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The genetic determinants of resistance to mefloquine in malaria parasites are unclear. Some studies have implied that amplification of, or mutations in, the multidrug resistance gene pfmdr1 in Plasmodium falciparum may be involved. Using the rodent malaria model Plasmodium chabaudi, we investigated the role of the orthologue of this gene, pcmdr1, in a stable mefloquine-resistant mutant, AS(15MF/3), selected from a sensitive clone. pcmdr1 exists as a single copy gene on chromosome 12 of the sensitive clone. In AS(15MF/3), the gene was found to have undergone duplication, with one copy translocating to chromosome 4. mRNA levels of pcmdr1 were higher in the mutant than in the parent sensitive clone. A partial genetic map of the translocation showed that other genes in addition to pcmdr1 had been cotranslocated. The sequences of both copies of pcmdr1 of AS(15MF/3) were identical to that of the parent sensitive clone. A cross was made between AS(15MF/3) and an unrelated mefloquine-sensitive clone, AJ. Phenotypic and molecular analysis of progeny clones showed that duplication and overexpression of the pcmdr1 gene was an important determinant of resistance. However, not all mefloquine-resistant progeny contained the duplicated gene, showing that at least one other gene was involved in resistance.

Here we examined the impact of host immunity on relationships between parasite virulence, transmission rate, intrinsic growth rate and host recovery rate in the rodent malaria parasite, Plasmodium chabaudi. Groups of naïve and immunized mice were infected with 1 of 10 cloned lines of parasites and their infection dynamics were monitored for 19 days. We found that (1) host immunity reduced the growth rate, virulence, transmission rate and infection length, with a consequent 3-fold reduction in life-time transmission potential, (2) clone means for these traits ranked similarly across naïve and immunized mice, (3) regression slopes of transmission potential on growth rate, virulence and infection length were similar in naive and immunized mice, (4) virulence and infection length were positively correlated in immunized but not naïve mice, and (5) for a similar level of parasite growth rate and virulence, transmission potential and infection length were lower in immunized than naïve mice. Thus host immunity reduced all these fitness traits in a manner consistent with direct parasite-driven biological links among them. These results support the basic assumption underlying our theory that predicts that anti-disease vaccines will select for higher virulence in those microparasites for which virulence is integrally linked to transmission.

Production of parasite-specific antibodies is an important component of immunity to blood stage malaria infection, as shown by several previous studies in rodent models. However, no study has addressed the induction of humoral immunity by different parasites in a genetically homogeneous host population. Here, levels of parasite-specific immunoglobulin isotypes were measured during primary infections of Plasmodium chabaudi and of Plasmodium yoelii in inbred NIH mice inoculated with cloned lines of either avirulent or virulent erythrocytic parasites. Non-lethal infections were characterized by early and late significant upregulation of IgG2a and IgG1, respectively. In contrast, for lethal infections, a slower, reduced IgG2a response correlated with a rapidly fatal outcome prior to any significant synthesis of IgG1. It is proposed that the sequential upregulated synthesis of parasite-specific IgG2a (cytophilic) and IgG1 (non-cytophilic) is associated with protective immunity to blood stage malaria infections in mice. This may provide an immunological framework for examining humoral immunity to malaria in humans.

We have used the method of amplified fragment length polymorphism (AFLP) to identify genetic polymorphisms between two cloned isolates of the rodent malaria parasite Plasmodium chabaudi chabaudi. The method employs polymerase chain reaction (PCR)-amplification of genomic DNA fragments cut with specific combinations of restriction endonucleases; we used EcoRI and Tru1I (isoschizomer of MseI). We have identified 819 parasite clone-specific AFLPs between P. c. chabaudi clones AS and AJ. Of these, 403 fragments were specific to AS and 416 to AJ. In preparing blood stage parasites for DNA, nucleated host cells were removed by successive filtration of infected blood through powdered cellulose and Plasmodipur filters. This reduced nucleated host cell contamination to around 1-10 per million parasite nuclei and reduced host DNA to below the limit of detection by the AFLP method. Analysis of our results showed that the total number of PCR-amplified fragments of parasite DNA was consistent with the predicted number of EcoRI sites in the parasite genome. 19.4% of all amplified fragments were P. c. chabaudi clone-specific. From this figure we estimated that the diversity between clones AS and AJ, measured as the probability of a sequence difference, was between about 8 x 10(-3) and 4.6 x 10(-4) per base pair. This is consistent with the sequence diversity found between alleles of candidate drug resistance genes from P. c. chabaudi clones AS and AJ identified and sequenced in this laboratory., (Copyright 2002 Elsevier Science B.V.)

We have studied resistance to sulfadoxine-pyrimethamine (S/P) in the rodent malaria parasite Plasmodium chabaudi. A stable S/P-resistant mutant, AS(50S/P), was selected by drug treatment of a clone, AS(PYR), already resistant to pyrimethamine. The sequences of the P. chabaudi dhfr and dhps genes were obtained and found to be identical in AS(50S/P) and AS(PYR), showing that resistance to S/P in AS(50S/P) was not due to additional mutations in either gene. AS(50S/P) was crossed with a drug-sensitive clone, AJ, and 16 independent recombinant progeny were obtained. These clones were phenotyped for their susceptibility to S/P and to sulfadoxine and pyrimethamine separately. Pyrimethamine resistance was invariably associated with S/P resistance, but no correlation was found between resistance to S/P and resistance to sulfadoxine. Quantitative trait locus analysis of the progeny with 31 chromosome-specific markers showed that mutant P. chabaudi dhfr, or one or more genes closely linked to it, was a major determinant of S/P resistance. In addition, the inheritance of genes on chromosomes 5 and 13 from the sensitive parent appeared to contribute to the level of resistance observed. These results demonstrate that the S/P resistance of the AS(50S/P) mutant of P. chabaudi does not involve mutation in dhps and is not due simply to a combination of two genes determining resistance to pyrimethamine and sulfadoxine separately.

In an effort to understand what limits the virulence of malaria parasites, we infected inbred mice of three genotypes (C57Bl/6J, CBA/Ca and DBA/2) with one of two parasite lines of the rodent malaria Plasmodium chabaudi. One of these parasite lines had been serially passaged through C57Bl/6J mice and had evolved higher asexual growth rate, virulence and transmission in the process. The other parasite line was the unadapted ancestral line which had low virulence. In all three host genotypes, the C57Bl/6J-adapted parasite line was more virulent than the ancestral line thus indicating that trade-offs in virulence between alternative host genotypes had not placed strong constraints on the evolution of high virulence in this system. By examining the infection dynamics for fitness-related components-asexual parasite population growth, transmission and virulence-we revealed alternative possible explanations for what sets the upper limit to virulence in nature. The total number of transmission forms (gametocytes) produced during the infection, a measure of parasite Darwinian fitness, was four-fold higher in mice that survived the infection than those which died. Among mice that survived, total gametocyte production was greatest in the host genotype that suffered intermediate levels of morbidity (anaemia and weight loss). Thus, there were transmission costs of high virulence that were partly due to host death (as most theoretical models of virulence evolution assume), but perhaps partly due to some factor related to high morbidity. Both mortality and morbidity-related factors might therefore influence the upper limit on virulence of malaria parasites.

Models of malaria epidemiology and evolution are frequently based on the assumption that vector-parasitic associations are benign. Implicit in this assumption is the supposition that all Plasmodium parasites have an equal and neutral effect on vector survival, and thus that there is no parasite genetic variation for vector virulence. While some data support the assumption of avirulence, there has been no examination of the impact of parasite genetic diversity. We conducted a laboratory study with the rodent malaria parasite, Plasmodium chabaudi and the vector, Anopheles stephensi, to determine whether mosquito mortality varied with parasite genotype (CR and ER clones), infection diversity (single versus mixed genotype) and nutrient availability. Vector mortality varied significantly between parasite genotypes, but the rank order of virulence depended on environmental conditions. In standard conditions, mixed genotype infections were the most virulent but when glucose water was limited, mortality was highest in mosquitoes infected with CR. These genotype-by-environment interactions were repeatable across two experiments and could not be explained by variation in anaemia, gametocytaemia, blood meal size, mosquito body size, infection rate or oocyst burden. Variation in the genetic and environmental determinants of virulence may explain conflicting accounts of Plasmodium pathogenicity to mosquitoes in the malaria literature.

It is well recognized that drug resistance is the most significant obstacle to gaining effective malaria control. Despite the enormous advances in the knowledge of the biochemistry and molecular biology of malaria parasites, only a few genes determining resistance to the commonly used drugs have been identified. The idea that rodent malaria parasites should be exploited more widely for such work, in view of the practical problems of studying this subject experimentally in human malaria, is presented.

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

Apical membrane Ag 1 (AMA1) is a leading malaria vaccine candidate. Homologues of AMA1 can induce protection in mice and monkeys, but the mechanism of immunity is not understood. Mice immunized with a refolded, recombinant, Plasmodium chabaudi AMA1 fragment (AMA1B) can withstand subsequent challenge with P. chabaudi adami. Here we show that CD4+ T cell depletion, but not gammadelta T cell depletion, can cause a significant drop in antiparasite immunity in either immunized normal or immunized B cell KO mice. In normal mice, this loss of immunity is not accompanied by a decline in Ab levels. These observations indicate a role for AMA1-specific Ab-independent T cell-mediated immunity. However, the loss of immunity in normal CD4+ T cell-depleted mice is temporary. Furthermore, immunized B cell KO mice cannot survive infection, demonstrating the absolute importance of B cells, and presumably Ab, in AMA1-induced immunity. CD4+ T cells specific for a cryptic conserved epitope on AMA1 can adoptively transfer protection to athymic (nu/nu) mice, the level of which is enhanced by cotransfer of rabbit anti-AMA1-specific antisera. Recipients of rabbit antisera alone do not survive. Some protected recipients of T cells plus antisera do not develop their own AMA 1-specific Ab response, suggesting that AMA 1-specific CMI alone can protect mice. These data are the first to demonstrate the specificity of any protective CMI response in malaria and have important implications for developing a malaria vaccine.

Although several candidate vaccine antigens have been developed for malaria, there is as yet no effective single vaccine available. There is a growing consensus that the ultimate malaria vaccine will be multivalent, requiring the identification of a suitable cocktail of antigens. However, evaluation of the multitude of potential malaria vaccine antigens in suitable combinations is a daunting task. Here we describe the validation of expression library immunization (ELI) as a tool for the discovery of sequences protective against malaria infection. A genomic Plasmodium chabaudi expression library was constructed comprising ten separate pools of 3000 plasmids. Over three vaccine trials using biolistic delivery of pools composed of 616 to 30,000 plasmids we report up to 63% protection of mice from a challenge with P. chabaudi adami DS, a highly virulent strain. Overall, ELI protected 36% of vaccinated mice against virulent challenge compared with only 3.2% survival of control mice. These results demonstrate that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

Previous studies of Plasmodium falciparum have identified a region of chromosome 2 in which are clustered three genes for glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins, MSP2, MSP5, and MSP4, arranged in tandem. MSP4 and MSP5 both encode proteins 272 residues long that contain hydrophobic signal sequences, GPI attachment signals, and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. The locations and similar structural features of these genes suggest that they have arisen from a gene duplication event. Here we describe the identification of the syntenic region of the genome in the murine malaria parasite, Plasmodium chabaudi adami DS. Only one open reading frame is present in this region, and it encodes a protein with structural features reminiscent of both MSP4 and MSP5, including a single EGF-like domain. Accordingly, the gene has been designated PcMSP4/5. The homologue of the P. falciparum MSP2 gene could not be found in P. chabaudi; however, the amino terminus of the PcMSP4/5 protein shows similarity to that of MSP2. The PcMSP4/5 gene encodes a protein with an apparent molecular mass of 36 kDa, and this protein is detected in mature stages of the parasite. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites and developing and free merozoites. The PcMSP4/5 gene is transcribed in both ring and trophozoite stages but appears to be spliced in a stage-specific manner such that the central intron is spliced from the mRNA in the parasitic stage in which the protein is expressed.

Several highly antigenic proteins containing tandem repeats rich in glutamic acid residues have been described in Plasmodium falciparum. However, relatively little information is available about analogous genes in rodent parasites. This report describes a 4.2-kb genomic DNA fragment from P. chabaudi with a deduced amino acid sequence that is predominantly glutamate-rich tandem repeats. Several different monoclonal antibodies raised against a 93-kDa P. chabaudi protein, which does not correspond to the cloned DNA fragment, recognize a recombinant protein expressed from the 4.2-kb DNA fragment. The only sequence similarities between these two genes are tandem repeats with a predominance of glutamate pairs followed by a hydrophobic residue. This repetitious-sequence motif may be the basis for the observed cross-reactivity. A similar motif has been demonstrated to be the basis for antibody cross-reactivity between glutamate-rich proteins of P. falciparum. The expression of multiple glutamate-rich proteins with cross-reacting epitopes may be a general phenomenon in Plasmodium species.

The malarial parasite dramatically affects the structure and function of the erythrocyte membrane by exporting proteins that specifically interact with the host membrane. This report describes the complete sequence and some biochemical properties of a 93-kDa Plasmodium chabaudi chabaudi protein that interacts with the host erythrocyte membrane. Approximately 40% of the deduced protein sequence consists of tandem repeats of 14 amino acids that are rich in glutamic acid residues. Comparison of the repeat sequences from two different P. c. chabaudi strains derived from the same initial isolate revealed an exact duplication of 294 nucleotides suggesting a recent unequal crossing-over event. However, in spite of this potentially high level of intragenic recombination activity, the repeat sequences from P. c. adami are rather conserved suggesting structural or functional constraints on the protein and tandem repeats. The 93-kDa protein exists in an oligomeric form as revealed by gel filtration chromatography and non-denaturing gel electrophoresis. A predominantly alpha-helical predicted secondary structure and a discrepancy between the estimated molecular sizes determined from non-denaturing gel electrophoresis and gel filtration chromatography suggest that the protein is a long rod-shaped or fibrillar, protein. Attributes shared between the 93-kDa protein, some P. falciparum proteins with glutamate-rich tandem repeats, and cytoskeletal proteins suggest that these parasite proteins function as cytoskeletal proteins that possibly stabilize the erythrocyte membrane.

Detection and analysis of pathogens by PCR plays an important role in infectious disease research. The value of these studies would be diminished if nuclear material from dead parasites were found to remain in circulation for extended periods and thus result in positive amplification. This possibility was tested in experimental rodent malaria infections. Blood samples were obtained from infected mice during and following drug or immune clearance of Plasmodium chabaudi chabaudi parasitemias. Detection of parasite DNA by a sensitive Plasmodium-specific PCR amplification assay was associated with the presence of viable parasites, as detected by subinoculation. No parasite DNA could be detected by PCR 48 h after the injection of killed parasites into mice. Nuclear material from parasites removed by drug or immune responses is rapidly cleared from the circulation and does not contribute significantly to amplification. Thus, results from PCR analysis of malaria-infected blood accurately reflect the presence of live parasites.

cDNA and genomic DNA clones covering the entire open reading frame (ORF) for a Plasmodium chabaudi 96V protein were isolated. From the first ATG codon the intronless gene codes for a 229-kDa protein. Antisera raised against recombinant polypeptides coded by two different regions of the gene reacted with a 240/225-kDa doublet on Western blots of parasite extracts. In immunofluorescence studies the same sera detected the antigen at the apical end of the merozoite, possibly in rhoptry organelles. In Western blotting experiments the recombinant polypeptides were recognised by antibodies induced by natural infection. A 364-amino acid residue repetitive region, based on 32 11-mer repeats divided by two 6-mer repeats into three blocks, is located in the centre of the protein. Analysis of this repetitive region led us to propose a model in which each of the three units forms an alpha-helical coiled-coil triple-helix containing a possible leucine-histidine zipper. Each unit resembles in structure the units present in spectrin. The repeat region is flanked by predicted heptad based alpha-helical coiled-coil regions, and we propose that the protein forms a dimer. The 229-kDa protein has the overall character of a cytoskeletal protein. We have named the 229-kDa protein repetitive organellar protein (ROPE) and suggest that ROPE may be involved in the process of invasion, possibly by interacting with the erythrocyte cytoskeleton, and that the leucine histidine-zipper may be involved in molecular mimicry of spectrin.

We have attempted to establish the degree of linkage conservation between different species of the malaria parasite Plasmodium. Initially, the chromosome locations of 42 homologous genes were established in parasites from a rodent malaria species and the human malaria parasite P. falciparum. Of these genes, 26 appeared to be conserved within ten synteny groups between the two genomes. Several synteny groups were analysed further by long-range restriction mapping of digested chromosomes. Finally, a fine restriction map of one of the linkage groups was made from the rodent malaria parasites P. berghei and from P. falciparum and from the simian malaria parasite P. knowlesi. The fine-scale organisation of this linkage group appears to have remained intact among the three species, despite the evolutionary distance between them. This provides the first example of linkage conservation between the rodent, simian and human malaria species, which represent three different branches of the inferred phylogenetic tree of the genus Plasmodium.

We have located a possible chloroquine resistance locus in the genome of the rodent malaria parasite Plasmodium chabaudi. Two genetically distinct clones of the parasite were grown in vivo and allowed to undergo genetic crossing. The clones differed from each other in their susceptibility to chloroquine; AS(3CQ) had been selected for a low level of resistance to the drug whereas AJ is chloroquine-sensitive. Independent recombinant progeny (20) were cloned from the products of two crosses, phenotyped for their susceptibility to chloroquine, and genotyped for their inheritance of 46 chromosome-specific markers. No association was found between chloroquine susceptibility and the inheritance of pcmdr1, the P. chabaudi homologue of the pfmdr1 multi-drug resistance gene of P. falciparum. Also, there was no association between chloroquine susceptibility and the inheritance of a marker linked to a putative chloroquine resistance locus in a P. falciparum cross. However, 16 of the progeny clones showed co-segregation of four linked markers on chromosome 11 with their resistance phenotype. This result suggests that a locus for chloroquine resistance exists on this chromosome in P. chabaudi.

Interactions between malaria parasite clones within mixed infections can have a profound effect on transmission and therefore the epidemiology of the disease. However, factors which determine the relative transmission success of individual clones from mixed infections are unknown. We have used two clones of the rodent malaria Plasmodium chabaudi to investigate changes in the clonal composition of asexual parasites over the course of mixed-clone infections in mice and how these relate to the clonal composition of transmission (oocyst) populations in mosquitoes. Clonal composition was determined using monoclonal antibody analyses for the asexual blood stage populations and PCR analysis of single oocysts for the transmission populations in mosquitoes. The relative frequency of the two clones changed dramatically during the course of the infection in mice, depending on their ratio in the inoculum. The clonal composition of parasites within mosquitoes most closely resembled that in the asexual infection at the time of transmission rather than that at any point earlier in the infection. These results provide no evidence that clones increase rates of gametocytogenesis in response to competitive suppression. Most likely, transmission success follows from asexual success in the later parts of the infection. The clone which dominated the earlier part of the infection, when most parasites are produced, did not necessarily dominate the transmission from the infection. The two clones differed in competitive ability and the data suggest that interactions with the host immune system may be a major factor in determining transmission success from mixed-clone infections.

Interactions between parasite genotypes sharing a host are poorly understood, but have important consequences for the epidemiology and evolution of the parasite. In mixed-genotype malaria infections, patterns of asexual replication and transmission favoured by natural selection may be different from those in single-genotype infections. The infectivity to mosquitoes of mixed-genotype and single-genotype infections were compared using 2 clones of Plasmodium chabaudi inoculated into mice either together or alone. Mice given mixed-clone infections received the sum of the inocula given to the single-clone controls. Mosquitoes were fed on the mice and the numbers of oocysts which developed were counted to assess transmission intensity. For 3 combinations of starting inocula and feed days, mixed-clone infections produced more oocysts per mosquito than the sum of the 2 single-clone infections. This effect was correlated with an increase in gametocyte density, but was less clearly related to asexual infection parameters. The results show that interactions between clones in mixed-clone infections can profoundly affect transmission.

Mixed-genotype infections of microparasites are common, but almost nothing is known about how competitive interactions within hosts affect the subsequent transmission success of individual genotypes. We investigated changes in the composition of mixed-genotype infections of the rodent malaria Plasmodium chabaudi clones CR and ER by monoclonal antibody analysis of the asexual infection in mice, and by PCR amplification of clone-specific alleles in oocysts sampled from mosquitoes which had fed on these mice. Mixed-clone infections were initiated with a 9:1 ratio of the two clones, with ER as the minority in the first experiment and CR as the minority in the second experiment. When beginning as the majority, clones achieved parasite densities in mice comparable to those achieved in control (single-clone) infections. When they began as the minority, clones were suppressed to less than 10% of control parasitaemias during the early part of the infections. However, in mosquitoes, the frequency of the initially rare clone was substantially greater than it was in mice at the start of the infection or four days prior to the feed. In both experiments, the minority clone in the inocula produced as many, or more, oocysts than it did as a single-clone infection. These experiments show that asexual dominance during most of the infection is poorly correlated to transmission probability, and therefore that the assumption that within-host population size correlates to transmission probability may not be warranted. They also raise the fundamental question of why transmission rates of individual genotypes are often higher from mixed than single-clone infections.

Apical membrane antigen 1 (AMA-1), an asexual blood-stage antigen of Plasmodium falciparum, is an important candidate for testing as a component of a malaria vaccine. This study investigates the nature of diversity in the Plasmodium chabaudi adami homolog of AMA-1 and the impact of that diversity on the efficacy of the recombinant antigen as a vaccine against challenge with a heterologous strain of P. chabaudi. The nucleotide sequence of the AMA-1 gene from strain DS differs from the published 556KA sequence at 79 sites. The large number of mutations, the nonrandom distribution of both synonymous and nonsynonymous mutations, and the nature of both the codon changes and the resulting amino acid substitutions suggest that positive selection operates on the AMA-1 gene in regions coding for antigenic sites. Protective immune responses induced by AMA-1 were strain specific. Immunization of mice with the refolded ectodomain of DS AMA-1 provided partial protection against challenge with virulent DS (homologous) parasites but failed to protect against challenge with avirulent 556KA (heterologous) parasites. Passive immunization of mice with rabbit antibodies raised against the same antigen had little effect on heterologous challenge but provided significant protection against the homologous DS parasites.

During the course of chronic malaria infection antigenic variants of a parasite antigen are expressed and exposed on the surface of infected erythrocyte membranes. There also exists a number of apparently invariant single gene copy blood-stage antigens, exposed or non-exposed, which have been shown to afford immunity under experimental conditions. To determine why the host, presented with invariant 'protective' antigens, is unable to control infections effectively, immunity to a representative single gene copy antigen, the merozoite surface protein 1 (MSP1) was investigated in Plasmodium chabaudi chabaudi AS, a murine model of chronic malaria. Immunization with monoclonal antibody affinity purified native MSP1 resulted in enhanced control of parasitaemia on challenge, irrespective of the parasite inoculum size; challenge with a single parasite, however, suggested that expansion of resistant parasite subpopulations was not occurring. Challenge of mice immunized with recombinant fusion proteins encoding N- or C-terminal regions of the P.c. chabaudi AS MSP1 produced inconsistent effects, often parasitaemias were indistinguishable from controls despite significant anti-MSP1 antibody responses. The not unlikely contamination of MSP1 native preparations with erythrocyte (E) components was considered. Immunization with a mixture of the MSP1 C-terminus recombinant polypeptide and a Triton X-100 solubilized lysate of normal E resulted in enhanced control of parasitaemia, however, no effect was seen after administration of either component on its own. Co-immunization of E with the N-terminus polypeptide reversed the inhibition seen, on this occasion with this construct alone.

The number of chromosomes and the chromosomal location and linkage of more than 50 probes, mainly of genes, have been established in four species of Plasmodium which infect African murine rodents. We expected that the location and linkage of genes would not be conserved between these species of malaria parasites since extensive inter- and intraspecific size differences of the chromosomes existed and large scale internal rearrangements and chromosome translocations in parasites from laboratory lines had been reported. Our study showed that all four species contained 14 chromosomes, ranging in size between 0.5 and 3.5 Mb, which showed extensive size polymorphisms. The location and linkage of the genes on the polymorphic chromosomes, however, was conserved and nearly identical between these species. These results indicate that size polymorphisms of the chromosomes are more likely due to variation in non-coding (subtelomeric, repeat) sequences and show that a high plasticity of internal regions of chromosomes that may exist does not frequently affect chromosomal location and linkage of genes.

We have determined the complete genomic sequence of the Plasmodium chabaudi erythrocyte membrane antigen (PcEMA1) in 4 different parasite strains. The gene structure consisted of a short region encoding a signal sequence separated from the main coding region by an intervening sequence. The overall identity of the three P. chabaudi adami deduced protein sequences to their consensus was 100%, 99.8% and 88% for 556KA, DK and DS respectively, with a general pattern of increasing divergence from the N- to the C-terminus. The P. chabaudi chabaudi strain CB was 72% homologous to the P. chabaudi adami consensus sequence. A gene related to PcEMA1, designated PcEMA1-R, has been identified in the genome of P. chabaudi adami but not in P. chabaudi chabaudi. The partial sequence for this gene in P. chabaudi adami strain DS predicts that it could encode a truncated form of PcEMA1, but its status as a pseudogene or an independent, expressed gene has not been resolved.

The rates of switching between expression of variable antigen types (VATS) have been investigated in vivo in a cloned line of Plasmodium chabaudi chabaudi. VAT-specific hyperimmune sera combined with an immunogold-silver staining technique were used to detect VATS, and five estimates of VAT-specific switching rates were determined for three of them. VAT-specific switching rates were consistent for each VAT and differed between VATS in the range 1.3 x 10(-2) to 4.3 x 10(-4) switches per schizont per generation. This variation suggests that hierarchical expression of VATS in an infection may be determined by switch rates. A minimum estimate of the overall switching rate was determined by summation of the VAT-specific rates in each of two experiments. In both cases the results showed that at least 1 in every 80 schizonts switched VAT expression every generation. This is the first report of antigenic switching rates for malaria parasites measured in vivo in the presence of minimal specific immune pressure, and is the first to show that VAT-specific switching rates vary between VATS. We conclude that switching is rapid and spontaneous, and is regulated, at least in part, by the VATS involved.

The molecular karyotypes of the African murine malaria parasites P. berghei (3 strains, 2 lines) P. yoeli (2 strains) P. chabaudi (3 strains, 1 line) and P. vinckei (4 strains) have been studied using orthogonal field alternation gel electrophoresis (OFAGE). The genome of each species was resolved into 9 to 11 distinct chromosomal DNA banas molecules of varying intensities which seem to represent 14 chromosomes ranging in size from 600 kb to 3500 kb. The position of certain chromosomes allowed the identification of a unique karyotype for each of the strains and lines under study. P. yoelii appears by criteria of chromosome size, chromosome numbers and localisation of DNA probes to differ considerably from the other three rodent malaria species. The chromosomal location of 5 DNA probes allowed the identification of corresponding chromosomes in rodent malaria parasites and the differentiation between species and strains. Assignment of the "PMMSA" gene of P. c. chabaudi IP-PC 1 enables the distinction of the four rodent malaria species. The molecular karyotype combined to chromosomal assignment of DNA probes provides a useful tool for a more precise characterization by a genetic definition of malaria parasites.

The complete nucleotide sequence of the gene encoding the merozoite surface protein 1 (MSP-1) from the rodent malaria parasite Plasmodium chabaudi chabaudi AS has been determined by direct sequencing of overlapping PCR derived fragments. Comparison of the P. c. chabaudi AS nucleotide sequence with the previously published P. c. chabaudi IP-PC1 sequence indicates that although the MSP-1 gene of these two P. c. chabaudi strains is highly conserved, with sequence identity often approaching 100%, interspersed throughout the molecule are 5 regions of divergence. This is at variance with published data which suggested that the P. c. chabaudi AS and P. c. chabaudi IP-PC1 MSP-1 sequences are largely identical. Epitope mapping studies with a panel of anti-P. c. chabaudi AS MSP-1 monoclonal antibodies demonstrate that whilst most of these mAbs recognise epitopes at the N-terminus of the MSP-1 molecule, two mAbs, including one capable of inhibiting challenge infections in mice in an in vivo passive transfer assay, recognise epitopes which map to the C-terminus.

The in vivo inhibitory action of NIMP23, a monoclonal antibody raised against the rodent parasite Plasmodium chabaudi chabaudi AS, has previously been shown to be strain-specific, capable of delaying significantly the onset of P. c. chabaudi AS but not a P. c. chabaudi CB challenge parasitaemia. The epitope to which this mAb binds has been mapped to the second of two epidermal growth factor-like domains located at the C-terminus of the merozoite surface protein 1 (MSP-1) of P. c. chabaudi AS. The C-terminus region of the MSP-1 of P. c. chabaudi is a region of heterogeneity with AS and CB strain parasites showing only 78% identity at the amino acid level. The critical amino acid substitution which accounts for the strain specificity of the NIMP23 monoclonal antibody has now been identified. Polymerase chain reaction directed mutagenesis experiments demonstrate that a single proline to asparagine substitution at position 1722 in the primary amino acid sequence is sufficient to convert NIMP23-negative P. c. chabaudi CB expression constructs into NIMP23-positive clones whilst the converse substitution of an asparagine for a proline residue converts P. c. chabaudi AS expression constructs into NIMP23-negative clones.

We identify phosphoproteins that are associated to the infected erythrocyte membrane of Plasmodium chabaudi. The specific antibodies that recognize the erythrocyte membrane-associated antigens, namely PcEMA1 and PcLEMA, are studied. According to sequencing analysis, neither the gene structure nor the predicted properties of these two antigens are completely distinct. The PcEMA1 is a 50 kDa acidic protein, with a pI of about 4.4, that contains 25 phosphorylation sites principally located in the repetitive sequence. Almost all of this molecule is hydrophilic. The predicted (predictment ability) of this protein is, hence, associated to the host's erythrocyte cytoskeleton and it is synthesized through an entire erythrocyte cycle. PcLEMA, instead, is a 74 kDa basic protein, with a pI of about 9.8, that contains 11 possible phosphorylation sites. This gene contains two exons: exon 1 shows transmembrane characteristics while exon 2 contains part of the hydrophilic repetitive sequences. Thus this protein is predicted as a transmembrane-associated protein and is synthesized only at the last stages of the erythrocytic cycle. In in-vitro phosphorylation experiments, both PcEMA1 and PcLEMA are phosphorylated by endogenous kinases activities. However, the degree of phosphorylation differs between the two in that PcEMA1 reveals a higher phosphorylation intensity than PcLEMA.

Screening of a cDNA library (prepared in lambda gt11) of the blood stages of Plasmodium chabaudi chabaudi (AS) with immune serum has revealed an antigen the elicits a strong antibody response in infected mice. The clone (clone 6) expressing that antigen contains a 0.7 kb insert and produces a beta-galactosidase fusion protein of about 150 kDa. In Western blot analysis performed on parasite extracts, monoclonal antibodies and polyclonal sera prepared against the fusion protein revealed that the fusion protein contains part of a malarial protein of 93 kDa. Northern hybridization with clone 6 insert as probe detected a plasmodial RNA of about 3.2 kb, which could well code for a protein of this size. The insert hybridized to a single EcoRI fragment and a single HindIII fragment in genomic Southern blotting, suggesting that the gene is present in one copy in the P. chabaudi genome. The DNA sequence of clone 6 insert predicts a hydrophilic, acidic polypeptide consisting of seven repeats of 23-34 amino acids rich in lysine (24%) and aspartic acid (17.5%).

Cloned lines of the four rodent Plasmodium species can be differentiated by the RFLP pattern generated following Southern blotting and probing with PCsv4.1, a probe derived from a P. chabaudi chabaudi genomic library. Groups of CBA/Ca mice were inoculated simultaneously with cloned lines from two parasite species or strains. Six mixed species and three mixed strain infections using rodent malaria lines were initiated. The composition of the parasite population in each group was determined qualitatively and semi-quantitatively by analysis of the DNA purified from daily blood samples, thereby providing a dynamic representation of each mixed infection. Effects on the course of parasitaemias are presented and discussed.

We have characterized by molecular cloning and sequencing a Plasmodium chabaudi antigen that is associated with the membrane of the infected erythrocyte throughout the entire intraerythrocytic cycle. The protein (PcEMA1) has a predicted size of 50 kDa and contains a major tandem repeat array of 16 octapeptides that constitutes almost 30% of the protein. At its amino-terminus, PcEMA1 has a string of hydrophobic residues characteristic of a secreted protein, but does not contain a hydrophobic membrane-spanning segment. The antigen appears to reside on the cytoplasmic face of the erythrocytic membrane. PcEMA1 has a predicted pI of 4.4 and is a potential phosphoprotein.

A DNA probe PCsv4 and a subclone thereof PCsv4.1, hybridize specifically to rodent malaria DNA. DNA purified from a small volume (10 microliters) of infected mouse blood was used to determine the composition of the parasite population present. The hybridization signal following PCsv4 probing of slot-blotted DNA correlated directly with parasitaemia. The hybridization pattern and intensity, resulting from probing restriction enzyme digested and Southern-blotted genomic DNA, determined the identity of the infecting parasite line(s), and provided a semi-quantitative measure of parasite burden. Fifteen parasite lines representative of all four Plasmodium species infecting rodents can be differentiated in this way.

Diversity in a major merozoite surface antigen (MSA-1) of Plasmodium chabaudi has been examined using a panel of monoclonal antibodies (MoAbs). The antigen was found to be different in each of twelve cloned isolates, as shown by its reactivity with the MoAbs in immunofluorescence tests. In genetic crossing experiments, the diverse forms of this antigen were shown to be determined by allelic variation of a single gene. Recombination occurred between the MSA-1 gene, a second blood stage antigen and enzyme markers.