I – INTRODUCTION
Neospora caninum is a protozoon that was originally described as a parasite in dogs, in which it causes myositis and encephalitis. Bovine neosporosis is now recognised as a major cause of spontaneous abortion in cattle. It is highly suspected on 20% of the farms with repeated abortions and a cow that is seropositive for Neospora caninum has a threefold greater risk of aborting than a cow that is Neospora-negative. Neospora is responsible for 21% of spontaneous abortions occurring in an individual animal. This percentage rises to 33% for the herd as a whole. Verticle transmission is the rule (at least 80% of the calves born to seropositive cows are infected). Serotesting before the calf’s first colostrum intake will reveal prenatal infection. As it is a blocking test, it can be used in all animal species.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates sensitised by SRS2 Neospora caninum E. coli recombinant protein. The whole plate is coated with the recombinant protein. The operator deposits the previously diluted test sera and plasma in the microplate’s wells. After 2 hours’ incubation and a rinse step, the operator adds the conjugate, which is a specific monoclonal antibody against SRS2 protein coupled to peroxidase. After incubating and washing the preparation, the operator adds the chromogen tetramethylbenzidine (TMB). This chromogen has the advantage of being more sensitive than the other peroxidase chromogens and not being carcinogenic. The intensity of the colour is inversely proportionate to the sample’s serum titre. Positive and negative control sera are provided with the kit to be able to validate the test results.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plate (12 x 8). The entire surface of each microplate has been sensitised with SRS2 protein.
– Washing solution: One 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.
-Dilution buffer: One bottle of 5X colored, concentrated buffer for diluting samples and conjugate. The bottle’s content is to be diluted with distilled or demineralised water. If a deposit forms at the bottom of the container filter the solution on Whatman filter paper.
– Conjugate: One vial of anti- SRS2 protein peroxidase conjugate (horseradish peroxidase-labelled anti- SRS2 protein monoclonal antibody).
– Positive reference: One bottle of positive serum. Store this reagent between +2°C and +8°C.
– Negative reference: One bottle of negative serum. Store this reagent between +2°C and +8°C.
– Single component TMB: One bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from light. This solution is ready to use.
– Stop solution: One bottle of the 1 M phosphoric acid stop solution.