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The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus-all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Abstract: Milk is an ancient, fundamental mammalian adaptation that provides nutrition and biochemical communication to offspring. Microbiomes have been detected in milk of all species studied to date. In this review, we discuss: (a) routes by which microbes may enter milk; (b) evidence for proposed milk microbiome adaptive functions; (c) variation in milk microbiomes across mammals; and (d) future research directions, including suggestions for how to address outstanding questions on the viability and functionality of milk microbiomes. Milk microbes may be sourced from the maternal gastrointestinal tract, oral, skin, and mammary gland microbiomes and from neonatal oral and skin microbiomes. Given the variety of microbial sources, stochastic processes strongly influence milk microbiome assembly, but milk microbiomes appear to be influenced by maternal evolutionary history, diet, environment, and milk nutrients. Milk microbes have been proposed to colonize the neonatal intestinal tract and produce gene and metabolic products that influence physiology, metabolism, and immune system development. Limited epidemiological data indicate that early-life exposure to milk microbes can result in positive, long-term health outcomes. Milk microbiomes can be modified by dietary changes including providing the mother with probiotics and prebiotics. Milk replacers (i.e. infant formula) may benefit from supplementation with probiotics and prebiotics, but data are lacking on probiotics' usefulness, and supplementation should be evidence based. Overall, milk microbiome literature outside of human and model systems is scarce. We highlight the need for mechanistic studies in model species paired with comparative studies across mammals to further our understanding of mammalian milk microbiome evolution. A broader study of milk microbiomes has the potential to inform animal care with relevance to ex situ endangered species., Lay Summary: Milk is an ancient adaptation that supports the growth and development of mammalian neonates and infants. Beyond its fundamental nutritional function, milk influences all aspects of neonatal development, especially immune function. All kinds of milks so far studied have contained a milk microbiome. In this review, we focus on what is known about the collection of bacterial members found in milk microbiomes. Milk microbiomes include members sourced from maternal and infant microbiomes and they appear to be influenced by maternal evolutionary history, diet, milk nutrients, and environment, as well as by random chance. Once a neonate begins nursing, microbes from milk colonize their gut and produce byproducts that influence their physiology, metabolism, and immune development. Empirical data on milk microbiomes outside of humans and model systems are sparse. Greater study of milk microbiomes across mammals will expand our understanding of mammalian evolution and improve the health of animals under human care.

Microbiome science has provided groundbreaking insights into human and animal health. Similarly, evolutionary medicine - the incorporation of eco-evolutionary concepts into primarily human medical theory and practice - is increasingly recognised for its novel perspectives on modern diseases. Studies of host-microbe relationships have been expanded beyond humans to include a wide range of animal taxa, adding new facets to our understanding of animal ecology, evolution, behaviour, and health. In this review, we propose that a broader application of evolutionary medicine, combined with microbiome science, can provide valuable and innovative perspectives on animal care and conservation. First, we draw on classic ecological principles, such as alternative stable states, to propose an eco-evolutionary framework for understanding variation in animal microbiomes and their role in animal health and wellbeing. With a focus on mammalian gut microbiomes, we apply this framework to populations of animals under human care, with particular relevance to the many animal species that suffer diseases linked to gut microbial dysfunction (e.g. gut distress and infection, autoimmune disorders, obesity). We discuss diet and microbial landscapes (i.e. the microbes in the animal's external environment), as two factors that are (i) proposed to represent evolutionary mismatches for captive animals, (ii) linked to gut microbiome structure and function, and (iii) potentially best understood from an evolutionary medicine perspective. Keeping within our evolutionary framework, we highlight the potential benefits - and pitfalls - of modern microbial therapies, such as pre- and probiotics, faecal microbiota transplants, and microbial rewilding. We discuss the limited, yet growing, empirical evidence for the use of microbial therapies to modulate animal gut microbiomes beneficially. Interspersed throughout, we propose 12 actionable steps, grounded in evolutionary medicine, that can be applied to practical animal care and management. We encourage that these actionable steps be paired with integration of eco-evolutionary perspectives into our definitions of appropriate animal care standards. The evolutionary perspectives proposed herein may be best appreciated when applied to the broad diversity of species under human care, rather than when solely focused on humans. We urge animal care professionals, veterinarians, nutritionists, scientists, and others to collaborate on these efforts, allowing for simultaneous care of animal patients and the generation of valuable empirical data., (© 2023 Cambridge Philosophical Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Host-associated microbiomes are influenced by evolutionary history and proximate factors such as diet and environment. Zoos house animals in relatively standardized and manipulatable environments, making zoo populations valuable for studying microbiomes. Using a small population of five, closely related primate species housed under nearly identical environments, we investigated gut microbiome variation regarding (a) congruence between host evolutionary history and gut bacterial composition (i.e. phylosymbiosis), (b) a longitudinal reduction in dietary sugar intake, and (c) ingestion of bacteria from dietary sources. We found that the primate gut microbiomes varied across individuals and showed phylosymbiosis. When animals were fed diets with reduced sugar and increased fibre, we found host-specific changes in taxonomically distinct microbes (Phascolarctobacterium, Megasphaera, and Sharpea). Yet, these bacterial genera share similar functional potential (fibre degradation), indicating that the distinct bacterial communities may fulfill similar functions. Although all individuals received the same diet, the diet-associated bacteria in primate gut microbiomes were distinct across individuals of different species, suggesting a mechanism that selects for unique dietary microbes to persist in animal guts. Our findings show that the microbiomes of a small, captive primate population housed under uniform environmental conditions still show patterns congruent with combined influences of evolutionary history and diet., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Milk production is an ancient adaptation that unites all mammals. Milk contains a microbiome that can contribute to offspring health and microbial-immunological development. We generated a comprehensive milk microbiome dataset (16S rRNA gene) for the class Mammalia, representing 47 species from all placental superorders, to determine processes structuring milk microbiomes. We show that across Mammalia, milk exposes offspring to maternal bacterial and archaeal symbionts throughout lactation. Deterministic processes of environmental selection accounted for 20% of milk microbiome assembly processes; milk microbiomes were similar from mammals with the same host superorder (Afrotheria, Laurasiathera, Euarchontoglires, and Xenarthra: 6%), environment (marine captive, marine wild, terrestrial captive, and terrestrial wild: 6%), diet (carnivore, omnivore, herbivore, and insectivore: 5%), and milk nutrient content (sugar, fat, and protein: 3%). We found that diet directly and indirectly impacted milk microbiomes, with indirect effects being mediated by milk sugar content. Stochastic processes, such as ecological drift, accounted for 80% of milk microbiome assembly processes, which was high compared to mammalian gut and mammalian skin microbiomes (69% and 45%, respectively). Even amid high stochasticity and indirect effects, our results of direct dietary effects on milk microbiomes provide support for enteromammary trafficking, representing a mechanism by which bacteria are transferred from the mother's gut to mammary gland and then to offspring postnatally. The microbial species present in milk reflect both selective pressures and stochastic processes at the host level, exemplifying various ecological and evolutionary factors acting on milk microbiomes, which, in turn, set the stage for offspring health and development.

The microbes inhabiting an animal's gastrointestinal tracts, collectively known as the gut microbiome, are vital to animal health and wellbeing. For animals experiencing gut distress or infection, modulation of the gut microbiome, for example, via fecal microbiota transplant (FMT), provides a possible disease prevention and treatment method. The beneficial microbes present in the donor's transplanted feces can help combat pathogens, assist in digestion, and rebalance the recipient's microbiota. Investigating the efficacy of FMTs in animal health is a crucial step toward improving management strategies for species under human care. We present a case study of the use of FMTs in a two-toed sloth experiencing abnormally large, clumped, and frequent stools. We used 16 S rRNA amplicon sequencing of fecal samples to (a) compare the microbiomes of the FMT donor, a healthy, cohoused conspecific, and the FMT recipient and (b) assess the influence of multiple rounds of FMTs on the recipient's microbiome and stool consistency and frequency over time. In response to the FMTs, we found that the recipient's microbiome showed trends toward increased diversity, shifted community composition, and altered membership that more resembled the community of the donor. FMT treatment was also associated with marked, yet temporary, alleviation of the recipient's abnormal bowel movements, suggesting a broader impact on gut health. Our results provide valuable preliminary evidence that FMT treatments can augment the recipient's gut microbiome, with potential implications for animal health and management., (© 2023 Wiley Periodicals LLC.)

Microbial rewilding, whereby exposure to naturalistic environments can modulate or augment gut microbiomes and improve host-microbe symbiosis, is being harnessed as an innovative approach to human health, one that may also have significant value to animal care and conservation. To test for microbial rewilding in animal microbiomes, we used a unique population of wild-born ring-tailed lemurs (Lemur catta) that were initially held as illegal pets in unnatural settings and, subsequently, relocated to a rescue center in Madagascar where they live in naturalistic environments. Using amplicon and shotgun metagenomic sequencing of lemur and environmental microbiomes, we found multiple lines of evidence for microbial rewilding in lemurs that were transitioned from unnatural to naturalistic environments: A lemur's duration of exposure to naturalistic settings significantly correlated with (a) increased compositional similarly to the gut communities of wild lemurs, (b) decreased proportions of antibiotic resistance genes that were likely acquired via human contact during pethood, and (c) greater covariation with soil microbiomes from natural habitats. Beyond the inherent psychosocial value of naturalistic environments, we find that actions, such as providing appropriate diets, minimizing contact with humans, and increasing exposure to natural environmental consortia, may assist in maximizing host-microbe symbiosis in animals under human care., (© 2022. The Author(s).)

For captive primates, greater provisioning of leafy greens or foliage can promote natural foraging behavior while boosting fiber intake. Recalcitrant fiber, although minimally available to endogenous metabolism, is readily fermented into nutrients by gut microbes. Whereas most primates in captivity consume fiber-limited diets and harbor imbalanced gut microbiota compared to their wild conspecifics, the importance of fiber provisioning to primate gut microbiota has predominately been studied in folivores. We, therefore, determined if commercial lettuce could be used to encourage foraging behavior and modify the gut microbiota of captive frugivores. We provisioned ruffed lemurs (Varecia rubra and V. variegata) with romaine lettuce, on top of the standard dietary fare, for 10 consecutive days. Before and across the period of lettuce supplementation, we collected observational data of animal feeding and fecal samples for microbiome analysis, determined via amplicon sequencing. The ruffed lemurs and their gut microbes responded to lettuce provisioning. In particular, younger animals readily ate lettuce and showed no decline in consumption across study days. When controlling for the effects of host species and social-group membership, lettuce consumption shifted the composition of the gut microbiome away from each lemur's own baseline, an effect that became stronger as the study progressed. In the final study days, Ruminococcaceae UCG-008 and Akkermansia, microbes typically and respectively associated with fiber metabolism and host health, were significantly enriched in the consortia of lettuce-provisioned subjects. Ultimately, the routine offering of lettuce, leafy greens, or foliage to captive frugivores may benefit animal wellbeing., (© 2020 Wiley Periodicals LLC.)

Feeding strategy and diet are increasingly recognized for their roles in governing primate gut microbiome (GMB) composition. Whereas feeding strategy reflects evolutionary adaptations to a host's environment, diet is a more proximate measure of food intake. Host phylogeny, which is intertwined with feeding strategy, is an additional, and often confounding factor that shapes GMBs across host lineages. Nocturnal strepsirrhines are an intriguing and underutilized group in which to examine the links between these three factors and GMB composition. Here, we compare GMB composition in four species of captive, nocturnal strepsirrhines with varying feeding strategies and phylogenetic relationships, but nearly identical diets. We use 16S rRNA sequences to determine gut bacterial composition. Despite similar husbandry conditions, including diet, we find that GMB composition varies significantly across host species and is linked to host feeding strategy and phylogeny. The GMBs of the omnivorous and the frugivorous species were significantly more diverse than were those of the insectivorous and exudativorous species. Across all hosts, GMBs were enriched for bacterial taxa associated with the macronutrient resources linked to the host's respective feeding strategy. Ultimately, the reported variation in microbiome composition suggests that the impacts of captivity and concurrent diet do not overshadow patterns of feeding strategy and phylogeny. As our understanding of primate GMBs progresses, populations of captive primates can provide insight into the evolution of host-microbe relationships, as well as inform future captive management protocols that enhance primate health and conservation., (© 2019 Wiley Periodicals, Inc.)