Chapter 3 - Testing

3.1 Tuberculin skin testing for bovine tuberculosis (bTB)

This section deals with tuberculin skin testing procedures only. Information about the disease itself may be found in module 8.3 and on the CFIA website.

Tuberculin skin testing of livestock has been the cornerstone of on-farm active surveillance for the National Bovine Tuberculosis (TB) Eradication Program (NBTEP) for many years.

  • testing is also applied for the purpose of import, export, entry into semen production centres, and to support disease investigations
  • the basis of tuberculin testing is the induction of delayed-type hypersensitivity reaction to the intradermal injection of antigenic substances derived from a laboratory strain of Mycobacterium bovis (M. bovis)
  • the hypersensitivity response results in the accumulation of cells and fluid in the dermis which creates swelling at the injection site
  • in test positive animals, the intradermal skin reaction develops within 24 hours post-injection, reaching a maximum by 48 to 72 hours, characterized by erythema and swelling, but rarely to the point of induration or ulceration

There are 2 limitations associated with intradermal tuberculin tests:

  • some animals infected with M. bovis fail to respond to the test (false negatives)
    • the number of animals in this category is a measure of the sensitivity of the test; the probability of the test correctly identifying as reactors those animals that are truly infected
    • false negative responses may be observed for a variety of reasons, including compromised tuberculin quality, improper testing technique, the chronicity and extent of the disease process, or variations in the time of testing in relation to calving and previous tuberculin tests
  • some animals that are not infected with M. bovis react to the test (false positives)
    • the number of animals in this category is a measure of the specificity of the test; the probability of the test correctly identifying as negative those animals that are truly not infected
    • most false positive responses are due to the animal having been exposed to antigens similar to, or shared with, those in tuberculin

Sensitivity and specificity are characteristics of the tuberculin skin test itself and are not influenced by the population on which the test is applied (that is if the population is TB-free or actually has TB at a given prevalence).

However, what is influenced by the prevalence of TB in a population is the probability that a reactor animal is truly infected with M. bovis (the positive predictive value of a positive test result).

  • positive and negative predictive values are influenced by the prevalence of disease in the population that is being tested
  • the higher the prevalence in a population, the more likely that an animal that reacts to the tuberculin test actually has the disease, than if the test were performed in a population with a low prevalence

Tuberculin procurement, handling, storage, use, and disposal

All tuberculin used in Canada is produced by the Biologics Production Unit of the Canadian Food Inspection Agency (CFIA) Ottawa Laboratory Fallowfield (OLF).

Bovine purified protein derivative (PPD) tuberculin, produced by CFIA, contains 3250 IU in each 0.1 mL.

Tuberculin is distributed by district offices to accredited veterinarians.

  • accredited veterinarians that wish to obtain tuberculin are to contact their local district office who will provide tuberculin upon request free of charge

In light of the short turnaround time for the production of tuberculin, it is unnecessary to stockpile an inventory in anticipation of future needs.

  • stockpiling leads to unnecessary and increased wastage of tuberculin and contributes to supply shortages

Tuberculin has an expiry date of 1 year from the time of potency testing (when stored and handled appropriately) but ideally should be used within a few months of receipt.

  • manage tuberculin inventories carefully to avoid its expiration before use

Always check the expiry date of tuberculin before use.

  • do not use expired tuberculin
  • a tuberculin test is valid only if carried out prior to, or on the same date as, the expiration date on the bottle
  • if expired tuberculin is used inadvertently to conduct an official TB test, the test is invalid and will have to be repeated after the appropriate period of time has passed, as required for the particular tuberculin test procedure
  • contact the district office immediately for further instructions

To be reliable, tuberculin must be handled and used according to strict parameters:

  • store tuberculin in a cool environment (4°C), protected from light
    • tuberculin exposed to extreme temperatures or to sunlight for more than a few hours is compromised and should not be used
  • always pack tuberculin on ice or frozen cold packs during transit
  • use an aseptic technique when handling tuberculin
    • to prevent contamination of tuberculin, disinfect the stopper of the vial after peeling back the aluminum seal
  • immediately dispose of any expired tuberculin as follows:
    • discard directly as biological waste; or
    • return to your local district office for disposal (only full and unopened bottles)
  • any full / unopened vials of valid/unexpired tuberculin that is no longer intended for field use should be returned to the local district office

Be aware that tuberculin is for use only by trained and qualified accredited veterinarians.

Tuberculin testing notes

Testing may involve considerable travel time and many hours to process all animals to be tested. To facilitate this process, it is important to prepare in advance to ensure that all materials will be available at the test site.

Syringes and needles

Use 1 ml tuberculin syringe. Only a single dose may be drawn at a time. Multiple doses in the same syringe are not allowed.

Use a new needle for each animal: 26 gauge, ⅜" long.

Be aware that tuberculin syringes may be re-loaded, with a new needle for each animal.

Do not pre-load syringes with tuberculin prior to testing under any circumstances.

Tests available

A tuberculin skin test (TST) may only be performed by trained and qualified accredited veterinarians (AV):

Note concerning TST: The CFT test in yak and Asian water buffalo, post axillary test in camelids, and the base-of-the-ear tuberculin test for swine are not official tests for bovine TB under the NBTEP and are only performed under export programs testing for live animals and semen (that is, they are test ineligible species).

An accredited veterinarian may only perform a tuberculin skin test in any of the above listed species provided that it is authorized in their accredited veterinarian agreement.

  • for tuberculin skin testing in any test ineligible species not listed in this section, prior consultation with and permission from the accredited veterinarian's supervising district office is required BEFORE testing is performed (for example, to determine the appropriate test).

For accredited veterinarians, training and qualification entails having successfully completed:

  • a review of this section of the Manual with a district veterinarian; and
  • on farm demonstration and practice on live animals with a CFIA veterinary inspector
    • the district veterinarian must keep a record in the accredited veterinarian's file regarding the date and place where the injection technique was practised on farm

For accuracy of test results conducted under the NBTEP, it is essential that the same person conduct both the injection-day and reading-day measurements for these tests, other than for exceptional circumstances that make this impossible (unplanned and unavoidable absence).

Information owners require before testing

Before conducting a tuberculin skin test, the accredited veterinarian should ensure the owner is aware of relevant information, such as the consequences of a non-negative result. Disclosure statements vary depending upon the species tested. Regardless of the species tested, the owner should be advised that:

  • the animal should not be moved from the current premises until the final results have been reported
  • negative test results do not provide any exemption from existing food safety program requirements, including those for raw and unpasteurized milk
  • their personal information is collected by CFIA under the authority of Health of Animals Act for the purpose of supporting reportable disease management in Canada
  • their personal information will be protected under the provisions of the Privacy Act and will be stored in Personal Information Bank CFIA PPU 050 / CFIA CON 025
  • there is implied consent to share their personal information with approved third parties when testing is requested

If the animal tested is a domestic bovine (Bos taurus/indicus), bison, domestic sheep, domestic goat, or cervid (that is, a test eligible species in which the tuberculin skin test is considered an official test for Bovine TB under the NBTEP), advise the owner that:

  • a reactor animal will trigger disease investigation measures that may include:
    • additional testing of the reactor animal,
    • an epidemiological investigation,
    • testing of additional animals in the reactor's herds of residence,
    • legal movement controls (that is, quarantine), and
    • humane destruction of the animal(s) to facilitate confirmatory tissue testing
    • additional information

If the animal tested is a yak, Asian water buffalo, camelid, swine, non-domestic sheep or goat species or other species not mentioned elsewhere (that is, a test ineligible species in which the tuberculin skin test is NOT considered an official test for Bovine TB under the NBTEP), advise the owner that:

  • these species are considered test ineligible under the NBTEP and, as such, there are no available live-animal ancillary tests in the case of a reactor (i.e. non-negative tuberculin skin test result) to clear the animal of suspicion of disease
  • a reactor animal will trigger disease investigation measures that may include:
    • an epidemiological investigation that extends from the current premises of residence to include all previous herds of residence of the reactor animal,
    • testing of test-eligible animals in the current and/or pervious herds of residence,
    • legal movement controls (that is, quarantine), and
    • humane destruction of the animal(s) to facilitate confirmatory tissue testing

Adjunct activities

If the owner or operator plans to medicate the animals (for example vaccines, anti-parasitic) while the animals are being handled for TB testing, all medications shall be applied at the time of the tuberculin testing reading, not at the time of tuberculin injection.

This facilitates the salvage of carcasses for human food, if animals must be destroyed due to test results or injury and ensures that the medication does not interfere with the test results.

Test date

The date of the tuberculin test is to be recorded on the report of intradermal test, including both the tuberculin injection date and the reading date.

  • for export purpose and entry into a semen production centre, the test date recorded is the reading date
  • to calculate the interval that has elapsed since a tuberculin test was performed, use the tuberculin injection date

Caudal fold tuberculin (CFT) test

Preparation

The CFT test is the standard official screening test for bovine TB in cattle, bison, domestic sheep and domestic goats.

The CFT test can be conducted by accredited veterinarians in yak and Asian water buffalo for the purpose of satisfying export requirements to the U.S (see module 5.2C)

  • the CFT test in yak and Asian water buffalo is NOT recognized as an official screening test under the NBTEP, as these species are considered test ineligible.
  • ancillary testing of reactors in these species using the CCT test is not recognized under the NBTEP.
    • although a CCT may be performed to clear the remainder of animals for the purposes of export to the US, a negative CCT result does NOT clear a reactor animal of suspicion of disease for the purposes of a domestic disease investigation.

The test involves an intradermal injection of 0.1 mL of bovine PPD tuberculin into either caudal fold, with observation and palpation at 72 hours ± 6 hours.

Do not apply the CFT test to an animal that has had a tuberculin test of any kind during the preceding 60 days.

Prepare the following equipment:

tuberculin – bovine PPD tuberculin (store in dark at 4°C)
syringes – 1 mL tuberculin syringes
needles – 26 gauge, ⅜" long
absorbent cotton
Form CFIA/ACIA 1524 – Report of Intradermal Test
headlamp or flashlight, if poor lighting

Adequately restrain each animal for testing to ensure correct animal identification, proper preparation of the injection site, careful tuberculin injection, and reading of the test result. Do not apply or observe the test without having the animal adequately restrained.

Record on Form CFIA/ACIA 1524 the individual animal identification of each animal tested.

The injection site is the caudal fold, distal to the base of the tail, well away from the hairline, in the center of the fold.

  • use either the left or right fold, but be consistent in which side you use for each test
  • if a lesion is encountered on the caudal fold use the opposite fold and make a note of this change

Injection

Clean the caudal fold with cotton (moistened with water, if necessary). Do not use alcohol to clean the injection site.

Note any abnormalities, lesions, or scabs observed in the vicinity of the injection site, recording on Form CFIA/ACIA 1524 to avoid being mistaken for tuberculin responses.

Check tuberculin label to confirm type – M. bovis tuberculin has an orange label.

Check/confirm the tuberculin expiry date against current date.

Disinfect rubber stopper with alcohol after opening aluminium seal.

Fill tuberculin syringe to 0.1 mL volume. Only a single dose can be drawn.

Raise tail and locate the caudal tail folds.

Insert needle into the dermis of the skin on the center line of either the left or right caudal tail fold, distal to the tail base and away from the hair line.

  • be consistent and use either the right or left caudal fold for all test animals on the farm
  • lift the tip of the needle slightly after insertion to ensure it is clearly outlined under the skin
  • inject 0.1 mL of tuberculin intradermally and withdraw the needle slowly

View injection site to ensure that the injected tuberculin is visible and palpable as a definite bleb.

  • if not, this indicates that either the needle was inserted too deep or a portion of the tuberculin was not successfully injected.
  • if this occurs, the administration of tuberculin should be repeated on the opposite side; ensure the new/final injection site is noted on Form CFIA/ACIA 1524 to prevent any confusion at reading

Discard needle immediately into a sharps container.

Reading

After 72 hours ± 6 hours, restrain the animal and confirm its identification.

Raise the tail with 1 hand to slightly stretch the caudal tail folds.

Observe and palpate the length of the caudal fold injection site with the thumb and index finger of the other hand.

Record the response to the CFT test for each animal on Form CFIA/ACIA 1524) as follows:

  • negative (Neg.): there is no visible or palpable change in tissue at the site of injection; or
  • reactor: there is a visible or palpable disturbance in tissue at the site of injection
    • when in doubt regarding the nature of the response to the CFT test, classify the animal as a reactor
    • in the case of reactor(s), contact the district office immediately

Animals classified as reactors on the CFT test are retested by a CFIA veterinarian, using the Comparative Cervical Tuberculin (CCT) Test (refer to the CCT test section).

  • Yak and Asian water buffalo are considered test ineligible species under the NBTEP, and therefore cannot be retested using the CCT test unless required to clear the remainder of animals in a group for export (however, the CCT result will not be recognized for the purposes of the triggered domestic disease investigation)

All tuberculin tests are official and must be recorded and reported to your local district office by 1 of the following methods:

  • fully completed Form CFIA/ACIA 1524, including animal information for each animal tested
    • a copy of the completed Form CFIA/ACIA 1524 is forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animals were tested) or the certificate of health for entry into a semen production centre (Form CFIA/ACIA 1634 – Certificate of Health for Entry into a Semen Production Centre or Isolation worksheet and Authorization for Release); or
  • partially completed Form CFIA/ACIA 1524 as follows and attach an Animal List
    • 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
    • attach to Form CFIA/ACIA 1524 a list of animals that were TB tested and found negative
      • in the animal information section of the Form CFIA/ACIA 1524, insert a statement that reads "The (insert the number 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
    • any animals with non-negative results must be included in the animal information section of Form CFIA/ACIA 1524
    • a copy of the completed Form CFIA/ACIA 1524 and the attached list are forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animals were tested) or the certificate for entry into a semen production centre (Form CFIA/ACIA 1634 or Isolation worksheet and Authorization for Release)

Monitoring CFT testing: performance standard

The CFT test has less than perfect specificity (ranges from 97 to 99%), resulting in a false positive reactor rate in a disease-free livestock population of 1% to 3% per year.

  • false positive reactors are an indicator that the test is being performed correctly as they are expected when applying this test in a group of animals without disease

A minimum performance standard exists for all inspectors and veterinarians, including accredited veterinarians who conduct CFT testing.

The reactor rate of each accredited veterinarian trained to conduct CFT test shall be monitored and calculated based on the number of cattle or bison tested for each 1-year period.

  • if a reactor rate less than the minimum performance standard of 1% is identified, this will be addressed and appropriate actions taken (that is additional training or supervision, audit) and documented
  • if insufficient cattle/bison are tested in 1 year for evaluation of compliance with the standard, the period for review will be extended

District veterinarians involved in the evaluation of official services performed by accredited veterinarians are responsible for monitoring reactor rates of accredited veterinarians.

  • failure to meet the minimum performance standard (that is non-conformance) noted during evaluation of accredited veterinarian services will be addressed according to the Procedures Manual – Management of Accredited Veterinary Services – to improve the services provided

Authorization to perform CFT test may be withdrawn for accredited veterinarians where non-conformance to the minimum performance standard cannot be resolved.

Mid-cervical tuberculin (MCT) test

Preparation

The MCT test is the standard official screening test for tuberculosis in cervids. The test involves an intradermal injection of 0.1 mL of bovine PPD tuberculin in the mid-cervical area, with observation and palpation at 72 hours ± 6 hours.

Do not apply the MCT test to an animal that has had a tuberculin test of any kind during the preceding 60 days.

Prepare the following equipment:
tuberculin – bovine PPD tuberculin (store in dark at 4°C)
syringes – 1 mL tuberculin syringes
needles – 26 gauge, ⅜" long
absorbent cotton
electric clippers with number 40 head
Form CFIA/ACIA 1524 – Report of Intradermal Test
headlamp or flashlight, if poor lighting

Adequately restrain each cervid for testing to ensure correct animal identification, proper preparation of the injection site, careful tuberculin injection, and proper reading of the test result. Do not apply or observe the test without having the animal adequately restrained.

Record on Form CFIA/ACIA 1524 the individual animal identification of each animal tested.

  • acceptable animal identification is as follows: tag approved (or considered equivalent) under the Livestock Identification and Traceability (TRACE) program, a HofA ear tag, a registered tattoo, or an ear tag approved by a provincial regulatory authority for farmed cervids

Injection

Use clippers to clip an area in the mid-cervical region that is approximately 8cm square.

Clean the injection site with cotton (moistened with water, if necessary). Do not use alcohol to clean the injection site.

  • note any abnormalities, lesions or scabs observed in the vicinity of the injection site, recording on Form CFIA/ACIA 1524 to avoid being mistaken for tuberculin responses

Check tuberculin label for type – M. bovis tuberculin has an orange label.

Check/confirm expiry date against current date.

Disinfect rubber stopper with alcohol after opening aluminium seal.

Fill tuberculin syringe to 0.1 mL volume.

Insert needle into the dermis of the skin. Lift the tip of the needle slightly after insertion to ensure it is clearly outlined under the skin. Inject 0.1 mL of tuberculin intradermally and withdraw the needle slowly.

View injection site to ensure that the injected tuberculin is visible and palpable as a definite bleb.

  • if not, this indicates that either the needle was inserted too deep or a portion of the tuberculin was not successfully injected
  • if this occurs, the administration of tuberculin should be repeated on the other side of the neck, if possible, or at least 15 cm rostral or caudal from the previous injection site; ensure the new/final injection site is noted on Form CFIA/ACIA 1524 to prevent any confusion at reading.

Discard needle immediately into a sharps container.

Reading

Observe and palpate the injection site at 72 hours ± 6 hours post-injection. Good lighting is essential.

Record the responses to the MCT test for each animal on Form CFIA/ACIA 1524 as follows:

  • negative (Neg.): there is no visible or palpable change in tissue at the site of injection; or
  • reactor: there is a visible or palpable disturbance in tissue at the site of injection
    • when in doubt with respect to the nature of the response to the MCT test, classify the animal as a reactor
    • in the case of reactor(s), contact the district office immediately

Any animal that is classified as reactor on the MCT test is retested by a CFIA veterinarian with the Comparative Cervical Tuberculin (CCT) test (refer to the CCT test section).

All tuberculin tests are official and must be recorded and reported to your local district office by 1 of the following methods:

  • fully completed Form CFIA/ACIA 1524, including animal information for each animal tested
    • a copy of the completed Form CFIA/ACIA 1524 is forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animal was tested); or
  • partially completed Form CFIA/ACIA 1524 as follows and attach an Animal List
    • 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
    • attach to Form CFIA/ACIA 1524 a list of animals that were TB tested and found negative
      • in the animal information section of the Form CFIA/ACIA 1524, insert a statement that reads "The (insert number 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.
    • any animals with non-negative results must be included in the animal information section of Form CFIA/ACIA 1524
    • a copy of the completed Form CFIA/ACIA 1524 and the attached list are forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animal was tested)

Comparative cervical tuberculin (CCT) test (for information purpose only. Not to be performed by accredited veterinarians)

It is recognized that the majority of screening test CFT/MCT reactions in Canada are not due to actual bovine TB infection.

Follow-up comparative cervical tuberculin (CCT) testing serves to rule out the vast majority of false positive MCT reactions and identify any truly infected animals.

  • the CCT test is performed by a trained and qualified CFIA veterinary inspectors and only on test eligible species (that is, cattle, bison, domestic sheep, domestic goats, and cervids)

The CCT test involves an intradermal injection of 0.1 mL of bovine PPD tuberculin and 0.1 mL of avian PPD tuberculin in the cervical region

  • this test is based on the premise that M. bovis (the agent of bovine TB) is somewhat antigenically distinct from M. avium and other related environmental mycobacterial species.  The difference in size of reaction at both sites is used to determine if an animal's reaction to the CFT/MCT test was more likely due to exposure to M. bovis or to the antigens from M. avium or other cross-reactive mycobacteria
    • an animal exposed to M. bovis will have greater reactivity at the ovine PPD injection site relative to the avian PPD injection site

Animals that are negative to the CCT test are not subject to further disease investigation activities.

Any animal that is classified as positive to the CCT test is considered to be a TB suspect and ordered destroyed (with compensation paid) for confirmatory testing.

  • the animal is subject to a detailed post-mortem examination, followed by the laboratory examination of any abnormalities that may be observed, as well as a selection of normal tissues
  • if the laboratory analyses result in the isolation and identification of M. bovis, disease eradication activities are initiated on the affected premises

For export purposes, CFT/MCT reactor animals that are subsequently negative to the CCT test are not eligible for exportation.

  • their herd mates with a negative CFT/MCT test result may be eligible for exportation. Contact the district veterinarian for more information

For the CCT test to work properly, it must be applied at specific times after a screening CFT/MCT test, which in some circumstances requires a waiting period.  Although this can be inconvenient for some individual producers, the delay is preferable to the possibility of missing infected animals which could spread disease.

The CCT test may be applied as follows:

  • up to 10 days following the injection of tuberculin for the CFT test (cattle and bison only)
  • for the purpose of exportation to the USA only, the CCT test may also be performed on a cervid reactor to the MCT test up to 10 days following the injection of tuberculin for the MCT test, in order to qualify herd mates for export if the CCT is negative
  • at least 60 days following the injection of tuberculin for the CFT test (cattle, bison, goats, sheep); or
  • at least 60 days following the injection of tuberculin for the MCT test (cervid)

All subsequent CCT test re-tests must be conducted at intervals of not fewer than 60 days.

Single post-axillary test for camelids

Preparation

A post-axillary intradermal tuberculin test is a standard test for tuberculosis in New World (llama, alpaca) and Old World (camels) Camelidae.

Although this test is not recognized as an official test under the NBTEP, the post-axillary intradermal tuberculin test is performed in Canada under live animal export and import programs.

  • use the test procedures in this section, unless the export health certificate stipulates otherwise

The test involves intradermal injections of 0.1 mL of bovine PPD tuberculin into the skin, posterior to the axilla and elbow of the animal, with observation, palpation, and measurement at 72 hours ± 6 hours post-injection.

Response to this test is determined by a relatively small change in skin thickness; therefore, it is imperative that a regimented approach is taken in applying the test.

  • measurements are influenced by the skin tension (related to animal positioning and restraint); by the amount of pressure applied to the skin by the callipers, and by the size of the skin fold picked up for measurement
  • make every effort to standardize these conditions for both injection and reading

Note: do not apply this test to an animal that has had a tuberculin test of any kind during the preceding 90 days.

Ensure that this test is performed by a trained and qualified accredited veterinarian, subject to the requirements of the importing country.

Prepare the following equipment:

tuberculin – bovine PPD tuberculin (store in dark at 4°C)
syringes – 1 mL tuberculin syringes
needles – 26 gauge, ⅜" long
absorbent cotton
electric clippers with number 40 head
callipers
Form CFIA/ACIA 1524 – Report of Intradermal Test
headlamp or flashlight, if poor lighting

Record the individual animal identification of each animal tested on Form CFIA/ACIA 1524.

acceptable animal identification is as follows: a HofA ear tag, a registered tattoo, or a microchip number, if a reader is available

Injection

Adequately restrain each animal for testing to ensure correct animal identification, the proper preparation of the injection site, accurate pre-injection measurement, careful tuberculin injection, careful palpation, and accurate post-injection measurement.

Prepare the injection site by clipping an 8-cm square area on the lateral aspect of the chest wall in the axillary region near the point of the elbow.

Clean the injection site with cotton (moistened with water, if necessary). Do not use alcohol to clean the injection site.

Inspect and palpate the intended injection site, recording any abnormalities, lesions, or scabs that are observed in the vicinity of the injection site on Form CFIA/ACIA 1524 to avoid being mistaken for tuberculin responses.

Prior to injecting the tuberculin, lift the fold of skin in the center of the injection area, apply the callipers to the fold, adjust the callipers just until they grip the skin but can still slip off the fold, slip the callipers off the fold, and read the calliper measurement to the nearest 0.5 mm.

  • record the measurement on Form CFIA/ACIA 1524

Note: perform a single calliper measurement at the injection site. Only if a problem is encountered with this measurement is the first measurement discarded and the entire process repeated. Repeated measurements will "flatten" the skin.

Draw 0.1 mL of bovine PPD tuberculin into the syringe.

Inject 0.1mL of tuberculin intradermally into the center of the injection area, lifting the tip of the needle slightly after insertion to ensure it is clearly outlined under the skin.  Withdraw the needle slowly.

View injection site to ensure that the injected tuberculin is visible and palpable as a definite bleb.

  • If not, this indicates that either the needle was inserted too deep or a portion of the tuberculin was not successfully injected.
  • if this occurs, the administration of tuberculin should be repeated on the opposite side; ensure the new/final injection site is noted on Form CFIA/ACIA 1524 to prevent any confusion at reading.

Discard needle immediately into a sharps container.

Reading

Observe and palpate the injection site in good light at 72 hours ± 6 hours after the injection to locate the point of greatest response. Note that, in camelids, response at the injection site is frequently diffuse. The response can also be highly variable.

Lift the fold of skin in the area of greatest response or, if there is no area of greatest response, in the centre of the injection area and measure the thickness of the fold with the same callipers used on the injection date and in the same manner as on the injection date, to the nearest 0.5 mm.

  • record the measurement to the nearest 0.5 mm, on Form CFIA/ACIA 1524

Note: perform a single calliper measurement at the injection site. Only if a problem is encountered with this measurement is the first measurement discarded and the entire process repeated. Repeated measurements will "flatten" the skin.

Subtract the pre-injection measurement from the post-injection measurement and record the difference on Form CFIA/ACIA 1524. This value is the "skin thickness difference value."

Interpret the "skin thickness difference value," according to applicable export or import requirements. If interpretation is not specified, classify the animal's TB test result as follows and record on Form CFIA/ACIA 1524:

  • negative (Neg): there is no visible or palpable reaction at the injection site, and "skin thickness difference value" is less than 1.5 mm; or
  • reactor: there is a visible or palpable reaction at the injection site, or "skin thickness difference value" is equal to or greater than 1.5 mm
    • contact the CFIA district office immediately

All tuberculin tests must be recorded and reported to your local district office by 1 of the following methods:

  • fully completed Form CFIA/ACIA 1524, including animal information for each animal tested
    • a copy of the completed Form CFIA/ACIA 1524 is forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animals were tested); or
  • partially completed Form CFIA/ACIA 1524 as follows and attach an Animal List
    • 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
    • attach to Form CFIA/ACIA 1524 a list of animals that were TB tested and found negative
      • in the animal information section of the Form CFIA/ACIA 1524, insert a statement that reads "The (insert number of animals) (insert species) listed on attached list were tested on the date shown and at the location shown with negative results"
      • the accredited veterinarian should sign and date the list
    • any animals with non-negative results must be included in the animal information section of Form CFIA/ACIA 1524
    • a copy of the completed Form CFIA/ACIA 1524 and the attached list are forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animals were tested)

Base-of-the-ear tuberculin test for swine

Preparation

The base-of-the-ear intradermal tuberculin test is the standard test for tuberculosis in swine.

Although this test is not recognized as an official test under the NBTEP, the base-of-the-ear intradermal tuberculin test is performed in Canada under the Artificial Insemination Program and for the purposes of live-animal export.

  • use the test procedures in this section, unless the export health certificate stipulates otherwise

The test involves an intradermal injection of 0.1 mL of bovine PPD tuberculin into the skin at the base of the ear, with observation and palpation at 48 hours post-injection.

Note: do not apply this test to an animal that has had a tuberculin test of any kind during the preceding 60 days.

Ensure that this test is performed by a trained and qualified accredited veterinarian, subject to the requirements of the importing country.

Prepare the following equipment:

tuberculin – bovine PPD tuberculin (store in dark at 4°C)
syringes – 1 mL tuberculin syringes
needles – 26 gauge, ⅜" long
absorbent cotton
Form CFIA/ACIA 1524 – Report of Intradermal Test
headlamp or flashlight, if poor lighting

Sufficiently restrain each pig for testing to ensure, correct reading of the animal identification, careful tuberculin injection, and proper reading of the test result.

The method of restraint used can be modified according to the temperament and the weight of the pig. Avoid applying or observing the test without having the animal adequately restrained.

Record the individual animal identification of each animal tested on Form CFIA/ACIA 1524.

Injection

The tuberculin injection site in swine is the dorsal surface of the ear, slightly distal to/above the ear base. Use either the left or right ear but be consistent.

Clean the dorsal surface of the base of the ear with cotton (moistened with water as necessary). Do not use alcohol to clean the injection site.

Note any abnormalities, lesions, or scabs observed in the vicinity of the injection site, recording on Form CFIA/ACIA 1524 to avoid being mistaken for tuberculin responses.

Check tuberculin label to confirm type – M. bovis tuberculin has an orange label.

Check/confirm the tuberculin expiry date against the current date.

Disinfect rubber stopper with alcohol after opening aluminium seal.

Fill tuberculin syringe to 0.1 mL volume.

Standing to 1 side of the pig, insert needle into the dermis of skin at the base of the ear. Lift the tip of the needle slightly after insertion to ensure it is clearly outlined under the skin. Inject 0.1 mL of tuberculin intradermally and withdraw the needle slowly.

View injection site to ensure that the injected tuberculin is visible and palpable as a definite bleb.

  • If not, this indicates that either the needle was inserted too deep or a portion of the tuberculin was not successfully injected.
  • if this occurs, the administration of tuberculin should be repeated on the other ear; ensure the new/final injection site is noted on Form CFIA/ACIA 1524 to prevent any confusion at reading.

Discard needle immediately into a sharps container.

Reading

Observe and palpate the injection site at 48 hours post-injection. Good lighting is essential.

Unless the importing country has stipulated a different interpretation, record the response to the test on Form CFIA/ACIA 1524 as follows:

  • negative (Neg.): there is no visible or palpable change in tissue at the site of injection; or
  • reactor: there is a visible or palpable disturbance in tissue at the site of injection. Notify the CFIA district office

No ancillary comparative skin testing is available in pigs.

Whenever a porcine animal is classified as a reactor, an investigation must be undertaken by CFIA to identify any contact that the reactor may have had with test eligible species (that is, cattle, bison, domestic sheep, domestic goats, and cervids) since its birth.

  • if any such contact is identified, the test eligible species (for example, their herd/flock) must be investigated and tested by CFIA employees

The fate of a reactor porcine animal is determined by the standards of the program under which the animal was tested and may include retesting the reactor after at least 60 days or removing the animal from the semen production centre or export shipment.

All tuberculin tests are official and must be recorded and reported to your local district office by 1 of the following methods

  • fully completed Form CFIA/ACIA 1524 including animal information for each animal tested
    • a copy of the completed Form CFIA/ACIA 1524 is forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animals were tested) or the certificate of health for entry into a semen production centre (Form CFIA/ACIA 1634 or isolation release document); or
  • partially completed Form CFIA/ACIA 1524 as follows and attach an Animal List
    • 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
    • attach to Form CFIA/ACIA 1524 a list of animals that were TB tested and were found
      In the animal information section of the Form CFIA/ACIA 1524, insert a statement that reads "The (insert number of animals) (insert species) listed on attached list were tested on the date shown and at the location shown with negative results"
    • the accredited veterinarian should sign and date the list
    • any animals with non-negative results must be included in the animal information section of Form CFIA/ACIA 1524
    • a copy of the completed Form CFIA/ACIA 1524 and the attached list are forwarded to the district office responsible of the area where the animals were tested
    • a copy must also accompany the export health certificate sent for endorsement to the district office (if different from the district where the animals were tested) or the certificate of health for entry into a semen production centre (Form CFIA/ACIA 1634 or isolation release document)

3.2 Serologic Testing

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

Information on the specific tests required for each activity can be found in the applicable module(s) of this manual.

Objectives

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

  • the collection of body tissue or fluid from an animal (for example, serum)
  • the injection of an animal for the purpose of determining that animal's freedom from infection with disease (for example, tuberculin testing as outlined in module 3.1)

The reliability of any diagnostic laboratory procedure depends directly on the type of specimens or samples received and on their condition. The laboratory must receive a sample of good quality for diagnostic purposes.

Collection, materials and supplies

It is extremely important that animals are identified correctly.

Unless otherwise indicated, a minimum of a 1 ml sample of serum is required for each test.

A separate sample must be submitted for each diagnostic test required even if the requested tests 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 1 mL serum samples are required

For serum samples:

  • blood can be collected in either silicone coated tubes (red stopper) or serum separator tubes (SSTs)
    • *Notes regarding use of serum separator tubes (SSTs):  A centrifuge that produces a minimum relative centrifugal force (RCF) of 1100-1300 RCF is required if the accredited veterinarian intends to use serum separator tubes (SST) for blood sampling.  Do not store whole blood in SSTs for extended periods of time, particularly if exposed to warmer temperatures, as this will lead to leaching of the serum separator gel into the sample, which interferes with serological tests.  Also, the centrifuged SST tube alone will not prevent hemolysis and leakage of blood cell components back across the plug into the serum fraction.  Samples should be decanted without delay into new red top serum tubes and never be shipped to the laboratory in SSTs.
  • use disposable needles
  • use a waterproof marker to number the vials
  • centrifuge tubes and aliquot into individual red top tubes for each test required
    • 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
  • serum should be kept refrigerated until shipping.  If a delay in shipment to the lab is anticipated, serum should be frozen where suitable given the test
    • always verify specific sample handling and shipping requirements for each sample type and test

The costs associated with material and supplies are the responsibility of the accredited veterinarian.

Preparation and packaging of serum samples

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

When submitting replicate samples (that is, the same sample collected from one animal on one day) for multiple tests on the same animal, label each vial with the same vial number as indicated on the submission form

  • if samples are being sent to multiple labs, the vial numbers will all still be identical for replicate samples

When submitting paired samples, label with the same vial number for the same animal.

  • the acute sample (the oldest one) will be identified A, and the convalescent sample (the most recent) will be identified C (for example: 001A, 001C, 002A 002C, 003A, 003C, etc.)

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

  • the majority of samples collected under the Accredited Veterinarian Program will be classified as "Exempt Animal Specimens" under the Transportation of Dangerous Goods Regulations
    • Exempt Animal Specimens are defined as "Animal specimens not believed to contain infectious substances (for example: samples taken for surveillance or export purposes) or specimens where the pathogens have been neutralized/inactivated"
    • for samples not meeting this definition, consult with your District Veterinarian as to the proper classification and shipping procedures required
  • refer to the following figure for the standards required when shipping Exempt Animal Specimen

Outer package markings checklist

  • name, address, phone number of the shipper and consignee
  • the words – "Exempt Animal Specimen" on the packaging

Statements on Shipper's Waybill

  • statement – Exempt Animal Specimen

Note: if multiple fragile primary receptacles are placed in a single secondary packaging, they must either be individually wrapped or separated to prevent contact between receptacles.

Long description

Pictorial representation of the minimum packaging shipping standards required for Exempt Animal Specimens

Outer packaging

Secondary packaging leakproof or siftproof (for example sealed plastic bag)

Primary receptacle leakproof or siftproof

Specimen

Absorbent packing material (for liquids)

Vials should be packed in boxes designed for this purpose in a manner to prevent breakage (for example, a New York box or styrofoam vial shipper).

  • the use of an appropriate container is a requirement when submitting samples to a laboratory
  • 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 laboratory submission forms should be placed in an envelope and attached to the outside of the protective containers.

Laboratories

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 CFIA laboratories or a CFIA-approved laboratory, unless specified otherwise (see Module 3.6).

  • "CFIA-approved" labs are private laboratories are specifically approved by us to conduct official tests for Equine Infectious Anemia (EIA) and brucellosis submitted by accredited veterinarians.

In cases where required tests are not available in a CFIA or CFIA-approved lab, the accredited veterinarian manual will specify that the test may be performed in a private laboratory that meets the requirements of the Policy on the Use of External Laboratories

  • the accredited veterinarian must verify with their local district office that the laboratory meets the specified requirements before sending a sample

The accredited veterinarian is responsible for the shipping and laboratory fees.

Unless otherwise stated, all specimens sent to a CFIA or CFIA-approved laboratory must be submitted with a CFIA-approved submission form for the official test being requested.

  • please contact the CFIA-approved laboratory directly to confirm what documentation or notification is required for sample submission (see Module 3.6)

Regardless of the type of submission being done, it is the responsibility of the accredited veterinarian to provide all information required.

  • laboratories may refuse to conduct analysis of samples submitted with forms that are not fully complete
  • please refer to your district veterinarian for guidance on the completion of forms and for instructions on the distribution of forms for samples being sent to CFIA laboratories

Submission forms and procedures

Submission forms for samples sent to CFIA laboratories are available at your local CFIA district office.

For the details on submission forms for EIA, brucellosis, or enzootic bovine leucosis, refer to the relevant sections.

Export and Artificial Insemination testing in CFIA laboratories

  • submitted using Form CFIA/ACIA 5473 – Animal Health Import, Export and Artificial Insemination Specimen Submission
  • instructions for submission of samples are printed on that form
    • it should be noted that each CFIA Form 5473 bears a unique reference number and can only be used once
    • do not make photocopies of these forms for use in other submissions.
    • do make photocopies of completed forms when sending samples from the same submission to multiple labs for testing
  • a laboratory notification number, which is available from your district veterinarian, must be entered in the "Reason for Test" section of Form CFIA/ACIA 5473
    • ensure the correct notification number for the samples being tested is used
    • for samples collected under the AI program, also check the box that corresponds to the appropriate stage of testing (that is; FAI - on farm, IAI - pre-entry isolation, AAI - annual or SAI - semi annual resident tests)
  • the accredited veterinarian's submitter ID is the Registration Number as indicated on Page 1 of the AV's authorized CFIA/ACIA Form 1625 and
    • this registration number corresponds to the province where the supervising district office for the submitter is located (that is, where the accredited veterinarian signed their Form 1625)
    • it is always the same regardless of the location where the samples were collected
  • for each test requested, a serum sample and a copy of the appropriate completed submission form must be sent to each laboratory
    • test codes for samples submitted by accredited veterinarian's to a CFIA laboratory can be found in module 10.1A
  • a copy of the submission form must be kept on file and a copy must be sent to your local CFIA district office when the sample is submitted to a CFIA laboratory

Digital submission forms (EIA)

Currently, a digital submission pathway exists only for EIA testing – refer to section 3.2A (EIA testing) .

Shipping samples

Include all pertinent/required forms with the samples.

  • when sending samples to multiple labs as part of the same submission, include a copy of the applicable submission form to each lab
  • please contact the CFIA-approved laboratory directly to confirm what documentation or notification is required when using a CFIA-approved digital EIA certification system for sample submission (contact information available in Module 3.6)

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 the laboratories
  • samples to be shipped may be grouped at an intermediary location into a larger shipment provided that the samples are not opened and/or repackaged after leaving the accredited veterinarian's possession

After collection, samples are processed, refrigerated and shipped to the laboratory without delay.

  • in case of delayed shipment, consult with the applicable lab whether samples can be frozen or not given the type of test being requested

Ship samples only during the week to avoid having them delayed in transit or held over at the laboratory on weekends or holidays.

  • it is advised to only ship samples Mon-Wed, unless receipt at the lab can be guaranteed prior to close of business for the weekend
  • pay particular attention to holiday closures

Vial breakage will require re-bleeding an animal and significantly prolong laboratory test times.
The accredited veterinarian is responsible to resubmit if a specimen is unfit for testing.

  • refer to Module 3.7 for common causes of "unfit" samples.

The accredited veterinarian is responsible for shipping costs.

  • if samples are sent by mistake to the wrong CFIA laboratory, the laboratory may as a courtesy resend it to the appropriate laboratory, if the accredited veterinarian has agreed to provide their courier account number to cover the shipping cost

3.2A – Equine Infectious Anemia (EIA) testing

In this section:

Refer to module 8.4 of this manual for disease-specific information on EIA.

Information owners require before sampling

Before taking the blood sample advise the owner,

  • of the consequences of a final positive result (for more information refer to EIA ‒ What to expect if your animals may be infected)
  • that a sample with a non-negative screening result will require further testing to be completed at the CFIA's EIA National Reference Laboratory (NRL) and that reporting of final results can take up to 10 days from the time of the original submission
  • not to move the equine from the current premises until final results have been reported
  • that their personal information is collected by CFIA under the authority of Health of Animals Act for the purpose of supporting the control of EIA in Canada
  • that there is implied consent to share their personal information with approved third parties when testing is requested
  • that their personal information will be protected under the provisions of the Privacy Act and will be stored in Personal Information Bank CFIA PPU 050/CFIA CON 025

Testing for domestic activities and export to the United States (U.S.) and Mexico:

EIA accredited veterinarians are authorized to collect blood samples for EIA testing and submit them only to CFIA EIA-approved laboratories listed in Module 3.6. Testing may be required:

  • for export to the U.S. and/or Mexico
  • to meet domestic health requirements that have been established by industry (for example, attendance at racetracks, shows, fairs or for sales/transfers of ownership)
  • as part of an owner's or stable's biosecurity plan (for example, for new introductions)
  • when disease is suspected

Note:  It is important to use good biosecurity practices when sampling equines in order to prevent the transfer of contaminated blood between animals and samples. The following guidelines should be used when examining equines or procuring samples:

  • wear clean gloves at all times and change gloves between animals
  • sample those assumed or suspected of being negative for EIA first
  • sample those suspected of being infected with EIA last
  • ensure that all blood collection equipment is single use only and appropriately discarded and disposed of

Shipping samples

CFIA-approved laboratories will only accept submissions that are accompanied by a CFIA-approved (digital or paper) submission document that is complete and accurate. See the following sections for details pertaining to approved paper and digital submission methods.

After collection, samples are kept refrigerated and shipped to the laboratory without delay. Use the safest and quickest means of shipment available. Ship samples only during the week to avoid having them delayed in transit or held over at the laboratory on weekends or holidays.

In case of delayed shipment, samples may be frozen and sent later if the following requirements are met:

  • serum samples are frozen within 5 days of sampling date
  • samples are maintained at –15°C or colder until shipment
  • cycles of freeze/thaw must be avoided so do not place the samples in the freezer door
  • samples must be sent to the laboratory to allow for the analysis to be performed within 3 weeks of the sampling date

Note: a test result will only be valid if the test is performed within 3 weeks of the sampling date. Approved laboratories should not perform analysis on samples that do not comply with this timeline.

Submission

Accredited veterinarians (AV) must use a CFIA-approved digital EIA test certification system to submit samples and create EIA test certificates. Note that paper submission forms for EIA testing are available, however, their use is limited as described under Paper Form CFIA/ACIA 3937.

CFIA-approved digital EIA test certification system:

A CFIA-approved digital EIA test certification system, listed below, is available in both official languages, although the main homepage is only available in English. Once you have registered to use the system (fees will apply), you will be provided with a username, a password, and a link to a login page. The user's browser language will be detected by the system and will determine which language is displayed on the Canadian platform. The login page may also be accessed from the main homepage by selecting "login".

To use a CFIA-approved digital EIA test certification system, the accredited veterinarian must:

  • register with the digital service provider and receive the necessary training from them (refer to link above for provider contact information)
  • send samples to a CFIA-approved laboratory which is registered with the same provider (see Module 3.6)

The data fields in the digital system are based on paper Form CFIA/ACIA 3937, and the following information requirements apply to both:

Name & Address of Owner: record the legal name of the person, company or organization and all contact information.

Animal Location: record the precise legal description of the land location of the equine at the time of sampling and the district office serving that location.

Reason for Testing: check-off only 1 box to indicate why the test is being requested. If more than 1 applies, choose the 1 that is most appropriate at the time of sampling.

Description of Horse:

  • complete all fields as completely as possible
    • use section 2.2 Identification of Horses as a reference
    • the description must match the horse
    • if the description is not considered accurate, the form may be rejected as an official supporting document
    • date of birth – YYYY-MM-DD or if exact date is not known, use January 1 of the birth year as default
    • Note: if a more accurate birthdate is later determined or the description changes (e.g. new scar, coat change), the corresponding fields can be updated in the animal's profile in the digital platform, however, be aware of any incongruencies between the EIA test certificate and any valid export certificates.
  • if the animal being sampled is a non-horse equid (such as a donkey, mule, zebra or crossbreed), indicate this in the "breed" field
  • if applicable, the following data must also be entered:
    • registration number: enter breed registration number
    • tattoo and brands: self-explanatory
    • identification method: enter the form of identification (for example, microchip, iris scan)
    • identification number: enter the identification number associated with the identification method

Submitter Information: record the submitter code (accreditation number), submitter name and signature, sampling date, and all contact information.

If a digital submission is created and a mistake is noted before the approved lab completes testing, an accredited veterinarian can ask the approved laboratory to reject the incorrect submission.

  • please contact the approved laboratory to confirm the details for this process.

If a mistake is noted after the sample has been tested, the accredited veterinarian may, at the owner's request, re-sample the equine and provide a new submission.

Digital photographs

Ensure that the digital photographs are of good quality by taking into consideration lighting conditions, cleanliness, image resolution, proper positioning of the equine and the absence of objects that could obstruct the animal (for example, saddle, blanket, structures). The only animal that should be visible in the photograph is the one being tested. The only exceptions to this would be if other animals are partially visible in the distant background or if a foal is with its mare. In the latter case, the animal being tested must not be obstructed by the other. Every effort should be made to exclude human handlers from the picture.

It is the responsibility of the accredited veterinarian to ensure that the photographs in the equine's digital profile are representative of the animal's appearance at the time of sampling. If significant changes have occurred since the original photos were taken, new photos are required and the physical description text amended to reflect the changes. Examples include newly acquired marks such as brands, scars, or age-related colour changes.

Getting good quality photographs is usually possible although sometimes it can be challenging due to the animal's temperament and/or the environmental conditions. It's recommended that when it's known that photographs need to be taken at an upcoming appointment, that the veterinary practice notify clients of the need to have the animal adequately cleaned so that their markings are visible, as is necessary for the paper form, and that they are in an area with sufficient lighting. If it is known that an equine will not allow for proper photographs to be taken during the appointment, it's possible to ask the owner to take their own photographs, send them to the veterinary practice and have the practice upload them onto the digital platform. The photographs must conform with the requirements listed in this module and the accredited veterinarian must be able to confirm that the submitted pictures match the animal they are sampling.

Failure to produce good quality photographs may result in the digital EIA test certificate being rejected by an inspector. If needed, please reach out to the digital service provider for tips on how to get the best photographs.

Note that all white markings need to be visible on digital photographs and that the full length of all appendages needs to appear in the photographs. For frontal photographs, if the only markings are on the head, the photograph can be limited to the head. If there are white markings on the front legs, the full length of the legs must be included in the photographs. All descriptor fields are required to be filled. Please use "N/A" or "None" if appropriate. If a digital submission system is used for equines with very detailed markings (for example, Appaloosas), written descriptions of the white markings are not required and "N/A" can be used in the descriptor fields.

Accredited veterinarians are responsible for the security and proper use of their digital signature and shall take reasonable care to prevent its misuse. Only accredited veterinarians are authorized to sign documents using their password protected electronic signature. Other clinic staff do not have the authority to be part of the signing process. Utilization of your digital signature by anyone other than yourself would be considered a breach of your agreement as an accredited veterinarian and would render the form invalid. Please note that provisions of article 10 from your Accredited Veterinarian Agreement also apply to the digital submission of documents through a digital system.

Final digital EIA test certificates are recognized as official EIA test results if they are produced using a CFIA-approved digital EIA test certification system. Black and white or colour printed copies of the CFIA-approved electronic EIA test certificate are both acceptable and can be used for domestic use within Canada and as proof of a negative EIA test result for export purposes.

Result reporting (Digital Certificates)

For a negative EIA test result, the CFIA-approved digital EIA test certificate is available online in real-time by the owner (optional), the accredited veterinarian, the CFIA-approved laboratory and us.

For a non-negative EIA test result, the system will generate a CFIA-approved digital EIA test certificate that is watermarked with the word "inconclusive". The owner of the equine will not have access to the digital inconclusive test certificate on the CFIA-approved digital EIA test certification system. The submitting accredited veterinarian will have access to the digital inconclusive test certificate but is NOT to provide the client with a copy of the certificate or report the result to the owner until after confirmatory testing at the EIA NRL is complete. If the results are confirmed to be positive for EIA, the CFIA district office will contact the accredited veterinarian and the animal's owner to inform them of next steps. If the results are confirmed to be negative, the CFIA district office will forward the results to the accredited veterinarian who will forward the negative documentation to the owner.

Note concerning non-negative screening test results: a sample with a non-negative screening test result will be automatically forwarded to the NRL by the CFIA-approved laboratory for confirmatory testing. The equine is considered a suspect case pending confirmatory testing and should not be retested by an accredited veterinarian while awaiting the final test results.

Voiding certificates

If an error is identified after a certificate has been issued, a new sample and new submission form will be required. This may occur when an error in the certificate is identified by the CFIA-approved laboratory, district veterinarian, accredited veterinarian (AV), or horse owner.

If this happens, the accredited veterinarian should:

  • access the online platform and void the erroneous digital certificate from the system by following the instructions provided by the service provider
    • the reason for voiding the certificate should be entered into the field provided
  • advise the owner of the horse to destroy any existing paper copies of the voided certificate
  • advise the district veterinarian of the actions taken, along with the laboratory number (that is: accession number) of the voided certificate

Paper Form CFIA/ACIA 3937 – Submission of Samples and Communication of Results

Paper submission forms still exist for EIA testing, however, their use is limited to the following exceptions:

  1. the accredited veterinarian submits samples on behalf of a practice to which all the following apply:
    • 10 or less samples are submitted for EIA testing per calendar year
    • the practice previously identified themselves as a low-volume tester to the CFIA by submitting a digital exemption application form (available annually in November)
    • the practice is approved and remains in good-standing for a digital exemption and they have not received communications from us to the contrary
  2. the AV's practice is registered to use a CFIA-approved digital EIA test certification system but the accredited veterinarian is forced to use a paper submission form due to circumstances outside of their control
    • For example, there is a technical malfunction with the digital system and they are unable to complete the sample submission, or capturing digital photographs is not possible. This is referred to as emergency use. If a practice is expected to exceed 10 emergency use submissions in a calendar year, first contact your local animal health district office. Failure to do so will result in the practice becoming designated as unapproved for use of the paper submission form.

If either (1) or (2) apply, an accredited veterinarian is permitted to submit samples for testing using paper Form CFIA/ACIA 3937 – Equine Infectious Anemia (EIA) Serum Test Report and Certificate.

In addition, paper form CFIA/ACIA 4679 – Equine Infectious Anemia (EIA) Domestic Serum Test Report and Certificate for multiple horses is no longer accepted.

Digital exemption application process

Practices must apply for an exemption from using the digital certification system as described below:

  • the exemption application period runs from November 1 to November 30 every calendar year
  • a practice is only required to apply for the digital exemption ONCE (not annually) – a new application would only be needed if the practice information changes significantly (for example, new practice name, new location, new ownership)
  • practices that have applied for a digital exemption will not receive a response from the CFIA. The exceptions to this would be if we require additional information, clarification, or if it is determined during the review of annual laboratory submission data that the practice does not meet the exemption criteria. Therefore, unless informed otherwise, an applicant is approved for the digital exemption.
  • any practice (not individual accredited veterinarian) that is going to exceed 10 paper submissions during the year needs to follow the instructions outlined in this module to start using the digital EIA test certification system.  Such practices will be designated as unapproved.
    • any practice designated as unapproved (as a result of exceeding 10 paper submissions in a calendar year or failing to apply for the digital exemption following a CFIA reminder/request) will not be able to submit the paper form to approved laboratories and doing so will result in the submission being rejected by the lab.
    • the CFIA will contact all practices designated as unapproved via email prior to revoking the practice's ability to use the paper form
  • for any questions or to obtain copies of Form CFIA/ACIA 3937, reach out to your local animal health district office.

All fields on Form CFIA/ACIA 3937 must be completed in full unless the information does not exist; strike-out all unused fields. Please refer to the digital system submission section above for a description of the required information.

The top 3 copies (1-white, 2-light yellow and 3-pink) of Form CFIA/ACIA 3937 must be submitted to the CFIA-approved laboratory with the blood sample. The submitter (accredited veterinarian) retains the fourth copy (dark yellow). This copy is not to be marked with results or used as a duplicate.

Result reporting (Paper Form CFIA/ACIA 3937)

For a negative EIA test result at the CFIA-approved laboratory, the form will be marked with the test results and signed by an authorized person. Copy 1 (white) of the form will be returned to the submitter (accredited veterinarian). Copy 2 (light yellow) will be forwarded by the CFIA-approved laboratory to the district office that serves the sampling location. Test results will not be disclosed by the CFIA-approved laboratory over the telephone. Under unique circumstances, the accredited veterinarian can request that the laboratory send a copy of the original certificate via fax or email to the district office prior to the district office receiving the mailed copy.

Under no circumstances is an accredited veterinarian or a CFIA-approved laboratory to make any alterations to the form once laboratory results have been entered. If an error is noted, for example the animal description is not accurate or if required information is missing, the accredited veterinarian may, at the owner's request, re-sample the equine for testing and provide a new submission form. Failure to re-test and re-submit an accurate/complete form may result in the form being rejected by a CFIA veterinarian as an official supporting document.

In the case of a non-negative EIA test result at a CFIA-approved laboratory, the district office that serves the sampling location will be notified of the result within 24 hours by the laboratory. Copy 1 (white) and copy 2 (light yellow) of the form will be mailed to the district office. The serum sample will be forwarded to the NRL where confirmatory testing will be conducted.

Non-negative screening test results

A sample with a non-negative screening test result will be automatically forwarded to the NRL by the CFIA-approved laboratory for confirmatory testing. The equine is considered a suspect case pending confirmatory testing and should not be retested by an accredited veterinarian while awaiting the final test results.

If the final result is confirmed to be negative, a CFIA veterinarian will send the original submitter (accredited veterinarian) copy 1 (white) of the form along with a Client Information Sheet (CIS) that includes the NRL test results.

If the confirmatory results are non-negative (inconclusive or positive), those will be reported to the accredited veterinarian, as well as the owner of the equine, by a CFIA veterinarian.

3.2B – Serologic testing for Brucellosis

In this section:

Refer to module 8.2 of this manual for disease-specific information on brucellosis.

Information owners require before sampling

Before taking a sample for brucellosis testing, advise the animal owner:

  • a sample with a non-negative screening result will require further (ancillary) testing at a CFIA laboratory
  • a non-negative ancillary test result will trigger a CFIA-led disease investigation which may include:
    • an epidemiological investigation,
    • legal movement controls (that is, quarantine)
    • further testing of the non-negative animal and/or exposed livestock, and
    • possible destruction of the non-negative animal to facilitate confirmatory tissue testing
  • of the consequences of a confirmed positive case of brucellosis (see fact sheet).

Additionally, inform the owner:

  • Canada considers its domestic livestock population free from brucellosis
  • Animals should not be moved from the current premises until final results have been reported
  • negative test results do not provide any exemption from existing food safety program requirements, including those for raw and unpasteurized milk
  • there is implied consent to share their personal information with approved third parties when testing is requested
  • that their personal information will be protected under the provisions of the Privacy Act and will be stored in Personal Information Bank CFIA PPU 050 / CFIA CON 025
  • for more information pertaining to the CFIA brucellosis program, see the Brucellosis Fact Sheet or contact your local CFIA district office

Reasons for brucellosis testing

Accredited veterinarians may submit samples for brucellosis testing:

  • to participate in the Artificial Insemination Program
  • to meet export certification requirements (for example, to satisfy the federal/state testing requirements of the U.S. or Mexico)
  • upon request of an animal owner.

These samples are sent either to CFIA-approved laboratories (BPAT only) or CFIA laboratories (for FPA, ELISA, or BPAT), depending on the reason for testing,  type of test required, and/or the species being tested.

Note that if brucellosis is suspected by an accredited veterinarian, it must be reported to a CFIA district office as per the Health of Animals Act

  • samples may not be submitted by accredited veterinarians to approved laboratories if brucellosis is suspected
  • the overseeing district office will provide guidance on how to proceed in this situation
Artificial Insemination Program testing

Accredited veterinarians may submit samples for brucellosis testing to a CFIA lab to meet requirements under the Artificial Insemination Program.

These samples are submitted using Form CFIA/ACIA 5473 – Animal Health Import, Export and Artificial Insemination Specimen Submission, which can be obtained from your local CFIA district office.

When submitting samples for bovines in the isolation facility of a semen production center, refer to the Special Procedure for Submission to a CFIA Laboratory.

For specific details regarding brucellosis testing required under the Artificial Insemination Program, refer to the relevant sections of module 10 of this manual.

Live animal export to the United States (U.S.) and Mexico

When determining the testing requirements for live animal export, accredited veterinarians are to consult the applicable export certificate and/or import permit for the specific type of test required as well as the analysing laboratory (CFIA lab versus CFIA-approved lab).

  • current export certificates are available from your local district office

Accredited veterinarians will submit samples for brucellosis testing to a CFIA-approved lab where the Buffered Plate Agglutination Test (BPAT) is required (unless otherwise stated on the export certificate).

When submitting samples to a CFIA-approved laboratory follow the guidance outlined below:

  • a notification number is not required on Form CFIA/ACIA 5473, but the destination should be identified as the "USA" or "Mexico"
  • it is acceptable to complete the first 2 pages of Form CFIA/ACIA 5473 in full and mark the third page (Sample Information) with "See attached list"
    • the list can be a spreadsheet that records the animals' identification, description, and the corresponding vial numbers
    • the list should indicate the "Form 5473 reference number 20##IEAB-00000#####-4" and be signed – the form reference number is the number which appears in the bottom centre of all 3 pages of Form CFIA/ACIA 5473 and must begin with the current calendar year

When tests for Brucellosis other than the BPAT are required to meet export requirements (for example, the Fluorescence Polarizing Assay (FPA) or enzyme-linked immunosorbent assay (ELISA)) accredited veterinarians will submit samples for brucellosis testing to a CFIA lab (and/or when indicated in the applicable export certificate).

  • these samples are also submitted using Form CFIA/ACIA 5473 – Animal Health Import, Export and Artificial Insemination Specimen Submission, which can be obtained from your local CFIA district office.
  • when submitting samples to a CFIA laboratory (e.g. for export to Mexico), refer to the Special Procedure for Submission to a CFIA Laboratory.
Owner-requested brucellosis testing

Testing conducted for purposes other than export or the Artificial Insemination Program is considered owner request testing (for example, provincial or premises requirement related to a domestic movement; excludes wild animal surveillance/testing).

Samples collected for owner requested testing are sent to CFIA-approved laboratories for the Buffered Plate Antigen Test (BPAT).

Submission requirements:
  • The accredited veterinarian is responsible for the following before sampling:
  • verify that the approved laboratory can:
  • test the requested species
  • accommodate the number of samples, and
  • meet the required timeline
  • samples must be submitted to a CFIA-approved laboratory using the laboratory's submission form
  • on the laboratory's form, indicate the CFIA district office location where the sampled animal(s) is(are) located, and the reason for test (e.g. under comments or history field) as such: "Owner request testing under <insert name> CFIA district office"
Species approved for testing at CFIA-approved laboratories:

CFIA-approved laboratories are only authorized to analyze samples from the following farmed/domesticated animal species:

  • Cattle (Bos taurus/indicus) and American bison (Bison[Bos] bison)
  • Domestic yak (Bos grunniens) and water buffalo (Bubalus bubalis)
  • Domestic sheep (Ovis aries) and domestic goats (Capra hircus)
  • Farmed cervids:
    • Elk, red deer (including hybrids) and Sika deer of the genus Cervus
    • White-tailed deer and mule deer of the genus Odocoileus
    • Reindeer / caribou (Rangifer tarandus)
    • Fallow deer (Dama dama)
  • Domestic pigs (Sus domesticus) and farmed wild boar (Sus scrofa)
  • Camelids (family Camelidae)

For brucellosis testing of species other than those listed above or any wild animals, the accredited veterinarian must consult with their local CFIA district office to determine test eligibility and the proper submission procedures.

3.2C – Serologic testing for Enzootic bovine leucosis (EBL)

EBL samples collected under the Canadian Health Accredited Herd (CHAH) program.

  • submitted to a CFIA laboratory using Form CFIA/ACIA 3841 – Canada Health Accredited Herd – Serum Test Report.
    • use test code BLV-ELI
  • an electronic template for submission of EBL-CHAH samples must be used in lieu of filling out the animal information table of the CFIA/ACIA 3841 (see Special Procedure for Submission to a CFIA Laboratory")

3.3 Testing for bovine genital campylobacteriosis and trichomonosis

This part of the Accredited Veterinarian's Manual will provide the following information necessary for testing for campylobacteriosis and trichomonosis.

Objectives

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

  • the collection of body tissue or fluid from an animal (for example serum, preputial smegma)
  • the injection of an animal for the purpose of determining that animal's freedom from infection with disease (for example, tuberculin testing)

The sampling procedure described in this section may be used to submit samples for export purposes (for example, to qualify cattle to Mexico when applicable) or to meet testing requirements for both the National Artificial Insemination and Embryo Export Approval Programs. Testing to meet State (that is, non federal) requirements or any other owner requested testing is outside the scope of the AV program must be done in non CFIA labs that can provide media and offer testing.

General information

Tritrichomonas foetus (also referred as Trichomonas foetus or T. foetus) is a flagellated protozoan parasite that lives in the reproductive tract of cattle and is only transmitted through venereal contact. Bulls that become infected tend to become carriers for life and they are the main reservoir for the disease. In bulls, the organism is found in the crypts of the penile or preputial mucosa. Tritrichomonas foetus may be sparsely and unevenly distributed in the preputial cavity.

The highest concentration of the organism is thought to occur at the glans penis. Cows that become infected may experience embryonic death, abortion, or pyometra. Some cows may remain infected throughout a successful gestation and transmit the infection at the start of the next season. Although the prevalence of this carrier cow phenomenon is unknown, it is believed to be low.

Campylobacter fetus is divided into 2 subspecies:

  • C. fetus subsp. venerealis and
  • C. fetus subsp. fetus

By definition, C. fetus subsp. venerealis is the cause of Bovine Genital Campylobacteriosis (BGC), a venereally transmitted disease of cattle causing infertility, early embryonic death and abortion with considerable economic losses in endemic regions. While bovine infections with C. fetus subsp. fetus can also occur, they are only associated with sporadic abortion. C. fetus ssp. venerealis is the agent of interest for the National Artificial Insemination and Embryo Export Approval Programs and the export certification for bovines to Mexico.

Materials and supplies

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

Materials required:

  • disposable gloves
  • scissors
  • 53 cm (up to 68 cm in larger bulls) infusion/insemination pipette, individually wrapped
  • disposable 20 ml syringe
  • disposable 3 ml syringe with small gauge needle (not greater than 20 gauge)
  • collection and transport media for each organism

Note on transport media requirements: each organism (Campylobacter and Tritrichomonas) has specific transport media requirements. The transport and collection media itself are currently provided at no charge however, there is a shipping fee for transporting the media from the laboratory to the requesting client (accredited veterinarian or artificial insemination centre). A courier service account number must be provided when ordering the material.

  • Campylobacter: transport enrichment medium (TEM) and saline vials are ordered from the Animal Health Microbiology Laboratory located at our Ottawa Laboratory (Fallowfield)
    • a specific order form is available through CFIA district offices
    • the completed form must be emailed to the laboratory (by the weekly deadline indicated on the form)
    • TEM and saline vials are shipped from the laboratory together in 1 foam container covered by a corrugated cardboard sleeve and placed in a larger polystyrene foam chest with ice packs
    • media is not sent out on Fridays
    • if the TEM vials happen to warm up during transport to the clinic, this is not critical for their subsequent use
  • However, once received, the TEM and collection vials must be stored at 4°C until use
    • the foam container carries 10 TEM vials and 5 or 6 saline collection vials
      • duplicate TEM vials are required for each animal tested
      • the outer sleeve on each foam container holds the unit together
      • has red arrows indicating the "up" position of the vials
      • slip the cardboard sleeve off the foam container (the top of each foam container is stamped in both English and French)
    • remove the top from the foam container to reveal both the TEM and saline collection vials
    • check for a media label (attached to the outside of each vial) and the expiry date which is written on the media label
      • all TEM vials must have a media label and be inoculated on or before the expiry date to be accepted for culture
    • TEM vials have a shelf life of 90 days from date of production
  • Tritrichomonas foetus transport media (TFTM): transport media is sent from the Centre for Food-borne and Animal Parasitology at our Saskatoon Laboratory
    • a specific order form is available through CFIA district offices
    • the completed form must be emailed to the laboratory (by the weekly deadline indicated on the form) to order the media
    • the transport media must be stored at 4°C as well until use and
      • has a shelf life of 14 days from date of production
    • media can be used up to and including, the date of expiry

Note on ordering of transport media: contact your local district office well in advance of any planned sampling. While media can usually be shipped to the requester within 5 days of receiving the order, the state of current supplies (for example, short expiry date) or large orders may require production of a new lot of media which may take up to 6 weeks.

Sample collection

Good sample collection technique as well as care in storage and shipping are very important to achieve the highest possible sensitivity. A video produced by the CFIA's Saskatoon Laboratory, Center for Food Borne and Animals Parasitology is available at the district office to view the collection procedure for Tritrichomonas sampling.

Sampling dates should be selected taking into account shipping time to the laboratory. Samples must arrive at the lab no later than Thursday and should not be shipped over the weekend to as they must be received by the lab within a certain time after inoculation of the media (48 hours for Campylobacter; 5 days for Tritrichomonas).

For sampling, all transport media (TEM and TFTM) and saline vials should be brought to room temperature (18 to 37°C) prior to inoculation with sample.

  • the expiry date must be verified before sampling procedure begins
  • 2 separate samples should be collected if testing for both organisms is required
  • the animal must be restrained to avoid injury to the collector and to the animal
  • Separate sampling materials must be used for each animal collected.

Male animals:

  • preputial fluid (smegma) is collected
  • sampling should not be conducted until at least 7 days have elapsed following natural service or semen collection by means of an artificial vagina
  • use a separate pair of disposable gloves for each animal
  • long hairs and fecal balls at the prepuce orifice should be cut with scissors to reduce contamination
    • care must be taken to properly disinfect and rinse the scissors between different animals
    • washing the prepuce is not advisable as this may introduce contaminants and/or flush out existing target organisms
    • disinfectant must be avoided
      • if the preputial area is excessively dirty, the external part only can be carefully cleaned with water and dried with paper towel
  • insert the pipette into the prepuce about halfway
  • connect a 20 ml syringe to the pipette and apply negative pressure
    • the pipette should be held parallel to and along the length of the prepuce
    • in situations where introducing contamination into the sheath is a concern, the plastic cover of the pipette may be left in place until the end of the pipette extends beyond the dirtiest part of the preputial orifice
  • advance the pipette until it reaches the fornix
  • using back and forth movements from the fornix to within 5 cm of the preputial orifice, slide the pipette 10 to 15 times to gently scrape the preputial mucosa
    • it is important that the pipette contacts the glans penis
  • the scrapings will be collected by the negative pressure in the pipette
  • retract the pipette and syringe out of the prepuce

Female animals:

  • cervical mucus or vaginal discharge must be sampled
  • use a separate pair of disposable gloves for each animal
  • debris should be cleaned from the external vulva with a lightly wetted dry paper towel
    • no disinfectant should be used
  • immobilize the cervix per rectum and insert the pipette in the cranial third of the vagina
  • the tip of the pipette is advanced to the cervix
    • negative pressure is applied to collect cervical mucus via a syringe
    • vaginal discharge may also be collected from the anterior vagina
  • retract the pipette and remove it from the vulva

Sample preparation and packaging instructions

The reliability of any diagnostic laboratory procedure depends directly on the type of specimens or samples received and on their condition. It is extremely important that animals are correctly identified and the information entered on the forms is complete and accurate.

Each vial must be marked indelibly with the number corresponding to the one entered on the submission form in the column under vial number for the corresponding animal being tested.

Duplicate vials for Campylobacter must be identified with the same vial number followed by suffix A or B for the corresponding animal

Tritrichomonas:

  • properly label the TFTM vial (label the tube with the identifier, not just the lid)
  • expel all collected sample material into the TFTM media
    • gently aspirate some transport media into the pipette and repeatedly rinse any remaining contents of the pipette and syringe into the media
  • place TFTM vials containing samples back into the insulated container box from which they were shipped from the lab
  • inoculated material must be packed to exclude light and minimize temperature fluctuation
    • to prevent temperature fluctuation during shipping, samples placed in insulated container should be packed with newspaper or alternative insulating material to fill any air space
  • samples must be kept between 10 to 37°C
    • when the samples are stored or shipped at 4°C or below, the viability of the organisms decreases and thus the sensitivity of the test decreases
    • use of insulated containers is essential
    • samples frozen or shipped on ice will not provide reliable results and will require re-collection
    • in summer months, care should be taken to make sure that the samples are not exposed to temperatures exceeding 37°C
      • do not add ice packs as this will drop the temperature of the samples below 10°C
    • in winter months, gel packs may be warmed to a maximum of 37°C and added to the shipment to keep samples above 10°C
      • do not place these materials in direct contact with the samples and clarify on the submission form that these additional insulating packs were not frozen, but included with the packaging at room temperature or below the maximum temperature stated above
    • samples must reach the Saskatoon laboratory within 5 days of collection or they will not be tested and will be reported as "unfit"

Campylobacter: For each animal sampled,

  • label 2 TEM vials with the sample number and suffix (one vial labelled A and the duplicate vial labelled B), using the provided label space on the side of the vials; and
  • label 1 saline vial

Uncap the saline collection vial

  • collect a preputial sample as per previous instructions and transfer the sample into the saline vial by submerging the collection pipette tip into the liquid and expel any material collected in the pipette and syringe
    • repeatedly rinse any remaining contents by pulling back and forth the syringe plunger very gently several times in the vial
    • the sample must not be left in the saline for more than 15 minutes before the transfer and inoculation of the TEM vials
  • using a 3 ml disposable sterile syringe and a small gauge needle (should be no larger than 20 gauge), carefully aspirate 2 ml of the upper layer from the saline collection vial
    • exclude all air from the syringe before transfer
  • inject 1 ml of the aspirated saline through the rubber membrane into the TEM vial labelled with suffix A (DO NOT unscrew the top of the TEM vials as this will disrupt the special gas mixture within)
  • repeat this step for the second TEM vial, labelled with suffix B, using the remaining 1 ml aspirated saline in the syringe
    • mix the contents by gently shaking the TEM vials
  • place the TEM vials and used saline collection vials back into their placement slots in the bottom portion of the foam container from which they originated
    • when all slots in the foam container are filled with the TEM and saline vials, place the foam top on the container and slide the corrugated sleeve over the unit with the red arrows pointed up
    • do not cut, mark or alter foam containers or cardboard sleeves
  • place the foam containers into a larger polystyrene foam chest without ice packs and ship back to our Ottawa laboratory (Fallowfield) without delay
    • temperatures during transport should be between 18 to 37°C
    • use of warmed gel packs should be considered during colder months as described above for Tritrichomonas
  • 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
    • TEM vials must be received in the diagnostic laboratory within 48 hours after the vials are inoculated
    • samples that reach the laboratory after this time period will be considered unfit for analysis
  • samples 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
    • if samples arrive at the laboratory cold or frozen, they will not be analyzed and the result will be reported as unfit

Submission forms

Samples are to be submitted using the CFIA/ACIA Form 5473 and the corresponding Excel template.

A copy of the appropriate completed submission form must be sent to the district office and for each test requested, a copy of the submission form must be sent to the laboratory along with the samples.

Care must be taken when filling out the forms. All copies must be legible.

All information should be recorded, including ear tag numbers, tattoos or other types of identification.

The type of tests 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.

A laboratory notification number, which is available from a CFIA district veterinarian, must be entered in the "Reason for Test" section of the form.

  • our laboratory will not perform the analysis if the notification number is missing.

It is the responsibility of the accredited veterinarian to provide all information required, or to re-submit if a specimen is unfit for testing.

  • laboratories may refuse to conduct analysis of samples submitted with forms that are not properly completed.

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

It is essential that samples be shipped in a timely fashion to the appropriate laboratory to avoid unnecessary impacts on the integrity of the samples and potential re-sampling.

  • if uncertain, check with a CFIA district veterinarian before shipping.

Vial breakage can result in re-sampling an animal and significantly prolong laboratory test result turnaround times.

  • 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 the laboratory.

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

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

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 the laboratories.

The accredited veterinarian is responsible for shipping costs.

Laboratories

Samples for Campylobacter testing must be sent to our Ottawa laboratory (Fallowfield).

Samples for Tritrichomonas testing must be sent to our Saskatoon Laboratory.

Our laboratory information may be found in module 3.6.

Test results

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.

The accredited veterinarian is responsible for advising the owner of the results.

Laboratory fees

The accredited veterinarian is responsible for laboratory fees.

The district office will recover applicable fees for all animals that had tests performed in CFIA laboratories, regardless of the test result.

The Canadian Food Inspection Agency Fees Notice can be consulted for more information.

3.4 - Special procedure for submission to a CFIA laboratory

When an accredited veterinarian needs to send samples to be analyzed in CFIA laboratories from:

  • bovine in isolation under the AI program (isolation notification IAI),
  • sheep and goats, wild boar or cervids for exportation to Mexico,
  • bovines under the EBL-CHAH program

an Excel spreadsheet template must be completed and forwarded electronically in addition to the paper form submission (CFIA/ACIA Form 5473 – bovine isolation testing, export to Mexico; CFIA/ACIA Form 3841 – EBL-CHAH testing) that will be attached to the samples to be sent.

  • the template for each activity listed is available through your local district office

The spreadsheet will be used by CFIA laboratories to import data into the Laboratory Sample Tracking System (LSTS), preventing transcription errors and saving time, especially on larger submissions.

The template must be completed as follows:

  • for each submission, a new template will need to be completed
  • no column can be deleted or added to the template and the mandatory format is to be followed
  • mandatory fields in the template include sample number, date sampled, sample type, animal identification (ID) (at least 1 of the 6 fields about ID is mandatory) and breed
    • enter the age in column N
    • if the age is unknown this column may be left blank as long as you select from the drop down menu "estimate" for column L and "not provided" for column M
    • some fields do not apply for isolation testing or for export to Mexico. They may be left blank, but they cannot be deleted
  • the spreadsheet is saved under a new name containing the reference number of the CFIA Form 5473 form it will refer to (for example 2014IEAB-0000079706-4)
    • for EBL-CHAH submissions, the file name should contain the name of the herd sampled and date of testing
  • the electronic submission of the template does not preclude the requirement to send a paper copy of the submission form along with the sample.
    • it is acceptable to complete the first 2 pages of Form CFIA/ACIA 5473 in full and mark the third page with "See attached list" to be acceptable
    • the attached list must show all the information for each animal on the same page (use the hide function from the spreadsheet to remove unnecessary columns)
    • the reference number must be written on every page, and each page needs to be initialed by the accredited veterinarian

For all samples, the properly named Excel file must be sent to a generic email address: cfia.labtemplate-gabaritlab.acia@inspection.gc.ca

  • a copy must be sent as well to the local CFIA district office where the animals were located when tested
  • each email must contain only 1 attached template
  • in the email subject line, provide the CFIA Form 5473 reference number or name of the EBL-CHAH herd only
  • in the body of the message, you may provide info on the date the samples are shipped, if possible. 
  • the generic laboratory email address is not assigned to a specific laboratory and cannot be used to submit questions or requests.  Any questions will need to be directed to your local CFIA district office

3.5 Genotype Testing for Scrapie Susceptibility

Sheep

Genotype analysis of the prion gene has been determined to be a significant factor in susceptibility to infection with classical scrapie in sheep. One is most likely to discover the disease in sheep in genotypes with the highest susceptibility (See table 1).

The genetic makeup of sheep, as with all mammals, are diploid organisms. All cells contain 2 copies of each chromosome, and thus, 2 copies of the gene that codes for the prion protein. Genes are made up of codons, a stretch of DNA that determines which particular amino acid will be included at a particular location of a protein (in this case, the prion protein). The prion protein is comprised of 256 amino acids, so there are 256 codons determining these amino acids in the prion protein gene (PRNP).

Studies from various geographic areas and different breeds of sheep have found 3 codons to be most significantly related to susceptibility to classical scrapie: 136, 154, and 171. The genotypes of sheep in Canada are referred to using these codon number and the corresponding 2 alleles associated with the codon. For example, 136 AV; 154 RR; 171 QR (also referred to as ARQ/VRR in short hand). In North America, codons 136 and 171 are given primary importance in association with susceptibility to classical scrapie.

The profile of the sheep's prion genotype varies among countries, flocks, and breeds. While there is no genotype that confers absolute resistance to scrapie infection, the presence of arginine (R) at codon 171 confers a high level of resistance to classical scrapie. Conversely, the presence of glutamine (Q) at codon 171 is associated with susceptibility. This is supported by the fact that of all sheep diagnosed with classical scrapie in Canada from 1998 to 2008, 93% were of the genotype ARQ/ARQ, and over 98% were homozygous 171QQ. Similarly, the majority of classical scrapie cases reported worldwide have been in 171QQ sheep.

Although rare in the Canadian sheep population, histidine (H) or lysine (K) may also be found at codon 171, but both are currently considered equivalent to 171Q in term of susceptibility. The valine (V) amino acid at codon 136, confers greater susceptibility among the 171QR population. Table 1 summarizes the relative susceptibility of sheep genotypes to classical scrapie.

Table 1 Summary of the relative susceptibility of sheep genotypes to classical scrapie
Genotype (136, 171) Susceptibility to classical scrapie
AA, RR Negligible
AA, QR very low
AV, QR intermediate
AA, QQ
AV, QQ
VV, QQ		 high

A significant finding, as it relates to the transmission of scrapie, is that the genotype of the fetus influences the migration and accumulation of abnormal prion in the placenta of an infected ewe. A 171QQ-infected ewe carrying a 171QQ fetus results in the accumulation of large quantities of abnormal prion, which is subsequently shed during birth or abortion. However, when a scrapie-infected ewe carries a 171QR or 171RR fetus, the abnormal prion does not cross to the fetal side of the placenta. The use of a 171RR ram for breeding, therefore, can prevent the shedding of abnormal prion at lambing from infected ewes.

The knowledge of genetic resistance to classical scrapie has led to the implementation of breeding programs in several countries that are aimed at reducing national flock susceptibility. Although the heavy promotion of the use of 171RR rams across Canada may be effective in minimizing the spread of scrapie, it would also result in a significant change in the genotype profile of the national flock. Currently, Canada's sheep population demographics show considerable variation in scrapie genotype profiles between breeds and flocks.

Breeding for scrapie resistance is an effective disease control tool that may be applied by owners. To date, the selection for resistant scrapie genotypes has not been associated with negative production traits.

Note that atypical scrapie does not follow the same association with genotype as described for classical scrapie and has been detected in numerous countries, primarily through the testing of deadstock and active scrapie surveillance programs. Although now referred to as atypical scrapie, cases were initially referred to as Nor98, as the first case was identified in Norway in 1998. Atypical scrapie appears to be clinically, pathologically, biochemically, and epidemiologically unrelated to classical scrapie and therefore not a known risk for spread as it is limited to that individual animal.

Goats

Over the last several years, CFIA evaluated the evidence for genetic resistance to classical scrapie in goats. Genotype data obtained from several scrapie-infected goat herds in Canada, combined with ongoing research findings from North American and European studies, provide convincing evidence for genetic resistance in goats.

The CFIA is currently working towards the incorporation of the use of genetic resistance alleles S146 and K222 in goats in Canada's domestic National Scrapie Eradication Program (NSEP). This is consistent with our trading partners, the United States (U.S.) and the European Union (EU). Internationally, other scrapie-affected countries (including the U.S.) with eradication programs have started to use in their domestic programs.

Further research in the Canadian context is still needed for goat genotypes and resistance to scrapie and will be done via a pilot project to assess for scrapie resistance in positive goat herds. The hope is that goat genotyping can be fully incorporated into the NSEP in the future, along with the goal of ultimately being able to also use goat genotyping for scrapie resistance for import and export purposes.

Sampling procedures for genotyping

Samples taken for genotyping for the purposes of the National Scrapie Flock Certification Program (SFCP) or for export the U.S. must be taken by an accredited veterinarian. Approved identifiers are required on sheep being genotyped and the official identification number (15 digits) included with the sample submission.

Jugular venipuncture

Jugular 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.

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 DNA.

Collect a full 7 to 10 ml of blood in an ethylenediaminetetraacetate (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.

Module 3.2 of this manual proves detailed instructions on the packaging, shipping and submission of serological samples.

Samples collected under the Scrapie Flock Certification Program should be submitted to a lab listed in Annex G of Module 7.5 of this manual.

Samples collected in support of export to the U.S., should be submitted to the National Veterinary Services Laboratory (NVSL) or another laboratory designated by NVSL to verify genotype. Select "Ovine PrP Genotyping" at the link to access lab information.

Alternate tissues samples

Genotype analysis may be performed on tissues other than whole blood provided that the approved lab has been evaluated and approved for testing of the type of tissue submitted (for example, skin biopsy). It is the responsibility of the accredited veterinarian to confirm what tissue testing is offered under the approved lab's scope of duties.

3.6 Laboratories

CFIA-approved laboratories

Laboratories approved to test Equine Infectious Anemia (EIA) and brucellosis – Buffered plate antigen test (BPAT).

Simply select the disease and/or test type in the filter option to obtain the most current list of labs available.

Canadian Food Inspection Agency (CFIA) laboratories

Our 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 CFIA laboratories:

CFIA Lethbridge Laboratory
(Animal Diseases Research Institute)

CFIA Saskatoon Laboratory
(Centre for Food-borne and Animal Parasitology)

CFIA Winnipeg Laboratory
(National Centre for Foreign Animal Disease, Canadian Science Centre for Human and Animal Health)

CFIA Ottawa Laboratory

CFIA Saint-Hyacinthe Laboratory
(Health of Animals and Food Laboratory)

3.7 - Criteria for rejection of samples and submission forms in CFIA animal health laboratories

Our labs and CFIA-approved labs must adhere to quality assurance standards in the receipt and testing of all samples received.

CFIA-approved labs set their own internal criteria for the rejection of samples received and have protocols in place to notify the submitters of samples rejection and the reasons for the rejection.

  • these criteria can be obtained directly from the specific CFIA-approved lab.

For samples submitted to CFIA labs, criteria for rejection are listed in the following tables.

  • reasons for rejection may be related to the sample itself or the sample submission form.
  • notification of rejection of samples will come to the accredited veterinarian through your local CFIA district office and will consist of the submission reference number, vial(s) number(s) of the rejected samples (if applicable) and the reason for rejection.
  • based on the reason for rejection, the accredited veterinarian will be required to undertake the appropriate corrective measures for the sample(s) and/or the submission form in order to complete the testing.

A – General for all sample types

No. Criteria of rejection for samples Result on Record of Analysis (ROA) Comments
A1 Contamination Unfit
  • New sample is needed (with a new specimen submission form (SSF) unless extra serum from the same sample (same collection) can be provided by the submitter)
  • Contamination: sample that shows 1 or more of the following conditions: turbidity, cloudy, opacity, mucoid, gelatinous, bad smell, brownish colour, evidence of bacterial growth or of foreign material in the sample (for example feces, hair, dirt)
A2 Incorrect type of sample or container Unfit
  • New sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
  • Example: blood instead of serum or green cap instead of red cap, swab sent in inappropriate type of media, swab sent in media when it should not, etc.
A3 No ID on sample's container (vial/bag) Unfit
  • New sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
A4 Error in ID Unfit
  • New sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
A5 Duplicate samples Unfit
  • If 2 samples bear the same ID in a unique set of samples, the lab cannot assume which one is the good one (for instance pick 1 of the 2 for test)
  • The lab has to consider both duplicate samples "Unfit"
A6 Missing sample No sample received (NSR)
  • A new sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
  • If a lab has to perform 2 analyses and receives only 1 sample where the document "Animal Health Test and Analyses Performed in CFIA Lab" states that 1 sample per test is needed, the lab receives 1 sample for 1 analysis and authorizes the second analysis as NSR
A7 Extra sample Sample is not tested
  • No prior communication with the submitter before the sample is put aside
  • Example: a lab receives a SSF that lists samples 1 to 6 and samples identified 1 to 7 are in the box
  • Sample number 7 will not be tested
A8 Broken vial Broken on arrival
  • New sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
A9 Insufficient volume/quantity Insufficient sample
  • New sample is needed (with a new SSF unless extra portion from the same sample (same collection) can be provided by the submitter). Includes serum, blood, straw of semen, gram of tissue, etc.
  • Refer to the AH Test and Analyses performed in CFIA Lab document to see the volume/quantity required
A10 Sets of samples with the same IDs in a unique box Unfit
  • If 2 sets of samples or more are sent in a unique box, and if some samples bear the same IDs, these samples will be reported as unfit

B – Serology samples

No. Criteria of rejection for samples Result on Record of Analysis (ROA) Comments
B1 Lipaemia Unfit
  • Not suitable for test
  • A new sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
  • Lipaemia: sample that has a cloudy aspect
  • Note: aliquots may not be equivalent in terms of lipaemia and lipaemia may be an issue for some assays and not for others
B2 Haemolysis Unfit
  • Not suitable for test
  • A new sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
  • Haemolysis: serum sample that is red to burgundy due to red cell lysis
  • Note: aliquots may not be equivalent in terms of haemolysis and haemolysis may be important for some assays or for others, not
B3 Fibrin clot (avian samples) Unfit
  • Not suitable for test
  • The clots makes it impossible to collect serum for analysis
  • A new sample is needed (with a new SSF unless extra serum from the same sample (same collection) can be provided by the submitter)
  • Note: the fibrin clot may appear during the transport of the samples

C – Bovine Spongiform Encephalopathy (BSE) samples

Note: BSE testing is not done under the Accredited Veterinarian Program, but accredited veterinarians may be involved in the collection of these sample on behalf of CFIA

No. Criteria of rejection for samples Result on Record of Analysis (ROA) Comments
C1 Damaged sample Not tested – No sample
  • The brainstem around the medulla oblongata/obex area has been sliced/severed
  • When the damage is so extensive that the obex cannot be recognized, the sample is called "Damaged, No Obex (DNO)" and the result is entered as "Not tested – No Sample"
C2 Autolyzed sample Not tested – Unsuitable
  • Tissue degradation by intrinsic enzymes
  • When the autolysis is so extensive that no brain areas can be identified the sample is called "Autolyzed-Unfit"
C3 Incorrect type of sample Not tested – No sample
  • When incorrect region of the nervous system is sent as: spinal cord, cerebellum, cerebral cortex, etc.
  • None are acceptable, even if a clinical history is provided

D – Other specific sample types

Note: these specifications apply to samples taken for routine tests and are an excerpt from the document Animal Health Tests and Analyses performed in CFIA laboratories

No. Criteria of rejection for samples Result on Record of Analysis (ROA) Comments
D1 Avian Influenza Unfit
  • Pool: serum pooled
  • Swabs: Universal Transport Medium (UTM) containing swabs, mix of cloacal and tracheal in the same UTM.
D2 Avian Paramyxovirus (Newcastle Disease) Unfit
  • Swabs: UTM containing swabs, mix of cloacal and tracheal in the same UTM
D3 Bovine Viral Diarrhea Unfit
  • State: serum frozen for virus isolation (ISOL) test, semen not frozen and not shipped in liquid nitrogen dry shipper
D4 Campylobacter fetus Unfit
  • Age: sample older than 48 hours
  • Temperature: sample temperature outside of 18 to 37°C
D5 Contagious Equine Metritis Unfit
  • Age: sample older than 48 hours
  • State: sample frozen for culture test
D6 Cysticercosis Unfit
  • State: sample frozen
D7 Elaphostrongylus cervi Unfit
  • State: sample frozen
D8 In-vitro fertilized embryo – virus isolation Unfit
  • State: sample not frozen and not shipped in liquid nitrogen dry shipper
D9 Infectious Bovine Rhinotracheitis Unfit
  • State: semen not frozen and not shipped in liquid nitrogen dry shipper
D10 Mycoplasma Unfit
  • Age: sample older than 48 hours for culture test
  • State: semen not frozen and not shipped in liquid nitrogen dry shipper
D11 Salmonella Unfit
  • Age: sample older than 48 hours for culture test
D12 Tritrichomonas (Trichomonosis) Unfit
  • Age: sample older than 120 hours for culture test
  • Temperature: sample temperature outside of 10°C to 37°C
D13 Turkey Rhinotracheitis (TRT) Unfit
  • Pool: serum pooled
  • Swabs: UTM containing swabs, mix of cloacal and tracheal in the same UTM
D14 All Histopathology tests (for example Cysticercosis, TB) Unfit
  • State: samples frozen

3.8 - Test results

The accredited veterinarian is responsible for advising the owner of the results of testing.

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 CFIA district office

Test results from CFIA-approved laboratories are usually reported directly to the submitter and the local CFIA district office.

For additional information on distribution of test results when using a CFIA-approved digital EIA certification system, refer to section 3.2A.

Test results from non CFIA-approved private laboratories are usually reported directly to the submitter only.

  • the accredited veterinarian will provide a copy of such test results to CFIA upon request.

Schedule testing as to allow sufficient time to receive results.

  • turn around times for samples tested are relative to the day the samples are received by the laboratory
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