Mature Schizonts Research Articles

Morphological features and differential counts of Plasmodium knowles i parasites in naturally acquired human infections

BackgroundHuman infections with Plasmodium knowlesi, a simian malaria parasite, are more common than previously thought. They have been detected by molecular detection methods in various countries in Southeast Asia, where they were initially diagnosed by microscopy mainly as Plasmodium malariae and at times, as Plasmodium falciparum. There is a paucity of information on the morphology of P. knowlesi parasites and proportion of each erythrocytic stage in naturally acquired human infections. Therefore, detailed descriptions of the morphological characteristics and differential counts of the erythrocytic stages of P. knowlesi parasites in human infections were made, photographs were taken, and morphological features were compared with those of P. malariae and P. falciparum.MethodsThick and thin blood films were made prior to administration of anti-malarial treatment in patients who were subsequently confirmed as having single species knowlesi infections by PCR assays. Giemsa-stained blood films, prepared from 10 randomly selected patients with a parasitaemia ranging from 610 to 236,000 parasites per μl blood, were examined.ResultsThe P. knowlesi infection was highly synchronous in only one patient, where 97% of the parasites were at the late trophozoite stage. Early, late and mature trophozoites and schizonts were observed in films from all patients except three; where schizonts and early trophozoites were absent in two and one patient, respectively. Gametocytes were observed in four patients, comprising only between 1.2 to 2.8% of infected erythrocytes. The early trophozoites of P. knowlesi morphologically resemble those of P. falciparum. The late and mature trophozoites, schizonts and gametocytes appear very similar to those of P. malariae. Careful examinations revealed that some minor morphological differences existed between P. knowlesi and P. malariae. These include trophozoites of knowlesi with double chromatin dots and at times with two or three parasites per erythrocyte and mature schizonts of P. knowlesi having 16 merozoites, compared with 12 for P. malariae.ConclusionPlasmodium knowlesi infections in humans are not highly synchronous. The morphological resemblance of early trophozoites of P. knowlesi to P. falciparum and later erythrocytic stages to P. malariae makes it extremely difficult to identify P. knowlesi infections by microscopy alone.

Experimental conditions affect the outcome of Plasmodium falciparum platelet-mediated clumping assays

BackgroundPlatelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IE) is a parasite adhesion phenotype that has been associated with severe malaria in some, but not all, field isolate studies. A variety of experimental conditions have been used to study clumping in vitro, with substantial differences in parasitaemia (Pt), haematocrit (Ht), and time of reaction between studies. It is unknown whether these experimental variables affect the outcome of parasite clumping assays.MethodsThe effects of Pt (1, 4 and 12%), Ht (2, 5 and 10%) and time (15 min, 30 min, 1 h, 2 h) on the clumping of P. falciparum clone HB3 were examined. The effects of platelet freshness and parasite maturity were also studied.ResultsAt low Ht (2%), the Pt of the culture has a large effect on clumping, with significantly higher clumping occurring at 12% Pt (mean 47% of IE in clumps) compared to 4% Pt (mean 26% IE in clumps) or 1% Pt (mean 7% IE in clumps) (ANOVA, p = 0.0004). Similarly, at low Pt (1%), the Ht of the culture has a large effect on clumping, with significantly higher clumping occurring at 10% Ht (mean 62% IE in clumps) compared to 5% Ht (mean 25% IE in clumps) or 2% Ht (mean 10% IE in clumps) (ANOVA, p = 0.0004). Combinations of high Ht and high Pt were impractical because of the difficulty assessing clumping in densely packed IE and the rapid formation of enormous clumps that could not be counted accurately. There was no significant difference in clumping when fresh platelets were used compared to platelets stored at 4°C for 10 days. Clumping was a property of mature pigmented-trophozoites and schizonts but not ring stage parasites.ConclusionThe Pt and Ht at which in vitro clumping assays are set up have a profound effect on the outcome. All previous field isolate studies on clumping and malaria severity suffer from potential problems in experimental design and methodology. Future studies of clumping should use standardized conditions and control for Pt, and should take into account the limitations and variability inherent in the assay.

Rhoptry neck protein RON2 forms a complex with microneme protein AMA1 in Plasmodium falciparum merozoites

Erythrocyte invasion is an essential step in the establishment of host infection by malaria parasites, and is a major target of intervention strategies that attempt to control the disease. Recent proteome analysis of the closely-related apicomplexan parasite, Toxoplasma gondii, revealed a panel of novel proteins (RONs) located at the neck portion of the rhoptries. Three of these proteins, RON2, RON4, and RON5 have been shown to form a complex with the microneme protein Apical Membrane Protein 1 (AMA1). This complex, termed the Moving Junction complex, localizes at the interface of the parasite and the host cell during the invasion process. Here we characterized a RON2 ortholog in Plasmodium falciparum. PfRON2 transcription peaked at the mature schizont stage and was expressed at the neck portion of the rhoptry in the merozoite. Co-immunoprecipitation of PfRON2, PfRON4 and PfAMA1 indicated that the complex formation is conserved between T. gondii and P. falciparum, suggesting that co-operative function of the rhoptry and microneme proteins is a common mechanism in apicomplexan parasites during host cell invasion. PfRON2 possesses a region displaying homology with the rhoptry body protein PfRhopH1/Clag, a component of the RhopH complex. However, here we present co-immunoprecipitation studies which suggest that PfRON2 is not a component of the RhopH complex and has an independent role. Nucleotide polymorphism analysis suggested that PfRON2 was under diversifying selective pressure. This evidence suggests that RON2 appears to have a fundamental role in host cell invasion by apicomplexan parasites, and is a potential target for malaria intervention strategies.

In vitro drug sensitivity testing in Plasmodium vivax

The possibility of short-term in vitro cultivation, i.e. growth of asexual erythrocytic stages up to the stage of mature schizonts, permits in principle the development of drug sensitivity tests also for Plasmodium vivax. In the absence of a sequestration of erythrocytes carrying the advanced stages of schizogony, asexual parasites of all stages may be seen in the peripheral blood of patients infected with P. vivax. This precludes schizont maturation tests since schizont development will be unduly influenced by the number of advanced trophozoites. A test system reflecting the age composition of the parasite population and its progression without and under the influence of inhibitors was found to yield precise results also in the higher IC ranges. The population-based test procedure would also permit the identification of any stage-specific impact of antimalarial agents.

Crystal Structure of Soluble Domain of Malaria Sporozoite Protein UIS3 in Complex with Lipid

Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-Å resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3130-229) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3130-229 is a novel, compact, and all α-helical structure bound to one molecule of PE. The PfUIS3130-229-PE complex structure reveals a novel binding site with specific interactions between PfUIS3130-229 and the PE head group. One acyl chain of PE wraps around part of PfUIS3130-229 and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3130-229 interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3130-229 with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.

Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites

We report two improved assays for in vitro and in vivo screening of chemicals with potential anti-malarial activity against the blood stages of the rodent malaria parasite Plasmodiumberghei. These assays are based on the determination of luciferase activity (luminescence) in small blood samples containing transgenic blood stage parasites that express luciferase under the control of a promoter that is either schizont-specific (ama-1) or constitutive (eef1αa). Assay 1, the in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC50 values comparable to those obtained with standard in vitro drug-susceptibility assays. The second assay, the in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1αa promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. Both tests are suitable for use in larger-scale in vitro and in vivo screening of drugs. The standard methodology of anti-malarial drug screening and validation, which includes testing in rodent models of malaria, can be improved by the incorporation of such assays.

Light and Electron Microscopic Study on a Haemogregarine Species Infecting the Viper Cerastes cerastes gasperitti from Saudi Arabia

A Haemogregarine sp. infecting the Viper Cerastes cerastes gasperitti (35%) from K.S.A. was recorded for the 1st time. Parasites were recorded exclusively in the red blood cells of the Vipers. Erythrocytic parasites measured 17.5 x 3 microm with a nucleus of 8.1 x 2.5 microm. Mature Schizonts of 30 x 2.5 microm and producing 18-30 merozoites by ectomerogony were reported in the endothelial cells of blood capillaries of different internal organs (liver, spleen and lung). The merozoites measured 13 x 3.5 microm and these were similar to the stages in the red blood cells. The motile stages of the parasite recorded in the present study showed all the general architectures of the Apicomplexa with some pecularities such as presence of micronemes and rhoptries at both sides of the nucleus and the occurance of large number of subpellicular microtubules (80-90) extending to the posterior end of the parasite.

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear ( Ursus thibetanus japonicus)

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear ( Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7 ± 4.6 × 42.7 ± 4.5 µm in size. Each mature schizont contained approximately 80–130 merozoites and 0–5 residual bodies. The merozoites were 7.0 ± 0.7 × 1.8 ± 0.3 µm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9 ± 0.3 × 3.3 ± 0.2 µm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks ( Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 × 234.0 µm and 331.8 × 231.7 µm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2 ± 2.5 × 27.0 ± 2.9 µm in size. Each sporocyst contained at least 8–16 sporozoites, with the sporozoites being 12.2 ± 1.4 × 3.5 ± 0.5 µm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.

Development and validation of real-time polymerase chain reaction assays specific to four species of Eimeria

The development of quantitative real-time polymerase chain reaction (PCR) assays specific to Eimeria acervulina, Eimeria maxima, Eimeria necatrix and Eimeria tenella is described and validated. The PCR templates adopted include a fragment of a gene encoding a microneme protein and previously characterized species-specific random amplified polymorphic DNA (RAPD) sequences. The sensitivity of each assay allowed the consistent detection of between one and 10 parasite genomes, equivalent to between one or two sporulated oocysts or a fraction of a single mature schizont, unaffected by the presence of chicken (host) or other Eimeria species DNA. Regression coefficients in excess of 0.99 over linear ranges of at least six orders of magnitude, together with comparable PCR efficiencies, demonstrated the robust reproducibility of each assay and suggest that two or more may be successfully multiplexed. The species-specific assays described here, combined with a previously published generic Eimeria species real-time PCR, provide valuable components in a “tool box” to accurately quantify the presence of specific Eimeria species in environmental or within-host phases of the lifecycle with little specialist knowledge. The application of these assays may benefit chicken husbandry, veterinary practice, quality control of live vaccine production and scientific research.

Cross-reactivity Studies of an Anti- Plasmodium vivax Apical Membrane Antigen 1 Monoclonal Antibody: Binding and Structural Characterisation

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.

Prophylactic potential of liposomized integral membrane protein of Plasmodium yoelii nigeriensis against blood stage infection in BALB/c mice

Triton X-114 phase separated integral membrane proteins (IMPs) of a multidrug resistant strain of Plasmodium yoelii nigeriensis ( P. yoelii) were screened for their potential to impart protection against malaria infection in BALB/c mice. As revealed by immunoblotting, antibodies present in parasite specific sera from convalescent (protected) as well as immunized (partially protected) animals recognized different membrane proteins. A thorough investigation reveals that P. yoelii specific convalescent sera recognized IMPs with molecular masses ranging from 21 to 81 kDa. Among various membrane proteins, the IMPs corresponding to 81 and 66 kDa molecular weight were highly prominent in the immunoblots probed with the sera from convalescent animals, whereas sera from immunized animals failed to produce impressive band pattern. Immunofluorescence assay revealed that the 66-kDa IMP specific antibodies reacted with fixed smears of mature schizonts and merozoites. Further immunization with 66 kDa IMP (PyIMP) purified through polyclonal IgG sepharose 4B affinity did not impart effective immune response (in its free form) and could provided partial protection only. On the other hand, animals immunized with 66 kDa PyIMP entrapped in phosphatidyl-choline/cholesterol (PC/chol) liposomes protected BALB/c mice against lethal P. yoelii challenge.

Extensive proteolytic processing of the malaria parasite merozoite surface protein 7 during biosynthesis and parasite release from erythrocytes

In Plasmodium falciparum, merozoite surface protein 7 (MSP7) was originally identified as a 22kDa protein on the merozoite surface and associated with the MSP1 complex shed during erythrocyte invasion. MSP7 is synthesised in schizonts as a 351-amino acid precursor that undergoes proteolytic processing. During biosynthesis the MSP1 and MSP7 precursors form a complex that is targeted to the surface of developing merozoites. In the sequential proteolytic processing of MSP7, N- and C-terminal 20 and 33kDa products of primary processing, MSP720 and MSP733 are formed and MSP733 remains bound to full length MSP1. Later in the mature schizont, MSP720 disappears from the merozoite surface and on merozoite release MSP733 undergoes a secondary cleavage yielding the 22kDa MSP722 associated with MSP1. In free merozoites, both MSP722 and a further cleaved product, MSP719 present only in some parasite lines, were detected; these two derivatives are shed as part of the protein complex with MSP1 fragments during erythrocyte invasion. Primary processing of MSP7 is brefeldin A-sensitive while secondary processing is resistant to both calcium chelators and serine protease inhibitors. Primary processing of MSP7 occurs prior to that of MSP1 in a post-Golgi compartment, whereas the secondary cleavage occurs on the surface of the developing merozoite, possibly at the time of MSP1 primary processing and well before the secondary processing of MSP1.

ARE PLASMODIUM FALCIPARUM PARASITES PRESENT IN PERIPHERAL BLOOD GENETICALLY THE SAME AS THOSE SEQUESTERED IN THE TISSUES?

Since quinine does not inhibit the growth of Plasmodium falciparum ring stages or mature schizonts, parasites may continue to emerge from sequestration sites after starting treatment. We used polymerase chain reaction amplification of P. falciparum merozoite surface protein 1 (MSP-1) and MSP-2 alleles to distinguish genotypes infecting 58 children with severe malaria. To examine changes in parasite populations in peripheral blood over time, we compared changes in number and spectrum of genotypes in samples on admission to a hospital to those obtained up to 24 hours later. Thirty-four children lost genotypes, 21 retained genotypes, and 3 gained an extra P. falciparum genotype at one locus but not the other. The lack of novel genotypes emerging suggests that among children with severe malaria the dominant clones sequestered in deep organs are usually the same as those in peripheral circulation.

Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion.

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.

Hepatozoonosis in Two Species of Japanese Wild Cat

Hepatozoon sp. infections were detected in two species of Japanese wild cat, Iriomote wild cat (Felis iriomotensis) and Tsushima leopard cat (Felis bengalensis euptilura), between April 1993 and October 2005. The prevalence was 56.7% (17/30) and 14.3% (6/42), respectively. The most affected organ was the heart; all infected animals had organisms in their hearts. The parasitizing form was schizont and various developmental stages were observed. The size of schizont and merozoite was 22.3 +/- 3.1 x 15.3 +/- 2.2 mum and 6.1 +/- 0.6 x 2.3 +/- 0.2 mum, respectively. Few inflammatory reactions against the parasites were observed. Electron microscopically, organisms were located in parasitophorous vacuoles of unidentified host cells, and mature schizonts consisted of numerous merozoites. This is the first report of hepatozoonosis in Japanese felids.

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.

Clinical and molecular aspects of severe malaria

The erythrocytic cycle of Plasmodium falciparum presents a particularity in relation to other Plasmodium species that infect man. Mature trophozoites and schizonts are sequestered from the peripheral circulation due to adhesion of infected erythrocytes to host endothelial cells. Modifications in the surface of infected erythrocytes, termed knobs, seem to facilitate adhesion to endothelium and other erythrocytes. Adhesion provides better maturation in the microaerophilic venous atmosphere and allows the parasite to escape clearance by the spleen which recognizes the erythrocytes loss of deformability. Adhesion to the endothelium, or cytoadherence, has an important role in the pathogenicity of the disease, causing occlusion of small vessels and contributing to failure of many organs. Cytoadherence can also describe adhesion of infected erythrocytes to uninfected erythrocytes, a phenomenon widely known as rosetting. Clinical aspects of severe malaria, as well as the host receptors and parasite ligands involved in cytoadherence and rosetting, are reviewed here. The erythrocyte membrane protein 1 of P. falciparum (PfEMP1) appears to be the principal adhesive ligand of infected erythrocytes and will be discussed in more detail. Understanding the role of host receptors and parasite ligands in the development of different clinical syndromes is urgently needed to identify vaccination targets in order to decrease the mortality rates of this disease.

Eimeria tenella microneme protein EtMIC3: identification, localisation and role in host cell infection

The gene coding for Eimeria tenella protein EtMIC3 was cloned by screening a sporozoite cDNA library with two independent monoclonal antibodies raised against the oocyst stage. The deduced sequence of EtMIC3 is 988 amino acids long. The protein presents seven repeats in tandem, with four highly conserved internal repeats and three more divergent external repeats. Each repeat is characterised by a tyrosine kinase phosphorylation site, WRCY, and a reminiscent motif of the thrombospondin1 (TSP1)-type I domain, CXXXCG. The protein EtMIC3 is localised at the apex of free parasite stages. It is not detected in the early intracellular parasite stage but is synthesised in mature schizonts. Secretion of the protein is induced when sporozoites are incubated in complete medium at 41 °C. Strangely enough, the two independent mAb that allow cloning of EtMIC3 interfere with parasitic growth in different ways. One is able to inhibit parasite invasion whereas the other inhibits development. Expression and localisation of the protein EtMIC3 are consistent with a protein involved in the invasion process as is expected for a microneme protein.

Activity of benzothiazoles and chemical derivatives onPlasmodium falciparum

Malaria is a major health concern particularly in Africa which has about 90% of the worldwide annual clinical cases. The increasing number of drug-resistant Plasmodium falciparum justifies the search for new drugs in this field. Antimalarial activity of 2-substituted 6-nitro- and 6-amino-benzothiazoles and their anthranilic acids has been tested. An in vitro study has been performed on W2 and 3D7 strains of P. falciparum and on clinical isolates from malaria-infected patients. Toxicity has been assessed on THP1 human monocytic cells. For the most active drug candidates, the in vitro study was followed by in vivo assays on P. berghei-infected mice and by in vitro assays in order to determine the stage-dependency and the mechanism of action. Of 39 derivatives tested in vitro, 2 had specific antimalarial properties. Each compound was active on all stages of the parasite, but one was markedly active on mature schizonts, while the other was more active on young schizont forms. Both drugs were also active on mitochondrial membrane potential. In vivo data confirmed efficiency with a sustained decrease of parasitaemia. Products A12 and C7 may be considered as potential antimalarial worthy of further chemical and biological research.

Expression of anti-apoptotic factors in cells parasitized by second-generation schizonts of Eimeria tenella and Eimeria necatrix

Intracellular infections by parasites require a functional anti-apoptotic mechanism for parasite survival within the host cell. The intracellular cycle of Eimeria tenella and Eimeria necatrix in chicken intestinal cells involves the maturation of schizonts within the epithelial cells lining the crypt lumen of the ceca ( E. tenella) and jejunum ( E. necatrix). After invasion, these cells detach from the epithelial layer and migrate into the underlying connective tissue, where maturation of second-generation schizonts takes place. However, the detached epithelial cells that harbour the parasite and localize in the lamina propia do not undergo apoptosis despite the fact that they are parasitized cells and are located in an inappropriate microenvironment. In this study we consider the hypothesis that E. tenella and E. necatrix may inhibit the host cell apoptosis that accompanies parasite-mediated transformation during late schizogony. To that end, the expression of both NF-κB, a transcriptional factor that blocks parasite-induced apoptosis, and bcl-x L, an anti-apoptotic protein induced by NF-κB, were studied in the host cell during the maturation of second-generation schizonts. In addition, the expression of the phosphorylated inhibitor of NF-κB, p-IkB α, was also studied to further confirm NF-κB activation. Immunocytochemical techniques, flow cytometric and blott analysis were applied by using polyclonal antibodies that specifically react with bcl-x L, p-IkB α, and NF-κB to detect these anti-apoptotic proteins in the parasitized cell. Our results offer evidence that both these coccidial species first induce NF-κB activation to protect the transformed parasitized cells from apoptosis, allowing the second-generation schizonts to mature, and later, after complete schizonts maturation, cause NF-κB inhibition to trigger host cell apoptosis in order to facilitate the escape of merozoites. To determine whether inhibition of the NF-κB pathway would induce apoptosis of the host cell, a protease inhibitor (TPCK), which induces apoptosis by mediating inhibition of IkB phosphorylation, was administered to parasitized chickens.