猪链球菌4型分离株生物学特性研究

Study on Biological Characteristics of Streptococcus suis Type 4 Isolates

为分析猪链球菌4型(Streptococcus suis type 4,SS4)分离株的病原生物学特性,试验对来自中国不同地区的10株猪链球菌4型进行了多位点序列分型,通过PCR方法对猪链球菌7个保守管家基因aroA、gki、dpr、mutS、recA、thrA、cpn60进行扩增,测序后将结果上传至MLST数据库查找序列型,然后制作聚类分析图来阐明菌株之间的亲缘关系;采用PCR方法对7种主要的毒力基因gdh、mrp、epf、sly、fbps、gapdh与orf2进行鉴定,通过毒力因子谱来分析猪链球菌4型毒力因子的分布;以纯化的10株细菌对BALB/c小鼠进行动物致病性试验,根据小鼠致死数量筛选出最强毒株,并进行新西兰兔致病性试验。结果显示,10株猪链球菌4型经多位点序列分型,6株为ST850型,3株为ST1006型,1株为ST94型;结合菌株分离地区分析发现,广东和江苏地区菌株有较高的同源性,江沪地区菌株出现分化现象,表现为遗传多样性;10株菌株均检测到了gdh、gapdh和orf2毒力基因,7株检测到sly基因,4株检测到fbps基因,根据毒力因子谱发现共有3个毒力基因型,gdh+sly+gapdh+orf2+型有6株,gdh+fbps+gapdh+orf2+型有3株,gdh+sly+fbps+gapdh+orf2+型仅有1株。动物致病性试验表明,10株细菌均能使BALB/c小鼠死亡,其中SH1510的半数致死量低至1×108 CFU,对小鼠的致病性最强,将纯化的SH1510菌液接种新西兰兔,可使新西兰兔出现典型的神经症状并死亡。以上结果为猪链球菌的遗传进化、毒力研究提供了新的数据,丰富了猪链球菌病的研究。

In order to analyze the pathogenic biological characteristics of Streptococcus suis type 4(SS4).10 strains of SS4 from different regions in China were subjected to multilocus sequence typing.Seven conserved housekeeping genes aroA,gki,dpr,mutS,recA,thrA and cpn60 of Streptococcus suis were amplified by PCR.The sequencing results were uploaded to MLST database to find the sequence type,and the clustering analysis map was constructed to clarify the genetic relationship in strains.The seven major virulence genes gdh,mrp,epf,sly,fbps,gapdhand orf2 were identified by PCR,and the distribution of virulence factors of SS4 was analyzed by virulence factor spectrum.BALB/c mice were subjected to animal pathogenicity test with 10 strains ofpurified bacteria,and the strongest strains were selected according to the lethal number of mice and subjected to New Zealand rabbit pathogenicity test.The results of multilocus sequence typing showed that 6strains were ST850,3strains were ST1006,and 1strain was ST94.According to the location analysis,the strains in Guangdong and Jiangsu had high homology,and the strains in Jiangsu and Shanghai showed differentiation,which showed genetic diversity.gdh,gapdh and orf2 genes were detected in 10 strains,slygene was detected in 7strains,and fbps gene were detected in 4strains.According to the virulence factor spectrum,a total of 3virulence genotypes were found,6strains were gdh+sly+gapdh+orf2+type,3strains were gdh+fbps+gapdh+orf2+type,and 1strain was gdh+sly+fbps+gapdh+orf2+type.Animal pathogenicity test results showed that 10 strains could all cause the death of BALB/c mice.The LD50 of SH1510was as low as 1×10^8 CFU,which was the most pathogenic to mice,and SH1510 could cause typical neurological symptoms and death in New Zealand rabbits.The above results provided new data for the genetic evolution and virulence studies of Streptococcus suis,which had enriched the study of Streptococcus suis.