Mycobacterial determinants of survival and fitness within the bovine host

Bovine tuberculosis (BTB) is a persistent problem in certain areas of the UK. The current control strategy is to test herds for the presence of BTB at timely intervals and slaughter test-positive animals. However, the observation that BTB is spreading in both geographical area and prevalence suggests that the existing control strategy is not working effectively. The cost of the control programme is £100 million annually, therefore not only is the policy ineffective, it is also a significant economic burden. In addition, BTB can cause tuberculosis in humans, and while this is currently rare, the escalating prevalence of BTB has the potential to develop into a zoonotic (the passage of disease from animals to humans) risk.

BTB is caused by a bacterium called Mycobacterium bovis. This is very closely related to Mycobacterium tuberculosis, a bacterium that commonly causes tuberculosis. Tuberculosis in cattle and humans show great similarities and, like the human disease, vaccination is an alternative strategy to control BTB. The vaccine against human tuberculosis is a strain of Mycobacterium bovis called Mycobacterium bovis BCG, however this provides limited protection against the disease in both cattle and humans.

Mycobacterium bovis is transmitted by aerosol, once within the animal it enters into cells called macrophages. These cells, normally dedicated to the killing and removal of bacterial pathogens, are unable to kill the bacteria. Instead, Mycobacterium bovis adapts and survives within the lungs in structures called granulomas. However, we do not know much about the genes that allow the bacteria to survive and cause disease in the host.

We will knock-out the function of every single gene in the genome of Mycobacterium bovis (approximately 4000) and determine those genes that are required for survival in bovine macrophages and in the whole cow. We will achieve this by using a technique called Transposon Directed Insertion Seqeuncing (TraDIS). This technique has been applied successfully to the study of other bacterial pathogens of medical and veterinary importance, but this will be the first time it has been used to study the genetic determinants of disease in Mycobacterium bovis. We will make libraries of mutants where the function of every gene in the genome has been knocked-out. We will then put the libraries into "screens". Mutants that are recovered after being through a "screen" are compared to the original pre-screened mutant pool by DNA sequencing. Mutants that are unable to survive the screen are identified and as a result we will identify all the genes necessary for survival in the host. Those mutants that are unable to survive in the host represent potential vaccine candidates.

We will be able to assess the function of essential genes using a combination of computational analysis, literature searching and comparisons to screens performed on different types of bacteriological media (in vitro). The functions that are essential for survival in the host also reflects the conditions within the host. At the end of this project we will have filled in key gaps in the knowledge of BTB in the area of host pathogen interactions and have identified several potential vaccine candidates to be considered for future development.

Bovine tuberculosis (BTB), a disease of cattle caused by Mycobacterium bovis, is a persistent and longstanding problem in the UK. Despite the presence of an expensive test and slaughter control programme (estimated to cost £100 million annually), the disease is spreading in prevalence and geographic area. Vaccination is a promising alternative control strategy but the only available vaccine in the short to medium term is BCG, an attenuated strain of M. bovis. This has variable and low efficacy, therefore a greater understanding of host pathogen interactions are needed in order to design novel and effective control strategies for BTB.
The overall goal of this proposal is to identify the bacterial genetic determinants for survival of M. bovis in disease relevant models of natural infection. These include in vitro, ex vivo in bovine alveolar macrophages and in the bovine host. In order to do this we will use Transposon Directed Insertion Seqeuncing (TraDIS methodology). This approach, which has been used successfully in other bacteria, involves the generation of a saturated transposon library of mutants of M. bovis which are then put through selective pressures (e.g. the bovine host). Sequence counts derived from an input pool (no selective pressure) are compared to an output pool (exposed to selective pressure) allowing for the estimation of fitness for each mutant. This work will allow us to identify the genetic requirements for survival of M. bovis in one of its natural hosts thereby providing a fuller understanding of the physiological adaptations M. bovis makes in the host and the nature of the selective pressure. Additionally, identification of the genes that are essential for survival in the bovine host provides an excellent unbiased starting point for the rationale design of new and improved vaccines.

The primary aim of this work is to identify the genetic determinant of survival for M. bovis in the bovine host. This will give us a greater understanding of the metabolic and physiological adaptations made by M. bovis and of the selective pressures in the host. The information (i.e. genes essential for survival) has direct relevance to the design of novel, live attenuated vaccines for the treatment of bovine tuberculosis. As the information also has relevance to our understanding of human tuberculosis, benefits to both animal and human health are anticipated.
Therefore we envisage

Economic benefit to the UK cattle farming (meat and milk) industry: Poor cattle health, especially through the increase in bovine tuberculosis is, however, a major issue negatively impacting on the efficiency and economics of food production. A better understanding of bovine tuberculosis and novel vaccination advances will benefit this industry thereby providing overall benefit to the UK

Economic benefit to the UK through commercial exploitation of discoveries: Human TB is a global health concern with more than 2 billion people infected. Therefore in addition to companies with an animal health focus (e.g. Zooetis) commercial ventures with a focus on human health will also benefit from this work.

Societal and economic benefits to the UK through the provision of a skilled workforce: The project involves the training of two PDRAs in both project specific and transferable skills. Both the RVC and LSHTM recognise the importance of the training element for their PDRAs and have a programe of training in generic skills that the PDRAs will utilise. The RVC have adapted the "Concordat to Support the Career Development of Researchers" and created a "Concordat Code of Practice and Guide" which demonstrates their commitment to the provision of effective support for Research Staff. The RVC has also established an internal Researcher Association led by PDRAs. The Researcher Association meet regularly and ensure PDRAs at the RVC get opportunities in training, personal development and networking, both within the college and with external bodies

Societal benefits through the improved sustainability of food production systems: A sustainable industry is required in order for consumers to benefit from inexpensive but high quality food. Animal health, welfare and public health are closely and inextricably linked. The optimization of animal health and well-being will improve the quality of animal based products. Poor welfare of food producing animals is another topic of increasing concern to the British public that will be addressed by this proposal. Societal impacts through the general public will also be realised through public engagement activities detailed in the pathways to impact attachment.

Societal benefits through improved animal and human health: The over-all average culling rate in the UK dairy herd is currently 22% due to infertility, poor health and low milk production, with animals only averaging 3 lactations before culling. The expectation is that all farm livestock should have "a life worth living - from their point of view" (Farm Animal Welfare Council, 2009). Welfare would benefit if health status was improved. This proposal will provide new information into the abilities of M. bovis to survive within the host which may help to identify new vaccine strategies. Finally, the outputs of the proposal will also be applicable to human tuberculosis, there are potential direct impacts on human health.