猪链球菌2型安徽分离株多位点序列分型与毒力特征研究

Study on multilocus sequence typing and virulence characteristics of clinical Streptococcus suis type 2 isolated from Anhui province

为了解安徽地区猪链球菌2型(SS2)临床分离株毒力基因分布、分子分型特征及其与致病性的相关性,本研究收集了58株源自临床患病猪的SS2,应用PCR技术检测其毒力基因:胞外蛋白因子基因(epf)、溶菌酶释放蛋白基因(mrp)和溶血素基因(sly),应用多位点序列分型方法(MLST)对其进行基因分型,并分析其与动物致病性之间的相关性。结果显示,58株分离菌,epf^+/mrp^+/sly^+型为44.83%(26/58),epf^+/mrp^-/sly^+型为25.86%(15/58),epf^-/mrp^+/sly^+型为18.97%(11/58),epf^-/mrp^-/sly^+和epf^-/mrp^-/sly^-型均为5.17%(3/58)。共分出10种ST型,其中ST1 26株(44.83%),ST7 18株(31.03%),ST28 6株(10.34%),ST117、ST156和ST308均为1株,各占1.72%,另有4种新发现的ST型,即ST958 2株(3.45%),ST957、ST959、ST970各1株(1.72%);分为4种克隆复合物(ST1 complex、ST27 complex、ST87 complex、ST188 complex)。ST1以epf^+/mrp^+/sly^+型为主(69.23%),其中23株(88.46%)分离自全身感染病猪;ST7以epf^+/mrp^-/sly^+型为主(55.56%),其次为epf^+/mrp^+/sly^+型(38.89%),其中13株(72.22%)分离自全身感染病猪。通过对斑马鱼、昆明鼠感染试验和LD50的测定,筛选出的6株强毒菌株分为ST1型(2株)和ST7型(4株)。结果表明,epf^+/mrp^+/sly^+是安徽地区SS2的优势毒力基因型,其次为epf^+/mrp^-/sly^+型。获得10种ST型,其中4种为首次发现,并且ST1和ST7为优势型。具有ST1/epf^+/mrp^+/sly^+遗传特征的菌株在临床上均倾向于导致全身感染。本研究结果揭示了安徽地区SS2毒力基因型、MLST遗传特征与毒力、动物临床症状之间的相关性,为开展SS2流行病学研究,区分不同菌株间毒力差异的研究提供参考依据。

In order to investigate the virulence gene distribution, molecular typing characteristics and their association with pathogenicity of clinical isolates of Streptococcus suis serotype 2(SS2) in Anhui province, a total of 58 isolates of SS2 from clinically infected pigs were collected in this study. We utilized PCR to detect the epf, mrp, and sly virulence genes. In addition,the multiocus sequence typing(MLST) was used for molecular typing analysed by combining with clinical characterization and artificial infection test, comprehensively. Our results showed that the virulence genotypes were divided into ten types of ST, totally.These virulence genotypes included 44.83% of epf^+/mrp^+/sly^+(26 in 58), 25.86% of epf^+/mrp^-/sly^+(15 in 58), 18.97% of epf^-/mrp^+/sly^+(11 in 58) and 5.17% of epf^-/mrp^-/sly(3 in 58). Moreover, these ten types of ST included 26 isolates of ST1(44.83%), 18 ST7(31.03%),6 ST28(10.34%),and 1 ST117(1.72%),1 ST156(1.72%),1 ST308(1.72%),2 ST958(3.45%),1 ST957(1.72%),1 ST959(1.72%)and 1 ST970(1.72%).The last 4 kinds of ST types were newly discovered.In addition,these ten types of ST were divided into 4 cloning complexes:ST1 complex,ST27 complex,ST87 complex and ST188 complex.ST1 was mainly epfrepresenting 69.23%,and 23 in 26 stains of ST1(88.46%)were isolated from systemic infected pigs;ST7 was mainly epfand about 13 in 18 isolates(72.22%)were isolated from systemic infected pigs.Six virulent SS2 isolates,divided into 2 ST1 and 4 ST7,were sifted by artificial infection test,reference strains(HA9801 and ZY05719)comparison.The results showed that epfwas the dominant virulence genotype of SS2 in Anhui,followed by epf^+/In these ten types of ST we have identified,ST1 and ST7 were dominant types,and four types were identified for the first time.From previous statistical conclusions,SS2 isolates with ST1/epfgenetic characteristics were clinically prone to causing systemic infection,which was also proved in this study.Our data revealed the relationship between the virulence genotype,MLST genetic characteristics of SS2 and its virulence and clinical characterization in Anhui province,which provide a basis for the further SS2 epidemiological studies and the vaccine development.