Membrane Feeding Experiments Research Articles

Infectivity of patent Plasmodium falciparum gametocyte carriers to mosquitoes: establishing capacity to investigate the infectious reservoir of malaria in a low-transmission setting in The Gambia.

BackgroundUnderstanding the human malaria infectious reservoir is important for elimination initiatives. Here, we implemented mosquito membrane feeding experiments to prepare for larger studies to quantify the transmission potential and relative contribution of the human infectious reservoir.MethodsPatients with clinical malaria attending four health facilities with at least 16 Plasmodium falciparum gametocytes per μL were recruited during the 2018 transmission season. Infectiousness to mosquitoes was assessed by direct membrane feeding assay (DMFA). We compared our results with a Bayesian predictive model to investigate the relationship between infectiousness and gametocyte density and explore the impact of fever on gametocyte infectivity.ResultsA total of 3177 suspected malaria cases were screened; 43.3% (1376) had microscopically patent P. falciparum parasites and 3.6% (114) of them had gametocytes. Out of 68 DMFAs, 38 (55.9%) resulted in at least one infected mosquito, with a total of 15.4% (1178/7667) of mosquitoes infected with 1–475 oocysts per gut. The relationship between mosquito infection prevalence and gametocytaemia was similar to other African settings and negatively associated with fever (OR: 0.188, 95% CI 0.0603 to 0.585, p=0.0039).ConclusionsAmong symptomatic malaria patients, fever is strongly associated with transmission failure. Future studies can use DMFA to better understand the human malaria reservoir in settings of low endemicity in The Gambia and inform malaria elimination initiatives.

Optimization of Plasmodium vivax sporozoite production from Anopheles stephensi in South West India

BackgroundEfforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here.MethodsMembrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively.ResultsLaboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load.ConclusionsOptimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

Transmission of molecularly undetectable circulating parasite clones leads to high infection complexity in mosquitoes post feeding

Plasmodium falciparum malaria infections often comprise multiple distinct parasite clones. Few datasets have directly assessed infection complexity in humans and mosquitoes they infect. Examining parasites using molecular tools may provide insights into the selective transmissibility of isolates. Using capillary electrophoresis genotyping and next generation amplicon sequencing, we analysed complexity of parasite infections in human blood and in the midguts of mosquitoes that became infected in membrane feeding experiments using the same blood material in two West African settings. Median numbers of clones in humans and mosquitoes were higher in samples from Burkina Faso (4.5, interquartile range 2–8 for humans; and 2, interquartile range 1–3 for mosquitoes) than in The Gambia (2, interquartile range 1–3 and 1, interquartile range 1–3, for humans and mosquitoes, respectively). Whilst the median number of clones was commonly higher in human blood samples, not all transmitted alleles were detectable in the human peripheral blood. In both study sample sets, additional parasite alleles were identified in mosquitoes compared with the matched human samples (10–88.9% of all clones/feeding assay, n = 73 feeding assays). The results are likely due to preferential amplification of the most abundant clones in peripheral blood but confirm the presence of low density clones that produce transmissible sexual stage parasites.

Dynamics of Plasmodium vivax sporogony in wild Anopheles stephensi in a malaria-endemic region of Western India

BackgroundIn global efforts to track mosquito infectivity and parasite elimination, controlled mosquito-feeding experiments can help in understanding the dynamics of parasite development in vectors. Anopheles stephensi is often accepted as the major urban malaria vector that transmits Plasmodium in Goa and elsewhere in South Asia. However, much needs to be learned about the interactions of Plasmodium vivax with An. stephensi. As a component of the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA), a series of membrane-feeding experiments with wild An. stephensi and P. vivax were carried out to better understand this vector-parasite interaction.MethodsWild An. stephensi larvae and pupae were collected from curing water in construction sites in the city of Ponda, Goa, India. The larvae and pupae were reared at the MESA ICEMR insectary within the National Institute of Malaria Research (NIMR) field unit in Goa until they emerged into adult mosquitoes. Blood for membrane-feeding experiments was obtained from malaria patients at the local Goa Medical College and Hospital who volunteered for the study. Parasites were counted by Miller reticule technique and correlation between gametocytaemia/parasitaemia and successful mosquito infection was studied.ResultsA weak but significant correlation was found between patient blood gametocytaemia/parasitaemia and mosquito oocyst load. No correlation was observed between gametocytaemia/parasitaemia and oocyst infection rates, and between gametocyte sex ratio and oocyst load. When it came to development of the parasite in the mosquito, a strong positive correlation was observed between oocyst midgut levels and sporozoite infection rates, and between oocyst levels and salivary gland sporozoite loads. Kinetic studies showed that sporozoites appeared in the salivary gland as early as day 7, post-infection.ConclusionsThis is the first study in India to carry out membrane-feeding experiments with wild An. stephensi and P. vivax. A wide range of mosquito infection loads and infection rates were observed, pointing to a strong interplay between parasite, vector and human factors. Most of the present observations are in agreement with feeding experiments conducted with P. vivax elsewhere in the world.

The gametocytocidal efficacy of primaquine in malaria asymptomatic carriers treated with dihydroartemisinin-piperaquine in The Gambia (PRINOGAM): study protocol for a randomised controlled trial.

BackgroundFinding efficacious tools to decrease and interrupt malaria transmission is essential to sustain the gains in malaria control and contain the emergence of artemisinin resistance. Primaquine is effective against Plasmodium falciparum gametocytes and recommended for treatment campaigns in (pre-)elimination settings. Safety concerns preclude its use in endemic African countries with variable proportions of glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. The efficacy of the current recommended dose needs to be evaluated, particularly in individuals with an asymptomatic malaria infection.Methods/designThis is a four-arm, open label, randomized controlled trial that aims to determine and compare the effect of three different single doses of primaquine combined with dihydroartemisinin-piperaquine, an artemisinin-based combination therapy, on gametocyte carriage in asymptomatic, malaria infected, G6PD-normal individuals. Approximately 1,200 participants are enrolled and followed for 42 days, with the primary endpoint being the prevalence of Plasmodium falciparum gametocyte carriage at day 7 of follow-up determined by quantitative nucleic acid sequence based amplification assay. Direct membrane feeding experiments to determine infectiousness to mosquitoes are conducted as a biological secondary endpoint.DiscussionSub-Saharan Africa, with a relatively high but poorly characterized G6PD prevalence, could potentially benefit from the use of primaquine to further reduce or interrupt malaria transmission. However, G6PD screening may not be feasible given the cost and difficulties in interpreting test results in terms of risk of haemolysis. Because the haemolytic effect of primaquine is dose-dependent, determining the minimal gametocytocidal and transmission-blocking dose of primaquine is extremely important to help address public health concerns over its safety and validate the efficacy of lower than recommended dosages. By including infectiousness to mosquitoes, the trial provides complementary evidence for the potential of the drug to interrupt transmission to mosquitoes.Trial registrationClinicalTrials.gov: NCT01838902 (12 April 2013).

A Quantum Dot-Immunofluorescent Labeling Method to Investigate the Interactions between a Crinivirus and Its Whitefly Vector.

Successful vector-mediated plant virus transmission entails an intricate but poorly understood interplay of interactions among virus, vector, and plant. The complexity of interactions requires continually improving/evaluating tools and methods for investigating the determinants that are central to mediating virus transmission. A recent study using an organic fluorophore (Alexa Fluor)-based immunofluorescent localization assay demonstrated that specific retention of Lettuce infectious yellows virus (LIYV) virions in the anterior foregut or cibarium of its whitefly vector is required for virus transmission. Continuous exposure of organic fluorophore to high excitation light intensity can result in diminished or loss of signals, potentially confounding the identification of important interactions associated with virus transmission. This limitation can be circumvented by incorporation of photostable fluorescent nanocrystals, such as quantum dots (QDs), into the assay. We have developed and evaluated a QD-immunofluorescent labeling method for the in vitro and in situ localization of LIYV virions based on the recognition specificity of streptavidin-conjugated QD605 (S-QD605) for biotin-conjugated anti-LIYV IgG (B-αIgG). IgG biotinylation was verified in a blot overlay assay by probing SDS-PAGE separated B-αIgG with S-QD605. Immunoblot analyses of LIYV using B-αIgG and S-QD605 resulted in a virus detection limit comparable to that of DAS-ELISA. In membrane feeding experiments, QD signals were observed in the anterior foregut or cibarium of virion-fed whitefly vectors but absent in those of virion-fed whitefly non-vectors. Specific virion retention in whitefly vectors corresponded with successful virus transmission. A fluorescence photobleaching assay of viruliferous whiteflies fed B-αIgG and S-QD605 vs. those fed anti-LIYV IgG and Alexa Fluor 488-conjugated IgG revealed that QD signal was stable and deteriorated approx. seven- to eight-fold slower than that of Alexa Fluor.

Acquisition of Lettuce infectious yellows virus by Bemisia tabaci perturbs the transmission of Lettuce chlorosis virus

Viruses in the genus Crinivirus infect diverse plant species and are transmitted by specific whitefly vectors, but the basis for vector specific transmission remains poorly understood. Here, we demonstrated that purified virion preparations of Lettuce chlorosis virus (LCV) contained filamentous particles that were consistently transmitted to plants by whiteflies (Bemisia tabaci biotypes A and B) following membrane feeding, suggesting that the preparations contained biologically active virions with all the components essential for specific vector transmission. We also demonstrated in sequential membrane feeding experiments that B. tabaci biotype A pre-fed with high concentrations of Lettuce infectious yellows virus (LIYV) virions followed by decreasing concentrations of LCV virions either abolished or interfered with the transmission of the latter. However, in the reverse treatment, an abolishment/interference in transmission of LIYV was not observed. These results suggest that both viruses share a common transmission pathway in B. tabaci biotype A, and factors other than virion quality and quantity may additionally influence their transmission. To begin investigating the viral determinants that are involved in mediating the whitefly transmission of LCV, virions were analyzed by Western immunoblotting. Our results showed that virions were positively identified by antisera produced against three E. coli expressed recombinant LCV capsid proteins—the major coat protein [CP], minor CP [CPm], and P60.

The Dynamics of Naturally Acquired Immune Responses to Plasmodium falciparum Sexual Stage Antigens Pfs230 & Pfs48/45 in a Low Endemic Area in Tanzania

BackgroundNaturally acquired immune responses against sexual stages of P. falciparum can reduce the transmission of malaria from humans to mosquitoes. These antigens are candidate transmission-blocking vaccines but little is known about the acquisition of sexual stage immunity after exposure to gametocytes, or their longevity and functionality. We conducted a longitudinal study on functional sexual stage immune responses.Methodology/Principal FindingsParasitaemic individuals (n = 116) were recruited at a health centre in Lower Moshi, Tanzania. Patients presented with gametocytes (n = 16), developed circulating gametocytes by day 7 (n = 69) or between day 7 and 14 (n = 10) after treatment or did not develop gametocytes (n = 21). Serum samples were collected on the first day of gametocytaemia and 28 and 84 days post-enrolment (or d7, 28, 84 after enrolment from gametocyte-negative individuals). Antibody responses to sexual stage antigens Pfs230 and Pfs48/45 were detected in 20.7% (72/348) and 15.2% (53/348) of the samples, respectively, and were less prevalent than antibodies against asexual stage antigens MSP-119 (48.1%; 137/285) and AMA-1 (52.4%; 129/246)(p<0.001). The prevalence of anti-Pfs230 (p = 0.026) and anti-Pfs48/45 antibodies (p = 0.017) increased with longer duration of gametocyte exposure and had an estimated half-life of approximately 3 months. Membrane feeding experiments demonstrated a strong association between the prevalence and concentration of Pfs230 and Pfs48/45 antibodies and transmission reducing activity (TRA, p<0.01).Conclusions/SignificanceIn a longitudinal study, anti-Pfs230 and Pf48/45 antibodies developed rapidly after exposure to gametocytes and were strongly associated with transmission-reducing activity. Our data indicate that the extent of antigen exposure is important in eliciting functional transmission-reducing immune responses.

Substantial contribution of submicroscopical Plasmodium falciparum gametocyte carriage to the infectious reservoir in an area of seasonal transmission.

BackgroundMan to mosquito transmission of malaria depends on the presence of the sexual stage parasites, gametocytes, that often circulate at low densities. Gametocyte densities below the microscopical threshold of detection may be sufficient to infect mosquitoes but the importance of submicroscopical gametocyte carriage in different transmission settings is unknown.Methodology/Principal FindingsMembrane feeding experiments were carried out on 80 children below 14 years of age at the end of the wet season in an area of seasonal malaria transmission in Burkina Faso. Gametocytes were quantified by microscopy and by Pfs25-based quantitative nucleic acid sequence-based amplification assay (QT-NASBA). The children's infectiousness was determined by membrane feeding experiments in which a venous blood sample was offered to locally reared Anopheles mosquitoes. Gametocytes were detected in 30.0% (24/80) of the children by microscopy compared to 91.6% (65/71) by QT-NASBA (p<0.001). We observed a strong association between QT-NASBA gametocyte density and infection rates (p = 0.007). Children with microscopically detectable gametocytes were more likely to be infectious (68.2% compared to 31.7% of carriers of submicroscopical gametocytes, p = 0.001), and on average infected more mosquitoes (13.2% compared to 2.3%, p<0.001). However, because of the high prevalence of submicroscopical gametocyte carriage in the study population, carriers of sub-microscopical gametocytes were responsible for 24.2% of the malaria transmission in this population.Conclusions/SignificanceSubmicroscopical gametocyte carriage is common in an area of seasonal transmission in Burkina Faso and contributes substantially to the human infectious reservoir. Submicroscopical gametocyte carriage should therefore be considered when implementing interventions that aim to reduce malaria transmission.

Immunogenicity in rhesus of the Plasmodium vivax mosquito stage antigen Pvs25H with Alhydrogel and Montanide ISA 720

Pvs25 is an ookinete surface protein from Plasmodium vivax that is the target of transmission-blocking antibodies. Two immunogenicity trials in rhesus monkeys with a recombinant form of the protein, Pvs25H, were undertaken. Monkeys were vaccinated with Pvs25H adsorbed to Alhydrogel or emulsified in Montanide ISA 720 at 0, 4 and 27 weeks (study 1) or in Montanide ISA 720 at 0 and 18 weeks (study 2) with 1.5 or 15 microg Pvs25H in 0.1 or 0.5 mL of emulsion (four combinations). Immunogenicity was assessed by ELISA and by membrane-feeding experiments using P. vivax-infected blood from human volunteers (studies 1 and 2) or from chimpanzees (study 1). Both vaccine trials generated antibodies that blocked transmission of P. vivax to mosquitoes. Antibody titres and transmission blocking were higher with Montanide ISA 720 than with Alhydrogel in the first trial and with the 15 microg Pvs25H/0.5 mL ISA 720 combination in the second trial.

THE PRESENCE OF PLASMODIUM FALCIPARUM GAMETOCYTES IN HUMAN BLOOD INCREASES THE GRAVIDITY OF ANOPHELES GAMBIAE MOSQUITOES

We conducted a field study in an area of endemic malaria transmission in western Kenya to determine whether mosquitoes that feed on gametocyte-infected blood but do not become infected have reduced or enhanced fecundity in comparison to mosquitoes fed on uninfected blood. Fifteen paired membrane-feeding experiments were conducted in which two strains of Anopheles gambiae mosquitoes were simultaneously fed on either Plasmodium falciparum-infected blood from children or uninfected control blood from adults. The presence of noninfecting gametocytes in blood increased the probability that An. gambiae would produce eggs after one blood meal by sixfold (odds ratio for control relative to infected blood group 0.16; 95% CI 0.10-0.23). This result could not be explained by variation in blood meal size or hemoglobin content between hosts. When children cleared their infections, the difference in gravidity between mosquitoes fed on their blood and uninfected adults disappeared, suggesting this phenomenon is due to the presence of Plasmodium gametocytes in blood and not to host-specific factors such as age. This result was observed in two mosquito strains that differ in their innate fecundity, suggesting it may apply generally. To our knowledge, this is the first time that Plasmodium has been implicated as enhancing vector gravidity.

Phagocytosis does not play a major role in naturally acquired transmission-blocking immunity to Plasmodium falciparum malaria.

Phagocytosis of Plasmodium falciparum sexual stages in vitro and within the mosquito midgut was assayed in order to assess its role in transmission-blocking immunity to malaria. Both monocytes/macrophages (MM) and polymorphonuclear neutrophils (PMN) phagocytosed malarial gametes in vitro, but levels of phagocytosis were low. Intraerythrocytic gametocytes were not susceptible to phagocytosis. In vitro phagocytosis was positively correlated with levels of antibodies against the gamete surface proteins Pfs230 and Pfs48/45. Immunoglobulin G (IgG) subclass analysis revealed that phagocytosis was correlated with levels of antigamete IgG1. In vivo membrane-feeding experiments were performed in the presence of both pooled and individual malaria immune sera. The phagocytic process proceeded less efficiently in vivo than in vitro, which may be related to the lower ambient temperature (26 degrees C, compared with 37 degrees C). Finally, although we found a correlation between the ability of a serum to promote phagocytosis in vitro and the presence of antibodies against transmission-blocking target antigens, we were unable to demonstrate a role for MM- or PMN-mediated phagocytosis in reduction of infectivity of the malarial parasite to mosquitoes.

Host haematological factors influencing the transmission of Plasmodium falciparum gametocytes to Anopheles gambiae s.s. mosquitoes.

We investigated the relationship between selected host haematological and parasitological parameters and the density and infectivity of Plasmodium falciparum gametocytes. 143 individuals (age range 1-62 years) attending an outpatient clinic in Farafenni, The Gambia, who had peripheral blood gametocytaemia were recruited (mean gametocyte density 123.7/microl, range 5-17,000/microl). Of the parameters measured, packed cell volume (PCV), reticulocyte count (RetC) and lymphocyte count (LyC) were significantly associated with gametocyte density (r = - 0.17, P < 0.05; r = 0.21, P < 0.01; r = 0.18, P < 0.05, respectively). Data from membrane feeding experiments in which 15 or more mosquitoes were dissected showed that 60.7% (53/87) of gametocyte carriers infected one or more mosquitoes. Gametocyte density was strongly correlated with transmission success (TS) (r = 0.3, P < 0.005) and, in successful infections, with both mosquito prevalence (MP) (r = 0.36, P < 0.005) and mean oocyst burden (MOB) (r = 0.65, P < 0.0001). None of the other factors measured were significantly associated with any of these indices in bivariate analysis. Regression modelling showed that both gametocyte density and PCV were positively associated with gametocyte carrier infectivity to mosquitoes (LRchi2 = 100.7 and 47.2, respectively) and, in successful infections, with MOB (beta = 0.16, t = 4.9, P < 0.001; beta = 0.02, t = 2.3, P < 0.05, respectively). The positive association with PCV suggests that blood meal quality influences infection probably as a nutritional requirement, however, as this effect was most apparent at high gametocyte densities, its epidemiological significance is questionable. Though the haematological parameters associated with gametocyte density are a direct consequence of asexual infection, they may also represent an adaptive mechanism for optimization of sexual stage development.

Stylet activity of cowpea aphid (Homoptera: Aphididae) on leaf extracts of resistant and susceptible cowpea cultivars

Electrical penetration graph recordings using direct current (DC-EPGs) were used to analyze aspects of the probing behavior of cowpea aphid,Aphis craccivora Koch, on intact plants and on hexane, ethyl acetate, and methanol extracts of leaves of aphid-resistant (ICV-12) and aphid-susceptible (ICV-1) cultivars of cowpeaVigna unguiculata (L.) Walp. In one set of experiments, recordings were done on plants with or without parafilm wrapping, or on plants painted with raw leaf juice and extracts of the two cultivars. In another study, recordings were done on leaf extracts homogenized in water or in 0.5M sucrose solution and then placed in parafilm membrane sachets. Electrodes were inserted into soil mix for the experiments on potted plants or into extract fractions and raw juice for the membrane feeding experiments on leaf extracts in parafilm sachets. Waveform signals were recorded from resistance fluctuations from interactions between aphids and substrates, and electromotive forces generated within each preparation. ICV-12 plants with or without parafilm wrapping, and ethyl acetate extracts and raw juice of that cultivar significantly (P≤0.05) reduced stylet penetration behavior. Thus, antixenosis as manifested by disruption of aphid stylet activity on host substrates, appeared to be a governing modality of aphid resistance in ICV-12.

Leukocytes in a Plasmodium falciparum-infected blood meal reduce transmission of malaria to Anopheles mosquitoes

Mosquitoes are infected with Plasmodium falciparum by taking a blood meal from a gametocyte carrier. Since a mosquito takes a volume of 1 to 2 microl, a blood meal may contain 1 x 10(4) to 3 x 10(4) leukocytes (WBC). The majority of WBC are composed of neutrophils which may phagocytose and kill developing gametes inside the mosquito midgut. Phagocytosis was measured in vitro by a luminol-dependent chemiluminescence (CL) assay. In the presence of P. falciparum gametes, sera from areas of endemicity had an increased CL response compared to controls. In mosquito membrane feeding experiments some such sera showed a transmission reduction which was related to the presence of viable WBC. The results of this study suggest that phagocytosis of opsonized gametes inside the mosquito midgut occurs and can contribute to a reduction in the transmission of P. falciparum parasites.

Transmission blockade of Plasmodium falciparum malaria by anti-Pfs230-specific antibodies is isotype dependent.

By use of the parental hybridoma cell line 63F2A2 that produces specific antibodies of immunoglobulin isotype G1 (IgG1; 63F2A2.1) against Pfs230, we attempted to enrich for the synthesis of the downstream switch variant IgG2b and IgG2a monoclonal antibodies (MAbs) of the hybridoma cell line (63F2A2.2b and 63F2A2.2a, respectively). The parental IgG1 did not reduce the Plasmodium falciparum transmission in a bioassay irrespective of the presence of complement. MAbs 63F2A2.2b and 63F2A2.2a were effective in reducing the infectivity of P. falciparum parasites to Anopheles gambiae mosquitoes in membrane-feeding experiments. A transmission reduction of 91% was accomplished by the 63F2A2.2b switch variant, and a reduction of greater than 99% was accomplished by the 63F2A2.2a switch variant, but only in the presence of active human complement. Subsequently, the transmission-reducing effect of MAb 63F2A2.2b or 63F2A2.2a was confirmed in vitro by the rapid lysis of newly formed macrogametes or zygotes in the presence of active complement. MAb 63F2A2.1 did not lyse the newly formed macrogametes or zygotes irrespective of the presence of complement.

Transmission blocking immunity may provide clues that antimalarial immunity is largely T-independent

Humans infected with malaria produce antibodies against the sexual stages of the parasite (Mendis et al., 1987; Graves et ai., 1988), as they do against pre-erythrocytic (Nardin et al., 1979) and asexual blood stages (Cohen et al., 1961). These antibodies are evoked by gametocytes which are precursors of the sexual stages that are presented to the host immune system during a blood infection. The antibodies take effect in the midgut of a mosquito when it takes a blood meal from an infected person, where they react either with surface antigens of male and female gametes that are formed in the mosquito midgut and prevent fertilization, or with postfertilization stages such as the zygote, and prevent their further development (reviewed by Carter et ai., 1988). A potent vaccine-induced immunity against these sexual stages could reduce malar'~. ~:ansmission in endemic areas (De Zoysa et ai., i988, i99i). Parasite molecules that are targets of transmissionblocking antibodies have been defined in both of the major human malaria parasites P. falciparum (Carter et al., 1988; Rener et al., 1983; Vermulen et al., 1985) and P. vivax (Premawansa et al., 1990), and the genes coding for two of them, Pfs25, an ookinete antigen of P. falciparum (Kaslow et al., 1988) and GAM-1 of P. vivax (personal communication, P.H. David) have been cloned. The Pfs25 ookinete antigen has been successfully expressed in the vaccinia virus, and immunization of mice with recombinant vaccinia has been . . . . . . s a t ~ , to induce antibodies which blocked the infectivity of P. falciparum gametocytes to mosquitoes in membrane feeding experiments (Kaslow et al., 1991). Thus transmission blocking malaria vaccines may soon be ready for clinical testing.

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.

Target antigens of transmission blocking immunity of Plasmodium vivax malaria. Characterization and polymorphism in natural parasite isolates.

A panel of 20 anti-Plasmodium vivax female gamete mAb has been established and was characterized with respect to their transmission-blocking properties in membrane-feeding experiments and their target Ag identified. Seven mAb suppressed the infectivity of P. vivax parasites to Anopheles tesselatus mosquitoes. The m.w. of the Ag recognized by these mAb were ascertained by SDS-PAGE and Western blots. Three sets of polypeptides of low Mr--20, 24, and a doublet of 37/42 kDa--have been defined as target Ag of transmission-blocking antibodies of P. vivax. All epitopes of these target Ag were found to be dependent on the tertiary conformational structure of the Ag. Polymorphism of target Ag of transmission-blocking immunity was investigated in over 30 natural isolates of P. vivax in Sri Lanka based on the reactivity of a mAb with an isolate as assessed by the indirect immunofluorescent test with the use of live extracellular female gametes, and in Western blots with the use of extracted gametes. The functional consequences of antigenic polymorphism on immunity was investigated in transmission-blocking assays by using membrane-feeding experiments. A majority of target Ag of transmission-blocking immunity were found to be polymorphic, exhibiting size as well as epitope polymorphism. Results indicate that failure of a mAb to affect the infectivity of a parasite isolate of P. vivax to mosquitoes can be caused by polymorphism of the target Ag among isolates.

Mortality in adult tsetse, Glossina morsitans morsitans, caused by entomopathogenic bacteria

Mortality in adult tsetse, Glossina morsitans morsitans, caused by Pseudomonas aeruginosa, Serratia marcescens, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis H-14, B. thuringiensis 1, B. thuringiensis 5, B. thuringiensis var. insraelensis, and Providentia rettgeri was determined. When bacteria were smeared on rabbit skin and tsetse allowed to feed only once on the contaminated area, mortality 8 days postingestion was significantly higher ( P < 0.01) in tsetse fed on P. aeruginosa, S. marcescens, B. thuringiensis 1, and P. rettgeri and increased when tsetse were allowed to feed for the second time on the contaminated skin. With this smear technique, however, mortalities were generally not remarkable. In artificial membrane feeding experiments using low concentrations of bacteria (−10 6/ml of blood), the B. thuringiensis strains caused low mortality, except B. thuringiensis H-14, which caused 59% mortality. However, at this concentration, P. aeruginosa, S. marcescens, B. cereus, and P. rettgeri caused highly significant ( P < 0.01) mortality (64–96%). When higher concentrations of bacteria (10 7/ml) were used, all the bacteria tested, except B. sphaericus, caused high mortality ranging from 70 to 98%. Thus, mortality depended on the species of bacteria, the dose ingested, and time postingestion.