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 pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

• MeSH
• azidy farmakologie   MeSH
• biotest MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mikrobiální viabilita MeSH
• Mycobacterium avium subsp. paratuberculosis * genetika   MeSH
• palladium farmakologie   MeSH
• paratuberkulóza * mikrobiologie   MeSH
• propidium farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy MeSH
• palladium MeSH
• propidium MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.

• Klíčová slova
• Mycobacterium avium subsp. paratuberculosis, live-dead discrimination, mycobacteria, platinum, propidium monoazide, qPCR, viability,
• 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