Course of parasitemia in pregnant versus non-pregnant mice re-infected with a heterologous strain of <i>P</i>. <i>chabaudi</i>.

<p>Mice were inoculated intravenously with <i>Pcc-</i>AS sporozoites, randomly assigned to be paired or not with males 36 days post-inoculation, and then injected with <i>Pcc-</i>CB iRBCs 46 days post-inoculation. Parasitemia was monitored by daily blood smears throughout the study. Data are presented as means with 95% confidence intervals. Similar results were obtained in two repeats of this experiment.</p

Course of <i>P</i>. <i>chabaudi</i> infection differs between pups born to infected versus uninfected dams.

<p>(A) Pups, born to dams with different infection histories (indicated by icons in the figure), were infected with <i>Pcc-</i>CB sporozoites, and peripheral parasitemia followed for up to 32 days; (A, inset) parasite burden between days 22 and 32. (B) Peak parasite burden in pups born to dams that were given HI versus pups born to mothers that were not given HI or were never infected. Data are presented as means with 95% confidence intervals. Differences between groups in the mean values of peak parasitemia were compared using Welch’s unequal variances t-test.</p

Heterologous Infection of Pregnant Mice Induces Low Birth Weight and Modifies Offspring Susceptibility to Malaria

<div><p>Pregnancy malaria (PM) is associated with poor pregnancy outcomes, and can arise due to relapse, recrudescence or a re-infection with heterologous parasites. We have used the <i>Plasmodium chabaudi</i> model of pregnancy malaria in C57BL/6 mice to examine recrudescence and heterologous infection using <i>CB</i> and <i>AS</i> parasite strains. After an initial course of patent parasitemia and first recrudescence, <i>CB</i> but not <i>AS</i> parasites were observed to recrudesce again in most animals that became pregnant. Pregnancy exacerbated heterologous <i>CB</i> infection of <i>AS-</i>experienced mice, leading to mortality and impaired post-natal growth of pups. Parasites were detected in placental blood without evidence of sequestration, unlike <i>P</i>. <i>falciparum</i> but similar to other malaria species that infect pregnant women. Inflammatory cytokine levels were elevated in pregnant females during malaria, and associated with intensity of infection and with poor outcomes. Pups born to dams during heterologous infection were more resistant to malaria infections at 6–7 weeks of age, compared to pups born to malaria-experienced but uninfected dams or to malaria-naïve dams. In summary, our mouse model reproduces several features of human PM, including recrudescences, heterologous infections, poor pregnancy outcomes associated with inflammatory cytokines, and modulation of offspring susceptibility to malaria. This model should be further studied to explore mechanisms underlying PM pathogenesis.</p></div

Peripheral cytokine responses in <i>P</i>. <i>chabaudi</i> re-infected or never-infected pregnant mice, and in non-pregnant re-infected mice.

<p>Cytokines were measured at sequential time-points before (“Naïve”) and during infection (“Active Infection”), prior to pregnancy (“Before Pairing”), and then at gestational day 18 for all pregnant mouse groups (“Uninfected”, “HI_low”, “HI_high” and “No HI”) and simultaneously in re-infected non-pregnant control mice (Non Pregnant HI). (A-F) Cytokines IFN-γ, IL-12, IL-6, IL-10, TNF-α and MCP-1 were measured using Milliplex Map Mouse cytokine kit. Data were analyzed by one-way ANOVA using Bonferroni test to correct for multiple comparisons, and are presented as box and whiskers plots showing min and max values. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 and NS = not significant.</p

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

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

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

Weight gain in pups born to dams that received <i>Pcc-</i>AS before and <i>Pcc-</i>CB during pregnancy, dams that received <i>Pcc-</i>AS before pregnancy and no other infection during pregnancy, or dams that were never infected.

<p>Weights of pups were documented for up to 42 days after birth. (A) Weight in grams throughout the pre-weaning period. (B) Weight in grams throughout the post-weaning period. Data are presented as means with 95% confidence intervals. Differences in weight were analyzed by creating a generalized linear model while taking into account the correlation due to repeated measures within pups and due to multiple pups born to the same dam. β = GLM regression coefficient comparing weight across groups.</p

Recrudescence of <i>P</i>. <i>chabaudi</i> in mice during pregnancy.

<p>After a primary infection initiated by inoculation of <i>Pcc-</i>CB sporozoites, mice were paired with males at 35 days post-inoculation (“Pregnant Mice”, n = 16) or remained unpaired (“Non Pregnant Mice”, n = 12). Parasitemia was monitored by daily blood smears throughout the study. Data are presented as means with 95% confidence intervals. Experiment was repeated with similar results.</p