养殖刺参腐皮综合症病灶处七株优势菌生理生化特性及其群体感应信号分子活性检测
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摘要
随着国内刺参养殖业的迅速发展,养殖过程中相继出现了一系列病害问题,给刺参养殖业带来了巨大的损失。腐皮综合症因其传染性高,死亡率高而成为刺参养殖病害的研究重点。该病发病特征为:染病初期,先从口部出现感染症状,表现为围口膜松弛,触手对外界刺激反应迟钝,继而大部分刺参会出现排脏现象。中期感染的刺参身体收缩、僵直,一般口腹部出现小面积溃疡,形成蓝白色小斑点,渐渐丧失摄食能力。感染末期,病灶变大,溃疡处增多,最后导致刺参死亡,溶化成鼻涕状胶体。
关于刺参疾病的研究目前国内外均处于初级阶段,一是因为刺参病害是近几年才出现的;二是因为病害发生情况随机,没有规律可循;三是发病原因和过程多种多样,有种质抗病力差、机械损伤、养殖环境不良等内外因素,使问题更加复杂化。
为研究患病刺参腐皮综合症的发病机理及病灶处优势菌的性质和群体感应系统,本文以青岛某养殖场的养殖刺参为材料,对从患病刺参病灶部位分离纯化得到的七株优势菌,即:C6、4、TB、BP2、BP3、BP4、BP6,进行理化性质研究,结果表明,这七株菌生长较为迅速,TB、BP2和BP3在培养13小时左右便进入平台期;4、BP4和BP6培养19小时左右生长进入平台期;C6生长最慢,需要培养22小时后生长进入平台期。最佳培养条件为:盐度33～36%o,温度32℃左右,pH值7～8左右。抗生素敏感实验及生化指标测定的结果表明,这七株菌对氟嗪酸、氯霉素、先锋必、复方新诺明、红霉素、庆大霉素、氟哌酸等抗生素有广泛的抗性;在测试的20项生化指标中精氨酸双水解酶、赖氨酸脱羧酶、鸟氨酶脱羧酶、脲酶、色氨酸脱氨酶、3-羟基丁酮酶均为阳性反应。
通过对七株优势菌进行群体感应信号分子的提取、纯化、鉴定等方面的研究发现,菌株C6、4、TB、BP2、BP3、BP4和BP6的群体感应信号分子确实存在,但是其产生量很少,与阳性对照有鲜明的对比。AHLs是由一个可变的乙酰基链尾部与一个稳定的高丝氨酸内酯头部相连,该结构在酸性条件下最稳定,强碱可能导致高丝氨酸内酯开环,从而破坏其结构。实验表明,七株优势菌株群体感应信号分子的活性与培养基pH值的变化基本上成反比,由此推测,在偏碱性的液体中,很可能会造成信号分子的分解而降低其活性。根据相关资料显示,刺参养殖池中移植大叶藻能够大幅减少腐皮综合症的发病几率。由此推测,大叶藻中很可能含有能够拮抗AHLs的活性物质,如果能将这种活性物质提取出来,将对防治刺参腐皮综合症有积极的意义。本研究为进一步探索养殖刺参腐皮综合症的发病原因、过程和新型抗菌手段以及其它致病菌的抗感染研究提供重要的参考,在水产养殖业的抗菌感染中具有理论价值和实际意义。
Along with the domestic rearing industry of Stichopus japonica rapidly expand, a series of diseases are booming, which bring severe economic loss. Reared Stichopus japonica in China suffers from a kind of skin ulceration disease, which has higher mortality rate and infectivity, become to be the research focus. The infection involves a head shaking and viscera expelling at early time, and is followed with mouth and cloacal opening tumidity, then the body of the diseased individuals will be shrinking and rigidity together with white or blue lesion spots on the body wall. In the later period, these small lesion spots grow more and bigger to large ulceration and finally cause the death of individuals. This disease can often cause all individuals reared in the same pool die in a very short time. And the pathogens are supposed to be bacterial by most researchers. However, the relevant research has in the primary stage at present, for the diseases was random occurrence and much more environmental factors which led it is more difficult to solve.
In order to study the pathogenesis and the Quorum sensing research of reared Stichopus japonica suffers from a kind of skin ulceration disease, this article takes the reared Stichopus japonica from Qingdao as the material, separate 7 strains: C6, 4, TB, BP2, BP3, BP4 and BP6. The physics and chemistry research indicated that these strains grow rapidly: after 22 hours, C6 grows in its platform stage, 4, BP4 and BP6 need 19 hours, and then, TB, BP2 and BP3 only need 13 hours. The best cultivate conditions are: Salinity 33-36‰, temperature about 32℃, pH value about7~ 8. The antibiotic sensitive experiments show that these bacterium strains have widespread resistance on Chloromycetin, Trimethoprim and sulphame-thoxazole, Erythromycin, Gentamycin, Fluperacid and so on. In the API-20E experiments, arginine double hydrolytic enzyme, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase and 3- hydroxy-butanone enzymes are the positive reactions.
Through research on the extraction, purification, appraisal and so on of the 7 strains' quorum sensing auto-inducer, it is indicated that the auto-inducers of the 7 strains are really existed, but have the low quantity and show the sharp contrast with the positive control. The molecular structure of AHLs is connected by invariable acetazolamide chain and stable L-homoserine lactones. This structure is more stable under the acidic condition and strong base will led the L-homoserine lactones split-ring, then destroy its structure. The experiments indicated that the activeness of the 7 strains' quorum sensing auto-inducers and the culture medium pH value change basically is in reverse proportion. So it can be believed that in the higher pH value liquid will possibly cause the auto-inducer breakdown to reduce its activeness. According to the correlation data, transplants the eelgrass may reduce the disease morbidity. It seems that there is a promising alternative for antibiotics in fighting bacterial infections by using techniques that disrupt quorum sensing systems of pathogenic bacteria. This new approach might also have value in aquaculture since a link between quorum sensing and virulence factor expression in several aquatic pathogens has been demonstrated. Although the datas about the impact of quorum sensing on virulence of aquatic pathogens are still lacking, fundamental research in the quorum sensing's domain will provide much more information to help people find the directed search for antagonists.
关于刺参疾病的研究目前国内外均处于初级阶段,一是因为刺参病害是近几年才出现的;二是因为病害发生情况随机,没有规律可循;三是发病原因和过程多种多样,有种质抗病力差、机械损伤、养殖环境不良等内外因素,使问题更加复杂化。
为研究患病刺参腐皮综合症的发病机理及病灶处优势菌的性质和群体感应系统,本文以青岛某养殖场的养殖刺参为材料,对从患病刺参病灶部位分离纯化得到的七株优势菌,即:C6、4、TB、BP2、BP3、BP4、BP6,进行理化性质研究,结果表明,这七株菌生长较为迅速,TB、BP2和BP3在培养13小时左右便进入平台期;4、BP4和BP6培养19小时左右生长进入平台期;C6生长最慢,需要培养22小时后生长进入平台期。最佳培养条件为:盐度33～36%o,温度32℃左右,pH值7～8左右。抗生素敏感实验及生化指标测定的结果表明,这七株菌对氟嗪酸、氯霉素、先锋必、复方新诺明、红霉素、庆大霉素、氟哌酸等抗生素有广泛的抗性;在测试的20项生化指标中精氨酸双水解酶、赖氨酸脱羧酶、鸟氨酶脱羧酶、脲酶、色氨酸脱氨酶、3-羟基丁酮酶均为阳性反应。
通过对七株优势菌进行群体感应信号分子的提取、纯化、鉴定等方面的研究发现,菌株C6、4、TB、BP2、BP3、BP4和BP6的群体感应信号分子确实存在,但是其产生量很少,与阳性对照有鲜明的对比。AHLs是由一个可变的乙酰基链尾部与一个稳定的高丝氨酸内酯头部相连,该结构在酸性条件下最稳定,强碱可能导致高丝氨酸内酯开环,从而破坏其结构。实验表明,七株优势菌株群体感应信号分子的活性与培养基pH值的变化基本上成反比,由此推测,在偏碱性的液体中,很可能会造成信号分子的分解而降低其活性。根据相关资料显示,刺参养殖池中移植大叶藻能够大幅减少腐皮综合症的发病几率。由此推测,大叶藻中很可能含有能够拮抗AHLs的活性物质,如果能将这种活性物质提取出来,将对防治刺参腐皮综合症有积极的意义。本研究为进一步探索养殖刺参腐皮综合症的发病原因、过程和新型抗菌手段以及其它致病菌的抗感染研究提供重要的参考,在水产养殖业的抗菌感染中具有理论价值和实际意义。
Along with the domestic rearing industry of Stichopus japonica rapidly expand, a series of diseases are booming, which bring severe economic loss. Reared Stichopus japonica in China suffers from a kind of skin ulceration disease, which has higher mortality rate and infectivity, become to be the research focus. The infection involves a head shaking and viscera expelling at early time, and is followed with mouth and cloacal opening tumidity, then the body of the diseased individuals will be shrinking and rigidity together with white or blue lesion spots on the body wall. In the later period, these small lesion spots grow more and bigger to large ulceration and finally cause the death of individuals. This disease can often cause all individuals reared in the same pool die in a very short time. And the pathogens are supposed to be bacterial by most researchers. However, the relevant research has in the primary stage at present, for the diseases was random occurrence and much more environmental factors which led it is more difficult to solve.
In order to study the pathogenesis and the Quorum sensing research of reared Stichopus japonica suffers from a kind of skin ulceration disease, this article takes the reared Stichopus japonica from Qingdao as the material, separate 7 strains: C6, 4, TB, BP2, BP3, BP4 and BP6. The physics and chemistry research indicated that these strains grow rapidly: after 22 hours, C6 grows in its platform stage, 4, BP4 and BP6 need 19 hours, and then, TB, BP2 and BP3 only need 13 hours. The best cultivate conditions are: Salinity 33-36‰, temperature about 32℃, pH value about7~ 8. The antibiotic sensitive experiments show that these bacterium strains have widespread resistance on Chloromycetin, Trimethoprim and sulphame-thoxazole, Erythromycin, Gentamycin, Fluperacid and so on. In the API-20E experiments, arginine double hydrolytic enzyme, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase and 3- hydroxy-butanone enzymes are the positive reactions.
Through research on the extraction, purification, appraisal and so on of the 7 strains' quorum sensing auto-inducer, it is indicated that the auto-inducers of the 7 strains are really existed, but have the low quantity and show the sharp contrast with the positive control. The molecular structure of AHLs is connected by invariable acetazolamide chain and stable L-homoserine lactones. This structure is more stable under the acidic condition and strong base will led the L-homoserine lactones split-ring, then destroy its structure. The experiments indicated that the activeness of the 7 strains' quorum sensing auto-inducers and the culture medium pH value change basically is in reverse proportion. So it can be believed that in the higher pH value liquid will possibly cause the auto-inducer breakdown to reduce its activeness. According to the correlation data, transplants the eelgrass may reduce the disease morbidity. It seems that there is a promising alternative for antibiotics in fighting bacterial infections by using techniques that disrupt quorum sensing systems of pathogenic bacteria. This new approach might also have value in aquaculture since a link between quorum sensing and virulence factor expression in several aquatic pathogens has been demonstrated. Although the datas about the impact of quorum sensing on virulence of aquatic pathogens are still lacking, fundamental research in the quorum sensing's domain will provide much more information to help people find the directed search for antagonists.
引文
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