The trade of ornamental tropical fish is a business of considerable economic concern. The m ain importers of aquarium fish in Europe are Asian and Latin American countries . Maintenance conditions and intercontinental travels are stress factors for fish, which can lead to diseases onset. The potential health risk related to the importation of ornamental exotic species is an aspect that must be strongly considered, especially for potentially zoonotic diseases. I n this context, fish mycobacteriosis represent a risk for health of fish, aquaculture operators and aquarium holders. T his work aims to elucidate on the presence of mycobacteria in ornamental fish imported in Italy from non-EU countries .
Healt monitoring was carried out in the two-year period 2017-2018: in the first year, 33 fish were analyzed, in the second period 70 , for a total of 103 specimens . A ll fish were sampled at importation time before selling to pet stores. Each fish was euthanized according to the current legislation , subjected to necropsy and liver collection for mycobacterial research. Liver has been homogenized, decontaminated and inoculated in selective media (Löwenstein-Jensen and Stonebrinks medium). G rown colonies were tested with Ziehl-Neelsen (ZN) staining and, if positive, identified at species level using PCR and sequencing of a portion of heat shock protein 65 (hsp65 ) gene.
Typical lesions of mycobacteriosis (miliary nodules) have not been found following autopsy; however, different non-specific clinical signs have been highlighted. The culture test showed the growth of colonies on selective media for a total of 42 specimens (40,8%) . A total of 48 isolates were tested with ZN , because some fish had morphologically different colonies (coinfections) : all staining were positive . Biomolecular analysis led to the identification of all isolates at genus level; for 38 (79,1%) strains it was also possible to determine the species. Thirteen different species of nontuberculous mycobacteria have been recognized : M. abscessus (3) , M. arceuilense (1) , M. chelonae (5) , M. conceptionense (1) , M. fortuitum (4) , M. gordonae (1) , M. immunogenum (3) , M. marinum (2) , M. mucogenicum (5) , M. peregrinum (5) , M. phocaicum (2) , M. senegalense (5) and M. septicum (1).
Literature indicates that M . marinum , M. fortuitum and M. chelonae are the most implicated species in fish mycobacteriosis episodes: the present work confirms this statement , as these species have all been isolated . Besides these, lesser known species with doubt pathogenic value have also been isolated , confirming the complexity of g enus Mycobacterium . In relation to these data, further studies regarding such species are suggested to clarify the real importance as fish and human pathogens . Given the importance of the ornamental fish market and the growing number of people approaching to aquarium species , the comparison between isolates of fish and humans should be necessary , in particular for species in which human infections are reported.