山东省霍乱弧菌的时空分布及变化
发布时间：2018-08-08 21:36
【摘要】：[研究背景] 霍乱(Cholera)是一古老的急性肠道传染病,全球发生的七次霍乱大流行都严重危害着人们的生命和健康,特别是对于发展中国家而言更是重大的公共卫生问题。霍乱的病原体霍乱弧菌(Vibrio cholerae, V. cholerae)是一革兰氏阴性弧菌,可分为200多个血清型,其中O1群和O139群是两种最为重要的能引起严重临床症状且能引起霍乱暴发流行的血清型,O1群主要包括小川型(Ogawa)和稻叶型(Inaba)两种血清型以及古典型(Classical biotype)和El Tor型(El Tor biotype)两种生物型。 在全球前六次的霍乱大流行中,病原体主要是O1群古典型霍乱弧菌,而自1961年起第七次霍乱流行中,O1群El Tor型取代了古典型成为优势菌型,并与1964年传入中国广东,同年在山东检出,此后波及全省各地区。1992年,O139群霍乱弧菌作为新的血清型在印度出现并迅速向邻国引起暴发疫情,第二年在中国新疆出现了O13群霍乱病例,进而于1997年在烟台莱阳检出山东省首株O139群霍乱弧菌。自1964年以来,霍乱疫情在山东省一直没有间断,经历了多次暴发流行且累及各个地区,血清型也经过了O1群小川型、O1群稻叶型和O139群多次变化。 由于历史原因,1964～1975年期间霍乱疫情报告工作不完善,有关的霍乱疫情报告及记录均为空白。本研究对山东省自1976年至今霍乱疫情流行病学特征及不同时间和地区代表菌株的毒力基因、脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis, PFGE)基因分型等分子流行病学研究能够细致的分析霍乱在局部地区的流行特征时空变异规律,对霍乱疫情的防控有着更加现实的指导意义,同时山东霍乱的流行特征也能反映全国乃至全球的态势,对大范围的研究有着补充作用。 [研究目的] 1、系统分析山东省霍乱疫情的时间、地区、人群等三间分布流行病学特征； 2、细致了解山东省霍乱弧菌血清型、毒力基因及PFGE基因分型的时空分布及变化规律,同时探讨三者之间的关系； 3、初步建立山东省霍乱弧菌的PFGE基因分型数据库,便于以后霍乱疫情的溯源及比对分析。 [研究方法] 1、流行病学资料整理：对山东省自1976年以来登记的所有霍乱疫情菌株血清型、分离时间、分离地点、病人临床资料、流行强度等流行病学资料进行整理。 2、菌株选择及复核：根据流行病资料选取时间和空间两个维度的代表菌株,按照以暴发为主、兼顾散发的原则选取代表菌株,同时增菌分离培养及血清凝集和生化鉴定。 3、脉冲场凝胶电泳(PFGE)分型：按照PulseNet霍乱弧菌PFGE分型的标准方案进行操作,细菌裂解时间为1h,Not酶切时间为4h,电泳条件Blockl初始和最终转换时间为2s和10s,电泳时间为13h,Block2中时间分别为20s、25s和6h。 4、毒力基因检测：提取代表菌株核酸,PCR检测ctxAB、 tcpA、 tcp1、toxR、 rtxA和hlyA等6种毒力基因。 5、数据分析：利用SAS9.1软件对山东省霍乱弧菌血清型毒力基因的分布差异等分类数据进行卡方检验；利用Bionumerics6.0软件对代表菌株的PFGE条带进行聚类分析,结合流行病学资料分析时空变化规律。 [结果] 1、流行病学特征： (1)自1976年以来,山东省各地区均有霍乱疫情出现,其中鲁西南和鲁北地区最为严重,鲁中地区患者相对较少； (2)自1976年以来,山东省霍乱病例几乎每年均有出现,其中在1983年～1984年和1994年出现了两个流行高峰； (3)从1976年至今山东省霍乱疫情分离菌株依次经历了小川型、稻叶型、和0139群的更替变迁过程； (4)小川型霍乱弧菌疫情主要集中在鲁北地区,而稻叶型霍乱弧菌则主要集中在鲁西南地区； (5)01群霍乱疫情患者年龄分布较为均匀,而0139群霍乱患者多为老年人。 2、毒力基因分布： (1)毒力基因hlyA和toxR,rtxA在环境株和临床分离株阳性率均接近100%,而临床分离株rtxAB、 tcp I和tcpA三种毒力基因的阳性率则远高于环境分离株； (2)临床分离株ctxAB、 tcp I和tcpA三种毒力基因的阳性率有随时间上升的趋势,但2000年之后优势菌群0139群霍乱弧菌毒力基因的携带率有所降低； (3)毒力基因tcp I和tcpA表达较为一致,且在暴发疫情分离株的阳性率高于散发疫情菌株,而ctxAB没有明显差异。 3、PFGE基因分型： (1)250株代表菌株整体上主要聚为PⅠ、PⅡ、PⅢ和PⅣ4类,前3类和血清血型相吻合,依次为临床分离株O1群稻叶型、0139群和01群小川型,而PⅣ主要为毒力基因缺失的菌株,来源及血清型较为混杂； (2)PⅠ可进一步聚类为PⅠa和PⅠb亚类,其中PⅠa主要为1980年～1983年鲁西南地区菌株,PⅠb主要为2001年鲁中和鲁北地区菌株；PⅡ可分为PⅡa和PⅡb亚类,PⅡa为2009年之前的0139群菌株,PⅡb为2013年济宁霍乱疫情菌株；PⅢ可分为PⅢa、PⅢb和PⅢc亚类,其中PⅢa为1998年～1999年分散全省的菌株形成的亚类,PⅢb为1986年～1997年主要分布于鲁东和鲁北地区菌株形成的亚类,PⅢc主要是1978年济宁和日照分离菌株；PⅣ类的菌株来源较为混乱,但毒力基因缺失为其共同特征； (3)250株霍乱弧菌形成了179个PFGE型,在每个亚类中均存在多菌株形成的同一PFGE型别,这主要见于三种情况：①来自同一霍乱暴发疫情；②不同时间相同地域的霍乱疫情；③同一时间相近地域的霍乱疫情。 [结论] 1、在第七次全球霍乱大流行中,山东省霍乱疫情持续存在了半个世纪,流行强度发生着变化,不同年份和地区的优势血清型出现多次更替； 2、山东省临床分离的霍乱弧菌多为产毒株,且毒力基因ctxAB、 tcp I和tcpA的阳性率存在时间和地区差异。而毒力基因缺失的临床分离株,可能与hlyA、toxR和rtxA等其他较为保守的毒力基因有关；另外,相对于ctxAB, tcp I和tcpA对霍乱疫情的流行强度可能更为重要； 3、PFGE基因分型更深入地显示了山东省霍乱弧菌在不同的时间和地区细微变化,与血清型和毒力基因均存在密切的关系,其聚类的基因相似性可为霍乱疫情传统流行病学关联提供线索。
[Abstract]:[research background]
Cholera (Cholera) is an ancient and acute intestinal infectious disease. Seven cholera pandemics worldwide are seriously endangering people's lives and health, especially for developing countries. The pathogen of Cholera (Vibrio cholerae, V. cholerae) is a gram-negative Vibrio, which can be divided into 2 More than 00 serotypes, of which the O1 group and the O139 group are the two most important serotypes that can cause serious clinical symptoms and cause cholera outbreak. The O1 group mainly includes two serotypes of the Sichuan type (Ogawa) and the rice leaf type (Inaba), and the two biological forms of the classical (Classical biotype) and El Tor (El Tor biotype).
Among the first six cholera pandemics worldwide, the main pathogen is the O1 group of the classical Vibrio cholerae, and in the seventh cholera epidemic in 1961, the O1 group El Tor replaced the classical type as the dominant bacteria type, and was introduced into Guangdong in China in 1964, and was detected in the same year in the same year in all regions of the province, and the O139 group of Vibrio cholerae is a new one. The serotype appeared in India and caused a rapid outbreak to neighbouring countries. Second years in Xinjiang, China, a group of O13 cholera appeared in Xinjiang, China, and the first O139 group of Vibrio cholerae was detected in Shandong Province in Laiyang, Yantai in 1997. Since 1964, the cholera epidemic has not been interrupted in Shandong Province. It also passed through O1 group of Ogawa type, O1 group rice leaf type and O139 group.
Due to historical reasons, the report and record of cholera epidemic situation in 1964~1975 years were not perfect, and the reports and records of cholera epidemic were blank. The epidemiological characteristics of cholera epidemic in Shandong province and the virulence genes of different time and regional representative strains from 1976 to date in Shandong Province, pulse field gel electrophoresis (Pulsed Field Gel Electrophoresis, PF) GE) molecular epidemiological studies, such as genotyping, can carefully analyze the temporal and spatial variability of cholera epidemic in local areas, and have more practical guiding significance for the prevention and control of cholera epidemic. Meanwhile, the epidemic characteristics of cholera in Shandong can also reflect the national and global situation, and have a complementary role to the wide range of research.
[research purposes]
1, we systematically analyzed the epidemiological characteristics of three cholera outbreaks in Shandong province.
2. To understand the temporal and spatial distribution and variation of serotypes, virulence genes and PFGE genotypes of Vibrio cholerae in Shandong Province, and to explore the relationship among them.
3, PFGE genotyping database of Vibrio cholerae in Shandong province was initially established to facilitate tracing and comparative analysis of cholera epidemic situation.
[research methods]
1, epidemiological data were arranged: the epidemiological data of all cholera epidemic strains, such as serotype, separation time, separation place, clinical data and epidemic intensity of all cholera epidemic strains registered since 1976 in Shandong Province, were collected.
2, the selection and reexamination of the strains: according to the epidemiological data to select the representative strains of the two dimensions of time and space, according to the outbreak of the strain mainly, taking into account the principle of sporadic selection of the representative strains, at the same time, increase the isolation and culture and serum agglutination and biochemical identification.
3, pulse field gel electrophoresis (PFGE) typing: according to the standard scheme of PulseNet cholera vibrio PFGE typing, the time of bacterial cracking is 1H, the time of Not enzyme cutting is 4h, the initial and final conversion time of Blockl is 2S and 10s, and the time of electrophoresis is 13h.
4, virulence gene detection: extract the nucleic acid of the representative strain, and detect 6 virulence genes such as ctxAB, tcpA, TCP1, toxR, rtxA and hlyA by PCR.
5, data analysis: using SAS9.1 software to carry out a chi square test on the distribution difference of Vibrio cholerae serotype virulence genes in Shandong Province, and using Bionumerics6.0 software to cluster analysis on the PFGE bands of the representative strains and analyze the temporal and spatial variation rules according to the epidemiological data.
[results]
1, epidemiological characteristics:
(1) Since 1976, cholera epidemics have occurred in all regions of Shandong Province, the most serious in the southwest and north of Shandong Province, and relatively few patients in the central part of Shandong Province.
(2) Since 1976, cholera cases have occurred almost every year in Shandong Province, with two epidemic peaks from 1983 to 1984 and 1994.
(3) From 1976 to now, strains isolated from cholera epidemic situation in Shandong Province have undergone successive changes of Ogawa type, rice leaf type and 0139 group.
(4) the outbreak of Vibrio cholerae in Ogawa is mainly concentrated in the north of Shandong Province, while the rice leaf type Vibrio cholerae is mainly concentrated in Southwest Shandong.
(5) the age distribution of 01 groups of cholera patients is more homogeneous, while the 0139 group of cholera patients are mostly elderly people.
2, the distribution of virulence genes:
(1) the positive rates of virulence genes hlyA and toxR, rtxA in both environmental and clinical isolates were close to 100%, while the positive rates of three virulence genes of rtxAB, TCP I and tcpA in clinical isolates were much higher than those of environmental isolates.
(2) the positive rates of three virulence genes of clinical isolates ctxAB, TCP I and tcpA were increasing with time, but the carrying rate of Vibrio cholerae virulence genes of 0139 groups of dominant groups decreased after 2000.
(3) The expression of virulence genes TCP I and tcpA was consistent, and the positive rate of isolates in outbreak was higher than that in sporadic outbreak, but there was no significant difference in ctxAB.
3, PFGE genotyping:
(1) the 250 representative strains were mainly composed of P I, P II, P III and P IV 4. The first 3 types were in accordance with the serum blood groups, which were followed by the clinical isolates of O1 group rice leaf type, 0139 group and 01 group of small Sichuan type, while P IV was mainly a strain of virulence gene deletion, and the source and serotype were mixed;
(2) P I can be further clustered into P I a and P I B subclass, in which P I a mainly is from 1980 to 1983 in southwestern Shandong Province, P I B is mainly in 2001 of Shandong and Northern Shandong Province; P II can be divided into P A and subordinate class of 2009. III C subclass, of which P III A is a subclass of strain formation in the province from 1998 to 1999, P III B is a subclass of strains formed mainly from 1986 to 1997 in Shandong and Northern Shandong, P III C is mainly in 1978 Jining and sunshine isolates, and P IV strains are more chaotic, but the loss of virulence genes is the common characteristic.
(3) 250 strains of Vibrio cholerae formed 179 PFGE types, and there were the same PFGE types of multiple strains in each subclass. This was mainly seen in three cases: (1) from the same cholera outbreak; (2) the cholera epidemic in the same region at different time; and the cholera epidemic in the same area at the same time.
[Conclusion]
1, in the seventh global cholera pandemic, the cholera epidemic in Shandong lasted for half a century, the intensity of the epidemic changed, and the dominant serotypes in different years and regions had been replaced by many times.
2, the clinical isolates of Vibrio cholerae in Shandong province are mostly strains of Vibrio cholerae, and the positive rates of virulence genes ctxAB, TCP I and tcpA have time and regional differences. However, the clinical isolates with the lack of virulence genes may be related to other more conservative virulence genes such as hlyA, toxR and rtxA, and TCP I and tcpA are prevalent in cholera epidemic relative to ctxAB. Strength may be more important.
3, the PFGE genotyping shows the subtle changes in the time and area of Vibrio cholerae in Shandong province. There is a close relationship with the serotypes and virulence genes. The genetic similarity of the cluster can provide clues for the traditional epidemiological association of cholera epidemic.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.5
本文编号：2173105
[Abstract]:[research background]
Cholera (Cholera) is an ancient and acute intestinal infectious disease. Seven cholera pandemics worldwide are seriously endangering people's lives and health, especially for developing countries. The pathogen of Cholera (Vibrio cholerae, V. cholerae) is a gram-negative Vibrio, which can be divided into 2 More than 00 serotypes, of which the O1 group and the O139 group are the two most important serotypes that can cause serious clinical symptoms and cause cholera outbreak. The O1 group mainly includes two serotypes of the Sichuan type (Ogawa) and the rice leaf type (Inaba), and the two biological forms of the classical (Classical biotype) and El Tor (El Tor biotype).
Among the first six cholera pandemics worldwide, the main pathogen is the O1 group of the classical Vibrio cholerae, and in the seventh cholera epidemic in 1961, the O1 group El Tor replaced the classical type as the dominant bacteria type, and was introduced into Guangdong in China in 1964, and was detected in the same year in the same year in all regions of the province, and the O139 group of Vibrio cholerae is a new one. The serotype appeared in India and caused a rapid outbreak to neighbouring countries. Second years in Xinjiang, China, a group of O13 cholera appeared in Xinjiang, China, and the first O139 group of Vibrio cholerae was detected in Shandong Province in Laiyang, Yantai in 1997. Since 1964, the cholera epidemic has not been interrupted in Shandong Province. It also passed through O1 group of Ogawa type, O1 group rice leaf type and O139 group.
Due to historical reasons, the report and record of cholera epidemic situation in 1964~1975 years were not perfect, and the reports and records of cholera epidemic were blank. The epidemiological characteristics of cholera epidemic in Shandong province and the virulence genes of different time and regional representative strains from 1976 to date in Shandong Province, pulse field gel electrophoresis (Pulsed Field Gel Electrophoresis, PF) GE) molecular epidemiological studies, such as genotyping, can carefully analyze the temporal and spatial variability of cholera epidemic in local areas, and have more practical guiding significance for the prevention and control of cholera epidemic. Meanwhile, the epidemic characteristics of cholera in Shandong can also reflect the national and global situation, and have a complementary role to the wide range of research.
[research purposes]
1, we systematically analyzed the epidemiological characteristics of three cholera outbreaks in Shandong province.
2. To understand the temporal and spatial distribution and variation of serotypes, virulence genes and PFGE genotypes of Vibrio cholerae in Shandong Province, and to explore the relationship among them.
3, PFGE genotyping database of Vibrio cholerae in Shandong province was initially established to facilitate tracing and comparative analysis of cholera epidemic situation.
[research methods]
1, epidemiological data were arranged: the epidemiological data of all cholera epidemic strains, such as serotype, separation time, separation place, clinical data and epidemic intensity of all cholera epidemic strains registered since 1976 in Shandong Province, were collected.
2, the selection and reexamination of the strains: according to the epidemiological data to select the representative strains of the two dimensions of time and space, according to the outbreak of the strain mainly, taking into account the principle of sporadic selection of the representative strains, at the same time, increase the isolation and culture and serum agglutination and biochemical identification.
3, pulse field gel electrophoresis (PFGE) typing: according to the standard scheme of PulseNet cholera vibrio PFGE typing, the time of bacterial cracking is 1H, the time of Not enzyme cutting is 4h, the initial and final conversion time of Blockl is 2S and 10s, and the time of electrophoresis is 13h.
4, virulence gene detection: extract the nucleic acid of the representative strain, and detect 6 virulence genes such as ctxAB, tcpA, TCP1, toxR, rtxA and hlyA by PCR.
5, data analysis: using SAS9.1 software to carry out a chi square test on the distribution difference of Vibrio cholerae serotype virulence genes in Shandong Province, and using Bionumerics6.0 software to cluster analysis on the PFGE bands of the representative strains and analyze the temporal and spatial variation rules according to the epidemiological data.
[results]
1, epidemiological characteristics:
(1) Since 1976, cholera epidemics have occurred in all regions of Shandong Province, the most serious in the southwest and north of Shandong Province, and relatively few patients in the central part of Shandong Province.
(2) Since 1976, cholera cases have occurred almost every year in Shandong Province, with two epidemic peaks from 1983 to 1984 and 1994.
(3) From 1976 to now, strains isolated from cholera epidemic situation in Shandong Province have undergone successive changes of Ogawa type, rice leaf type and 0139 group.
(4) the outbreak of Vibrio cholerae in Ogawa is mainly concentrated in the north of Shandong Province, while the rice leaf type Vibrio cholerae is mainly concentrated in Southwest Shandong.
(5) the age distribution of 01 groups of cholera patients is more homogeneous, while the 0139 group of cholera patients are mostly elderly people.
2, the distribution of virulence genes:
(1) the positive rates of virulence genes hlyA and toxR, rtxA in both environmental and clinical isolates were close to 100%, while the positive rates of three virulence genes of rtxAB, TCP I and tcpA in clinical isolates were much higher than those of environmental isolates.
(2) the positive rates of three virulence genes of clinical isolates ctxAB, TCP I and tcpA were increasing with time, but the carrying rate of Vibrio cholerae virulence genes of 0139 groups of dominant groups decreased after 2000.
(3) The expression of virulence genes TCP I and tcpA was consistent, and the positive rate of isolates in outbreak was higher than that in sporadic outbreak, but there was no significant difference in ctxAB.
3, PFGE genotyping:
(1) the 250 representative strains were mainly composed of P I, P II, P III and P IV 4. The first 3 types were in accordance with the serum blood groups, which were followed by the clinical isolates of O1 group rice leaf type, 0139 group and 01 group of small Sichuan type, while P IV was mainly a strain of virulence gene deletion, and the source and serotype were mixed;
(2) P I can be further clustered into P I a and P I B subclass, in which P I a mainly is from 1980 to 1983 in southwestern Shandong Province, P I B is mainly in 2001 of Shandong and Northern Shandong Province; P II can be divided into P A and subordinate class of 2009. III C subclass, of which P III A is a subclass of strain formation in the province from 1998 to 1999, P III B is a subclass of strains formed mainly from 1986 to 1997 in Shandong and Northern Shandong, P III C is mainly in 1978 Jining and sunshine isolates, and P IV strains are more chaotic, but the loss of virulence genes is the common characteristic.
(3) 250 strains of Vibrio cholerae formed 179 PFGE types, and there were the same PFGE types of multiple strains in each subclass. This was mainly seen in three cases: (1) from the same cholera outbreak; (2) the cholera epidemic in the same region at different time; and the cholera epidemic in the same area at the same time.
[Conclusion]
1, in the seventh global cholera pandemic, the cholera epidemic in Shandong lasted for half a century, the intensity of the epidemic changed, and the dominant serotypes in different years and regions had been replaced by many times.
2, the clinical isolates of Vibrio cholerae in Shandong province are mostly strains of Vibrio cholerae, and the positive rates of virulence genes ctxAB, TCP I and tcpA have time and regional differences. However, the clinical isolates with the lack of virulence genes may be related to other more conservative virulence genes such as hlyA, toxR and rtxA, and TCP I and tcpA are prevalent in cholera epidemic relative to ctxAB. Strength may be more important.
3, the PFGE genotyping shows the subtle changes in the time and area of Vibrio cholerae in Shandong province. There is a close relationship with the serotypes and virulence genes. The genetic similarity of the cluster can provide clues for the traditional epidemiological association of cholera epidemic.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.5
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