Because mycobacteria grow very slowly and are challenging to culture, the current test for M. bovis is based on a skin test that detects the animal’s immunological response to either past or present infection with the bacterium. Bacteriophage amplification technology is an alternative approach, which exploits bacteriophages that infect and replicate in mycobacterial cells as a surrogate for growth of the bacterium itself. The species-specific bacteriophages infect only viable mycobacterial cells in a sample, replicate in them and then lyse the bacterial cells, releasing virus and bacterial DNA, target sequences of which can be amplified, traditionally using PCR, and detected.
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The University of Nottingham’s Drs. Cath Rees and Benjamin Swift have now adapted this technology to develop an RPA-bacteriophage amplification assay for detecting M. bovis in the peripheral blood of cattle. It’s the first time this approach has been used to detect BTB, explains Dr. Rees, Associate Professor of Microbiology at the University of Nottingham’s School of Biosciences. “Not only is RPA isothermal, and so negates the need for a thermal cycler or other sophisticated instrumentation, but the lyophilised reagents are stable at ambient temperatures, so there is no need for a cold chain. The RPA bacteriophage assay provides results in just 48 hours, and achieves good sensitivity because using phage to lyse the cells is at least 100-fold more sensitive than standard DNA extraction methods for mycobacteria, which can’t easily break open their cell walls. The RPA step is then as specific as any PCR-based assay, and unlike PCR, RPA is not inhibited by blood components, so the technique proved to be more reliable when used to amplify DNA from blood samples.”
The team’s initial tests on artificially inoculated bovine blood found that RPA-bacteriophage amplification could detect just 10 infected cells per ml. The assay was then applied to peripheral blood from cattle in a herd that had been slaughtered as a result of positive immunological skin tests. The results surprisingly showed that bacterial cells were present in the blood of even early-infected cattle. “This indicates that the test could be used to detect M. bovis infection even sooner than the skin test in individual animals,” Dr. Rees suggests. “Our initial development is focused on two main areas. Firstly, use of the technology as a rapid diagnostic for identifying all the infected animals in a herd in which some animals have skin-tested positive. This could help to stop the spread of the disease and unnecessary slaughter. Secondly, the versatility and portability of RPA is ideal for adapting the technology for the development of portable, field-based diagnostics for mycobacterial species that infect other domesticated animals, in major agricultural regions and in rural settings where there may be a lack of utilities.”
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1 Benjamin M. C. Swift, Thomas W. Convery & Catherine E. D. Rees (2016): Evidence of Mycobacterium tuberculosis Complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA, Virulence, DOI: 10.1080/21505594.2016.1191729