2009-2015年贵州省致病性钩端螺旋体分离株PFGE分型分析
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摘要
目的了解贵州省2009-2015年分离自鼠类宿主动物的钩端螺旋体(简称钩体)PFGE带型特征,为当地钩体病的预防和控制提供科学依据。方法对贵州省近年分离自鼠类宿主动物的56株钩体采用致病性钩体特异引物进行PCR扩增鉴定,应用血清分群PCR对其进行血清群鉴定,采用包埋法提取钩体染色体DNA,利用核酸内切酶NotI对DNA进行酶切,酶切片段经PFGE电泳后采用凝胶成像系统拍照获得DNA指纹图谱,采用国家病原体分子分型实验数据分析与传输系统进行处理和聚类分析。结果致病性钩体特异性PCR和血清分群PCR鉴定显示56株钩体均为黄疸出血群钩体株,PFGE分型将其分为38个带型,聚类分析显示多数钩体株与黄疸出血群赖型具有较高的相似度,为主要流行优势带型,少部分钩体株与爪哇群和波摩那群代表菌株具有较高相似性,聚类关系相对较近。结论黄疸出血群赖型是贵州省近年流行的优势型,钩体株PFGE带型具有多样性,可为当地钩体病的监测、暴发调查和传染源追踪提供参考依据。
Objectives To ascertain the molecular epidemiologic characteristics of pathogenic Leptospira isolated from rats in Guizhou Province from 2009 to 2015 and to provide a scientific basis for the control and prevention of leptospirosis in Guizhou Province. Methods Pathogen-specific PCR was used to identify Leptospira isolates, and serogroup-specific PCR was used to identify the serogroup of the isolates. Chromosomal DNA extracted from leptospiral isolates was digested with the restriction endonuclease Not I, and the DNA segments were separated using PFGE. The Chinese National System for Molecular Typing of Pathogens(http://101.200.213.193:8080/pulsenet) was used to manage PFGE images obtained from leptospiral isolates, and the PFGE maps of leptospiral isolates were compared with those of representative strains belonging to L. interrogans for cluster analysis. Results Leptospira-specific PCR identified the 56 Leptospira isolates as pathogenic Leptospira, and serogroup-specific PCR indicated that all 56 isolates belonged to the Icterohaemorrhagiae serogroup of Leptospira. PFGE indicated that the 56 strains of L. interrogans from Guizhou Province could be classified into 38 PFGE types. Cluster analysis indicated that most of the isolates from Guizhou Province were relatively closely clustered with representative strain 56601 of the Icterohaemorrhagiae serogroup. A few isolates were most closely clustered with representative strain 56608 of the Pomona serogroup and a representative strain of the Java serogroup. Conclusion PFGE revealed that the Icterohaemorrhagiae serogroup has been the prevalent serogroup of L. interrogans in Guizhou Province over the past few years. PFGE also revealed the molecular diversity of the isolates in Guizhou Province. These findings should help to control and prevent leptospirosis in Guizhou Province.
Objectives To ascertain the molecular epidemiologic characteristics of pathogenic Leptospira isolated from rats in Guizhou Province from 2009 to 2015 and to provide a scientific basis for the control and prevention of leptospirosis in Guizhou Province. Methods Pathogen-specific PCR was used to identify Leptospira isolates, and serogroup-specific PCR was used to identify the serogroup of the isolates. Chromosomal DNA extracted from leptospiral isolates was digested with the restriction endonuclease Not I, and the DNA segments were separated using PFGE. The Chinese National System for Molecular Typing of Pathogens(http://101.200.213.193:8080/pulsenet) was used to manage PFGE images obtained from leptospiral isolates, and the PFGE maps of leptospiral isolates were compared with those of representative strains belonging to L. interrogans for cluster analysis. Results Leptospira-specific PCR identified the 56 Leptospira isolates as pathogenic Leptospira, and serogroup-specific PCR indicated that all 56 isolates belonged to the Icterohaemorrhagiae serogroup of Leptospira. PFGE indicated that the 56 strains of L. interrogans from Guizhou Province could be classified into 38 PFGE types. Cluster analysis indicated that most of the isolates from Guizhou Province were relatively closely clustered with representative strain 56601 of the Icterohaemorrhagiae serogroup. A few isolates were most closely clustered with representative strain 56608 of the Pomona serogroup and a representative strain of the Java serogroup. Conclusion PFGE revealed that the Icterohaemorrhagiae serogroup has been the prevalent serogroup of L. interrogans in Guizhou Province over the past few years. PFGE also revealed the molecular diversity of the isolates in Guizhou Province. These findings should help to control and prevent leptospirosis in Guizhou Province.
引文
[1] 张金龙. 钩端螺旋体分子特性分析研究[D]. 长春: 长春工业大学, 2015.
[2] 杨会棉, 蒋秀高. 钩端螺旋体的分子分型方法[J]. 中国人兽共患病学报, 2011, 27(11): 1024-7.
[3] Bharti AR, Nally JE, Ricaldi JN, et al. Leptospirosis a zoonotic disease of global importance[J]. Lancet Infect Dis, 2003, 3(12): 757.
[4] Levett PN. Leptospirosis[J]. Chin Microbiol Rev, 2001, 14(5): 296.
[5] Abela-Ridder B, Sikkema R, Hartsree RA. Estimating the burden of human leptospirosis[J]. Int J Antimicrob Agents 2010, 36(Suppl 1): S5-7.
[6] 顾坚未, 蒋秀高, 郭晓奎. 我国钩端螺旋体血清型及其更迭[J]. 中华医学实践杂志, 2005, 4(1): 22-3.
[7] 严杰, 戴保民, 余恩庶. 钩端螺旋体病学[M]. 3版. 北京: 人民卫生出版社, 2006: 1-3.
[8] 刘英, 陈红, 李沛丽, 等. 贵州省钩端螺旋体血清群特异性PCR鉴定结果与分析[J]. 中国人兽共患病学报, 2015, 31(12): 1146-50, 1156.
[9] 张艳, 郭宗琪, 李秀文, 等. 钩端螺旋体脉冲场凝胶电泳标准化技术的建立及谱型特征初步分析[J]. 中华流行病学杂志, 2007, 28(8): 772-5.
[10] Li SJ, Wang DM, Zhang CC, et al. Molecular typing of Leptospira spp. strains isolated from field mice confirms a link to human leptospirosis[J]. Epidemiol Infect, 2013, 14(11): 2278-85.
[11] Herrmann JL, Bellenger E, Perolat P, et al. Pulsed-field gel electrophoresis of Not I digests of Leptospiral DNA: a rapid method of serovar identification[J]. Clin Microbiol, 1992, 11(30): 1696-720.
[12] 蒋秀高, 肖玉春, 聂一新, 等. 致病性钩端螺旋体脉冲场凝胶电泳图谱分型研究[J]. 中华微生物学和免疫学杂志, 2001, 21(4): 378-81.
[13] 郭宗琪, 黄自英, 陈娜, 等. 四川省致病性钩端螺旋体脉冲场电泳分型分析[J]. 预防医学情报杂志, 2007, 23(6): 644-6.
[14] Galloway RL, Levett PN. Evaluation of a modified pulsed-field gel electrophoresis approach for the identification of Leptospira serovars [J]. Am J Trop Med Hyg, 2008, 78(4): 628-32.
[15] 李世军, 张翠彩, 李秀文, 等. 贵州省3株钩端螺旋体分离株PFGE分型与基因种鉴定[J].中国人兽共患病学报, 2011, 27(7): 587-90.
[16] 刘英, 李世军, 马青, 等. 贵州省黑线姬鼠钩端螺旋体分离株MLVA分型研究[J]. 中国病原生物学杂志, 2017, 12(1): 20-4.
[17] 黄荷, 刘英, 李世军, 等. 贵州省黎平县钩端螺旋体病危险因素调查[J]. 中国人兽共患病学报, 2017, 33(12): 1136-43.
[2] 杨会棉, 蒋秀高. 钩端螺旋体的分子分型方法[J]. 中国人兽共患病学报, 2011, 27(11): 1024-7.
[3] Bharti AR, Nally JE, Ricaldi JN, et al. Leptospirosis a zoonotic disease of global importance[J]. Lancet Infect Dis, 2003, 3(12): 757.
[4] Levett PN. Leptospirosis[J]. Chin Microbiol Rev, 2001, 14(5): 296.
[5] Abela-Ridder B, Sikkema R, Hartsree RA. Estimating the burden of human leptospirosis[J]. Int J Antimicrob Agents 2010, 36(Suppl 1): S5-7.
[6] 顾坚未, 蒋秀高, 郭晓奎. 我国钩端螺旋体血清型及其更迭[J]. 中华医学实践杂志, 2005, 4(1): 22-3.
[7] 严杰, 戴保民, 余恩庶. 钩端螺旋体病学[M]. 3版. 北京: 人民卫生出版社, 2006: 1-3.
[8] 刘英, 陈红, 李沛丽, 等. 贵州省钩端螺旋体血清群特异性PCR鉴定结果与分析[J]. 中国人兽共患病学报, 2015, 31(12): 1146-50, 1156.
[9] 张艳, 郭宗琪, 李秀文, 等. 钩端螺旋体脉冲场凝胶电泳标准化技术的建立及谱型特征初步分析[J]. 中华流行病学杂志, 2007, 28(8): 772-5.
[10] Li SJ, Wang DM, Zhang CC, et al. Molecular typing of Leptospira spp. strains isolated from field mice confirms a link to human leptospirosis[J]. Epidemiol Infect, 2013, 14(11): 2278-85.
[11] Herrmann JL, Bellenger E, Perolat P, et al. Pulsed-field gel electrophoresis of Not I digests of Leptospiral DNA: a rapid method of serovar identification[J]. Clin Microbiol, 1992, 11(30): 1696-720.
[12] 蒋秀高, 肖玉春, 聂一新, 等. 致病性钩端螺旋体脉冲场凝胶电泳图谱分型研究[J]. 中华微生物学和免疫学杂志, 2001, 21(4): 378-81.
[13] 郭宗琪, 黄自英, 陈娜, 等. 四川省致病性钩端螺旋体脉冲场电泳分型分析[J]. 预防医学情报杂志, 2007, 23(6): 644-6.
[14] Galloway RL, Levett PN. Evaluation of a modified pulsed-field gel electrophoresis approach for the identification of Leptospira serovars [J]. Am J Trop Med Hyg, 2008, 78(4): 628-32.
[15] 李世军, 张翠彩, 李秀文, 等. 贵州省3株钩端螺旋体分离株PFGE分型与基因种鉴定[J].中国人兽共患病学报, 2011, 27(7): 587-90.
[16] 刘英, 李世军, 马青, 等. 贵州省黑线姬鼠钩端螺旋体分离株MLVA分型研究[J]. 中国病原生物学杂志, 2017, 12(1): 20-4.
[17] 黄荷, 刘英, 李世军, 等. 贵州省黎平县钩端螺旋体病危险因素调查[J]. 中国人兽共患病学报, 2017, 33(12): 1136-43.