. Sequencing analysis indicated that the Flemish cap isolates grouped in Genotype 3, which also includes isolates from wild fish caught in the North Sea and coastal waters of the UK and Ireland, isolates derived from outbreaks of VHS in turbot farms in the British Isles, and an isolate from European eel Anguilla anguilla caught in northern France. Characterisation using RFLPs resulted in the development of a simple and reliable method of typing VHSV at the genotype level using a 2-step restriction analysis (2-SRA) assay.

Experimental susceptibility of Atlantic cod, Gadus morhua (L.), and Atlantic halibut, Hippoglossus hippoglossus (L.), to different genotypes of viral haemorrhagic septicaemia virus.

Viral haemorrhagic septicaemia (VHS) is a well-characterized disease of rainbow trout, Oncorhynchus mykiss, which has also caused economic losses in marine turbot farms in the British Isles. We have previously demonstrated that turbot, Scophthalmus maximus, are susceptible to isolates of viral haemorrhagic septicaemia virus (VHSV) that are endemic in the marine environment, highlighting a potential risk to marine aquaculture. Given the increasing interest in the intensive rearing of additional aquaculture species such as Atlantic cod, Gadus morhua, and Atlantic halibut, Hippoglossus hippoglossus, this study aimed at investigating the susceptibility of these species to VHSV. Both species were found to be largely resistant to VHS following immersion challenge with a selection of 18 isolates, representing the known marine VHSV genotypes. Only one and two VHSV-associated mortalities occurred out of a total of 1710 and 1254 halibut and cod, respectively. These findings suggest that there is a low direct risk to the development of commercial cod and halibut aquaculture from the existing endemic reservoir of VHSV. This study, coupled to field observations has, however, highlighted the fact that both species can become infected with VHSV. The known adaptability of RNA viruses, together with the selection pressures associated with intensive aquaculture would thus advocate a cautious approach to VHSV surveillance within these emerging industries.

Sequential pathology after experimental infection with marine viral hemorrhagic septicemia virus isolates of low and high virulence in turbot (Scophthalmus maximus L).

Three marine viral hemorrhagic septicemia virus isolates were used to bath challenge turbot with the purpose of studying mortality and the pathology and antigen distribution over time. Two high-virulence isolates, 860/94, 4p168 and a low-virulence isolate 1p3 from a Baltic Sea herring were used. Organ samples were collected sequentially at 2, 4, 7, 10, 15, 20, 25, and 45 days postinfection. Specimens were processed for virology, histopathology, and immunohistochemistry. Organs during the early stages of infection (from 2 to 7 days) had virus isolation from all three groups only on day 7. Virus titer in kidney and heart sampled at day 25 was higher for the two virulent isolates compared with the low-virulence isolate. The viral distribution in situ of the two more virulent isolates from turbot (860/94) and herring (4p168) resembled viral hemorrhagic septicemia in rainbow trout with regard to the target organs. Early infection of endothelial cells in both kidney and heart was observed. Accumulated mean mortality was 41.5% for the turbot isolate 860/94, 48% for the herring isolate 4p168, and 3.5% for the herring isolate 1p3. This study revealed that the isolates from turbot (860/94) and herring (4p168) induced significantly higher mortality compared with the virus-free control and the herring isolate (1p3). The onset of mortality is markedly later in turbot compared with what is seen in rainbow trout.

Comparative susceptibility of turbot Scophthalmus maximus to different genotypes of viral haemorrhagic septicaemia virus.

Viral haemorrhagic septicaemia (VHS) disease has exerted a significant impact on the development of turbot aquaculture in the British Isles. The source of such outbreaks is believed to be naturally occurring marine isolates of viral haemorrhagic septicaemia virus (VHSV), which are endemic in the marine environment of Northern Europe. Genetic studies have classified these marine VHSV isolates into genotypes based on their geographic rather than host-species origin. This study set out to explore the hypothesis that susceptibility of turbot to VHSV might be genotype specific. Immersion infection of turbot with a range of isolates, selected according to genotype, identified significant differences between susceptibility and genotype. Viruses belonging to Genotypes Ib (Baltic marine isolates) and III (North Sea/E. Atlantic marine isolates) caused significantly higher mortality than isolates from Genotypes Ia (isolates associated with rainbow trout aquaculture) and II (Baltic marine isolates). This study serves to highlight the importance of thoroughly investigating the susceptibility of any given species to the range of pathogens to which they might be exposed prior to considering them resistant to any disease. Furthermore, it highlights different risk factors that might be associated with turbot aquaculture undertaken in different environments. Finally, an increased knowledge of the relative virulence of different isolates in turbot will assist in understanding virulence determinants, which could lead to advances in disease control.

Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV).

The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups of isolates associated with rainbow trout aquaculture (Genotype Ia) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also confirmed. Genotype III included marine isolates from around the British Isles in addition to those associated with turbot mariculture, highlighting a continued risk to the development of this industry. Genotype IV consisted of isolates from the marine environment in North America. Taken together, these findings suggest a marine origin of VHSV in rainbow trout aquaculture. The implications of these findings with respect to the future control of VHSV are discussed. The capacity for molecular phylogenetic analysis to resolve complex epidemiological problems is also demonstrated and its likely future importance to disease management issues highlighted.

An evaluation of current diagnostic tests for the detection of infectious salmon anaemia virus (ISAV) following experimental water-borne infection of Atlantic salmon, Salmo salar L.

Four commonly used diagnostic tests [reverse transcription polymerase chain reaction (RT-PCR), indirect fluorescent antibody test (IFAT), virus culture and light microscopy] were evaluated for their ability to detect infectious salmon anaemia virus (ISAV) or tissue pathology following experimental infection of Atlantic salmon. Fish were infected with ISAV by water-borne exposure which mimics the route of natural infection. Forty-five per cent of pre-clinical fish tested yielded positive results by RT-PCR for at least one of the organs tested (kidney, heart, gill, liver, blood). No significant difference was detected between organs in the number or time of first occurrence of positive result. Virus culture identified a total of 14% of pre-clinical fish as ISAV-infected. The presence of ISAV in heart tissue was particularly notable (13% of fish sampled) as was the inability to culture virus from spleen tissue. In the case of IFAT, 15% of fish sampled were positive, although tissue other than kidney proved unsuitable for use in this method. Only limited ISAV-specific pathology was detectable by histological examination of fish prior to the onset of clinical disease. These findings reveal important information regarding the optimal choice of both tissue sample and diagnostic test for the routine diagnosis of ISAV.

Infectious salmon anaemia virus in wild fish from Scotland.

Following the outbreak of infectious salmon anaemia (ISA) at salmon farms in Scotland, UK, a survey was established to determine the extent of infection in wild fish. All fish tested were free from the clinical symptoms of ISA. Isolations of ISAV were made from 5 sea trout within areas where ISA affected salmon farms were located. Evidence for ISAV in other sea trout was provided by ISA RT-PCR diagnostic tests. Results from ISA RT-PCR tests reveal evidence for ISAV being present in salmon parr, adult salmon and juvenile brown trout in rivers distant from salmon farms and indicate that, at the time of the survey (1998-1999), ISAV may have been widely distributed. Nucleotide sequence analysis of segments 2 and 8 showed that for most sequences from wild fish there was 100% homology with ISAV isolated from clinically affected farmed fish although evidence is presented which indicates variability in ISAV sequences from wild fish. Modelling the RT-PCR findings indicates that ISAV among salmonid fish was spatially non-random. Brown trout, sea trout and salmon (adult and parr) show a pattern of occasionally large numbers of positive samples against a background of very low numbers.

Experimental infection models and susceptibility of Atlantic salmon Salmo salar to a Scottish isolate of infectious salmon anaemia virus.

Infection models for both fresh and seawater salmon were established using a Scottish isolate of infectious salmon anaemia virus (ISAV). Modes of infection were intra-peritoneal injection, cohabitation and immersion exposure, and a range of doses was tested. Development of these models using a Scottish isolate of ISAV provided an approximation of the minimum infective dose leading to mortality under different infection regimens. The models also allow prediction of the time to first mortality and an estimation of expected total mortality following the various routes of infection. Such knowledge is important to the development of anti-ISAV vaccines and to future studies aimed at understanding the biology of ISAV in general.

Distribution of viral haemorrhagic septicaemia virus in wild fish species of the North Sea, north east Atlantic Ocean and Irish Sea.

A surveillance programme was initiated on the occurrence and distribution of viral haemorrhagic septicaemia virus (VHSV) in wild marine fish. Six research cruises were undertaken in an 18 mo period during 1997 and 1998, covering the North Sea, the Atlantic waters off the north and west coasts of Scotland and the Irish Sea. A total of 19,293 fish were sampled from 23 different species including cod, haddock, Norway pout, herring and sprat. Individual fish lengths were recorded and the fish were checked for lesions, haemorrhaging and other signs of disease. Pools of organ samples were taken for virus assay. The majority of fish sampled did not display clinical signs indicative of viral haemorrhagic septicaemia. A small number of cod were found with skin lesions and haddock with skin haemorrhaging. Of the 2081 organ and skin sample pools collected, 21 tested positive for VHSV by tissue culture and enzyme-linked immunosorbent assay. Seventeen of the isolates originated from Norway pout Trisopterus esmarkii, one from cod Gadus morhua (skin lesion), one from herring Clupea harengus, one from whiting Merlangius merlangus, and one from a previously unreported host species, poor cod Trisopterus minutus.

Experimental susceptibility of Atlantic salmon Salmo salar and turbot Scophthalmus maxim us to European freshwater and marine isolates of viral haemorrhagic septicaemia virus.

A number of viral haemorrhagic septicaemia (VHS) virus isolates of European marine origin were shown to be of low pathogenicity or non-pathogenic to Atlantic salmon parr by water-borne infection. A reference freshwater VHS virus isolate known to be highly pathogenic to rainbow trout was also of low pathogenicity to Atlantic salmon. Virus was detected in some mortalities, however, demonstrating viral entry and replication. European marine VHS virus isolates do not appear to pose an imminent threat to the Atlantic salmon culture industry. Turbot were found to be refractive or of low susceptibility to marine VHS virus isolates of sprat origin and to a reference freshwater isolate, with mortalities of 0 to 13.5%. Conversely, turbot were susceptible by varying degrees to a number of VHS virus isolates taken from herring, with mortalities ranging from 16 to 68%. These results emphasise the vulnerability of turbot culture to the VHS virus isolates that are enzootic to the European marine environment.

Digestive enzyme levels and histopathology of pancreas disease in farmed Atlantic salmon (Salmo salar).

1. The pancreatic digestive enzyme activities of trypsin and chymotrypsin were assessed in vitro and were found to correlate well with the histological changes characteristic of pancreas disease (PD) in farmed Atlantic salmon (Salmo salar). 2. Pancreatic enzyme activity was assessed in vivo using the chymotrypsin specific substrate N-benzoyl-L-tyrosyl-p-aminobenzoic acid. In all cases, significantly less p-aminobenzoic acid was excreted by fish later found to be suffering from PD. 3. It is concluded that the in vitro digestive enzyme assay was effective in diagnosing PD with the in vivo method, indicating further promise for assessing exocrine pancreatic function.

Effects of increasing dietary linoleic acid on phospholipid fatty acid composition and eicosanoid production in leucocytes and gill cells of Atlantic salmon (Salmo salar).

Diets containing linoleic acid at 10, 25 and 45% of total dietary fatty acids were fed to three groups of post-smolt Atlantic salmon (Salmo salar) for 18 weeks. Incorporation of linoleic acid into membrane phospholipids of leucocytes and gills increased in response to dietary intake. In general, there was an increase in arachidonic acid and a decrease in eicosapentaenoic acid in the individual phospholipids of both cell types in response to increasing dietary linoleic acid. These changes in eicosanoid precursors were reflected in significantly increased plasma concentrations of 6-keto-PGF1 alpha and TXB2 in salmon given the highest dietary linoleic acid. In whole blood stimulated with the calcium ionophore A23187, LTB4, 12-HETE and TXB2 were significantly increased and 12-HEPE significantly decreased in response to increasing dietary linoleic acid. In isolated gill cells stimulated with A23187, 12-HEPE, 12-HETE, 14-HDHE and TXB2 were all decreased in response to increasing dietary linoleic acid, although the ratio of 12-HEPE/12-HETE was also decreased.

Homeoviscous adaptation and thermal compensation of sodium pump of trout erythrocytes.

The effects of thermal acclimation of trout on the transport activity and turnover number of the erythrocyte Na+ pump have been determined. Na+ pump activity was estimated by measuring the ouabain-sensitive K+ influx in Na(+)-loaded cells and the number of active pumps determined by Scatchard analysis of [3H]ouabain binding and by correlation of ouabain binding with pump inhibition. Cold acclimation was associated with an increase in pump activity of up to 60%, although the furosemide-sensitive and residual fluxes were unaffected. The number of ouabain binding sites was similar in both acclimation groups at approximately 21,000-23,000 sites/cell. This means that cold acclimation induced an increase in the transport turnover number of pump molecules from approximately 6 to 9 s-1. Cold acclimation was also associated with a decrease in membrane order as indicated by steady-state fluorescence polarization of the membrane probe, 1,3-diphenyl-1,3,5-hexatriene, with a homeoviscous efficacy of 25-41%. That membrane order may influence pump transport activity is supported by experiments on cholesterol supplementation, which caused both an increase in membrane order and a decrease in pump turnover number. The degree of pump compensation was dependent on the season, with greatest responses in the late spring and declining responses through to winter. By contrast, changes in membrane order were observed throughout the year. Expression of pump activity and erythropoiesis may vary throughout the seasonal cycle in complex ways that confuse the direct comparison study of cellular properties in a heterogeneous population of cells.