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

Abstract

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) 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 Actiphagemethod was 95 % (95 % CI; 0.84-0.99) and specificity was 100 % (95% CI; 0.92-1). We further used Actiphageto 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.