Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis

The high sensitivity of PCR compared to the difficulties of fecal culture i n sheep prompted the development of PCR protocols for detection of Mycobact erium avium subsp. paratuberculosis DNA in sheep feces. Although the PCR it self is well developed, and does nor pose large technical problems, concent rating the bacteria from samples that may contain low numbers of bacilli us ing practical methods is still the main difficulty for the use of this tech nique. In this study, we describe an extraction protocol for the concentrat ion and purification of M. avium subsp, paratuberculosis DNA from fecal sam ples and we compare it with other methods. The diagnostic performance of th e freeze-boiling method was evaluated using a reference method [Vet. Rec. 1 34 (1994) 95] on fecal samples from a group of selected sheep from differen t flocks of known individual serological, pathological, and cultural paratu berculosis status. Using, as a reference, a combination of results in those conventional methods, the freeze-boiling PCR protocol showed a sensitivity of 94.1%, and a specificity of 92.3%. (C) 2000 Elsevier Science B.V. All r ights reserved.