Plasmodium sporozoites trickle out of the injection site

Abstract

Plasmodium sporozoites make a remarkable journey from the skin, where they are deposited by an infected Anopheline mosquito, to the liver, where they invade hepatocytes and develop into exoerythrocytic stages. Although much work has been done to elucidate the molecular mechanisms by which sporozoites invade hepatocytes, little is known about the interactions between host and parasite before the sporozoite enters the blood circulation. It has always been assumed that sporozoites rapidly exit the injection site, making their interactions with the host at this site, brief and difficult to study. Using quantitative PCR, we determined the kinetics with which sporozoites leave the injection site and arrive in the liver and found that the majority of infective sporozoites remain in the skin for hours. We then performed sub-inoculation experiments which confirmed these findings and showed that the pattern of sporozoite exit from the injection site resembles a slow trickle. Last, we found that drainage of approximately 20% of the sporozoite inoculum to the lymphatics is associated with a significant enlargement of the draining lymph node, a response not observed after intravenous inoculation. These findings indicate that there is ample time for host and parasite to interact at the inoculation site and are of relevance to the pre-erythrocytic stage malaria vaccine effort.

Fig. 1

Kinetics with which sporozoites disappear from the inoculation site. Mice were injected intradermally in the ear with 5000 P. yoelii sporozoites, the ears were removed at the indicated time points and the number of sporozoites in each ear was quantified by PCR. There were four mice per group and the means ± standard deviations are shown.

Fig. 2

Kinetics of sporozoite arrival in the liver. Mice were inoculated intradermally in the ear with 4000 P. yoelii sporozoites (A), exposed to 20 infective mosquito bites, 10 in each ear (B), or 20 infective mosquito bites in the back (C) and the injection site was removed at the indicated time points. Control mice had the uninjected ear removed at 3 h (A), no ears removed (B) or an adjacent uninjected site removed at 3 h (C). Forty hours after sporozoite inoculation, mice were sacrificed and liver parasite burden was quantified by RT-PCR. There were five (A) or eight (B and C) mice per group and the means ± standard deviations are shown.

Fig. 3

Intradermally injected sporozoites are slowly released from the injection site into the bloodstream. Donor mice were injected with 25 000 P. yoelii sporozoites either intravenously (A) or intradermally (B). At the indicated time points after injection, donor mice were exsanguinated by cardiac puncture and one-third of the recovered blood (∼300 μl) was injected intravenously into each recipient mouse, which was then monitored for the presence of blood stage parasites beginning on the third day after inoculation. x-axis, the time of blood transfer from the donor; y-axis, the proportion of recipient mice that became positive for erythrocytic stage parasites; z-axis, the pre-patent period for the mice that became positive. Shown are the combined data from three experiments. There were four to six donors and 10–18 recipients per time point.

Fig. 4

Sporozoite infectivity after intradermal versus intravenous inoculation. Mice were inoculated with 5000 P. yoelii sporozoites by intravenous or intradermal injection and 40 h later they were sacrificed and liver parasite burden was quantified by RT-PCR. There were five mice per group and shown are the means ± standard deviations.

Fig. 5

A proportion of intradermally injected sporozoites go to the draining lymph node. A. Five thousand P. yoelii sporozoites were injected into one ear of each mouse and at the indicated time points the ipsilateral auricular lymph node was removed and parasite numbers were quantified by PCR. There were five mice per group and shown are the means ± standard deviations. B. Mice were injected in the left ear either intradermally with medium alone (Naive), 5000 P. yoelii sporozoites intravenously (IV Spz), salivary gland homogenates intradermally (ID Uninf), or 5000 P. yoelii sporozoites intradermally (ID Spz) or fed upon by 10 uninfected mosquitoes (MB Uninf) or 10 infected mosquitoes (MB Inf) and 6 days later the left auricular lymph node was removed, fixed and photographed. Bar = 1 μm. C. Lymph nodes from the mice in B were weighed. There were five mice per group and shown are the means ± standard deviations. Student's t-test for unpaired data showed that the weights of the lymph nodes of mice injected intradermally with sporozoites was significantly different from those of mice injected intradermally with uninfected salivary glands (*P < 0.001) and the weights of lymph nodes of mice bitten by infected mosquitoes was significantly different from those bitten by uninfected mosquitoes (**P < 0.01).