Phage-Based Test Could Improve TB Detection

Mycobacterial diseases in cattle such as Johne’s disease and bovine tuberculosis are notoriously difficult and time-consuming to diagnose. UK researchers have, however, developed a phage-based diagnostic biotechnology (PBD) that can quickly detect small numbers of mycobacterial cells in a blood sample and can differentiate between live and dead cells. A California branch of the company that developed the technology, Pandion Biotech, now is working to commercialize the test in the United States.

Researchers have used the system to study Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis or MAP), the pathogen that causes Johne’s disease, and Mycobacterium bovis, which causes bovine tuberculosis.

The PBD technology uses specific bacteriophage (phage) to infect mycobacterial bacterial cells in a sample. The phage only infects a specific mycobacteria, and only infects live, viable cells of that species. If it finds a live cell of the target species, the phage replicates and breaks open the infected cell. According to PBA research, the test is sensitive enough to detect a single live bacterium.

Researchers from University of Nottingham recently published a report in the journal Virulence, outlining their tests using the PBD technology to detect M. bovis in samples from cattle. The article is titled “Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive

cattle detected using phage-RPA.”

The researchers note that bacteriophage amplification technology was developed 20 years ago as a method to rapidly detect and enumerate slow-growing pathogenic mycobacteria. A combined phage-PCR method has previously been shown to detect and enumerate MAP in a range of matrices such as milk, cheese and blood, but detection of M. bovis in clinical samples using this approach has not been described before.

The researchers adapted the previous method for detection of M. bovis in blood by developing a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA). They found the method could detect M. bovis BCG within 48 hours, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When the researchers tested blood samples from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)-negative beef herd, Mycobacterium tuberculosis complex (MTC) cells were not detected from any of the 45 blood samples. However when they tested blood samples from SCCIT-positive animals, they found viable MTC bacteria in 66 %, or 27 of 41 samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % had detectable levels of MTC whereas only 57 % of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. The researchers say these results indicate this simple, rapid method can be applied for the study of M. bovis infections. “The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease,” they say.