J Microbiol Methods. 2013 Sep;94(3):175-9.

Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h.

Swift BM, Denton EJ, Mahendran SA, Huxley JN, Rees CE.

School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK. stxbs@nottingham.ac.uk

 

ABSTRACT

The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne’s disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne’s disease. © 2013. Published by Elsevier B.V.

KEYWORDS: Bacteriophage, FASTplaqueTB assay, FPTB, Johne’s disease, MP, Magnetic separation, Media Plus, PMMS, Paratuberculosis, Peptide mediated magnetic separation, Rapid detection

PMID: 23811207

 

SUPPLEMENT:

The slow growth of some pathogenic mycobacteria makes detection by traditional culture extremely difficult. For instance, culture results for M bovis can take up to eight weeks. Similarly, Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease, can take up to 16 weeks to culture. The long incubation times and poor levels of sensitivity achieved when culturing mycobacteria from blood limits its diagnostic power.

Methods such as PCR, are often used to detect bacterial DNA as an alternative to culture. Blood assays based on PCR amplification of genomic signature sequences from Mycobacterium have been described, but a major drawback of these methods are that they do not differentiate between living and dead cells. It is vital that only viable cells are detected rather than residual DNA from cells that have been inactivated, either by the host immune system or by treatment. Unfortunately, PCR-based bovine TB detection methods have been found to be limited both by specificity and sensitivity [1]. Thus methods of detecting viable cells are needed, that are faster and more sensitive that the current tests available.

Bacteriophages are viruses that infect bacterial cells. They can either be lytic (infect and lyse their host) or lysogenic (can infect and integrate with their hosts genome; for a schematic see Fig. 1). Phage have a specific host range and will only replicate within a viable cell. The ability of bacteriophage to infect and replicate within the slow growing mycobacteria’s own generation time has been the basis of many phage based detection methods [2]. A commercial phage-based detection platform has been developed for the detection of M. tuberculosis in sputum samples as a diagnostic test in people [3]. As the lytic phage used in the assay can infect a wide range of mycobacteria, this has led to a range of assay formats that can report on the presence of other viable mycobacterial cells in a sample within two days [4-6]. Rather than waiting for the growth of the mycobacterial cells, the assay monitors the replication of the bacteriophage in a viable host cell. The identity of the cell detected is then confirmed by PCR (Fig. 2). This can be done as a species-specific test [6] or multiplex PCR assays have been developed that will simultaneously report on the presence of a range of different pathogenic mycobacteria (bovine TB and MAP) [4]. These phage-based assays are low cost and do not need investment in specialist equipment or expensive reagents, and results are available within 48 hours.

RapidMAP (PBD Biotech Ltd.) uses mycobacteriophage D29 that has a broad host range within the Mycobacterium genus, including the faster growing non-pathogens. The assay has been successfully used to detect viable MAP in the blood of cattle with Johne’s disease. The results show that it is able to reproducibly detect low numbers of viable cells with better sensitivity than direct PCR and it even detected bacteria in the early stages of infection in blood ELISA-negative animals. The use of the broad-host range phage in the RapidMAP test means that other pathogenic mycobacteria, such as M. bovis, can be detected by simply changing the endpoint PCR. Thus the developed blood test is applicable to MAP and bTB in a veterinary setting.

 

Acknowledgements

B.M.C.S was funded by a Lab21/University of Nottingham PhD studentship. E.J.D. and S.M. were supported a BBSRC and Wellcome Trust Summer Studentships, respectively.

 

References
1. Parra A, Garcia N, Garcia A, Lacombe A, Moreno F, et al. (2008) Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis. Veterinary Microbiology 127: 315-324.
2. Monk AB, Rees CD, Barrow P, Hagens S, Harper DR (2010) Bacteriophage applications: where are we now? Lett Appl Microbiol 51: 363-369.
3. Albert H, Heydenrych A, Brookes R, Mole RJ, Harley B, et al. (2002) Performance of a rapid phage-based test, FASTPlaqueTB, to diagnose pulmonary tuberculosis from sputum specimens in South Africa. Int J Tuberc Lung Dis 6: 529-537.
4. Stanley EC, Mole RJ, Smith RJ, Glenn SM, Barer MR, et al. (2007) Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours. Applied and Environmental Microbiology 73: 1851-1857.
5. Botsaris G, Slana I, Liapi M, Dodd C, Economides C, et al. (2010) Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese. International Journal of Food Microbiology 141: S87-S90.
6. Swift BM, Denton EJ, Mahendran SA, Huxley JN, Rees CE (2013) Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h. J Microbiol Methods 94: 175-179.

Contact

Ben Swift – stxbs@nottingham.ac.uk

Cath Rees – cath.rees@nottingham.ac.uk

 

phage-life-cycles
Fig 1. Bacteriophage life cycle

phage-assay
Fig 2. Schematic of the RapidMAP phage assay.

 

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