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Microb Biotechnol. 2019 Dec 3. doi: 10.1111/1751-7915.13518. [Epub ahead of print]

The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne's disease-infected animals.

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Pathobiology and Population Sciences, Royal Veterinary College, Hawkshead, Herts, AL9 7TA, UK.
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK.
School of Veterinary and Medicine Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK.
Moredun Research Institute, Pentlands Science Park, Penicuik, EH26 0PZ, UK.


Here, we describe the development of a method that exploits bacteriophage D29 as a lysis agent for efficient DNA extraction from low numbers of mycobacterial cells. This method (Actiphage® ) used in combination with PCR achieved rapid and sensitive (LOD ≤ 10 cell ml-1 ) detection and identification of viable, pathogenic mycobacteria in blood samples within 6 h. We demonstrate that mycobacteriophage D29 can be used to detect a range of mycobacteria from clinical blood samples including both Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis without the need for culture and confirms our earlier observations that a low-level bacteraemia is associated with these infections in cattle. In a study of M. bovis-infected cattle (n = 41), the sensitivity of the Actiphage® method was 95 % (95 % CI; 0.84-0.99) and specificity was 100 % (95% CI; 0.92-1). We further used Actiphage® to demonstrate viable Mycobacterium avium subsp. paratuberculosis is present in the blood of Johne's infected cattle. This method provides a revolutionary new tool for the study of infections caused by these difficult to grow pathogens.

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