David J Wilson, Thomas J Baldwin, Amanda Wilhelm, Chelsea H Whitehouse, Jennifer L Bunnell, Kerry A Rood and Arnaud Van Wettere J
The primary objective was assessment of effect of sample pooling on Tritrichomonas foetus detection using qPCR in beef bulls within 5 logs of trichomonad concentration in spiked preputial samples. The secondary objective was to use qPCR Ct values to estimate the percentage of beef bulls in the field population within each log of trichomonad concentration. Solutions containing none, 1,10,100,1000 and 10,000 trichomonads (T)/ml were made and tested by qPCR. Test sensitivities for single positive samples were: 1 T/ml, 20/40 (50%); 10 T/ml, 40/40 (100%); 100 T/ml, 40/40 (100%); 1000 T/ml, 40/40 (100%); 10,000 T/ml, 40/40 (100%). For 5-sample pools (1 positive and 4 negative), sensitivities were: 1 T/ml, 6/40 (15%), 10 T/ml, 26/40 (65%); 100 T/ml 40/40 (100%); 1000 T/ml, 40/40 (100%); 10,000 T/ml, 40/40 (100%). Specificity for both single and pooled negative samples was 40/40 negative (100%). Diagnostic field samples (n = 130) in which T. foetus was detected by qPCR were classified by Ct to estimate the proportion of positive bulls within each trichomonad concentration: 1 T/ml 10 (7.7%); 10 T/ml 17 (13.1%); ≥ 100 T/ml 103 (79.2%). Accounting for the above sensitivities and proportions of bulls, sensitivity for trichomoniasis detection in the bull population was 96.2% using single preputial samples and 88.9% using 5-sample pools. Therefore 7.3% of all T. foetus-positive bulls were not detected using 5-sample pools compared with using single samples. Detection of bovine trichomoniasis by qPCR is minimally affected by using 5-sample pools, considering the trichomonad concentration of most positive bulls.
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