Transgenic Plasmodium berghei sporozoites expressing β-galactosidase for quantification of sporozoite transmission

Abstract

Malaria transmission occurs during a blood-meal of an infected Anopheles mosquito. Visualization and quantification of sporozoites along the journey from the mosquito midgut, where they develop, to the vertebrate liver, their final target organ, is important for understanding many aspects of sporozoite biology. Here we describe the generation of Plasmodium berghei parasites that express the reporter gene lacZ as a stable transgene, under the control of the sporozoite-specific CSP promoter. Transgenic sporozoites expressing β-galactosidase can be simply visualized and quantified in an enzymatic assay. In addition, these sporozoites can be used to quantify sporozoites deposited in subcutaneous tissue during natural infection.

Direct Microscopic Quantification of Dynamics of Plasmodium berghei Sporozoite Transmission from Mosquitoes to Mice

“…Results indicated that mosquitoes allowed to feed for 3 min deposited a mean of 123 sporozoites, which was less than 1% of the calculated salivary gland load. Comparable results were obtained with transgenic P. berghei sporozoites expressing ␤-galactosidase, quantified after homogenization of the biopsy specimen, followed by a colorimetric assay (8). The reduced sensitivity of this assay required that 10 to 15 mosquitoes feed on the ear to allow detection of sporozoites.…”

“…This led to more-sophisticated approaches to quantifying transmission during mosquito feeding on live rodent hosts (8,16). A study to estimate P. yoelii sporozoite numbers injected into a mouse ear used quantitative PCR measurement of sporozoite 18S rRNA extracted from a biopsy specimen (16).…”

“…The relatively large number of studies attempting to quantify the dynamics of this process attests to the importance with which it has been viewed by malariologists; the history and rationale for such attempts have been reviewed previously (6,24,26). The approaches used have included assessment of numbers of sporozoites released by mosquitoes into liquid media (reviewed in reference 6) or onto glass slides (12), counting of EEF after mosquito feeding on rodent hosts (26), and determination of sporozoite 18S rRNA (16) or ␤-galactosidase expressed by transgenic sporozoites (8) within the skin of a mouse after sporozoite introduction by mosquitoes.…”

CD8+T Cells Eliminate Liver-Stage Plasmodium berghei Parasites without Detectable Bystander Effect

“…We used large numbers of parasites in all experiments (1 ϫ 10 4 to 1 ϫ 10 5 ), i.e., several orders of magnitude larger than the number of parasites injected by an infected mosquito, which is in the range of 1 ϫ 10 1 to 1 ϫ 10 3 (21)(22)(23). Moreover, the use of mice, which have small livers, further increased the density of parasite infection.…”

Getting infectious: formation and maturation of Plasmodium sporozoites in the Anopheles vector

“…What signal triggers the developmental switch in salivary glands is again unknown. Quantification of CSP expression using transgenic parasites that express a reporter gene under the stage‐specific control of the CSP promoter (Engelmann et al ., 2006) confirmed an earlier observation that CSP levels are strictly inversely correlated with sporozoite numbers in salivary glands (Beier et al ., 1992). This finding may be explained by a feedback regulation in response to extracellular CSP levels pointing again to a potential role of CSP in signalling to the parasite interior.…”