Chapter 3 - Testing
3.4 Testing for Bovine Genital Campylobacteriosis and Trichomonosis

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

  1. general information
  2. materials and supplies
  3. sample collection
  4. preparation and packaging
  5. submission forms
  6. shipping
  7. laboratories
  8. test results
  9. laboratory fees

Objectives

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

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

2. 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 test requirements for the National Artificial Insemination Program.

General information

3. Tritrichomonas foetus (also referred as Trichomonas foetus or T. foetus) is a flagellated protozoan parasite that lives in the reproductive track 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.

4. 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 Program and the export certification for bovines to Mexico.

Materials and Supplies

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

Material required:

  1. disposable gloves
  2. scissors
  3. 53 cm (up to 68 cm in larger bulls) infusion/insemination pipette, individually wrapped
  4. disposable 20 ml syringe
  5. disposable 3 ml syringe with small gauge needle (not greater than 20G)
  6. collection and transport media for each organism:
    Each organism (Campylobacter and Tritrichomonas) has specific transport media requirements. The transport and collection media itself is provided at no charge at the moment. 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
    1. Campylobacter: transport enrichment medium (TEM) and saline vials are ordered from the Animal Health Microbiology Laboratory located at the CFIA Ottawa Laboratory (Fallowfield). A specific order form is available through the district office. TEM transport and liquid sample collection (saline) vials are shipped from the laboratory together in one foam container covered by a corrugated cardboard sleeve and placed in a larger polystyrene foam chest with ice packs. 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 transport vials and 6 or 7 saline collection vials. Duplicate TEM vials are required for each animal tested. The outer sleeve on each foam container holds the unit together and 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.
    2. Tritrichomonas foetus transport media (TFTM): the transport media is sent from the centre for Food-borne and Parasitology at the CFIA Saskatoon Laboratory. A specific order form is available through the district office. The form must be faxed to the laboratory 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.

Sample Collection

6. Good sample collection technique as well as care in storage and shipping are very important to achieve the highest possible sensitivity. For sampling, all transport media (TEM and TFTM) and saline vials should be brought to room temperature (18-37°C) prior to inoculation with sample. Expiry date must be verified before sampling procedure begins. A video (DVD) produced by the CFIA Saskatoon Laboratory is available at the district office to view the collection procedure for Tritrichomonas sampling.

7. Two separate samples should be collected if testing for both organisms is required.

8. 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.

  1. Male:
    1. 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.
    2. 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 actually introduce contaminants and/or flush out available 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.
    3. Insert the pipette into the prepuce about half way. Connect a 20 ml syringe to the pipette and apply negative pressure. The pipette should be held parallel along the length of the prepuce. In situations where introducing contamination into the sheaths 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 10-15 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.
  2. Female animals:
    1. Cervical mucus or vaginal discharge must be sampled.
    2. Use a separate pair of disposable gloves for each animal. Debris should be cleaned from the vulva with dry paper towel. No disinfectant should be used.
    3. 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.

Sample preparation and packaging instructions

9. 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 identified correctly and the information entered on the forms is complete and accurate.

10. Each vial must be marked indelibly with the number corresponding to the one entered on the submission form in the column under VIAL # for the corresponding animal being tested. Duplicate vials for Campylobacter must be identified with the same vial number for the corresponding animal.

11. Tritrichomonas:

  1. Properly label the TFTM vial. 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.
  2. Transport media for Tritrichomonas foetus are sent in an insulated container that should be reused to send the samples back to the laboratory. Place TFTM samples back into the box from which they originated. Inoculated material must be packed to exclude light and 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 10-37°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 39°C. Samples must reach the Saskatoon laboratory within 5 days of collection or they will not be tested and will be reported as "Unfit".

12. Campylobacter:

  1. Properly label 2 TEM vials and one 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.
    Note: the sample must not be left in the saline for more than 15 minutes before the transfer and inoculation of the TEM vials.
  2. 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. TEM vials are provided with a piece of adhesive tape over the top. Lift the free end of the adhesive tape to expose the rubber membrane in the top (Do not unscrew the top of the TEM vials as this will disrupt the special gas mixture within).
  3. Inject 1 ml of the aspirated saline (step b. above) through the rubber membrane into the TEM vial. Replace the adhesive tape over the rubber membrane. Do not inject the aspirated sample directly through the adhesive tape.
  4. Repeat this step for the second TEM vial using the remaining 1 ml aspirated saline in the syringe. Mix the contents by gently shaking the TEM vials.
  5. 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 transport and saline vials, place the foam top on the container, and slide the corrugated sleeve over the unit with the red arrows pointed up.
  6. Place the foam containers into a larger polystyrene foam chest without ice packs (temperatures during transport should be between 18-37°C) and ship back to the CFIA Ottawa laboratory (Fallowfield) without delay. Use the safest and quickest means of shipment available and ship samples only during the week to avoid having them delayed in transit or held over at the laboratory on weekends or holidays. 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.
  7. 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.

Special consideration

13. For security reasons, in the case of an intractable animal where it is not possible to take a second sample when testing for both organisms, the single sample taken may be processed as follows. Please note however that following this procedure may negatively impact the sensitivity of the respective tests. The first half of the sample is inoculated into the TFTM media from the Saskatoon laboratory by expressing sample material onto the inside wall of the tube directly above the TFTM media. The tube should be re-capped and inverted several times to mix the sample into the media. The second half of the sample is inoculated in the saline vial provided by the Ottawa laboratory for Campylobacter testing. For further processing of each sample, refer to the above-mentioned procedures (11 and 12 above). Do not transfer the entire sample into the saline vial, and then split it between the TFTM and TEM media. The sample for Tritrichomonas must not have been diluted in saline before inoculation in the TFTM media, as this can further reduce the sensitivity of the test.
Note: This procedure must be reserved for exceptional cases only where the collection of two separate samples would be unsafe for the collector.

Submission Forms

14. Submission forms are available at the CFIA district office. 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.

15. Care must be taken when filling out the forms. 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 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.

16. CFIA/ACIA Form 5473 - Animal Health Import, Export and Artificial Insemination Specimen Submission must be used. 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 the form. The CFIA laboratory will not perform the analysis if the notification number is missing.

17. It is the responsibility of the accredited veterinarian to provide all information required, or to resubmit if a specimen is unfit for testing. Laboratories may refuse to conduct analysis of samples submitted with forms that are not fully 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

18. It is essential that samples be shipped in a timely fashion to the appropriate laboratory to avoid unnecessary re-sampling. If uncertain, check with a CFIA district veterinarian before shipping.

19. 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.

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

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

22. 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.

23. The accredited veterinarian is responsible for shipping costs.

Laboratories

24. Samples for Campylobacter testing must be sent to the CFIA Ottawa laboratory (Fallowfield). Samples for Tritrichomonas testing must be sent to the CFIA Saskatoon Laboratory. CFIA laboratory information may be found in module 3.3.

Test Results

25. 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

26. The accredited veterinarian is responsible for laboratory fees. The district office will recover applicable fees for all animals that had tests performed in a CFIA laboratory, regardless of the test result. The Canadian Food Inspection Agency Fees Notice can be consulted for more information.

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