title: An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces.
abstract: There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk Adiapure and accompanying PCR detection kit Adiavet alongside 'in-house' molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml(-1) raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for 'in-house' Map detection were observed when the Adiapure extraction kit was combined with 'in-house' detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900ml(-1) raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml(-1) raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.
authors.affiliation: Agriculture, Food and Environmental Science Division, Food Microbiology Branch, Agri-Food and Biosciences Institute (AFBI), Newforge Lane, Belfast, BT9 5PX N. Ireland, UK. john.donaghy@afbini.gov.uk
authors.forename: J A
authors.initials: JA
authors.lastname: Donaghy
authors.forename: M T
authors.initials: MT
authors.lastname: Rowe
authors.forename: J L W
authors.initials: JL
authors.lastname: Rademaker
authors.forename: P
authors.initials: P
authors.lastname: Hammer
authors.forename: L
authors.initials: L
authors.lastname: Herman
authors.forename: V
authors.initials: V
authors.lastname: De Jonghe
authors.forename: B
authors.initials: B
authors.lastname: Blanchard
authors.forename: K
authors.initials: K
authors.lastname: Duhem
authors.forename: E
authors.initials: E
authors.lastname: Vindel
mesh_terms: D000818:Animals; D002417:Cattle; D019411:Clinical Laboratory Techniques / Q000592:standards*; D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis; D005243:Feces / Q000382:microbiology; D005506:Food Contamination / Q000032:analysis*; D005784:Gene Amplification; D006801:Humans; D008892:Milk / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D011933:Reagent Kits, Diagnostic / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic