Appendix 1D - Rectal Biopsy Procedures

As potentially infective lymphoid tissue is being harvested, and as the site of harvesting is theoretically a possible route of introduction (lymphoid tissue) of the scrapie agent, precautions to prevent potential transmission of scrapie are required in sampling potential source flocks. Instruments can be handled in one of the following ways:

• Single use instruments may be used and discarded after use on a single animal.
• Multi-use instruments may be disinfected with a product and protocol known to be priocidal (see appendix 10.2 - Sanitary Precautions/Disinfectants)
• Alternatively, the forceps and scissors used on an individual sheep are placed in a bag accompanied by the individual sheep identification. These instruments are then stored until the results of the tests are known. If the test results for the individual sheep on which the particular set of instruments were used are positive, it is recommended to discard those instruments. If the test results for the individual sheep on which a particular set of instruments were used are negative, those instruments may be routinely disinfected and returned to normal use.

Technique

One person is required to restrain the sheep (e.g. by holding them against a wall or in a corner).

Verify the individual identification of the animal. Sheep of all genotypes can be screened using this method. This method can also be applied to goats.

Anaethestic/analgesic cream (lidocaine/prilocaine) can be applied to the distal, rectal mucosa with a gloved finger. An example would be EMLA (Eutectic Mixture of Local Anesthetics), a 1:1 oil/water emulsion of a eutectic mixture of lidocaine and prilocaine bases cream produced by Astrazeneca Canada Inc. Lidocaine and prilocaine, both solids at room temperature, form a eutectic that is an oil with a 16°C melting point.

If using a rectal speculum, insert into rectum after liberal application of analgesic cream which also doubles as a lubricant.

Palpate/visualize the rectoanal junction. Samples should be taken approx 1-2 cm anterior (aboral) to the rectoanal junction. Preferred locations for sampling include the dorsolateral (10 and 2 o'clock positions) and ventrolateral (4 and 8 o'clock positions) surfaces of the mucosa. Samples should be taken at 10, 2 and, if necessary, 4 and 8 o'clock positions. Avoid sampling the roof or the floor of the mucosa (the 6 and 12 o'clock positions). Using forceps, pick up the rectal mucosa/crypt creating a "tent" of tissue. Using scissors, excise the tent of tissue. Spread the sample on a sponge in a small cassette and place the cassette in a small vial of formalin.

For each LSTS submission, complete CFIA/ACIA 5420 Animal Health Transmissible Spongiform Encephalopathy (TSE) Specimen Submission Form.

Submission Information Section:

• Reason for Test: select disease investigation;
• Disease Category: select scrapie;
• Sample type: select lymphoid tissue;
• Preservative: indicate if the tissue is being submitted as fresh and/or frozen and/or formalin fixed;
• Test Codes: if a selection is required, select code for immunohistochemistry;
• Laboratory: all samples from animals involved in a disease investigation should be submitted to the Ottawa Laboratory Fallowfield (OLF).
• Case Number: A number assigned by the network program specialist for scrapie is to be assigned upon the confirmation of a new index case/premises. This number is to be used on every laboratory submission of animals epidemiologically associated with this index case.
• All other parts of the form should be completed to the best of the inspector's ability.

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