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

2. Overview of my talk Background on bacterial communities and mastitis Aims and objectives of my research 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 Harmon, 1994

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, 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. PhD Aims 1.To obtain a clearer understanding of the 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 if pathogen strain load is limited 4. To determine whether the sequence of colonisation of bacteria influences the health of the mammary gland and sheep

10. Objectives 1.To use traditional microbiological culturing methods and biochemical diagnostic tests to obtain a clearer understanding of the microbial community in the sheep udder 2. To develop and optimise a bacterial DNA extraction method from sheep milk for the investigation of the microbial community of sheep udders 3. 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

11. Hypotheses to be tested 1. With increasing number of lactations, the number, species and strains of bacteria colonising the udder increase 2. Pathogen load and presence of specific virulence markers in sheep with either clinical or sub-clinical mastitis is related to sheep age (number of lactations) 3. The bacterial load of pathogens and the ratio of pathogenic to benign strains of microbes affect weight gain in lambs

12. Current work- DNA extraction 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 did an in-depth literature search of DNA extraction methods used on cow and sheep milk for the investigation of mastitis I then combined aspects of these different methods into my own version.

13. 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 37 O C for one hour. 5. Add 80µl of proteinase K. 6. Incubate at 56 O C for one and a half hours. 7. Add 200µl buffer AL and vortex (lysis buffer from Qiagen DNeasy kit). 8. Incubate at 56 O C 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.

14. DNA quality Sample IDDNA concentration [ng/µl] 260/280 Milk sample 1 (T11M1)76.781.64 Milk sample 2 (T11M2)22.081.65 Milk sample 3 (T11M3)21.181.68 Spiked milk sample (T11 +) 65.921.77 Negative control (T11 -) 0.832.20

15. DNA extraction trial results

16. Contamination issues As the PCR results on the previous slide show, many of the DNA extraction process negative controls have been producing a positive PCR result To combat this, possible sources of contamination have been investigated thoroughly

17. Changing primers As results of the extraction process were viewed using PCR, it was decided to try changing the general bacterial primers being used and see if this had any effect.

18. DNA extractions with PRBAF/R primers 1 PRBA338F/PRUN518R 236BP (Øvreas et al., 1997)

19. DNA extraction with PRBAF/R primers 2

20. DNA extraction with PRBAF/R primers 3

21. Moving forward Further discussion of the steps in the DNA extraction method to identify areas of improvement. A few more trials of the method to assess if contamination, PCR consistency issues have been resolved Processing of sheep milk samples!

22. Acknowledgements Professor Laura Green Dr Kevin Purdy Dr Ed Smith Selene Huntley and Selin Cooper English Beef and Lamb Executive (EBLEX) BBSRC Biosciences KTN Quality milk management services (QMMS)