Commission Regulation (EU) No 206/2010Show full title

[X1 Foot-and-mouth disease (FMD) U.K.

A. Collecting oesophageal/pharyngeal samples and testing shall be carried out according to the following protocol: U.K.

Reagents

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Prior to sampling, transport medium is prepared. Two ml volumes are dispensed in as many containers as there are animals to be sampled. The containers used must withstand freezing over solid CO ₂ or liquid nitrogen. Samples are obtained by the use of a specially-designed sputum collector or ‘probang’. To obtain a sample the probang cup is passed through the mouth, over the dorsum of the tongue and down into the upper part of the oesophagus. Attempts are made to scrape the surface epithelium of the upper oesophagus and pharynx by movements directed laterally and dorsally. The probang is then withdrawn, if possible after the animal has swallowed. The cup must be full and contain a mixture of mucus, saliva, oesophageal fluid and cellular debris. Care must be taken to ensure that each specimen contains some visible cellular material. Very rough handling which causes bleeding must be avoided. Samples from some animals may be heavily contaminated with ruminal contents. Such samples must be discarded and the mouth of the animal flushed with water, or preferably physiological saline, before repeat sampling.

Treatmentof samples:

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Each sample collected in the probang cup is examined for quality and 2 ml added to an equal volume of transport medium in a container which can withstand freezing. The containers are tightly closed, sealed, disinfected and labelled. The samples are kept cool (+ 4 °C) and examined within three to four hours or placed over dry ice (- 69 °C) or liquid nitrogen and kept frozen until examined. Between animals the probang is disinfected and washed in three changes of clean water.

Testing for FMD virus:

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Samples are inoculated into cultures of primary bovine thyroid cell cultures using at least three tubes per sample. Other susceptible cells such as primary bovine or porcine kidney cells can be used but it must be kept in mind that for some strains of FMD virus they are less sensitive. The tubes are incubated at 37 °C on a roller apparatus and examined daily for 48 hours for the presence of a cytopathic effect (CPE). If negative, cultures are blind passaged onto new cultures and re-examined for 48 hours. The specificity of any CPE must be confirmed.

Recommended transport media:

1.

0,08M phosphate buffer pH 7,2 containing 0,01 % bovine serum albumin, 0,002 % phenol red and antibiotics.

2.

Tissue culture medium (such as Eagle's MEM) containing 0,04 M Hepes buffer, 0,01 % bovine serum albumin and antibiotics, pH 7,2.

3.

Antibiotics (per ml final) must be added to the transport medium such as penicillin 1 000 IU, neomycin sulphate100 IU, polymyxin B sulphate50 IU, mycostatin100 IU.

B. The virus neutralisation test shall be carried out according to the following protocol: U.K.

Reagents

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Stock FMDV antigen is prepared in cell cultures or on cattle tongues and stored at - 70 °C or less or at - 20 °C after the addition of 50 % glycerol. This is the stock antigen. FMDV is stable under these conditions and titres vary little over a period of months.

Procedure

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The test is carried out in flat-bottomed tissue culture grade microtitre plates using susceptible cells such as IB-RS-2, BHK-21 or calf kidney cells. Sera for the test are diluted 1/4 in serum-free cell culture medium with the addition of 100 IU/ml neomycin or other suitable antibiotics. Sera are inactivated at 56 °C for 30 minutes and 0.05 ml amounts are used to prepare a twofold series on microtitre plates using 0,05 ml diluting loops. Pre-titrated virus also diluted in serum-free culture medium and containing 100 TCID50/0.05 ml is then added to each well. Following incubation at 37 °C for one hour to allow neutralisation to take place, 0,05 ml of suspension cells containing 0,5 to 1.0 × 10 ⁶ cells per 1 ml in cell culture medium containing serum free of FMD antibody is added to each well and the plates are sealed. Plates are incubated at 37 °C. Monolayers are normally confluent within 24 hours. CPE is usually sufficiently advanced at 48 hours for a microscopic reading of the test. At this time a final microscopic reading may be made or the plates may be fixed and stained for macroscopic reading, for instance using 10 % formol-saline and 0,05 % methylene blue.

Controls

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Controls in each test include homologous antiserum of known titre, a cell control, a serum toxicity control, a medium control, and a virus titration from which the actual amount of virus in the test is calculated.

Interpretation

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Wells with evidence of CPE are considered to be infected and neutralisation titres are expressed as the reciprocal of the final dilution of serum present in the serum/virus mixtures at the 50 % end point estimated according to the Spearman-Karber method. (Karber, G., 1931, Archiv fuer Experimentelle Pathologie und Pharmokologie, 162, 480.). Tests are considered to be valid when the actual amount of virus used per well in the test is between 101,5 and 102,5 TCID50 and when the titre of the reference serum is within twofold of its expected titre, estimated from the mode of previous titrations. When the controls are outside these limits the tests are repeated. An end point titre of 1/11 or less is taken as negative.

C. The detection and quantification of antibody by ELISA shall be carried out according to the following protocol: U.K.

Reagents

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Rabbit antisera to 146S antigen of seven types of foot-and-mouth disease virus (FMDV) used at a predetermined optimum concentration in carbonate/bicarbonate buffer, pH 9,6. Antigens are prepared from selected strains of virus grown on monolayers of BHK-21 cells. The unpurified supernatants are used and pretitrated according to the protocol but without serum, to give a dilution which after the addition of an equal volume of PBST (phosphate buffered saline containing 0,05 % Tween-20 and phenol red indicator) would give an optical density reading of between 1,2 and 1,5. The viruses can be used inactivated. PBST is used as a diluent. Guinea-pig antisera are prepared by inoculating guinea pigs with 146S antigen of each serotype. A predetermined optimum concentration is prepared in PBST containing 10 % normal bovine serum and 5 % normal rabbit serum. Rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is used at a predetermined optimum concentration in PBST containing 10 % normal bovine serum and 5 % normal rabbit serum. Test sera are diluted in PBST.

Procedure: U.K.

1.

ELISA plates are coated with 50 μl of rabbit antiviral sera overnight in a humidity chamber at room temperature.

2.

Fifty microlitres of a duplicate, twofold series of each test serum starting at 1/4 are prepared in U-bottomed multiwell plates (carrier plates). Fifty microlitres of a constant dose of antigen are added to each well and the mixtures are left overnight at 4 °C. The addition of the antigen reduces the starting serum dilution to 1/8.

3.

The ELISA plates are washed five times with PBST.

4.

Fifty microlitres of serum/antigen mixtures are then transferred from the carrier plates to the rabbit-serum-coated ELISA plates and incubated at 37 °C for one hour on a rotary shaker.

5.

After washing, 50 μl of guinea-pig antiserum to the antigen used in point 4 is added to each well. The plates are incubated at 37 °C for one hour a rotary shaker.

6.

The plates are washed and 50 μl of rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is added to each well. The plates are incubated at 37 °C for one hour on a rotary shaker.

7.

The plates are washed and 50 μl of orthophenylene diamine containing 0,05 % H ₂ O ₂ (30 %) w/v is added to each well.

8.

The reaction is stopped after 15 minutes with 1,25M H ₂ SO ₄ .

The plates are read spectrophotometrically at 492 nm on an ELISA reader linked to a microcomputer.

Controls

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For each antigen used 40 wells contain no serum but contain antigen diluted in PBST. A duplicated twofold dilution series of homologous bovine reference antiserum. A duplicate twofold dilution series of negative bovine serum.

Interpretation

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Antibody titres are expressed as the final dilution of tests serum giving 50 % of the mean OD value recorded in the virus control wells where test serum is absent. Titres in excess of 1/40 are considered positive.

References

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Hamblin C, Barnett ITR and Hedger RS (1986) ‘ A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA. ’ Journal of Immunological Methods, 93, 115 to 121.11. ]

Editorial Information

X1 Substituted by Corrigendum to Commission Regulation (EU) No 206/2010 of 12 March 2010 laying down lists of third countries, territories or parts thereof authorised for the introduction into the European Union of certain animals and fresh meat and the veterinary certification requirements (Official Journal of the European Union L 73 of 20 March 2010).