For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9% in Cluster R and 12.6% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis, M. fortuitum and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.

• Klíčová slova
• decontamination, mycobacteria other than tuberculosis (MOTT), non-tuberculous mycobacteria (NTM), quaternary ammonium compounds, saprophytic environmental mycobacteria,
• Publikační typ
• časopisecké články MeSH

The objective of the present study was to evaluate the suitability of environmental sampling to screen Czech dairy herds to detect Mycobacterium avium ssp. paratuberculosis (MAP) and to find the most convenient location for the MAP detection in the lactating cow area. Environmental samples (ES, n = 72) from milking parlour holding pens (n = 19), milking alleyways (n = 19) and free-stall alleyways (n = 34) from 19 herds were simultaneously tested to detect MAP by a quantitative PCR (qPCR) and bacterial culture. Eight and thirteen samples from the milking parlour holding pens, twelve and eleven samples from the milking alleyways and eleven and eighteen samples from the free-stall alleyways were qPCR and culture positive, respectively. A 4.6 times higher probability of being culture positive than qPCR positive was detected for the assessable MAP detection results from the free-stall alleyways [P = 0.008 6, odds ration (OR) = 4.572 8)] and no association was found between the results from the milking parlour holding pens (P = 0.191 4) and the milking alleyways (P > 0.999 9) and the diagnostic method used. The percentage of qPCR-positive samples in the tested locations was detected for the milking alleyways (63.2%), free-stall alleyways and milking parlour holding pens. The herd infectious status was in agreement with 16 (84.2%), 14 (73.7%) and 12 (63.2%) qPCR results from the milking alleyways, free-stall alleyways (32.4%) and milking parlour holding pens (42.1%), respectively. No statistically significant differences were detected for these results (P = 0.396 1). MAP was detected by the qPCR and bacterial culture in all three locations where the ES were collected. We suggest an environmental sampling followed by MAP detection by qPCR as an easy-to-perform time-saving protocol for MAP screening in Czech dairy herds. Although the milking alleyways seem to be the most convenient location for the environmental sampling, this assumption was not statistically supported.

• Klíčová slova
• Johne’s disease, faecal culture, lactating cow, quantitative PCR, sampling location,
• Publikační typ
• časopisecké články 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

Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pathogens in a sample is derived. Spectrophotometric measurement of absorbance is a routine method for estimating nucleic acid concentration, however, it may be affected by presence of other potentially contaminating nucleic acids or proteins and salts. Therefore, absorbance measurement is not reliable for estimating the concentration of stock solutions of quantification standards, based on which they are subsequently diluted. In this study, we utilized digital PCR (dPCR) for absolute quantification of qPCR plasmid standards and thus detecting possible discrepancies in the determination of the plasmid DNA number of standards derived from UV spectrophotometry. The concept of dPCR utilization for quantification of standards was applied on 45 qPCR assays using droplet-based and chip-based dPCR platforms. Using dPCR, we found that spectrophotometry overestimated the concentrations of standard stock solutions in the majority of cases. Furthermore, batch-to-batch variation in standard quantity was revealed, as well as quantitative changes in standards over time. Finally, it was demonstrated that droplet-based dPCR is a suitable tool for achieving defined quantity of quantification plasmid standards and ensuring the quantity over time, which is crucial for acquiring homogenous, reproducible and comparable quantitative data by qPCR.

• Klíčová slova
• absolute quantification, digital PCR, qPCR, quantification plasmid standard, quantity verification, real time PCR,
• Publikační typ
• časopisecké články MeSH

Cell-free supernatants (CFSs) extracted from various lactic acid bacteria (LAB) cultures were applied to Mycobacterium avium subsp. paratuberculosis (MAP) cells to determine their effect on MAP viability. In addition, 5% lactic acid (LA; pH 3) and commercially synthetized nisin bacteriocin were also tested. This procedure was chosen in order to mimic the influence of LAB compounds during the production and storage of fermented milk products, which can be contaminated by MAP. Its presence in milk and milk products is of public concern due to the possible ingestion of MAP by consumers and the discussed role of MAP in Crohn's disease. Propidium monoazide real-time PCR (PMA qPCR) was used for viability determination. Although all CFS showed significant effects on MAP viability, two distinct groups of CFS - effective and less effective - could be distinguished. The effective CFSs were extracted from various lactobacilli cultures, their pH values were mostly lower than 4.5, and their application resulted in >2 log10 reductions in MAP viability. The group of less effective CFS were filtered from Lactococcus and enterococci cultures, their pH values were higher than 4.5, and their effect on MAP viability was <2 log10. LA elicited a reduction in MAP viability that was similar to that of the group of less effective CFS. Almost no effect was found when using commercially synthetized nisin at concentrations of 0.1-1000 μg/ml. A combination of the influence of the type of bacteriocin, the length of its action, bacteriocin production strain, and pH are all probably required for a successful reduction in MAP viability. However, certain bacteriocins and their respective LAB strains (Lactobacillus sp.) appear to play a greater role in reducing the viability of MAP than pH.

• Klíčová slova
• bacteriocin, cell-free supernatant, lactic acid, pH, paratuberculosis, propidium monoazide, real-time PCR, viability,
• 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

Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety. Although the concept of PCR is relatively simple, there are specific issues in qPCR that developers and users of this technology must bear in mind. These include the use of correct terminology and definitions, understanding of the principle of PCR, difficulties with interpretation and presentation of data, the limitations of qPCR in different areas of microbial diagnostics and parameters important for the description of qPCR performance. It is not our intention in this review to describe every single aspect of qPCR design, optimization, and validation; however, it is our hope that this basic guide will help to orient beginners and users of qPCR in the use of this powerful technique.

• Klíčová slova
• accuracy, limit of detection, limit of quantification, precision, quantitative PCR, trueness,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

There is no European legislation concerning paratuberculosis that requires that imported animals be kept in quarantine and commonly they are directly released into areas with other animals. In this study, detection of latent infection of paratuberculosis in healthy mouflons previously diagnosed as paratuberculosis-free, but originating from a real time quantitative PCR- (qPCR-) positive herd, occurred after their transport to a new farm. During a twelve-day quarantine period, all mouflons irregularly shed Mycobacterium avium subsp. paratuberculosis (MAP) in faeces, and in a small number of cases also in milk. After the animals were released from quarantine, MAP was detected for a further two days, after which, testing was negative, except in one case. Therefore, the stress connected with transport, novel environment, dietary change, or limited area with high density of animals might have contributed to the induction of paratuberculosis and the shedding of MAP from the animals, previously diagnosed as MAP-negative. According to these results, the keeping of imported animals in quarantine and their examination for MAP presence not only before the transport but also afterwards should be recommended. The designation of a particular area of a farm as a quarantine enclosure could help to mitigate the impact of stress caused by a confined space with a high density of animals.

• Publikační typ
• časopisecké články MeSH

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.

• MeSH
• bakteriologické techniky metody   MeSH
• bydlení zvířat MeSH
• DNA bakterií genetika   MeSH
• hospodářská zvířata MeSH
• kontrola infekce metody   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mikrobiologie životního prostředí * MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   růst a vývoj   izolace a purifikace   MeSH
• nemoci skotu diagnóza   mikrobiologie   MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• rostliny mikrobiologie   MeSH
• skot MeSH
• transpozibilní elementy DNA MeSH
• veterinární lékařství metody   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• transpozibilní elementy DNA MeSH

In this study, products from all steps of anaerobic digestion at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle were examined and quantified for the presence of the causal agent of paratuberculosis, Mycobacterium avium subsp. paratuberculosis, using culture and quantitative real-time PCR (qPCR). Viable M. avium subsp. paratuberculosis cells were detected using culture in fermentors for up to 2 months; the presence of M. avium subsp. paratuberculosis DNA (10(1) cells/g) was demonstrated in all anaerobic fermentors and digestate 16 months after initiation of work at a biogas plant, using IS900 qPCR. F57 qPCR was able to detect M. avium subsp. paratuberculosis DNA (10(2) cells/g) at up to 12 months. According to these results, a fermentation process that extended beyond 2 months removed all viable M. avium subsp. paratuberculosis cells and therefore rendered its product M. avium subsp. paratuberculosis free. However, M. avium subsp. paratuberculosis DNA was found during all the examined periods (more than 1 year), which could be explained by either residual DNA being released from dead cells or by the presence of viable cells whose amount was under the limit of cultivability. As the latter hypothesis cannot be excluded, the safety of the final products of digestion used for fertilization or animal bedding cannot be defined, and further investigation is necessary to confirm or refute this risk.

• MeSH
• anaerobióza MeSH
• bakteriální nálož metody   MeSH
• biopaliva * MeSH
• bioreaktory mikrobiologie   MeSH
• časové faktory MeSH
• DNA bakterií genetika   izolace a purifikace   MeSH
• hnůj mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   růst a vývoj   izolace a purifikace   MeSH
• nemoci skotu mikrobiologie   MeSH
• paratuberkulóza mikrobiologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• skot MeSH
• transpozibilní elementy DNA MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biopaliva * MeSH
• DNA bakterií MeSH
• hnůj MeSH
• transpozibilní elementy DNA MeSH