Transcript No Slide Title

Oct 2013
The development of a DIVA
test: differentiation of infected
and vaccinated animals
Dr Cath Rees
School of Biosciences
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Mycobacterial disease

Mycobacterium tuberculosis (Mtb)
 Causes tuberculosis in humans ; more than 1 million deaths annually
Mycobacterium bovis (Btb)
 Causes TB in animals
 Defra estimates cost of £1 billion for England alone over the next decade

Mycobacterium avium subsp. paratuberculosis
(MAP)
Oct 2013

◦ Johne’s disease
 Inflammatory bowel disease of ruminants (cows, sheep, goats)
 Results in loss of productivity
 National cost estimated at £12.1 million annually
◦ Crohn’s disease
 Very similar aetiology
 MAP has been linked to Crohn’s disease in humans
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Problem of Mycobacteria detection
• Group divided into fast and slow growers
• M. avium subsp. paratuberculosis (MAP)
• M. tuberculosis (Mtb)
• M. bovis (Btb)
Oct 2013
• Major pathogens are all slow growers including
• Slow growing group require 8-18 weeks to form
colonies
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Culture results too slow as a diagnostic test
Contamination of samples leads to high failure rate
Chemical decontamination reduces sensitivity
Long periods of incubation – space issue
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Use of bacteriophage to detect
bacteria
• Host range determines the type of cell infected
• Evolved to specifically bind to structures on the
surface of its own host cell type
• Viruses replicate inside the cell and produce 50+
phage per infection
Oct 2013
• Bacteriophage are viruses that specifically
infect bacteria
Head
Tail
Fibers
Base
Plate
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Using bacteriophage to detect bacteria
• Bacteria doubling time: 20 min – days
• Bacteriophage replicate within the doubling time of the host
• Reduces time to reach detectable levels of particles
Number of Bacteria or Bacteriophage
1.00E+09
Bacterial
growth
1.00E+08
Oct 2013
• Bacteriophage replicate more rapidly than
bacteria
1.00E+07
1.00E+06
Phage
Burst
size =
100
1.00E+05
1.00E+04
1.00E+03
Detection limit
1.00E+02
1.00E+01
1.00E+00
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No. of Generations/Rounds of Replication
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The FASTPlaqueTB Assay
• A phage growth (amplification) assay
• Initially developed by UoN spin-out company for the
detection of TB in human sputum samples
Oct 2013
• Low cost test using standard microbiological techniques
• Designed for developing world markets
• Able to detect low numbers of cells
• Needed for early detection of disease
• Only live cells detected
• Advantage of culture but with speed of indirect detection
methods
• Results gained in 48 h c.f 14 days for most rapid culture
method
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FAST-Plaque
Phage Amplification Assay
INFECTION
PHAGE
DESTROYED
USING
SELECTIVE
VIRUCIDE
BACTERIOPHAGE D29
(BROAD HOST RANGE)
PLAQUES ON
AGAR PLATE:
GENUS
IDENTIFICATION
Oct 2013
Mycobacterial
cell
NEUTRALISATION
& ADDITION OF
FAST GROWING
CELLS TO
FORM LAWN
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Plating out
Incubation
Plaques
form
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Control
bTB
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MAP
PCR Amplification of
genomic “signature
sequences”
Oct 2013
PCR assay developed to identify cell
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bTB
+ MAP
DNA extraction and
PCR for genotype
determination
Initial target cell DNA
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New Applications: Milk Assay
• Standard milk analysis methods used to
prepare sample
• Used by industry for somatic cell count & TVC
• good reproducibility and sensitivity demonstrated
• Test now being
developed for Btb in
raw milk
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Oct 2013
• Test developed for MAP
Specific application for
artisan cheese producers
Botsaris et al., (2013) Int J Food Micro 164: 76-80
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All new assays need specific
sample preparation methods
• Detection and identification of Mycobacteria
has been carried out in:
• Decontamination and centrifugation
• Albert et al., (2002) Int J Tuberc Lung Dis, 6: 529–537
• Milk (MAP and Btb)
Oct 2013
• Sputum (Mtb)
• Centrifugation and fat removal
• Stanley et al., (2007) Appl Env Micro, 73: 1851–1857
• Cheese (MAP)
• Homogenizing and centrifugation
• Botsaris et al., (2010) Int. J. Food Micro, 141: S87–S90
• Blood (MAP)
• Centrifugation and magnetic bead separation
• Swift et al., (2013) J Micro Meth, 94: 175–179
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MAP Blood assay
Results
gained using
1 ml blood
samples
Oct 2013
• Blood assay developed for detection of MAP in
blood
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Can Mtb be found in blood?
• Many publications in literature describe detection of Mtb
from peripheral blood mononuclear cells (PBMC) by PCR
• PCR detection often more frequent that positive culture
• Meaning ambiguous due to lack of ability to confirm result by
culture
• Chemical decontamination kills some Mtb leading to underreporting
Oct 2013
• Difficulties of culture methods mean that this is
not routinely performed
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Can Btb be found in cattle blood?
• Reports in literature of culture of Btb from
bovine blood
• Number of studies limited by difficulty of method
• Btb detected in both reactor and non-reactor animals
Oct 2013
• Difficulties of culture methods mean that this
is not routinely performed
• Potential for phage assay to be used to replace
culture results
• Aid understanding of other test results
• Increase speed of studies required to develop vaccine
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Automation of Assay
• For routine analysis of large numbers of
samples, plate assay has limitations
• Automated 5 h tube test currently being
patented by UoN
Oct 2013
• High throughput assay and automation required
• Applicable for bTB diagnosis (DIVA test)
• Need to fully develop methodology and evaluate
performance
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400 bp
Detection of viable MAP cells
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Summary
Phage Amplification Assay – Viable/Genus level
PCR Assay - Speciation
M. smegmatis
M. smegmatis
Sample
Processing
M. smegmatis
MAP Bacillus
M. smegmatis
M. smegmatis
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Phage-based detection method has an established
record of use for Mtb
Rapid, quantitative detection of MAP in bovine blood
demonstrated
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5h
Oct 2013
Day 1
Sample preparation is key to success of assay
Detects very low numbers of cells
Provides Live/Dead differentiation
DNA preserved for molecular identification
Equally applicable for detection of Btb
Rapid, automated format possible for practical
application
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Acknowledgements
Dr Emma Stanley
Dr George Botsaris
Ben Swift
Sophie Mahendran
Emily Denton
School of Biociences
Oct 2013
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 Dr Jon Huxley
– University of Nottingham
Dr Irene Grant
Queen’s University, Belfast
Sutton Bonington Campus
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