The development of a DIVA test: differentiation of infected and vaccinated animals

1. The development of a DIVA test: differentiation of infected and vaccinated animals Oct 2013 Dr Cath Rees School of Biosciences

2. 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) • 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 Oct 2013

3. Problem of Mycobacteria detection • Group divided into fast and slow growers • Major pathogens are all slow growers including • M. aviumsubsp.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 Oct 2013

4. Use of bacteriophage to detect bacteria • 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 Oct 2013 Head Tail Fibers Base Plate

5. Using bacteriophage to detect bacteria • 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 Oct 2013 Detection limit

6. 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 Oct 2013 6

7. FAST-Plaque Phage Amplification Assay Mycobacterial cell INFECTION PHAGE DESTROYED USING SELECTIVE VIRUCIDE BACTERIOPHAGE D29 (BROAD HOST RANGE) Oct 2013 NEUTRALISATION & ADDITION OF FAST GROWING CELLS TO FORM LAWN PLAQUES ON AGAR PLATE: GENUS IDENTIFICATION

8. PCR assay developed to identify cell Incubation Plating out Plaques form Oct 2013 1 2 3 4 Control bTB MAP bTB + MAP DNA extraction and PCR for genotype determination PCR Amplification of genomic “signature sequences” Initial target cell DNA

9. 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 • Test now being developed for Btbin raw milk • Specific application for artisan cheese producers Oct 2013 Botsariset al., (2013) Int J Food Micro 164: 76-80

10. 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) ApplEnvMicro, 73: 1851–1857 • Cheese (MAP) • Homogenizing and centrifugation • Botsariset 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 Oct 2013

11. MAP Blood assay • Blood assay developed for detection of MAP in blood Results gained using 1 ml blood samples Oct 2013

12. Can Mtbbe 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 Oct 2013

13. 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 Oct 2013

14. 1 2 3 4 5 6 Automation of Assay 400 bp • 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 Oct 2013 Detection of viable MAP cells

15. Summary M. smegmatis M. smegmatis M. smegmatis M. smegmatis M. smegmatis Sample Processing • 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 Oct 2013 Phage Amplification Assay – Viable/Genus levelPCR Assay - Speciation MAP Bacillus Day 15 h

16. School of Biociences Sutton Bonington Campus Acknowledgements • Dr Emma Stanley • Dr George Botsaris • Ben Swift • Sophie Mahendran • Emily Denton • Dr Jon Huxley • University of Nottingham Dr Irene Grant Queen’s University, Belfast Oct 2013