Isolation ofMycobacterium paratuberculosisfrom Milk by Immunomagnetic Separation

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Abstract
An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation ofMycobacterium paratuberculosiscells from milk. Rabbit polyclonal antibodies against radiation-killed intactM. paratuberculosiscells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosisIgG-coated beads (IMB) reacted strongly with laboratory strains ofM. paratuberculosisas determined by slide agglutination, and microscopic examination confirmed thatM. paratuberculosiscells attached to the IMB. The IMB were found to have a maximum binding capacity of 104to 105CFU ofM. paratuberculosis. Studies showed that IMS selectively recoveredM. paratuberculosisfrom inoculated milk containing as few as 10 CFU ofM. paratuberculosisper ml when 10 μl of IMB (ca. 106beads) 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 ofM. paratuberculosisfrom 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 ofM. paratuberculosisin milk when it is used in conjunction with end point detection methods, such as IS900PCR or an enzyme-linked immunosorbent assay.