title: Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and Propidium Monoazide qPCR.
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.
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, National Reference Centre for Paratuberculosis, Gariga, Podenzano (PC), Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Ricchi
authors.forename: Caterina
authors.initials: C
authors.lastname: De Cicco
authors.forename: Petr
authors.initials: P
authors.lastname: Kralik
authors.forename: Vladimir
authors.initials: V
authors.lastname: Babak
authors.forename: Maria B
authors.initials: MB
authors.lastname: Boniotti
authors.forename: Roberto
authors.initials: R
authors.lastname: Savi
authors.forename: Giulia
authors.initials: G
authors.lastname: Cerutti
authors.forename: Giuliana
authors.initials: G
authors.lastname: Cammi
authors.forename: Chiara
authors.initials: C
authors.lastname: Garbarino
authors.forename: Norma
authors.initials: N
authors.lastname: Arrigoni
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D005516:Food Microbiology; D005798:Genes, Bacterial; D006801:Humans; D007364:Intercalating Agents / Q000737:chemistry; D057230:Limit of Detection; D050296:Microbial Viability; D008892:Milk / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification / Q000502:physiology*; D009842:Oligopeptides / Q000737:chemistry; D011419:Propidium / Q000031:analogs & derivatives / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D001386:Azides; D004269:DNA, Bacterial; D007364:Intercalating Agents; D009842:Oligopeptides; C533957:propidium monoazide; D011419:Propidium
keywords: Mycobacterium avium subsp. paratuberculosis; Propidium Monoazide; milk; qPCR; viability