黄河口地区致病性猪链球菌分离鉴定与主要毒力因子的PCR检测
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摘要
猪链球菌(Streptococcus suis, SS)是危害现代养猪业的一种重要人兽共患病病原,该菌血清型多、抗原结构复杂,在临床上可呈现不同的特征,如败血症、脑膜炎、心内膜炎、肺炎、关节炎等,还常常与其他疾病合并感染,发病率和死亡率较高。由于目前对猪链球菌病的发生规律、病原特性以及防治措施还不是十分清楚,单纯靠药物又不能有效地控制该病的发生和蔓延。2005—2009年,黄河口地区的规模化养猪场所饲养的猪有不同程度的猪链球菌病发生。该试验从病原学着手研究,在对黄河口地区分离的猪链球菌菌株进行培养、鉴定基础上,建立PCR方法对其进行毒力基因检测,以便为该病的免疫防治提供理论依据。本课题分为两个部分进行研究。
1黄河口地区致病性猪链球菌的分离鉴定及耐药性状况调查收集2005年—2009年黄河口地区规模化养猪场疑似猪链球菌患病猪,根据临床诊断、病理学观察,结合病原的分离培养与鉴定等,确诊为猪链球菌病。通过革兰氏染色镜检、生化试验、药敏试验,对临床分离的猪源链球菌进行流行病学的初步研究。从该地区的发病猪分离到的15株猪源链球菌,其形态、染色、培养特征及理化特性符合猪链球菌的特点,药敏试验结果显示,黄河口地区猪链球菌仅对氨苄西林、阿米卡星、头孢噻肟、头孢唑啉敏感,而对其他常规药物已产生了不同程度的耐药性。
2猪链球菌两种主要毒力因子的双重PCR检测根据猪链球菌两种主要毒力因子基因Sly和MRP的基因序列,设计和合成了2对特异性引物,通过体系和条件优化,建立双重PCR检测方法,对15株背景明确的猪链球菌保存菌株进行检测。结果显示,Sly检出率为4/15,MRP检出率为2/15,阳性、阴性对照均成立。分析结果表明该双重PCR检测方法,特异性和敏感性较好,可用于快速诊断以及猪链球菌相关毒力因子的分子流行病学调查。
Streptococcus suis is one of the primary diseases that threaten modern pig industry. Because of the great varity of streptothrix serum type and the complex structure of antigen, it presents different clinical symptom, such as septicemia, meningoencephalitis, endocarditis, pneumonitis, porodenia arthritis and so on. Now we are not very clear with occurrence regulation and the original characteristic of streptococcosis disease, and its infection and spread can not be effectively controlled simply by medicines. There occurred swine streptococcosis at different degree in large-scale piggery in Huanghe River Mouth Area from 2005 to 2009. In order to provide theoretical basis for immune protection of Swine Streptococcosis, we study it from pathogen, on the foundation of culture and identification of Streptococcus suis from Huanghe River Mouth Area, PCR method was established to detecte the virulence gen. Three experiments were carried out to obtain the study.
1 Isolation and identification and investigation on serotypes and drug resistance of pathogenic Streptococcus suis of Huanghe River Mouth Area. Molecular epidemiology of Streptococcus suis isolates were primarily studied through morphology, gramstain, biochemical test, drug sensitivity test. Fifteen Streptococcus suis strains were isolated from infected pigs of different areas in Huanghe River Mouth Area, which morphology, dyeing, cultural characteristics and physicochemical properties are coincidence with Streptococcus suis. The drug sensitivity test indicated that Huanghe River isolates are only sensitive to ampicillin, amikacin, cefotaxime, cefazolin, and have produced drug resistance at different degree to other routine drug.
2 Detection of virulence-associated factors of Streptococcus suis by duplex PCR assay. Design and synthesis two distinct primers based on the main virulence factors Sly and MRP of Streptococcus suis, establish a duplex PCR detection method by optimizing the condition and system, the method was used to analyse the experimental strains and control strains. With the identified 15 Streptococcus suis strains, the positive detection rates of Sly, MRP were 4/15 , 2/15 , there were no specific amplification products in negative control strains. The results demonstrated that duplex PCR was a highly specific and sensitive diagnostic tool for the detection of virulence-associated factors of Streptococcus suis.
1黄河口地区致病性猪链球菌的分离鉴定及耐药性状况调查收集2005年—2009年黄河口地区规模化养猪场疑似猪链球菌患病猪,根据临床诊断、病理学观察,结合病原的分离培养与鉴定等,确诊为猪链球菌病。通过革兰氏染色镜检、生化试验、药敏试验,对临床分离的猪源链球菌进行流行病学的初步研究。从该地区的发病猪分离到的15株猪源链球菌,其形态、染色、培养特征及理化特性符合猪链球菌的特点,药敏试验结果显示,黄河口地区猪链球菌仅对氨苄西林、阿米卡星、头孢噻肟、头孢唑啉敏感,而对其他常规药物已产生了不同程度的耐药性。
2猪链球菌两种主要毒力因子的双重PCR检测根据猪链球菌两种主要毒力因子基因Sly和MRP的基因序列,设计和合成了2对特异性引物,通过体系和条件优化,建立双重PCR检测方法,对15株背景明确的猪链球菌保存菌株进行检测。结果显示,Sly检出率为4/15,MRP检出率为2/15,阳性、阴性对照均成立。分析结果表明该双重PCR检测方法,特异性和敏感性较好,可用于快速诊断以及猪链球菌相关毒力因子的分子流行病学调查。
Streptococcus suis is one of the primary diseases that threaten modern pig industry. Because of the great varity of streptothrix serum type and the complex structure of antigen, it presents different clinical symptom, such as septicemia, meningoencephalitis, endocarditis, pneumonitis, porodenia arthritis and so on. Now we are not very clear with occurrence regulation and the original characteristic of streptococcosis disease, and its infection and spread can not be effectively controlled simply by medicines. There occurred swine streptococcosis at different degree in large-scale piggery in Huanghe River Mouth Area from 2005 to 2009. In order to provide theoretical basis for immune protection of Swine Streptococcosis, we study it from pathogen, on the foundation of culture and identification of Streptococcus suis from Huanghe River Mouth Area, PCR method was established to detecte the virulence gen. Three experiments were carried out to obtain the study.
1 Isolation and identification and investigation on serotypes and drug resistance of pathogenic Streptococcus suis of Huanghe River Mouth Area. Molecular epidemiology of Streptococcus suis isolates were primarily studied through morphology, gramstain, biochemical test, drug sensitivity test. Fifteen Streptococcus suis strains were isolated from infected pigs of different areas in Huanghe River Mouth Area, which morphology, dyeing, cultural characteristics and physicochemical properties are coincidence with Streptococcus suis. The drug sensitivity test indicated that Huanghe River isolates are only sensitive to ampicillin, amikacin, cefotaxime, cefazolin, and have produced drug resistance at different degree to other routine drug.
2 Detection of virulence-associated factors of Streptococcus suis by duplex PCR assay. Design and synthesis two distinct primers based on the main virulence factors Sly and MRP of Streptococcus suis, establish a duplex PCR detection method by optimizing the condition and system, the method was used to analyse the experimental strains and control strains. With the identified 15 Streptococcus suis strains, the positive detection rates of Sly, MRP were 4/15 , 2/15 , there were no specific amplification products in negative control strains. The results demonstrated that duplex PCR was a highly specific and sensitive diagnostic tool for the detection of virulence-associated factors of Streptococcus suis.
引文
[1]白静,猪链球菌病病原分离鉴定及防治研究[J].安徽农业科学,2007,35(7):1944,1947.
[2]白雪梅,张亚兰,孙娜,等.100株猪链球菌的生化检测及药物敏感性分析,中国人兽共患病学报,2006, 22(5):396.
[3]蔡宝祥,主编.家畜传染病学[M](第3版).北京:中国农业出版社,1996.
[4]常洪涛,姚雪静,陈陆,等.河南省致病性猪源链球菌部分微生物学特性及耐药性研究[J].安徽农业科学,2008,36(22):9533-9535, 9545.
[5]陈博文,罗宝正,陈竞帆,等.多重荧光PCR检测Ⅱ型猪链球菌方法的建立及应用[J].中国兽医学报,2007,27(3):355-358.
[6]陈炜,刘平,郭什坤,等.六安地区猪链球菌分离鉴定及药敏学试验,[J]畜牧与饲料科学,2009,30(2):183-184.
[7]陈泽祥,吴礼杰,许力干,等.广西猪源链球菌分离株的分群鉴定[J].中国畜牧兽医,2005,32(12):38-39.
[8]杜文金,孙融冰.Ⅱ型猪链球菌的致病因子研究进展[J].中国动物检疫,1998,15(3):47-48.
[9]段正赢,徐涤平,土红琳,等.猪链球菌病单价与多价灭活苗的免疫效果[J].安徽农业科学,2007,35(31):9931,9995.
[10]樊国燕,臧金灿.猪链球菌河南株的分离鉴定与耐药性监测,黑龙江畜牧兽医科技版,2009年10月(上)95.
[11]甘孟侯,杨汉春.中国猪病学[M].北京:中国农业出版社,2005:333-336.
[12]高尚庆,雷连成,韩文瑜.猪链球菌2型基因工程疫苗研究进展[J].生物技术通报,2007,(2):33-35.
[13]顾永远,汤赛冬,成建忠,等.威胁养猪业三大细菌性传染病的诊治[J].现代农业科技,2009(4):220-221.
[14]郭伶,陈宁利,陈艳会.锦州地区猪链球菌病病原的分离鉴定[J],畜牧与兽医,2007,39(10):46-48.
[15]韩正田,李芬,辛眷兰,等.猪链球菌病的发病特点与诊治[J].现代农业科技,2007(19):198,205.
[16]江南,杨兴祥.四川省83例人感染猪链球菌病患者的临床特征[J].中华急诊医学杂志,2005,(14):891-894.
[17]江定丰,陈灵芝。猪链球菌2型感染猪和人的现状及研究进展,动物医学进展,2008(5):82-85.
[18]江定丰,曹晋蓉,詹松鹤,等.猪链球菌2型安徽株的分离鉴定与药物敏感试验[J].畜牧与兽医,2007,39(6):48-50.
[19]姜艳华,孙乾,王楷宬,等.猪链球菌35个血清型标准抗血清的制备及应用[J],中国动物检疫,2008,25(9):19-21
[20]李建鑫,猪链球菌病的诊断及防治,China Swine Industry,2009(10):56-57.
[21]李立虎,猪链球菌病的研究进展,畜牧与饲料科学,2009,(2):173-174.
[22]李茂林,黄健强.2型猪链球菌毒力因子概述[J].河南畜牧兽医,2005,26(11):9-11.
[23]李明,何孔旺,陆承平.检测两种猪源链球菌抗体间接ELISA方法的筛选与应用[J].中国人兽共患病杂志,2005,21(10):867-870.
[24]刘军,冯书章,尹铁勇,等.猪链球菌2型和9型菌株的多重PCR检测[J].中国人兽共患病杂志,2005,21(6):510-514.
[25]龙塔,白喜婷,董发明,等.猪链球菌病流行情况调查[J].河南科技大学学报:农学版,2004,24(1):10-12.
[26]陆承平,姚火春.猪链球茵致病性研究及其公共卫生意义[J].中国预防医学杂志,2006.
[27]卢耀娟,梁炯明,刘义威,等.五起人感染猪链球菌病病例的流行病学调查及控制效果分析[J].公共卫生与预防医学,2009,20(1):70.
[28]罗隆泽,刘莉,李燕春,等.四川省人感染猪链球菌病的病原分离与鉴定[J].预防医学情报杂志,2005,21(4):386-387.
[29]罗隆泽,李燕春,郭宗琪,等.猪链球菌选择性培养基研究[J],预防兽医学情报杂志,2007,23(5):511-514.
[30]罗隆泽,王鑫,等.四川资阳地区健康猪2型猪链球菌分离与分子生物学特征分析[J].中国人兽共患病学报,2009,25(9):842-845.
[31]吕立新,何孔旺,倪艳秀,等.从正常屠辛猪扁桃体中分离到致病性猪链球菌2型[J].中国人兽共患病杂志.2008, 24(4):379-383.
[32]吕强,吴建林,袁巧,等.四川省人感染猪链球菌病流行病学调查分析[J].预防医学情报杂志,2005,21(4):379-383.
[33]马清霞何孔旺陆承平.猪链球菌2型及其毒力因子检测多重PCR的建立及应用[J].中国兽医学报,2006,26(1):28-31.
[34]马跃军,李玉荣,刘斌,等.猪链球菌病的综合防治与公共卫生安全,畜牧与饲料科学,2009,30(7-8):157-163.
[35]明光君,王辉.猪链球菌病的病症及防治,养殖技术顾问,2009(12):96.
[36]潘劲草,叶榕,王满琴,等.O139群霍乱弧菌的核糖体基因分型及其与药物抗性相关性研究[J].国外医学·流行病学传染病学分册,2005,32(4):196-200.
[37]沈萍,黎满香.猪链球菌2型及7型分离株对16种抗生素的耐药性分析[J].安徽农业科学,2009,37(1):140-141.
[38]万彬,吕春容.人一感染猪链球菌病患者心理状态分析及护理对策[J].2009,24(6):1574-1576.
[39]王海丽,王长军,潘秀珍,等.猪链球菌2型人源分离株截短的溶菌酶释放蛋白基因的克隆及原核表达[J].细胞与分子免疫学杂志,2006,22(2):178-180.
[40]王华,赵云玲,王君玮,等.猪链球菌病流行病学及其疫苗研究现状[J].动物医学进展,2006,27(9):27-31.
[41]王君玮,王志亮,赵永刚,等.人-猪链球菌2型2005四川分离株的鉴定和耐药性研究[C].中国畜牧兽医学会家畜传染病学分会第11次学术研讨会,2005:179-182.
[42]王显清,猪链球菌病的诊断与防治[J].现代农业科技,2008 (24): 246-248.
[43]韦俊超,朱水荣,杨婷婷,等.东阳市一例人感染猪链球菌病的病原学及分子生物学鉴定分析[J].中国卫生检验杂志,2009,19(7): 1641-1646.
[44]肖萍,张海燕,陈莉.1例猪链球菌的分离与鉴定[J].养殖与饲料,2008(10):23-24.
[45]徐梅芳,付臣,等。浅谈猪链球菌病的流行特点,中国畜禽种业,2009(9):81-82.
[46]杨鸿,林奕,苏子珩,等.珠三角部分地区猪链球菌的药物敏感性调查与分析,养猪,2009(6):66-67.
[47]杨霞,杜艳芬,陈丽影,等.猪链球菌的分离与鉴定[J]。河南科技大学学报(自然科学版).2007,28(5):60-63.
[48]杨珍,王楷宬,范伟兴,等.表观健康猪群携带猪链球菌情况流行病学调查[J].中国人兽共患病学报,2009,25(10):977-979,999.
[49]姚开虎,杨永弘.人感染猪链球菌病[J].中华传染病杂志,2006,24:64-66.
[50]姚龙涛.猪的链球菌病[J].动物科学与动物医学,2005,(10):32-35.
[51]尤玉民,沈健.人-猪链球菌感染性综合征的流行病学研究[J].中国初级卫生保健,2001,15(7):20-21.
[52]曾巧英陆承平.猪链球菌Ⅱ型溶血酶释放蛋白诱导内皮细胞融合[J].中国农业科学,2005,34(4):821-825.
[53]张宝华.猪链球菌病的诊断与防治[J].现代农业科技, 2008(12): 258.
[54]张燚,李义,猪链球菌病的病原分离鉴定及防制措施研究[J].中国畜牧兽医,2009,36(11):151-153.
[55]章祥友,丁俊琪,秦海蓓.猪链球菌引起的人猪共患病22例临床分析[J].热带医学杂志,2002,2 (4):361-363.
[56]赵华梅,潘秀珍,唐家琪.猪链球菌毒力因子和鉴定方法的研究进展[J].微生物学杂志,2006,26(1):77-80.
[57]赵瑞宏,潘孝成.猪链球菌LJ株的分离与鉴定[J].安徽农业科学,2007,35(23):7068,7070.
[58]周俊霞.17例人感染猪链球菌病患者的护理干预[J].吉林医学,2009,30(4):314-315.
[59]Allgaier A, Goethe R, Wisselink H J, et al. Relatedness of Streptococcus suis isolates of various serotypes and clinical backgrounds as evaluated by macro-restriction analysis and expression of potential virulence traits [J]. J Clin Microbiol, 2001, 39: 445-453.
[60]Astrid D G. Contribution of fibronectin-binding protein to pathogenesis of Streptococcus suis serotype 2[J]. Inefct Immun, 2002, 70(3):1319-1325.
[61]Berthelot-Herault F, Gottschalk M, Labbe A, et a1. Experimental airborne transmission of Streptococcus suis capsular type 2 in pigs [J]. Veterinary microbiology, 2001, 821: 69-80.
[62]Berthelot-Herault F, Morvan H, Keribin AM, et a1. Production of muraminidase released protein (MRP), extracellular factor (EF) and Suilysin by field isolates of Streptococcus suis capsular types 2, 1/2, 9, 7 and 3 isolated from swine in France [J]. Vet Res, 2000, 31(5): 473-479.
[63]Blumberg M J, Kiehlbauch A, Wachsmuth I K. Molecular epidemiology of Yersinia enterocolitica O: 3 infections: use of chromosomal DNA restriction fragment length polymorphisms of rRNA genes [J]. J Clin Microbiol, 2003, 29: 2368-2374.
[64]Brassard J, Gottschalk M, Quessy S, et a1. Cloning and purification of the Streptococcus suis serotype 2 glyceraldehyde-3-phosphatedehydrogenase and its involvement as an adhesion [J]. Vet Microbiol, 2004, 102(12):87-94.
[65] Carland N, Nizet V, Rubens C, et a1. Streptococcus suis serotype 2 interactions with human brain microvascular endothelial cells [J]. Infect lmmun, 2000, 68(2):637-643.
[66] Chan YC, Wilder-Smith A, Ong B K, et a1. Adult community acquired bacterial meningitis in a Singaporean teaching hospita1 [J]. A seven year overview (1993-2000). Singapore Med J, 2002, 43(12): 632-636.
[67]Costa A T, Lobato F C, Abreu V L, et a1. Serotyping and evaluation of the virulence in mice of Streptococcus suis strains insolated from diseased pigs[J]. Rev Inst Med Trop Sal Paulo, 2005, 47(2): 113-115.
[68]Donsakul K, Dejthevapon C, Witoonpanich R. Streptococcus suis infection: clinical features and diagnostic pitfalls [J]. Southeast Asian J Trop Med Public Health, 2003, 34(1): 154-158.
[69]Elliott SD, Clifton-Hadley F, Tai J. Streptococcus infection in young pigs.V.An immunologic from streptococcus suis type 2 with particular reference to vaccination against streptococcus infection in pigs [J]. J Hyg, 1980, 85: 275-285.
[70] Florence B H, Marois C, Gottschalk M, et al. Geneti diversity of Streptococcus suis strains isolated from pigs and humans as revealed by pulsed-field gel electrophoresis[J].Clin Microbiol,2002,(40)615-619.
[71]Fongcom A. Streptococcus suis infection in Northern Thailand [J]. J Med Assoc Thail, 2001(84): 1502-1508.
[72]Gottschalk M, Segura. The pathogenesis of the menmgitis caused by Streptococcus suis: the unresolved questions [J]. Veterinary microbiology, 2000, 196: 1-14.
[73]Grimont F, Grimont A D. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools [J]. Ann Inst Pasteur Microbiol, 1999, 137B: 165-175.
[74]Halaby T, Hoitsma E R. Streptococcus suis meningitis: a poache’s risk [J]. Eur J Clin Microbiol Infect Dis, 2000, 19: 943-945.
[75]Harel J, Martinez G, Nassar A, et a1. Identification of an inducible bacteriophage in a virulent strain of Streptococcns suis serotype 2[J]. Infect andlmmun, 2003, 71(10): 6104-6108.
[76]Hoofar J, Malorny B, Abdulmawjood A, et a1. Practical considerations in design of internal amplification controls for diagnostic PCR assays [J]. J Clin Microbiol, 2004, 42(5): 1863-1868.
[77]Ilangovan U, Ton-That H, Iwahara J, et a1. Structure of sortase: the transpeptidase that anchors proteins to the cel wall of Staphylococcus aurens [J]. Proc Natl Acad Sci USA, 2001, 98(11): 6056-6061.
[78] Kopic J, Paradzik MT, pandak N. Streptococcus suis infection as a cause of severe illness: 2 cases from Croatia [J]. Scand J Infect Dis, 2002, 34(9): 683-684.
[79]Mamianian S K, Liu G, Jensen E R, et a1. Staphylococcns aurens sottase mutants defective in the display of surface proteins and in the pathogenesis of animal infections [J]. Proc Natl Acad Sci USA, 2000, 97(10): 5510-5515.
[80]Marcelo G, Marida S. The pathogenesis of the meningitis caused by Streptococcus suis: the unresolved questions [J]. Vet Microbio1, 2000, 76: 259-272.
[81]Marois C, Bougeard S, Gottschalk M, et a1. Multiplex PCR assay for detection of Streptococcus suis species and serotypes 2 and 1/2 in tonsils of live and dead pigs [J]. J Clin Microbiol, 2004, 42(7): 3169-3175.
[82]Norton P M, Polph C, ward P N, et a1. Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin [J]. FEMS Immunol Med Microbiol, 1999, 26: 25-35.
[83]Numata S, Nakamura Y, Imamura Y, et a1. Rapid quantitative analysis of human cytomegalovirus DNA by the real-time polymerase chain reaction method [J]. Arch Pathol Lab Med, 2005, 129(2): 200-204.
[84]Okwumabua O, Persaud J S, Reddy P G. Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2[J]. Clin Diagn Lab Immunol, 2001, 8: 251-257.
[85]Osaki M, Takamatsu D, Shimoji Y, et a1. Characterization of Streptocuccus suis genes encoding proteins homologous to sortase of gram-positive bacteria [J]. Bacteriol, 2002, 184(4): 971-982.
[86] Pang Y G, Jiang J Y,Wang L Z, et al.Effects of immunological stress on immune response in different breeds of piglets[J].Animal Husbandry and Feed Science, 2009,1 (2):28-31.
[87]Saulnier P. Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant staphylococcus aureus [J]. J Clin Microbiol, 2003, 31:9821.
[88]Silva L M, Baums C G, Rehm T, et a1. Virulence-associated gene profiling of Streptococcus suis isolates by PCR [J]. Vet Microbiol, 2006, 104(1): 87-94.
[89]Smith H E, Martine R, Norbert S Z, et al. Virulent Strains of Streptococcus suis Serotype 2 and Highly Virulent Strains of Streptococcus suis Serotype 1 Can Be Recognized by a Unique Ribotype Profile[J]. J Clin Microbiol, 1997, (5): 1049-1053.
[90]Smith H E, Damman M, Vander Velde J, et a1. Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor [J]. Infect Immun, 1999, 67(4): 1750-1756.
[91]Smith H E, Vecht U, Wissdink H J, et a1. Mutants of Streptococcus suis types l and 2 impaired in expression of muramidase-released protein and extracelluhr protein induce disease in newborn germfree pigs [J]. Infect Immun, 1996, 64(10): 4409-4412.
[92]Smith H E, Veenbergen V, Vander Veled J, et a1. The cps genes of Streptococcus suis serotypes 1, 2 and 9: development of rapid serotype-specific PCR assays [J ]. J Clin Microbiol,1999,37(10): 3146-3152.
[93]Spackman E, Senne D A, Bulaga L L, et a1. Development of real-time RT-PCR for the detection of avian influenza virus [J]. Avian Dis, 2003, 47(3): 1079-1082.
[94]Staats J J, Feder I, Okwumabua O, et a1. Streptococcus suis: past and present presence [J]. Vet Res Commun, 1997, 21(6): 381-407.
[95]Staats J J, Plattner B L, Nietfeld J, et al. Use of Ribotyping and Hemolysin Activity To Identify Highly Virulent Streptococcus suis Type 2 Isolates[J]. J Clin Microbiol, 1998, 2: 15-19.
[96]Strangmnn E, Froleke H, Kohse K P. Septic shock caused by Streptococcus suis: case report and investigation of a risk group [J]. Int J Hyg Environ Health, 2002, 205(5): 385-392.
[97]Swildens B, Wisselink H J, Engel B, et a1. Detection of extracellular factor-positive Streptococcus suis serotype 2 strains in tonsillar swabs of live sows by PCR [J]. Vet Microbiol, 2005, 109(3/4): 223-228.
[98]Tang J Q, Wang C J, Feng YJ, et a1. Streptococcal toxic shock syndrome caused by streptococcus suis serotype2 [J]. PLoS Med, 2006, 3(5): 151.
[99]Tian Y, Aarestrup F M, Lu C P. Characterization of Streptococcus suis serotype 7 isolates from diseased pigs in Denmark [J]. Veterinary Microbiology, 2004, 103: 55–62.
[100]Ton-That H, Mazmanian S K, Faull K F, et a1. Anchoring of surface proteins to the cell wall of Staphylococcus aurens Sortase-catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-G1y3 substrates [J]. J Biol Chem, 2000, 275(13): 9876-9881.
[101]Vanier G, Segura M, Friedl P, et al. Invasion of porcine brain microvascular endothelial cells by Streptococcus suis serotype 2[J]. Infect lmmun, 2004, 72(3): 1441-1449.
[102]Vela A I, Goyache J, Tarradas C, et a1. Analysis of genetic diversity of Streptococcus suis clinical isolates from pigs in Spain by pulsed-field gel electrophoresis[J]. J Clin Microbio, 2003, 41(6): 2498- 2502.
[103]Vilaichone R K, Vilaichone W, Nunthapisud P, et a1. Streptococcus suis infection in Thailand [J]. J Med Assoc Thai, 2002, 85(1): S109-117.
[104]Wisselink H J, Joosten J J, Smith H E. Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs [J]. J Clin Microbiol, 2002, 40(8): 2922-2929.
[105]Wisselink H J, Smith H E, Stockhofe-Zurwieden, et a1. Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries [J]. Vet Microbiol, 2000, 74(3): 237-248.
[106]Yam W C, Chan K H, Chow K H, et a1. Clinical evaluation of real-time PCR assays for rapid diagnosis of SARS coronavirus during outbreak and post-epidemic periods [J]. J Clin Virol, 2005, 33(1): 19-24.
[107] Zhang C P,NiNg Y B,Zhang Z Q,et al.Prevalence of Streptococcus suis isolated from clinically healthy sows in China[J].Agricultural Sciences in China,2009,8(5):638-642.
[108]Zink S D, Burns D L. Importance of srtA and srtB for growth of Bacillus anthracis in maerophages[J]. Infect lmmun, 2005, 73(8): 5222-5228.
[2]白雪梅,张亚兰,孙娜,等.100株猪链球菌的生化检测及药物敏感性分析,中国人兽共患病学报,2006, 22(5):396.
[3]蔡宝祥,主编.家畜传染病学[M](第3版).北京:中国农业出版社,1996.
[4]常洪涛,姚雪静,陈陆,等.河南省致病性猪源链球菌部分微生物学特性及耐药性研究[J].安徽农业科学,2008,36(22):9533-9535, 9545.
[5]陈博文,罗宝正,陈竞帆,等.多重荧光PCR检测Ⅱ型猪链球菌方法的建立及应用[J].中国兽医学报,2007,27(3):355-358.
[6]陈炜,刘平,郭什坤,等.六安地区猪链球菌分离鉴定及药敏学试验,[J]畜牧与饲料科学,2009,30(2):183-184.
[7]陈泽祥,吴礼杰,许力干,等.广西猪源链球菌分离株的分群鉴定[J].中国畜牧兽医,2005,32(12):38-39.
[8]杜文金,孙融冰.Ⅱ型猪链球菌的致病因子研究进展[J].中国动物检疫,1998,15(3):47-48.
[9]段正赢,徐涤平,土红琳,等.猪链球菌病单价与多价灭活苗的免疫效果[J].安徽农业科学,2007,35(31):9931,9995.
[10]樊国燕,臧金灿.猪链球菌河南株的分离鉴定与耐药性监测,黑龙江畜牧兽医科技版,2009年10月(上)95.
[11]甘孟侯,杨汉春.中国猪病学[M].北京:中国农业出版社,2005:333-336.
[12]高尚庆,雷连成,韩文瑜.猪链球菌2型基因工程疫苗研究进展[J].生物技术通报,2007,(2):33-35.
[13]顾永远,汤赛冬,成建忠,等.威胁养猪业三大细菌性传染病的诊治[J].现代农业科技,2009(4):220-221.
[14]郭伶,陈宁利,陈艳会.锦州地区猪链球菌病病原的分离鉴定[J],畜牧与兽医,2007,39(10):46-48.
[15]韩正田,李芬,辛眷兰,等.猪链球菌病的发病特点与诊治[J].现代农业科技,2007(19):198,205.
[16]江南,杨兴祥.四川省83例人感染猪链球菌病患者的临床特征[J].中华急诊医学杂志,2005,(14):891-894.
[17]江定丰,陈灵芝。猪链球菌2型感染猪和人的现状及研究进展,动物医学进展,2008(5):82-85.
[18]江定丰,曹晋蓉,詹松鹤,等.猪链球菌2型安徽株的分离鉴定与药物敏感试验[J].畜牧与兽医,2007,39(6):48-50.
[19]姜艳华,孙乾,王楷宬,等.猪链球菌35个血清型标准抗血清的制备及应用[J],中国动物检疫,2008,25(9):19-21
[20]李建鑫,猪链球菌病的诊断及防治,China Swine Industry,2009(10):56-57.
[21]李立虎,猪链球菌病的研究进展,畜牧与饲料科学,2009,(2):173-174.
[22]李茂林,黄健强.2型猪链球菌毒力因子概述[J].河南畜牧兽医,2005,26(11):9-11.
[23]李明,何孔旺,陆承平.检测两种猪源链球菌抗体间接ELISA方法的筛选与应用[J].中国人兽共患病杂志,2005,21(10):867-870.
[24]刘军,冯书章,尹铁勇,等.猪链球菌2型和9型菌株的多重PCR检测[J].中国人兽共患病杂志,2005,21(6):510-514.
[25]龙塔,白喜婷,董发明,等.猪链球菌病流行情况调查[J].河南科技大学学报:农学版,2004,24(1):10-12.
[26]陆承平,姚火春.猪链球茵致病性研究及其公共卫生意义[J].中国预防医学杂志,2006.
[27]卢耀娟,梁炯明,刘义威,等.五起人感染猪链球菌病病例的流行病学调查及控制效果分析[J].公共卫生与预防医学,2009,20(1):70.
[28]罗隆泽,刘莉,李燕春,等.四川省人感染猪链球菌病的病原分离与鉴定[J].预防医学情报杂志,2005,21(4):386-387.
[29]罗隆泽,李燕春,郭宗琪,等.猪链球菌选择性培养基研究[J],预防兽医学情报杂志,2007,23(5):511-514.
[30]罗隆泽,王鑫,等.四川资阳地区健康猪2型猪链球菌分离与分子生物学特征分析[J].中国人兽共患病学报,2009,25(9):842-845.
[31]吕立新,何孔旺,倪艳秀,等.从正常屠辛猪扁桃体中分离到致病性猪链球菌2型[J].中国人兽共患病杂志.2008, 24(4):379-383.
[32]吕强,吴建林,袁巧,等.四川省人感染猪链球菌病流行病学调查分析[J].预防医学情报杂志,2005,21(4):379-383.
[33]马清霞何孔旺陆承平.猪链球菌2型及其毒力因子检测多重PCR的建立及应用[J].中国兽医学报,2006,26(1):28-31.
[34]马跃军,李玉荣,刘斌,等.猪链球菌病的综合防治与公共卫生安全,畜牧与饲料科学,2009,30(7-8):157-163.
[35]明光君,王辉.猪链球菌病的病症及防治,养殖技术顾问,2009(12):96.
[36]潘劲草,叶榕,王满琴,等.O139群霍乱弧菌的核糖体基因分型及其与药物抗性相关性研究[J].国外医学·流行病学传染病学分册,2005,32(4):196-200.
[37]沈萍,黎满香.猪链球菌2型及7型分离株对16种抗生素的耐药性分析[J].安徽农业科学,2009,37(1):140-141.
[38]万彬,吕春容.人一感染猪链球菌病患者心理状态分析及护理对策[J].2009,24(6):1574-1576.
[39]王海丽,王长军,潘秀珍,等.猪链球菌2型人源分离株截短的溶菌酶释放蛋白基因的克隆及原核表达[J].细胞与分子免疫学杂志,2006,22(2):178-180.
[40]王华,赵云玲,王君玮,等.猪链球菌病流行病学及其疫苗研究现状[J].动物医学进展,2006,27(9):27-31.
[41]王君玮,王志亮,赵永刚,等.人-猪链球菌2型2005四川分离株的鉴定和耐药性研究[C].中国畜牧兽医学会家畜传染病学分会第11次学术研讨会,2005:179-182.
[42]王显清,猪链球菌病的诊断与防治[J].现代农业科技,2008 (24): 246-248.
[43]韦俊超,朱水荣,杨婷婷,等.东阳市一例人感染猪链球菌病的病原学及分子生物学鉴定分析[J].中国卫生检验杂志,2009,19(7): 1641-1646.
[44]肖萍,张海燕,陈莉.1例猪链球菌的分离与鉴定[J].养殖与饲料,2008(10):23-24.
[45]徐梅芳,付臣,等。浅谈猪链球菌病的流行特点,中国畜禽种业,2009(9):81-82.
[46]杨鸿,林奕,苏子珩,等.珠三角部分地区猪链球菌的药物敏感性调查与分析,养猪,2009(6):66-67.
[47]杨霞,杜艳芬,陈丽影,等.猪链球菌的分离与鉴定[J]。河南科技大学学报(自然科学版).2007,28(5):60-63.
[48]杨珍,王楷宬,范伟兴,等.表观健康猪群携带猪链球菌情况流行病学调查[J].中国人兽共患病学报,2009,25(10):977-979,999.
[49]姚开虎,杨永弘.人感染猪链球菌病[J].中华传染病杂志,2006,24:64-66.
[50]姚龙涛.猪的链球菌病[J].动物科学与动物医学,2005,(10):32-35.
[51]尤玉民,沈健.人-猪链球菌感染性综合征的流行病学研究[J].中国初级卫生保健,2001,15(7):20-21.
[52]曾巧英陆承平.猪链球菌Ⅱ型溶血酶释放蛋白诱导内皮细胞融合[J].中国农业科学,2005,34(4):821-825.
[53]张宝华.猪链球菌病的诊断与防治[J].现代农业科技, 2008(12): 258.
[54]张燚,李义,猪链球菌病的病原分离鉴定及防制措施研究[J].中国畜牧兽医,2009,36(11):151-153.
[55]章祥友,丁俊琪,秦海蓓.猪链球菌引起的人猪共患病22例临床分析[J].热带医学杂志,2002,2 (4):361-363.
[56]赵华梅,潘秀珍,唐家琪.猪链球菌毒力因子和鉴定方法的研究进展[J].微生物学杂志,2006,26(1):77-80.
[57]赵瑞宏,潘孝成.猪链球菌LJ株的分离与鉴定[J].安徽农业科学,2007,35(23):7068,7070.
[58]周俊霞.17例人感染猪链球菌病患者的护理干预[J].吉林医学,2009,30(4):314-315.
[59]Allgaier A, Goethe R, Wisselink H J, et al. Relatedness of Streptococcus suis isolates of various serotypes and clinical backgrounds as evaluated by macro-restriction analysis and expression of potential virulence traits [J]. J Clin Microbiol, 2001, 39: 445-453.
[60]Astrid D G. Contribution of fibronectin-binding protein to pathogenesis of Streptococcus suis serotype 2[J]. Inefct Immun, 2002, 70(3):1319-1325.
[61]Berthelot-Herault F, Gottschalk M, Labbe A, et a1. Experimental airborne transmission of Streptococcus suis capsular type 2 in pigs [J]. Veterinary microbiology, 2001, 821: 69-80.
[62]Berthelot-Herault F, Morvan H, Keribin AM, et a1. Production of muraminidase released protein (MRP), extracellular factor (EF) and Suilysin by field isolates of Streptococcus suis capsular types 2, 1/2, 9, 7 and 3 isolated from swine in France [J]. Vet Res, 2000, 31(5): 473-479.
[63]Blumberg M J, Kiehlbauch A, Wachsmuth I K. Molecular epidemiology of Yersinia enterocolitica O: 3 infections: use of chromosomal DNA restriction fragment length polymorphisms of rRNA genes [J]. J Clin Microbiol, 2003, 29: 2368-2374.
[64]Brassard J, Gottschalk M, Quessy S, et a1. Cloning and purification of the Streptococcus suis serotype 2 glyceraldehyde-3-phosphatedehydrogenase and its involvement as an adhesion [J]. Vet Microbiol, 2004, 102(12):87-94.
[65] Carland N, Nizet V, Rubens C, et a1. Streptococcus suis serotype 2 interactions with human brain microvascular endothelial cells [J]. Infect lmmun, 2000, 68(2):637-643.
[66] Chan YC, Wilder-Smith A, Ong B K, et a1. Adult community acquired bacterial meningitis in a Singaporean teaching hospita1 [J]. A seven year overview (1993-2000). Singapore Med J, 2002, 43(12): 632-636.
[67]Costa A T, Lobato F C, Abreu V L, et a1. Serotyping and evaluation of the virulence in mice of Streptococcus suis strains insolated from diseased pigs[J]. Rev Inst Med Trop Sal Paulo, 2005, 47(2): 113-115.
[68]Donsakul K, Dejthevapon C, Witoonpanich R. Streptococcus suis infection: clinical features and diagnostic pitfalls [J]. Southeast Asian J Trop Med Public Health, 2003, 34(1): 154-158.
[69]Elliott SD, Clifton-Hadley F, Tai J. Streptococcus infection in young pigs.V.An immunologic from streptococcus suis type 2 with particular reference to vaccination against streptococcus infection in pigs [J]. J Hyg, 1980, 85: 275-285.
[70] Florence B H, Marois C, Gottschalk M, et al. Geneti diversity of Streptococcus suis strains isolated from pigs and humans as revealed by pulsed-field gel electrophoresis[J].Clin Microbiol,2002,(40)615-619.
[71]Fongcom A. Streptococcus suis infection in Northern Thailand [J]. J Med Assoc Thail, 2001(84): 1502-1508.
[72]Gottschalk M, Segura. The pathogenesis of the menmgitis caused by Streptococcus suis: the unresolved questions [J]. Veterinary microbiology, 2000, 196: 1-14.
[73]Grimont F, Grimont A D. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools [J]. Ann Inst Pasteur Microbiol, 1999, 137B: 165-175.
[74]Halaby T, Hoitsma E R. Streptococcus suis meningitis: a poache’s risk [J]. Eur J Clin Microbiol Infect Dis, 2000, 19: 943-945.
[75]Harel J, Martinez G, Nassar A, et a1. Identification of an inducible bacteriophage in a virulent strain of Streptococcns suis serotype 2[J]. Infect andlmmun, 2003, 71(10): 6104-6108.
[76]Hoofar J, Malorny B, Abdulmawjood A, et a1. Practical considerations in design of internal amplification controls for diagnostic PCR assays [J]. J Clin Microbiol, 2004, 42(5): 1863-1868.
[77]Ilangovan U, Ton-That H, Iwahara J, et a1. Structure of sortase: the transpeptidase that anchors proteins to the cel wall of Staphylococcus aurens [J]. Proc Natl Acad Sci USA, 2001, 98(11): 6056-6061.
[78] Kopic J, Paradzik MT, pandak N. Streptococcus suis infection as a cause of severe illness: 2 cases from Croatia [J]. Scand J Infect Dis, 2002, 34(9): 683-684.
[79]Mamianian S K, Liu G, Jensen E R, et a1. Staphylococcns aurens sottase mutants defective in the display of surface proteins and in the pathogenesis of animal infections [J]. Proc Natl Acad Sci USA, 2000, 97(10): 5510-5515.
[80]Marcelo G, Marida S. The pathogenesis of the meningitis caused by Streptococcus suis: the unresolved questions [J]. Vet Microbio1, 2000, 76: 259-272.
[81]Marois C, Bougeard S, Gottschalk M, et a1. Multiplex PCR assay for detection of Streptococcus suis species and serotypes 2 and 1/2 in tonsils of live and dead pigs [J]. J Clin Microbiol, 2004, 42(7): 3169-3175.
[82]Norton P M, Polph C, ward P N, et a1. Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin [J]. FEMS Immunol Med Microbiol, 1999, 26: 25-35.
[83]Numata S, Nakamura Y, Imamura Y, et a1. Rapid quantitative analysis of human cytomegalovirus DNA by the real-time polymerase chain reaction method [J]. Arch Pathol Lab Med, 2005, 129(2): 200-204.
[84]Okwumabua O, Persaud J S, Reddy P G. Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2[J]. Clin Diagn Lab Immunol, 2001, 8: 251-257.
[85]Osaki M, Takamatsu D, Shimoji Y, et a1. Characterization of Streptocuccus suis genes encoding proteins homologous to sortase of gram-positive bacteria [J]. Bacteriol, 2002, 184(4): 971-982.
[86] Pang Y G, Jiang J Y,Wang L Z, et al.Effects of immunological stress on immune response in different breeds of piglets[J].Animal Husbandry and Feed Science, 2009,1 (2):28-31.
[87]Saulnier P. Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant staphylococcus aureus [J]. J Clin Microbiol, 2003, 31:9821.
[88]Silva L M, Baums C G, Rehm T, et a1. Virulence-associated gene profiling of Streptococcus suis isolates by PCR [J]. Vet Microbiol, 2006, 104(1): 87-94.
[89]Smith H E, Martine R, Norbert S Z, et al. Virulent Strains of Streptococcus suis Serotype 2 and Highly Virulent Strains of Streptococcus suis Serotype 1 Can Be Recognized by a Unique Ribotype Profile[J]. J Clin Microbiol, 1997, (5): 1049-1053.
[90]Smith H E, Damman M, Vander Velde J, et a1. Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor [J]. Infect Immun, 1999, 67(4): 1750-1756.
[91]Smith H E, Vecht U, Wissdink H J, et a1. Mutants of Streptococcus suis types l and 2 impaired in expression of muramidase-released protein and extracelluhr protein induce disease in newborn germfree pigs [J]. Infect Immun, 1996, 64(10): 4409-4412.
[92]Smith H E, Veenbergen V, Vander Veled J, et a1. The cps genes of Streptococcus suis serotypes 1, 2 and 9: development of rapid serotype-specific PCR assays [J ]. J Clin Microbiol,1999,37(10): 3146-3152.
[93]Spackman E, Senne D A, Bulaga L L, et a1. Development of real-time RT-PCR for the detection of avian influenza virus [J]. Avian Dis, 2003, 47(3): 1079-1082.
[94]Staats J J, Feder I, Okwumabua O, et a1. Streptococcus suis: past and present presence [J]. Vet Res Commun, 1997, 21(6): 381-407.
[95]Staats J J, Plattner B L, Nietfeld J, et al. Use of Ribotyping and Hemolysin Activity To Identify Highly Virulent Streptococcus suis Type 2 Isolates[J]. J Clin Microbiol, 1998, 2: 15-19.
[96]Strangmnn E, Froleke H, Kohse K P. Septic shock caused by Streptococcus suis: case report and investigation of a risk group [J]. Int J Hyg Environ Health, 2002, 205(5): 385-392.
[97]Swildens B, Wisselink H J, Engel B, et a1. Detection of extracellular factor-positive Streptococcus suis serotype 2 strains in tonsillar swabs of live sows by PCR [J]. Vet Microbiol, 2005, 109(3/4): 223-228.
[98]Tang J Q, Wang C J, Feng YJ, et a1. Streptococcal toxic shock syndrome caused by streptococcus suis serotype2 [J]. PLoS Med, 2006, 3(5): 151.
[99]Tian Y, Aarestrup F M, Lu C P. Characterization of Streptococcus suis serotype 7 isolates from diseased pigs in Denmark [J]. Veterinary Microbiology, 2004, 103: 55–62.
[100]Ton-That H, Mazmanian S K, Faull K F, et a1. Anchoring of surface proteins to the cell wall of Staphylococcus aurens Sortase-catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-G1y3 substrates [J]. J Biol Chem, 2000, 275(13): 9876-9881.
[101]Vanier G, Segura M, Friedl P, et al. Invasion of porcine brain microvascular endothelial cells by Streptococcus suis serotype 2[J]. Infect lmmun, 2004, 72(3): 1441-1449.
[102]Vela A I, Goyache J, Tarradas C, et a1. Analysis of genetic diversity of Streptococcus suis clinical isolates from pigs in Spain by pulsed-field gel electrophoresis[J]. J Clin Microbio, 2003, 41(6): 2498- 2502.
[103]Vilaichone R K, Vilaichone W, Nunthapisud P, et a1. Streptococcus suis infection in Thailand [J]. J Med Assoc Thai, 2002, 85(1): S109-117.
[104]Wisselink H J, Joosten J J, Smith H E. Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs [J]. J Clin Microbiol, 2002, 40(8): 2922-2929.
[105]Wisselink H J, Smith H E, Stockhofe-Zurwieden, et a1. Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries [J]. Vet Microbiol, 2000, 74(3): 237-248.
[106]Yam W C, Chan K H, Chow K H, et a1. Clinical evaluation of real-time PCR assays for rapid diagnosis of SARS coronavirus during outbreak and post-epidemic periods [J]. J Clin Virol, 2005, 33(1): 19-24.
[107] Zhang C P,NiNg Y B,Zhang Z Q,et al.Prevalence of Streptococcus suis isolated from clinically healthy sows in China[J].Agricultural Sciences in China,2009,8(5):638-642.
[108]Zink S D, Burns D L. Importance of srtA and srtB for growth of Bacillus anthracis in maerophages[J]. Infect lmmun, 2005, 73(8): 5222-5228.