Effects of Biological Materials and Collection Media on PCR Detection of <i>Tritrichomonas foetus</i>

Abstract

Tritrichomonas foetus is an important pathogen of the bovine reproductive tract causing early embryonic death and abortion in cows and persistent, asymptomatic infection in bulls. PCR detection methods have greatly enhanced diagnostic accuracy over culture; however, pre-analytical sample handling is just as critical as technical performance in detecting this pathogen and is not well studied. The purpose of this study was to investigate the effects of biological materials present in the prepuce on PCR detection of T. foetus in a variety of collection media. Simulated preputial samples were created using InPouch TM (IP) media, lactated ringers solution (LRS), or sterile saline (SAL); inoculated with low numbers of one of three T. foetus strains; and spiked with either blood, semen, urine, or sham treatment. Samples were transported to the lab, placed in growth media (LRS and SAL samples), incubated, and tested for T. foetus by PCR. Samples containing urine had statistically significantly greater mean Ct values (P = 0.008) than samples containing other materials, seen most dramatically in IP (P < 0.0001.) Urine contamination resulted in significantly (P = 0.037) fewer samples being identified as "positive" for T. foetus. Overall, SAL collections also had significantly higher mean Ct than IP or LRS (P < 0.001), and were less likely (P = 0.018) to results in classification as a "positive" sample. Results of this study indicate that collection media and biological materials can affect T. foetus PCR detection. The presence of urine in preputial samples can result in false negative results, while blood had no detrimental effects.

Bovine Trichomonosis Cases in the United States 2015–2019

“…There are widely accepted sampling techniques, particularly for bulls ( 23 , 34 ). Proper sampling techniques are important as the presence of urine has been shown to decrease PCR sensitivity ( 35 ). According to Code of Federal regulations, when testing is required for interstate movement, a Category II Accredited Veterinarian must be responsible for collecting and submitting samples to an approved diagnostic laboratory.…”