Diagnosis and official regulatory testing for bovine trichomoniasis in Washington

Major highlights of bovine trichomoniasis testing

  • WADDL offers a Trich Direct Realtime PCR test that uses phosphate buffered saline (PBS) or lactated ringers solution (LRS) as the primary transport medium and is more sensitive than the currently used PCR test.
  • Bovine trichomoniasis is a disease that causes reduced reproductive performance in cattle herds.
  • Bovine trichomoniasis is a disease regulated by the Washington State Department of Agriculture (WSDA- State Veterinarian).
  • Control of trichomoniasis in cattle herds requires culling of infected carrier bulls.
  • The required sample for “official” detection of trichomoniasis in bulls is a smegma sample from either a preputial scraping or preputial wash taken by an accredited veterinarian registered by the WSDA and inoculated into 2mL PBS (phosphate buffered saline) in a 15mL screw cap tube, 2mL lactated ringers solution (LRS) in a 15mL screw cap tube, or other acceptable transport media.
  • 15mL screw-cap conical tubes can be purchased from most veterinary supply companies, including MWI Animal Health or Amazon.
  • WADDL will still accept samples in commercial Trichomonas test media (TF transport tubes) that are in date.
  • Detection of infected carrier bulls requires laboratory testing and the official laboratory test in the state of Washington for bovine trichomoniasis is the polymerase chain reaction (PCR) test.
  • WSU-WADDL is an official testing laboratory for bovine trichomoniasis in the state of Washington.
  • Samples for bovine trichomoniasis testing should be submitted on a “WADDL Trichomoniasis PCR Form.”

What’s new in trichomonas testing?

Trichomonas testing by pooled PCR (up to five bulls from the same owner) is now allowed by WSDA and available at WSU-WADDL.

Two methods of sample collection and submission are now allowed by WSDA and available at WSU-WADDL.

New easier handling trichomonas test media tube container is now available from BioMed Diagnostics Inc.

What is trichomoniasis?

Bovine venereal trichomoniasis is caused by Tritrichomonas foetus, a flagellate protozoan parasite. T. foetus lives in the reproductive tracts of bulls and cows and has worldwide distribution. The widespread use of artificial insemination in many areas of the world has helped to reduce its prevalence. Trichomoniasis is still of importance in herds where artificial insemination is not used. 

How is trichomoniasis transmitted?

Venereal transmission can occur from an infected bull to an uninfected cow (or heifer), or from an infected cow (or heifer) to an uninfected bull. Bulls are the main reservoir of trichomoniasis, and bulls greater than 4 years of age tend to be long-term carriers. For this reason, samples from bulls are preferred for diagnosing and controlling the disease in cattle herds. 

What are the clinical signs of trichomoniasis in individual cattle?

Chronically infected bulls show no lesions or clinical disease. Infected bulls appear normal, breed normally, and can infect an entire herd through natural service. In the newly infected cow or heifer (in those never before infected with T. foetus), there is inflammation of the reproductive tract (vagina, cervix, uterus), which may result in a discharge from the vagina or, in severe cases, pyometra (uterus distended with pus and the cow’s inability to cycle). If the cow is pregnant, the infection results in placentitis (inflammation of the birth membranes) and causes early abortion (1-16 weeks of gestation). 

How do I know if my cattle herd has trichomoniasis?

A tentative diagnosis of trichomoniasis as a cause of reproductive failure in a herd is based upon clinical history (presence of clinical signs in individual cows in a bull bred herd), signs of early abortion, repeated returns to service, high percentage of unbred cows, and irregular estrus cycles. Confirmation of trichomoniasis requires demonstration of T. foetus parasites by laboratory testing. 

How do I get trichomoniasis out of my cattle herd or prevent trichomoniasis from entering my cattle herd?

Control of trichomoniasis in cattle herds requires identification of bulls infected with T. foetus by laboratory methods and removal of the infected bulls from the herd. Also, testing all purchased bulls for T. foetus before entering the herd will prevent introduction of trichomoniasis into your herd. Bulls become chronically infected with T. foetus, are long-term carriers, and can serve as the source of trichomoniasis in cattle herds from one breeding season to the next. Cows with trichomoniasis spontaneously clear infection in 90-95 days and are not a source of T. foetus infection from one breeding season to the next. There is no effective method for treating T. foetus-infected bulls. Infected bulls are generally culled from the herd and sold for slaughter. 

What samples do I take for laboratory identification of trichomoniasis?

In bulls, preputial smegma (preputial scraping) is the optimal sample. In cows and heifers, T. foetus parasites are most reliably identified from placental fluids, placenta, stomach contents of aborted fetuses, uterine washings, pyometra discharge, or vaginal mucus. Herd-based diagnosis is most reliably made from preputial scrapings of bulls or vaginal scrapings/fluids from cows. 

How are samples collected for laboratory identification of trichomoniasis?

Appropriate sample collection for accurate diagnosis of trichomoniasis is critical. In infected bulls, T. foetus parasites live deep in the preputial folds (microscopic crevices inside the prepuce), requiring a preputial scraping or preputial washing. The scraping is generally accomplished with an artificial insemination pipette or special metal brush (dry pipette technique). Preputial washing can be accomplished using Phosphate Buffered Saline (PBS) or Lactated Ringers Solution (LRS) transport medium. The required sample for “official” detection of trichomoniasis in bulls is a smegma sample from either a preputial scraping or preputial wash inoculated into 2mL PBS in a 15mL screw cap tube or 2mL LRS in a 15mL screw cap tube. For official regulatory testing, sampling must be done by a veterinarian certified and registered with the Washington State Department of Agriculture (WSDA).

How should I store samples for trichomoniasis testing prior to shipping, and how should they be shipped?

Preputial scraping or wash samples submitted to WSU-WADDL should be collected into 15 ml screw cap tubes containing either 2ml of Phosphate Buffered Saline (PBS) or 2ml of Lactated Ringers Solution (LRS) using either method shown below. 15mL screw-cap conical tubes can be purchased from most veterinary supply companies, including MWI Animal Health or Amazon.

  • Method 1: Samples collected into either PBS or LRS refrigerated at 4°C and received at the laboratory within 5 days post-collection.
  • Method 2: Sample collected into either PBS or LRS frozen at -20°C and received at the laboratory within 7 days post-collection.

Samples should be shipped by overnight courier or mail with ice packs, in an insulated container (i.e., Styrofoam cooler), that has been placed inside of another shipping container (i.e., cardboard box).

WADDL will still accept samples in commercial trichomonas test media (TF transport tubes) that are in date. The samples in TF tubes can also use either method shown above, as well as the same shipping instructions.

Samples using either method should also be submitted on the WADDL Trichomoniasis PCR Accession Form. Please fill out the form completely and check either individual PCR or Pooled PCR as appropriate (see criteria for Pooled PCR testing below).

What are the laboratory tests for trichomoniasis?

Official regulatory tests for trichomoniasis vary state to state but generally are restricted to culture or polymerase chain reaction (PCR) methods. The T. foetus PCR used in WADDL beginning April 2022 is more sensitive than the PCR test used prior to April 2022.

This “Trich Direct Realtime PCR” test recently published in 2018 has been accepted by multiple states for official “Trich” testing. The test utilizes phosphate buffered saline (PBS) or lactated ringer’s solution (LRS) as the transport medium, which also is more convenient for submitting veterinarians. The new test also is accepted as the official test for trichomoniasis by the WSDA if submitted to an official laboratory using the methods and shipping times described in this article (5 days for refrigerated samples and 7 days for frozen samples). WSU-WADDL is the only Washington laboratory recognized by the state Veterinarian for official trichomoniasis testing and conducts testing using a quantitative PCR (qPCR) method. WADDL has validated the direct smega PCR assay in collection media described in this article.

What are the Washington State Department of Agriculture (WSDA) regulations regarding trichomoniasis?

Because bulls with trichomoniasis do not show clinical signs of infection and because trichomoniasis can cause economic losses to Washington cattle herds through significant reproductive failure, the WSDA and state veterinarian require all breeding bulls entering the state be shown free of trichomoniasis. Some brief highlights of the WAC Bovine Trichomoniasis Requirements are listed below.

  • Breeding bulls may be imported into state of Washington if:
    • Originate from a herd wherein all bulls have tested negative for trichomoniasis since removal from female cattle; or
    • Tested negative to bovine trichomoniasis quantitative PCR (qPCR) test within 30 days of import and had no contact with female cattle; or
    • Tested negative to bovine trichomoniasis culture test, if from state that recognizes a culture test as an official test.
  • If the bulls originate from a herd where one or more bulls or cows have been found infected with bovine trichomoniasis within the past 12 months or have unknown status, the bulls must have two negative PCR tests one week apart. The samples for each test must be collected within 30 days before cattle are imported into Washington state, and an import permit must be obtained from the director and include a certifying statement that the bulls originated from an infected herd.
  • Virgin bulls are exempt from bovine trichomoniasis test requirements. If sold, virgin bulls must be officially identified and accompanied by a certificate signed by the owner or the owner’s designee that they have had no breeding contact with female cattle.
  • Registered veterinarians shall only utilize official laboratories recognized by the state veterinarian for testing of trichomoniasis samples.
  • Polymerase chain reaction is accepted as an official test when completed by a qualified laboratory approved by the director and when the sample is collected, shipped, and received as indicated by the qualified laboratory.
    • Other tests for trichomoniasis may be approved as official tests by the state veterinarian after the tests have been proven effective by research, have been evaluated sufficiently to determine efficacy, and a protocol for use of the test has been established.
    • For testing done at WSU-WADDL, an official test is one in which the sample is received in the laboratory in good condition as described in this article. Samples in transit for more than the shipping time described in above will not be accepted for official testing and will be discarded.

What are the WSDA requirements for pooled PCR testing?

WSDA has had Trichomoniasis regulations since 2008. Testing protocol advancements since the implementation of the regulations has allowed WSDA to revisit the issue of pooling with low-risk situations or for non-regulatory purposes. This action still complies with the Trichomoniasis import regulations for import in WAC 16-54-086.

The criteria where pooling of up to five bulls will be allowed are:

  • Bulls that are over 18 months old cannot be exempted as virgin bulls and have had no breeding contact with females.
  • Bulls originate from a herd where there is no history of trichomoniasis infection and are part of a single herd, not assembled cattle.
  • As part of a disease investigation, pooling may be allowed if the herd does not have a history of infection or in the judgment of the herd veterinarian Trichomoniasis infection is unlikely. 

Samples must not be pre-pooled within tubes by the submitting veterinarian but are pooled after receipt of in the laboratory so that individual animals within a pool can be accurately identified (individuals in positive pools are re-tested individually).

Pooling will not be allowed for:

  • Multiple owners for a single pool. This effectively eliminates pooling as an option at livestock markets.
  • Infected herds or herds with a history of infection within the past 12 months.

Some states will not accept pooled samples for importation testing into their state. Check the state of destination requirements before requesting pooled samples.

Categories: Cattle