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.

Tim J Bull,Tulika Munshi,Paula M Lopez-Perez,Sofie B Hartmann,Sven Hofmann,Joanna S Garcia,Heidi Mikkelsen,Maria R Sørensen,Kai Hilpert,Gregers Jungersen

doi:10.3389/fmicb.2016.02112

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.

Highlights

MATERIALS AND METHODSMycobacterium avium subspecies paratuberculosis (MAP) is an economically important pathogen (McAloon et al, 2016) causing Johne’s disease in wide range of wild and domestic animals that has been linked as a zoonotic agent involved in the progression of Crohn’s disease in humans (Gitlin et al, 2012)
The remainder, comprising 34 vaccinated and 13 sham-vaccinated (Saline) individuals were successfully challenged 3 weeks post vaccination with an oral dose of MAP and maintained in appropriate housing for 28 weeks post challenge
Final MAP loads adjusted for sample weight and collated as cfu, cfl, or genome equivalents (Geq) per 100 mg of sample tissue (Supplementary Table S1) showed all animals had at least 70% of samples positive for MAP by at least one method with 3% (16/470) being negative by all three methods

Summary

Introduction

MATERIALS AND METHODSMycobacterium avium subspecies paratuberculosis (MAP) is an economically important pathogen (McAloon et al, 2016) causing Johne’s disease in wide range of wild and domestic animals that has been linked as a zoonotic agent involved in the progression of Crohn’s disease in humans (Gitlin et al, 2012). The ability of MAP to exist in a variety of phenotypes, some with a high resistance to killing (Grant and Rowe, 2004), has increased the importance of providing accurate quantitative estimates of viable counts when testing for the presence of this pathogen in food (Botsaris et al, 2016; Galiero et al, 2016; Ricchi et al, 2016), animal and human samples (Timms et al, 2016). MAP is widely accepted as a difficult organism to culture reproducibly and accurately, at low loads (Hines et al, 2007). This is relevant in early stages of MAP disease pathogenesis which are often interspersed with periods of low MAP shedding, presumably as a result of diminutive loads in tissues (Kalis et al, 2001). Whilst detection is not necessarily indicative of clinical disease, identifying the presence and quantity of viable MAP provides an important marker of disease and infectious spreading potential, relevant to optimizing strategies of disease control at herd level and for individual assessments of treatment efficacies

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