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

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.

The primary objectives of this project are to develop air-interface respiratory organ culture models to investigate the adherence of bovine and ovine M. haemolytica isolates to different regions of the upper respiratory tract (URT) of cattle and sheep and to evaluate the role of the OmpA outer membrane protein in adherence and host-specificity. The project will be carried out in three phases: Phase 1. Bovine and ovine air-interface respiratory organ culture models will be established using fresh tissues from abattoir material and techniques that have already been devised for other species. Air-interface cultures will be established using nasal, nasopharyngeal and tracheal tissues. The longevity and structural integrity of the models will be determined and experiments will be conducted with the different tissue types to standardise the adherence assays using well-characterised bovine and ovine M. haemolytica isolates. Phase 2. The organ culture models will be used to study the specificity of adherence of bovine and ovine M. haemolytica isolates to different regions of the URT of cattle and sheep. An extensive and well-characterised culture collection is available but a core group of 32 reference isolates will be used initially. These isolates represent the major evolutionary lineages and 11 capsular serotypes, and have defined lipopolysaccharide and outer membrane protein types. Phase 3. The role of OmpA in the adherence of bovine and ovine M. haemolytica isolates to the URT of cattle and sheep will be evaluated indirectly by comparing isolates with known OmpA types (complete nucleotide and inferred amino acid sequences are available for all 32 isolates). Direct analyses of OmpA function will be carried out by inhibition assays using purified OmpA incorporated into proteoliposomes and anti-OmpA antibodies. In addition, OmpA will be cloned and expressed on the cell-surface of E. coli and its affect on adherence determined.