Are microbiomes important to mammary gland health in dairy cows?

Mastitis is a bacterial infection of the udder (mammary gland). Each year ~ 50% of cows suffer from mastitis and about 1 in 4 of these die or are culled. It is the most common cause of disease and death in dairy cows. Mastitis is managed by good hygiene along with treatment and/or prevention by antibiotics administered via the teat. Over 150 types of bacteria can cause mastitis with 5 bacterial types believed to cause most cases.

Research and management is currently focused on individual strains with the idea that understanding and developing vaccines and treatments for each strain will lead to better control of mastitis. To date such an approach has not led to a reduction in the occurrence of mastitis and mastitis remains a very common disease in dairy cows and other lactating farm animals.

Antibiotics are given routinely when a cow stops milking 6-8 weeks before calving (the dry period) to clear existing infection. Ironically, there is good evidence that udders that are already colonised by certain bacterial species are the least likely to suffer from mastitis. In these udders the bacteria present cause very little inflammation and do not reduce milk quality. We do not yet understand what happens that leads to cows changing from carrying low levels of apparently harmless bacteria in its udder and suffering no disease to then going on to develop mastitis.

We now know that many species of bacteria living together (a community) are found in many sites of an animal's body, including the gut, mouth and, most recently, both human and cow mammary glands. In the gut it appears that disease can occur when the balanced bacterial community is disturbed allowing one bacterial strain to dominate, e.g. causing diarrhoea. In this grant we hypothesise that there is a natural community of bacteria in the cow udder that is present before the cow is first suckled by its calf and that this community plays an important part in preventing mastitis. We aim to:

1. Investigate when bacteria can first be detected in the udders of young calves and heifers.
2. Investigate how the bacteria in the udder change over time from when a cow is treated with antibiotics at the beginning of the dry period through to the birth of its next calf and for the following 4 weeks, the period when cows are most likely to get mastitis.

New technologies have made it possible for us to detect and identify all the bacterial species present in a milk sample without having to grow them. We will take milk samples from the mammary gland quarters of 200 cows from 2 farms on 12 occasions from drying off to 4 weeks after calving to give 800 mammary gland quarter sample sets in total. We will select at least 65 quarter sample sets where mastitis occurred and 65 sets where it did not and use modern molecular tools to identify all the bacteria in these cows' quarters and investigate how the microbial community forms and changes over time at drying off, with calving, milking, mastitis, and treatment with antibiotics and whether it remains stable if not disturbed by disease and treatment. We will use statistical analysis to determine whether specific bacteria or combinations of bacteria help protect against mastitis, how antibiotics affect the management and control of the disease and produce ideas for new strategies to develop and maintain cow health and milk output and quality.

Mastitis, caused by a wide range of bacteria, is the most economically important endemic disease of dairy cows. Intramammary infections (IMI) result in ~1 in 6 cows dying/being culled per year, reducing food production efficiency and food security. Recent studies have shown there are microbiomes in the gut, mouth, skin and human mammary gland (MG). We propose to define the MG microbiome, its dynamics and relationship with changing intramammary health and disease leading to a paradigm shift in our understanding of IMI. We will test the following hypotheses:

There is a functional microbiome in the bovine MG which develops from birth
The MG microbiome has a direct role in bovine MG health
Pathogens in the MG do not always cause disease, due in part to the microbiome
Deeper culturing from milk than the current standard will improve IMI management

We will analyse mammary tissue of ~30 immature cows and foetuses using culture and molecular techniques described below to show when bacteria begin to colonise the MG.

In a prospective, longitudinal study we will collect 12 repeat samples from 800 quarters (giving quarter sample sets) from 200 cows on 2 farms from drying off to 4 weeks post calving. We will select at least 65 quarter sample sets where IMI occurred and 65 uninfected sets for analysis. We will use qPCR to estimate total bacterial load and pyrosequencing to identify members of the microbiome. We will model these data to investigate how the microbiome forms and changes over time at drying off, with calving, milking, mastitis and antibiotic treatment. We will determine whether specific bacteria or combinations of bacteria help protect against mastitis, how antibiotics impact disease management and control and produce ideas for new strategies to develop and maintain health and milk output and quality. We will use enhanced culture of 20% of the above samples to test whether industry could refine its current standards to improve treatment/prevention of IMI.

Endemic and chronic bacterial diseases are a major health and welfare issue for both humans and animals and many appear to be associated with the disturbance of naturally existing microbiomes. In our project, we aim to understand the role the bovine mammary gland microbiome plays in intramammary infections (IMI) in dairy cattle and gain insights into its pathogenesis and management. IMI have major economic, environmental and welfare impacts. Therefore the results from this project have potential to impact across a number of animal (cattle, sheep and pig) and also human sectors, from fundamental and applied science to the actual management of mammary gland health on farms. The 'Pathways to Impact' of this proposal describes how end-users and other stakeholders will be engaged.

Understanding chronic bacterial diseases.
If our hypotheses prove to be correct and the MG is similar to other epithelial sites (which seems reasonable) then our results will have a direct impact on the dairy industry (vets, agricultural advisors and farmers), the veterinary pharmaceutical companies and all parties interested in diseases where the causative bacteria live in communities e.g. bacterial causes of lameness, abortion, pneumonia, dental disease, urogenital disease, gut diseases for example.

Current management of intramammary infections
This project will provide evidence that explains how the current management of IMI perturbs the MG microbiome and challenges the current approach that one pathogen causes disease in an otherwise 'sterile' udder. Current management and strategies for prevention of IMI may have to be reviewed. Our immediate message might appear negative; that current strategies for management and prevention will not lead to a significant reduction in the incidence of IMI. However, this knowledge will have a positive impact, because new management and treatment strategies should be possible and our results will open the way for new approaches to improve the prevention of IMI. Particular attention will be paid to possible novel treatments should the data suggest these may be possible and suitable partners within the ARC (Dairy Co, pharmaceutical companies) will be sought to exploit these ideas.

Our project will produce results that will help define how microbiomes function within individuals and how microbiomes respond to perturbations. In addition we will develop statistical models incorporating microbiome, host and environmental data. Such data and concepts will be directly relevant to organisations seeking to understand and exploit microbiomes or microbial communities in other environments, including humans and engineered systems. KJP has an ongoing collaboration with researchers investigating wound biofilm development and its treatment, which represents a similar poly-microbial environment.

This project, while based in the strategic science of understanding and controlling IMI, has the potential to impact both fundamental and strategic science across many fields. It will provide a model for investigating, understanding and potentially exploiting microbiomes and in the use of cutting-edge scientific approaches to tackling real-world problems in a coherent multidisciplinary fashion.