LABORATORY DIAGNOSIS OF Mycobacterium bovis INFECTION AND
STRAIN TYPING OF ISOLATES
• Eamon Costello
• Central Veterinary Research Laboratory, Backweston, Celbridge, Co. Kildare
Central Veterinary Research Laboratory Mycobacteriology section
• National reference laboratory for tuberculosis in animals
• DiagnosticsHistopathology and culture
of tissue samples
Anamnestic antibody ELISA
• Research
• Tuberculin potency assays
Samples submitted for TB Diagnostic tests
• Lymph node lesions detected at abattoir post mortem examination of non-reactor cattle: - approx. 5,000 per year
• Selected ‘singleton reactor’ cattle: - approx. 600 per year
• Reactors from herds where DVO is undertaking epidemiological investigation: - approx. 800 per year
Diagnostic methods
• Macroscopic examination
• Histopathology» Rapid diagnosis» Good correlation with culture (94%)» 85% of lesions diagnosed by histopathology
• Culture» Solid media (Stonebrinks + LJ)» Liquid media (MGIT 960)» Blood agar
Accuracy of histopathological examination
1000Negative by
Histopathology
(N = 100)
12 (6%)188 (94%)Positive by
Histopathology
(N = 200)
Negative by culturePositive by
culture
MGIT 960 Liquid Culture System
• Liquid medium
• Growth of mycobacteria leads to depletion of oxygen
• Oxygen sensitive fluorescent chemical indicator in the culture tube
• System for simultaneous incubation and monitoring of culture tubes
Isolate identification proceduresGrowth characteristics
• Growth rate and effect of temperature on growth
• Colony colour and morphology
• Cording
DNA typing methods
45 4
45 5
45 6
45 12
45 14
45 20
45 21
40 7
40 22
44 12
44 16
44 25
44 32
38 32
35 24
43 25
•RFLP analysis
•2000 isolates typed
•Good differentiation of strains
•Spoligotyping
•PCR based method
50% of isolates SB140
•VNTR
•Results can be expressed in a
digital format
Direct repeat locus 1 Direct repeat locus 2
Strain A 1 2 3 4 5 1 2 5 2
Strain B 1 2 3 1 2 3 4 3 4
Genetic relationship between strains in different countries
Based on spoligotyping
• SB 140 clonal complex• 1101101000001110111111111111111111111100000• 1101101000001110111111111111111100111100000
• Ireland• Great Britain• Australia • Canada• Argentina
• SB120 clonal complex• 1101111101111110111111111111111111111100000
• 1101111101111110111101111111111111111100000
• Continental Europe
• Brazil
• Cameroon
• Chad
Acknowledgements• Kevin Kenny• Frances Quigley• Don O’Grady• Orla Flynn• Tony Gogarty• Gillian Madigan• Jack McGuirk• Joanne McLernon• Damien O’Meara• Jane O’Rourke• Maureen Padden• Elvira Sovsic• Marion Teeling• Dermot Yearsley