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Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals.

Bull, TJ; Munshi, T; Mikkelsen, H; Hartmann, SB; Sørensen, MR; Garcia, JS; Lopez-Perez, PM; Hofmann, S; Hilpert, K; Jungersen, G (2017) Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals. Frontiers in Microbiology, 7. p. 2112. ISSN 1664-302X https://doi.org/10.3389/fmicb.2016.02112
SGUL Authors: Bull, Timothy John

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Abstract

The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase in recoverability and an improved sensitivity of up to three logs when compared with conventional culture. Using TiKa culture, MAP clumping was minimal and produced visible colonies in half the time required by standard culture methods. Parallel quantitative evaluation of the TiKa culture approach and qPCR on MAP loads in tissue and gut mucosal samples from a MAP vaccine-challenge study, showed good correlations between colony counts (cfu) and qPCR derived genome equivalents (Geq) over a large range of loads with a 30% greater sensitivity for TiKa culture approach at low loads (two logs). Furthermore, the relative fold changes in Geq and cfu from the TiKa culture approach suggests that non-mucosal tissue loads from MAP infected animals contained a reduced proportion of non-viable MAP (mean 19-fold) which was reduced significantly further (mean 190-fold) in vaccinated "reactor" calves. This study shows TiKa culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin.

Item Type: Article
Additional Information: Copyright © 2017 Bull, Munshi, Mikkelsen, Hartmann, Sørensen, Garcia, Lopez-Perez, Hofmann, Hilpert and Jungersen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Keywords: Mycobacterium avium subspecies paratuberculosis, TiKa culture, improved culture, qPCR, quantification, Mycobacterium avium subspecies paratuberculosis, improved culture, quantification, qPCR, TiKa culture
SGUL Research Institute / Research Centre: Academic Structure > Infection and Immunity Research Institute (INII)
Journal or Publication Title: Frontiers in Microbiology
ISSN: 1664-302X
Language: eng
Dates:
DateEvent
14 December 2016Accepted
4 January 2017Published
PubMed ID: 28101082
Web of Science ID: WOS:000391153100001
Go to PubMed abstract
URI: http://openaccess.sgul.ac.uk/id/eprint/108548
Publisher's version: https://doi.org/10.3389/fmicb.2016.02112

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