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.
Persistence of Tritrichomonas foetus in naturally infected cows and heifers in Argentina.
Tritrichomonas foetus infection was investigated in 76 pregnant and 64 non-pregnant cows slaughtered in the local abbattoir and in two different lots of first-service heifers that were found to be non-pregnant 60 days post breeding (PB). In live and slaughtered animals, mucus samples were obtained from the vagina and from the vagina and uterus, respectively, using a "screw-head scraper rod". In pregnant cows, samples of amniotic and allantoid fluid were also collected, as well as samples from the stomach contents of the fetuses. All samples were cultured in Modified Plastridge Medium. T. foetus was isolated from three pregnant and two non-pregnant slaughtered cows. Parasites were recovered from the vagina of these five cows, as well as from the uterus in two cases and from the fetus in one case. Lot I of first-service heifers consisted of 323 females from eight different farms. Bulls infected with T. foetus from these farms were culled or treated, and heifers found empty at diagnosis of pregnancy were culled. Lot II consisted of 120 heifers from a single farm where T. foetus was controlled only in bulls. All heifers from Lot I were T. foetus negative. In Lot II, 12 of 120 heifers (10%) were T. foetus positive. In ten of these the parasites were observed once, in one at 60 days PB, in seven at 160 days PB and in two at 240 days PB; in the remaining two infected heifers, an irregular pattern of isolation persisted during 300 days PB. On the basis of these results, control methods are discussed and analysed.
Comparison of the Diagnostic Sensitivity of a Commercially Available Culture Kit and a Diagnostic Culture Test Using Diamond's Media for Diagnosing Tritrichomonas Foetus in Bulls
A number of different culture media have been described for use in the diagnosis of Tritrichomonas foetus infection in bulls, and recently, a commercial culture kit has become available. The objective of this study was to compare the sensitivity of 2 culture-based diagnostic tests for T. foetus in bulls. One test used a commercial kit for transport and culture of the samples. The other test used a thioglycollate transport medium (TFTM) for transport and a modified Diamond's medium (MDM) for culture of the samples. Twenty-one bulls infected with T. foetus were sampled repeatedly. On each sampling day, samples collected from the left and right sides of the bull were tested with one of the 2 diagnostic tests being compared. The effect of the type of diagnostic test on the outcome of the test was evaluated with a chi-square test for the calculated odds ratio. Because repeated tests from the same bull cannot be considered independent measures, unadjusted chi-square tests were adjusted for the effect of clustering by bull. Samples tested using the commercial kit were 6.95 times as likely to be positive as samples tested with a diagnostic test using MDM (P < 0.001).