Improvements in Tritrichomonas foetus molecular testing
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.
Detection of Trichomoniasis in cattle in Nineveh province
Article history: Received March 11, 2020 Accepted April 22, 2020 Available online February 22, 2021 The current study was conducted on the detection of Tritrichomonas foetus in vaginal mucus of infected cows using conventional polymerase chain reaction technique (cPCR) in Nineveh province, Iraq. A total of 87 vaginal mucus samples were collected randomly from the vagina of Heifer cows of different ages (2-4, >4-6, >6 years old) and stages of pregnancy with different clinical status (early embryonic death, pyometra, abortion and healthy animals once) by washing cow’s vagina using artificial insemination pipette, DNA extraction of T. foetus was done from vaginal mucus samples, cPCR was attempt using TFR3 and TFR4 primers, Results indicated that 11 cows (12.6 %) were positive for T. foetus. The clinical status of cows demonstrated statistically significant (P<0.05) a higher percentage of Early Embryonic Death at (6.9%) compared to pyometra, abortion, and healthy cows. Furthermore, the percentage of T. foetus infection was significantly (P<0.05) elevated among cows (>2-4 years old) at (8%) compared to (>4-6 years old) and (>6 years old) cows. This study concluded that T. foetus infection was an elevated percentage of infection in cows with early embryonic death and in cows (>2-4) years old. This study is the first detected T. foetus in cattle in Nineveh province.
Effects of temperature on the survival of Tritrichomonas foetus in transport, Diamond's and InPouch TF media
The abilities of two isolates of Trtrichomonas foetus to survive and replicate in transport and Diamond's medium or in the InPouch TF system (Bio-Med Diagnostics) when exposed to different temperatures for different periods were determined in a series of experiments. Tubes containing thioglycollate transport medium or pouches were inoculated with 4000 to 5000 organisms and kept for up to seven days at 37°C, 22°C, 4°C, or -20°C. When the holding time had elapsed, the numbers of motile T foetus were counted. Samples in transport medium were transferred to Diamond's medium, and both the pouches and tubes containing Diamond's medium were incubated at 37°C. The cultures were examined and counted four or five times during the 10 to 14 day culture period. The sensitivity of the test under the different conditions, expressed as the number of positive cultures/the total number of samples × 100, varied from zero to 100 per cent depending upon the combination of variables considered. In each medium, with both isolates of T foetus, all samples kept for up to four days at 22°C or 37°C were positive. All cultures of samples kept more than five days at 4°C were negative. No positive cultures were detected when samples were kept more than three hours at -20°C. The day on which the cultures reached mean peak concentration varied with the temperature at which the samples had been kept before they were cultured.