Canadian Food Inspection Agency - Animal Products - Accredited Veterinarian's Manual - Module 3

3.0 Testing

3.1 Tuberculosis Testing

Tuberculin Tests-Background Information

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.

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

a) type of tuberculin, (bovine, avian, or human);
b) site of injection (cervical or caudal);
c) quantity of tuberculin used;
d) late pregnancy/postpartum;
e) state of nutrition; and,
f) whether or not the animal is infected.

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.

Similarly some cattle, which are 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 being the probability of the test correctly identifying as negative those individuals which are 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.

Caudal Fold Tuberculin Test

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.

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.

Equipment and supplies required:

a) bovine PPD tuberculin provided by the CFIA district office. Store in a cool (4ºC) dark place and do not use after the expiry date;
b) 1 mL disposable tuberculin syringe with a needle calibre 26 of 3/8";
c) absorbent cotton;
d) CFIA/ACIA 1524 on which to record the test and the animal identification.

Note: Only fill the syringes with tuberculin at the time of injection, never in advance.

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.

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 CFIA/ACIA 1524 form as follows:

a) no change in the tissue at the point of injection - "no reaction," decision "negative."
b) any change in the tissue at the point of injection, 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.

Equipment: as in paragraph 7 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:

a) no change in the tissue at the point of injection - "no reaction," decision "negative."
b) any visible or palpable change in the tissue at the point of injection, notify the district veterinarian immediately.

Post-Axillary Test

18.

For export testing of New World camelidae only, 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.

Equipment: 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 the test report form as follows:

a) if no change in the tissue at the injection site, or less than 1.5 mm increase in average thickness, record "no reaction" decision "negative;"
b) if there is a visible or palpable reaction including 1.5 mm or greater increase in average skin thickness, record the decision as "suspicious" and notify the district veterinarian immediately.

3.2 Serologic Testing

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

a. material and supplies
b. serum samples - preparation and packaging
c. submission forms
d. shipping
e. test results
f. laboratory fees
g. laboratories

Objectives

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

a. the collection of body tissue or fluid from an animal (e.g. serum);
b. 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

The costs associated with needles, vials, packaging and shipping charges for transporting samples must be paid by the accredited veterinarian.

Collection, Preparation and Packaging Serum Samples

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.

Regardless of the type of specimen, the laboratory must receive a sample of good quality for diagnostic purposes. 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.

For clotted blood samples:

a. use silicone-treated vials (red stopper), or serum separator tubes (SST), which should be centrifuged before shipping and the clot removed;
b. use disposable needles;
c. use a waterproof marker to number the vials;
d. use appropriate containers to ship the samples;
e. include all pertinent/required forms with the samples.

Each vial must be marked indelibly with the number corresponding to the one entered on the submission form in the column under VIAL NO. Label the vial, not the stopper.

Unless otherwise indicated, a minimum of a one mL sample of serum or 5 mL centrifuged SST sample is required for each test even if various tests are requested and are done at the same laboratory. Laboratories prefer serum samples to maintain sample integrity. For example, if brucellosis and leptospirosis tests are requested, two samples are required.

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.

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 federal or accredited laboratory, use CFIA/ACIA 5159. Complete one form for each test requested. A laboratory notification number available from the district veterinarian must be included.

13.

To send EIA samples to an accredited laboratory, use CFIA/ACIA 3937.

14.

If more than one horse is being tested for EIA, use CFIA/ACIA 4679; however, CFIA/ACIA 4679 can not be used for export purposes.

15.

To send EBL-CHAH samples to an accredited laboratory, use CFIA/ACIA 3841.

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 federal laboratories.

Shiping 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 accredited private laboratories 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 federal 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 federal laboratory providing diagnostic services for animal and plant health programs and would usually be submitted by the district veterinarian.

27.

Private laboratories are accredited 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. Module 3.3 Approved Laboratories lists current accredited laboratories. Update as necessary.

3.3 Laboratories

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Laboratories Accredited for Equine Infectious Anemia, Enzootic Bovine Leucosis, and Brucellosis - BPAT

EIA/BLV/Brucellosis Approved Laboratories
Updated: 2006-05-11

Approved Laboratory	EIA	BLV	Brucellosis
Animal Health Monitoring Laboratory 1767 Angus Campbell Rd. Abbotsford, BC V3G 2M3 Tel: (604) 556-3135; Fax.: (604) 556-3133 Contact: Letitia Curley Submitter Code: AHML004	Yes	Yes	Yes
Atlantic Veterinary College Regional Diagnostic Virology Laboratory 550 University Avenue Charlottetown, PEI C1A 4P3 Tel: (902) 566-0877; Fax.: (902)566-0723 Contact: Dr. Gerry Johnson (902)566-0854 Dr. Carmencita V. Yason (902)566-0752 Submitter Code: AVC005	Yes	Yes
Biovet Inc. 4375 Beaudry Avenue St-Hyacinthe, QC J2S 8W2 Tel: (450) 771-7291; Fax.: (450) 771-4158 Contact : Dr. Pierre Hébert ext 230 Contact for Brucellosis: Annie St.- Germain Contact: Isabel Cayer Submitter Code: BVI006 Laboratory ext. 232 Fax.: 771-2996	Yes	Yes	Yes
Central Laboratory for Veterinarians Ltd. 5645, 199th St. Langley, BC V3A 1H9 Tel: (604) 533-4884; Fax.: (604) 533-0289 Contact: Dr. Sally Lester Submitter Code: CLV002 (800) 663-1425 ext. 224	Yes
International Bio-Institute 6589 First Ligne, R.R. 3 Fergus, ON N1M 2W4 Tel: (519) 787-0294; Fax.: (519) 787-9371 Contact: Dr. Donal McKeown Contact for Brucellosis: Donna McKeown Submitter code: IBI008	Yes	Yes	Yes
Laboratoire d'expertise en pathologie animale du Québec (LEPAQ) (Sainte-Foy - M.A.P.A.Q.) 2700, Einstein Street Sainte-Foy, QC G1P 3W8 Tel: (418) 643-6140; Fax.: (418) 644-6327 Contact Person: Dr. Yasmine-Djaouida Chalal * Irène Bouffard Dr. Marie Nadeau (Ext. 206) Submitter code: STFOY010	Yes	Yes
Manitoba Veterinary Diagnostic Laboratory Veterinary Services Branch 545 University Crescent Winnipeg, MB R3T 5S6 Tel: (204) 945-7632; Fax.: (204) 948-2654 Contact for Brucellosis: Chris Link-Muirhead			Yes
Meyer Laboratory Services 119 Warner Drive, RR # 1 Long Sault, ON K0C 1P0 Tel: (613) 938-2185: Fax.: (613) 933-8726 Contact: Janet Tessier Director: John Meyer	Yes
Palliser Animal Health Laboratories Ltd. #6, 320 W.T. Hill Blvd South Lethbridge, AB T1J 4W9 Tel: (403) 328-1844; Fax.: (403) 328-1798 Contact: Brenda Logan-Handsaeme Submitter code: PAL013	Yes		Yes
Prairie Diagnostic Services - Regina 4840 Wascana Parkway Regina, SK S4S 7J6 Tel: (306) 787-6435; Fax.: (306) 787-6439 Contact: Gail Krohn Gail Krohn (306) 787-3056 or (306) 787-6431 Laboratory Director/Manager: Al Perry, (Alt) Dr. Choon Yong	Yes	Yes	Yes
University of Guelph Animal Health Laboratory. P.O. Box 3612, Guelph, ON N1H 6R8 Tel: (519) 824-4120, Laboratory ext. 54502 Fax.: (519) 821-8072 Contact: Dr. Grant Maxie ext. 54544 * Dr. Davor Ojkic, ext. 54524 Submitter Code: AHL1825	Yes
Veterinarians Diagnostic Laboratory 2355 Derry Rd East Mississauga, ON L5S 1V6 Tel: (905) 405-9400 Fax.: as for telephone. Contact: Mr. Kader P. Prasad Submitter code: VDL016	Yes
Vita-Tech Canada Inc. Veterinary Laboratory Services 1345 Denison Street Markham, ON L3R 5V2 Tel: (905) 475-6499; Fax.: (905) 475-7309 Contact: Dr. Alfred Choo ext. 228 Contact for Brucellosis: Grace Rejano, Ext. 250 *Frederick Wong Jacklyn: laboratory ext. 237 Submitter code: VT019	Yes	Yes	Yes

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 Laboratory - Township Rd 9-1, P.O. Box 640 Lethbridge AB T1J 3Z4
Tel.:403-382-5500 Fax.:403-381-1202
(Animal Diseases Research Institute)

Indigenous Bovine and Equine Viral Diseases Centre of Expertise
Leptospirosis and Anaplasmosis Centre of Expertise
Non-traditional Livestock Centre of Expertise
Reference Laboratory for Anthrax

CFIA Laboratory - 116 Veterinary Road, Saskatoon, SK S7N 2R3
Tel.: 306-975-4071 Fax.: 306-975-5711
(Health of Animals Laboratory)

Centre for Animal Parasitology

CFIA Laboratory - 1015 Arlington Street, Winnipeg, MB R3E 3M4
Tel.: 204-789-2001 Fax.: 204-789-2038
(Canadian Science Centre for Human and Animal Health)

National Centre for Foreign Animal Diseases

CFIA Ottawa Laboratory - 3851 Fallowfield Rd. Ottawa ON K2H 8P9
Tel.: 613-228-6698 Fax.: 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

CFIA Laboratory -3400 Casavant Blvd West, St. Hyancinthe QC J2S 8E3
Tel.: 450-773-7730 Fax.: 450-773-8152
(Health of Animals and Food Laboratory)

Indigenous Porcine Diseases Centre of Expertise
Retrovirology Centre of Expertise