A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk.

National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden.
Journal of Microbiological Methods (Impact Factor: 2.1). 11/2008; 75(2):335-40. DOI: 10.1016/j.mimet.2008.07.009
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Ann Lindberg, Jul 02, 2015
  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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 · 2.55 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.
    Preventive Veterinary Medicine 05/2009; 89(3-4):223-6. DOI:10.1016/j.prevetmed.2009.02.020 · 2.51 Impact Factor