We would like to thank Prof. Dr. Niels Olsen Saraiva C&#226;mara from the Animal Center of the Biomedical Sciences Institute of the University of S&#227;o 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&#39; grant 2017/23933-3).

All experimental procedures were approved by the Animal Care and Use Committee at the Institute of Bioscience of the University of S&#227;o Paulo (CEUA 342/2019), and were conducted in accordance with the recommendations and the policies for the Care and Use of Laboratory Animals of S&#227;o 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 Amastigotes15,20,35,36,37,38
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.
<ol>
	<li>Grow La promastigotes in a 25 cm2 cell culture flask containing 10 mL of medium for promastigotes (pro-medium) (pH = 7.0).</li>
	<li>Incubate at 25 &#176;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 parasites39,40,41,42,43,44.</li>
	<li>Pipette 5 mL of logarithmic growth phase promastigote culture into a new 25 cm2 flask.</li>
	<li>Add 5 mL of medium for axenic amastigotes (ama-medium) (pH = 5.2).</li>
	<li>Incubate at 34 &#176;C for 3-4 days.</li>
	<li>Split the culture by diluting with ama-medium at a ratio of 1:3 into a new 25 cm2 flask.</li>
	<li>Incubate at 34 &#176;C for 3-5 days. 
	NOTE: Incubate the axenic amastigotes for up to 5 days, as it represents the end of maturation37.</li>
</ol>
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 S&#227;o Paulo. Animals received food and water ad libitum.
<ol>
	<li>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).</li>
	<li>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 &#181;L of PBS is recommended).</li>
	<li>Load a tuberculin syringe with a 27 G needle with the prepared parasite suspension.</li>
	<li>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&#39;s response to a toe pinch.</li>
	<li>Inoculate 50 &#181;L 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).</li>
</ol>
3. Mouse Footpad Lesion Development
<ol>
	<li>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.</li>
	<li>Calculate the difference of the thickness between the left and right hind footpads weekly to evaluate lesion progression.</li>
	<li>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&#39; 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.</li>
</ol>
4. Mice Footpad Lesion Extraction and Parasite Limiting Dilution
<ol>
	<li>Prepare a 96 well plate (flat bottom) for the limiting dilution assay46,47 by adding 180 &#181;L 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).</li>
	<li>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.</li>
	<li>Sacrifice the animal in a CO2 chamber, following the Animal Care and Use Committees&#39; recommendations. Disinfect the animal by spraying with 70% ethanol.</li>
	<li>Excise the animal&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Homogenize the tissue 10x using the grinder for complete tissue disruption.</li>
	<li>Allow the mixture to sediment for 10 min, then collect 20 &#181;L of the supernatant.</li>
	<li>Load 20 &#181;L of the supernatant in the 1st column of the 1st 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 &#181;L to each well and label A1, B1, C1, and D1).</li>
	<li>Homogenize each well in the 1st column 10x using a multichannel pipette. Then, transfer 20 &#181;L of the diluted samples from the 1st column to the 2nd column. 
	NOTE: It is important to homogenize each well 10x from one column to the next.</li>
	<li>Repeat step 4.10 to all remaining columns until the last column (12th) and discard the final 20 &#181;L of diluted sample. 
	NOTE: Change the tips after each dilution to avoid cross-contamination.</li>
	<li>Seal the plates with a film and incubate at 25 &#176;C for 7 days in a humid chamber.</li>
</ol>
5. Lesion Parasite Load Determination
<ol>
	<li>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.</li>
	<li>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 &#181;L of supernatant (the non-diluted sample in step 4.7). Because this 20 &#181;L was diluted with 180 &#181;L 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).</li>
	<li>Plot the average of the quadruplicated result for each animal with a log Y-scale.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Represent the data as average &#177; standard deviation using at least five animals per experimental group as replicates.</li>
	<li>Perform statistical analysis using unpaired two-tailed test, considering a p value &#60; 0.05 as significant.</li>
</ol>

The authors declare they have no competing financial interests.

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 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 bioimaging50,51,52. 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&#39; 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&#39; studies may be then compared with other Leishmania species to determine whether a specific pathway is evolutionarily conserved or divergent21,53,54.
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&#39;s saliva. In experimental in vivo infections, the most frequent site of inoculation is the animal&#39;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 types24,56. Therefore, some research groups use laboratory-infected sand flies to infect the animal&#39;s ear dermis to mimic the natural transmission56,57,58,59. 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&#39; 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 cases19,20,35,37,38,64. 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&#39; LPG expression18,66. The efficiency of the differentiation protocol can be evaluated by determining the expression levels of amastin-family genes64,67. Amastins are members of a conserved gene family that are differentially modulated during the Leishmania life cycle68 and are associated with parasite virulence and pathogenesis67,69,70. 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&#39;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 background29,30,55. 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 humans72,73. Experimental murine infections with L. amazonensis have been described to cause moderate lesions in C57BL/6 mice in comparison to other mice strains28,34,74. 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 strain29,26,60,61,62,63,75. 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.

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 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 leishmaniasis4,5. There is no available vaccine due to the diversity among Leishmania species and host genetic backgrounds6,7. Treatment options are also limited as most of the currently available drugs are either expensive, toxic, and/or may require long-term treatment8,9. Besides, there have been reports of drug resistance against the available treatments10,11.
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 macrophages12,13. Amastigotes&#39; ability to invade, survive, and replicate despite the defense mechanisms of the vertebrate host&#39;s macrophages are subject to many studies14,15,16,17. 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 studies14,19,20,21. 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 outcome24,25,26,27. Furthermore, susceptibility and resistance to the infection in humans and mice are highly regulated by the genetic backgrounds of the host and parasite4,5,22,28,29,30,31. BALB/c mice are highly susceptible to L. amazonensis cutaneous infection, showing a rapid disease progression with the parasites&#39; 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 model5,34.
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.

<table><tbody><tr><td>96-well plate</td><td>Greiner bio-ne</td><td>655180</td><td>A flat-bottom plate for limiting dilution assay</td></tr><tr><td>adenine</td><td>Sigma</td><td>A8626</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>caliper</td><td>Mitutoyo</td><td>700-118-20</td><td>A caliper to measure the thickness of footpad</td></tr><tr><td>cell culture flask</td><td>Corning</td><td>353014</td><td>A 25 cm&lt;sup&gt;2&lt;/sup&gt; volume cell culture flask to cultivate Leishmania parasite</td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5804R</td><td>An equipament used for separating samples based on its density</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator 34 &amp;deg;C</td><td>Thermo Scientific</td><td>3110</td><td>An incubator for amastigotes differentiation</td></tr><tr><td>ethanol</td><td>Merck</td><td>K50237083820</td><td>A disinfectant for general items</td></tr><tr><td>fetal bovine serum</td><td>Gibco</td><td>12657-029</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>glass tissue grinder tube</td><td>Thomas Scientific</td><td>3431 E04</td><td>A tube to collect and disrupt infected footpad tissue</td></tr><tr><td>glucose</td><td>Synth</td><td>G1008.01.AH</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>GraphPad Prism Software</td><td>GraphPad</td><td></td><td>A software used to plot the data and calculate statistical significance</td></tr><tr><td>hemin</td><td>Sigma</td><td>H-2250</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>HEPES</td><td>Promega</td><td>H5303</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>incubator 25 &amp;deg;C</td><td>Fanem</td><td>347CD</td><td>An incubator for promastigotes cultivation</td></tr><tr><td>inverted microscope</td><td>Nikon</td><td>TMS</td><td>An equipament used to visual analyze the promastigote and amastigote cultures</td></tr><tr><td>isoflurane</td><td></td><td></td><td>An inhalant anesthetics for mice (3-5%)</td></tr><tr><td>laminar flow cabinet</td><td>Veco</td><td>VLFS-09</td><td>A biosafety cabinet used for aseptical work area</td></tr><tr><td>M199 cell culture media</td><td>Gibco</td><td>31100-035</td><td>A cell culture media for Leishmania cultivation</td></tr><tr><td>microcentrifuge tube</td><td>Axygen</td><td>MCT150C</td><td>A microtube used for sample collection, processing and storage</td></tr><tr><td>multichanel pipette</td><td>Labsystems</td><td>F61978</td><td>A multichannel pipette used for limiting dilution assay</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>6329</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>NaOH</td><td>Sigma</td><td>S8045</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>Neubauer chamber</td><td>HBG</td><td>2266</td><td>A hemocytometer to count the parasite suspension</td></tr><tr><td>optical microscope</td><td>Nikon</td><td>E200</td><td>An optical equipament used to count parasite</td></tr><tr><td>parafilm</td><td>Bemis</td><td>349</td><td>A flexible and resistant plastic to seal the plate</td></tr><tr><td>penicillin/streptomycin</td><td>Gibco</td><td>15140122</td><td>Supplement added to M199 cell culture media</td></tr><tr><td>Petri dishes</td><td>TPP</td><td>93100</td><td>A sterile dish to dissect the footpad tissue</td></tr><tr><td>pipetman kit</td><td>Gilson</td><td>F167360</td><td>A micropipette kit containing four pipettors (P2 P20 P200 P1000)</td></tr><tr><td>scale</td><td>Quimis</td><td>BG2000</td><td>An equipament used to weigh collected footpad lesions</td></tr><tr><td>scalpel</td><td>Solidor</td><td>10237580026</td><td>A scalpel to cut and collect footpad tissue</td></tr><tr><td>serological pipette 10 mL</td><td>Nest</td><td>327001</td><td>A sterile pipette used for transfering mililiter volumes</td></tr><tr><td>tips</td><td>Axygen</td><td></td><td>A pipette tip used for transfering microliter volumes</td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250-061</td><td>A dye used to count viable parasites</td></tr><tr><td>trypticase peptone</td><td>Merck</td><td></td><td>Supplement added to M199 cell culture media</td></tr><tr><td>tuberculin syringe</td><td>BD</td><td>305945</td><td>A syringe with 27G needle to inoculate the parasite suspension</td></tr></tbody></table>

in vivo infection with leishmania amazonensis to evaluate parasite virulence in mice

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&#39;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.

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 &#181;m wide and 20 &#181;m long, with a flagellum typically emerging from the anterior extremity. Amastigotes have a rounded or ovoid body ranging in size from 2-6 &#181;m in length and 1.5-3 &#181;m in width, and possess an inapparent flagellum12,13 (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 Leishmania12,13.
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&#39;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 &#176;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 &#176;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&#39;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-LIR1AB), which is the La-LIR1-/- with the LIR1 ORF integrated back into the ribosomal locus14. When La-LIR1AB was injected into a mouse&#39;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-LIR1AB 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60617/60617fig1.jpg" /> 
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 &#956;m. (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 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60617/60617fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60617/60617fig2.jpg" /> 
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 &#177; 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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60617/60617fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60617/60617fig3.jpg" /> 
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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60617/60617fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/60617/60617suppfig1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60617/60617suppfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/60617/60617suppfig2.jpg" /> 
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-LIR1AB). (A) Footpad cutaneous lesion progression of La-WT, La-LIR1-/-, and La-LIR1AB infected mice analyzed weekly. The data represent the average &#177; 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-LIR1AB 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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60617/60617suppfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice at JoVE.com

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

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.

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

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.2K Views.  University of Sao Paulo. Dit protocol is geschikt voor het bestuderen van Leishmania virulentie, omdat het een systemisch beeld geeft van de gewervelde gastheer van de cutane infectie. Het belangrijkste voordeel van de vaststelling van dit protocol is dat het onderzoekers in staat stelt om de host ontstekingsreactie en parasiet replicatie gelijktijdig te evalueren. Deze methode kan worden gebruikt voor het karakteriseren van verschillende doelen en behandelingen met betrekking tot virulentie, die nieuwe inzichten in ...

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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

Biology

Assessment of Leishmania Virulence within Cultured Macrophages

Source: Sarkar, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of&#160;Leishmania&#160;Parasites.</a>&#160;J. Vis. Exp.&#160;(2018)
This video investigates Leishmania&#39;s virulence by treating macrophages with metacyclic promastigotes, resulting in the formation of intracellular amastigotes. The confirmation of Leishmania&#39;s virulence is achieved through nucleic acid fluorescent dye staining. This insight can contribute to the development of new therapies for leishmaniasis.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

Source: Sarkar, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the macrophage and parasite nuclei.

1. Plating BMMs on Coverslips for Infection 

	Prepare 6-well tissue culture plates by placing four (4) sterile 12 mm glass coverslips (autoclaved and ...

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

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