Metagenomics in One Health — from standardization to targeted application

This thesis is meant to further the applicability of metagenomics in One Health by discussing key forms of standardization, as well as targeted applications. Metagenomics is a powerful pathogen-agnostic tool, allowing for broad-range non-targeted detection of microorganisms. This is especially useful for application in One Health investigations to detect known and unknown microorganisms in animals, humans and the environment. In this thesis, I highlight the benefits and limitations of metagenomics in One Health and urge for the continual adaptation to new forms of standardization, evolving technology, and novel areas of research. Standardization of terms and techniques is a key challenge in One Health investigations as the field is highly interdisciplinary with many cross-continental collaborations and large-scale consortiums. This thesis addresses two forms of standardization to improve effective communication and interdisciplinary exchange. First, it highlights the need for an empirical framework to determine a ‘disease reservoir’, a central topic for pathogens of zoonotic or anthropozoonotic origin (Chapter B1). To exemplify the use of the proposed criteria and avoid misunderstandings, the established framework is applied to a variety of known zoonotic diseases. In addition to standardizing terminology, this thesis establishes appropriate handling conditions for swab samples for metagenomic studies (Chapter B2). Here, I emphasize the need for appropriate controls (blank controls and mock communities), standardized procedures and continual monitoring of contamination. The presented handling conditions were used for all subsequent procedures (Chapter B3 and B4) to ensure compatibility between datasets. Based on the standardized procedures, this thesis demonstrates the applicability of target metagenomics in two studies investigating different aspects of the One Health triad. In both studies, the bacterial diversity was examined by sequencing a single hypervariable region of the 16S rRNA gene under controlled laboratory conditions and thus provide important baseline data for future investigations. The first study focused on examining the urogenital microbiota of rhesus monkeys (Macaca mulatta), a commonly-used translational animal model (Chapter B3). Endocrine-regulated processes, cage-mate relationships and group associations were found to impact the urogenital microbiota of rhesus monkeys at the German Primate Center. Based on the observed plasticity of the urogenital microbiota, an increased awareness of microbiota considerations of translational animal models is essential. Additionally, the identified microbiota provides baseline data for One Health investigations on wild non-human primates in health and disease. Here, Treponema species have been previously identified in multiple taxa of wild non-human primates. To further explore the diversity of these medically and ecologically-relevant bacteria, the second application of metagenomics in this thesis focused on establishing and validating a metataxonomic tool to identify Treponema (Chapter B4). The incorporation of a spirochete-specific enrichment step and the modular amplicon approach allowed us to detect Treponema to a near species-level using only a small amount of DNA. The presented data from the in-silico and in-vitro experiments using mock communities and clinical samples provided confidence in the applicability of the metataxonomic approach. Further characterization of different microbiota with a focus on Treponema will provide informative data about microorganism-host relationships in various animals and ecosystems within the One Health triad