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Bovine trichomoniasis is a venereally transmitted infection by the protozoan,
Tritrichomonas foetus. The major pathological manifestation of this infection is early embryonic death or abortion in an impregnated cow. The cow shows few other signs of infection. An infected bull is asymptomatic. The organism can only infrequently be found in direct microscopic examination of clinical specimens and serological methods of diagnosis are not reliable.
PRINCIPLES OF THE PROCEDURE
The InPouch™ TF is a self-contained system for the detection of
T. foetus from bovine preputial or vaginal samples. The proprietary medium is selective for the transport and growth of the trichomonad, while inhibiting the growth of yeast, mold and bacteria which might interfere with a reliable diagnosis.
The InPouch consists of a high barrier, oxygen resistant, plastic pouch with two V-shaped chambers connected by a narrow passage. This two-compartment system allows direct observation (
wet mount) of a newly collected specimen in the upper chamber before expressing the contents into the lower chamber for culture.
The InPouch™ is sensitive enough that
an inoculum containing as little as 1 organism is sufficient to potentially result in a presumptive positive test.
Presumptive positive pouches for
T. foetus can be tested via a PCR procedure to verify the result. Transport and off-site testing can be performed easily due to the flexible packaging and integral design of the pouch.
The InPouch medium contains the following: trypticase, protease, peptone, yeast extract, maltose and other nutrients, amino acids, salts, antifungal and antimicrobial agents in a normal saline phosphate buffer.
in vitro diagnostic use only. The pouch is labeled for veterinary microbial identification and test results only.
State Departments of Agriculture may require that only state certified veterinarians collect and read bovine cultures. Only state certified laboratories may read bovine cultures and perform PCR confirmation testing.
All specimens should be handled according to CDC-NIH recommendations for potentially infectious organisms at BIOHAZARD LEVEL 2.
STORAGE AND SHELF LIFE
Never refrigerate or freeze
the InPouches. Store uninoculated InPouches at room temperature (18ºC to 25ºC) horizontally, away from direct sunlight. Product shelf life is 12 months from the date of manufacture. Do not use an InPouch if it appears to be cloudy, leaky, dark brown, dry, or if the medium has thick syrup-like consistency.
InPouch™ TF pouches
Package Insert (this document)
Microscope clips (optional and sold separately)
20 ml syringes
Wooden applicator sticks/sterile cotton-tipped swabs
The specimen collection technique is important regardless of the nutrient medium used for culturing.
Use a clean, dry infusion pipette with a 20mL syringe on each bull or cow,
OR, a satisfactory sample can also be obtained using a long, sterile cotton swab for collection.
Clip the hair around the prepucial orifice in bulls.
If necessary, rinse out the prepucial cavity with sterile saline (not water) to clean out mud and manure. This will help prevent contamination from non-pathogenic intestinal trichomonads and coliform bacteria.
Direct the pipette to the distal penis in the sheath. Obtain the specimen by scraping the mucosa of the distal penis and the fornix area while applying suction with the syringe.
For cows, advance the pipette gently to the floor of the vaginal fornix, and patiently aspirate mucus. This may take 20-60 seconds, due to the viscosity of mucus. Some persistence may be required to get a thick, slightly yellow material that tends to stick to the pipette.
The specimen should NOT be refrigerated or frozen.
Remove the pouch from the bag, and manually express the liquid so that there is approximately 1 ml in the upper chamber.
Be sure that the liquid in the upper chamber is below the closure tape to prevent fluid from leaking upon opening.
Tear the pouch open at the notch just above the closure. Open the pouch by pulling the closure tape's middle tabs apart.
Bovine infusion pipette (for swab, see below)
- insert the specimen pipette tip into the liquid of the pouch's upper chamber and expel 0.5 -1.0 cc of the sample into the pouch. If the collected material adheres to the wall of the pipette, rinse the pipette by flushing a small amount of the liquid medium back and forth into the pouch. Minimize the production of bubbles. Dispose of both pipette and syringe.
- insert the swab into the liquid of the pouch's upper chamber. The swab can be ''milked'' out by pressing the tip of the swab between the fingers through the flexible walls of the pouch. Dispose of the swab.
For wet mount examination, squeeze the top to close, roll the top edge down twice and fold over the end tabs. For culture or transport, express the media into the lower chamber and roll the pouch down until the tape is at the top of the label. Fold the wire tape's end tabs to lock the roll.
Use the BioMed label to write animal identification
InPouch TF is an excellent device for the transport of
samples. The plastic pouch resists damage in transport and
maintains viability. Inoculated InPouches should be
maintained between 15ºC - 37ºC.
Wet Mount -
Immediate microscopic examination. Place the viewing clip horizontally over the upper chamber and close the clip. (Using the clip is optional). Observe with a microscope under low power 10x, using 20x or 40x if necessary for confirmation.
Immediate Specimen Concentration
- It is possible to concentrate
the cellular material by standing the pouch vertically or by placing
it in a shirt pocket for at least 15 minutes prior to microscopic
evaluation. The trichomonads will concentrate at the bottom of the
Incubate the pouch vertically at 35ºC - 37ºC for 18-24 hours.
- before reading the pouch, pull the pouch up and down across the edge of a table approximately 3-4 times.
- Place the viewing clip horizontally over the lower chamber and close the clip. (Using the clip is optional). Observe with a microscope under low power (10x). Use a higher power (20x or 40x) if necessary for confirmation.
READING TIP: Trichomomads gravitate to the edges of the
InPouch. If none are seen along the edges, briefly scan
the liquid. Make sure to focus on the liquid and not the textured
plastic of the pouch. Do not mistake Brownian motion of small
debris particles for evidence of trichomonad activity.
1 to 10 trichomonads observed are enough for a presumptive positive.
Repeat the evaluations daily for the presence of motile trichomonads for
6 days (after inoculation).
Field studies indicate that if
is present, 98% of InPouches will be positive at 5 days,
2% of InPouches will be positive at 6 days.
InPouches should be autoclaved or disposed of by equivalent means at
BIOSAFETY LEVEL 2.
The InPouch diagnostic medium is manufactured in small lots. Each lot is released only after a QC test of viability, doubling time, and sterility. QC testing is repeated through the end of shelf life by BioMed. Refer to BioMed Diagnostics Quality Control Procedure
(Doc.# 100- 135).
BioMed Diagnostics maintains a live culture of
T. foetus (clinical isolate) for QC purposes. This live culture (positive control) can be purchased from BioMed Diagnostics (Catalog #11-1015).
To maintain an active culture, inoculate a new pouch with one drop (approximately 40μl) of the actively growing culture and incubate at 37ºC for 24 hours. They can then be moved to a 32°C incubator or to room temperature. Subculture every 3-4 days when the organisms reach a concentration of 1 x 10
InPouch TF medium is known to be effective in culturing
T. foetus, T. suis, T. galliniae and P. hominis.
Limitations to the Procedure
In bovine samples
P. hominis, is a contaminant.
While differential staining can sometimes distinguish trichomonad species based on the number of flagellae, PCR testing is the best way to distinguish among trichomonads.
The InPouch medium suppresses but does not eliminate yeast and bacterial growth. A build-up of gas from bacterial growth can be vented by opening the pouches inside a
BIOHAZARD LEVEL 2 HOOD.
Too much fecal material can make the medium too cloudy to examine. Subculture, if necessary, into another InPouch.
WARNING: This product contains a chemical known to the State of California to cause cancer, birth defects and other reproductive harm.
Thomas, et al., Agri-Practice Journal, 11:13-17,1990.
Borchardt, Norman, Thomas, Harmon, Veterinary Medicine, Feb: 104-112, 1992.
Document No. 100-005 Rev. H 6/2008
Tritrichomonas foetus Test
Catalog No. 11-1001 20 Test Kit
Catalog No. 11-1003 100 Test Kit
A SELECTIVE CULTURE SYSTEM FOR
THE DIAGNOSIS OF
For Veterinary Use Only
In Vitro Diagnostic Use Only
1388 Antelope Road
White City, OR 97503
fax: (541) 830-3001
Incubation and Examination
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