3. Testing

3.1 Tuberculosis Testing

Tuberculin Tests - Background Information

1. For many decades, intradermal tuberculin testing has been the most commonly used diagnostic procedure for detecting bovine tuberculosis in live animals. The basis of tuberculin testing is the induction of a delayed hypersensitivity reaction to the intradermal injection of tuberculin, a protein purified derivative of a laboratory culture of Mycobacterium bovis (M. bovis). A positive reaction is manifested by erythema and induration at the injection site appearing at 8-12 hours post injection, and peaking between two (2) and seven (7) days.

2. Factors which may influence the sensitivity and specificity of the test include:

1. type of tuberculin, (bovine, avian, or human);
2. site of injection (cervical or caudal);
3. quantity of tuberculin used;
4. late pregnancy/postpartum;
5. state of nutrition; and
6. whether or not the animal is infected.

3. Some cattle infected with M. bovis fail to respond to the injection of tuberculin (false negatives). The number of cattle in this category is a measure of the sensitivity of the particular test, sensitivity being the probability of the test correctly identifying as positive those cattle which are truly positive. False negative responses may be observed in cattle which are debilitated, in the very early or late stages of the disease, postpartum, or otherwise stressed, or cattle desensitized by tuberculin injected within the previous 8-60 days.

4. Similarly, some cattle not infected with M. bovis respond to the injection of tuberculin (false positive). The number of cattle in this category is a measure of the specificity of the test, being the probability of the test correctly identifying as negative those individuals not infected. Most false positive responses in cattle are due to the animal having been exposed to antigens similar to or shared with those in the tuberculin used in the test.

Equipment and Supplies

1. Bovine PPD tuberculin provided by the CFIA district office - Store in a cool (4°C) dark place and do not use after the expiry date.
2. 1 ml disposable tuberculin syringe with a needle calibre 26 of 3/8" - Multiple-dose syringes are not to be used. To preserve tuberculin potency and maximise test accuracy, syringes are to be loaded with 0.1 ml of tuberculin just prior to making the injection, and are not to be pre-loaded. A new needle is required for each injection.
3. Absorbent cotton.
4. Form CFIA/ACIA 1524-Report of Intradermal Test on which to record the test and the animal identification.

Recording and Reporting Tuberculin Tests

5. All tuberculin tests are official and must be recorded and reported to a CFIA District office by one of the following methods:

1. Fully completed Form CFIA/ACIA 1524, including animal information for each animal tested. A copy of the completed Form CFIA/ACIA 1524 accompanies the health certificate or AI Unit Entry Form to a CFIA District Office where the certificate is to be endorsed.
2. Partially completed Form CFIA/ACIA 1524 as follows and attach an Animal List.
   1. Complete all parts of Form CFIA/ACIA 1524 (except the animal information section) including the signature block where the accredited veterinarian certified that he/she, tested the animals described on date and at location shown.
   2. Attach to Form CFIA/ACIA 1524 a list of animals that were TB tested, were found negative and are being presented for export certification. In the animal information section of the Form CFIA/ACIA 1524, insert a statement that reads The (insert # of animals) (insert species) listed on attached list were tested on the date shown above and at the location shown above with negative results. The accredited veterinarian should sign and date the list.
   3. Any animals with non-negative results must be included in the animal information section of Form CFIA/ACIA 1524.
   4. A copy of the completed Form CFIA/ACIA 1524 with its completed attached list accompanies health certificate of AI Unit Entry Form to the District Office where the certificate is to be endorsed.

Caudal Fold Tuberculin Test

6. The caudal fold tuberculin test is used for routine screening and diagnostic or export tests of cattle, bison, sheep, or goats at low risk of having tuberculosis.

7. The test is an intradermal injection of 0.1 ml of bovine purified protein derivative (PPD) tuberculin in the caudal fold of cattle, bison, sheep, or goats with observation and palpation at 72 ± 6 hours. Animals cannot be tested if they have been injected with tuberculin within the previous 60 days.

8. Do not conduct the test without having the animal adequately restrained. Restrain all animals sufficiently to permit the accurate identification of the animal and proper injection of tuberculin. Both confirmation of identification of the animal and palpation of the injection site are required to interpret the test results. The veterinarian performing the injection must interpret the tuberculin test result.

9. The injection site is the caudal fold, distal to the base of the tail, well away from the hairline, in the centre of the fold. Note any abnormalities found near the injection site on the test record so that they will not be mistaken for tuberculin responses. Either left or right fold may be used, but be consistent and always record which fold was injected.

10. Clean the caudal fold with dry cotton or cotton dipped in saline water. (Never use alcohol). Inject 0.1 ml of tuberculin intradermally, lifting the tip of the needle slightly after insertion to ensure that it is clearly outlined just under the skin. Following the injection, the injected tuberculin should be visible as a palpable bleb. Use a fresh sterile needle for each animal tested.

11. Observe and palpate the injection site at 72 ± 6 hours post injection. Raise the tail to stretch the caudal fold slightly and palpate the length of the caudal fold with the thumb and index finger of the other hand. Record test results on the Form CFIA/ACIA 1524 as follows:

1. If there is no change in the tissue at the point of injection record the result as negative.
2. If there is any change in the tissue at the point of injection, record the result as Reactor and notify the district veterinarian immediately.

Mid-cervical Intradermal Test in Cervids

12. The mid-cervical intradermal test is the standard test for tuberculosis in cervids (deer, elk).

13. Inject 0.1 ml of bovine PPD tuberculin in the mid-cervical area of cervids with observation and palpation at 72 ± 6 hours. In the case of animals for export to the U.S., do not repeat this test on the same animal at intervals of less than 90 days.

14. The equipment is the same as in paragraph 7 above, plus electric clipper with a #40 head and an indelible felt marker.

15. Adequately restrain all cervids to be tested to ensure adequate preparation of the injection site, proper application of the tuberculin injection, correct recording of animal identification and correct reading of the test.

16. Use clippers to clip an area in the mid-cervical region approximately 8 cm square. Mark the site of injection. Inject 0.1 ml of tuberculin intradermally, lifting the tip of needle slightly after insertion to ensure it is clearly outlined under the skin. Following the injection, the injected tuberculin should be visible and palpable as a definite bleb.

17. Observe and palpate the injection site at 72 ± 6 hours post injection. The response at the skin injection site is frequently diffuse as opposed to the circumscribed response on caudal fold test in cattle. Good lighting and restraint is essential. Record responses to the test on Form CFIA/ACIA 1524 as follows:

1. If there is no change in the tissue at the point of injection, record the result as negative.
2. If there is any visible or palpable change in the tissue at the point of injection, record the result as Reactor. Note: If there is a visible or palpable change in the tissue at the point of injection, notify the district veterinarian immediately.

Post-Axillary Test

18. The post-axillary test for export testing of New World camelidae only and is done at the request of the importing country.

19. The intradermal test is performed at the post-axillary site in camelidae. Responders to the test are determined by relatively small changes in skin thickness, and therefore it is imperative that a regimented approach be taken in applying this test. Measurements will be influenced by skin tension (related to restraint), by the amount of pressure placed on the skin by the calipers, and by the amount of skin picked up for measurement. Make every effort to standardize the conditions for both injections and readings.

20. Do not repeat this test on the same animal at intervals of less than 90 days.

21. The equipment is the same as in paragraph 7 above, plus a caliper capable of accurately measuring to less than 0.1 mm.

22. Adequately restrain all camelidae to be tested to ensure adequate preparation of the injection site, proper tuberculin injection, correct recording of animal identification, and proper reading of test. Use clippers to clip an area in the axillary region approximately 8 cm square. Mark the site of injection, and palpate and record any unusual findings.

23. Using the dial caliper measure the marked spot three times. Record the average of the measurements.

24. Inject 0.1 ml of tuberculin intradermally, lifting the tip of the needle slightly after injection to ensure it is clearly outlined under the skin. Following the injection, the injected tuberculin should be visible and palpable as a definite bleb.

25. Observe and palpate the injection site at 72 ± 6 hours. The response at the skin injection site is frequently diffuse as opposed to the circumscribed response on the caudal fold test in cattle. Using the dial caliper, measure the marked spot three (3) times. Record the average of the measurements. Record responses to the test on Form CFIA/ACIA 1524 as follows:

1. If there is no change in the tissue at the injection site, or less than 1.5 mm increase in average thickness, record result as negative.
2. if there is a visible or palpable reaction including 1.5 mm or greater increase in average skin thickness, record the result as Reactor and notify the district veterinarian immediately.

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3.2 Serologic Testing

This part of the Accredited Veterinarian's Manual will provide the following information necessary for serologic testing:

1. material and supplies
2. serum samples-preparation and packaging
3. submission forms
4. shipping
5. test results
6. laboratory fees
7. laboratories

Objectives

1. Section 2 of the Health of Animals Regulations interprets test as including:

1. the collection of body tissue or fluid from an animal (e.g. serum);
2. the injection of an animal for the purpose of determining that animal's freedom from infection with disease (e.g. tuberculin testing).

Materials and Supplies

2. The costs associated with needles, vials, packaging and shipping charges for transporting samples must be paid by the accredited veterinarian. Small (3 ml) vials with or without internal thread closures should be avoided as these are prone to leakage and may cause technical problems for the laboratory.

Collection, Preparation and Packaging of Serum Samples

3. The reliability of any diagnostic laboratory procedure depends directly on the type of specimens or samples received and on their condition. A separate sample must be submitted for each diagnostic test required. It is extremely important that animals are identified correctly and the information entered on the forms is complete and accurate.

4. Regardless of the type of specimen, the laboratory must receive a sample of good quality for diagnostic purposes. After collection, samples must be shipped to the laboratory without delay. Use the safest and quickest means of shipment available and ship samples only during the week to avoid having them delayed in transit or held over at the laboratory on weekends or holidays. Samples for virus isolation tests such as BVD-IP must not be frozen prior to submission, and must be protected from extremes of temperature during transit. Any factor that affects the viability of the agent in question will affect the sensitivity of the test and therefore the reliability of the test result.

5. For clotted blood samples:

1. Use silicone-treated vials (red stopper), which should be centrifuged before shipping and the clot removed.
2. Use disposable needles.
3. Use a waterproof marker to number the vials.
4. Use appropriate containers to ship the samples.
5. Include all pertinent/required forms with the samples.

6. Each vial must be marked indelibly with the number corresponding to the one entered on the submission form in the column under VIAL N^o. Label the vial, not the stopper.

7. Unless otherwise indicated, a minimum of a 1 ml sample of serum or 5 ml centrifuged serum separator tubes (SST) sample is required for each test even if various tests are requested and are done at the same laboratory. Laboratories prefer separate serum samples for each test in order to maintain sample integrity. For example, if brucellosis and leptospirosis tests are requested, two samples are required.

8. All samples for each test must be alpha-numerically identified and/or numbered sequentially to coincide with the identification recorded on the test chart(s). For a large number of samples, advise the laboratory of the shipment.

9. Vials should be shipped in boxes designed for this purpose.

Submission Forms

10. Submission forms are available at the CFIA district office. For each test requested, a serum sample and a copy of the appropriate completed submission form must be sent to the laboratory.

11. Forms should be typed or printed and all copies must be legible. All information should be recorded on the form including ear tag numbers, tattoos or other types of identification. The type of test being requested, and the date and location where the specimens were collected, should be recorded. Accredited veterinarians must include their name, address (including postal code) and telephone number, and must sign at the bottom of the form. The submitter code must also be indicated, as well as the identification code of the CFIA district office in which the animals are located.

12. For samples submitted to a CFIA or CFIA approved laboratory, use Form CFIA/ACIA 5473-Animal Health Import, Export and Artificial Insemination Specimen Submission. Instructions for submission of samples are printed on that form. Except for the reason identified below a laboratory notification number, which is available from a CFIA district veterinarian, must be entered in the Reason For Test section of Form CFIA/ACIA 5473.

When submitting samples for brucellosis testing to an accredited laboratory, it is acceptable to complete the first two pages of Form CFIA/ACIA 5473 in full and mark the third page with See attached list. The list can be a spreadsheet that records the animals' identification, description and the vial numbers, and should identify form reference number. That reference number is the number which appears in the bottom center of all three pages of Form CFIA/ACIA 5473.

If animals are being tested for brucellosis for export to the USA, a notification number is not required on Form CFIA/ACIA 5473, but the destination should be identified as USA.

13. To send Equine Infectious Anemia (EIA) samples to a CFIA-approved laboratory, use Form CFIA/ACIA 3937-Equine Infectious Anemia (EIA) Serum Test Report and Certificate.

14. If more than one horse is being tested for EIA, use Form CFIA/ACIA 4679-Equine Infectious Anemia (EIA) Domestic Serum Test Result and Certificate; however, Form CFIA/ACIA 4679 cannot be used for export purposes.

15. To send Enzootic Bovine Leukosis-Canadian Health Accredited Herd (EBL-CHAH) samples to CFIA-approved laboratory, use Form CFIA/ACIA 3841-Canada Health Accredited Herd Serum Test Report.

16. It is the responsibility of the accredited veterinarian to provide all information required, or to resubmit if a specimen is unfit for testing. Laboratories may refuse to conduct analysis of samples submitted with forms that are not fully complete. Refer to a CFIA district veterinarian for examples of completed forms, and for instructions on distribution of forms for samples being sent to CFIA laboratories.

Shipping Samples

17. All the laboratories do not perform tests for all diseases. Therefore, it is essential that samples be shipped in a timely fashion to the appropriate laboratory to facilitate testing and avoid unnecessary delays. If uncertain, check with a CFIA district veterinarian before shipping.

18. Vial breakage can result in re-bleeding an animal and significantly prolong laboratory test times. When shipping more than one container, ensure that they are individually identified and packaged to prevent breakage. In addition to a legal responsibility, a submitter has a moral obligation to ensure the safety of transportation industry workers and laboratory staff handling the specimen. The use of an appropriate container is a requirement when submitting samples to a laboratory.

19. Accredited veterinarians must make sure that the packaging and shipping procedure used complies with regulations concerning the transportation of such samples.

20. The laboratory submission forms should be placed in an envelope and attached to the outside of the protective containers.

21. The shipment of samples must never be entrusted to the animal owner or exporter. The accredited veterinarian must be able to maintain a chain of custody for samples shipped to their laboratories.

22. The accredited veterinarian is responsible for shipping costs.

Test Results

23. Test results from private laboratories approved by the CFIA are usually reported directly to the submitter and the CFIA district office. The accredited veterinarian is responsible for advising the owner of the results. Schedule testing so as to allow sufficient time to receive results (time required will vary from one region to another).

24. Test results reported from CFIA laboratories are generated through the Laboratory Sample Tracking System (LSTS). The test results will be provided to the accredited veterinarian by the local district office.

Laboratory Fees for Testing

25. The accredited veterinarian is responsible for laboratory fees.

Laboratories

26. Most testing, mandated by the Health of Animals Act and Regulations or conducted in response to a specific request by an importing country, is conducted in a CFIA laboratory providing diagnostic services for animal and plant health programs and would usually be submitted by the district veterinarian.

27. Certain private laboratories are approved by the CFIA to conduct various tests for accredited veterinarians. The nature of the tests performed at these laboratories and the list of approved laboratories changes significantly from month to month. Ask a CFIA district veterinarian for the most up-to-date list. Similarly, the laboratories maintain a list of accredited veterinarians for whom they are authorized to perform official tests. All specimens must be received on the appropriate submission form for the official test being requested. For the current list of laboratories approved for Equine infectious anemia (EIA), Bovine leucosis virus (BLV) and Brucellosis testing, contact your CFIA District Veterinarian. Module 3.3 Laboratories lists the CFIA laboratories for your reference.

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3.3 Laboratories

Laboratories Approved to Test EIA, BLV and Brucellosis - Buffered Plate Antigen Test (BPAT)

For the current list of laboratories approved for EIA, BLV and Brucellosis testing, please contact your CFIA District Veterinarian.

Canadian Food Inspection Agency Laboratories

CFIA's Animal Health Laboratory Services are delivered by a network of Centres of Expertise, each of which is the national centre for its area of specialization.

The Centres are located at the following CFIA laboratories:

CFIA Lethbride Laboratory
Township Road 9-1, Postal Box 640
Lethbridge, Alberta T1J 3Z4
Telephone: 403-382-5500
Facsimile: 403-381-1202
(Animal Diseases Research Institute)

• Indigenous Bovine and Equine Viral Diseases Centre of Expertise
• Anthrax, Leptospirosis and Anaplasmosis Centre of Expertise
• Non-traditional Livestock Centre of Expertise
• OIE Reference Laboratory for Anthrax, BSE, IBR and BVD

CFIA Saskatoon Laboratory
116 Veterinary Road
Saskatoon, Saskatchewan S7N 2R3
Telephone: 306-975-4071
Facsimile: 306-975-5711

• Centre for Food-Borne and Animal Parasitology
• Parasitic Diseases of Livestock and Farmed Game Animals Centre of Expertise
• OIE Reference Laboratory for Trichinellosis
• OIE Collaborating Centre for Food-Borne Zoonotic Parasites

CFIA Winnipeg Laboratory
1015 Arlington Street
Winnipeg, Manitoba R3E 3M4
Telephone: 204-789-2001
Facsimile: 204-789-2038
(Canadian Science Centre for Human and Animal Health)

• National Centre for Foreign Animal Diseases
• OIE Reference Laboratory for AI and CSF

CFIA Ottawa Laboratory
3851 Fallowfield Road
Ottawa, Ontario K2H 8P9
Telephone: 613-228-6698
Facsimile: 613-228-6668
(Animal Diseases Research Institute/Center for Plant Quarantine Pests)

• Avian Diseases Centre of Expertise
• Brucellosis Centre of Expertise
• Germplasm Centre of Expertise
• Mycobacterial Diseases Centre of Expertise
• Rabies Centre of Expertise
• OIE Reference Laboratory for Rabies, Brucellosis, Scrapie and CWD

CFIA Saint-Hyancinthe Laboratory
3400 West Casavant boulevard
Saint-Hyancinthe, Quebec J2S 8E3
Telephone: 450-773-7730
Facsimile: 450-773-8152
(Health of Animals and Food Laboratory)

• Indigenous Porcine Diseases Centre of Expertise
• Retrovirology Centre of Expertise

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