Abstract
Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.
Highlights
Infectious salmon anaemia (ISA) is a systemic infectious disease of farmed Atlantic salmon (Salmo salar L.), which has had a significant economic impact on salmon farming, in particular in Norway and Chile [1]
The six Monoclonal antibodies (mAbs) that reacted with all the Infectious salmon anaemia virus (ISAV) isolates tested in enzyme-linked immunosorbent assay (ELISA), recognised the nucleoprotein (NP) of the virus, represented by the band present at 66–71 kDa in immunoblotting (Fig 1)
All of these mAbs detected the virus in infected SHK1 cells, by indirect fluorescent antibody technique (IFAT) using cells grown on coverslips and infected with virus and examined by confocal microscopy
Summary
Infectious salmon anaemia (ISA) is a systemic infectious disease of farmed Atlantic salmon (Salmo salar L.), which has had a significant economic impact on salmon farming, in particular in Norway and Chile [1]. The first cases of ISAV were reported in Norway in the 1980s [4] and cases have since been reported from Canada (1996, 1998, 2012), Scotland (1998), Faroe Islands (2000), USA (2001) and Chile (2007, 2013) [3,5]. Mortality levels are variable during ISA outbreaks and can be as low as 0.5–1.0% per day, but without intervention cumulative mortality in infected populations can reach as high as 90% [3], emphasising the need for early diagnosis to control the spread of the virus. Vaccination has been carried out in Norway, Canada and Chile, complete protection has not been achieved with these vaccines to date [3], a recently developed DNA vaccine has been shown to provide good protection in laboratory-based experiments [7]
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