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.
The influence of temperature and simulated transport conditions of diagnostic samples on real-time polymerase chain reaction for the detection of Tritrichomonas foetus DNA
Bovine trichomoniasis is a sexually transmitted disease in cattle that causes considerable economic loss due to abortions and infertility. In vitro culture of the organisms is the traditional method for diagnosis. However, culture cannot differentiate Tritrichomonas foetus from other, closely related nonpathogenic protozoa. Recently, a quantitative real-time polymerase chain reaction (qPCR) was developed for the differential diagnosis of trichomoniasis. The objective of the current work was to evaluate the effect of different simulated transport conditions on samples containing T. foetus for the diagnosis of trichomoniasis using culture and qPCR. Results indicate that transport temperatures of 4–20°C for 1–3 days before culture will reduce or temporarily inhibit parasite replication but maintain viability. Testing of samples by either culture or qPCR would be expected to give positive results. However, diagnosis of trichomonads by both methods was negatively affected when specimens were maintained at transport temperatures of 42°C for 24 hr or more. The current study stresses the importance of ensuring that clinical samples arrive to the diagnostic laboratory within 24–48 hr and of avoiding temperature transport conditions above 37°C in order to achieve an accurate diagnosis of trichomoniasis in cattle.
Synergistic Effect of High Temperature and High pH on the Destruction of Salmonella enteritidis and Escherichia co1i O157:H7.
This study was undertaken to determine if high temperature and high pH interact synergistically to enhance the rate of destruction of two important gram-negative foodborne pathogens, Escherichia coli O157:H7 and Salmonella enteritidis . The rates of destruction in NaHCO3-NaOH buffers at pH 7.0, 10.0, and 11.0 were determined at 35, 40, 45, 50, 55, 60, and 65°C. Use of an improved heating protocol eliminated a "tailing effect" at longer exposure times. The present study demonstrated that the combination of high pH and high temperature resulted in a highly significant synergistic interaction (P > F = 0.0001), which caused rapid death of both E. coli O157:H7 and S. enteritidis . This "alka-therm" technology might be used commercially to destroy gram-negative foodborne pathogens on various raw agricultural commodities.