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.
II – PRINCIPLE OF THE TEST The test uses 96-well microtitration plates sensitised by a purified Neospora caninum protein. The plate’s odd columns (1, 3, 5, 7, 9 and 11) contain the purified protein, whereas the even columns (2, 4, 6, 8, 10 and 12) contain a control antigen. We thus have a genuine negative control. Using such a control reduces the number of false positives considerably. The test blood sera, plasma or milks are diluted in the buffer for dilution. The plate is incubated and washed, then the conjugate, a peroxidase-labelled anti-bovine IgG1 monoclonal antibody, is added to the wells. The plate is then incubated a second time at 21°C +/- 3°C washed again and the chromogen tetramethylbenzidine (TMB) is added. This chromogen has the advantages of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If specific anti-Neospora caninum immunoglobulins are present in the test sera or milks the conjugate remains bound to the microwell that contains the protozoon and the enzyme catalyses the transformation of the colorless chromogen into a pigmented compound. The intensity of the resulting blue colour is proportionate to the titre of specific antibody in the sample. The signal read off the negative control microwell is subtracted from that of the positive microwell sensitised by the protozoon protein.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plates (6 strips of 16 wells). The odd columns (1, 3, 5, 7, 9 and 11) are sensitised by purified protein from Neospora caninum and the even columns (2, 4, 6, 8, 10 and 12) by the control antigen. – 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 all crystals have disappeared. 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 the blood sera, plasma milks and conjugate. The bottle’s content is to be diluted with distilled or demineralised water. If a deposit forms at the bottom of the receptacle filter the solution on Whatman filter paper. – Conjugate: 1 bottle of anti-bovine immunoglobulin-peroxidase conjugate (horseradish peroxidase-labelled anti-bovine IgG1 monoclonal antibody). – Positive serum: One bottle of positive serum. Store this reagent between +2°C and +8°C – Negative serum: One bottle of negative serum. Store this reagent between +2°C and +8°C. – Tracer: One bottle of tracer. Store this reagent between +2°C and +8°C. The tracer is a reference sample that can be used to check the intra-laboratory reproducibility of the kit’s batch. Intra-laboratory reproducibility: Degree of agreement between the results of reiterated tests on the same sample with an identical technical protocol in a given laboratory under variable working conditions. – 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.