Fish ulcers are spread worldwide affecting a large number of fish species in both fresh and marine water. It has been known as: ulcerative disease syndrome (UDS), ulcerative mycosis, ulcerative disease (UD), goldfish ulcer (GUD), Carp erythrodermatitis(CE) and furunculosis. Aetiology and pathogen agents are controversial, however the correlation between ulcerative outbreak and pollution are common point within the researchers. Aeromonas salmonicida has frequently been identified as the causative agent for fish ulcer, especially furunculosis. Unfortunately, bacteriologic identification of Aeromonas is time-consuming and subtyping of strain is based on complex biochemical typing schemes. The Polymerase Chain Reaction (PCR) is able to amplify very small amounts of DNA, even in biological samples such as blood. Based on this sensitive test we tried to develop a diagnostic method for Aeromonas salmonicida and Aeromonas sobria, the latter being less pathogenic for fish. In a first part, methods of different DNA preparation and PCR protocols were used to optimize the identification of Aeromonas in pure cultures. Sensitivity of the PCR test was compared with the classical bacteriologic culture. In the last part, we tried to use the optimized lysis protocol and the optimized PCR reaction on fish ulcers, sea sediment and water. As a general conclusion, it can be stated that the PCR method we developed, is a much faster method when compared to the bacteriologic culture. Furthermore, it is a sensitive technique giving positive results with Aeromonas in concentrations which gave negative culturing results. Finally, the PCR technique using specific Aeromonas salmonicida and sobria primers is specific for the relevant bacteria tested in our trials. It should also be stressed that this PCR technique can be easily applied in third world countries since only a limited set of primers have to be synthesized, simple bacterial fragmentation is used and no expensive radioactivity nor restriction enzymes are necessary. |
