Mixed-genotype infections of malaria parasites: within-host dynamics and transmission success of competing clones.

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.     

VIRULENCE OF MIXED-CLONE AND SINGLE-CLONE INFECTIONS OF THE RODENT MALARIA PLASMODIUM CHABAUDI

Most evolutionary models treat virulence as an unavoidable consequence of microparasite replication and have predicted that in mixed-genotype infections, natural selection should favor higher levels of virulence than is optimal in genetically uniform infections. Increased virulence may evolve as a genetically fixed strategy, appropriate for the frequency of mixed infections in the population, or may occur as a conditional response to mixed infection, that is, a facultative strategy. Here we test whether facultative alterations in replication rates in the presence of competing genotypes occur and generate greater virulence. An important alternative, not currently incorporated in models of the evolution of virulence, is that host responses mounted against genetically diverse parasites may be more costly or less effective than those against genetically uniform parasites. If so, mixed clone infections will be more virulent for a given parasite replication rate. Two groups of mice were infected with one of two clones of Plasmodium chabaudi parasites, and three groups of mice were infected with 1:9, 5:5, or 9:1 mixtures of the same two clones. Virulence was assessed by monitoring mouse body weight and red blood cell density. Transmission stage densities were significantly higher in mixed- than in single-clone infections. Within treatment groups, transmission stage production increased with the virulence of the infection, a phenotypic correlation consistent with the genetic correlation assumed by much of the theoretical work on the evolution of virulence. Consistent with theoretical predictions of facultative alterations in virulence, we found that mice infected with both parasite clones lost more weight and had on average lower blood counts than those infected with single-clone infections. However, there was no consistent evidence of the mechanism invoked by evolutionary models that predict this effect. Replication rates and parasite densities were not always higher in ???mixed-clone infections, and for a given replication rate or parasite density, mixed-clone infections were still more virulent. Instead, prolonged anemia and increased transmission may have occured because genetically diverse infections are less rapidly cleared by hosts. Differences in maximum weight loss occured even when there were comparable parasite densities in mixed- and single-clone infections. We suggest that mounting an immune response against more that one parasite genotype is more costly for hosts, which therefore suffer higher virulence.     

Immune-Mediated Competition in Rodent Malaria Is Most Likely Caused by Induced Changes in Innate Immune Clearance of Merozoites

Malarial infections are often genetically diverse, leading to competitive interactions between parasites. A quantitative understanding of the competition between strains is essential to understand a wide range of issues, including the evolution of virulence and drug resistance. In this study, we use dynamical-model based Bayesian inference to investigate the cause of competitive suppression of an avirulent clone of Plasmodium chabaudi (AS) by a virulent clone (AJ) in immuno-deficient and competent mice. We test whether competitive suppression is caused by clone-specific differences in one or more of the following processes: adaptive immune clearance of merozoites and parasitised red blood cells (RBCs), background loss of merozoites and parasitised RBCs, RBC age preference, RBC infection rate, burst size, and within-RBC interference. These processes were parameterised in dynamical mathematical models and fitted to experimental data. We found that just one parameter , the ratio of background loss rate of merozoites to invasion rate of mature RBCs, needed to be clone-specific to predict the data. Interestingly, was found to be the same for both clones in single-clone infections, but different between the clones in mixed infections. The size of this difference was largest in immuno-competent mice and smallest in immuno-deficient mice. This explains why competitive suppression was alleviated in immuno-deficient mice. We found that competitive suppression acts early in infection, even before the day of peak parasitaemia. These results lead us to argue that the innate immune response clearing merozoites is the most likely, but not necessarily the only, mediator of competitive interactions between virulent and avirulent clones. Moreover, in mixed infections we predict there to be an interaction between the clones and the innate immune response which induces changes in the strength of its clearance of merozoites. What this interaction is unknown, but future refinement of the model, challenged with other datasets, may lead to its discovery.     

Parasite-mediated selection in experimental Daphnia magna populations

Abstract It has been suggested that parasites are a strong selecting force for their hosts and therefore may alter the outcome of competition among host genotypes. We tested the extent to which parasite-mediated selection by different parasite species influenced competition among clones of the cyclic parthenogen Daphnia magna . We monitored clone frequency changes in laboratory microcosm populations consisting of 21 D. magna clones. Parasite treatments (two microsporidians, Glugoides intestinalis and Ordospora colligata ) and a parasite-free control treatment were followed over a nine-month period. A further treatment with the bacterium Pasteuria ramosa failed. We found significant differences in clonal success among the treatments: the two parasite treatments differed from the control treatment and from each other. Additionally, we measured the clone-specific population carrying capacity, competitive ability against tester clones, and reproductive success of infected and uninfected females to test whether they correlate with clonal success in the microcosms. The clone-specific competitive ability was a good predictor of clonal success in the microcosms, but clonal carrying capacity and host reproductive success were not. Our study shows that parasite-mediated selection can strongly alter the outcome of clonal competition. The results suggest that parasites may influence microevolution in Daphnia populations during periods of asexual reproduction.     

A lotka-volterra model of coexistence between a sexual population and multiple asexual clones

At carrying capacity, small advantages in competitive ability can compensate a sexual population for its two-fold disadvantage in growth capacity when facing invasion by asexual mutants. In this paper, we develop a generic analytical model to consider the ecology of a sexual population comprising equal numbers of males and females, competing for shared prey resources with multiple female-only clones. We assume that the clones arise from the sexual population and are distinguished from it only by having narrower resource niches and twice the growth capacity. For sexual populations, at density-dependent carrying capacity, intra-specific competition between clonal individuals prevents them from realizing their two-fold advantage in intrinsic growth. This prediction leads to three novel outcomes: (i) a sexual population can coexist with any number of clones, provided their combined competitive impact remains less than the impact of the clones on each other; (ii) a sexual species can immediately exclude asexual invaders if it is a fast growing and strong competitor of shared resources and also has refuge in an abundant alternative resource; (iii) the rate of accumulation of clones in a sexual population will be slowed by intra and inter-specific competition amongst the clones themselves, in addition to the competitive impact from the original sexual population.     

TWO MOTILE STAGES OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE): MORPHOLOGY,GROWTH, AND TOXICITY1

Eight months after the 1988 bloom of Chrysochromulina polylepis Manton et Parke in Skagerrak and Kattegat, off the coasts of Norway, Sweden, and Denmark, an alternate cell type carrying a scale complement different from that of authentic C. polylepis appeared in some clones isolated from the bloom. The cultures were recloned, and the development of the new clones was monitored. In clones with 100% cells of the alternate type, authentic cells reappeared, suggesting that the alternate cell type is a stage in the life cycle of C. polylepis and that transition between cell types occurs in both directions. Growth rates of clone cultures (termed a cultures) producing exclusively authentic cells, and of clone cultures (termed β cultures), capable of producing the alternate cell type, were compared at various combinations of temperature and photon fluence rate. The β cultures were less tolerant of high temperatures and photon fluence rates (≤20° C, 570 μmol photons·m^?2·s^?1) than were the α cultures. At lower temperatures and photon fluence rates (≤16° C, ≤90 μmol photons·m^?2·s^?1ss), β cultures grew better than α cultures. Relative abundance of the two cell types in β cultures changed in an apparently random manner during these experiments. Preliminary results from flow cytometric analyses indicated that cells of the alternate type were about twice the size and contained an equal or smaller amount of DNA per cell compared to the authentic cells. The β cultures were less toxic to Artemia nauplii than were the a cultures. Three other Chrysochromulina species tested were apparently nontoxic.     

Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses.

We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.     

Relative clonal density of malaria parasites in mixed-genotype infections: validation of a technique using microsatellite markers for Plasmodium falciparum and P. mexicanum

Quantifying the relative proportion of coexisting genotypes (clones) of a malaria parasite within its vertebrate host's blood would provide insights into critical features of the biology of the parasite, including competition among clones, gametocyte sex ratio, and virulence. However, no technique has been available to extract such data for natural parasite-host systems when the number of clones cycling in the overall parasite population is likely to be large. Recent studies find that data from genetic analyzer instruments for microsatellite markers allow measuring clonal proportions. We conducted a validation study for Plasmodium mexicanum and Plasmodium falciparum by mixing DNA from single-clone infections to simulate mixed infections of each species with known proportions of clones. Results for any mixture of DNA gave highly reproducible results. The relationship between known and measured relative proportions of clones was linear, with high regression r2 values. Known and measured clone proportions for simulated infections followed over time (mixtures) were compared with 3 methods: using uncorrected data, with uncorrected data and confidence intervals constructed from observed experimental error, and using a baseline mixture of equal proportions to calibrate all other results. All 3 methods demonstrated value in studies of mixed-genotype infections sampled a single time or followed over time. Thus, the method should open new windows into the biology of malaria parasites.     

Ecological differences among clones of Daphnia pulex Leydig

Summary Natural populations of Daphnia pulex that reproduce by obligate parthenogenesis include a number of clones. Studies on two common and two rare clones from southwestern Ontario revealed significant differences in their intrinsic rates of increase, competitive abilities, rates of ephippial egg production, and lifespans. Environmental factors such as temperature and food type had large inluences on the rate of increase of each clone. Differences in rates of increase among clones were most pronounced at temperatures higher than those encountered in nature. In general, the covariance of life history traits among clones was high. The outcome of competitive encounters between clones was deterministic and in most cases was unaffected by temperature. Clones with high rates of increase tended to be better competitors than those with low rates of increase.     

Premature Salmonella Typhimurium growth inhibition in competition with other Gram-negative organisms is redox potential regulated via RpoS induction

AIMS: To identify the role of oxidation-reduction (redox) potential in the premature growth inhibition and RpoS induction in Salmonella serotype Typhimurium in competitive growth experiments. METHODS AND RESULTS: Oxidation-reduction potential was measured throughout the growth of a minority population of Salm. Typhimurium in mixed cultures with other Gram-negative and Gram-positive organisms. A lux-based reporter was also used to evaluate RpoS activity in Salm. Typhimurium in competitor studies. In a mixed culture, the multiplication of a minority population of Salm. Typhimurium was inhibited when competing Gram-negative organisms entered the stationary phase. This was not seen when the competing flora was Gram-positive. The change in redox potential during growth in mixed cultures was closely linked to the inhibition of Salm. Typhimurium growth by Gram-negative competitors. An artificially induced drop in redox potential earlier during growth in mixed cultures with Gram-negative organisms reduced the time to RpoS induction in Salm. Typhimurium and thus inhibited its multiplication prematurely. In contrast, RpoS induction and growth inhibition were prevented under high redox potential conditions. CONCLUSIONS: This work shows that the inhibitory activity of competitive organisms can be mediated through their effect on redox potential-regulated RpoS induction. SIGNIFICANCE AND IMPACT OF THE STUDY: Redox potential is shown to be an important determinant of Salm. Typhimurium growth, an observation with practical implications both for its control and detection.     

初始克隆分株数对大米草表型可塑性及生物量分配的影响

克隆植物大米草 (Spartina anglica) 目前在我国出现了严重的自然衰退 (Dieback),为了阐明大米草衰退的机理,分析影响大米草形态可塑性的因素与自然衰退之间的相关性,以期为近缘植物互花米草 (S. alterniflora) 这一爆发种群的生物控制提供借鉴,对3种不同初始克隆分株数 (单克隆、三克隆和五克隆) 大米草的克隆生长、生物量累积与分配和异速生长特征进行了野外栽培试验。研究结果表明,初始克隆分株数对间隔子长度影响较弱;初始多克隆的分支强度高于初始单克隆;初始三克隆和五克隆在总生物量 (7.921 5~10.431 7 g 和 8.903 9~10.431 7 g)、地上生物量 (3.396 1~4.255 8 g 和3.618 4~4.338 9 g)、地下生物量 (4.286 9~5.206 6 g 和 5.298 8~6.079 3 g)和根状茎生物量 (1.318 6~1.767 7 g 和 1.499 1~2.038 7 g) 积累上均显著高于初始单克隆,不同初始克隆分株数条件下根生物量差异不显著;初始多克隆倾向于将资源更多地分配给根状茎,而初始单克隆倾向于将更多的资源分配给根系。由此推断,在不同初始克隆分株数条件下,大米草的形态可塑性和生物量分配格局的差异显示出在同样资源格局下,初始多克隆的克隆生殖能力较初始单克隆强。初始多克隆生长的大米草较初始单克隆生长的大米草更能占据优势生境,选择生境“觅养”的能力与克隆繁殖能力更强。     

Selection of chemically defined media for CHO cell fed-batch culture processes

Two CHO cell clones derived from the same parental CHO^BC® cell line and producing the same monoclonal antibody (BC-G, a low producing clone; BC-P, a high producing clone) were tested in four basal media in all possible combinations with three feeds (=12 conditions) in fed-batch cultures. Higher amino acid feeding did not always lead to higher mAb production. The two clones showed differences in cell physiology, metabolism and optimal medium-feed combinations. During the phase transitions of all cultures, cell metabolism showed a shift represented by lower specific consumption and production rates, except for the specific glucose consumption rate in cultures fed by Actifeed A/B. The BC-P clone fed by Actifeed A/B showed a threefold cell volume increase and an increase of the specific consumption rate of glucose in the stationary phase. Since feeding was based on glucose this resulted in accumulation of amino acids for this feed, while this did not occur for the poorer feed (EFA/B). The same feed also led to an increase of cell size for the BC-G clone, but to a lesser extent.     

Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence

During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.     

GENETIC RELATIONSHIPS BETWEEN PARASITE VIRULENCE AND TRANSMISSION IN THE RODENT MALARIA PLASMODIUM CHABAUDI

Many parasites evolve to become virulent rather than benign mutualists. One of the major theoretical models of parasite virulence postulates that this is because rapid within-host replication rates are necessary for successful transmission (parasite fitness) and that virulence (damage to the host) is an unavoidable consequence of this rapid replication. Two fundamental assumptions underlying this so-called evolutionary trade-off model have rarely been tested empirically: (1) that higher replication rates lead to higher levels of virulence; and (2) that higher replication rates lead to higher transmission. Both of these relationships must have a genetic basis for this evolutionary hypothesis to be relevant. These assumptions were tested in the rodent malaria parasite, Plasmodium chabaudi, by examining genetic relationships between virulence and transmission traits across a population of eight parasite clones isolated from the wild. Each clone was injected into groups of inbred mice in a controlled laboratory environment, and replication rate (measured by maximum asexual parasitemia), virulence (measured by live-weight loss and degree of anemia in the mouse), and transmission (measured by density of sexual forms, gametocytes, in the blood and proportion of mosquitoes infected after taking a blood-meal from the mouse) were assessed. It was found that clones differed widely in these traits and these clone differences were repeatable over successive blood passages. Virulence traits were strongly phenotypically and genetically (i.e., across clones) correlated to maximum parasitemia thus supporting the first assumption that rapid replication causes higher virulence. Transmission traits were also positively phenotypically and genetically correlated to parasitemia, which supports the second assumption that rapid replication leads to higher transmission. Thus, two assumptions of the parasite-centered trade-off model of the evolution of virulence were shown to be justified in malaria parasites.     

Variable reproductive output among clones of Spartina alterniflora (Poaceae) invading San Francisco Bay,California: the influence of herbivory,pollination, and establishment site

Spartina alterniflora has recently been introduced to San Francisco Bay, California, and is rapidly invading open mud flats, growing in circular patches that we found to be individual genetic clones. We collected spikelet samples from more than 200 clones and observed germination rates ranging from 0% to 59%, indicating substantial variation in reproductive output among clones. Several experiments were performed to explore the cause of variation. Pollination manipulations showed that S. alterniflora is outcrossing, but pollen supplements did not increase spikelet germination rates. Exclusion of the only insect herbivore (a phloem feeder, Prokelisia marginata) from developing inflorescences increased the proportion of spikelets containing seed, but failed to increase germinations per spikelet. Spikelets from Willapa Bay, Washington, grown free of insect herbivores, had germination rates similar to San Francisco Bay. These results suggest herbivory is not limiting reproductive output of S. alterniflora. Spikelet viability was not related to clone size; however, clones located lower in the intertidal or far up a drainage slough averaged fewer germinations per spikelet, suggesting clones in areas with lower genet density may have lower spikelet viabilities. Spikelet samples from different sections of clones growing across wide environmental ranges had similar rates of germination, suggesting some genetic influence on spikelet viability. Differential reproductive output among clones and the novel selective environment of San Francisco Bay are expected to cause gene frequency changes in this rapidly expanding population.     

Transmission stage investment of malaria parasites in response to in-host competition

Conspecific competition occurs in a multitude of organisms, particularly in parasites, where several clones are commonly sharing limited resources inside their host. In theory, increased or decreased transmission investment might maximize parasite fitness in the face of competition, but, to our knowledge, this has not been tested experimentally. We developed and used a clone-specific, stage-specific, quantitative PCR protocol to quantify Plasmodium chabaudi replication and transmission stage densities in mixed-clone infections. We co-infected mice from two strains with an avirulent and virulent parasite clone and found competitive suppression of in-host (blood-stage) parasite densities and generally corresponding reductions in transmission stage production, with the virulent clone obtaining overall competitive superiority. In response to competitive suppression, there was little evidence of any alteration in transmission stage investment, apart from a small reduction by one of the two clones in one of the two host strains. This alteration did not result in a competitive advantage, although it might have reduced the disadvantage. This study supports much of the current literature, which predicts that conspecific in-host competition will result in a competitive advantage and positive selection for virulent clones and thus the evolution of higher virulence.     

Differential regulation of activation, clonal expansion, and antibody secretion in human B cells

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.     

Clonal interactions in fibroblast proliferation: recognition of self vs. non-self

The proliferation and aging of fibroblast populations has been postulated to include a process of clonal selection. Using carbocyanine dyes to label clonal fibroblast populations, we were able to follow their growth in mixed cultures. Individual fibroblast clones seeded as the minority population (20%) with either another clone or the parent line (differentially labeled) always demonstrated increase relative growth so that, by the end of 4 weeks, approximately equal numbers of both populations were present. Labeled cells of the same clone mixed as the minority population with differentially labeled cells of the same clone maintained their minority status. The results indicate that clonal populations of fibroblasts are able to recognize "self" as different from "non-self" and that this recognition leads to alterations in cellular proliferation.     

The role of immune-mediated apparent competition in genetically diverse malaria infections

Competitive interactions between coinfecting genotypes of the same pathogen can impose selection on virulence, but the direction of this selection depends on the mechanisms behind the interactions. Here, we investigate how host immune responses contribute to competition between clones in mixed infections of the rodent malaria parasite Plasmodium chabaudi. We studied single and mixed infections of a virulent and an avirulent clone and compared the extent of competition in immunodeficient and immunocompetent mice (nude mice and T cell-reconstituted nude mice, respectively). In immunocompetent mice, the avirulent clone suffered more from competition than did the virulent clone. The competitive suppression of the avirulent clone was alleviated in immunodeficient mice. Moreover, the relative density of the avirulent clone in mixed infections was higher in immunodeficient than in immunocompetent mice. We conclude that immune-mediated interactions contributed to competitive suppression of the avirulent clone, although other mechanisms, presumably competition for resources such as red blood cells, must also be important. Because only the avirulent clone suffered from immune-mediated competition, this mechanism should contribute to selection for increased virulence in mixed infections in this host-parasite system. As far as we are aware, this is the first direct experimental evidence of immune-mediated apparent competition in any host-parasite system.