Abstract
BackgroundDisseminated 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.ResultsUsing 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.ConclusionsThis 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.
Highlights
Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne’s disease
Faecal and blood samples were collected in the same month for faecal culture, PCR and serum antibody ELISA testing
The unexposed cohort remained Mycobacterium avium subsp. paratuberculosis (MAP)-negative as determined by faecal culture, serum Ab ELISA and faecal PCR (Table 1) by the end of the experiments
Summary
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. 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. A common test for Johne’s disease is the serum antibody ELISA test which monitors the humoral immune response of the animal following MAP exposure. The long periods of time required for culture of this organism (even using automated systems) means that to date very few studies of blood samples have been completed so that the incidence, intensity and duration of mycobacteraemia is not known in any animal species
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