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On the basis of DNA-DNA hybridization and numerical taxonomy analysis, the mycobacterial species Mycobacterium avium is subdivided into three subspecies, M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, which share extensive sequence identity (8). Nevertheless, these subspecies can be differentiated from each other on the basis of host range, mycobactin dependence, and the presence of specific insertion elements (17). M. avium subsp. paratuberculosis causes Johne's disease, a severe gastroenteritis in ruminants, with a significant impact on the agricultural economy, particularly the dairy industry (17). In the Belgian cattle population, paratuberculosis prevalence was determined by a serological survey, conducted from December 1997 to March 1998. This approach resulted in an estimated true herd prevalence of M. avium subsp. paratuberculosis infection of 6% (6). Dairy cattle usually start fecal shedding at 2 years of age and develop clinical symptoms around 4 years of age. Infection with M. avium subsp. paratuberculosis is commonly acquired early in life via the fecal-oral route through the ingestion of contaminated colostrum, milk, water, or feed (46) and possibly through intrauterine transmission (42). M. avium subsp. paratuberculosis is extremely robust, and bacteria were reported to survive up to 250 days in water and feces and on pastures (27).Cell-mediated immune responses seem to control the initial infection for a sustained period of time, and clinical symptoms only appear in cows after a number of years, often after the first or second calving, possibly because of enhanced intracellular multiplication of M. avium subsp. paratuberculosis organisms caused by alterations in the hormonal milieu (15). Decreased cell-mediated responses are likely related to a loss of antigen-specific CD4 ϩ T cells, which is most prominent in the ileum lesions from symptomatic animals (24). Also, Khalifeh et al. demonstrated that transforming growth factor ␤ and interleukin-10 (IL-10) mRNA levels are higher in cows that have progressed to the clinical stage of the disease, compared to subclinically infected or healthy cows (23). It is not clear for the moment, whether this reflects a shift from a Th1-to a Th2-biased immune response or rather the development of a regulatory T-cell circuit (10). Vaccines consisting of whole killed or attenuated live M. avium subsp. paratuberculosis bacilli can provide partial protection by delaying fecal shedding and reducing the number of clinically affected animals, but they do not protect against infection. In the context of bovine tuberculosis (M. bovis) control and eradication programs, it is worth mentioning that animals immunized with these paratuberculosis vaccines develop positive reactions in the tuberculin skin test (the reference bovine tuberculosis detection method), and therefore paratuberculosis vaccination is subject to approval by

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Paratuberculosis is a chronic enteritis caused in domestic and wild ruminant species by Mycobacterium avium subsp. paratuberculosis (MAP) that is responsible for major economic losses to the agricultural industry. To date, no satisfactory therapeutic, vaccine, or diagnostic tools are available, globally impairing all control programs. In this study, we have undertaken a large-scale postgenomic analysis of MAP proteins, to identify specific antigens that could potentially improve the diagnosis of paratuberculosis. Two complementary approaches were implemented, the first one consisting in the systematic proteomic identification of proteins present in MAP culture filtrates (CFs), followed by the selection of MAP-specific proteins by BLAST query on available mycobacterial genomes. The resulting database represents the first established secretome of MAP and a useful source of potentially specific antigens. The second approach consisted in the immunoproteomic analysis of both MAP extracts and CFs, using sera from MAP-infected and uninfected cattle. Combining results obtained with both approaches resulted in the identification of 25 candidate diagnostic antigens. Five of these were tested in an ELISA assay for their diagnostic potential, on a limited panel of field sera, and the combination of three of them competed in performance with available commercial assays, reaching a test sensitivity of 94.74% and specificity of 97.92%.

There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an efficacious BU vaccine.