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) 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

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

• MeSH
• DNA bakterií klasifikace   genetika   MeSH
• feces chemie   mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce normy   MeSH
• lyofilizace MeSH
• Mycobacterium avium subsp. paratuberculosis klasifikace   genetika   izolace a purifikace   MeSH
• nemoci skotu diagnóza   MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• referenční standardy MeSH
• senzitivita a specificita MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH

Timely and reliable detection of animals shedding Mycobacterium avium subsp. paratuberculosis (MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using milk and faecal samples artificially contaminated by MAP with a focus on modern instrumental automatic DNA isolation procedures based on magnetic separation. In parallel, an automatic and manual version of magnetic separation and two methods of faecal samples preparation were compared. Commercially available DNA isolation kits were evaluated, and the selected kits were used in a trial of automatic magnetic beads-based isolation and compared with the manual version of each kit. Detection of the single copy element F57 was performed by qPCR to quantify MAP and determine the isolation efficiency. The evaluated kits showed significant differences in DNA isolation efficiencies. The best results were observed with the silica column Blood and Tissue kit for milk and Zymo Research for faeces. The highest isolation efficiency for magnetic separation was achieved with MagMAX for both matrices. The magnetic separation and silica column isolation methods used in this study represent frequently used methods in mycobacterial diagnostics.

• Klíčová slova
• DNA isolation, F57 PCR, Johne’s disease, Mycobacterium avium subsp. paratuberculosis, faeces, magnetic separation, milk, paratuberculosis, silica columns,
• Publikační typ
• časopisecké články MeSH

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

• Klíčová slova
• Camelid, Cattle, Control, Deer, Goat, Paratuberculosis, Prevalence, Review, Sheep, Wildlife,
• MeSH
• chov zvířat MeSH
• divoká zvířata mikrobiologie   MeSH
• hlášení nemocí normy   MeSH
• incidence MeSH
• Mycobacterium avium subsp. paratuberculosis izolace a purifikace   MeSH
• paratuberkulóza ekonomika   epidemiologie   prevence a kontrola   MeSH
• přežvýkavci mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The main reasons to improve the detection of Mycobacterium avium subsp. paratuberculosis (MAP) are animal health and monitoring of MAP entering the food chain via meat, milk, and/or dairy products. Different approaches can be used for the detection of MAP, but the use of magnetic separation especially in conjunction with PCR as an end-point detection method has risen in past years. However, the extraction of DNA which is a crucial step prior to PCR detection can be complicated due to the presence of inhibitory substances. Magnetic separation methods involving either antibodies or peptides represent a powerful tool for selective separation of target bacteria from other nontarget microorganisms and inhibitory sample components. These methods enable the concentration of pathogens present in the initial matrix into smaller volume and facilitate the isolation of sufficient quantities of pure DNA. The purpose of this review was to summarize the methods based on the magnetic separation approach that are currently available for the detection of MAP in a broad range of matrices.

• MeSH
• DNA bakterií izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• mléko mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis izolace a purifikace   patogenita   MeSH
• nemoci skotu diagnóza   genetika   mikrobiologie   MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• potravinářská mikrobiologie MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA bakterií MeSH