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.
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.
School of Veterinary Medicine and Science, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK. benjamin.swift@nottingham.ac.uk.
Benjamin M C
BM
Swift
School of Veterinary Medicine and Science, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK.
Jonathan N
JN
Huxley
The University of Sydney, Farm Animal and Veterinary Public Health, Faculty of Veterinary Science, Camden, Australia.
Karren M
KM
Plain
The University of Sydney, Farm Animal and Veterinary Public Health, Faculty of Veterinary Science, Camden, Australia.
Douglas J
DJ
Begg
The University of Sydney, Farm Animal and Veterinary Public Health, Faculty of Veterinary Science, Camden, Australia.
Kumudika
K
de Silva
The University of Sydney, Farm Animal and Veterinary Public Health, Faculty of Veterinary Science, Camden, Australia.
Auriol C
AC
Purdie
The University of Sydney, Farm Animal and Veterinary Public Health, Faculty of Veterinary Science, Camden, Australia.
Richard J
RJ
Whittington
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK.
Catherine E D
CE
Rees
D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods / Q000662:veterinary; D001435:Bacteriophages; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D008280:Magnetics; D008297:Male; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000097:blood / Q000382:microbiology*
Bacteraemia; Bacteriophage; Blood test; Detection method; Johne’s disease; Mycobacteria; Paratuberculosis