Plasmodium berghei

Plasmodium berghei used for the transfection express GFP and luciferase and was obtained through BEI Resources, NIAID, NIH: P. berghei, strain ANKA 676m1c11, MRA-868, contributed by Chris J Janse and Andrew P Waters [8 (link)]. To generate P. berghei expressing full-length P. falciparum CSP and GFP-luciferase, the same transfection strategy described in detail by Espinosa et al. was followed [4 (link)]. Sporozoites were generated by infecting Anopheles stephensi mosquitoes by allowing 5-days old mosquitoes to feed on parasite-infected mice at approximately 1–3% parasitaemia. Prior to feeding mosquitoes, the blood of each infected mouse was examined for the presence of gametocyte exflagellation to ensure mosquito infection. This is done by microscopic examination of blood in ookinete medium [7 (link)]. After infection, mosquitoes were maintained in an incubator at 19–20 °C. On top of the cages, mosquitoes were supplied with a sterile cotton pad soaked in 10% sucrose, which was changed every 48 h.
Sporozoites were generally harvested at days 20–22 post infection, unless otherwise described. In experiments to evaluate the effect of sporozoite age on infectivity, sporozoites were harvested between 18 and 28 days.

The parasite strain Plasmodium berghei ANKA used in these studies was supplied by MR4 (uncloned line MRA-311, depositor Thomas McCutchan). A known CM-susceptible mouse strain was used: 4–6 week old female C57BL/6 mice (C57BL/6NCRL from Charles River Breeding Laboratories, Portage, Michigan, USA), which were acclimated in the Research Resource Center of Northwestern University for 2 weeks, per protocol, with food and water ad libitum. Any mouse with obvious physical deformities or size discrepancies was not included in the experiment. When the mice reached 6–8 weeks of life, 16 experimental mice were inoculated intraperitoneally (IP) with 1×10⁶ PbA (0.1mL) taken from a homologue donor, which had been infected from frozen stock. Four control mice were injected IP with 0.1mL of sterile PBS. Each cage contained four experimental mice with one control mouse. One control mouse was discarded due to congenital growth retardation, leaving three control mice. Mice were assessed via the RMCBS every 24 hours. Attempts were made to assess the mice at the same time each day, usually the evening hours, for every experiment; however, this was not always achieved, and mice needed to be assessed frequently, at variable times of the day. Blood was sampled daily from tail tip phlebotomy, and parasitemia was determined by tabulating the number of Giemsa-stained parasitized-RBCs out of a total of 500 RBCs, counted by a blinded operator using 40× light microscopy. Early experiments revealed that a mouse with a RMCBS of 5, or less, would die within 1–4 hours, and such mice were labeled moribund and were anesthetized with 10% pentobarbital IP. Mice were exsanguinated via heart puncture and/or inferior vena cava sever, and the brains were carefully excised and placed in pH-balanced 10% formalin for 1–2 weeks.
We further assessed the effectiveness and specificity of the RMCBS by comparing the trajectories of illness in subjects of a non-MCM model and symptomatic and asymptomatic MCM mice. We examined the RMCBS in PbA-infected C57BL/6 (known MCM-susceptible) mice and PbA-infected balb/c (known MCM-resistant) mice. Experimental and control mice were inoculated, assessed, and euthanized as described above. Intracerebral hemorrhages were evaluated by an operator blinded to the subject phenotype, viewing 5 H&E-stained sagittal sections under 40×.

In this study, male and female inbred BALB/c mice aged 8–9 weeks were used. The rodent facility at IPR provided the animals. They were housed in standard Macron type II cages within 12 hours’ dark/light cycle at 23 °C. Food and water was provided ad libitum according the IPR’s Animal Science Department standard operating procedures.
Plasmodium berghei, strain ANKA (sourced from KEMRI) was retrieved from the IPR repository and maintained in mice [50 (link)] for use in this study. This parasite strain was utilized because it causes severe disease in BALB/c mice with clinical characteristics similar to P. falciparum infection [51 (link)]. Despite the phylogenetic distance between rodent and human malaria parasites, P. berghei and the human malaria parasites possess conserved genes that have over time allowed their use in Peters’ model of antimalarial drug efficacy testing [52 (link)].

Both IndCh and IndInt isofemale lines were maintained as two separate isofemale colonies after 20 generations and 23 generations, respectively, instead of single female progeny [29 ]. These two lines are currently being maintained in TIGS insectary. In both the colonies, males and females were allowed to inbreed within the filial generation, blood-fed and allowed to lay eggs in masses. All the key fitness parameters were monitored and recorded. These two isofemale colonies were utilized for insecticide-susceptibility assays and vectorial competence studies with Plasmodium berghei and Plasmodium falciparum.

Microtubules and SPM3 localization of fixed Plasmodium berghei sporozoites were visualized using a Zeiss Airyscan 2 LSM900 laser scanning confocal microscope. Both midgut and salivary gland sporozoites were isolated as described above but without subsequent Accudenz purification. Instead, sporozoites were directly pelleted for 3 min at 13,000 rpm (Biofuge Primo; Thermo Fisher Scientific). The supernatant was removed, and sporozoites were resuspended in RPMI containing 3% bovine serum albumin (Carl Roth). For imaging, 30,000 to 50,000 sporozoites were transferred into a well of a Labtek slide (μ-slide, 8-well ibiTreat, no. 80826). Sporozoites were pelleted for 3 to 4 min at 800 rpm (Multifuge S1-R; Heraeus) and then incubated for 10 min at room temperature, allowing sporozoites to attach and start gliding while leaving the supernatant on top. Sporozoites were washed three times with RPMI before being fixed in 4% paraformaldehyde (PFA) in PBS at room temperature. After 1 h of incubation, fixative was removed by washing sporozoites three times with RPMI followed by permeabilization with 0.5% Triton X-100 in PBS for 1 h at room temperature. Before incubation with primary antibody, sporozoites were again washed three times with RPMI. Sporozoites were incubated for 1 h at room temperature with mouse antitubulin (1:500; Sigma-Aldrich, no. T5168) to stain microtubules, followed by three washing steps with RPMI. Alexa Fluor 546-conjugated goat anti-mouse immunoglobulin (1:500; Invitrogen, no. A11030) was used as the secondary antibody and incubated for 30 min up to 1 h at RT before incubation of sporozoites for 15 min in RPMI containing 1:1,000-diluted Hoechst 33342 from a 10 μM Hoechst 33342 stock solution (Thermo Fisher Scientific). Sporozoites were washed three times in RPMI and subsequently imaged. Z-stacks were acquired with a z-spacing of 0.5 μm and a total of 29 layers being taken. Images were directly 3D processed by the internal 3D Airyscan processing option.