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Abstract: The gastrointestinal tract is a balanced ecosystem that can get out of balance and predisposed to clostridial diseases or other pathological conditions. The objective of the present study was to evaluate the gut microbiota in dairy cows suffering from chronic botulism. Cows were investigated for Clostridium (C.) botulinum in faeces and rumen fluids. In order to study the relationship between botulism and gastrointestinal microbiota, faeces and rumen fluid were tested for bacterial composition using conventional microbiological culture techniques and fluorescence in situ hybridization (FISH). Protozoa were analyzed in rumen fluid microscopically. The presence of C. botulinum was associated with specific changes in the faecal microbiota, especially a significant reduction of total aerobic bacteria, total anaerobic bacteria, enterococci, Clostridium perfringens and yeast and fungi. Also C. botulinum positive rumen fluid had significantly more Bacteroides spp., C. histolyticum group, Alfa- proteobacteria, Gammaproteobacteria, and sulfate-reducing bacteria; as well as significantly fewer Euryaracheota, and the protozoa Epidinium spp. Dasytricha spp., Diplodiniinae spp. and Ophryoscolex spp. In conclusion, C. botulinum is common in dairy cows in Germany but the incidence of botulism is associated with microbial changes and composition in the gastrointestinal tract. Bacteria, yeast and protozoa appear to be crucial in the colonization process; however, the chronology of these events and role of each microbial group needs further evaluation. [Copyright &y& Elsevier]