Acute diarrhoea in horses is still a major challenge in veterinary practice and is associated with significant morbidities and mortalities. Numerous pathogens have been suggested in the pathogenesis of this clinical syndrome, including bacteria, viruses and parasites, but definitive proof of the association is often lacking due to the absence of large-scale control-case studies. An early and reliable diagnosis of the causative agent is critical to implement an appropriate treatment and improve the survival rate of the diseased animal. To achieve this goal, field veterinarians need a sensitive, fast and multipathogen diagnostic tool. The emergence of new laboratory methods such as next-generation sequencing opened new avenues for the identification of the pathogens involved. The use of such methods on large numbers of high-quality clinical cases and appropriate controls followed by a robust statistical analysis would allow the identification of the infectious agents significantly associated with diarrhoea in horses. In addition, these methods will permit an accurate evaluation of local geographic variants of the most important pathogens in Western Europe, while the current published data mostly concern the American continent. The work plan of the project is multidisciplinary and proposes to establish and validate multiplex real time assays to target the most significant pathogens associated with the syndrome of equine acute diarrhoea, adapted to the situation in Western Europe, to the benefit of equine veterinarians.