Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

• Published in: Veterinary parasitology 2016-08, Vol.226, p.57-64
• Main Authors: Losada-Barragán, Monica, Cavalcanti, Amanda, Umaña-Pérez, Adriana, Porrozzi, Renato, Cuervo-Escobar, Sergio, Vallejo, Andrés Felipe, Sánchez-Gómez, Myriam, Cuervo, Patricia
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Cricetinae; DNA Primers - chemistry; DNA, Protozoan - chemistry; Dog Diseases - parasitology; Dogs; Leishmania infantum - genetics; Leishmania infantum - growth & development; Leishmania infantum - isolation & purification; Leishmaniasis, Visceral - parasitology; Leishmaniasis, Visceral - veterinary; Liver - parasitology; Mice; Mice, Inbred BALB C; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Spleen - parasitology
• ISSN: 0304-4017; 1873-2550
• DOI: 10.1016/j.vetpar.2016.05.022

•QPCR strategy for specific detection of L. infantum parasites in animal samples.•Specific primer-probe for distinguishing between L. infantum and L. donovani species.•Approach for the diagnosis of canine visceral leishmaniasis due to L. infantum.•Parasite load quantification in symptomatic and asymptomatic animals by qPCR. Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05–0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p