Detection of Mycobacterium avium subsp. paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture |
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| Authors: | R Sting M Hrubenja J Mandl G Seemann A Salditt S Waibel |
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| Institution: | 1. Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/3, Fellbach D 70736, Germany;2. Rindergesundheitsdienst Stuttgart, Tierseuchenkasse Baden-Württemberg, Schaflandstr. 3/3, Fellbach D 70736, Germany;3. Staatliches Tierärztliches Untersuchungsamt Aulendorf – Diagnostikzentrum, Löwenbreitestr. 20, Aulendorf D 88326, Germany;2. Department of Community Health Sciences, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada;1. SRUC, West Mains Road, Edinburgh EH9 3JG, UK;2. Biomathematics and Statistics Scotland, King’s Buildings, West Mains Road, Edinburgh EH9 3JZ, UK;1. Clinic for Ruminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria;2. Texas A&M AgriLife Research, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University System, Amarillo, TX, USA;3. Veterinary Medicine Pathology, Department of Infectious Diseases and Pathology, Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL, USA;4. Food Animal Reproduction and Medicine Service, Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL, USA;1. Department of Food Science and Technology, Faculty of Agriculture Science, Tabriz Branch, Islamic Azad University, Tabriz, Iran;2. Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran;3. Department of Veterinary Medicine, Shabestar Branch, Islamic Azad University, Shabestar, Iran;1. Department of Animal Sciences, Institute of Veterinary Medicine, Division of Microbiology and Animal Hygiene, Faculty of Agricultural Sciences, Georg-August-University, Burckhardtweg 2, D-37077 Göttingen, Germany;2. Veterinary Clinic, Paschenaustrasse 51, D-48432 Rheine, Germany |
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| 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. |
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| Keywords: | Johne’s disease Paratuberculosis Cattle Faeces Real-time PCR Culture |
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