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.     

Competitive suppression in mixed-clone parasite cultures

Mixed-genotype infections occur frequently in natural populations. Parasite genotypes are expected to interact within a host: competing for shared nutrients and being affected by the host's immune response to each other. Theoretically, competing parasites can be expected to exhibit increased rates of replication. Here, we investigate whether interactions between clones of Theileria annulata, a protozoan parasite of cattle, affect clones' replication rates in mixed cultures in vitro. Intrinsic replication rates and carrying capacities estimated from single-clone control cultures were used to predict replication rates of mixed cultures under different competitive assumptions. Mixed-culture dynamics deviated significantly from expectations in five out of six different clone combinations tested. Contrary to expectation, mixed cultures often replicated more slowly than predicted from single-clone control cultures. Competition coefficients were calculated from the mixed-culture data and a competitive hierarchy of clones determined. The results suggest that inherent competitive ability may be greater in clones with lower carrying capacities-those clones which would otherwise be excluded in a genetically diverse environment. Moreover, significant negative deviations from expected replication rates corresponded with successful out-competing of a higher carrying capacity clone by a lower carrying capacity clone.     

Prevalence of blood parasites in cattle from wilayates of Annaba and El Tarf east Algeria

Between June and September 2002, a preliminary study was conducted to assess the prevalence of blood parasites of cattle in eastern Algeria. Fifty-four bovines of different genotypes were submitted to clinical examination. From each animal, blood smears were made and stained by Giemsa. Four species of parasites, namely Theileria annulata, T. orientalis, Babesia bovis and Anaplasma marginale were encountered. Fifty animals carried single or multiple infections with blood parasites and four were found negative. The rate of single infections (72.3%, n = 39) was almost three times higher than multiple infections (20.3%, n = 11). The high percentage of single infections was recorded with T. annulata (53.7%). However single infection with Anaplasma marginale (7.4 %), B. bovis (5.6%) and T orientalis (5.6%) were very low compared to T. annulata infection. The rates of mixed infection were as follows: T. annulata/A. marginale 9.3%, T. annulata/T. orientalis 5.6%, A. marginale/T. orientalis 3.7% and B. bovis/A. marginale 1.9%.     

Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii.     

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.     

The effects of multiple infections on the expression and evolution of virulence in a Daphnia-endoparasite system

Multiple infections of a host by different strains of the same microparasite are common in nature. Although numerous models have been developed in an attempt to predict the evolutionary effects of intrahost competition, tests of the assumptions of these models are rare and the outcome is diverse. In the present study we examined the outcome of mixed-isolate infections in individual hosts, using a single clone of the waterflea Daphnia magna and three isolates of its semelparous endoparasite Pasteuria ramosa . We exposed individual Daphnia to single- and mixed-isolate infection treatments, both simultaneously and sequentially. Virulence was assessed by monitoring host mortality and fecundity, and parasite spore production was used as a measure of parasite fitness. Consistent with most assumptions, in multiply infected hosts we found that the virulence of mixed infections resembled that of the more virulent competitor, both in simultaneous multiple infections and in sequential multiple infections in which the virulent isolate was first to infect. The more virulent competitor also produced the vast majority of transmission stages. Only when the less virulent isolate was first to infect, the intrahost contest resembled scramble competition, whereby both isolates suffered by producing fewer transmission stages. Surprisingly, mixed-isolate infections resulted in lower fecundity-costs for the hosts, suggesting that parasite competition comes with an advantage for the host relative to single infections. Finally, spore production correlated positively with time-to-host-death. Thus, early-killing of more competitive isolates produces less transmission stages than less virulent, inferior isolates. Our results are consistent with the idea that less virulent parasite lines may be replaced by more virulent strains under conditions with high rates of multiple infections.     

Clinical and laboratory studies on theileriosis outbreaks in Sicily, Italy

A total of 153 blood samples were collected all over the Sicilian island in 15 farms. 29.4% and 25.5% were PCR positive for Theileria annulata and T. buffeli/orientalis respectively. 15.7% of cattle blood samples showed a mixed infection with both Theileria species. Each farm showed an infection rate ranging from 0% to 100%. Hyalomma marginatum and Boophilus annulatus were the most common ticks collected in April and November 1998 respectively. The aim of the present work was to investigate on haemoparasite species spread in Sicily. Ticks vectors involved in the transmission, distribution, risk areas and seasons, state of healthy carrier, are also discussed in this paper.     

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.     

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.     

Condition-dependent virulence in a horizontally and vertically transmitted bacterial parasite

Virulence is a key component of parasite fitness. Its expression and selective value may not only depend on the features of the parasite's life cycle, but also on host genotype or environmental conditions. Using the freshwater ciliate Paramecium caudatum and its endonuclear bacterial parasite Holospora undulata , we measured variation in virulence (reduction in host division and survival), parasite load and fidelity of vertical transmission for (i) different stages of infection (associated with different opportunities for vertical and horizontal transmission), (ii) different host clones, and (iii) two food conditions. Later stages of infection dedicated to horizontal transmission were more virulent than earlier stages which rely on vertical transmission only. Besides, investment in horizontal transmission decreased the efficacy of vertical transmission, indicating a tradeoff between the two pathways. This may explain the phenotypic plasticity of transmission mode of this parasite. To some extent, virulence, parasite load and transmission fidelity varied with host clone identity and food treatment (higher virulence at low food). These results suggest that virulence is not a constant property of the parasite, and that a single (and simple) relationship between virulence and transmission does not exist.     

Macrophages behaving badly: infected cells and subversion of immune responses to Theileria annulata

The protozoan parasite Theileria annulata is the causative agent of the tick-borne disease tropical theileriosis, responsible for morbidity and mortality of cattle in many developing countries. Here, John Campbell and Roger Spooner discuss how the parasite might evade immune destruction during an acute primary infection. Theileria annulata macroschizont-infected macrophages act as over-efficient antigen-presenting cells within the infected draining lymph node. Infected cells activate CD4+ and CD8+ T cells abnormally, giving rise to a cascade of cytokine production. This altered immune response does not reject the parasitized cells, and might actively participate in the growth of the developing parasite.     

Reduction of transmission stages concomitant with increased host immune responses to hypervirulent Sarcocystis singaporensis, and natural selection for intermediate virulence.

Parasite virulence (pathogenicity depending on inoculum size) and host immune reactions were examined for the apicomplexan protozoan Sarcocystis singaporensis. This parasite is endemic in southeastern Asia and multiplies as a proliferation (merozoite) and transmission stage (bradyzoite) in rats. Virulence in wild brown rats of parasites freshly isolated in the wild (wild-type) was surprisingly constant within the endemic area and showed an intermediate level. In contrast, serially passaged parasites either became avirulent or virulence increased markedly (hypervirulence). Production of transmission stages was maximal for the wild-type whereas numbers were significantly reduced for hypervirulent and avirulent (shown in a previous study) parasites. Analyses of B and T cell immunity revealed that immune responses of WKY rats to the transmission stage were significantly higher for hypervirulent than for wild-type parasites. These results suggest that it is the immune system of the host that is not only responsible for reduction of transmission stages in individual rats, but also could act as a selective force that maintains intermediate virulence at the population level because reduction of muscle stages challenges transmission of S. singaporensis to the definitive host. Collectively, the presented data support evolutionary theory, which predicts intermediate rates of parasite growth in nature and an ‘arms race’ between host immunity and parasite proliferation.     

Reduction of transmission stages concomitant with increased host immune responses to hypervirulent Sarcocystis singaporensis, and natural selection for intermediate virulence

Parasite virulence (pathogenicity depending on inoculum size) and host immune reactions were examined for the apicomplexan protozoan Sarcocystis singaporensis. This parasite is endemic in southeastern Asia and multiplies as a proliferation (merozoite) and transmission stage (bradyzoite) in rats. Virulence in wild brown rats of parasites freshly isolated in the wild (wild-type) was surprisingly constant within the endemic area and showed an intermediate level. In contrast, serially passaged parasites either became avirulent or virulence increased markedly (hypervirulence). Production of transmission stages was maximal for the wild-type whereas numbers were significantly reduced for hypervirulent and avirulent (shown in a previous study) parasites. Analyses of B and T cell immunity revealed that immune responses of WKY rats to the transmission stage were significantly higher for hypervirulent than for wild-type parasites. These results suggest that it is the immune system of the host that is not only responsible for reduction of transmission stages in individual rats, but also could act as a selective force that maintains intermediate virulence at the population level because reduction of muscle stages challenges transmission of S. singaporensis to the definitive host. Collectively, the presented data support evolutionary theory, which predicts intermediate rates of parasite growth in nature and an ‘arms race’ between host immunity and parasite proliferation.     

Co-occurrences of parasite clones and altered host phenotype in a snail-trematode system

The frequent co-occurrence of two or more genotypes of the same parasite species in the same individual hosts has often been predicted to select for higher levels of virulence. Thus, if parasites can adjust their level of host exploitation in response to competition for resources, mixed-clone infections should have more profound impacts on the host. Trematode parasites are known to induce a wide range of modifications in the morphology (size, shell shape or ornamentation) of their snail intermediate host. Still, whether mixed-clone trematode infections have additive effects on the phenotypic alterations of the host remains to be tested. Here, we used the snail Potamopyrgus antipodarum-infected by the trematode Coitocaecum parvum to test for both the general effect of the parasite on host phenotype and possible increased host exploitation in multi-clone infections. Significant differences in size, shell shape and spinosity were found between infected and uninfected snails, and we determined that one quarter of naturally infected snails supported mixed-clone infections of C. parvum. From the parasite perspective, this meant that almost half of the clones identified in this study shared their snail host with at least one other clone. Intra-host competition may be intense, with each clone in a mixed-clone infection experiencing major reductions in volume and number of sporocysts (and consequently multiplication rate and cercarial production) compared with single-clone infections. However, there was no significant difference in the intensity of host phenotype modifications between single and multiple-clone infections. These results demonstrate that competition between parasite genotypes may be strong, and suggest that the frequency of mixed-clone infections in this system may have selected for an increased level of host exploitation in the parasite population, such that a single-clone is associated with a high degree of host phenotypic alteration.     

A Theileria annulata DNA binding protein localized to the host cell nucleus alters the phenotype of a bovine macrophage cell line

The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (SuAT1) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected 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.     

Antagonistic competition moderates virulence in Bacillus thuringiensis

Classical models of the evolution of virulence predict that multiple infections should select for elevated virulence, if increased competitiveness arises from faster growth. However, diverse modes of parasite competition (resource-based, antagonism, immunity manipulation) can lead to adaptations with different implications for virulence. Using an experimental evolution approach we investigated the hypothesis that selection in mixed-strain infections will lead to increased antagonism that trades off against investment in virulence. Selection in mixed infections led to improved suppression of competitors in the bacterial insect pathogen Bacillus thuringiensis. Increased antagonism was associated with decreased virulence in three out of four selected lines. Moreover, mixed infections were less virulent than single-strain infections, and between-strain competition tended to decrease pathogen growth in vivo and in vitro. Spiteful interactions among these bacteria may be favoured because of the high metabolic costs of virulence factors and the high risk of mixed infections.     

Life history and virulence are linked in the ectoparasitic salmon louse Lepeophtheirus salmonis

Models of virulence evolution for horizontally transmitted parasites often assume that transmission rate (the probability that an infected host infects a susceptible host) and virulence (the increase in host mortality due to infection) are positively correlated, because higher rates of production of propagules may cause more damages to the host. However, empirical support for this assumption is scant and limited to microparasites. To fill this gap, we explored the relationships between parasite life history and virulence in the salmon louse, Lepeophtheirus salmonis, a horizontally transmitted copepod ectoparasite on Atlantic salmon Salmo salar. In the laboratory, we infected juvenile salmon hosts with equal doses of infective L. salmonis larvae and monitored parasite age at first reproduction, parasite fecundity, area of damage caused on the skin of the host, and host weight and length gain. We found that earlier onset of parasite reproduction was associated with higher parasite fecundity. Moreover, higher parasite fecundity (a proxy for transmission rate, as infection probability increases with higher numbers of parasite larvae released to the water) was associated with lower host weight gain (correlated with lower survival in juvenile salmon), supporting the presence of a virulence–transmission trade‐off. Our results are relevant in the context of increasing intensive farming, where frequent anti‐parasite drug use and increased host density may have selected for faster production of parasite transmission stages, via earlier reproduction and increased early fecundity. Our study highlights that salmon lice, therefore, are a good model for studying how human activity may affect the evolution of parasite virulence.     

A comparison of two different techniques for the detection of blood parasite, Theileria annulata, in cattle from two districts in Khyber Pukhtoon Khwa Province (Pakistan)

The present study was carried out to determine the prevalence of Theileria annulata in large ruminants from two districts, Peshawar and Kohat, in Khyber Pukhtoon Khwa (Pakistan). Blood samples were collected from 95 cattle. Data on the characteristics of animals and herds were collected through questionnaires. No significant risk factors were found associated with the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa stained slides, were compared and it was found that PCR amplification is a more sensitive tool (33.7% parasite detection), as compared to smear scanning (5.2% parasite detection) for the detection of Theileria annulata. 32 out of 95 animals, from both districts, produced the 721-bp fragment specific for Theileria annulata.     

THE EFFECT OF ELEVATED MUTATION RATES ON THE EVOLUTION OF COOPERATION AND VIRULENCE OF PSEUDOMONAS AERUGINOSA

Within-host competition between parasite genotypes can play an important role in the evolution of parasite virulence. For example, competition can increase virulence by imposing selection for parasites that replicate at a faster absolute rate within the host, but may also decrease virulence by selecting for faster relative growth rates through social exploitation of conspecifics. For many parasites, both outcomes are possible. We investigated how competition affected the evolution of virulence of the opportunistic pathogen Pseudomonas aeruginosa in caterpillar hosts, over the course of an approximately 60 generation selection experiment. We initiated infections with clonal populations of either wild-type bacteria or an isogenic mutant with an approximately 100-fold higher mutation rate, resulting in low and high between-genotype competition, respectively. We observed the evolution of increased virulence, growth rate, and public goods cheating (exploitation of extracellular iron scavenging siderophores produced by ancestral populations) in mutator but not wild-type, populations. We conclude increases in absolute within-host growth rates appear to be more important than social cheating in driving virulence evolution in this experimental context.