BIO K 264
Viral haemorrhagic septicaemia (VHS) is a disease of farmed rainbow trout, farmed turbot, farmed Japanese flounder as well as several wild freshwater and marine species caused by VHSV rhabdovirus. Disease generally occurs at temperature between 4°C and 14°C. At water temperature between 15°C and 18°C, the disease generally takes a short course with a modest accumulated mortality. Disease rarely occurs at higher temperatures. VHS outbreaks occur during all seasons, but are most common in spring when water temperatures are rising or fluctuating. The clinical signs of the disease are high mortality (which can reach up to 100% in fry), especially during the young trout’s first winter. The subjects exhibit lethargy, melanosis and exophthalmia. The paleness of their gills reflects their anaemic condition. An autopsy will reveal the presence of numerous sites of haemorrhages in the viscera and muscle mass, distended abdomen due to oedema in the peritoneal cavity. VHS can also occur in a nervous form, characterised by severe abnormal swimming behaviour, such as constant flashing and/or spiralling. IHN is a viral disease caused by a rhabdovirus. It affect most salmonid species, especially the fry and young fish. Susceptible species include: rainbow or steelhead trout (Oncorhynchus mykiss), 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 clinical disease generally occurs in water at temperature 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 distended abdomen. Enteritis is evidenced by long, whitish excrement. Autopsy reveals exophtalmia, 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 VHS on the basis of clinical evidence alone. A differential diagnosis obtained by laboratory investigation thus appears to be indispensable. The VHS-IHN ELISA test confirms the virus’s growth on a susceptible cell line.
II – PRINCIPLE OF THE TEST
The gold standard for detection of IHNV and VHSV 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 specific antibodies for the VHS and IHN viruses. Rows A and E have been sensitised with anti-VHSV, rows C and G with anti-IHNV 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 conjugates, peroxidase labelled anti-VHSV and anti-IHNV specific monoclonal antibodies. After this second incubation, the plate is washed again and the chromogen (tetramethylbenzidine) is added. This chromogen has the advantages of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If VHSV or IHNV are present in the cell culture supernatant, the conjugates remain 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 VHSV or 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. Control antigen is provided with the kit so as to validate the test results.
III – COMPOSITION OF THE KIT
– Microplates: Two 96-well microtitration plates. The rows A, C, E, G are sensitised by specific antibodies, the rows B, D, F, H by non specific antibodies.
Row A: anti-VHSV
Row B: control
Row C: anti-IHNV Row D: control
Row E: anti-VHSV Row F: control
Row G: anti-IHNV
Row H: control
– Washing solution: One 100 ml bottle of 20x 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.
– Conjugates: Two 12 ml vials of colored conjugates. These solutions are ready to use. VHSV (red), IHNV (blue).
– Positive Controls: 2 vials of 2 ml colored controls. VHSV (red), IHNV (blue). These solutions are ready to use.
– Single component TMB One 25-ml bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from light. This solution is ready to use.
– Stopping solution: One 15-ml bottle of the 1 M phosphoric acid stop solution.