Use of pooled protozoal cultures of preputial scraping samples obtained from bulls for the detection of Tritrichomonas foetus by means of a real-time polymerase chain reaction assay

• Localización: JAVMA: Journal of the American Veterinary Medical Association, ISSN-e 0003-1488, Vol. 244, Nº. 3, 2014, págs. 352-356
• Idioma: inglés
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• Resumen

  • Objective�To determine the sensitivity of a real-time PCR assay for the detection of Tritrichomonas foetus in protozoal cultures of preputial scraping samples pooled from up to 25 bulls and to determine the specificity of that assay for detection of T foetus in cultures for individual animals.

    Design�Cross-sectional study.

    Animals�188 bulls and 150 steers.

    Procedures�Preputial scraping samples were collected, placed in a culture kit, and incubated at 37°C for 7 days. Cultures for individual animals were tested for T foetus by means of a real-time PCR assay. Pools of protozoal cultures were made by including fixed aliquots of samples with known positive and negative results in ratios of 1:2, 1:3, 1:5, 1:10, 1:15, 1:20, and 1:25. Specificities of the real-time PCR assay and culture for detection of T foetus in samples obtained from individual animals and sensitivity of real-time PCR assay for each evaluated pool ratio were determined.

    Results�Specificity estimates for culture and the real-time PCR assay for detection of T foetus in preputial scraping samples for individual animals were not significantly different (98.8% and 100%, respectively). Sensitivities of the real-time PCR assay for the various pooled samples with known positive and negative T foetus results were not significantly different; overall sensitivity of the assay was 94%.

    Conclusions and Clinical Relevance�Results indicated the evaluated real-time PCR assay had high specificity and good sensitivity for the detection of T foetus in pooled protozoal cultures of preputial scraping samples obtained from up to 25 animals.

    Reproductive performance of animals is one of the most important factors affecting the economic success of cow-calf operations.1�3 Early pregnancy loss and failure to conceive are the primary causes of poor reproductive performance of cows.1 Trichomoniasis is a venereal disease of cattle caused by Tritrichomonas foetus; this disease is commonly identified in herds with prolonged calving seasons and low pregnancy4,5 and calving6 rates. Trichomoniasis has a substantial effect on the profitability of cow-calf operations, with an estimated loss of 5% to 35% return/cow that undergoes breeding.7 Methods for the control of trichomoniasis typically include identification and subsequent elimination of infected carrier bulls from a herd.8 Identification of carrier bulls is commonly accomplished by means of collection of preputial scraping samples, protozoal culture, and microscopic identification. Although protozoal culture is the gold-standard method, the sensitivity of that method for identification of T foetus is variable (range, 67.8% to 98.9%),8�13 depending on factors such as sample collection variables, transport conditions, and culture method. Polymerase chain reaction assays have been developed to replace14 or complement15 culture-based methods for detection of T foetus.

    Polymerase chain reaction assays (both conventional and real-time methods) have a lower detection limit than culture methods for T foetus.14 This has led to the use of testing strategies such as the analysis of pooled preputial scraping samples. Results of other studies16,a indicate that the sensitivities of conventional and real-time PCR assays of pooled preputial scraping samples obtained from groups of 5 bulls (1 sample with positive results included in each pool) for detection of T foetus are 100%. However, there is a need for additional information regarding the analysis of pooled samples from a larger number of animals to optimize this testing method. Given the economic consequences of culling a healthy bull from a herd, further studies regarding the potential for false-positive real-time PCR assay results are warranted. The objectives of the study reported here were to determine the specificity of a real-time PCR assay for the detection of T foetus in preputial scraping samples collected by use of a commercially available culture kit and the sensitivity of a real-time PCR assay for detection of that organism after preputial scraping sample collection, protozoal culture, and pooling of culture media for groups of samples obtained from up to 25 bulls.