Protocol for Trichomonas Diagnosis in Cattle

Diagnosis of trichomoniasis is made when Trichomonas organisms are observed in the smegma or preputial flush samples of bulls, or the uterine or vaginal fluid of cows. The organisms may be observed by direct microscopic examination of the fresh samples, by examination of culture media inoculated with infected material, or by the detection of T. foetus DNA through Polymerase Chain Reaction (PCR).

• Culture: Collected samples shall be introduced into the InPouch® TF and incubated at 37 ºC (98.6 ºF). Samples should be microscopically observed every other day for a period of 7 days. Culture testing can be done at ISDA certified facilities by ISDA certified readers. Culture testing is no longer performed at the ISDA – Animal Health Laboratory.
• qPCR: Also known as quantitative real-time PCR, a molecular biology method of detecting targeted DNA or RNA sequences. PCR is believed to have several advantages over traditional culture testing, including the following:
  • qPCR is generally considered to have greater sensitivity and specificity.
  • qPCR results are typically generated within 2-3 days.
  • qPCR can distinguish between Tritrichomonas foetusand other trichomonads.
  • In some instances, one qPCR test may be accepted versus three separate culture tests.
  • Inoculated pouches for qPCR are shipped cold (frozen), so there is no need for hand-warmers to maintain adequate organism growth/survival temperatures.
• The collected sample is introduced into the InPouch® TF system, a TF-Transit tube, or a 15 mL centrifuge tube (screw top) with 3-5 mL of Phosphate Buffered Saline (PBS). Pouches and tubes for qPCR testing do not need incubation. All samples should be frozen and shipped on ice-packs to the ISDA – Animal Health Laboratory or another accredited laboratory.
• The ISDA – Animal Health Laboratory does offer pooled Tritrichomonas foetus PCR testing, with a maximum of 5 samples per pool. Submit TF-Transit Tubes, PBS tubes, or pouches as you would for individual testing. Pooling of samples must be completed by the ISDA – Animal Health Laboratory; samples arriving pre-pooled will NOT be accepted.  In the event that a pooled sample tests positive, the samples are then tested individually at the individual PCR test price.

• The preferred sample from the male is smegma from the glans penis. This can be obtained by performing a vigorous back and forth scraping motion along the glans using a sterile insemination pipette while applying negative pressure with an attached 20 or 30 mL syringe. This material is then inoculated into a medium suitable for transport to the laboratory.
• The preferred sample from the female is cervical mucus or uterine secretions. These samples can easily be collected by applying negative pressure with a syringe attached to a sterile insemination pipette, while the pipette is positioned within the open cervix or positioned to collect fluid from the vaginal floor. This material is then inoculated into a medium suitable for transport to the laboratory.

Transportation media for T. foetus PCR:

There are three recognized media for transportation of T. foetus samples for PCR testing in the state of Idaho: (1) TF-Transit Tubes (2) 15 mL centrifuge tubes with 3-5 mL of PBS (3) BioMed’s Bovine InPouch® TF (additional fee for pouches will be charged). Samples submitted in other media, such as Diamonds (MDM), can contain PCR inhibitors and will be rejected.

InPouch® TF:

Inoculate the sample into the small upper chamber of the pouch. Incorporate the sample contents of the upper chamber into the media by squeezing the liquid into the lower chamber and mix gently. Express any air bubbles out of the lower chamber. This action helps to maintain the anaerobic quality of the medium. Roll the wire strips the rest of the way down (like a Whirl-Pak®) to seal the lower chamber. Make sure you roll the wire strips to the lower chamber.

Remember that at least 5 mm of sample should be collected at the bottom of the pouch. It may take some time for the material to settle to the bottom of the pouch to accurately measure. Samples that contain less than 5 mm of material should be rejected.

TF-Transit or PBS Tube:

Inject the 0.5 – 1.0 mL sample into the tube and replace cap securely.

Shipping for Culture Testing:

Note: The shipping and handling of the inoculated medium sample for culture is one of the most critical steps in trichomoniasis diagnosis. It is important to arrange shipping so that the samples arrive at the testing laboratory within 48 hours of collection. Only those samples which are received at a certified diagnostic facility within 48 hours from time of collection will be considered for a valid culture test. Samples received after 48 hours from the time of collection are not eligible for culture testing, however these samples can be submitted for PCR testing.

Also note the Trichomoniasis Test and Report Form must have the “Date of Collection” completed or the sample(s) will not be tested.

• Inoculated media should be kept as close as possible to general room temperature (65 ºF – 75 ºF) until it is incubated. It is especially important to avoid overheating or freezing.
• If the samples are to be shipped to another laboratory or clinic for examination, ship the inoculated pouches in insulated containers (no ice) that will protect the samples from extreme temperatures. Trichomonads are very susceptible to either freezing or overheating. Chemical hand warmers or micro-waved gel packs should be placed in the shipping containers to maintain room-temperature during summer and winter months.

Shipping for real-time PCR (qPCR) Testing:

• All samples submitted to ISDA – Animal Health Laboratory for qPCR testing should be collected into the TF-Transit tube, PBS Tube, or InPouch® TF.
• No incubation is required before shipping.
• Samples should be frozen and shipped with gel ice-packs to ISDA – Animal Health Laboratory.

Note: If a T. foetus positive culture pouch is identified, the ISDA – Animal Health Laboratory can perform a confirmation qPCR on the sample. The pouch should be frozen, packed with gel icepacks, and shipped or sent by courier to the ISDA – Animal Health Laboratory.

Note: All Trichomoniasis culture tests are considered official tests. There are no “unofficial tests.” Only laboratories approved by ISDA Division of Animal Industries shall test official Trichomoniasis samples per IDAPA 02.04.29. Lab personnel must be trained and certified by ISDA AHL personnel following the most current revision of SOP PRST1 T. foetus Identification and Training to examine samples for Tritrichomonas foetus per IDAPA 02.04.29. Clinics/Veterinarians are required to recertify every 3 years by completing the ISDA AHL proficiency test.

A clinic must also have an initial, one time only, facility inspection to read Trichomonas samples. This inspection can be completed by ISDA field staff following the most current revision of SOP PRSTWKS2 Certification of Facilities for T. foetus Microscopy Testing.

InPouch® TF Pouch:

For microscopic examination, place the bottom of the lower chamber of the pouch on the raised platform of the open viewing frame. Close and lock the frame over the pouch. Trichomonads generally will first be found slightly above the bottom border of the chamber.

Note: If samples are opaque, mix and try to read in the clear spaces. The pouch and frame are then placed on the microscope under a low power (10X) and examined for typical motile organisms (see Interpretation of Results below) in the following manner:

• The day samples are received is Day 0 (zero). If samples have been in shipment for more than a few hours, it is suggested to examine the samples the day they arrive. Incubate the samples vertically (upright) at 37 ºC (98 – 99 ºF).
• Samples should be closely examined on days 1, 3, 5 and 7. The results for that day’s reading are recorded on the Official “Trichomonas Test and Report Form” (ADMFRM8) and the “Trichomonas Test and Report Form Continuation Sheet (ADMFRM9), if needed. Record the date read at the top of the column above the column number. Then record the results for each sample in the column for that day’s reading. The final results are recorded at the end of day 7, or earlier for those samples that have already turned positive. If positive, notify the submitter of the pouch or the owner of the bull in addition to the state veterinarian and or the AHL director. Note: Report positive results as soon as they are found, i.e., if any pouch of a multi-pouch submission from one herd is found positive, it should be reported immediately.
• Upon completion of the 7 days of testing, the laboratory performing the test fills out the summary information and the “certified reader” signs the forms. The forms are then forwarded according to the distribution labels at the bottom of the form.

Positive: A sample is considered positive when viable, motile trichomonad organisms are observed either upon direct microscopic examination of the sample when collected, or in the culture medium on any of the reading days. Samples may be sent for T. foetus PCR confirmation. Trichomoniasis is a reportable disease in the state of Idaho, and it is the responsibility of the reader/clinic to report a positive sample to the state veterinarian.

Negative: A sample is considered negative when no viable/motile trichomonads are observed in the culture medium during any of the reading days and after the full 7 days incubation has been completed.

Note: A sample must be cultured for the full 7 days before it can be resulted as negative.

All pouches should be retained for 7 days at room temperature after the final read. This allows samples to be tested by qPCR if questions arise.

Re-Test: When the Trichomonas organism dies, it immediately loses its motility. Its morphology, however, will degenerate more slowly. With a rapidly growing or heavily inoculated sample, the trichomonad organisms can sometimes overgrow the medium and die off within 36–48-hours. That is the reason for the every-other-day reading schedule; you should catch the organisms sometime during their growth phase. However, if you encounter a culture sample with an abundance of non-motile organisms of typical trichomonad size, mark the test chart as “RE-TEST” for that sample and request that a second specimen be submitted for that animal as soon as possible. Alternately, the sample can be submitted for qPCR confirmation.

Reasoning: Some yeasts and spores will be of similar size and morphology as a dead (non-motile) trichomonad. Thus, rather than incorrectly calling the animal “Positive,” a second sample should be immediately requested and read every day (in case it’s a rapidly growing trichomonad) to observe if actual, motile trichomonad organisms are present. If no viable, motile trichomonad organisms are found upon the re-culture, the animal is negative.

Interpretation of qPCR Results can be found in the most current revision of SOP MOL5 Tritrichomonas foetus Real-Time PCR on file at ISDA – Animal Health Laboratory.

Pouches may be discarded at the end of the required 7-day retention period and the contents should be sterilized (regardless of whether the final results were positive or negative) in accordance with the EPA and OSHA requirements for disposal of biological wastes. This is best accomplished by autoclaving the pouches prior to discarding. If an autoclave is not available or if autoclaving is not practical, inactivate by adding Clorox, Nolvasan, or some other disinfectant to the pouches and shaking vigorously prior to disposal.