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