BIO K 369
I – INTRODUCTION
Respiratory disorders are of major concern for bovidae, given the frequency of such infections and the high number of animals affected. These infections occur in all countries that practice intensive livestock farming in which large numbers of animals are confined to small areas. Treatment and diagnosis are both complicated due to the multifactorial character of this diseases etiology. Viruses and bacteria combined with stress due either to transport in overcrowded vans or dirty or poorly ventilated stabling, for instance, play a key role in triggering acute respiratory infections. These infections are particularly common among young animals, although they also affect adult animals. In most cases the animals that show signs of respiratory ailments harbour several pathogens, some of which may act synergistically. So, it is generally recognised that viruses are the first pathogens to intervene, whereas bacteria act as second invaders to worsen the animal’s condition. Shipping fever is a good example of the synergism that can exist between a virus (PI3) and a bacterium, such as Mannheimia haemolytica, in the respiratory tract. The BIO-X RESPIRATORY ELISA kit consequently enables one to evaluate the humoral immune response of cattle to five pathogens commonly implicated in bovine respiratory infections. These are bovine respiratory syncytial virus (BRSV), parainfluenza 3 virus (BPI3), adenovirus type 3, Mycoplasma bovis and Mannheimia haemolytica.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates sensitised by monoclonal antibodies specific to the three pathogens (BRSV, BPI3 and adenovirus). These antibodies are used to trap the pathogens as well as to purify them from lysates of the cells in which the viruses were grown. For Mycoplasma bovis, the plate sensitised by a recombinant protein from Mycoplasma bovis expressed by E. coli. A gene from Mycoplasma bovis is expressed by this recombinant E.coli culture. For Mannheimia haemolytica the plate sensitised by purified LipoPolySaccharide (LPS). The distribution of these pathogens on the microtitration plate is as follows:
Columns 1 & 7: BRSV
Columns 2 & 8: BPI3
Columns 3 & 9: Mycoplasma bovis
Columns 4 & 10: Mannheimia haemolytica
Columns 5 & 11: Adenovirus 3
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.