Studies on survival strategy of Flexibacter sp., the causative bacterium of spotting disease of sea urchin Strongylocentrotus intermedius

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The survival in low water temperature, detection method in VBNC state and resuscitation from VBNC condition of the bacterium Flexibacter sp., the pathogen of spotting disease of sea urchin Strongylocentrotus intermedius, were examined. The important results obtained were as follwos:1. The pathogen lost its colony forming ability in 75% ASW at 5℃ after 15 days of incubation. This state was clarified as VBNC state. In VBNC state the bacterial cells became short rod or round in shape and lost its pathogenicity to sea urchin.2. A primer set was constructed based on the 16S rRNA sequences for detection of the bacterium. The primer set was specific for the bacterium.3. The VBNC cells resuscitated by temperature up-shift from 5° to 25℃ in 75% ASW in the presence of 0.0017% (approximately 60μM Fe) ferric chloride solution and in 75% ASW containing homogenated sea urchin. The morphology of the resuscitated cell was similar to that of the original cell, they showed positive reaction in agglutination test against rabbit antiserum moreover, the cells regained pathogenicity to sea urchin.From the above results it is clear that during low water temperature the cells enter into VBNC state, but survive in that condition by adhering to surface fo sea urchin. The findings also clearly indicate that when the water temperature increases the VBNC cells resuscitate and cause disease to the sea urchin.