两株鹅源鸭疫里默氏杆菌的分离鉴定及生物学特性研究

发布时间：2018-04-12 15:18

本文选题：鸭疫里默氏杆菌 + 分离　；参考：《安徽农业大学》2015年硕士论文

【摘要】：鸭疫里默氏杆菌病又称鸭传染性浆膜炎(Duck infectious serositis),是由鸭疫里默氏杆菌(Riemerella anatipestifer,RA)引起的一种主要侵害鸭的细菌性传染病,该病发病率、死亡率都很高,严重影响全球养鸭业的经济效益。2013年7月和2014年4月安徽省内两鹅养殖场先后爆发雏鹅急性死亡的病例,患病雏鹅出现拉白色或绿色拉稀、脚掌发黄无血色、头颈扭曲震颤、共济失调等症状,甚至有部分病鹅未表现症状就急性死亡。对病死雏鹅剖检可见心脏、肝脏覆盖纤维素性渗出物,脑膜严重出血。根据临床的表现和剖检所见病理变化初步怀疑为鸭疫里默氏杆菌病。从患病雏鹅的脑部取样进行细菌分离,获得的单个菌落直径约1mm,凸起,湿润,光滑,呈露珠状,镜检为革兰氏染色阴性短小杆菌。通过使用PCR方法扩增分离菌株的16S rRNA后进行测序,序列比对结果显示,两株分离菌株与鸭疫里默氏杆菌同源性为99%,证实引起这两起雏鹅急性死亡疫病的病原菌为RA,分别命名为LAG1株和HFG1株。两株RA分离株的生化特性试验结果一致,二者均为触酶试验结果和脲酶试验结果阳性,不还原硝酸盐,不能液化明胶,多种糖微量反应试验、醇酵解试验、V-P试验、MR试验、吲哚试验结果均为阴性。通过血清凝集试验鉴定这两株鹅源RA均为血清15型。根据RA ompA基因序列的保守区设计引物,建立检测RA的PCR方法,敏感性试验表明所建立的PCR检测方法可检出213ng的RA的DNA。对抗生素的耐药性检测结果表明LAG1株和HFG1株RA对β-内酰胺类药物(d18mm),四环素类药物(d16mm),大环内酯类药物高度敏感(d23mm);对氯霉素中度敏感(17mmd13mm);对氨基糖苷类药物耐药(d11mm)。LAG1和HFG1对不良环境的抵抗力略有不同,在酸性条件下HFG1的耐受能力较强,而在碱性条件下LAG1的耐受力较强;在抵抗氧化剂方面H2O2对LAG1和HFG1的最小杀菌浓度(MBC)分别为40mM和20mM;在抵抗洗涤剂TritonX-100对LAG1和HFG1的最小杀菌浓度(MBC)均为0.004%。采用10倍倍比稀释的方法将菌液计数并稀释到含菌量为109、108、107、106、105、104和103CFU/mL的7个稀释度,采用腿部肌肉注射的方法攻毒7日龄樱桃谷鸭,测定LAG1和HFG1的半数致死量(LD50),结果显示:LAG1株的LD50为7.75×103CFU;HFG1株的LD50为5.91×105CFU。将这两株RA分别制备油乳剂灭活苗免疫樱桃谷鸭测定其对血清1型WJ4、血清2型Yb2、血清10型HXb2及原菌株的免疫保护效率,结果显示LAG1株制备的灭活油乳剂疫苗在二次免疫14d后,对原菌株和10型HXb2的攻击可以提供较高的保护,保护率分别可以达到85.7%(6/7)和71.43%(5/7),对血清1型WJ4、血清2型Yb2攻击的保护率很低均为14.2%(1/7);。使用HFG1株所制备的灭活油乳剂疫苗对原菌株具有很好的保护率为85.7%(6/7),对血清10型HXb2的保护力欠佳,为和57.14%(4/7)。对血清1型WJ4无保护力,对血清2型Yb2的保护率仅为14.2%(1/7)。结果对RA疫苗的研制提供一定的指导意义。
[Abstract]:Riemer's disease, also known as Duck infectious serositis, is a major bacterial infectious disease which is caused by Riemerella anatipestifera. The incidence and mortality of the disease are very high.In July 2013 and April 2014, the two goose farms in Anhui Province have experienced acute goose deaths. The sick chicks appear to be white or green, their feet are yellow and bloodless, and their heads and necks are twisted and tremors.Ataxia and other symptoms, and even some sick geese did not show symptoms on the acute death.The heart and liver were covered with cellulose exudate and meningeal hemorrhage was observed in dead goose.According to the clinical manifestation and the pathological changes of autopsy, it was suspected to be Riemer's disease of duck.The isolated bacteria were isolated from the brain of the infected goose. The single colony was about 1 mm in diameter, protruding, moist, smooth and dewdrop.The 16s rRNA of the isolated strain was amplified by PCR and sequenced.The homology between the two isolates and Riemer Duck strain was 99. It was confirmed that the pathogen causing the acute death blight of Gosling was RAA, named LAG1 strain and HFG1 strain, respectively.The biochemical characteristics of the two RA isolates were identical. Both of them were positive results of thixozyme test and urease test, no reduction of nitrate, no liquefaction of gelatin, multi-sugar micro-reaction test, alcohol fermentation test and V-P test.The results of indole test were negative.The seroagglutination test identified the two geese RA as serotype 15.According to the conserved region of RA ompA gene primer was designed to establish a PCR method for detecting RA. The sensitivity test showed that the established PCR detection method could detect 213ng RA.Resistance to adverse environments is slightly different,The tolerance of HFG1 in acidic condition was stronger than that of LAG1 in alkaline condition.The minimum bactericidal concentration of H2O2 to LAG1 and HFG1 were 40mM and 20mM, respectively, and the minimum bactericidal concentration of TritonX-100 to LAG1 and HFG1 were 0.004.The bacterial fluid was counted and diluted to 7 dilutions containing 109108107106105104 and 103CFU/mL by 10 times dilution, and the 7 day old Cherry Valley duck was poisoned by leg intramuscular injection.Half lethal dose (LD50) of LAG1 and HFG1 were determined. The results showed that the LD50 of the strain was 7.75 脳 10 ~ 3 CFU / F _ 1 and the LD50 of the strain was 5.91 脳 10 ~ 5 CFU.Cherry Valley Ducks were immunized with oil emulsion inactivated vaccine prepared from these two strains of RA, respectively. The immune protection efficiency of these two strains against serotype 1 WJ4, serotype 2 Yb2, serotype 10 HXb2 and original strain were determined. The results showed that the inactivated oil emulsion vaccine prepared by LAG1 strain was immunized for 14 days after secondary immunization.The protective rates of the original strain and type 10 HXb2 were 85.7% and 71.43%, respectively. The protective rates of WJ4 and type 2 Yb2 were 14.2 / 7 / 7 respectively.The inactivated oil emulsion vaccine prepared with HFG1 strain had a good protective rate of 85.7% to the original strain, and had poor protection to serotype 10 HXb2 (57.14% / 7%).There was no protective effect on serum type 1 WJ4, but the protective rate of serum type 2 Yb2 was only 14.2g / 7.The results provide some guidance for the development of RA vaccine.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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