BIO K 362
I – INTRODUCTION
Diarrhoea or scours in piglets can be very common at both the neonatal and the post-weaning stage. It is a common cause of mortality in piglets and is often closely associated with poor hygiene, inappropriate husbandry (e.g., early weaning), stressful environments, and inappropriate feeding factors. Diarrhoea in newborn and weaned piglets is caused mainly by enterotoxigenic Escherichia coli (ETEC) with fimbriae F4 (formerly called K88), F5 (formerly K99), F18, and F41. The diagnosis of the aetiological agents of diarrhoea must be performed in the laboratory because the different micro-organisms cannot be differentiated by the clinical signs. Of these laboratory methods, the ELISA technique is easy to implement, requires little equipment, and is particularly suitable for analysing a large number of samples. The test is fast and reliable and can even be evaluated directly with the naked eye if spectrophotometric equipment is not available. E. coli bacteria can be isolated from faecal samples on an appropriate growth medium. Minca medium is often used because it allows optimal expression of attachment factors F4, F5, F18, and F41. However, culturing may be unsuccessful if the animal has previously been given antibiotics. In this case, the ELISA test can be very useful because it will detect the attachment factors even on dead bacteria. Moreover, when the ELISA test for detecting attachment factors F4, F5, F18, and F41 is performed directly on faecal samples, it will give more reliable results than isolating the bacteria on a culture medium because it allows quantification of the attachment factors F4, F5, F18, and F41 in the sample rather than simply indicating the presence or absence of the factors in a limited number of isolated strains. Be that as it may, identifying E. coli bacteria on a growth medium is not sufficient on its own. It must be coupled with a detection test for attachment factors F4, F5, F18, F41 or toxins. The ELISA test can be used to detect the attachment factors F4, F5, F18, and F41 produced by E. coli bacteria in culture.
II – PRINCIPLE OF THE TEST
The test uses 96-well microtitration plates in which rows A, C, E, and G have been sensitised by specific antibodies for the pathogens sought. These antibodies allow specific capture of these pathogens in the samples (faecal material) being tested. Rows B, D, F, and H have been sensitised with antibodies that are not specific for these pathogens. These negative controls enable you to determine specific binding on the microplate. Using them limits greatly the proportion of false positive results. The faecal material is diluted in dilution buffer and incubated on the microplate for 1 hour at 21 +/- 3 °C. After this first incubation step, the plate is washed, and then conjugates are added to the wells. These conjugates are peroxidase-labelled monoclonal antibodies that are specific for the pathogenic agents. The plate is then incubated a second time for 1 hour at 21 +/- 3 °C and washed once more, after which the detection solution, single component TMB (tetramethylbenzidine), is added. This chromogen has the advantage of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If one or more of the specific pathogens sought are present in the sample, the corresponding conjugate or conjugates remain bound to the corresponding microwells and the enzyme catalyses the transformation of the colourless chromogen into a blue compound. The intensity of the colour is proportionate to the titre of the specific pathogen(s) in the sample. The signals recorded for the negative control microwells are subtracted from the corresponding positive microwells’ optical densities. A positive control is provided with the kit to validate the test results.
III – COMPOSITION OF THE KIT
– Microplates: Two 96-well microtitration plates. The capture antibodies are distributed on the plates as follows:
Row A: anti- E. coli F4
Row B: control
Row C: anti- E. coli F5
Row D: control
Row E: anti-E. coli F18
Row F: control
Row G: anti- E. coli F41
Row H: control
– Washing solution: bottle of 20-fold concentrated washing solution. The solution crystallises spontaneously when cold. If only part of the solution is to be used, bring the bottle to 21 +/- 3 °C until all crystals disappear, mix the solution well, and remove the necessary volume. Dilute the buffer twentyfold with distilled or demineralised water.
– Dilution buffer: bottle of coloured, 5-fold concentrated buffer for diluting samples. If only part of the solution is to be used, mix it well and remove the necessary volume. Dilute this buffer fivefold with distilled or demineralised water. If a deposit forms at the bottom of the container, filter the solution on Whatman filter paper.
– Conjugates: vials of conjugates, each identified by a specific colour: E. coli F4 (yellow), E. coli F5 (Blue), E. coli F18 (purple), and E. coli F41 (orange).
– Each conjugate’s specificity is shown on the bottle. These solutions are ready to use.
– Positive Control: The reagent is ready to use.
– Single component TMB solution: bottle of the chromogen tetramethylbenzidine (TMB). This reagent must be stored in the dark at a temperature between +2 and +8 °C. It is ready to use.
– Stopping solution: bottle of stop solution containing 1 M phosphoric acid .