Title: Whole Genome sequencing of cattle and badger samples
Funder: Department for Environment, Food and Rural Affairs

Abstract: Description The project will assess the validity and utility of whole genome sequencing (WGS) of isolates of Mycobacterium bovis from infected cattle and badgers in Great Britain. Evidence to date suggests WGS provides better differentiation of different strains of M. bovis than the current system of genetic analysis (spoligotyping and variable number tandem repeat (VNTR) typing). Studies of human, and of cattle, tuberculosis show that WGS provides more detail of the source of infection than older technology and may allow inference of the direction of transmission between cases e.g. between different herds of cattle, and between cattle and badger populations. The M. bovis genome slowly but consistently acquires mutations over time, and hence it may be possible to determine the progression of infections based on genetic variation between isolates. In order to support an application for accreditation of WGS technology, the reproducibility of WGS will be measured through comparison of duplicate samples of M. bovis isolates from British cattle. Differentiation of M. bovis strains using WGS will also be compared to spoligotyping and VNTR typing results. Samples will be obtained from infected cattle from all bovine tuberculosis (TB) breakdowns that occur over a six month period throughout England, Wales and Scotland. The samples will be sent for culture, and if M. bovis is isolated, spoligotying and VNTR typing will be conducted according to usual procedures and WGS will be conducted in parallel. M. bovis isolates from badgers, where available will also be analysed using WGS technology. Cattle-isolate WGS data will be also be obtained to estimate the variation of M. bovis strains that occur in herds with multiple infected cattle. Currently only one culture positive sample is genotyped using spoligotypes and VNTR. Herds with TB breakdowns in the HRA and Edge area of England will be enrolled into a study cohort and remain in it until the end of the restrictions. Samples will be taken from all skin test reactors (up to a limit of around 10 per breakdown) that have visible lesions. Historical isolates from these herds, where available will also be sequenced. Analysis of additional isolates using WGS should increase differentiation of strains, assist in delineating transmission pathways and help clarify reasons for recurrence and persistence of TB in cattle herds. The data obtained will contribute to a long-term objective of the creation of a database of the distribution and diversity of the population of M. bovis strains to support TB control in Great Britain Objective This study aims to develop a validated genotyping system based on Whole Genome Sequencing (WGS); for improved TB surveillance, better determination of the source of TB breakdowns, and better understanding of local TB spread and persistence. This will help meet the policy objective to reduce bTB in England and Wales over the next 25 years, and reach Official TB free status for England by 2038. APHA will collect samples from current GB surveillance activities and undertake WGS. This method provides a high level of genetic information per sample and allows greater granularity in identifying the location of specific strains of TB, whether herds have more than one strain present and where TB infection may have come from/ spread to. This powerful methodology is currently being used to great effect in a current breakdown in the England Low Incidence Area and is providing a vast amount of data to clarify local spread. This methodology will be incorporated into an automated pipeline for sample analysis and integrated into TB surveillance systems to improve on the current Homerange mapping systems which are used by local vets to investigate TB breakdowns. As WGS data builds up APHA will be able to provide an extremely detailed picture of TB across GB with high resolution at a local level. Specific study objectives will be delivered via two work packages comprising; 1. Validation and accreditation of M. bovis WGS to support typing of M. bovis isolated as part of routine surveillance and for research into the control of TB (WP1).2. Development of further analytical procedures to provide a higher resolution picture of the genetic diversity of these organisms to understand the genetic differences within the currently identified genotypes using spoligotyping and VNTR typing (WP1). 3. To describe the variation of M. bovis strains measured by WGS in herds with multiple reactors with visible lesions and compare this variation to that ascribed by spoligotyping and VNTR typing (WP2).


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION