INTRODUCTION: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a chronic infectious intestinal disease occurring in domestic and wild ruminants. Early diagnosis of infected herds enabling timely adoption of control measures is tremendously important in view of the fact that the disease has a significant economic impact on farmers. The aim of this study was to evaluate the possibility of rapid detection of viable MAP on small ruminant farms based on environmental sample examination using a novel phage-based test named Actiphage. MATERIAL AND METHODS: A total of 9 fresh and 28 frozen (8 or 11 years at -70°C) environmental samples originating from paratuberculosis-affected farms were analysed for the presence of MAP by four different diagnostic methods: Actiphage combined with real-time PCR targeting insertion sequence 900 (IS900 qPCR), conventional phage amplification assay, culture (frozen samples only), and direct ĪS900 qPCR. RESULTS: Viable MAP was detected in one fresh environmental sample using Actiphage-IS900 qPCR. None of the frozen samples tested positive using this diagnostic approach, which was consistent with the results of culture examination also providing information on viability. CONCLUSION: This study describes other possible and innovative uses of phage-based methods in paratuberculosis control strategies. The Actiphage-qPCR was found to be less laborious than culture and provided results within six hours, suggesting that it may be a valuable tool for rapid initial determination of the infectious status of farmed animals based on environmental sample examination.

• Klíčová slova
• phage amplification assay, Actiphage-qPCR, Mycobacterium avium subsp. paratuberculosis, environmental samples, small ruminants.,
• Publikační typ
• časopisecké články MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) is a well-known causative agent of paratuberculosis, a chronic infectious granulomatous enteritis of ruminants contributing to significant economic losses worldwide. Current conventional diagnostic tools are far from being sufficient to manage and control this disease. Therefore, increased attention has been paid to alternative approaches including phage-based assays employing lytic bacteriophage D29 to detect MAP cells. The aim of the present study was to assess the applicability and efficiency of the recently developed phage-based kit termed Actiphage® combined with IS900 real-time PCR (qPCR) for rapid detection and quantification of viable MAP in milk samples. We demonstrated that Actiphage® in combination with IS900 qPCR allows for rapid and sensitive detection and identification of viable MAP in milk samples with a limit of detection of 1 MAP per 50 ml milk. Using this method, the presence of viable MAP cells was successfully determined in 30.77% of fresh goat, sheep and cow milk samples originating from paratuberculosis-affected herds. We further used Actiphage assay to define the time-lapse aspect of testing naturally contaminated milk and milk filters frozen for various lengths of time by phage-based techniques. Viable MAP was detected in 13.04% of frozen milk samples and 28.57% of frozen milk filters using Actiphage-qPCR. The results suggest the ability to detect viable MAP in these samples following freezing for more than 1 year. The obtained results support the views of the beneficial role of this technology in the control or monitoring of paratuberculosis.

• Klíčová slova
• Actiphage, Mycobacterium avium subsp. paratuberculosis, frozen samples, milk, milk filter, paratuberculosis, phage-based detection, viability determination,
• Publikační typ
• časopisecké články MeSH