Development of a novel phage‐mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis

• Published in: Journal of applied microbiology Vol. 115; no. 3; pp. 808 - 817
• Main Authors: Stewart, L.D., Foddai, A., Elliott, C.T., Grant, I.R.
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01-09-2013; Oxford University Press
• Subjects: Animals; Biological and medical sciences; burst size; Enzyme-Linked Immunosorbent Assay - methods; enzyme‐linked immunosorbent assay; Feces; Feces - microbiology; Fundamental and applied biological sciences. Psychology; Immunoassay; immunomagnetic separation; Johne's disease; Microbiology; Milk; Milk - microbiology; Mycobacteriophages - immunology; Mycobacterium avium; Mycobacterium avium subsp. paratuberculosis; Mycobacterium avium subsp. paratuberculosis - isolation & purification; phage amplification assay; burst size; Immunomagnetic method; Mycobacterium avium subsp. paratuberculosis; Disease; Applied microbiology; Paratuberculosis; Size; Mycobacterial infection; phage amplification assay; Immunological method; Rapid technique; Bacteria; Mycobacterium avium; immunomagnetic separation; Infection; Virus; Amplification; Mycobacteriales; Johne's disease; enzyme-linked immunosorbent assay; Bacteriosis; Mycobacteriaceae; Actinomycetes; Detection; ELISA assay; Viability; Phage
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.12275

Aims
The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture.
Methods and Results
The new method couples Map‐specific peptide‐mediated magnetic separation technique with an optimized phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method, and the dynamic range of the assay was 3 × 102–6 × 108 phage ml−1. When low numbers of Map were present (102 CFU ml−1), the burst size of a single host Map cell was maximal (103 phage per cell) resulting in a highly sensitive screening assay.
Conclusion
A rapid, sensitive immuno‐based screening method suitable for the detection of viable Map in milk and faeces was developed.
Significance and Impact of the Study
The novel PMS‐phage‐ELISA permits sensitive, qualitative detection of viable Map in milk or faeces samples within 48 h, representing a substantial decrease in time to detection compared with current culture methods for Map.