Occurrence of Mycobacterium sp. in euryhaline fishes in Greek aquaculture farms

Objective: Fish mycobacteriosis is a worldwide problem with an economic impact on fish industry as it involves about 150 of marine and fresh-water fish species. The development of the disease may be asymptomatic with long incubation period resulting to weight loss and mortalities in fish stocks. In Greece, aquaculture is an important economic activity with concern on fish health related problems. Under the frame of EPAL project funded by the Hellenic Ministry of Rural Development and Food, our effort was to estimate the prevalence of mycobacteriosis in fish samples collected from different sea farms. Method & Materials: Twenty one specimens (Sparus aurata), five of them asymptomatic, were randomly collected from various sea farms and subjected to mycobacteria isolation and identification analysis. For these purposes, classical and PCR techniques were involved. For classical analysis the Lowenstein-Jensen broth was used with an incubation period of 30 days at 37 degrees Celsius. For PCR analysis, a portion of internal organs was homogenized, diluted in PBS, sonificated, boiled, centrifuged and the supernatant was used as a template for PCR amplification. Primers were specific for the IS2404 sequence of M. ulcerans which was by 99.7% analogue to M. marinum. Results & Conclusion: In classical culture technique, one (4.8%) out of twenty one fish samples was positive to Mycobacterium sp., while in PCR, two samples (9.5%) were found positive. These results indicated that there was a level of agreement between the two methods and that the prevalence of mycobacteria in random symptomatic and asymptomatic samples lies between 5 and 10%. It seems that most of the Mycobacterium appears to be genetically diverse than generally assumed. This genetic diversity seems to be associated with the environment of origin and translates into significant phenotypic differences between wild isolates. Isolation and identification of mycobacteria to the species level is a laborious and time consuming task especially when conventional microbiological or biochemical methods are employed. Given the importance of mycobacteriosis in fish industry, time and cost efficient methods of identification should be adopted by the fish aquaculture industry.