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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.
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.
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....
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.......
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 .......
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 .......
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).
....Name | Company | Catalog Number | Comments |
96-well plate | Greiner bio-ne | 655180 | A flat-bottom plate for limiting dilution assay |
adenine | Sigma | A8626 | Supplement added to M199 cell culture media |
caliper | Mitutoyo | 700-118-20 | A caliper to measure the thickness of footpad |
cell culture flask | Corning | 353014 | A 25 cm2 volume cell culture flask to cultivate Leishmania parasite |
centrifuge | Eppendorf | 5804R | An equipament used for separating samples based on its density |
CO2 incubator 34 °C | Thermo Scientific | 3110 | An incubator for amastigotes differentiation |
ethanol | Merck | K50237083820 | A disinfectant for general items |
fetal bovine serum | Gibco | 12657-029 | Supplement added to M199 cell culture media |
glass tissue grinder tube | Thomas Scientific | 3431 E04 | A tube to collect and disrupt infected footpad tissue |
glucose | Synth | G1008.01.AH | Supplement added to M199 cell culture media |
GraphPad Prism Software | GraphPad | A software used to plot the data and calculate statistical significance | |
hemin | Sigma | H-2250 | Supplement added to M199 cell culture media |
HEPES | Promega | H5303 | Supplement added to M199 cell culture media |
incubator 25 °C | Fanem | 347CD | An incubator for promastigotes cultivation |
inverted microscope | Nikon | TMS | An equipament used to visual analyze the promastigote and amastigote cultures |
isoflurane | An inhalant anesthetics for mice (3-5%) | ||
laminar flow cabinet | Veco | VLFS-09 | A biosafety cabinet used for aseptical work area |
M199 cell culture media | Gibco | 31100-035 | A cell culture media for Leishmania cultivation |
microcentrifuge tube | Axygen | MCT150C | A microtube used for sample collection, processing and storage |
multichanel pipette | Labsystems | F61978 | A multichannel pipette used for limiting dilution assay |
NaHCO3 | Merck | 6329 | Supplement added to M199 cell culture media |
NaOH | Sigma | S8045 | Supplement added to M199 cell culture media |
Neubauer chamber | HBG | 2266 | A hemocytometer to count the parasite suspension |
optical microscope | Nikon | E200 | An optical equipament used to count parasite |
parafilm | Bemis | 349 | A flexible and resistant plastic to seal the plate |
penicillin/streptomycin | Gibco | 15140122 | Supplement added to M199 cell culture media |
Petri dishes | TPP | 93100 | A sterile dish to dissect the footpad tissue |
pipetman kit | Gilson | F167360 | A micropipette kit containing four pipettors (P2 P20 P200 P1000) |
scale | Quimis | BG2000 | An equipament used to weigh collected footpad lesions |
scalpel | Solidor | 10237580026 | A scalpel to cut and collect footpad tissue |
serological pipette 10 mL | Nest | 327001 | A sterile pipette used for transfering mililiter volumes |
tips | Axygen | A pipette tip used for transfering microliter volumes | |
Trypan blue | Gibco | 15250-061 | A dye used to count viable parasites |
trypticase peptone | Merck | Supplement added to M199 cell culture media | |
tuberculin syringe | BD | 305945 | A syringe with 27G needle to inoculate the parasite suspension |
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