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

‘The bacterial ecology of the ruminant udder with particular reference to ewes’. Emma Monaghan. Talk Outline. Background on my research Hypotheses Work completed to date Future plans. Intramammary infection.

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

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

  2. Talk Outline • Background on my research • Hypotheses • Work completed to date • Future plans

  3. Intramammary infection • Inflammation of the mammary gland usually caused by a bacterial infection is termed mastitis • The presentation of mastitis can be defined by severity, clinical signs and type of bacterial infection • Identified from Somatic cell count (SCC) from the milk and/or clinical signs • Treated with antibiotic/anti-inflammatory

  4. Mastitis- what it can look like.....

  5. Consequences of 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 • Welfare issues

  6. Persistence of bacterial pathogens? • DNA fingerprinting used to discriminate between E.coli strains present in the bovine udder • ~20% of recurrent cases of E.coli mastitis in cows studied caused by same genotype suggesting persistence of the organism within the mammary gland • Are bacterial species associated with clinical mastitis evolving to be more capable of causing persistent infections?

  7. Microbial communities GI Tract Respiratory system Skin

  8. Research hypotheses Overall hypotheses: • A natural microbial community forms in the mammary gland • Disease is caused when the community is perturbed Understanding of the bacterial genera in microbial community and how this changes with time and age of sheep Determine whether microbial colonisation of the udder is inevitable, detrimental or potentially beneficial Determine whether the bacterial species colonising the mammary gland influence the health of the mammary gland • Using culture-independent, whole community approaches

  9. Current research aim • To obtain an understanding of the bacterial genera in the microbial community in the sheep mammary gland

  10. How? DNA extraction PCR amplification DGGE analysis

  11. How? DNA extraction PCR amplification DGGE analysis

  12. How? 1 2 3 4 5 6 7 8 ++ DNA extraction PCR amplification DGGE analysis

  13. DNA extraction

  14. DNA extraction procedure Lysis stage – SDS, phenol, bead beating, freeze-thaw

  15. DNA extraction procedure Removal of proteins – hydroxyapaptite columns

  16. DNA extraction procedure DNA purification- sephadex columns

  17. PCR amplification Investigations indicated either a single or double round PCR (depending on the samples) amplified sufficient DNA for DGGE analysis

  18. Mini-trial of techniques • Selected two ewes aged 2 (A48) and 4 (A17) years • Milk samples collected from each udder half over eight consecutive weeks • Bacteriology and somatic cell count (SCC) information available Left half Right half Mastitis research at Warwick

  19. DGGE Analysis Ewe A17 Ewe A48 2 3 4 5 6 7 8 + + 1 2 3 4 5 6 7 8 + +

  20. Mini-trial findings Mini-trial samples No product in 1 round of PCR So used double round PCR and nested approach False positive problem Faint PCR product even after two rounds Inconsistent results

  21. Summary of challenges Milk quality and storage effects Small amounts of bacterial DNA Components of milk PCR primers and variation in results

  22. Challenge identified in the mini-trial! 2 rounds of PCR required to produce sufficient product One round of PCR Two rounds of PCR

  23. What to do? Changed aspects of the PCR programme Changed PCR reagents Altered magnesium concentrations Used additives such as DMSO and BSA Used nested approach of a general bacterial PCR followed by the DGGE PCR Changing primer sets

  24. Success! 341f-GC/518R (Muyzer and Schafer, 2001) Amplified DNA from milk samples in two rounds of PCR without false positive generation BUT....... DNA extraction negative controls remained positive Maybe controls now contaminated?

  25. Fresh DNA extractions Extracted DNA from sets of milk samples from three different ewes (A7, A32, A37) Why? Mini-trial samples deteriorated in quality Have undergone multiple freeze-thaw cycles, many used up completely Could be contaminated from frequency of use Processed milk samples for ewes in question may contain levels of bacteria below limit of detection of extraction method (~102)

  26. Bacterial primers tested

  27. Bacterial primers tested

  28. 357f-GC/518R (Muyzer et al 1993) Ewe A7 Ewe A37 Ewe A32

  29. 27F/338r-GC (Hunt et al 2011) Ewe A37 Ewe A7 Ewe A32

  30. Mini-trial DNA extractions with 27F/338r-Gc Ewe A17 Ewe A48

  31. Results from PCR on fresh DNA extractions....... 27F/338r-GC amplify bacterial DNA from milk sample DNA with no detection of DNA in extraction or PCR negative controls 357f-GC/518R amplify bacterial DNA without any false positive generation or contamination detection, but amplification was weaker than 27F/338r-GC for the same samples

  32. DGGE- Second time lucky? 27F/338r-GC Hunt et al 2011 Right half of udder Left half of udder 1 2 3 4 5 6 7 8 + - 1 2 3 4 5 + -

  33. Moving forward • Part one: Optimisation of 27F/338r-GC PCR: • Vary cycle number • Increase DNA template added • Purify PCR product • Part two: Optimisation of DGGE • Alter gradient to increase separation of multiple bands • Increase amount of PCR product added • Decrease amount of DNA ladder added

  34. Part one: Optimisation of 27F/338r-GC PCR (1): • Increasing cycle number: 35 cycles 40 cycles

  35. Part one: Optimisation of 27F/338r-GC PCR (2): • Increasing DNA template: 2µl DNA template 4µl DNA template

  36. Freeze-thaw effects… Run (1) Run (2) Run (3)

  37. Part two: DGGE optimisation • Experimented with the gradient (20-80%) • Optimisation of positive control samples • Changed staining from ethidium bromide to SYBR Gold • Optimised amount of PCR product and DNA ladder added to each DGGE gel

  38. Part two: DGGE optimisation Left half Right half 35 cycles 40 cycles 1 2 3 4 5 6 7 8 + - 1 2 3 4 5 + - 1 2 3 4 5 1 2 3 4 5 + 20-80% 30-80%

  39. Conclusions to date • DNA extraction method needs to be carefully selected • Controls are important • Cannot be confident of results without them • Milk is a difficult sample type • Contains proteins, fats • Variable consistency • Careful handling • PCR primer variation • Different primers produce different results • One round of PCR only • Ewe sample set variation • Some milk samples may contain levels of bacteria below the limit of detection

  40. Future work • DNA extraction and PCR of sets of milk samples from three ewes of parity one

  41. Future work • DNA extraction and PCR of sets of milk samples from three ewes of parity one • DGGE optimisation and processing of above samples • Identification of any patterns/changes in community within ewes across the sampling period • Pyrosequencing and expansion of study

  42. Acknowledgements Funders People Professor Laura Green Dr Kevin Purdy Barbara Payne Dr Ed Smith Selene Huntley Participating farmers Thank you for listening! Mastitis research at Warwick

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