Abstract
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.
Highlights
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s Disease (JD) in ruminants
We have previously demonstrated that transposon Tn5367 inserts relatively random into the MAP genome (Harris et al, 1999)
The full library or a subset of strains were subjected to various individual screens to find strains of interest for studying MAP physiology and pathogenesis, and www.frontiersin.org principally to search for attenuated strains that could be used as first-generation vaccine candidates in animal trials
Summary
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s Disease (JD) in ruminants. Paratuberculosis (MAP) is the etiologic agent of Johne’s Disease (JD) in ruminants. This enteritis has significant economic impact and worldwide distribution (Sweeney, 1996). In the United States, annual losses to the dairy industry have been estimated from 250 million (Ott et al, 1999) to $1.5 billion (Stabel, 1998). Vaccination is one of the most cost effective disease control measures. Though there are JD vaccines that reduce clinical disease and shedding, their efficacies are limited and none afford long-term protective immunity. For example in the United States, Mycopar®
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