林麝肺源致病性大肠杆菌的分离鉴定及毒力基因的检测

发布时间：2018-05-09 16:19

本文选题：林麝 + 大肠杆菌　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：林麝是国家一级保护动物,麝香具有较高的药用价值。为保护和发展林麝资源,增加麝香产量,我国自1958年开始进行人工养殖林麝的研究,但是由于各种疾病和环境等因素的影响,养殖规模一直受到限制。尤其是大肠杆菌导致的林麝肺炎发病率及死亡率均较高,为林麝养殖业造成了很大的危害。2016年2月陕西宝鸡某林麝养殖基地人工繁殖林麝突然发病,自行用青霉素、链霉素等药物治疗后效果不明显并且出现死亡病例。对死亡林麝剖检观察,发现主要病变在肺脏。为确定导致林麝发病的病原菌,从死亡林麝肺脏进行细菌分离,对分离所得细菌进行生化特性、耐药性、致病性、16SrRNA序列分析以及毒力基因的检测。主要获得以下结果:1.从林麝的病变组织中分离到2株细菌,菌株1和菌株2。在血平板上菌株1为表面光滑,半透明,直径3mm左右的灰白色菌落,镜检呈革兰氏阴性短杆菌,两端钝圆,成对或散在;菌株2为表面光滑,边缘整齐,半透明,直径1mm左右的灰白色菌落,镜检呈革兰氏阴性杆菌,聚集成堆。2.经糖发酵试验(葡萄糖、麦芽糖、乳糖、阿拉伯糖)、M.R试验、V-P试验、靛基质试验、柠檬酸盐利用试验共5项生化试验鉴定,根据《伯杰氏细菌鉴定手册》判断标准,可以确定分离所得菌株1和菌株2均为大肠杆菌。3.对分离所得菌株进行16SrRNA序列测定,并与标准参考菌株进行序列比对分析。结果表明,两株细菌的核苷酸序列与大肠杆菌核苷酸的同源性为100%,菌株1与Escherichia coli O104:H4序列相似性较高、菌株2与Escherichia coli O78序列相似性较高。由此可将菌株1和菌株2确定为大肠杆菌。4.用药敏试验检测了两株分离菌对抗生素的耐药性,发现头孢类(头孢唑啉、头孢曲松钠)和单环β-内酰胺类(氨曲南)对2株林麝肺部致病性大肠杆菌均敏感;氨基糖苷类(链霉素)对其均中度敏感;对其他类别抗生素均呈现不同程度的耐药性。将治疗药物由青霉素、链霉素改成头孢曲松钠和氨曲南后,效果较好。5.致病性试验结果显示菌株1和菌株2均有较强的毒性,腹腔注射72 h之后,试验组小白鼠死亡率为75%。死亡小白鼠肺脏中可以分别分离到菌株1和菌株2。6.毒力基因检测试验中,两株分离菌株均检出了ompA基因,其他的粘附素类(afa/draB、iha、sfa/focCD)、铁转运系统类(sitD ep.a、sitD chr.、iucD.a)、血清类(neuC)和毒素类(sat、vat)9种毒力基因均未检出。综上所述,本研究中的病例是由于大肠杆菌引起的林麝肺炎,且分离出的大肠杆菌具有很强的耐药性和致病性,经药敏试验结果指导治疗及以分离菌株为抗原制成疫苗免疫后病情得到控制。
[Abstract]:Forest musk deer is a national first-class protected animal, musk has high medicinal value. In order to protect and develop the resources of forest musk deer and increase the production of musk musk, the study on artificial cultivation of forest musk deer has been carried out in China since 1958. However, due to the influence of various diseases and environmental factors, the scale of cultivation has been restricted. In particular, the incidence and mortality of pneumonia in forest musk deer caused by Escherichia coli were high, which caused great harm to the forest musk deer breeding industry. In February 2016, the artificially propagated forest musk deer in a forest musk deer breeding base in Baoji, Shaanxi Province, suddenly developed a disease and used penicillin on their own. The effect of streptomycin and other drugs after treatment is not obvious and there are death cases. The main pathological changes of the dead forest musk deer were found in the lungs. In order to identify the pathogenic bacteria causing the disease of the forest musk deer, the bacteria were isolated from the lungs of the dead forest musk deer. The biochemistry characteristics, drug resistance, pathogenicity of 16s rRNA sequence and virulence gene were analyzed. The main results are as follows: 1. Two strains of bacteria, strain 1 and strain 2. On the blood plate, strain 1 was a grayish white colony with a smooth surface, translucent and 3mm diameter. Microscopic examination showed Gram-negative short bacilli with obtuse circles at both ends in pairs or scattered, and strain 2 had a smooth surface, neat edges and translucent surfaces. The grayish white colonies about the diameter of 1mm showed Gram-negative bacilli by microscope and gathered in piles of. 2. 2. The sugar fermentation tests (glucose, maltose, lactose, arabinosaccharide) M.R test V-P test, indigo matrix test, citrate utilization test were identified by 5 biochemical tests. It can be confirmed that both strain 1 and strain 2 are Escherichia coli. The isolated strains were sequenced by 16SrRNA and compared with standard reference strains. The results showed that the nucleotide sequence of the two strains was 100% homology with that of Escherichia coli, the similarity between strain 1 and Escherichia coli O104:H4 sequence was high, and the similarity between strain 2 and Escherichia coli O78 sequence was higher. Therefore, strain 1 and strain 2 can be identified as Escherichia coli. The antibiotic resistance of two isolates to antibiotics was tested by drug sensitivity test. It was found that both cefazolin and ceftriaxone sodium and monocyclic 尾-lactam (aztreonam) were sensitive to two strains of pathogenic Escherichia coli in the lung of musk deer forest. The aminoglycosides (streptomycin) were moderately sensitive to them and showed resistance to other antibiotics to varying degrees. After changing the therapeutic drugs from penicillin, streptomycin to ceftriaxone sodium and aztreonam, the effect was better. 5. The pathogenicity test showed that both strain 1 and strain 2 had strong toxicity. After 72 hours of intraperitoneal injection, the mortality of mice in the experimental group was 75. Strain 1 and strain 2.6 could be isolated from the lungs of dead mice respectively. In virulence gene test, ompA gene was detected in two isolated strains, but 9 virulence genes were not detected in other adhesin class, such as afa / drab Bhaha / fa / foci, iron transport system, sitD ep.asitD chr. neuc, serum neuC) and toxin satvatanus. To sum up, the case in this study is caused by the forest musk deer pneumonia caused by Escherichia coli, and the isolated Escherichia coli has strong resistance and pathogenicity. The treatment was guided by the results of drug sensitivity test and the disease was controlled after the vaccine was immunized with the isolated strain as antigen.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.94

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