title: Detection of Mycobacterium avium subsp. paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture.
abstract: One of the most relevant aspects in the diagnosis of paratuberculosis (Johne's disease) in cattle is the availability of a method for the rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis (MAP) in order to facilitate the prompt removal of pathogen-shedding animals from a herd. To meet this requirement, methods for pre-treatment of bovine faecal samples and subsequent extraction of DNA for detection of MAP by real-time PCR were compared with MAP culture results. A total of 116 bovine faecal samples that showed weak (64.7%), moderate (18.1%) or strong (17.2%) growth of MAP on solid HEY medium were investigated. For PCR, supernatants, sediments or bacterial pellets were obtained from faecal samples by pre-treatment before extraction of MAP DNA based on silica membranes or magnetic particles. Samples then were tested by MAP IS900 and ISMav2 real-time PCR with an analytical sensitivity of 6 and 28 genome equivalents (GE) per mL, respectively. The best results were obtained by including a microfiltration step in the sample pre-treatment in combination with silica membrane-based mini-columns or magnetic particles for DNA extraction. This approach enhanced the detection rate of MAP in IS900 real-time PCR from 58.6% to 84.5% using silica membrane mini-columns and from 61.2% to 64.7% using magnetic particles.
authors.affiliation: Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/3, Fellbach D 70736, Germany. Electronic address: reinhard.sting@cvuas.bwl.de.
authors.forename: R
authors.initials: R
authors.lastname: Sting
authors.affiliation: Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/3, Fellbach D 70736, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Hrubenja
authors.affiliation: Rindergesundheitsdienst Stuttgart, Tierseuchenkasse Baden-Württemberg, Schaflandstr. 3/3, Fellbach D 70736, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Mandl
authors.affiliation: Rindergesundheitsdienst Stuttgart, Tierseuchenkasse Baden-Württemberg, Schaflandstr. 3/3, Fellbach D 70736, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Seemann
authors.affiliation: Staatliches Tierärztliches Untersuchungsamt Aulendorf - Diagnostikzentrum, Löwenbreitestr. 20, Aulendorf D 88326, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Salditt
authors.affiliation: Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/3, Fellbach D 70736, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Waibel
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000662:veterinary*; D002417:Cattle; D005243:Feces / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis* / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*
keywords: Cattle; Culture; Faeces; Johne’s disease; Paratuberculosis; Real-time PCR