發(fā)布時間：2018-03-10 04:37

  本文選題：豬丹毒絲菌　切入點：廣西流行株　出處：《吉林農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：豬丹毒絲菌(Erysipelothrix rhusiopathiae)俗稱豬丹毒桿菌或丹毒絲菌,屬丹毒絲菌屬(Erysipelothrix),是一種纖細的小桿菌,革蘭染色陽性,人獸共患病原菌。該菌于1882年被Pasteur首次分離到,1886年Loffler首次精確的描述了引起豬丹毒病的病原。該菌血清型復雜,目前已發(fā)現(xiàn)1、1a、2、2a、2b、3~24及N型等26種之多。豬丹毒在我國被認為是一種古老的疫病,上世紀80-90年代豬丹毒與豬肺疫、豬瘟被稱為養(yǎng)豬業(yè)的三大疾病,曾給我國養(yǎng)豬業(yè)帶來嚴重的經(jīng)濟損失。該病已經(jīng)有二十余年極少見到,幾乎處于沉寂狀態(tài),臨床上基本上沒有大面積的發(fā)生與流行,成為被從業(yè)人員“遺忘”多年的疾病。然而,近年來豬丹毒又時有發(fā)生,在我國南方的廣西、廣東、湖南、四川、福建、江西、湖南、安徽等地日趨活躍,頻頻爆發(fā),造成不同生長階段的豬群發(fā)病死亡。隨著時間的推移,該病有從南向北發(fā)展,呈現(xiàn)日趨嚴重的態(tài)勢。被“遺忘”多年的豬丹毒疫情突然爆發(fā),讓不少養(yǎng)殖戶措手不及,造成豬群發(fā)生大面積急性感染死亡,損失慘重。因此,對于豬丹毒絲菌的研究再一次引起學者和從業(yè)人員的關注與重視。廣西獸醫(yī)研究所自2012年5月至2012年10月,從廣西壯族自治區(qū)的貴港市、賓陽縣、陸川縣、巴馬縣4個地市縣送檢的疑似豬丹毒病料中,通過細菌分離培養(yǎng)、生理生化特性測定、16Sr DNA序列測定與分析、動物致病性試驗、血清型鑒定等系統(tǒng)的鑒定與分析,共分離到12株豬丹毒絲菌。其中,貴港市7株、賓陽縣1株、陸川縣1株、巴馬縣3株,分別命名為: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,血清型均為1a型,能致死小白鼠。按照不同分離地區(qū),選取Er.GXGG-1、Er.GXBY-1、Er.GXLC-1、Er.GXBM-1菌株作為代表菌株,與4株上世紀50年代至80年代豬丹毒絲菌參考菌株(CVCC124、CVCC131、CVCC134、CVCC1246),通過觀察菌株的生長形態(tài)特征,繪制生長曲線,測定生理生化特性、藥物敏感特性、半數(shù)致死量(LD50)及疫苗免疫保護作用,同時用PCR方法擴增8株豬丹毒絲菌4個毒力因子的7個毒力基因片段,進行細菌生物學特性的比較研究。結果8株菌表現(xiàn)出一致的形態(tài)特征、生長曲線和生理生化特性;LD50結果顯示,新近分離到的4株菌半數(shù)致死量高于4株參考菌株,表明新近分離菌株毒力有所下降;免疫保護試驗結果表明,免疫GC42株活疫苗的昆明小白鼠在注射不同年代的豬丹毒絲菌后仍能保持80%-100%存活;對8株菌進行30種常用抗菌藥物的藥物敏感試驗,結果顯示,8株菌對β-內(nèi)酰胺類、氯霉素類、大環(huán)內(nèi)酯類、硝基呋喃類和雙萜類藥物仍保持高敏,但對四環(huán)素、林可霉素、諾氟沙星敏感性出現(xiàn)差異;4株新近臨床分離菌株對上述3種藥物的敏感性顯著低于4株老菌株,表現(xiàn)為耐藥。多數(shù)菌株對氨基糖苷類、多肽類、磺胺類、硝基咪唑類、利福霉素家族藥物表現(xiàn)為耐藥。8株菌均能擴增出4個毒力因子的7個毒力基因片段。毒力基因遺傳變異分析表明,與參考菌株相比,新近臨床分離菌株毒力基因中僅CPS-A、CPS-C發(fā)生變異,而Spa A、Sialidase、CPS-B、Rsp A、Rsp B為保守基因,同源性近100%。依據(jù)以上實驗結果,選取GXBY-1株作為代表菌株,提取其全基因組,利用新一代高通量測序技術Illumina Hi Seq2000平臺,對GXBY-1株進行全基因組數(shù)據(jù)的采集、組裝與分析。結果顯示,GXBY-1株基因組大小為1 888 332bp,GC含量36.52%,共2個scaffold,11個contig�；�因組序列分析后發(fā)現(xiàn),GXBY-1株的基因組含有1 766個基因,總長度為1 668 564 bp,平均長度945 bp,占基因組全長的88.36%。串聯(lián)重復序列共62個,總長為9 057 bp,占基因組全長的0.4796%。小衛(wèi)星序列44個,微衛(wèi)星序列1個。t RNA 57個,r RNA 28個。本研究在機體和分子水平上,揭示了流行于廣西壯族自治區(qū)豬丹毒絲菌的生物學特性和基因組特征,為該病的科學防控及開展深入研究奠定了基礎。
[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.

【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

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本文編號：1591819