The bacterial ecology of the ruminant udder with particular reference to ewes Emma Monaghan

1. The bacterial ecology of the ruminant udder with particular reference to ewes Emma Monaghan

2. Summary of talk content • Brief background on bacterial communities and mastitis to introduce my research • Aims and objectives • Hypotheses and experimental design • Current work • Moving forward

3. Bacterial communities • The composition and development of colonising bacterial communities is an important determinant of the health of all higher-order organisms. • Investigation of the interactions between animal hosts and their bacterial communities is necessary to understand them and potentially manipulate them with the aim of improving the health status of animals and minimising costs incurred as a result of disease

4. Bacterial communities • Sometimes bacterial communities are only harmful when the balance of organisms changes e.g. gum disease in humans (Marsh, 1995) and footrot in sheep (Kaler, et al., 2010). • Other bacterial communities might always be detrimental to the host. One example of this might be mastitis which can be caused by one or more species of bacteria that might be part of a bacterial community.

5. Definition of mastitis • Mastitis is an inflammation of the mammary gland, usually as a result of infection with pathogenic microorganisms, most often of a bacterial origin

6. Mastitis background • The presentation of mastitis can be defined by severity, clinical signs and type of bacterial infection • When severity is used to describe mastitis, subclinical, clinical are commonly used • When duration is used to describe mastitis, acute and chronic are commonly used

7. Consequences of mastitis infection • Temporary or permanent loss of milk production • Reduction in milk quality • Reduction in lamb weight • Increased costs from purchase of milk replacements and treatments

8. Bacterial species associated with mastitis • S.aureus • M.haemolytica • E.coli • Strep. uberis • Strep. dysgalactiae • Strep. agalactiae • Coagulase negative staphylococci (CNS)

9. Overall hypothesis • A natural community forms in the udder and disease is caused when the community is perturbed. • An alternative hypothesis is that bacteria in the udder may always be detrimental to host health.

10. PhD aims • To obtain an understanding of the total bacterial species in the microbial community in the sheep udder 2. To investigate how the microbial community structure changes with age of sheep 3. To determine whether microbial colonisation of the udder is inevitable, always detrimental or potentially beneficial 4. To determine whether the bacterial species colonising the mammary gland influence the health of the mammary gland

11. Objectives • To develop/identify and optimise a bacterial DNA extraction method from sheep milk for the investigation of the microbial community of sheep udders • To use molecular-based whole community approaches including Q-PCR, genetic fingerprinting techniques such as DGGE and DNA sequencing techniques such as pyrosequencing to analyse the whole bacterial community in sheep milk

12. Hypotheses to be tested • With increasing number of lactations, the number, species and strains of bacteria colonising the udder increase • The bacterial load of pathogens and the ratio of pathogenic to benign strains of microbes will affect weight gain in lambs 3. Colonising bacterial species will be present during periods where there are no observable signs of disease

13. Current work – DNA extractions • I have recently been working on developing an efficient and consistent method for the extraction of bacterial DNA from sheep milk for the molecular based whole community approach • I have conducted an in-depth literature search of DNA extraction methods used on cow and sheep milk for the investigation of mastitis • I have combined aspects of these different methods into my own version in addition to testing a number of potentially suitable methods.

14. Tested DNA extraction methods • Own developed method (detailed on next slide) • Combinations of Nucleospin Blood and Tissue kits • Norgen bacterial DNA isolation from milk kit • Kevin Purdy DNA extraction from environmental samples • Combination of Purdy method and phenol-chloroform

15. Own DNA extraction method • 1. Bead beat 500µl of each sample in a collection tube. • 2. Transfer 200µl of the sample to a fresh 1.5ml microcentrifuge tube. • 3. Add 200µl lysis buffer [0.04M Tris-HCl pH 8, 0.004M EDTA pH8, 2% Triton X-100, 0.05mg/ml lysozyme, 0.167mg/ml lysostaphin]. • 4. Incubate at 37OC for one hour. • 5. Add 80µl of proteinase K. • 6. Incubate at 56OC for one and a half hours. • 7. Add 200µl buffer AL and vortex (lysis buffer from Qiagen DNeasy kit). • 8. Incubate at 56OC for 30 minutes. • 9. Add 320µl of 100% ethanol and vortex. • 10. Apply to DNeasy columns at ~500µl at a time • 11. Wash in 500µl buffer AW1 (wash buffer from Qiagen DNeasy kit) via centrifugation at 11,000 x g for 1 minute. • 12. Wash in 500µl buffer AW2 via centrifugation at 14,000 x g for 3 minutes. (The extra centrifugation is to dry the membrane) • 13. Place in fresh collection tube and centrifuge at 11,000 x g for 1 minute. • 14. Place the column in a fresh 1.5ml microcentrifuge tube and elute in 50µl elution buffer (Tris-HCl and water) via centrifugation at 11,000 x g for 1 minute.

16. DNA extraction results – own method

17. DNA extraction results -Nucleospin blood kit

18. Contamination problems • Contamination problems have been experienced with the Nucleospin Blood kit and the in house developed method • To combat this, possible sources of contamination have been investigated thoroughly

19. DNA extraction results – Norgen kit

20. DNA extraction results –Purdy method

21. Purdy and Phenol-chloroform

22. Moving forward- DGGE work • Using primers 341F-GC and 907R (Schäfer and Muyzer, 2001) • Currently optimising the PCR and DGGE conditions to test a range of the DNA extraction method samples

23. Acknowledgements • Professor Laura Green • Dr Kevin Purdy • Dr Ed Smith • Selene Huntley, Selin Cooper, Simon Williams • English Beef and Lamb Executive (EBLEX) • BBSRC • Biosciences KTN

24. Any questions or suggestions?