Info r mati o n p r o vided b y S ENASA for the r eco g niti o n o f Ar g e n t i n a  ́s Patagonia as a Re g ion com p rised in Article 9 2

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# NATIONAL ANIMAL HEALTH AND AGRIFOOD QUALITY SERVICE



# APPENDIX II : WHAT ARE THE REQUIRED PROCEDURES FOR SPECIMEN COLLECTION?

April 200 3Info r mati o n p r o vided b y S ENASA for the r eco g niti o n o f Ar g e n t i n a  ́s Patagonia as a Re g ion com p rised in Article 9 2

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### For each relevant hazard, is the pest or disease agent known to exist in the region? The required procedures for specimen collection are described in the following section:
Epithelium sample submission:

• Samples must be obtained from the most recent lingual or podal vesicles; if the vesicle has to be broken is even better. Check the necessary animals in order to obtain enough samples. The better the quality and quantity of the remitted material, the better the chances for a quick diagnosis.

• Once the sample was obtained, place it into the provided plastic jar with Vallee medium; label it indicating the Sample Remission Protocol number. When sending more than one jar, please identify each one of them with the protocol number and correlative jar number.

• Vallee medium must be pink. If it turned yellow, discard it.

• Close the jar and seal it with adhesive or masking tape.

• After the jar is closed, disinfect the external part of the jar using a proper disinfectant (0.2% citric acid), if not available, use pure vinegar.

• Then, wrap the jar in cotton or any other absorbent material.

• Place the jar into a rigid and unbreakable container with a thread or pressure lid (powdered milk can or similar, plastic container, tupperware, etc)

• Use cotton or paper to fill any remaining empty space to avoid movement of the sample.Info r mati o n p r o vided b y S ENASA for the r eco g niti o n o f Ar g e n t i n a as a Re g ion com p rised in Article 9 2

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### • Put the container into a Styrofoam box with enough ice-packs to keep it cold for the shipping time.

• Close the box, seal the top using masking or adhesive tape.

• In the external part of the box paste a plastic envelope with the pertinentprotocol. Place the label with the “BIOHAZARD” sign and indicating: “EPITHELIUM”, its destination (Virología - Fiebre Aftosa) and the sender data.

Do not place into the same box any other type of sample (serum).
Serum sample remission
The serum sample remitted must be placed into a Styrofoam box, with the tubes correctly identified and placed in such way to avoid damage and leaks.
Put into the box enough ice-packs to keep it cold during the shipping time, close it and seal it. In the external part of the box paste the plastic envelope with a protocol photocopy indicating the species . Label the box with the “BIOHAZARD” sign, indicating that it is “ SERUM ” and its destination (Virología- Fiebre Aftosa) as well as the sender data.

Each sample must be sent in its pertinent box. Send serum and epithelium in separated boxes, attaching the protocols to the box using plastic envelopes. Never send serum with epithelium fo r FMD diagnosis in the same box.Info r mati o n p r o vided b y S ENASA for the r eco g niti o n o f Ar g e n t i n a as a Re g ion com p rised in Article 9 2

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### Please indicate in all cases: District (town, place) from where the sample is shipped. Transport company. Number of shipping document. Estimated day and time of arrival.

Use “BIOHAZARD” labels only when deliverin g samples of vesi cular disease suspect, or when the remitted material needs to be so identified.

The computerized sample reception syst em will reject any remission that does not comply with these requirements.

Oesophageal-pharyngeal liquid (OPL) remission Equipment:

• Eagle medium.

• Probang cups.

• 2 plastic buckets.

• Sterile plastic jars for sample collection (one for each animal).

• Styrofoam coolers.

• Freezed ice packs, -20° C (not from refrigerator or dry ice).

• Coveralls.

• Boots

• Washcloths.Info r mati o n p r o vided b y S ENASA for the r eco g niti o n o f Ar g e n t i n a as a Re g ion com p rised in Article 9 2

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### • Protocolized sample remission forms.

• Disinfectant (1% acetic acid or sodium carbonate).

Animals Preparation Animals to be sampled must fast 12 hours (food and water). Supply them water one hour before sample collection to avoid food presence in the sample and to lubricate the digestive tract. It is recommended to collect samples in the morning. Sample collectionThe animal must be restrained. Get the sample of pharynge and the anterior portion of the esophagus with the adequate size Probang. To do this, put the cup in the mouth and, making a slight pressure, allow the animal to swallow it by moving the cup up and down, three or four times (more times can stimulate regurgitation, which is not advisable). Remove the cup avoiding any trauma and bleeding. Keepthe animal’s mouth open gently without spoiling its content. If regurgitation happens, a small amount of water can be used to stimulate swallowing before a tryingto get a new sample. The use of disinfectant is not recommended because residues can remain in the cup that may inactivate the virus. After sampling disinfect all the equipment (cups, boots, etc, as well as the vehicle before leaving the premises) using 1% acetic acid. Preparation of the sampleInfo r mati o n p r o vided b y S ENASA for the r eco g niti o n o f Ar g e n t i n a as a Re g ion com p rised in Article 9 2

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### Mix the sample collected with an equivalent volume of the transport solution (Eagle medium); close it and seal it to avoid leaking and identify the jar. Shake vigorously and freeze using a jar with dry ice moving the material for a uniform freezing.

VERY IMPORTANT: If the period elapsed from sample collection to the arrival to the laboratory is NOT more than 24 hs the sample can go refrigerated (use -20° C freeze ice-packs). On the contrary dry ice must always be used.

Boxes prepared to be sentPrepare the boxes just before sending them. If the sample goes refrigerated, include several ice packs. Close the box with adhesive tape. In the external part of the box, attach the protocol in a plastic envelope, place a Biohazard label indicating that “IT CONTAINS OESOPHAG EAL-PHARYNGEAL LIQUID SAMPLE” as well as the destination address (Virología- Fiebre Aftosa) and sender information. OPL samples to perform PCR Put into tubes with 5 ml of Trizol (monophasic solution of phenol and guanidine isothiocyanate), only 2 ml of the OPL sample. Put it in Styrofoam box with enough ice-packs. Seal the tube lids with adhesive tape and place them in a vertical position to avoid leaking. Send samples for Probang and samples for PCR in separated boxes.

Warning: Trizol is toxic, handle it using latex gloves.Info r mati o n p r o vided b y S ENASA for the r eco g niti o n o f Ar g e n t i n a as a Re g ion com p rised in Article 9 2

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### In the external part of the box, attach the protocol in a plastic envelope, place a biohazard label indicating that “IT CONTAINS OESOPHAG EAL-PHARYNGEAL LIQUID SAMPLE WITH TRIZOL” as well as the destination address (Virología Fiebre Aftosa) and sender information.