Identification of avian pathogenic Escherichia coli genes required for carriage and virulence in poultry

大肠杆菌通常是动物和人类肠道中无害的居民。然而，一些大肠杆菌菌株具有使它们具有致病潜力的特性。致病性大肠杆菌是全世界腹泻病的主要病因，但某些菌株可引起肠道外疾病，如尿路感染、败血症和脑膜炎。使大肠杆菌能够穿过粘膜屏障并在全身部位持续存在的细菌因素尚不清楚。其中一组肠道外病原体包括禽致病性大肠杆菌（APEC），它会引起鸟类严重的全身性疾病。亚太经合组织是世界范围内家禽业的主要经济和福利问题。亚太经合组织感染所有年龄的鸟类，并引起大肠杆菌病，可导致突然死亡，蛋孵化减少和屠宰时尸体被谴责。虽然鸟类可能因病毒感染、压力和环境因素而易患大肠杆菌病，但人们普遍认为，以亚太经合组织为目标可以缓解这一问题。然而，影响APEC在家禽中携带和毒力的细菌因素尚不清楚。此外，从大肠杆菌病家禽中分离出的APEC通常是不相关的，并且在毒力强的分离株中发现很少存在保守性状。由于死亡前缺乏疾病迹象以及对新的抗生素耐药性的演变和传播的担忧，抗生素治疗变得复杂。虽然已经开发出针对亚太经合组织的疫苗，但这些疫苗的价值有限，因为它们只能保护鸟类免受其衍生毒株的侵害。迫切需要开发具有广泛交叉保护作用的亚太经合组织疫苗。我们相信，通过剖析APEC在家禽呼吸道定植并传播到全身部位的机制，我们可以确定可能适合纳入疫苗的细菌因子。为了使此类疫苗对所有亚太经合组织有效，有必要确认这些因素存在于各种各样的亚太经合组织分离株中。我们建议采用一种称为标记转座子诱变（STM）的技术来确定火鸡大肠杆菌病诱导所需的APEC因子。STM使得追踪独特标记的细菌突变体在与宿主相互作用过程中的命运成为可能。从接种的鸟类中恢复的突变体池的组成可以与接种物的组成进行比较，这样，突变体无法在鉴定的宿主中存活（第1部分）。图1)。在这种弱化的突变体中被破坏的因素可以被识别出来。我们已经成功地使用STM鉴定了家畜定植所需的沙门氏菌和大肠杆菌O157基因。根据从这些研究中收集到的信息，我们随后寻求开发控制这些病原体的疫苗。认识到拟议研究对家禽业的价值，英国家禽协会和Aviagen火鸡（原英国联合火鸡有限公司）已同意分别以现金和实物支持该项目。该项目的目的是：1。找出使亚太经合组织能在火鸡的呼吸道定居并传播到其他器官的因素。2. 为了证实这些因素存在于广泛的APEC分离株中。3. 为了确认这些因素在疾病中发挥重要作用，通过破坏它们并描述火鸡的突变菌株。然后将在若干实验室实验中评估丧失毒力的原因。4. 目的探讨纯化的APEC毒力因子是否能诱导对APEC感染的保护作用。

Escherichia coli is typically a harmless inhabitant of intestines of animals and humans. However, some E. coli strains possess traits that equip them with the potential to cause disease. Pathogenic E. coli are a major cause of diarrhoeal disease world-wide, but some strains can cause extra-intestinal disorders such as urinary tract infections, septicaemia and meningitis. The bacterial factors that enable E. coli to cross mucosal barriers and persist at systemic sites are poorly understood. One such group of extra-intestinal pathogens comprises avian pathogenic E. coli (APEC), which cause severe systemic disease in birds. APEC are a major economic and welfare problem for the poultry industry world-wide. APEC affect birds of all ages and cause colibacillosis which can result in sudden death, reduced egg hatching and condemnation of carcasses at slaughter. Whilst birds may be prediposed to colibacillosis by viral infection, stress and environmental factors, it is widely agreed that the problem could be alleviated by targeting APEC. However, the bacterial factors that influence the carriage and virulence of APEC in poultry are poorly understood. Furthermore, APEC isolated from poultry with colibacillosis are often unrelated and few conserved traits have been found to exist among virulent isolates. Antibiotic treatment is complicated by the lack of signs of disease prior to death and concern about the evolution and spread of new antibiotic resistances. Whilst vaccines against APEC have been developed, these are of limited value since they protect birds only against the strain from which they were derived. There is a pressing need to develop broadly cross-protective APEC vaccines. We believe that by dissecting the mechanisms by which APEC colonise the respiratory tract of poultry and spread to systemic sites we can identify bacterial factors that may be suitable for inclusion into a vaccine. For such vaccines to be effective against all APEC, it will be necessary to confirm that the factors are present in a wide variety of field APEC isolates. We propose to employ a technique called signature-tagged transposon mutagenesis (STM) to identify APEC factors required for the induction of colibacillosis in turkeys. STM makes it possible to track the fate of uniquely tagged bacterial mutants during their interaction with the host. The composition of a pool of mutants recovered from inoculated birds can be compared to the composition of the inoculum and in this way, mutants unable to survive in the host identified (Part 1. Fig. 1). The factors disrupted in such weakened mutants can then be identified. We have successfully used STM to identify genes of Salmonella and E. coli O157 required for colonisation of farm animals. With the information gleaned from such studies, we have then sought to develop vaccines for the control of these agents. In recognition of the value of the proposed studies to the poultry industry the British Poultry Council and Aviagen Turkeys (formerly British United Turkeys Ltd) have agreed to support the project in cash and in kind, respectively. The aims of the project are: 1. To identify factors that enable APEC to colonise the respiratory tract of turkeys and spread to other organs. 2. To confirm that such factors are found in a wide variety of field APEC isolates. 3. To confirm that such factors play an important role in disease by disrupting them and characterising the mutant strains in turkeys. The reasons for the loss of virulence will then be assessed in a number of laboratory-based experiments. 4. To determine whether purified APEC virulence factors can induce protection against APEC infection.