title: An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds.
abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne's disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold's egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne's) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.
authors.affiliation: GD Animal Health Service, Amsbergstraat 7, Postbus 9, 7400 AA, Deventer, The Netherlands.
authors.forename: Annet
authors.initials: A
authors.lastname: Heuvelink
authors.affiliation: Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq.|Food Sciences, Institute of Veterinary Food Science, Justus Liebig University Giessen, Frankfurter Str. 92, 35392, Giessen, Germany.
authors.forename: Abdulwahed Ahmed
authors.initials: AA
authors.lastname: Hassan
authors.affiliation: GD Animal Health Service, Amsbergstraat 7, Postbus 9, 7400 AA, Deventer, The Netherlands.
authors.forename: Hilmar
authors.initials: H
authors.lastname: van Weering
authors.affiliation: GD Animal Health Service, Amsbergstraat 7, Postbus 9, 7400 AA, Deventer, The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: van Engelen
authors.affiliation: Food Sciences, Institute of Veterinary Food Science, Justus Liebig University Giessen, Frankfurter Str. 92, 35392, Giessen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Bülte
authors.affiliation: Dairy Sciences, Institute of Veterinary Food Science, Justus Liebig University Giessen, Ludwigstr. 21, 35390, Giessen, Germany. oemer.akineden@vetmed.uni-giessen.de.
authors.forename: Ömer
authors.initials: Ö
authors.lastname: Akineden
mesh_terms: D000818:Animals; D058345:Asymptomatic Infections; D001431:Bacteriological Techniques / Q000379:methods*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis*; D005243:Feces / Q000382:microbiology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D009426:Netherlands; D010283:Paratuberculosis / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods