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 protectiv...

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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 cm2 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>CO2 incubator 34 &#176;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 &#176;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>NaHCO3</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 ...

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 ...

This protocol is suitable for studying Leishmania virulence, because it provides a systemic view of the vertebrate host to the cutaneous infection. The main advantage of adopting this protocol is that it allows researchers to evaluate the host inflammatory response and parasite replication simultaneously. This method can be used for characterizing several targets and treatments related to virulence, which could bring new insights into cutaneous leishmaniasis control.It can also be applied to other mice strains and Leishmania species that cause cutaneous leishmaniasis. This assay has some critical steps, such as requiring trained personnels that are comfortable working with mice and have experience in performing subplantar injections to avoid accidental exposure to infectious material. Demonstrating the procedure will be Dr.Ricardo Zampieri, a technician from my laboratory.Start by growing L.amazonensis promastigotes in a 25 centimeter squared cell culture flask, containing 10 milliliters of pro-medium, for three days at 25 degrees Celsius. After the incubation, pipette five milliliters of logarithmic growth phase promastigote culture into a new flask. Add five milliliters of ama-medium to the flask, and incubate it at 34 degrees Celsius for three to four days.Split the culture by diluting it at a ratio of one to three with fresh ama-medium in a new flask. Then incubate it for another three to five days. Transfer an aliquot of the axenic amastigotes suspension diluted in PBS to a Neubauer chamber and count the non-flagellated parasites.Then, dilute the suspension in PBS according to the desired inoculum dose and the number of inoculums intended. Load a tuberculin syringe with a 27 gauge needle with the prepared parasite suspension. Ensure that the mouse is fully anesthetized with a toe pinch and inoculate 50 microliters of the suspension into the subplantar tissue on the left hind footpad.Record the progression of the lesion once a week, by measuring the thickness of the infected and noninfected footpads using a caliper, and calculate the difference in thickness between the two footpads to evaluate lesion progression. Add one milliliter of pro-medium in a glass tissue grinder tube per lesion and weigh the tube. Spray 70%ethanol on the footpad for disinfection and excise the animal's foot at its heel to extract the infected footpad.Ensure that the scissors and forceps are sterilized by keeping them soaked in 70%ethanol. Place the footpad in a sterile Petri dish and dissect it with the forceps and scalpel. Collect all soft tissue and discard the bones.Transfer the collected tissues to the glass tissue grinder tube and weigh the tube again to determine the lesion weight. Homogenize the tissue 10 times with the grinder for complete tissue disruption, and allow the mixture to settlement. Collect 20 microliters of the supernatant and load it in the first column and first lane of the 96 well plate, prepared according to the manuscript directions.Repeat this step for the next three lanes to have quadruplicate results for each animal. Homogenize each well 10 times using a multichannel pipette, and transfer 20 microliters of the diluted sample from the first to the second column. Continue the serial dilution until the last column is reached, and discard the final 20 microliters of the diluted sample.Seal the plate with film, and incubate it at 25 degrees Celsius for seven days in a humid chamber. After the incubation, analyze the plate under an inverted microscope to determine the last parasite-containing well for each lane. Leishmania protozoan parasites exist in two developmental forms during their life cycle in invertebrate and vertebrate hosts:promastigotes and amastigotes.Axenic conditions can simulate different host environments in vitro, maintaining the parasite morphology and viability. Axenic conditions for amastigotes mimic the acidic environment and increased temperature of the vertebrate hosts. Promastigotes will differentiate into amastigotes under these conditions.When transferred to neutral pH and incubated at 25 degrees Celsius, axenic amastigotes transform back to promastigotes. The virulence difference of wild type and Leishmania Iron Regulator 1 knockout strains was observed on infected mouse footpads. LIR1 regulates intracellular iron levels in Leishmania, mediating iron export and preventing its intracellular accumulation to toxic levels.The infection assay that includes the difference in swelling and lesion progression revealed that LIR1 is essential for L.amazonensis in vivo virulence. Parasite load was determined by performing a limiting dilution assay from the infected lesions. The lesions of the LIR1 knockout infected mice contain 10 to the six fold fewer parasites than those of the wild type Leishmania infected mice, revealing that the absence of LIR1 prevents intracellular replication of the amastigotes.One of the most important things to remember when you are performing this procedure is to use Leishmania cultures that were passaged in vitro less than ten times to avoid the loss of parasite virulence. In addition to this procedure, you could use in vitro infection assays. However, this is a limited method, because it does not provide a systemic physiological overview of the host-parasite interaction.This in vivo method is the best approach to evaluate systemic responses for cutaneous leishmaniasis, such as quantifying serum biomarkers in recovered host tissues for further immunological studies.

in-vivo-infection-with-leishmania-amazonensis-to-evaluate-parasite

Research

JoVE Journal

Immunology and Infection

8.0K visualizações.  University of Sao Paulo. Este protocolo é adequado para estudar a virulência de Leishmania, pois fornece uma visão sistêmica do hospedeiro vertebrado para a infecção cutânea. A principal vantagem de adotar esse protocolo é que ele permite que os pesquisadores avaliem a resposta inflamatória hospedeira e a replicação de parasitas simultaneamente. Este método pode ser usado para caracterizar vários alvos e tratamentos relacionados à virulência, o que poderia trazer novos insights sobre o controle da leis...