Identification of mycobacteria from unhealthy and apparently healthy aquarium fish using both conventional and PCR analyses of hsp65 gene

Abstract

Seventy-eight unhealthy and forty-two apparently healthy looking freshwater aquarium fish belonging to twenty-six different species were collected from local aquarium stores in different cities and were investigated for the presence of mycobacteria in north-western Iran by culture and molecular methods. Using the culture method, Mycobacterium sp. was isolated from 31.6% of the fish (38 cases), while it was obtained in 30.8% (37 cases) by polymerase chain reaction (PCR) assay for a 439 bp fragment of the heat shock protein 65kD gene (hsp65). Acid-fast bacilli (AFB) were seen on the smears of 27 samples (22.5%) by direct microscopic examination method of Ziehl-Neelsen (ZN, for staining Mycobacterium spp. isolates). Based on all three methods, 38 purified bacterial isolates, which are well-known pathogens in humans as well as in fish, were identified: nine isolates of M. fortuitum, nine M. marinum, seven M. smegmatis, four M. terrea, four M. flavescens, three M. gordonae and two isolates of M. asiaticum. Mycobacterium marinum, M. fortuitum, M. smegmatis and M. flavescens were common in both diseased and healthy fish groups. In conclusion, considering diagnostic challenge for both culture and molecular methods of bacterial identification and their importance, especially in apparently healthy looking freshwater aquarium fish, the results of this study demonstrates that, in terms of reliable sensitivity, conventional microbial identification is superior to molecular techniques.