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An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds

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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.

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Authors and Affiliations

  1. GD Animal Health Service, Amsbergstraat 7, Postbus 9, 7400 AA, Deventer, The Netherlands

    Annet Heuvelink, Hilmar van Weering & Erik van Engelen

  2. Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

    Abdulwahed Ahmed Hassan

  3. Food Sciences, Institute of Veterinary Food Science, Justus Liebig University Giessen, Frankfurter Str. 92, 35392, Giessen, Germany

    Abdulwahed Ahmed Hassan & Michael Bülte

  4. Dairy Sciences, Institute of Veterinary Food Science, Justus Liebig University Giessen, Ludwigstr. 21, 35390, Giessen, Germany

    Ömer Akineden

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  1. Abdulwahed Ahmed Hassan
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  2. Hilmar van Weering
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  3. Erik van Engelen
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  4. Michael Bülte
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  5. Ömer Akineden
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Correspondence to Ömer Akineden.

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Heuvelink, A., Hassan, A.A., van Weering, H. et al. An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds. Folia Microbiol 62, 197–205 (2017). https://doi.org/10.1007/s12223-016-0488-1

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