Simple Guidelines for the Interpretation of Tri-trichomonas Foetus PCR Results from Preputial Washes/Scrapings from Bulls

Dr Rick Last – BVSc; M.Med.Vet (Pathology)

Specialist Veterinary Pathologist

 

Introduction

The polymerase chain reaction (PCR) has become the preferred laboratory test for the diagnosis of Tritrichomonas foetus infection in bulls (preputial scrapings or washings) and cows (lochial testing). Multiple experimental trials have demonstrated that to identify all infected bulls in a particular herd, at least three consecutive PCR tests on preputial scrapings / washes collected at 7-10 intervals is required. There are apparently no statistically significant differences in the PCR sensitivity on samples collected by scraping compared to those collected by washing.

 

Test Sensitivity

The sensitivity of a single test is ±73%, two tests at weekly intervals ±90%, three tests at weekly intervals ±96% and four tests at weekly intervals ±99%. The current RuVASA recommendations for bull certification, is three consecutive negative PCR tests on preputial scrapings / washes collected at 7-10 intervals. One should allow at least 2 weeks sexual rest before the implementation of this bull testing procedure. A major reason behind the three consecutive negative test requirement, is that the protozoan is not persistently present in the preputium and therefore a positive bull can produce a scraping or washing sample which is devoid of Tritrichomonas foetus nucleic acid and test negative.

 

Transportation

Phosphate Buffered Saline, Steve’s Media, skimmed Milk Powder and Trichomonas Culture Medium all serve as suitable transport media for the PCR assay. One of the major advantages of the PCR technique in Tritrichomonas foetus diagnostics is that the time restraints for getting samples to the laboratory are far more extended than those required for culture. Various studies have shown that samples maintained at room temperature or below for 3 – 4 days, showed no significant decrease in PCR test sensitivity.

Transport temperature of samples to the laboratory should be maintained at between 4-20°C. With transport at temperatures above 37°C the accuracy of the PCR analysis is compromised.

The sample quality should be assessed before further processing. The sample should be free of contamination with feces or dirt, free from large amounts of blood and should contain adequate amounts of cellular material from the lamina interna.

 

Interpreting the POSITIVE PCR Result

If one or more samples from a group of bulls tests positive for Tritrichomonas foetus nucleic acid on PCR analysis, then this should be considered a positive herd. Under such circumstances all bulls in the herd should be subjected to the three consecutive PCR tests run at 7-10 intervals routine, to identify all infected bulls in the herd.

False positive results are extremely rare but can occur due to cross contamination during sample collection, contamination of collection utensils and / or sample containers, contamination of PCR equipment or reagents with T.foetus DNA from previous reactions that had positive samples or from positive controls.

False positive reactions might be considered where logical epidemiological principles suggest a false positive result for example when large numbers of bulls in a group (where no infection was suspected) test positive, or when a single bull out of a large group tests positive where there is no clinical indication of Trichomoniasis.

Under these circumstances positive bulls should be isolated and re-tested under the three consecutive PCR test system at 7-10 day intervals. Only if all 3 consecutive tests are negative should they be considered for re-introduction to the breeding herd.

 

Interpreting the NEGATIVE PCR Result

A negative result for Tritrichomonas foetus nucleic acid on PCR analysis indicates there was no nucleic acid present in the sample tested. False negative results can occur with inadequate sampling, biological variation in the presence of the protozoan or when the organisms are present in sub-detectable quantities, incorrect storage and transportation, plus there are likely unidentified inhibitory substances and/or hydrolytic enzymes produced by the normal preputial flora which may affect PCR assay efficacy.

A negative result is not a guarantee of pathogen absence, particularly in pooled samples. A single negative PCR result should not replace 3 consecutive negative PCR tests, especially in herds where the true prevalence of disease is high. Avoid collecting preputial samples immediately after the bull leaves the cow herd, as this will increase the chance of a false negative result. Allow at least 2 weeks sexual rest before sample collection.

 

Pooling of Samples

Pooling of samples for Tritrichomonas foetus PCR testing is a cost-effective alternative for routine bull testing, but should only be considered in herds where infection is not suspected i.e. to confirm the negative status of the herd. Samples from up to 5 bulls can be pooled prior to submission or at the laboratory, with the sensitivity being similar to that of a single test (±73%). Pooling of samples should be avoided at all costs if there is any level of suspicion of an infected herd.

Further Reading

1. Holm D. 2015. Choosing the right trichomonosis diagnostic protocol. In publication.

2. Holm D. 2015. Trichomonosis in beef cattle in South Africa: An update. In publication.

3. McMillen L, Lew A E. 2006. Improved detection of Tri-trichomonas foetus in bovine diagnostic specimens using a novel probe based real time PCR assay. Veterinary Parasitology 141:204- 215.

4. Mukhufhi N, Irons P C, Michel A, Peta F. 2003. Evaluation of a PCR test for the diagnosis of Tritrichomonas foetus infection in bulls: Evaluation of sample collection method, storage and transport medium on the test. Theriogenology 60:1269-1278.

5. Ondrak J D et al. 2010. Repeat testing by the use of culture and PCR assay to detect Tritrichomonas foetus carrier bulls in an infected Nebraska herd. Journal of the American Veterinary Medical Association 237:1068-1073.

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