AVIS - Foot and Mouth Disease

Laboratory Tests

Virus/Antigen Detection

Virus Isolation in Tissue Culture

Disinfectant solution 0.2% (w/v) citric acid
Dissolve 2 g of citric acid in 1 litre of water.

Eagle's Minimum Essential Medium (Eagle's MEM)
This can be formulated according to standard tissue culture protocols or, alternatively, can be supplied complete as Minimum Essential Medium Eagle (HEPES Modification), from Sigma Ltd (500 ml).

Sample buffer
0.04 M phosphate buffer, pH 7.4 +/-0.05

NaH₂PO₄.2H₂O 6.1 g
KH₂PO₄ 0.78 g
make up to 1 litre with sterile distilled water and check pH

Tissue culture
Appropriate cell cultures, grown as monolayers in tubes or flasks, should be prepared as required in a dedicated tissue culture facility. Description of tissue culture methodology and procedures is beyond the scope of this module.

Preparation of test samples

Prepare a 10% suspension of epithelium as follows.

1. Place approximately 3 ml of sample buffer in a small bottle and weigh accurately to two decimal places.

2. Remove a piece of epithelium (approximately 0.5 g) from the transport medium, dab dry on tissue paper and then place in the pre-weighed bottle.

3. Measure and record the pH of the original transport medium using either pH strips or by adding a few drops into an indicator medium. This is a useful indication as to whether or not the medium was suitable for maintaining the viability of any FMD virus that might be present.

4. Calculate the weight of the piece of epithelium by subtracting the weight of the bottle containing medium alone from the weight of the bottle plus epithelium. If necessary remove or add more epithelium until approximately 0.5 g is present.

5. Remove the epithelium, dab dry, and grind in the mortar with a little sand.

6. Add sufficient sample buffer to make a 10% suspension e.g., 0.5 g of tissue in 5 ml of buffer.

7. Transfer ground suspension to a small bottle and centrifuge in a bench centrifuge at approximately 1000 g (maximum rpm) for 10 minutes.

8. Harvest the clarified supernatant fluid into a small glass bottle. Place 1.5 ml (minimum) of this original sample suspension into a bottle for subsequent testing in the antigen detection ELISA.