Epigenetic signatures of social status in wild female spotted hyenas (Crocuta crocuta).

In mammalian societies, dominance hierarchies translate into inequalities in health, reproductive performance and survival. DNA methylation is thought to mediate the effects of social status on gene expression and phenotypic outcomes, yet a study of social status-specific DNA methylation profiles in different age classes in a wild social mammal is missing. We tested for social status signatures in DNA methylation profiles in wild female spotted hyenas (Crocuta crocuta), cubs and adults, using non-invasively collected gut epithelium samples. In spotted hyena clans, female social status influences access to resources, foraging behavior, health, reproductive performance and survival. We identified 149 differentially methylated regions between 42 high- and low-ranking female spotted hyenas (cubs and adults). Differentially methylated genes were associated with energy conversion, immune function, glutamate receptor signalling and ion transport. Our results provide evidence that socio-environmental inequalities are reflected at the molecular level in cubs and adults in a wild social mammal.

Early-life adversity predicts performance and fitness in a wild social carnivore.

Studies on humans indicate that encountering multiple sources of adversity in childhood increases the risk of poor long-term health and premature death. Far less is known about cumulative effects of adversity during early life in wildlife. Focusing on the spotted hyena Crocuta crocuta, a social mammal with small litters, extensive maternal care, slow development and access to resources determined by social rank, we determined the contribution of ecological, maternal, social and demographic factors during early life on performance and fitness, and tested whether the impact of early-life adversity is cumulative. Using longitudinal data from 666 female hyenas in the Serengeti National Park, we determined the early growth rate, survival to adulthood, age at first reproduction (AFR), lifetime reproductive success (LRS) and longevity. We fitted multivariate models in which we tested the effects of environmental factors on these performance measures. We then constructed a cumulative adversity index and fitted models to test the effect of this index on each performance measure. Finally, the value of cumulative adversity models was tested by comparing them to multivariate and single-effect models in which the effect of each environmental factor was considered separately. High maternal rank decreased the AFR of daughters. Singleton and dominant cubs had higher growth rate than subordinate cubs, and singletons also had a higher survival chance to adulthood than subordinates. Daughters of prime age mothers had a higher growth rate, longevity and LRS. Little and heavy rainfall decreased survival to adulthood. Increasing numbers of lactating female clan members decreased growth rate, survival to adulthood and LRS. Cumulative adversity negatively affected short-term performance and LRS. Multivariate models outperformed cumulative adversity and single-effect models for all measures except for AFR and longevity, for which single-effect models performed better. Our results suggest that in some wildlife populations the combination of specific conditions in early life may matter more than the accumulation of adverse conditions as such.

Des études menées chez l'homme indiquent que le fait de rencontrer de multiples sources d'adversité pendant l'enfance augmente le risque de troubles de la santé à long terme et de décès prématuré. On en sait beaucoup moins sur les effets cumulatifs de l'adversité en début de vie chez les animaux sauvages. En nous concentrant sur la hyène tachetée Crocuta crocuta, un mammifère social avec des portées de petite taille, des soins maternels intensifs, un développement lent et un accès aux ressources déterminé par le rang social, nous avons déterminé la contribution des facteurs écologiques, maternels, sociaux et démographiques en début de vie sur la performance et le succès reproducteur, et nous avons testé si l'adversité précoce a un effet cumulatif. En utilisant les données longitudinales de 666 hyènes femelles dans le parc national du Serengeti, nous avons calculé le taux de croissance précoce, la survie à l'âge adulte, l'âge à la première reproduction, le succès reproducteur et la longévité. Nous avons utilisé des modèles multivariés dans lesquels nous avons testé les effets des facteurs environnementaux sur ces mesures de performance. Nous avons ensuite construit un indice de risque cumulatif et utilisé des modèles pour tester l'effet de cet indice sur chaque mesure de performance. Enfin, nous avons testé la valeur de ces modèles en les comparant aux modèles multivariés et simples dans lesquels l'effet de chaque facteur environnemental était considéré séparément. Un rang maternel élevé diminue l'âge à la première reproduction des filles. Les femelles sans frère ou sœur ou dominantes dans une portée de jumeaux ont un taux de croissance plus élevé que les femelles subordonnées, et les femelles sans frère ou sœur ont également une plus grande chance de survie à l'âge adulte que les subordonnées. Les filles de mères dans la fleur de l'âge montrent de meilleurs taux de croissance, longévité et succès reproducteur. Des précipitations faibles ou fortes réduisent la survie à l'âge adulte. L'augmentation du nombre de femelles allaitantes dans le clan réduit le taux de croissance, la survie à l'âge adulte et le succès reproducteur. L'adversité précoce cumulée a un effet négatif sur la performance à court terme et le succès reproducteur. Les modèles multivariés sont toutefois plus performants que les modèles avec l'indice de risque cumulatif et plus performants que les modèles simples pour toutes les mesures, à l'exception de l'âge à la première reproduction et de la longévité, pour lesquelles les modèles simples sont plus performants. Nos résultats suggèrent que chez certaines populations sauvages, la combinaison de conditions spécifiques rencontrées en début de vie peut avoir plus d'importance que l'accumulation de conditions défavorables en tant que telles.

Noninvasively measured immune responses reflect current parasite infections in a wild carnivore and are linked to longevity.

Host immune defenses are important components of host-parasite interactions that affect the outcome of infection and may have fitness consequences for hosts when increased allocation of resources to immune responses undermines other essential life processes. Research on host-parasite interactions in large free-ranging wild mammals is currently hampered by a lack of verified noninvasive assays. We successfully adapted existing assays to measure innate and adaptive immune responses produced by the gastrointestinal mucosa in spotted hyena (Crocuta crocuta) feces, including enzyme-linked immunosorbent assays (ELISAs), to quantify fecal immunoglobulins (total IgA, total IgG) and total fecal O-linked oligosaccharides (mucin). We investigated the effect of infection load by an energetically costly hookworm (Ancylostoma), parasite richness, host age, sex, year of sampling, and clan membership on immune responses and asked whether high investment in immune responses during early life affects longevity in individually known spotted hyenas in the Serengeti National Park, Tanzania. Fecal concentrations of IgA, IgG, and mucin increased with Ancylostoma egg load and were higher in juveniles than in adults. Females had higher mucin concentrations than males. Juvenile females had higher IgG concentrations than juvenile males, whereas adult females had lower IgG concentrations than adult males. High IgA concentrations during the first year of life were linked to reduced longevity after controlling for age at sampling and Ancylostoma egg load. Our study demonstrates that the use of noninvasive methods can increase knowledge on the complex relationship between gastrointestinal parasites and host local immune responses in wild large mammals and reveal fitness-relevant effects of these responses.

Genetic diversity of vector-borne pathogens in spotted and brown hyenas from Namibia and Tanzania relates to ecological conditions rather than host taxonomy.

BACKGROUND: Improved knowledge on vector-borne pathogens in wildlife will help determine their effect on host species at the population and individual level and whether these are affected by anthropogenic factors such as global climate change and landscape changes. Here, samples from brown hyenas (Parahyaena brunnea) from Namibia (BHNA) and spotted hyenas (Crocuta crocuta) from Namibia (SHNA) and Tanzania (SHTZ) were screened for vector-borne pathogens to assess the frequency and genetic diversity of pathogens and the effect of ecological conditions and host taxonomy on this diversity. METHODS: Tissue samples from BHNA (n = 17), SHNA (n = 19) and SHTZ (n = 25) were analysed by PCRs targeting Anaplasmataceae, Rickettsia spp., piroplasms, specifically Babesia lengau-like piroplasms, Hepatozoidae and filarioids. After sequencing, maximum-likelihood phylogenetic analyses were conducted. RESULTS: The relative frequency of Anaplasmataceae was significantly higher in BHNA (82.4%) and SHNA (100.0%) than in SHTZ (32.0%). Only Anaplasma phagocytophilum/platys-like and Anaplasma bovis-like sequences were detected. Rickettsia raoultii was found in one BHNA and three SHTZ. This is the first report of R. raoultii from sub-Saharan Africa. Babesia lengau-like piroplasms were found in 70.6% of BHNA, 88.9% of SHNA and 32.0% of SHTZ, showing higher sequence diversity than B. lengau from South African cheetahs (Acinonyx jubatus). In one SHTZ, a Babesia vogeli-like sequence was identified. Hepatozoon felis-like parasites were identified in 64.7% of BHNA, 36.8% of SHNA and 44.0% of SHTZ. Phylogenetic analysis placed the sequences outside the major H. felis cluster originating from wild and domestic felids. Filarioids were detected in 47.1% of BHNA, 47.4% of SHNA and 36.0% of SHTZ. Phylogenetic analysis revealed high genetic diversity and suggested the presence of several undescribed species. Co-infections were frequently detected in SHNA and BHNA (BHNA median 3 pathogens, range 1-4; SHNA median 3 pathogens, range 2-4) and significantly rarer in SHTZ (median 1, range 0-4, 9 individuals uninfected). CONCLUSIONS: The frequencies of all pathogens groups were high, and except for Rickettsia, multiple species and genotypes were identified for each pathogen group. Ecological conditions explained pathogen identity and diversity better than host taxonomy.

'Keeping the kids at home' can limit the persistence of contagious pathogens in social animals.

Social networks are considered to be 'highly modular' when individuals within one module are more connected to each other than they are to individuals in other modules. It is currently unclear how highly modular social networks influence the persistence of contagious pathogens that generate lifelong immunity in their hosts when between-group interactions are age dependent. This trait occurs in social species with communal nurseries, where juveniles are reared together for a substantial period in burrows or similar forms of containment and are thus in isolation from contact with individuals in other social groups. Our main objective was to determine whether, and to what extent, such age-dependent patterns of between-group interactions consistently increased the fade-out probability of contagious pathogens that generate lifelong immunity in their hosts. We hypothesised that in populations of species where juveniles are raised in communal nurseries, a high proportion of recovered adults in a group would form a 'protective barrier' around susceptible juveniles against pathogen transmission, thereby increasing the probability of epidemic fade-out in the population. To test this idea, we implemented a spatially implicit individual-based susceptible-infected-recovered (SIR) model for a large range of generic host and pathogen traits. Our results indicated that (a) the probability of epidemic fade-out was consistently higher in populations with communal nurseries, especially for highly contagious pathogens (high basic reproduction number, R0 ) and (b) communal nurseries can counteract the cost of group living in terms of infection risk to a greater extent than variation in other traits. We discuss our findings in relation to herd immunity and outline the importance of considering the network structure of a given host population before implementing management measures such as vaccinations, since interventions focused on individuals with high between-group contact should be particularly effective for controlling pathogen spread in hosts with communal nurseries.

The virus-host interface: Molecular interactions of Alphacoronavirus-1 variants from wild and domestic hosts with mammalian aminopeptidase N.

The Alphacoronavirus-1 species include viruses that infect numerous mammalian species. To better understand the wide host range of these viruses, better knowledge on the molecular determinants of virus-host cell entry mechanisms in wildlife hosts is essential. We investigated Alphacoronavirus-1 infection in carnivores using long-term data on Serengeti spotted hyenas (Crocuta crocuta) and molecular analyses guided by the tertiary structure of the viral spike (S) attachment protein's interface with the host receptor aminopeptidase N (APN). We sequenced the complete 3'-end region of the genome of nine variants from wild African carnivores, plus the APN gene of 15 wild carnivore species. Our results revealed two outbreaks of Alphacoronavirus-1 infection in spotted hyenas associated with genetically distinct canine coronavirus type II (CCoVII) variants. Within the receptor binding domain (RBD) of the S gene the residues that directly bind to the APN receptor were conserved in all variants studied, even those infecting phylogenetically diverse host taxa. We identified a variable region within RBD located next to a region that directly interacts with the APN receptor. Two residues within this variable region were under positive selection in hyena variants, indicating that both sites were associated with adaptation of CCoVII to spotted hyena APN. Analysis of APN sequences revealed that most residues that interact with the S protein are conserved in wild carnivores, whereas some adjacent residues are highly variable. Of the variable residues, four that are critical for virus-host binding were under positive selection and may modulate the efficiency of virus attachment to carnivore APN.

Seasonal host and ecological drivers may promote restricted water as a viral vector.

In climates with seasonally limited precipitation, terrestrial animals congregate at high densities at scarce water sources. We hypothesize that viruses can exploit the recurrence of these diverse animal congregations to spread. In this study, we test the central prediction of this hypothesis - that viruses employing this transmission strategy remain stable and infectious in water. Equid herpesviruses (EHVs) were chosen as a model as they have been shown to remain stable and infectious in water for weeks under laboratory conditions. Using fecal data from wild equids from a previous study, we establish that EHVs are shed more frequently by their hosts during the dry season, increasing the probability of water source contamination with EHV. We document the presence of several strains of EHVs present in high genome copy number from the surface water and sediments of waterholes sampled across a variety of mammalian assemblages, locations, temperatures and pH. Phylogenetic analysis reveals that the different EHV strains found exhibit little divergence despite representing ancient lineages. We employed molecular approaches to show that EHVs shed remain stable in waterholes with detection decreasing with increasing temperature in sediments. Infectivity experiments using cell culture reveals that EHVs remain infectious in water derived from waterholes. The results are supportive of water as an abiotic viral vector for EHV.

Effects of life history stage and climatic conditions on fecal egg counts in plains zebras (Equus quagga) in the Serengeti National Park.

In wildlife, endoparasite burden can be affected by host life history stage, environmental conditions, host abundance, and parasite co-infections. We tested the effects of these factors on gastrointestinal parasite infection in plains zebras (Equus quagga) in the Serengeti ecosystem, Tanzania, using fecal egg counts of two nematode families (Strongylidae and Ascarididae) and the presence/absence of cestode (Anoplocephalidae) eggs. We predicted higher egg counts of Strongylidae and Ascarididae, and increased likelihood of Anoplocephalidae infection in individuals (1) during energetically costly life history stages when resource allocation to immune processes may decrease and in young zebras after weaning because of increased uptake of infective stages with forage, (2) when climatic conditions facilitate survival of infective stages, (3) when large zebra aggregations increase forage contamination with infective stages, and (4) in individuals co-infected with more than one parasite group as this may indicate reduced immune competence. Strongylidae egg counts were higher, and the occurrence of Anoplocephalidae eggs was more likely in bachelors than in band stallions, whereas Ascarididae egg counts were higher in band stallions. Strongylidae and Ascarididae egg counts were not increased in lactating females. Strongylidae egg counts were higher in subadults than in foals. Regardless of sex and age, Ascarididae infections were more likely under wet conditions. Co-infections did not affect Strongylidae egg counts. Ascarididae egg counts in adult females were higher when individuals were co-infected with Anoplocephalidae. We present evidence that parasite burdens in plains zebras are affected by life history stage, environmental conditions, and co-infection.

Parasite infections in a social carnivore: Evidence of their fitness consequences and factors modulating infection load.

There are substantial individual differences in parasite composition and infection load in wildlife populations. Few studies have investigated the factors shaping this heterogeneity in large wild mammals or the impact of parasite infections on Darwinian fitness, particularly in juveniles. A host's parasite composition and infection load can be shaped by factors that determine contact with infective parasite stages and those that determine the host's resistance to infection, such as abiotic and social environmental factors, and age. Host-parasite interactions and synergies between coinfecting parasites may also be important. We test predictions derived from these different processes to investigate factors shaping infection loads (fecal egg/oocyte load) of two energetically costly gastrointestinal parasites: the hookworm Ancylostoma and the intracellular Cystoisospora, in juvenile spotted hyenas (Crocuta crocuta) in the Serengeti National Park, in Tanzania. We also assess whether parasite infections curtail survival to adulthood and longevity. Ancylostoma and Cystoisospora infection loads declined as the number of adult clan members increased, a result consistent with an encounter-reduction effect whereby adults reduced encounters between juveniles and infective larvae, but were not affected by the number of juveniles in a clan. Infection loads decreased with age, possibly because active immune responses to infection improved with age. Differences in parasite load between clans possibly indicate variation in abiotic environmental factors between clan den sites. The survival of juveniles (<365 days old) to adulthood decreased with Ancylostoma load, increased with age, and was modulated by maternal social status. High-ranking individuals with low Ancylostoma loads had a higher survivorship during the first 4 years of life than high-ranking individuals with high Ancylostoma loads. These findings suggest that high infection loads with energetically costly parasites such as hookworms during early life can have negative fitness consequences.

Evidence of high exposure to Toxoplasma gondii in free-ranging and captive African carnivores.

Toxoplasma gondii is an ubiquitous intracellular protozoan parasite. Mammals and birds are intermediate hosts and felid species are definitive hosts. In most human altered habitats the domestic cat is the predominant definitive host. Current knowledge of T. gondii infection in African ecosystems is limited. This study aimed to assess exposure to T. gondii in wild carnivores in the Serengeti ecosystem in East Africa. Carnivores can be infected by the consumption of tissue cysts when feeding on infected animals and by incidental ingestion of oocysts from environmental contamination. Incidental ingestion should occur regardless of a species' diet whereas the consumption of cysts should increase the chance of infection in carnivorous species. This predicts higher seropositivity in carnivorous than in insectivorous carnivores and lower seropositivity in juvenile carnivores with a long dependency on milk than in adults. We found high seropositivity in carnivorous species: 100% (15 of 15 samples) in adult African lions, 93% (38 of 41 samples) in adult spotted hyenas and one striped hyena sample was positive, whereas all four samples from the insectivorous bat-eared fox were negative. Juvenile hyenas (11 of 19 sera) had significantly lower seropositivity than adults (38 of 41 sera). Long-term monitoring of spotted hyenas revealed no significant difference in seropositivity between two periods (1988-1992 and 2000 to 2016). Identical results were produced in lion and hyena samples by a commercial multi-species ELISA (at serum dilution 1:10) and an in-house ELISA based on a recombinant T. gondii protein (at serum dilution 1:100), making the latter a useful alternative for small amounts of serum. We suggest that diet, age and lifetime range are factors determining seropositivity in carnivores in the Serengeti ecosystem and suggest that the role of small wild felids in the spread of T. gondii in the African ecosystem warrants investigation.

Slow recovery from a disease epidemic in the spotted hyena, a keystone social carnivore.

Predicting the impact of disease epidemics on wildlife populations is one of the twenty-first century's main conservation challenges. The long-term demographic responses of wildlife populations to epidemics and the life history and social traits modulating these responses are generally unknown, particularly for K-selected social species. Here we develop a stage-structured matrix population model to provide a long-term projection of demographic responses by a keystone social predator, the spotted hyena, to a virulent epidemic of canine distemper virus (CDV) in the Serengeti ecosystem in 1993/1994 and predict the recovery time for the population following the epidemic. Using two decades of longitudinal data from 625 known hyenas, we demonstrate that although the reduction in population size was moderate, i.e., the population showed high ecological 'resistance' to the novel CDV genotype present, recovery was slow. Interestingly, high-ranking females accelerated the population's recovery, thereby lessening the impact of the epidemic on the population.

Robustness of Eco-Epidemiological Capture-Recapture Parameter Estimates to Variation in Infection State Uncertainty.

Estimating eco-epidemiological parameters in free-ranging populations can be challenging. As known individuals may be undetected during a field session, or their health status uncertain, the collected data are typically "imperfect". Multi-event capture-mark-recapture (MECMR) models constitute a substantial methodological advance by accounting for such imperfect data. In these models, animals can be "undetected" or "detected" at each time step. Detected animals can be assigned an infection state, such as "susceptible" (S), "infected" (I), or "recovered" (R), or an "unknown" (U) state, when for instance no biological sample could be collected. There may be heterogeneity in the assignment of infection states, depending on the manifestation of the disease in the host or the diagnostic method. For example, if obtaining the samples needed to prove viral infection in a detected animal is difficult, this can result in a low chance of assigning the I state. Currently, it is unknown how much uncertainty MECMR models can tolerate to provide reliable estimates of eco-epidemiological parameters and whether these parameters are sensitive to heterogeneity in the assignment of infection states. We used simulations to assess how estimates of the survival probability of individuals in different infection states and the probabilities of infection and recovery responded to (1) increasing infection state uncertainty (i.e., the proportion of U) from 20 to 90%, and (2) heterogeneity in the probability of assigning infection states. We simulated data, mimicking a highly virulent disease, and used SIR-MECMR models to quantify bias and precision. For most parameter estimates, bias increased and precision decreased gradually with state uncertainty. The probabilities of survival of I and R individuals and of detection of R individuals were very robust to increasing state uncertainty. In contrast, the probabilities of survival and detection of S individuals, and the infection and recovery probabilities showed high biases and low precisions when state uncertainty was >50%, particularly when the assignment of the S state was reduced. Considering this specific disease scenario, SIR-MECMR models are globally robust to state uncertainty and heterogeneity in state assignment, but the previously mentioned parameter estimates should be carefully interpreted if the proportion of U is high.

Physiological costs of infection: herpesvirus replication is linked to blood oxidative stress in equids.

Viruses may have a dramatic impact on the health of their animal hosts. The patho-physiological mechanisms underlying viral infections in animals are, however, not well understood. It is increasingly recognized that oxidative stress may be a major physiological cost of viral infections. Here we compare three blood-based markers of oxidative status in herpes positive and negative individuals of the domestic horse (Equus ferus caballus) and of both captive and free-ranging Mongolian khulan (Equus hemionus hemionus) and plains zebra (Equus quagga). Herpes positive free-ranging animals had significantly more protein oxidative damage and lower glutathione peroxidase (antioxidant enzyme) than negative ones, providing correlative support for a link between oxidative stress and herpesvirus infection in free-living equids. Conversely, we found weak evidence for oxidative stress in herpes positive captive animals. Hence our work indicates that environment (captive versus free living) might affect the physiological response of equids to herpesvirus infection. The Mongolian khulan and the plains zebra are currently classified as near threatened by the International Union for Conservation of Nature. Thus, understanding health impacts of pathogens on these species is critical to maintaining viable captive and wild populations.

Social status mediates the fitness costs of infection with canine distemper virus in Serengeti spotted hyenas.

The extent to which the fitness costs of infection are mediated by key life-history traits such as age or social status is still unclear. Within populations, individual heterogeneity in the outcome of infection is the result of two successive processes; the degree of contact with the pathogen (exposure) and the immune response to infection. In social mammals, because individuals holding high social status typically interact more frequently with group members, they should be more often in contact with infected individuals than those of low social status. However, when access to resources is determined by social status, individuals with a high social status are often better nourished, have a greater opportunity to allocate resources to immune processes and therefore should have a smaller chance of succumbing to infection than individuals with low social status.We investigated the risk and fitness costs of infection during a virulent epidemic of canine distemper virus (CDV) in a social carnivore, the spotted hyena, in the Serengeti National Park. We analysed two decades of detailed life-history data from 625 females and 816 males using a multi-event capture-mark-recapture model that accounts for uncertainty in the assignment of individual infection states.Cubs of mothers with a high social status had a lower probability of CDV infection and were more likely to survive infection than those with low social status. Subadult and adult females with high social status had a higher infection probability than those with low social status. Subadult females and pre-breeder males that had recovered from CDV infection had a lower survival than susceptible ones.Our study disentangles the relative importance of individual exposure and resource allocation to immune processes, demonstrates fitness costs of infection for juveniles, particularly for those with low social status, shows that patterns of infection can be driven by different mechanisms among juveniles and adults and establishes a negative relationship between infection and fitness in a free-ranging mammal. A http://onlinelibrary.wiley.com/doi/10.1111/1365-2435.13059/suppinfo is available for this article.

Equine behavioral enrichment toys as tools for non-invasive recovery of viral and host DNA.

Direct collection of samples from wildlife can be difficult and sometimes impossible. Non-invasive remote sampling for the purpose of DNA extraction is a potential tool for monitoring the presence of wildlife at the individual level, and for identifying the pathogens shed by wildlife. Equine herpesviruses (EHV) are common pathogens of equids that can be fatal if transmitted to other mammals. Transmission usually occurs by nasal aerosol discharge from virus-shedding individuals. The aim of this study was to validate a simple, non-invasive method to track EHV shedding in zebras and to establish an efficient protocol for genotyping individual zebras from environmental DNA (eDNA). A commercially available horse enrichment toy was deployed in captive Grévy's, mountain, and plains zebra enclosures and swabbed after 4-24 hr. Using eDNA extracted from these swabs four EHV strains (EHV-1, EHV-7, wild ass herpesvirus and zebra herpesvirus) were detected by PCR and confirmed by sequencing, and 12 of 16 zebras present in the enclosures were identified as having interacted with the enrichment toy by mitochondrial DNA amplification and sequencing. We conclude that, when direct sampling is difficult or prohibited, non-invasive sampling of eDNA can be a useful tool to determine the genetics of individuals or populations and for detecting pathogen shedding in captive wildlife.

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.

Canine distemper virus in the Serengeti ecosystem: molecular adaptation to different carnivore species.

Was the 1993/1994 fatal canine distemper virus (CDV) epidemic in lions and spotted hyaenas in the Serengeti ecosystem caused by the recent spillover of a virulent domestic dog strain or one well adapted to these noncanids? We examine this question using sequence data from 13 'Serengeti' strains including five complete genomes obtained between 1993 and 2011. Phylogenetic and haplotype network analyses reveal that strains from noncanids during the epidemic were more closely related to each other than to those from domestic or wild canids. All noncanid 'Serengeti' strains during the epidemic encoded: (1) one novel substitution G134S in the CDV-V protein; and (2) the rare amino acid combination 519I/549H at two sites under positive selection in the region of the CDV-H protein that binds to SLAM (CD 150) host cell receptors. Worldwide, only a few noncanid strains in the America II lineage encode CDV-H 519I/549H. All canid 'Serengeti' strains during the epidemic coded CDV-V 134G, and CDV-H 519R/549Y, or 519R/549H. A functional assay of cell entry revealed the highest performance by CDV-H proteins encoding 519I/549H in cells expressing lion SLAM receptors, and the highest performance by proteins encoding 519R/549Y, typical of dog strains worldwide, in cells expressing dog SLAM receptors. Our findings are consistent with an epidemic in lions and hyaenas caused by CDV variants better adapted to noncanids than canids, but not with the recent spillover of a dog strain. Our study reveals a greater complexity of CDV molecular epidemiology in multihost environments than previously thought.

Divergent Sapovirus Strains and Infection Prevalence in Wild Carnivores in the Serengeti Ecosystem: A Long-Term Study.

The genus Sapovirus, in the family Caliciviridae, includes enteric viruses of humans and domestic animals. Information on sapovirus infection of wildlife is limited and is currently lacking for any free-ranging wildlife species in Africa. By screening a large number of predominantly fecal samples (n = 631) obtained from five carnivore species in the Serengeti ecosystem, East Africa, sapovirus RNA was detected in the spotted hyena (Crocuta crocuta, family Hyaenidae), African lion (Panthera leo, family Felidae), and bat-eared fox (Otocyon megalotis, family Canidae), but not in golden or silver-backed jackals (Canis aureus and C. mesomelas, respectively, family Canidae). A phylogenetic analysis based on partial RNA-dependent RNA polymerase (RdRp) gene sequences placed the sapovirus strains from African carnivores in a monophyletic group. Within this monophyletic group, sapovirus strains from spotted hyenas formed one independent sub-group, and those from bat-eared fox and African lion a second sub-group. The percentage nucleotide similarity between sapoviruses from African carnivores and those from other species was low (< 70.4%). Long-term monitoring of sapovirus in a population of individually known spotted hyenas from 2001 to 2012 revealed: i) a relatively high overall infection prevalence (34.8%); ii) the circulation of several genetically diverse variants; iii) large fluctuations in infection prevalence across years, indicative of outbreaks; iv) no significant difference in the likelihood of infection between animals in different age categories. The likelihood of sapovirus infection decreased with increasing hyena group size, suggesting an encounter reduction effect, but was independent of socially mediated ano-genital contact, or the extent of the area over which an individual roamed.

Zebra Alphaherpesviruses (EHV-1 and EHV-9): Genetic Diversity, Latency and Co-Infections.

Alphaherpesviruses are highly prevalent in equine populations and co-infections with more than one of these viruses' strains frequently diagnosed. Lytic replication and latency with subsequent reactivation, along with new episodes of disease, can be influenced by genetic diversity generated by spontaneous mutation and recombination. Latency enhances virus survival by providing an epidemiological strategy for long-term maintenance of divergent strains in animal populations. The alphaherpesviruses equine herpesvirus 1 (EHV-1) and 9 (EHV-9) have recently been shown to cross species barriers, including a recombinant EHV-1 observed in fatal infections of a polar bear and Asian rhinoceros. Little is known about the latency and genetic diversity of EHV-1 and EHV-9, especially among zoo and wild equids. Here, we report evidence of limited genetic diversity in EHV-9 in zebras, whereas there is substantial genetic variability in EHV-1. We demonstrate that zebras can be lytically and latently infected with both viruses concurrently. Such a co-occurrence of infection in zebras suggests that even relatively slow-evolving viruses such as equine herpesviruses have the potential to diversify rapidly by recombination. This has potential consequences for the diagnosis of these viruses and their management in wild and captive equid populations.

Correction: Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

[This corrects the article DOI: 10.1371/journal.pone.0128706.].