Comparative adherence of bovine and ovine Mannheimia haemolytica strains to air-interface respiratory organ culture models from cattle and sheep

溶血性曼氏杆菌是牛羊重要的呼吸道感染病原。这些感染对动物健康产生了重大影响，并给英国和全球的养殖业造成了相当大的经济损失。抗生素和疫苗被用来对抗这些感染，但需要更好的控制方法，因为抗药性菌株的发生率正在增加，而疫苗并不能提供完全的保护。迫切需要开发更有效的疫苗来对抗溶血分枝杆菌，但其致病机制尚不清楚，保护性抗原的定义也不明确。溶血分枝杆菌由基因上不同的亚群组成，这些亚群专门适应牛或羊，并在牛或羊身上引起疾病。此外，牛和羊的分离株有一种截然不同的细胞表面蛋白(OmpA)，在其他细菌物种中，这种蛋白参与了粘附性。这些数据表明，溶血分枝杆菌OmpA蛋白可能参与了牛和羊的上呼吸道黏附，并在宿主适应中发挥作用。病原菌具有广泛的毒力机制，但黏附于粘膜表面是疾病过程中的第一个关键阶段。如果可以阻止坚持，从理论上讲，它应该可以防止殖民和疾病进展。不幸的是，人们对溶血分枝杆菌附着在牛和羊的URT上的机制知之甚少。一个主要原因是没有合适的实验室方法来代表牛和绵羊URT的组织和生理条件。在活牛羊身上进行实验是非常困难的，在伦理上也是不合理的。最近，空气界面呼吸器官培养方法已经在其他物种中得到开发，这些物种使用来自死亡动物URT的组织，以及与活动物中遇到的相似的生理条件。本项目拟利用这项技术来研究牛和绵羊溶血分枝杆菌分离株对牛和绵羊URT的粘附性，并研究OmpA在粘附性和宿主特异性中的作用。本项目的具体目的和目标如下：(1)利用牛和绵羊空气界面呼吸道器官培养模型，详细描述溶血支原体与呼吸道上皮细胞之间的相互作用，以研究细菌在牛和绵羊URT中的黏附和定植。空气界面培养将使用来自URT不同区域的组织建立，包括鼻腔(鼻甲)、鼻咽(扁桃体)和气管，并感染具有良好特征的致病牛羊田间分离株。(2)研究牛、羊溶血分枝杆菌分离株在代表牛、羊不同URT部位器官培养模型上的粘附性。通过这种方式，我们将确定牛和绵羊溶血分枝杆菌分离物是否以宿主特异性的方式附着在URT上，并有助于该物种的宿主适应。(3)探讨OmpA蛋白在牛、羊溶血分枝杆菌分离株与牛、羊器官培养模型黏附中的作用。通过这种方式，我们将检验这一假设，即OmpA具有配体样功能，以宿主特异性的方式参与与牛和羊URT的结合，并在定植和宿主特异性中发挥作用。在项目过程中开发的空气界面呼吸器官培养模型在牛羊其他病原体的研究中具有重要的潜在应用。因此，该项目不仅将促进溶血分枝杆菌的研究，也将促进相关领域的研究。这些模型的广泛使用将导致在动物实验中使用牛和羊的数量大幅减少。

Mannheimia haemolytica is responsible for important respiratory tract infections of cattle and sheep. These infections have a major impact on animal health and cause considerable economic losses to the farming industry in the UK and globally. Antibiotics and vaccines are used to combat these infections but better methods of control are required because the incidence of antibiotic resistant strains is increasing and vaccines do not provide complete protection. The development of more efficient vaccines against M. haemolytica is urgently required but the mechanisms of pathogenesis of this pathogen are poorly understood and protective antigens are ill-defined. M. haemolytica consists of genetically distinct sub-populations that are specifically adapted to, and cause disease in, either cattle or sheep. Furthermore, bovine and ovine isolates have very different forms of a cell-surface protein (OmpA) which, in other bacterial species, is involved in adherence. These data suggest that the OmpA protein of M. haemolytica might be involved in adherence to the upper respiratory tract (URT) of cattle and sheep and play a role in host-adaptation. Pathogenic bacteria possess a wide range of virulence mechanisms but adherence to mucosal surfaces is the first critical stage in the disease process. If adherence can be blocked it should, in theory, be possible to prevent colonisation and progression to disease. Unfortunately, very little is known about the mechanisms used by M. haemolytica to adhere to the URT of cattle and sheep. A major reason for this is the unavailability of suitable laboratory methods that represent the tissues and physiological conditions of the bovine and ovine URT. It is very difficult and ethically unjustifiable to carry out experiments in live cattle and sheep. Recently, air-interface respiratory organ culture methods have been developed in other species which use tissue derived from the URT of dead animals and physiological conditions that are similar to those encountered in the living animal. This project proposes to use this technology to investigate the adherence of bovine and ovine M. haemolytica isolates to the URT of cattle and sheep and to study the role of OmpA in adherence and host-specificity. The specific aims and objectives of the project are as follows: (1) To characterise in detail the interactions between M. haemolytica and respiratory epithelium by using bovine and ovine air-interface respiratory organ culture models to investigate bacterial adherence and colonisation of the URT of cattle and sheep. Air-interface cultures will be established using tissue from different regions of the URT including the nasal cavity (turbinates), the nasopharynx (tonsils), and trachea and infected with a well-characterised pathogenic bovine and ovine field isolate. (2) To study the adherence of bovine and ovine M. haemolytica isolates to organ culture models representing different regions of the URT of cattle and sheep. In this way we will determine whether adherence of bovine and ovine M. haemolytica isolates to the URT occurs in a host-specific manner and contributes to host adaptation in this species. (3) To evaluate the role of the OmpA protein in the adherence of bovine and ovine M. haemolytica isolates to organ culture models of cattle and sheep. In this way we will test the hypothesis that OmpA has a ligand-like function, is involved in binding in a host-specific manner to the bovine and ovine URT, and plays a role in colonisation and host-specificity. The air-interface respiratory organ culture models developed during the course of the project have significant potential applications in the study of other pathogens of cattle and sheep. Therefore, the proposed project will lead to the more rapid progression not only of M. haemolytica research but also research in related fields. The wider use of these models will lead to a significant reduction in the use of cattle and sheep in animal experiments.