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, Bacterial classification   genetics   MeSH
• Feces chemistry   microbiology   MeSH
• Real-Time Polymerase Chain Reaction standards   MeSH
• Freeze Drying MeSH
• Mycobacterium avium subsp. paratuberculosis classification   genetics   isolation & purification   MeSH
• Cattle Diseases diagnosis   MeSH
• Paratuberculosis diagnosis   microbiology   MeSH
• Reference Standards MeSH
• Sensitivity and Specificity MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Asymptomatic Infections * MeSH
• Bacteriological Techniques methods   MeSH
• Molecular Diagnostic Techniques methods   MeSH
• Feces microbiology   MeSH
• Real-Time Polymerase Chain Reaction methods   MeSH
• Mycobacterium avium subsp. paratuberculosis classification   genetics   growth & development   isolation & purification   MeSH
• Cattle Diseases diagnosis   MeSH
• Specimen Handling methods   MeSH
• Paratuberculosis diagnosis   MeSH
• Sensitivity and Specificity MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Comparative Study MeSH
• Geographicals
• Netherlands MeSH

The main reasons to improve the detection ofMycobacterium aviumsubsp.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, Bacterial isolation & purification   MeSH
• Feces microbiology   MeSH
• Milk microbiology   MeSH
• Mycobacterium avium subsp. paratuberculosis isolation & purification   pathogenicity   MeSH
• Cattle Diseases diagnosis   genetics   microbiology   MeSH
• Paratuberculosis diagnosis   microbiology   MeSH
• Food Microbiology MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review 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
• Enzyme-Linked Immunosorbent Assay MeSH
• Goats MeSH
• Milk microbiology   MeSH
• Mycobacterium avium subsp. paratuberculosis genetics   immunology   isolation & purification   MeSH
• Goat Diseases diagnosis   epidemiology   microbiology   MeSH
• Paratuberculosis diagnosis   epidemiology   microbiology   MeSH
• Polymerase Chain Reaction MeSH
• Cheese microbiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Italy epidemiology   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, Bacterial genetics   MeSH
• Multiplex Polymerase Chain Reaction MeSH
• Mycobacterium avium subsp. paratuberculosis classification   genetics   isolation & purification   MeSH
• Sheep Diseases diagnosis   microbiology   MeSH
• Sheep MeSH
• Paratuberculosis diagnosis   microbiology   MeSH
• Polymorphism, Restriction Fragment Length MeSH
• DNA Transposable Elements MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Argentina MeSH

The aim of this study was to assess the suitability of real-time quantitative PCR (qPCR) for the detection of Mycobacterium avium ssp. paratuberculosis (MAP) in milk filters as a herd level indicator of paratuberculosis infection. Seventy-nine samples from textile or metal milk filters from 15 herds with defined MAP prevalence (infection status = noninfected, 0-5%, 5-10%, or >10% of animals with clinically confirmed paratuberculosis) were analyzed. The MAP DNA was isolated by a modified commercially available protocol for feces, and detection and quantification of the pathogen was performed by the IS900 qPCR. Mycobacterium avium ssp. paratuberculosis DNA was detected in 63 (79.7%) samples. Determination of MAP infection established by fecal and tissue culture was correctly confirmed by the analysis of milk filters on 11 of 12 infected farms; MAP was not detected in filters from 3 farms where paratuberculosis was never diagnosed. Statistical analysis of the data supports the evidence that milk filters can be used as a template for the direct detection of MAP on the herd level. The probability of successful MAP detection in milk filters in a herd with MAP-infected cows is at least 94.3%. Absolute numbers of MAP detected on the milk filter can be used for a rough estimation of paratuberculosis prevalence >10% in the herd. Analysis of milk filters for the presence of MAP can be a useful tool for the detection of paratuberculosis on the herd level before any individual control strategies.

• MeSH
• DNA, Bacterial genetics   MeSH
• Filtration veterinary   MeSH
• Real-Time Polymerase Chain Reaction methods   veterinary   MeSH
• Milk microbiology   MeSH
• Mycobacterium avium subsp. paratuberculosis genetics   MeSH
• Cattle Diseases diagnosis   microbiology   MeSH
• Paratuberculosis diagnosis   MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Different approaches are used for determining the number of Mycobacterium avium subsp. paratuberculosis (MAP) cells in a suspension. The majority of them are based upon culture (determination of CFU) or visual/instrumental direct counting of MAP cells. In this study, we have compared the culture method with a previously published F57 based quantitative real-time PCR (F57qPCR) method, to determine their relative abilities to count the number of three different MAP isolates in suspensions with the same optical densities (OD). McFarland turbidity standards were also compared with F57qPCR and culture, due to its frequent inclusion and use in MAP studies. FINDINGS: The numbers of MAP in two-fold serial dilutions of isolates with respective OD measurements were determined by F57qPCR and culture. It was found that culture provided lower MAP CFU counts by approximately two log10, compared to F57qPCR. The McFarland standards (as defined for E. coli) showed an almost perfect fit with the enumeration of MAP performed by F57qPCR. CONCLUSIONS: It is recommended to use culture and/or qPCR estimations of MAP numbers in experiments where all subsequent counts are performed using the same method. It is certainly not recommended the use of culture as the standard for qPCR experiments and vice versa.

• MeSH
• Bacterial Load methods   MeSH
• Densitometry MeSH
• DNA, Bacterial analysis   MeSH
• Escherichia coli cytology   MeSH
• Feces microbiology   MeSH
• Culture Media MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Mycobacterium avium subsp. paratuberculosis cytology   genetics   isolation & purification   MeSH
• Cattle Diseases diagnosis   microbiology   MeSH
• Paratuberculosis diagnosis   microbiology   MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Bacteriological Techniques methods   MeSH
• Housing, Animal MeSH
• DNA, Bacterial genetics   MeSH
• Animals, Domestic MeSH
• Infection Control methods   MeSH
• Real-Time Polymerase Chain Reaction methods   MeSH
• Environmental Microbiology MeSH
• Mycobacterium avium subsp. paratuberculosis genetics   growth & development   isolation & purification   MeSH
• Cattle Diseases diagnosis   microbiology   MeSH
• Paratuberculosis diagnosis   microbiology   MeSH
• Plants microbiology   MeSH
• Cattle MeSH
• DNA Transposable Elements MeSH
• Veterinary Medicine methods   MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis in ruminants, has a lipid-rich cell wall which facilitates its survival and persistence in the environment. This property of the organism is exploited when it is cultured as decontaminating agents and antibiotics are used to suppress the growth of contaminating microflora, but such treatments can also negatively affect the isolation of MAP itself. The objective of this study was to assess the effect of the 'VAN' antibiotics (vancomycin, amphotericin B and nalidixic acid) on the viability of MAP using a propidium monoazide real time quantitative PCR (PMA qPCR) and culture. Long-term (5 week) treatment with VAN antibiotics resulted in a larger decrease in bacterial numbers compared to short-term (3 day) exposure. The PMA qPCR assay indicated that 50 μg/mL of vancomycin, 50 μg/mL of nalidixic acid, and 200 μg/mL of amphotericin B were 'threshold' concentrations, respectively, above which the decline in the viability of MAP was statistically significant. Using culture, these threshold concentrations were 100 μg/mL of vancomycin, 50-100 μg/mL of nalidixic acid, and 100 μg/mL of amphotericin B, respectively. Given that the two methods were found to be comparable, the PMA qPCR is a potentially more convenient and effective alternative to culture in detecting MAP.

• MeSH
• Amphotericin B pharmacology   MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Azides metabolism   MeSH
• Time Factors MeSH
• Real-Time Polymerase Chain Reaction methods   MeSH
• Nalidixic Acid pharmacology   MeSH
• Mycobacterium avium subsp. paratuberculosis drug effects   growth & development   isolation & purification   metabolism   MeSH
• Paratuberculosis diagnosis   microbiology   MeSH
• Colony Count, Microbial methods   MeSH
• Ruminants microbiology   MeSH
• Propidium analogs & derivatives   metabolism   MeSH
• Vancomycin pharmacology   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

This study focused on the development of a reliable and cost-efficient DNA isolation procedure for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces by previously developed IS900 and F57 quantitative real time PCR (qPCR) and their comparison with culture. The recovery of MAP DNA from the spiking experiments ranged from 29.1 to 102.4% of the input amount of MAP with median 37.9%. The limit of detection was determined to be 1.03 × 10(4) for F57 qPCR and 6.87 × 10(2)MAP cells per gram of faeces for IS900 qPCR, respectively. The developed technique for DNA isolation was coupled with IS900 qPCR and compared to traditional MAP culture using a cohort of 1906 faecal samples examined from 12 dairy cattle farms in our laboratory. From those 1906 original faecal samples, 875 were positive by IS900 qPCR and 169 by culture. None of the culture positive samples was negative by IS900 qPCR. This data facilitated development of a predictive model capable of estimating the probability of being culture positive by estimating the absolute number of MAP per gram of faeces as determined IS900 qPCR without performing the culture.

• MeSH
• DNA, Bacterial analysis   isolation & purification   MeSH
• Feces microbiology   MeSH
• Limit of Detection MeSH
• Logistic Models MeSH
• Mycobacterium avium subsp. paratuberculosis genetics   isolation & purification   MeSH
• Cattle Diseases diagnosis   microbiology   MeSH
• Paratuberculosis diagnosis   microbiology   MeSH
• Polymerase Chain Reaction methods   veterinary   MeSH
• Sensitivity and Specificity MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH