Defining early entry mechanisms of Mycobacterium avium paratuberculosis into the host

Lead Research Organisation: Moredun Research Institute
Department Name: Vaccines and Diagnostics

Abstract

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.

Technical Summary

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.

Planned Impact

Johne's disease (JD) is a fatal infectious enteritis principally of ruminants caused by Mycobacterium avium paratuberculosis (MAP). The disease impacts on animal health and welfare, food security and is responsible for significant economic losses to the livestock industries. JD is very difficult to diagnose and control. There is no single test that can reliably detect all stages of the disease and subclinically infected animals can spread infection via intermittent shedding of MAP in faeces and milk. Diagnosis and control could be significantly improved if it were possible to detect animals early in infection and determine which animals would progress to clinical disease. This project aims to study the early infection events and identify key cellular responses and concurrent changes in bacterial function and gene expression. As such, the research and academic communities are the primary beneficiaries of our project outputs. In the long term, the new knowledge could lead to the identification of specific targets for diagnostics or vaccine development or novel strategies for intervening and reducing early infection such as dietary supplements. This would ultimately improve animal health and welfare, food security and economic competitiveness of the livestock industries.

The project will enhance interaction and communication between academics and research workers and build research capacity by encouraging the next generation of research workers to embrace and expand the new technologies developed during this project. There will be training opportunities within the project for students, post-doctoral scientists and technicians in molecular microbiology, cellular immunology, pathology, surgery and anaesthetics. Knowledge exchange to researchers out with the project team and other stakeholders such as policy makers will be achieved through conferences, peer reviewed publications, agricultural shows and the workshop planned for year three.

Information from the project will help farmers and veterinarians to understand the disease process and the rationale behind current management and control recommendations. We will engage with these beneficiaries via the Moredun Foundation and Moredun Scientific Limited, which are well connected to the farming, veterinary, animal health and livestock industries and host events and focus groups across the UK to raise awareness of JD. This will encourage more farmers to carry out JD testing and control and reduce the impacts of JD.

The project results will have both societal and economic benefits. The information will be available to dairy and beef producers, associated food industries, supermarkets and policy makers to design and implement effective JD management and control measures. This will improve food quality and safety for society and increase the economic competitiveness of these industries. Through organisation of public events at shows and festivals we will improve public awareness of the potential impacts of JD on the economies of farming and livestock associated industries, the quality and safety of our food supply and the possible effect of MAP on human health.

Publications

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