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

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

Emma Monaghan


Talk outline
Talk Outline particular reference to ewes’

  • Background on my research

  • Hypotheses

  • Work completed to date

  • Future plans


Intramammary infection
Intramammary infection particular reference to ewes’

  • 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


Mastitis what it can look like
Mastitis- what it can look like..... particular reference to ewes’


Consequences of infection
Consequences of infection particular reference to ewes’

  • 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


Persistence of bacterial pathogens
Persistence of bacterial pathogens? particular reference to ewes’

  • 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?


Microbial communities
Microbial communities particular reference to ewes’

GI Tract

Respiratory system

Skin


Research hypotheses
Research hypotheses particular reference to ewes’

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


Current research aim
Current research aim particular reference to ewes’

  • To obtain an understanding of the bacterial genera in the microbial community in the sheep mammary gland


How? particular reference to ewes’

DNA extraction

PCR amplification

DGGE analysis


How? particular reference to ewes’

DNA extraction

PCR amplification

DGGE analysis


How? particular reference to ewes’

1 2 3 4 5 6 7 8 ++

DNA extraction

PCR amplification

DGGE analysis


Dna extraction
DNA extraction particular reference to ewes’


Dna extraction procedure
DNA extraction procedure particular reference to ewes’

Lysis stage – SDS, phenol, bead beating, freeze-thaw


Dna extraction procedure1
DNA extraction procedure particular reference to ewes’

Removal of proteins – hydroxyapaptite columns


Dna extraction procedure2
DNA extraction procedure particular reference to ewes’

DNA purification- sephadex columns


Pcr amplification
PCR amplification particular reference to ewes’

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


Mini trial of techniques
Mini-trial of techniques particular reference to ewes’

  • 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


Dgge analysis
DGGE Analysis particular reference to ewes’

Ewe A17

Ewe A48

2 3 4 5 6 7 8 + +

1 2 3 4 5 6 7 8 + +


Mini trial findings
Mini-trial findings particular reference to ewes’

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


Summary of challenges
Summary of challenges particular reference to ewes’

Milk quality and storage effects

Small amounts of bacterial DNA

Components of milk

PCR primers and variation in results


Challenge identified in the mini trial
Challenge identified in the mini-trial! particular reference to ewes’

2 rounds of PCR required to produce sufficient product

One round of PCR

Two rounds of PCR


What to do
What to do? particular reference to ewes’

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


Success
Success! particular reference to ewes’

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?


Fresh dna extractions
Fresh DNA extractions particular reference to ewes’

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)


Bacterial primers tested
Bacterial primers tested particular reference to ewes’


Bacterial primers tested1
Bacterial primers tested particular reference to ewes’


357f gc 518r muyzer et al 1993
357f-GC/518R (Muyzer et al 1993) particular reference to ewes’

Ewe A7

Ewe A37

Ewe A32


27f 338r gc hunt et al 2011
27F/338r-GC (Hunt et al 2011) particular reference to ewes’

Ewe A37

Ewe A7

Ewe A32


Mini trial dna extractions with 27f 338r gc
Mini-trial DNA extractions with 27F/338r-Gc particular reference to ewes’

Ewe A17

Ewe A48


Results from pcr on fresh dna extractions
Results from PCR on fresh DNA extractions....... particular reference to ewes’

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


Dgge second time lucky
DGGE- Second time lucky? particular reference to ewes’

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 + -


Moving forward
Moving forward particular reference to ewes’

  • 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


Part one optimisation of 27f 338r gc pcr 1
Part one: Optimisation of 27F/338r-GC PCR (1): particular reference to ewes’

  • Increasing cycle number:

35 cycles

40 cycles


Part one optimisation of 27f 338r gc pcr 2
Part one: Optimisation of 27F/338r-GC PCR (2): particular reference to ewes’

  • Increasing DNA template:

2µl DNA template

4µl DNA template


Freeze thaw effects
Freeze-thaw effects… particular reference to ewes’

Run (1)

Run (2)

Run (3)


Part two dgge optimisation
Part two: DGGE optimisation particular reference to ewes’

  • 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


Part two dgge optimisation1
Part two: DGGE optimisation particular reference to ewes’

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%


Conclusions to date
Conclusions to date particular reference to ewes’

  • 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


Future work
Future work particular reference to ewes’

  • DNA extraction and PCR of sets of milk samples from three ewes of parity one


Future work1
Future work particular reference to ewes’

  • 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


Acknowledgements
Acknowledgements particular reference to ewes’

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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