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.

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.

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.

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.

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.

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.

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.

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.

Monoclonal antibody against the Plasmodium falciparum chitinase, PfCHT1, recognizes a malaria transmission-blocking epitope in Plasmodium gallinaceum ookinetes unrelated to the chitinase PgCHT1.

To initiate invasion of the mosquito midgut, Plasmodium ookinetes secrete chitinases that are necessary to cross the chitin-containing peritrophic matrix en route to invading the epithelial cell surface. To investigate chitinases as potential immunological targets of blocking malaria parasite transmission to mosquitoes, a monoclonal antibody (MAb) was identified that neutralized the enzymatic activity of the sole chitinase of Plasmodium falciparum, PfCHT1, identified to date. This MAb, designated 1C3, previously shown to react with an apical structure of P. falciparum ookinetes, also reacts with a discrete apical structure of P. gallinaceum ookinetes. In membrane feeding assays, MAb 1C3 markedly inhibited P. gallinaceum oocyst development in mosquito midguts. MAb 1C3 affinity isolated an approximately 210-kDa antigen which, under reducing conditions, became a 35-kDa antigen. This isolated 35-kDa protein cross-reacted with an antiserum raised against a synthetic peptide derived from the P. gallinaceum chitinase active site, PgCHT1, even though MAb 1C3 did not recognize native or recombinant PgCHT1 on Western blot. Therefore, this affinity-purified 35-kDa antigen appears similar to a previously identified protein, PgCHT2, a putative second chitinase of P. gallinaceum. Epitope mapping indicated MAb 1C3 recognized a region of PfCHT1 that diverges from a homologous amino acid sequence conserved within sequenced chitinases of P. berghei, P. yoelii, and P. gallinaceum (PgCHT1). A synthetic peptide derived from the mapped 1C3 epitope may be useful as a component of a subunit transmission-blocking vaccine.

Identification of novel Plasmodium gallinaceum zygote- and ookinete-expressed proteins as targets for blocking malaria transmission.

The development of transmission-blocking vaccines is one approach to malaria control. To identify novel Plasmodium zygote- and ookinete-secreted proteins as targets of blocking malaria transmission, monoclonal antibodies (MAbs) were produced against parasite-secreted proteins found in Plasmodium gallinaceum ookinete culture supernatants. Four MAbs-1A6, 2A5, 2B5, and 4B6-were identified that bound to P. gallinaceum zygotes and ookinetes in diverse patterns in terms of spatial localization on parasites, time course of antigen expression, and Western immunoblot patterns. MAbs 2A5 and 4B6 recognized more than one protein band as detected by Western immunoblot of P. gallinaceum ookinete supernatants. Beginning at 0 h postfertilization, MAb 2A5 recognized a diverse set of antigens; at 10 h postfertilization, MAb 4B6 recognized several antigens as well. MAb 1A6 recognized a single approximately 17-kDa protein, and 2B5 recognized a single approximately 32-kDa protein at 15 h postfertilization. In membrane feeding assays to assess the effect of these MAbs on P. gallinaceum infectivity for Aedes aegypti mosquitoes, the addition of MAbs 1A6 and 2B5 to infectious blood meals significantly inhibited oocyst development in the mosquito midgut. In contrast, MAb 2A5 seemed to enhance infectivity. These results demonstrate that Plasmodium ookinetes secrete proteins (in addition to previously characterized chitinases) that may be targets for blocking malaria transmission. Future investigation of ookinete-secreted neutralization-sensitive molecules should provide valuable insight into mechanisms by which ookinetes exit the blood meal, penetrate and transverse the peritrophic matrix, and invade the mosquito midgut epithelium.

Susceptibilidade e resposta imune humoral do hospedeiro natural Gallus gallus domesticus a esporozoítas de Plasmodium gallinaceum (Brumpt, 1935)

Investigou-se a susceptibilidade da galinha doméstica, hospedeiro natural do Plasmodium gallinaceum (Pg), aos esporozoítas inoculados por diferentes vias, bem como a sua capacidade de produzir anticorpos anti-esporozoítas. Aves de uma ou três semanas de idade foram inoculadas com esporozoítas pela picada de Aedes Fluviatilis, pela via subcutânea (s.c.) ou intravenosa(i.v.) e as parasitemias acompanhadas através de esfregaços sanguíneos diários. Nas aves de uma semana inoculadas pela picada de Aedes ocorreu 100 por cento de infecção, enquanto nas aves de três semanas ocorreu variação de 40 a 100 por cento, proporcional ao número de fêmeas alimentadas. A inoculação de esporozoítas pela seringa resultou em 100 por cento de infecção. O período pré-patente (PPP) da malária foi menor na inoculação i.v., no entanto, a idade da ave não influenciou o PPP. Aves mais jovens tiveram maiores parasitemias e mortalidades. Nos soros coletados após a primoinoculação, anticorpos anti-esporozoítas foram detectados pela imunofluorescência indireta (IFI) nos dias 7 e 14 após inoculação, declinando na terceira semana.Na Elisa com esporozoítas isolados de mosquitos, os soros foram positivos a partir da terceira semana. Nas três reinoculações de esporozoítas, os títulos de anticorpos atingiram um platô após o segundo inóculo. As aves inoculadas pelas tr~es vias apresentaram títulos similares ao final das reinoculações, embora as que foram inoculadas pela picada apresentaram maior aumento no título de anticorpos. Pelas análises de western-blot com sedimento ou antígenos solúveis de esporozoítas, a proteína circumesporozoíta (cs) foi reconhecida pelos soros de aves inoculadas por qualquer das três vias utilizadas, a partir da segunda semana após inoculação. Outras proteínas de variados pesos moleculares foram também reconhecidas, algumas pertencentes a antígenos obtidos de Aedes não infectados, entre elas uma proteína de 7 kda.

No modelo aviário, o hospedeiro natural produziu anticorpos contra os antígenos de esporozoítas com elevados títulos e prevalência, tendo a proteína CS confirmado a sua imunogenicidade. A possível presença de anticorpos protetores nos soros de aves expostas aos esporozoítas e o estudo das diferenças qualitativas entre os esporozoítas inoculados pela picada e seringa, suas conseqüencias na interação parasita-hospedeiro e resposta imune subseqüente necessitam ser melhor estudadas no futuro.

Susceptibilidade e resposta imune humoral do hospedeiro natural Gallus gallus domesticus a esporozoítas de Plasmodium gallinaceum (Brumpt, 1935) / Susceptibility and humoral immune response of the natural host Gallus gallus domesticus the sporozoite of Plasmodium gallinaceum (Brumpt, 1935)

Investigou-se a susceptibilidade da galinha doméstica, hospedeiro natural do Plasmodium gallinaceum (Pg), aos esporozoítas inoculados por diferentes vias, bem como a sua capacidade de produzir anticorpos anti-esporozoítas. Aves de uma ou três semanas de idade foram inoculadas com esporozoítas pela picada de Aedes Fluviatilis, pela via subcutânea (s.c.) ou intravenosa (i.v.) e as parasitemias acompanhadas através de esfregaços sanguíneos diários. Nas aves de uma semana inoculadas pela picada de Aedes ocorreu 100 por cento de infecção, enquanto nas aves de três semanas ocorreu variação de 40 a 100 por cento, proporcional ao número de fêmeas alimentadas. A inoculação de esporozoítas pela seringa resultou em 100 por cento de infecção. O período pré-patente (PPP) da malária foi menor na inoculação i.v., no entanto, a idade da ave não influenciou o PPP. Aves mais jovens tiveram maiores parasitemias e mortalidades. Nos soros coletados após a primoinoculação, anticorpos anti-esporozoítas foram detectados pela imunofluorescência indireta (IFI) nos dias 7 e 14 após inoculação, declinando na terceira semana. Na Elisa com esporozoítas isolados de mosquitos, os soros foram positivos a partir da terceira semana. Nas três reinoculações de esporozoítas, os títulos de anticorpos atingiram um platô após o segundo inóculo. As aves inoculadas pelas três vias apresentaram títulos similares ao final das reinoculações, embora as que foram inoculadas pela picada apresentaram maior aumento no título de anticorpos. Pelas análises de western-blot com sedimento ou antígenos solúveis de esporozoítas, a proteína circumesporozoíta (cs) foi reconhecida pelos soros de aves inoculadas por qualquer das três vias utilizadas, a partir da segunda semana após inoculação. Outras proteínas de variados pesos moleculares foram também reconhecidas, algumas pertencentes a antígenos obtidos de Aedes não infectados, entre elas uma proteína de 7 kda. No modelo aviário, o hospedeiro natural produziu anticorpos contra os antígenos de esporozoítas com elevados títulos e prevalência, tendo a proteína CS confirmado a sua imunogenicidade. A possível presença de anticorpos protetores nos soros de aves expostas aos esporozoítas e o estudo das diferenças qualitativas entre os esporozoítas inoculados pela picada e seringa, suas conseqüencias na interação parasita-hospedeiro e resposta imune subseqüente necessitam ser melhor estudadas no futuro.

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.

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.

Antisporozoite antibodies with protective and nonprotective activities: in vitro and in vivo correlations using Plasmodium gallinaceum, an avian model.

A correlation was observed between in vivo and in vitro activity of six monoclonal antibodies (mAb) against the major circumsporozoite protein of the avian malaria Plasmodium gallinaceum as follows. (1) Two mAb were protective, totally abrogating sporozoite infectivity to chicks, its natural host, in vivo; they caused 100% inhibition of sporozoite invasion (ISI) in vitro to SL-29 chicken fibroblasts and intense ISI to cultured chicken macrophages, as well as inhibited the exoerythrocytic development of sporozoites taken up by macrophages, the initial cell host of P. gallinaceum sporozoites. (2) Two mAb were partially protective in that they reduced sporozoite infectivity to chicks, caused partial ISI to SL-29 and macrophage cells and partial inhibition to the exoerythrocytic development of sporozoites in macrophages in vitro. (3) Two mAb were totally inactive in vivo although they both bound to the sporozoite antigens as detected by indirect immunofluorescence, western blot, and ELISA; they both failed to induce ISI or inhibit the exoerythrocytic development in macrophages. The possible participation of macrophages as the initial cell type involved in sporozoite destruction in the presence of anti-circumsporozoite antibodies is discussed.

Plasmodium gallinaceum: sporozoite invasion of Aedes aegypti salivary glands is inhibited by anti-gland antibodies and by lectins.

There is evidence which suggests that malaria sporozoites recognize mosquito salivary glands by specific receptor-ligand interactions. We are interested in identifying the putative salivary gland receptor(s) for sporozoite invasion. We used an in vivo bioassay for sporozoite invasion of salivary glands. In this assay, purified sporozoites from mature oocytes of Plasmodium gallinaceum were injected into Aedes aegypti mosquitoes and salivary glands were dissected at different time points after injection. One half of the maximum invasion of salivary glands by sporozoites occurred by 6 hr, and salivary gland sporozoite load did not increase further after 24 hr postinjection. This assay was used to determine the effect of experimental treatments with antibodies and lectins at 24 hr postinjection. We raised a rabbit polyclonal antiserum against female Ae. aegypti salivary glands which recognized tissue-specific determinants in the basal lamina of salivary glands. Purified IgG antibody fraction of the immune serum blocked sporozoite invasion in vivo. We tested a panel of 19 lectins and found 7 which bound to salivary glands. Of these 7, succinylated wheat germ agglutinin and wheat germ agglutinin completely blocked sporozoite invasion; Pisum sativum agglutinin and soybean agglutinin partially blocked; and concanavalin A, Dolichos biflorus agglutinin, and Phaseolus vulgaris erythroagglutinin did not block. Our results suggest that sporozoites interact with glycosylated salivary gland surface molecules which serve as receptors for invasion, and which may be in the salivary gland basal lamina. Because the putative sporozoite receptors contain immunogenic determinants, it is feasible to identify them by an immunological strategy.

Developmental changes in the circumsporozoite proteins of Plasmodium berghei and P. gallinaceum in their mosquito vectors.

The circumsporozoite (CS) protein covers the surface of the sporozoite of plasmodia. Its role in the development of the malaria parasite in mosquito vectors remains unknown. CS-epitope-containing proteins appear on undifferentiated oocysts on day 7 in Plasmodium berghei and on day 5 in P. gallinaceum as demonstrated by indirect fluorescence antibody tests using monoclonal antibodies directed against the CS-protein repeats. The three-dimensional distribution of the CS-epitope-containing proteins on oocysts was analyzed by confocal scanning laser microscopy. A strong antibody binding was found in patches around the oocysts of P. berghei and P. gallinaceum, and an accumulation of labeled proteins was found at the base of the oocysts of both species. In Western blots of infected midguts and salivary glands the antibodies recognized two peptides in the salivary glands but up to ten peptides in midgut extracts. The larger number of peptides recognized in midgut preparations might indicate breakdown products during the escape of the sporozoites from the oocyst and their migration on the midgut in the mosquito vector. The data indicate a possible involvement of the CS protein in an active migration process of the sporozoites in the mosquito vector.