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Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle

Swift, Benjamin M.C.; Huxley, J.N.; Plain, Karen M.; Begg, Douglas J.; de Silva, Kumidika; Purdie, Auriol C.; Whittington, Richard J.; Rees, Catherine

Authors

Benjamin M.C. Swift sbzbms@nottingham.ac.uk

J.N. Huxley jon.huxley@nottingham.ac.uk

Karen M. Plain

Douglas J. Begg

Kumidika de Silva

Auriol C. Purdie

Richard J. Whittington

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CATH REES cath.rees@nottingham.ac.uk
Associate Professor



Abstract

Background

Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne’s disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne’s disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture.

Results

Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step.

Conclusions

This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne’s disease progression by warranting further research on the presence of MAP in blood.

Citation

Swift, B. M., Huxley, J., Plain, K. M., Begg, D. J., de Silva, K., Purdie, A. C., …Rees, C. (2016). Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle. BMC Veterinary Research, 12, https://doi.org/10.1186/s12917-016-0728-2

Journal Article Type Article
Acceptance Date Jun 7, 2016
Publication Date Jun 16, 2016
Deposit Date Oct 27, 2016
Publicly Available Date Oct 27, 2016
Journal BMC Veterinary Research
Electronic ISSN 1746-6148
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 12
Article Number 115
DOI https://doi.org/10.1186/s12917-016-0728-2
Keywords Johne’s disease, Paratuberculosis, Mycobacteria, Bacteriophage, Bacteraemia, Blood test, Detection method
Public URL http://eprints.nottingham.ac.uk/id/eprint/37976
Publisher URL http://bmcvetres.biomedcentral.com/articles/10.1186/s12917-016-0728-2
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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