In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

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 Organization^1,2. 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.......

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 groups^{22,24,27,29,31,32,34,49} and .......

Materials

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