A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis

Abstract

Eukaryotic parasites in the genus Leishmania place approximately 350 million people per year at risk of disease. In addition to their global health significance, Leishmania spp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR-based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we developed a sybr-green based quantitative touchdown PCR assay for a highly conserved and single-copy putative RNA-binding protein, DRBD3. With primers that share greater than 90% sequence identity across all sequenced Leishmania spp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA for Leishmania major, L. donovani, L. venezuelensis, and L. panamensis. Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains of L. major (Seidman and Friedlin), and L. venezeuelensis. These characteristics rival the sensitivity of previously reported qPCR based methods of parasite quantitation while amplifying a stable, single copy gene. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.

Keywords: DRBD3; Leishmania; Leishmaniasis; Mouse; Parasite burden; Quantitative real-time PCR.

Figure 1. Primer design and optimization of DRBD3 based qPCR for parasite quantification.

(A) Multisequence alignment based on 11 homologous sequences to L. major Sd. DRBD3 found using NCBI Blast. (B) Primer sequences and cycling parameters used. (C) DRBD3 primers amplify a 140 bp product specifically. Product visualized with ethidium bromide staining of a 1% agarose gel run and compared to New England BioLabs 100 bp ladder. (D) DRBD3 primers amplify diverse Leishmania spp. A representative plot of C_t value vs log dilution of parasite burden is shown. The average primer efficiency (±standard error of the mean) is indicated in parentheses for the following species: L. major Seidman (n = 6), L. major Friedlin (n = 5), L. venezuelensis (n = 5), L. panamensis (n = 3), and L. donovani (n = 3).

Figure 2. DRBD3 primers are able to assess parasite burden from infected mouse tissue.

(A and B) Representative amplification plots and melt curve analysis from the draining lymph nodes of mice at 34 days post infection. Parasites amplified from infected tissue had melting temperatures corresponding with the expected 84.7 °C. DNA from uninfected tissue resulted in non-specific amplification at later cycles with melt temps outside of 84.7 ± 0.5 °C. Samples that generated any product outside of this range were classified as having no Leishmania specific amplification. (C) Quantification of total parasites in the draining popliteal lymph nodes and infected footpads at 1 and 34 days post infection with L. major Seidman (Sd.). At 1 day post infection, five lymph node samples had no Leishmania specific amplification and are not plotted. All samples from 34 days post infection had detectable Leishmania specific amplification. (D) DRBD3 quantification provides insights to disease pathogenesis in L. major Freidlin (Fd.) and L. venezuelensis infections. Comparison of footpad thickness and parasite burden in the draining lymph nodes and infected footpads at 36 days post infection. Four lymph node samples and one footpad samples had no Leishmania specific amplification and are therefore not plotted. P-values calculated by parametric Students T-test.