Epigenetic age estimation of wild mice using faecal samples.

Age is a key parameter in population ecology, with a myriad of biological processes changing with age as organisms develop in early life then later senesce. As age is often hard to accurately measure with non-lethal methods, epigenetic methods of age estimation (epigenetic clocks) have become a popular tool in animal ecology and are often developed or calibrated using captive animals of known age. However, studies typically rely on invasive blood or tissue samples, which limit their application in more sensitive or elusive species. Moreover, few studies have directly assessed how methylation patterns and epigenetic age estimates compare across environmental contexts (e.g. captive or laboratory-based vs. wild animals). Here, we built a targeted epigenetic clock from laboratory house mice (strain C57BL/6, Mus musculus) using DNA from non-invasive faecal samples, and then used it to estimate age in a population of wild mice (Mus musculus domesticus) of unknown age. This laboratory mouse-derived epigenetic clock accurately predicted adult wild mice to be older than juveniles and showed that wild mice typically increased in epigenetic age over time, but with wide variation in epigenetic ageing rate among individuals. Our results also suggested that, for a given body mass, wild mice had higher methylation across targeted CpG sites than laboratory mice (and consistently higher epigenetic age estimates as a result), even among the smallest, juvenile mice. This suggests wild and laboratory mice may display different CpG methylation levels from very early in life and indicates caution is needed when developing epigenetic clocks on laboratory animals and applying them in the wild.

Social and environmental transmission spread different sets of gut microbes in wild mice.

Gut microbes shape many aspects of organismal biology, yet how these key bacteria transmit among hosts in natural populations remains poorly understood. Recent work in mammals has emphasized either transmission through social contacts or indirect transmission through environmental contact, but the relative importance of different routes has not been directly assessed. Here we used a novel radio-frequency identification-based tracking system to collect long-term high-resolution data on social relationships, space use and microhabitat in a wild population of mice (Apodemus sylvaticus), while regularly characterizing their gut microbiota with 16S ribosomal RNA profiling. Through probabilistic modelling of the resulting data, we identify positive and statistically distinct signals of social and environmental transmission, captured by social networks and overlap in home ranges, respectively. Strikingly, microorganisms with distinct biological attributes drove these different transmission signals. While the social network effect on microbiota was driven by anaerobic bacteria, the effect of shared space was most influenced by aerotolerant spore-forming bacteria. These findings support the prediction that social contact is important for the transfer of microorganisms with low oxygen tolerance, while those that can tolerate oxygen or form spores may be able to transmit indirectly through the environment. Overall, these results suggest social and environmental transmission routes can spread biologically distinct members of the mammalian gut microbiota.

Gut microbiota of the critically endangered Saiga antelope across two wild populations in a year without mass mortality.

The Saiga are migratory antelopes inhabiting the grasslands of Eurasia. Over the last century, Saiga have been pushed to the brink of extinction by mass mortality events and intense poaching. Yet, despite the high profile of the Saiga as an animal of conservation concern, little is known of its biology. In particular, the gut microbiota of Saiga has not been studied, despite its potential importance in health. Here, we characterise the gut microbiota of Saiga from two geographically distinct populations in Kazakhstan and compare it with that of other antelope species. We identified a consistent gut microbial diversity and composition among individuals and across two Saiga populations during a year without die-offs, with over 85% of bacterial genera being common to both populations despite vast geographic separation. We further show that the Saiga gut microbiota resembled that of five other antelopes. The putative causative agent of Saiga mass die-offs, Pasteurella multocida, was not detected in the Saiga microbiota. Our findings provide the first description of the Saiga gut microbiota, generating a baseline for future work investigating the microbiota's role in health and mass die-offs, and supporting the conservation of this critically endangered species.

Maternal transmission gives way to social transmission during gut microbiota assembly in wild mice.

BACKGROUND: The mammalian gut microbiota influences a wide array of phenotypes which are relevant to fitness, yet knowledge about the transmission routes by which gut microbes colonise hosts in natural populations remains limited. Here, we use an intensively studied wild population of wood mice (Apodemus sylvaticus) to examine how vertical (maternal) and horizontal (social) transmission routes influence gut microbiota composition throughout life. RESULTS: We identify independent signals of maternal transmission (sharing of taxa between a mother and her offspring) and social transmission (sharing of taxa predicted by the social network), whose relative magnitudes shift as hosts age. In early life, gut microbiota composition is predicted by both maternal and social relationships, but by adulthood the impact of maternal transmission becomes undetectable, leaving only a signal of social transmission. By exploring which taxa drive the maternal transmission signal, we identify a candidate maternally-transmitted bacterial family in wood mice, the Muribaculaceae. CONCLUSION: Overall, our findings point to an ontogenetically shifting transmission landscape in wild mice, with a mother's influence on microbiota composition waning as offspring age, while the relative impact of social contacts grows.

Experimental temperatures shape host microbiome diversity and composition.

Global climate change has led to more extreme thermal events. Plants and animals harbour diverse microbial communities, which may be vital for their physiological performance and help them survive stressful climatic conditions. The extent to which microbiome communities change in response to warming or cooling may be important for predicting host performance under global change. Using a meta-analysis of 1377 microbiomes from 43 terrestrial and aquatic species, we found a decrease in the amplicon sequence variant-level microbiome phylogenetic diversity and alteration of microbiome composition under both experimental warming and cooling. Microbiome beta dispersion was not affected by temperature changes. We showed that the host habitat and experimental factors affected microbiome diversity and composition more than host biological traits. In particular, aquatic organisms-especially in marine habitats-experienced a greater depletion in microbiome diversity under cold conditions, compared to terrestrial hosts. Exposure involving a sudden long and static temperature shift was associated with microbiome diversity loss, but this reduction was attenuated by prior-experimental lab acclimation or when a ramped regime (i.e., warming) was used. Microbial differential abundance and co-occurrence network analyses revealed several potential indicator bacterial classes for hosts in heated environments and on different biome levels. Overall, our findings improve our understanding on the impact of global temperature changes on animal and plant microbiome structures across a diverse range of habitats. The next step is to link these changes to measures of host fitness, as well as microbial community functions, to determine whether microbiomes can buffer some species against a more thermally variable and extreme world.

Seasonal dynamics of the wild rodent faecal virome.

Viral discovery studies in wild animals often rely on cross-sectional surveys at a single time point. As a result, our understanding of the temporal stability of wild animal viromes remains poorly resolved. While studies of single host-virus systems indicate that host and environmental factors influence seasonal virus transmission dynamics, comparable insights for whole viral communities in multiple hosts are lacking. Utilizing noninvasive faecal samples from a long-term wild rodent study, we characterized viral communities of three common European rodent species (Apodemus sylvaticus, A. flavicollis and Myodes glareolus) living in temperate woodland over a single year. Our findings indicate that a substantial fraction of the rodent virome is seasonally transient and associated with vertebrate or bacteria hosts. Further analyses of one of the most common virus families, Picornaviridae, show pronounced temporal changes in viral richness and evenness, which were associated with concurrent and up to ~3-month lags in host density, ambient temperature, rainfall and humidity, suggesting complex feedbacks from the host and environmental factors on virus transmission and shedding in seasonal habitats. Overall, this study emphasizes the importance of understanding the seasonal dynamics of wild animal viromes in order to better predict and mitigate zoonotic risks.

Synchronous Seasonality in the Gut Microbiota of Wild Mouse Populations.

The gut microbiome performs many important functions in mammalian hosts, with community composition shaping its functional role. However, the factors that drive individual microbiota variation in wild animals and to what extent these are predictable or idiosyncratic across populations remains poorly understood. Here, we use a multi-population dataset from a common rodent species (the wood mouse, Apodemus sylvaticus), to test whether a consistent "core" gut microbiota is identifiable in this species, and to what extent the predictors of microbiota variation are consistent across populations. Between 2014 and 2018 we used capture-mark-recapture and 16S rRNA profiling to intensively monitor two wild wood mouse populations and their gut microbiota, as well as characterising the microbiota from a laboratory-housed colony of the same species. Although the microbiota was broadly similar at high taxonomic levels, the two wild populations did not share a single bacterial amplicon sequence variant (ASV), despite being only 50km apart. Meanwhile, the laboratory-housed colony shared many ASVs with one of the wild populations from which it is thought to have been founded decades ago. Despite not sharing any ASVs, the two wild populations shared a phylogenetically more similar microbiota than either did with the colony, and the factors predicting compositional variation in each wild population were remarkably similar. We identified a strong and consistent pattern of seasonal microbiota restructuring that occurred at both sites, in all years, and within individual mice. While the microbiota was highly individualised, some seasonal convergence occurred in late winter/early spring. These findings reveal highly repeatable seasonal gut microbiota dynamics in multiple populations of this species, despite different taxa being involved. This provides a platform for future work to understand the drivers and functional implications of such predictable seasonal microbiome restructuring, including whether it might provide the host with adaptive seasonal phenotypic plasticity.

Bifidobacterium castoris strains isolated from wild mice show evidence of frequent host switching and diverse carbohydrate metabolism potential.

Members of the gut microbiota genus Bifidobacterium are widely distributed human and animal symbionts believed to exert beneficial effects on their hosts. However, in-depth genomic analyses of animal-associated species and strains are somewhat lacking, particularly in wild animal populations. Here, to examine patterns of host specificity and carbohydrate metabolism capacity, we sequenced whole genomes of Bifidobacterium isolated from wild-caught small mammals from two European countries (UK and Lithuania). Members of Bifidobacterium castoris, Bifidobacterium animalis and Bifodobacterium pseudolongum were detected in wild mice (Apodemus sylvaticus, Apodemus agrarius and Apodemus flavicollis), but not voles or shrews. B. castoris constituted the most commonly recovered Bifidobacterium (78% of all isolates), with the majority of strains only detected in a single population, although populations frequently harboured multiple co-circulating strains. Phylogenetic analysis revealed that the mouse-associated B. castoris clades were not specific to a particular location or host species, and their distribution across the host phylogeny was consistent with regular host shifts rather than host-microbe codiversification. Functional analysis, including in vitro growth assays, suggested that mouse-derived B. castoris strains encoded an extensive arsenal of carbohydrate-active enzymes, including putative novel glycosyl hydrolases such as chitosanases, along with genes encoding putative exopolysaccharides, some of which may have been acquired via horizontal gene transfer. Overall, these results provide a rare genome-level analysis of host specificity and genomic capacity among important gut symbionts of wild animals, and reveal that Bifidobacterium has a labile relationship with its host over evolutionary time scales.

Social networks strongly predict the gut microbiota of wild mice.

The mammalian gut teems with microbes, yet how hosts acquire these symbionts remains poorly understood. Research in primates suggests that microbes can be picked up via social contact, but the role of social interactions in non-group-living species remains underexplored. Here, we use a passive tracking system to collect high resolution spatiotemporal activity data from wild mice (Apodemus sylvaticus). Social network analysis revealed social association strength to be the strongest predictor of microbiota similarity among individuals, controlling for factors including spatial proximity and kinship, which had far smaller or nonsignificant effects. This social effect was limited to interactions involving males (male-male and male-female), implicating sex-dependent behaviours as driving processes. Social network position also predicted microbiota richness, with well-connected individuals having the most diverse microbiotas. Overall, these findings suggest social contact provides a key transmission pathway for gut symbionts even in relatively asocial mammals, that strongly shapes the adult gut microbiota. This work underlines the potential for individuals to pick up beneficial symbionts as well as pathogens from social interactions.

A 16S rRNA Gene and Draft Genome Database for the Murine Oral Bacterial Community.

A curated murine oral microbiome database to be used as a reference for mouse-based studies has been constructed using a combination of bacterial culture, 16S rRNA gene amplicon, and whole-genome sequencing. The database comprises a collection of nearly full-length 16S rRNA gene sequences from cultured isolates and draft genomes from representative taxa collected from a range of sources, including specific-pathogen-free laboratory mice, wild Mus musculus domesticus mice, and formerly wild wood mouse Apodemus sylvaticus At present, it comprises 103 mouse oral taxa (MOT) spanning four phyla-Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes-including 12 novel undescribed species-level taxa. The key observations from this study are (i) the low diversity and predominantly culturable nature of the laboratory mouse oral microbiome and (ii) the identification of three major murine-specific oral bacterial lineages, namely, Streptococcus danieliae (MOT10), Lactobacillus murinus (MOT93), and Gemella species 2 (MOT43), which is one of the novel, still-unnamed taxa. Of these, S. danieliae is of particular interest, since it is a major component of the oral microbiome from all strains of healthy and periodontally diseased laboratory mice, as well as being present in wild mice. It is expected that this well-characterized database should be a useful resource for in vitro experimentation and mouse model studies in the field of oral microbiology.IMPORTANCE Mouse model studies are frequently used in oral microbiome research, particularly to investigate diseases such as periodontitis and caries, as well as other related systemic diseases. We have reported here the details of the development of a curated reference database to characterize the oral microbial community in laboratory and some wild mice. The genomic information and findings reported here can help improve the outcomes and accuracy of host-microbe experimental studies that use murine models to understand health and disease. Work is also under way to make the reference data sets publicly available on a web server to enable easy access and downloading for researchers across the world.

Effects of laboratory domestication on the rodent gut microbiome.

The domestication of the laboratory mouse has influenced the composition of its native gut microbiome, which is now known to differ from that of its wild ancestor. However, limited exploration of the rodent gut microbiome beyond the model species Mus musculus has made it difficult to interpret microbiome variation in a broader phylogenetic context. Here, we analyse 120 de novo and 469 public metagenomically-sequenced faecal and caecal samples from 16 rodent hosts representing wild, laboratory and captive lifestyles. Distinct gut bacterial communities were observed between rodent host genera, with broadly distributed species originating from the as-yet-uncultured bacterial genera UBA9475 and UBA2821 in the families Oscillospiraceae and Lachnospiraceae, respectively. In laboratory mice, Helicobacteraceae were generally depleted relative to wild mice and specific Muribaculaceae populations were enriched in different laboratory facilities, suggesting facility-specific outgrowths of this historically dominant rodent gut family. Several bacterial families of clinical interest, including Akkermansiaceae, Streptococcaceae and Enterobacteriaceae, were inferred to have gained over half of their representative species in mice within the laboratory environment, being undetected in most wild rodents and suggesting an association between laboratory domestication and pathobiont emergence.

Identifying Microbiome-Mediated Behaviour in Wild Vertebrates.

Recent research in laboratory animals has illuminated how the vertebrate gut microbiome can have diverse and powerful effects on the brain and behaviour. However, the ecological relevance of this microbiome-gut-brain (MGB) axis outside the laboratory remains unexplored. Here we argue that understanding behavioural and cognitive effects of the gut microbiome in natural populations is an important goal for behavioural ecology that may shed light on the mechanisms and evolution of behavioural plasticity. We outline a toolkit of approaches that could be applied in this endeavour and argue that beyond collecting observational data on the microbiome and behaviour from free-living animals, the incorporation of manipulative approaches tailored to such systems will be a key next step to progress understanding in this area.

The evolution of ecological facilitation within mixed-species biofilms in the mouse gastrointestinal tract.

The eco-evolutionary interactions among members of the vertebrate gut microbiota that ultimately result in host-specific communities are poorly understood. Here we show that Lactobacillus reuteri coexists with  species that belong to the Lactobacillus johnsonii cluster (L. johnsonii, L. gasseri, and L taiwanensis) in a taxonomically wide range of rodents, suggesting cohabitation over evolutionary times. The two dominant Lactobacillus species found in wild mice establish a commensalistic relationship in gastric biofilms when introduced together into germ-free mice in which L. reuteri facilitates colonization of L. taiwanensis. Genomic analysis revealed allopatric diversification in strains of both species that originated from geographically separated locations (Scotland and France). Allopatry of the strains resulted in reduced formation of mixed biofilms in vitro, indicating that interspecies interactions in gastric Lactobacillus-biofilms are the result of an adaptive evolutionary process that occurred in a biogeographical context. In summary, these findings suggest that members within the vertebrate gut microbiota can evolve inter-dependencies through ecological facilitation, which could represent one mechanism by which host-specific bacterial communities assemble across vertebrate species and an explanation for their spatial and biogeographic patterns.

Parasite-Microbiota Interactions With the Vertebrate Gut: Synthesis Through an Ecological Lens.

The vertebrate gut teems with a large, diverse, and dynamic bacterial community that has pervasive effects on gut physiology, metabolism, and immunity. Under natural conditions, these microbes share their habitat with a similarly dynamic community of eukaryotes (helminths, protozoa, and fungi), many of which are well-known parasites. Both parasites and the prokaryotic microbiota can dramatically alter the physical and immune landscape of the gut, creating ample opportunities for them to interact. Such interactions may critically alter infection outcomes and affect overall host health and disease. For instance, parasite infection can change how a host interacts with its bacterial flora, either driving or protecting against dysbiosis and inflammatory disease. Conversely, the microbiota can alter a parasite's colonization success, replication, and virulence, shifting it along the parasitism-mutualism spectrum. The mechanisms and consequences of these interactions are just starting to be elucidated in an emergent transdisciplinary area at the boundary of microbiology and parasitology. However, heterogeneity in experimental designs, host and parasite species, and a largely phenomenological and taxonomic approach to synthesizing the literature have meant that common themes across studies remain elusive. Here, we use an ecological perspective to review the literature on interactions between the prokaryotic microbiota and eukaryotic parasites in the vertebrate gut. Using knowledge about parasite biology and ecology, we discuss mechanisms by which they may interact with gut microbes, the consequences of such interactions for host health, and how understanding parasite-microbiota interactions may lead to novel approaches in disease control.

Interpreting ambiguous 'trace' results in Schistosoma mansoni CCA Tests: Estimating sensitivity and specificity of ambiguous results with no gold standard.

BACKGROUND: The development of new diagnostics is an important tool in the fight against disease. Latent Class Analysis (LCA) is used to estimate the sensitivity and specificity of tests in the absence of a gold standard. The main field diagnostic for Schistosoma mansoni infection, Kato-Katz (KK), is not very sensitive at low infection intensities. A point-of-care circulating cathodic antigen (CCA) test has been shown to be more sensitive than KK. However, CCA can return an ambiguous 'trace' result between 'positive' and 'negative', and much debate has focused on interpretation of traces results. METHODOLOGY/PRINCIPLE FINDINGS: We show how LCA can be extended to include ambiguous trace results and analyse S. mansoni studies from both Côte d'Ivoire (CdI) and Uganda. We compare the diagnostic performance of KK and CCA and the observed results by each test to the estimated infection prevalence in the population. Prevalence by KK was higher in CdI (13.4%) than in Uganda (6.1%), but prevalence by CCA was similar between countries, both when trace was assumed to be negative (CCAtn: 11.7% in CdI and 9.7% in Uganda) and positive (CCAtp: 20.1% in CdI and 22.5% in Uganda). The estimated sensitivity of CCA was more consistent between countries than the estimated sensitivity of KK, and estimated infection prevalence did not significantly differ between CdI (20.5%) and Uganda (19.1%). The prevalence by CCA with trace as positive did not differ significantly from estimates of infection prevalence in either country, whereas both KK and CCA with trace as negative significantly underestimated infection prevalence in both countries. CONCLUSIONS: Incorporation of ambiguous results into an LCA enables the effect of different treatment thresholds to be directly assessed and is applicable in many fields. Our results showed that CCA with trace as positive most accurately estimated infection prevalence.

Optimising cluster survey design for planning schistosomiasis preventive chemotherapy.

BACKGROUND: The cornerstone of current schistosomiasis control programmes is delivery of praziquantel to at-risk populations. Such preventive chemotherapy requires accurate information on the geographic distribution of infection, yet the performance of alternative survey designs for estimating prevalence and converting this into treatment decisions has not been thoroughly evaluated. METHODOLOGY/PRINCIPAL FINDINGS: We used baseline schistosomiasis mapping surveys from three countries (Malawi, Côte d'Ivoire and Liberia) to generate spatially realistic gold standard datasets, against which we tested alternative two-stage cluster survey designs. We assessed how sampling different numbers of schools per district (2-20) and children per school (10-50) influences the accuracy of prevalence estimates and treatment class assignment, and we compared survey cost-efficiency using data from Malawi. Due to the focal nature of schistosomiasis, up to 53% simulated surveys involving 2-5 schools per district failed to detect schistosomiasis in low endemicity areas (1-10% prevalence). Increasing the number of schools surveyed per district improved treatment class assignment far more than increasing the number of children sampled per school. For Malawi, surveys of 15 schools per district and 20-30 children per school reliably detected endemic schistosomiasis and maximised cost-efficiency. In sensitivity analyses where treatment costs and the country considered were varied, optimal survey size was remarkably consistent, with cost-efficiency maximised at 15-20 schools per district. CONCLUSIONS/SIGNIFICANCE: Among two-stage cluster surveys for schistosomiasis, our simulations indicated that surveying 15-20 schools per district and 20-30 children per school optimised cost-efficiency and minimised the risk of under-treatment, with surveys involving more schools of greater cost-efficiency as treatment costs rose.

The impact of albendazole treatment on the incidence of viral- and bacterial-induced diarrhea in school children in southern Vietnam: study protocol for a randomized controlled trial.

BACKGROUND: Anthelmintics are one of the more commonly available classes of drugs to treat infections by parasitic helminths (especially nematodes) in the human intestinal tract. As a result of their cost-effectiveness, mass school-based deworming programs are becoming routine practice in developing countries. However, experimental and clinical evidence suggests that anthelmintic treatments may increase susceptibility to other gastrointestinal infections caused by bacteria, viruses, or protozoa. Hypothesizing that anthelmintics may increase diarrheal infections in treated children, we aim to evaluate the impact of anthelmintics on the incidence of diarrheal disease caused by viral and bacterial pathogens in school children in southern Vietnam. METHODS/DESIGN: This is a randomized, double-blinded, placebo-controlled trial to investigate the effects of albendazole treatment versus placebo on the incidence of viral- and bacterial-induced diarrhea in 350 helminth-infected and 350 helminth-uninfected Vietnamese school children aged 6-15 years. Four hundred milligrams of albendazole, or placebo treatment will be administered once every 3 months for 12 months. At the end of 12 months, all participants will receive albendazole treatment. The primary endpoint of this study is the incidence of diarrheal disease assessed by 12 months of weekly active and passive case surveillance. Secondary endpoints include the prevalence and intensities of helminth, viral, and bacterial infections, alterations in host immunity and the gut microbiota with helminth and pathogen clearance, changes in mean z scores of body weight indices over time, and the number and severity of adverse events. DISCUSSION: In order to reduce helminth burdens, anthelmintics are being routinely administered to children in developing countries. However, the effects of anthelmintic treatment on susceptibility to other diseases, including diarrheal pathogens, remain unknown. It is important to monitor for unintended consequences of drug treatments in co-infected populations. In this trial, we will examine how anthelmintic treatment impacts host susceptibility to diarrheal infections, with the aim of informing deworming programs of any indirect effects of mass anthelmintic administrations on co-infecting enteric pathogens. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02597556 . Registered on 3 November 2015.

One health - an ecological and evolutionary framework for tackling Neglected Zoonotic Diseases.

Understanding the complex population biology and transmission ecology of multihost parasites has been declared as one of the major challenges of biomedical sciences for the 21st century and the Neglected Zoonotic Diseases (NZDs) are perhaps the most neglected of all the Neglected Tropical Diseases (NTDs). Here we consider how multihost parasite transmission and evolutionary dynamics may affect the success of human and animal disease control programmes, particularly neglected diseases of the developing world. We review the different types of zoonotic interactions that occur, both ecological and evolutionary, their potential relevance for current human control activities, and make suggestions for the development of an empirical evidence base and theoretical framework to better understand and predict the outcome of such interactions. In particular, we consider whether preventive chemotherapy, the current mainstay of NTD control, can be successful without a One Health approach. Transmission within and between animal reservoirs and humans can have important ecological and evolutionary consequences, driving the evolution and establishment of drug resistance, as well as providing selective pressures for spill-over, host switching, hybridizations and introgressions between animal and human parasites. Our aim here is to highlight the importance of both elucidating disease ecology, including identifying key hosts and tailoring control effort accordingly, and understanding parasite evolution, such as precisely how infectious agents may respond and adapt to anthropogenic change. Both elements are essential if we are to alleviate disease risks from NZDs in humans, domestic animals and wildlife.