猪链球菌2型检测方法的研究及其在出入境检疫中的应用
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摘要
猪链球菌(Streptococcus suis,SS)是一种重要的人畜共患病病原菌,能引起猪的败血症、脑膜炎、肺炎、多发性关节炎和多发性浆膜炎等；该菌在特定情况下也能感染人,引起人的急性败血症、脑膜炎、心内膜炎等疾病。猪链球菌病呈世界性分布。目前,美国、荷兰、英国、加拿大、澳大利亚、新西兰、比利时、巴西、丹麦、挪威、芬兰、西班牙、德国、爱尔兰、中国、日本等几乎所有养猪国家和地区均有发生猪链球菌病的报道,这不仅给养猪业造成了巨大的经济损失,同时还对公共卫生尤其是相关从业人员的健康构成严重威胁。在中国,SS2最早于90年代初发生于广东,但未见感染人的报道；1998～1999年江苏省部分地区猪群爆发该病,同时造成25人感染发病,其中14人死亡；尤其是2005年夏季在四川猪群爆发该病,造成214人感染发病,其中39人死亡.此外,重庆、香港、江西、广东等地也有人感染猪链球菌的零星病例发生。因此,加强猪链球菌检测技术的研究,开展猪链球菌的检测,防止猪链球菌2型传入传出,为进出口贸易提供技术保障,具有重要的意义。
1.猪源动物产品中猪链球菌2型检测方法的建立
建立了猪源动物产品中猪链球菌2型的选择性增菌和选择性平板分离方法。样品与Todd-Hewitt肉汤(THB)按1：10的比例均质后,加入终浓度分别为0.2g/L叠氮钠、0.0002g/L结晶紫和0.1g/L亚硫酸钠,于37℃静置培养18h后,在含有0.2g/L叠氮钠、0.0002g/L结晶紫和0.1g/L亚硫酸钠的5%脱纤维羊血哥伦比亚琼脂的选择性平板上进行平板分离。与非选择性增菌液和分离平板相比,猪链球菌典型菌落显著增加,杂菌显著减少,分离更加容易,分离率显著提高。
2.猪链球菌2型分型血清的制备
将猪链球菌2型(SS2)参考菌株ATCC43765在Todd-Hewitt肉汤中连续传代三代,每次培养6小时,然后按照1：10的比例接种于Todd-Hewitt肉汤仅培养2小时,经灭活处理后获得含较多荚膜物质的全菌菌体抗原。该抗原按照每周三次,除第一周按2.0x109cfu/次,其余按8.0x109cfu/次静脉免疫家兔制备全菌抗血清。经测定,该血清仅与SS2发生凝集反应,凝集价为128,不与其它链球菌发生凝集反应,表明该血清具有较高的特异性,可作为SS2的分型血清。在全国检验检疫系统广泛应用,仅在江苏口岸对125株疑似菌株进行鉴定,共鉴定出17株猪链球菌2型菌株。
3.猪链球菌2型溶血素基因的检测
根据GenBank发表的猪链球菌2型(SS2)溶血素基因(sly)全序列,利用oligo(7.0)软件设计和合成了可扩增长度为495bp的引物,建立了的SS2溶血素基因的PCR检测方法。应用该方法可从SS2参考菌株ATCC43765、SS2江苏分离株HA9801中扩增出495bp的条带,利用Hind Ⅲ限制性内切酶对具有相应单一酶切位点的PCR扩增产物就进行酶切,获得预期的314bp和181bp的2个DNA片段,检测灵敏度达到2.5×102cfu/mL;但不能从马链球菌兽疫亚种ATCC35246、大肠埃希菌ATCC25922、金黄色葡萄球菌ATCC25923.单增李斯特杆菌ATCC19115/413、粪肠球菌ATCC29212中扩增出相应条带。PCR扩增产物的测序结果与GenBank的猪链球菌sly基因(Z36907)核苷酸序列完全一致。表明本方法可快速、敏感、特异地检测SS2的溶血素基因。对江苏口岸分离的125株疑似猪链球菌菌株进行溶血素基因检测,其中27株猪链球菌均检出了猪链球菌溶血素基因,阳性率为100%,其它菌株均未检出猪链球菌溶血素基因。
4.江苏出入境猪源动物产品中猪链球菌的检测
2005年～2010年,对江苏出入境猪源动物产品中进行了猪链球菌检测,通过选择性增菌和平板分离,从920份样品中分离到125链球菌菌株。通过生化鉴定、血清学鉴定和分子鉴定,共检出27株猪链球菌,阳性率为2.93%(27/920),其中猪链球菌2型为17株,阳性率为1.85%(17/920)。结果表明,江苏出口养殖场及进出境猪副产品中有猪链球菌存在,但流行率不很高。上述工作为江苏地区猪源动物产品的国际贸易提供了技术支撑,为猪链球菌病防治提供了可靠的科学依据。
相关研究内容已经纳入质检总局制定的重大动物疫情防控猪链球菌2型实施方案和猪链球菌2型检测的系列国家标准。
Streptococcus suis(SS) infections are considered a major worldwide problem in the swine industry. It is associated with a variety of conditions including meningitis, septicaemia, polyserisitis, arthritis, endocarditis and pneumonia. It has also been isolated from cases of rhinitis and abortion. Some types, mainly2,can occasionally cause meningitis in people as well as pigs. Fortunately human cases are rare. An important pig and human S.suis outbreak in China in summer2005, leading to39human death cases. So, it is very urgent and important to establish and apply detection methods for Streptococcus suis.
1. Detection methods for S. suis type2in pig products
New selective and differential media were developed for detection of S. suis type2in pig products. After homogenized with Todd-Hewitt broth(dilution factor of1:10), samples were cultured in THB containing sodium azide (400micrograms/ml), crystal violet (0.4micrograms/ml) and sodium sulfite((200micrograms/ml) for18hours at37℃.Then,it was isolated on the differential agar consisted of Colombia agar,5%defibrinated sheep blood with addition of sodium azide (200micrograms/ml), crystal violet (0.2micrograms/ml) and. sodium sulfite((100micrograms/ml).The isolates were identified by molecular,serolgcial and chemmical method.
2. Production of typing antisera for S. suis type2
ATCC43765was used for the production of antisera. To obtain antigens containing much capsular material for immunization, the strain was subcultured three times in Todd-Hewitt broth with6h incubation,5ml of the last6-h subculture was inoculated into50ml of a prewarmed broth, which was incubated for only2h. For the production of antisera, rabbits weighing3kg were given three injections per week of increasing concentrations of bacteria for4weeks. Antisera were titrated by using the tube agglutination test, and the agglutination titer of the antisera was128. The antisera did not agglutinated with other streptococci except S.suis type2. So, the antisera can be used to type S. suis type2。
3. Detection of suilysin gene of Streptococcus suis type2by PCR
Streptococcus suis type2is an important pathogen of swine which occasionally infects humans as well, and suilysin is one of the most important virulence factor. PCR assays were developed for the rapid and sensitive detection of suilysin gene (sly) of Streptococcus suis type2. PCR primers based on the sequence of sly can amplify a495bp PCR product from S.suis type2Strains ATCC43765and Strain HA9801isolated in Jiangsu Province in1998, however, no PCR product was detected from other bacterias。The sensitivity of the PCR assay was250cfuml-1of the bacteria. Digested with Hind Ⅲ, the PCR products could produce314bp and181bp DNA fragments as expected. The sequences of sly PCR products were the same as Z36907showed in GenBank. The results demonstrated that this PCR assay is a rapid, sensitive and specific diagnostic tool for detection of suilysin gene of type2.
4. Detection of Streptococcus suis type2from entry-exit pig products in Jiangsu
Abstract:Entry-exit pig products in Jiangsu were investigated for the presence of Streptococcus suis from2005to2010. Streptococcus suis was found in31of920samples (3.37%), and Streptococcus suis type2was found in17of920samples (1.85%). It suggested that Streptococcus suis was present in pig products, however,the prevelance is low.
1.猪源动物产品中猪链球菌2型检测方法的建立
建立了猪源动物产品中猪链球菌2型的选择性增菌和选择性平板分离方法。样品与Todd-Hewitt肉汤(THB)按1：10的比例均质后,加入终浓度分别为0.2g/L叠氮钠、0.0002g/L结晶紫和0.1g/L亚硫酸钠,于37℃静置培养18h后,在含有0.2g/L叠氮钠、0.0002g/L结晶紫和0.1g/L亚硫酸钠的5%脱纤维羊血哥伦比亚琼脂的选择性平板上进行平板分离。与非选择性增菌液和分离平板相比,猪链球菌典型菌落显著增加,杂菌显著减少,分离更加容易,分离率显著提高。
2.猪链球菌2型分型血清的制备
将猪链球菌2型(SS2)参考菌株ATCC43765在Todd-Hewitt肉汤中连续传代三代,每次培养6小时,然后按照1：10的比例接种于Todd-Hewitt肉汤仅培养2小时,经灭活处理后获得含较多荚膜物质的全菌菌体抗原。该抗原按照每周三次,除第一周按2.0x109cfu/次,其余按8.0x109cfu/次静脉免疫家兔制备全菌抗血清。经测定,该血清仅与SS2发生凝集反应,凝集价为128,不与其它链球菌发生凝集反应,表明该血清具有较高的特异性,可作为SS2的分型血清。在全国检验检疫系统广泛应用,仅在江苏口岸对125株疑似菌株进行鉴定,共鉴定出17株猪链球菌2型菌株。
3.猪链球菌2型溶血素基因的检测
根据GenBank发表的猪链球菌2型(SS2)溶血素基因(sly)全序列,利用oligo(7.0)软件设计和合成了可扩增长度为495bp的引物,建立了的SS2溶血素基因的PCR检测方法。应用该方法可从SS2参考菌株ATCC43765、SS2江苏分离株HA9801中扩增出495bp的条带,利用Hind Ⅲ限制性内切酶对具有相应单一酶切位点的PCR扩增产物就进行酶切,获得预期的314bp和181bp的2个DNA片段,检测灵敏度达到2.5×102cfu/mL;但不能从马链球菌兽疫亚种ATCC35246、大肠埃希菌ATCC25922、金黄色葡萄球菌ATCC25923.单增李斯特杆菌ATCC19115/413、粪肠球菌ATCC29212中扩增出相应条带。PCR扩增产物的测序结果与GenBank的猪链球菌sly基因(Z36907)核苷酸序列完全一致。表明本方法可快速、敏感、特异地检测SS2的溶血素基因。对江苏口岸分离的125株疑似猪链球菌菌株进行溶血素基因检测,其中27株猪链球菌均检出了猪链球菌溶血素基因,阳性率为100%,其它菌株均未检出猪链球菌溶血素基因。
4.江苏出入境猪源动物产品中猪链球菌的检测
2005年～2010年,对江苏出入境猪源动物产品中进行了猪链球菌检测,通过选择性增菌和平板分离,从920份样品中分离到125链球菌菌株。通过生化鉴定、血清学鉴定和分子鉴定,共检出27株猪链球菌,阳性率为2.93%(27/920),其中猪链球菌2型为17株,阳性率为1.85%(17/920)。结果表明,江苏出口养殖场及进出境猪副产品中有猪链球菌存在,但流行率不很高。上述工作为江苏地区猪源动物产品的国际贸易提供了技术支撑,为猪链球菌病防治提供了可靠的科学依据。
相关研究内容已经纳入质检总局制定的重大动物疫情防控猪链球菌2型实施方案和猪链球菌2型检测的系列国家标准。
Streptococcus suis(SS) infections are considered a major worldwide problem in the swine industry. It is associated with a variety of conditions including meningitis, septicaemia, polyserisitis, arthritis, endocarditis and pneumonia. It has also been isolated from cases of rhinitis and abortion. Some types, mainly2,can occasionally cause meningitis in people as well as pigs. Fortunately human cases are rare. An important pig and human S.suis outbreak in China in summer2005, leading to39human death cases. So, it is very urgent and important to establish and apply detection methods for Streptococcus suis.
1. Detection methods for S. suis type2in pig products
New selective and differential media were developed for detection of S. suis type2in pig products. After homogenized with Todd-Hewitt broth(dilution factor of1:10), samples were cultured in THB containing sodium azide (400micrograms/ml), crystal violet (0.4micrograms/ml) and sodium sulfite((200micrograms/ml) for18hours at37℃.Then,it was isolated on the differential agar consisted of Colombia agar,5%defibrinated sheep blood with addition of sodium azide (200micrograms/ml), crystal violet (0.2micrograms/ml) and. sodium sulfite((100micrograms/ml).The isolates were identified by molecular,serolgcial and chemmical method.
2. Production of typing antisera for S. suis type2
ATCC43765was used for the production of antisera. To obtain antigens containing much capsular material for immunization, the strain was subcultured three times in Todd-Hewitt broth with6h incubation,5ml of the last6-h subculture was inoculated into50ml of a prewarmed broth, which was incubated for only2h. For the production of antisera, rabbits weighing3kg were given three injections per week of increasing concentrations of bacteria for4weeks. Antisera were titrated by using the tube agglutination test, and the agglutination titer of the antisera was128. The antisera did not agglutinated with other streptococci except S.suis type2. So, the antisera can be used to type S. suis type2。
3. Detection of suilysin gene of Streptococcus suis type2by PCR
Streptococcus suis type2is an important pathogen of swine which occasionally infects humans as well, and suilysin is one of the most important virulence factor. PCR assays were developed for the rapid and sensitive detection of suilysin gene (sly) of Streptococcus suis type2. PCR primers based on the sequence of sly can amplify a495bp PCR product from S.suis type2Strains ATCC43765and Strain HA9801isolated in Jiangsu Province in1998, however, no PCR product was detected from other bacterias。The sensitivity of the PCR assay was250cfuml-1of the bacteria. Digested with Hind Ⅲ, the PCR products could produce314bp and181bp DNA fragments as expected. The sequences of sly PCR products were the same as Z36907showed in GenBank. The results demonstrated that this PCR assay is a rapid, sensitive and specific diagnostic tool for detection of suilysin gene of type2.
4. Detection of Streptococcus suis type2from entry-exit pig products in Jiangsu
Abstract:Entry-exit pig products in Jiangsu were investigated for the presence of Streptococcus suis from2005to2010. Streptococcus suis was found in31of920samples (3.37%), and Streptococcus suis type2was found in17of920samples (1.85%). It suggested that Streptococcus suis was present in pig products, however,the prevelance is low.
引文
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