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East Africa has one of the world’s fastest growing human populations—many of whom are dependent on livestock—as well as some of the world’s largest wildlife populations. Humans, livestock, and wildlife often interact closely, intimately linking human, animal, and environmental health. The concept of One Health captures this interconnectedness, including the social structures and beliefs driving interactions between species and their environments. East African policymakers and researchers are recognising and encouraging One Health research, with both groups increasingly playing a leading role in this subject area. One Health research requires interaction between scientists from different disciplines, such as the biological and social sciences and human and veterinary medicine. Different disciplines draw on norms, methodologies, and terminologies that have evolved within their respective institutions and that may be distinct from or in conflict with one another. These differences impact interdisciplinary research, both around theoretical and methodological approaches and during project operationalisation. We present experiential knowledge gained from numerous ongoing projects in northern Tanzania, including those dealing with bacterial zoonoses associated with febrile illness, foodborne disease, and anthrax. We use the examples to illustrate differences between and within social and biological sciences and between industrialised and traditional societies, for example, with regard to consenting procedures or the ethical treatment of animals. We describe challenges encountered in ethical approval processes, consenting procedures, and field and laboratory logistics and offer suggestions for improvement. While considerable investment of time in sensitisation, communication, and collaboration is needed to overcome interdisciplinary challenges inherent in One Health research, this can yield great rewards in paving the way for successful implementation of One Health projects. Furthermore, continued investment in African institutions and scientists will strengthen the role of East Africa as a world leader in One Health research.