An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces

• Published in: Food microbiology Vol. 25; no. 1; pp. 128 - 135
• Main Authors: Donaghy, J.A., Rowe, M.T., Rademaker, J.L.W., Hammer, P., Herman, L., De Jonghe, V., Blanchard, B., Duhem, K., Vindel, E.
• Format: Journal Article
• Language: English
• Published: London Elsevier Ltd 01.02.2008; Elsevier
• Subjects: accuracy; Animals; bacterial contamination; Biological and medical sciences; Cattle; cattle diseases; Clinical Laboratory Techniques - standards; Colony Count, Microbial; Consumer Product Safety; culture media; DNA, Bacterial - analysis; feces; Feces - microbiology; Food Contamination - analysis; Food industries; Food microbiology; food safety; Fundamental and applied biological sciences. Psychology; Gene Amplification; General aspects; Humans; in vitro culture; inoculum; Methods of analysis, processing and quality control, regulation, standards; microbial detection; Milk; Milk - microbiology; molecular epidemiology; molecular genetics; Mycobacterium avium; Mycobacterium avium subsp. paratuberculosis; Mycobacterium avium subsp. paratuberculosis - isolation & purification; paratuberculosis; PCR detection; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; raw milk; Reagent Kits, Diagnostic - standards; Reproducibility of Results; reverse transcriptase polymerase chain reaction; ring tests; safety testing; Sensitivity and Specificity; Mycobacterium avium subsp. paratuberculosis; PCR detection; Milk; Paratuberculosis; Dairy product; Mycobacterium avium; Mycobacterial infection; Laboratory; Infection; Raw milk; Polymerase chain reaction; Analysis method; Mycobacteriales; Bacteriosis; Mycobacteriaceae; Bacteria; Isolation; Actinomycetes; Control method; Feces; Molecular biology; Detection
• ISSN: 0740-0020; 1095-9998
• DOI: 10.1016/j.fm.2007.06.007

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 IS900 ml −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.