The effect of chloroquine treatment on the infectivity of Plasmodium chabaudi gametocytes.

The antimalarial drug chloroquine has been reported to increase the infectivity of the forms of blood-stage malaria parasites (gametocytes) that are capable of infecting mosquito vectors. This effect has been demonstrated convincingly in the short term (12 h post treatment), although several authors have suggested infectivity enhancement a week or more after treatment. We carried out experiments to investigate the effects of chloroquine on the longer-term infectivity of gametocytes of the rodent malaria parasite, Plasmodium chabaudi, to Anopheles stephensi mosquitoes. Gametocytes of chloroquine-treated infections were significantly more infectious than untreated infections 6 and 7 days post-treatment, although not on days 8 and 9. However, this effect was most likely the result of a reduction in infectivity in untreated infections, caused by immune activity which was not so pronounced in chloroquine-treated infections. Gametocytaemia (gametocytes per r.b.c.) showed a strong positive and linear relationship with infectivity. Infectivity was not influenced by either asexual parasitaemia, asexual density or anaemia. Parsimonious interpretations of the effect of chloroquine on gametocyte infectivity are discussed.     

Plasmodium falciparum gametocytes: their longevity and infectivity.

The longevity and infectivity of isolated populations of Plasmodium falciparum gametocytes were studied. Following chloroquine treatment gametocyte numbers fell with a constant rate of loss over a period of 16-24 days; the populations had a half-life of 2-4 days. The sex ratio stayed constant throughout at 4 female: 1 male. The ability of the microgametocytes to exflagellate and the infectivity of the population to mosquitoes persisted for 3 weeks. Antibodies to the gametocytes were detected but not in every patient studied. It was concluded that the gametocytes of P. falciparum are both long-lived and show persistent infectivity to mosquitoes. They can stimulate antibody production but the immune response appears to play no part in their elimination, which probably takes place in the spleen as a part of the normal process of removing old, damaged and malformed red cells.     

Human immune responses that reduce the transmission of Plasmodium falciparum in African populations

Malaria-infected individuals can develop antibodies which reduce the infectiousness of Plasmodium gametocytes to biting Anopheles mosquitoes. When ingested in a bloodmeal together with gametocytes, these antibodies reduce or prevent subsequent parasite maturation in the insect host. This transmission-blocking immunity is usually measured in human sera by testing its effect on the infectivity of gametocytes grown in vitro. Here we evaluate evidence of transmission-blocking immunity in eight studies conducted in three African countries. Plasmodium falciparum gametocytes isolated from each individual were fed to mosquitoes in both autologous plasma collected with the parasites, and permissive serum from non-exposed donors. Evidence of transmission reducing effects of autologous plasma was found in all countries. Experiments involving 116 Gambian children (aged 0.5-15 years) were combined to determine which factors were associated with transmission reducing immune responses. The chances of infecting at least one mosquito and the average proportion of infected mosquitoes were negatively associated with recent exposure to gametocytes and sampling late in the season. These results suggest that effective malaria transmission-reducing antibodies do not commonly circulate in African children, and that recent gametocyte carriage is required to initiate and/or boost such responses.     

Plasmodium cynomolgi: serum-mediated blocking and enhancement of infectivity to mosquitoes during infections in the natural host, Macaca sinica

The infectivity of Plasmodium cynomolgi in its natural host, the toque monkey, Macaca sinica, to Anopheles tessellatus mosquitoes was studied in relation to the evolution of anti-sexual-stage immunity in the host during the course of a blood-induced infection. The effects of serum on the infectivity of gametocytes and the intrinsic infectivity of gametocytes to mosquitoes on each day were assessed in membrane feeding experiments. Mosquitoes were also directly fed on the animal on each day. Our results demonstrate that during the very early patent period, before the peak of gametocytemia, the infection serum enhanced the infectivity of gametocytes up to two to three times above their infectivity in normal monkey serum. Subsequently, serum drawn post-peak of parasitemia ceased to enhance, and began to suppress, infectivity. After 2-3 months, long after parasitemias ceased patency, the serum no longer suppressed and between 3 and 4 months the serum again tended to enhance gamete infectivity before losing any significant effect. Serum infectivity enhancing effects were consistent with low indirect immunofluorescence test antibody titers against blood stage parasites first during the very early days of a blood infection before reaching blocking levels, and again during convalescence when antibodies were declining. The serum infectivity blocking effects on gametocytes were seen at the peak of antibody titers from about Days 9 to 23 of an infection. From 78 to 95% of the total infectivity of the parasite to mosquitoes during an infection occurred when infectivity enhancing activity was present in the serum. Hence, the infectivity of the parasite to mosquitoes was largely dependent on infectivity enhancing antibodies in host serum.     

Short-term effect of chloroquine on the infectivity of Plasmodium chabaudi gametocytes.

The short-term enhancing effect of chloroquine on gametocyte infectivity was investigated with Plasmodium chabaudi chabaudi, a synchronous parasite which is highly sensitive to chloroquine. In comparison with control groups, oocyst numbers increased in mosquitoes fed on mice 12 hours after the injection of 5 mg/kg chloroquine (180% of controls) although it was not statistically significant. No effect was seen with 1 mg/kg chloroquine. The authors interpretation is that chloroquine impaired the schizogony, thus reducing also the release of toxic material of parasite origin which blocks gametocytes infectivity. Results of similar experiments with other rodent species of Plasmodium are compared and discussed in relation with the chronobiological characteristics of these parasites.     

Chronosexuality of Plasmodium species of Central African Muridae.

A host harbouring many parasite species of the same genus is a phenomenon frequently observed in numerous parasitic infections. This is the case for the Plasmodium parasites of Muridae in Central Africa, where three different parasite species are found in the same rodent host species. It is highly likely that these three Plasmodium species are transmitted simultaneously by the same vector. We and others have shown that the maturation periods of the various asexual and sexual stages in the rodent, differ amongst the three parasites. In this article we propose that these differences are the product of complex adaptations which result, for all three Plasmodium species, in a maximum peak of infectivity to the insect vector occurring around 3 a.m., the period of highest activity of the nocturnal host rodent.     

The novel putative transporter NPT1 plays a critical role in early stages of Plasmodium berghei sexual development

Transmission of Plasmodium species from a mammalian host to the mosquito vector requires the uptake, during an infected blood meal, of gametocytes, the precursor cells of the gametes. Relatively little is known about the molecular mechanisms involved in the developmental switch from asexual development to sexual differentiation or the maturation and survival of gametocytes. Here, we show that a gene coding for a novel putative transporter, NPT1, plays a crucial role in the development of Plasmodium berghei gametocytes. Parasites lacking NPT1 are severely compromised in the production of gametocytes and the rare gametocytes produced are unable to differentiate into fertile gametes. This is the earliest block in gametocytogenesis obtained by reverse genetics and the first to demonstrate the role of a protein with a putative transport function in sexual development. These results and the high degree of conservation of NPT1 in Plasmodium species suggest that this protein could be an attractive target for the development of novel drugs to block the spread of malaria.     

Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Sulfadoxine-pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.     

Effect of xanthurenic acid on infectivity of Plasmodium falciparum to Anopheles stephensi.

Terminally differentiated malarial gametocytes remain in the vertebrate circulation in a developmentally arrested state until they are taken up by the mosquito. The gametocytes then undergo gametogenesis in the mosquito mid-gut within minutes after ingestion of the infected blood meal. The male gametogenesis (exflagellation) can be triggered by the combination of a decrease in temperature of at least 5 degrees C and a simultaneous increase in pH between 8.0 and 8.3. Xanthurenic acid, which is present in mosquito mid-gut as well as in mosquito head, had been shown to induce exflagellation in vitro at a non-permissible pH. Here we report for the first time that with the increasing concentration of exogenous xanthurenic acid, there is a gradual increase in the number of oocysts in the mid-gut of infected mosquitoes. The concentration of xanthurenic acid for optimum infection in the membrane feeding assay was determined to be 100 microM. Three different strains of Plasmodium falciparum, viz. 3D7, 7G8 and W2 were tested in different experiments and similar findings hold true for all of them. These results demonstrate that xanthurenic acid not only induces exflagellation of male gametocytes but also promotes infectivity of Plasmodium falciparum to mosquito vectors.     

Plasmodium gallinaceum: transmission-blocking immunity in chickens. I. Comparative immunogenicity of gametocyte- and gamete-containing preparations.

Immunization of chickens by intravenous inoculation of preparations derived from blood infected with Plasmodium galinaceum led to reduced infectivity to mosquitoes (Aedes aegypti) during subsequently induced blood infection but had little effect on the course of asexual parasitemia. Immunization with preparations containing intracellular gametocytes was much less effective than immunization with preparations in which the extracellular gametes had been released during gametogenesis (emergence and exflagellation) prior to inoculation. By far the most effective material were preparations of partially purified gametes of both sexes. Three weekly inoculations of this material resulted in 99.99% suppression of infectivity to mosquitoes during subsequently induced blood infection. Preparations of purified gamete material from which gametes of one or another sex were absent were considerably less effective as transmission-blocking immunogens than the mixed gamete preparation. It is possible that the two sexes of gamete act synergistically to induce transmission-blocking immunity.     

Malaria's Missing Number: Calculating the Human Component of R0 by a Within-Host Mechanistic Model of Plasmodium falciparum Infection and Transmission

Human infection by malarial parasites of the genus Plasmodium begins with the bite of an infected Anopheles mosquito. Current estimates place malaria mortality at over 650,000 individuals each year, mostly in African children. Efforts to reduce disease burden can benefit from the development of mathematical models of disease transmission. To date, however, comprehensive modeling of the parameters defining human infectivity to mosquitoes has remained elusive. Here, we describe a mechanistic within-host model of Plasmodium falciparum infection in humans and pathogen transmission to the mosquito vector. Our model incorporates the entire parasite lifecycle, including the intra-erythrocytic asexual forms responsible for disease, the onset of symptoms, the development and maturation of intra-erythrocytic gametocytes that are transmissible to Anopheles mosquitoes, and human-to-mosquito infectivity. These model components were parameterized from malaria therapy data and other studies to simulate individual infections, and the ensemble of outputs was found to reproduce the full range of patient responses to infection. Using this model, we assessed human infectivity over the course of untreated infections and examined the effects in relation to transmission intensity, expressed by the basic reproduction number R₀ (defined as the number of secondary cases produced by a single typical infection in a completely susceptible population). Our studies predict that net human-to-mosquito infectivity from a single non-immune individual is on average equal to 32 fully infectious days. This estimate of mean infectivity is equivalent to calculating the human component of malarial R₀. We also predict that mean daily infectivity exceeds five percent for approximately 138 days. The mechanistic framework described herein, made available as stand-alone software, will enable investigators to conduct detailed studies into theories of malaria control, including the effects of drug treatment and drug resistance on transmission.     

The effect of partial host immunity on the transmission of malaria parasites.

Experiments were carried out to determine the effect of partial host immunity against the rodent malaria parasite Plasmodium chabaudi on the transmission success of the parasite. There was a fourfold reduction in both the blood-stage, asexually replicating parasite density and the gametocyte (transmissable stage) density in immunized hosts. Some of the reduction in asexual parasite densities was due to strain-specific immunity, but there was no evidence that strain-specific immunity affected gametocyte densities. However, immunity did affect transmission in a strain-specific manner, with a fivefold reduction in gametocyte infectivity to mosquitoes in homologous challenges compared with heterologous challenges or non-immunized controls. This implies the existence of a mechanism of strain-specific infectivity-reducing immunity that does not affect the density of gametocytes circulating in peripheral blood. The proportion of asexual parasites that produced gametocytes increased during the course of infection in both non-immunized and in immunized hosts, but immunity increased gametocyte production early in the infection.     

The development of exoerythrocytic stages of Plasmodium inui shortti in New World monkeys

Attempts are being made to adapt Old World monkey malarial parasites to New World monkeys for vaccine and molecular studies. Several of these (Plasmodium cynomolgi Berok, Plasmodium fragile, and Plasmodium knowlesi) grow readily but have failed to produce infective gametocytes. Plasmodium gonderi and Plasmodium fieldi develop in the liver after sporozoite inoculation but have failed to establish infection in the erythrocyte. Anopheles dirus mosquitoes infected with Plasmodium inui shortti by feeding on infected macaques transmitted the infection to Saimiri boliviensis monkeys. Infective gametocytes were produced, and sporozoite transmission from Saimiri to Saimiri monkey was obtained. Exoerythrocytic stages have also been observed in the liver tissue of Saimiri monkeys. The availability of the complete transmission cycle provides an additional resource for immunologic and vaccine studies.     

A developmental defect in Plasmodium falciparum male gametogenesis

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha- tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.     

Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement

Gametocytes are the intraerythrocytic stages of malaria parasites that infect mosquitoes. When gametocytes of the chicken malaria parasite Plasmodium gallinaceum are ingested by a mosquito they become extracellular in the mosquito midgut, form gametes, and fertilize within 10 to 15 min after the insect has taken a blood meal. Gametocytes of P. gallinaceum were infectious when fed to Aedes aegypti mosquitoes in blood meals containing native serum from chickens or from the non-host species, man or sheep. Gametocytes stimulated to undergo gametogenesis and to fertilize in vitro were also infectious when fed to mosquitoes in native chicken serum. However, native serum from most non-host species, including sheep and man, suppressed the infectivity of newly fertilized zygotes to mosquitoes and lysed the zygotes in vitro. These effects were shown to be due to the activity of the alternative pathway of complement (APC) in the serum of the non-host species. After mild trypsin treatment, the zygotes of P. gallinaceum no longer infected mosquitoes in the presence of native chicken serum, although in heat-inactivated chicken serum their infectivity was normal. We conclude that trypsin-sensitive components on the zygotes surface protect them from destruction by the APC of their native host. The ability of gametocytes of P. gallinaceum to infect mosquitoes in the presence of native human serum is probably due to proteases that inactivate the APC of human serum before the gametes and zygotes emerge as extracellular parasites in the blood meal.     

A malarial parasite of Australian skinks, Plasmodium mackerrasae sp. n.

Plasmodium mackerrasae sp. n. parasitizes the Australian lizards Egernia cunninghami and E. striolata (Sauria: Scincidae). Described from an experimental host, E. whitei, it produces mature schizonts containing 6--12 nuclei arranged peripherally as a rosette, and round to oval gametocytes which are equal to or slightly smaller than host cell nuclei. Both schizonts and gametocytes parasitize all cells in the erythrocyte series. Presence of pigment in both asexual and sexual stages is correlated with maturity of the host cell. Asexual forms contain a single large vacuole, whereas mature gametocytes may show 1--4 vacuoles. Plasmodium mackerrasae resembles most closely P. sasai of Japan and P. tropiduri of tropical America. It differs from P. sasai by lacking fan-shaped schizonts and by having less heavily pigmented gametocytes, and from P. tropiduri by less variability in shape and greater vacuolation of the gametocytes. Host and geographic differences further support its distinction.     

Malaria transmission,sex ratio and erythrocytes with two gametocytes

Transmission of haemospororin parasites (phylum Apicomplexa) needs the fertilization of at least one female by one male gamete within the bloodmeal of a suitable vector. Male and female gamete precursors (gametocytes) in Plasmodium and Haemoproteus parasites are normally alone inside the erythrocytes of the vertebrate host, but they also occur in male-female pairs in single erythrocytes. These paired gametocytes could enhance transmission success by facilitating the encounter between the female and male gametes when inside the midgut of the vector. Further study of these particular infections could provide new insights into the biology of and control strategies for haemospororin parasites.