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Oct 2013 The development of a DIVA test: differentiation of infected and vaccinated animals Dr Cath Rees School of Biosciences 1 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 2 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 • • • • 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 3 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 4 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 1 2 3 4 5 6 7 8 9 No. of Generations/Rounds of Replication 10 5 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 6 6 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 7 Plating out Incubation Plaques form 1 2 Control bTB 3 MAP PCR Amplification of genomic “signature sequences” Oct 2013 PCR assay developed to identify cell 4 bTB + MAP DNA extraction and PCR for genotype determination Initial target cell DNA 8 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 • Oct 2013 • Test developed for MAP Specific application for artisan cheese producers Botsaris et al., (2013) Int J Food Micro 164: 76-80 9 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 10 MAP Blood assay Results gained using 1 ml blood samples Oct 2013 • Blood assay developed for detection of MAP in blood 11 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 12 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 13 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 1 2 3 4 5 6 400 bp Detection of viable MAP cells 14 Summary Phage Amplification Assay – Viable/Genus level PCR Assay - Speciation M. smegmatis M. smegmatis Sample Processing M. smegmatis MAP Bacillus M. smegmatis M. smegmatis Phage-based detection method has an established record of use for Mtb Rapid, quantitative detection of MAP in bovine blood demonstrated ◦ ◦ ◦ ◦ 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 15 Acknowledgements Dr Emma Stanley Dr George Botsaris Ben Swift Sophie Mahendran Emily Denton School of Biociences Oct 2013 • • • • • Dr Jon Huxley – University of Nottingham Dr Irene Grant Queen’s University, Belfast Sutton Bonington Campus 16