猪牛源非结核分枝杆菌分离鉴定及其主要基因的分析研究
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摘要
非结核分枝杆菌(Nontuberculous mycobacterium,NTM)与结核杆菌、麻风杆菌一起被列为分枝杆菌,由于其能引起与结核类似的病症而得名,为革兰氏阳性菌,抗酸染色阳性。该菌截止到2003年共发现有95种,其中大部分为致病菌或条件致病菌。除了能引起肺部病变以外,还能引起动物体多种其它组织病变,其临床诊断时多有误诊为结核杆菌的报道。NTM与结核分枝杆菌虽同属于分枝杆菌属,但其耐药性不同,所以误诊后按结核杆菌给药的病例久治不愈,另外,大部分NTM对结核菌素变态反应(PPD)呈阴性,难以发现,这都给畜牧生产业带来极大损失,所以对动物NTM的分离鉴定及进一步研究是十分必要的。
关于NTM的研究报道中,国内外已经发表的文献大都是关于人NTM的报道,对动物NTM的报道很少,本实验对动物NTM展开研究,实验共分以下四部分：1、非结核分枝杆菌的分离鉴定：从吉林省境内多个地区采集经PPD检验呈阴性的猪、牛颌下淋巴结与肠系膜淋巴结样品共计2100份,经酸处理后研磨成匀浆,接种于新配制的改良罗杰氏斜面培养基中,每个样品接种三管,37℃培养,每3d观察一次,培养8周以上,抗酸染色可见菌株,用改良罗杰氏斜面培养基扩增抗酸染色阳性菌,应用多种方法对扩增的抗酸菌进行鉴定分类工作,所采取的方法有实验室常规方法：观察记录其生长时间、菌落形态等特征：生长特性实验：将该菌于改良罗杰氏培养基中置于28℃、37℃、45℃条件下观察其生长情况；生物化学鉴定：应用耐热触酶、硝酸盐还原、吐温80水解、尿素酶、芳香硫酸酯酶、铁离子吸收、亚碲酸盐还原实验分别鉴定分离菌；鉴别培养基生长实验：应用PNB培养基、TCH培养基、氯化钠培养基、苦味酸培养基、谷氨酸钠葡萄糖培养基、麦康凯琼脂培养基对分离菌进行鉴定；分子生物学方法：应用热休克蛋白hsp65基因的聚合酶链式反应-限制性片段长度多态性分析(PCR-RFLP)技术鉴定分离菌。联合分析多种方法所得结果,最终对所分离到的菌株做出鉴别分类,其中共计得到菌株28个,分别为：浅黄分枝杆菌4株,瘰疬分枝杆菌2株,胞内分枝杆菌2株,蟾蜍分枝杆菌6株,牛分枝杆菌1株,偶发分枝杆菌3株,苏加分枝杆菌2株,新金分枝杆菌4株,龟分枝杆菌1株,戈登分枝杆菌1株,母牛分枝杆菌2株。结果表明虽然被检样品经过PPD变态反应筛选,但仍然有非结核分枝杆菌被分离出来,说明非结核分枝杆菌猪、牛群中最新的流行情况并不乐观,加之近年来在饲养管理和监测控制措施实行和完善的前提下,非结核分枝杆菌仍有较高的流行,说明该菌有较强的适应性,为了防止其广泛流行产生大的影响,应在防治结核分枝杆菌的同时加强非结核分枝杆菌的监测和防控工作。2、非结核分枝杆菌血清学分组及抗原性分析：对分离到的NTM血清学分组,将NTM转移接种到Middle brook 7H10抗原培养基中,生长后用0.3%甲醛灭菌生理盐水制备免疫用死菌制剂,用灭菌生理盐水制备免疫用活菌制剂,分四次免疫健康兔,每次间隔一周,一、二次用死菌制剂免疫,三、四次用活菌制剂免疫,并于第四次免疫一周后心脏采血制备抗血清。用含石炭酸的磷酸盐缓冲液洗下抗原培养基上的NTM,经过处理后,用PBS缓冲液制备成抗原。用抗血清和抗原进行凝集反应和凝集素吸收试验,最终对分离到的NTM做出血清学分组及抗原性分析,结果将NTM分离株分成五个血清型：NH1、NH2、NH5、ZH1、ZC4、ZC14为一种血清型,ZC2、ZC3、ZH9、ZH10为一种血清型,NH9、NC11、NC12、NC13、NC14、ZC11、ZC12为一种血清型,NH3、NH6、NC4、NC7、NC10、ZH5、ZH6、ZH13、ZC7、ZC8为一种血清型,NH8单独为一种血清型,说明吉林省地区分布的NTM血清型分类结构较具体,有进一步分类研究,并根据其分型制定监测和防制措施价值。3、分离菌株中偶发分枝杆菌全基因组文库的构建：从分离NTM中选取已经鉴定确认为偶发分枝杆菌的分离株,改良核酸提取方法提取全基因组,用限制性内切酶Sau3A I随机酶切全基因组,通过体系中酶浓度调节控制酶切后基因片段主要集中在1kb到7kb之间,把酶切片段通过随机连接的方式连入经过BamH I单酶切和去磷酸化处理过的pUC18载体中,再转化入自制的感受态细菌DH5a,经蓝白斑筛选、PCR、双酶切等方法检测连接效率并得到阳性连接,刮取培养基表面阳性菌,加甘油保存于-80℃,实验得到阳性克隆4×104个,根据统计学计算达到基因组文库要求,该文库的建立为以后对该菌特殊基因方面的研究及基因结构和调控等研究打下良好基础,同时也为其它NTM的进一步研究工作打下了实验基础。4、基因组文库的应用即hsp65基因片段的分析：从已经制备的偶发分枝杆菌的全基因组文库中克隆出长413bp的hsp65基因片段,经测序后,采用Blast、DNAStar软件,对其进行分析,得到该偶发分枝杆菌的基因特征和进化特征。
本实验从NTM的分离培养到分离鉴定和血清学分型,再到全基因组文库的构建和基因组文库的应用及NTM基因序列的测序分析,为NTM实验室研究工作制订了一整套流程,为实验室就分离到的NTM进一步研究工作打下基础,同时实验所得各数据也为撑控NTM最新流行情况、针对NTM特点提出合理有效的防治措施提供了科学依据。
Mycobacterium Including Nontuberculous mycobacterium and Mycobacterium tuberculosis and so on. Because of Nontuberculous mycobacterium's similar symptoms with tuberculosis, it is named. Nontuberculous mycobacterium is a Gram-positive bacteria,its Acid-fast staining is positive. To 2003, there are 95 kinds Nontuberculous mycobacterium have been found, most of them are pathogenic or opportunistic pathogen. In addition lung disease, it can cause a variety of animal other tissues disease, the clinical diagnosises are often reported misdiagnosed as tuberculosis. Although NTM and Mycobacterium tuberculosis are Mycobacterium, but the drug resistance are different, so can not be cured after the misdiagnosis as Mycobacterium tuberculosis. Meanwhile, most of the NTM on the PPD was negative, they are difficult to be found, that cause great losses to the livestock industry, so isolation and further research on the animal's NTM is necessary.
NTM reports on the research, domestic and foreign literature, mostly published reports on human NTM, NTM few reports of animal. In this study,the research on animals NTM experiment is divided into four parts.1. Isolation and identification of non-tuberculosis mycobacteria:Collected 2,100 submandibular lymph nodes and mesenteric lymph nodes from pigs and cattle whose PPD test is negative in many parts of Jilin Province.These samples are grinded into a homogenized after treated with acid and are inoculated in L-J culture medium which is fresh, each sample was inoculated with three, cultured at 37℃, observed once every 3 days, continued 8 weeks. Acid-fast staining tests bacterials after growthing. Amplificed bacterias with L-J medium which are positive by acid-fast stain. Classified bacteria which are amplificed in several ways. The approaches taken have laboratory conventional methods, Biochemical methods, growth characteristics experiment and Molecular biology methods. Laboratory conventional methods:Bacteria are cultured at 28℃, 37℃,45℃on L-J medium and observe the growth of its. Biochemical methods:Heat-resistant catalase, nitrate reduction, Tween 80 hydrolysis, urease, aromatic sulfatase, iron uptake, tellurite reduction test bacteria isolated. Growth characteristics experiment:Application PNB medium, TCH medium, sodium chloride medium, medium picric acid, sodium glutamate glucose medium, MacConkey medium for identificating of bacteria isolated. Molecular biology methods:Application of heat shock protein hsp65 gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) Identification of bacteria isolated. Synthesis and analysis of the results of various methods, make the final identification. A total of 28 strains has been obtained. These mycobacteria were identified as follow:4 strains as Mycobacterium gilvum,2 strains as Mycobacterium scrofulaceum,2 strains as Mycobacterium intracellulare,6 strains as Mycobacterium senopi,1 strains as Mycobacterium bovis,3 strains as Mycobacterium fortuitum,2 strains as Mycobacterium szulgai,4 strains as Mycobacterium neoaurum.1 strains as Mycobacterium testudinis，1 strains as Mycobacterium gordonae，2 strains as Mycobacterium vaccae. The results show that although the samples were tested by PPD, but there are still non-tuberculous mycobacteria were isolated. Non-tuberculosis mycobacteria's epidemic situation in pigs, cattle is not optimistic. It shows the strong adaptability of non-tuberculosis mycobacteria that non-tuberculous mycobacteria have a higher epidemic undering the premise of feeding management and monitoring of the implementation and improvement of control measures,In recent years. In order to prevent a big impact by widespreading, we should be strengthening the monitoring of non-tuberculous mycobacteria, and prevention and control when preventionning and controlling Mycobacterium tuberculosis.2, Non-tuberculosis mycobacteria serologily group and antigenic analysis:Grouped non-tuberculosis serologily. Non-tuberculosis is inoculated on the Middlebrook 7H10 antigen medium. After growning of bacterial, using 0.3%formaldehyde in sterile saline preparars of immune preparations with dead bacteria and prepared immunization with viable preparations with sterile saline. Immune health rabbits in fourth, one week intervals, the first and second immunization with dead bacteria preparations, three or four times immunization with viable preparations. In the week after the forth immunization, antiserum with heart blood. Using phosphate buffer Containing carbolic acid washed non-tuberculosis on the antigen medium. After processing, is prepared antigen with PBS buffer. Make agglutination and agglutinin absorption test With anti-serum and antigen. Ultimately makeing non-tuberculosis serological grouping and analysis of antigens. The results is that non-tuberculosis were divided into five groups of serotype:NH1,NH2,NH5,ZH1,ZC4,ZC14 are one group,ZC2,ZC3,ZH9,ZH10 are one group,NH9,NC11,NC12,NC13,NC14,ZC11,ZC12 are one group,NH3,NH6,NC4,NC7,NC10,ZH5,ZH6,ZH13,ZC7,ZC8 are one group,NH8 are one group. Shows the distribution of serotypes of non-tuberculosis in Jilin Province is more specific classification structure.There is the value of Classification of further development and making control measures. Construction of DNA Library of Mycobacterium fortuitum that is a Nontuberculous Mycobacteria:Select Mycobacterium fortuitum from non-tuberculosis which has been recognized. Extracte whole genome by improving method and digeste genome with the restriction enzyme Sau3A I randomly. Through regulateing and controlling concentration of enzyme in system of gene fragments digested to digest gene in between 1000bp to 7000bp. The fragments are connected by way of random connections into pUC18 vector digested by BamHⅠ,then transform the prouduct in to DH5a made by ourself. The blue-white screening, PCR, restriction enzyme were used to detect the result of connection and get the positive connection efficiency. Scraping the positive bacteria from the surface of the medium. Stored at 80℃with glycerol.There are 4×104 positive have been gain in experiment.The genomic library is sucessful according to statistical calculation. The establishment of the library establish a good basis for the study of strain specific genes and gene structure and regulation. At the same time, to lay a foundation for the further study of other NTM.4 Application of genomic library and analysis of the hsp65 gene:Cloned the hsp65 gene fragment length of 413bp from Mycobacterium fortuitum's genomic library which has been prepared. After sequencing, using Blast, DNAStar software to analyze hsp65 gene to get the gene and evolutionary characteristics of Mycobacterium fortuitum. In this study,from the isolation and identification of the non-tuberculosis to serotyping.and to the construction of whole genome library and the application of genomic library, analysis of gene sequence.This study sets a processes for laboratory research on non-tuberculosis, lays the foundation for further research on non-tuberculosis in laboratory. At the same time,the date catched from the experiment provides the scientific basis for obtainning and controlling the latest epidemic of non-tuberculosis and make a rdasonable and effective control measures of non-tuberculosis.
关于NTM的研究报道中,国内外已经发表的文献大都是关于人NTM的报道,对动物NTM的报道很少,本实验对动物NTM展开研究,实验共分以下四部分：1、非结核分枝杆菌的分离鉴定：从吉林省境内多个地区采集经PPD检验呈阴性的猪、牛颌下淋巴结与肠系膜淋巴结样品共计2100份,经酸处理后研磨成匀浆,接种于新配制的改良罗杰氏斜面培养基中,每个样品接种三管,37℃培养,每3d观察一次,培养8周以上,抗酸染色可见菌株,用改良罗杰氏斜面培养基扩增抗酸染色阳性菌,应用多种方法对扩增的抗酸菌进行鉴定分类工作,所采取的方法有实验室常规方法：观察记录其生长时间、菌落形态等特征：生长特性实验：将该菌于改良罗杰氏培养基中置于28℃、37℃、45℃条件下观察其生长情况；生物化学鉴定：应用耐热触酶、硝酸盐还原、吐温80水解、尿素酶、芳香硫酸酯酶、铁离子吸收、亚碲酸盐还原实验分别鉴定分离菌；鉴别培养基生长实验：应用PNB培养基、TCH培养基、氯化钠培养基、苦味酸培养基、谷氨酸钠葡萄糖培养基、麦康凯琼脂培养基对分离菌进行鉴定；分子生物学方法：应用热休克蛋白hsp65基因的聚合酶链式反应-限制性片段长度多态性分析(PCR-RFLP)技术鉴定分离菌。联合分析多种方法所得结果,最终对所分离到的菌株做出鉴别分类,其中共计得到菌株28个,分别为：浅黄分枝杆菌4株,瘰疬分枝杆菌2株,胞内分枝杆菌2株,蟾蜍分枝杆菌6株,牛分枝杆菌1株,偶发分枝杆菌3株,苏加分枝杆菌2株,新金分枝杆菌4株,龟分枝杆菌1株,戈登分枝杆菌1株,母牛分枝杆菌2株。结果表明虽然被检样品经过PPD变态反应筛选,但仍然有非结核分枝杆菌被分离出来,说明非结核分枝杆菌猪、牛群中最新的流行情况并不乐观,加之近年来在饲养管理和监测控制措施实行和完善的前提下,非结核分枝杆菌仍有较高的流行,说明该菌有较强的适应性,为了防止其广泛流行产生大的影响,应在防治结核分枝杆菌的同时加强非结核分枝杆菌的监测和防控工作。2、非结核分枝杆菌血清学分组及抗原性分析：对分离到的NTM血清学分组,将NTM转移接种到Middle brook 7H10抗原培养基中,生长后用0.3%甲醛灭菌生理盐水制备免疫用死菌制剂,用灭菌生理盐水制备免疫用活菌制剂,分四次免疫健康兔,每次间隔一周,一、二次用死菌制剂免疫,三、四次用活菌制剂免疫,并于第四次免疫一周后心脏采血制备抗血清。用含石炭酸的磷酸盐缓冲液洗下抗原培养基上的NTM,经过处理后,用PBS缓冲液制备成抗原。用抗血清和抗原进行凝集反应和凝集素吸收试验,最终对分离到的NTM做出血清学分组及抗原性分析,结果将NTM分离株分成五个血清型：NH1、NH2、NH5、ZH1、ZC4、ZC14为一种血清型,ZC2、ZC3、ZH9、ZH10为一种血清型,NH9、NC11、NC12、NC13、NC14、ZC11、ZC12为一种血清型,NH3、NH6、NC4、NC7、NC10、ZH5、ZH6、ZH13、ZC7、ZC8为一种血清型,NH8单独为一种血清型,说明吉林省地区分布的NTM血清型分类结构较具体,有进一步分类研究,并根据其分型制定监测和防制措施价值。3、分离菌株中偶发分枝杆菌全基因组文库的构建：从分离NTM中选取已经鉴定确认为偶发分枝杆菌的分离株,改良核酸提取方法提取全基因组,用限制性内切酶Sau3A I随机酶切全基因组,通过体系中酶浓度调节控制酶切后基因片段主要集中在1kb到7kb之间,把酶切片段通过随机连接的方式连入经过BamH I单酶切和去磷酸化处理过的pUC18载体中,再转化入自制的感受态细菌DH5a,经蓝白斑筛选、PCR、双酶切等方法检测连接效率并得到阳性连接,刮取培养基表面阳性菌,加甘油保存于-80℃,实验得到阳性克隆4×104个,根据统计学计算达到基因组文库要求,该文库的建立为以后对该菌特殊基因方面的研究及基因结构和调控等研究打下良好基础,同时也为其它NTM的进一步研究工作打下了实验基础。4、基因组文库的应用即hsp65基因片段的分析：从已经制备的偶发分枝杆菌的全基因组文库中克隆出长413bp的hsp65基因片段,经测序后,采用Blast、DNAStar软件,对其进行分析,得到该偶发分枝杆菌的基因特征和进化特征。
本实验从NTM的分离培养到分离鉴定和血清学分型,再到全基因组文库的构建和基因组文库的应用及NTM基因序列的测序分析,为NTM实验室研究工作制订了一整套流程,为实验室就分离到的NTM进一步研究工作打下基础,同时实验所得各数据也为撑控NTM最新流行情况、针对NTM特点提出合理有效的防治措施提供了科学依据。
Mycobacterium Including Nontuberculous mycobacterium and Mycobacterium tuberculosis and so on. Because of Nontuberculous mycobacterium's similar symptoms with tuberculosis, it is named. Nontuberculous mycobacterium is a Gram-positive bacteria,its Acid-fast staining is positive. To 2003, there are 95 kinds Nontuberculous mycobacterium have been found, most of them are pathogenic or opportunistic pathogen. In addition lung disease, it can cause a variety of animal other tissues disease, the clinical diagnosises are often reported misdiagnosed as tuberculosis. Although NTM and Mycobacterium tuberculosis are Mycobacterium, but the drug resistance are different, so can not be cured after the misdiagnosis as Mycobacterium tuberculosis. Meanwhile, most of the NTM on the PPD was negative, they are difficult to be found, that cause great losses to the livestock industry, so isolation and further research on the animal's NTM is necessary.
NTM reports on the research, domestic and foreign literature, mostly published reports on human NTM, NTM few reports of animal. In this study,the research on animals NTM experiment is divided into four parts.1. Isolation and identification of non-tuberculosis mycobacteria:Collected 2,100 submandibular lymph nodes and mesenteric lymph nodes from pigs and cattle whose PPD test is negative in many parts of Jilin Province.These samples are grinded into a homogenized after treated with acid and are inoculated in L-J culture medium which is fresh, each sample was inoculated with three, cultured at 37℃, observed once every 3 days, continued 8 weeks. Acid-fast staining tests bacterials after growthing. Amplificed bacterias with L-J medium which are positive by acid-fast stain. Classified bacteria which are amplificed in several ways. The approaches taken have laboratory conventional methods, Biochemical methods, growth characteristics experiment and Molecular biology methods. Laboratory conventional methods:Bacteria are cultured at 28℃, 37℃,45℃on L-J medium and observe the growth of its. Biochemical methods:Heat-resistant catalase, nitrate reduction, Tween 80 hydrolysis, urease, aromatic sulfatase, iron uptake, tellurite reduction test bacteria isolated. Growth characteristics experiment:Application PNB medium, TCH medium, sodium chloride medium, medium picric acid, sodium glutamate glucose medium, MacConkey medium for identificating of bacteria isolated. Molecular biology methods:Application of heat shock protein hsp65 gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) Identification of bacteria isolated. Synthesis and analysis of the results of various methods, make the final identification. A total of 28 strains has been obtained. These mycobacteria were identified as follow:4 strains as Mycobacterium gilvum,2 strains as Mycobacterium scrofulaceum,2 strains as Mycobacterium intracellulare,6 strains as Mycobacterium senopi,1 strains as Mycobacterium bovis,3 strains as Mycobacterium fortuitum,2 strains as Mycobacterium szulgai,4 strains as Mycobacterium neoaurum.1 strains as Mycobacterium testudinis，1 strains as Mycobacterium gordonae，2 strains as Mycobacterium vaccae. The results show that although the samples were tested by PPD, but there are still non-tuberculous mycobacteria were isolated. Non-tuberculosis mycobacteria's epidemic situation in pigs, cattle is not optimistic. It shows the strong adaptability of non-tuberculosis mycobacteria that non-tuberculous mycobacteria have a higher epidemic undering the premise of feeding management and monitoring of the implementation and improvement of control measures,In recent years. In order to prevent a big impact by widespreading, we should be strengthening the monitoring of non-tuberculous mycobacteria, and prevention and control when preventionning and controlling Mycobacterium tuberculosis.2, Non-tuberculosis mycobacteria serologily group and antigenic analysis:Grouped non-tuberculosis serologily. Non-tuberculosis is inoculated on the Middlebrook 7H10 antigen medium. After growning of bacterial, using 0.3%formaldehyde in sterile saline preparars of immune preparations with dead bacteria and prepared immunization with viable preparations with sterile saline. Immune health rabbits in fourth, one week intervals, the first and second immunization with dead bacteria preparations, three or four times immunization with viable preparations. In the week after the forth immunization, antiserum with heart blood. Using phosphate buffer Containing carbolic acid washed non-tuberculosis on the antigen medium. After processing, is prepared antigen with PBS buffer. Make agglutination and agglutinin absorption test With anti-serum and antigen. Ultimately makeing non-tuberculosis serological grouping and analysis of antigens. The results is that non-tuberculosis were divided into five groups of serotype:NH1,NH2,NH5,ZH1,ZC4,ZC14 are one group,ZC2,ZC3,ZH9,ZH10 are one group,NH9,NC11,NC12,NC13,NC14,ZC11,ZC12 are one group,NH3,NH6,NC4,NC7,NC10,ZH5,ZH6,ZH13,ZC7,ZC8 are one group,NH8 are one group. Shows the distribution of serotypes of non-tuberculosis in Jilin Province is more specific classification structure.There is the value of Classification of further development and making control measures. Construction of DNA Library of Mycobacterium fortuitum that is a Nontuberculous Mycobacteria:Select Mycobacterium fortuitum from non-tuberculosis which has been recognized. Extracte whole genome by improving method and digeste genome with the restriction enzyme Sau3A I randomly. Through regulateing and controlling concentration of enzyme in system of gene fragments digested to digest gene in between 1000bp to 7000bp. The fragments are connected by way of random connections into pUC18 vector digested by BamHⅠ,then transform the prouduct in to DH5a made by ourself. The blue-white screening, PCR, restriction enzyme were used to detect the result of connection and get the positive connection efficiency. Scraping the positive bacteria from the surface of the medium. Stored at 80℃with glycerol.There are 4×104 positive have been gain in experiment.The genomic library is sucessful according to statistical calculation. The establishment of the library establish a good basis for the study of strain specific genes and gene structure and regulation. At the same time, to lay a foundation for the further study of other NTM.4 Application of genomic library and analysis of the hsp65 gene:Cloned the hsp65 gene fragment length of 413bp from Mycobacterium fortuitum's genomic library which has been prepared. After sequencing, using Blast, DNAStar software to analyze hsp65 gene to get the gene and evolutionary characteristics of Mycobacterium fortuitum. In this study,from the isolation and identification of the non-tuberculosis to serotyping.and to the construction of whole genome library and the application of genomic library, analysis of gene sequence.This study sets a processes for laboratory research on non-tuberculosis, lays the foundation for further research on non-tuberculosis in laboratory. At the same time,the date catched from the experiment provides the scientific basis for obtainning and controlling the latest epidemic of non-tuberculosis and make a rdasonable and effective control measures of non-tuberculosis.
引文
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