Search

Experimental studies for understanding the relationship between Plasmodium vivax and its vector hosts are difficult, because of to the lack of a long-term, in vitro continuous culture system unavailability of infected blood samples, seasonality of the disease, and the concentration of most cases in remote areas. This study evaluates the duration of the infectivity of P. vivax to Anopheles aquasalis after collecting blood from malaria-infected patients. Blood was collected from patients and stored at 4 °C and 37 °C. Every day, for 4 days, the blood was fed to An. aquasalis adult females, and a Giemsa-stained thick blood smear was mounted to account for sexual (gametocytes) and asexual (trophozoites and schizonts) stages and calculate parasitemia. Oocysts in the midgut of the mosquitoes were counted on the seventh day after feeding. Kruskal-Wallis test was used to compare the mean number of oocysts (MO) and the parasite density (PD) in each storage condition and post-infection time-points. The Mann-Whitney test was used to compare the number of oocysts for each day between temperatures. The results show that P. vivax stored at 4 °C and at 37 °C has its infectivity to An. aquasalis preserved for 2 days and 3 days, respectively. Infection rate (IR), PD and MO were higher on the day of blood collection and decreased gradually over time. The parasite density (number of parasites/μL) diminished faster at 4 °C than at 37 °C. In this study, a preservation protocol is shown for long-lasting infectivity of P. vivax in a blood sample taken from malaria-infected patients. These results show that infectivity of P. vivax stored at 4 °C and at 37 °C to An. aquasalis persist until 3 days after blood collection, but parasite density, infection rate, and mean of oocysts decreased 24h after blood collection. Since the malaria cases are increasingly far from the urban areas these results indicate that is possible, losing some infectivity, to realize experimental infections several dozen hours after the blood collection. However, it is necessary to improve the procedures for preserving P. vivax gametocytes for mosquito infection in the laboratory.

The evaluation of transmission reducing interventions (TRI) to control malaria widely uses membrane feeding assays. In such assays, the intensity of Plasmodium infection in the vector might affect the measured efficacy of the candidates to block transmission. Gametocyte density in the host blood is a determinant of the infection success in the mosquito, however, uncertain estimates of parasite densities and intrinsic characteristics of the infected blood can induce variability. To reduce this variation, a feasible method is to dilute infectious blood samples. We describe the effect of diluting samples of Plasmodium -containing blood samples to allow accurate relative measures of gametocyte densities and their impact on mosquito infectivity and TRI efficacy. Natural Plasmodium falciparum samples were diluted to generate a wide range of parasite densities, and fed to Anopheles coluzzii mosquitoes. This was compared with parallel dilutions conducted on Plasmodium berghei infections. We examined how blood dilution influences the observed blocking activity of anti-Pbs28 monoclonal antibody using the P. berghei/Anopheles stephensi system. In the natural species combination P. falciparum/An. coluzzii , blood dilution using heat-inactivated, infected blood as diluents, revealed positive near linear relationships, between gametocyte densities and oocyst loads in the range tested. A similar relationship was observed in the P. berghei/An. stephensi system when using a similar dilution method. In contrast, diluting infected mice blood with fresh uninfected blood dramatically increases the infectiousness. This suggests that highly infected mice blood contains inhibitory factors or reduced blood moieties, which impede infection and may in turn, lead to misinterpretation when comparing individual TRI evaluation assays. In the lab system, the transmission blocking activity of an antibody specific for Pbs28 was confirmed to be density-dependent. This highlights the need to carefully interpret evaluations of TRI candidates, regarding gametocyte densities in the P. berghei/An. stephensi system.

Actin has important roles in Plasmodium parasites but its exact function in different life stages is not yet fully elucidated. Here we report the localization of ubiquitous actin I in gametocytes of the rodent model parasite P. berghei. Using an antibody specifically recognizing F-actin and deconvolution microscopy we detected actin I in a punctate pattern in gametocytes. 3D-Structured Illumination Microscopy which allows sub-diffraction limit imaging resolved the signal into structures of less than 130 nm length. A portion of actin I was soluble, but the protein was also found complexed in a stabilized form which could only be completely solubilized by treatment with SDS. An additional population of actin was pelleted at 100 000 × g, consistent with F-actin. Our results suggest that actin in this non-motile form of the parasite is present in short filaments cross-linked to other structures in a cytoskeleton.

Species of Haemoproteus (Haemoproteidae) are cosmopolitan haemosporidian parasites, some of which cause severe diseases in birds. Numerous recent studies address molecular characterization, distribution and genetic diversity of haemoproteids. However, the information about their vectors is scarce. We investigated sporogonic development of two widespread species of Haemoproteus (Haemoproteus minutus and Haemoproteus belopolskyi) in the experimentally infected biting midge Culicoides impunctatus. Wild-caught flies were allowed to take blood meals on naturally infected common blackbirds Turdus merula and icterine warblers Hippolais icterina harboring mature gametocytes of H. minutus (lineage hTURDUS2) and H. belopolskyi (hHIICT1), respectively. The engorged flies were collected, transported to the laboratory, held at 15–18 °C, and dissected daily in order to obtain ookinetes, oocysts and sporozoites. Mature ookinetes of H. minutus developed blisteringly rapidly; they were numerous in the midgut content between 1 and 4 h post exposure. Ookinetes of H. belopolskyi developed slower and were reported 1 day post exposure (dpe). Oocysts of both parasites were seen in the midgut wall 3–4 dpe. Sporozoites of H. minutus and H. belopolskyi were first observed in the salivary glands preparations 7 dpe. The percentage of experimentally infected flies with sporozoites of H. minutus was 82.1% and 91.7% with H. belopolskyi. In accordance with microscopy data, polymerase chain reaction amplification and sequencing confirmed presence of the corresponding parasite lineages in experimentally infected biting midges. Sporogonic stages of these parasites were described and illustrated. This study indicates that C. impunctatus is involved in the transmission of deadly H. minutus, which kills captive parrots in Europe. This biting midge is an important vector of avian haemoproteids and worth more attention in epidemiology research of avian haemoproteosis.

In vitro culture of Plasmodium falciparum gametocytes is essential for studying sexual development of the parasite. Here we describe a simple method for producing synchronous gametocyte culture without contamination of asexual stages. This method employs heparin’s activity in blocking merozoite invasion of erythrocytes to eliminate asexual stage parasites from gametocyte culture. We show that following induction of gametocyte formation, addition of heparin in culture medium for four days effectively eliminates asexual stages and produces pure, synchronous cultures of gametocytes. Compared with the commonly used N-acetylglucosamine treatment method, heparin treatment requires shorter time to eliminate asexual stages and causes significantly less hemolysis in late stage gametocyte cultures.

Genetic cross is a powerful tool for studying malaria genes contributing to drug resistance, parasite development, and pathogenesis. Cloning and identification of recombinant progeny (RP) is laborious and expensive, especially when a large proportion of progeny derived from self-fertilization are present in the uncloned progeny of a genetic cross. Since the frequency of cross-fertilization affects the number of recombinant progeny in a genetic cross, it is important to optimize the procedure of a genetic cross to maximize the cross-fertilization. Here we investigated the factors that might influence the chances of obtaining RP from a genetic cross and showed that different Plasmodium yoelii strains/subspecies/clones had unique abilities in producing oocysts in a mosquito midgut. When a genetic cross is performed between two parents producing different numbers of functional gametocytes, the ratio of parental parasites must be adjusted to improve the chance of obtaining RP. An optimized parental ratio could be established based on oocyst counts from single infection of each parent before crossing experiments, which may reflect the efficiency of gametocyte production and/or fertilization. The timing of progeny cloning is also important; cloning of genetic cross progeny from mice directly infected with sporozoites (vs. frozen blood after needle passage) at a time when parasitemia is low (usually

Proteases are crucial enzymes with varying roles in living organisms. In the malaria parasite Plasmodium falciparum, the role of proteases has been deciphered mainly in the asexual blood stages and shown to represent promising drug targets. However, little is known about their functions in the sexual blood stages, which are important for transmission of the disease from the human to the mosquito vector. Determination of their stage-specific expression during the malaria life-cycle is crucial for the effective design of multi-stage anti-malaria drugs aimed at eradicating the disease. In this study, we screened the P. falciparum genome database for putative proteases and determined the transcript and protein expression profiles of selected proteases in the plasmodial blood stages using semi-quantitative RT-PCR and indirect immunofluorescence assay. Database mining identified a total of 148 putative proteases, out of which 18 were demonstrated to be expressed in the blood stages on the transcript level; for 12 of these proteins synthesis was confirmed. While three of these proteases exhibit gametocyte-specific expression, two are restricted to the asexual blood stages and seven are found in both stages, making them interesting multi-stage drug targets.

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per microL of blood for asexual stage and 88-264 parasites per microL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.

Mature gametocytes, the sexual stage of Plasmodium falciparum, ensure the continued transmission of malaria from the human host to the mosquito vector. Even if gametocytes are not implicated in the malaria physiopathology it is crucial to the spread of malaria. Gametocytes are to be a key target for drugs used against Plasmodium in public health. The expression levels of 4 sexual-stage specific genes, Pfs 16, Pfs 25, Pfg 27 and S 18S rRNA, during gametocytogenesis of various P. falciparum strains were analyzed by a real time PCR assay. The strains showed different capacities to produce mature gametocytes and in parallel different patterns of sexual gene expression. There was a correlation only between Pfs 16 cDNA overexpression in the first 48h of the culture and the production of mature gametocytes. Pfs 16 is an early marker of the development of mature gametocytes in cultures and is therefore a potential target for new antimalarial drugs.

Genes encoding Plasmodium falciparum proteins Pfs16 and Pfpeg3/mdv1, specifically appearing in the parasitophorous vacuole of the early gametocytes, are upregulated at the onset of sexual differentiation. Analysis of asexual development in gametocyte producing and non-producing clones of P. falciparum indicated that these genes are also transcribed at a low level in asexual parasites, although their protein products are not detectable in these stages by immunofluorescence. Immunoelectron microscopic analysis of stage II gametocytes indicated that Pfs16 and Pfpeg3/mdv1 proteins co-localise in the parasitophorous vacuole membrane and in all derived membranous structures (such as the multi-laminate membrane whorls of the circular clefts in the infected erythrocyte cytoplasm and the membranes of the gametocyte food vacuoles). In this analysis both proteins were also observed for the first time in the membrane and in the lumen of distinct cleft-like structures in the erythrocyte cytoplasm.

While seeking strategies for interfering with Plasmodium development in vertebrate/invertebrate hosts, we tested the activity of gomesin, an antimicrobial peptide isolated from the hemocytes of the spider Acanthoscurria gomesiana. Gomesin was tested against asexual, sexual and pre-sporogonic forms of Plasmodium falciparum and Plasmodium berghei parasites. The peptide inhibited the in vitro growth of intraerythrocytic forms of P. falciparum. When gomesin was added to in vitro culture of P. berghei mature gametocytes, it significantly inhibited the exflagellation of male gametes and the formation of ookinetes. In vivo, the peptide reduced the number of oocysts of both Plasmodium species in Anopheles stephensi mosquitoes, and did not appear to affect the mosquitoes. These properties make gomesin an excellent candidate as a transmission blocking agent for the genetic engineering of mosquitoes.

In Plasmodium falciparum, a family of six secreted proteins having a conserved architecture of multiple adhesive domains was recently identified by genome annotation. Three of the proteins, termed PfCCp1, PfCCp2, and PfCCp3 due to a common LCCL domain, are expressed inside the gametocyte parasitophorous vacuole and released during gamete emergence, where they relocate extracellularly surrounding exflagellation complexes. In this study we show that the three PfCCp proteins are co-expressed at the gametocyte surface. Abrogation of PfCCp3 in gene disruptant parasites leads to the loss of PfCCp1 and PfCCp2 protein but not transcript, indicating a co-dependent protein expression. In view of these data we propose that the three PfCCp proteins interact during gametocytogenesis by formation of a protein complex.

The molecular mechanisms underlying gametocytogenesis in malaria parasites are not understood. Plasmodium falciparum cdc2-related kinase 1 (pfcrk-1), a gene that is expressed predominantly in gametocytes, bears homology to the PITSLRE subfamily of cyclin-dependent kinases and has been hypothesized to function as a negative regulator of the cell cycle. We attempted to knock-out pbcrk-1, the P. berghei orthologue of pfcrk-1, but were unable to recover P. berghei parasites with a disrupted pbcrk-1 locus. In contrast, an integration event at this locus that did not result in a loss-of-function of the pbcrk-1 gene was readily observed. This strongly suggests that a functional pbcrk-1 gene product is essential to intraerythrocytic asexual multiplication.

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 μM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l -cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1 mM), and exflagellation center numbers were restored by the addition of Zn2+. A reduction of ookinete production was observed when TPCK/TLCK (100 μM) was added at 2 h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1 mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.

Cui, L., Rzomp, K. A., Fan, Q., Martin, S. K., and Williams, J. 2001. Plasmodium falciparum: Differential display analysis of gene expression during gametocytogenesis. Experimental Parasitology 99 , 244–254. With the Plasmodium falciparum genome sequencing near completion, functional analysis of individual parasite genes has become the major task of the postgenomic era. Understanding the expression patterns of individual genes is the initial step toward this goal. In this report, we have examined gene expression during gametocytogenesis of the malaria parasite, P. falciparum , using a modified differential display (DD) method. The modifications of this method include adjusting the dNTP mix, using upstream primers with higher AT contents, and reducing the extension temperature of the polymerase chain reaction (PCR). With a combination of 16 arbitrary upstream primers and 3 one-base-anchored oligo(dT) primers, we have successfully cloned 80 unique cDNA tags from stage IV–V gametocytes. Further analysis by dot blots and semiquantitative reverse transcriptase-PCR showed that at least 49 cDNAs had induced or elevated levels of expression in gametocytes. These results indicate that this modified DD procedure is suitable for large-scale identification of developmentally regulated genes in the AT-rich Plasmodium genome.

Both early gametocytes (I-II) and asexual trophozoite stages of Plasmodium falciparum digest hemoglobin and detoxify haem by polymerizing it into parasite pigment called hemozoin. The mechanism of polymerization is unclear but it has been proposed that histidine-rich protein II may facilitate transport of hemoglobin to the food vacuole and catalyze the polymerization in asexual stages. We describe the transcription of histidine-rich protein II in gametocytes by Northern blot analysis and the expression of the protein in these stages by immunoprecipitation and Western blotting. Localization of histidine-rich protein II within the gametocyte by immunofluorescence assay and immunoelectron microscopy clearly illustrated the presence of this molecule in the infected red cell cytosol in the early stages of gametocyte development and internalization in the later gametocyte as it matures. There is a strong correlation between the stage-specific trafficking of histidine-rich protein II in gametocytes and the susceptibility of early but not late gametocytes to the antimalarial drug chloroquine.

Concentrates of late stage parasites of the gametocyte-forming clone HB-3 were mixed with blood rich in reticulocytes from anemic patients, or with normal control blood, and kept under culture conditions for 4 days. Significantly more gametocytes were always formed in the reticulocyte-rich blood than in the control. This was true whether the anemic blood supported a larger asexual parasitemia than the control, or a lower one, or the same and without regard to the cause of the anemia. Gametocytes as a percentage of asexual forms were up to 10 times higher in reticulocyte-rich blood than in normal blood.

Item does not contain fulltext 4 p.

The correlation of P. falciparum lactate dehydrogenase (pLDH) activities and patent infections was evaluated for monitoring therapeutic responses and drug resistance in 70 patients with microscopically confirmed P. falciparum malaria in Nigeria. Each patient was treated with standard dosages of artemether (53 patients), chloroquine (7 patients), sulfadoxine-pyrimethamine (6 patients), or halofantrine (4 patients). Response of infection to treatment was monitored by microscopic examination of thick and thin blood smears, clinical symptoms, and levels of pLDH activities in blood products. pLDH activity was determined using an antibody capture technique and 3-acetyl pyridine adenine dinucleotide developed to enhance sensitivity of the enzyme detection. All patients treated with artemether were cured while 5 patients treated with chloroquine, 1 treated with sulfadoxine-pyrimethamine, and 2 treated with halofantrine suffered recrudescent infections after treatment. pLDH activity was detected in blood products obtained from patients with patent or recrudescent infections determined by microscopy and clinical symptoms. Levels of pLDH activities in whole blood and packed cells from the patients correlated with qualitative detection of parasites in blood smears and in patients with high gametocyte counts. Gametocyte counts in the patients after treatment ranged from 40 gametocytes/microliter of blood to 4923 gametocytes/microliter of blood. There is a consistent relationship between patent infection and pLDH activities that could easily be determined in whole blood and packed cells from the patients. Further development of the procedure will enhance its valuable application in clinical management of drug-resistant malaria in the endemic areas.

Some molecular characteristics of Plasmodium falciparum lines which do not produce gametocytes are described. Parasites carrying a subtelomerically deleted chromosome 9 cannot form even the earliest forms of gametocytes, detectable with antibodies against the gametocyte-specific antigen Pfg27. In a parasite culture of clone HB3, in which both intact and deleted forms of chromosome 9 are present, full-length chromosome 9 molecules are retained mainly in gametocytes. These data suggest that the subtelomeric portion of chromosome 9 is required at an early stage of gametocytogenesis. Parasite subclones derived from gametocyte producing clone 3D7, which completely lost ability to produce gametocytes, are also described. Unlike the previous gametocyteless lines, these parasites stably maintain a full-length chromosome 9 and the ability to cytoadhere to C32 melanoma cells after prolonged asexual propagation. Their defect in sexual development is therefore genetically and functionally distinct from that of parasites carrying a deleted chromosome 9. Gametocyteless subclones derived from 3D7 do not produce any Pfg27 mRNA, while this gene is anomalously expressed in asexual stage parasites of two lines of a different genetic background, 1776sel8 and C10, one able and the other unable to produce gametocytes.

Plasmodium relictum is a widespread invasive agent of avian malaria, responsible for acute, chronic and debilitating diseases in many species of birds. Recent PCR-based studies revealed astonishing genetic diversity of avian malaria parasites (genus Plasmodium), with numerous genetic lineages deposited in GenBank. Many studies addressed distribution and evolutionary relationships of avian Plasmodium lineages, but information about patterns of development of different lineages in mosquito vectors remains insufficient. Here we present data on sporogonic development of 2 widespread mitochondrial cytochrome b lineages (cyt b) of P. relictum (pSGS1 and pGRW11) in mosquito Culex pipiens pipiens. Genetic distance between these lineages is 0.2%; they fall in a well-supported clade in the phylogenetic tree. Three P. relictum strains were isolated from common crossbill (Loxia curvirostra, lineage pSGS1), domestic canary (Serinus canaria domestica, pSGS1) and house sparrow (Passer domesticus, pGRW11). These strains were multiplied in domestic canaries and used as donors of malarial gametocytes to infect C. p. pipiens. Mosquitoes were allowed to take blood meal on infected canaries and then dissected on intervals to study development of sporogonic stages. All 3 strains developed synchronously and completed sporogony in this vector, with infective sporozoites reported in the salivary glands on the day 14 after infection. Ookinetes, oocysts and sporozoites of all strains were indistinguishable morphologically. This study shows that patterns of sporogonic development of the closely related lineages pSGS1 and pGRW11 and different strains of the lineage pSGS1 of P. relictum are similar indicating that phylogenetic trees based on the cyt b gene likely can be used for predicting sporogonic development of genetically similar avian malaria lineages in mosquito vectors.

International audience; Babesiosis is a tick-transmitted disease of mammalian hosts, caused by the intraerythrocytic protozoan parasites of the genus Babesia. Transmission of Babesia parasites from the vertebrate host to the tick is mediated by sexual stages, the gametocytes which are the only intraerythrocytic stages that survive and develop inside the vector. Very few data are available concerning these parasite stages and some markers are needed in order to refine our knowledge of Babesia life cycle inside the tick and to permit the monitoring of parasite transmission from vertebrate to vector. We previously identified some potential markers of the Babesia divergens gametocytes using an in silico post-genomic approach based on sequence identity between the available genomes of Plasmodium and Babesia spp. Here, one of the identified proteins, BdCCp2, was validated as a marker of sexual stages of B. divergens, in infected ticks challenged with antisera directed against recombinant BdCCp2 protein. The BdCCp2 protein was detected by Western blot in some infected ticks, as a discrete band of approximately 171 kDa, while no signal was detected in the laboratory-reared non-infected tick. BdCCp2 was also detected, by immunohistochemical analyses, in piriform or ovoid bodies, measuring 2.5-4.5 μm in diameter, in the gut of partially engorged ticks that were experimentally infected. This molecular marker can then be used in the future to characterize and analyze the biology of B. divergens gametocytes.

The transmission of Plasmodium berghei-infected mice to Anopheles stephensi mosquitoes showed a peak number of oocysts early in the infection prior to the peak of gametocytaemia. This was followed by a precipitous decline on Days 4 and 5 (see also Dearsley et al., Parasitology, 100, 359-368, 1990). By measuring percentage relative infectivity (using membrane feeds with viable gametocytes), we have shown that serum collected daily during the course of a blood-induced infection blocked infectivity from Day 6 postinfection onward. Although there was a correlation between anti-blood stage antibody levels and the loss of infectivity, a comparison of the infectivity pattern of P. berghei-infected BALB/c and SCID mice (the latter being incapable of antibody production) revealed the same pattern of inhibition in both mouse strains, suggesting that antibody alone is not responsible for this suppression. Sera taken from SCID mice late in the infection tested in vitro demonstrated a decline in infectivity similar to that observed in vivo, suggesting that a non-antibody serum factor(s) is responsible for the sustained decline in infectivity of P. berghei to A. stephensi mosquitoes.

The sporogonic cycle of Plasmodium falciparum was obtained in vitro. Mature gametocytes, from blood-stage cultures, produced gametes that underwent fertilization at elevated pH and ambient temperatures. Wheat germ agglutinin stimulated transformation of zygotes into retorts and ookinetes. Twenty-four hours thereafter, 18-49% mature ookinetes and 10-20% intermediate retort forms were counted. Cultures were seeded onto a basement membrane-like gel (Matrigel) in coculture with Drosophila melanogaster cells. Both ookinetes and retorts attached to Matrigel and transformed into oocysts. Mature oocysts and sporozoites expressed circumsporozoite protein. The entire life cycle of Plasmodium falciparum, the most important malaria pathogen of humans, can now be studied in vitro.

We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events.

Mature exoerythrocytic forms (EEF) of Plasmodium falciparum from the chimpanzee were examined by light- and transmission electron microscopy from a liver biopsy taken on Day 6 after sporozoite inoculation. Infectivity of the sporozoites obtained from whole mosquitoes which were membrane fed on cultured gametocytes was about 4-6%. In comparison, salivary gland sporozoites added to human hepatocytes in vitro had only a developmental percentage of 0.02 to 0.05% at Day 5. The EEF found in the liver biopsy were not all at the same stage of development. Immature compact parasites were seen simultaneously with stages with fully formed merozoites, indicating a rapid final maturation or asynchrony. At Day 7.5, large numbers of rings were already seen in the peripheral blood, indicating a duration of the liver development of P. falciparum in the chimpanzee of about 5.5-6 days. The process of merogony at the fine structural level was comparable to that described for rodent and other primate parasites in vivo. Compared to the fine structure of EEF in vitro in cultured human hepatocytes, the parasites described here were much more advanced in development. There appeared to be some cell infiltration with collagen deposition around the intracellular parasite; however, no marked degeneration of EEF was observed.

We have developed two reverse transcription polymerase chain reaction (RT-PCR) techniques to detect and quantify the transmission stages (gametocytes) of Plasmodium chabaudi malaria parasites. Both the qualitative and quantitative techniques are based on the amplification of mRNA coding for the P. chabaudi protein Pcs230, which is expressed exclusively in gametocytes. The quantitative RT-PCR (qRT-PCR) technique was developed and validated by examining serial dilutions of known gametocyte densities. The method generated a high correlation between calibration curves of blind samples (R(2)=0.86). The technique was found to be specific, reproducible, and time efficient for quantification of both patent and sub-patent gametocytemia with a sensitivity level 100-1000 times greater than microscopy. The qualitative RT-PCR (RT-PCR) technique was used to monitor the persistence and dynamics of P. chabaudi gametocytes following acute infection. Mice in two independent experiments were sampled for up to 87 days post-infection. RT-PCR showed that gametocytes can persist for up to 8 weeks, post-infection, whereas microscopy could only detect gametocytes up to 6 weeks. Potential applications of the above techniques for studying the ecology, evolution, and epidemiology of malaria transmission are discussed.

The gametocytocidal action of a new enamine analogue of primaquine, elubaquine (compound CDRI 80/53, bulaquine), has been evaluated against Plasmodium cynomolgi B in rhesus monkeys. Colony bred Anopheles stephensi mosquitoes were fed on gametocyte carrying rhesus monkeys prior to and at varying intervals after oral administration of a single dose of elubaquine at doses ranging between 0.63 and 5.00 mg/kg. Complete loss of oocyst development and mosquito infectivity was observed within 24 h after administering a single 1.25 mg/kg dose, while higher dose of 3.75 mg/kg inhibited oocyst development within 5 h, indicating gametocytocidal action of the compound. Elubaquine did not show any action against developing oocysts in the vector.

Despite significant progress in the identification of the genetic basis of the refractory phenotype, little is known about the physiological mechanism of refractoriness. This study therefore examined the physiological basis of mosquito refractoriness in the Aedes aegypti/P. gallinaceum system, in which a selected refractory strain does not permit Plasmodium oocyst formation. We examined the kinetics of two major proteolytic enzymes involved in blood meal digestion and the dynamics of ookinete formation for two refractory populations (strains Moyo-R and Formosus) and one susceptible population (strain Red). Healthy ookinetes were observed in both the susceptible and the refractory populations, although the susceptible population generally exhibited higher enzymatic activity for trypsin and aminopeptidase than the refractory populations. Parasite numbers in the susceptible Red population showed a 4- to 7-fold decrease in abundance during the transition from the ookinete stage to the oocyst stage, far less than the refractory populations (30- to 92-fold reduction). Due to its smaller body size, Moyo-R individuals generally ingest a smaller blood meal and thus intake fewer gametocytes than Red individuals. Thus, the possibility that refractoriness in the Moyo-R population results from fewer gametocytes being ingested is examined. We found that the Red population remained highly susceptible and the Moyo-R population stayed refractory when those individuals with similar blood meal size were compared. We conclude that failure of oocyst development in the refractory mosquitoes is not due to ookinete damage by proteolytic enzymes or to fewer gametocytes being ingested, but rather is due to a midgut barrier or to some other mechanism.

The proportion of asexual blood-stage malaria parasites that develop into transmission stages (gametocytes) can increase in response to stress. We investigated whether stress imposed by a variety of antimalarial drugs administered before or during infection increased gametocyte production (gametocytogenesis) in vivo in the rodent malaria parasite, Plasmodium chabaudi. All methods of drug treatment greatly reduced the numbers of asexual parasites produced during an infection but resulted in either no reduction in numbers of gametocytes or a smaller reduction than that experienced by asexuals. We used a simple model to estimate temporal variation in gametocyte production. Temporal patterns of gametocytogenesis did not greatly differ between untreated and prophylaxis infections, with rates of gametocytogenesis always increasing as the infection progressed. In contrast, administration of drugs 5 days after infection stimulated increased rates of gametocytogenesis early in the infection, resulting in earlier peak gametocyte densities relative to untreated infections. Given the correlation between gametocyte densities and infectivity to mosquito vectors, and the high frequency of subcurative drug therapy and prophylaxis in human populations, these data suggest that antimalarial drugs may frequently have only a small effect on reducing malaria transmission and may help to explain the rapid spread of drug-resistant geno-types.

The effect of natural malaria transmission-blocking factors in the blood of Plasmodium falciparum gametocyte carriers was assessed in two types of functional bioassays. In the direct membrane feeding assay (DMFA), a comparison is made between the infectivity of gametocytes from a naturally infected gametocyte carrier in the presence of autologous plasma and the infectivity in the presence of replacement plasma from nonimmune donors. In the standard membrane feeder assay (SMFA), cultured NF54 gametocytes are used to measure the capacity of endemic sera to block transmission. In the DMFA, 18 out of 48 sera (37.5%) from Cameroonian gametocyte carriers reduced transmission significantly, while in the SMFA 22 out of 48 sera (45.8%) produced transmission reduction. There was a positive correlation between both assays (r + 0.41, P < 0.05). Antibodies against epitopes of transmission-blocking target antigens Pfs48/45 and Pfs230 were measured in competition ELISAs and compared with the results of DMFA and SMFA. Serological reactivity in competition ELISAs against three epitopes of Pfs48/45 was significantly higher in the group of transmission-reducing sera in both the DMFA and the SMFA, especially for epitope III. No significant difference was found for Pfs230 antibodies (epitope I). Sensitivity of the serological assays was approximately 60%, with a specificity of around 70%. Serological tests cannot replace the functional bioassay in field situations as yet, but can contribute in the selection of sera for SMFA evaluation.

The infectivity of P. berghei-infected TO mice to mosquitoes declines rapidly 2 to 5 days after blood inoculation, in spite of rising numbers of gametocytes in the blood. This pattern is typical of many malaria infections and various factors, particularly specific and nonspecific immune responses, have previously been implicated in the decline. Here we report that (1) simple physiological changes in the mouse blood, namely, falling pH and bicarbonate levels induced by high parasitaemias, are responsible for the sustained inhibition of infectivity; (2) the inhibition is reversible in vivo by the addition of sodium bicarbonate alone; (3) the inhibition occurs at the point of exflagellation; (4) contrary to previous observations (Kawamoto et al. 1992), exflagellation in P. berghei, like that in P. gallinaceum (Bishop and McConnachie 1956; Nijhout and Carter 1978; Nijhout 1979) and P. falciparum (Ogwan'g et al. 1993), is dependent on extracellular bicarbonate; and (5) induction of exflagellation by a mosquito factor is bicarbonate dependent. These new observations are critical to the design and interpretation of experiments on other transmission blocking phenomena.

The water-soluble artemisinin analogue sodium beta-artelinate, a fast-acting blood schizontocide, was evaluated for gametocytocidal action against simian malaria Plasmodium cynomolgi B, and a single dose of the compound has been found to be an effective gametocytocide by both oral and intravenous routes. The compound was able to sterilize the circulating gametocytes in rhesus monkey, resulting in loss of mosquito infectivity and oocyst development in the Anopheles stephensi. However, no sporontocidal action has been observed with this compound.

A Plasmodium falciparum malaria blood stage antigen was detected using a human monoclonal antibody (MAb A52A6) obtained from a clinically immune donor. Immunofluorescence analysis showed that the MAb reacted with the intracellular parasite throughout the asexual blood stage cycle as well as with gametocytes. The MAb also reacted with the surface of erythrocytes containing late stage P. falciparum parasites. The antigen seen by the MAb was species- but not strain- or isolate-specific. At rupture of the infected erythrocytes, antigenic material was deposited on the membrane of uninfected cells surrounding the parasite. At merozoite invasion MAb reactive material was present on the invaginating erythrocyte membrane, indicating an involvement of the antigen in the invasion process. This was also indicated by the high capacity of the MAb to inhibit merozoite invasion in vitro. The antigen appears to be a phosphoglycolipid, sensitive to phospholipase and present in lipid extracts of P. falciparum-infected erythrocytes.

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.

Freeze-fracturing has been used to study the architecture of the pellicular complex of the gametocytes of Plasmodium falciparum. The gametocyte is surrounded by three membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes is split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on their surfaces. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. In gametocytes, unlike in sporozoites, the intramembranous particles are always distributed randomly and lack any definite pattern or orientations. A unique feature of gametocytes revealed by the freeze-fracturing technique is the presence of several transverse sutures on the middle membrane that encircle the gametocyte and give it a segmented appearance.

Midgut tissue from Aedes aegypti stimulated exflagellation of gametocytes of Plasmodium gallinaceum in the absence of bicarbonate, a factor necessary for in vitro exflagellation. Exflagellation was also stimulated when washed infected red cells in a buffered saline (pH 7.4) not containing bicarbonate were introduced into the midgut by enema. The exflagellation-stimulating activity was neither sex nor species specific. Preparations of a mosquito exflagellation factor (MEF) were obtained without tissue disruption by collecting the fluid excreted by Anopheles stephensi females while they were feeding on warm saline. MEF was dialyzable and stable to boiling and decarbonation. Thus, MEF is not bicarbonate.

The occurrence of zygotic nuclear reduction and the haploid state of postzygotic stages were demonstrated in Eimeria tenella (Coccidia, Eimeriina) and Hepatozoon domerguei (Coccidia, Adeleina) by direct measurement of DNA by microdensitometry. Nuclear divisions, which were probably mitotic, were demonstrated in the macrogametocytes of a species of Hepatocystis (Coccidia, Haemosporina). The macrogametocytes of E. tenella were Feulgen negative but gametocytes of both sexes of H. domerguei were Feulgen positive and had DNA values estimated at about twice the haploid amount. This latter finding was interpreted as DNA synthesis preceding a maturation process which involved mitotic divisions and led to micro- and macrogamete formation. In macrogametogenesis this may be an atavistic trait, recapitulating the evolution of sexuality or a method for increased RNA synthesis.

An electron microscopic study of Plasmodium simium infections in the squirrel monkey has supplied information on the ultrastructure of erythrocytic trophozoites, schizonts, merozoites, and gametocytes, in addition to an unusual form of host cell pathology. In general, the structural features, as well as certain specialized functions, e.g., hemoglobin ingestion and utilization, nuclear and cytoplasmic division, were found to be similar to those described for other malarial parasites. Some striking features were noted, however. A highly asynchronous mode of merozoite production was observed within single segmenting parasites in spite of the overall developmental synchrony displayed by the population as a whole. Secondly, during parasite segmentation, newly formed merozoites are connected to one another, as well as to the parasitophorous membrane, by periodic surface strands. It is speculated that these interparasite bridges serve as structural support to the segmenting parasite. When merozoites are matured fully, these interconnections break, leaving a uniform array of short surface bristles. In addition, a number of different pathological changes in host cell structure have been noted. Localized surface discontinuities appear in region of infected cells where apical regions of developing or fully mature merozoites are abutted against the plasma membrane. These profiles suggest that these specialized apical regions of the merozoite function in release as well as in host cell penetration. More generalized surface pathology occurs within parasitized erythrocytes in the form of surface blebs, surface clefts, and associated cytoplasmic microvesicles. The severity of this pathology increases as the intraerythrocytic parasite matures. Topographically these altered cells have a “berry-like” surface texture which makes them quite distinctive when viewed by scanning electron microscopy.

Heat-inactivated serum from chickens with transmission-blocking immunity to Plasmodium gallinaceum prevented the in vitro development of ookinetes from gametocytes of P. gallinaceum only when present during the period between the initiation of gametogeriesis and the release of the microgametes. When added after this time immune serum failed to suppress ookinete development. Immune serum did not prevent the formation of gametes from gametocytes. These results are interpreted to indicate that immune serum contains factors which prevent fertilization of the malarial gametes but which do not affect the development of the zygote once fertilization has taken place. Two distinct reactions of malarial gametes with serum from chickens with transmission-suppressing immunity are described—the gamete-agglutination (AG) reaction and the microgamete surface-fixation (SF) reaction. Both reactions were associated with the immunoglobulin fraction of immune serum. The presence of SF antibodies during a blood infection correlated closely with effective transmission-blocking immunity in vivo ; AG antibodies, on the other hand, were present in various circumstances in the absence of transmission-blocking immunity. AG and SF antibodies occurred not only in birds immunized with P. gallinaceum -gamete preparations but also during or following infections in unimmunized birds; SF antibodies appeared only following the peak of asexual infection in unimmunized birds and were of low titer. In immunized birds blood infections following live challenge invariably boosted low levels of SF antibodies. The results of immunization of chickens and Rhesus monkeys with gametes of their respective malaria parasites, P. gallinaceum and P. knowlesi , are compared.

In acute, blood-induced infections of chickens, the malarial parasite Plasmodium gallinaceum is most infective to the mosquito Aedes aegypti 1 day before gametocyte numbers peak. In an effort to account for this disynchrony , daily changes in parasite infectivity, parasitemia, hematocrit, and hemoglobin were measured during the course of infections. Three events were correlated with the loss of infectivity: (1) In the 24 hr between park infectivity and peak gametocytemia , schizont-induced hemolysis reduced the red blood cell volume 22%. (2) P. gallinaceum zygotes, fertilized in vitro and mixed with heavily infected red blood cells from which all viable, mature gametocytes had been removed, produced 67% fewer oocytes than when combined with uninfected red blood cells. (3) Zygotes fertilized in vitro on the day of peak parasitemia produced 47% fewer oocysts than zygotes prepared 24 hr earlier. It appears that high parasite density reduces infectiousness by destroying, through hemolysis and intraerythrocytic metabolism, a substance necessary to the sporogonic stages, and that there is also an intrinsic loss of infectivity, possibly due to decreased efficiency of fertilization.

There has long been a need for a laboratory host of human plasmodia. Although certain of the anthropoid apes, especially Pan troglodytes (the chimpanzee) are susceptible to one or more species of these malarias, there are numerous difficulties in utilizing such animals as experimental subjects. Of the Old World monkeys, only macaques have shown some susceptibility to Plasmodium falciparum. With the demonstration in 1966 that P. vivax would develop in Aotus trivirgatus (the night monkey), New World monkeys became established as models for investigation. Later it was determined that vivax lines could be maintained in Saimiri, Saguinus, and Ateles spp. Adaptation of P. falciparum directly from man has been achieved in Aotus, with subsequent passage in Saimiri and Cebus; other Panamanian monkeys show only transitory parasitemias. Thus far, P. malariae has been infective only to Aotus. Infectivity of the gametocytes to mosquitoes has been retained in monkeys, with transmission back to man or to other monkeys, thus providing all stages of the parasites for biologic, chemotherapeutic, pathologic or immunologic studies.

The antimalarial activity of the dihydrofolate reductase inhibitors, chlorguanide and pyrimethamine, against gametocytes and sporogony of the simian parasite, Plasmodium cynomolgi was tested. Plasmodium cynomolgi infected rhesus monkeys (Macaca mulatta) were treated orally with multiple doses of chlorguanide (5.6 or 11.3 mg base/kg/Day X5) or a single dose of pyrimethamine (3 mg base/kg). Batches of mosquitoes (Anopheles maculatus) were allowed to feed immediately prior to and at appropriate intervals after drug administration. The effects of the drugs on the developmental stages of the parasite were assessed within the mosquito host. The results indicated that sporogony was interrupted in mosquitoes fed 1 hr after initial dosing with chlorguanide. With pyrimethamine, a clear-cut sporontocidal action was shown as early as 15 min after the drug was given. Both compounds inhibited further development of young oocysts and successive batches of mosquitoes fed 1 hr or more after medication ware consistently negative for sporozoites. Administration of either compound in the indicated doses did not prevent exflagellation, zygote or ookinete formation. However, the behavior of the latter forms was apparently altered by the action of the drug. There was a marked retarding effect in the formation of ookinetes in mosquitoes fed 24 hr after initial medication and thereafter. The schizontocidal and gametocytocidal action of chlorguanide was more evident than it was in pyrimethamine.

The relative susceptibility of Aedes aegypti and Anopheles quadrimaculatus to Plasmodium gallinaceum infection was determined in experiments in which paired groups of the two species were fed on chicks with different levels of infection. Mosquito infections were described by the geometric mean of oocyst numbers in infected individuals and the percentage infection in a sample. These parameters were positively related in both species. When the geometric mean of oocyst numbers exceeded 40, A. aegypti manifested total susceptibility; infection incidence in A. quadrimaculatus was always lower than in a comparable group of A. aegypti and a portion of the anopheline population appeared refractory to infection. The changes in incidence, intensity and range of infection in mosquito samples fed at intervals during the course of patent chick infection are described. A. aegypti always had the higher incidence of infection and, in samples fed when gametocyte numbers were low, the infected culicines also had the greater range and intensity of infection. In samples fed at, or near, the peak of gametocytaemia, however, the range and mean of oocyst numbers in infected A. quadrimaculatus equaled or exceeded those in A. aegypti . Gametocyte infectivity declined for both species as gametocyte numbers increased in a developing chick infection and, for A. aegypti only, increased as gametocyte numbers declined in infections progressing to recovery. Transfer of a volume of heavily infected blood, by intravenous injection, to an uninfected chick immediately restored gametocyte infectivity for both species of mosquito. Increase and decrease in infectivity were both more markedly evidenced by changes in the level of A. aegypti infection.

Gametocytes of P. gallinaceum were irradiated in vivo at dosages between 500 and 20,000 r. Oocysts which developed from these gametocytes exhibited slower growth, abnormal development and increased mortality in proportion to dosage. A significant decrease in mean oocyst size was apparent at 1,000 r. Oocyst mortality, attributed to the action of dominant lethals, was 60% at 4,000 r and 95% at 20,000 r. Sporozoites from oocysts which developed from gametocytes receiving 8,000 r or more appeared to be non-infective. Aedes aegypti mosquitoes which had been irradiated as larvae (500–1,500 r) or pupae (3,000–5,000 r) usually showed an increased resistance to infection by non-irradiated parasites. Their F1 progeny had a two to six-fold increase in susceptibility to infection as compared with unexposed controls.