Infection rates after exposure to the bite of one to eight <i>P. yoelii</i>-infected mosquitoes.

<p>BALB/c mice were exposed to one, two, four, or five to eight bites by <i>A. stephensi</i> mosquitoes infected with <i>P. yoelii</i> sporozoites. 7 and 14 days following bites, mice were assessed for parasitemia by blood smear. Data from individual mice from five separate experiments (experiments 1–5) are shown. A single strain of <i>A. stephensi</i> (NIJ.SAN01) was used in experiments 1–4 and a second strain (SXK.SAN02) in experiment 5.</p

Sporozoite density in infected mosquitoes.

<p>The numbers of sporozoites was determined in salivary glands of mosquitoes from the same container as the mosquitoes used in the experiments. Salivary glands from the indicated number of mosquitoes were pooled, sporozoites were isolated from the salivary glands, the total numbers of sporozoites were determined, and the mean numbers of sporozoites/mosquito were calculated.</p

Salivary gland scores of individual mosquitoes in Experiments 1–5.

<p>After feeding, the mosquitoes were dissected to demonstrate that they had taken a blood meal and establish the salivary gland score (1+ to 3+, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008947#s2" target="_blank">Methods</a>). Nine of the 17 mosquitoes that fed on mice that did not develop parasitemia (negatives) had the highest salivary gland score of 3+. The geometric mean salivary gland scores of the mosquitoes were not significantly different between the two groups (p = 0.2856, Wilcoxon Two Sample Test).</p

Infectivity of PySPZ inoculated IV.

<p>A sample of 15 mosquitoes was randomly taken from the same container in which the mosquitoes used to bite mice in experiment #5 were taken. Salivary glands were dissected from the 15 mosquitoes and sporozoites isolated. The indicated numbers (first column) were injected IV into mice. 7 days and 14 days later, the presence of parasitemia was determined by microscopic evaluation of thin blood smears. The ID₅₀ was calculated, and determined to be 1.09 PySPZ.</p

Are these hypnozoites?

<p>HepG2-A16 cells were infected, maintained for 9 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#pone-0014275-g004" target="_blank">Fig 4</a> and stained with PvCSP mAb. Approximately 10% of the Pv liver stage parasites that expressed PvCSP were similar in size to 3-day trophozoites, and much smaller than the 9-day schizonts. Are these hypnozoites?</p

<i>In vitro</i> development of hepatocyte stage parasites.

<p>25,000 Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 3 days and 50,000 Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 9 days, and the numbers of early liver stage trophozoites (3 day assay) or late liver stage schizonts (9 day assay) were counted by immunofluorescence microscopy after staining with the anti-PvCSP mAb, NVS3 (20 µg/mL).</p

<i>In-vitro</i> development of late liver stage schizonts expressing PvMSP1.

<p>20,000 HepG2-A16 cells were infected with 50,000 Pv spz. Three hrs later uninfected Pv spz were washed off and the culture was maintained for 9 days with daily media changes. Mature Pv liver stage schizonts (400 X magnification) in HepG2-A16 cells were stained with (A) the mAb to the PvCSP, NVS3 (20 µg/ml) or (B) with a mAb against Pv merozoite surface protein 1(PvMSP1), 3F8.A2 (1∶50 dilution). As a negative control, uninfected HepG2-A16 cells were incubated with the individual mAbs and labeled secondary antibodies. There was no evidence of staining in these negative control cultures (data not shown).</p

Multiple Pv liver stage parasites were seen in single hepatocyte in a 3 Day culture.

<p>HepG2-A16 cells were infected with Pv spz and the liver stage trophozoites were stained with the anti-PvCSP mAb, NVS3. Some HepG2-A16 cells were seen with multiple liver stage parasites (400X magnification). <b>N</b>: Nucleus of hepatocyte, C: Cytoplasm of hepatocyte, White Arrows: Individual 3 Day hepatocyte stage Pv.</p

Hepatotoxic changes in HepG2-A16 cells after treatment with high dose primaquine for 9 days.

<p>Culturing HepG2-A16 cells with high concentrations of primaquine (10 µg/mL) induced hepatotoxic changes. <b>P</b>: Parasite; <b>White arrows</b>: Dense bodies in cytoplasm; <b>E</b>: Empty spaces due to focal cell loss in the cell culture well.</p

Immunofluorescence Assay.

<p>Air dried Pv spz were immunostained with monoclonal antibody against PvCSP (NVS3) at 0.98 ng/mL as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>. The end point titer is 1∶1,024,000.</p