本文選題：水貂 + 小鼠��； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：肺炎克雷伯氏菌(Klebsiella Pneumoniae,K.Pneumoniae),是一種可引起多種動(dòng)物患病的條件性致病菌,也是常見(jiàn)的人獸共患病病原。本研究對(duì)貂源肺炎克雷伯氏菌血清型和毒力基因進(jìn)行了檢測(cè)并對(duì)其致病性進(jìn)行了研究;其對(duì)水貂克雷伯氏菌的防控具有重要的實(shí)踐意義,更具有重要的公共衛(wèi)生學(xué)意義。1.血清型的檢測(cè)采用克雷伯氏菌血清型檢測(cè)PCR方法對(duì)本實(shí)驗(yàn)室分離保存的23株水貂肺炎克雷伯氏菌進(jìn)行了常見(jiàn)血清型(K1、K2、K5、K20、K54和K57)的檢測(cè)。并對(duì)其中15株菌(K.pneumoniae-SD-1~K.pneumoniae-SD-13,K.pneumoniae-SD-15和K.pneumoniae-SD-21)的陽(yáng)性血清型進(jìn)行了克隆測(cè)序分析,且于GenBank中收錄的肺炎克雷伯氏菌菌株作為參考菌株進(jìn)行比較,制作了系統(tǒng)發(fā)生樹(shù)和同源性的分析。其結(jié)果除了K.pneumoniae-SD-15不是上面血清型中的任何一個(gè)血清型之外,其他22株都是K2血清型;15株代表菌株中的14株陽(yáng)性血清型K2基因與參考菌株23(分離于人的器官)、wzy-K2(分離于人的痰液)都在一個(gè)分支上,親緣關(guān)系很近;與參考菌株23、HK787、ATCC43816和VGH52523的同源性為99.7%-100%。表明山東地區(qū)以K2血清型為主的強(qiáng)毒株肺炎克雷伯氏菌能感染水貂并且在水貂上流行。2.毒力基因的檢測(cè)采用PCR方法對(duì)主要毒力基因rmpA、菌素、iroNB、iucB、wcaG、uge、magA、wabG、all S、ureA、fim和ybtA進(jìn)行檢測(cè),結(jié)果顯示ureA基因的檢出率100%(23/23);菌素、rmpA、uge和wabG的檢出率均為96%(22/23);毒力基因Iuc B檢出率87%(20/23)、毒力基因ybt的檢出率為13%(3/23),其他毒力基因fim、wcaG、allS、magA和iroNB均未檢出,其中有16株菌株均有ureA、IucB、rmpA、菌素、uge和wab 6種主要毒力基因,1株菌株均有ureA、IucB、rmpA、菌素、uge、wab和ybtA 7種毒力基因。這表明同一菌株有好幾種不同的毒力基因。并對(duì)其中15株菌(K.pneumoniae-SD-1~K.pneumoniae-SD-13,K.pneumoniae-SD-15和K.pneumoniae-SD-21)的陽(yáng)性毒力基因進(jìn)行了克隆測(cè)序分析,且于GenBank中收錄的肺炎克雷伯氏菌菌株作為參考菌株進(jìn)行比較,制作了系統(tǒng)發(fā)生樹(shù)和同源性的分析。系統(tǒng)發(fā)生樹(shù)結(jié)果顯示:15株代表菌株中的14株陽(yáng)性rmpA基因與參考菌株TMV-5(分離于人的器官)的血緣關(guān)系很近;15株代表菌株中的14株陽(yáng)性菌素基因與參考菌株cg43(分離于人的血液)、ed23(分離于人的血液)都在一個(gè)分支上;15株代表菌株的陽(yáng)性wabg基因與參考菌株cg43(分離于人的血液)、ntuh-k2044(分離于人的肝臟和血液)都在一個(gè)分支上;15株代表菌株中的12株陽(yáng)性iucb基因與參考菌株rjf999(分離于人的血液)、t69(分離于人的痰液)親緣關(guān)系很近;15株代表菌株中的2株陽(yáng)性ybta基因與參考菌株ntuh-k2044(分離于人的肝臟和血液)親緣關(guān)系很近。同源性分析結(jié)果顯示:15株代表菌株中的14株陽(yáng)性rmpa基因與參考菌株ntuh-k2044、kctc2242、ed23和rjf293的同源性為99.1%-100%;15株代表菌株中的14株陽(yáng)性菌素基因與參考菌株atcc43816、cg43和ed23的同源性為98.2%-100%;15株代表菌株的陽(yáng)性wabg基因與參考菌株ntuh-k2044、kctc2242、ed23、cav1217和cav1042的同源性為98.4%-100%;15株代表菌株的陽(yáng)性u(píng)rea基因與參考菌株cg43、rjf293、cav1042和u41的同源性為98.2%-100%;15株代表菌株中的14株陽(yáng)性u(píng)ge基因與參考菌株kctc2242、rjf293、cav1453和kp52.145的同源性為97.8%-100%;15株代表菌株中的12株陽(yáng)性iucb基因與參考菌株rjf293和sb227的同源性為94.6-99.7%;15株代表菌株中的2株代表菌株的ybta基因與參考菌株ntuh-k2044、kp36、11和ed23的同源性99.3-99.7%。結(jié)果表明貂源肺炎克雷伯氏菌與人源肺炎克雷伯氏菌的親緣關(guān)系很近。3.致病性研究本研究根據(jù)分離菌株所攜帶的血清型、毒力基因的不同選擇3株菌株,建立貂源肺炎克雷伯氏菌對(duì)小鼠和水貂的致病性研究。分別給小鼠和水貂攻毒后,結(jié)果顯示,在2—8天均表現(xiàn)精神沉郁、食欲減退,結(jié)膜蒼白,呼吸淺速,站立不穩(wěn)且抽搐,被毛逆亂,體溫升高,偶爾咳嗽,鼻腔內(nèi)有分泌物,第4—7天死亡達(dá)到高峰,剖檢死亡小鼠和水貂發(fā)現(xiàn)腹腔內(nèi)均有明顯的黏液,且易拉絲,肝有壞死灶、發(fā)硬、發(fā)黑;肺腫大出血;病理組織學(xué)觀察,肺、肝,腦都有不同程度的出血,充血現(xiàn)象。ld50結(jié)果顯示,k.pneumoniae-sd-12和k.pneumoniae-sd-21菌株對(duì)小鼠的ld50分別為5.0×102cfu和3.2×100cfu;對(duì)水貂的ld50分別為1.6×103cfu和3.2×101cfu;然而k.pneumoniae-sd-15對(duì)小鼠和水貂都沒(méi)有致病性。由上述血清型和毒力基因檢測(cè)可知,k.pneumoniae-sd-12和k.pneumoniae-sd-21菌株均有莢膜血清型k2、菌素和rmpa毒力基因,而k.pneumoniae-sd-15不是血清型k2,且沒(méi)有菌素和rmpa毒力基因。本研究的結(jié)果證實(shí),貂源肺炎克雷伯氏菌含有莢膜血清型k2、菌素和rmpa毒力基因的菌株具有較高的致病性。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.61

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