LABORATORY DIAGNOSIS OF Mycobacterium bovis INFECTION AND STRAIN TYPING OF ISOLATES Eamon Costello Central Veterinary Research Laboratory, Backweston, Celbridge, Co. Kildare 1

Central Veterinary Research Laboratory Mycobacteriology section National reference laboratory for tuberculosis in animals Diagnostics Histopathology 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 Culture 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 100 Negative by Histopathology (N = 100) 12 (6%) 188 (94%) Positive by (N = 200) Negative by culture 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 procedures Growth characteristics Growth rate and effect of temperature on growth Colony colour and morphology Cording

DNA typing methods RFLP analysis Spoligotyping VNTR 2000 isolates typed Good differentiation of strains Spoligotyping PCR based method 50% of isolates SB140 VNTR Results can be expressed in a digital format

Genetic relationship between strains in different countries Based on spoligotyping SB120 clonal complex 1101111101111110111111111111111111111100000 1101111101111110111101111111111111111100000 Continental Europe Brazil Cameroon Chad SB 140 clonal complex 1101101000001110111111111111111111111100000 1101101000001110111111111111111100111100000 Ireland Great Britain Australia Canada Argentina

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