BIO K 368
I – INTRODUCTION
The task of determining the cause of an abortion in cattle is generally a rather difficult one because, most of the time, it is the consequence of an event which happened weeks to months earlier. Often also, the foetus is maintained in the uterus for hours and even days after its death, and, when it is finally evacuated, it has undergone autolysis, in such a way that it is difficult to do any type of analysis. Also, many causes of abortion in cattle are to this day still unknown. Moreover, many pathogens are rarely looked for because they are difficult or dangerous to handle (Coxiella burnetii, Chlamydia abortus ..). Pathogens directly or indirectly responsible for abortions are numerous and varied, which complicates the diagnosis. Amongst the major pathogens responsible for abortions, one can find viruses (BoHV-1, BVDV, BoHV-4), bacteria (Brucella abortus, Trueperella pyogenes, colibacille, streptocoque, Coxiella burnetii, Leptospira hardjo, Ureaplasma diversum, Campylobacter foetus, Borrelia coriaceae, Yersinia pseudotuberculosis, Chlamydia abortus, Salmonella, Listeria monocytogenes et Haemophilus somnus), parasites (Distoma hepatica, Trichomonas, Sarcocystis, Neospora), fungi (Aspergillus fumigatus, Mortierella wolfii, as well as Mucor, Absidia, Rhizopus) and yeasts (Candida). The kit aims at demonstrating the existence of a seroconversion toward the pathogenic agents mentioned above, in adult cattle, that it is, in the animal who aborted, but especially in the other animals of the herd, ideally in 10% of the livestock or the cowshed. Indeed, when the abortion occurs, the serological titer of the cow has often reached its maximum and it is not possible to show a seroconversion. It is thus preferable to test the other animals of the herd in order to verify whether the suspected infection is still active. If many animals show a clear seroconversion toward one of the five pathogens of the kit, one can attribute the abortion to this pathogen.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates sensitised by a monoclonal antibody specific to Neospora caninum This antibody is used to trap Neospora caninum as well as to purify it from cultures of this protozoan. For Salmonella dublin and Leptospira hardjo, the plate sensitised by purified LipoPolySaccharide (LPS). For BoHV-4 the plate sensitised by purified virus. For Coxiella burnetii the plate sensitised by phase I and phase II antigenic extract from Coxiella burnetii cells. The distribution of these pathogens on the microtitration plate is as follows:
Columns 1 & 7: BoHV-4
Columns 2 & 8: Neospora caninum
Columns 3 & 9: Coxiella burnetii phase I + II
Columns 4 & 10: Salmonella dublin Columns 5 & 11: Leptospira hardjo
Columns 6 & 12: negative control
Columns 6 & 12 contain one monoclonal antibody.
Using such a control reduces the number of false positives considerably. The test sera and plasma are diluted 1:100 in an appropriate buffer and incubated on the plate for one hour at 21°C +/- 3°C. The plate is washed and the conjugate, a peroxidase-labelled anti-bovine IgG1 monoclonal antibody, is added to the wells. The plate is reincubated at 21°C +/- 3°C for 1 hour. After this second incubation, the preparation is washed and the chromogen (tetramethylbenzidine) is added. This chromogen has the advantage of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If specific immunoglobulins are present in the test sera the conjugate remains bound to the corresponding microwell 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 specific antibody in the sample. The signals recorded for the negative control microwells are subtracted from the corresponding positive microwells. . It is possible to quantify the reactivity of an unknown sample on a scale ranging from 0 to +++++.
III – COMPOSITION OF THE KIT
– Microplates: 96-well microtitration plates. The distribution of the different valencies is indicated on the aluminium wrapper. – 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 the blood sera, plasma and conjugate. Dilute this concentrated dilution buffer 1:5 with distilled or demineralised water. If a deposit forms at the bottom of the container filter the solution on Whatman filter paper. – Conjugate: One bottle of anti-bovine immunoglobulin-peroxidase conjugate (horseradish peroxidaselabelled 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. – Single component TMB: One bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from the light. – Stop solution: One bottle of the 1 M phosphoric acid stop solution.