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的无害居民。然而，一些E.大肠杆菌菌株具有使它们具有致病潜力的特性。致病性大肠大肠杆菌是全球性肠道疾病的主要致病菌，但有些菌株可引起肠道外疾病，如尿路感染、败血症和脑膜炎。使E.对大肠杆菌如何穿过粘膜屏障并在全身部位持续存在还知之甚少。一组这样的肠外病原体包括禽致病性大肠杆菌。大肠杆菌（APEC），导致鸟类严重的全身性疾病。亚太经合组织是世界家禽业的一个主要经济和福利问题。亚太经合组织影响所有年龄的鸟类，并导致大肠杆菌病，可导致突然死亡，减少鸡蛋孵化和屠宰时的尸体报废。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，针对亚太经合组织可以缓解这一问题。然而，对影响禽APEC携带和毒力的细菌因素知之甚少。此外，从患有大肠杆菌病的家禽中分离的APEC通常是不相关的，并且已经发现在强毒株之间存在很少的保守性状。抗生素治疗由于缺乏死亡前的疾病迹象以及对新抗生素耐药性的演变和传播的担忧而变得复杂。虽然已经开发了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来源的菌株的侵害。迫切需要开发广泛交叉保护的APEC疫苗。我们相信，通过解剖APEC定植于家禽呼吸道并扩散至全身部位的机制，我们可以鉴定出可能适合纳入疫苗的细菌因子。为了使这些疫苗对所有APEC都有效，有必要确认这些因子存在于各种各样的田间APEC分离株中。我们建议采用一种技术，称为签名标记的转座子诱变（STM），以确定APEC因素所需的诱导火鸡大肠杆菌病。STM使得追踪独特标记的细菌突变体在与宿主相互作用期间的命运成为可能。从接种的禽类中回收的突变体库的组成可以与接种物的组成进行比较，并且以这种方式，鉴定出不能在宿主中存活的突变体（第1部分）。图1）。然后可以鉴定在这种弱化突变体中被破坏的因子。我们已经成功地利用扫描隧道显微镜鉴定了沙门氏菌和大肠杆菌的基因。大肠杆菌O157，用于农场动物的定殖。根据这些研究所收集的资料，我们设法研制控制这些病原体的疫苗。鉴于拟议研究对家禽业的价值，英国家禽理事会和Aviagen Turkeys（前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。该项目的目标是：1。确定使APEC能够殖民火鸡的呼吸道和传播到其他器官的因素。2.确认这些因子存在于多种田间APEC分离株中。3.通过破坏这些因子并对火鸡中的突变株进行表征，证实这些因子在疾病中起重要作用。然后将在一些实验室实验中评估毒力丧失的原因。4.确定纯化的APEC毒力因子是否能诱导针对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.