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

  • Background on my research

  • Hypotheses

  • Work completed to date

  • Future plans


Intramammary infection

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


Mastitis what it can look like

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


Consequences of infection

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


Persistence of bacterial pathogens

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?


Microbial communities

Microbial communities

GI Tract

Respiratory system

Skin


Research hypotheses

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


Current research aim

Current research aim

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


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

How?

DNA extraction

PCR amplification

DGGE analysis


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

How?

DNA extraction

PCR amplification

DGGE analysis


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

How?

1 2 3 4 5 6 7 8 ++

DNA extraction

PCR amplification

DGGE analysis


Dna extraction

DNA extraction


Dna extraction procedure

DNA extraction procedure

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


Dna extraction procedure1

DNA extraction procedure

Removal of proteins – hydroxyapaptite columns


Dna extraction procedure2

DNA extraction procedure

DNA purification- sephadex columns


Pcr amplification

PCR amplification

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

  • 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

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

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

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!

2 rounds of PCR required to produce sufficient product

One round of PCR

Two rounds of PCR


What to do

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


Success

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?


Fresh dna extractions

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)


Bacterial primers tested

Bacterial primers tested


Bacterial primers tested1

Bacterial primers tested


357f gc 518r muyzer et al 1993

357f-GC/518R (Muyzer et al 1993)

Ewe A7

Ewe A37

Ewe A32


27f 338r gc hunt et al 2011

27F/338r-GC (Hunt et al 2011)

Ewe A37

Ewe A7

Ewe A32


Mini trial dna extractions with 27f 338r gc

Mini-trial DNA extractions with 27F/338r-Gc

Ewe A17

Ewe A48


Results from pcr on fresh dna extractions

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


Dgge second time lucky

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 + -


Moving forward

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


Part one optimisation of 27f 338r gc pcr 1

Part one: Optimisation of 27F/338r-GC PCR (1):

  • 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):

  • Increasing DNA template:

2µl DNA template

4µl DNA template


Freeze thaw effects

Freeze-thaw effects…

Run (1)

Run (2)

Run (3)


Part two dgge optimisation

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


Part two dgge optimisation1

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%


Conclusions to date

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


Future work

Future work

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


Future work1

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


Acknowledgements

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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