空泡形成毒素的生物学特性分析及078血清型禽大肠杆菌病疫苗的研制

发布时间：2018-06-05 22:57

本文选题：禽致病性大肠杆菌 + 空泡转运毒素　；参考：《扬州大学》2015年硕士论文

【摘要】：禽致病性大肠杆菌(Avian pathogenic E. coli,APEC)可以导致鸡、火鸡及其他禽类发生肠道外疾病,如心包炎、肝周炎、脑膜炎等,甚至引起败血症从而急性死亡。7-30日龄雏禽易感,成年家禽亦可发病。该病发病率高(3.8%-42.6%)、死亡率高(40%-60%)；极少数恶劣的环境可导致极高的死亡率(90.3%),我国禽类养殖业饱受其害。最近有研究表明,APEC亦可引起人类及哺乳动物发病,甚至死亡。目前多数养殖厂主要通过添加大量抗生素防控该病,然而由于半个多世纪广泛应用抗生素,APEC已经演化出多种血清型的耐药性菌株,这使得我们承受着巨大的压力。本研究根据NCBI公布的序列,分别设计了18对、14对引物用以检测毒力基因及耐药基因的分布情况；同时对菌株的药物敏感性、生物被膜形成能力、致病性及血清型分布进行检测。毒力基因检测结果显示,分离株的fimC、mat、ibeB、yijp及ompA五种毒力基因的检出率分别为98%、96%、96%、90%、90%为APEC的保守性基因,vat和papC检出率仅为32%和10%。耐药基因检测结果显示,氨基糖苷类、磺胺类、喹诺酮类、四环素类抗生素的抗性基因strA、strB、dhps、sul Ⅱ及parC检出率分别为98%、98%、100%、98%及100%,β-内酰胺类抗性基因oxa1、oxa5及oxa31检出率分别为0%、0%及4%。药敏试验结果显示,所有APEC对四环素、克林霉素及利福平抗生素100%耐受,对头孢菌素类的菌必治耐药性最低仅为16%,对酰胺类的氯霉素耐受率其次为34%；大环内酯类的红霉素及阿奇霉素耐药率分别为90%、82%,喹若酮类的氟哌酸抗生素耐药率为82%,头孢菌素类的头孢噻分耐药率为84%。APEC分离株的多重耐药比较严重,66%分离株同时耐受10种抗生素,4% APEC分离株耐受本次检测的所有类型抗生素,表明APEC广泛耐药且存在交叉耐药现象。生物被膜形成能力检测结果显示,APEC分离株的生物被膜形成能力均较弱。血清型分型结果显示,078血清型为主要血清型,该群对10日龄樱桃谷鸭致病性差异较大；APEC的致病机理在于APEC首先利用黏附相关因子与宿主细胞表面特异性受体结合从而定殖于特定宿主细胞表面,然后通过侵袭相关毒力因子侵袭宿主细胞,继而进入血液循环系统扩散至各脏器,并在多种毒力因子的共同参与下导致宿主发病。空泡转运毒素(Vacuolating autotransporter toxin, Vat)是APEC的重要毒力因子之一,该蛋白属于V型分泌系统(T5SS),其可通过其自身的特异性结构直接分泌到膜外。本研究通过采用Red同源重组的方法构建vat缺失株,比较野生株、vat缺失株与互补株在生长特性、凝集沉淀能力、运动性、生物被膜形成能力、致病性等生物学特性的差异。结果表明,该基因能够抑制APEC泳动能力以及凝集能力,对生物被膜形成、体内存活、致病性具有促进作用,但不影响生长速度、环境耐受力以及黏附入侵能力。防控禽大肠杆菌病始终坚持防重于治,因此开发高效、安全的疫苗迫在眉睫。国内研究表明APEC血清型众多,因此开发各地区的主要血清型的多价苗是一个不错的选择。我们先前的研究表明江苏、安徽周边地区流行的078血清型约占64%,为主要血清型,其次为02、01血清型,因此对078血清型APEC的灭活苗进行研究。结果表明,选取的078血清型菌株APCE94制备的油乳剂灭活苗对同源血清型强毒株具有很好的保护性,该菌株制备的疫苗以5×108CFU/只免疫雏鸭,仅首免即可提供至少90%的保护率,首免14 d血清效价达到1：400,二免100%保护雏鸭免受078血清型强毒株的感染,二免10d血清效价达到1：1600,但该疫苗对血清01、02型强毒菌株不能提供交叉保护作用。
[Abstract]:Avian pathogenic Escherichia coli (Avian pathogenic E. coli, APEC) can cause intestinal diseases in chickens, turkeys and other birds, such as pericarditis, liver pericarditis, meningitis, and even septicemia, causing acute death of.7-30 days old fowl and adult poultry. The incidence of the disease is high (3.8%-42.6%), high death rate (40%-60%); very few (40%-60%); very few. A number of harsh environments can lead to extremely high mortality (90.3%). China's poultry breeding industry is suffering. Recent studies have shown that APEC can also cause human and mammal disease, even death. At present, most farms mainly use a large number of antibiotics to prevent and control the disease. However, antibiotics are widely used in more than half a century, and APEC has evolved. According to the sequence published by NCBI, 18 pairs were designed and 14 pairs of primers were used to detect the distribution of virulence genes and drug resistance genes, and the drug sensitivity, biofilm ability, pathogenicity and serotype distribution of the strains were detected. The detection of virulence gene showed that the detection rates of five virulence genes of fimC, mat, ibeB, yijp and ompA were 98%, 96%, 96%, 90% and 90%, respectively. The detection rates of VAT and papC were only 32% and 10%. resistance genes, and the resistance genes of aminoglycosides, sulfonamides, quinolones and tetracycline antibiotics, strA, were detected. The detection rates of strB, DHPs, Sul II and parC were 98%, 98%, 100%, 98% and 100% respectively. The beta lactam resistance gene Oxa1, oxa5 and oxa31 detection rates were 0%, 0% and 4%. drug sensitivity tests showed that all APEC tolerance to tetracycline, clindamycin and rifampicin was 100%, and the drug resistance of cephalosporins was only 16%, against amides. The tolerance rate of chloramphenicol was 34%, the resistance rate of erythromycin and azithromycin was 90%, 82%, and 82% of quinolones, and the cephalosporin resistance rate of cephalosporins was more severe in 84%.APEC isolates. 10 antibiotics were tolerated in 66% isolates and 4% APEC isolates were resistant. All types of antibiotics tested by this test showed that APEC was extensively drug-resistant and had cross resistance. The biological membrane formation ability test showed that the biofilm formation ability of APEC isolates was weak. Serotype results showed that 078 serotype was the main serotype, and the group had greater pathogenicity to 10 days old Cherry Valley ducks; APEC The pathogenic mechanism is that APEC first colonized the surface of a specific host cell by binding the adhesion related factors to the surface specific receptor of the host cell, and then invaded the host cell by invading the related virulence factors and then entered the blood circulation system and spread to the organs, and led to the host's hair under the joint involvement of various virulence factors. Vacuolating autotransporter toxin (Vat) is one of the important virulence factors of APEC. The protein belongs to the V secretory system (T5SS), which can be secreted directly to the membrane through its own specific structure. This study constructs a vat deletion strain by using the homologous recombination method of Red to compare the wild strain, VAT deletion and complementation. The results show that the gene can inhibit the APEC swimming ability and agglutination ability, which can promote the formation, survival and pathogenicity of the biofilm, but does not affect the growth rate, environmental tolerance and adhesion. Invasion prevention and control of avian colibacillosis has always persisted in the prevention of weight and the development of efficient and safe vaccines is imminent. Domestic studies have shown that there are many serotypes of APEC. Therefore, it is a good choice to develop the major serotype polyvalent vaccine in various regions. Our previous study showed that the 078 serotypes prevalent in the surrounding areas of Jiangsu and Anhui accounted for about 078 serotypes. 64%, for the main serotype, followed by the 02,01 serotype, the inactivated vaccine of the 078 serotype APEC was studied. The results showed that the selected 078 serotype strain APCE94 prepared by the oil emulsion inactivated vaccine had a good protective effect on the homologous serotype strain. The vaccine prepared by this strain was 5 * 108CFU/ only immunized ducks, only the first exemption could be provided to the vaccine. Less than 90% of the protection rate, the first free 14 d serum titer reached 1:400, two free 100% protected ducks from 078 serotype virulent strains, and two 10d serum titer reached 1:1600, but the vaccine could not provide cross protection to the serotype 01,02 virulent strain.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61;S859.797

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