Malaria parasite development in a Drosophila model.

Malaria is a devastating public health menace, killing over one million people every year and infecting about half a billion. Here it is shown that the protozoan Plasmodium gallinaceum, a close relative of the human malaria parasite Plasmodium falciparum, can develop in the fruit fly Drosophila melanogaster. Plasmodium gallinaceum ookinetes injected into the fly developed into sporozoites infectious to the vertebrate host with similar kinetics as seen in the mosquito host Aedes aegypti. In the fly, a component of the insect's innate immune system, the macrophage, can destroy Plasmodia. These experiments suggest that Drosophila can be used as a surrogate mosquito for defining the genetic pathways involved in both vector competence and part of the parasite sexual cycle.

Laminin and a Plasmodium ookinete surface protein inhibit melanotic encapsulation of Sephadex beads in the hemocoel of mosquitoes.

In refractory mosquitoes, melanotic encapsulation of Plasmodium ookinetes and oocysts is a commonly observed immune response. However, in susceptible mosquitoes, Plasmodium oocysts develop extracellularly in the body cavity without being recognized by the immune system. Like Plasmodium gallinaceum oocysts, negatively charged carboxymethyl (CM)-Sephadex beads implanted in the hemocoel of Aedes aegypti female mosquitoes were not usually melanized, but were coated with mosquito-derived laminin. Conversely, electrically neutral G-Sephadex beads were routinely melanized. Since mosquito laminin coated both CM-Sephadex beads and P. gallinaceum oocysts, we hypothesized that laminin prevents melanization of both. To test this hypothesis, we coated cyanogen-bromide-activated G-Sephadex beads with laminin, recombinant P. gallinaceum ookinete surface protein (PgS28) or bovine serum albumin (BSA). Beads were implanted into the abdominal body cavity of female Aedes aegypti and retrieved 4 days later. Uncoated controls as well as BSA-coated G-Sephadex beads were melanized in a normal manner. However, melanization of beads coated with mouse laminin, Drosophila L2-secreted proteins or PgS28 was markedly reduced. Fluorescent antibody labeling showed that PgS28-coated beads had adsorbed mosquito laminin on their surface. Thus, mosquito laminin interacting with Plasmodium surface proteins probably masks oocysts from the mosquito's immune system, thereby facilitating their development in the body cavity.

Comparison of the primary structure of the 25 kDa ookinete surface antigens of Plasmodium falciparum and Plasmodium gallinaceum reveal six conserved regions.

The gene encoding the 25 kDa ookinete surface antigen (Pgs25) of Plasmodium gallinaceum has been cloned using an oligonucleotide probe directed against one of the EGF-like domains of the P. falciparum 25 kDa ookinete surface antigen (Pfs25). The Pgs25 gene codes for a polypeptide of 215 amino acids, two amino residues less than Pfs25. The deduced amino acid sequence contains a putative signal sequence at the amino-terminus, four tandemly repeated EGF-like domains, and a hydrophobic region at the carboxyl-terminus. By comparing Pgs25 with Pfs25, six conserved regions, consisting of six or more amino acid residues, have been identified. Most of the conserved regions are outside EGF-like core consensus sequences. The most striking conservation is the spacing of the cysteines.

Alpha-tubulin II is a male-specific protein in Plasmodium falciparum.

The tubulin gene family in Plasmodium falciparum consists of one beta-tubulin and two alpha-tubulin genes (alpha-tubulin I and II). We present here data indicating that alpha-tubulin II is expressed only in male sexual stage parasites. An IgM mAb, 5E7, specifically reacted with stage III (day 4-5) through mature (day 10-11) male gametocytes and with emerging, exflagellating, or freely moving male gametes. No reactivity was detected in female gametocytes, female gametes, sporozoites, or asexual parasites. mAb 5E7 also specifically recognized male gametes of the avian parasite, Plasmodium gallinaceum, and immunoblotted a 50 kDa protein in extracts of male gametes from both species. This 50 kDa antigen was localized by immunoelectron microscopy to axonemes of male gametes in a pattern similar to that obtained with anti-alpha- and anti-beta-tubulin antibodies. Furthermore, mAb 5E7 specifically reacted with recombinant alpha-tubulin II protein obtained using the PCR-amplified alpha-tubulin II gene from a gametocyte-specific cDNA library. The sex-specific expression of alpha-tubulin II and its localization to axoneme of the male parasite suggest a role for this molecule in the morphologic changes that occur during exflagellation and in the motility of the parasite. alpha-Tubulin II and mAb 5E7 may prove useful tools in studies of the biology of sexual stage differentiation and development in P. falciparum in addition to the general understanding of post-translational modifications of tubulin isoforms.

Phase separation in Triton X-114 of antigens of transmission blocking immunity in Plasmodium gallinaceum.

The distribution of proteins of mosquito midgut forms of Plasmodium gallinaceum in the detergent-free (aqueous) and detergent-enriched phases was studied using a phase separation technique in Triton X-114. Of the three surface proteins on gametes and newly fertilized zygotes (240, 56, and 54 kDa) immunoprecipitated by transmission blocking monoclonal antibodies, 240 kDa protein was recovered in the aqueous phase, whereas 56 and 54 kDa proteins were found preferentially in the detergent phase. The hydrophobic properties of the 56 and 54 kDa proteins were also shown by their strong tendency to interact with the lipid bilayers and a hydrophobic matrix phenyl-Sepharose. Monoclonal antibody IID3B3 immunoprecipitated all the three proteins from the whole Triton extract but in the phase-separated extracts reacted only with the 240 kDa protein in the aqueous phase and not with the 56 and 54 kDa doublet in the detergent phase. In Western blot analysis also monoclonal antibody IID3B3 reacted only with the 240 kDa protein. The 240 kDa protein in the aqueous phase was retained by monoclonal antibody IID3B3 linked to Sepharose 4B beads and could be eluted either with 0.1 M acetic acid or 50 mM diethylamine. The 56 and 54 kDa doublet in the detergent phase could be bound to and eluted from Sepharose 4B beads-linked monoclonal antibody IID4 or rabbit anti-male P. gallinaceum gamete serum. Two stage-specific glycoproteins of 26 and 28 kDa on the surface of ookinetes of P. gallinaceum were also separated in the detergent phase following Triton X-114 extraction. Phase separation in Triton X-114 offers a simple approach to the separation of a select group of proteins from the bulk of the cellular proteins.

Direct and indirect immunosuppression by a malaria parasite in its mosquito vector.

Malaria parasites develop as oocysts within the haemocoel of their mosquito vector during a period that is longer than the average lifespan of many of their vectors. How can they escape from the mosquito's immune responses during their long development? Whereas older oocysts might camouflage themselves by incorporating mosquito-derived proteins into their surface capsule, younger stages are susceptible to the mosquito's immune response and must rely on other methods of immune evasion. We show that the malaria parasite Plasmodium gallinaceum suppresses the encapsulation immune response of its mosquito vector, Aedes aegypti, and in particular that the parasite uses both an indirect and a direct strategy for immunosuppression. Thus, when we fed mosquitoes with the plasma of infected chickens, the efficacy of the mosquitoes to encapsulate negatively charged Sephadex beads was considerably reduced, whether the parasite was present in the blood meal or not. In addition, zygotes that were created ex vivo and added to the blood of uninfected chickens reduced the efficacy of the encapsulation response. As dead zygotes had no effect on encapsulation, this result demonstrates active suppression of the mosquito's immune response by malaria parasites.

Engineering mosquito resistance to malaria parasites: the avian malaria model.

Genetic approaches to controlling the transmission of mosquito-borne diseases are being developed to augment the available chemical control practices and environmental manipulation methods. Much progress has been made in laboratory-based research that seeks to develop antipathogen or antivector effector genes and methods for genetically manipulating host vector strains. Research is summarized here in the development of a malaria-resistant phenotype using as a model system the avian parasite, Plasmodium gallinaceum, and the mosquito, Aedes aegypti. Robust transformation technology based on a number of transposable elements, the identification of promoter regions derived from endogenous mosquito genes, and the development of single-chain antibodies as effector genes have made it possible to produce malaria-resistant mosquitoes. Future challenges include discovery of methods for spreading antiparasite genes through mosquito populations, determining the threshold levels below which parasite intensities of infection must be held, and defining the circumstances in which a genetic control strategy would be employed in the field.

Virus-expressed, recombinant single-chain antibody blocks sporozoite infection of salivary glands in Plasmodium gallinaceum-infected Aedes aegypti.

Transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. We have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. Mouse monoclonal antibodies that recognize the circumsporozoite protein of Plasmodium gallinaceum can block sporozoite invasion of Aedes aegypti salivary glands. An anti-circumsporozoite monoclonal antibody, N2H6D5, whose corresponding heavy- and light-chain gene variable regions were engineered as a single-chain antibody construct, binds to P. gallinaceum sporozoites and prevents infection of Ae. aegypti salivary glands when expressed from a Sindbis virus. Mean intensities of sporozoite infections of salivary glands in mosquitoes expressing N2scFv were reduced as much as 99.9% when compared to controls.

Reinterpretation of the genetics of susceptibility of Aedes aegypti to Plasmodium gallinaceum.

Several studies have demonstrated a genetic basis for variation in susceptibility of Aedes aegypti to Plasmodium gallinaceum. Although 25 yr ago it was reported that P. gallinaceum susceptibility in Ae. aegypti is determined primarily by a single autosomal dominant gene, evidence for additional genetic factors has emerged. Two sublines, 1 refractory and 1 of intermediate susceptibility to P. gallinaceum, have been selected from the Moyo-In-Dry strain (MOYO) of Ae. aegypti. Prior to selection, the MOYO population was 20.3% refractory. Genetic crosses of the highly susceptible Rockefeller strain (ROCK) and the 2 selected sublines of the MOYO strain provide evidence for a complex mode of inheritance of Plasmodium susceptibility in Ae. aegypti.

Complement effects of the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. II. Changes in sensitivity to complement-like factors during zygote development.

During transformation into ookinetes, the zygotes of Plasmodium gallinaceum are initially resistant to lysis by heat-labile and EDTA-sensitive factors in the serum of their natural host, the chicken. Between 6 and 8 hr postgametogenesis, zygotes cultured in vitro lose their resistance to these factors. Loss of resistance to these factors in vitro is reflected by loss of infectivity of the zygotes to Aedes aegypti mosquitoes in the presence of native chicken serum. These factors are probably components of the alternative pathway of complement (APC) of chicken serum. Gametocytes of P. gallinaceum in chicken blood are able to infect A. aegypti mosquitoes apparently due to inactivation of the APC in a blood meal within 3-4 hr after ingestion, i.e., several hours before the zygotes lose their resistance to chicken APC. In addition to the heat-labile factors (APC) in chicken serum, the zygotes are transiently sensitive to other factor(s) in the mosquito blood meal. These factor(s) are not destroyed by prior heating of the chicken serum given in a blood meal and therefore cannot be complement components. The antiparasitic effects of the factors are neutralized by addition of EDTA to the blood meal and could be due to an EDTA-sensitive metalloprotease present in the mosquito midgut.

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti.

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.

Immunodiagnosis of malaria.

Different approaches to immunodiagnosis of malaria in an endemic country have been described in this paper. Demonstration of circulating malaria antigen may be done by gel-diffusion and counter-immunoelectrophoresis. Parasite associated antigen may be demonstrated by highly sensitive methods like radio-immunoassay or enzyme linked immuno sorbent assay. Malaria antibodies of the IgG type being long lasting do not appear to have any role in immuno-diagnosis. However, determination of malaria specific IgM antibodies by P. gallinaceum haemagglutination test or IgM immunofluorescence test may be simple and useful in immunodiagnosis of malaria. The tests though evaluated in different laboratories may not be applicable in the field for diagnosis of malaria at the present moment. However, it is envisaged that with the availability of different specificities of monoclonal antibodies by way of hybridoma technology and also with the help of recombinant DNA techniques immunodiagnosis of malaria in the field situation may become a reality.

Preliminary results of an anticircumsporozoite DNA vaccine trial for protection against avian malaria in captive African black-footed penguins (Spheniscus demersus).

Captive juvenile African black-footed penguins (Spheniscus demersus) housed in an outdoor enclosure at the Baltimore Zoo have an average 50% mortality from avian malarial (Plasmodium sp.) infection each year without intense monitoring for disease and chemotherapeutic intervention. During the 1996 malaria transmission season, the safety and efficacy of an anti-circumsporozoite (CSP) DNA vaccine encoding the Plasmodium gallinaceum CSP protein against P. relictum were studied. The goal was to reduce clinical disease and death without initiating sterile immunity after release into an area with stable, endemic avian malaria. The birds were monitored for adverse clinical signs associated with vaccination, the stimulation of an anti-CSP antibody response, and protection afforded by the vaccine. The presence of P. relictum in trapped culicine mosquitoes within the penguin enclosure was monitored to assess parasite pressure. Among the vaccinated penguins, the parasitemia rate dropped from approximately 50% to approximately 17% despite intense parasite pressure, as determined by mosquito infection rate. During the year of the vaccine trial, no mortalities due to malaria occurred and no undesirable vaccination side effects occurred. This is the first trial of an antimalarial vaccine in a captive penguin colony.

Pathogenic action of Plasmodium gallinaceum in chickens: brain histology and nitric oxide production by blood monocyte-derived macrophages.

Plasmodium infection causes major losses to animal and human populations. The characterization of experimental malaria models is needed for a better understanding of disease mechanisms and the development of new treatment protocols. Chickens infected with Plasmodium gallinaceum constitute an adequate malaria model due to the phylogenetic proximity of this parasite to human Plasmodium as well as similarities in disease manifestation, such as cerebral malaria. The aim of the present study was to further characterize the experimental chicken model with an emphasis on clinical manifestations, cerebral histology and nitric oxide (NO) produced by macrophages. The results revealed that mortality was correlated to higher parasitemia. Parasitemia was positively correlated to temperature and negatively correlated to haematocrit value. Brain histology of infected birds revealed inflammatory infiltrates and blocked microvasculature. Macrophages derived from blood monocytes produced NO after activation, with a higher production positively correlated to parasitemia. These results characterize histological aspects of chicken brain malaria and demonstrate the activation of the innate immune system caused by the infection in chickens.

Effect of the Aedes fluviatilis saliva on the development of Plasmodium gallinaceum infection in Gallus (gallus) domesticus.

Effect of Aedes fluviatilis saliva on the development of Plasmodium gallinaceum experimental infection in Gallus (gallus) domesticus was studied in distinct aspects. Chickens subcutaneously infected with sporozoites in the presence of the mosquito salivary gland homogenates (SGH) showed higher levels of parasitaemia when compared to those ones that received only the sporozoites. However, the parasitaemia levels were lower among chickens previously immunized by SGH or non-infected mosquito bites compared to the controls, which did not receive saliva. High levels of anti-saliva antibodies were observed in those immunized chickens. Moreover, 53 and 102 kDa saliva proteins were recognized by sera from immunized chickens. After the sporozoite challenge, the chickens also showed significant levels of anti-sporozoite antibodies. However, the ability to generate anti-sporozoites antibodies was not correlated to the saliva immunization. Our results suggest that mosquito saliva components enhance P. gallinaceum parasite development in naive chickens. However, the prior exposure of chickens to salivary components controls the parasitemia levels in infected individuals.

The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes.

We studied the point at which a monoclonal antibody (mAb C5) to a surface protein (Pgs25) on Plasmodium gallinaceum ookinetes blocked the infection of Aedes aegypti mosquitoes. The antibody did not block the development of zygotes to ookinetes in vitro. Development of ookinetes to oocysts in the mosquito was blocked to the same extent whether zygotes grew to ookinetes in the presence of mAb C5 or the antibody was added after the ookinetes had reached full development. When ookinetes developed in vitro in the presence of mAb C5, antibody remained on the surface of the parasite for the next 50 hr and did not block attachment to the peritrophic membrane. When ookinetes were fed to mosquitoes, two subpopulations of mosquitoes were observed (high numbers of oocysts per midgut and low numbers of oocysts per midgut). mAb C5 reduced the number of oocysts per midgut in the subpopulation that had low numbers of oocysts. The subpopulation that had high numbers of oocysts was unaffected by antibody, indicating that the antibody did not block invasion of the midgut epithelium. When mAb C5 was fed with gametocytes, the parasites invaded the epithelium at the same time (between 30 and 35 hr after the blood meal) as in controls, although at a markedly reduced rate. The ultrastructural observations were consistent with a block of parasites within the peritrophic membrane and not with a block at the epithelium, as parasites were not seen to accumulate within the space between the peritrophic membrane and the epithelium. The mechanism by which mAb C5 to Pgs25 of P. gallinaceum blocks the penetration of the peritrophic membrane remains undefined. We present evidence that the parasite modifies the peritrophic membrane during penetration, an observation first made for Babesia microti during penetration of the peritrophic membrane in Ixodes ticks. Ookinetes in the absence of antibodies appeared to disrupt the layers of the peritrophic membrane, suggesting an enzymatic mechanism for penetration.

Reduced efficacy of the immune melanization response in mosquitoes infected by malaria parasites.

Although the mosquito vectors of malaria have an effective immune system capable of encapsulating many foreign particles, they rarely encapsulate malaria parasites in natural populations. A possible reason for this apparent paradox is that infection by malaria reduces the capability of the mosquito to mount an effective immune response. To investigate this possibility, we blood-fed Aedes aegypti mosquitoes on an uninfected chicken or on one infected with Plasmodium gallinaceum, and compared the proportions of the infected and uninfected mosquitoes that melanized a negatively charged Sephadex bead injected into the thorax 1, 2 and 4 days after blood-feeding. About 40% of the uninfected mosquitoes, but less than 25% of the infected ones, melanized the bead. The difference between infected and uninfected mosquitoes was most obvious 1 day after infection (at the parasite's ookinete stage), while the difference diminished during the early oocyst stage (2 days after infection) and disappeared at the later oocyst stage (4 days after infection). These results suggest that the parasite can either actively suppress its vector's immune response or that it modifies the blood of its chicken host in away that reduces the efficacy of the mosquito's immune system. In either case, the reduction of immunocompetence can have important consequences for malaria control, in particular for the current effort being invested into the genetic manipulation of mosquitoes.