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Applied and Environmental Microbiology, September 1998, p. 3153-3158, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Isolation of Mycobacterium paratuberculosis from Milk by Immunomagnetic Separation

Irene R. Grant,1,* Hywel J. Ball,2 and Michael T. Rowe1

Department of Food Science (Food Microbiology), The Queen's University of Belfast, Belfast BT9 5PX,1 and Veterinary Sciences Division, Department of Agriculture for Northern Ireland, Belfast BT4 3SD,2 United Kingdom

Received 3 November 1997/Accepted 10 June 1998

An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium paratuberculosis cells from milk. Rabbit polyclonal antibodies against radiation-killed intact M. paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. paratuberculosis as determined by slide agglutination, and microscopic examination confirmed that M. paratuberculosis cells attached to the IMB. The IMB were found to have a maximum binding capacity of 104 to 105 CFU of M. paratuberculosis. Studies showed that IMS selectively recovered M. paratuberculosis from inoculated milk containing as few as 10 CFU of M. paratuberculosis per ml when 10 µl of IMB (ca. 106 beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline-0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation of M. paratuberculosis from a milk sample relies on chemical decontamination, followed by culturing on Herrold's egg yolk medium, which must be incubated at 37°C for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of M. paratuberculosis in milk when it is used in conjunction with end point detection methods, such as IS900 PCR or an enzyme-linked immunosorbent assay.

* Corresponding author. Mailing address: Department of Food Science (Food Microbiology), The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, United Kingdom. Phone: 44 1232 255299. Fax: 44 1232 668376. E-mail: I.Grant{at}qub.ac.uk.

Applied and Environmental Microbiology, September 1998, p. 3153-3158, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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