I – INTRODUCTION
Infectious haematopoietic necrosis (INH) is a viral disease caused by a rhabdovirus. It affects most salmonid species, especially the fry and young fish. Susceptible species include rainbow or steelhead trout (Oncorhynchus mykiss) and brown trout (Salmo trutta); Pacific salmon, including Chinook (O. tshawytscha), sockeye (O. nerka), chum (O. keta), masou (O. masou) and coho (O. kisutch); and Atlantic salmon (Salmo salar). The disease causes sometimes extremely high economic losses, whether in fresh water populations or seawater fisheries. The clinical disease generally occurs in water at temperatures between 8 and 15°C. It is characterized by nervous system and digestive disorders: alternating apathy and spasmodic movements, darkening of the skin, pale gills and a distended abdomen. Enteritis is evidenced by long, whitish excrement. Autopsy reveals exophthalmos, ascites and haemorrhages in the muscle mass and viscera. The liver, kidney and spleen are pale. The mortality rates associated with the virus can be high. It is almost impossible to distinguish IHN from viral haemorrhagic septicaemia (VHS), another Salmonidae viral infection likewise caused by a rhabdovirus, on the basis of clinical evidence alone. A differential diagnosis obtained by laboratory investigation thus appears to be indispensable.
II – PRINCIPLE OF THE TEST
The gold standard for detection of IHNV is the isolation of the virus in cell culture followed by its immunological or molecular identification. The infected specimens are completely homogenised (either by stomacher, blender or mortar and pestle with sterile sand) and subsequently suspended in an antibioticsupplemented culture medium. The preparation is centrifuged and a 24-well cell culture plate is inoculated with a serial dilution of the supernatant. After 1 hour’s incubation at optimal temperature culture medium is added to each well and the plate is incubated until a cytopathogenic effect is observed. At this point, the plate is frozen. It is ready to be tested by ELISA. The test uses 96-well microtitration plates sensitised by a monoclonal antibody specific for the IHN virus. Rows A, C, E, G have been sensitised with this antibody and rows B, D, F, H contain non specific antibodies. These control rows allow the differentiation between specific immunological reactions and non specific binding so as to eliminate false positives. The supernatants are incubated on the microplate for 1 hour at 21°C +/- 3°C. After this first incubation step, the plate is washed and incubated for 1 hour with the conjugate, a peroxidase labelled anti-IHNV specific monoclonal antibody. After this second incubation, the plate is washed again and thechromogen (tetramethylbenzidine) is added. This chromogen has the advantages of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If IHNV is present in the cell culture supernatant, the conjugate remains bound to the corresponding microwells and the enzyme catalyses the transformation of the colourless chromogen into a pigmented compound. The intensity of the resulting blue colour is proportionate to the titre of IHNV in the supernatant. The enzymatic reaction can be stopped by acidification and the resulting optical density at 450 nm recorded using a photometer. The signals recorded for the negative control microwells are subtracted from the corresponding positive microwells. A positive control is provided with the kit so as to validate the test results. This kit does not crossreact with VHSV.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plates. Rows A, C, E, G are sensitised by anti-IHNV specific antibodies, while rows B, D, F, H are sensitised by the non specific antibodies. – Washing solution: Bottle concentrated washing solution. The solution crystallises spontaneously when cold. If only part of the solution is to be used, bring the bottle to 21°C +/- 3°C until disappearance of all crystals. Mix the solution well and remove the necessary volume. Dilute the buffer 1:20 with distilled or demineralised water. – Conjugate: Vial of coloured conjugate. This solution is ready to use. – Positive Control: The reagent is ready to use. – Single component TMB: Bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from light. This solution is ready to use. – Stopping solution: Bottle of the 1 M phosphoric acid stop solution