Amplicon sequencing allows differential quantification of closely related parasite species: an example from rodent Coccidia (Eimeria).

BACKGROUND: Quantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended. METHODS: We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population. RESULTS: We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance. CONCLUSIONS: We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.

Guts within guts: the microbiome of the intestinal helminth parasite Ascaris suum is derived but distinct from its host.

BACKGROUND: Intestinal helminths are extremely prevalent among humans and animals. In particular, intestinal roundworms affect more than 1 billion people around the globe and are a major issue in animal husbandry. These pathogens live in intimate contact with the host gut microbiota and harbor bacteria within their own intestines. Knowledge of the bacterial host microbiome at the site of infection is limited, and data on the parasite microbiome is, to the best of our knowledge, non-existent. RESULTS: The intestinal microbiome of the natural parasite and zoonotic macropathogen, Ascaris suum was analyzed in contrast to the diversity and composition of the infected host gut. 16S sequencing of the parasite intestine and host intestinal compartments showed that the parasite gut has a significantly less diverse microbiome than its host, and the host gut exhibits a reduced microbiome diversity at the site of parasite infection in the jejunum. While the host's microbiome composition at the site of infection significantly determines the microbiome composition of its parasite, microbial signatures differentiate the nematodes from their hosts as the Ascaris intestine supports the growth of microbes that are otherwise under-represented in the host gut. CONCLUSION: Our data clearly indicate that a nematode infection reduces the microbiome diversity of the host gut, and that the nematode gut represents a selective bacterial niche harboring bacteria that are derived but distinct from the host gut. Video Abstract.

Shifting focus from resistance to disease tolerance: A review on hybrid house mice.

Parasites have been proposed to modulate the fitness of hybridizing hosts in part based on observations in the European house mouse hybrid zone (HMHZ), a tension zone in which hybrids show reduced fitness. We here review evidence (1) for parasite load differences in hybrid versus parental mice and (2) for health and fitness effects of parasites promoting or preventing introgression and hybridization. The question of relative resistance or susceptibility of hybrids to parasites in the HMHZ has long been controversial. Recent field studies found hybrids to be more resistant than mice from parental subspecies against infections with pinworms and protozoans (Eimeria spp.). We argue that the field studies underlying the contradictory impression of hybrid susceptibility have limitations in sample size, statistical analysis and scope, focusing only on macroparasites. We suggest that weighted evidence from field studies indicate hybrid resistance. Health is a fitness component through which resistance can modulate overall fitness. Resistance, however, should not be extrapolated directly to a fitness effect, as the relationship between resistance and health can be modulated by tolerance. In our own recent work, we found that the relationship between health and resistance (tolerance) differs between infections with the related species E. falciformis and E. ferrisi. Health and tolerance need to be assessed directly and the choice of parasite has made this difficult in previous experimental studies of house mice. We discuss how experimental Eimeria spp. infections in hybrid house mice can address resistance, health and tolerance in conjunction.

Whip- and pinworm infections elicit contrasting effector and distinct regulatory responses in wild house mice.

Infections with high doses of intestinal nematodes result in protective immunity based on robust type 2 responses in most mouse lines under laboratory conditions. Here, we report on cellular responses of wild house mice from northern Germany. We detected robust Th1 responses in wild house mice naturally infected with the whipworm Trichuris muris. In contrast, mice infected with pinworms (Syphacia, Aspiculuris) reported type-2 activity by elevated IgG1 levels and eosinophil counts, but also harbored high frequencies of Foxp3+ regulatory T cells, suggesting that natural whip- and pinworm infections induce distinct immunoregulatory as well as effector profiles.

DNA-based quantification and counting of transmission stages provides different but complementary parasite load estimates: an example from rodent coccidia (Eimeria).

BACKGROUND: Counting parasite transmission stages in faeces is the classical measurement to quantify "parasite load". DNA-based quantifications of parasite intensities from faecal samples are relatively novel and often validated against such counts. When microscopic and molecular quantifications do not correlate, it is unclear whether oocyst counts or DNA-based intensity better reflects biologically meaningful concepts. Here, we investigate this issue using the example of Eimeria ferrisi (Coccidia), an intracellular parasite of house mice (Mus musculus). METHODS: We performed an infection experiment of house mice with E. ferrisi, in which the intensity of infection correlates with increased health impact on the host, measured as temporary weight loss during infection. We recorded the number of parasite transmissive stages (oocysts) per gram of faeces (OPG) and, as a DNA-based measurement, the number of Eimeria genome copies per gram of faeces for 10 days post-infection (dpi). We assessed weight loss relative to the day of experimental infection as a proxy of host health and evaluated whether DNA or oocyst counts are better predictors of host health. RESULTS: Absolute quantification of Eimeria DNA and oocyst counts showed similar but slightly diverging temporal patterns during 10 dpi. We detected Eimeria DNA earlier than the first appearance of oocysts in faeces. Additionally, Eimeria OPGs within each dpi did not explain parasite DNA intensity. Early dpi were characterized by high DNA intensity with low oocyst counts, while late infections showed the opposite pattern. The intensity of Eimeria DNA was consistently a stronger predictor of either maximal weight loss (1 value per animal during the infection course) or weight loss on each day during the experiment when controlling for between-dpi and between-individual variance. CONCLUSIONS: Eimeria ferrisi oocyst counts correlate weakly with parasite intensity assessed through DNA quantification. DNA is likely partially derived from life-cycle stages other than transmissive oocysts. DNA-based intensities predict health outcomes of infection for the host more robustly than counts of transmissive stages. We conclude that DNA-based quantifications should not necessarily require validation against counts of transmissive stages. Instead, DNA-based load estimates should be evaluated as complementary sources of information with potential specific biological relevance for each host-parasite system.

Dual RNA Sequencing Meta-analysis in Plasmodium Infection Identifies Host-Parasite Interactions.

Dual RNA sequencing (RNA-Seq) is the simultaneous transcriptomic analysis of interacting symbionts, for example, in malaria. Potential cross-species interactions identified by correlated gene expression might highlight interlinked signaling, metabolic, or gene regulatory pathways in addition to physically interacting proteins. Often, malaria studies address one of the interacting organisms-host or parasite-rendering the other "contamination." Here we perform a meta-analysis using such studies for cross-species expression analysis. We screened experiments for gene expression from host and Plasmodium. Out of 171 studies in Homo sapiens, Macaca mulatta, and Mus musculus, we identified 63 potential studies containing host and parasite data. While 16 studies (1,950 samples) explicitly performed dual RNA-Seq, 47 (1,398 samples) originally focused on one organism. We found 915 experimental replicates from 20 blood studies to be suitable for coexpression analysis and used orthologs for meta-analysis across different host-parasite systems. Centrality metrics from the derived gene expression networks correlated with gene essentiality in the parasites. We found indications of host immune response to elements of the Plasmodium protein degradation system, an antimalarial drug target. We identified well-studied immune responses in the host with our coexpression networks, as our approach recovers known broad processes interlinked between hosts and parasites in addition to individual host and parasite protein associations. The set of core interactions represents commonalities between human malaria and its model systems for prioritization in laboratory experiments. Our approach might also allow insights into the transferability of model systems for different pathways in malaria studies.IMPORTANCE Malaria still causes about 400,000 deaths a year and is one of the most studied infectious diseases. The disease is studied in mice and monkeys as lab models to derive potential therapeutic intervention in human malaria. Interactions between Plasmodium spp. and its hosts are either conserved across different host-parasite systems or idiosyncratic to those systems. Here we use correlation of gene expression from different RNA-Seq studies to infer common host-parasite interactions across human, mouse, and monkey studies. First, we find a set of very conserved interactors, worth further scrutiny in focused laboratory experiments. Second, this work might help assess to which extent experiments and knowledge on different pathways can be transferred from models to humans for potential therapy.

Toxoplasma and Eimeria co-opt the host cFos expression for intracellular development in mammalian cells.

Successful asexual reproduction of intracellular pathogens depends on their potential to exploit host resources and subvert antimicrobial defense. In this work, we deployed two prevalent apicomplexan parasites of mammalian cells, namely Toxoplasma gondii and Eimeria falciformis, to identify potential host determinants of infection. Expression analyses of the young adult mouse colonic (YAMC) epithelial cells upon infection by either parasite showed regulation of several distinct transcripts, indicating that these two pathogens program their intracellular niches in a tailored manner. Conversely, parasitized mouse embryonic fibroblasts (MEFs) displayed a divergent transcriptome compared to corresponding YAMC epithelial cells, suggesting that individual host cells mount a fairly discrete response when encountering a particular pathogen. Among several host transcripts similarly altered by T. gondii and E. falciformis, we identified cFos, a master transcription factor, that was consistently induced throughout the infection. Indeed, asexual growth of both parasites was strongly impaired in MEF host cells lacking cFos expression. Last but not the least, our differential transcriptomics of the infected MEFs (parental and cFos-/- mutant) and YAMC epithelial cells disclosed a cFos-centered network, underlying signal cascades, as well as a repertoire of nucleotides- and ion-binding proteins, which presumably act in consort to acclimatize the mammalian cell and thereby facilitate the parasite development.

The P-glycoprotein repertoire of the equine parasitic nematode Parascaris univalens.

P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the major site of expression but individual gene expression patterns were not conserved between the two nematodes. In P. univalens, PunPgp-9, PunPgp-11.1 and PunPgp-16.2 consistently exhibited the highest expression level in two independent transcriptome data sets. Using RNA-Seq, no significant upregulation of any Pgp was detected following in vitro incubation of adult P. univalens with ivermectin suggesting that drug-induced upregulation is not the mechanism of Pgp-mediated ML resistance. Expression and functional analyses of PunPgp-2 and PunPgp-9 in Saccharomyces cerevisiae provide evidence for an interaction with ketoconazole and ivermectin, but not thiabendazole. Overall, this study established reliable reference gene models with significantly improved annotation for the P. univalens Pgp repertoire and provides a foundation for a better understanding of Pgp-mediated anthelmintic resistance.

Generalist Eimeria species in rodents: Multilocus analyses indicate inadequate resolution of established markers.

Intracellular parasites of the genus Eimeria are described as tissue/host-specific. Phylogenetic classification of rodent Eimeria suggested that some species have a broader host range than previously assumed. We explore whether Eimeria spp. infecting house mice are misclassified by the most widely used molecular markers due to a lack of resolution, or whether, instead, these parasite species are indeed infecting multiple host species.With the commonly used markers (18S/COI), we recovered monophyletic clades of E. falciformis and E. vermiformis from Mus that included E. apionodes identified in other rodent host species (Apodemus spp., Myodes glareolus, and Microtus arvalis). A lack of internal resolution in these clades could suggest the existence of a species complex with a wide host range infecting murid and cricetid rodents. We question, however, the power of COI and 18S markers to provide adequate resolution for assessing host specificity. In addition to the rarely used marker ORF470 from the apicoplast genome, we present multilocus genotyping as an alternative approach. Phylogenetic analysis of 35 nuclear markers differentiated E. falciformis from house mice from isolates from Apodemus hosts. Isolates of E. vermiformis from Mus are still found in clusters interspersed with non-Mus isolates, even with this high-resolution data.In conclusion, we show that species-level resolution should not be assumed for COI and 18S markers in coccidia. Host-parasite cospeciation at shallow phylogenetic nodes, as well as contemporary coccidian host ranges more generally, is still open questions that need to be addressed using novel genetic markers with higher resolution.

Coupling between tolerance and resistance for two related Eimeria parasite species.

Resistance (host capacity to reduce parasite burden) and tolerance (host capacity to reduce impact on its health for a given parasite burden) manifest two different lines of defense. Tolerance can be independent from resistance, traded off against it, or the two can be positively correlated because of redundancy in underlying (immune) processes. We here tested whether this coupling between tolerance and resistance could differ upon infection with closely related parasite species. We tested this in experimental infections with two parasite species of the genus Eimeria. We measured proxies for resistance (the (inverse of) number of parasite transmission stages (oocysts) per gram of feces at the day of maximal shedding) and tolerance (the slope of maximum relative weight loss compared to day of infection on number of oocysts per gram of feces at the day of maximal shedding for each host strain) in four inbred mouse strains and four groups of F1 hybrids belonging to two mouse subspecies, Mus musculus domesticus and Mus musculus musculus. We found a negative correlation between resistance and tolerance against Eimeria falciformis, while the two are uncoupled against Eimeria ferrisi. We conclude that resistance and tolerance against the first parasite species might be traded off, but evolve more independently in different mouse genotypes against the latter. We argue that evolution of the host immune defenses can be studied largely irrespective of parasite isolates if resistance-tolerance coupling is absent or weak (E. ferrisi) but host-parasite coevolution is more likely observable and best studied in a system with negatively correlated tolerance and resistance (E. falciformis).

Intensity of infection with intracellular Eimeria spp. and pinworms is reduced in hybrid mice compared to parental subspecies.

Genetic diversity in animal immune systems is usually beneficial. In hybrid recombinants, this is less clear, as the immune system could also be impacted by genetic conflicts. In the European house mouse hybrid zone, the long-standing impression that hybrid mice are more highly parasitized and less fit than parentals persists despite the findings of recent studies. Working across a novel transect, we assessed infections by intracellular protozoans, Eimeria spp., and infections by extracellular macroparasites, pinworms. For Eimeria, we found lower intensities in hybrid hosts than in parental mice but no evidence of lowered probability of infection or increased mortality in the centre of the hybrid zone. This means ecological factors are very unlikely to be responsible for the reduced load of infected hybrids. Focusing on parasite intensity (load in infected hosts), we also corroborated reduced pinworm loads reported for hybrid mice in previous studies. We conclude that intensity of diverse parasites, including the previously unstudied Eimeria, is reduced in hybrid mice compared to parental subspecies. We suggest caution in extrapolating this to differences in hybrid host fitness in the absence of, for example, evidence for a link between parasitemia and health.

Detection and quantification of house mouse Eimeria at the species level - Challenges and solutions for the assessment of coccidia in wildlife.

Detection and quantification of coccidia in studies of wildlife can be challenging. Therefore, prevalence of coccidia is often not assessed at the parasite species level in non-livestock animals. Parasite species - specific prevalences are especially important when studying evolutionary questions in wild populations. We tested whether increased host population density increases prevalence of individual Eimeria species at the farm level, as predicted by epidemiological theory. We studied free-living commensal populations of the house mouse (Mus musculus) in Germany, and established a strategy to detect and quantify Eimeria infections. We show that a novel diagnostic primer targeting the apicoplast genome (Ap5) and coprological assessment after flotation provide complementary detection results increasing sensitivity. Genotyping PCRs confirm detection in a subset of samples and cross-validation of different PCR markers does not indicate bias towards a particular parasite species in genotyping. We were able to detect double infections and to determine the preferred niche of each parasite species along the distal-proximal axis of the intestine. Parasite genotyping from tissue samples provides additional indication for the absence of species bias in genotyping amplifications. Three Eimeria species were found infecting house mice at different prevalences: Eimeria ferrisi (16.7%; 95% CI 13.2-20.7), E. falciformis (4.2%; 95% CI 2.6-6.8) and E. vermiformis (1.9%; 95% CI 0.9-3.8). We also find that mice in dense populations are more likely to be infected with E. falciformis and E. ferrisi. We provide methods for the assessment of prevalences of coccidia at the species level in rodent systems. We show and discuss how such data can help to test hypotheses in ecology, evolution and epidemiology on a species level.

A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection.

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-ß, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-ß was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-ß expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection.

Recolonizing gray wolves increase parasite infection risk in their prey.

The recent recolonization of Central Europe by the European gray wolf (Canis lupus) provides an opportunity to study the dynamics of parasite transmission for cases when a definitive host returns after a phase of local extinction. We investigated whether a newly established wolf population increased the prevalence of those parasites in ungulate intermediate hosts representing wolf prey, whether some parasite species are particularly well adapted to wolves, and the potential basis for such adaptations. We recorded Sarcocystis species richness in wolves and Sarcocystis prevalence in ungulates harvested in study sites with and without permanent wolf presence in Germany using microscopy and DNA metabarcoding. Sarcocystis prevalence in red deer (Cervus elaphus) was significantly higher in wolf areas (79.7%) than in control areas (26.3%) but not in roe deer (Capreolus capreolus) (97.2% vs. 90.4%) or wild boar (Sus scrofa) (82.8% vs. 64.9%). Of 11 Sarcocystis species, Sarcocystis taeniata and Sarcocystis grueneri occurred more often in wolves than expected from the Sarcocystis infection patterns of ungulate prey. Both Sarcocystis species showed a higher increase in prevalence in ungulates in wolf areas than other Sarcocystis species, suggesting that they are particularly well adapted to wolves, and are examples of "wolf specialists". Sarcocystis species richness in wolves was significantly higher in pups than in adults. "Wolf specialists" persisted during wolf maturation. The results of this study demonstrate that (1) predator-prey interactions influence parasite prevalence, if both predator and prey are part of the parasite life cycle, (2) mesopredators do not necessarily replace the apex predator in parasite transmission dynamics for particular parasites of which the apex predator is the definitive host, even if meso- and apex predators were from the same taxonomic family (here: Canidae, e.g., red foxes Vulpes vulpes), and (3) age-dependent immune maturation contributes to the control of protozoan infection in wolves.

Dual RNA-seq reveals no plastic transcriptional response of the coccidian parasite Eimeria falciformis to host immune defenses.

BACKGROUND: Parasites can either respond to differences in immune defenses that exist between individual hosts plastically or, alternatively, follow a genetically canalized ("hard wired") program of infection. Assuming that large-scale functional plasticity would be discernible in the parasite transcriptome we have performed a dual RNA-seq study of the lifecycle of Eimeria falciformis using infected mice with different immune status as models for coccidian infections. RESULTS: We compared parasite and host transcriptomes (dual transcriptome) between naïve and challenge infected mice, as well as between immune competent and immune deficient ones. Mice with different immune competence show transcriptional differences as well as differences in parasite reproduction (oocyst shedding). Broad gene categories represented by differently abundant host genes indicate enrichments for immune reaction and tissue repair functions. More specifically, TGF-beta, EGF, TNF and IL-1 and IL-6 are examples of functional annotations represented differently depending on host immune status. Much in contrast, parasite transcriptomes were neither different between Coccidia isolated from immune competent and immune deficient mice, nor between those harvested from naïve and challenge infected mice. Instead, parasite transcriptomes have distinct profiles early and late in infection, characterized largely by biosynthesis or motility associated functional gene groups, respectively. Extracellular sporozoite and oocyst stages showed distinct transcriptional profiles and sporozoite transcriptomes were found enriched for species specific genes and likely pathogenicity factors. CONCLUSION: We propose that the niche and host-specific parasite E. falciformis uses a genetically canalized program of infection. This program is likely fixed in an evolutionary process rather than employing phenotypic plasticity to interact with its host. This in turn might limit the potential of the parasite to adapt to new host species or niches, forcing it to coevolve with its host.

The Intestinal Eukaryotic and Bacterial Biome of Spotted Hyenas: The Impact of Social Status and Age on Diversity and Composition.

In mammals, two factors likely to affect the diversity and composition of intestinal bacteria (bacterial microbiome) and eukaryotes (eukaryome) are social status and age. In species in which social status determines access to resources, socially dominant animals maintain better immune processes and health status than subordinates. As high species diversity is an index of ecosystem health, the intestinal biome of healthier, socially dominant animals should be more diverse than those of subordinates. Gradual colonization of the juvenile intestine after birth predicts lower intestinal biome diversity in juveniles than adults. We tested these predictions on the effect of: (1) age (juvenile/adult) and (2) social status (low/high) on bacterial microbiome and eukaryome diversity and composition in the spotted hyena (Crocuta crocuta), a highly social, female-dominated carnivore in which social status determines access to resources. We comprehensively screened feces from 35 individually known adult females and 7 juveniles in the Serengeti ecosystem for bacteria and eukaryotes, using a set of 48 different amplicons (4 for bacterial 16S, 44 for eukaryote 18S) in a multi-amplicon sequencing approach. We compared sequence abundances to classical coprological egg or oocyst counts. For all parasite taxa detected in more than six samples, the number of sequence reads significantly predicted the number of eggs or oocysts counted, underscoring the value of an amplicon sequencing approach for quantitative measurements of parasite load. In line with our predictions, our results revealed a significantly less diverse microbiome in juveniles than adults and a significantly higher diversity of eukaryotes in high-ranking than low-ranking animals. We propose that free-ranging wildlife can provide an intriguing model system to assess the adaptive value of intestinal biome diversity for both bacteria and eukaryotes.

Population expansion and individual age affect endoparasite richness and diversity in a recolonising large carnivore population.

The recent recolonisation of the Central European lowland (CEL) by the grey wolf (Canis lupus) provides an excellent opportunity to study the effect of founder events on endoparasite diversity. Which role do prey and predator populations play in the re-establishment of endoparasite life cycles? Which intrinsic and extrinsic factors control individual endoparasite diversity in an expanding host population? In 53 individually known CEL wolves sampled in Germany, we revealed a community of four cestode, eight nematode, one trematode and 12 potential Sarcocystis species through molecular genetic techniques. Infections with zoonotic Echinococcus multilocularis, Trichinella britovi and T. spiralis occurred as single cases. Per capita endoparasite species richness and diversity significantly increased with population size and changed with age, whereas sex, microsatellite heterozygosity, and geographic origin had no effect. Tapeworm abundance (Taenia spp.) was significantly higher in immigrants than natives. Metacestode prevalence was slightly higher in ungulates from wolf territories than from control areas elsewhere. Even though alternative canid definitive hosts might also play a role within the investigated parasite life cycles, our findings indicate that (1) immigrated wolves increase parasite diversity in German packs, and (2) prevalence of wolf-associated parasites had declined during wolf absence and has now risen during recolonisation.

Transcriptome analyses of Anguillicola crassus from native and novel hosts.

Anguillicola crassus is a swim bladder nematode of eels. The parasite is native to the Asian eel Anguilla japonica, but was introduced to Europe and the European eel Anguilla anguilla in the early 1980s. A Taiwanese source has been proposed for this introduction. In the new host in the recipient area, the parasite appears to be more pathogenic. As a reason for these differences, genetically fixed differences in infectivity and development between Taiwanese and European A.crassus have been described and disentangled from plasticity induced by different host environments. To explore whether transcriptional regulation is involved in these lifecycle differences, we have analysed a "common garden", cross infection experiment, using deep-sequencing transcriptomics. Surprisingly, in the face of clear phenotypic differences in life history traits, we identified no significant differences in gene expression between parasite populations or between experimental host species. From 120,000 SNPs identified in the transcriptome data we found that European A. crassus were not a genetic subset of the Taiwanese nematodes sampled. The loci that have the major contribution to the European-Taiwanese population differentiation show an enrichment of synonymous and non-coding polymorphism. This argues against positive selection in population differentiation. However, genes involved in protein processing in the endoplasmatic reticulum membrane and genes bearing secretion signal sequences were enriched in the set of genes most differentiated between European and Taiwanese A. crassus. These genes could be a source for the phenotypically visible genetically fixed differences between European and Taiwanese A. crassus.

The genome of Eimeria falciformis--reduction and specialization in a single host apicomplexan parasite.

BACKGROUND: The phylum Apicomplexa comprises important unicellular human parasites such as Toxoplasma and Plasmodium. Eimeria is the largest and most diverse genus of apicomplexan parasites and some species of the genus are the causative agent of coccidiosis, a disease economically devastating in poultry. We report a complete genome sequence of the mouse parasite Eimeria falciformis. We assembled and annotated the genome sequence to study host-parasite interactions in this understudied genus in a model organism host. RESULTS: The genome of E. falciformis is 44 Mb in size and contains 5,879 predicted protein coding genes. Comparative analysis of E. falciformis with Toxoplasma gondii shows an emergence and diversification of gene families associated with motility and invasion mainly at the level of the Coccidia. Many rhoptry kinases, among them important virulence factors in T. gondii, are absent from the E. falciformis genome. Surface antigens are divergent between Eimeria species. Comparisons with T. gondii showed differences between genes involved in metabolism, N-glycan and GPI-anchor synthesis. E. falciformis possesses a reduced set of transmembrane transporters and we suggest an altered mode of iron uptake in the genus Eimeria. CONCLUSIONS: Reduced diversity of genes required for host-parasite interaction and transmembrane transport allow hypotheses on host adaptation and specialization of a single host parasite. The E. falciformis genome sequence sheds light on the evolution of the Coccidia and helps to identify determinants of host-parasite interaction critical for drug and vaccine development.

Eimeria falciformis infection of the mouse caecum identifies opposing roles of IFNγ-regulated host pathways for the parasite development.

Intracellular parasites reprogram host functions for their survival and reproduction. The extent and relevance of parasite-mediated host responses in vivo remains poorly studied, however. We utilized Eimeria falciformis, a parasite infecting the mouse intestinal epithelium, to identify and validate host determinants of parasite infection. Most prominent mouse genes induced during the onset of asexual and sexual growth of parasite comprise interferon γ (IFNγ)-regulated factors, e.g., immunity-related GTPases (IRGA6/B6/D/M2/M3), guanylate-binding proteins (GBP2/3/5/6/8), chemokines (CxCL9-11), and several enzymes of the kynurenine pathway including indoleamine 2,3-dioxygenase 1 (IDO1). These results indicated a multifarious innate defense (tryptophan catabolism, IRG, GBP, and chemokine signaling), and a consequential adaptive immune response (chemokine-cytokine signaling and lymphocyte recruitment). The inflammation- and immunity-associated transcripts were increased during the course of infection, following influx of B cells, T cells, and macrophages to the parasitized caecum tissue. Consistently, parasite growth was enhanced in animals inhibited for CxCr3, a major receptor for CxCL9-11 present on immune cells. Interestingly, despite a prominent induction, mouse IRGB6 failed to bind and disrupt the parasitophorous vacuole, implying an immune evasion by E. falciformis. Furthermore, oocyst output was impaired in IFNγ-R(-/-) and IDO1(-/-) mice, both of which suggest a subversion of IFNγ signaling by the parasite to promote its growth.