ISOLATION OF MYCOBACTERIUM-PARATUBERCULOSIS FROM MILK BY IMMUNOMAGNETIC SEPARATION

An immunomagnetic separation (IMS) technique was developed to facilita te selective isolation of Mycobacterium paratuberculosis cells from mi lk Rabbit polyclonal antibodies against radiation-killed intact M. par atuberculosis cells were produced and used to coat sheep anti-rabbit i mmunoglobulin G (IgG) type M-280 Dyna-beads. The rabbit anti-ill. para tuberculosis IgG-coated beads (IMB) reacted strongly with laboratory s trains of M. paratuberculosis as determined by slide agglutination, an d microscopic examination confirmed that M. paratberculosis cells atta ched to the IMB. The IMB were found to have a maximum binding capacity of 10(4) to 10(5) CFU of M paratuberculosis. Studies showed that IMS selectively recovered M paratuberculosis from inoculated milk containi ng as few as 10 CFU of M. paratuberculosis per mi when 10 mu l of IMB (ca. 10(6) beads) was added to 1 mi of milk and the preparation was in cubated for 30 min at room temperature with gentle agitation. Larger v olumes of milk (10 and 50 mi) were centrifuged and resuspended in 1 mi of phosphate-buffered saline-0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation o f M paratuberculosis from a milk sample relies on chemical decontamina tion, followed by culturing on Herrold's egg yolk medium, which must b e incubated at 37 degrees C for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of ill. paratuberculo sis in milk when it is used in conjunction with end point detection me thods, such as IS900 PCR or an enzyme-linked immunosorbent assay.