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 protective equipment should be utilized while handling live Leishmania parasites.

1. In Vitro Differentiation of L. amazonensis Promastigotes into Axenic Amastigotes^{15,20,35,36,37,38}

NOTE: L. amazonensis (MHOM/BR/1973/M2269) (La) parasite was used in this assay. Depending on the study purpose, the infective parasite form can be obtained either by purification of the metacyclic form using a density gradient, as previously described¹⁴, or by differentiation of promastigotes into axenic amastigotes, according to the following protocol.

1. Grow La promastigotes in a 25 cm² cell culture flask containing 10 mL of medium for promastigotes (pro-medium) (pH = 7.0).
2. Incubate at 25 °C for 3 days.
   NOTE: Use promastigote cultures that were passaged in vitro less than 10x to avoid the loss of virulence and aneuploidy changes of parasites^{39,40,41,42,43,44}.
3. Pipette 5 mL of logarithmic growth phase promastigote culture into a new 25 cm² flask.
4. Add 5 mL of medium for axenic amastigotes (ama-medium) (pH = 5.2).
5. Incubate at 34 °C for 3-4 days.
6. Split the culture by diluting with ama-medium at a ratio of 1:3 into a new 25 cm² flask.
7. Incubate at 34 °C for 3-5 days.
   NOTE: Incubate the axenic amastigotes for up to 5 days, as it represents the end of maturation³⁷.

2. C57BL/6 Footpad Infection with L. amazonensis

NOTE: Female C57BL/6 mice (6-8 weeks old) were obtained and maintained at the Animal Center of the Biomedical Sciences Institute of the University of São Paulo. Animals received food and water ad libitum.

1. Count the culture of axenic amastigotes by transferring an aliquot of the parasite suspension diluted in PBS to a Neubauer chamber (i.e., hemocytometer). Count the non-flagellated parasites, which represent amastigotes forms.
   NOTE: Alternatively, Trypan blue (1:1) can be used for counting the number of viable amastigotes (i.e., those not stained with Trypan blue).
2. Dilute the axenic amastigotes in PBS according to the desired inoculum dose and the number of inoculums intended (1 x 10⁶ axenic amastigotes in 50 µL of PBS is recommended).
3. Load a tuberculin syringe with a 27 G needle with the prepared parasite suspension.
4. Anesthetize a C57BL/6 mouse using 3-5% isoflurane, as recommended by Johns Hopkins University Animal Care and Use Committee⁴⁵. Assess the anesthetic depth by testing the mouse's response to a toe pinch.
5. Inoculate 50 µL of the homogenized parasite suspension (1 x 10⁶ axenic amastigotes or the desired inoculum dose) in the subplantar tissue of the left hind footpad, using the previously loaded tuberculin syringe (step 2.3).

3. Mouse Footpad Lesion Development

1. Measure the progression of the lesion once a week by measuring the thickness of the left (infected) and the right (noninfected) footpads using a caliper.
2. Calculate the difference of the thickness between the left and right hind footpads weekly to evaluate lesion progression.
3. Plot the calculated differences on the Y-axis and time of infection on the X-axis and calculate the statistical significance.
   NOTE: In accordance with the Animal Care and Use Committees' recommendations, animals must be sacrificed before the infected lesion becomes ulcerated, because skin ulcers can lead to secondary infection. As shown in Supplementary Figure 1, it takes approximately 10 weeks for the lesions to present signs of ulceration with the parasite dose (10⁶ axenic amastigotes) and host strain (C57BL/6) used in this protocol. The mice should be sacrificed for lesion extraction before the signs of ulceration are observed.

4. Mice Footpad Lesion Extraction and Parasite Limiting Dilution

1. Prepare a 96 well plate (flat bottom) for the limiting dilution assay^46,47 by adding 180 µL of pro-medium to all wells.
   NOTE: Use four plate lanes (half of the plate) for each animal, representing quadruplicate assays (i.e., animal 1 = lanes A-D; animal 2 = lanes E-H).
2. Add 1 mL of pro-medium in a glass tissue grinder tube per lesion and weigh the tube.
   NOTE: The tube must be kept sterile, so use a sterile lid and weigh the tube with the closed lid, if weighing outside the laminar flow cabinet.
3. Sacrifice the animal in a CO₂ chamber, following the Animal Care and Use Committees' recommendations. Disinfect the animal by spraying with 70% ethanol.
4. Excise the animal's foot at it heels to extract the infected footpad and spray 70% ethanol on the footpad for disinfection. Ensure the sterilization of scissors and forceps by keeping them soaked in 70% ethanol.
5. Place the infected footpad in a sterile Petri dish and dissect using sterile forceps and a scalpel to collect all soft tissues. Discard the bones.
   NOTE: Uniformity in the dissection step is recommended to avoid biased tissue recovery.
6. Transfer the collected tissues to the glass tissue grinder tube with pro-medium, then weigh the tube again to determine the lesion weight.
   NOTE: Avoid placing the forceps and scalpel directly into the medium, as small volumes may be removed, resulting in an underestimated tissue weight. The weight of the lesion is calculated by subtracting the weight of the tube containing pro-medium with the collected tissue from the weight of tube containing only pro-medium.
7. Homogenize the tissue 10x using the grinder for complete tissue disruption.
8. Allow the mixture to sediment for 10 min, then collect 20 µL of the supernatant.
9. Load 20 µL of the supernatant in the 1^st column of the 1^st lane of the 96 well plate prepared in step 4.1. Repeat this for the next three lanes to have quadruplicates for each animal (i.e., for animal 1 add 20 µL to each well and label A1, B1, C1, and D1).
10. Homogenize each well in the 1^st column 10x using a multichannel pipette. Then, transfer 20 µL of the diluted samples from the 1^st column to the 2^nd column.
    NOTE: It is important to homogenize each well 10x from one column to the next.
11. Repeat step 4.10 to all remaining columns until the last column (12^th) and discard the final 20 µL of diluted sample.
    NOTE: Change the tips after each dilution to avoid cross-contamination.
12. Seal the plates with a film and incubate at 25 °C for 7 days in a humid chamber.

5. Lesion Parasite Load Determination

1. Analyze the plates after 7 days of incubation using an inverted microscope to determine the last parasite-containing well for each lane.
   NOTE: It is also possible to have an indication of the growth of the parasites, or cell density, by visual analysis of the color changes and turbidity of the medium.
2. Calculate the parasite tissue load for each lane by dividing the dilution factor per lesion weight.
   NOTE: In a specific lane, if parasites are present only in columns 1-5 (i.e., wells A1-A5 are positive for parasite growth), this means that column 5 contained only 1 parasite, column 4 contained 10 parasites, and so on, in multiples of ten. Column 1 would contain 10⁴ parasites, which represents the number of parasites in the initial 20 µL of supernatant (the non-diluted sample in step 4.7). Because this 20 µL was diluted with 180 µL of pro-medium, the initial tube contained 5 x 10⁵ parasites: (1 x 10⁴) / (0.02 mL) = 5 x 10⁵ parasites/1 mL. Then, the parasite load in that lesion can be calculated by dividing the initial concentration of the parasite by the lesion weight: (5 x 10⁵) / lesion weight (see step 4.6).
3. Plot the average of the quadruplicated result for each animal with a log Y-scale.

6. Statistical Analysis

1. Represent the data as average ± standard deviation using at least five animals per experimental group as replicates.
2. Perform statistical analysis using unpaired two-tailed test, considering a p value < 0.05 as significant.

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 have a rounded or ovoid body ranging in size from 2-6 µm in length and 1.5-3 µm in width, and possess an inapparent flagellum^12,13 (Figure 1A). During the blood meal the invertebrate host, a hematophagous insect of the family Psychodidae, acquires macrophages infected with Leishmania amastigotes. Once these cells reach the sandfly digestive tube, amastigotes are released and differentiate to procyclic promastigotes (Figure 1A). These forms are noninfective and multiply intensively by binary division and colonize the digestive tube of the insect vector. The procyclic forms then differentiate to metacyclic forms, an infective and fast-moving form, presenting a thinner body and elongated flagellum (Figure 1A). The metacyclic forms invade the anterior portions of the esophagus and proventriculus of the sandfly, so that during its next blood meal, regurgitation ensures the inoculation of these infecting forms into a new vertebrate host. In the tegument of the vertebrate host, the parasites are phagocytosed by the macrophages and differentiate into amastigotes inside the parasitophorous vacuoles, where the amastigotes multiply by binary division and complete the life cycle of Leishmania^12,13.

Axenic conditions can simulate different host environments in vitro, maintaining the parasite morphology and viability. Axenic conditions for amastigotes were previously described simulating a macrophage's parasitophorous vacuole environment and triggering promastigote in vitro differentiation into the amastigote form³⁷. These conditions mimic the acidic environment (pH = 5.5) and the increased temperature of the vertebrate hosts (34 °C). Figure 1B illustrates promastigotes differentiated to amastigotes by changing these conditions in culture. The viability of these axenic amastigotes can be analyzed by Trypan blue staining, a method based on the principle that live cells possess intact membranes that exclude certain dyes, whereas dead cells do not⁴⁸. Alternatively, we analyzed the viability of axenic amastigotes verifying their ability to transform back to promastigotes when transferred to neutral pH and incubated at 25 °C (Figure 1B).

Here we propose an in vivo infection method to evaluate the virulence of different Leishmania strains. Figure 2A represents an in vivo infection assay showing the cutaneous lesion development of C57BL/6 mice footpads that were infected with wild type (La-WT) and Leishmania Iron Regulator 1 knockout (La-LIR1^-/-) L. amazonensis purified metacyclics. LIR1 regulates intracellular iron levels in Leishmania mediating iron export and preventing its intracellular accumulation to toxic levels¹⁴. Observing the progression of the thickness differences of the infected vs. noninfected footpads, we were able to demonstrate that the La-LIR1^-/- infected mice presented smaller lesions than La-WT infected mice (Figure 2A). Those findings revealed that LIR1 is essential for L. amazonensis in vivo virulence. This demonstrates the importance and efficacy of this method in assessing the differences of Leishmania-driven cutaneous diseases. Figure 2B illustrates the noninfected (right) and infected (left) footpads and lesion development 73 days postinfection, showing the differences in swelling and lesion progression of La-WT and La-LIR1^-/- infected mice.

The progression of the Leishmania infection consists not only of the lesion development, which represents the inflammatory response, but also the parasite's intracellular replication. To evaluate parasite replication, the parasite load of the lesions was determined by extracting the infected lesion, followed by a limiting dilution assay in a 96 well plate (Figure 3A). Figure 3B shows the parasite load analysis of the footpad lesions from La-WT and La-LIR1^-/- infected mice after 73 days of infection. From the limiting dilution assay, we detected 10⁶-fold fewer parasites in the lesion of the La-LIR1^-/- infected mice in comparison to La-WT, revealing that absence of LIR1 prevents intracellular replication of the amastigotes¹⁴.

One of the advantages of evaluating both lesion development and parasite load is to detect possible differences of parasite intracellular replication and the host inflammatory response. We observed differences between these two phenotypes using the add-back LIR1 (La-LIR1^AB), which is the La-LIR1^-/- with the LIR1 ORF integrated back into the ribosomal locus¹⁴. When La-LIR1^AB was injected into a mouse's footpad, we observed intermediate-sized lesions compared to La-LIR1^-/- and La-WT infections but a remarkable full parasite load rescue of the La-WT phenotype (Supplementary Figure 2). These results indicate that La-LIR1^AB parasites were able to replicate like La-WT parasites in a long-term in vivo infection. However, the mouse inflammatory response was not as exacerbated as La-WT infections because the lesions were significantly smaller.

Thereby, the method described here was shown to be essential for the identification and characterization of a L. amazonensis virulence factor required for the successful amastigote intracellular replication and cutaneous lesion development in the mammalian hosts.

Figure 1: Morphology of L. amazonensis promastigotes and amastigotes. (A) Illustrations of the different morphological forms of Leishmania: procyclic promastigote, metacyclic promastigote, and amastigote. Scale bar = 2 μm. (B) Pictures of in vitro L. amazonensis cultures. In vitro differentiation of promastigotes into axenic amastigotes, and of axenic amastigotes back to promastigotes by changing the pH and temperature conditions, as described in the step-by-step protocol. The pictures were taken using an inverted microscope. Scale bar = 50 μm. Please click here to view a larger version of this figure.

Figure 2: LIR1 knockout markedly reduces L. amazonensis in vivo lesion development. C57BL/6 mice were inoculated in the left hind footpad with 10⁶ purified metacyclics of L. amazonensis wild type (La-WT) and L. amazonensis LIR1 knockout (La-LIR1^-/-). (A) Footpad cutaneous lesion progression of La-WT and La-LIR1^-/- infected mice analyzed weekly. The data represent the average ± SEM of the infected footpad subtracted by noninfected footpad thickness from five different mice in each group (adapted from Laranjeira-Silva et al.¹⁴). (B) Pictures of the noninfected and infected footpads of La-WT and La-LIR1^-/- infected miceshowing the differences in swelling 73 days postinfection (adapted from Laranjeira-Silva et al.¹⁴). Please click here to view a larger version of this figure.

Figure 3: LIR1 knockout markedly reduces L. amazonensis in vivo intracellular replication. (A) An illustration of a 96 well plate representing the 10x serial dilutions of the recovered footpad tissues infected with L. amazonensis wild type (La-WT) and LIR1 knockout (La-LIR1^-/-). Rows A-D represent the quadruplicate of the serial dilutions of the La-WT-footpad lesion sample. Rows E-H represent the quadruplicate of the serial dilutions of the La-LIR1^-/--footpad lesion sample. The different shades of gray represent the observed cell densities per well (i.e., lighter color means fewer parasites). The wells marked in red represent the last wells that contained parasites per replicate. (B) Parasite load in recovered footpad tissues of C57BL/6 mice infected with 10⁶ purified metacyclics of L. amazonensis wild type (La-WT) and L. amazonensis LIR1 knockout (La-LIR1^-/-) determined 73 days postinfection. The data represent the average of the parasite load per mg of tissue from five different mice in each group (adapted from Laranjeira-Silva et al.¹⁴). Please click here to view a larger version of this figure.

Supplementary Figure 1: Skin ulcer as indicative of a secondary infection. Representative pictures of C57BL/6 mice footpads infected with L. amazonensis wild type (La-WT) taken 80 days postinfection. Red arrows point to signs of ulceration, indicating that the experiment should be terminated. Please click here to view a larger version of this figure.

Supplementary Figure 2: The role of LIR1 on in vivo lesion development and intracellular parasite replication. C57BL/6 mice were inoculated in the left hind footpad with 10⁶ purified metacyclics of L. amazonensis wild type (La-WT), L. amazonensis LIR1 knockout (La-LIR1^-/-), and L. amazonensis LIR1 add-back (La-LIR1^AB). (A) Footpad cutaneous lesion progression of La-WT, La-LIR1^-/-, and La-LIR1^AB infected mice analyzed weekly. The data represent the average ± SEM of the infected footpad subtracted by noninfected footpad thickness from five different mice in each group (adapted from Laranjeira-Silva et al.¹⁴). (B) Parasite load in recovered footpad tissues from La-WT, La-LIR1^-/-, or La-LIR1^AB infected mice determined 73 days postinfection. The data represent the average of the parasite load per mg of tissue from five different mice in each group (adapted from Laranjeira-Silva et al.¹⁴). Please click here to view a larger version of this figure.

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 here we compiled a step-by-step protocol to standardize this method while considering the infrastructure limitations that some groups may have. This protocol can also be used to evaluate virulence of transgenic Leishmania parasites by in vivo bioimaging^50,51,52. As any other experimental procedure, this assay has limitations and critical steps to execute, such as requiring trained personnel that are comfortable working with mice and have experience in performing subplantar injections to avoid accidental infections. Standardizing protocols is extremely important to avoid biased results and to produce comparable results among different research groups.

The main advantage of using L. amazonensis as a model for cutaneous leishmaniases is because the footpad lesion caused by this species can be easily assessed in mice. The swelling of the footpad determined by the method described here represents the sum of two infection phenotypes: host inflammatory response and parasite replication. Both phenotypes can be evaluated separately by associating the parasite tissue load method that reflects parasite intracellular replication with the determination of the lesion thickness progression. Another advantage is that L. amazonensis' promastigotes are easily cultivated in vitro. Considering these, any research group can manipulate this Leishmania species according to their needs. The findings from L. amazonensis' studies may be then compared with other Leishmania species to determine whether a specific pathway is evolutionarily conserved or divergent^21,53,54.

In the natural cycle, Leishmania transmission to the vertebrate host occurs by the bite of an infected sand fly during the blood meal. The sand fly usually inoculates a few hundred Leishmania metacyclic promastigote forms in the insect's saliva. In experimental in vivo infections, the most frequent site of inoculation is the animal's footpad²². Intradermal injection into the ear or intraperitoneal injection are alternative sites of inoculation depending on the study purpose because each site presents different phagocytic cell types^24,56. Therefore, some research groups use laboratory-infected sand flies to infect the animal's ear dermis to mimic the natural transmission^56,57,58,59. However, this protocol presents some restrictions, such as the maintenance of sand fly colonies, which requires facilities not available for most research groups.

The original work describing in vivo C57BL/6 infection with La-LIR1^-/- has used purified metacyclic promastigotes forms¹⁴. However, Leishmania genetic manipulation can impair either the promastigotes' differentiation into axenic amastigotes¹⁴ or into metacyclic infective forms²⁰. Hence, depending on the Leishmania strain, the researcher should determine the most adequate method to obtain viable infective parasite forms for their study. The protocol of axenic amastigotes differentiated from promastigote cultures described here can be an easier alternative, producing comparable results in many cases^{19,20,35,37,38,64}. This approach avoids the use of other methods that typically result in lower yields of infective parasites, such as incubating promastigotes with specific but not widely available antibodies⁶⁵ for metacyclic promastigotes purification, or by density gradient dependent of metacyclics' LPG expression^18,66. The efficiency of the differentiation protocol can be evaluated by determining the expression levels of amastin-family genes^64,67. Amastins are members of a conserved gene family that are differentially modulated during the Leishmania life cycle⁶⁸ and are associated with parasite virulence and pathogenesis^67,69,70. Other markers can also be used to distinguish amastigote from promastigote forms. For example, gp63 is downregulated in amastigotes, because its role is to protect the promastigotes from the insect's digestive enzymes⁷¹.

The choice of the mouse strain is another critical step to be considered when developing a standardized in vivo infection protocol. Susceptibility and resistance to Leishmania infection in mice are mainly regulated by genetic background^29,30,55. In this protocol, the C57BL/6 strain was chosen because its immune response to L. amazonensis is closely related to the mixed Th1-Th2 response in humans^72,73. Experimental murine infections with L. amazonensis have been described to cause moderate lesions in C57BL/6 mice in comparison to other mice strains^28,34,74. However, depending on the parasite strain, differences in the lesion size are only detectable in susceptible mice strains, like BALB/c³⁶. The time course of infection also needs to be considered and correlates with the chosen mice strain^{29,26,60,61,62,63,75}. Ulcerated footpads should always be avoided as it may represent secondary infections and are often observed at long periods of infection, especially in experiments with susceptible mice strains. Designating the time of the day to start the infection is another step to be considered. As demonstrated in previous studies with L. amazonensis, the time of the day of parasite inoculum affects lesion development because the host-parasite interaction is affected in a circadian manner by the pineal-released melatonin during the dark time of day⁷⁵.

The major disadvantage of the in vivo infection method is that the experiment requires the use of a substantial number of laboratory animals and takes longer time to obtain final results compared to the in vitro infection method¹⁸. However, this latter aspect can be also considered as an advantage since the in vivo results reflect the natural time course of disease progression more accurately than the results obtained from in vitro infection. More importantly, the findings from L. amazonensis in vivo infection cannot only reflect the transient changes in parasite virulence but also acknowledges the systemic status of the host and all its players. Therefore, considering the several factors mentioned above, the method described in this protocol can be adapted to meet specific experimental needs for characterization of other targets and treatments related to virulence allowing new insights for cutaneous leishmaniasis control.

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