Identifying novel Mycobacterium species in fish using multiple gene phylogenetic analysis

ABSTRACT

Mycobacteria have, for a long time, been suspected to be causing severe disease in ornamental and farmed fish in Trinidad and Tobago (T&T), however, up to now, these mycobacteria species have not been identified and characterised. Many piscine mycobacteria species are also known to be zoonotic, potentially affecting human health. Objective: To identify and characterize the species of mycobacteria affecting fish (and possibly man) in T&T. Design and Methodology: Homogenised internal organs were collected from a total of 13 fish showing clinical signs consistent with mycobacterial infection. Samples were analysed using Ziehl-Neelsen (acid-fast) staining and real-time Polymerase Chain Reaction (rPCR). The species of mycobacteria were further characterised using conventional PCR targeting the 16s rRNA (564 bp), rpoB (396 bp) and sod (408 bp) genes. PCR products were sequenced and the sequences were compared with those from known and recently identified mycobacteria species through phylogenetic analysis. Results: Acid-fast non-branching bacilli were detected in all samples. All samples were also positive for Mycobacterium sp. by real-time PCR. Multi-gene phylogenetic analysis revealed the presence of two distinct species of mycobacteria. One aligned closely with Mycobacterium marinum, a well known pathogen affecting fish and man, and a second aligned closely with a species also known to affect both fish and humans, Mycobacterium stomatepiae. Conclusions: Phylogenetic analysis demonstrated the presence of two mycobacterium species in organs from fish showing clinical signs of Piscine Mycobacteriosis in T&T. Further work is needed to characterise these mycobacteria species and investigate their zoonotic potential.