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The development of a DIVA test: differentiation of infected and vaccinated animals. Dr Cath Rees School of Biosciences. Mycobacterial disease. Mycobacterium tuberculosis ( Mtb ) Causes tuberculosis in humans ; more than 1 million deaths annually Mycobacterium bovis ( Btb ) - PowerPoint PPT Presentation
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Oct
2
01
3
The development of a DIVA test: differentiation of infected and vaccinated animals
Dr Cath ReesSchool of Biosciences
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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)◦ 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
Mycobacterial disease
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• Group divided into fast and slow growers• Major pathogens are all slow growers including
• M. avium subsp. paratuberculosis (MAP)• M. tuberculosis (Mtb)• M. bovis (Btb)
• Slow growing group require 8-18 weeks to form colonies• 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
Problem of Mycobacteria detection
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• Bacteriophage are viruses that specifically infect 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
Use of bacteriophage to detect bacteria
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Tail Fibers Base
Plate
Head
• Bacteriophage replicate more rapidly than bacteria• Bacteria doubling time: 20 min – days • Bacteriophage replicate within the doubling time of the host• Reduces time to reach detectable levels of particles
Using bacteriophage to detect bacteria
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1 2 3 4 5 6 7 8 9 101.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
Bacterial growth
Phage Burst size = 100
No. of Generations/Rounds of Replication
Num
ber o
f Bac
teria
or B
acte
rioph
age
Detection limit
The FASTPlaqueTB Assay• A phage growth (amplification) assay
• Initially developed by UoN spin-out company for the detection of TB in human sputum samples• 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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PHAGE DESTROYED
USING SELECTIVE VIRUCIDE
Mycobacterialcell
BACTERIOPHAGE D29(BROAD HOST RANGE)
INFECTION
NEUTRALISATION& ADDITION OFFAST GROWING
CELLS TO FORM LAWN
PLAQUES ON AGAR PLATE:
GENUS IDENTIFICATION
Phage Amplification AssayFAST-Plaque
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PCR assay developed to identify cell
Plating out IncubationPlaques
form
Initial target cell DNADNA extraction and PCR for genotype
determination
PCR Amplification of genomic “signature
sequences”
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Control bTB MAP bTB + MAP
New Applications: Milk Assay• Standard milk analysis methods used to
prepare sample• Used by industry for somatic cell count & TVC
• Test developed for MAP• good reproducibility and sensitivity demonstrated
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Botsaris et al., (2013) Int J Food Micro 164: 76-80
• Test now being developed for Btb in raw milk• Specific application for
artisan cheese producers
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All new assays need specific sample preparation methods• Detection and identification of Mycobacteria
has been carried out in:• Sputum (Mtb)
• Decontamination and centrifugation • Albert et al., (2002) Int J Tuberc Lung Dis, 6: 529–537
• Milk (MAP and Btb)• 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• Blood assay developed for detection of MAP in
blood
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Results gained using 1 ml blood samples
Can Mtb be found in blood?• Difficulties of culture methods mean that this is
not routinely performed• 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 under-reporting
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Can Btb be found in cattle blood?• Difficulties of culture methods mean that this
is not routinely performed• 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
• 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• High throughput assay and automation required
• Automated 5 h tube test currently being patented by UoN• Applicable for bTB diagnosis (DIVA test)
• Need to fully develop methodology and evaluate performance
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1 2 3 4 5 6
400 bp
Detection of viable MAP cells
Summary
Phage-based detection method has an established record of use for Mtb
Rapid, quantitative detection of MAP in bovine blood demonstrated◦ 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
Phage Amplification Assay – Viable/Genus level PCR Assay - SpeciationM. smegmatis
M. smegmatis
M. smegmatis
M. smegmatis
M. smegmatis
MAP Bacillus
Day 1 5 h
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Sample Processing
Sutton Bonington Campus
School of Biociences
Acknowledgements• Dr Emma Stanley• Dr George Botsaris• Ben Swift• Sophie Mahendran• Emily Denton
Dr Jon Huxley– University of Nottingham
Dr Irene GrantQueen’s University, Belfast
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