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Background: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse., Results: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly., Conclusion: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.