Suzanna Hawkey presentation on "B. anthracis dead or alive on your slide?" at Biology of Anthrax, Tampa 2016

1. B. anthracis
Dead or alive on your slide?
Dr Suzanna Hawkey, Senior Microbiology Trainer
Novel and Dangerous Pathogens Training, PHE Porton

2. 2
Overview
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- UK handling requirements
- Suspicion of anthrax in animals
- Tasked to produce control and proficiency slides
- Biosafety considerations
- Validation
- Summary

3. 3
UK handling requirements
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- Categorisation
- UK Animal Health Regulations
- Anti-Terrorism, Crime & Security
(ACTSA)
- Biocontainment
B. anthracis - RG3
Intact pathogen
GMO & attenuated pathogen
Nucleic acid

4. 4 © Crown Copyright - PHE
2006 2008 2009 – 2013 2015
Anthrax in the UK
Inhalational anthrax Injectional anthrax
1970s -
2006
Sporadic
animals
cases

5. 5
NADP Training
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6. B. anthracis microscopy
6 © Crown Copyright - PHE
‘

7. 7 © Crown Copyright - PHE
Viability of B. anthracis material on microscopy slides following treatments
Blood culture microscopy
Heat fixation 70˚ C
Heat fixation
(70˚ C) minimum
2 minutes
Heat fixation
(85˚ C) minimum
2 minutes
Alcohol fixation
(1 minute 95%
methanol)
100% (9/9) 11.1% (1/9) 0% (0/9 )
B. endophyticus B. anthracis
Gram 11.1% (1/9 ) 0% (0/9)
M’Fadyean 100% (9/9 ) 100% (9/9)
Methodology informed by Blackwood et al . 2005

8. 8 © Crown Copyright - PHE
Westbury cases
Veterinary procedures:
Sudden death of cow
Blood swabs and blood films taken
Suspicious stained blood film
Premises placed under restriction
Incineration of carcase
Referral of samples to Rare and
Imported Pathogens Laboratory
(RIPL):
Unstained and stained slides
Swabs extracted for PCR and cultured

9. 9
Control and proficiency slides
Large scale production of blood films representing:
- low and high concentrations of B. anthracis (x600)
- Low B. anthracis mixed with Cl. perfringens (x200)
- Negative slides (x650)
Cl. septicum, Cl. novyi, Cl. perfringens
- Blood no organisms present (x500)
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10. 10
Rendered safe?
Sterilisation – complete destruction or elimination of microbial viability
including spores
Previous methods involved:
- formaldehyde fumigation followed by
- dry heat 2 hours 160˚ C
Surrogates do not always predict the behaviour of target organisms
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11. Procedure
11
Blood culture Bacteria on gel plug Re-suspended in formalin
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Formalin fixation Re-suspend in blood Slide production
Formalin
overnight and
centrifugation to
remove formalin
Adjust for low
and high
concentrations
Large scale and
addition of Cl.
perfringens for
mixed slides
BSC III BSC III BSC III
BSC III BSC I BSC I

12. 12 © Crown Copyright - PHE
Biosafety - principles
Eliminate Biological, Chemical, Thermal, Ergonomic, Sharps
Reduce Volume / titre, delivery of chemical, time
Isolate Primary containment (BSC III & I), centrifugation
Control Equipment, procedure, staff
“The application of knowledge, techniques and equipment
to prevent personal, laboratory and environmental
exposure to potentially infectious agents or biohazards’.

13. Validation
13 © Crown Copyright - PHE
Confirmation of method suitability:
- Formalin fixed material provided capsule visualisation
Testing inactivation method:
- 1ml 108 CFU ml-1 B. anthracis
- Resuscitate formalin fixed concentrated bacteria in broth overnight
- Culture to plates and monitored for 1 week
- Residual formalin? (wash, tube transfer, dilution and broth dilution)
- Spiked formalin fixed material
Confidence in inactivation – 100% of material sterility tested
then 50% and now 10% on every batch

14. 14
Summary
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Biosafety principles used to re-examine methods
- Reduce risk of spore formation
- Inactivate concentrated pathogen prior to slide production
Successful production of control and proficiency slides
- Growth and capsule production from low inoculum blood culture
produced shorter chains
Validated procedure for distribution
- Reproducible method, validation agreed for distribution
- Control of subsequent handling

15. Acknowledgements
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References and images
Blackwood, K. S., Burdz, T. V., Turenne, C. Y., Sharma, M. K., Kabani, A. M., & Wolfe, J. N. (2005).
Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a
containment level-III laboratory as part of a Laboratory Risk Assessment Program. Bio Med Central
Infectious Diseases, 5, (4).
B. anthracis images – NADP Training, PHE Porton
Cow blood smears – RIPL, PHE Porton
PHE Biosafety Programme Lead
• Heather Sheeley
NADP Training
• Prof. Nigel Silman
• Dr Jane Shallcross
• Amber Lansley
• Ben Gannon
• Dr Christopher Logue
• Clare Shieber
• Sara Fraser
Biosafety
• Allan Bennett
• Simon Parks
Rare & Imported Pathogens Laboratory
• Dr Andy Simpson
• Jason Busuttil
• Daniel Carter
Diagnostic Support
• Angela Sweed
• Anthony Crook