A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of

National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden.
Journal of Microbiological Methods (Impact Factor: 2.03). 11/2008; 75(2):335-40. DOI: 10.1016/j.mimet.2008.07.009
Source: PubMed


A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis (MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk. Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was assessed to 100 organisms per ml milk (corresponding to 1-10 CFU per ml) for samples of 10 ml. The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive by environmental culture, while 30% were positive by milk PCR. Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.

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    • "MAP has been identified in many body locations, such as lymph nodes, spleen, liver and animal hide (Wells et al., 2009; Wu et al., 2007) as well as in semen (Sharifzadeh et al., 2010), drinking water (Gill et al., 2011), milk and milk products (Corti and Stephan, 2002; Djønne et al., 2003; Donaghy et al., 2004; Millar et al., 1996). With the progression of the disease, the animal begins shedding MAP in the feces (Herthnek et al., 2008). Therefore, milk could easily get contaminated with high numbers of MAP during teat preparation (Dundee et al., 2001). "
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    ABSTRACT: Prevalence of Mycobacterium avium paratuberculosis in commercially pasteurized milk was studied. A total of 300 commercially pasteurized milk samples were purchased from various parts of Eastern-Azerbaijan province of Iran. Two 50 ml from each sample were centrifuged. DNA extraction was performed on one of the pellets and extracted DNA was evaluated for the presence of Mycobacterium avium paratuberculosis specific IS900 by PCR assay. In order to detect viable cells of the bacterium, the second related pellet was treated with 0.75% HPC and decontaminated samples cultured on two Herrold's egg yolk medium (supplemented with mycobactin J and amphotericin B, nalidixic acid, and vancomycin). Isolated colonies by culture method were confirmed by IS900-based PCR. Although M. avium paratuberculosis DNA was detected in 32 (10.7%) samples by PCR assay, viable bacterium was not isolated by culture method in any sample.
    African journal of microbiology research 01/2012; 6(23):1453--1456. · 0.54 Impact Factor
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    • "For the present study we used a non-mechanical method of cell lysis which was successfully applied for real-time PCR-based detection of the Gram-positive bacterial species Listeria monocytogenes in milk [15]. This protocol includes multiple incubation and washing steps at 45°C in combination with solvents and detergents, and maybe permits the recovery of MAP from milk fat as well [25]. "
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    ABSTRACT: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (Johne's disease) in ruminants and is suggested to be one of the etiologic factors in Crohn's disease in humans. Contaminated milk might expose humans to that pathogen. The aim of the present study was to develop a novel real-time PCR assay providing the additional possibility to detect viable Mycobacterium avium subsp. paratuberculosis (MAP) based on the MAP-specific Mptb52.16 target. The design included an internal amplification control to identify false negative results. Inclusivity and exclusivity tested on 10 MAP strains, 22 non-MAP mycobacteria, and 16 raw milk microflora strains achieved 100%. The detection limit in artificially contaminated raw milk was 2.42 × 101 MAP cells/ml milk. In a survey of naturally contaminated samples obtained from dairy herds with a known history of paratuberculosis, 47.8% pre-milk and 51.9% main milk samples tested positive. Real-time PCR-derived MAP-specific bacterial cell equivalents (bce) ranged from 1 × 100 to 5.1 × 102 bce/51 ml; the majority of samples had less than one bce per ml milk. Expression of the chosen target was detected in artificially contaminated raw milk as well as inoculated Dubos broth, thus confirming the real-time PCR assay's potential to detect viable MAP cells. Concentrating the DNA of a large sample volume in combination with the newly developed real-time PCR assay permitted quantification of low levels of MAP cells in raw milk and pasteurized milk. The selected target - Mptb52.16 - is promising with regard to the detection of viable MAP. Future studies integrating quantitative DNA- and RNA-based data might provide important information for risk assessment concerning the presence of MAP in raw milk and pasteurized milk.
    BMC Research Notes 10/2010; 3(1):251. DOI:10.1186/1756-0500-3-251
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    • "For other pathogens such as MAP, sensitivity of tests conducted on bulk milk is limited (Jayarao et al., 2004). Adjustments in interpretative criteria for ELISA results (Van Weering et al., 2007) or use of real-time PCR (Herthnek et al., 2008) may increase the sensitivity of detection for Johne's disease. When a group of animals is found to be positive based on herd-or string-level tests, subsequent animal-level tests can be used to identify infected animals. "
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    ABSTRACT: Contagious diseases are a threat to animal health and productivity, both nationally and at the farm level. This makes implementation of biosecurity measures to prevent their introduction and spread within countries and farms a necessity. Mastitis is the most common and costly contagious disease affecting dairy farms in the western world. The major mastitis pathogens are endemic in most countries, and biosecurity measures to prevent introduction and transmission must therefore be implemented at farm level. The 40-yr-old mastitis control plan remains a solid foundation to prevent the spread of contagious intramammary infections. Contagious diseases that do not affect the mammary gland directly may have an indirect effect on mastitis. This is true for list A diseases such as foot and mouth disease, for which biosecurity measures may need to be taken at national level, and for other infections with nonmastitis pathogens such as bovine viral diarrhea virus and Mycobacterium avium ssp. paratuberculosis. Maintaining a closed herd decreases the risk of introduction of pathogens that affect udder health directly or indirectly. If animals are purchased, their udder health history should be evaluated and they should be examined and tested for contagious diseases. Transmission of infections by and to humans and nonbovine animals may occur. Contact with visitors and nonbovine animals should therefore be minimized. Because of globalization and heightened consumer awareness, the importance of biosecurity now supersedes individual farms, and increased pressure to control transmission of contagious diseases can be expected at industry or government levels in western countries and elsewhere.
    Journal of Dairy Science 10/2009; 92(10):4717-29. DOI:10.3168/jds.2009-2347 · 2.57 Impact Factor
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