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Applied and Environmental Microbiology, September 1998, p. 3153-3158, Vol. 64, No. 9
Department of Food Science (Food
Microbiology),
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.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Isolation of Mycobacterium
paratuberculosis from Milk by Immunomagnetic
Separation
*
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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