肠炎沙门氏菌的分离鉴定与特异性检测方法的建立
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摘要
沙门氏菌属于肠杆菌科细菌,由此类细菌引起的各种动物疾病称为沙门氏菌病,除初生动物,人畜感染后一般呈无症状带菌状态,长期排菌,污染肉蛋产品,同时它们也可表现为有临床症状的致死疾病,可能加重病情或者增加死亡率,或者降低动物生产力等,对养殖业的经济效益影响重大。目前,鸡肠炎沙门氏菌污染禽蛋肉产品在全球范围内已是很严重的食品卫生问题。日、美等发达国家发生的食物中毒事件中很多都是由沙门氏菌引起的,肠炎沙门氏菌感染渐渐超越鼠伤寒沙门氏菌成为其最主要的病原菌。肠炎沙门氏菌引起鸡群发病的症状和病理学变化与鸡白痢沙门氏菌非常相似,所以在生产中常常把鸡源肠炎沙门氏菌感染症误诊为鸡白痢或伤寒。但是肠炎沙门氏菌的贮藏宿主谱比白痢沙门氏菌广泛,该病的防治和普查要比鸡白痢困难得多,建立肠炎沙门氏菌快速特异性检测方法在养殖业,医学以及公共卫生界有重要意义。本研究中不仅成功从临床发病鸡场成功分离到肠炎沙门氏菌致病株,并根据肠炎沙门氏菌独特的染色体基因序列建立PCR方法特异性检测肠炎沙门氏菌,根据肠炎沙门氏菌独特的表面菌毛抗原蛋白建立间接ELISA方法特异性检测肠炎沙门氏菌感染血清。
1肠炎沙门氏菌的分离与鉴定
从江苏某发病鸡场发病死亡雏鸡体内分离出1株细菌,经形态学观察、生化特性、血清学鉴定以及PCR鉴定,该菌株被鉴定为D群沙门氏菌-肠炎沙门氏菌,并命名为GY09。动物试验表明,该株细菌以1×109cfu剂量腹腔注射小鼠,试验组小鼠在72小时内全部死亡,以1×109cfu皮下注射3日龄SPF雏鸡,雏鸡在48小时内全部死亡；而以3×109cfu剂量皮下注射17日龄SPF鸡,试验组鸡在3周试验期内全部存活。药物敏感性试验表明,该株细菌已对革兰氏阴性菌感染适用的常规药物,如环丙沙星、诺氟沙星、四环素、链霉素等产生完全耐药性,而对青霉素和头孢类药物保持较高敏感性。本试验研究表明,肠炎沙门氏菌早期感染是雏鸡高死亡率的一种重要病因。
2肠炎沙门氏菌SEF14菌毛的抽提、纯化及生物学活性检测
肠炎沙门氏菌在适合的体外培养条件下,抽提和纯化SEF14菌毛蛋白并进行生物学活性的检测。本试验发现肠炎沙门氏菌在CFA培养液中,静止培养50h左右,SEF14菌毛表达量相对较多,在机械高速匀浆后,菌体表面菌毛脱落后经进一步的透析纯化,从而得到较纯的SEF14菌毛蛋白,蛋白大小和相关报道一致,约14KDa左右。Western-blotting检测发现纯化的重组蛋白rSEFA(体外表达的SEF14菌毛主要亚单位蛋白SEFA)免疫小鼠得到的高免血清能识别标准株肠炎沙门氏菌SEF14菌毛蛋白以及纯化的重组蛋白rSEFA,说明体外表达的rSEFA蛋白和体外抽提的SEF14菌毛蛋白一样有较好的免疫原性和反应原性。
3肠炎沙门氏菌特异性检测方法的建立
本试验部分建立和优化了PCR方法特异性检测肠炎沙门氏菌以及重组蛋白rSEFA介导的间接ELISA方法特异性检测肠炎沙门氏菌血清抗体。Sdfl为肠炎沙门氏菌特有的一段基因,设计一对引物特异性检测肠炎沙门氏菌,以此PCR方法能成功检测2株肠炎沙门氏菌标准株,5株不同来源肠炎沙门氏菌临床分离株,而相同PCR条件下不能从都柏林沙门氏菌、鸡白痢沙门氏菌、鼠伤寒沙门氏菌等其他沙门氏菌和其他同属肠杆菌科的常见致病菌染色体上扩增相应片段；本试验部分确定了间接ELISA方法抗原最佳包被量为7.5μg/m1,血清最适稀释度1：100,作用时间为60min；酶标二抗最适稀释度为1：4000,作用时间为60min；判定标准为0D值≥0.346,基于rSEFA介导的间接ELISA有较好的特异性,能特异性识别肠炎沙门氏菌和都柏林沙门氏菌感染血清,不能识别相近的鸡白痢沙门氏菌感染血清。这一方法的建立对肠炎沙门氏菌特异性抗体检测有潜在的应用前景。
Salmonellae are gram-negative facultative rod-shaped bacteria and a member of the family of Enterobacteriaceae. Salmonellae are important enteric pathogens and they cause one of the most common foodborne diseases. Many types of Salmonella bacteria cause Salmonellosis in kinds of animal and people, and they live in the intestinal tracts of warm and cold blooded animals. The elderly, infants, and those with impaired immune systems may have a more severe illness, and the adults infected with Salmonella usually remain symptomless and unnoticed. Foods of animal origin, such as meat, dairy products, and eggs, have been implicated in outbreaks of human Salmonellosis. The prevalence of S. enteritidis has dramatically increased worldwide, and infection caused by S. enteritidis are similar or exceed the number of S. typhimurium, and it has been reported as the most common serotype in the United States and other developed countries, and many people die of the disease every year. It is similar in proceeding of disease in S. enteritidis and S. pullorum, and sometimes we confused these diseases. The sperum of S. enteritidis is wider than S. pullorum, and it is harder to detect and control of S. enteritidis infection. In this study, we isolated and identified S. enteritidis strain from diseased chickens, and developed assays to detect S. enteritidis by PCR and indirect ELISA.
1Isolation and identification of S. enteritidis from Young Chickens
In this study, we isoalated and identidicated a strain of S. enteritidis from diseased chickens in Jiangsu province by morphology observation, biochemical characterization, serological examination and PCR amplification. Animal tests were conducted in ICR mouse and SPF chickens to evaluate the virulence of the isolate S. enteritidis. Results showed that the isolate S. enteritidis was lethal to4-week-old mouse in72hours after injection with1×109cfu intraperitoneally, it also was lethal to3-day-old SPF chicklings with the same dose in48hours by subcutaneous injection, but all eight17-day-old SPF chickens survived after challenging with3×109cfu. Antibiotics susceptibility tests revealed that the isolate S. enteritidis was resistant to multiple antibiotics such as ciprofloxacin, norfloxacin, tetracycline, streptomycin, et al., which are ever efficacious to gram-negative bacteria, but retains high sensitivities to ampicillin, cephalosporins, et al. The result indicated that infection of S. enteritidis in early stage of breeding was an important factor of high mortality of young chickens.
2Extraction, purification and immune activity analysis of SEF14fimbriae of S. enteritidis
S. enteritidis could express SEF14fimbriae in CFA medium and statically in vitro. And fimbriae were separated from S. enteritidis at room temperature by shearing the bacteria in a blender, and in this study we extracted and purified the SEF14fimbriae in vitro successfully. Both the mouse sera with high titer of anti-rSEFA or anti-SEF14were raised and detected after being immunized with the purified rSEFA protein or the purified SEF14fimbriae, which could recognize the purified SEF14fimbriae from reference strain50336and rSEFA, respectively. This indicated that rSEFA protein was immunogenicity and reactogenicity.
3Development of assays for detection of S. enteritidis
In this study we developed a PCR assay for detection of S. enteritidis according the sequence sdfl gene. This PCR assay just successfully decteted the S. enteritidis strains, and there was no PCR products from S. doublin, S. pullorum et al and other pathogens belong to Enterobacteriaceae. An indirect ELISA assay was developed for detection of S. enteritidis using a recombinant fusion antigen rSEFA. This assay was optimized for antigen coating centration of7.5μg/ml and a serum dilutin of1:100, with a standard incubation time60min. A reading of OD490≥0.346was scored as positive. The recombinant antigen has no cross reaction with sera of other relative disease. The result also showed the indirect ELISA is highly sensitive, specific and reproducible. The application of indirect ELISA would provide a simple and rapid method for monitoring serum antibodies against S. enteritidis infection.
1肠炎沙门氏菌的分离与鉴定
从江苏某发病鸡场发病死亡雏鸡体内分离出1株细菌,经形态学观察、生化特性、血清学鉴定以及PCR鉴定,该菌株被鉴定为D群沙门氏菌-肠炎沙门氏菌,并命名为GY09。动物试验表明,该株细菌以1×109cfu剂量腹腔注射小鼠,试验组小鼠在72小时内全部死亡,以1×109cfu皮下注射3日龄SPF雏鸡,雏鸡在48小时内全部死亡；而以3×109cfu剂量皮下注射17日龄SPF鸡,试验组鸡在3周试验期内全部存活。药物敏感性试验表明,该株细菌已对革兰氏阴性菌感染适用的常规药物,如环丙沙星、诺氟沙星、四环素、链霉素等产生完全耐药性,而对青霉素和头孢类药物保持较高敏感性。本试验研究表明,肠炎沙门氏菌早期感染是雏鸡高死亡率的一种重要病因。
2肠炎沙门氏菌SEF14菌毛的抽提、纯化及生物学活性检测
肠炎沙门氏菌在适合的体外培养条件下,抽提和纯化SEF14菌毛蛋白并进行生物学活性的检测。本试验发现肠炎沙门氏菌在CFA培养液中,静止培养50h左右,SEF14菌毛表达量相对较多,在机械高速匀浆后,菌体表面菌毛脱落后经进一步的透析纯化,从而得到较纯的SEF14菌毛蛋白,蛋白大小和相关报道一致,约14KDa左右。Western-blotting检测发现纯化的重组蛋白rSEFA(体外表达的SEF14菌毛主要亚单位蛋白SEFA)免疫小鼠得到的高免血清能识别标准株肠炎沙门氏菌SEF14菌毛蛋白以及纯化的重组蛋白rSEFA,说明体外表达的rSEFA蛋白和体外抽提的SEF14菌毛蛋白一样有较好的免疫原性和反应原性。
3肠炎沙门氏菌特异性检测方法的建立
本试验部分建立和优化了PCR方法特异性检测肠炎沙门氏菌以及重组蛋白rSEFA介导的间接ELISA方法特异性检测肠炎沙门氏菌血清抗体。Sdfl为肠炎沙门氏菌特有的一段基因,设计一对引物特异性检测肠炎沙门氏菌,以此PCR方法能成功检测2株肠炎沙门氏菌标准株,5株不同来源肠炎沙门氏菌临床分离株,而相同PCR条件下不能从都柏林沙门氏菌、鸡白痢沙门氏菌、鼠伤寒沙门氏菌等其他沙门氏菌和其他同属肠杆菌科的常见致病菌染色体上扩增相应片段；本试验部分确定了间接ELISA方法抗原最佳包被量为7.5μg/m1,血清最适稀释度1：100,作用时间为60min；酶标二抗最适稀释度为1：4000,作用时间为60min；判定标准为0D值≥0.346,基于rSEFA介导的间接ELISA有较好的特异性,能特异性识别肠炎沙门氏菌和都柏林沙门氏菌感染血清,不能识别相近的鸡白痢沙门氏菌感染血清。这一方法的建立对肠炎沙门氏菌特异性抗体检测有潜在的应用前景。
Salmonellae are gram-negative facultative rod-shaped bacteria and a member of the family of Enterobacteriaceae. Salmonellae are important enteric pathogens and they cause one of the most common foodborne diseases. Many types of Salmonella bacteria cause Salmonellosis in kinds of animal and people, and they live in the intestinal tracts of warm and cold blooded animals. The elderly, infants, and those with impaired immune systems may have a more severe illness, and the adults infected with Salmonella usually remain symptomless and unnoticed. Foods of animal origin, such as meat, dairy products, and eggs, have been implicated in outbreaks of human Salmonellosis. The prevalence of S. enteritidis has dramatically increased worldwide, and infection caused by S. enteritidis are similar or exceed the number of S. typhimurium, and it has been reported as the most common serotype in the United States and other developed countries, and many people die of the disease every year. It is similar in proceeding of disease in S. enteritidis and S. pullorum, and sometimes we confused these diseases. The sperum of S. enteritidis is wider than S. pullorum, and it is harder to detect and control of S. enteritidis infection. In this study, we isolated and identified S. enteritidis strain from diseased chickens, and developed assays to detect S. enteritidis by PCR and indirect ELISA.
1Isolation and identification of S. enteritidis from Young Chickens
In this study, we isoalated and identidicated a strain of S. enteritidis from diseased chickens in Jiangsu province by morphology observation, biochemical characterization, serological examination and PCR amplification. Animal tests were conducted in ICR mouse and SPF chickens to evaluate the virulence of the isolate S. enteritidis. Results showed that the isolate S. enteritidis was lethal to4-week-old mouse in72hours after injection with1×109cfu intraperitoneally, it also was lethal to3-day-old SPF chicklings with the same dose in48hours by subcutaneous injection, but all eight17-day-old SPF chickens survived after challenging with3×109cfu. Antibiotics susceptibility tests revealed that the isolate S. enteritidis was resistant to multiple antibiotics such as ciprofloxacin, norfloxacin, tetracycline, streptomycin, et al., which are ever efficacious to gram-negative bacteria, but retains high sensitivities to ampicillin, cephalosporins, et al. The result indicated that infection of S. enteritidis in early stage of breeding was an important factor of high mortality of young chickens.
2Extraction, purification and immune activity analysis of SEF14fimbriae of S. enteritidis
S. enteritidis could express SEF14fimbriae in CFA medium and statically in vitro. And fimbriae were separated from S. enteritidis at room temperature by shearing the bacteria in a blender, and in this study we extracted and purified the SEF14fimbriae in vitro successfully. Both the mouse sera with high titer of anti-rSEFA or anti-SEF14were raised and detected after being immunized with the purified rSEFA protein or the purified SEF14fimbriae, which could recognize the purified SEF14fimbriae from reference strain50336and rSEFA, respectively. This indicated that rSEFA protein was immunogenicity and reactogenicity.
3Development of assays for detection of S. enteritidis
In this study we developed a PCR assay for detection of S. enteritidis according the sequence sdfl gene. This PCR assay just successfully decteted the S. enteritidis strains, and there was no PCR products from S. doublin, S. pullorum et al and other pathogens belong to Enterobacteriaceae. An indirect ELISA assay was developed for detection of S. enteritidis using a recombinant fusion antigen rSEFA. This assay was optimized for antigen coating centration of7.5μg/ml and a serum dilutin of1:100, with a standard incubation time60min. A reading of OD490≥0.346was scored as positive. The recombinant antigen has no cross reaction with sera of other relative disease. The result also showed the indirect ELISA is highly sensitive, specific and reproducible. The application of indirect ELISA would provide a simple and rapid method for monitoring serum antibodies against S. enteritidis infection.
引文
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[1]卡尔尼克主编.禽病学[M].第十版.高福,苏敬良等译.北京：中国农业出版社,1999.83-94.
[2]曹蔚,文明.都柏林沙门氏菌引起的一次雏鸭副伤寒感染[J].贵州畜牧兽医, 2000,24,(2)：25-26.
[3]程龙飞,彭春香,郑腾.种番鸭肠炎沙门氏菌的分离鉴定[J].福建畜牧兽医,2007,29(3)：24-25.
[4]冯国金,龙建勇.鸡肠炎沙门氏菌病的病原分离与鉴定[J].中国家禽,2004,26(23)：14-16.
[5]王建舫,何诚,查振林,杨汉春.禽源沙门氏菌的血清型分布与耐药性分析[J].养禽与禽病防治,2009,8：9-11.
[6]曹澍泽等主编.兽医微生物学与免疫学技术[M].北京农业出版社.1992年第1版.34-48,49-54.
[7]萨姆布鲁克,弗里奇,曼尼阿蒂斯著.分子克隆实验指南[M](第2版).金冬雁,黎孟枫译,科学出版社,1992.908.
[8]LeMinor. Genus Ⅲ.Salmonella. in Noel R. Kring and John G. Holt Ed Bergey's manual of systematic bacteriology [M]. Volume 1, Willams & Wilkines, Baltimore/London,1SD-2,427-458.
[9]徐耀辉,焦新安,胡青海等.鸡白痢和鸡伤寒沙门氏菌的PCR-RFLP分子鉴别[J].扬州大学学报(农业与生命科学版),2005,26(1)：1-4.
[10]徐耀辉,焦新安,胡青海等.酶标单抗阻断ELISA检测鸡白痢和鸡伤寒抗体[J].中国兽医学报,2006,26(2)：140-143.
[11]刘佑明.樱桃谷鸭感染肠炎沙门氏菌的病原分离鉴定[J].动物医学,2004,21(9)：54-55.
[12]丁孟建,高继业,唐妤等.肉鸡源肠炎沙门氏菌的分离与鉴定及生物学特性观察[J].中国畜牧兽医.2009,36(5)：203-205.
[13]Matthias Voss.欧共体关于家禽沙门氏菌和其它人畜共患病的现行法规状况[J].中国家禽,2007,29(3)：45-49.
[14]陈溥言主编.兽医传染病学[M].北京：中国农业出版社,2006：123-124.
[1]Gantois I. Ducatelle R, Pasmans F et al. Salmonella enterica serovar Enteritidis genes induced during oviduct colonization and egg contamination in laying hens. Appl Environ Microbiol,2008,74(21):6616-6622.
[2]Ukuku D O, Jin T, Zhang H. Membrane damage and viability loss of Escherichia coli K-12 and Salmonella enteritidis in liquid egg by thermal death time disk treatment. J Food Prot,2008,71(10):1988-1995.
[3]Clouthier SC, Miiller KH, Doran JL, et al. Characterization of three fimbrial genes, sefABC, of Salmonella enteritidis. J.Bacteriol,1993,175(9):2523-2533.
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