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Lab meeting 24 th Jan

Lab meeting 24 th Jan . ‘Microbial ecology of the ruminant udder with particular reference to ewes’ Emma Monaghan. Microbial communities. ‘Multi-species assemblages in which organisms live and interact in a shared environment’

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Lab meeting 24 th Jan

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  1. Lab meeting 24th Jan ‘Microbial ecology of the ruminant udder with particular reference to ewes’ Emma Monaghan

  2. Microbial communities • ‘Multi-species assemblages in which organisms live and interact in a shared environment’ • Changes in interactions can result in disease development e.g. human gut

  3. Microbial communities • ‘Multi-species assemblages in which organisms live and interact in a shared environment’ • Changes in interactions can result in disease development e.g. human gut • Previous research has shown bacterial species such as E. coli to persist in the mammary gland for up to 90 days with no clinical signs of disease- community present?

  4. Current Research aim • To obtain an understanding of the total bacterial genera in the microbial community in the sheep udder

  5. How? • DNA extraction • PCR amplification • DGGE analysis

  6. How? • DNA extraction • PCR amplification • DGGE analysis

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

  8. DNA extraction

  9. DNA extraction • Lysis stage – SDS, phenol, bead beating, freeze-thaw • Removal of proteins – hydroxyapaptite • DNA purification- sephadex columns • Ethanol precipitation

  10. Mini-trial of techniques • Selected two ewes aged two (A48) and 4 (A17) years • Processed milk samples collected from each udder half over eight consecutive weeks in duplicate • Bacteriology and SCC information already available

  11. Bacteriology and SCC • Left half of udder • Right half of udder

  12. PCR amplification • Previous investigations indicated either a single or double round PCR (depending on the samples) amplified sufficient DNA for DGGE analysis

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

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

  15. Challenge! 2 rounds of PCR required to produce sufficient product One round of PCR Two rounds of PCR

  16. 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

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

  18. 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?

  19. 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 maycontain levels of bacteria below limit of detection of extraction method

  20. Bacterial primers tested

  21. Bacterial primers tested

  22. Bacterial primers tested

  23. 27F/338r-GC (Hunt et al 2011) Ewe A37 Ewe A7 Ewe A32

  24. 357f-GC/518R (Muyzer et al 1993) Ewe A7 Ewe A37 Ewe A32

  25. Current findings • 27F/338r-GC amplify bacterial DNA from majority of milk samples with no detection of DNA in extraction or PCR negative controls for sets of milk samples from three different ewes • 357f-GC/518R amplify bacterial DNA without any false positive generation or contamination detection, but amplification is weaker than 27F/338r-GC for the same samples

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

  27. Moving forward • Part one: Optimisation of 27F/338r-GC PCR: • Increase DNA template added • Vary cycle number • Purify PCR product • Re-suspend DNA extractions in smaller volume to concentrate DNA template • 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 • Part three: Other primers • 341f-GC/518R

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