猪2型链球菌间接ELISA和间接血凝抗体检测方法的建立及应用
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摘要
猪链球菌(Streptococcus suis，S．suis)能引起猪脑膜炎、心内膜炎、关节炎、肺炎和败血症等，该菌有35个血清型，其中2型流行最广、且致病性最强，并能感染人，是一种重要的人畜共患病病原。但猪链球菌经常与其他一些病原如猪放线杆菌、猪丹毒杆菌和猪蓝耳病病毒等发生混合感染，给临床诊断和抗体水平的检测造成了很大困难。因此，本课题的研究目的：
1建立以猪2型链球菌荚膜多糖为抗原的间接ELISA检测方法。
2构建溶血素基因的原核表达质粒，并分析表达产物的免疫反应性，建立以溶血素为靶蛋白的猪链球菌间接血凝(IHA)抗体检测方法。
3以上述两种方法对临床上送检血清进行检测，验证建立的两方法的符合率，并分析最近一年内我国猪2型链球菌的流行病学特点。
猪2型链球菌能产生多种毒力因子，常见的有荚膜多糖(CPS)、溶菌酶释放蛋白(Muramidase-released protein，MRP)、胞外因子(Extracellular factor，EF)，溶血素(Suilysin，SLY)等，其中CPS是最早被证实的猪2型链球菌毒力因子，也是进行分型的依据，本试验用改良的kodana方法提取猪2型链球菌的荚膜多糖，并以此作为抗原建立间接ELISA抗体检测方法，对各方面条件进行优化，研制出猪2型链球菌间接ELISA抗体检测试剂盒。另外发病猪的猪2型链球菌中有相当高比例的菌株表现出溶血素活性，因而猪2型链球菌溶血素被认为是该菌主要的毒力因子。本研究根据猪2型链球菌溶血素基因序列设计一对特异性引物，采用PCR扩增猪2型链球菌LT-1分离株溶血素基因，将扩增产物克隆到pMD18-T质粒载体中，酶切和PCR鉴定后测序鉴定。结果显示，扩增的LT-1株溶血素基因长度为1494bp，编码497个氨基酸。将溶血素基因克隆入表达载体pET-28a中，提取重组质粒进行PCR和酶切鉴定，筛选阳性克隆，用SDS-PAGE电泳和Western blot进行溶血素的表达和鉴定。结果表明表达产物具有免疫反应性。然后以表达的溶血素蛋白为抗原致敏绵羊红细胞，并对各方面条件优化，建立猪链球菌间接血凝试验(IHA)。
用间接ELISA方法检测从河南、安徽、江西、湖南、湖北、福建、广东、广西和北京等地送检的血清样品共3991份，结果表明阳性率分别为33.75％，41.6％等，各省血清样品阳性率从0％到41.6％。本研究建立的两种检测方法操作简单、特异性强、敏感性高并适用于猪链球菌的抗体检测和流行病学调查。
Streptococcus suis is an important swine pathogen that causes a. number ofPathological conditions such as meningitis, endocarditis, arthritis, pneumonia andsepticemia. There are currently 35 seroypes of Streptococcus suis, among which type 2 isthe most popular and pathogenic. But Streptococcus suis type 2 often combined infectionwith some other etiological agents as Actinobacillus suis, Bacillus erysipelatous andPRRS virus, It results in tremendous difficulty by clinical diagnosis and antibodiesdetection. Thus the topic purpose of this study:
1 To establish indirect ELISA detection method of Streptococcus suis type 2 usedcapsular bolysaccharides as antigen.
2 To construct the prokaryotic expression vectors for suilysin gene and examine itsimmunoreactivity of expression product and establish indirect hemagglutination assay(IHA) detection method of Streptococcus suis used Suilysin protein as antigen.
3 Detect clinical serums by two above-mentioned kinds of method to check thecoincidence of two kinds of method and analyze the epidemiology characteristic ofStreptococcus suis type 2 in lately one year in our courtry.
Streptococcus suis type 2 can create many kinds of virulence factor, for examplecapsular bolysaccharides(CPS), Muramidase-released protein (MRP),Extracellular factor (EF), Suilysin (SLY) and so on. Among the total, capsularbolysaccharides is most early corroborative virulence factor of Streptococcus suis type 2,it is also the base of typing. This test extracts capsular bolysaccharides by modifiedkodana method to establish indirect ELISA detection method and optimizes the bestworking condition to manufacture indirect ELISA detection kit Otherwise, Streptococcussuis type 2 has a high proportional to display suilysin activity, so Suilysinis knownas the virulence factor. On the base of the suquence of suilysin gene, The suilysin gene ofStreptococcus suis type 2 isolated LT-1 was amplified by PCR from bacterial genomicDNA, cloned into pMD18-T vector and then sequenced. The sequencing result showedthat the PCR product was composed of 1494 nucleotides encoding a polypeptide of 497amino acids. Streptococcus suis suilysin gene was cloned into a prokaryotic expression plasmid pET-28a, and the recombinant plasmid was then identifid by PCR and restrictionenzyme digestion and screened the positive clones. Suilysin expression was analyzed bySDS-PAGE and Western blot. Suilysin was expressed in the recombinant strains andreactive to positive serum as shown by immnuno-blotting. The sheep erythrocyte(SE) wasallergized by the expressed SLY protein and optimized all round conditions, then theindirect hemagglutination assay was established of Streptococcus suis.
To detect 3 991 shares serum products by indirect ELISA detection method inHenan, Anhui, Jiangxi, Hunan, Hubei, Fujian, Guangdong, Guangxi and Beijing, the rateof positive was 33.75 %, 41.6% and so on. The two kinds of method established bythis research operate simplely, specificity highly, sensitivity highly and refered todetecting antibody and epidemiological survey agaist Streptococcus suis.
1建立以猪2型链球菌荚膜多糖为抗原的间接ELISA检测方法。
2构建溶血素基因的原核表达质粒，并分析表达产物的免疫反应性，建立以溶血素为靶蛋白的猪链球菌间接血凝(IHA)抗体检测方法。
3以上述两种方法对临床上送检血清进行检测，验证建立的两方法的符合率，并分析最近一年内我国猪2型链球菌的流行病学特点。
猪2型链球菌能产生多种毒力因子，常见的有荚膜多糖(CPS)、溶菌酶释放蛋白(Muramidase-released protein，MRP)、胞外因子(Extracellular factor，EF)，溶血素(Suilysin，SLY)等，其中CPS是最早被证实的猪2型链球菌毒力因子，也是进行分型的依据，本试验用改良的kodana方法提取猪2型链球菌的荚膜多糖，并以此作为抗原建立间接ELISA抗体检测方法，对各方面条件进行优化，研制出猪2型链球菌间接ELISA抗体检测试剂盒。另外发病猪的猪2型链球菌中有相当高比例的菌株表现出溶血素活性，因而猪2型链球菌溶血素被认为是该菌主要的毒力因子。本研究根据猪2型链球菌溶血素基因序列设计一对特异性引物，采用PCR扩增猪2型链球菌LT-1分离株溶血素基因，将扩增产物克隆到pMD18-T质粒载体中，酶切和PCR鉴定后测序鉴定。结果显示，扩增的LT-1株溶血素基因长度为1494bp，编码497个氨基酸。将溶血素基因克隆入表达载体pET-28a中，提取重组质粒进行PCR和酶切鉴定，筛选阳性克隆，用SDS-PAGE电泳和Western blot进行溶血素的表达和鉴定。结果表明表达产物具有免疫反应性。然后以表达的溶血素蛋白为抗原致敏绵羊红细胞，并对各方面条件优化，建立猪链球菌间接血凝试验(IHA)。
用间接ELISA方法检测从河南、安徽、江西、湖南、湖北、福建、广东、广西和北京等地送检的血清样品共3991份，结果表明阳性率分别为33.75％，41.6％等，各省血清样品阳性率从0％到41.6％。本研究建立的两种检测方法操作简单、特异性强、敏感性高并适用于猪链球菌的抗体检测和流行病学调查。
Streptococcus suis is an important swine pathogen that causes a. number ofPathological conditions such as meningitis, endocarditis, arthritis, pneumonia andsepticemia. There are currently 35 seroypes of Streptococcus suis, among which type 2 isthe most popular and pathogenic. But Streptococcus suis type 2 often combined infectionwith some other etiological agents as Actinobacillus suis, Bacillus erysipelatous andPRRS virus, It results in tremendous difficulty by clinical diagnosis and antibodiesdetection. Thus the topic purpose of this study:
1 To establish indirect ELISA detection method of Streptococcus suis type 2 usedcapsular bolysaccharides as antigen.
2 To construct the prokaryotic expression vectors for suilysin gene and examine itsimmunoreactivity of expression product and establish indirect hemagglutination assay(IHA) detection method of Streptococcus suis used Suilysin protein as antigen.
3 Detect clinical serums by two above-mentioned kinds of method to check thecoincidence of two kinds of method and analyze the epidemiology characteristic ofStreptococcus suis type 2 in lately one year in our courtry.
Streptococcus suis type 2 can create many kinds of virulence factor, for examplecapsular bolysaccharides(CPS), Muramidase-released protein (MRP),Extracellular factor (EF), Suilysin (SLY) and so on. Among the total, capsularbolysaccharides is most early corroborative virulence factor of Streptococcus suis type 2,it is also the base of typing. This test extracts capsular bolysaccharides by modifiedkodana method to establish indirect ELISA detection method and optimizes the bestworking condition to manufacture indirect ELISA detection kit Otherwise, Streptococcussuis type 2 has a high proportional to display suilysin activity, so Suilysinis knownas the virulence factor. On the base of the suquence of suilysin gene, The suilysin gene ofStreptococcus suis type 2 isolated LT-1 was amplified by PCR from bacterial genomicDNA, cloned into pMD18-T vector and then sequenced. The sequencing result showedthat the PCR product was composed of 1494 nucleotides encoding a polypeptide of 497amino acids. Streptococcus suis suilysin gene was cloned into a prokaryotic expression plasmid pET-28a, and the recombinant plasmid was then identifid by PCR and restrictionenzyme digestion and screened the positive clones. Suilysin expression was analyzed bySDS-PAGE and Western blot. Suilysin was expressed in the recombinant strains andreactive to positive serum as shown by immnuno-blotting. The sheep erythrocyte(SE) wasallergized by the expressed SLY protein and optimized all round conditions, then theindirect hemagglutination assay was established of Streptococcus suis.
To detect 3 991 shares serum products by indirect ELISA detection method inHenan, Anhui, Jiangxi, Hunan, Hubei, Fujian, Guangdong, Guangxi and Beijing, the rateof positive was 33.75 %, 41.6% and so on. The two kinds of method established bythis research operate simplely, specificity highly, sensitivity highly and refered todetecting antibody and epidemiological survey agaist Streptococcus suis.
引文
1．黄毓茂，黄引贤．2型猪链球菌的血清学鉴定．中国兽医学报，1995，15(1)：63-65
2．姚火春，陈国强，陆承平．猪链球菌1998分离株病原特性鉴定．南京农业大学学报，1999，22(2)：67-70
3．刘佩红，沈素芳，王永康等．上海地区猪源链球菌分离株的病原特性鉴定．中国兽医学报，2001，21(1)：42-46
4．欧瑜，陆承平．猪链球菌2型胞外蛋白因子基因片段的克隆与表达．中国兽医学报，2003，23(2)：163-166
5．蔡宝祥主编，家畜传染病学，第3版，北京：中国农业出版社，1996
6．黄静，张苏强，猪溶血性链球菌诊治报告，中国兽医杂志，1992，18(1)：22-23
7．陈国强，姚火春，陆承平．猪链球菌2型溶血毒素的产生条件及其特性分析．南京农业大学学报，2000，23(3)：71-75
8．萨姆布鲁克J，弗里奇EF，曼尼阿蒂斯T著，金冬雁，黎孟枫等译：分子克隆实验指南．北京：科学出版社，1999
9．查红波．2型猪链球菌研究进展．江西饲料，2005：(4)：27-29
10．黄金海，王英珍．三种新发细菌性猪病研究进展．动物科学与动物医学，2000：17(3)：45-47．
11．窦婕香．猪链球菌病的诊断和防治．动物科学与动物医学，2004：21(4)：61-62
12．杨百亮，王学娟．间接血凝抑制试验检测血清和卵黄中鸡毒支原体抗体的研究．中国家禽-2005：27(1)：19-21
13．林琳，江斌，吴胜会，吴南洋，张世忠，吴宗福．三种猪瘟抗体检测技术的应用比较．福建畜牧兽医-2004：26(3)：23-24
14．李纯德，郭长胜．间接血凝法检测伤寒、副伤寒抗体．中国公共卫生，2004：20(1)：93-93
15．张德林，李学瑞，杜重波．用弓形虫代谢分泌抗原制备间接血凝诊断试剂的研究．中国兽医科技，2003：33(9)：15-18
16．邹永新，李嘉爱，赖月辉，罗映霞，吴文福，巫月生等．检测鸡抗猪流行性腹泻抗体的微量间接血凝试验方法的建立．广东畜牧兽医科技，2003：28(6)：37-39
17．逯忠新，柳纪省等．猪传染性胸膜肺炎血清抗体检测．中国兽医科技，2001：31(5)：16-17
18．蒋启荣．正向间接血凝试验检测奶牛O型口蹄疫抗体．上海奶牛，2000：23(1)：41-41
19．金爱华，姚龙涛等．间接血凝抑制试验检测猪盖他病抗体方法的建立和应用．中国兽医杂志，2000：26(9)：17-18
20．蔡家利，蓝希嵌等．间接血凝试验检测猪繁殖与呼吸综合征病毒抗体．西南农业大学学报，2001：23(1)：45-46，50
21．胡小宏，聂绪明，何景，王汉成．正向间接血凝试验在检测猪瘟抗体中的应用．动物医学进展，2004：25(5)：131-131
22. Arends J, Zanen H. Meningitis caused by Streptococcus suis in humans. Rev Infect Dis, 1988, 10:131—137
23. Berthelot-Herault F, Morvan H, Keribin AM, et. al. Production of muraminidase-released protein(MRP), extracellular factor (EF) and haemolysin by field isolates of Streptococcus svis capsular types2, 1/2, 9, 7 and 3 isolated from swine in France. Vet Res, 2000, 31: 473-479
24. Burton JP, Chilcott CN, Moore CJ, Speiser G, Tagg JR. A preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodour parameters. J Appl Microbiol 2006:100(4):754-64
25. Charland N, Harel J, Kobisch, et al. Streptococcus suis serotype 2 mutants deficient in capsular expression. Microbiology, 1998, 144:325-332
26. Charland N, Jacques M, Lacouture S, et al.Characterization andprotective activity of a monoclonal antibody against a capsularepitope shared by Streptococcus suis serotypes 1, 2 and 1/2. Microbiology, 1997, 143: 3607-3614
27. Charland N, Kobisch M, Martineau-Doize B, et al.Role of capsular sialic acid in virulence and resistance to phagocytosis of Streptococcus suis capsular type 2. FEMS Irnmunol Med Microbiol, 1996, 14:195-203
28. Chatellier S, Gottschalk M, Higgins R, et al. Relatedness of Streptococcus suis serotype 2 isolates from different geographical origins as evaluated by molecular fingerprinting and phenotyping. J Clin Microbiol, 1999, 37:362-366
29. De Moor C E. Septicaemic infections in pigs caused by haemolytic Streptococci of new Lancefield groups designated R, S and T. Antonievan Leeuwenhoek,1963, 29:272-280
30. Del Campo sepulveda E, Altman E, Kobisch M, et al. Detection of antibodies against Streptococcus suis type 2 using a purified capsular polysaccharide antigen-based indirect ELISA. Vet Microbiol, 1996,52:113-125
31. Devriese L, Sustronck T, Maenhouth T, et al. Streptococcus svis meningitis in a horse. Vet Rec, 1990,127:68
32. Elliott S. Streptococcal infection in young pigs. I. An immunological study of the causative agent (PM streptococcus).J Hyg Camb, 1966, 64:205-212
33. Evriese L, Haesebrouck F, de Herdt P, et al. Streptococcus svis infections in birds. Avian Pathol, 1994,23:721-724
34. Gottschalk M, Lebrun A, Wisselink H J, et al.Production of virulence-related proteins by Canadian strains of Streptococcus suis capsular type 2. Can Vet J, 1998, 62:75-79
35. Gottschalk K, Higgins R, Jacques M, et al. Characterization of six new capsular types (23 through 28)of Streptococcus suss.J Clin Microbiol, 1993,31:2895-2900
36. Gottschalk K, Higgins R, QuessyS, et al. Dilemma of the virulence of Streptococcus suis. J Clin Microbiol, 1999, 37:4202-4203
37. Heath P, Hunt B, Duff J. Streptococcus suis serotype 14 as a cause of pig disease in the UK. Vet Rec, 1996,139:450-451
38. Higgins R, Gottschalk M. Distribution of Streptococcus suis capsular types in 1999. Can Vet J, 2000,41:414
39. Higgins R, Gottschalk M, Boudreau M, et al. Description of six new apsular types (29-34) of Streptococcus suis. J Vet Diagn Invest, 1995, 7:405-406
40. Higgins R,Gottschalk M,Beaudoin M. Streptococcus suis infection in swine: A sixteen-month study. Can J Vet Res, 1990, 54:170-173
41. Kilpper-Balz R, Schleifer K. Streptococcus suis sp. nov, nom. rev. Int J Syst Bacteriol, 1987,37:160-162
42. Smi th H E, Vecht U,L J, et al. Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of streptococcus suis type 2. Infect Immun, 1992, 60:2361-2367
43. Smith H E, Reek F H, Vecht U, et al. Repeats in an extracellular proteinof weakly pathogenic strains of streptococcus suss are absent in pathogenic strains. Infect Immun, 1993,61:3318-3326
44. Smith H E, Damman M, van der Velde, et al.Identification and characterization of the CPS locus of Streptococcus suss serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infect Immun, 1999, 67:1750-1756
45. Staats J J, Feder I, Okwumabua, et al. Streptococcus suis: past and present. Vet Res Commun, 1977,21:381-407
46. Staats, J.J, Feder, I, Okwumabua, O, Chengappa, M.M. Streptococcus suis: past and present. Vet Res Commun. 21(6), 1997. 381-407
47. Stoats J, Plattner B, Stewart G, et al. Presence of the Streptococcus suis Suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suistype 2 isolates. Vet Microbiol, 1999, 70:201-211
48. Touil, Higgin R, and Nadeau M. A isolation of Streptococcus suis from diseased pigs in Canada. Vet Microbiol, 1988,17:171-177
49. Vecht U, Wisselink H J, Jellema M C, et al. Identification of tw peoteins associated with virulence of Ssuis type 2. Infect Immun, 1991 59:3156-3162
50. Wessels M, Rubens C, Benedi V, et al, Definition of a bacterial virulence factor:sialylation of the group B streptococcal capsule Proc Natl Acad Sci USA, 1989, 86:8983-8987
51. Windsor R, Elliott S. Streptococcal infection in young pigs. IV. An outbreak of Streptococcal meningitis in weaned pigs. J Hyg Camb, 1975,75:69-78
2．姚火春，陈国强，陆承平．猪链球菌1998分离株病原特性鉴定．南京农业大学学报，1999，22(2)：67-70
3．刘佩红，沈素芳，王永康等．上海地区猪源链球菌分离株的病原特性鉴定．中国兽医学报，2001，21(1)：42-46
4．欧瑜，陆承平．猪链球菌2型胞外蛋白因子基因片段的克隆与表达．中国兽医学报，2003，23(2)：163-166
5．蔡宝祥主编，家畜传染病学，第3版，北京：中国农业出版社，1996
6．黄静，张苏强，猪溶血性链球菌诊治报告，中国兽医杂志，1992，18(1)：22-23
7．陈国强，姚火春，陆承平．猪链球菌2型溶血毒素的产生条件及其特性分析．南京农业大学学报，2000，23(3)：71-75
8．萨姆布鲁克J，弗里奇EF，曼尼阿蒂斯T著，金冬雁，黎孟枫等译：分子克隆实验指南．北京：科学出版社，1999
9．查红波．2型猪链球菌研究进展．江西饲料，2005：(4)：27-29
10．黄金海，王英珍．三种新发细菌性猪病研究进展．动物科学与动物医学，2000：17(3)：45-47．
11．窦婕香．猪链球菌病的诊断和防治．动物科学与动物医学，2004：21(4)：61-62
12．杨百亮，王学娟．间接血凝抑制试验检测血清和卵黄中鸡毒支原体抗体的研究．中国家禽-2005：27(1)：19-21
13．林琳，江斌，吴胜会，吴南洋，张世忠，吴宗福．三种猪瘟抗体检测技术的应用比较．福建畜牧兽医-2004：26(3)：23-24
14．李纯德，郭长胜．间接血凝法检测伤寒、副伤寒抗体．中国公共卫生，2004：20(1)：93-93
15．张德林，李学瑞，杜重波．用弓形虫代谢分泌抗原制备间接血凝诊断试剂的研究．中国兽医科技，2003：33(9)：15-18
16．邹永新，李嘉爱，赖月辉，罗映霞，吴文福，巫月生等．检测鸡抗猪流行性腹泻抗体的微量间接血凝试验方法的建立．广东畜牧兽医科技，2003：28(6)：37-39
17．逯忠新，柳纪省等．猪传染性胸膜肺炎血清抗体检测．中国兽医科技，2001：31(5)：16-17
18．蒋启荣．正向间接血凝试验检测奶牛O型口蹄疫抗体．上海奶牛，2000：23(1)：41-41
19．金爱华，姚龙涛等．间接血凝抑制试验检测猪盖他病抗体方法的建立和应用．中国兽医杂志，2000：26(9)：17-18
20．蔡家利，蓝希嵌等．间接血凝试验检测猪繁殖与呼吸综合征病毒抗体．西南农业大学学报，2001：23(1)：45-46，50
21．胡小宏，聂绪明，何景，王汉成．正向间接血凝试验在检测猪瘟抗体中的应用．动物医学进展，2004：25(5)：131-131
22. Arends J, Zanen H. Meningitis caused by Streptococcus suis in humans. Rev Infect Dis, 1988, 10:131—137
23. Berthelot-Herault F, Morvan H, Keribin AM, et. al. Production of muraminidase-released protein(MRP), extracellular factor (EF) and haemolysin by field isolates of Streptococcus svis capsular types2, 1/2, 9, 7 and 3 isolated from swine in France. Vet Res, 2000, 31: 473-479
24. Burton JP, Chilcott CN, Moore CJ, Speiser G, Tagg JR. A preliminary study of the effect of probiotic Streptococcus salivarius K12 on oral malodour parameters. J Appl Microbiol 2006:100(4):754-64
25. Charland N, Harel J, Kobisch, et al. Streptococcus suis serotype 2 mutants deficient in capsular expression. Microbiology, 1998, 144:325-332
26. Charland N, Jacques M, Lacouture S, et al.Characterization andprotective activity of a monoclonal antibody against a capsularepitope shared by Streptococcus suis serotypes 1, 2 and 1/2. Microbiology, 1997, 143: 3607-3614
27. Charland N, Kobisch M, Martineau-Doize B, et al.Role of capsular sialic acid in virulence and resistance to phagocytosis of Streptococcus suis capsular type 2. FEMS Irnmunol Med Microbiol, 1996, 14:195-203
28. Chatellier S, Gottschalk M, Higgins R, et al. Relatedness of Streptococcus suis serotype 2 isolates from different geographical origins as evaluated by molecular fingerprinting and phenotyping. J Clin Microbiol, 1999, 37:362-366
29. De Moor C E. Septicaemic infections in pigs caused by haemolytic Streptococci of new Lancefield groups designated R, S and T. Antonievan Leeuwenhoek,1963, 29:272-280
30. Del Campo sepulveda E, Altman E, Kobisch M, et al. Detection of antibodies against Streptococcus suis type 2 using a purified capsular polysaccharide antigen-based indirect ELISA. Vet Microbiol, 1996,52:113-125
31. Devriese L, Sustronck T, Maenhouth T, et al. Streptococcus svis meningitis in a horse. Vet Rec, 1990,127:68
32. Elliott S. Streptococcal infection in young pigs. I. An immunological study of the causative agent (PM streptococcus).J Hyg Camb, 1966, 64:205-212
33. Evriese L, Haesebrouck F, de Herdt P, et al. Streptococcus svis infections in birds. Avian Pathol, 1994,23:721-724
34. Gottschalk M, Lebrun A, Wisselink H J, et al.Production of virulence-related proteins by Canadian strains of Streptococcus suis capsular type 2. Can Vet J, 1998, 62:75-79
35. Gottschalk K, Higgins R, Jacques M, et al. Characterization of six new capsular types (23 through 28)of Streptococcus suss.J Clin Microbiol, 1993,31:2895-2900
36. Gottschalk K, Higgins R, QuessyS, et al. Dilemma of the virulence of Streptococcus suis. J Clin Microbiol, 1999, 37:4202-4203
37. Heath P, Hunt B, Duff J. Streptococcus suis serotype 14 as a cause of pig disease in the UK. Vet Rec, 1996,139:450-451
38. Higgins R, Gottschalk M. Distribution of Streptococcus suis capsular types in 1999. Can Vet J, 2000,41:414
39. Higgins R, Gottschalk M, Boudreau M, et al. Description of six new apsular types (29-34) of Streptococcus suis. J Vet Diagn Invest, 1995, 7:405-406
40. Higgins R,Gottschalk M,Beaudoin M. Streptococcus suis infection in swine: A sixteen-month study. Can J Vet Res, 1990, 54:170-173
41. Kilpper-Balz R, Schleifer K. Streptococcus suis sp. nov, nom. rev. Int J Syst Bacteriol, 1987,37:160-162
42. Smi th H E, Vecht U,L J, et al. Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of streptococcus suis type 2. Infect Immun, 1992, 60:2361-2367
43. Smith H E, Reek F H, Vecht U, et al. Repeats in an extracellular proteinof weakly pathogenic strains of streptococcus suss are absent in pathogenic strains. Infect Immun, 1993,61:3318-3326
44. Smith H E, Damman M, van der Velde, et al.Identification and characterization of the CPS locus of Streptococcus suss serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infect Immun, 1999, 67:1750-1756
45. Staats J J, Feder I, Okwumabua, et al. Streptococcus suis: past and present. Vet Res Commun, 1977,21:381-407
46. Staats, J.J, Feder, I, Okwumabua, O, Chengappa, M.M. Streptococcus suis: past and present. Vet Res Commun. 21(6), 1997. 381-407
47. Stoats J, Plattner B, Stewart G, et al. Presence of the Streptococcus suis Suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suistype 2 isolates. Vet Microbiol, 1999, 70:201-211
48. Touil, Higgin R, and Nadeau M. A isolation of Streptococcus suis from diseased pigs in Canada. Vet Microbiol, 1988,17:171-177
49. Vecht U, Wisselink H J, Jellema M C, et al. Identification of tw peoteins associated with virulence of Ssuis type 2. Infect Immun, 1991 59:3156-3162
50. Wessels M, Rubens C, Benedi V, et al, Definition of a bacterial virulence factor:sialylation of the group B streptococcal capsule Proc Natl Acad Sci USA, 1989, 86:8983-8987
51. Windsor R, Elliott S. Streptococcal infection in young pigs. IV. An outbreak of Streptococcal meningitis in weaned pigs. J Hyg Camb, 1975,75:69-78