7.9 Appendix 1: Sampling Procedures and Laboratories List

Appendix 1A: Brain Sampling Procedures

Entire heads may be submitted fresh or frozen to an approved laboratory.

Obex Harvesting Technique

Recommended tools:

• autopsy knife for disarticulation of the head (if required);
• obex removal spoon or knife; and
• rat tooth forceps.

Additional tools that you may choose to use:

• scalpel; and
• scissors.

The obex removal spoon or knife may be fashioned from the Zwilling J.A. Henckels – Jessica Grapefruit spoon or knife, respectively. Alternatively, any butter knife or suitably long and concave spoon (6 mm concavity) can be modified. The edges of the knife must be sharpened. The edges of the spoon should be cut down to measure 22-26 mm at the widest point and sharpened to a blade.

With the aid of an autopsy knife, disarticulate the head at the level of the atlanto-occipital articulation. Place the head dorsal side down on a table with the foramen magnum (the opening of the spinal canal) facing you.

Using the forceps with your left hand, grasp the dura mater (the thick lining around the spinal cord) and with the scissors in your right hand, make a single cut down the centre line to form two flaps. (This step is not mandatory.) Remove the congealed blood from around the cord.

With the forceps in your left hand holding the dura, sever the cranial nerves from the cord. This can be done with scissors or with the obex spoon/knife by passing the spoon/knife around the cord, keeping the flat side of the spoon/knife against the cord. (See next picture for position of obex spoon/knife in relation to the cord.) This is the most important step in freeing up the cord. The cord must be completely free from attachments (by cranial nerves) in all directions. If you do not sufficiently free up the cord, you will not be able to sample anteriorly enough to get the obex, or one side will be missing.

Brain Sampling Procedures - Image 5

Insert the obex spoon/knife face down into the vertebral canal and move it forward, lodging the tip as far cranially as it will go. This should position the tip of the spoon/knife over the part of the cord just anterior to the obex and just behind the cerebellar attachments.

Brain Sampling Procedures - Image 7

Pick up the head and (if right-handed) hold in left hand, dorsal side down. While pushing down with your index finger, manoeuvre the tip of the obex spoon/knife back and forth over the cord to sever it from the rest of the brain just rostral to the obex and caudal to the cerebellar attachments. Rotate the skull with your other hand in the opposite direction of the spoon/knife to facilitate the cutting of the cord.

Use the obex spoon/knife to gently pull the severed portion of the brain from the canal. The obex is recognizable as a V-shaped depression. This portion is the single most important site for diagnostic testing.

Contact the laboratory to which you will be submitting the samples, and request information on preparing the sample for submission (fresh frozen or forminalized).

Freeze samples and batch ship in a frozen state to an approved laboratory for enzyme-linked immunosorbent assay (ELISA) testing.

Alternatively, place the brain stem and cerebellum in a 500 ml glass jar containing 10% buffered formalin fixative. The ratio of the volume of fixative to tissue should be 10:1. Fix the specimen for 24 hours to five days, depending on the size of the sample (penetration of formalin is at least 5 mm of tissue thickness/day), and then ship to an approved laboratory. These tissues will be subject to histopathology and immunohistochemistry.

Tissues for scrapie surveillance testing may be packaged and shipped as exempt animal specimens in accordance with the Transportation of Dangerous Goods Regulations. The basic four-part packaging system may be used. Following fixation, the specimen must be packaged in a primary watertight receptacle (Whirl-Pak bag) with labelling. Enclose this in a second durable watertight packaging (Whirl-Pak bag) with adequate absorbent material to absorb all fluid in case of breakage. Outer packaging, such as a fibreboard box, protects the contents from external physical damage. The description of the contents on the shipper's waybill should include the notation "exempt animal specimen."

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Appendix 1B: Sampling Procedures for Genotyping

Venipuncture

To facilitate the rapid collection of blood samples from multiple sheep in as little time as possible, it is suggested to not waste time upending the sheep. Restrain sheep for venipuncture in a standing position. Do not clip wool.

Occlude thoracic inlet, palpate or approximate the location of the jugular vein, and insert Vacutainer needle.

A single-use needle is required for each animal, to prevent cross-contamination of deoxyribonucleic acid (DNA).

Collect a full 7-10 ml of blood in an ethylenediaminetetraacetate acid (EDTA) tube (lilac or purple top).

Make sure that the sample and individual animal identification are irrefutably connected.

Samples should be stored at 4°C until shipped. Ship to laboratory as soon as possible.

Check with individual laboratories for preferences regarding sample submission, including preferred day of submission.

Tissue Samples for Genotyping of Deadstock

Fresh or frozen tissues such as brain, liver, spleen, and kidney can be used. Check with individual laboratories for preferences regarding tissue selection. As the head or brain sample is being forwarded for scrapie testing, it would be easiest for the laboratory to forward a sample of brain for genotyping.

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Appendix 1C: Third Eyelid Lymphoid Follicle Sampling Procedures

As potentially infective lymphoid tissue is being harvested and the site of harvesting (the eye) is a potential route of introduction of the scrapie agent, precautions are required to prevent the potential transmission of scrapie when 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 prionicidal. (See 7.9 Appendix 2.)
• Alternatively, the forceps and scissors used on an individual sheep may be placed in a bag, accompanied by the individual sheep identification. These instruments can then be 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.

Verify by checking individual identification that this animal has been genotyped as a 171 QQ.

Instill several drops of a local ophthalmic anaesthetic agent (proparacaine, lidocaine, or xylocaine) into the selected eye.

Apply a drop of 1% histamine solution to the bulbar surface of the third eyelid. This will dramatically facilitate the visualization of the now hyperemic, slightly raised/irregular area that represents the lymphoid follicles.

Note: The histamine solution can be compounded by a veterinary pharmacy, such as the following:

• Veterinary Pharmacy, Imperial Road, Guelph, Ontario, 519-824-7887; or
• Strathcona Prescription Centre 8225-105 Street, Edmonton, Alberta, T6E 4H2, 780-432-0394 or 888-433-2334.

Allow 5-6 minutes for applied drops to take effect.

Grasp the edge of the third eyelid with tissue forceps, evert, and visualize the ventral (bulbar) surface of the third eyelid.

Using scissors (Metzenbaum preferred), blades flush with the third eyelid, cut identified lymphoid follicles from the third eyelid. Place sample in a test tube or a small vial of formalin. Forward sample to a laboratory capable of testing lymphoid tissues for scrapie.

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Appendix 1D: RAMALT Biopsy Sampling Procedures

As potentially infective lymphoid tissue is being harvested, and as the site of harvesting (recto-anal tissue) is theoretically a possible route of introduction 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 prionicidal (7.9 Appendix 2).
• Alternatively, the forceps and scissors used on an individual animal may be placed in a bag accompanied by the individual animal's identification. These instruments may then be stored until the results of the tests are known. If the test results for the individual animal 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 animal 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 animal by holding it against a wall or in a corner.

Verify the individual identification of the animal (sheep or goat). For sheep, also verify by checking individual identification that this animal has been genotyped as a QQ171.

Note: Sheep of all genotypes can be screened using this method.

Anesthetic/analgesic cream (lidocaine and prilocaine) can be applied to the distal rectal mucosa with a gloved finger. An example would be Eutectic Mixture of Local Anesthetics (EMLA), a 1:1 oil/water emulsion of a eutectic mixture of lidocaine and prilocaine based 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.

Allow approximately five minutes for the analgesic/anaesthetic cream to take effect.

Palpate/visualize the recto-anal junction. Take samples approx 1-2 cm anterior (aboral) to the recto-anal 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. Take samples 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.

Note: For more information visit the Veterinary Instrumentation website.

Forward the sample to a laboratory that is capable of testing lymphoid tissues for scrapie.

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Appendix 1E: List of Laboratories

Canadian Laboratories Providing Scrapie Genotyping, Not Currently CFIA-Approved

GenServe Laboratories
Saskatchewan Research Council
125-15 Innovation Boulevard
Saskatoon, Saskatchewan, S7N 2X8
Telephone: 306-933-7700; Toll-free: 1-866-420-2293
Facsimile: 306-933-5505

TransBIOTech
201 Monseigneur-Bourget Drive
Levis, Quebec, G6V 9V6
Telephone: 418-833-8876
Facsimile: 418-833-8867

Idexx Laboratories
1345 Denison Street
Markham, Ontario, L3R 5V2
Telephone (Toll-free): 1-800-667-3411
Facsimile: 905-475-7309

CFIA-Approved Laboratories for Scrapie Genotyping

Animal Health Laboratory
Laboratory Services (University of Guelph)
Ontario Veterinary College
Building 49, Box 3612
Guelph, Ontario, N1H 6R8
Telephone: 519-824-4120, ext. 54544
Facsimile: 519-821-8072

Laboratories approved by the United States Department of Agriculture (USDA) for scrapie genotyping are acceptable to the CFIA Terrestrial Animal Health Division for export and flock certification testing. The current list of USDA-approved labs for scrapie genotyping can be found on the USDA Animal and Plant Health Inspection Service website under Approved Laboratories for Genotype Testing.

Orchid Cellmark (Testing done in United Kingdom)
635 Columbia Street
New Westminster, British Columbia, V3M 1A7
Telephone: 604-523-2945; toll-free: 800-563-4363
Facsimile: 604-523-2974

LABOGENA (Available in French only)
Domaine de Vilvert
78352 Jouy-en-Josas
Cedex, France
Telephone: 01 34 65 21 21
Facsimile: 01 34 65 20 51

CFIA-Approved Laboratories for Scrapie Testing Using the BioRad ELISA

Alberta Agriculture and Rural Development
Agri-Food Laboratories Branch
TSE Laboratory
6909 - 116^th Street
Edmonton, Alberta, T6H 4P2
Telephone: 780-422-4830
Facsimile: 780-415-4527

Animal Health Laboratory
Laboratory Services (University of Guelph)
Ontario Veterinary College
Building 49, Box 3612
Guelph, Ontario, N1H 6R8
Telephone: 519-824-4120, ext. 54544
Facsimile: 519-821-8072

Prairie Diagnostic Services Inc.
52 Campus Drive
Saskatoon, Saskatchewan, S7N 5B4
Telephone: 306-966-7316

Manitoba Agriculture, Food and Rural Initiatives
Veterinary Services Branch
Veterinary Diagnostic Services
545 University Crescent
Winnipeg, Manitoba, R3T 5S6
Telephone: 204-945-8220

Ministère de l'Agriculture des Pêcheries et de l'Alimentation du Québec (MAPAQ) (Available in French only)
Laboratoire d'épidémiosurveillance animale du Québec
3220 Sicotte Street
Saint-Hyacinthe, Quebec, J2S 7X9
Telephone: 450-778-6542
Facsimile: 450-778-6535

Note: Samples may also be submitted through a CFIA district office to CFIA laboratories in Ottawa (Fallowfield), Lethbridge, or Saint-Hyacinthe as part of the National Scrapie Surveillance Program.

CFIA-Approved Laboratories for Scrapie Third Eyelid and RAMALT Biopsy Testing Using Immunohistochemistry

The CFIA Ottawa Laboratory (Fallowfield) is conducting the immunohistochemistry (IHC) test. Arrange for submission and payment for this test through a local district office.

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