Are microbiomes important to mammary gland health in dairy cows?

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Significant research resource.
We have collected ~10,000 milk samples from 200 cows from 2 farms over 28 days.
From these we have measured Somatic Cell Counts for each
Selected samples for celture-based work
~2000 samples were analysed using amplicon sequencing, manuscript in preparation.

SCC analysed for all samples which showed distinct categories of change in SCC in different quarters. Also showed SCC is highly dynamic in the first few days after calving and then generally becomes more stable.

A bacterial marker (16S rRNA gene) was amplified from DNA from 2000 selected samples (full detests for 48 cows) plus all associated controls. These have been sequence and analysed.

All 2000 sequenced samples were analysed by qPCR
Exploitation Route These data show how udder microbiomes vary in a wide range of ways.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC MIBTP REP programme
Amount £2,500 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2014 
End 09/2014
 
Description BT MBio
Amount £4,000 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 06/2018
 
Description Harry Smith Vacation Studentship
Amount £1,560 (GBP)
Organisation Society of General Microbiology 
Sector Charity/Non Profit
Country European Union (EU)
Start 06/2014 
End 08/2014
 
Description KW MBio
Amount £4,000 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 05/2015
 
Description Undergraduate Research Support Scheme
Amount £850 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 07/2015 
End 08/2015
 
Description ARC Mastitis 1st meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation to the First Animal Health Research Club Dissemination Meeting, London 2014

Purdy KJ, Green MJ, Bradley AJ, Green LE.

Are microbiomes important to mammary gland health in dairy cows?
Year(s) Of Engagement Activity 2014
 
Description ARC Mastitis 2nd Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation to the 2nd Animal Health Research Club Dissemination Meeting, Edinburgh 2015

Smith E, Green MJ, Bradley AJ, Green LE, Purdy KJ.

Are microbiomes important to mammary gland health in dairy cows?
Year(s) Of Engagement Activity 2015
 
Description ARC Mastitis 3rd Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation to the 3rd Animal Health Research Club Dissemination Meeting, Edinburgh 2016

Smith E, Green MJ, Bradley AJ, Green LE, Purdy KJ.

Are microbiomes important to mammary gland health in dairy cows?
Year(s) Of Engagement Activity 2016
 
Description Animal Microbiome Congress, Europe 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation called Investigating the Bovine Mammary Gland microbiome.
Year(s) Of Engagement Activity 2018
URL https://www.animalmicrobiomecongress.com
 
Description BBSRC Fringe Meeting, Oxford Farming Conference, Oxford 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited presentation (The importance of microbial communities in animal health: Mastitis as a model disease) at a BBSRC Fringe Meeting at this major farming conference. Intended purpose was to help showcase BBSRC-funded warning related work and how blue-skies research can have a direct impact.
Year(s) Of Engagement Activity 2015
 
Description BSAS Microbiome 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Bristish Association of Animal Science Annual Meeting; Dublin 2018
Are microbiomes important to mammary gland health in cows?
Year(s) Of Engagement Activity 2018
 
Description CRWAD 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation at the Conference of Research Workers in Animal Disease (CRWAD), Chicago, 2017. This is a major US/International conference on animal disease and was attended by more than 500 researchers from around the world.
Year(s) Of Engagement Activity 2017
 
Description ISME 15 Cooper 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster Presentation, International Symposium of Microbial Ecology 15, Seoul, South Korea

Cooper, S, Payne, B, Bradley AJ, Purdy KJ, Green LE: MALDI-ToF-MS of bacterial reservoirs and transmission pathways of intramammary infections
Year(s) Of Engagement Activity 2014
 
Description ISME 17 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Preentaion of a poster: Absence of a core microbiome in the bovine mammary gland: a challenge to the mutualistic microbiome concept? 
Year(s) Of Engagement Activity 2018
URL https://isme17.isme-microbes.org
 
Description ISME16 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 16th ISME Conference, Montreal, Canada. Presentation of overall ideas from project.
Year(s) Of Engagement Activity 2016
 
Description Participation in an activity, workshop or similar - ARC Mastitis 4th Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Meeting of the BBSRC Animal Health Research Club. Presentation of results from the ARC grant and discussion of possible exploitation or further study.
Year(s) Of Engagement Activity 2017
 
Description Public engagement science evening University of Warwick 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A 6 - 8pm evening meeting at the School of Life Sciences where the theme was microbiomes. One of five evening events in 2016-17
Year(s) Of Engagement Activity 2017