01159-A: Detection of Brucellosis canis DNA in Canine Blood Via qPCR Analysis
Grant Status: Closed
Project SummaryPrimer and probe sets for quantitative real-time Polymerase Chain Reaction analysis (qPCR) were optimized to be specific to Brucella canis and abortus and are sufficiently sensitive enough to detect as few as 1 bacterium in 1mL of blood. These primers and probes also detect B. canis and abortus in urine, semen, vaginal swabs and tissue, specifically reproductive tissues or samples where the bacterium is found in higher numbers during disease, when the bacterium appears to sequester into the reproductive tissue and not be present in the blood. The results suggest that bacteria may only be in the blood for a short time (acute infection and later chronic infection) before localizing in reproductive tissue thus making the window for qPCR testing of blood at the beginning and end of the spectra of disease and perhaps less clinically useful. The results suggest that the qPCR assay on urine may be more clinically diagnostic than whole blood samples in male dogs due to prostatic infection and shedding of bacteria into the urine. There is initial evidence that this may also be true for semen. There is also evidence that qPCR assay on vaginal swabs may be more clinically diagnostic than whole blood samples in female dogs due to vaginal infection and presence of bacteria in this area. It has been additionally identified, using a novel qPCR analysis that the standard operating procedure of canine semen freezing for artificial insemination is not sufficient to kill Brucella. This fact is critically important to the dog breeding community as perhaps testing via culture/qPCR should be mandated prior to use for artificial insemination. Further studies are necessary to determine the best, least invasive, samples to submit in order to diagnose Brucellosis early and with high sensitivity (decrease false negatives). The new qPCR assay has a very low false positive rate when using whole blood or other samples, a vast improvement over the previous screening standard for canine Brucellosis; RSAT.
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