Plasmodium Falciparum Sporozoites Research Articles

A shift from Indoor Residual Spraying (IRS) with bendiocarb to Long-Lasting Insecticidal (mosquito) Nets (LLINs) associated with changes in malaria transmission indicators in pyrethroid resistance areas in Benin

BackgroundIndoor residual spraying (IRS) was implemented in the department of Ouémé-Plateau, southern Benin, in 2008 and withdrawn in 2011, when long lasting insecticidal nets (LLINs) were distributed to the communities that were previously targeted by IRS. Did the LLIN strategy provide a better level of protection against malaria transmission than IRS?MethodsEntomological surveillance was carried out to assess indicators of transmission risk during the last year of IRS and the first year after the LLIN intervention was put in place (2010–2011). Mosquito biting density was sampled by human landing collection (HLC). Females of Anopheles gambiae s.l. were dissected to estimate the parity rates and the blood meal index. A subsample of the An. gambiae s.l. collection was tested for presence of Plasmodium falciparum sporozoites. In addition, window exit traps and pyrethrum spray catches were performed to assess exophagic behavior of Anopheles vectors.ResultsThere were significant increases in all the indicators following withdrawal of IRS. Vector biting density (p<0.001) and longevity (OR=3.81[3.01-4.82] 95% CI; p<0.001) of the An. gambiae s.l. increased significantly; so too did the blood meal index (OR=1.48 [1.1-1.99] 95% CI; p<0.001). Entomological inoculation rate, after IRS withdrawal at one surveillance site, Adjohoun, rose two fold (9.0 infected bites/person/9 months (Apr-Dec 2011) versus 3.66 infective bites/person during the 9 months preceding IRS (Apr-Dec 2010). A second site, Missérété, experienced a six-fold increase after IRS cessation (15.1 infective bites/person/9 months versus 2.41 during IRS). Exophily after IRS cessation decreased significantly in all areas (p<0.001) suggesting that mosquitoes were more likely to rest in houses with LLINs, than in houses subjected to IRS.ConclusionLLINs did not impact on indicators of transmission to the same levels as did IRS after IRS withdrawal.

Taking a Bite out of Malaria: Controlled Human Malaria Infection by Needle and Syringe

Taking a Bite out of Malaria: Controlled Human Malaria Infection by Needle and Syringe

Entomological indices of malaria transmission in Chikhwawa district, Southern Malawi

BackgroundAlthough malaria is highly prevalent throughout Malawi, little is known of its transmission dynamics. This paper describes the seasonal activity of the different vectors, human biting indices, sporozoite rates and the entomological inoculation rate in a low-lying rural area in southern Malawi.MethodsVectors were sampled over 52 weeks from January 2002 to January 2003, by pyrethrum knockdown catch in two villages in Chikhwawa district, in the Lower Shire Valley.ResultsIn total, 7,717 anophelines were collected of which 55.1% were Anopheles gambiae sensu lato and 44.9% were Anopheles funestus. Three members of the An. gambiae complex were identified by PCR: Anopheles arabiensis (75%) was abundant throughout the year, An. gambiae s.s. (25%) was most common during the wet season and Anopheles quadriannulatus occurred at a very low frequency (n=16). An. funestus was found in all samples but was most common during the dry season.Anopheles gambiae s.s. and An. funestus were highly anthropophilic with human blood indices of 99.2% and 96.3%, respectively. Anopheles arabiensis had fed predominantly on humans (85.0%) and less commonly on cattle (10.9%; 1.2% of blood meals were of mixed origin). Plasmodium falciparum (192/3,984) and Plasmodium malariae (1/3,984) sporozoites were detected by PCR in An. arabiensis (3.2%) and An. funestus (4.5%), and in a significantly higher proportion of An. gambiae s.s. (10.6%)(p<0.01). All three vectors were present throughout the year and malaria transmission occurred in every month, although with greatest intensity during the rainy season (January to April). The combined human blood index exceeded 92% and the P. falciparum sporozoite rate was 4.8%, resulting in estimated inoculation rates of 183 infective bites/ person per annum, or an average rate of ~15 infective bites/person/month.ConclusionsThe results demonstrate the importance of An. gambiae s.s., An. arabiensis and An. funestus in driving the high levels of malaria transmission in the south of Malawi. Sustained and high coverage or roll out of current approaches to malaria control (primarily insecticide-treated bed nets and indoor residual house spraying) in the area are likely to reduce the observed high malaria transmission rate and consequently the incidence of human infections, unless impeded by increasing resistance of vectors to insecticides.

Controlled Human Malaria Infections by Intradermal Injection of Cryopreserved Plasmodium falciparum Sporozoites

Controlled human malaria infection with sporozoites is a standardized and powerful tool for evaluation of malaria vaccine and drug efficacy but so far only applied by exposure to bites of Plasmodium falciparum (Pf)-infected mosquitoes. We assessed in an open label Phase 1 trial, infection after intradermal injection of respectively 2,500, 10,000, or 25,000 aseptic, purified, vialed, cryopreserved Pf sporozoites (PfSPZ) in three groups (N = 6/group) of healthy Dutch volunteers. Infection was safe and parasitemia developed in 15 of 18 volunteers (84%), 5 of 6 volunteers in each group. There were no differences between groups in time until parasitemia by microscopy or quantitative polymerase chain reaction, parasite kinetics, clinical symptoms, or laboratory values. This is the first successful infection by needle and syringe with PfSPZ manufactured in compliance with regulatory standards. After further optimization, the use of such PfSPZ may facilitate and accelerate clinical development of novel malaria drugs and vaccines.

The high immunogenicity induced by modified sporozoites’ malarial peptides depends on their phi (ϕ) and psi (ψ) angles

The importance of CSP- and STARP-derived ϕ and ψ dihedral angles in mHABP structure was analysed by 1H NMR in the search for molecules which can be included as components of a first-line-of-defence Plasmodium falciparum sporozoite multi-epitope vaccine against the most lethal form of human malaria. Most of the aforementioned dihedral angles were left-hand-like polyproline type II (PPIIL) structures whilst others had right-hand-like α-helix (αR), thus allowing mHABPS to fit better into MHCII molecules and thereby form an appropriate pMHCII complex and also establish the H-bonds which stabilise such complex and by this means induce an appropriate immune response. This information has great implications for vaccine development, malaria being one of them.

Reconsideration of Anopheles rivulorum as a vector of Plasmodium falciparum in western Kenya: some evidence from biting time, blood preference, sporozoite positive rate, and pyrethroid resistance

BackgroundAnopheles gambiae, An. arabiensis, and An. funestus are widespread malaria vectors in Africa. Anopheles rivulorum is the next most widespread species in the An. funestus group. The role of An. rivulorum as a malaria vector has not been fully studied, although it has been found to be a minor or opportunistic transmitter of Plasmodium falciparum.MethodsMosquitoes were collected indoors over a 12-hour period using a light source attached to a rotating bottle collector in order to determine peak activity times and to provide DNA for meal source identification. Gravid female mosquitoes were collected indoors via an aspirator to generate F1 progeny for testing insecticidal susceptibility. Blood meal sources were identified using a multiplexed PCR assay for human and bovine cytochrome-B, and by matching sequences generated with primers targeting vertebrate and mammalian cytochrome-B segments to the Genbank database.ResultsAnopheles rivulorum fed on human blood in the early evening between 18:00 and 20:00, when insecticide-treated bed nets are not in use, and the presence of Plasmodium falciparum sporozoites in 0.70% of the An. rivulorum individuals tested was demonstrated. Susceptibility to permethrin, deltamethrin, and DDT is higher in An. rivulorum (84.8%, 91.4%, and 100%, respectively) than in An. funestus s.s. (36.8%, 36.4%, and 70%, respectively), whereas mortality rates for propoxur and fenitrothion were 100% for both species. Resistance to pyrethroids was very high in An. funestus s.s. and the potential of the development of high resistance was suspected in An. rivulorum.ConclusionGiven the tendency for An. rivulorum to be active early in the evening, the presence of P. falciparum in the species, and the potential for the development of pyrethroid resistance, we strongly advocate reconsideration of the latent ability of this species as an epidemiologically important malaria vector.

Plasmodium liver load following parenteral sporozoite administration in rodents

One of the bottlenecks in the development of a whole sporozoite malaria vaccine is the route and method of sporozoite administration. Immunization and challenge of human volunteers by mosquito bites is effective, but cannot be used as a vaccine. Intravenous immunization with sporozoites is effective in rodents and non-human primates, and being studied in humans, but is not yet used for licensed vaccines for infectious diseases. Intradermal and subcutaneous immunization regimens show a strong decrease in protective efficacy, which in rodents, is associated with a decreased degree of parasite liver infection during immunization. The objective of this study was to explore alternative routes of sporozoite administration to increase efficiency of liver infection. Using in vivo imaging, we found that IM injection of sporozoites resulted in a greater parasite liver load compared to ID and SC injection. The use of small inoculation volumes and multiple injections further increased the subsequent liver load. These observations were corroborated in a Plasmodium yoelii model using cryopreserved sporozoites administered ID. Our findings provide a rationale for the design of clinical trials to optimize needle and syringe administration of Plasmodium falciparum sporozoites.

Estimation of malaria transmission intensity in Sennar state, central Sudan

Understanding the behaviour of malaria vectors is crucial for planning mosquito control programmes. The aim of this study was to estimate the malaria transmission intensity in 2 different ecological zones in a highly endemic malaria area of Sennar state in central Sudan over the main transmission period. Species confirmation by PCR indicated that Anopheles arabiensis was the only malaria vector in the study area, with high anthropophilic behaviour (84.9% human-feeding). ELISA studies showed Plasmodium falciparum sporozoite rates rose from 1.8% to 4.5% and the average entomological inoculation rates rose from 2.4 to 4.2 infectious bites per person per night in September (the beginning) to November (the end) of the 3-month transmission season. The proportion of malaria-positive slides ranged from 50.1% to 57.0%. The proportion of human-blood positive mosquitoes was significantly higher in the irrigated area (El Booster) compared with the non-irrigated area (Rahal).

Impact of entomological interventions on malaria vector bionomics in low transmission settings in Zambia

Entomological interventions for malaria control are being scaled up in the context of the integrated vector management strategy in Zambia. This paper reports the continuous entomological monitoring of the operational impact of indoor residual insecticide spraying (IRS) and distribution of about 6 million insecticide-impregnated bed-nets (ITN) over two peak malaria transmission seasons. Mosquitoes were captured daily using exit window traps at monitoring sentinel sites and analyzed for species identification, densities, and sporozoite rates to assess the efficacy of the vector control tools. All the three major malaria vectors; Anopheles gambiae senso stricto (s.s.), Anopheles arabiensis andAnopheles funestus were collected and identified. The intervention effect of IRS and ITNs was more pronounced on A. gambiae s.s. and A. funestus than A. arabiensis (c2 = 0.003, df = 1, P = 0.956), indicating that A. gambiae s.s. and A. funestus are amenable to control by IRS and ITNs. None of these vectors tested positive for Plasmodium falciparumsporozoites, thus, signifying their lack of transmission potential. This study demonstrates that entomological monitoring and evaluation is an indispensable underpinning for rational insecticide based malaria vector control. It provides compelling evidence for the need to integrate entomological parameters into routine surveillance systems, and also strongly substantiates the deployment of the integrated vector management strategy. Key words: Zambia, malaria, impact, indoor residual spraying, insecticide treated nets, transmission.

Ag-specific recall of cytokine producing CD4 and CD8 T cells in humans immunized with genetically attenuated Plasmodium falciparum sporozoite vaccine (166.12)

Abstract A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by deletion of 2 pre-erythrocytic genes, was administered to 6 subjects by bites from Anopheles mosquitoes. Previous studies of immune responses elicited by recombinant malaria vaccines show that protection is linked to Ag-specific T cells producing IFN-γ, TNF, and/or IL-2. In this study we asked if Pf GAP vaccine induced Ag-specific T cell responses characterized by inflammatory cytokines. We analyzed pre- and post vaccination PBMC for Ag-specific T cell responses to well characterized pre-erythrocytic and erythrocytic P. falciparum protein and peptide Ags. Additionally, we examined responses to several novel Pf liver-stage Ag (Pf LSA) because Pf LSA-dependent immunity might contribute to GAP-induced protection. IFN-γ and TNF responses to multiple antigens were significantly enhanced post vaccination; IFN-γ responses were primarily attributed to CD8+ T cells while TNF was secreted primarily by CD4+ T cells. Notably, a subject with a breakthrough peripheral parasitemia did show positive IFN-γ responses to erythrocytic-stage Ags, but no responses were recalled with the novel Pf LSA. In summary, two exposures to a Pf GAP vaccine induced persisting T cell responses specific for Pf LSA in humans. Future trials that assess the efficacy of Pf GAP vaccines should examine the role of LSA-specific responses in protection.

Unexpected fold in the circumsporozoite protein target of malaria vaccines.

Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an "αTSR" domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

Artemisinin-based combination therapy does not measurably reduce human infectiousness to vectors in a setting of intense malaria transmission

BackgroundArtemisinin-based combination therapy (ACT) for treating malaria has activity against immature gametocytes. In theory, this property may complement the effect of terminating otherwise lengthy malaria infections and reducing the parasite reservoir in the human population that can infect vector mosquitoes. However, this has never been verified at a population level in a setting with intense transmission, where chronically infectious asymptomatic carriers are common and cured patients are rapidly and repeatedly re-infected.MethodsFrom 2001 to 2004, malaria vector densities were monitored using light traps in three Tanzanian districts. Mosquitoes were dissected to determine parous and oocyst rates. Plasmodium falciparum sporozoite rates were determined by ELISA. Sulphadoxine-pyrimethamine (SP) monotherapy was used for treatment of uncomplicated malaria in the contiguous districts of Kilombero and Ulanga throughout this period. In Rufiji district, the standard drug was changed to artesunate co-administered with SP (AS + SP) in March 2003. The effects of this change in case management on malaria parasite infection in the vectors were analysed.ResultsPlasmodium falciparum entomological inoculation rates exceeded 300 infective bites per person per year at both sites over the whole period. The introduction of AS + SP in Rufiji was associated with increased oocyst prevalence (OR [95%CI] = 3.9 [2.9-5.3], p < 0.001), but had no consistent effect on sporozoite prevalence (OR [95%CI] = 0.9 [0.7-1.2], p = 0.5). The estimated infectiousness of the human population in Rufiji was very low prior to the change in drug policy. Emergence rates and parous rates of the vectors varied substantially throughout the study period, which affected estimates of infectiousness. The latter consequently cannot be explained by the change in drug policy.ConclusionsIn high perennial transmission settings, only a small proportion of infections in humans are symptomatic or treated, so case management with ACT may have little impact on overall infectiousness of the human population. Variations in infection levels in vectors largely depend on the age distribution of the mosquito population. Benefits of ACT in suppressing transmission are more likely to be evident where transmission is already low or effective vector control is widely implemented.

Gliding Motility of Babesia bovis Merozoites Visualized by Time-Lapse Video Microscopy

Background Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.Methodology/Principal FindingsGliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.Conclusions/SignificanceThis is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.

Controlled Human Blood Stage Malaria Infection: Current Status and Potential Applications

Controlled human malaria infection by blood stage parasite (BSP) inoculation is an alternative to the well-established model of infection with Plasmodium falciparum sporozoites delivered by mosquito bites. The BSP model has been utilized less frequently, but its use is increasing. Advantages of BSP challenge include greater ease of administration, better standardization of the infecting dose per volunteer, and good inter-study reproducibility of in vivo parasite dynamics. Recently, a surprising reduction in clinical symptoms at microscopic patency in the BSP model has been identified, which has an undefined and intriguing pathophysiologic basis, but may make this approach more acceptable to volunteers. We summarize clinical, parasitologic, and immunologic data from all BSP challenges to date, explore differences between the BSP and sporozoite models, and propose future applications for BSP challenge.

Cross-Species Immunity Following Immunization With a Circumsporozoite Protein–Based Vaccine for Malaria

Malaria continues to be a major public health concern, and there are concerted efforts to eliminate it. The quest for a vaccine remains a top priority, and vaccines based on the circumsporozoite protein (CSP) are among the lead candidates, with the RTS,S vaccine currently undergoing phase 3 testing in Africa. Previous studies have reported anti-CSP antibody-mediated enhancement of in vitro invasion of homologous sporozoites. This effect has been shown to be concentration dependent; high-level antibodies are inhibitory, whereas low-level antibodies lead to enhancement of invasion. Nondominant shared epitopes may lead to the generation of low titers of cross-reactive antibodies that may prove to be detrimental. We report cross-species recognition of Plasmodium falciparum and Plasmodium berghei sporozoites by anti-Plasmodium vivax CSP serum samples. In addition, we report that vaccination of mice with VMP001, a P. vivax CSP vaccine candidate, reduces, not enhances, P. berghei infection in mice.

Malaria entomological profile in Tanzania from 1950 to 2010: a review of mosquito distribution, vectorial capacity and insecticide resistance

In Sub Saharan Africa where most of the malaria cases and deaths occur, members of the Anopheles gambiae species complex and Anophelesfunestus species group are the important malaria vectors. Control efforts against these vectors in Tanzania like in most other Sub Saharan countries have failed to achieve the set objectives of eliminating transmission due to scarcity of information about the enormous diversity of Anopheles mosquito species and their susceptibility status to insecticides used for malaria vector control. Understanding the diversity and insecticide susceptibility status of these vectors and other factors relating to their importance as vectors (such as malaria transmission dynamics, vector biology, ecology, behaviour and population genetics) is crucial to developing a better and sound intervention strategies that will reduce man-vector contact and also manage the emergency of insecticide resistance early and hence .a success in malaria control. The objective of this review was therefore to obtain the information from published and unpublished documents on spatial distribution and composition of malaria vectors, key features of their behaviour, transmission indices and susceptibility status to insecticides in Tanzania. All data available were collated into a database. Details recorded for each data source were the locality, latitude/longitude, time/period of study, species, abundance, sampling/collection methods, species identification methods, insecticide resistance status, including evidence of the kdr allele, and Plasmodium falciparum sporozoite rate. This collation resulted in a total of 368 publications, encompassing 806,273 Anopheles mosquitoes from 157 georeferenced locations being collected and identified across Tanzania from 1950s to 2010. Overall, the vector species most often reported included An. gambiae complex (66.8%), An. funestus complex (21.8%), An. gambiae s.s. (2.1%) and An. arabiensis (9%). A variety of sampling/ collection and species identification methods were used with an increase in molecular techniques in recent decades. Only 32.2% and 8.4% of the data sets reported on sporozoite analysis and entomological inoculation rate (EIR), respectively which highlights the paucity of such important information in the country. Studies demonstrated efficacy of all four major classes of insecticides against malaria vectors in Tanzania with focal points showing phenotypic resistance. About 95% of malaria entomological data was obtained from northeastern Tanzania. This shows the disproportionate nature of the available information with the western part of the country having none. Therefore it is important for the country to establish entomological surveillance system with state of the art to capture all vitally important entomological indices including vector bionomics in areas of Tanzania where very few or no studies have been done. This is vital in planning and implementing evidence based malaria vector control programmes as well as in monitoring the current malaria control interventions.

Assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates

The critical first step in the clinical development of a malaria vaccine, based on live-attenuated Plasmodium falciparum sporozoites, is the guarantee of complete arrest in the liver. We report on an approach for assessing adequacy of attenuation of genetically attenuated sporozoites in vivo using the Plasmodium berghei model of malaria and P. falciparum sporozoites cultured in primary human hepatocytes. We show that two genetically attenuated sporozoite vaccine candidates, Δp52+p36 and Δfabb/f, are not adequately attenuated. Sporozoites infection of mice with both P. berghei candidates can result in blood infections. We also provide evidence that P. falciparum sporozoites of the leading vaccine candidate that is similarly attenuated through the deletion of the genes encoding the proteins P52 and P36, can develop into replicating liver stages. Therefore, we propose a minimal set of screening criteria to assess adequacy of sporozoite attenuation necessary before advancing into further clinical development and studies in humans.

Development of a quantitative flow cytometry-based assay to assess infection by Plasmodium falciparum sporozoites

The human malaria parasite Plasmodium falciparum causes the most deadly parasitic disease worldwide, necessitating the development of interventions that block infection. Yet, preclinical assays to measure inhibition of infection date from the 1980s and are based on microscopy. Here, we describe the development of a simple flow cytometric assay that can be used to quantitatively assess P. falciparum sporozoite infection in vitro in low and medium throughput. We demonstrate the utility of this assay for assessing both drug inhibition of infection and measuring efficacy of antibodies in blocking parasite infection. This methodology will aid in assessing functional antibody responses to vaccination and novel drugs that prevent mosquito-to-man transmission of malaria.

First clinical trial of purified, irradiated malaria sporozoites in humans

Evaluation of: Epstein JE, Tewari K, Lyke KE et al. Live attenuated malaria vaccine designed to protect through hepatic CD8 T cell immunity. Science 334(6055), 475–480 (2011).Purified, irradiated, metabolically active sporozoites of Plasmodium falciparum (PfSPZ) have been administered to 80 volunteers through subcutaneous or intradermal routes. This PfSPZ vaccine has been shown to be safe but of low immunogenicity and hence protective efficacy as shown after challenge with infected mosquitoes. Intravenous delivery of PfSPZ in nonhuman primates induced memory CD8+ and CD4+ T cells specific for PfSPZ that reside in the liver. Further studies using PfSPZ and the intravenous route in humans are warranted to fully explore the potential of purified, attenuated sporozoites for malaria vaccine development.

Reproductive success in Anopheles arabiensis and the M and S molecular forms of Anopheles gambiae: Do natural sporozoite infection and body size matter?

Malaria parasites stages prior to sporozoite formation are known to affect the fecundity of several species of mosquitoes in the laboratory, but little is known about this phenomenon in natural conditions especially with sporozoite-infected anophelines. The reproductive success of wild-caught Anopheles arabiensis and the M and S molecular forms of Anopheles gambiae was investigated by comparing females infected with Plasmodium falciparum sporozoites to females free of sporozoites. Association between sporozoite-infected females’ body size and their egg batch size was also measured. There was no significant reduction in egg production due to sporozoite infection among wild females An. arabiensis and the M and S form of An. gambiae. The infected groups and the controls laid similar numbers of eggs. A positive association was found between body size of females infected with P. falciparum and mean egg production. Infected females of the molecular forms of An. gambiae and their sibling species An. arabiensis invest similarly in egg batch size regardless of their body size although the expected egg batch size may differ among them because of differences in their mean body size. A reduction of egg production related to infection status was not observed among females harboring sporozoites. Therefore for the gonotrophic cycles that occur once sporozoites are present, natural infection of all three vectors we studied has no or minimal effect on their densities or their reproductive outputs.