猪链球菌2型溶血素基因缺失菌株的构建及生物学特性的研究

溶血素(SLY)在猪链球菌2型(SS2)侵入和裂解细胞的过程中发挥重要作用,被认为是SS2的一种重要的毒力相关因子。为了探讨SLY在2005年中国四川资阳分离强毒株SS205ZY致病过程中的作用,作者利用同源重组基因敲除法成功构建了SS205ZY的sly基因敲除突变菌株(Δsly)。并比较了菌株的溶血能力以及对小鼠的致病力。结果表明sly基因敲除后可导致猪链球菌裂解红细胞的能力显著下降,对小鼠的致病力也有一定程度的减弱,但仍表现较高的致病力。本研究结果提示SLY是猪链球菌裂解细胞的重要毒力相关因子,但造成SS205ZY的高致病性很显然与多种毒力相关因子的协同作用有关。     

猪链球菌2型溶血素基因缺失株构建及其生物学特性分析

猪链球菌溶血素(SLY)是较早确定的猪链球菌毒力因子之一,具有细胞毒性,在猪链球菌致病过程中发挥重要作用。利用同源重组技术成功构建了猪链球菌2型(SS2)sly基因缺失的突变株SS2-Δsly,缺失株的体外溶血活性消失,对小鼠脑血管内皮细胞的细胞毒性显著低于亲本菌株(P<0.01),但不影响猪链球菌2型在巨噬细胞中的存活。该菌株可以用于研究SS2溶血素在感染细胞中的作用机制。     

双点突变对猪链球菌2型溶血素活性的影响

猪链球菌2型作为一种人兽共患病病原,日益受到关注。而溶血素是其分泌的外毒素,是公认的重要毒力因子之一,具有良好的免疫原性。本试验通过PCR方法获得猪链球菌2型野生型溶血素基因sly和463、464双点突变的溶血素突变体基因slym。将sly和slym克隆至表达载体,构建了2个重组载体并在大肠杆菌中表达了野生型溶血素rSLY和突变型溶血素rSLYm。经过蛋白杂交试验,证明表达的rSLY和rSLYm与提取的猪链球菌的天然野生型SLY分子量完全一致。以提取的野生型SLY为对照,通过溶血试验证明,突变型溶血素rSLYm失去了溶血活性;通过接种PK15、RK13、SUVEC细胞单层,证明突变型溶血素rSLYm失去细胞毒性;通过小鼠试验,证明突变型SLYm对小鼠没有毒力。本试验通过溶血试验、细胞接种和小鼠实验,证明双点突变灭活了野生型溶血素的溶血活性、细胞毒性和小鼠毒力。该溶血素突变体经免疫实验证实后,可作为猪链球菌2型亚单位疫苗的候选抗原。     

猪链球菌2型江苏分离株溶血素基因的检测及序列分析

根据猪链球菌2型(strep tococcus su is type 2)溶血素基因(sly)设计和合成了一对可扩增其完整阅读框的引物,对HA 9801等6株猪链球菌2型江苏分离株、1株德国分离株SS2-D及猪链球菌C群参考株ATCC 35246的核酸进行PCR扩增,结果显示HA 9801等6株江苏分离株及德国株SS2呈阳性,ATCC 35246呈阴性。HA 9801株PCR产物纯化后测序,序列分析结果表明该DNA片段与猪链球菌2型1933株的sly基因同源性为99%。     

猪链球菌2型江苏分离株溶血素基因检测及序列分析

根据猪链球菌2型（Streptococcus suis type 2）溶血素基因（sly）设计和合成了一对可扩增其完整阅读框的引物，对HA9801等6株猪链球菌2型江苏分离株，1株德国分离株SS2-D及猪链球菌C群参考株ATCC35246的核酸进行PCR扩增，结果显示HA9801等6株江苏分离株及德国株SS2呈阳性．ATCC35246呈阴性。HA9801株PCR产物纯化后测序．序列分析表明该DNA片段与猪链球菌2型1933株的sly基因同源性为99％。     

猪链球菌2型新毒力相关基因lin缺失菌株的构建

利用温度敏感型穿梭自杀质粒pSET4s,定点敲除猪链球菌2型野生型强毒株ZY458的lin基因,构建基因缺失突变菌株458△lin,利用家兔感染模型对ZY458及其突变菌株的生物学特性进行比较研究.家兔感染试验表明,接种ZY458菌株的家兔体质量下降,出现明显的临床症状,5只家兔4 d内全部死亡;而458△lin感染组家兔生长正常,未出现任何明显临床症状.结果表明,lin基因是猪链球菌2型新发现的一种毒力相关基因,在猪链球菌2型的致病过程中具有重要作用.     

天津地区猪链球菌3型菌株的致病性及耐药特性研究

试验旨在对分离自天津地区发病猪场的7株猪链球菌3型菌株(Streptococcus suis type 3,SS 3)进行致病性和耐药特性研究。应用剂量为1.0×10~7、1.0×10~8和1.0×10~9 CFU/只的细菌对小鼠进行致病力研究,用PCR方法检测7株SS 3型菌株的毒力基因mrp、ef、sly、gdh、gapdh、fbps和orf2,并对这7株SS 3型菌株进行药物敏感试验。致病力试验结果显示,接种剂量为1.0×10~9和1.0×10~8 CFU/只时分别有6和3株SS 3型菌株可使小鼠100.0%(5/5)发病死亡,接种剂量1.0×10~7 CFU时仍有1株SS 3型菌株可使80.0%(4/5)小鼠发病死亡,这7株SS 3型菌株对小鼠的致病力依次为R15056R15042=S15030Y12024=Y09011Y13125Y13164。毒力基因检测结果表明,共有3种毒力基因型,R12024、Y13164、R15042和S15030株为gdh、gapdh、fbps和orf2毒力基因阳性,Y13125和R15056株为sly、gdh、gapdh、fbps和orf2毒力基因阳性,Y09011株为gdh、fbps和orf2毒力基因阳性。药物敏感性试验结果显示,7株SS 3型对头孢喹肟相对最敏感,其次为阿莫西林、环丙沙星和磺胺间甲氧嘧啶钠,但对强力霉素100.0%耐药,且所有菌株均呈现3重以上的耐药性。本研究为进一步开展天津地区SS 3型流行特点及致病机理研究奠定了基础,可为天津地区SS 3型菌株的综合防控提供理论指导。     

猪链球菌2型重组suilysin的高效表达及其生物学特性

根据猪链球菌2型(SS2)HA9801株溶血素(suilysin,SLY)基因全长序列设计引物,扩增SLY基因,与原核表达载体pET-28a连接,转入大肠杆菌Transetta (DE3)中诱导表达,收集表达菌体,超声破壁、镍柱纯化获得可溶性rSLY蛋白,电泳鉴定后测定rSLY的溶血活性及其溶血谱,系统研究温度、酸碱度、离子强度等理化因素对rSLY溶血活性的影响.结果表明,本试验建立的rSLY原核表达体系能高效可溶性表达具有溶血活性的rSLY,纯度达电泳纯,相对分子质量为54 000左右,100 mL菌液诱导后能纯化出约8 mg电泳纯的rSLY.rSLY具有高溶血活性,溶血比活为4 057 HU/mg.rSLY对多种动物的红细胞有溶血作用,其中对猪、兔和豚鼠的作用最强.rSLY在4℃保存时溶血活性最稳定,保存48h后溶血活性不降低.在pH 6～7和离子强度为500 mmol/L时溶血活性最高.本试验获得了高表达、可溶性、高溶血活性的rSLY,首次系统研究了不同理化因素对rSLY生物学活性的影响,为SLY致病机理深入研究及SS2亚单位疫苗和诊断试剂的研制奠定了基础.     

江苏省某猪场猪链球菌分离鉴定及致病特性分析

为了探明2018年3月江苏省某规模化猪场发病猪的病原,本研究对发病猪进行采样和细菌分离,并对细菌进行了PCR鉴定、多位点序列分型(MLST)及小鼠致病力分析。结果发现:在该场4份发病猪组织中分离到猪链球菌2型(SS2)、猪链球菌9型(SS9)菌株各2株,MLST分析显示分离的SS2菌株属于ST7,SS9菌株属于ST243,SS2、SS9分离菌株的毒力基因表现型分别为mrp^+epf^+sly^+orf2^+sao^+fbps^+gdh^+和mrp^-epf^-sly^-orf2^+sao^-fbps^+gdh^+。动物试验显示其中3株猪链球菌分离菌株对BALB/c小鼠有较强致病力。研究结果为该养殖场猪链球菌感染防控措施的制定提供了重要参考。     

猪链球菌2型调控因子Rex缺失株的构建及其致病性鉴定

为鉴定猪链球菌2型强毒株SS2-1调控因子Rex基因的缺失对细菌毒力的影响,本研究利用同源重组方法构建猪链球菌Rex缺失株,命名为SS2-1△Rex,并通过缺失株的生物学特性及其小鼠致病性试验进行鉴定。结果显示亲本株、缺失株及互补株在溶血能力、增殖特性和革兰染色形态无显著差异;在对小鼠致病性试验中,接种SS2-1组小鼠死亡率为87.5%,而SS2-1△Rex组死亡率为12.5%,SS2-1△Rex/p Rex对小鼠的毒力有所恢复,死亡率为50%;Log-Rank(Mantel-Cox)对数秩检验分析结果显示亲本株和缺失株对小鼠致病性存在显著差异。本研究结果表明调节因子Rex是猪链球菌2型的毒力相关因子。本实验为深入研究Rex调控毒力因子的转录和表达及其具体的机制奠定了实验基础。     

致病性猪链球菌2型的病原分离鉴定及毒力因子的PCR检测

通过对广西某猪场急性死亡的猪进行病原分离,分离到革兰氏阳性球菌,用链球菌快速鉴定试剂条Rapid ID 32 Strep鉴定为猪链球菌2型;并对分离菌进行猪链球菌2型荚膜多糖抗原（cps2J）及其重要毒力因子溶菌酶释放蛋白（MRP）、细胞外蛋白因子（EF）和溶血素（SLY）的多重PCR检测,同时对cps2J基因进行序列测定,与GenBank发表的猪链球菌2型相比,同源性为98.8%,毒力因子MRP、EF和SLY检测均为阳性,证实广西某猪场急性死亡的猪为高毒力猪链球菌2型感染所致。动物试验中,该菌可引起小白鼠部分死亡,家兔体温最高升至40.3℃,最后败血而死。     

Distribution of capsular serotypes and virulence markers of Streptococcus suis isolated from pigs with polyserositis in Korea

The objective of this study was to determine the capsular serotypes and potential virulence factors of Streptococcus suis isolated from pigs with polyserositis. Among the 24 isolates evaluated, serotype 3 [7 (29%) of the isolates] and serotype 4 [5 (21%)] were the most common. The isolates were also studied for the presence of the genes mrp, epf, and sly, which encode muramidase-released protein (MRP), extracellular factor (EF), and suilysin (SLY), respectively. Of the 24 isolates, 8 carried mrp: 4 of serotype 3, 2 of serotype 2, and 2 of serotype 4. One mrp(+) isolate (serotype 2) also carried the epf gene. All 24 isolates carried the sly gene. The serotype and genotype distribution greatly differed from that reported for isolates from pigs with other clinical manifestations of S. suis infection in other countries.     

Putative mannose-specific phosphotransferase system component IID represses expression of suilysin in serotype 2 Streptococcus suis

In this study, we generated a genomic mutant library from a North American strain of serotype 2 Streptococcus suis using the pGh9:ISS1 transposition vector. Suilysin is the hemolysin made by S. suis. A hyper-hemolytic mutant was identified by screening for hemolytic phenotype using media with human blood. The hyper-hemolytic phenotype was characterised by a quantitative hemolysis microplate method. The use of green fluorescent protein (GFP) as a reporter also showed that suilysin gene expression was greater in the mutant. DNA sequence analysis of 3.8 kb surrounding the ISS1 insertion site revealed four open reading frames (ORFs) with three consecutive ORFs that belong to a putative mannose-specific phosphotransferase system (PTS). The S. suis gene homologous to mannose permease IID, manN, was interrupted by the transposon. A complementation test showed that manN repressed the expression of suilysin and the absence of manN was responsible for the hyper-hemolytic phenotype. However, both wild type and isogenic hyper-hemolytic mutant S. suis fermented mannose, glucose and lactose. Thus, despite its potential roles in carbohydrate transport, phosphorylation and metabolism, the manN homologue in the putative mannose-specific PTS regulates gene expression in S. suis.     

猪链球菌2型HA0609的分离及致病性研究

从江苏省某屠宰场猪的扁桃体中分离到1株细菌,通过培养特性、菌体形态、菌落形态、染色特性、生化试验以及荚膜多糖(cps)基因的PCR检测,确定为猪链球菌2型,命名为HA0609。本试验针对猪链球菌7种主要毒力因子——谷氨酸脱氢酶(gdh)、溶菌酶释放蛋白(mrp)、胞外因子(epf)、溶血素(sly)、纤连蛋白/血纤蛋白原结合蛋白(fpbs)、次黄嘌呤核苷酸脱氢酶(impdh)及毒力相关序列orf2,进行PCR检测。与已知强毒株比较,该菌株2种主要毒力因子sly和epf均为阴性。动物试验显示HA0609对猪、兔和Balb/c鼠均无致病性。     

猪链球菌2型溶血素B细胞表位预测

目的预测猪链球菌2型(SS2)溶血素(SLY)B细胞表位。方法以DNAstar分析为主,综合分析二级结构、亲水性、表面可及性及抗原性指数,辅以吴玉章氨基酸抗原指数计算方法进行SS2溶血素B细胞表位预测。结果推测最有可能的B细胞表位位于溶血素N端第74～85、231～244区域位。结论应用多参数预测SS2溶血素的特征,为表位疫苗的研制奠定了基础。     

猪链球菌4型分离株生物学特性研究

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。     

Inactivation of the sodA gene of Streptococcus suis type 2 encoding superoxide dismutase leads to reduced virulence to mice

Superoxide dismutase (SOD) is a virulence factor of certain pathogenic bacteria by diminishing the effect of oxidative burst of phagocytic cells. Earlier reports indicated the presence of manganese-cofactored SOD in Streptococcus suis type 2 (SS2). However, the biological role of SOD and its coding sequence in SS2 has not yet been characterized. The SSU1356-ORF of a clinical SS2 strain ZJ081101 encodes a protein of 201 amino acids with 81-88% identity to SodA of other Streptococcus spp. A sod deletion mutant (Δsod) from the clinical strain was constructed. SOD activity was absent in the cell extract from the Δsod mutant, but present in that from the wild-type or the sod-complemented (CΔsod) strain. The Δsod mutant was more susceptible to oxidative stresses induced by hydrogen peroxide or paraquat. Survival of the sod deletion mutant in RAW264.7 macrophages was only half of that of the wild-type strain. Deletion of sod significantly attenuated virulence of SS2 to mice. Effects of such genetic deletion were complementable using the strain CΔsod. The co-inoculation experiment in mice revealed that the Δsod mutant was far more easily cleared from the body than the wild-type strain as shown by about 3-log reduction of its infection potential in blood and tissues. In summary, we reveal an important role of SOD in pathogenesis of S. suis type 2, most probably by scavenging reactive oxygen species from macrophages.     

Prevalence of the suilysin gene in Streptococcus suis strains isolated from diseased and healthy carrier pigs

Knowledge of virulence factors of Streptococcus suis is limited. Several virulence factor candidates have been proposed, among them suilysin, which is responsible for a toxic effect on epithelial cells. The aim of this study was to detect the suilysin gene sequence in Streptococcus suis strains of various origin. In total 63 Streptococcus suis isolates were investigated. Forty four of them originated from tissues of streptococcosis affected animals. The remaining 19 strains were isolated from tonsils of healthy carrier pigs. Suilysin gene specific sequence was detected in 79% of the strains tested. In isolates obtained from pigs with signs of streptococcosis this gene sequence was recorded in 85% of cases. In Streptococcus suis strains isolated from healthy carrier pigs the suilysin gene was detected in 63% of the isolates. It seems that suilysin toxic activity is only one of the many steps involved in the pathogenesis of Streptococcus suis infection and that strain's virulence cannot be stated only on the basis of suilysin gene sequence presence.