BIO K 275
I – INTRODUCTION
Spring viraemia of carp is a contagious viral disease of the Cyprinidae. Other species, such as the sheatfish (Silurus glanis), are also sensitive to this virus. The cause of the disease is a rhabdovirus. Generally, young fish up to one year old are most susceptible to clinical disease, but all groups can be affected. The disease carries with it a high mortality rate. The clinical signs of contamination are petechial haemorrhages of the skin and gills, dark coloring of the tegument, exophthalmia and a distended abdomen. Loss of balance is also seen in diseased fish. The internal lesions are characterised by petechial haemorrhages of the viscera, fibrinous peritonitis, and catarrhal or necrotic enteritis. While the serological traces of a Rhabdovirus infection indicate that serology may be a valid alternative for studying the health status of a carp population, laboratory diagnosis of the disease usually involves identification of the virus in cell cultures.
II – PRINCIPLE OF THE TEST
The infected specimens are ground up in a mortar with the help of sand, then put in solution in an antibioticsupplemented culture medium. It is also possible to use stomacher or blender. 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 SVC virus. Rows A, C, E, G have been sensitised with these antibodies 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-SVCV specific monoclonal antibody. 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 SVCV 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 SVCV 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 antigen is provided with the kit so as to validate the test results.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plates (12 X 8). Rows A, C, E, G are sensitised by anti-SVCV 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.