【作者】 陈涛；

【导师】 姚火春；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 猪链球菌(Streptococcus suis,SS)为革兰氏阳性菌,根据菌体荚膜抗原特性的不同,可以分成35个血清型,其中以SS2流行最广、致病性最强。SS2不仅可引起猪脑膜炎、关节炎、败血症、肺炎和突然死亡,严重危害养猪业的发展,而且可感染特定人群并致死,是一种重要的人畜共患病病原菌。SS2的致病机制目前尚不明确,Egfp-HA9801重组菌株带有增强型绿色荧光蛋白基因(egfp)和壮观霉素抗性基因(spcr),通过同源重组方法构建而成,希望以此菌株作为绿色荧光蛋白标记菌株来研究SS2的致病机制。然而对Egfp-HA9801重组菌株的生物学特性、动物体内感染动态及荧光特性的考察是利用其研究SS2致病机制的重要前提。通过菌落形态观察、溶血性检测、生化试验和生长曲线测定等,比较Egfp-HA9801重组菌株与HA9801野生菌株的生物学特性。结果显示Egfp-HA9801的各项生物学特性稳定且和HA9801基本相同,表明在SS2基因组中插入egfp和spcr基因未对其生物学特性产生显著影响,这为应用Egfp-HA9801重组菌株研究SS2的动态感染机制奠定了基础。以BALB/C小鼠为动物感染模型,进行S.suis2Egfp-HA9801重组菌株和HA9801野生菌株的动物体内感染动态的比较试验。采用腹腔注射的方式,两菌株分别以不同稀释度的菌液接种各试验组小鼠,观察小鼠临床症状,统计72h内小鼠死亡数量,在接种1h、2h、4h、6h、8h、10h和12h时进行感染小鼠组织的活菌计数,绘制各组织器官内细菌含量动态变化曲线,同时对从感染小鼠体内分离的细菌进行鉴定。结果显示,Egfp-HA9801和HA9801接种小鼠后,小鼠均出现急性感染症状,并能从感染小鼠体内分离到相应菌株。两菌株的LD50分别为3.200×108CFU和3.259×108CFU,且各组织器官内细菌含量动态变化曲线也基本相同,统计学分析结果表明毒力和感染后各组织器官内细菌含量两菌株差异不显著(P>0.05)。这表明Egfp-HA9801重组菌株在毒力、感染后临床症状、感染进程方面与HA9801野生株基本相同,提示其可以作为标记菌株来研究自然状态下SS2的动态感染机制。应用荧光显微镜对Egfp-HA9801的荧光特性进行初步检测。细菌分别在28℃、30℃和37℃条件下进行体外培养,将培养12h、24h、36h和48h的菌液涂片镜检,同时以6.0×108CFU的细菌量腹腔注射BALB/C小鼠,于攻毒1h、2h、4h、6h、8h、10h和12h后处死小鼠取组织制作冰冻切片镜检。结果表明,37℃、36h条件下培养的Egfp-HA9801菌体相比较其他条件,绿色荧光更明显,而感染后的小鼠组织冰冻切片镜检,Egfp-HA9801组也可观察到明显绿色荧光。综上所述,Egfp-HA9801重组菌株的生物学特性和在BALB/C小鼠体内感染动态与HA9801野生株基本相同,且绿色荧光明显,易观察,可作为示踪菌株来研究SS2的致病机制。更多还原

【Abstract】 Streptococcus suis is a Gram-positive coccus that has been considered an important swine pathogen and a zoonotic agent causing meningitis, arthritis,septicaemia, pneumonia and even sudden death in swine and humans. So far,35different serotypes of S. suis have been described. Among them, serotype2is the most virulent and isolated from both diseased swine and humans frequently. There has been increasing numbers of studies on the virulence factors and pathogenesis of S. suis2, but the pathogenesis of SS2is not very clear by now. The S.suis2Egfp-HA9801recombinant with egfp and spcr gene, was constructed for pathogenesis studies as a EGFP labeled strain. However, firstly it is very significant to study on the biological, infection ttrends and fluorescence characteristics of the recombinant before using it on the SS2pathogenesis research.The biological characteristics were compared between the recombinant strain (Egfp-HA9801) and the wild strain (HA9801) by observing the characteristics of morphology, hemolysis, biochemistry, and the growth curve. The results showed that the biological characteristics of Egfp-HA9801were stable and similar to HA9801,and had not been clearly influenced by inserting egfp and spcr gene into SS2genome. So this study laid the foundation for using the EGFP labeled Egfp-HA9801on pathogenesis research.BALB/C mice were used as model animal to compare the characteristics of infection trends in infected animal between the S.suis2recombinant (Egfp-HA9801) and the wild strain (HA9801). The BALB/C mice were intraperitoneally inoculated with Egfp-HA9801and HA9801respectively which were diluted in sterile THB, and observed for clinical symptoms. Meanwhile, the numbers of dead mice within72hours were recorded and the dynamic curve of bacteria in different tissue also were drew according to the results of viable colony count. Finally, the bacteria isolated from the infected mice was also identified. All the infected mice had showed clear acute infectious symptoms and the inoculated bacteria could be isolated from the corresponding infected mice. Moreover, LD50of Egfp-HA9801and HA9801were3.200×108CFU and3.259×108CFU respectively and the dynamic curve of the two strains were extremely similar but no significant difference between them (P>0.05). The results also indicated that Egfp-HA9801was similar with HA9801for virulence, symptoms and infection process. Therefore, the recombinant strain can be used as EGFP labeled strain for SS2pathogenesis research under natural conditions.The fluorescence of Egfp-HA9801was detected initially with epifluorescence microscopy in this study. The bacteria cells grown at28℃,30℃and37℃for12h、24h、36h and48h were detected respectively. And the cryosections of the tissue, obtained from the dead BALB/C mice which were intraperitoneally inoculated with6.0×108CFU bacteria, were also detected. The results of this study indicated that the Egfp-HA9801cells grown at37℃for36hours showed more obvious green fluorescence than the others. Furthermore the tissue cryosections fluorescence of Egfp-HA9801-injected mice were also observed.In summary, Egfp-HA9801is extremely similar with HA9801on the characteristics of biological and infection trends, and the fluorescence of Egfp-HA9801is obvious and can be observed easily. Consequently, Egfp-HA9801can be used as a tracer on SS2pathogenesis research.更多还原