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

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

2. Talk content • Background information • Research aims and techniques • Summary of work completed to date • Future work

3. Mastitis • Defined as any swelling of the sheep udder usually caused by a bacterial infection • Consequences include: reduction in milk quality and lamb weight, temporary/permanent loss of milk production • Frustrating disease for farmers to manage as more than 120 species of bacteria have been linked to mastitis Winter, 2001

4. Persistence of environmental bacteria E. coli Isolates: 4 weeks pre-calving 2 weeks pre-calving 1 weeks pre-calving Mastitis: Week 1 (M1) Week 7 (M2) Week 10 (M3) 4 2 1 M1 M2 M3 Bradley, A.J. and Green, M.J. (2001). Adaptation of Escherichia coli to the bovine mammary gland. J. CLIN. Microbiol 39; 1845-9.

5. Research aims and techniques Overall hypothesis: A natural microbial community forms in the meat sheep udder and disease is caused when the community is perturbed • Main aims: • To obtain an understanding of the total bacterial genera in the microbial community in the sheep udder and how the community structure changes with age of sheep . • To determine whether microbial colonisation of the udder is inevitable, always detrimental or potentially beneficial. • To determine whether the bacterial species colonising the mammary gland influence the health of the mammary gland • Using molecular-based whole community approaches including PCR, denaturing gradient gel electrophoresis (DGGE) and DNA sequencing techniques such as pyrosequencing

6. Bacterial DNA extraction from sheep milk Fat Calcium ions Bacteria inside cells Milk as a substrate Proteins

7. Bacterial DNA extraction methods tested • Own developed method • Combinations of the 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 extraction

8. Contamination issues

9. Purdy DNA extraction method

10. Mini-trial • Mini-trial investigating two ewes • Identify practical/technical issues • Selected milk samples from two ewes- A17 and A48 which were collected over eight consecutive weeks in 2009

13. Mini-trial DGGE Results 1- A17

14. Mini-trial Results 2 –A48

15. Practical/Technical issues • Practical issues centred on the DNA extraction method and preparing columns and reagents • Technical issues : • Loading of DGGE gels • Post DGGE PCR – first round PCR negative controls generating positive response after second round of PCR

16. DGGE PCR

17. Investigating technical issues • Changing PCR programme (temp/annealing/extension time/cycles) • Changing PCR reagents, amount of template added • Altering Mg concentration • Using additives DMSO and BSA • Nested PCR approach with general bacterial primers then DGGE primers

18. Future work • Testing further general bacterial primer combinations with the current DGGE primers n a nested PCR approach • Testing different DGGE primer sets both individually and with general bacterial primers in a nested approach in case the DGGE primers are the source of false positives

19. Acknowledgements • Professor Laura Green and Dr Kevin purdy • Simon Williams • Ed Smith • Selin Cooper and Selene Huntley • Funding: BBSRC, BioSciences KTN, EBLEX