Improvements in Tritrichomonas foetus molecular testing

Abstract

Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing for Tritrichomonas foetus and culling of infected males. Challenges associated with testing include cost of culture medium, time and labor burden of sample incubation and processing, and adverse effects of bacterial growth on detection sensitivity. To overcome these challenges, we developed a direct reverse-transcription quantitative real-time PCR (direct RT-qPCR) utilizing smegma, eliminating the use of culture medium. In an analysis of 166 field samples (56 positives and 110 negatives as determined using microscopic reading of cultures as the reference test), the direct RT-qPCR exhibited 100% diagnostic sensitivity and 100% specificity, whereas the currently employed qPCR (culture qPCR), which utilizes cultured samples, exhibited 95% diagnostic sensitivity and 100% specificity. Agreement between direct RT-qPCR and culture qPCR was 98%. Moreover, direct RT-qPCR identified 3 more positive samples and exhibited lower quantification cycle (Cq) values among positives by culture reading than did culture qPCR (direct RT-qPCR Cq range = 14.6-32.3 vs. culture qPCR Cq range = 18.7-37.4). The direct RT-qPCR enables simplified sample collection, elimination of culture medium, faster results, applicability in cows, and lower cost than culture qPCR.

Investigation of Tritrichomonas foetus in cryopreserved bovine semen by culture and polymerase chain reaction

“…A North American study showed that quantitative real-time PCR (RT-qPCR) presented 100% sensitivity and specificity for smegma samples. In comparison, PCR analysis of culture aliquots showed 5% less sensitivity (Ginter Summarell et al, 2018). Therefore, new techniques and methodologies may obtain a different result than the one obtained in the present study.…”

“…The samples for the present study were chosen because they had not yet been surveyed for T. foetus. In addition, other studies have shown that PCR can have better results when performed directly with pure samples rather than with culture samples (Ginter Summarell et al, 2018). Since it was not possible to use the parabasalid in vivo, we employed a previously extracted DNA sample as positive control.…”

Detection of Trichomoniasis in cattle in Nineveh province

“…Another method is by inoculating the parasite in different types of culture media., this method takes from two to seven days and does not differentiate between the different types of Tritrichomnas spp. (6). Further, the serological test can be used for diagnosis of the parasite such as mucus agglutination test and ELISA test, but these tests have limited use because of low sensitivity, the specificity of tests, and in bulls do not develop tangible immune responses (7).…”

Detection of Tritrichomonas foetus by RT-rtPCR in pooled bovine preputial washings

“…Samples had been stored at −20°C prior to use in our study. To evaluate the updated workflow, 20 individual T. foetus– positive samples were extracted, in triplicate, and tested using both the previously described workflow 8 and the new workflow, which utilized a commercial extraction kit (MagMAX CORE nucleic acid purification kit) and a newer master mix chemistry (TaqMan fast virus 1-step master mix). Total nucleic acid was extracted following the manufacturer’s instructions.…”