Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections

BACKGROUND: Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections. RESULTS: Whereas there was a trade-off between transmission success and virulence as defined by host mortality, contradictory clone-specific patterns occurred when defining virulence by anaemia. A negative relationship between anaemia and transmission success was found for one of the parasite clones, whereas there was no relationship for the other. Notably the two parasite clones also differed in a transmission phenotype (gametocyte sex ratio) that has previously been shown to respond adaptively to a changing blood environment. In addition, as predicted by evolutionary theory, mixed infections resulted in increased anaemia. The increased anaemia was, however, not correlated with any discernable parasite trait (e.g. parasite density) or with increased transmission. CONCLUSIONS: We found some evidence supporting the hypothesis that there is an adaptive basis correlating virulence (as defined by host mortality) and transmission success in malaria parasites. This confirms the validity of applying evolutionary virulence theory to biomedical research and adds support to the prediction that partially effective vaccines may select for increasingly virulent malaria parasite strains. By contrast, there was no consistent correlation between transmission and sub-lethal anaemia, a more common outcome of malaria infection. However, overall, the data are not inconsistent with the recent proposal that sub-lethal effects may impose an upper limit on virulence. Moreover, clone specific differences in transmission phenotypes linked to anaemia do suggest that there is considerable adaptive potential relating anaemia and transmission that may lead to uncertain consequences following intervention strategies

Age-structured gametocyte allocation links immunity to epidemiology in malaria parasites

Background Despite a long history of attempts to model malaria epidemiology, the over-riding conclusion is that a detailed understanding of host-parasite interactions leading to immunity is required. It is still not known what governs the duration of an infection and how within-human parasite dynamics relate to malaria epidemiology. Presentation of the hypothesis Immunity to Plasmodium falciparum develops slowly and requires repeated exposure to the parasite, which thus generates age-structure in the host-parasite interaction. An age-structured degree of immunity would present the parasite with humans of highly variable quality. Evolutionary theory suggests that natural selection will mould adaptive phenotypes that are more precise (less variant) in "high quality" habitats, where lifetime reproductive success is best. Variability in malaria parasite gametocyte density is predicted to be less variable in those age groups who best infect mosquitoes. Thus, the extent to which variation in gametocyte density is a simple parasite phenotype reflecting the complex within-host parasite dynamics is addressed. Testing the hypothesis Gametocyte densities and corresponding infectiousness to mosquitoes from published data sets and studies in both rural and urban Cameroon are analysed. The mean and variation in gametocyte density according to age group are considered and compared with transmission success (proportion of mosquitoes infected). Across a wide range of settings endemic for malaria, the age group that infected most mosquitoes had the least variation in gametocyte density, i.e. there was a significant relationship between the variance rather than the mean gametocyte density and age-specific parasite transmission success. In these settings, the acquisition of immunity over time was evident as a decrease in asexual parasite densities with age. By contrast, in an urban setting, there were no such age-structured relationships either with variation in gametocyte density or asexual parasite density. Implications of the hypothesis Gametocyte production is seemingly predicted by evolutionary theory, insofar as a reproductive phenotype (gametocyte density) is most precisely expressed (i.e. is most invariant) in the most infectious human age group. This human age group would thus be expected to be the habitat most suitable for the parasite. Comprehension of the immuno-epidemiology of malaria, a requisite for any vaccine strategies, remains poor. Immunological characterization of the human population stratified by parasite gametocyte allocation would be a step forward in identifying the salient immunological pathways of what makes a human a good habitat

Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India

<p>Abstract</p> <p>Background</p> <p>Molecular techniques have facilitated the studies on genetic diversity of <it>Plasmodium </it>species particularly from field isolates collected directly from patients. The <it>msp-1 </it>and <it>msp-2 </it>are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the <it>msp-1 </it>gene and the central repetitive domain (block3) of the <it>msp-2 </it>gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among <it>Plasmodium falciparum </it>population of India, this report evaluates the extent of genetic diversity in the field isolates of <it>P. falciparum </it>in eastern and north-eastern regions of India.</p> <p>Methods</p> <p>A study was designed to assess the diversity of <it>msp-1 </it>and <it>msp-2 </it>among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa.</p> <p>Results</p> <p><it>P. falciparum </it>isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of <it>msp-1 </it>and <it>msp-2</it>. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the <it>P. falciparum </it>populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of <it>msp-1 </it>was observed to be monomorphic with an allele size of 150/160 bp. The observed 80–90% sequence identity of Indian isolates with data of other regions suggests that Indian <it>P. falciparum </it>population is a mixture of different strains.</p> <p>Conclusion</p> <p>The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of <it>msp-1 </it>(block 2) and <it>msp-2 </it>(central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.</p

“Sexual” Population Structure and Genetics of the Malaria Agent P. falciparum

The population genetics and structure of P. falciparum determine the rate at which malaria evolves in response to interventions such as drugs and vaccines. This has been the source of considerable recent controversy, but here we demonstrate the organism to be essentially sexual, in an area of moderately high transmission in the Lower Shire Valley, Malawi. Seven thousand mosquitoes were collected and dissected, and genetic data were obtained on 190 oocysts from 56 infected midguts. The oocysts were genotyped at three microsatellite loci and the MSP1 locus. Selfing rate was estimated as 50% and there was significant genotypic linkage disequilibrium (LD) in the pooled oocysts. A more appropriate analysis searching for genotypic LD in outcrossed oocysts and/or haplotypic LD in the selfed oocysts found no evidence for LD, indicating that the population was effectively sexual. Inbreeding estimates at MSP1 were higher than at the microsatellites, possibly indicative of immune action against MSP1, but the effect was confounded by the probable presence of null mutations. Mating appeared to occur at random in mosquitoes and evidence regarding whether malaria clones in the same host were related (presumably through simultaneous inoculation in the same mosquito bite) was ambiguous. This is the most detailed genetic analysis yet of P. falciparum sexual stages, and shows P. falciparum to be a sexual organism whose genomes are in linkage equilibrium, which acts to slow the emergence of drug resistance and vaccine insensitivity, extending the likely useful therapeutic lifespan of drugs and vaccines

Information use and plasticity in the reproductive decisions of malaria parasites

BACKGROUND: Investment in the production of transmissible stages (gametocytes) and their sex ratio are malaria parasite traits that underpin mosquito infectivity and are therefore central to epidemiology. Malaria parasites adjust their levels of investment into gametocytes and sex ratio in response to changes in the in-host environment (including red blood cell resource availability, host immune responses, competition from con-specific genotypes in mixed infections, and drug treatment). This plasticity appears to be adaptive (strategic) because parasites prioritize investment (in sexual versus asexual stages and male versus female stages) in manners predicted to maximize fitness. However, the information, or ‘cues’ that parasites use to detect environmental changes and make appropriate decisions about investment into gametocytes and their sex ratio are unknown. METHODS: Single genotype Plasmodium chabaudi infections were exposed to ‘cue’ treatments consisting of intact or lysed uninfected red blood cells, lysed parasitized RBCs of the same clone or an unrelated clone, and an unmanipulated control. Infection dynamics (proportion of reticulocytes, red blood cell and asexual stage parasite densities) were monitored, and changes in gametocyte investment and sex ratio in response to cue treatments, applied either pre- or post-peak of infection were examined. RESULTS AND CONCLUSIONS: A significant reduction in gametocyte density was observed in response to the presence of lysed parasite material and a borderline significant increase in sex ratio (proportion of male gametocytes) upon exposure to lysed red blood cells (both uninfected and infected) was observed. Furthermore, the changes in gametocyte density and sex ratio in response to these cues depend on the age of infection. Demonstrating that variation in gametocyte investment and sex ratio observed during infections are a result of parasite strategies (rather than the footprint of host physiology), provides a foundation to investigate the fitness consequences of plasticity and explore whether drugs could be developed to trick parasites into making suboptimal decisions

Transformation of the rodent malaria parasite Plasmodium chabaudi and generation of a stable fluorescent line PcGFPCON

<p>Abstract</p> <p>Background</p> <p>The rodent malaria parasite <it>Plasmodium chabaudi </it>has proven of great value in the analysis of fundamental aspects of host-parasite-vector interactions implicated in disease pathology and parasite evolutionary ecology. However, the lack of gene modification technologies for this model has precluded more direct functional studies.</p> <p>Methods</p> <p>The development of <it>in vitro </it>culture methods to yield <it>P. chabaudi </it>schizonts for transfection and conditions for genetic modification of this rodent malaria model are reported.</p> <p>Results</p> <p>Independent <it>P. chabaudi </it>gene-integrant lines that constitutively express high levels of green fluorescent protein throughout their life cycle have been generated.</p> <p>Conclusion</p> <p>Genetic modification of <it>P. chabaudi </it>is now possible. The production of genetically distinct reference lines offers substantial advances to our understanding of malaria parasite biology, especially interactions with the immune system during chronic infection.</p

Sex and Death: The Effects of Innate Immune Factors on the Sexual Reproduction of Malaria Parasites

Malaria parasites must undergo a round of sexual reproduction in the blood meal of a mosquito vector to be transmitted between hosts. Developing a transmission-blocking intervention to prevent parasites from mating is a major goal of biomedicine, but its effectiveness could be compromised if parasites can compensate by simply adjusting their sex allocation strategies. Recently, the application of evolutionary theory for sex allocation has been supported by experiments demonstrating that malaria parasites adjust their sex ratios in response to infection genetic diversity, precisely as predicted. Theory also predicts that parasites should adjust sex allocation in response to host immunity. Whilst data are supportive, the assumptions underlying this prediction – that host immune responses have differential effects on the mating ability of males and females – have not yet been tested. Here, we combine experimental work with theoretical models in order to investigate whether the development and fertility of male and female parasites is affected by innate immune factors and develop new theory to predict how parasites' sex allocation strategies should evolve in response to the observed effects. Specifically, we demonstrate that reactive nitrogen species impair gametogenesis of males only, but reduce the fertility of both male and female gametes. In contrast, tumour necrosis factor-α does not influence gametogenesis in either sex but impairs zygote development. Therefore, our experiments demonstrate that immune factors have complex effects on each sex, ranging from reducing the ability of gametocytes to develop into gametes, to affecting the viability of offspring. We incorporate these results into theory to predict how the evolutionary trajectories of parasite sex ratio strategies are shaped by sex differences in gamete production, fertility and offspring development. We show that medical interventions targeting offspring development are more likely to be ‘evolution-proof’ than interventions directed at killing males or females. Given the drive to develop medical interventions that interfere with parasite mating, our data and theoretical models have important implications

Does the drug sensitivity of malaria parasites depend on their virulence?

<p>Abstract</p> <p>Background</p> <p>Chemotherapy can prompt the evolution of classical drug resistance, but selection can also favour other parasite traits that confer a survival advantage in the presence of drugs. The experiments reported here test the hypothesis that sub-optimal drug treatment of malaria parasites might generate survival and transmission advantages for virulent parasites.</p> <p>Methods</p> <p>Two <it>Plasmodium chabaudi </it>lines, one derived from the other by serial passage, were used to establish avirulent and virulent infections in mice. After five days, infections were treated with various doses of pyrimethamine administered over 1 or 4 days. Virulence measures (weight and anaemia), parasite and gametocyte dynamics were followed until day 21.</p> <p>Results</p> <p>All treatment regimes reduced parasite and gametocyte densities, but infections with the virulent line always produced more parasites and more gametocytes than infections with the avirulent line. Consistent with our hypothesis, drug treatment was disproportionately effective against the less virulent parasites. Treatment did not affect the relative transmission advantage of the virulent line. Neither of the lines contained known mutations conferring classical drug resistance.</p> <p>Conclusion</p> <p>Drug-sensitivity of malaria parasites can be virulence-dependent, with virulent parasites more likely to survive sub-optimal treatment. If this proves to be general for a variety of drugs and parasite species, selection imposed by sub-optimal drug treatment could result in the evolution of more aggressive malaria parasites.</p

The Relationship of Within-Host Multiplication and Virulence in a Plant-Virus System

Background. Virulence does not represent any obvious advantage to parasites. Most models of virulence evolution assume that virulence is an unavoidable consequence of within-host multiplication of parasites, resulting in trade-offs between within-host multiplication and between-host transmission fitness components. Experimental support for the central assumption of this hypothesis, i.e., for a positive correlation between within-host multiplication rates and virulence, is limited for plant-parasite systems. Methodology/Principal Findings. We have addressed this issue in the system Arabidopsis thaliana-Cucumber mosaic virus (CMV). Virus multiplication and the effect of infection on plant growth and on viable seed production were quantified for 21 Arabidopsis wild genotypes infected by 3 CMV isolates. The effect of infection on plant growth and seed production depended of plant architecture and length of postembryonic life cycle, two genetically-determined traits, as well as on the time of infection in the plant's life cycle. A relationship between virus multiplication and virulence was not a general feature of this host-parasite system. This could be explained by tolerance mechanisms determined by the host genotype and operating differently on two components of plant fitness, biomass production and resource allocation to seeds. However, a positive relationship between virus multiplication and virulence was detected for some accessions with short life cycle and high seed weight to biomass ratio, which show lower levels of tolerance to infection. Conclusions/Significance. These results show that genotype-specific tolerance mechanisms may lead to the absence of a clear relationship between parasite multiplication and virulence. Furthermore, a positive correlation between parasite multiplication and virulence may occur only in some genotypes and/or environmental conditions for a given host-parasite system. Thus, our results challenge the general validity of the trade-off hypothesis for virulence evolution, and stress the need of considering the effect of both the host and parasite genotypes in analyses of host-parasite interactions. © 2007 Pagán et al.Ministerio de Educación y Ciencia, Spain.Peer Reviewe