马链球菌兽疫亚种类M蛋白亚单位疫苗小鼠免疫试验

为获得控制猪链球菌病安全高效的疫苗，本试验进行了马链球菌兽疫亚种类M蛋白亚单位疫苗的研制。应用热酸法提取马链球菌兽疫亚种ATCC35246株类M蛋白，并分别采用羟基磷灰石（HAT）层析和冷酒精沉淀法进行纯化，结果得到较纯的类M蛋白。用热酸提取的粗制类M蛋白、2种纯化的类M蛋白及全菌苗免疫小鼠，结果保护率分别为92％（11／12）、100％（12／12）、100％（12／12）和83％（10／12），表明类M蛋白亚单位疫苗有进一步开发的必要。     

马链球菌兽疫亚种ATCC35246酮基转移酶的原核表达及其免疫保护试验

根据已发表的马链球菌兽疫亚种MGCS10565酮基转移酶(transketolase)的基因序列,设计并合成引物。以ATCC35246株基因组DNA为模板,通过PCR技术,扩增出目的基因并定向克隆至表达载体pET-28a(+)中,然后将重组质粒转化入大肠杆菌BL21(DE3)中,分析并纯化表达产物。选用ICR小鼠作为实验动物模型,以纯化的重组融合蛋白通过皮下注射途径免疫小鼠,并用间接ELISA法监测小鼠血清中的抗体效价。结果表明重组蛋白免疫小鼠后能产生有效的免疫应答,血清中抗体水平有明显的升高。加强免疫2周后,以5LD50的ATCC35246强毒株攻击免疫组及对照组,结果免疫组小鼠的保护率可达37.5%。表明原核表达产物免疫ICR小鼠,可使其对同源菌株攻击产生一定的保护作用,在亚单位疫苗研制中具有潜在的应用价值。     

马链球菌兽疫亚种的保护性抗原SzP蛋白的鉴定与评价

马链球菌兽疫亚种是引起猪链球菌病的主要病原之一。该菌的致病机理尚不清楚,且缺乏合适的疫苗,使猪链球菌病很难得到有效的控制。本实验通过构建SzP重组表达载体,纯化重组蛋白,对其的免疫效力进行了评价。结果表明该蛋白可以诱导高滴度的血清IgG抗体,并且可提供一定的免疫保护效力。进一步研究表明该蛋白是一个重要的体内诱导抗原,可诱导高水平的Thl和Th2型免疫应答。揭示了SzP蛋白在致病过程中起到重要作用,为新型疫苗的研制及致病机制的研究奠定了基础。     

豚鼠源马链球菌兽疫亚种的分离鉴定

为了查明河北省唐山市某豚鼠养殖场发病豚鼠感染的病原菌,本试验采集发病豚鼠的眼角脓性分泌物,采用细菌分离纯化、革兰染色、生化鉴定、16S rRNA基因PCR扩增方法对分离菌进行鉴定,通过K-B试纸法检测分离菌株对24种抗菌药的敏感性,试管二倍稀释法测定19种中药对该分离菌株的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并通过动物回归试验检测该分离菌株的致病性。结果显示,分离菌株在血琼脂培养基培养24 h生长为针尖状透明菌落、呈β溶血,革兰染色为阳性圆球状、3～5个连接呈短链状,生化特性与马链球菌兽疫亚种相符,将其命名为MLQJ-1;16S rRNA序列比对结果显示,MLQJ-1与马链球菌兽疫亚种同源性高达99.72%,在系统进化树中处于同一分支,鉴定为马链球菌兽疫亚种;MLQJ-1对24种抗菌药的敏感性不同,对青霉素、头孢曲松和阿莫西林等8种抗菌药敏感,对阿米卡星、新霉素和卡那霉素等10种抗菌药耐药;苦地丁、苦参、黄芩和千里光对MLQJ-1的MIC值介于1.9～15.6 mg/mL,MBC值介于1.9～31.2 mg/mL;小鼠腹腔接种MLQJ-1菌液后24 h内全部死亡。结果表明,...     

类M蛋白亚单位疫苗的佐剂筛选

将铝胶、ISA206和CpG三种佐剂分别与热盐酸提取的马链球菌兽疫亚种(Streptococcus equi subsp. zooepidemicus)ATCC35246株类M蛋白配合,制成疫苗接种20日龄的小鼠,并设不加佐剂的类M蛋白亚单位疫苗免疫对照组.二次免疫后用ATCC35246株活菌攻击,铝胶、ISA206、CpG和对照组的保护率分别为75%(12/16)、81.25%(13/16)、68.75%(11/16)和56.25%(9/16).试验结果表明ISA206的免疫增强作用高于其他两种佐剂,可望与类M蛋白亚单位疫苗配合,用于预防猪链球菌病.     

内肽酶O对马链球菌兽疫亚种突破血脑屏障的影响

运用同源重组法构建马链球菌兽疫亚种(SEZ) ATCC35246株(SEZ WT)pepO基因缺失株(SEZ△pepO)及互补株(SEZ C△pepO),比较分析pepO基因对SEZ形态特征、生长性能、感染小鼠的能力、小鼠全血存活率及小鼠脑组织定殖能力的影响;观察感染后小鼠脑组织的病理变化;选取人脑微血管内皮细胞(hBMEC)为体外血脑屏障模型细胞,测定pepO基因对hBMEC细胞的黏附侵袭能力以及对体外血脑屏障模型的穿透能力的影响。结果显示,pepO基因缺失后不影响SEZ在培养基中的生长速率,也不影响SEZ在小鼠全血中的存活率,但缺失株感染小鼠的能力显著降低(P<0.05),在小鼠脑组织细菌载量显著降低(P<0.05);SEZ WT株和SEZ C△pepO株感染组小鼠脑组织出现明显充血、出血、水肿的脑膜炎症状,组织切片可见大量出血及炎性细胞浸润,SEZ△pepO株感染组小鼠脑组织未见明显病变;SEZ△pepO对hBMEC细胞的黏附侵袭率以及对体外血脑屏障模型的穿透率较SEZ WT和SEZ C△pepO均显著降低(P<0.001)。本研究初步证明内肽酶O与SEZ毒力密...     

断奶仔猪马链球菌兽疫亚种感染的诊断及防治

针对湖南某猪场出现的断奶仔猪体温升高、关节肿大并死亡的疫情进行了实验室诊断并提出了防治措施。通过对送检病死猪四肢关节解剖、涂片镜检、细菌分离鉴定表明该场的断奶仔猪为马链球菌兽疫亚种感染。用分离所得菌进行药物敏感性试验表明该细菌对头孢类药物比较敏感,用头孢类药物治疗能取得很好的疗效。随后用分离细菌制备的灭活疫苗接种母猪和哺乳仔猪取得了良好的预防作用。     

马链球菌兽疫亚种毒力因子研究进展

马链球菌兽疫亚种(Streptococcus equi ssp.Zooepidemicus,SEZ)属于兰氏分类C群,旧称兽疫链球菌,普遍存在于动物的黏膜,尤其是马属动物的皮肤、上呼吸道黏膜、扁桃体以及生殖道等处。可致多种家畜的炎症及败血病。毒力因子是指构成细菌毒力的物质。马链球菌兽疫亚种具有类M蛋     

快速鉴定猪链球菌和马链球菌兽疫亚种双重PCR方法的建立

本试验旨在建立一种快速、特异、敏感的双重PCR鉴定猪链球菌和马链球菌兽疫亚种病原检测方法。根据猪链球菌GDH蛋白和马链球菌类 M 蛋白的基因保守区分别设计引物,优化了该双重PCR检测方法的引物浓度及比例,并筛选了其最佳退火温度;用该双重PCR反应体系以其他几株阴性菌株为对照,检测了该反应体系的特异性。以新鲜培养的猪链球菌倍比稀释后进行菌落计数,对该检测方法的敏感性进行了鉴定。M-like和GDH引物的加入量均为1 μL(20 pmol/L),最佳退火温度为52.3℃;该双重PCR反应体系有较高敏感性,检测马链球菌兽疫亚种和猪链球菌的敏感度分别达100和10 CFU;特异性试验结果显示,常见的5种病原菌在该双重PCR体系中无特异性条带出现;临床应用该方法分离鉴定了1株猪链球菌和2株马链球菌兽疫亚种。本试验建立了一种能同时检测猪链球菌和马链球菌兽疫亚种的双重PCR方法,且该方法应用快速、特异且敏感。     

马源马链球菌兽疫亚种新疆株的分离鉴定及遗传特性分析

为了解马源马链球菌兽疫亚种（S.zooepidemicus）新疆分离株马链球菌兽疫亚种类M蛋白（SzM）基因的分子进化与变异情况,为该菌引起的感染性疾病的防控提供依据,本试验对新疆地区某马场采集的病马淋巴结样品进行病原菌的分离培养和生化鉴定,并对分离菌株进行药物敏感性试验。根据已发表的马源SzM基因序列设计引物,对其SzM基因进行PCR扩增及序列测定。将获得的SzM序列与GenBank中不同动物源马链球菌兽疫亚种分离株序列进行同源性比对和遗传进化分析。结果显示,分离得到了一株革兰氏阳性链球菌,将其命名为马链球菌兽疫亚种ZMSY15-1。药物敏感性试验结果表明,分离菌株对青霉素、磺胺嘧啶钠耐药,对其他14种药物均敏感。序列分析结果显示,马链球菌兽疫亚种ZMSY15-1与国内外不同动物源分离株SzM基因氨基酸同源性为56.0%~70.0%。遗传进化分析结果显示,这些菌株可分为4个群。马链球菌兽疫亚种ZMSY15-1与猪源分离株SzM蛋白的氨基酸同源性为59.9%,分别属于2个不同的群,其与美国马源分离株NH55426亲缘关系最近。本试验结果可丰富国内马源马链球菌兽疫亚种SzM基因的信息数据,为马链球菌兽疫亚种的致病机制研究和预防控制提供参考依据。     

2种猪源链球菌菌体细胞表面疏水性及体外结合纤连蛋白的检测

采用盐析法测定了马链球菌兽疫亚种ATCC35246株（简称35246）和猪链球菌2型9801株（简称HA9801）的表面疏水性。35246在浓度为1．0mol／L的硫酸铵中2min内析出，HA9801在0．6mol／L的硫酸铵中析出。用不同浓度的人纤连蛋白（Fn）包被ELISA板，采用ELISA方法检测了对数生长后期的35246和HA9801体外结合固定Fn的情况。35246的D415随细菌浓度的升高而增大，而9801的D415与对照相比，经t检验差异不显著（P〉0．05）。采用间接免疫荧光试验，对二者体外结合游离的人Fn的情况进行了检测，结果在2种细菌周围均见绿色荧光。结果表明，二菌均为表面疏水菌；35246株既可结合固定的Fn，又可结合游离的Fn；而HA9801株仅与游离的Fn结合。     

致病性马兽疫链球菌的分离鉴定、生物学特性及其M-like基因的序列分析

为鉴定锦州某猪场引起关节炎病疫情的病原,本研究将患猪关节液在血平板培养基上培养进行细菌分离;对分离细菌进行形态学观察、生化反应、致病性试验及药物敏感性试验;并对其M-like基因进行克隆和测序分析.结果在显微镜下观察到周围具有透明的β溶血环的革兰氏阳性中长链球菌,经生化反应及M-like基因鉴定为马兽疫链球菌(命名为10JZ12);分离株10JZ12对小鼠的致死剂量为1.75× 108 cfu;并对苯唑西林、青霉素G、红霉素、克拉霉素、克林霉素、米诺环素、环丙沙星、左氟沙星、多粘菌素B、呋喃妥因敏感,对氯霉素、四环素、诺氟沙星、复方新诺明不敏感.     

Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected.     

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.     

Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi

The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA-seeI multiplex polymerase chain reaction.     

黑豚C群兽疫链球菌病的诊断

湖南省长沙市某黑豚养殖场发生疫情,通过对发病黑豚临床和病理学检查,从病料中分离到成链状的革兰氏阳性细菌,通过鲜血琼脂平板培养菌落具β溶血,PCR扩增和测序结果显示与C群兽疫链球菌有很高同源性,在小鼠致病性试验中,发病致死的小鼠组织内可再次分离到该菌。临床与实验室检测结果表明:该黑豚确系C群兽疫链球菌感染。     

Peritonitis in a llama caused by Streptococcus equi subsp. zooepidemicus.

A 7-month-old, male llama was diagnosed with peritonitis caused by Streptococcus equi subsp. zooepidemicus. Clinical findings, medical treatment, and case outcome are described. Hematogenous dissemination from suspected pneumonia is proposed as the route of infection in this case. Possible transmission of the organism through contact with horses is discussed.     

Dissemination of the superantigen encoding genes seeL, seeM, szeL and szeM in Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Bacterial superantigens are one of the major virulence factors produced by Streptococcus pyogenes and Staphylococcus aureus. The two novel superantigen encoding genes seeM and seeL were described for S. equi subsp. equi which is known as the causative agent of strangles in equids. In the present study previously characterized S. equi subsp. equi strains and strains of various other animal pathogenic streptococcal species and subspecies were investigated for the presence of the superantigen encoding genes seeM and seeL by polymerase chain reaction. According to these studies seeL and seeM appeared to be a constant characteristic of all investigated S. equi subsp. equi strains. Surprisingly, one S. equi subsp. zooepidemicus strain (S.z. 122) was also positive for both genes. The species identity of this S. equi subsp. zooepidemicus strain could additionally be confirmed by sequencing the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. The superantigen encoding genes could not be found among additionally investigated S. equi subsp. zooepidemicus strains or among strains of seven other streptococcal species. The seeL and seeM genes of the S. equi subsp. equi strain S.e. CF32 and the genes szeL and szeM of the S. equi subsp. zooepidemicus strain S.z. 122 were cloned and sequenced. A sequence comparison revealed a high degree of sequence homology between seeL, szeL, speL and seeM, szeM and speM, respectively. The superantigenic toxins L and M seemed to be widely distributed virulence factors of S. equi subsp. equi, rare among S. equi subsp. zooepidemicus but did not occur among a number of other animal pathogenic streptococcal species.