| Summary: | Background: Mycobacteria of the <i>Mycobacterium avium</i> complex (MAC) pose a significant risk to zoological collections. <i>Mycobacterium avium</i> subspecies <i>paratuberculosis</i> (MAP) is a member of MAC and the causative agent of Johne’s disease. Despite many reports in animals kept in zoological gardens, systemic surveillance has rarely been reported. Methods: In this study, archived serum samples collected from animal species at the Wilhelma Zoological and Botanical Gardens in Stuttgart, Germany, were screened for the presence of antibodies against MAC and MAP. In addition, molecular investigations were performed on necropsy, fecal, and environmental samples. Results: In total, 30/381 serum samples of various mammalian species were positive for MAC antibodies in ELISA, while one sample of a reticulated giraffe (<i>Giraffa camelopardalis reticulata</i>) was positive in MAP-specific ELISA. Samples from many species were positive in pan-<i>Mycobacterium</i> real-time PCR (40/43 fecal samples, 27/43 environmental samples, and 31/90 necropsy samples). Surprisingly, no sample was positive in the MAP-specific molecular assays. However, two environmental samples from primate enclosures were positive in <i>Mycobacterium avium</i> subspecies <i>hominissuis</i> (MAH)-specific real-time PCR. Conclusions: The results reveal serological indications of MAC infections in the zoological collection. However, the presence of a MAP-contaminated environment by a high-shedding individual animal or MAP-infected population is unlikely.
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