Control of trichomoniasis: Control at the state and farm levels (Proceedings)

Trichomoniasis, or "trich," is a disease that can cause devastating reductions in the percentage of cows exposed to a bull that successfully calve.1,2,3 The disease is caused by a protozoan parasite, Tritrichomonas foetus and the organism is transmitted by the act of mating.4 Infected bulls seldom exhibit any symptoms, while infected cows will most commonly have early embryonic loss (generally day 20 to 90 of gestation), and occasionally pyometra or abortion of larger fetuses. In infected herds, ranchers will often notice that a high percentage of cattle are still showing estrus 60 days or more into the breeding season. The number of cows that calve can be reduced by 20% to 40% and the mean calving date will be later than in non-infected herds.

Infection in the cow occurs primarily by exposure to an infected bull at breeding. Initial infection of the female does not cause rapid conception failure, rather the pregnancy progresses to about 20 to 90 days, at which time the embryo/fetus dies and is resorbed or aborted. A period of infertility may last for another two to six months as a result of the infection. Cows that are infected with Trichomonas foetus typically clear the infection within a few months. Immunity, however, is not permanent and the cow is subject to re-infection in subsequent breeding periods. Occasionally an open cow will fail to clear the infection or a pregnant cow will remain infected through pregnancy and deliver a normal calf. These chronic carrier conditions in females are rare, but of concern, because these females can serve as a source of infection to bulls in the following breeding season; or be a route of introduction to a herd that purchases a chronically-infected cow.

Bulls contract the organism by breeding an infected cow. Because most cows will clear the infection within a few months, the female most likely became infected during the present breeding season from exposure to an infected bull. The trich organism colonizes the penile crypts and prepuce, but does not produce any symptoms in infected bulls. Bulls less than 4 years of age tend to clear the infection, while those 4 years of age and older are often infected for life; and no legal treatment is available to clear infected bulls. Although young bulls tend not to become permanent carriers, they can still spread the infection to susceptible females during the period that they are infected.

Testing for trichomoniasis: Sample collection and handling

Because adult bulls are chronically infected with the organism (vs. females and young bulls that are temporarily infected), smegma samples obtained from the prepuce/penis are most commonly used to establish a herd diagnosis (infected or non-infected herd) and to classify individual bulls as infected or non-infected. A positive test result from cervical mucus samples taken from females with pyometra can also be used to establish a herd as being infected. The samples are cultured in a growth media that supports the growth of flagellated protozoa. The transport and culture media commonly used in the U.S. today is the InPouch™ TF (biomed Diagnostics Inc., San Jose, CA). Historically, a variety of media have been used with Diamond's media being used in many earlier investigations of Trichomoniasis.

In bulls, the preferred sample is from the glans penis. This can be obtained by using a sterile insemination pipette and performing a vigorous back and forth scraping motion along the glans while applying negative pressure with an attached 10 ml syringe. A separate pipette and syringe should be used for each animal. The preferred sample from females is the cervical mucous or uterine secretions. These samples can 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.

The handling and/or shipping of the inoculated media samples is one of the most critical steps in Trichomoniasis diagnosis. When using the InPouch™ TF, inoculate the sample into the small upper chamber of the pouch, flush out the pipette. If the pouches are to be shipped, the samples should be left in the upper chamber and not pressed into the lower chamber until they arrive at the lab. This allows them to be handled freely but then once at the lab, the inoculation of the lower chamber can be completed and the pouch maintained upright from then on. In-Pouch TF media can be transported by commercial carrier, but this must be by overnight express/one-day delivery and the inoculated media should be kept at 65° F to 75° F until it is incubated. It is especially important to avoid overheating or freezing. Ship the inoculated pouches in insulated containers (no ice) that will protect the samples from extreme temperatures because Trichomonads are very susceptible to either freezing or overheating. It is important to arrange shipping so the samples arrive at the laboratory or clinic that will perform the testing within 30 hours of collection. For both culture and PCR, preputial samples should be cultured as soon after collection as possible. Storing preputial samples for even 24 hours may result in a loss of approximately 10% in diagnostic sensitivity, with longer delays causing more drastic declines. If the culture will be performed on-site, squeeze or "squeegee" the liquid down into the lower chamber. Carefully express air bubbles out of the lower chamber to maintain the anaerobic environment. Roll the top of the plastic pouch down to the top of the lower chamber and fold the wire strips across to hold and seal it. Don't stir or mix the content and keep the packet upright at all times once the lower chamber is inoculated.

Testing for trichomoniasis: Diagnostic laboratory tests

Culture of smegma samples from bulls (and to a lesser extent cervical mucus from cows) that have had at least seven days of sexual rest has been the most commonly used diagnostic test for identifying infected herds and individuals. Because the population of protozoa appears to fluctuate over time and because the organism is difficult to collect, transport, and culture, a single test has poor sensitivity (many false-negative classifications). For samples collected in field settings, sensitivities of 70-90% are often reported. In order for a bull to be considered negative for trichomoniasis, he must have three samples taken at weekly intervals be test-negative. All positive bulls should be sent to slaughter.

Another problem with culture tests to identify T. foetus carriers is that a number of other organisms (e.g. Trichomonads, Tetratrichomonads spp. and Pentatrichomonas hominis) can be present on the penis/sheath that look similar to T. foetus under light microscopic examination.

Because of these limitations, polymerase chain reaction (PCR)-based diagnosis has been proposed as an alternate or additional test for T. foetus because it relies on the amplification of DNA from the organism and not on the successful culture of the live organism. However, sensitivity and specificity of PCR on smegma taken directly from bulls may be affected by inhibitory factors and the DNA may be easily disrupted as both components of both urine and blood can inhibit PCR sensitivity. Experiments have shown PCR to be similar in sensitivity and specificity to culture.

Sensitivity and Specificity of culture and PCR of smegma samples from experimentally-infected bulls17

When using a test with imperfect to poor sensitivity to screen symptom-free cattle in order to keep a devastating disease out of a herd, more than one test must be used. Running two or more tests in parallel, where they are submitted simultaneously or taken sequentially from essentially the same population, is used to find positive animals when test sensitivity is low. Parallel tests are interpreted as positive if any test results in a positive response. Trich testing (whether using culture or PCR) should utilize a strategy where bulls with a negative test are tested again at least 7 days later for three successive tests for herd bulls and six successive tests for bulls used for A.I. If expertise and care are exercised while collecting, transporting, and culturing samples for either culture or PCR evaluation, three tests should provide a diagnostic sensitivity of about 85 to 99% (correctly identify about 85 to 99% of infected bulls) and will misclassify a few non-infected bulls as being infected. If sample collection is difficult because of limitations of facilities or bull behavior, or if transport and culture environments are not optimum (i.e. sample stored before shipping, or arriving at lab with more than a 24 hour delay, or being exposed to hot or cold temperatures between collection and arrival at lab) sensitivity will be decreased and fewer than 85 to 99% of positive bulls will be identified.

Vaccination

A vaccine is available and may help infected animals clear the infection more rapidly than non-vaccinates. All the cows and bulls may be vaccinated before breeding; but efficacy in bulls has not been established. The first vaccination should be given 8 weeks prior to breeding and the second 4 weeks prior to breeding. Previously vaccinated cows should be revaccinated annually 4 weeks prior to breeding. Vaccination may be a helpful adjunct to other Trich control measures, but vaccination without institution of other controls has virtually no value.

Management of infected herds

For herds where Trich is identified in at least one bull or cow, a rigorous control program should be initiated immediately. Control strategies in infected herds will vary based on a number of factors such as herd size, number of breeding groups, number of breeding seasons, and frequency of importing bulls or females.

Summary recommendations for positive trichomonas herds

Bulls

     1. Test all bulls in the herd using at least three tests at weekly intervals and after at least a 7 day period of sexual rest before the first test. Send all Trichomonas test positive bulls to slaughter

     2. Retest bulls (3 negative tests) for Trichomonas prior to each breeding season

     3. Test all imported bulls regardless of age (3 negative tests to enter herd)

     4. Keep the average bull age as young as possible. Some experts recommend removing bulls greater than 3 years of age – others suggest that aggressive annual testing of bulls plus removal of bulls greater than 5 years of age is adequate.

     5. Sexually rest bulls a minimum of three months between breeding seasons.

Cows

     1. Cull all open cows

     2. Remove bulls after no more than a four month breeding season. Examine the herd for pregnancy and cull all open heifers and cows.

     3. Watch calving for abortions, or weak calves. Submit to KSU diagnostic lab as needed to confirm Trichomonas abortions. Cull cows that suffer Trichomonas abortions

     4. Cull all cows that have a Trichomonas positive abortion

     5. At pregnancy palpation watch for pyometras in cows, cull any cows with a pyometra.

     6. Increase efforts to keep neighbor cows and bulls out, and inform neighbors of the situation.

     7. Optional: Vaccinate cows 8 weeks and 4 weeks prior to breeding with approved vaccine

Prevention

If trichomoniasis has been identified in the geographic vicinity or the ranch routinely buys open cows and aged bulls, the herd is at risk of becoming infected with Trich. Since trichomoniasis is a venereal disease and can only be transmitted by sexual contact, the best prevention is to never allow infected bulls or cows to come into contact with a susceptible herd. Specific measures include the following:

     • Keep fences in good repair to prevent accidental contact with potentially infected cattle. Monitor traffic of cattle in and out of the herd.

     • Replacement females should either be pregnant (more than 120 days) or less than six months of age.

     • Replacement bulls should be known virgins. If the sexual history is unknown or questionable, replacement bulls should be cultured three times before they enter the herd.

     • Sexually rest bulls a minimum of three months between breeding seasons. The longer the rest period, the better, as this allows more time for an infected young bull to potentially clear itself of the infection.

     • Use only virgin bulls on heifers. Remove the bulls after three months of breeding. Examine heifers for pregnancy and cull all non-pregnant animals.

     • Remove bulls from the adult cows after three to four months of breeding. Examine cows for pregnancy and cull all non-pregnant cows. In herds with split breeding seasons, (spring and fall), do not move non-pregnant cows from one herd to the other.

     • Use artificial insemination when practical (e.g., dairies and intensively managed beef operations).

     • Use of a killed vaccine specific for T. foetus have been developed and is an optional adjunct preventive measure. Test trials indicate vaccination is capable of inducing an immune response earlier than that which occurs naturally in infected cows. Vaccination has been shown to be effective in reducing losses in T. foetus infected herds and can be used as an additional tool in controlling the disease.

State regulations

Because of the potential for devastating losses due to trichomoniasis and the asymptomatic nature of the primary reservoir (bulls), many states are instituting regulations for the importation of bulls (and in some cases cows/heifers) into their states. These regulations often times provide an incomplete barrier in order to remain feasible, and are not adequate to prevent introduction of Trich into client herds. Regulations differ by state in their definition of a virgin bull, the regulations on importing females, and the type and number of negative tests required. In order to be feasible, most states only require one negative test for entry of a bull. Because of the poor sensitivity of our current tests, this leaves a reasonable expectation that some imported bulls will be Trich carriers; and bulls at risk of being previously exposed to Trich should not be allowed to breed cows until they have had at least two more negative tests.

Texas: 2009 (expanded 2010)

     • Testing of non-virgin (raised away from cows after weaning and less than 24 months of age – extended to 30 months if signed by breeder's accredited veterinarian) bulls prior to importation into the state

     • Texas bulls that change ownership (except to slaughter) must be either certified as a virgin bull or be tested for trichomoniasis.

          o Bulls older than 30 months or bulls that were maintained with cows after weaning must have a negative Trichomoniasis test within 30 days prior to change in ownership.

          o A certified, accredited veterinarian must collect the sample for testing at the Texas Veterinary Medical Diagnostic Laboratory.

     • A single PCR or three weekly cultures are acceptable as tests

     • Positive bulls must go to slaughter within 30 days of test confirmation

     • Bulls from positive herds will each be tested twice with a PCR test seven days apart, or three times via culture also from samples collected seven days apart.

Nebraska: 2008

     • All bulls over 24 months of age and all non-virgin bulls less than 24 months of age shall have either three consecutive official negative Trichomoniasis culture tests at least one week apart; or one negative PCR test within 30 days prior to importation into the state.

     • Samples submitted for testing shall be collected by an accredited veterinarian and conducted by a laboratory accredited by the American Association of Veterinary Laboratory Diagnosticians.

     • Bull must not be used for breeding between the time of testing and the time the bull is imported into Nebraska.

     • A Certificate of Veterinary Inspection (CVI) signed by an accredited veterinarian for bulls covered under this order shall bear the following statement:

     • "The bull(s) represented on this CVI are not from a known positive T. foetus herd and have had three consecutive official negative T. foetus culture tests which were at least a week apart; or one negative PCR test within 30 days prior to importation, and there has been no breeding activity since the first culture test or the PCR test."

     • Exceptions to testing requirements for bulls:

          o Virgin bulls aged 24 months of age or less must be accompanied by a CVI containing a signed statement from an accredited veterinarian that the bulls have had no contact with breeding females.

          o Bulls imported to Nebraska for slaughter

          o Bulls imported to Nebraska not to be used for breeding purposes (i.e. exhibitions, rodeos, bull test stations)

          o Nebraska-origin breeding bulls going into another state for seasonal grazing purposes may return to Nebraska without testing if accompanied by a CVI with the following statement "these breeding bulls have not commingled with any other herd." The CVI must be issued within 30 days of import into Nebraska.

     • Breeding females originating from a known positive T. foetus herd will not be allowed to enter Nebraska unless they are consigned to slaughter.

     • Breeding females imported into Nebraska and not consigned to an approved Nebraska Livestock Auction Market, must have one or more of the following statements placed on the CVI and signed by an accredited veterinarian of the herd of origin:

          o "The heifers listed on this CVI are known virgin heifers."

          o "Heifers listed on this CVI were exposed for their first breeding only to a known negative T. foetus bull or artificially inseminated, and are not yet 120 days pregnant."

          o "Cows or heifers listed on this CVI are at least 120 days pregnant."

          o "The cows listed on this CVI did not originate from a know positive T. foetus herd, have a calf at side, and have not had any exposure to known positive T. foetus bull(s) or bull(s) of unknown T. foetus status since calving.

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