貂源绿脓杆菌的分离鉴定及其致病性研究
发布时间：2018-09-12 18:05
【摘要】：随着经济的发展,近年来,山东省毛皮动物养殖业发展迅速,目前已成为我国毛皮动物养殖第一大省,毛皮动物饲养量约占全国的二分之一。但是随着我省毛皮动物养殖业集约化程度的提高,毛皮动物的发病率持续上升,疫病防控形势日趋严峻,疫病成为制约毛皮动物产业发展的重要因素,给毛皮动物养殖业造成了很大的经济损失。水貂出血性肺炎是严重妨碍水貂养殖业的重要疫病之一,且疫情日趋严重。绿脓杆菌(Pseudomonas aeruginosa,P.aeruginosa)又称铜绿假单胞菌,是引起水貂出血性肺炎的病原之一,为了有效的防控水貂出血性肺炎,应揭示阐明绿脓杆菌对水貂的致病性。本研究采用常规细菌分离鉴定技术,对43份患出血性肺炎的水貂肺组织进行了绿脓杆菌的分离鉴定,同时选择绿脓杆菌代表菌株,建立绿脓杆菌致病性研究动物模型,来阐明绿脓杆菌的致病性。1.绿脓杆菌的分离鉴定2013年,在山东省诸城、文登、临沂等地采集了43份患出血性肺炎的水貂肺组织。通过常规细菌分离技术对这43份病料进行了细菌分离培养,并采用形态学观察、生化试验、药敏试验与16SrDNA测序等方法,对分离到的细菌进行鉴定。结果,分离到的5株细菌,均为绿脓杆菌,分别命名为F1、F5、F6、F8、F10,分离率为11.6%。药敏试验结果表明,5株绿脓杆菌均对青霉素、红霉素、卡那霉素耐药。16SrDNA序列同源性分析结果表明,5株绿脓杆菌与14株参考菌株间的核苷酸同源性为80.9%~100%,5株绿脓杆菌之间的核苷酸同源性为98.9%~99.7%;系统发育分析显示,F5、F6、F8、F10与F1均在同一个大的分支上。2.貂源绿脓杆菌的致病性研究首先以F1株作为代表菌株,对小鼠进行腹腔接种,建立绿脓杆菌对小鼠的致病性研究动物模型。将30只健康小鼠随机分成6组,隔离饲养,每组5只。第1-5组小鼠分别腹腔接种菌落数为6.4×107CFU、6.4×106CFU、6.4×105CFU、6.4×104CFU、6.4×103CFU的F1菌液,第6组小鼠作为阴性对照。攻毒后对实验小鼠连续观察7天,记录小鼠的临床症状,对在实验期间死亡的小鼠进行剖检,采集心、肝、脾、肺、肾进行细菌的分布检测及病理组织学检查。结果,第1、2组小鼠在接种后12h-18h内全部死亡,小鼠的心、肝、脾、肺、肾均有不同程度地出血,尤以肺部病变更为严重;病理组织学检查可见小鼠心肌出血、肝细胞坏死、脾脏有大量炎性细胞、肺脏出血、肺泡壁增厚、肾间质有红细胞;在上述器官中均分离到了绿脓杆菌,在小鼠肺部含量最高,脾脏中含量最低;测得F1对小鼠的LD50为1.6×106CFU/mL。小鼠致病性试验结果表明绿脓杆菌对小鼠具有较强的致病力。以F1株作为代表菌株,对水貂进行鼻腔接种,建立绿脓杆菌对水貂的致病性研究动物模型。将20只健康3月龄水貂分成5组,隔离饲养,每组4只。第1-4组分别鼻腔接种菌落数为1.6×108CFU、1.6×106CFU、1.6×104CFU、1.6×102CFU的F1菌液,第5组水貂为阴性对照,攻毒后连续观察14天,对实验期间死亡的水貂和实验结束时扑杀的所有水貂进行剖检,采集其心、肝、脾、肺、肾进行病理组织学检查及细菌分离。结果,第1组水貂在人工感染后20h-44h内全部死亡,剖检可见死亡水貂的肺部严重出血、脾脏肿大,在其心、肝、脾、肺、肾中均分离到了绿脓杆菌。病理组织学检查可见心肌纤维出血、肝细胞变性、脾脏出血坏死、肺出血、间质增厚。测得F1对水貂的LD50为3.2×107CFU/mL;其他人工感染组在接种后的第1-3d表现精神不振、食欲下降等临床症状,但自攻毒后第4d恢复正常,直至实验结束,病理组织学观察未见异常,也均未从相关脏器中分离到绿脓杆菌。实验结果表明,绿脓杆菌能够感染水貂并引起发病,但致病性不强,应该还有其他引起水貂出血性肺炎的病因。本研究阐明了绿脓杆菌对水貂的致病性,为水貂出血性肺炎的防治提供了实验数据,奠定了理论基础。
[Abstract]:With the development of economy, the fur animal breeding industry in Shandong Province has developed rapidly in recent years, and now it has become the largest fur animal breeding Province in China. The amount of fur animal breeding accounts for about half of the whole country. The epidemic is becoming more and more serious. Pseudomonas aeruginosa (P. aeruginosa), also known as Pseudomonas aeruginosa, is one of the important epidemic diseases that seriously hinder mink breeding. In order to prevent and control mink haemorrhagic pneumonia effectively, the pathogenicity of Pseudomonas aeruginosa should be clarified. In this study, 43 mink lung tissues with haemorrhagic pneumonia were isolated and identified by routine bacterial isolation and identification techniques, and the representative strains of Pseudomonas aeruginosa were selected. Pseudomonas aeruginosa pathogenicity animal model was established to clarify the pathogenicity of Pseudomonas aeruginosa. 1. Isolation and identification of Pseudomonas aeruginosa in 2013, 43 mink lung tissues suffering from hemorrhagic pneumonia were collected in Zhucheng, Wendeng, Linyi, Shandong Province. Bacterial isolation and culture of these 43 samples were carried out by routine bacterial isolation technology, and morphological observation was adopted. Results: Five strains of Pseudomonas aeruginosa were isolated, named F1, F5, F6, F8, F10, and the isolation rate was 11.6%. The results of drug susceptibility test showed that five strains of Pseudomonas aeruginosa were resistant to penicillin, erythromycin and kanamycin. The results showed that the nucleotide homology between 5 Pseudomonas aeruginosa strains and 14 reference strains was 80.9%-100%, and the nucleotide homology between 5 Pseudomonas aeruginosa strains was 98.9%-99.7%. Phylogenetic analysis showed that F5, F6, F8, F10 and F1 were all in the same large branch. Thirty healthy mice were randomly divided into 6 groups and fed in isolation with 5 mice in each group. The number of colonies inoculated into the abdominal cavity of mice in groups 1-5 was 6.4 x 107 CFU, 6.4 x 106 CFU, 6.4 x 105 CFU, 6.4 x 104 CFU, 6.4 x 103 CFU. The F1 bacterial solution of mice in group 6 was used as negative control. The clinical symptoms of the mice were observed continuously for 7 days. The heart, liver, spleen, lungs and kidneys of the dead mice were dissected and the distribution of bacteria and pathological histology were examined. The pathological examination showed that myocardial hemorrhage, hepatocyte necrosis, a large number of inflammatory cells in the spleen, pulmonary hemorrhage, alveolar wall thickening, renal interstitial red blood cells; Pseudomonas aeruginosa was isolated from the above organs, the highest content in the lungs of mice, the lowest content in the spleen; the LD50 of F1 in mice was 1.6 *10. The pathogenicity of Pseudomonas aeruginosa in 6 CFU/mL. mice showed that Pseudomonas aeruginosa had strong pathogenicity to mice. The pathogenicity of Pseudomonas aeruginosa to minks was studied by inoculating F1 strain into nasal cavity. Twenty healthy minks aged 3 months were divided into 5 groups and fed in isolation with 4 in each group. The F1 bacterium solution of 1.6 *108 CFU, 1.6 *106 CFU, 1.6 *104 CFU, 1.6 *102 CFU was negative control. The minks in the fifth group were observed continuously for 14 days after poisoning. The minks died during the experiment and all minks killed at the end of the experiment were dissected and their hearts, livers, spleens, lungs and kidneys were collected for pathological examination and bacterial isolation. Pseudomonas aeruginosa was isolated from the heart, liver, spleen, lung and kidney of dead minks. Myocardial fibrous hemorrhage, hepatocyte degeneration, splenic hemorrhage and necrosis, pulmonary hemorrhage and interstitial thickening were observed by histopathology. The LD50 of F1 was 3.2 *107 CFU/mL. Pseudomonas aeruginosa could infect mink and cause disease, but it was pathogenic. This study clarifies the pathogenicity of Pseudomonas aeruginosa to minks and provides experimental data for the prevention and treatment of mink hemorrhagic pneumonia.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92
本文编号：2239818
[Abstract]:With the development of economy, the fur animal breeding industry in Shandong Province has developed rapidly in recent years, and now it has become the largest fur animal breeding Province in China. The amount of fur animal breeding accounts for about half of the whole country. The epidemic is becoming more and more serious. Pseudomonas aeruginosa (P. aeruginosa), also known as Pseudomonas aeruginosa, is one of the important epidemic diseases that seriously hinder mink breeding. In order to prevent and control mink haemorrhagic pneumonia effectively, the pathogenicity of Pseudomonas aeruginosa should be clarified. In this study, 43 mink lung tissues with haemorrhagic pneumonia were isolated and identified by routine bacterial isolation and identification techniques, and the representative strains of Pseudomonas aeruginosa were selected. Pseudomonas aeruginosa pathogenicity animal model was established to clarify the pathogenicity of Pseudomonas aeruginosa. 1. Isolation and identification of Pseudomonas aeruginosa in 2013, 43 mink lung tissues suffering from hemorrhagic pneumonia were collected in Zhucheng, Wendeng, Linyi, Shandong Province. Bacterial isolation and culture of these 43 samples were carried out by routine bacterial isolation technology, and morphological observation was adopted. Results: Five strains of Pseudomonas aeruginosa were isolated, named F1, F5, F6, F8, F10, and the isolation rate was 11.6%. The results of drug susceptibility test showed that five strains of Pseudomonas aeruginosa were resistant to penicillin, erythromycin and kanamycin. The results showed that the nucleotide homology between 5 Pseudomonas aeruginosa strains and 14 reference strains was 80.9%-100%, and the nucleotide homology between 5 Pseudomonas aeruginosa strains was 98.9%-99.7%. Phylogenetic analysis showed that F5, F6, F8, F10 and F1 were all in the same large branch. Thirty healthy mice were randomly divided into 6 groups and fed in isolation with 5 mice in each group. The number of colonies inoculated into the abdominal cavity of mice in groups 1-5 was 6.4 x 107 CFU, 6.4 x 106 CFU, 6.4 x 105 CFU, 6.4 x 104 CFU, 6.4 x 103 CFU. The F1 bacterial solution of mice in group 6 was used as negative control. The clinical symptoms of the mice were observed continuously for 7 days. The heart, liver, spleen, lungs and kidneys of the dead mice were dissected and the distribution of bacteria and pathological histology were examined. The pathological examination showed that myocardial hemorrhage, hepatocyte necrosis, a large number of inflammatory cells in the spleen, pulmonary hemorrhage, alveolar wall thickening, renal interstitial red blood cells; Pseudomonas aeruginosa was isolated from the above organs, the highest content in the lungs of mice, the lowest content in the spleen; the LD50 of F1 in mice was 1.6 *10. The pathogenicity of Pseudomonas aeruginosa in 6 CFU/mL. mice showed that Pseudomonas aeruginosa had strong pathogenicity to mice. The pathogenicity of Pseudomonas aeruginosa to minks was studied by inoculating F1 strain into nasal cavity. Twenty healthy minks aged 3 months were divided into 5 groups and fed in isolation with 4 in each group. The F1 bacterium solution of 1.6 *108 CFU, 1.6 *106 CFU, 1.6 *104 CFU, 1.6 *102 CFU was negative control. The minks in the fifth group were observed continuously for 14 days after poisoning. The minks died during the experiment and all minks killed at the end of the experiment were dissected and their hearts, livers, spleens, lungs and kidneys were collected for pathological examination and bacterial isolation. Pseudomonas aeruginosa was isolated from the heart, liver, spleen, lung and kidney of dead minks. Myocardial fibrous hemorrhage, hepatocyte degeneration, splenic hemorrhage and necrosis, pulmonary hemorrhage and interstitial thickening were observed by histopathology. The LD50 of F1 was 3.2 *107 CFU/mL. Pseudomonas aeruginosa could infect mink and cause disease, but it was pathogenic. This study clarifies the pathogenicity of Pseudomonas aeruginosa to minks and provides experimental data for the prevention and treatment of mink hemorrhagic pneumonia.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92
【参考文献】
相关期刊论文 前7条
1 牛钟相,李雅林;动物绿脓杆菌病研究进展[J];动物医学进展;2003年01期
2 李绍红;;某三级中医医院铜绿假单胞菌的耐药性[J];中国感染控制杂志;2011年03期
3 潘■生,贺天笙,孟令新,房晓荣,郑丽敏;水貂假单胞菌脂多糖菌苗研制初报[J];毛皮动物饲养;1984年01期
4 张道永,胡景韶,王文贵,林毅;绿脓杆菌研究进展[J];四川畜牧兽医;1995年03期
5 蔡正求,宋未,东秀珠;细菌种特异性的16SrDNA寡核苷酸探针数据库的初步构建[J];首都师范大学学报(自然科学版);2004年S1期
6 卢东升,杨学海,卢红光;防治绿脓杆菌感染研究进展(续)[J];生物技术;1992年06期
7 崔红英,王健诚,吴秀友;水貂出血性肺炎的诊治[J];中国兽医杂志;2002年07期
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