Sensitivity of a real-time polymerase chain reaction for Tritrichomonas fetus in direct individual and pooled preputial samples

The objective of this study was to evaluate the sensitivity of a commercially available real-time polymerase chain reaction (PCR) test for the detection of Tritrichomonas fetus in individual and pooled direct preputial samples. Two samples were collected and processed once a week from nine T. fetus–infected bulls (n = 121) and placed into either an InPouch TF or 2 mL of PBS. Preputial samples were also collected into both media and PBS from 1016 other bulls. All pouches were cultured and evaluated as per manufacturer's instructions. The prepuce samples collected directly into PBS were individually evaluated using real-time PCR by a commercial diagnostic laboratory. Direct preputial samples were then randomly divided for pooling into groups of 5 and 10 samples, ensuring that every pool had one sample from a known infected bull before testing using real-time PCR. Sensitivity was estimated for culture and real-time PCR of the 121 direct and culture-enriched individual samples from nine infected bulls. There were no differences (P = 0.12) among the sensitivity estimates for culture, 95.0% (95% confidence interval [CI]: 89.6%–97.7%); real-time PCR of culture-enriched samples, 95.9% (95% CI: 90.7–98.2); and direct preputial samples, 90.1% (95% CI: 83.5–94.2). There was also no significant difference (P = 0.08) between the sensitivity of real-time PCR for direct preputial samples in 110 pools of 5 (83.6%, 95% CI: 75.6–89.4) or 10 samples (77.3%, 95% CI: 68.6–84.1). The use of three sequential direct samples, collected in PBS at weekly intervals and tested by real-time PCR, increased the sensitivity to 100% for pools of 5 and 96% for pools of 10. In conclusion, direct preputial samples collected in PBS and tested by real-time PCR individually have comparable sensitivity to culture and real-time PCR in enriched samples. The use of pooled direct preputial samples appears to be relatively sensitive. However, this strategy requires repeated sampling to optimize sensitivity. Real-time PCR testing of preputial samples collected directly into PBS with the option of pooling would decrease the cost associated with screening bulls, and increase the feasibility of large epidemiological studies and active surveillance.