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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

Abstract

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism's response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

Introduction

Leishmaniases are worldwide prevalent parasitic infectious diseases representing important challenges in developing countries and are recognized as one of the most important neglected tropical diseases by the World Health Organization1,2. The leishmaniases are characterized by cutaneous, mucosal, and/or visceral manifestations. Cutaneous leishmaniasis is usually caused by L. amazonensis, L. mexicana, L. braziliensis, L. guyanensis, L. major, L. tropica and L. aethiopica3. This form of the disease is often self-healing in hum....

Protocol

All experimental procedures were approved by the Animal Care and Use Committee at the Institute of Bioscience of the University of São Paulo (CEUA 342/2019), and were conducted in accordance with the recommendations and the policies for the Care and Use of Laboratory Animals of São Paulo State (Lei Estadual 11.977, de 25/08/2005) and the Brazilian government (Lei Federal 11.794, de 08/10/2008). All steps described in sections 1-5 should be carried out aseptically inside laminar flow cabinets. Personal protectiv.......

Representative Results

Leishmania protozoan parasites exist in two developmental forms during their life cycle in invertebrate and vertebrate hosts: promastigotes, the proliferative forms found in the lumen of the female sandfly; and amastigotes, the proliferative forms found in the parasitophorous vacuoles of the mammalian host cells. Promastigotes have an elongated body of approximately 1.5 µm wide and 20 µm long, with a flagellum typically emerging from the anterior extremity. Amastigotes .......

Discussion

The in vivo infection assay described in this protocol allows any researcher to evaluate in vivo cutaneous leishmaniasis considering the host-parasite interaction in a systemic scenario. These assays have been used by many groups22,24,27,29,31,32,34,49 and .......

Acknowledgements

We would like to thank Prof. Dr. Niels Olsen Saraiva Câmara from the Animal Center of the Biomedical Sciences Institute of the University of São Paulo for the support and Prof. Dr. Silvia Reni Uliana for providing the glass tissue grinder. This work was supported by Sao Paulo Research Foundation (FAPESP - MFLS' grant 2017/23933-3).

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Materials

NameCompanyCatalog NumberComments
96-well plateGreiner bio-ne655180A flat-bottom plate for limiting dilution assay
adenineSigmaA8626Supplement added to M199 cell culture media
caliperMitutoyo700-118-20A caliper to measure the thickness of footpad
cell culture flaskCorning353014A 25 cm2 volume cell culture flask to cultivate Leishmania parasite
centrifugeEppendorf5804RAn equipament used for separating samples based on its density
CO2 incubator 34 °CThermo Scientific3110An incubator for amastigotes differentiation
ethanolMerckK50237083820A disinfectant for general items
fetal bovine serumGibco12657-029Supplement added to M199 cell culture media
glass tissue grinder tubeThomas Scientific3431 E04A tube to collect and disrupt infected footpad tissue
glucoseSynthG1008.01.AHSupplement added to M199 cell culture media
GraphPad Prism SoftwareGraphPadA software used to plot the data and calculate statistical significance
heminSigmaH-2250Supplement added to M199 cell culture media
HEPESPromegaH5303Supplement added to M199 cell culture media
incubator 25 °CFanem347CDAn incubator for promastigotes cultivation
inverted microscopeNikonTMSAn equipament used to visual analyze the promastigote and amastigote cultures
isofluraneAn inhalant anesthetics for mice (3-5%)
laminar flow cabinetVecoVLFS-09A biosafety cabinet used for aseptical work area
M199 cell culture mediaGibco31100-035A cell culture media for Leishmania cultivation
microcentrifuge tubeAxygenMCT150CA microtube used for sample collection, processing and storage
multichanel pipetteLabsystemsF61978A multichannel pipette used for limiting dilution assay
NaHCO3Merck6329Supplement added to M199 cell culture media
NaOHSigmaS8045Supplement added to M199 cell culture media
Neubauer chamberHBG2266A hemocytometer to count the parasite suspension
optical microscopeNikonE200An optical equipament used to count parasite
parafilmBemis349A flexible and resistant plastic to seal the plate
penicillin/streptomycinGibco15140122Supplement added to M199 cell culture media
Petri dishesTPP93100A sterile dish to dissect the footpad tissue
pipetman kitGilsonF167360A micropipette kit containing four pipettors (P2 P20 P200 P1000)
scaleQuimisBG2000An equipament used to weigh collected footpad lesions
scalpelSolidor10237580026A scalpel to cut and collect footpad tissue
serological pipette 10 mLNest327001A sterile pipette used for transfering mililiter volumes
tipsAxygenA pipette tip used for transfering microliter volumes
Trypan blueGibco15250-061A dye used to count viable parasites
trypticase peptoneMerckSupplement added to M199 cell culture media
tuberculin syringeBD305945A syringe with 27G needle to inoculate the parasite suspension

References

  1. Alvar, J., et al. Leishmaniasis worldwide and global estimates of its incidence. PloS One. 7 (5), e35671 (2012).
  2. Ashford, R. W. The leishmaniases as emerging and reemerging zoonoses. International Journ....

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