ABSTRACT
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.
Subject(s)
Fish Diseases/transmission , Fish Diseases/virology , Isavirus , Orthomyxoviridae Infections/veterinary , Salmo salar , Virus Shedding/physiology , Animals , Oligonucleotides/genetics , Orthomyxoviridae Infections/transmission , Polymerase Chain Reaction/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary , SeawaterABSTRACT
A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.
Subject(s)
Birnaviridae Infections/veterinary , Fish Diseases/diagnosis , Fish Diseases/virology , In Situ Hybridization, Fluorescence/methods , Infectious pancreatic necrosis virus/isolation & purification , Salmo salar/virology , Animals , Birnaviridae Infections/diagnosis , Birnaviridae Infections/virology , Diazonium Compounds , Fluorescent Dyes/metabolism , In Situ Hybridization/methods , Infectious pancreatic necrosis virus/genetics , Kidney/cytology , Liver/virology , Naphthalenes , Paraffin EmbeddingABSTRACT
Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot.