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Chapter 3 - Testing
3.2 Serologic Testing

This page is part of the Guidance Document Repository (GDR).

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

  1. collection, material and supplies
  2. preparation and packaging of serum samples
  3. laboratories
  4. submission forms
    • paper submissions
    • electronic submissions with a Canadian Food Inspection Agency-approved electronic Equine Infectious Anemia (EIA) certification system
  5. special procedure for submission to the CFIA laboratories, applicable to samples from bovine tested in isolation (IAI notification), and from sheep and goats for export to Mexico
  6. shipping samples
  7. criteria for rejection of samples and submission forms in CFIA animal health laboratories
  8. test results

Objectives

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

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

2. 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

3. It is extremely important that animals are identified correctly. Animals which are tested must have a unique identification number. Refer to Module 2.1 Identification of Livestock or Module 2.2 Identification of horses for more information.

4. Unless otherwise indicated, a minimum of a 1 ml sample of serum or 5 ml centrifuged serum separator tubes (SST) sample is required for each test.

5. A separate sample must be submitted for each diagnostic test required even if various tests are requested and are done at the same laboratory. Laboratories prefer separate serum samples for each test in order to maintain sample integrity. For example, if brucellosis and leptospirosis tests are requested, 2 samples are required.

6. For clotted blood samples:

  1. use silicone-treated vials (red stopper), which should be centrifuged before shipping and the clot removed. Serum should be kept refrigerated until shipment.
  2. use disposable needles.
  3. use a waterproof marker to number the vials.

7. 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.

8. The costs associated with material and supplies must be paid by the accredited veterinarian.

Preparation and packaging of serum samples

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

10. Paired samples must be identified with the same vial number for the same animal. In addition, 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.)

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

12. Vials should be packed in boxes designed for this purpose.

13. The use of an appropriate container is a requirement when submitting samples to a laboratory. When shipping more than 1 container, ensure that they are individually identified and packaged to prevent breakage. In addition to a legal responsibility, a submitter has a moral obligation to ensure the safety of transportation industry workers and laboratory staff handling the specimen.

14. The laboratory submission forms should be placed in an envelope and attached to the outside of the protective containers. Please contact the CFIA-approved laboratory directly to confirm what documentation or notification is required when using a CFIA-approved electronic EIA certification system for sample submission. (Contact information available in Module 3.3 Laboratories).

Laboratories

15. Most testing, mandated by the Health of Animals Act and regulations or conducted in response to a specific request by an importing country, is conducted in a CFIA laboratory or a CFIA-approved laboratory, unless specified otherwise.

16. Certain private laboratories are specifically approved by the CFIA to conduct various official tests submitted by accredited veterinarians. Those laboratories are approved to test for EIA, Bovine leucosis virus (BLV, for herd certification purpose) and Brucellosis. They are listed in Module 3.3 Laboratories. These CFIA-approved laboratories maintain a list of accredited veterinarians for whom they are authorized to perform these tests. The submission will be rejected if the submitter is not accredited for that function.

17. Certain private laboratories, although not specifically approved by CFIA, may be authorized to perform test for export purpose. In such case, the accredited veterinarian manual will specify that the test may be performed in a private or authorized laboratory that meets the requirement of the Policy on the Use of External Laboratories. Before sending a sample to a private laboratory, please verify with the district office that the laboratory meets the specified requirements.

18. The accredited veterinarian is responsible for the laboratory fees. Applicable fees for test submitted to CFIA laboratories are available in Module 4.2 CFIA Fees.

19. 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 when using a CFIA-approved electronic EIA certification system for sample submission. (Contact information available in Module 3.3).

Paper submission forms

20. Submission forms are available at the CFIA district office. For each test requested, a serum sample and a copy of the appropriate completed submission form must be sent to the laboratory. A copy of the submission form must be kept on file, and a copy must be sent to the CFIA district office when the sample is submitted to a CFIA laboratory.

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

22. 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 a CFIA district veterinarian for examples of completed forms, and for instructions on the distribution of forms for samples being sent to CFIA laboratories.

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

24. To submit brucellosis samples to a CFIA-approved laboratory, use Form CFIA/ACIA Form 5473 as well. When samples are tested for export purpose, a notification number is not required on Form CFIA/ACIA 5473, but the destination should be identified as "USA or Mexico".

25. When submitting samples for brucellosis testing to a CFIA-approved laboratory, it is acceptable to complete the first 2 pages of Form CFIA/ACIA Form 5473 in full and mark the third page with "See attached list". The list can be a spreadsheet that records the animals' identification, description and the vial numbers, and should identify "form reference #_______________" and be signed . That reference number is the number which appears in the bottom centre of all 3 pages of Form CFIA/ACIA Form 5473.

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

27. EIA samples can be sent to a CFIA-approved laboratory using Form CFIA/ACIA 3937 – Equine Infectious Anemia (EIA) Serum Test Report and Certificate, or Form CFIA/ACIA 4679 – Equine Infectious Anemia (EIA) Domestic Serum Test Report and Certificate when more than 1 horse is being tested. Please note that Form CFIA/ACIA 4679 cannot be used for export purposes.

Electronic submissions with a CFIA-approved electronic EIA certification system

28. A CFIA-approved electronic EIA certification system can be used to submit samples for EIA testing to a CFIA-approved laboratory. Data fields of the electronic system are based on Form CFIA/ACIA 3937. For contact information, please refer to the List of CFIA-approved Electronic EIA Certification Systems (Module 8.4 Equine infectious Anemia).

29. The accredited veterinarian must:

  1. register with the service provider and receive the necessary training
  2. send samples to a CFIA-approved laboratory which is registered with the same provider (laboratory contact information available in Module 3.3)
  3. if the service provider is a U.S. based company, informed consent must be obtained from each client prior to the first time their personal information is input into the CFIA-approved electronic EIA certification system. A Consent to Disclose Client Information form will be made available to you by the service provider and will only be required to be filled out once per client. The client's information will be protected under the provisions of the applicable U.S. laws and regulations. Information may be accessible or protected as required pursuant to applicable U.S. laws and regulations. A copy of the completed consent form must be maintained in the client's record

30. For more details on the required information and digital photographs, please refer to Module 8.4 Equine Infectious Anemia.

31. 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 does not have the authority to be part of the signing process. Utilization of your electronic 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 electronic submission of documents through an electronic system.

32. Please contact the CFIA-approved laboratory directly to confirm what documentation or notification is required when using a CFIA-approved electronic EIA certification system for sample submission. (Contact information available in Module 3.3).

33. Final CFIA-approved electronic EIA test certificates are recognized as official EIA test results, and can be accessed online in real-time by the owner (optional), the accredited veterinarian, the CFIA-approved laboratory and CFIA. Black and white or colour printed copies of the 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.

34. For additional information regarding the use of an approved electronic EIA certification system, please contact the CFIA district office.

Special procedure for submission to CFIA laboratories, applicable to samples from bovine tested in isolation (IAI notification), and from sheep and goats, wild boar or cervids for export to Mexico

35. When an accredited veterinarian needs to send samples to be analyzed in CFIA laboratories from bovine under isolation notification (IAI) and from sheep and goats, wild boar or cervids for exportation to Mexico, an Excel spreadsheet will need to be completed and forwarded electronically in addition to the paper form CFIA/ACIA 5473 that will be attached to the samples to be sent.

36. This mandatory template will be used by the CFIA laboratories to import data into the Laboratory Sample Tracking System (LSTS). This will prevent transcription errors and will save time, especially on larger submissions.

37. The template must be completed as follows:

  1. for each submission, a new template will need to be completed. Make sure to save it under a new name containing the reference number of the LSTS form it will refer to (e.g. 2014IEAB-0000079706-4)
  2. the properly named Excel file must be sent to a generic e-mail address: cfia.labtemplate-gabaritlab.acia@inspection.gc.ca. A copy must be sent as well to the local district office where the animals were located when tested. Each e-mail must contain only one attached template.
  3. in the subject, provide the reference number only (2014IEAB-0000079706-4). In the body of the message, you may provide info on the date the samples are shipped, if possible.
  4. no column can be deleted or added to the template, and the mandatory format is to be followed. Otherwise, the importation of data at the laboratory level will fail leading to a delay in the analysis until a new template is forwarded.
  5. mandatory fields in the template include sample No., date sampled, sample type, animal identification (ID) (at least one 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.
  6. some fields do not apply for isolation testing or for export to Mexico. They can't be deleted but may be left blank: column C (time sampled), column O (collection date, for semen analysis only), column Q (individual sample location) or column R (number in pool).

38. The generic laboratory e-mail 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 the district office.

39. The electronic submission of the template does not preclude the requirement to send a paper copy of Form CFIA/ACIA 5473 along with the sample. However, 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". The list can be a copy of the electronic spreadsheet that records the vial numbers, the animal's identification, identify the "form reference #_______________" and be signed. The reference number is the number which appears in the bottom centre of all 3 pages of Form CFIA/ACIA 5473. 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.

40. The template is available from the district office.

Shipping samples

41. Include all pertinent/required forms with the samples. Please contact the CFIA-approved laboratory directly to confirm what documentation or notification is required when using a CFIA-approved electronic EIA certification system for sample submission. (contact information available in Module 3.3).

42. 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.

43. After collection, samples are 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.

44. In case of delayed shipment, serum samples for EIA may be frozen and sent at a later date. Consult Module 8.4 Equine Infectious Anemia for detailed instructions regarding storage and timing of analysis to ensure validity of results.

45. Samples for virus isolation tests such as Bovine viral diarrhea – Immunoperoxidase (BVD-IP) must not be frozen prior to submission, and must be protected from extremes of temperature during transit. Any factor that affects the viability of the agent in question will affect the sensitivity of the test and therefore the reliability of the test result.

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

47. It is the responsibility of the accredited veterinarian to resubmit if a specimen is unfit for testing.

48. 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.

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

CFIA 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 will have protocols in place for the notification of submitters when samples are rejected 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 tables below.

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 the CFIA District Office and will consist of the submission reference number, vial(s) number(s) of the rejected samples 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 #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 18-37°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 Tritrichomonosis (Trichomonosis) Unfit
  • Age: sample older than 120 hours for culture test
  • Temperature: sample temperature outside of 10-37C
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

Test results

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

50. 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.

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

52. For additional information on distribution of test results when using a CFIA-approved electronic EIA certification system, please refer to Module 8.4 Equine Infectious Anemia.

53. Test results from private laboratories are usually reported directly to the submitter.

54. Schedule testing so as to allow sufficient time to receive results. Time required will vary from 1 region to another.

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