Sporozoite Formation Research Articles

Identification of Inhibitors against P.Falciparum GAP50 and Human Complement Factor H Interaction in the Mosquito

Malaria is an infectious disease caused by obligate intracellular parasites of the genus Plasmodium. Severe malaria cases result in more than half a million deaths every year (World Malaria Report, 2013). Mosquitoes transmit the parasite from human to human during their blood meals. With the blood meal, mosquitoes ingest female and male gametocytes present in the blood of a Plasmodium infected human. Gametocytes develop into gametes (Aly et al, 2009) that undergo fertilization inside the mosquito's gut and later result in the formation of infectious sporozoites that are transmitted during a bite. This uptake, maturation and fertilization, represents a bottleneck in the parasite's life cycle, and a prime target for transmission blocking. In the mosquito's gut, macrogametes (female gametes) bind the human complement control regulator protein, hFactor H, via PfGAP50 to avoid complement-mediated lysis (Simon et al, 2013). A potential transmission-blocking small molecule inhibitor would disrupt the PfGAP50-hFactor H interaction, exposing the parasite to the human alternate pathway of complement for destruction.The goal of this project is to characterize the PfGAP50-hFactor H interaction in vitro, and to screen the Medicines for Malaria Venture Malaria Box library for small molecule protein-protein interaction (PPI) inhibitors using a surface plasmon resonance (SPR)-based assay. Several PPI inhibitors were identified and the inhibitory affect was further validated in a concentration-dependent SPR screen. Promising hits will be further tested in mosquitos. In vitro characterization of this interaction will provide a better understanding of pathogen-host interactions at a molecular level and allow the development of specific inhibitors of PfGAP50-hFactor H interaction in the mosquito.

The repeat region of the circumsporozoite protein is critical for sporozoite formation and maturation in Plasmodium.

The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH2-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.

Plasmodium yoelii vitamin B5 pantothenate transporter candidate is essential for parasite transmission to the mosquito.

In nearly all non-photosynthetic cells, pantothenate (vitamin B5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages.

Effect of ingested human antibodies induced by RTS, S/AS01 malaria vaccination in children on Plasmodium falciparum oocyst formation and sporogony in mosquitoes.

BackgroundThe circumsporozoite protein (CS protein) on the malaria parasites in mosquitoes plays an important role in sporogony in mosquitoes. The RTS,S/AS01 malaria vaccine candidate, which has shown significant efficacy against clinical malaria in a large Phase 3 trial, targets the Plasmodium falciparum CS protein, but the ability of serum from vaccinated individuals to inhibit sporogony in mosquitoes has not been evaluated.MethodsPreviously a double-blind, randomized trial of RTS,S/AS01 vaccine, as compared with rabies vaccine, in five- to 17-month old children in Tanzania was conducted. In this study, polyclonal human antibodies were purified from the pools of sera taken one month after the third vaccination. IgGs were purified from four pools of sera from 25 RTS,S/AS01 vaccinated children each, and two pools of sera from 25 children vaccinated with rabies vaccine each. The ability of antibodies to inhibit P. falciparum oocyst formation and/or sporogony in the mosquito host was evaluated by a standard membrane-feeding assay. The test antibodies were fed on day 0 (at the same time as the gametocyte feed), or on days 3 or 6 (serial-feed experiments). The oocyst and sporozoite counts were performed on days 8 and 16, respectively. In addition, two human anti-CS monoclonal antibodies (mAb) and a control mAb were also evaluated.ResultsPolyclonal anti-CS IgG preparations from RTS,S-vaccinated children tested at concentrations of 149-210 ELISA units (EU)/ml did not show significant inhibition in oocyst and sporozoite formation when the antibodies were fed with gametocytes at the same time, or later (serial-feed experiments). Similarly, anti-CS mAbs tested at 6,421 or 7,122 EU/ml did not show reduction in oocyst and sporozoite formation.ConclusionsThis study does not support the concept that anti-CS antibodies induced by the RTS,S/AS01 vaccines in humans noticeably reduce malaria transmission by blocking P. falciparum sporozoite development or salivary gland invasion in mosquitoes when taken up during feeding.

Taking a Bite Out of Mosquitoes: A New Drug to Block Transmission of Malaria

Despite concerted control efforts and decades of research, malaria remains a global scourge and the most important parasitic disease of humans. Infections caused by the parasite Plasmodium falciparum exact a huge toll, largely in subSaharan Africa, where nearly a million children die annually from complications of malaria. Integrated campaigns such as The Malaria Control and Evaluation Program in Africa (MACEPA) have adopted comprehensive strategies including insecticide-impregnated bed nets, artemesininbased combination therapies, aggressive indoor insecticide spraying, and advanced diagnostics to reduce mortality in children by nearly 20 percent [1]. Such recent advances coupled with evolving prospects for a vaccine against malaria [2] suggest that the goal of malaria eradication may soon be attainable, using an arsenal of preventive practices. For now, chemotherapeutic measures remain the mainstay of malaria management. Currently, combination therapy with artemesinin derivatives and agents such as mefloquine, lumefantrine, or amodiaquine are first-line therapies for uncomplicated malaria in most endemic regions of the world. Such treatments can be highly effectivewithresolutionofparasitemiawhen administered soon after onset. Although artemesinin-based therapies have proven successful in many trials across Africa and SoutheastAsia, there are growing concerns about evolving resistance of P. falciparum to these agents. Recently, Phyo et al [3] reported an alarming increase in rates of artemesinin resistance amongst P. falciparum isolates frompatients seen in clinics along the Thailand-Myanmar border.Furthermore,resistanceratesinWestern Cambodia were reported to be greater than 40percent.Assays of parasite clearancewere correlated with genotypic changes in parasite populations and nearly two-thirds of the variation in parasite clearance over a 3-year period was attributable to genetic polymorphisms in populations of P. falciparum. Indeed, emergence of widespread artemesinin resistance in this region of the world underscores the pressing need to develop novel anti-malarial agents. In this issue of the Journal, Ojo et al [4] report a very exciting new prospect for drug treatment of P. falciparum. The investigators describe a new class of antimalarial compounds that target the sexual stage of Plasmodium (Figure 1), specifically the parasite’s calcium-dependent protein kinase 4 (CDPK4), a signaling molecule that is essential for exflagellation of male gametocytes. This step is a precursor to the fusion ofmale and female gametocytes, which results in zygote formation in the mosquito. The sexual phase of the Plasmodium life cycle is critical and represents a potential target for drugs that would ultimately abort transmission of parasites by mosquitoes to humans. Compound 1294, reported by Ojo et al is a synthetic inhibitor of Plasmodium CDPK4. The authors demonstrate convincingly that the synthetic CDPK4 inhibitor inhibits exflagellation of gametocytes and, by acting on a specific serine residue of the parasite, avoids cross-reactivity with mammalian kinase substrates. Furthermore, the mechanism by which this compound acts on parasites is cleverly elucidated, using transgenic lines of Plasmodium. By generating a mutant parasite with a modified target site for the enzyme, the authors demonstrate that EC50 exflagellation values are shifted relative to wild-type parasites exposed to the same inhibitor. Thus, both efficacy of compound 1294 and its mode of action are presented in this seminal article. Gametocytes of P. falciparum begin maturation in the mammalian host and are taken up during a mosquito’s blood meal (Figure 1). Fusion of the gametocytes and formation of a zygote occur in the arthropod. Therefore, a CDPK4 inhibitor, taken as a drug by a human, would disrupt the parasite life cycle within the insect and prevent formation of infectious sporozoites. For this strategy to be effective, the drug would have to be widely bioavailable and have a long half-life, because Received and accepted 15 July 2013. Correspondence: Ravi Durvasula, MD, Chief of Medicine, Raymond G. Murphy VA Medical Center, 1501 San Pedro Drive SE, Albuquerque, NM 87108 (ravi.durvasula@va.gov). The Journal of Infectious Diseases Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2013. This work is written by (a) US Government employee(s) and is in the public domain in the US. DOI: 10.1093/infdis/jit523

4-(1H)-Quinolones and 1,2,3,4-Tetrahydroacridin-9(10H)-ones prevent the transmission of Plasmodium falciparum to Anopheles freeborni.

Malaria kills approximately 1 million people a year, mainly in sub-Saharan Africa. Essential steps in the life cycle of the parasite are the development of gametocytes, as well as the formation of oocysts and sporozoites, in the Anopheles mosquito vector. Preventing transmission of malaria through the mosquito is necessary for the control of the disease; nevertheless, the vast majority of drugs in use act primarily against the blood stages. The study described herein focuses on the assessment of the transmission-blocking activities of potent antierythrocytic stage agents derived from the 4(1H)-quinolone scaffold. In particular, three 3-alkyl- or 3-phenyl-4(1H)-quinolones (P4Qs), one 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), and one 1,2,3,4-tetrahydroacridin-9(10H)-one (THA) were assessed for their transmission-blocking activity against the mosquito stages of the human malaria parasite (Plasmodium falciparum) and the rodent parasite (P. berghei). Results showed that all of the experimental compounds reduced or prevented the exflagellation of male gametocytes and, more importantly, prevented parasite transmission to the mosquito vector. Additionally, treatment with ICI 56,780 reduced the number of sporozoites that reached the Anopheles salivary glands. These findings suggest that 4(1H)-quinolones, which have activity against the blood stages, can also prevent the transmission of Plasmodium to the mosquito and, hence, are potentially important drug candidates to eradicate malaria.

Serological responses to a soluble recombinant chimeric Plasmodium vivax circumsporozoite protein in VK210 and VK247 population

BackgroundCircumsporozoite protein (CSP) is essential for sporozoite formation and sporozoite invasion into human hepatocyte. Previously, a recombinant P. vivax CSP based on chimeric repeats (rPvCSP-c) representing two major alleles VK210 and VK247 within central region has been designed. Naturally acquired humoral immune responses study show that antigenicity of rPvCSP-c was much higher than that of native strain. However, the serologic reactivity of rPvCSP-c was still unclear in detail.MethodsIn present study, recognition of rPvCSP-c in vivax malaria typed VK210 and VK247 alleles was assessed. VK210 typed and VK247 typed sera from adult residents reacted specifically with rPvCSP-c using protein array and immunoblot assay. Additionally, anti-rPvCSP-c serum recognized the fixed VK210 and VK247 sporozoites by immunofluorescence assay. Furthermore, statistic analysis was performed for correlational detection.ResultsThe rPvCSP-c reacted with both VK210 typed and VK247 typed P. vivax infected patient sera and anti-rPvCSP-c immune serum also reacted with VK210 and VK247 sporozoite parasites of P. vivax specifically. There was a positive correlation between increased antibody level, age of patients and also associated with pvcsp repeat number, although the level of responses did vary considerably in their reactivity to the rPvCSP-c from negative to very high level within each age group.ConclusionsThese data confirmed the serologic reactivity of the novel rPvCSP-c in exposed both VK210 and VK247 populations. These results strongly suggested that this recombinant CSP was biologically active and potently immunogenic across major strains and raised the prospect that this protein could be used as serologic marker.

Aberrant Sporogonic Development of Dmc1 (a Meiotic Recombinase) Deficient Plasmodium berghei Parasites

BackgroundIn Plasmodium, meiosis occurs in diploid zygotes as they develop into haploid motile ookinetes inside the mosquito. Further sporogonic development involves transformation of ookinetes into oocysts and formation of infective sporozoites.Methodology/Principal FindingsReverse genetics was employed to examine the role of the meiotic specific recombinase Dmc1, a bacterial RecA homolog during sporogony in Plasmodium berghei. PbDmc1 knockout (KO) parasites showed normal asexual growth kinetics compared to WT parasites; however oocyst formation in mosquitoes was reduced by 50 to 80%. Moreover, the majority of oocysts were retarded in their growth and were smaller in size compared to WT parasites. Only a few Dmc1 KO parasites completed maturation resulting in formation of fewer sporozoites which were incapable of infecting naive mice or hepatocytes in vitro. PbDmc1 KO parasites were shown to be approximately 18 times more sensitive to Bizelesin, a DNA alkylating drug compared to WT parasites as reflected by impairment of oocyst formation and sporogonic development in the mosquito vector.Conclusions/SignificanceOur findings suggest that PbDmc1 plays a critical role in malaria transmission biology.

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.

Arrested Oocyst Maturation in Plasmodium Parasites Lacking Type II NADH:Ubiquinone Dehydrogenase

The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.

Transcription factor AP2‐Sp and its target genes in malarial sporozoites

The malarial sporozoite is the stage that infects the liver, and genes expressed in this stage are potential targets for vaccine development. Here, we demonstrate that specific gene expression in this stage is regulated by an AP2-related transcription factor, designated AP2-Sp (APETALA2 in sporozoites), that is expressed from the late oocyst to the salivary gland sporozoite. Disruption of the AP2-Sp gene did not affect parasite replication in the erythrocyte but resulted in loss of sporozoite formation. The electrophoretic mobility-shift assay showed that the DNA-binding domain of AP2-Sp recognizes specific eight-base sequences, beginning with TGCATG, which are present in the proximal promoter region of all known sporozoite-specific genes. Promoter assays demonstrated that these sequences act as cis-acting elements and are critical for the expression of sporozoite-specific genes with different expression profiles. In transgenic parasites that express endogenous AP2-O (APETALA2 in ookinetes), but whose AP2 domain had been swapped with that of AP2-Sp, several target genes of AP2-Sp were induced in the ookinete stage. These results indicate that AP2-Sp is a major transcription factor that regulates gene expression in the sporozoite stage.

Disruption of Plasmodium Sporozoite Transmission by Depletion of Sporozoite Invasion-Associated Protein 1

Accumulation of infectious Plasmodium sporozoites in Anopheles spp. salivary glands marks the final step of the complex development of the malaria parasite in the insect vector. Sporozoites are formed inside midgut-associated oocysts and actively egress into the mosquito hemocoel. Traversal of the salivary gland acinar cells correlates with the sporozoite's capacity to perform continuous gliding motility. Here, we characterized the cellular role of the Plasmodium berghei sporozoite invasion-associated protein 1 (SIAP-1). Intriguingly, SIAP-1 orthologs are found exclusively in apicomplexan hemoprotozoa, parasites that are transmitted by arthropod vectors, e.g., Plasmodium, Babesia, and Theileria species. By fluorescent tagging with mCherry, we show that SIAP-1 is expressed in oocyst-derived and salivary gland-associated sporozoites, where it accumulates at the apical tip. Targeted disruption of SIAP-1 does not affect sporozoite formation but causes a partial defect in sporozoite egress from oocysts and abolishes sporozoite colonization of mosquito salivary glands. Parasites with the siap-1(-) mutation are blocked in their capacity to perform continuous gliding motility. We propose that arthropod-transmitted apicomplexan parasites specifically express secretory factors, such as SIAP-1, that mediate efficient oocyst exit and migration to the salivary glands.

Analysis of mutant Plasmodium berghei parasites lacking expression of multiple PbCCp genes

Plasmodium encodes a family of six secreted multi-domain adhesive proteins, termed PCCps, which are released from gametocytes during emergence within the mosquito midgut. The expression and cellular localization of PCCp proteins predict a role either in gametocyte development or within the mosquito midgut during the transition from gametes into the ookinete stage. However, mutant parasites lacking expression of any single PCCp protein show a phenotype at the oocyst stage with a failure of oocyst maturation and sporozoite formation. In this study we investigated the stage-specific transcription of the PCCp genes of the rodent malaria parasite, Plasmodium berghei, and analyzed their promoter activities. Transcript expression analysis by quantitative real time RT-PCR showed that as in the human malaria parasite, Plasmodium falciparum, all PbCCp genes are predominantly transcribed in the gametocyte stage with a low level of transcription in the oocyst stage. Transgenic P. berghei parasites that contain the reporter protein GFP driven by the promoter regions of PbCCps showed pronounced GFP expression exclusively in gametocytes, in agreement with the RT-PCR data. To determine whether functional redundancies of different PCCp family members could explain the lack of a phenotype in gametocytes or gametes in single knockout mutant parasites, double gene null mutant P. berghei parasites were generated lacking either PCCp1 and PCCp3, or PCCp1 and PCCp4. The phenotype of these double knockout mutants was similar to that observed for single gene knockout mutants and manifest at the oocyst rather than the gametocyte or other stages within the mosquito midgut lumen.

Female Inheritance of Malarial lap Genes Is Essential for Mosquito Transmission

Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite–mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Δpblap sporozoites, but not if the individual Δpblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Δpbs47) and male-deficient (Δpbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Δpblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages.

Getting infectious: formation and maturation of Plasmodium sporozoites in the Anopheles vector

Research on Plasmodium sporozoite biology aims at understanding the developmental program steering the formation of mature infectious sporozoites - the transmission stage of the malaria parasite. The recent identification of genes that are vital for sporozoite egress from oocysts and subsequent targeting and transmigration of the mosquito salivary glands allows the identification of mosquito factors required for life cycle completion. Mature sporozoites appear to be equipped with the entire molecular repertoire for successful transmission and subsequent initiation of liver stage development. Innovative malaria intervention strategies that target the early, non-pathogenic phases of the life cycle will crucially depend on our insights into sporozoite biology and the underlying molecular mechanisms that lead the parasite from the mosquito midgut to the liver.

Mutational analysis of the GPI-anchor addition sequence from the circumsporozoite protein of Plasmodium

The plasma membrane of Plasmodium sporozoites is uniformly covered by the glycosylphosphatidylinositol (GPI)-anchored circumsporozoite (CS) protein. Sporozoites form in the mosquito midgut through a budding process that occurs within a multinucleate oocyst underneath the basal lamina of the gut. Earlier genetic studies established that normal sporozoite development requires CS. Mutant parasites lacking CS [CS (-)] do not form sporozoites. Ultrastructural analysis of the oocysts from these parasites revealed that there is an early block in the cytokinesis that occurs within the multinucleate oocysts to generate individual sporozoites. Parasites that are hypomorphic for CS expression gave rise to sporozoites with abnormal morphology. These results proved that CS plays a direct role in the maturation of oocysts and in the normal budding of sporozoites. In this article, we examined if the membrane localization of CS via a GPI-anchor, is crucial for its function during sporozoite formation. We generated three mutants in Plasmodium berghei CS, CS-DeltaGPI, CS-TM1 and CS-TM2. In CS-DeltaGPI, we deleted the signal sequence required for the addition of a GPI-anchor to CS. The resulting protein was found only in the cytoplasm of the oocyst. In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was substituted by the transmembrane domain and truncated (to different degrees) cytoplasmic tail of Plasmodium thrombospondin-related anonymous protein (TRAP). The resulting CS protein was detected on the plasma membrane of the oocysts. The amount of CS in the mutants was similar to that of wild type. The sporozoite budding and development were abrogated in both CS-DeltaGPI and CS-TM mutants. The ultrastructure of the mutant oocysts was indistinguishable from that of the CS (-) parasites. Our results suggest that the GPI-anchor of the CS protein is required for sporogenesis.

Expression of equi merozoite antigen 2 during development of Babesia equi in the midgut and salivary gland of the vector tick Boophilus microplus.

Equi merozoite antigens 1 and 2 (EMA-1 and EMA-2) are Babesia equi proteins expressed on the parasite surface during infection in horses and are orthologues of proteins in Theileria spp., which are also tick-transmitted protozoal pathogens. We determined in this study whether EMA-1 and EMA-2 were expressed within the vector tick Boophilus microplus. B. equi transitions through multiple, morphologically distinct stages, including sexual stages, and these transitions culminate in the formation of infectious sporozoites in the tick salivary gland. EMA-2-positive B. equi stages in the midgut lumen and midgut epithelial cells of Boophilus microplus nymphs were identified by reactivity with monoclonal antibody 36/253.21. This monoclonal antibody also recognized B. equi in salivary glands of adult Boophilus microplus. In addition, quantification of B. equi in the mammalian host and vector tick indicated that the duration of tick feeding and parasitemia levels affected the percentage of nymphs that contained morphologically distinct B. equi organisms in the midgut. In contrast, there was no conclusive evidence that B. equi EMA-1 was expressed in either the Boophilus microplus midgut or salivary gland when monoclonal antibody 36/18.57 was used. The expression of B. equi EMA-2 in Boophilus microplus provides a marker for detecting the various development stages and facilitates the identification of novel stage-specific Babesia proteins for testing transmission-blocking immunity.

Potential for Anopheles campestris (Diptera: Culicidae) to transmit malaria parasites in Pa Rai subdistrict (Aranyaprathet, Sa Kaeo Province), Thailand.

Member(s) of the Anopheles barbirostris group Reid, particularly Anopheles barbirostris and Anopheles campestris Reid are the suspected vectors of Plasmodium vivax in Pa Rai (Aranyaprathet, Sa Kaeo province). To determine if An. barbirostris, An. campestris, or both, are present in Pa Rai and to determine their potential to transmit malaria, a field and laboratory study was conducted. Isofemale colonizations of wild caught mosquitoes captured by landing catches were made for species confirmation and to determine the mosquito life cycle. Pupal morphology indicated all mosquitoes were An. campestris. During the late rainy season (October and November), An. campestris populations comprised 78.6% of all females captured by human landing catches and 7.1% of mosquitoes in a cow-baited trap. The biting activity cycle peaked between 2000 and 0100 hours and was highest (17.6 bites per person per hour) at 2300 hours. More An. campestris bit people indoors (nine bites per person per hour) than outdoors (four bites per person per hour). Immature An. campestris were found in ponds, swamps, rice-fields, puddles, marshes, ground pools, and pits with open sunlight to partial shade. The time from egg hatch to adult was 18-47 d and 14-22 d under laboratory (25.0-27.0 degrees C) and ambient (26-32 degrees C) conditions, respectively. The fecundity of An. campestris ranged from 173 to 311 eggs. Based on experimental infections, An. campestris was able to support the sporogonic cycle of P. vivax with 76.2 and 23.8% oocyst and sporozoite formation rate, respectively. An. campestris shows high potential as a malaria vector in Pa Rai.

Plasmodium knowlesi provides a rapid in vitro and in vivo transfection system that enables double-crossover gene knockout studies.

Transfection technology for malaria parasites provides a valuable tool for analyzing gene function and correlating genotype with phenotype. Transfection models are even more valuable when appropriate animal models are available in addition to complete in vitro systems to be able to fully analyze parasite-host interactions. Here we describe the development of such a model by using the nonhuman primate malaria Plasmodium knowlesi. Blood-stage parasites were adapted to long-term in vitro culture. In vitro-adapted parasites could re-adapt to in vivo growth and regain wild-type characteristics after a single passage through an intact rhesus monkey. P. knowlesi parasites, either in vitro adapted or in vivo derived, were successfully transfected to generate circumsporozoite protein (CSP) knockout parasites by double-crossover mechanisms. In vitro-transfected and cloned CSP knockout parasites were derived in a time span of only 18 days. Microscopic evaluation of developing oocysts from mosquitoes that had fed on CSP knockout parasites confirmed the impairment of sporozoite formation observed in P. berghei CSP knockout parasites. The P. knowlesi model currently is the only malaria system that combines rapid and precise double-crossover genetic manipulation procedures with complete in vitro as well as in vivo possibilities. This allows for full analysis of P. knowlesi genotype-phenotype relationships and host-parasite interactions in a system closely related to humans.

Eimeria tenella: hsp70 expression during sporogony.

There is an increasing interest in identifying the parasite components involved in the maturation, development, and infectivity of intracellular protozoan parasites. In the present study, a heat shock protein (hsp) of the family of 70 kDa hsp (hsp70), which play important roles in the stage conversion and virulence of these parasites, was examined. Whereas hsp70 expression has been examined in Eimeria tenella within host tissues, in the present study, oocysts of E. tenella were used to investigate the expression of hsp70 during sporulation without interference from the host; hsp70 expression during excystation was induced by incubating sporulated oocysts under various experimental conditions to produce the stimuli necessary for sporozoites to become active and to excyst in vitro. Hsp70 was detected by immunohistochemical techniques; quantitative flow cytometric analysis was also been carried out using specific monoclonal antibodies against hsp70. Hsp70 was expressed during sporulation but was not found in sporulated oocysts after the completion of sporulation. Oocysts re-expressed hsp70 when excystation was induced. The presence of hsp70 prior to infection may preadapt the parasite for additional stress in the host and may be involved in the formation of sporozoites.