2型猪链球菌天津分离株的鉴定及其遗传特征分析

Isolation,identification and genetic characteristics analysis of Tianjin isolate of Streptococcus suis serotype 2

为了解天津市某猪场引起育肥猪神经症状的病原及其特性和遗传特征,本研究采集8份患病猪脑组织样品经细菌分离、形态学观察、gdh基因和cps2J基因的PCR扩增及序列分析,确定8份样品中的分离株均为同一2型猪链球菌(SS2),命名为TJS75。测定其生长曲线、并按照文献方法进行溶血性试验、黏附/侵入试验和对小鼠的致病性试验,分析该SS2分离株特性。结果显示,TJS75菌株经TSB培养6 h~15 h可达对数生长期,对绵羊血红细胞(RBC)具有崩解效果,可黏附并侵入PK-15细胞(黏附率和侵袭率分别为5.16%和7.90%),且感染TJS75株的BALB/c小鼠出现不同程度的行动迟缓、呼吸急促、精神萎靡和神经症状等,经测定其LD50为2.15×107cfu/mL。进一步采用高通量测序,并预测其毒力基因,分析该菌株的遗传特征,结果显示,TJS75菌株基因组全长2368195 bp,GC含量40.88%,含有2299个编码基因,其中有1822、1830和1077个基因分别注释于GO、COG和KEGG数据库,携带的16S r RNA基因序列与国内分离的SS强毒株98HAH33和05ZYH33的亲缘性较近,毒力基因的预测结果显示该菌株携带499种毒力相关基因(编码173种毒力相关因子),依据功能分类,这些毒力基因参与SS2的黏附和侵袭、溶血、促进铁转运、自溶酶的合成、降解胶原酶、唾液酸的合成等。本研究首次分离到携带MRP毒力因子的ST25型SS2,为深入开展SS2 TJS75株的致病机制研究提供了科学依据。

In order to clarify the pathogen which caused the neurological symptoms in fattening pigs from a pig farm of Tianjin as well as its characteristics and genetic characteristics,in this study,8 brain tissue samples of diseased pigs were inoculated for bacterial isolation.After morphological observation,PCR amplification of gdh gene and cps2J gene and sequence analysis were performed on the isolated strains.Finally,the isolated strain of all 8 samples was identified as the same SS2,named TJS75.The pathogenic characteristics of the SS2 isolates were analyzed using various methods,including growth curve determination,hemolytic test,adhesion and invasion experiments,and animal pathogenicity experiments.The results showed that the TJS75 strain could reach the logarithmic growth phase after TSB culture for 6 to 15 hours of in vitro cultivation and had a disintegrating effect on sheep red blood cells(RBC),and could adhere to and invade PK-15 cells(the adhesion rate and invasion rate were 5.16%and 7.90%,respectively).The pathogenicity test results showed that each group of BALB/c mice inoculated with TJS75 exhibited varying degrees of clinical symptoms such as delayed movement,shortness of breath,lethargy and nerves.Moreover,the LD50 of TJS75 strain on BALB/c mice was 2.15×107cfu/mL.Using high-throughput sequencing,analysis and virulence gene prediction to determine the genetic characteristics of strains.The results showed that the total genome length of TJS75 strain was 2368195bp,with a GC content of 40.88%and containing 2229 coding genes.A total of 18221830,and 1077 genes were annotated in the GO,COG and KEGG databases.The 16S rRNA gene sequence of TJS75 strain was highly related to the highly virulent SS strains 98HAH33 and 05ZYH33,which were isolated in China.The predicted results of virulence genes showed that TJS75 strain contained 499 virulence related genes(encoding 173 virulence related factors).According to its functional classification,the functions of these virulence related factors included adhesion and invasion,hemolysis,promoting iron transport,participating in the synthesis of autolytic enzymes,degrading collagenase,and participating in sialic acid synthesis.This study would provide a scientific basis for further research on the pathogenic mechanism of SS serotype 2 strain TJS75.