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) 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

We here identified for the first time the presence of Mycobacterium avium paratuberculosis (MAP) sheep (S) strain in Argentina. IS900 polymerase chain reaction (PCR) was positive. The S strain was compared with MAP cattle (C) strains by using IS1311 PCR-restriction endonuclease analysis (PCR-REA), multiplex PCR and restriction fragment length polymorphism (RFLP) analysis.

• MeSH
• DNA bakterií genetika   MeSH
• multiplexová polymerázová řetězová reakce MeSH
• Mycobacterium avium subsp. paratuberculosis klasifikace   genetika   izolace a purifikace   MeSH
• nemoci ovcí diagnóza   mikrobiologie   MeSH
• ovce MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• transpozibilní elementy DNA MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Argentina MeSH

A rapid, cheap and sensitive detection method of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk was needed for routine usage. We developed two duplex real time qPCR systems specific for MAP detection. These real time qPCR assays amplify the multicopy element IS900 for qualitative analysis and the single copy element F57 for quantitative analysis. Both assays incorporate an internal amplification control amplified with the same primers as the targets and the same probes are used in both assays. The specificity of the assays was confirmed by the testing of 6 different MAP isolates, 12 isolates of other mycobacteria or bacterial species and 4 different mammalian DNAs. The sensitivity of the developed assays and isolation efficiency were demonstrated through the analysis of raw milk samples artificially contaminated with MAP cells and with plasmids containing cloned fragments of the targets (IS900 and F57). The developed assays for milk analysis were applied to samples from one farm with two faecal shedding cows. Three hundred and forty five individual milk samples were tested by real time qPCR assays and by cultivation. Hundred and eleven (32.5%) individual milk samples were positive by the real time qPCR, no milk sample was culture positive. The spread of MAP in individual, tank and bulk tank milk samples was also monitored.

• MeSH
• DNA bakterií MeSH
• druhová specificita MeSH
• kontaminace potravin analýza   MeSH
• lidé MeSH
• mléko mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   klasifikace   MeSH
• počet mikrobiálních kolonií metody   MeSH
• polymerázová řetězová reakce metody   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• skot MeSH
• spotřebitelská bezpečnost produktů MeSH
• transpozibilní elementy DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• finanční podpora výzkumu jako téma MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   klasifikace   MeSH
• Mycobacterium avium genetika   izolace a purifikace   klasifikace   MeSH
• paratuberkulóza epidemiologie   mikrobiologie   patologie   MeSH
• tuberkulóza epidemiologie   mikrobiologie   veterinární   MeSH
• vysoká zvěř mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Geografické názvy
• Česká republika MeSH

A survey of 22 farms confirmed the presence of paratuberculosis in wild rabbits in Scotland. Regional differences were apparent in the prevalence of the disease in rabbits, with a significantly higher incidence occurring in the Tayside region. Statistical analysis showed a significant relationship between a previous history or current problem of paratuberculosis in cattle and the presence of paratuberculosis in rabbits on the farms. Molecular genetic typing techniques could not discriminate between selected rabbit and cattle isolates from the same or different farms, suggesting that the same strain may infect and cause disease in both species and that interspecies transmission may occur. The possibility of interspecies transmission and the involvement of wildlife in the epidemiology of paratuberculosis have important implications for the control of the disease.

• MeSH
• divoká zvířata MeSH
• feces mikrobiologie   MeSH
• hospodářská zvířata MeSH
• incidence MeSH
• kozy MeSH
• králíci MeSH
• Mycobacterium avium subsp. paratuberculosis klasifikace   genetika   izolace a purifikace   MeSH
• ovce MeSH
• paratuberkulóza epidemiologie   přenos   MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• skot MeSH
• zeměpis MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Skotsko MeSH