J. H. E. T. Meuwissen

Absence of seasonal variation in malaria parasitaemia in an area of intense seasonal transmission.

Parasitological surveys carried out in two villages of the Kilombero district of Tanzania indicated a very high prevalence of Plasmodium falciparum parasitaemia throughout the year (all ages mean prevalence = 69.2%) and a low, unstable prevalence of P. malariae (all ages mean prevalence = 4.5%). Fevers (temperature > or = 37.5 degrees C) in both children and adults showed irregular changes in prevalence over time, but there was no seasonal pattern. Neither was there seasonal variation in either P. falciparum parasite prevalence or parasite densities. This was despite marked seasonality in vectors caught in CDC light-traps and in estimated sporozoite inoculations determined by ELISA. The estimated mean annual inoculation rate was extremely high, over 300 infectious bites per person per year, the main vectors being members of the A. gambiae complex and Anopheles funestus. There was considerable variation between houses but even in houses with relatively low mosquito numbers the inoculation rate was sufficient to maintain a maximal P. falciparum prevalence. Heterogeneities in exposure cannot explain why the parasite prevalence is not always 100%. In areas of such high transmission, parasitaemias are likely to be determined mainly by the interaction of schizogony and anti-blood stage immunity, since parasites arising from new inoculations generally comprise only a small proportion of the total in the circulation. In any one individual, this will lead to periodic fluctuations in levels of parasitaemia. These are unlikely to show a close relationship to either seasonal variation in inoculations or to differences between households in the local inoculation rate.

Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures

Synchronised gametocyte cultures were used to study the biosynthesis of the sexual stage target antigens (Mr 230 000, 48 000 and 25 000) for anti gamete/zygote antibodies. These antigens were shown to be synthesized during gametocyte development from day 2-3 onwards until gametogenesis occurred. After gametogenesis a 25 kDa protein was predominantly synthesized, whereas synthesis of the other target proteins was hardly detectable. The 48, 45, and 25 kDa proteins appeared to be glycosylated, in addition the 25 kDa was also acylated in that it bound [3H]palmitic acid covalently. The iso-electric point (pI) of these proteins was assessed as being 6.0 +/- 0.1 (for both 48 and 45 kDa) and 5.6 +/- 0.1 (for 25 kDa).

Survival and infection probabilities of anthropophagic anophelines from an area of high prevalence of Plasmodium falciparum in humans

Delayed and immediate oocyst rates; parous rates and sporozoite rates were obtained in Anopheles gambiae Giles, A. arabiensis Patten and A. funestus Giles from two villages in the Kilombero Valley, southern Tanzania during the wet season of 1991. Collection methods included light trap, indoor resting collection and nets with holes cut in their side. Mosquito survival estimates from parous rates obtained from light trap collections, were compared with estimates from capture-recapture experiments and from that obtained during a population decline. Methods of estimating the proportion of feeds infectious to mosquitoes, K, were also compared. This proportion varied between villages and species and was highest in the village with the greatest proportion of A. gambiae. We propose that absolute estimates of K should be obtained by determining the immediate oocyst rate and measuring the parous rate using the same host seeking mosquitoes. This estimate was only available from one village and ranged from 1.9% for A. gambiae s.l. to 3.4% for A. funestus.

Plasmodium falciparum ookinetes migrate intercellularly through Anopheles stephensi midgut epithelium.

The migration ofPlasmodium falciparum andP. berghei ookinetes through the midgut epithelium inAnopheles stephensi was studied by transmission electron microscopy. With ruthenium red (RR) staining, the results of previous studies were confirmed:P. falciparum ookinetes take an intercellular route through the midgut epithelium. In the same mosquito species, the rodent parasiteP. berghei appeared to take an intracellular position, as previously suggested by other authors. The intra- or intercellular ookinete migration ofP. berghei orP. falciparum, respectively, can perhaps be related to the higher mortality ofP. berghei-infected mosquitoes within the first 2 days of infection. Evidence is presented that oocyst capsule formation begins as early as during the migration of the ookinete. After localization between the epithelial cells and the midgut basal lamina, the rapidly expanding oocyst stretches the overlying layer of the latter at the haemocoelic surface while a new basal lamina is generated between the oocyst and epithelial cell.

Absence of relationships between selected human factors and natural infectivity of Plasmodium falciparum to mosquitoes in an area of high transmission

The effects of sex, age of the human host, patency of asexual and sexual stages and seasonality on infectiousness of Plasmodium falciparum to mosquitoes were investigated in a rural village in southern Tanzania between 1992 and 1994. Villagers from randomized subgroups of households were surveyed for malaria parasites. Gametocyte and trophozoite prevalences were age dependent and fluctuated without any clear pattern of seasonality. A sample of 107 participants, selected to include an excess of gametocyte carriers, slept under bednets with holes cut into the sides for 3 weeks. A total of 3837 Anopheles gambiae s.l. and 5403 A. funestus recovered from these bednets, was examined for all oocysts 5-7 days after feeding or for oocysts less than 17.5 microns in diameter 2-3 days after feeding. Additional blood slides from participants were taken twice weekly. The 5-7 day oocyst rates were 12.1% in A. gambiae s.l. and 10.9% in A. funestus and 2-3 day rates were 3.6 and 4.9%, respectively. The higher rates using the former method were attributed to previous infection. There were strong correlations in the levels of infection in both vectors when they fed on the same hosts. However, patent gametocytaemia was only weakly associated with the development of oocysts in the mosquito. Infectiousness was not related to host age, sex, or the season.

Transmission blocking antibody of the Plasmodium falciparum zygote/ookinete surface protein Pfs25 also influences sporozoite development

Summary The Plasmodium falciparum zygote ookinete surface protein, Pfs25, persists in the oocyst wall throughout its development. Anti‐25 kD transmission blocking antibody, given to infected Anopheles stephensi or A. gambiae mosquitoes in an additional bloodmeal, 3–6 days after being fed gametocyte infected blood, penetrated the oocyst and reacted with the 25 kD protein within it. This reaction caused a significant reduction in the number of developing sporozoites. Mouse serum containing antibodies raised by immunization with a recombinant 25 kD yeast product showed a similar effect.

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

SummaryRecent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm2, grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.

Biosynthesis of the 25-kDa protein in the macrogametes/zygotes of Plasmodium falciparum

Synthesis of the 25-kDa protein in the early midgut stages of Plasmodium falciparum was studied, using metabolic inhibitors (colchicine and actinomycin D) and pulse-labeling experiments. Experiments with colchicine showed that, immediately after induction of macrogametogenesis, 25-kDa protein synthesis occurs in both fertilized and nonfertilized macrogametes. The amount of 25-kDa protein synthesized increased slowly during time. Experiments with actinomycin D revealed that the slow increase of synthesis may be dependent on de novo messenger RNA synthesis.

Immunization against cerebral pathology in Plasmodium berghei-infected mice.

The development of cerebral lesions in Plasmodium berghei-infected mice was dependent on the strain of mice and the size of the infectious inoculum. In particular, C57Bl/6J mice develop cerebral lesions when infected with low numbers of parasitized erythrocytes. By increasing the number of parasites in the infectious inoculum, the percentage of animals that develop cerebral malaria is decreased. Varying degrees of protection against the development of cerebral malaria can be obtained by several methods of immunization. (1) Injection of mice with large numbers of disrupted parasitized erythrocytes 1 or 2 weeks before the challenge infection (protection up to 70%). (2) A 2-day immunizing infection given 9 or 14 days before the challenge infection (protection up to 85%). (3) Injection of mice with plasmodial exoantigen preparations 1 week before the challenge infection (variable protection-rate, up to 100%). In all mice protected against cerebral malaria, parasitaemia is not affected by the immunizing treatment, indicating that protective mechanisms against cerebral malaria and parasitaemia are independent.

Estimation of the infectious reservoir of Plasmodium falciparum in natural vector populations based on oocyst size

A method for determining the infectious reservoir of malaria (K) and vector survival rate (P) by measuring oocyst size and discriminating between the most recent and other infections is described. In the laboratory the mean diameter of 3 d oocysts in Anopheles gambiae, kept at 26 degrees C, was 11.5 microns and the mean diameter at day 5 was 24.5 microns. Oocyst sizes in wild caught mosquitoes from southern Tanzania, that had fed on the occupants of bed nets with holes in the sides, were more variable. 2060 A. gambiae s.l. and 1982 A. funestus were examined for oocysts 3 d after feeding; 796 and 654 oocysts from the 153 and 170 infected females, respectively, were measured. Because of misclassification errors, the use of a simple cut-off model, in which all oocysts less than 17.5 microns in diameter were considered to have arisen from the most recent feed, was thought to overestimate K and underestimate P. A statistical model which allows for overlap in the oocyst size distributions is described. Estimates of the infectious reservoir derived from this model were 2.8% for A. gambiae s.l. and 4.2% for A. funestus, and the estimated survival rates per gonotrophic cycle were 65.5% and 52.9%, respectively. The utility of measuring oocyst size in naturally infected mosquitoes is discussed.