Figure 1.
Luciferase activity of the Py-GFP-luc parasite.
(A, B) Luminescence during erythrocytic stages. (A) Py-GFP-luc infected red blood cells were serially diluted 1∶4 in RPMI and were mixed with an equal volume of luciferin. Luminescence (relative light units, RLU) was measured with a Centro microplate reader. Dashed line indicates background luminescence as determined by measuring uninfected RBCs. (B) Rainbow images of luminescence in BALB/cJ mice with Py-GFP-luc blood stage parasitemia. Mice were injected with luciferin and imaged. Bioluminescence was measured in total flux (p/s). (C, D) Luminescence of Py-GFP-luc oocysts. (C) Midguts from Py-GFP-luc infected mosquitoes were isolated 10 days after an infectious blood meal. The number of oocysts per midgut was estimated and midguts were categorized as follows: Uninfected (0), between 1 and 10 oocysts/midgut (1<10), between 11 and 100 oocysts/midgut (11<100), and greater than 100 oocysts/midgut (>100). Luciferase activity from individual midguts was determined using the Centro microplate reader (RLU) (C) and the IVIS animal imager (total flux) (D). (E) Luminescence of Py-GFP-luc sporozoites. Sporozoites were isolated from salivary glands of infected mosquitoes and serially diluted 1∶10 in RPMI. Luminescence (RLU) was determined as in (A). Dashed line indicates background luminescence of RPMI.
Figure 2.
Luminescence is proportional to Py-GFP-luc liver stage burden in vivo.
(A–E) Representative rainbow images of luminescence in livers of live mice 44 h following injection with (A) 1×105, (B) 1×104, (C) 1×103, (D) 100 or (E) 10 Py-GFP-luc sporozoites. Rainbow scales are expressed in radiance (p/s/cm2/sr). (F) Quantification of total flux from mice in (A–E) (n = 5). (G) Quantification of Py-GFP-luc liver stage burden from dissected livers of mice in (A–E) by qPCR. Ratios of P. yoelii 18S rRNA to murine GAPDH RNA were calculated and normalized to that of uninfected mice. (H-I) Liver stage burden following mosquito bite infection. (H) Representative rainbow images of luminescence in livers of BALB/cJ mice 44 h after being fed on by 5, 15, or 40 mosquitoes infected with Py-GFP-luc. Rainbow scales are expressed in radiance (p/s/cm2/sr). (I) Quantification of total flux from mice in (H). n = 5 mice per group.
Figure 3.
Luminescence correlates to Py-GFP-luc liver stage growth in vivo.
(A–C) Representative rainbow images of luminescence in livers of live mice injected i.v. with 1×105 Py-GFP-luc salivary gland sporozoites at (A) 16, (B) 24 and (C) 44 hpi. Rainbow scales are expressed in radiance (p/s/cm2/sr). (D) Quantification of total flux from mice in (A–C) (n = 5). (E) Quantification of parasite burden in the livers by qPCR of mice injected with 1×105 Py-GFP-luc salivary gland sporozoites at various time points post infection (n = 3). Ratios of P. yoelii 18S rRNA to murine GAPDH RNA were calculated and normalized to that of uninfected mice. Statistics (B, C) Student’s t test, **indicates a P value of 0.01>0.001.
Figure 4.
Use of bioluminescence to compare Py-GFP-luc liver stage burden in two mouse strains.
(A) BALB/cJ or C57BL/6 mice (n = 4) were infected with 5×104 Py-GFP-luc sporozoites i.v. The bellies of all the mice were shaved before imaging bioluminescence. Representative rainbow images are shown. Scale bar is in radiance (p/s/cm2/sr). (B) Quantification of parasite liver burden as determined by bioluminescence. (C) Quantification of parasite liver burden as determined by qPCR. Ratios of P. yoelii 18S rRNA to murine GAPDH RNA were calculated and normalized to that of BALB/cJ mice. (D, E) Days to patency comparisons between BALB/cJ and C57BL/6 mice. (D) BALB/cJ or C57BL/6 mice (n = 10) were infected i.v. with 100 (left), 20 (center) or 4 (right) P. yoelii sporozoites and blood stage patency was monitored in mice by daily thin smears. Graphs depict the percentage of mice free of blood stage malaria at each day post infection. (E) BALB/cJ or C57BL/6 mice were infected via the bites of either 15 (left) or 5 (right) P. yoelii infected mosquitoes. Blood stage patency was monitored and depicted as in (D). Statistics (B, C) Student’s t test ns = p>0.05. (D, E) Log rank Mantel-Cox test. P values are depicted in the figure.
Figure 5.
Use of bioluminescence to evaluate the efficacy of GAP immunization.
BALB/cJ mice were immunized with either 1 or 2 doses of the GAP Py-fabb/f-. Immunizations were spaced 3 weeks apart in multiply immunized mice. Four weeks after the final immunization naïve and immunized mice were challenged with 5×104 Py-GFP-luc sporozoites by i.v. injection. Bioluminescence was measured 24 hpi and 44 hpi. Mice were then monitored for blood stage patency. (A) Representative rainbow images of naïve, or immunized mice challenged with Py-GFP-luc (n = 6). Uninfected mice were also imaged as a control (n = 5). Scale bars are in radiance (p/s/cm2/sr). (B) Quantification of parasite liver burden by bioluminescence. (C) Blood stage patency in challenged mice was monitored from days 3 to 14 post infection by daily thin smears. Graph depicts the percentage of mice free of blood stage malaria at each day post infection.