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Rapid and sensitive detection of viable mycobacteria in bovine faeces and blood by a novel phagomagnetic separation (PhMS)-qPCR assay

K Meek(1) B Gilbride(2) H Dane(2) M Thomas(2) I R Grant(1,2)

1:Queen's University Belfast; 2:Rapid-Myco Technologies Limited

Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium bovis are slow-growing mycobacterial pathogens that cause Johne’s disease (Paratuberculosis) and Bovine Tuberculosis, respectively. Previously we developed a rapid and sensitive phage-based PhMS-qPCR assay to detect viable MAP in bovine milk within 24 h. The potential of this new test to detect viable MAP and/or M. bovis in bovine faeces and blood was investigated. Following careful optimization of sample preparation protocols, faeces samples (n=376) from calves and adult cattle on several Johne’s affected farms, and blood samples (n=174) from TB skin test reactor and routine slaughter <30 month old cattle collected at point of slaughter, were tested by PhMS-qPCR and culture. Results of faecal testing clearly demonstrate that the novel PhMS-qPCR assay can detect calves or cattle shedding viable MAP, i.e. infectious animals. Overall, 119 (31.6%) faeces samples tested PhMS-qPCR positive and there was slight agreement (Kappa 0.160) with culture results. Results of blood testing revealed that viable MAP and/or M. bovis mycobacteraemias were readily detectable in cattle at slaughter in Northern Ireland, with co-infections (both MAP and M. bovis detected in same animal) frequently encountered. Detection sensitivity by PhMS-qPCR was also greater than culture when blood was tested (78 and 45 of 174 blood samples positive for viable M. bovis, respectively). Collectively, these results indicate that the novel PhMS-qPCR assays rapidly and sensitively detects viable MAP or M. bovis in bovine faeces and blood. Therefore, they may represent new diagnostic tests for Johne’s disease and bovine Tuberculosis in the future. 

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