Parasite Burden Measurement in the Leishmania major Infected Mice by Using the Direct Fluorescent Microscopy, Limiting Dilution Assay, and Real-Time PCR Analysis

BACKGROUND: We aimed to compare parasite burden in BALB/c mice, using three methods including the direct fluorescent microscopic using recombinant Leishmania major expressing an enhanced green fluorescent protein, limiting dilution assay, and real-time PCR technique. METHODS: The current study was carried out in 2018, to induce stable enhanced green fluorescent protein (EGFP) production. Initially, the linearized DNA expression cassette (pLEXSY-egfp-sat2) was integrated into the ssu locus of L. major. The expression of EGFP in recombinant parasite was analyzed using direct fluorescent microscopy. Afterward, BALB/c mice were infected with the L. major( EGFP), and the infection was evaluated in the foot-pads and inguinal lymph-nodes using an in vivo imaging system. Subsequently, eight BALB/c mice were infected with L. major( EGFP), and the results of evaluating parasite burden by a SYBR-Green based real-time PCR analysis and the limiting dilution assays were compared to the results obtained from the direct fluorescent microscopy. RESULTS: The results of the direct fluorescent microscopy showed that EGFP gene stably was expressed in parasites. Moreover, the in vivo imaging analysis of foot-pad lesions revealed that the infection caused by L. major( EGFP) was progressing over time. Additionally, significant correlations were observed between the results of parasite burden assay using the direct fluorescent microscopy and either limiting dilution assay (r=0.976, P<0.0001) or quantitative real-time PCR assay (r=0.857, P<0.001). CONCLUSION: Ultimately, the utilization of the direct fluorescent microscopy by employing a stable EGFP-expressing L. major is a suitable substitution for the existing methods to quantify parasite burden.