猪丹毒丝菌广西分离株生物学特性研究及其基因组生物信息学分析

发布时间：2018-03-10 04:37

  本文选题：猪丹毒丝菌　切入点：广西流行株　出处：《吉林农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪丹毒丝菌(Erysipelothrix rhusiopathiae)俗称猪丹毒杆菌或丹毒丝菌,属丹毒丝菌属(Erysipelothrix),是一种纤细的小杆菌,革兰染色阳性,人兽共患病原菌。该菌于1882年被Pasteur首次分离到,1886年Loffler首次精确的描述了引起猪丹毒病的病原。该菌血清型复杂,目前已发现1、1a、2、2a、2b、3~24及N型等26种之多。猪丹毒在我国被认为是一种古老的疫病,上世纪80-90年代猪丹毒与猪肺疫、猪瘟被称为养猪业的三大疾病,曾给我国养猪业带来严重的经济损失。该病已经有二十余年极少见到,几乎处于沉寂状态,临床上基本上没有大面积的发生与流行,成为被从业人员“遗忘”多年的疾病。然而,近年来猪丹毒又时有发生,在我国南方的广西、广东、湖南、四川、福建、江西、湖南、安徽等地日趋活跃,频频爆发,造成不同生长阶段的猪群发病死亡。随着时间的推移,该病有从南向北发展,呈现日趋严重的态势。被“遗忘”多年的猪丹毒疫情突然爆发,让不少养殖户措手不及,造成猪群发生大面积急性感染死亡,损失惨重。因此,对于猪丹毒丝菌的研究再一次引起学者和从业人员的关注与重视。广西兽医研究所自2012年5月至2012年10月,从广西壮族自治区的贵港市、宾阳县、陆川县、巴马县4个地市县送检的疑似猪丹毒病料中,通过细菌分离培养、生理生化特性测定、16Sr DNA序列测定与分析、动物致病性试验、血清型鉴定等系统的鉴定与分析,共分离到12株猪丹毒丝菌。其中,贵港市7株、宾阳县1株、陆川县1株、巴马县3株,分别命名为:Er.GXGG-1、Er.GXGG-2、Er.GXGG-3、Er.GXGG-4、Er.GXGG-5、Er.GXGG-6、Er.GXGG-7、Er.GXBY-1、Er.GXLC-1、Er.GXBM-1、Er.GXBM-2、Er.GXBM-3,血清型均为1a型,能致死小白鼠。按照不同分离地区,选取Er.GXGG-1、Er.GXBY-1、Er.GXLC-1、Er.GXBM-1菌株作为代表菌株,与4株上世纪50年代至80年代猪丹毒丝菌参考菌株(CVCC124、CVCC131、CVCC134、CVCC1246),通过观察菌株的生长形态特征,绘制生长曲线,测定生理生化特性、药物敏感特性、半数致死量(LD50)及疫苗免疫保护作用,同时用PCR方法扩增8株猪丹毒丝菌4个毒力因子的7个毒力基因片段,进行细菌生物学特性的比较研究。结果8株菌表现出一致的形态特征、生长曲线和生理生化特性;LD50结果显示,新近分离到的4株菌半数致死量高于4株参考菌株,表明新近分离菌株毒力有所下降;免疫保护试验结果表明,免疫GC42株活疫苗的昆明小白鼠在注射不同年代的猪丹毒丝菌后仍能保持80%-100%存活;对8株菌进行30种常用抗菌药物的药物敏感试验,结果显示,8株菌对β-内酰胺类、氯霉素类、大环内酯类、硝基呋喃类和双萜类药物仍保持高敏,但对四环素、林可霉素、诺氟沙星敏感性出现差异;4株新近临床分离菌株对上述3种药物的敏感性显著低于4株老菌株,表现为耐药。多数菌株对氨基糖苷类、多肽类、磺胺类、硝基咪唑类、利福霉素家族药物表现为耐药。8株菌均能扩增出4个毒力因子的7个毒力基因片段。毒力基因遗传变异分析表明,与参考菌株相比,新近临床分离菌株毒力基因中仅CPS-A、CPS-C发生变异,而Spa A、Sialidase、CPS-B、Rsp A、Rsp B为保守基因,同源性近100%。依据以上实验结果,选取GXBY-1株作为代表菌株,提取其全基因组,利用新一代高通量测序技术Illumina Hi Seq2000平台,对GXBY-1株进行全基因组数据的采集、组装与分析。结果显示,GXBY-1株基因组大小为1 888 332bp,GC含量36.52%,共2个scaffold,11个contig。基因组序列分析后发现,GXBY-1株的基因组含有1 766个基因,总长度为1 668 564 bp,平均长度945 bp,占基因组全长的88.36%。串联重复序列共62个,总长为9 057 bp,占基因组全长的0.4796%。小卫星序列44个,微卫星序列1个。t RNA 57个,r RNA 28个。本研究在机体和分子水平上,揭示了流行于广西壮族自治区猪丹毒丝菌的生物学特性和基因组特征,为该病的科学防控及开展深入研究奠定了基础。
[Abstract]:Erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae) commonly known as erysipelas or e.rhusiopathiae, genus Erysipelothrix (Erysipelothrix), is a kind of tiny bacteria, gram positive bacteria, zoonosis. The pathogen was first isolated in 1882 by Pasteur Loffler in 1886 for the first time, accurate description of swine erysipelas. The pathogenic bacterium serotype complex, it has been found that the 1,1a, 2,2a, 2b, 3~24 and N etc. more than 26 kinds of swine erysipelas in China is considered to be an ancient disease, the last century 80-90's swine erysipelas and lung disease, swine fever is known as the three major diseases in pig industry that brings the serious economic losses to the swine industry in China. The disease has been more than twenty years rarely see, almost in a quiet state, basically no clinical occurrence and epidemic area, become practitioners of "forgotten" years of disease. However, in recent years, swine erysipelas and Have occurred in southern China, Guangxi, Guangdong, Hunan, Sichuan, Fujian, Jiangxi, Hunan, Anhui and other places have become increasingly active, the frequent outbreak of swine disease, caused by the different growth stages of death. Over time, the disease has developed from the south to the north, becoming more and more serious situation. The "forgotten" the swine erysipelas epidemic outbreak, so many farmers to be taken by surprise, resulting in a large area of acute infection of pigs died and suffered heavy losses. Therefore, for the study of Erysipelothrix rhusiopathiae once again aroused the attention and attention of academics and practitioners. The Guangxi Veterinary Research Institute from May 2012 to October 2012, from the Guangxi Zhuang Autonomous Region Guigang City, Binyang County, Lu Chuan County, Bama County in 4 cities and counties a total of suspected swine erysipelas disease in cultured by bacteria isolation and determination of physiological and biochemical characteristics, determination and analysis of 16Sr DNA sequence, animal pathogenicity test, serum Identification and analysis of type identification system, 12 strains of Erysipelothrix rhusiopathiae were isolated. Among them, 7 strains of Guigang City, Binyang County, Lu Chuan County, 1 strains, 1 strains, 3 strains of Bama County, named Er.GXGG-1, Er.GXGG-2, Er.GXGG-3, Er.GXGG-4, Er.GXGG-5, Er.GXGG-6, Er.GXGG-7, Er.GXBY-1, Er.GXLC-1, Er.GXBM-1 Er.GXBM-2, Er.GXBM-3, and serotypes were 1A type, can be lethal to mice. According to the different separation area, select Er.GXGG-1, Er.GXBY-1, Er.GXLC-1, Er.GXBM-1 strains as representative strains, and 4 strains in 50s to 80s Erysipelothrix rhusiopathiae strains (CVCC124, CVCC131, CVCC134, CVCC1246), the growth morphology strain, draw the growth curve, determination of physiological and biochemical characteristics, drug sensitivity, median lethal dose (LD50) and immunity function, and amplified by PCR 8 strains of Erysipelothrix rhusiopathiae 4 virulence factors of 7 virulence gene fragments were fine Comparative study on the biological characteristics of bacteria. Results 8 strains showed similar morphology, growth curve and physiological and biochemical characteristics; LD50 results showed that the newly isolated 4 strains were higher than the median lethal dose of 4 reference strains, showed that the newly isolated strains decreased virulence; immune protection test results showed that the mice strains in Kunming live vaccine GC42 can still keep 80%-100% alive in Erysipelothrix rhusiopathiae in different times after injection; 30 kinds of antibiotic drug sensitivity test of 8 strains showed that 8 strains of beta lactam, Chloramphenicols, macrolides, nitrofurans and diterpene the drug remains Gao Min, but for lincomycin, tetracycline, norfloxacin sensitivity difference; 4 strains of recent clinical isolates of the 3 kinds of drug sensitivity of 4 strains was significantly lower than that of the old strains showed resistance. Most strains of aminoglycosides, Polypeptide, sulfonamides, nitromidazoles, rifamycin family of drugs is resistant.8 strains were amplified 4 virulence factors of 7 virulence gene fragments. Genetic variation of virulence analysis showed that, compared with the reference strains, recent clinical isolates of virulence genes in only CPS-A, CPS-C mutation, and Spa A, Sialidase, CPS-B, Rsp A, Rsp B gene is conservative, homology near 100%. on the basis of the above experimental results, GXBY-1 strain was selected as the representative strain, the whole genome extraction, the use of a new generation of high-throughput sequencing technology Illumina Hi Seq2000 platform, the whole genome of GXBY-1 strain data acquisition, assembly and analysis. Show that the GXBY-1 genome size is 1888 332bp, the content of GC 36.52%, a total of 2 scaffold, 11 contig. genomic sequence analysis showed that GXBY-1 strain genome contains 1766 genes, a total length of 1668564 BP, the average length of 945 B P, accounting for full-length 88.36%. tandem repeat sequences were 62, with a total length of 9057 BP, accounting for 0.4796%. of small satellite genome sequence of 44 microsatellite sequences, 1.T RNA 57, R RNA 28. In this study, the body and the molecular level, biological characteristics and genomic characteristics reveals popular in the Guangxi Zhuang Autonomous Region Erysipelothrix rhusiopathiae, laid the foundation for further study and carry out scientific prevention and control of the disease.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【参考文献】

中国期刊全文数据库 前5条

1 郭良兴;陈克研;赵魁;高巍;李志萍;赵传博;王丽;高丰;;猪丹毒杆菌的分离鉴定及耐药性试验[J];中国畜牧兽医;2011年04期

2 田新民;王定发;李琼;;猪常见细菌性疾病及中草药预防和控制[J];安徽农业科学;2013年25期

3 万培伟;熊莉娟;储玉双;胡善辉;黄小红;蒋新华;邓舜洲;张文波;;猪丹毒杆菌的分离鉴定及药敏试验[J];动物医学进展;2014年06期

4 邱美珍;杜丽飞;杨爱梅;王慧;杨俊;周望平;;猪丹毒杆菌临床分离鉴定与药敏试验[J];养猪;2011年06期

5 胡晓芬;王静;熊剑锋;吴晓明;张金林;秦兴保;马立保;;一起猪丹毒杆菌病例的临床分离鉴定及诊治[J];养猪;2014年02期

，

本文编号：1591819