Predicting the effects of parasite co-infection across species boundaries.

It is normal for hosts to be co-infected by parasites. Interactions among co-infecting species can have profound consequences, including changing parasite transmission dynamics, altering disease severity and confounding attempts at parasite control. Despite the importance of co-infection, there is currently no way to predict how different parasite species may interact with one another, nor the consequences of those interactions. Here, we demonstrate a method that enables such prediction by identifying two nematode parasite groups based on taxonomy and characteristics of the parasitological niche. From an understanding of the interactions between the two defined groups in one host system (wild rabbits), we predict how two different nematode species, from the same defined groups, will interact in co-infections in a different host system (sheep), and then we test this experimentally. We show that, as predicted, in co-infections, the blood-feeding nematode Haemonchus contortus suppresses aspects of the sheep immune response, thereby facilitating the establishment and/or survival of the nematode Trichostrongylus colubriformis; and that the T. colubriformis-induced immune response negatively affects H. contortus This work is, to our knowledge, the first to use empirical data from one host system to successfully predict the specific outcome of a different co-infection in a second host species. The study therefore takes the first step in defining a practical framework for predicting interspecific parasite interactions in other animal systems.

The comparative immunology of wild and laboratory mice, Mus musculus domesticus.

The laboratory mouse is the workhorse of immunology, used as a model of mammalian immune function, but how well immune responses of laboratory mice reflect those of free-living animals is unknown. Here we comprehensively characterize serological, cellular and functional immune parameters of wild mice and compare them with laboratory mice, finding that wild mouse cellular immune systems are, comparatively, in a highly activated (primed) state. Associations between immune parameters and infection suggest that high level pathogen exposure drives this activation. Moreover, wild mice have a population of highly activated myeloid cells not present in laboratory mice. By contrast, in vitro cytokine responses to pathogen-associated ligands are generally lower in cells from wild mice, probably reflecting the importance of maintaining immune homeostasis in the face of intense antigenic challenge in the wild. These data provide a comprehensive basis for validating (or not) laboratory mice as a useful and relevant immunological model system.

Extinction of an introduced warm-climate alien species, Xenopus laevis, by extreme weather events.

Invasive, non-native species represent a major threat to biodiversity worldwide. The African amphibian Xenopus laevis is widely regarded as an invasive species and a threat to local faunas. Populations originating at the Western Cape, South Africa, have been introduced on four continents, mostly in areas with a similar Mediterranean climate. Some introduced populations are also established in cooler environments where persistence for many decades suggests a capacity for long-term adaptation. In these cases, recent climate warming might enhance invasion ability, favouring range expansion, population growth and negative effects on native faunas. In the cool temperate UK, populations have been established for about 50 years in Wales and for an unknown period, probably >20 years, in England (Lincolnshire). Our field studies over 30 and 10 years, respectively, show that in favourable conditions there may be good recruitment, fast individual growth rates and large body size; maximum longevity exceeds 23 years. Nevertheless, areas of distribution remained limited, with numbers <500 in each population. In 2010, only a single individual was captured at each locality and further searching failed to record any others in repeated sampling up to 2014. We conclude that both populations are now extinct. The winters of 2009-2010 and 2010-2011 experienced extreme cold and drought (December 2010 was the coldest in 120 years and the third driest in 100 years). The extinction of X. laevis in these areas indicates that even relatively long-established alien species remain vulnerable to rare extreme weather conditions.

Copy number variation and genetic diversity of MHC Class IIb alleles in an alien population of Xenopus laevis.

Xenopus laevis (the African clawed frog), which originated through hybridisation and whole genome duplication, has been used as a model for genetics and development for many years, but surprisingly little is known about immune gene variation in natural populations. The purpose of this study was to use an isolated population of X. laevis that was introduced to Wales, UK in the past 50 years to investigate how variation at the MHC compares to that at other loci, following a severe population bottleneck. Among 18 individuals, we found nine alleles based on exon 2 sequences of the Class IIb region (which includes the peptide binding region). Individuals carried from one to three of the loci identified from previous laboratory studies. Genetic variation was an order of magnitude higher at the MHC compared with three single-copy nuclear genes, but all loci showed high levels of heterozygosity and nucleotide diversity and there was not an excess of homozygosity or decrease in diversity over time that would suggest extensive inbreeding in the introduced population. Tajima's D was positive for all loci, which is consistent with a bottleneck. Moreover, comparison with published sequences identified the source of the introduced population as the Western Cape region of South Africa, where most commercial suppliers have obtained their stocks. These factors suggest that despite founding by potentially already inbred individuals, the alien population in Wales has maintained substantial genetic variation at both adaptively important and neutral genes.

The biology of Strongyloides spp.

Strongyloides is a genus of parasitic nematodes that, unusually, has a free-living adult generation. Here we introduce the biology of this genus, especially the fascinating but complex life-cycle, together with an overview of the taxonomy, morphology, genetics, and genomics of this genus.

Patterns and processes in parasite co-infection.

Co-infection of individual hosts by multiple parasite species is a pattern that is very commonly observed in natural populations. Understanding the processes that generate these patterns poses a challenge. For example, it is difficult to discern the relative roles of exposure and susceptibility in generating the mixture and density of parasites within hosts. Yet discern them we must, if we are to design and deliver successful medical interventions for co-infected populations. Here, we synthesise an emergent understanding of how processes operate and interact to generate patterns of co-infection. We consider within-host communities (or infracommunities) generally, that is including not only classical parasites but also the microbiota that are so abundant on mucosal surfaces and which are increasingly understood to be so influential on host biology. We focus on communities that include a helminth, but we expect similar inferences to pertain to other taxa. We suggest that, thanks to recent research at both the within-host (e.g. immunological) and between-host (e.g. epidemiological) scales, researchers are poised to reveal the processes that generate the observed distribution of parasite communities among hosts. Progress will be facilitated by using new technologies as well as statistical and experimental tools to test competing hypotheses about processes that might generate patterns in co-infection data. By understanding the multiple interactions that underlie patterns of co-infection, we will be able to understand and intelligently predict how a suite of co-infections (and thus the host that bears them) will together respond to medical interventions as well as other environmental changes. The challenge for us all is to become scholars of co-infections.

Comparison of the distal gut microbiota from people and animals in Africa.

The gut microbiota plays a key role in the maintenance of healthy gut function as well as many other aspects of health. High-throughput sequence analyses have revealed the composition of the gut microbiota, showing that there is a core signature to the human gut microbiota, as well as variation in its composition between people. The gut microbiota of animals is also being investigated. We are interested in the relationship between bacterial taxa of the human gut microbiota and those in the gut microbiota of domestic and semi-wild animals. While it is clear that some human gut bacterial pathogens come from animals (showing that human--animal transmission occurs), the extent to which the usually non-pathogenic commensal taxa are shared between humans and animals has not been explored. To investigate this we compared the distal gut microbiota of humans, cattle and semi-captive chimpanzees in communities that are geographically sympatric in Uganda. The gut microbiotas of these three host species could be distinguished by the different proportions of bacterial taxa present. We defined multiple operational taxonomic units (OTUs) by sequence similarity and found evidence that some OTUs were common between human, cattle and chimpanzees, with the largest number of shared OTUs occurring between chimpanzees and humans, as might be expected with their close physiological similarity. These results show the potential for the sharing of usually commensal bacterial taxa between humans and other animals. This suggests that further investigation of this phenomenon is needed to fully understand how it drives the composition of human and animal gut microbiotas.

The relative contribution of co-infection to focal infection risk in children.

Co-infection is ubiquitous in people in the developing world but little is known regarding the potential for one parasite to act as a risk factor for another. Using generalized linear mixed modelling approaches applied to data from school-aged children from Zanzibar, Tanzania, we determined the strength of association between four focal infections (i.e. Ascaris lumbricoides, Trichuris trichiura, hookworm and self-reported fever, the latter used as a proxy for viral, bacterial or protozoal infections) and the prevalence or intensity of each of the helminth infections. We compared these potential co-infections with additional risk factors, specifically, host sex and age, socioeconomic status and physical environment, and determined what the relative contribution of each risk factor was. We found that the risk of infection with all four focal infections was strongly associated with at least one other infection, and that this was frequently dependent on the intensity of that other infection. In comparison, no other incorporated risk factor was associated with all focal infections. Successful control of infectious diseases requires identification of infection risk factors. This study demonstrates that co-infection is likely to be one of these principal risk factors and should therefore be given greater consideration when designing disease-control strategies. Future work should also incorporate other potential risk factors, including host genetics which were not available in this study and, ideally, assess the risks via experimental manipulation.

Wild mice provide insights into natural killer cell maturation and memory.

The immune system has evolved, and continues to evolve, in response to the selection pressure that infections exert on animals in their natural environments, yet much of our understanding about how the immune system functions comes from studies of model species maintained in the almost complete absence of such environmental selection. The scientific discipline of immunology has among its aims the improvement of human and animal health by the application of immunological knowledge. As research on humans and domesticated animals is highly constrained-ethically, logistically and financially-experimental animal models have become an invaluable tool for dissecting the functioning of the immune system. The house mouse (Mus musculus) is by far the most widely used animal model in immunological research but laboratory-reared mice provide a very narrow view of the immune system-that of a well-fed and comfortably housed animal with minimal exposure to microbial pathogens. Indeed, so much of our immunological knowledge comes from studies of a very few highly inbred mouse strains that-to all intents and purposes-our immunological knowledge is based on enormously detailed studies of very small numbers of individual mice. The limitations of studies in inbred strains of laboratory mice are well-recognized (Pedersen & Babayan 2011), but serious attempts to address these limitations have been few and far between. However, the emerging field of 'ecological immunology' where free-living populations are studied in their natural habitat is beginning to redress this imbalance (Viney et al. 2005; Martin et al. 2006; Owen et al. 2010; Abolins et al. 2011). As demonstrated in the work by Boysen et al. (2011) in this issue of Molecular Ecology, studies in wild animal populations-especially free-living M. musculus-represent a valuable bridge between studies in humans and livestock and studies of captive animals.

Wild immunology: converging on the real world.

Recently, the Centre for Immunity, Infection and Evolution sponsored a one-day symposium entitled "Wild Immunology." The CIIE is a new Wellcome Trust-funded initiative with the remit to connect evolutionary biology and ecology with research in immunology and infectious diseases in order to gain an interdisciplinary perspective on challenges to global health. The central question of the symposium was, "Why should we try to understand infection and immunity in wild systems?" Specifically, how does the immune response operate in the wild and how do multiple coinfections and commensalism affect immune responses and host health in these wild systems? The symposium brought together a broad program of speakers, ranging from laboratory immunologists to infectious disease ecologists, working on wild birds, unmanaged animals, wild and laboratory rodents, and on questions ranging from the dynamics of coinfection to how commensal bacteria affect the development of the immune system. The meeting on wild immunology, organized by Amy Pedersen, Simon Babayan, and Rick Maizels, was held at the University of Edinburgh on 30 June 2011.

Transgenesis in the parasitic nematode Strongyloides ratti.

Strongyloides and related genera are advantageous subjects for transgenesis in parasitic nematodes, primarily by gonadal microinjection as has been used with Caenorhabditis elegans. Transgenesis has been achieved in Strongyloides stercoralis and in Parastrongyloides trichosuri, but both of these lack well-adapted, conventional laboratory hosts in which to derive transgenic lines. By contrast, Strongyloides ratti develops in laboratory rats with high efficiency and offers the added advantages of robust genomic and transcriptomic databases and substantial volumes of genetic, developmental and immunological data. Therefore, we evaluated methodology for transgenesis in S. stercoralis as a means of transforming S. ratti. S. stercoralis-based GFP reporter constructs were expressed in a proportion of F1 transgenic S. ratti following gonadal microinjection into parental free-living females. Frequencies of transgene expression in S. ratti, ranged from 3.7% for pAJ09 to 6.8% for pAJ20; respective frequencies for these constructs in S. stercoralis were 5.6% and 33.5%. Anatomical patterns of transgene expression were virtually identical in S. ratti and S. stercoralis. This is the first report of transgenesis in S. ratti, an important model organism for biological investigations of parasitic nematodes. Availability of the rat as a well-adapted laboratory host will facilitate derivation of transgenic lines of this parasite.

Measures of immune function of wild mice, Mus musculus.

The immune function of wild animals has been rather little studied. Wild animals' immune function may differ from that of laboratory bred animals because of their different environments. This idea follows from the concept of resource partitioning in which animals distribute scarce resources to all aspects of life, including to costly immune responses. A logical extension of this idea is that there may be substantial interindividual variation in the immune function of wild animals. To begin to investigate this, we compared the immune function of a laboratory bred mouse strain (C57BL/6, a widely used mouse strain that makes potent immune responses) and wild caught Mus musculus. We found that by most measures of immune function, the wild caught mice had greater immune function. Specifically, wild mice had greater concentrations and more avid antigen-specific IgG responses, as well as higher concentrations of total IgG and IgE, compared with those laboratory bred mice. Moreover, flow cytometric analysis showed a comparatively greater overall level of activation of the cells of the immune system in wild mice. Lastly, we observed that immune function was substantially more variable among wild caught mice than among the laboratory bred mice. The next research challenge is to understand which aspects of an individual animal's life determine its immune function.

Response of the Strongyloides ratti transcriptome to host immunological environment.

The immunological environment experienced by parasitic nematodes varies greatly between hosts and is particularly influenced by whether or not a host has been previously infected. How a parasitic nematode responds to these different environments is poorly understood, but may allow a parasite to ameliorate the adverse effects of host immunity on parasite fitness. Here we use a microarray approach to identify genes in the parasitic nematode Strongyloides ratti that exhibit differential transcription between different rat host immunological environments, and between replicate lines of S. ratti selected for either early or late reproduction. We hypothesise that such genes may be used by this species to cope with and respond to its host environment. Our results showed that, despite large phenotypic differences between S. ratti adults from different immunological environments, the S. ratti transcriptome exhibited a relatively stable pattern of expression. Thus, differential expression amongst treatments was limited to a small proportion of transcripts and generally involved only modest fold changes. These transcripts included a group of collagen genes up-regulated in parasites early in an infection, and in immunised host environments, which may be related to protection against the damage caused to a parasite by host immune responses. We found that later in an infection, a number of genes associated with muscle function and repair were up-regulated in immunised host environments; these may help parasites maintain their position in the host intestine. Differences in transcription between selection lines of S. ratti were only observed in immunised hosts and included genes associated with the response to the host's immunological environment.

Detecting interspecific macroparasite interactions from ecological data: patterns and process.

There is great interest in the occurrence and consequences of interspecific interactions among co-infecting parasites. However, the extent to which interactions occur is unknown, because there are no validated methods for their detection. We developed a model that generated abundance data for two interacting macroparasite (e.g., helminth) species, and challenged the data with various approaches to determine whether they could detect the underlying interactions. Current approaches performed poorly - either suggesting there was no interaction when, in reality, there was a strong interaction occurring, or inferring the presence of an interaction when there was none. We suggest the novel application of a generalized linear mixed modelling (GLMM)-based approach, which we show to be more reliable than current approaches, even when infection rates of both parasites are correlated (e.g., via a shared transmission route). We suggest that the lack of clarity regarding the presence or absence of interactions in natural systems may be largely attributed to the unreliable nature of existing methods for detecting them. However, application of the GLMM approach may provide a more robust method of detection for these potentially important interspecific interactions from ecological data.

A genetic map of the animal-parasitic nematode Strongyloides ratti.

Classical genetic approaches are rarely used with metazoan endo-parasites, largely because the adult stages are usually hidden within hosts, making controlled crosses difficult. The nematode Strongyloides ratti is a parasite of the small intestine of rats, and is a relative of the parasite of humans S. stercoralis. The life-cycle of Strongyloides spp. has a facultative free-living adult generation. Here we describe procedures for genetic mapping, and a genetic map, for S. ratti. This is, as far as we are aware, the first genetic map of an animal parasitic nematode. This significantly improves the usefulness of S. ratti as experimentally tractable system for parasitological investigations and for comparative studies with the model nematode Caenorhabditis elegans.

Natural variation in gene expression in the early development of dauer larvae of Caenorhabditis elegans.

BACKGROUND: The free-living nematode Caenorhabditis elegans makes a developmental decision based on environmental conditions: larvae either arrest as dauer larva, or continue development into reproductive adults. There is natural variation among C. elegans lines in the sensitivity of this decision to environmental conditions; that is, there is variation in the phenotypic plasticity of dauer larva development. We hypothesised that these differences may be transcriptionally controlled in early stage larvae. We investigated this by microarray analysis of different C. elegans lines under different environmental conditions, specifically the presence and absence of dauer larva-inducing pheromone. RESULTS: There were substantial transcriptional differences between four C. elegans lines under the same environmental conditions. The expression of approximately 2,000 genes differed between genetically different lines, with each line showing a largely line-specific transcriptional profile. The expression of genes that are markers of larval moulting suggested that the lines may be developing at different rates. The expression of a total of 89 genes was putatively affected by dauer larva or non-dauer larva-inducing conditions. Among the upstream regions of these genes there was an over-representation of DAF-16-binding motifs. CONCLUSION: Under the same environmental conditions genetically different lines of C. elegans had substantial transcriptional differences. This variation may be due to differences in the developmental rates of the lines. Different environmental conditions had a rather smaller effect on transcription. The preponderance of DAF-16-binding motifs upstream of these genes was consistent with these genes playing a key role in the decision between development into dauer or into non-dauer larvae. There was little overlap between the genes whose expression was affected by environmental conditions and previously identified loci involved in the plasticity of dauer larva development.

How did parasitic worms evolve?

Nematodes are important parasites of humans and other animals. Nematode parasitism is thought to have evolved by free-living, facultatively developing, arrested larvae becoming associated with animals, ultimately becoming parasites. The formation of free-living arrested larvae of the nematode Caenorhabditis elegans is controlled by the environment, and involves dafachronic acid (DA) and transforming growth factor (TGF)-beta signalling. Recent data have shown that DA acid signalling plays a conserved role in controlling larval development in both free-living and parasitic species. In contrast, TGF-beta signalling does not seem to be conserved; this difference perhaps points to how nematode parasitism did evolve.

Transcript profiles of long- and short-lived adults implicate protein synthesis in evolved differences in ageing in the nematode Strongyloides ratti.

The nematode Strongyloides ratti shows remarkable phenotypic plasticity in ageing, with parasitic adults living at least 80-times longer than free-living adults. Given that long- and short-lived adults are genetically identical, this plasticity is likely to be due to differences in gene expression. To try and understand how this inter-morph difference in longevity evolved, we compared gene expression in long- and short-lived adults. DNA microarray analysis of long- and short-lived adults identified 32 genes that were up-regulated in long-lived adults, and 96 genes up-regulated in short-lived adults. Strikingly, 38.5% of the genes expressed more in the short-lived morph are predicted to encode ribosomal proteins, compared with only 9% in the long-lived morph. Among the 32 longevity-associated genes there was very little enrichment of genes linked to cellular maintenance. Overall, we have therefore observed a negative correlation between expression of ribosomal protein genes and longevity in S. ratti. Interestingly, engineered reduction of expression of ribosomal protein genes increases lifespan in the free-living nematode Caenorhabditis elegans. Our study therefore suggests that differences in levels of protein synthesis could contribute to evolved differences in animal longevity.

Immunological responses elicited by different infection regimes with Strongyloides ratti.

Nematode infections are a ubiquitous feature of vertebrate life. In nature, such nematode infections are acquired by continued exposure to infective stages over a prolonged period of time. By contrast, experimental laboratory infections are typically induced by the administration of a single (and often large) dose of infective stages. Previous work has shown that the size of an infection dose can have significant effects on anti-nematode immune responses. Here we investigated the effect of different infection regimes of Strongyloides ratti, comparing single and repeated dose infections, on the host immune response that was elicited. We considered and compared infections of the same size, but administered in different ways. We considered infection size in two ways: the maximum dose of worms administered and the cumulative worm exposure time. We found that both infection regimes resulted in Th2-type immune response, characterised by IL4 and IL13 produced by S. ratti stimulated mesenteric lymph node cells, anti-S. ratti IgG(1) and intestinal rat mast cell protease II (RMCPII) production. We observed some small quantitative immunological differences between different infection regimes, in which the concentration of IL4, IL13, anti-S. ratti IgG(1) and IgG(2a) and RMCPII were affected. However, these differences were quantitatively relatively modest compared with the temporal dynamics of the anti-S. ratti immune response as a whole.

Microarray analysis of gender- and parasite-specific gene transcription in Strongyloides ratti.

The molecular mechanisms by which parasitic nematodes reproduce and have adapted to life within a host are unclear. In the present study, microarray analysis was used to explore differential transcription among the different stages and sexes of Strongyloides ratti, a parasitic nematode of brown rats. Specifically, gender-biased transcription between free-living females and free-living males, and parasitic-biased transcription between parasitic females and free-living females was determined. Of the estimated 3,688 distinct transcripts represented on the microarray, 743 (20%) exhibited male-biased transcription of >1.4-fold (2(0.5)), 689 (19%) female-biased transcription, 418 (11%) parasitic-biased transcription and 305 (8%) free-living-biased transcription. Among those transcripts that exhibited the highest levels of differential transcription, an orthologue of major sperm protein was identified in males, distinct aspartic protease orthologues in either parasitic or in free-living females, and orthologues of hsp-17 chaperone in parasitic females. These 3,688 transcripts were separated into 12 clusters, such that the pattern of transcription between life-stages and biological replicates was similar among transcripts within a cluster and dissimilar between clusters. Using annotation inferred from Caenorhabditis elegans, gene ontology terms over-represented in one or more clusters were identified and showed that female-biased transcription was associated with genes involved in reproductive processes and larval development, male-biased transcription was linked to genes involved in metabolism, and free-living-biased transcription related to genes involved in the regulation of body fluids and response to external stimulus. The association of gene ontology with parasite-biased transcription was less clear. The present findings for S. ratti provide a basis for a detailed exploration of differentially regulated molecules and might assist in the search for novel drug or vaccine targets in parasitic nematodes.