摘要
猪链球菌2型(Streptococcus suis type2,SS2)是猪链球菌病的重要病原,能导致仔猪患脑膜炎、败血症、关节炎、心内膜炎、肺炎,且是一种重要的人畜共患病病原,对养猪业及公共卫生均构成严重威胁。本文研究了重组猪链球菌2型MRP诱导猪肺巨噬细胞凋亡,以阐明猪链球菌2型的分子致病机理；建立了基于16S～23S rDNA区间序列(Intergenic Spacer region, ISR)的鉴定猪源链球菌的PCR方法；对猪链球菌1998年江苏分离株进行了病原学研究。
以纯化的重组猪链球菌2型MRP蛋白与猪肺巨噬细胞作用,研究MRP对猪巨噬细胞的致病作用,以探讨MRP的致病机制。通过光学显微镜形态及电子显微镜形态观察,DNA琼脂凝胶电泳和流式细胞仪检测等手段,研究MRP对猪巨噬细胞的作用。结果表明,50μg/mL和100μg/mL的重组MRP能诱导猪肺巨噬细胞发生凋亡,在作用6h后,光学显微镜和电子显微镜下可见感染细胞呈现典型的细胞凋亡形态,DNA琼脂凝胶电泳呈“梯带”图谱,流式细胞仪检测结果显示明显的亚二倍体DNA峰,凋亡率分别为10.8%和13.32%。证实MRP是SS2的重要毒力因子。
猪链球菌2型与马链球菌兽疫亚种是我国猪源链球菌病的两种主要病原,对养猪业构成严重威胁。本试验利用16S-23S rDNA区间序列(ISR)的多态性,结合16S rDNA和23S rDNA的保守性,分别在16S rDNA末端和23S rDNA前端保守区设计上下游引物,PCR结果为23株猪源和8株人源猪链球菌2型参考株及分离株扩增长度约为1180bp,1株猪链球菌2型弱毒株(T15)为1070bp,25株马链球菌兽疫亚种约为1390bp,因此由PCR产物长度可区分猪链球菌和马链球菌兽疫亚种,从而达到一对引物区分两种细菌的目的。另外,针对猪链球菌2型和马链球菌兽疫亚种,分别在16S-23S rDNA区间序列内设计两对引物,结果显示能特异性地扩增出230和190bp的目的片段。由此,基于16S-23S rDNA ISR的PCR技术能鉴定猪链球菌2型和马链球菌兽疫亚种。对三株代表菌(HA9801, T15, ATCC35246)的16S-23S rDNA ISR进行测序发现,种的差异表现为碱基的插入和缺失,此对引物为区分猪链球菌2型强毒和弱毒株提供了可能。
取1998年在江苏分离的3株猪源链球菌,进行了病原学鉴定。其形态、染色均符合链球菌的特点,具α或β溶血,生化试验结果不稳定。用国际通用的链球菌乳胶分型诊断液检测上述分离株,均不在其检测范围,即不属于链球菌兰氏分群的A～G群。但它们凝集猪链球菌2型抗血清,人工接种小鼠无致病性,兔有致病性。表明此次流行的猪败血症的病原不同于以往曾在我国广泛流行的马腺疫链球菌兽疫亚种,而是猪链球菌2型。
Streptococcus suis type2is an important pathogen of swine steptococcosis. It is responsible for a wide variety of porcine disease syndromes, such as septicaema, meningitis, arthritis and endocarditis. It also recognized as a human pathogen, causing mainly septicaema, meningitis and endocarditis. In this study, Apoptosis in porcine pulmonary alveolar macrophages is induced by recombinant MRP of Streptococcus suis type2, rapid identification of streptoccus suis type2and Streptococcus equi subsp. Zooepidemicus based on16S-23S rDNA were studied and the isolates of swine Streptococcus in Jiangsu Province during1998were identificated.
Porcine pulmonary alveolar macrophages (PAMs) were treated with recombinant MRP of Streptococcus suis type2, for studying the pathogenesis of S. suis type2. The results showed that50μg/mLand100μg/mL recombinant MRP of Streptococcus suis type2was able to induce the PAMs apoptosis after treated6h. Under light and electron microscopes, morphological changes in recombinant MRP of Streptococcus suis type2treated cells were observed, including cell shrinkage, nuclear condensation, plasma membrane blebbing, and cytoplasm vacuolization. DNA laddering was showed in agarose electrophoresis. And the subdiploid DNA peaks were observed in flow cytometry analysis, the apoptotic rates were10.8%and13.32%respectively. It's could be confirmed that MRP is the mainly virulent of Streptococcus suis type2.
Swine Streptococcicosis is an important pathogen of zoonosis mainly caused by Streptococcus suis serotype2(SS2) or S. equi subsp. zooepidemicus and generates grave threat to human health and pig-breeding. We developed a new rapid molecular method of identifying these pathogens by PCR using a pair of primers derived from the highly conserved flanking region of the3'end of16S and the5'end of23S rDNA genes. The size of PCR products showed polymorphism that the lengh of20virulent SS2strains were all1180bp, all of25S. equi subsp. zooepidemicus strains were1390bp, and1low virulent SS2(T15) strains was1070bp. The PCR method based on16S-23S rDNA intergenic spacer region (ISR) were successful in rapid identifying these two pathogen species of Swine Streptococcicosis. The sequencing results of the PCR products of the three strains, SS2(HA9801, T15) and S. equi subsp. zooepidemicus (ATCC35246), revealed that the base delection in some regions of16S-23S rDNA ISR induced the variance between these two species. In addition, the forward PCR result of SS9was almost as the same as SS2, so a pair of primer from the16S-23S rDNA ISR of SS2strains was used in order to distinguish SS2from other streptococci, and all the virulent SS2trains were definited (Lengh≈230bp).
Three isolates were obtained from diseased pigs with septicemia in Jiangsu Province in summer1998. The morphological, cultural and biochemistry characters indicated that the isolated bacteria belonged to genus Streptococcus. In addition, the API-20Strep system was reconfirmed the above result. Furthermore, the isolates could be agglutinated by the antiserum of Strep, suis type2, but not by the Lancefild group A～G latex diagnostic kit. The isolates demonstrated pathogenecity to rabbit but not to mouse. It concluded that Strep. suis type2was the pathogen of the pig disease occurred in Jiangsu during1998.
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