ABSTRACT Anthropogenic land-use change influences multiple dimensions of infectious disease transmission among humans and other animals. Under parent grant RO1 TW011493, we are taking an ecological and evolutionary approach to investigate zoonotic disease emergence in rural Madagascar, where several zoonotic diseases represent significant public health challenges. To capture the complexity of dynamics at the human-animal interface, we are studying multiple host and pathogen species along gradients of land use change. An overarching goal of the research is to build epidemiologically relevant networks that represent social, close-contact, and environmental overlap among humans, domesticated mammals, and small mammals, and to test whether these “transmission potential networks” (TPNs) capture variation in infection patterns. This supplemental request addresses this important goal by funding research to screen for gastrointestinal bacterial pathogens, which are significant contributors to diarrheal diseases, one of the leading causes of death worldwide. Co-investigators in our partner lab at the University of California Santa Barbara (UCSB) will screen for a wide range of pathogenic and non- pathogenic bacteria using 16S rRNA metabarcoding, which will be supplemented with high-throughput approaches to differentiate pathogenic and non-pathogenic strains of Escherichia coli. This work will complete our characterization of major parasite groups (viruses, helminths, protozoa, and bacteria) that are spread via different transmission pathways among multiple host species. Supplemental funding is essential to this effort to extend the laboratory research at UCSB, where parent grant funding was originally focused on helminth screening. The relative ease and low cost of additional bacterial pathogen screening provides an unprecedented opportunity to enrich the multi-host, multi-pathogen dataset to achieve the Specific Aims in the parent grant. Supplemental funds will also support additional lab work to complete helminth screening, which was interrupted due to COVID-related complications of shipping, limited time in the lab, and personnel shortages. The parent RO1 grant supports all fieldwork, DNA extractions, and data analysis as part of the current sampling plan.