Altered expression of Plasmodium knowlesi variant antigen on the erythrocyte membrane in splenectomized rhesus monkeys

Variant antigens are present on the surface of Plasmodium knowlesi malaria-infected erythrocytes as detected by the schizont-infected cell agglutination (SICA) assay. We found that parasitized erythrocytes passaged in splenectomized monkeys did not agglutinate with immune sera. On the first passage from intact to splenectomized monkeys, the SICA titers decreased 4- to 16-fold; after the second and subsequent passages in splenectomized monkeys, the infected cells became nonagglutinable to all sera tested, including sera from animals infected with the nonagglutinating parasites. This loss of agglutinability could have resulted from selection of a genetically distinct subpopulation of the original parasites or the ability of the original parasites to alter their phenotypic expression. We have designated the new nonagglutinable phenotype, SICA [-], and the agglutinable phenotype, SICA [+]. The loss of agglutinability indicates that the variant antigen normally expressed on the erythrocyte membrane of infected cells is altered or absent. Because SICA [-] parasites developed in the absence of the spleen, the major organ of host defense against malaria, then this organ may in some manner influence or modulate antigenic expression in P. knowlesi and possibly other malaria parasites.     

Absence of erythrocyte sequestration in a case of babesiosis in a splenectomized human patient

Background

Recent studies have highlighted the over-diagnosis of malaria in clinical settings in Africa. This study assessed the impact of a training programme implemented as part of an intervention trial on diagnostic behaviour of clinicians in a rural district hospital in a low-moderate malaria transmission setting.

Methods

From the beginning of 2005, a randomized controlled trial (RCT) of intermittent preventive treatment for malaria in infants (IPTi) has been conducted at the study hospital. As part of the RCT, the study team offered laboratory quality assurance, and supervision and training of paediatric ward staff using information on malaria epidemiology in the community. Data on clinical and blood slide confirmed cases of malaria from 2001 to 2005 were extracted from the hospital records.

Results

The proportion of blood slides positive for malaria parasites had decreased from 21% in 2001 to 7% in 2005 (p <.01). The proportion of outpatient and inpatient cases diagnosed as malaria ranged between 34% and 28% from 2001 to 2004 and this decreased substantially to 17% after the introduction of the package of training and support in 2005 (p <.01). There was no clear trend in the ratio of blood slide examined versus total diagnosis of malaria.

Conclusion

It may be possible to change the diagnostic behaviour of clinicians by rigorous training using local malaria epidemiology data and supportive supervision.     

Lipidic vacuoles in Plasmodium knowlesi erythrocytic schizonts

Electron microscopy of schizont development in erythrocytic Plasmodium knowlesi has revealed that spheroidal vacuoles 250 nm in diameter with semi-dense contents appear at the periphery of the parasite prior to the budding of merozoites. When treated with non-polar solvents, their contents are completely extracted, and after fixation in tannic-glutaraldehyde they contain regular lamellae with a periodicity of 5.5 nm. Both of these reactions are typical of lipids. Some of these structures are associated with phagosomal vacuoles which may contribute to their lamellae. They disappear at the onset of merozoite formation, but membranous whorls of various sizes continue to be associated with the schizont surface during budding of merozoites. It is suggested that the lipidic vacuoles are a source of preformed lipid which can be utilized rapidly during the generation of merozoites.     

Plasmodium knowlesi as a Threat to Global Public Health

Malaria is a tropical disease caused by protozoans of the Plasmodium genus. Delayed diagnosis and misdiagnosis are strongly associated with higher mortality. In recent years, a greater importance is attributed to Plasmodium knowlesi, a species found mainly in Southeast Asia. Routine parasitological diagnostics are associated with certain limitations and difficulties in unambiguous determination of the parasite species based only on microscopic image. Recently, molecular techniques have been increasingly used for predictive diagnosis. The aim of the study is to draw attention to the risk of travelling to knowlesi malaria endemic areas and to raise awareness among personnel involved in the therapeutic process.     

Lipidic Vacuoles in Plasmodium knowlesi Erythrocytic Schizonts

ABSTRACT. Electron microscopy of schizont development in erythrocytic Plasmodium knowlesi has revealed that spheroidal vacuoles 250 nm in diameter with semi-dense contents appear at the periphery of the parasite prior to the budding of merozoites. When treated with non-polar solvents, their contents are completely extracted, and after fixation in tannic-glutaraldehyde they contain regular lamellae with a periodicity of 5.5 nm. Both of these reactions are typical of lipids. Some of these structures are associated with phagosomal vacuoles which may contribute to their lamellae. They disappear at the onset of merozoite formation, but membranous whorls of various sizes continue to be associated with the schizont surface during budding of merozoites. It is suggested that the lipidic vacuoles are a source of preformed lipid which can be utilized rapidly during the generation of merozoites.     

Limitations of microscopy to differentiate Plasmodium species in a region co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi

Background

The question whether Plasmodium falciparum infection affects the fitness of mosquito vectors remains open. A hurdle for resolving this question is the lack of appropriate control, non-infected mosquitoes that can be compared to the infected ones. It was shown recently that heating P. falciparum gametocyte-infected blood before feeding by malaria vectors inhibits the infection. Therefore, the same source of gametocyte-infected blood could be divided in two parts, one heated, serving as the control, the other unheated, allowing the comparison of infected and uninfected mosquitoes which fed on exactly the same blood otherwise. However, before using this method for characterizing the cost of infection to mosquitoes, it is necessary to establish whether feeding on previously heated blood affects the survival and fecundity of mosquito females.

Methods

Anopheles gambiae M molecular form females were exposed to heated versus non-heated, parasite-free human blood to mimic blood meal on non-infectious versus infectious gametocyte-containing blood. Life history traits of mosquito females fed on blood that was heat-treated or not were then compared.

Results

The results reveal that heat treatment of the blood did not affect the survival and fecundity of mosquito females. Consistently, blood heat treatment did not affect the quantity of blood ingested.

Conclusions

The study indicates that heat inactivation of gametocyte-infected blood will only inhibit mosquito infection and that this method is suitable for quantifying the fitness cost incurred by mosquitoes upon infection by P. falciparum.     

Protective antigens of bloodstage Plasmodium knowlesi parasites

The simian malaria Plasmodium knowlesi provides many favourable features as an experimental model; it can be grown in vivo or in vitro. Parasites of defined variant specificity and stage of development are readily obtained and both the natural host and a highly susceptible host are available for experimental infection and vaccination trials. Proteins synthesized by erythrocytic P. knowlesi parasites are characteristic of the developmental stage, as are the alterations that the parasite induces in the red cell surface. Erythrocytic merozoites are anatomically and biochemically complex, their surface alone is covered by at least eight distinct polypeptides. Immune serum from merozoite-immunized rhesus recognizes many parasite components, especially those synthesized by schizonts. All of the merozoite surface components and some of the schizont-infected red cell surface antigens are recognized by such immune sera. Rhesus monkeys rendered immune by repeated infection may by contrast recognize comparatively few antigens; a positive correlation was established for these 'naturally' immunized monkeys between protection and antibody directed against a 74 000 molecular mass antigen. Immunization with this purified antigen confers partial protection. Other putative protective antigens have been identified by monoclonal antibodies that inhibit merozoite invasion of red cells in vitro. The antigens recognized by inhibitory monoclonal antibodies are synthesized exclusively by schizonts and are processed, at the time of schizont rupture and merozoite release, to smaller molecules that are present on the merozoite surface. The multiplicity of protective antigens is clearly demonstrated by the fact that seven distinct merozoite surface antigens are recognized by three different inhibitory monoclonals. None of the protective antigens identified are variant or strain specific.     

A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium

The 66-kDa merozoite surface antigen (PK66) of Plasmodium knowlesi, a simian malaria, possesses vaccine-related properties that are thought to originate from a receptor-like role in parasite invasion of erythrocytes. We report the complete sequence of PK66 which allowed the demonstration that highly conserved analogues exist throughout Plasmodium including a recently reported gene from P. falciparum (Peterson, M. G., Marshall, V. M., Smythe, J. A., Crewther, P. E., Lew, A., Silva, A., Anders, R. F., and Kemp, D. J. (1989) Mol. Cell. Biol. 9, 3151-3155). These analogues are highly promising vaccination candidates. The distribution of PK66 changes after schizont rupture in a coordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies suggest that, PK66 is excluded from the erythrocyte at, and behind, the invasion interface.     

Transmission and Control of Plasmodium knowlesi: A Mathematical Modelling Study

Introduction

Plasmodium knowlesi is now recognised as a leading cause of malaria in Malaysia. As humans come into increasing contact with the reservoir host (long-tailed macaques) as a consequence of deforestation, assessing the potential for a shift from zoonotic to sustained P. knowlesi transmission between humans is critical.

Methods

A multi-host, multi-site transmission model was developed, taking into account the three areas (forest, farm, and village) where transmission is thought to occur. Latin hypercube sampling of model parameters was used to identify parameter sets consistent with possible prevalence in macaques and humans inferred from observed data. We then explore the consequences of increasing human-macaque contact in the farm, the likely impact of rapid treatment, and the use of long-lasting insecticide-treated nets (LLINs) in preventing wider spread of this emerging infection.

Results

Identified model parameters were consistent with transmission being sustained by the macaques with spill over infections into the human population and with high overall basic reproduction numbers (up to 2267). The extent to which macaques forage in the farms had a non-linear relationship with human infection prevalence, the highest prevalence occurring when macaques forage in the farms but return frequently to the forest where they experience higher contact with vectors and hence sustain transmission. Only one of 1,046 parameter sets was consistent with sustained human-to-human transmission in the absence of macaques, although with a low human reproduction number (R_0H = 1.04). Simulations showed LLINs and rapid treatment provide personal protection to humans with maximal estimated reductions in human prevalence of 42% and 95%, respectively.

Conclusion

This model simulates conditions where P. knowlesi transmission may occur and the potential impact of control measures. Predictions suggest that conventional control measures are sufficient at reducing the risk of infection in humans, but they must be actively implemented if P. knowlesi is to be controlled.     

Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.