Native hosts for the bacterial agent that causes Johne's disease are ruminants, which include cattle, sheep and goats among others. These large animals are often too costly to be used in testing experimental vaccines. In this chapter, we provide detailed methods to use an inexpensive and more manageable animal host, the ferret, to test efficacy and immunogenicity of live-attenuated Mycobacterium avium subspecies paratuberculosis (MAP) mutant strains prior to consideration as vaccine candidates.