The Laboratory Diagnosis of Brucellosis: Past, Present and Future

1
The Laboratory Diagnosis of Brucellosis Past,
Present and Future
by John McGiven OIE Reference Laboratory for
Brucellosis WHO/FAO Collaborating Centre for
Brucellosis
2
Brucellosis

• Brucellosis is caused by the intracellular
  pathogen Brucella
• First discovered by Bruce in 1887. First
  diagnostic test developed in 1897 (SAT)
• Facultative intracellular Gram negative
  cocobacillary rods with a cell wall including
  LPS, peptidoglycans and OMPs
• The Genus includes six classical species with
  rough and smooth strains (B.abortus,
  B.melitensis, B.suis, B.canis, B.ovis
  B.neotomae), and marine mammal species
• Brucella species have primary animal hosts but
  can cross species boundaries and Brucella is
  zoonotic
• Symptoms of infection are variable, dependent on
  infective strain and host species but include
  abortion in cattle and recurrent fever in humans

3
Prevalence of Human Brucellosis
4
Brucellosis in Northern Ireland
5
(No Transcript)
6
DiagnosisBacterial Culture

• Gold Standard (sensitivity)
• Very expensive

7
Cellular Immunity

• Brucellin Skin Test (on farm)
• Laboratory cell stimulation IFNg test

Molecular Diagnosis

• RFLP
• PCR
• SNP
• VNTR

8
Classical Serodiagnosis of Brucellosis

• Serological diagnosis based on whole cell antigen
  (e.g. SAT, CFT, RBT)
• sLPS is dominant epitope
• Importance of standardisation (OIEISS,
  International Units OIE Manual for Diagnostic
  tests and Vaccines)

9
Serodiagnosis of Brucellosis by ELISA

• Serum (indirect, competitive), milk
• Validated Standardised (OIEELISASP/WP/NSS)
• Quality Controlled

10
Automation of Brucellosis Diagnosis
11
The Future of Brucellosis Serodiagnosis

• Aims
• Reduce false positives (i.e. improve specificity)
• Diagnose latent infection (i.e. improve
  sensitivity)
• Discriminate between vaccinated and
  non-vaccinated animals
• Improve testing efficiency
• Develop point of care (penside) tests

12
Brucella AlphaLISA
Excitation 680nm
Emission 520-620nm
O2
Streptavidin coated Donor Bead
Acceptor Bead
Biotin
Mab BM40
Brucella sLPS (16M)
Homogeneous version of the VLA cELISA
13
Luminex xMAP Technology

• Multiplex Assays
• LPS (smooth rough)
• Native antigens (cellular/periplasmic)
• Recombinant Proteins

14
Electro-Chemi-Luminescence

• Rapid Multiplex Assays
• Homogeneous Multiplex Assays?

15
Summary

• Brucellosis is most common global zoonosis
• Serology is the mainstay of diagnosis
• Classical tests are well standardised and
  effective
• ELISAs have improved efficiency and are well
  standardised
• Novel antigens may add value to standard test
  results
• Multiplexing antigens will provide efficient data
  gathering
• Homogeneous multiplex assays are on the horizon

16
Acknowledgements
VLA Brucella Research Judy Stack Lorraine
Perrett Nicola Commander Andrew Taylor Racheal
Thirlwall Emma-Jane Dale Lucy Duncombe Iain
Thompson Nassira Bouzelmat VLA Technology
Transfer Unit Jason Sawyer Contact
j.mcgiven_at_vla.defra.gsi.gov.uk www.defra.gov.uk/co
rporate/vla