Effect of sampling site on the diagnosis of canine parvovirus infection in dogs using polymerase chain reaction.

BACKGROUND: Accurate diagnosis is imperative in dogs with clinical signs of parvovirus infection (CPV-2). OBJECTIVES: To assess quantitative real-time PCR (qRT-PCR) for the diagnosis of CPV-2 infection, and determine the optimal sampling site. Secondarily, to compare qRT-PCR with a point-of-care PCR kit (PCRun), and to assess sensitivity of serology for CPV diagnosis. ANIMALS: Sixty dogs with naturally acquired parvovirus infection, 44 unvaccinated puppies, of which 16 were followed after first and second vaccination, 15 adult dogs, of which 10 were followed also after a booster vaccine, and 9 dogs with distemper virus infection. METHODS: Prospective study. Samples from the rectum, blood, and pharynx were obtained for PCR. RESULTS: All dogs with a clinical diagnosis of parvovirus infection were positive by qRT-PCR in at least 1 sampling site (ie, rectum, blood, pharynx), and 50 (83%) of 60 were positive in all sites. qRT-PCR was negative in 67 (99%) of 68 healthy puppies (before-vaccination), puppies with distemper, and healthy adult dogs. Ten days after initial vaccination of puppies, 62% (fecal), 31% (blood), and 12% (pharyngeal) of samples were positive for CPV-2 on qRT-PCR. The proportion of positive pharyngeal samples decreased 20 days after vaccination and all sites were negative 12-28 days after second vaccination. Vaccinated adults were negative before and after booster vaccination. CONCLUSIONS AND CLINICAL IMPORTANCE: Molecular detection of CPV is sensitive, but specificity is hampered temporarily during the vaccination period. Blood, feces, and pharynx are suitable sampling sites. Fecal samples had the lowest sensitivity in sick dogs and highest positivity in puppies after vaccination.

A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

BACKGROUND: Ehrlichiosis is an important emerging infectious disease of the canid family and humans worldwide. To date, no extensive evaluation or validation of a molecular diagnostic test for ehrlichiosis has been published. Here, we present data for a newly designed TaqMan assay and compare its performance to a commercial technology (PCRun®). Both of these real-time methods of analysis were evaluated using a comprehensive number of prospective and retrospective samples collected from dogs exhibiting symptoms of ehrlichiosis. RESULTS: Whole blood samples collected from dogs, retrospectively in the United Kingdom and prospectively in Israel, were analysed for the presence of Ehrlichia canis and Ehrlichia minasensis DNA using the TaqMan PCR, developed specifically for this study. The results were compared to those of a real time commercial isothermal amplification method (PCRun® system developed by Biogal Galed Labs ACS, Galed, Israel). The sensitivity and specificity (CI: 95%) of the TaqMan PCR and PCRun® were both determined to be 100% and absolute, for all of the samples tested. Interestingly, both tests were demonstrated to be highly comparable, irrespective of differences in amplification chemistry or sequences targeted. Host differences, incidence of disease and geographical location of the isolates had little impact on the positivity recorded by each of the diagnostic methods. CONCLUSIONS: It was evident that both amplification methods were equally suited for diagnosing canine ehrlichiosis and while the PCRun® clearly amplified all clinically relevant Ehrlichia species known to infect dogs and humans, the TaqMan method was more specific for E. canis and E. minasensis. This work demonstrates that despite good analytical sensitivities and specificities for Ehrlichia spp. neither method could fully account for the clinical diagnosis of thrombocytopenia.

Amplification of Mycoplasma haemofelis DNA by a PCR for point-of-care use.

We compared a qualitative in-clinic (IC)-PCR for the detection of Mycoplasma haemofelis DNA with the results of a commercial qualitative laboratory-based, conventional (c)PCR. In order to determine the specificity of both tests, Bartonella spp. samples were included. Forty-three previously tested blood samples with known PCR results for hemoplasmas and Bartonella spp. were selected. The samples were split between 2 laboratories. At the first laboratory, DNA was purified and run on 2 cPCR assays for the detection of hemoplasmas and Bartonella spp. At the second laboratory, DNA was purified using 2 purification protocols and both run in the IC-PCR assay. The cPCR results confirmed that 18 samples were positive for M. haemofelis, 5 for ' Candidatus M. haemominutum', 8 for Bartonella henselae, 2 for Bartonella clarridgeiae, and 10 were negative for both genera. No mixed infections were observed. The IC-PCR assay for the detection of M. haemofelis had a sensitivity of 94.4% and specificity of 96%, when using the same DNA purification method as the first laboratory. Using the second purification method, the sensitivity of the IC-PCR assay was 77.8% and specificity was 96%. Bartonella species were not detected by the IC-PCR M. haemofelis assay. The IC-PCR assay decreased the amount of time to final result compared to a cPCR assay.