Real-Time Quantitative PCR Analysis of Leishmania DNA in Canine Bone Marrow and Peripheral Blood Samples
*Hospital Clínic Veterinari, Facultat de Veterinària. Universitat Autònoma de Barcelona
Diagnosis of canine leishmaniasis can be extremely challenging due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical and asymptomatic dogs in endemic areas. As a result, diagnosis requires identification of the organism in dogs with clinical signs compatible with the disease. The experiences of a veterinary research and diagnostic laboratory are summarized on the development and application of the PCR to diagnose canine leishmaniasis. However, since canine leishmaniasis is associated with tissue loads of residual, latent parasites after treatment, non-quantitative PCR tests are of little value in indicating a positive or negative result. At present, the classical PCR and nested PCR methods are being replaced by real-time PCR detection. The purpose of this study was to evaluate the ability of a real-time TaqMan PCR procedure for rapid detection and quantitative analysis of Leishmania parasites in canine bone marrow and peripheral blood samples.
Clinical specimens were obtained from 30 dogs with confirmed leishmaniasis, either before or after treatment with meglumine antimonate and allopurinol. DNA was prepared from 1 ml bone marrow and blood samples using a lysis buffer with proteinase K. Serial dilutions of L. infantum DNA were obtained from a culture (5x106 promastigotes/ml) by direct lysis of the parasites using the same lysis buffer.
The quantitative analysis of the Leishmania DNA amplification was accomplished by comparing the TaqMan system developed for Leishmania kinetoplast and a TaqMan system of the 18S RNA as internal reference of canine genomic DNA amplification. For the Leishmania TaqMan system we amplified a 120 bp fragment of the Leishmania kinetoplast minicircle DNA. Both the primers and the probe were designed to target sequences corresponding to conserved regions in different Leishmania species.
Each amplification was performed in triplicate, in a 25 ul reaction mixture using the components of the TaqMan Universal PCR Master Mix (Applied Biosystems). Primers and probe were added at 900 nM each and 200 nM respectively. The thermal cycling profile was 50°C 2', 95°C 10' and 40 cycles at 95°C 15'' and 60°C 60''. Amplification data were collected at every cycle for each one of the samples and analysed by the 7900 HT Sequence Detection System.
We determined the standard curve obtained with the TaqMan Leishmania PCR test from serial dilution of L. infantum DNA. An optimum correlation was obtained between Ct (threshold cycle) and DNA serial dilutions from 105 to 101 promastigotes (R2: 0.994; Slope: -3.34) The presence of Leishmania DNA in the different samples was quantitatively analysed in relation to both the standard curve and the 18S RNA amplification in each one of them. Sensitivity was greater in bone marrow samples, and positive samples previously analysed by end-point PCR were amplified at Ct 14-16. Peripheral blood samples were also evaluated, although Ct was higher in all the cases.
The present work is the first development of TaqMan probes for canine leishmaniasis. We conclude that the real-time PCR test allows the detection and relative quantification of the number of parasites present in the samples with more sensitivity than classical PCR tests. The possibility to detect DNA in a quantitative manner could contribute significantly to the diagnosis and management of Leishmania infections, as well as to the understanding of parasitic-host interactions.