This study investigates the infection process of Plasmodium berghei, a malaria-causing parasite, using female mosquito salivary glands. The research outlines the method of isolating sporozoites and their subsequent injection into mice, leading to the development of cerebral malaria.
Begin with a tube containing salivary glands from female mosquitoes infected with Plasmodium berghei — a malaria-causing parasite.
Mechanically disrupt the glands, releasing the sporozoites — an infective form of the parasite into the medium.
Pellet the gland fractions and debris. Collect the sporozoite-containing supernatant and inject them into the tail vein of a restrained mouse.
The injected sporozoites travel through the bloodstream and reach the liver, where they infect hepatocytes and multiply, forming merozoites — an invasive form of the parasite.
Over time, hepatocytes release merozoites into the bloodstream, invading the red blood cells, or RBCs.
Inside the RBCs, the merozoites replicate asexually, developing into mature form and expressing parasite-derived antigens on the RBCs' surface.
These antigens bind to endothelial receptors, including those in brain blood vessels, leading to the sequestration of infected and non-infected RBCs.
This abnormal RBC accumulation disrupts the blood-brain barrier, causing fluid accumulation in the brain's parenchyma and developing cerebral malaria in the mouse.
Begin by collecting female mosquitoes from their cage 17 to 22 days after the blood meal. Use forceps to place 3 to 4 mosquitoes on a glass slide covered with a drop of cold RPMI medium. Then, place the slide under a microscope.
Carefully stretch the mosquito between the head and body with forceps, and use a syringe and needle to isolate the salivary gland. Next, collect the salivary glands from the glass slide by sucking them up with a glass pipette, and transferring them into a 1.5-milliliter centrifuge tube. Then, use a small plastic stick to smash the isolated salivary glands within the centrifuge tube for three minutes in order to isolate the sporozoites from the salivary gland tissue.
Centrifuge for three minutes at 1,000 times g in 4 degrees Celsius to purify the sporozoites from the remaining tissue. Pipette the supernatant, which contains the sporozoites, to a new centrifuge tube, and count the purified sporozoites in a Neubauer hemocytometer. Sporozoites show a typical counter-clockwise movement.
Next, adjust the concentration of the purified sporozoites to 10,000 per milliliter by adding phosphate-buffered saline. Finally, place a C57BL6 mouse in a restrainer, and put its tail into warm water to better visualize the tail vein. Then, inject a total of 1,000 sporozoites, or 0.1 milliliters, into the tail vein to initiate infection.
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