Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Due to the striking similarities of the lifecycle and biology of rodent malaria parasites, the human malaria parasites, rodent malaria models have become indispensable for malaria research. These standard methods can help answer key questions in the malaria research field about how to perform a phenotypic analysis of wild type and transgenic rodent malaria species. Demonstrating the procedures will be graduate students Gozde Deveci and Ilknur Yilmaz from my laboratory.

Begin by quickly thawing cryo preserved frozen parasite stocks and immediately using a one milliliter syringe equipped with a 26 gauge needle to inject at least two donor mice per genotype intraperitoneally with approximately 200 microliters of stock. Twenty-four hours after the injection, use a 24 or 26 gauge needle to prick the tail of the first mouse and collect the blood droplet on a microscope slide. Use the short edge of another slide to quickly smear the blood across the first slide.

When the blood has completely dried, fix the slides in 100%methanol for at least one minute. Stain the fixed samples in Giemsa for 10 to 15 minutes followed by a single or double distilled water wash. When the slides have dried, view the samples under a 100X objective and oil immersion on a light microscope for counting of the total number of infected erythrocytes per grid.

Two to three days after the infection, secure an anesthetized mouse in which the parasitemia has reached between 0.1 to 1%in the supine position and use forceps to lift the skin near the base of the sternum to facilitate the creation of a skin incision. Pull the skin apart at the site of the incision to expose the diaphragm and the top of the abdominal cavity and use the forceps to hold the base of the ribcage while cutting through the diaphragm to enter the thoracic cavity. Pin back the ribcage to expose the heart and use a one milliliter syringe containing at least 20 microliters of heparin and equipped with a 26 gauge needle to gently the puncture the heart tissue.

Slowly retract the plunger to collect approximately one milliliter of blood and dispense the blood into a microcentrifuge tube. When all of the blood has been harvested, warm the recipient mice under a red heat lamp and load one insulin syringe equipped with a 27 gauge needle per recipient with the appropriate dose of parasite. Then place the first recipient into a restrainer and inject one full dose of infected erythrocytes into a dilated tail vein.

For mosquito feeding, allow adult four to seven-day-old female Anopheles stephensi or A.gambiae mosquitoes starved for eight to 12 hours to feed more at least 15 minutes on anesthetized infected mice with the highest exflagellation rate. To determine the number of oocyst sporozoites per mosquito, euthanize 20 to 30 mosquitoes in the freezer for not less than 10 minutes and use a binocular dissection scope and two 26 or 27 gauge needles to dissect the midguts of mosquitoes on a glass slide containing the appropriate dissection solution. To dissect the midgut, first hold the lower part of the abdomen in place with one needle and carefully push the thorax very lightly at the junction between the abdomen and thorax in an upward direction until it separates the thorax and a white colored midgut is exposed dangling down from the thorax.

Now cut the midgut from the esophagus end using the same needle that was used to push the thorax upward thus separating and spreading out the midgut. Raise the dissected midgut from the dissection medium using a needle and transfer it to a tube filled with 200 microliters of RPMI. When all midguts have been dissected and harvested, place the tube containing the midguts into the centrifuge for one minute at 700 times g.

Then grind the harvested tissues with a pestle two times. Transfer 12 microliters of the diluted tissue solution to a hemacytometer and incubate the hemacytometer at room temperature for five minutes to allow the contents to settle. Then determine the average number of oocyst sporozoites on a light microscope under phase contrast and a 40X magnification.

To determine the number of salivary gland sporozoites per mosquito, at the appropriate time point post feeding, freeze euthanized 50 to 100 female mosquitoes as demonstrated and use a dissecting microscope and the side of a 26 or 27 gauge needle to isolate the salivary glands. To dissect salivary glands, first hold the upper abdomen or thorax in place with the beveled side of one needle and carefully push the head very lightly and very short pushes at the junction between the head and thorax in an upward direction. Push until the head is carefully separated from the thorax without tearing up the glassy salivary gland.

Use a short glass Pasteur pipette to raise the dissected salivary gland from the dissection medium and place it into a 1.5 milliliter tube. When all salivary glands have been dissected and harvested, place the tube containing the glands into the centrifuge for one minute at 700 times g. Grind the harvested tissues with a pestle two times.

Then determine the average number of salivary gland sporozoites as previously done. Transgenic reporter malaria parasites engineered to express EGFP under the control of a strong and constitutive promoter exhibit the fluorescence signal in blood stages, ookinetes, young oocysts on Anopheles stephensi midguts, and in sporozoites isolated from the salivary glands of Anopheles stephensi females. Flow cytometry parasitemia percentage values correspond directly to the estimated parasitemia percentages obtained by monitoring Giemsa stained thin blood smears as demonstrated confirming the validity of the latter quantification method.

In addition, estimation of the percentages of each of the different asexual and sexual stages depends on the morphological evaluation of the erythrocytes which cannot be assessed by flow cytometry. Comparison of the intraperitoneal versus intravenous routes of infection reveals a statistically significant decrease in the IP infected group parasitemia percentage compared to the IV infected group parasitemia percentage during the first four days of infection demonstrating that the IV route of infection is a more quantitatively accurate route for assays with the malaria parasite blood stages. Of note, phenylhydrazine treatment significantly increases the number of gametocytes which increases the rate of male gamete formation which in turn increases the fertilization rate and the number of all subsequent mosquito stages.

There is a scarcity of standardized methods for the phenotypical analysis of rodent malaria blood stage parasites and their transmission to the mosquito vector. These methods will help to provide standardized and simplified protocols for studying these pathogens and their lifecycle stages.