Most malaria vaccines work only between 20 and 50 percent of the time. Thus, scientists are eager to understand more about how the malaria parasites infect their host. New imaging data has revealed a previously unrecognized step on the parasites’ transmission path, which could aid in the development of better vaccination strategies. Mouse malaria parasites (green) glide in circles on a glass surface (red) because they have a curved shape. Malaria is transmitted by an infected mosquito into the skin of a mammal and travels to the liver to infect red blood cells and cause disease. Friedrich Frischknecht from the department of parasitology at the University of Heidelberg Medical School in Germany and his colleagues, working at that time at Pasteur Institute in Paris, have studied how the malaria parasites move once inside the host. Using wide-field imaging and confocal microscopy, they found that parasites seem to experience various fates inside their mammalian hosts. Using wide-field imaging and confocal microscopy, researchers have discovered that parasites move in various paths in their host. Each color represents a different parasite’s path inside the skin of a living mouse. Images courtesy of Sylvia Münter, University of Heidelberg Medical School. As reported in the February issue of Nature Medicine, the researchers let a mosquito infected with green fluorescent clones of the mouse malaria parasite Plasmodium berghei bite the ear of a hairless mouse. They observed the site using a Zeiss or an Olympus inverted microscope and found that once the mosquito bites, it distributes the parasites over an area of about 400 x 600 μm. The parasites in the skin displayed a forward-gliding motion, which seemed like a random path. “The parasites move in a peculiar way because they are shaped in a slightly curved manner,” Frischknecht said. He explained that, in vitro, the parasites always move forward in a circle because they are curved like bananas, but that, inside the skin, they appear to move randomly, probably because of all the dense tissue. The researchers initially captured video images of the parasites’ journey using an imaging system from Till Photonics GmbH of Gräfelfing, Germany, and later upgraded to a spinning disk confocal microscope from PerkinElmer Inc. of Wellesley, Mass. They tracked the blood vessels using either transmission light microscopy or by injecting red fluorescent bovine serum albumin into them. As expected, some parasites invaded the blood vessels, usually within the first 20 minutes after the bite. They glided into the skin and attached to the blood vessel walls, which they penetrated until they were suddenly carried away at the same speed of the circulating red blood cells, indicating their invasion into the vessel. To determine just how many of the parasites were actually invading the blood vessels, the scientists, in 12 experiments, counted about 20 parasites on average in the mouse’s skin just after the mosquito bite. They also observed that some didn’t move in a forward motion but instead drifted in a sideward and low-velocity movement. They carefully watched more of the video imaging and realized that the parasites were drifting because they were in the lymphatic vessels. They confirmed this using confocal microscopy and by injecting more parasites with red fluorescent dextran. The images showed that, after about an hour, half of the parasites left the skin site and about 70 percent of them invaded the blood vessels, while 30 percent invaded the lymphatic vessels. They were surprised by these results. The researchers then changed the model system from the ear to the footpad of the mouse — where it is easier to extract the draining lymph nodes — to further analyze the fate of the parasites that entered the lymphatic system. After allowing 10 infected mosquitoes to bite the footpad, they dissected the two draining lymph nodes and found about 50 parasites inside the first lymph node, which represented about 25 percent of the injected parasites, but found none in the second. To assess the fate of the parasites in the lymph node, they labeled them with antibodies and performed time-lapse imaging using a spinning disk confocal microscope with a Hamamatsu CCD camera. Image acquisition time varied between 20 ms and 2 s, depending on the tissue depth, and they recorded images for up to 25 minutes at a time. Image files were processed using the National Institutes of Health’s ImageJ and Adobe software. They found that, after one hour, most of the parasites still had their characteristic “banana” shape, but that after four hours, they appeared damaged, and at eight hours, most of them had lost their fluorescence. The researchers then compared how parasites develop in the lymph node as opposed to how they develop in the liver (the final stop for those that traveled through blood vessels). After about 15 hours, a small proportion of the lymph node parasites appeared the same as those developing in the liver. But after 24 hours, the liver parasites had grown much more than the ones in the lymph nodes. And after 48 hours, the researchers could no longer detect any parasites in the lymphatic vessels. “We don’t know whether they are eaten up or whether they deteriorate — or what happens to them,” Frischknecht said. But he stressed that just knowing that some of them enter the lymphatic vessels shows that the parasites seem to meet various fates in their host, which could be important for future vaccination development. The researchers would like to understand more about the parasite’s movement and outcome inside mammals. They would like to combine light microscopy with cryoelectron tomography to observe them more closely. Frischknecht said that they will image the parasites using a light microscope and then will freeze them and look at them with the cryoelectron microscope to see if they can learn more about their three-dimensional structures implicated in parasite movement.