Real Time PCR Development to Quantify the Parasite Load in Golden Hamsters

Infected with Leishmania infantum

Oliveira, P.N.M.A.1, Cappato, M.S.2, Muniz, F.R.2, Da-Cruz, A.M.2, Ribeiro-Romão2, R.P. Moreira, O.C.3,
Santos-Oliveira, J.R.1,2

1
Programa Multicêntrico de Bioquímica e Biologia Molecular – Instituto Federal de Educação, Ciência e
Tecnologia, IFRJ, Rio de Janeiro, Brazil; 2Laboratório Interdisciplinar de Pesquisas Médicas – Instituto
Oswaldo Cruz, IOC-Fundação Oswaldo Cruz; 3 Laboratório de Biologia Molecular e Doenças Endêmicas –
Instituto Oswaldo Cruz, IOC-Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

Introduction: In Americas, visceral leishmaniaisis (VL) is caused by Leishmania

(Leishmania) infantum. VL is a systemic disease of chronic evolution, characterized
by intense immunosuppression and inflammatory response. Experimental models are
essential to provide information about disease's mechanisms. Golden hamster
(Mesocricetus auratus) reproduces the human VL disease and qPCR could monitor
drug’s efficacy, vaccine candidates and the disease evolution, since it's an accurate,
cheaper and rapid method to quantify the parasite load (PL) in infected hamsters.

Objectives: Standardize and validate a real time PCR assay to quantify with great
sensitivity and reproducibility the parasite load in hamsters infected by L. infantum.
Material and Methods: Absolute quantification by qPCR assay was performed using
SYBR Green®. A standard curve was produced from DNA samples extracted by
hamsters tissues spiked with promastigotes forms of L. infantum and constructed
using a 5-fold serially diluted sample of L. infantum DNA, with 10∧7–2,048x10∧−1
parasites/reaction tube. Kinetoplast DNA (kDNA) was compared with other gene
targets. To validate this method, hamsters were intraperitoneally infected with 2x107
amastigotes of L. infantum. After 60 days post-infection and 75,150 and 180 days
post anti-Leishmania treatment, spleen and blood were removed for DNA extraction
and quantification.

Results and Discussion: Optimal primers concentrations and annealing

temperature were standardized using primers for kDNA target (Pita-Pereira, 2012)
Fw:250 nM and Rv:250 nM,62°C; for kDNA target (Mary, 2004) Fw:200nM and
Rv:200nM,62°C and for polymerase target (Bretagne,2001) Fw:400nM and
Rv:400nM,66°C. After primers comparation, the kDNA target (Mary, 2004) was
chosen for its better sensitivity. Primer specificity showed suitable for viscerotropic
species, although it have amplified two dermotropic species among eight tested: L.
major and L. amazonensis.

Conclusion: KDNA target (Mary et al., 2004) was chosen to quantify the parasite
load by qPCR DNA from hamsters infected by L. infantum before and after anti-
Leishmania treatment in blood and spleen, which will enable to follow up the clinical
evolution of the animal.