貂源肺炎克雷伯氏菌血清型和毒力基因的检测及其致病性研究

发布时间：2018-06-21 04:59

本文选题：水貂 + 小鼠　；参考：《山东农业大学》2017年硕士论文

【摘要】：肺炎克雷伯氏菌(Klebsiella Pneumoniae,K.Pneumoniae),是一种可引起多种动物患病的条件性致病菌,也是常见的人兽共患病病原。本研究对貂源肺炎克雷伯氏菌血清型和毒力基因进行了检测并对其致病性进行了研究;其对水貂克雷伯氏菌的防控具有重要的实践意义,更具有重要的公共卫生学意义。1.血清型的检测采用克雷伯氏菌血清型检测PCR方法对本实验室分离保存的23株水貂肺炎克雷伯氏菌进行了常见血清型(K1、K2、K5、K20、K54和K57)的检测。并对其中15株菌(K.pneumoniae-SD-1~K.pneumoniae-SD-13,K.pneumoniae-SD-15和K.pneumoniae-SD-21)的阳性血清型进行了克隆测序分析,且于GenBank中收录的肺炎克雷伯氏菌菌株作为参考菌株进行比较,制作了系统发生树和同源性的分析。其结果除了K.pneumoniae-SD-15不是上面血清型中的任何一个血清型之外,其他22株都是K2血清型;15株代表菌株中的14株阳性血清型K2基因与参考菌株23(分离于人的器官)、wzy-K2(分离于人的痰液)都在一个分支上,亲缘关系很近;与参考菌株23、HK787、ATCC43816和VGH52523的同源性为99.7%-100%。表明山东地区以K2血清型为主的强毒株肺炎克雷伯氏菌能感染水貂并且在水貂上流行。2.毒力基因的检测采用PCR方法对主要毒力基因rmpA、菌素、iroNB、iucB、wcaG、uge、magA、wabG、all S、ureA、fim和ybtA进行检测,结果显示ureA基因的检出率100%(23/23);菌素、rmpA、uge和wabG的检出率均为96%(22/23);毒力基因Iuc B检出率87%(20/23)、毒力基因ybt的检出率为13%(3/23),其他毒力基因fim、wcaG、allS、magA和iroNB均未检出,其中有16株菌株均有ureA、IucB、rmpA、菌素、uge和wab 6种主要毒力基因,1株菌株均有ureA、IucB、rmpA、菌素、uge、wab和ybtA 7种毒力基因。这表明同一菌株有好几种不同的毒力基因。并对其中15株菌(K.pneumoniae-SD-1~K.pneumoniae-SD-13,K.pneumoniae-SD-15和K.pneumoniae-SD-21)的阳性毒力基因进行了克隆测序分析,且于GenBank中收录的肺炎克雷伯氏菌菌株作为参考菌株进行比较,制作了系统发生树和同源性的分析。系统发生树结果显示:15株代表菌株中的14株阳性rmpA基因与参考菌株TMV-5(分离于人的器官)的血缘关系很近;15株代表菌株中的14株阳性菌素基因与参考菌株cg43(分离于人的血液)、ed23(分离于人的血液)都在一个分支上;15株代表菌株的阳性wabg基因与参考菌株cg43(分离于人的血液)、ntuh-k2044(分离于人的肝脏和血液)都在一个分支上;15株代表菌株中的12株阳性iucb基因与参考菌株rjf999(分离于人的血液)、t69(分离于人的痰液)亲缘关系很近;15株代表菌株中的2株阳性ybta基因与参考菌株ntuh-k2044(分离于人的肝脏和血液)亲缘关系很近。同源性分析结果显示:15株代表菌株中的14株阳性rmpa基因与参考菌株ntuh-k2044、kctc2242、ed23和rjf293的同源性为99.1%-100%;15株代表菌株中的14株阳性菌素基因与参考菌株atcc43816、cg43和ed23的同源性为98.2%-100%;15株代表菌株的阳性wabg基因与参考菌株ntuh-k2044、kctc2242、ed23、cav1217和cav1042的同源性为98.4%-100%;15株代表菌株的阳性urea基因与参考菌株cg43、rjf293、cav1042和u41的同源性为98.2%-100%;15株代表菌株中的14株阳性uge基因与参考菌株kctc2242、rjf293、cav1453和kp52.145的同源性为97.8%-100%;15株代表菌株中的12株阳性iucb基因与参考菌株rjf293和sb227的同源性为94.6-99.7%;15株代表菌株中的2株代表菌株的ybta基因与参考菌株ntuh-k2044、kp36、11和ed23的同源性99.3-99.7%。结果表明貂源肺炎克雷伯氏菌与人源肺炎克雷伯氏菌的亲缘关系很近。3.致病性研究本研究根据分离菌株所携带的血清型、毒力基因的不同选择3株菌株,建立貂源肺炎克雷伯氏菌对小鼠和水貂的致病性研究。分别给小鼠和水貂攻毒后,结果显示,在2—8天均表现精神沉郁、食欲减退,结膜苍白,呼吸浅速,站立不稳且抽搐,被毛逆乱,体温升高,偶尔咳嗽,鼻腔内有分泌物,第4—7天死亡达到高峰,剖检死亡小鼠和水貂发现腹腔内均有明显的黏液,且易拉丝,肝有坏死灶、发硬、发黑;肺肿大出血;病理组织学观察,肺、肝,脑都有不同程度的出血,充血现象。ld50结果显示,k.pneumoniae-sd-12和k.pneumoniae-sd-21菌株对小鼠的ld50分别为5.0×102cfu和3.2×100cfu;对水貂的ld50分别为1.6×103cfu和3.2×101cfu;然而k.pneumoniae-sd-15对小鼠和水貂都没有致病性。由上述血清型和毒力基因检测可知,k.pneumoniae-sd-12和k.pneumoniae-sd-21菌株均有荚膜血清型k2、菌素和rmpa毒力基因,而k.pneumoniae-sd-15不是血清型k2,且没有菌素和rmpa毒力基因。本研究的结果证实,貂源肺炎克雷伯氏菌含有荚膜血清型k2、菌素和rmpa毒力基因的菌株具有较高的致病性。
[Abstract]:Klebsiella Pneumoniae (K.Pneumoniae) is a conditional pathogenic bacteria that can cause a variety of animal diseases and is a common pathogeny of zoonosis. In this study, the serotype and virulence genes of Klebsiella pneumoniae were detected and the pathogenicity of Klebsiella pneumoniae, and the prevention of Klebsiella in mink. Control has important practical significance and more important public health significance.1. serotype detection using Kleber's serotype PCR method to detect 23 strains of Klebsiella pneumoniae isolated and preserved in our laboratory (K1, K2, K5, K20, K54 and K57). And 15 strains of bacteria (K.pneumoniae-SD-1~K.pn) The positive serotypes of eumoniae-SD-13, K.pneumoniae-SD-15 and K.pneumoniae-SD-21 were cloned and sequenced, and the strains of Klebsiella pneumoniae included in GenBank were compared as reference strains, and the phylogenetic tree and homology analysis were made. The results were not K.pneumoniae-SD-15 in any of the above serotypes. In addition to the serotypes, the other 22 were K2 serotypes, 14 of the 15 representative strains were positive for serotype K2 and 23 (separated from human organs), and wzy-K2 (isolated from human sputum) was on a branch and was closely related. The homology of HK787, ATCC43816 and VGH52523 with reference strains 23, 99.7%-100%. showed that the Shandong region Klebsiella pneumoniae, a strong strain of K2 serotype, can infect mink and detect the.2. virulence gene in mink. The PCR method was used to detect the main virulence genes rmpA, bacteria, iroNB, iucB, wcaG, uge, magA, wabG, all S. The detection rates were 96% (22/23), the detection rate of virulence gene Iuc B was 87% (20/23), the detection rate of virulence gene YBT was 13% (3/23), and other virulence genes were not detected in FIM, wcaG, allS, magA and iroNB, of which 16 strains had 6 main virulence genes, 1 strains, 7 species. Virulence genes. This indicates that there are several different virulence genes in the same strain. The positive virulence genes of 15 strains (K.pneumoniae-SD-1~K.pneumoniae-SD-13, K.pneumoniae-SD-15 and K.pneumoniae-SD-21) were cloned and sequenced, and the strains of Klebsiella pneumoniae included in GenBank were compared as reference strains. The phylogenetic tree and homology analysis were made. The phylogenetic tree showed that 14 positive rmpA genes of 15 representative strains were closely related to the reference strain TMV-5 (isolated from human organs); 14 of the 15 strains of positive bacteria and reference strain cg43 (separated from human blood), and Ed23 (isolated from human blood). The positive wabg gene of 15 representative strains and the reference strain cg43 (separated from the human blood), ntuh-k2044 (isolated from the human liver and blood) were all on one branch, and 12 of the 15 representative strains of the strains were closely related to the reference strain rjf999 (separated from the human blood) and T69 (isolated from human sputum). The 2 positive ybta genes of the 15 representative strains were closely related to the reference strain ntuh-k2044 (separated from the human liver and blood). The homology analysis showed that 14 positive rmpA genes of the 15 representative strains were identical to the reference strain ntuh-k2044, kctc2242, Ed23 and rjf293 as 99.1%-100%, and 14 of the 15 strains of the strain were positive. The homology of the bacteriocin gene with the reference strain atcc43816, cg43 and Ed23 was 98.2%-100%, and the homology of the positive wabg gene of the 15 representative strains and the reference strain ntuh-k2044, kctc2242, Ed23, cav1217 and cav1042 was 98.4%-100%, and the positive urea gene and the reference strain of the 15 strains were homologous to the strain; 15 The homology of 14 positive uge genes in the strain of the strain and the reference strain kctc2242, rjf293, cav1453 and kp52.145 was 97.8%-100%, and the homology of 12 positive iucb genes in the 15 representative strains was 94.6-99.7% with the reference strain rjf293 and sb227, and the ybta gene of the 2 representative strains of the 15 strains of the strain was similar to the reference strain. The homologous 99.3-99.7%. results of 1 and Ed23 showed that the relationship between Klebsiella pneumoniae and Klebsiella pneumoniae was very close to.3. pathogenicity. The study on the pathogenicity of Klebsiella pneumoniae to mice and mink based on the serotypes and virulence genes of 3 strains carried by the isolated strains. The results showed that the mice and minks were not infected, and the results showed that they showed depression in the 2 to 8 days, paleness of the conjunctiva, shallow conjunctiva, shallow breathing, unstable standing and convulsion, the inverse disorder of the body, the rise of body temperature, the occasional coughing, the secretion in the nasal cavity, the peak of fourth to 7 days, and the apparent mucus in the abdominal cavity of the dead mice and minks. The liver has necrotic foci, hard and black; pulmonary swelling and bleeding; histopathological observation, lung, liver and brain have different levels of bleeding, and the.Ld50 results show that the LD50 of k.pneumoniae-sd-12 and k.pneumoniae-sd-21 strains to mice are 5 x 102CFU and 3.2 x 100cfu respectively; the LD50 of mink is 1.6 x 103cfu and 3.2 x 101cfu respectively; however K., however, K., respectively. Pneumoniae-sd-15 had no pathogenicity to both mice and mink. From the above serotypes and virulence genes, the k.pneumoniae-sd-12 and k.pneumoniae-sd-21 strains had a capsule serotype K2, bacteriin and rmpA virulence genes, and k.pneumoniae-sd-15 was not a serotype K2, and no bacteria and rmpA virulence genes. The results of this study confirmed the source of mink. Klebsiella pneumoniae containing capsular serotype K2, pathogenic bacterium and rmpA strains with high virulence genes.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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