# 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 (Text with EEA relevance), Division A

Extent: E+W+S+N.I.

Collecting oesophageal/pharyngeal samples and testing shall be carried out according to the following protocol:
Reagents 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 The probang is then withdrawn, if possible after the animal has swallowed.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: 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: 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.

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Source: https://www.legislation.gov.uk/eur/2010/206/annex/I/part/6/division/8/division/A (the official version)
Page: https://openlegislation.uk/eu-regulation-2010-206/annex-I/part-6/division-8/division-A
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.