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

海胆中间型圆斑病病原菌Flexibacter sp.生存策略的研究

基本信息

批准号：
12660159
负责人：
TAJIMA Kenichi
金额：
$ 2.18万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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.

对中间球海胆斑点病病原菌Flexibacter sp.在低温水温下的存活情况、VBNC状态下的检测方法和VBNC条件下的复苏进行了研究。主要研究结果如下：1.病原菌在75%ASW中培养15天后，在5℃时失去了集落形成能力。该状态被澄清为VBNC状态。在VBNC状态下，细菌细胞变短、杆状或圆形，对海胆失去致病性。根据该细菌的16S rRNA序列，构建了一套检测该细菌的引物。所设计的引物是针对该细菌而设计的。在含0.0017%(约60℃M Fe)三氯化铁溶液和含海胆匀浆的75%ASW中，将温度从5°升高到25μ，复苏VBNC细胞。复苏后的细胞形态与原始细胞相似，与兔抗血清凝集试验呈阳性反应，且对海胆恢复了致病能力，说明在低水温条件下，细胞进入VBNC状态，但在此状态下通过对海胆表面的黏附而存活。研究结果还清楚地表明，当水温升高时，VBNC细胞复苏，并对海胆造成疾病。