A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk

Sepideh Hosseiniporgham,Simone Russo,Lucio Rebechesu,Stefano Lollai,Pierangela Pintore,Maria Dattena,Leonardo A Sechi,Angelo Ruiu

doi:10.1038/s41598-021-04451-w

Abstract

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

Highlights

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium
In order to compare the status of viable MAP in milk samples taken from 21 MAP shedder sheep animals in two instances of sampling, a parallel study via the two phage assays was carried out on sheep milk samples collected within a 2-week gap and the results were compared with other MAP-diagnostic approaches
As qPCR insertion sequence 900 (IS900) on DNAs extracted from retrieved phage beads depicted that the threshold cycle (TC) corresponding each concentration of MAP in phosphate-buffered saline (PBS) was at the same range as its milk counterpart (Table 1)

Summary

Introduction

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. In a modern one-day phage assay (PhMS-qPCR), no more peptides were used as ligands in the capturing structure and mycobacteriophage D29 was directly and covalently (via amine groups) conjugated to the surface of paramagnetic beads This modification improved the limit of detection ( LOD50%) of MAP via phage assay to only 10 viable cells in 50 mL milk sample, and reduced the length of procedure to almost 7 h30. The following study aimed to optimize a conventional and novel magnetic separation phage qPCR assay that respectively works with (PMS-phage assay) and without (phage-bead qPCR) intervention of MAP specific complementary peptides of aMp3 and aMptD evaluating the functionality of each assay in detection of viable MAP in sheep and goat milk samples. The specificity and sensitivity of both phage assays were computed through ROC curve analysis

Methods

Results

Conclusion