Infections frequently consist of more than one strain of a given pathogen. Experiments have shown that co-infecting strains often compete, so that the infection intensity of each strain in mixed infections is lower than in single strain infections. Such within-host competition can have important epidemiological and evolutionary consequences. However, the extent of competition has rarely been investigated in wild, naturally infected hosts, where there is noise in the form of varying inoculation doses, asynchronous infections and host heterogeneity, which can potentially alleviate or eliminate competition. Here, we investigated the extent of competition between Borrelia afzelii strains (as determined by ospC genotype) in three host species sampled in the wild. For this purpose, we developed a protocol for 454 amplicon sequencing of ospC, which allows both detection and quantification of each individual strain in an infection. Each host individual was infected with one to six ospC strains. The infection intensity of each strain was lower in mixed infections than in single ones, showing that there was competition. Rank-abundance plots revealed that there was typically one dominant strain, but that the evenness of the relative infection intensity of the different strains in an infection increased with the multiplicity of infection. We conclude that within-host competition can play an important role under natural conditions despite many potential sources of noise, and that quantification by next-generation amplicon sequencing offers new possibilities to dissect within-host interactions in naturally infected hosts.
Borrelia afzelii; ospC; mixed infections; virulence; within-host competition
The dynamics and consequences of host–parasite coevolution depend on the nature of host genotype-by-parasite genotype interactions (G × G) for host and parasite fitness. G × G with crossing reaction norms can yield cyclic dynamics of allele frequencies (“Red Queen” dynamics) while G × G where the variance among host genotypes differs between parasite genotypes results in selective sweeps (“arms race” dynamics). Here, we investigate the relative potential for arms race and Red Queen coevolution in a protist host–parasite system, the dinoflagellate Alexandrium minutum and its parasite Parvilucifera sinerae. We challenged nine different clones of A. minutum with 10 clones of P. sinerae in a fully factorial design and measured infection success and host and parasite fitness. Each host genotype was successfully infected by four to ten of the parasite genotypes. There were strong G × Gs for infection success, as well as both host and parasite fitness. About three quarters of the G × G variance components for host and parasite fitness were due to crossing reaction norms. There were no general costs of resistance or infectivity. We conclude that there is high potential for Red Queen dynamics in this host–parasite system.
Alexandrium minutum; dinoflagellate; fluctuating selection; frequency-dependent selection; Parvilucifera sinerae
This Primer examines the phenomenon of infection tolerance and discusses three recent PLOS Biology research articles about the causes and consequences of variation in tolerance in natural populations.
How do we defend ourselves against pathogenic microbes and other parasites infecting us? Research on defence against parasites has traditionally focused on resistance—the ability to prevent infection or limit parasite replication. The genetics, physiology, and evolutionary ecology of such traits are now relatively well understood. During the last few years it has been realized that another, conceptually different type of defence also plays an important role in animal host–parasite interactions. This type of defence is called tolerance, and can be defined as the ability to limit the health effects of parasites without preventing infection or controlling parasite replication. Our understanding of the causes and consequences of variation in tolerance is, however, still rudimentary. Three recent studies shed light on these questions. In a study of HIV in humans, Regoes et al. show that an MHC class I gene affects not only resistance (as previously known) but also tolerance. In a study of voles, Jackson et al. identify a transcription factor mediating age differences in tolerance to macroparasites. Finally, Hayward et al. demonstrate that tolerance to intestinal parasites in sheep is under positive directional selection, but that most of the variation is environmentally induced rather than heritable. These studies increase our knowledge of the genetic and physiological sources of variation in tolerance, and how this variation affects Darwinian fitness. In addition, they illustrate different approaches to untangle tolerance from other factors determining the health effects of infectious disease.
The tick-borne bacterium “Candidatus Neoehrlichia mikurensis” has recently been recognized as a human pathogen. Together with Borrelia afzelii, it is one of the most common pathogens found in the tick Ixodes ricinus. Here, we compared the epidemiologies of “Ca. Neoehrlichia mikurensis” and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole (Myodes glareolus), using real-time PCR for detection and quantification. The prevalences of “Ca. Neoehrlichia mikurensis” and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “Ca. Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “Ca. Neoehrlichia mikurensis” was significantly higher than that of B. afzelii. We found a high frequency of double infections; 46% of all samples infected with “Ca. Neoehrlichia mikurensis” also had a coinfection with B. afzelii. The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.
The discovery of the key role of Toll-like receptors (TLRs) in initiating innate immune responses and modulating adaptive immunity has revolutionized our understanding of vertebrate defence against pathogens. Yet, despite their central role in pathogen recognition and defence initiation, there is little information on how variation in TLRs influences disease susceptibility in natural populations. Here, we assessed the extent of naturally occurring polymorphisms at TLR2 in wild bank voles (Myodes glareolus) and tested for associations between TLR2 variants and infection with Borrelia afzelii, a common tick-transmitted pathogen in rodents and one of the causative agents of human Lyme disease. Bank voles in our population had 15 different TLR2 haplotypes (10 different haplotypes at the amino acid level), which grouped in three well-separated clusters. In a large-scale capture–mark–recapture study, we show that voles carrying TLR2 haplotypes of one particular cluster (TLR2c2) were almost three times less likely to be Borrelia infected than animals carrying other haplotypes. Moreover, neutrality tests suggested that TLR2 has been under positive selection. This is, to our knowledge, the first demonstration of an association between TLR polymorphism and parasitism in wildlife, and a striking example that genetic variation at innate immune receptors can have a large impact on host resistance.
wildlife disease; host–parasite interactions; Borrelia; innate immune defence; Toll-like receptors; disease resistance
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.
Malaria infections often consist of more than one strain of the same parasitic species. Understanding the within-host competition between these various strains is essential to understanding the evolution and epidemiology of drug resistance in malarial infections. The infection process and the competition between strains involve complicated biological processes that are explained by various hypotheses. Mathematical models tested against experimental data provide quantitative measures to compare these hypotheses and enable us to discern the actual biological processes that contribute to the observed dynamics. We use a group of models against experimental data on rodent malaria to test various hypotheses. Such quantitative measures, in understanding rodent malaria, can be considered as a step towards understanding within-host parasite dynamics. Our work presented here demonstrates how confronting mathematical models with data allows the discovery of subtle and novel interactions between hosts and parasites that would be impractical to do in an experiment and allows the rejection of hypotheses that are incorrect. It is our contention that understanding the forces controlling within-host parasite dynamics in well-defined experimental model is a necessary step towards understanding these features in natural infections.
Host resistance against parasites depends on three aspects: the ability to prevent, control and clear infections. In vertebrates the immune system consists of innate and adaptive immunity. Innate immunity is particularly important for preventing infection and eradicating established infections at an early stage while adaptive immunity is slow, but powerful, and essential for controlling infection intensities and eventually clearing infections. Major Histocompatibility Complex (MHC) molecules are central in adaptive immunity, and studies on parasite resistance and MHC in wild animals have found effects on both infection intensity (parasite load) and infection status (infected or not). It seems MHC can affect both the ability to control infection intensities and the ability to clear infections. However, these two aspects have rarely been considered simultaneously, and their relative importance in natural populations is therefore unclear. Here we investigate if MHC class I genotype affects infection intensity and infection status with a frequent avian malaria infection Haemoproteus majoris in a natural population of blue tits Cyanistes caeruleus. We found a significant negative association between a single MHC allele and infection intensity but no association with infection status. Blue tits that carry a specific MHC allele seem able to suppress H. majoris infection intensity, while we have no evidence that this allele also has an effect on clearance of the H. majoris infection, a result that is in contrast with some previous studies of MHC and avian malaria. A likely explanation could be that the clearance rate of avian malaria parasites differs between avian malaria lineages and/or between avian hosts.
We examined small mammals as hosts for Anaplasmataceae in southern Sweden. Of 771 rodents, 68 (8.8%) were infected by Candidatus Neoehrlichia mikurensis, but no other Anaplasmataceae were found. Candidatus N. mikurensis has recently been found in human patients in Germany, Switzerland, and Sweden, which suggests that this could be an emerging pathogen in Europe.
Anaplasmataceae; Candidatus Neoehrlichia mikurensis; Anaplasma phagocytophilum; Bartonella; bacteria; tick-borne disease; zoonotic disease; wild rodents; Rickettsia; Sweden; dispatch
Malarial infection is associated with complex immune and erythropoietic responses in the host. A quantitative understanding of these processes is essential to help inform malaria therapy and for the design of effective vaccines. In this study, we use a statistical model-fitting approach to investigate the immune and erythropoietic responses in Plasmodium chabaudi infections of mice. Three mouse phenotypes (wildtype, T-cell-deficient nude mice, and nude mice reconstituted with T-cells taken from wildtype mice) were infected with one of two parasite clones (AS or AJ). Under a Bayesian framework, we use an adaptive population-based Markov chain Monte Carlo method and fit a set of dynamical models to observed data on parasite and red blood cell (RBC) densities. Model fits are compared using Bayes' factors and parameter estimates obtained. We consider three independent immune mechanisms: clearance of parasitised RBCs (pRBC), clearance of unparasitised RBCs (uRBC), and clearance of parasites that burst from RBCs (merozoites). Our results suggest that the immune response of wildtype mice is associated with less destruction of uRBCs, compared to the immune response of nude mice. There is a greater degree of synchronisation between pRBC and uRBC clearance than between either mechanism and merozoite clearance. In all three mouse phenotypes, control of the peak of parasite density is associated with pRBC clearance. In wildtype mice and AS-infected nude mice, control of the peak is also associated with uRBC clearance. Our results suggest that uRBC clearance, rather than RBC infection, is the major determinant of RBC dynamics from approximately day 12 post-innoculation. During the first 2–3 weeks of blood-stage infection, immune-mediated clearance of pRBCs and uRBCs appears to have a much stronger effect than immune-mediated merozoite clearance. Upregulation of erythropoiesis is dependent on mouse phenotype and is greater in wildtype and reconstitited mice. Our study highlights the informative power of statistically rigorous model-fitting techniques in elucidating biological systems.
Malaria is a disease caused by a protozoan parasite of the genus Plasmodium. Every year there are around 250 million human cases of malaria, resulting in around a million deaths. Most of the severe cases and deaths are due to Plasmodium falciparum, which is endemic in much of sub-Saharan Africa and other tropical areas. The pathology of malaria is related to the asexual stage of the parasite. Understanding the infection dynamics during this stage is therefore essential to inform malaria treatment and vaccine design. Experimental infections of rodents represent an important first step towards understanding the more complicated human infections. We developed a series of models representing different hypotheses about the main processes regulating the infection dynamics during the asexual stage. Models were fit to data on Plasmodium chabaudi infections of mice, using a Bayesian statistical framework. The accuracy of different models in explaining the RBC and parasite densities was quantified. We identify the role of different types of immune-mediated mechanism, and show that RBC production (erythropoiesis) increases during infection. Differences between mouse phenotypes are explained. Our study highlights the informative power of model-fitting techniques in explaining biological systems.
Plant biologists have long recognized that host defence against parasites and pathogens can be divided into two conceptually different components: the ability to limit parasite burden (resistance) and the ability to limit the harm caused by a given burden (tolerance). Together these two components determine how well a host is protected against the effects of parasitism. This distinction is useful because it recognizes that hosts that are best at controlling parasite burdens are not necessarily the healthiest. Moreover, resistance and tolerance can be expected to have different effects on the epidemiology of infectious diseases and host–parasite coevolution. However, studies of defence in animals have to date focused on resistance, whereas the possibility of tolerance and its implications have been largely overlooked. The aim of our review is to (i) describe the statistical framework for analysis of tolerance developed in plant science and how this can be applied to animals, (ii) review evidence of genetic and environmental variation for tolerance in animals, and studies indicating which mechanisms could contribute to this variation, and (iii) outline avenues for future research on this topic.
immunopathology; infectious disease; resistance; tolerance; virulence
The ability of hosts to withstand a given number of pathogens is a critical component of health. Now playing catch-up with plant biologists, animal biologists are starting to formally separate this form of defense from classical resistance.
Although parasitism has been acknowledged as an important selective force in the evolution of host life histories, studies of fitness effects of parasites in wild populations have yielded mixed results. One reason for this may be that most studies only test for a linear relationship between infection intensity and host fitness. If resistance to parasites is costly, however, fitness may be reduced both for hosts with low infection intensities (cost of resistance) and high infection intensities (cost of parasitism), such that individuals with intermediate infection intensities have highest fitness. Under this scenario one would expect a non-linear relationship between infection intensity and fitness.
Using data from blue tits (Cyanistes caeruleus) in southern Sweden, we investigated the relationship between the intensity of infection of its blood parasite (Haemoproteus majoris) and host survival to the following winter. Presence and intensity of parasite infections were determined by microscopy and confirmed using PCR of a 480bp section of the cytochrome-b-gene. While a linear model suggested no relationship between parasite intensity and survival (F = 0.01, p = 0.94), a non-linear model showed a significant negative quadratic effect (quadratic parasite intensity: F = 4.65, p = 0.032; linear parasite intensity F = 4.47, p = 0.035). Visualization using the cubic spline technique showed maximum survival at intermediate parasite intensities.
Our results indicate that failing to recognize the potential for a non-linear relationship between parasite infection intensity and host fitness may lead to the potentially erroneous conclusion that the parasite is harmless to its host. Here we show that high parasite intensities indeed reduced survival, but this effect was masked by reduced survival for birds heavily suppressing their parasite intensities. Reduced survival among hosts with low parasite intensities suggests costs of controlling parasite infections; however, the nature of such costs remains to be elucidated.
Ecological interactions between microparasite populations in the same host are an important source of selection on pathogen traits such as virulence and drug resistance. In the rodent malaria model Plasmodium chabaudi in laboratory mice, parasites that are more virulent can competitively suppress less virulent parasites in mixed infections. There is evidence that some of this suppression is due to immune-mediated apparent competition, where an immune response elicited by one parasite population suppress the population density of another. This raises the question whether enhanced immunity following vaccination would intensify competitive interactions, thus strengthening selection for virulence in Plasmodium populations. Using the P. chabaudi model, we studied mixed infections of virulent and avirulent genotypes in CD4+T cell-depleted mice. Enhanced efficacy of CD4+T cell-dependent responses is the aim of several candidate malaria vaccines. We hypothesized that if immune-mediated interactions were involved in competition, removal of the CD4+T cells would alleviate competitive suppression of the avirulent parasite. Instead, we found no alleviation of competition in the acute phase, and significant enhancement of competitive suppression after parasite densities had peaked. Thus, the host immune response may actually be alleviating other forms of competition, such as that over red blood cells. Our results suggest that the CD4+-dependent immune response, and mechanisms that act to enhance it such as vaccination, may not have the undesirable affect of exacerbating within-host competition and hence the strength of this source of selection for virulence.
malaria; CD4+T cells; competition
One of the central tenets in life-history theory is that there is a trade-off between current and future reproduction (i.e. a cost of reproduction). The mechanism for this cost of reproduction is, however, largely unknown. One hypothesis is that the high workload during reproduction compromises resistance to parasites and that the resulting increase in parasitaemia has negative effects on the prospects of future survival. Although empirical evidence for a negative relationship between reproductive effort and parasite resistance exists, the causal relationships between reproductive effort, parasite resistance and future reproduction are still unclear. We use a path analytical approach to investigate whether a change in parasite resistance (as measured by intensities of infections by the blood parasite Haemoproteus) after manipulation of reproductive effort, translates into altered survival in female blue tits. Our results show a negative relationship between reproductive effort and parasite resistance, although evident only in first-year breeders. Moreover, we found survival costs of reproduction in first-year breeders. These costs were, however, not mediated by the blood parasite studied.
Vertebrates have evolved an adaptive immune system in addition to the ancestral innate immune system. It is often assumed that a trade-off between costs and benefits of defence governs the evolution of immunological defence, but the costs and benefits specific to the adaptive immune system are poorly known. We used genetically engineered mice lacking lymphocytes (i.e. mice without adaptive, but with innate, immunity) as a model of the ancestral state in the evolution of the vertebrate immune system. To investigate if the magnitude of adaptive defence is constrained by the energetic costs of producing lymphocytes etc., we compared the basal metabolic rate of normal and lymphocyte-deficient mice. We found that lymphocyte-deficient mice had a higher basal metabolic rate than normal mice with both innate and adaptive immune defence. This suggests that the evolution of the adaptive immune system has not been constrained by energetic costs. Rather, it should have been favoured by the energy savings associated with a combination of innate and adaptive immune defence.
Differences in growth conditions during early life have been suggested to cause long-lasting effects on morphology and quality of adult birds. We experimentally investigated the effect of early growth conditions on the expression of sexual ornaments later in life in male ring-necked pheasants (Phasianus colchicus). We also investigated the effects on immune function, as it could be a functional link between early nutrition and ornament expression. We manipulated the dietary protein intake during the first eight weeks post hatching. Males receiving fodder with 27% protein during the first three weeks of life grew larger and more colourful wattles when sexually mature than males receiving a low-protein diet (20.5% protein). Spur length was unaffected by diet treatment. Manipulation of food protein levels during weeks 4-8 after hatching had no effect on the development of ornaments. The different protein treatments had no long-term effect on either humoral or cell-mediated immune responses. There was, however, a positive relationship between spur length and cell-mediated immune responsiveness. Our study shows that expression of a sexual ornament in adult pheasants reflects nutritional conditions early in life. Because the expression of secondary sexual ornaments is affected by conditions during early growth, by selecting more ornamented males, females would choose mates that are superior at handling early nutritional stress. If the susceptibility to early nutritional stress also has a hereditary basis, females may benefit by obtaining 'good genes'.