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Published on: October 7, 2025
Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and
Matteo Ricchi1, Caterina De Cicco, Petr Kralik
1Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Gariga, Podenzano (PC), Italy.
Abstract:
The causative agent of paratuberculosis in ruminants, Mycobacterium avium subsp. paratuberculosis (MAP), although still a matter of debate, has been linked with Crohn's and other human diseases. The availability of rapid methods for assessing the viability of MAP cells in food, in particular milk, could be of great use for risk management in food safety. MAP viability is generally assessed using culture techniques that require prolonged incubation periods for the growth of MAP. To differentiate between viable and nonviable MAP cells in milk samples, this study explores the combination of two already described techniques: peptide magnetic bead separation followed by Propidium Monoazide qPCR. Using an Ordinal Multinomial Logistic Regression model to analyze the results obtained after spiking milk samples with mixtures containing different percentages of viable/dead cells, we were able to assess the probability of the viability status of MAP found in milk. This model was applied to contaminated pasteurized milk to ascertain the efficacy of heat treatment in MAP killing. The method reported herein can potentially be used for direct detection of MAP viability in milk.
Insights
This study presents a rapid method to detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in milk. The new technique combines magnetic bead separation with qPCR, offering a faster alternative to traditional culture methods for food safety.
Area of Science:
- Food Safety
- Microbiology
- Veterinary Medicine
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) is linked to ruminant paratuberculosis and potentially human diseases.
- Current MAP viability assessment in food relies on slow culture techniques.
- Rapid detection of MAP viability in milk is crucial for food safety risk management.
Purpose of the Study:
- To develop and validate a rapid method for differentiating viable from nonviable MAP cells in milk.
- To assess the efficacy of heat treatment in eliminating MAP in pasteurized milk.
Main Methods:
- Combined peptide magnetic bead separation with Propidium Monoazide qPCR.
- Utilized an Ordinal Multinomial Logistic Regression model to analyze viability.
- Applied the method to spiked milk samples and pasteurized milk.
Main Results:
- Successfully differentiated viable and nonviable MAP cells in milk samples.
- The developed method demonstrated the probability of MAP viability status.
- Assessed the effectiveness of heat treatment in killing MAP in contaminated milk.
Conclusions:
- The combined technique offers a potentially rapid and accurate method for MAP viability detection in milk.
- This method can aid in assessing the effectiveness of food processing, like pasteurization, in eliminating viable MAP.
- The findings contribute to improved food safety strategies for MAP-contaminated products.
