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 humans due to the induction of protective cellular immune response. However, the cellular immune response may fail, and the disease can progress to disseminated cutaneous leishmaniasis^4,5. There is no available vaccine due to the diversity among Leishmania species and host genetic backgrounds^6,7. Treatment options are also limited as most of the currently available drugs are either expensive, toxic, and/or may require long-term treatment^8,9. Besides, there have been reports of drug resistance against the available treatments^10,11.

The causative agent of leishmaniases is the protozoan parasite Leishmania. The parasite presents two distinct morphological forms in its life cycle: promastigotes, the flagellated form found in sandflies; and amastigotes, the intracellular form found in the parasitophorous vacuoles of the mammalian host macrophages^12,13. Amastigotes' ability to invade, survive, and replicate despite the defense mechanisms of the vertebrate host's macrophages are subject to many studies^14,15,16,17. Consequently, several research groups have been describing in vitro macrophage infection assays to evaluate the impact of specific environmental factors, as well as parasite and host genes on parasite infectivity. This assay presents several advantages, such as the ability to adapt studies to a high throughput format, relatively shorter time period to obtain results, and reduced number of laboratory animals sacrificed¹⁸. However, the findings of in vitro assays are limited because they do not always replicate in vivo studies^14,19,20,21. In vivo assays provide a systemic physiological overview of the host-parasite interaction, which cannot be fully mimicked by in vitro assays. For instance, immunological studies can be performed through immunohistochemical assays from collected footpad tissue sections or even from popliteal lymph nodes for analysis of the recovered immune cells²².

Animals are often used as a model for human diseases in biological and biomedical research to better understand the underlying physiological mechanisms of the diseases²³. In the case of leishmaniasis, the route, site, or dose of inoculation influence the disease outcome^24,25,26,27. Furthermore, susceptibility and resistance to the infection in humans and mice are highly regulated by the genetic backgrounds of the host and parasite^{4,5,22,28,29,30,31}. BALB/c mice are highly susceptible to L. amazonensis cutaneous infection, showing a rapid disease progression with the parasites' dissemination to the lymph nodes, spleen, and liver³². As the disease may progress to cutaneous metastases, the infection can be fatal. In contrast, C57BL/6 mice often develop chronic lesions with persistent parasite loads in L. amazonensis infection assays³³. Thereby, L. amazonensis infection with this particular mouse species has been considered an excellent model to study chronic forms of cutaneous leishmaniasis in humans, because it mimics the disease progression better than the BALB/c mice infection model^5,34.

Hence, we propose that the murine in vivo infection is a useful method for Leishmania virulence physiological studies applicable to human disease, allowing a systemic view of the host-parasite interaction. Revisiting well-established assays²², we present here a compiled step-by-step protocol of the in vivo infection of C57BL/6 mice with L. amazonensis that comprises the parasite differentiation into axenic amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. This protocol can be adapted to other mice strains and Leishmania species that cause cutaneous leishmaniases. In conclusion, the method presented here is crucial in identifying new anti-Leishmania drug targets and vaccines, as well as in physiological studies of the host immune and metabolic responses to Leishmania infection.

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

Materials

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