the development of a diva test: differentiation of infected and vaccinated animals

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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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