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.
Molecular detection of Tritrichomonas foetus in bovine samples: a novel real-time polymerase chain reaction (PCR) assay targeting EF1-alpha-Tf1 and a comparative study of published PCR techniques
The parasite T. foetus causes trichomonosis in cattle but is generally asymptomatic in males. Thus, many bulls carrying the disease go unnoticed, making the detection of T. foetus in bulls an important aspect for its control. Due to drawbacks posed by its cultivation, PCR is a preferred option for diagnostic laboratories. Most published PCR protocols target the genomic region compring the 18S, 5.8S, and 28S rRNA genes and internal transcribed spacers 1 and 2 (rRNA-ITS region), homologous to that of other Tritrichomonas species. There is minimal information on alternative genetic targets and no comparative studies have been published. We compared a protocol based on the microsatellite TfRE (called H94) and five protocols based on the rRNA-ITS region (called M06, M15, G02, G05, and N02). We also designed and evaluated a novel PCR-based assay on the EF1-alpha-Tf1 gene (called V21). The analytical sensitivity and specificity assays for the PCR protocols were performed according to the World Organisation for Animal Health (OIE) directives and the comparative study was performed with a widely used PCR (M06) on clinical samples from 466 breeding bulls. V21 showed a high degree of agreement with our reference M06 (kappa = 0.967), as well as M15 (kappa = 0.958), G05 (kappa = 0.948), and H94 (kappa = 0.986). Protocols H94 and V21 appear to be good approaches for confirming clinical cases in preputial bull samples when genomic regions alternative to rRNA-ITS are required. By contrast, N02 gave false negatives and G02 false positives.
Evaluation of Analgesic Effect of Caudal Epidural Tramadol, Tramadol-Lidocaine, and Lidocaine in Water Buffalo Calves (Bubalus bubalis)
Aim of this study was to compare the analgesic effect of tramadol and a combination of tramadol-lidocaine with that produced by lidocaine administration in the epidural space in buffalo calves. In a prospective randomized crossover study, ten male buffalo calves were used to compare the epidural analgesic effect of tramadol (1 mg/kg) and tramadol-lidocaine combination (0.5 mg/kg and 0.11 mg/kg, resp.) with that produced by 2% lidocaine (0.22 mg/kg). Loss of sensation was examined by pin-prick test. Onset time, duration, and degree of analgesia and ataxia were recorded after each treatment. Heart rate (HR), respiratory rate (RR), rectal temperature, and haematobiochemical parameters were recorded after all treatments. Time to onset and duration of analgesia, respectively, were as follows: tramadol 11 ± 2 min and 208 ± 15 min; tramadol-lidocaine 6 ± 2 min and 168 ± 9 min; lidocaine 4 ± 1 min and 67 ± 13 min. Onset time and duration were significantly longer with tramadol than the other treatments. Duration was significantly longer with tramadol-lidocaine than lidocaine. Ataxia was mildly observed in tramadol-lidocaine and was moderate in lidocaine. HR, RR, and rectal temperature did not differ significantly from baseline after any treatment. Haematobiochemical parameters returned to basal levels by 24 h after all treatments. This combination might be clinically useful to provide analgesia in buffalo for long-duration surgical procedures.