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A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk
Sepideh Hosseiniporgham1, Lucio Rebechesu2, Pierangela Pintore2
1Dipartimento di Scienze Biomediche, Università di Sassari, Sassari, Italy.
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.
Insights
A new phage-bead qPCR (PBQ) assay detects viable Mycobacterium avium subsp. paratuberculosis (MAP) in milk with superior sensitivity. This method offers a faster, more accurate tool for identifying MAP in ruminant milk, crucial for controlling paratuberculosis.
Area of Science:
- Veterinary Microbiology
- Food Safety
- Diagnostic Assays
Background:
- Paratuberculosis is a chronic, incurable gastroenteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP).
- Accurate and rapid detection of viable MAP in milk is essential for disease control and prevention in dairy herds.
- Current diagnostic methods may have limitations in sensitivity and speed for detecting viable MAP.
Purpose of the Study:
- To optimize and compare a novel phage-bead qPCR (PBQ) assay with a conventional peptide-mediated magnetic separation (PMS)-phage assay for detecting viable MAP in ruminant milk.
- To determine the prevalence of viable MAP in unpasteurized sheep and goat milk samples.
- To evaluate the diagnostic performance of PBQ and PMS-phage assays in identifying MAP shedders.
Main Methods:
- Optimization of a phage-based quantitative polymerase chain reaction (qPCR) assay using magnetic beads (PBQ).
- Comparison of PBQ with a conventional peptide-mediated magnetic separation (PMS)-phage assay.
- Testing of unpasteurized sheep and goat milk samples (n=41) and serial milk samples from sheep (n=21) for viable MAP.
Main Results:
- The PBQ assay demonstrated a superior limit of detection (LOD) of 10 MAP cells/10 mL milk compared to 100 cells/10 mL for the PMS-phage assay.
- Viable MAP was detected in 48.78% of unpasteurized sheep and goat milk samples using the PBQ assay.
- While both assays detected no viable MAP in serial samples from recently sampled sheep, MAP DNA and antibodies were found, indicating MAP shedders.
Conclusions:
- The PBQ assay is a promising method for assessing MAP viability in milk due to its lower LOD, rapidity, and higher sensitivity.
- PBQ offers advantages over the PMS-phage assay, including the elimination of M. smegmatis culture and virucidal treatment steps.
- Both PBQ and PMS-phage assays are valuable tools, but PBQ shows greater potential for efficient and sensitive detection of viable MAP in milk.
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