SUMMARY: Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures.

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log ₁₀ CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log ₁₀ CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log ₁₀ CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log ₁₀ CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas , Clavibacter , Curtobacterium , and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum . These microorganisms may be useful for biocontrol and other applications.

D- Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes d- alanine racemase (Alr) and d- alanine: d- alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis . We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of d- alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of l- and d- alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of l- over d- alanine. In all strains, DCS treatment led to a significant accumulation of l- alanine and a concomitant decease of d- alanine, with approximately a 20-fold excess of l- alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the d- alanine branch of mycobacterial peptidoglycan biosynthesis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants causing chronic diarrhea, malnutrition and muscular wasting. Neonates and young animals are infected primarily by the fecal-oral route. MAP attaches to, translocates via the intestinal mucosa, and is phagocytosed by macrophages. The ensuing host cellular immune response leads to granulomatous enteritis characterized by a thick and corrugated intestinal wall. We review various tissue culture systems, ileal loops, and mice, goats and cattle used to study MAP pathogenesis. MAP can be detected in clinical samples by microscopy, culturing, PCR and an enzyme-linked immunosorbent assay. There are commercial vaccines that reduce clinical disease and shedding, unfortunately, their efficacies are limited and may not engender long-term protective immunity. Moreover, the potential linkage with Crohn’s disease and other human diseases makes MAP a concern as a zoonotic pathogen. Potential therapies with anti-mycobacterial agents are also discussed. The completion of the MAP K-10 genome sequence has greatly improved our understanding of MAP pathogenesis. The analysis of this sequence has identified a wide range of gene functions involved in virulence, lipid metabolism, transcriptional regulation and main metabolic pathways. We also review the transposons utilized to generate random transposon mutant libraries and the recent advances in the post-genomic era. This includes the generation and characterization of allelic exchange mutants, transcriptomic analysis, transposon mutant banks analysis, new efforts to generate comprehensive mutant libraries and the application of transposon site hybridization mutagenesis (TraSH) and transposon sequencing (Tn-Seq) for global analysis of the MAP genome. Further analysis of candidate vaccine strains development is also provided with critical discussions on their benefits and shortcomings, and strategies to develop a highly efficacious live-attenuated vaccine capable of differentiating infected from vaccinated animals (DIVA).

Mycobacterium aviumsubsp.paratuberculosis, the agent of Johne's disease, infects ruminant hosts by translocation through the intestinal mucosa. A number of studies have suggested thatM. aviumsubsp.paratuberculosisinteracts with M cells in the Peyer's patches of the small intestine. The invasion of the intestinal mucosa byM. aviumsubsp.paratuberculosisandMycobacterium aviumsubsp.hominissuis, a pathogen known to interact with intestinal cells, was compared.M. aviumsubsp.paratuberculosiswas capable of invading the mucosa, but it was significantly less efficient at dissemination thanM. aviumsubsp.hominissuis. B-cell knockout (KO) mice, which lack Peyer's patches, were used to demonstrate thatM. aviumsubsp.paratuberculosisenters the intestinal mucosa through enterocytes in the absence of M cells. In addition, the results indicated thatM. aviumsubsp.paratuberculosishad equal abilities to cross the mucosa in both Peyer's patch and non-Peyer's patch segments of normal mice.M. aviumsubsp.paratuberculosiswas also shown to interact with epithelial cells by an α₅β₁integrin-independent pathway. Upon translocation, dendritic cells ingestM. aviumsubsp.paratuberculosis, but this process does not lead to efficient dissemination of the infection. In summary,M. aviumsubsp.paratuberculosisinteracts with the intestinal mucosa by crossing both Peyer's patches and non-Peyer's patch areas but does not translocate or disseminate efficiently.