Introduction
The study of Mycobacterium avium subsp. paratuberculosis (MAP) pathogenesis and the identification and functional characterization of virulence determinants requires the use of molecular genetic approaches.
This task is challenging since MAP is one of the slowest-growing mycobacterial species, requiring 3 weeks or more to observe colonies on Middlebrook agar supplemented with mycobactin J. In the previous edition of this book, we described the early development of genetic systems to introduce specific gene mutations to define pathogenic determinants. It is now possible to inactivate any non-essential gene by allelic exchange or to generate libraries of transposon mutants for genome-wide analysis. The identification of replicating vectors also allows the introduction of the wild-type gene into mutant strains to fulfil molecular Koch's postulates (Falkow, 2004). In this chapter, after briefly reviewing the molecular tools developed prior to 2009, we will focus on the main advances in the field to finetune the strategies for the genetic manipulation of MAP.7.2
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