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.
