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 InPouchTM (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.
Highlights
Tritrichomonas foetus is an important pathogen of the bovine reproductive tract causing early embryonic death and abortion [1] [2]
Each sample was inoculated with approximately 150 organisms of an individual strain to simulate low levels of T. foetus colonization followed by one type of biological material (0.1 mL freshly-collected whole blood [BLOOD], 0.5 mL of freshly collected urine [URINE], 0.1 mL of freshly-collected semen [SEMEN], or 0.1 mL sterile saline [SHAM].) Volumes of biological materials were chosen to simulate the quantities that are consistent with what has been obtained during a collection of a preputial scraping or wash, at the time of a breeding
The effects were most evident in samples inoculated into IP media, in which mean cycles to threshold (Ct) values in urine-containing samples were five cycles greater than samples containing blood, semen, or no biological treatment Urine significantly inhibited detection (P = 0.002) of T. foetus in SAL samples (P = 0.002) compared with other biological materials
Summary
Tritrichomonas foetus is an important pathogen of the bovine reproductive tract causing early embryonic death and abortion [1] [2]. This protozoal organism colonizes the epithelium of the penis and prepuce in the bull and has been identified in the distal urethra; not in the penile or prostatic urethra, accessory sex glands, nor epididymis [1] [3]. No approved treatments exist to clear the infection; identification and removal of infected bulls is the best way to minimize T. foetus impacts [8] [9]
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