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

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

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is considered to be a potential zoonotic pathogen and meat is one of the sources of MAP exposure for humans. MAP has been shown to be relatively resistant to different food processing methods, but there is a lack of information about the effects of ripening and fermentation processes on MAP survival in meat. Our results demonstrate that a short ripening process during teewurst production did not reduce MAP counts and viable mycobacteria were detected even during 4 weeks of storage. Although no viable MAP was recovered during the dry fermented sausage production process, there was no reduction in MAP count detected by real time PCR during production and storage of both sausages. Although the impact of foodborne exposure to viable MAP and/or mycobacterial components has not yet been clearly determined, the consumption of raw fermented meat products may be considered as a possible route of MAP transmission to humans.

• MeSH
• DNA bakterií izolace a purifikace   MeSH
• fermentace MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• manipulace s potravinami metody   MeSH
• masné výrobky mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   růst a vývoj   izolace a purifikace   MeSH
• paratuberkulóza mikrobiologie   MeSH
• prasata MeSH
• skladování potravin MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne's disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold's egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne's) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.

• MeSH
• asymptomatické infekce * MeSH
• bakteriologické techniky metody   MeSH
• diagnostické techniky molekulární metody   MeSH
• feces mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• Mycobacterium avium subsp. paratuberculosis klasifikace   genetika   růst a vývoj   izolace a purifikace   MeSH
• nemoci skotu diagnóza   MeSH
• odběr biologického vzorku metody   MeSH
• paratuberkulóza diagnóza   MeSH
• senzitivita a specificita MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• srovnávací studie MeSH
• Geografické názvy
• Nizozemsko MeSH

Mycobacterium avium paratuberculosis (Map) is a pathogen which causes a chronic progressive granulomatous enteritis known as paratuberculosis or Johne's disease and it primarily affects wild and domestic ruminants. The aim of this research was to examine a flock which consisted of 294 goats and was located in Garfagnana district (Tuscany, Italy) performing ELISA tests, culture and IS900 PCR assay; direct diagnostic methods were carried out not only on bulk tank milk and cheese samples but also on individual milk and tissue specimens collected from nine subjects positive to ELISA tests. Out of 294 animals, 20 goats (6.8%) were positive to ELISA surveys. Bulk tank milk samples were negative to culture and to PCR assay carried out on the DNA extracted directly from them, while, with respect to cheese, Map was detected by culture in 2/12 (16.66%) cheeses ripened for 3-7 days, and by PCR in 2/12 (16.66%) cheeses ripened for 3-7 days and in 3/12 (25%) cheeses ripened for 45 days. Regarding individual milk samples, Map was detected by culture in 2/9 (22.22%) specimens and by PCR in 5/9 (55.55%) samples. Furthermore, Map was isolated from the intestine in 9/9 (100%) animals, from the mesenteric lymph nodes in 8/9 (88.88%) subjects, from the liver in 4/9 (44.44%) goats, from the spleen in 5/9 (55.55%) animals, while Map DNA was found in all the tissue samples analyzed.The results demonstrated the presence of paratuberculosis in a goat flock located in Garfagnana district (Tuscany, Italy).

• MeSH
• ELISA MeSH
• kozy MeSH
• mléko mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   imunologie   izolace a purifikace   MeSH
• nemoci koz diagnóza   epidemiologie   mikrobiologie   MeSH
• paratuberkulóza diagnóza   epidemiologie   mikrobiologie   MeSH
• polymerázová řetězová reakce MeSH
• sýr mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Itálie epidemiologie   MeSH

Surveys from different parts of the world have reported that viable Mycobacterium avium subsp. paratuberculosis (MAP) can be cultured from approximately 2% of samples of retail pasteurised milk samples. Pasteurised milk is used for the production of powdered infant formula (PIF) and therefore there is a concern that MAP may also be present in these products. Several studies have previously reported the detection of MAP in PIF using PCR-based assays. However, culture-based surveys of PIF have not detected viable MAP. Here we describe a phage amplification assay coupled with PCR (page-PCR) that can rapidly detect viable MAP in PIF. The results of a small survey showed that the phage-PCR assay detected viable MAP in 13% (4/32) of PIF samples. Culture detected viable MAP in 9% (3/32) PIF samples, all of which were also phage-PCR positive. Direct IS900 PCR detected MAP DNA in 22% (7/32) of PIF samples. The presence of viable MAP in PIF indicates that MAP either survived PIF manufacturing or that post-production contamination occurred. Irrespective of the route of MAP contamination, the presence of viable MAP in PIF is a potential public health concern.

• MeSH
• bakteriofágy genetika   MeSH
• DNA bakterií genetika   MeSH
• kojenec MeSH
• lidé MeSH
• mléko mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   MeSH
• náhražky mateřského mléka mikrobiologie   MeSH
• novorozenec MeSH
• polymerázová řetězová reakce metody   MeSH
• zvířata MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• novorozenec MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to determine possible differences in the faecal microbiota of dairy cows infected with Mycobacterium avium subsp. paratuberculosis (Johne's disease) in comparison with noninfected cows from the same herds. Faecal samples from cows in 4 herds were tested for M. avium subsp. paratuberculosis by real-time PCR, and faecal bacterial populations were analysed by 454 pyrosequencing of the 16S rRNA gene. The most notable differences between shedding and nonshedding cows were an increase in the genus Psychrobacter and a decrease in the genera Oscillospira, Ruminococcus, and Bifidobacterium in cows infected with M. avium subsp. paratuberculosis. The present study is the first to report the faecal microbial composition in dairy cows infected with M. avium subsp. paratuberculosis.

• MeSH
• feces mikrobiologie   MeSH
• mlékárenství MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   fyziologie   MeSH
• nemoci skotu mikrobiologie   MeSH
• paratuberkulóza mikrobiologie   MeSH
• sekvenční analýza DNA veterinární   MeSH
• skot mikrobiologie   MeSH
• vylučování bakterií z těla MeSH
• zvířata MeSH
• Check Tag
• skot mikrobiologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

INTRODUCTION AND OBJECTIVE: Mycobacteria have been isolated from almost all types of natural waters, as well as from man-made water distribution systems. Detection of mycobacteria using PCR has been described in different types of water; however, currently, there is no standardised protocol for the processing of large volumes of water. MATERIAL AND METHODS: In the present study, different filtering methods are tested and optimised for tap or river water filtration up to 10 L, as well as filter processing and DNA isolation using four commercially available kits. RESULTS: The PowerWater DNA isolation kit (MoBio, USA), together with a kit used for soil and other environmental samples (PowerSoil DNA isolation kit, MoBio), had the highest efficiency. Filtration of 10 L of water and elution of the filter in PBS with the addition of 0.05% of Tween 80 is suggested. CONCLUSIONS: The described protocol for filter elution is recommended, and the use of the PowerWater DNA isolation kit for the highest mycobacterial DNA yield from water samples. The described protocol is suitable for parallel detection of mycobacteria using cultivation.

• MeSH
• DNA bakterií analýza   MeSH
• filtrace metody   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• mikrobiologie vody * MeSH
• monitorování životního prostředí metody   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   MeSH
• sladká voda mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The aims of this study were to describe spatial contamination of the environment on a mouflon pasture, as well as to assess the contamination of grass and roots after surface contamination and in depth contamination with feces and buried tissues from animals infected with Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). Samples of soil, roots, and aerial parts of plants were collected from different locations inside the mouflon pasture, and one control sample site was chosen outside the area where the animals are living. M. a. paratuberculosis DNA was present in all the examined sites and was more often detected in roots than in soil. DNA was detected at up to 80 cm of depth and was spatially more widespread than the initial hypothesis of M. a. paratuberculosis leaching vertically into deeper layers of soil. This study broadens our knowledge of the spread and persistence of M. a. paratuberculosis in an environment with highly infected animals.

• MeSH
• krmivo pro zvířata analýza   mikrobiologie   MeSH
• lipnicovité mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   fyziologie   MeSH
• nemoci skotu mikrobiologie   přenos   MeSH
• paratuberkulóza mikrobiologie   přenos   MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

It has been suggested that passive shedding of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces may occur, but reliable data are missing. Passive shedding assumes the ingestion of MAP in contaminated feed and passive passage through the gastrointestinal tract without causing infection. In this study the presence of MAP in faeces in a closed herd of Limousin cattle was monitored for 53 months using quantitative real time PCR (qPCR) and culture. The initial prevalence of MAP in the herd was determined to be 63.4% and 4.9% using qPCR and culture, respectively. After the removal of two culture- and qPCR-positive (>10(4) MAP cells/g) cows, the prevalence of MAP using qPCR decreased to 42.1% and later to 15.6% and 6.7%. The continuous removal of suspected animals from the herd during the monitoring period minimised the presence of MAP in faeces to sporadic, which may have resulted from a decrease in the environmental infectious pressure. The findings suggest that the presence of low numbers of MAP in bovine faeces may not necessarily be caused by real infection, but rather by passive passage of MAP. This phenomenon should therefore be considered when interpreting MAP qPCR data.

• MeSH
• ELISA veterinární   MeSH
• feces mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce veterinární   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   MeSH
• nemoci skotu epidemiologie   mikrobiologie   MeSH
• paratuberkulóza epidemiologie   mikrobiologie   MeSH
• prevalence MeSH
• roční období MeSH
• skot MeSH
• vylučování bakterií z těla * MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Francie MeSH