9型猪链球菌广东株的分离鉴定和基因序列分析

doi:10.16431/j.cnki.1671-7236.2018.04.023

摘要/Abstract

摘要：

为了解9型猪链球菌广东分离株毒力因子基因型、基因变异和进化情况，本试验采集发病猪脑脊液、关节液、脾脏、肝脏、淋巴结进行细菌分离培养和生化鉴定，采用PCR方法鉴定其血清型及部分毒力基因，对cps9D、gdh、gapdh与orf2基因进行序列测定和分析，并构建系统发育树。结果显示，分离菌镜检为革兰氏阳性链状球菌，在鲜血琼脂平板中呈β溶血，可发酵大多数糖类，能成功扩增cps9D基因，含重要的保守基因gdh及毒力基因gapdh和orf2，表明分离菌株为9型猪链球菌。分离菌株cps9D、gdh、gapdh、orf2基因核苷酸序列与GenBank上国内外参考菌株的同源性分别为95.9%~100.0%、98.6%~99.8%、98.7%~99.6%和95.5%~99.9%，说明分离菌株与国内外其他9型猪链球菌毒株核苷酸同源性都较高，且与国内外的流行毒株基本一致，未发生太大的变异。系统发育树表明，分离株与国内外来源不同的猪链球菌分离株同源性高，亲缘关系密切。

关键词: 9型猪链球菌; 分离鉴定; 毒力基因; 序列分析

Abstract:

In order to understand the virulence factor,gene mutation and evolution of Streptococcus suis serotype 9 strain from Guangdong,the cerebrospinal fluid,joint fluid,spleen,liver,lung and lymph node were collected for bacterium culture and biochemical identification,and PCR method was used to identify its serotype and the partial virulence genes.cps9D,gdh,gapdh and orf2 genes were sequenced and analyzed,and phylogenetic trees were constructed.The results showed that the isolate was gram-positive chain cocci,β-hemolysis in blood agar plate,and could ferment most sugars,and amplify cps9D gene successfully,including important conservative gene gdh and virulence genes gapdh and orf2,which proved that it was Streptococcus suis serotype 9.The nucleotide homologies of cps9D,gdh,gapdh and orf2 genes of the isolate with different strains at home and abroad in GenBank were 95.9% to 100.0%,98.6% to 99.8%,98.7% to 99.6% and 95.5% to 99.9%,respectively.It showed that the isolate had higher nucleotide homology with other Streptococcus suis serotype 9 strains at home and abroad,which was basically consistent with the epidemic strains at home and abroad,and there was not much variation.Phylogenetic tree showed that the isolate had high homology and close relationship with Streptococcus suis isolates from different sources at home and abroad,and they were closely related.

Key words: Streptococcus suis serotype 9; isolation and identification; virulence gene; sequence analysis

中图分类号:

S852.65

李丽, 黄良宗, 谢博, 张海龙, 顾万军. 9型猪链球菌广东株的分离鉴定和基因序列分析[J]. 《中国畜牧兽医》, 2018, 45(4): 1016-1026.

LI Li, HUANG Liangzong, XIE Bo, ZHANG Hailong, GU Wanjun. Isolation, Identification and Gene Sequence Analysis of Streptococcus suis Serotype 9 Strain from Guangdong[J]. , 2018, 45(4): 1016-1026.

参考文献

[1] FENG Y,ZHANG H,WU Z,et al.Streptococcus suis infection[J].Virulence,2014,5(4):447-497.
[2] 宋子杰,孙鑫鹏,王妍,等.猪链球菌的分离鉴定及药敏试验[J].山东畜牧兽医,2017,38(2):1-2. SONG Z J,SUN X P,WANG Y,et al.Isolation,identification and drug sensitivity test of Streptococcus suis[J].Shandong Journal of Animal Science and Veterinary Medicine,2017,38(2):1-2.(in Chinese)
[3] 谭仕旦,刘国平.湖北地区猪链球菌2型的鉴定及生物学特性研究[J].湖北农业科学,2017,56(14):2722-2724. TAN S D,LIU G P.Identification and biological characteristic of Streptococcus suis type 2 in Hubei[J].Hubei Agricultural Sciences,2017,56(14):2722-2724.(in Chinese)
[4] 付梦玉,林丽萍,郭道初,等.一株临床2型猪链球菌的分离鉴定及生物学特性分析[J].中国畜牧兽医,2015,42(7):1897-1904. FU M Y,LIN L P,GUO D C,et al.Isolation,identification and biological characteristics analysis of a strain of clinical Streptococcus suis serotype 2[J].China Animal Husbandry & Veterinary Medicine,2015,42(7):1897-1904.(in Chinese)
[5] 刘志科,张秋雨,陈创夫.猪链球菌病原的分离鉴定与药敏试验[J].养猪,2017,2:105-108. LIU Z K,ZHANG Q Y,CHEN C F.Isolation,identification and drug sensitivity test of pathogenic Streptococcus suis[J].Swine Production,2017,2:105-108.(in Chinese)
[6] STAATS J J,FEDER I,OKWUMABUA O,et al.Streptococcus suis:Past and present[J].Veterinary Research Communications,1997,21(6):381-407.
[7] NGA T V,NGHIA H D,TU LE T P,et al.Real-time PCR for detection of Streptococcus suis serotype 2 in cerebrospinal fluid of human patients with meningitis[J].Diagnostic Microbiology and Infectious Disease,2011,70(4):461-467.
[8] LIU Z J,ZHENG H,GOTTSCHALK M,et al.Development of multiplex PCR assays for the identification of the 33 serotypes of Streptococcus suis[J].PLoS One,2013,8(8):e72070.
[9] LUN Z R,WANG Q P,CHEN X G,et al.Streptococcus suis:An emerging zoonotic pathogen[J]. The Lancet Infectious Diseases,2007,7(3):201-209.
[10] 祝昊丹.猪链球菌9型毒力基因的筛选及鉴定[D].南京:南京农业大学,2011. ZHU H D.Screening and identification of putative virulent genes of Streptococcus suis type 9 strains[D].Nanjing:Nanjing Agricultural University,2011.(in Chinese)
[11] SMITH H E,VECHT U,GIELKENS A L,et al.Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein(muramidase-released protein) of Streptococcus suis type 2[J].Infection and Immunity,1992,60(6):2361-2367.
[12] VECHT U,WISSELINK H J,JELLEMA M L,et al.Identification of two proteins associated with virulence of Streptococcus suis type 2[J].Infection and Immunity,1991,59(9):3156-3162.
[13] DE GREEFF A,BUYS H,VERHAAR R,et al.Contribution of fibronectin-binding protein to pathogenesis of Streptococcus suis serotype 2[J].Infection and Immunity,2002,70(3):1319-1325.
[14] JACOBS A A,LOEFFEN P L,VAN DEN BERG A J,et al.Identification,purification and characterization of a thiol-activated hemolysin (suilysin) of Streptococcus suis[J].Infection and Immunity,1994,62(5):1742-1748.
[15] GOTTSCHALK M,SEGURA M.The pathogenesis of the meningitis caused by Streptococcus suis:The unresolved questions[J].Veterinary Microbiology,2000,76(3):259-272.
[16] 修福晓.猪链球菌9型国内分离株的毒力特性分析[D].南京:南京农业大学,2008. XIU F X.Virulence charicterization of Streptococcus suis serotype 9 isolated in China[D].Nanjing:Nanjing Agricultural University,2008.(in Chinese)
[17] 郑升博,华修国,陆伟芳,等.多重PCR方法检测猪链球菌主要致病血清型及其毒力[J].上海交通大学学报,2010,28(1):53-58. ZHENG S B,HUA X G,LU W F,et al.Detection of Streptococcus suis major serotypes and virulence-associated factors by multiplex PCR[J].Journal of Shanghai Jiaotong University(Agricultural Science),2010,28(1):53-58.(in Chinese)
[18] MAROIS C,BOUGEARD S,GOTTSCHALK M,et al.Multiplex PCR assay for detection of Streptococcus suis species and serotypes 2 and 1/2 in tonsils of live and dead pigs[J].Clinical Microbiol,2004,42(7):3169-3175.
[19] 胡杰,刘棋.致病性猪链球菌2型的病原分离鉴定及毒力因子的PCR检测[J].中国畜牧兽医,2009,36(1):49-52. HU J,LIU Q.Isolation and identification on pathogens of swine streptococcosis and PCR assays for detection of virulence factors of Streptococcus suis serotype 2[J].China Animal Husbandry & Veterinary Medicine,2009,36(1):49-52.(in Chinese)
[20] 彭凌,杨旭夫,朱必凤,等.猪链球菌广东株的分离鉴定及药敏试验[J].动物医学进展,2015,36(2):130-133. PENG L,YANG X F,ZHU B F.Isolation,identification and drug sensitivity test of Streptococcus suis strains from Guangdong[J].Progress in Veterinary Medicine,2015,36(2):130-133.(in Chinese)
[21] WISSELINK H J,SMITH H E,STOCKHOFE-ZURWIEDEN N,et al.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].Veterinary Microbiology,2000,74(3):237-248.
[22] COSTA A T,LOBATO F C,ABREU V L,et al.Serotyping and evaluation of the virulence in mice of Streptococcus strains isolated from diseased pigs[J].Revista do Instituto de Medicina Tropical de Sao Paulo,2005,47(2):113-115.
[23] 余炜烈,李春玲,王贵平,等.猪链球菌2型和9型广东分离株的病原特性[J].中国兽医科学,2007,37(8):650-654. YU W L,LI C L,WANG G P,et al.Pathogenic characteristics of Streptococcus suis type 2 and type 9 isolated from Guangdong province[J].Chinese Veterinary Science,2007,37(8):650-654.(in Chinese)
[24] 付薇,陈进喜,覃芳芸,等.猪链球菌9型cps9G基因的克隆与序列分析[J].动物医学进展,2009,30(1):1-4. FU W,CHEN J X,QIN F Y,et al.Cloning and sequence analysis of cps9G gene of Streptococcus suis serotype 9[J].Progress in Veterinary Medicine,2009,30(1):1-4.(in Chinese)
[25] OKWUMABUA O,PERSAUD J S,REDDY P G.Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2[J].Clinical and Diagnostic Laboratory Immunology,2001,8(2):251-257.
[26] 谭世君.重庆市猪链球菌流行病学调查及GDH基因的克隆和序列分析[D].重庆:西南大学,2007. TAN S J.Epidemiology of Streptococcus suis in Chongqing and cloning and sequence analysis of GDH gene[D].Chongqing:Southwest University,2007.(in Chinese)
[27] 白昀,覃伦,龙剑明,等.猪链球菌9型的分离与鉴定[J].上海畜牧兽医通讯,2007,1:36-37. BAI Y,QIN L,LONG J M,et al.Isolation and identification of Streptococcus suis type 9[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2007,1:36-37.(in Chinese)