Imaging malaria sporozoites in the dermis of the mammalian host

Abstract

The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. The steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo. INTRODUCTIONThe notion that Anopheles mosquitoes inject Plasmodium sporozoites into the dermis of the host, rather than directly into the blood circulation, was first suggested in the 1930s 1 and has since received experimental confirmation 2-4 . Sporozoites are ejected when the mosquito salivates 5 , especially when it probes the dermis, searching for a blood source. However, given the small number of sporozoites injected through its bite and the highly motile behavior of these sporozoites, the dermal phase of sporozoite infection is still poorly characterized. Only now, with the development of tools for studying the fast dynamics of sporozoites in real time, can their exact in vivo fate be analyzed.A fundamental feature of the malarial sporozoite is its motility and migratory behavior. Like other apicomplexan parasites, this needle-shaped cell (10 mm in length, 1 mm in width) moves by gliding over a substrate at very high speeds (up to 4 mm s À1 ). The sporozoite and its gliding properties have mainly been studied in species of Plasmodium that infect rodents (P. berghei and P. yoelii), which offer, over the human-infecting parasite species, the advantages of safety in handling the parasites and ease in manipulating their genome. The rodent systems offer an additional key advantage, the possibility of studying parasites in as near as possible to their natural environment. A number of wild-type fluorescent sporozoites expressing a fluorescent marker (GFP or RFP) through a variety of stage-specific or constitutive promoters are now available in both P. berghei 6,7 and P. yoelii 8 , which can now be imaged in the dermis or liver of rodent hosts 9-12 .Imaging techniques have been revolutionized by advancements in both microscope instrumentation and data collection/processing software. Although one-photon methods are more limited than two-phot...

The enhancement of arbovirus transmission and disease by mosquito saliva is associated with modulation of the host immune response

“…During blood-feeding an infected mosquito transmits virus in saliva secreted predominantly in the extravascular space of the skin (Amino et al 2007, Hurlbut 1966, Smith et al 2005, Styer et al 2007, Turell and Spielman 1992, Turell et al 1995. Depending on the virus, mosquito species, and method of quantification employed, the estimated level of virus inoculated within mosquito saliva ranges from approximately 10 1 to 10 7 pfu (Colton et al 2005, Colton and Nasci 2006, Gubler and Rosen 1976, Reisen et al 2005, Smith et al 2005, Styer et al 2007, Vanlandingham et al 2004).…”

Development of the malaria parasite in the skin of the mammalian host

“…All experiments were approved by the committee of Institut Pasteur and were performed in accordance with the applicable guidelines and regulations. A. stephensi (Sda500 strain) mosquitoes were reared using standard procedures (16). For intradermal injection of sporozoites into rodents, salivary gland sporozoites were dissected out and a small volume (0.2-10 μL) containing 5 × 10 3 to 3 × 10 5 sporozoites was deposited in the dermis of the ear by using a 35-to 36-gauge needle with a NanoFil syringe (World Precision Instruments).…”

“…Intravital imaging was performed as described (16). For immunolabelings, ears were excised, fixed with 4% paraformaldehyde/PBS for 2 h, and dehydrated in 10% and 30% sucrose/PBS before embedding in OCT. Ten-micrometer sections were cut on a CM3050S cryostat (Leica) and adhered to Superfrost Plus Slides (VWR).…”

“…We first analyzed the fate of the P. berghei sporozoites that remain in the skin of the host. Anopheles stephensi mosquitoes were allowed to transmit WT green fluorescent sporozoites (14) into the ear of SKH1 hairless mice (15), which display little autofluorescence in the skin, and parasites were imaged daily using spinning-disk confocal microscopy (16). Typically, inside hepatocytes, parasites round up and increase their size to become spherical exoerythrocytic forms (EEF) of ∼40-μm average diameter, undergo schizogony, and produce thousands of uninucleate merozoites in ∼50-70 h (4).…”

Oxidative Stress in Human Health and Disease

“…The major vector for parasite transmission is the female anopheline mosquito (107). During a blood meal, sporozoites are inoculated under the skin and travel through the blood stream, liver sinusoids to settle in a hepatocyte after traversing several (108)(109)(110)(111). This journey usually takes approximately 1 hour (112).…”