鸭疫里默氏杆菌特异性单克隆抗体的研制以及生物被膜和OmpA缺失株的研究
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摘要
鸭疫里默氏杆菌病(俗称鸭传染性浆膜炎)是由鸭疫里默氏杆菌(Riemerella anatipestifer,RA)引起的鸭、鹅、火鸡等多种其他鸟类的一种高致病性、接触传染性疾病。目前已报道的RA血清型有21个,各血清型之间缺乏交叉保护,给该病的疫苗防治带来了不便。国内养鸭集中地区的绝大多数鸭场都发生过RA感染,且存在持续性感染的问题,其机制到目前为止还未知。另外,目前对RA致病机制以及毒力因子的研究仍属空白。
本研究通过制备血清1型、2型RA的单抗来检测其与RA不同血清型分离株的交叉反应性,旨在寻找各血清型RA之间的共同抗原用于免疫诊断和疫苗研制。本研究共获得了14株针对血清1型RA的单抗和12株针对血清2型RA的单抗。ELISA试验表明14株血清1型单抗对相同血清1型菌株和血清6型、12型以及15型菌株均有反应性,说明血清1、6、12和15型RA之间存在共同的抗原表位。12株血清2型单抗中的3株与所有被检测的RA菌株都有反应性,说明不同血清型RA菌株具有共同的抗原表位。
生物被膜的形成通常与细菌的毒力相关。本试验测定了43株RA的生物被膜形成能力,并测定了CH3菌株形成生物被膜后生化特性的变化以及具有不同生物被膜形成能力的3株RA分离株的雏鸭致病性。结果表明RA菌株能形成生物被膜(18/43),葡萄糖能抑制生物被膜的形成但蔗糖对生物被膜的形成基本没有影响,生物被膜的形成增强了RA对抗生素和去污剂的抵抗。RA生物被膜的形成能力与致病性无直接相关性,但形成生物被膜后对环境抵抗力的增强能使该菌长期存活,因此也许是鸭场中RA感染持续性存在的原因之一。
本研究利用自杀性载体首次成功构建了RA Th4菌株的OmpA缺失株,为进一步研究OmpA在RA致病机制中的作用奠定基础。
Riemerella anatipestifer infection is a contagious disease of ducks, gose, turkeys and various other birds, and has caused the highest mortality among duck diseases. 21 serotypes had been identified by slide and tube agglutination tests with antisera. There was no cross-protection among the 21 serotypes. RA infection has been a continued problem in many duck farms.The precise mechanisms of RA persistence in duck farms are still unknown.
In this study, we prepared 14 or 12 monoclonal antibodies (mAbs) against serotype 1 or 2 Riemerella anatipestifer (RA). All 14 mAbs from serotype 1 reacted with all RA strains of serotype 1, 6, 12 and 15 using ELISA. It was suggested that common antigens existed among strains of those serotypes. Importantly, 3 mAbs from serotype 2 reacted with all the strains of different serotypes tested, suggesting common epitope existed among different serotype strains.
It was reported that bacterial biofilm was related to the bacterial virulence. In this study, we investigated the ability of 43 RA strains to produce biofilm, tested the effect of various chemical treatments on biofilmed versus planktonic isolates, and challenged ducklings with RA isolates to elucidate whether the ability to form biofilm plays a role in the persistence of RA infections. The result indicated that RA isolates produced biofilm (18 out of 43). Glucose, but not sucrose, was shown to inhibit biofilm formation, antimicrobial agents and detergent often fail to reach bacteria which are enclosed in a biofilm. Although we failed to demonstrate a directly correlation between biofilm-forming ability and virulence in RA, but successfully concluded that biofilmed RA isolates are more resistant than planktonic ones. This conclusion may explain the persistence of RA infections on duck farms, as infections by biofilmed bacteria are difficult to clear and viable biofilm-enclosed bacteria can cause a second infection when conditions are favorable.
Using suicide vector, we obtained OmpA deleted mutant from RA strain Th4. The mutant will be helpful for the further study of OmpA as a virulence factor.
本研究通过制备血清1型、2型RA的单抗来检测其与RA不同血清型分离株的交叉反应性,旨在寻找各血清型RA之间的共同抗原用于免疫诊断和疫苗研制。本研究共获得了14株针对血清1型RA的单抗和12株针对血清2型RA的单抗。ELISA试验表明14株血清1型单抗对相同血清1型菌株和血清6型、12型以及15型菌株均有反应性,说明血清1、6、12和15型RA之间存在共同的抗原表位。12株血清2型单抗中的3株与所有被检测的RA菌株都有反应性,说明不同血清型RA菌株具有共同的抗原表位。
生物被膜的形成通常与细菌的毒力相关。本试验测定了43株RA的生物被膜形成能力,并测定了CH3菌株形成生物被膜后生化特性的变化以及具有不同生物被膜形成能力的3株RA分离株的雏鸭致病性。结果表明RA菌株能形成生物被膜(18/43),葡萄糖能抑制生物被膜的形成但蔗糖对生物被膜的形成基本没有影响,生物被膜的形成增强了RA对抗生素和去污剂的抵抗。RA生物被膜的形成能力与致病性无直接相关性,但形成生物被膜后对环境抵抗力的增强能使该菌长期存活,因此也许是鸭场中RA感染持续性存在的原因之一。
本研究利用自杀性载体首次成功构建了RA Th4菌株的OmpA缺失株,为进一步研究OmpA在RA致病机制中的作用奠定基础。
Riemerella anatipestifer infection is a contagious disease of ducks, gose, turkeys and various other birds, and has caused the highest mortality among duck diseases. 21 serotypes had been identified by slide and tube agglutination tests with antisera. There was no cross-protection among the 21 serotypes. RA infection has been a continued problem in many duck farms.The precise mechanisms of RA persistence in duck farms are still unknown.
In this study, we prepared 14 or 12 monoclonal antibodies (mAbs) against serotype 1 or 2 Riemerella anatipestifer (RA). All 14 mAbs from serotype 1 reacted with all RA strains of serotype 1, 6, 12 and 15 using ELISA. It was suggested that common antigens existed among strains of those serotypes. Importantly, 3 mAbs from serotype 2 reacted with all the strains of different serotypes tested, suggesting common epitope existed among different serotype strains.
It was reported that bacterial biofilm was related to the bacterial virulence. In this study, we investigated the ability of 43 RA strains to produce biofilm, tested the effect of various chemical treatments on biofilmed versus planktonic isolates, and challenged ducklings with RA isolates to elucidate whether the ability to form biofilm plays a role in the persistence of RA infections. The result indicated that RA isolates produced biofilm (18 out of 43). Glucose, but not sucrose, was shown to inhibit biofilm formation, antimicrobial agents and detergent often fail to reach bacteria which are enclosed in a biofilm. Although we failed to demonstrate a directly correlation between biofilm-forming ability and virulence in RA, but successfully concluded that biofilmed RA isolates are more resistant than planktonic ones. This conclusion may explain the persistence of RA infections on duck farms, as infections by biofilmed bacteria are difficult to clear and viable biofilm-enclosed bacteria can cause a second infection when conditions are favorable.
Using suicide vector, we obtained OmpA deleted mutant from RA strain Th4. The mutant will be helpful for the further study of OmpA as a virulence factor.
引文
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