GCRF-BBR: Beyond the genome: Enabling tropical livestock EWAS of infectious diseases

Cattle are a vital component of the economies of low and middle income countries across the globe. They are a source of meat and dairy products, provide leather and other by-products and are often used as working animals. Cattle can be grazed where crops are not easily grown, provide manure as effective fertiliser and can convert forage into high-protein food. Often cattle are the most valuable possession individuals in low and middle income countries own, but are susceptible to a range of infectious diseases, which not only inflict a heavy economic burden on these countries but can also often be transmissible to humans.

Over two-thirds of the global cattle population are in low and middle income countries. Reducing the burden of cattle infectious diseases would consequently have major benefits to these areas. However for many diseases no vaccine or treatment is available. Where they are available, poor veterinary services generally mean the livestock keepers must pay for vaccines and treatments themselves. Consequently these diseases are major barriers to escaping poverty.

Due to the co-evolution of pathogens and cattle there are various examples of cattle breeds that show natural tolerance or resistance to important infectious diseases. Although these breeds are often less productive, making them less attractive to LMIC farmers, if the mechanisms underlying their resistance could be identified and harnessed, then breeds of both higher productivity and tolerance could potentially be developed.

The aim of this project is to develop the framework to enable the identification of genomic regions linked to tolerance/resistance via epigenome-wide association studies (EWAS). Differences in the epigenome, chemical modifications that overlay the genome, are linked to phenotypes through their association with gene regulation. By comparing the epigenomes of tolerant and susceptible animals it is possible to determine regions linked to disease resistance. However, at present we lack even baseline information on the epigenomes of LMIC cattle breeds, and the tools and resources for undertaking blood-based EWAS. In particular, as blood samples are a composite of various cell types, each with a distinct epigenome profile, it is necessary to disentangle any epigenetic alterations linked to disease tolerance from differences resulting purely from variation in cell-type composition between samples. We will address this by generating an epigenome atlas of major immune cell types across the two major sub-species of cattle prevalent in LMICs. Using this resource we will create a web portal to enable users to estimate the cell-type composition in their samples, allowing downstream EWAS not confounded by between sample composition variations, providing a greater ability to identify true epigenome differences linked to infectious diseases.

There is a range of cattle breeds in Africa and Asia that have co-existed with environment and pathogens for thousands of years, leading to adaptation and tolerance to local diseases. Notably some cattle breeds tolerate infection with pathogens that cause significant disease in others. The exploitation of this valuable resource of natural tolerance has been lacking, but efforts are beginning to characterise the genetic diversity of cattle across these regions. However, genetic variation in isolation does not explain all the heritability and inter-animal variation of diseases. To fully understand and exploit the mechanisms underlying natural resistance to tropical infections will require a holistic approach, studying contributions and interactions between both the genome and epigenome. Epigenome-wide association studies (EWAS) promise to be an invaluable component in defining genomic regions linked to disease tolerance, but at present the bovine methylome is largely undefined (& completely undefined for African & Asian breeds), meaning that the ability to identify methylation changes associated with phenotypic differences is limited. Human studies have particularly emphasised the importance of accounting for cellular heterogeneity in EWAS, but we currently lack the resources to do this in bovine studies.
The aim of this proposal is to address this lack of relevant tools and resources. This will involve first defining a resource of baseline bovine methylomes across key immune cell types that are important in the bovine response to pathogens, and beginning to capture diversity across cattle breeds relevant to LMIC agriculture. Using these reference sets we will then develop a portal for the deconvolution of cell-type specific signatures, enabling bovine EWAS from whole blood samples. The outputs of this project will significantly increase our knowledge base on LMIC cattle diversity and accelerate the potential of exploiting this valuable resource.

This pilot work will enable downstream EWAS for investigating cattle tropical infectious diseases. We therefore envisage a range of both short term and long term beneficiaries of this program of research.

- Enabling EWAS

Primary beneficiaries in the short term will be academics investigating the resistance/tolerance of cattle infectious diseases. The development of the web portal in this project will enable researchers to undertake EWAS from whole blood samples, dramatically simplifying the process of comparing methylation between animals than alternative approaches for removing the confounder of cellular heterogeneity such as flow cytometry.

- Reduced burden of infectious diseases

The primary long term target beneficiaries of this work are livestock holders in low and middle income countries through enabling the identification and exploitation of genomic regions linked to disease tolerance. The annual cost of treating cattle with an acaricide for example (to reduce tick-borne diseases) has been estimated at $6-$36 per animal, with treatment of an infected animal costing approximately $38. The development of naturally resistant and productive cattle breeds consequently has the promise of dramatic economic benefits to LMIC farmers and in particular the rural poor.

- Environmental benefits

Current use of insecticides and acaricides have significant impacts on the environment and soil fertility. Reducing their use through understanding alternative mechanisms of reducing disease burden could therefore have substantial longer term environmental benefits

- Informing methylation array development

We expect this work to feed into the development of bovine methylation arrays that would dramatically improve the ability to undertake EWAS, biomarker discovery analyses and investigation of gene by environment interactions. Importantly the identification of sites of methylation diversity in LMIC cattle breeds will ensure any methylation arrays are not restricted to just capturing sites of diversity in European breeds, a concern of current bovine genotyping arrays.

- Training of LMIC researchers

In conjunction with BecA (Biosciences Eastern and Central Africa) we will use this resource to develop EWAS training courses for African students and scientists. By providing not only the tools and resources but also the training to undertake EWAS from design to analysis we aim to enable local scientists to undertake EWAS targeted to local problems.

- Annotation of Indicine and African Taurine genomes

These methylation profiles will be used to inform accurate annotation of new Indicine and African taurine reference genomes being generated as part of a separate GCRF funded project. This will provide an appropriately tailored and accurate resource for researchers working on these cattle sub-species and will maximise the impact and use of this genomic information.