A simple method for separation of uninfected erythrocytes from those infected withPlasmodium berghei and for isolation of artificially released parasites

Rat erythrocytes infected withPlasmodium berghei were disrupted by gentle passage of Coneanavalin A (ConA) agglutinated cells through a 100 mesh stainless steel grid. The free parasites were separated from cell debris, unbroken infected cells, and from uninfected rat erythrocytes on a Percoll gradient. The free parasites remained morphologically intact, metabolically active, and infective to mice.The parasites were observed by light and electron microscopy. The incorporation of³H-isoleucine and³H-hypoxanthine was compared in intact and infected cells, and the infectivity was measured by the injection of parasites into susceptible mice. It seems that the combination of the two techniques used, produces a high yield of intact free parasites suitable for physiological or immunological studies.     

Comparative morphology of human and animal malaria parasites

Human and animal malaria parasites (Plasmodium falciparum, P. malariae, P. vivax, P. berghei, P. gallinaceum) were studied using special fixation and standardized methods, with special attention to their effects on host cells. Morphological alterations induced by the parasites in infected erythrocytes included knobs, invaginations, and caveola-vesicle complexes on the surface of the host cell and clefts, microvesicles, and small vesicles in the cytoplasm of the infected erythrocytes. ForP. malariae, the ultrastructural study revealed invaginations with associated microvesicles, but knobs did not occur on the surface of infected erythrocytes. The development of invaginations and microvesicles inP. malariae-infected erythrocytes corresponded to the morphological alterations induced byP. vivax. A new hypothesis concerning the origin of Schüffner's dots is discussed.Abbreviations bz budding zone - c cluster - cl cleft - cv caveolavesicle complex - e erythrocyte - g gametocyte - i mvagination - k knob - m merozoite - mv microvesicle - n nucleus - p pigment - pv parasitophorous vacuole - sm small vesicle - t trophozoite Supported by Deutsche Forschungsgemeinschaft (DFG)     

Surface properties of extracellular malaria parasites: electrophoretic and lectin-binding characteristics.

The surface charge and lectin-binding capacity of isolated malaria parasites and host erythrocytes were analyzed and compared by chromatographic, electrophoretic, and cytochemical methods. Results indicated that at physiological pH values both freshly prepared and glutaraldehyde-fixed parasites and erythrocytes possess a net negative surface charge. Both cell types were strongly bound to cation-exchange resins and underwent cathode-directed electrophoretic migration. The isoelectric points for erythrocyte-free parasites and uninfected erythrocytes were approximately 3.0 and 4.0, respectively. The different effects of selective enzymatic digestion and solvent extraction on the electrophoretic mobilities of free parasites and erythrocytes suggested that the chemical constituents responsibile for the net negative surface charges on each type of cell are different. The surface charge of the free parasites seemed mainly to be a function of ionized phospholipids rather than of the ionogenic sialic acid moieties, which are the major contributors to the negative charge on erythrocytes. Results of lectin-binding studies indicated that specific glycosidimoieties (i.e., glucose, galactose, mannose, and n-acetyglucosamine), common to the erythrocyte surface, were either absent or in low concentration at the parasite's surface. These observations suggest that the normally intracellular malaria parasites have surface characteristics, differing from those of the host cell, characterized by a scarcity of lectin-binding receptors and sialic acid residues and by the major contribution of lipids to their surface charge.     

Purification of the erythrocytic stages ofBabesia bigemina from cultures

Exposure of erythrocytes infected withBabesia bigemina to glycerol-enhanced osmotic shock yielded preparations containing infected erythrocyte ghosts, free parasites, and some intact erythrocytes. The released parasites were purified and concentrated by centrifugation in Percoll gradient. Recovered free parasites were shown by the fluorescein acetate technique to be metabolically active, but their infectivity in vitro was low. It was demonstrated by electron microscopy that most of the released parasites had intact plasma membranes. There was only slight contamination of the free-parasite preparation with host erythrocyte debris.     

Antibody-mediated elimination of malaria parasites (plasmodium berghei) in vivo.

An infective preparation of extracellular blood forms (FP) of Plasmodium berghei was used to study some aspects of the interaction between protective antibodies and malaria parasites. FP but not infected erythrocytes (IRBC) were shown by the fluorescent antibody technique to be coated by antibodies after in vitro incubation with immune serum. Preincubation of both FP and IRBC with immune serum followed by their washing did not result in enhanced elimination of the parasites in vivo. However, FP preincubated with immune serum and subsequently washed were eliminated more efficiently than FP preincubated with normal serum if the preparations were injected with some immune serum. Such an increase in the efficiency of elimination was not detected with similarly pretreated IRBC. It is thus probable that protective antibodies acted in vivo against extracellular parasites rather than against parasites in erythrocytes. The interaction between parasites and antibodies may be of a highly reversible nature, and washing of the in vitro-treated parasites may cause elution of antibody from the sensitized parasites so that the amount of antibody on the parasite falls below the critical level required for in vivo elimination.     

The distinct proteome of placental malaria parasites

Malaria proteins expressed on the surface of Plasmodium falciparum infected erythrocytes (IE) mediate adhesion and are targeted by protective immune responses. During pregnancy, IE sequester in the placenta. Placental IE bind to the molecule chondroitin sulfate A (CSA) and preferentially transcribe the gene that encodes VAR2CSA, a member of the PfEMP1 variant surface antigen family. Over successive pregnancies women develop specific immunity to CSA-binding IE and antibodies to VAR2CSA. We used tandem mass spectrometry together with accurate mass and time tag technology to study IE membrane fractions of placental parasites. VAR2CSA peptides were detected in placental IE and in IE from children, but the MC variant of VAR2CSA was specifically associated with placental IE. We identified six conserved hypothetical proteins with putative TM or signal peptides that were exclusively expressed by the placental IE, and 11 such proteins that were significantly more abundant in placental IE. One of these hypothetical proteins, PFI1785w, is a 42kDa molecule detected by Western blot in parasites infecting pregnant women but not those infecting children.     

Commonly recognised Plasmodium falciparum parasites cause cerebral malaria

Cerebral malaria (CM) is a devastating form of Plasmodium falciparum malaria, in which adherence and sequestration of infected red blood cells in cerebral blood vessels play a major role. In order to determine whether a distinct parasite phenotype favours the development of this severe complication, P. falciparum isolates from Gabonese children suffering from CM or uncomplicated malaria (UM) were analysed for their binding phenotypes and their recognition in flow cytometry. CM isolates exhibited the ability to form rosettes and to bind ICAM-1, in line with previous studies correlating these phenotypes with CM disease pathology. CM isolates were more reactive with plasma from our cohort than UM parasites. This observation, together with the finding that some CM isolates were highly correlated with each other in their immunoreactivities, confirms that common parasites bearing conserved epitopes, which are capable of inducing cross-reactive antibodies, can cause CM in children.     

Isolation and characterization of parasites and host cell ghosts from erythrocytes infected with Plasmodium chabaudi

A new procedure has been developed which allows the concomitant isolation of viable parasites and host cell plasma membranes from erythrocytes infected with Plasmodium chabaudi trophozoites. The average final yield of parasites is 56%. Free parasites reveal a well preserved ultrastructure, incorporate [14C]isoleucine for at least 3 h, and synthesize about the same proteins as parasites within erythrocytes as monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)-autoradiography. The host cell plasma membranes can be isolated in the form of ghosts with an average yield of 27%. The ghosts possess a structurally intact plasma membrane as revealed by freeze-etch electron microscopy. The ghosts are regularly associated with seven neo-proteins as identified by SDS-PAGE and isoelectric focusing (IEF)/SDS-PAGE. These neo-proteins have the following apparent molecular masses: 154 kDa, 145 kDa, 90 kDa, 72 kDa (pI 4.5), 67 kDa, 52 kDa, and 33 kDa (pI 5.7), respectively. The contamination of ghosts by parasite material and, conversely, the contamination of parasites by host cell plasma membranes is very low as demonstrated by light and electron microscopy, lactoperoxidase-mediated radioiodination and the distribution of the typical parasite marker enzymes such as choline kinase, cholinephosphotransferase and ethanolaminephosphotransferase.     

Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins

In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.     

Fractionation ofPlasmodium chabaudi-infected erythrocytes into parasites and ghosts

We have developed a new procedure for subfractionation ofPlasmodium chabaudi-infected erythrocytes into parasites and ghosts. Trophozoite-infected erythrocytes enriched over a percoll-step (: 1.10 g/cm³) are subjected to a glycerol-enhanced osmotic shock. This induces the release of parasites and the emergence of erythrocyte ghosts, which can be separated on a continuous percoll gradient (: 1.021.10 g/cm³). The parasites are intact in terms of ultrastructure and incorporation of¹⁴C-isoleucine. The erythrocyte ghosts are purified over a two-step percoll gradient (:1.01–1.02 g/cm³). Our method recovers about 40%–50% of the initial ghosts and 70%–95% of the initially freed parasites.     

A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain.

Microvascular sequestration was assessed in the brains of 50 Thai and Vietnamese patients who died from severe malaria (Plasmodium falciparum, 49; P. vivax, 1). Malaria parasites were sequestered in 46 cases; in 3 intravascular malaria pigment but no parasites were evident; and in the P. vivax case there was no sequestration. Cerebrovascular endothelial expression of the putative cytoadherence receptors ICAM-1, VCAM-1, E-selectin, and chondroitin sulfate and also HLA class II was increased. The median (range) ratio of cerebral to peripheral blood parasitemia was 40 (1.8 to 1500). Within the same brain different vessels had discrete but different populations of parasites, indicating that the adhesion characteristics of cerebrovascular endothelium change asynchronously during malaria and also that significant recirculation of parasitized erythrocytes following sequestration is unlikely. The median (range) ratio of schizonts to trophozoites (0.15:1; 0.0 to 11.7) was significantly lower than predicted from the parasite life cycle (P < 0.001). Antimalarial treatment arrests development at the trophozoite stages which remain sequestered in the brain. There were significantly more ring form parasites (age < 26 hours) in the cerebral microvasculature (median range: 19%; 0-90%) than expected from free mixing of these cells in the systemic circulation (median range ring parasitemia: 1.8%; 0-36.2%). All developmental stages of P. falciparum are sequestered in the brain in severe malaria.     

The possible use of arsine (AsH3) as therapy for malaria

Arsine has long been recognized as a selective toxin that targets the membrane of erythrocytes and causes leakage of the internal contents of these cells. In acute poisoning episodes, the victims usually sustain kidney malfunction resulting from collection of debris from the erythrocytes. The hypothesis presented here is very simple. Namely, the hypothesis is to evaluate the use of arsine (AsH3) against erythrocytes that have become infected with malaria parasites. Once malaria parasites have become established in erythrocytes, it is extremely difficult to displace them. Erythrocytes do not spontaneously undergo apoptosis, which is a mechanism that many types of cells would use to deny intracellular parasites an immune privileged domain. Moreover, antibiotics that are powerful and selective enough to defeat malaria parasites once they are established in the erythrocytes have not been found. Thus, approaches for selective hemolysis of malaria-infected erythrocytes and evicting the parasites may be attractive. It is not known, but should be easily determined experimentally, what the relative toxicity of arsine (AsH3) is to normal erythrocytes and malaria-infected erythrocytes.     

A semi-automatic method for quantification and classification of erythrocytes infected with malaria parasites in microscopic images

Visual quantification of parasitemia in thin blood films is a very tedious, subjective and time-consuming task. This study presents an original method for quantification and classification of erythrocytes in stained thin blood films infected with Plasmodium falciparum. The proposed approach is composed of three main phases: a preprocessing step, which corrects luminance differences. A segmentation step that uses the normalized RGB color space for classifying pixels either as erythrocyte or background followed by an Inclusion-Tree representation that structures the pixel information into objects, from which erythrocytes are found. Finally, a two step classification process identifies infected erythrocytes and differentiates the infection stage, using a trained bank of classifiers. Additionally, user intervention is allowed when the approach cannot make a proper decision. Four hundred fifty malaria images were used for training and evaluating the method. Automatic identification of infected erythrocytes showed a specificity of 99.7% and a sensitivity of 94%. The infection stage was determined with an average sensitivity of 78.8% and average specificity of 91.2%.     

Plasmodium (Haemamoeba) cathemerium gene sequences for phylogenetic analysis of malaria parasites

The DNA sequence information on avian malaria parasites of the genus Plasmodium is quite limited. At present, sequences of only 6 out of 34 valid species are available. However, sequence data of avian malaria parasites are particularly important with regard to the resolution of the phylogenetic relationships of the most virulent human malaria agent, Plasmodium falciparum. The question as to whether P. falciparum originates from avian or from mammalian parasites would contribute to our understanding of its biology and would probably facilitate the interpretation of experimental results. To add to the body of molecular data, we sequenced three genes (cytochrome b, 18 SSU rRNA, caseinolytic protease C) of different organellar origin of one of the most widespread avian malaria parasites, Plasmodium (Haemamoeba) cathemerium, which once used to be an important laboratory in vivo model in human malaria research. The analysis of the new P. cathemerium sequences in direct comparison with the rodent parasite P. berghei and the four human malaria parasites by pairwise distance calculation do not suggest a closer relationship of P. cathemerium to P. falciparum than to the other species involved.     

Time-course of synthesis,transport and incorporation of a protein identified in purified membranes of host erythrocytes infected with a knob-forming strain of Plasmodium falciparum

Membranes from host erythrocytes infected with a knob-positive strain of Plasmodium falciparum were purified by free-flow electrophoresis or gradient centrifugation. In these membranes the main parasite-derived protein was a 92 000 Da protein which was not present after infection of erythrocytes with the corresponding knob-negative strain. The protein is synthesized between 9–21 h after merozoite invasion and then synthesis ceases. At least 6 h elapses between the start of synthesis and the appearance of the protein in the erythrocyte membrane. No precursor proteins for the 92 000 Da protein were found. Since the purified erythrocyte membranes were free from contamination with whole parasites or parasite membranes, the 92 000 Da protein is clearly present in the host erythrocyte membrane.     

Automated image processing method for the diagnosis and classification of malaria on thin blood smears

Malaria is a serious global health problem, and rapid, accurate diagnosis is required to control the disease. An image processing algorithm to automate the diagnosis of malaria on thin blood smears is developed. The image classification system is designed to positively identify malaria parasites present in thin blood smears, and differentiate the species of malaria. Images are acquired using a charge-coupled device camera connected to a light microscope. Morphological and novel threshold selection techniques are used to identify erythrocytes (red blood cells) and possible parasites present on microscopic slides. Image features based on colour, texture and the geometry of the cells and parasites are generated, as well as features that make use of a priori knowledge of the classification problem and mimic features used by human technicians. A two-stage tree classifier using backpropogation feedforward neural networks distinguishes between true and false positives, and then diagnoses the species (Plasmodium falciparum, P. vivax, P. ovale or P. malariae) of the infection. Malaria samples obtained from the Department of Clinical Microbiology and Infectious Diseases at the University of the Witwatersrand Medical School are used for training and testing of the system. Infected erythrocytes are positively identified with a sensitivity of 85% and a positive predictive value (PPV) of 81%, which makes the method highly sensitive at diagnosing a complete sample provided many views are analysed. Species were correctly determined for 11 out of 15 samples.Nicholas E. Ross conducted research while at the University of the Witwatersrand School of Electrical and Information Engineering     

Alteration in host cell tropism limits the efficacy of immunization with a surface protein of malaria merozoites

Immunization with Plasmodium yoelii merozoite surface protein-8 (PyMSP-8) has been shown to protect mice against lethal P. yoelii 17XL malaria. Here we demonstrate that PyMSP-8-specific antibodies preferentially suppress P. yoelii 17XL growth in mature erythrocytes compared to growth in reticulocytes and do not suppress the growth of nonlethal P. yoelii 17X, a parasite that primarily replicates in reticulocytes. The protection against normocyte-associated P. yoelii malaria parasites is mediated by antibodies that recognize conformational epitopes of PyMSP-8 that are nonpolymorphic. We examined changes in gene expression in reticulocyte-restricted P. yoelii 17XL parasites that escaped neutralization by PyMSP-8-specific antibodies using P. yoelii DNA microarrays. Of interest, Pymsp-8 gene expression decreased, while the expression of msp-1, msp-7, and several rhoptry protein genes increased. Breakthrough parasites also exhibited increases in the expression of a subset of yir and Pyst-a genes that are predicted to encode polymorphic antigens expressed on the surface of infected erythrocytes. These data suggest that changes in the expression of parasite proteins expressed on the merozoite surface, as well as the surface of infected erythrocytes, may alter host cell tropism and contribute to the ability of malaria parasites to evade merozoite-specific, neutralizing antibodies.     

Identification of antigens diagnostic for European isolates ofBabesia equi by two-dimensional electrophoresis and Western blotting

A lysate ofBabesia equi-infected erythrocytes (USDA strain) was separated by two-dimensional electrophoresis and anlysed by Western blotting. Nine major antigens or antigen groups with mol. wts. ranging from 43 to 19 kDa were recognized by sera from horses experimentally infected with the USDA strain. Four antigens or antigen groups were also recognized by some or all sera from horses infected withB. caballi or not infected withBabesia spp. Of the remaining five antigens, four were recognized by all sera from field-infected horses from Europe. Thus four antigens with mol. wts. of 33, 31, 19 and 20 kDa were identified as diagnostic antigens for European isolates ofB. equi. None of the antigens diagnostic for European isolates was recognized by sera from field-infected horses from Brazil.     

Cerebral Plasmodium falciparum malaria: The role of PfEMP1 in its pathogenesis and immunity,and PfEMP1-based vaccines to prevent it

Malaria, a mosquito-borne infectious disease caused by parasites of the genus Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium-parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti-malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto-adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti-disease malaria vaccine. Here we review the role of ICAM-1 and EPCR receptor adhering falciparum-parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.