Original Article

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

Abstract

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. majorEGFP, 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. majorEGFP, 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. majorEGFP 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.

1. Aguilar Torrentera F, Lambot MA, et al. Parasitic load and histopathology of cutaneous lesions, lymph node, spleen, and liver from BALB/c and C57BL/6 mice infected with Leishmania mexicana. Am J Trop Med Hyg. 2002;66(3):273-9.
2. Berman JD, Lee LS. Activity of antileishmanial agents against amastigotes in human monocyte-derived macrophages and in mouse peritoneal macrophages. J Parasitol. 1984;70(2):220-5.
3. Sacks DL, Melby PC. Animal models for the analysis of immune responses to leishmaniasis. Curr Protoc Immunol. 2001;Chapter 19:Unit 19.2.
4. Titus RG, Marchand M, Boon T, et al. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol. 1985;7(5):545-55.
5. Taswell C. Limiting dilution assays for the separation, characterization, and quantitation of biologically active particles and their clonal progeny. Cell Separation.1987;4:109-45.
6. Ghotloo S, Haji Mollahoseini M, Najafi A, et al. Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishmania major Infected Mouse. Iran J Parasitol. 2015;10(4):571-6.
7. Mohammadiha A, Mohebali M, Haghighi A, et al. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol. 2013;133(1):89-94.
8. Mohammadiha A, Haghighi A, Mohebali M, et al. Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Vet Parasitol. 2013;192(1-3):83-90.
9. Dube A, Gupta R, Singh N. Reporter genes facilitating discovery of drugs targeting protozoan parasites. Trends Parasitol. 2009;25(9):432-9.
10. Stiner L, Halverson LJ. Development and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compounds. Appl Environ Microbiol. 2002;68(4):1962-71.
11. Wei T, Dai H. Quantification of GFP signals by fluorescent microscopy and flow cytometry. Methods Mol Biol. 2014;1163:23-31.
12. Chudakov DM, Matz MV, Lukyanov S, et al. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010;90(3):1103-63.
13. Misslitz A, Mottram JC, Overath P, et al. Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Mol Biochem Parasitol. 2000;107(2):251-61.
14. Dehghani F, Haji Molla Hoseini M, Memarnejadian A, et al. Immunomodulatory activities of chitin microparticles on Leishmania major-infected murine macrophages. Arch Med Res. 2011;42(7):572-6.
15. Kropf P, Kadolsky UD, Rogers M, et al. The Leishmaniasis Model. Methods in Microbiology. 2010; 37: 307-28.
16. Nicolas L, Prina E, Lang T, et al. Real-time PCR for detection and quantitation of Leishmania in mouse tissues. J Clin Microbiol. 2002;40(5):1666-9.
17. Singh N, Gupta R, Jaiswal AK, et al. Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening. J Antimicrob Chemother. 2009;64(2):370-4.
18. Mehta SR, Huang R, Yang M, et al. Real-time in vivo green fluorescent protein imaging of a murine leishmaniasis model as a new tool for Leishmania vaccine and drug discovery. Clin Vaccine Immunol. 2008;15(12):1764-70.
19. Reithinger R, Dujardin JC. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol. 2007;45(1):21-5.
20. Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71(3):267-75.
21. Jara M, Adaui V, Valencia BM, et al. Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: exploratory study of parasite load and clinical parameters. J Clin Microbiol. 2013;51(6):1826-33.
22. Mary C, Faraut F, Lascombe L, et al. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol. 2004;42(11):5249-55.
23. Rocha MN, Correa CM, Melo MN, et al. An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein. Diagn Microbiol Infect Dis. 2013;75(3):282-91.
24. Siqueira-Neto JL, Moon S, Jang J, et al. An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages. PLoS Negl Trop Dis. 2012;6(6):e1671.
25. Bastos MS, Souza LA, Onofre TS, et al. Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro. Mem Inst Oswaldo Cruz. 2017;112(2):155-9.
Files
IssueVol 15 No 4 (2020) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v15i4.4867
Keywords
Green fluorescent pro-tein Leishmania major Limiting dilution assay Parasite burden
Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
HAGHDOUST S, AZIZI M, HAJI MOLLA HOSEINI M, BANDEHPOUR M, MOHSENI MASOOLEH M, YEGANEH F. Parasite Burden Measurement in the Leishmania major Infected Mice by Using the Direct Fluorescent Microscopy, Limiting Dilution Assay, and Real-Time PCR Analysis. Iran J Parasitol. 2020;15(4):576-586.