Application of a Fluorescence Microscopy Technique for Detecting Viable Mycobacterium avium ssp . paratuberculosis Cells
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Application of a Fluorescence Microscopy Technique for Detecting Viable Mycobacterium avium ssp. paratuberculosis Cells in Milk Ö. Akineden & S. Weirich & A. Abdulmawjood & K. Failing & M. Bülte
Received: 10 April 2014 / Accepted: 11 June 2014 / Published online: 24 June 2014 # Springer Science+Business Media New York 2014
Abstract The aim of the present study was to develop a fast and reliable fluorescence staining technique in order to accurately determine the number of viable Mycobacterium avium ssp. paratuberculosis (MAP) cells in milk. Milk was artificially inoculated with known amounts of four different MAP strains. Different dilutions were tested by the combination of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and auramine orange (AO) staining. To validate this combined fluorescence staining technique, two additional methods were applied: culture and the FASTPlaqueTB™ assay. The detection limit of the combined CTC and AO staining was 102 colonyforming unit (CFU) mL−1 for spiked ultra-high-temperature (UHT) milk and 103 CFU mL−1for spiked raw milk (probability of detection >95 %). Combined CTC and AO staining provides the opportunity to determine the total cell count of acid fast bacteria as well as the number of respiratory active cells in milk within 8 h. Keywords Mycobacterium avium ssp. Paratuberculosis . Viable count . Auramine orange . Fluorescence staining . Real-time PCR . Rapid detection
Ö. Akineden : S. Weirich : M. Bülte (*) Institute of Veterinary Food Science, Justus Liebig University Giessen, Frankfurter Str. 92, 35392 Giessen, Germany e-mail: [email protected] A. Abdulmawjood Institute of Food Quality and Food Safety, University of Veterinary Medicine, Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany K. Failing Unit for Biomathematics and Data Processing, Justus Liebig University Giessen, Frankfurter Str. 95, 35392 Giessen, Germany
Introduction Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, also known as Johne’s disease, a chronic contagious enteritis of cattle, other ruminants, and many species of domestic and wild animals (Stevenson et al. 2009). Clinically and subclinically, paratuberculosis-infected animals shed the pathogen periodically in their feces, milk, colostrum, and semen (Larsen et al. 1981; Nielsen et al. 2008). Since the beginning of the twentieth century, there has been a controversial discussion in the literature whether MAP is involved in the pathogenesis of Crohn’s disease, a human inflammatory bowel disease. Despite direct contact with MAP-infected animals, humans may be exposed to MAP when consuming contaminated food products and water (Eltholth et al. 2009). In the past two decades, an increasing number of studies have focused on milk and dairy products which are considered to be a major source of exposure for humans (Slana et al. 2008; Anonymous 2010; Grant 2010). In milk, potentially not all MAP cells are completely inactivated by current pasteurization treatments, depending on the temperatur
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