Defining early entry mechanisms of Mycobacterium avium paratuberculosis into the host

Johne's disease (JD) is a common and chronic disease of the gut of ruminants caused by infection with the pathogen Mycobacterium avium subspecies paratuberculosis (MAP). At early stages following infection animals show little evidence of the disease, making it difficult to detect and easy for infection to spread unnoticed. JD has a significant impact on the health and welfare of cattle and sheep, with economic losses associated with reduced productivity and high mortality in the chronic stage. JD is an insidious and increasing problem in the UK and world-wide, with no effective control measures in cattle other than test and slaughter. This is partly because MAP is able to hide from and manipulate the immune system but also reflects the relative paucity and integration of researchers in the area, slow growth of MAP and high cost of experimental studies in target hosts.

We propose to investigate how MAP interacts with host cells in the gut at early stages following infection with strains of MAP of varying virulence. We will use a combination of methods to determine the critical cell types and mechanisms utilised by MAP for entry into the gut. These include tractable murine models, gut enteroids and surgical gut loops providing a unique overview of the MAP-host interaction. Moreover, we propose to define the role of MAP genes in survival and replication. These studies will address fundamental gaps in current knowledge and will facilitate the rational design of strategies to control and treat MAP infections.

Our proposal brings together researchers with complimentary expertise, established models of infection and a proven record of collaboration. We have three inter-linked objectives:

1) Define the cell types involved in transit of MAP across the gut mucosa using mouse models to accurately identify the cellular portal of entry

2) Characterise the mechanisms by which MAP transits the bovine gut using enteroids and surgical gut loop models to identify critical pathways and mechanisms of entry

3) Identify critical MAP genes required for host tissue invasion and survival using targeted and random mutagenesis approaches

This project brings together a unique team with expertise in MAP, bovine immunology, mucosal immune responses and intracellular bacteria. The major output will be the definition of key host, and pathogen factors that control entry and that could be targets for future intervention strategies for disease control.

Johne's disease of ruminants is a disease of major economic and animal health concern for which current control measures are inadequate. The disease is complex with divergent disease states, differences between host species and genetically diverse strains of the causative organism MAP. The ability of MAP to evade immune detection whilst persisting within gut and lymphatic tissues during the early subclinical phase of infection is likely to determine the nature and severity of disease. The effector mechanisms utilised by MAP are not yet fully understood.

The project will comprise comprehensive analyses of host responses, taking advantage of murine models where cell types can be depleted, or over-expressed, unique access to tissue culture systems (gut organoids) and surgical models enabling access to the gut. We will utilise a combination of cellular and molecular techniques including flow cytometry, confocal microscopy and bioimaging alongside transcriptomic analysis to define host cellular responses and gene expression profiles. We will use immunohistochemistry and culture to identify MAP and to assess bacterial survival and growth.

In parallel, we will identify the bacterial factors that determine intracellular net replication and pathogenesis using transposon-directed insertion-site sequencing and generation of specific mutants.

This project will produce an integrated analysis of host-pathogen interactions occurring in vivo following exposure to MAP and will facilitate identification of targets for disease intervention through improved diagnosis and/or vaccination.

Johne's disease (JD) of ruminants caused by Mycobacterium avium paratuberculosis (MAP) constrains animal health and welfare and impacts on economic performance and food security. Cattle and sheep infected with MAP suffer a protracted progression towards JD - whilst most animals are infected early in life, clinical signs of disease may not develop for a number of years, if at all. Thus, potentially large numbers of animals are significant sources of infection but remain undetected as current diagnostic measures are not adequately sensitive or specific. In order to devise better control strategies, detailed understanding of host and pathogen, and their interactions, is required. This project will yield valuable information on the early events that occur following infection, identifying key cellular responses and concurrent changes in bacterial function and gene expression. Such data will benefit those researchers studying JD or MAP and other intestinal diseases.

The project will also be of interest to industry as we will pinpoint areas for the development of new diagnostics, treatments and vaccines.

The project will enable training in diverse areas including molecular microbiology, cellular and molecular immunology, pathogenesis and in vivo research in large animals. These techniques will become embedded in the project team and as such the training will extend beyond those employed on this project to students, research assistants and post-doctoral scientists. Exchange of staff between institutions on the project, notably PDRA-1 who will work between the Roslin and Moredun Institutes will promote knowledge exchange.

Given the inter-disciplinary nature of this project, the impacts of the proposed work will go beyond direct academic beneficiaries. We will identify, protect and exploit intellectual property and engage with industry as appropriate (e.g., EBRCZoetis platform). Further, we will exploit existing links (e.g., Veterinary Surveillance Services) to those delivering knowledge and tools to the farming industry and will engage with veterinary surgeons responsible for delivery of JD diagnosis and management strategies. We will engage with policy makers and academics through publications, presentations at conferences, invited lectures, via our website pages and through links with relevant societies such as the British Society for Immunology and the Microbiology Society.

The results will have positive economic and societal impacts across a range of stakeholders. It will provide information to producers, dairy industry and policy makers on effective management plans and (new) preventative measures for the control of JD. Work carried out within the project will be of interest to the general public, not least because of the publicised but controversial link between MAP and Crohn's disease in humans, affecting approximately 1 in 1,000 people in the UK. Ultimately a reduction in JD will result in lower levels of human exposure to the bacterium in animal products.

The applicants have previously utilised short films, public presentations and lay articles to relay the importance of their work and we will use these methods to convey the purpose and projected outcomes of the project. We will engage with schools to promote education through a series of events organised through the host institutions (e.g., Edinburgh Science Festival).