猪链球菌流行病学调查和PCR-RFLP基因分型研究
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摘要
猪链球菌是一种条件性致病菌,通常定居在感染猪的上颚扁桃体,当感染猪的抵抗力降低时,可能引发猪链球菌病的流行,因此从猪扁桃体内分离猪链球菌对该病的流行病学监测具有重要的意义。研究发现相同血清型的不同菌株及不同血清型菌株间的毒力存在差异,而且相同血清型的菌株并不完全导致相同的疾病,但传统的血清分型方法不能很好反映菌株的特征。本试验尝试建立一新的基因分型方法PCR-RFLP对猪链球菌进行研究.
1.表观健康猪的猪链球菌流行病学调查：对采自杭州、重庆等20个不同地区屠宰场的248份扁桃体或淋巴结样品进行细菌分离培养,然后用GDH、CPS1i、CPS2j、CPS7h、CPS9g基因序列的PCR方法进行种型鉴定并用血清凝集试验复检,随后检测其毒力因子检测,应用多位点序列分型(Multilocus sequence typing,MLST)系统研究菌株间的遗传关系。结果：从248份样品中共检出14株猪链球菌(检出率为5.65%),其中3株为猪链球菌2型,2株为猪链球菌7型,1株为猪链球菌9型,8株不能定血清型；6株定型菌株血清凝集试验结果与PCR鉴定结果一致；对6株定型菌进行6个毒力因子(mrp、epf、sly、orf2、fbps、gapdh)检测发现5种毒力基因型：A1Cps2+mrp-epf+sly+orf2+fbps+gapdh+、A2 Cps2+mrp-epf+sly-orf2-fbps+gapdh-、A3Cps7+mrp+epf-sly-orf2-fbps-gapdh+、A4 Cps9+mrp-epf-sly-orf2-fbps+gapdh+、A5Cps7+mrp-epf-sly-orf2+fbps+gapdh+,A1和A2型的菌株为弱毒株；经MLST分析3株猪链球菌2型属于STl,1株7型属于ST29,1株9型(CQ15)属于一个新ST型,该型被猪链球菌MLST数据库(http://ssuis.mlst.net)收录并编号为ST128。
2.PCR-RFLP的建立及其在猪链球菌基因分型中的应用：本试验对32个血清型(1-31,1／2)猪链球菌参考株和47株背景明确的猪链球菌分离株的16S-23S ISR序列进行扩增,首先建立并优化PCR最佳反应体系、反应条件和酶切最佳反应体系、反应条件,扩增产物经酶切、凝胶电泳,最后在紫外灯下观察电泳图谱。结果：1上述菌株的PCR产物经双酶切后共得到22种RFLP型：其中Aa型有25株,占总菌株数的31.6%；Ib型有20株,占总菌株数的25.3%；Bf型有7株,占总菌株数8.9%；Ga型有4株,占总菌株数的5.1%；Be和Ka型各有3株,占总菌株数3.8%；Bh型有2株,占总菌株数2.5%；Eb、Bb、Gg、Di、Hc、Fj、Ca、Bd、Jk、Ll、Ie、Bm、Ag、Mn、Io型各有一株。2对酶切基因型进行统计分析,获得的PCR-RFLP方法的分辨指数为0.83。3猪链球菌1／2型和2型经PCR-RFLP分析,产生不同的RFLP型,可以区分这两种血清型,而其他的血清型无法用该法区分。4分离株中共有18株强毒株,其中7型一株,9型一株,2型16株,这些菌株经PCR-RFLP分析发现7型和2型的强弱毒株分别具有相同的RFLP型,所以无法用该法进行猪链球菌强弱毒株的区分。
Streptococcus suis (S. suis) is a conditional pathogen and usually settles in the upper tonsil of infected swine. When the immunity of infected swine falls, it will cause disease and subsequently lead to the prevalence of Streptococcus suis. Therefore, it is of great significance to detect and isolate S. suis from swine tonsil for the epidemiological surveillance of the disease. Some studies show that the virulence differs among the strains of different serotypes. But this phenomenon also exists in the same serotypes and some strains of the same serotype do not entirely cause the same disease. The current serotyping methods do not adequately reflect the characteristics of S. suis. So this work is sought to establish a new genotyping method for the study of Streptococcus suis by using PCR-RFLP.
1. Epidemiological investigation of Streptococcus suis in carried swine:248 tonsils or lymph were collected from 20 slaughterhouses in different regions of China Streptococcus suis were isolated from collected samples and purified in sheep blood plate. And then species and serotypes of suspected colony were identified by PCR assay on the basis of gene squences of GDH、CPSli、CPS2j、CPS7h、CPS9g, followed by serum agglutination test and virulence factors detection by PCR. Genetic relationships of isolates were studied by multilocus sequence typing (MLST). The results were as followed:14 strains of Streptococcus suis were identified by PCR assay, including 3 strains of ss2,2 strains of ss7, 1 strain of ss9 and 8 strains of non-serotype Streptococcus suis. The serum typing result of 6 isolates by serum agglutination test were consistent with the results by PCR assay.5 kinds of virulence genotypes were generated from 6 known serotype isolates during detecting 6 virulence factors(mrp、epf、sly、orf2、fbps、gapdh)by PCR:A1 Cps2+mrp-epf+sly+orf2+fbps+gapdh; A2 Cps2+mrp-epf+sly-orf2-fbps+gapdh-; A3 Cps7+mrp+epf-sly-orf2-fbps-gapdh+; A4 Cps9-mrp-epf-sly-orf2-fbps+gapdh+; A5 Cps7+mrp-epf-sly-orf2+fbps+gapdh+. Strains belonging to A1 and A2 were mild strains,and others weren't known. Analyzed by MLST, the results implied that 3 strains of ss2 belonged to ST1,1 strain of ss7 belonged to ST29 and 1 strain of ss9 (CQ15) belonged to a new ST type, which was not reported in previous publications. The new ST was submitted to MLST database of Streptococcus suis (http://ssuis.mlst.net) and assigned to ST128.
2. Development and application of PCR-RFLP in genotype research of Streptococcus suis:16S-23S ISR rDNA squences of 32 serotypes (1-31,1/2) reference strains and 47 isolates with clear background were amplified by PCR.
1) The establishment and optimization of system and condictions of PCR and enzymatic reaction.
2) The digestion of PCR products of reference strains with Rsal and HinfI.
3) The observation of different patterns gained by gel electrophoresis.
The results in this work were as followed:22 genotypes were generated after digestion with RsaI and HinfI, including 25 strains Aa (31.6%); 20 strains Ib (25.3%); 7 strains Bf (8.9%); 4 strains Ga (5.1%); 3 strains Be and 3 strains Ka (3.8%); 2 strains Bh (2.5%); 1 strain Eb、Bb、Gg、Di、Hc、Fj、Ca、Bd、Jk、Ll、Ie、Bm. Ag、Mn、Io, respectively. After analyzing enymatic patterns according to Simpsom's index of diversity (DI), DI of PCR-RFLP was 0.83. The patterns of serotype 1/2 and 2 of reference and isolate were different. So serotype 1/2 could be distinguished from serotype 2. But other serotypes could not be discriminated.18 virulent isolates included 1 strain SS7,1 strain SS9 and 16 strains SS2. The patterns of virulent and avirulent SS7 strains were the same, and so were SS2. Therefore, virulent or avirulent strains could not be discriminated by PCR-RFLP.
1.表观健康猪的猪链球菌流行病学调查：对采自杭州、重庆等20个不同地区屠宰场的248份扁桃体或淋巴结样品进行细菌分离培养,然后用GDH、CPS1i、CPS2j、CPS7h、CPS9g基因序列的PCR方法进行种型鉴定并用血清凝集试验复检,随后检测其毒力因子检测,应用多位点序列分型(Multilocus sequence typing,MLST)系统研究菌株间的遗传关系。结果：从248份样品中共检出14株猪链球菌(检出率为5.65%),其中3株为猪链球菌2型,2株为猪链球菌7型,1株为猪链球菌9型,8株不能定血清型；6株定型菌株血清凝集试验结果与PCR鉴定结果一致；对6株定型菌进行6个毒力因子(mrp、epf、sly、orf2、fbps、gapdh)检测发现5种毒力基因型：A1Cps2+mrp-epf+sly+orf2+fbps+gapdh+、A2 Cps2+mrp-epf+sly-orf2-fbps+gapdh-、A3Cps7+mrp+epf-sly-orf2-fbps-gapdh+、A4 Cps9+mrp-epf-sly-orf2-fbps+gapdh+、A5Cps7+mrp-epf-sly-orf2+fbps+gapdh+,A1和A2型的菌株为弱毒株；经MLST分析3株猪链球菌2型属于STl,1株7型属于ST29,1株9型(CQ15)属于一个新ST型,该型被猪链球菌MLST数据库(http://ssuis.mlst.net)收录并编号为ST128。
2.PCR-RFLP的建立及其在猪链球菌基因分型中的应用：本试验对32个血清型(1-31,1／2)猪链球菌参考株和47株背景明确的猪链球菌分离株的16S-23S ISR序列进行扩增,首先建立并优化PCR最佳反应体系、反应条件和酶切最佳反应体系、反应条件,扩增产物经酶切、凝胶电泳,最后在紫外灯下观察电泳图谱。结果：1上述菌株的PCR产物经双酶切后共得到22种RFLP型：其中Aa型有25株,占总菌株数的31.6%；Ib型有20株,占总菌株数的25.3%；Bf型有7株,占总菌株数8.9%；Ga型有4株,占总菌株数的5.1%；Be和Ka型各有3株,占总菌株数3.8%；Bh型有2株,占总菌株数2.5%；Eb、Bb、Gg、Di、Hc、Fj、Ca、Bd、Jk、Ll、Ie、Bm、Ag、Mn、Io型各有一株。2对酶切基因型进行统计分析,获得的PCR-RFLP方法的分辨指数为0.83。3猪链球菌1／2型和2型经PCR-RFLP分析,产生不同的RFLP型,可以区分这两种血清型,而其他的血清型无法用该法区分。4分离株中共有18株强毒株,其中7型一株,9型一株,2型16株,这些菌株经PCR-RFLP分析发现7型和2型的强弱毒株分别具有相同的RFLP型,所以无法用该法进行猪链球菌强弱毒株的区分。
Streptococcus suis (S. suis) is a conditional pathogen and usually settles in the upper tonsil of infected swine. When the immunity of infected swine falls, it will cause disease and subsequently lead to the prevalence of Streptococcus suis. Therefore, it is of great significance to detect and isolate S. suis from swine tonsil for the epidemiological surveillance of the disease. Some studies show that the virulence differs among the strains of different serotypes. But this phenomenon also exists in the same serotypes and some strains of the same serotype do not entirely cause the same disease. The current serotyping methods do not adequately reflect the characteristics of S. suis. So this work is sought to establish a new genotyping method for the study of Streptococcus suis by using PCR-RFLP.
1. Epidemiological investigation of Streptococcus suis in carried swine:248 tonsils or lymph were collected from 20 slaughterhouses in different regions of China Streptococcus suis were isolated from collected samples and purified in sheep blood plate. And then species and serotypes of suspected colony were identified by PCR assay on the basis of gene squences of GDH、CPSli、CPS2j、CPS7h、CPS9g, followed by serum agglutination test and virulence factors detection by PCR. Genetic relationships of isolates were studied by multilocus sequence typing (MLST). The results were as followed:14 strains of Streptococcus suis were identified by PCR assay, including 3 strains of ss2,2 strains of ss7, 1 strain of ss9 and 8 strains of non-serotype Streptococcus suis. The serum typing result of 6 isolates by serum agglutination test were consistent with the results by PCR assay.5 kinds of virulence genotypes were generated from 6 known serotype isolates during detecting 6 virulence factors(mrp、epf、sly、orf2、fbps、gapdh)by PCR:A1 Cps2+mrp-epf+sly+orf2+fbps+gapdh; A2 Cps2+mrp-epf+sly-orf2-fbps+gapdh-; A3 Cps7+mrp+epf-sly-orf2-fbps-gapdh+; A4 Cps9-mrp-epf-sly-orf2-fbps+gapdh+; A5 Cps7+mrp-epf-sly-orf2+fbps+gapdh+. Strains belonging to A1 and A2 were mild strains,and others weren't known. Analyzed by MLST, the results implied that 3 strains of ss2 belonged to ST1,1 strain of ss7 belonged to ST29 and 1 strain of ss9 (CQ15) belonged to a new ST type, which was not reported in previous publications. The new ST was submitted to MLST database of Streptococcus suis (http://ssuis.mlst.net) and assigned to ST128.
2. Development and application of PCR-RFLP in genotype research of Streptococcus suis:16S-23S ISR rDNA squences of 32 serotypes (1-31,1/2) reference strains and 47 isolates with clear background were amplified by PCR.
1) The establishment and optimization of system and condictions of PCR and enzymatic reaction.
2) The digestion of PCR products of reference strains with Rsal and HinfI.
3) The observation of different patterns gained by gel electrophoresis.
The results in this work were as followed:22 genotypes were generated after digestion with RsaI and HinfI, including 25 strains Aa (31.6%); 20 strains Ib (25.3%); 7 strains Bf (8.9%); 4 strains Ga (5.1%); 3 strains Be and 3 strains Ka (3.8%); 2 strains Bh (2.5%); 1 strain Eb、Bb、Gg、Di、Hc、Fj、Ca、Bd、Jk、Ll、Ie、Bm. Ag、Mn、Io, respectively. After analyzing enymatic patterns according to Simpsom's index of diversity (DI), DI of PCR-RFLP was 0.83. The patterns of serotype 1/2 and 2 of reference and isolate were different. So serotype 1/2 could be distinguished from serotype 2. But other serotypes could not be discriminated.18 virulent isolates included 1 strain SS7,1 strain SS9 and 16 strains SS2. The patterns of virulent and avirulent SS7 strains were the same, and so were SS2. Therefore, virulent or avirulent strains could not be discriminated by PCR-RFLP.
引文
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[12]Vecht U, Wisselink H J,Jellema M J,et al. Identification of two proteins associated with virulence of Streptococcus suis type 2[J].Infect Immun,1991,59:3156.
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[14]Vecht U, Wisselink H J, van Dijk JE, Smith HE. Virulence of Streptococcus suis type 2 strains in newborn germfree pigs depends on phenotype[J]. Infect Immun 1992,60:550～556.
[15]廖亚玲,邹全明.病原微生物基因多位点序列分型的研究进展[J].微生物免疫学进展,2007,35(4)： 65-68.
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[1]Perch B,Kristiansen P,Skadhauge K.Group R streptococci pathogenic for man Actapathol[J]. Microbiol Scand,1968,74:69-76.
[2]汪华,胡晓抒,朱凤才,等.人猪链球菌感染性综合征的流行病学调查[J].现代预防学,2000,27(3)：312-314.
[3]杨华富,朱凤才,史智扬,等.猪链球菌感染性综合症的病原特征分析[J].中国人兽共患病杂志,2001,17：92-93.
[4]吴国辉,王豫林,肖邦忠,等.重庆市人感染猪链球菌病的病原分离与鉴定[J].现代预防医学,2005,33(8)：1348-1350.
[5]Staats J J,Feder I,Okwumabua O,et al. Streptococcus suis:past and present[J]. Vet Res Communi, 1997,21:381-407.
[6]Ho Dang Trung Nghia,Ngo Thi Hoa,et al. Human Case of Streptococcus suis Serotype 16 Infection[J]. Emerg Infect Dis,2008,14(1):155-156.
[7]Rene Poggenborg,Shahin Gaini,Poul Kj(?)ldgaard,Jens J(?)rgen Christensen. Streptococcus suis:Mening-itis,spondylodiscitis and bacteraemia with a serotype 14 strain[J]. Scandinavian Journal of Infectious Diseases,40(4):346-349.
[8]Han D U, Choi C,Ham H J, Jung J H,Cho W S, Kim J,et al. Prevalence,capsular type and antimicro-bial susceptibility of Streptococcus suis isolated from slaughter pigs in Korea[J]. Can J Vet Res.2001,65: 151-155.
[9]何孔旺,倪艳秀,王继春,等.猪链球菌2型的分子流行病学研究[J].中国人兽共患病杂志,2002,18(5)：45-47.
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[12]Vecht U, Wisselink H J,Jellema M J,et al. Identification of two proteins associated with virulence of Streptococcus suis type 2[J].Infect Immun,1991,59:3156.
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[14]Vecht U, Wisselink H J, van Dijk JE, Smith HE. Virulence of Streptococcus suis type 2 strains in newborn germfree pigs depends on phenotype[J]. Infect Immun 1992,60:550～556.
[15]廖亚玲,邹全明.病原微生物基因多位点序列分型的研究进展[J].微生物免疫学进展,2007,35(4)： 65-68.
[16]King S J,Leigh J A,Heath P J,et al.Development of a multilocus sequence typing scheme for Strept-ococcus suis:Identification of virulent clones and potential capsular serotype exchange[J].J Clin Microbiol,2002,40:3671-3680.
[1]. Gottschalk M, Higgins R, Jacques M, et al.Characterization of six new capsular types (23 through 28) of Streptococcus suis[J]. Clin Microbiol,1991,29:2590-2594.
[2]. Gottschalk M, Higgins R, Jacques M, et al. Description of 14 new capsular types of Streptococcus suis [J]. Clin Microbiol,1989,27:2633-2636.
[3]. Higgins R, Gottschak M, Boudreau, et al. Description of six new capsular types (29-34) of Streptococcus suis [J]. Vet Diagn Investig,1995,7:405-406.
[4]. Perch B, Pedersen K B, Henrichsen J. Serology of capsulated streptococci pathogenic for pigs:six new serotypes of Streptococcus suis [J]. Clin Microbiol,1983,19:993-996.
[5].刘军,石谦,孙洋,等.四川资阳地区猪链球菌2型检测及其特性比较研究[J].中国人兽共患病学报,2006,22(12)：1095～1099.
[6]. Staats J J,Feder I, Okwumabua O, et al. Streptococcus suis:Past and Present[J]. Vet Res Commun,1997,21:381-407.
[7]. Samantha J. King, James A. Leigh, Peter J. Heath, et al. Development of a Multilocus Sequence Typing Scheme for the Pig Pathogen Streptococcus suis:Identification of Virulent Clones and Potential Capsular Serotype Exchange[J]. Clin Microbiol,2002,40(10):3671～3680.
[8].Florence Berthelot-Herault, Corinne Marois, Marcelo Gottschalk, et al. Diversity of Streptococcus suis Strains Isolated from Pigs and Humans as Revealed by Pulsed-Field Gel Electrophoresis [J]. Clin Microbio,2002,40(2):615～619.
[9]. Corinne Marois, Laetitia Le Devendec, Marcelo Gottschalk, Marylene Kobisch. Molecular characterization of Streptococcus suis strains by 16S-23Sintergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis[J]. The Canadian Journal of Veterinary Research,2006,70:94～104.
[10].Corinne Marois, Laetitia Le, Marcelo Gottschalk, et al. Detection and molecular typing of Streptococcus suis in tonsils from live pigs in France[J]. The Canadian Joural of Veterinary Research,2007,71:14～22.
[11].Hill JE, Gottschalk M,Brousseau R,et al. Biochemical analysis cpn60 and 16S rDNA sequence data indicate that Streptococcus suis serotypes 32 and 34 isolated from pigs are Streptococcus orisratti[J].Vet Microbiol,2005,107(1-2):63-69.
[12]. Sonia Chatellier, Jose Harel, Ying Zhang, et al. Phylogenetic diversity of streptococcus suis strains of various serotypes as revealed by 16S rRNA gene sequence comparison [J]. International Journal of systematic Bacteriology,1998,48:581-589.
[13]. Schlegel L, Grimont F, Grimont PA, et al. Identification of major streptococcal species by rrn-amplified ribosomal DNA restriction analysis[J]. J Clin Microbiol,2003,41:657-666.
[14]. Shaver YJ, Nagpal ML, Rudner R, Nakamura LK, Fox KF, Fox A. Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups[J]. J Microbiol Methods,2002,50:215～223.
[15]. P R Hunter, M A Gaston. Numerical Index of the Discriminatory Ability of Typing Systems:an Application of Simpson's Index of Diversity[J]. Clin Microbiol,1988,26(11):2465～2466.
[16]马俊英,刘健华,陈杖榴,等.细菌分型的分子生物学技术研究进展[J].中国兽医科学,2007,37(10)：914～918.
[17]Henk J Wisselink,Jeroen J, Joosten and Hilde E. Smith. Multiplex PCR Assays for Simultaneous Detection of Six Major Serotypes and Two Virulence-Associated Phenotypes of Streptococcus suis in Tonsillar Specimens from Pigs[J]. Journal of Clinical Microbiology,2002,40(8):2922～2929.