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The illegal trade of companion animals in the European Union poses several legal, ethical and health issues to the entire community. In the framework of the Biocrime Interreg project between Italy and Austria, we surveyed puppies and kittens confiscated at the borders to identify the most frequent pathogens associated with (i) the risk of spread within the shelter, (ii) the development of fatal disease and (iii) the zoonotic potential. From January 2018 to December 2020, we examined a total of 613 puppies and 62 kittens coming from 44 requisitions. Feces, skin specimens and blood sera from confiscated animals were tested to verify the presence of major infections and to assess the rabies post-vaccination immunity. Out of the total of individuals under investigation, necropsies and laboratory investigations were also performed on 79 puppies and three kittens that had died during the observation period. Results indicated a high prevalence of Canine Parvovirus (CPV) and Giardia spp. infections, CPV as the most likely cause of fatal gastroenteritis in puppies and Salmonella and Microsporum canis as major zoonotic pathogens. Conversely, both extended spectrum beta lactamases Escherichia coli and methicillin resistant Staphylococcus pseudintermedius strains as rare findings. Results highlighted that illegal animal trade could expose the human population to potential zoonotic risk and naïve animal population to potentially disrupting epidemic waves, both of these issues being largely underestimated when buying companion animals. [ABSTRACT FROM AUTHOR]