Host immune responses to ribosome, ribosomal proteins, and RNA from Mycobacterium bovis bacille de Calmette-Gúerin

The ribosomes from BCG strongly induced delayed type hypersensitivity (DTH) skin reactions in guinea pigs immunized with live BCG or heat killed Mycobacterium tuberculosis H37Rv, and also induced lymphocyte proliferative response in mice immunized with ribosomes. In contrast, neither ribosomal proteins nor RNA alone induced both DTH skin reactions and lymphocyte proliferative responses. Particle form consisted of ribosomal proteins and RNAs might be absolutely required for the activation of immune responses.     

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In 80% of cases, antituberculosis antibodies from the sera of patients with tuberculosis were ascertained to react in enzyme immunoassay (EIA) with antigens (ultrasound disintegrants (USDs)) of reverse mycobacteria isolated (initially) from patients with sarcoidosis. The USDs of reverse mycobacteria isolated from patients with sarcoidosis reacted in EIA with monoclonal antibodies (MAb) against M. tuberculosis complex antigens (unique and crossover). Both common and distinctive (unique) antigenic determinants were detected via MAb against different mycobacterial types by immunoblotting in the antigenic complexes of M. tuberculosis H37Rv and reverse strains isolated from patients with sarcoidosis.     

Growth of virulent and avirulent Mycobacterium tuberculosis strains in human macrophages

Mycobacterium tuberculosis H37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth of M. tuberculosis strains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics of M. tuberculosis strains in animal models are relevant to human tuberculosis infection.     

Characteristics and outcome of anti-glomerular basement membrane disease: a single-center experience

The relative virulence and avirulence of Mycobacterium tuberculosis strains H37Rv and H37Ra were previously defined using animal infection models. To investigate host species' specificity of mycobacterial virulence, growth of the 2 M. tuberculosis strains in human monocyte-derived macrophages in vitro was studied. Mycobacterial growth was evaluated by acid-fast staining, electron microscopy, and colony-forming units (cfu) assay. As expected, the 2 strains demonstrated significantly different growth rates in mouse macrophages in vitro (53 h for H37Rv, 370 h for H37Ra). In marked contrast, in human macrophages the average division times of the strains were nearly equal (80 h for H37Rv and 76 h for H37Ra by cfu measurement, and 96 h for H37Rv and 104 h for H37Ra by acid-fast staining). These findings indicate that observations of mycobacterial virulence in murine systems may not necessarily translate to the human system, in which different mechanisms to control mycobacterial growth may be expressed.     

Differences in cell-mediated immune responses of 'high-resistance' and 'low-resistance' mice to a nonpathogenic mycobacterium

The kinetics of the footpad response of bacillus Calmette-Guérin (BCG)-infected mice to soluble BCG antigens were compared in two strains of mice with different degrees of susceptibility to Mycobacterium lepraemurium. For the first 21 days the responses of the 'high-resistance' C57BL and the 'low-resistance' BALB/c to the nonpathogenic BCG were similar to that produced when the pathogenic mycobacterium was used. After 4 weeks the kinetics of the BALB/c mice changed and resembled that of the C57BL mice. The change in kinetics was compared with circulating antimycobacterial antibody levels and the response of draining lymph node cells in the antigen-specific lymphocyte transformation test. A dissociation was found between the kinetics of the delayed footpad response and the lymphocyte transformation response. Since both strains of mice are equally resistant to BCG, it appears that the delayed footpad response cannot be used as an indicator of host resistance in all mycobacterial infections.     

Genetic vaccination against Coccidioides immitis: comparison of vaccine efficacy of recombinant antigen 2 and antigen 2 cDNA

Previous studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls of Coccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathione S-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge with C. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses to C. immitis antigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge with C. immitis.     

Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis

Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.     

Dietary vitamin D affects cell-mediated hypersensitivity but not resistance to experimental pulmonary tuberculosis in guinea pigs

Outbred, Hartley strain guinea pigs were fed purified diets varying only in their levels of vitamin D. The amounts of vitamin D in the diets were adjusted to represent 0, 25, 50, 100, or 200% of the recommended level (1,180 IU/kg of body weight) for guinea pigs. In some experiments, half of the animals in each diet group were vaccinated with Mycobacterium bovis BCG vaccine at the time the diets were introduced. Six weeks later, all guinea pigs were infected by the respiratory route with a low dose of virulent M. tuberculosis H37Rv. Vitamin D-deficient animals exhibited marked reductions in levels of the major vitamin D metabolite, 25-hydroxyvitamin D3, in plasma. Altered vitamin D intake was accompanied by changes in antigen (purified protein derivative)-induced, cell-mediated immune responses both in vivo (tuberculin hypersensitivity) and in vitro (lymphoproliferation). Dermal tuberculin reactivity developed more slowly in vitamin D-deficient guinea pigs but eventually achieved normal levels. The proliferation of splenocytes cultured with purified protein derivative was suppressed by both deficiency and excess of dietary vitamin D. Vitamin D status did not affect the abilities of naive guinea pigs to control primary, pulmonary tuberculosis, nor did it influence the protective efficacy of BCG vaccination. We conclude that changes in dietary vitamin D are associated with alterations in some cellular immune functions but may not be an important determinant of disease outcome in pulmonary tuberculosis, as has been suggested previously.     

Antimycobacterial activity of substituted isosteres of pyridine- and pyrazinecarboxylic acids

Pyrazines and pyridines substituted with alkylated tetrazoles, esterified vinylogous carboxylic acids, and ketosulfides were synthesized as precursors of antimycobacterial agents which, after penetration of the mycobacterial cell wall, could be biotransformed by esterases or peroxidase-catalases. The expected products are tetrazoles, a vinylogous carboxylic acid, and CH-acidic ketosulfoxides, isosteres of pyrazinoic and nicotinic acids, which should inhibit mycobacterial growth when released inside the bacterial cell. The growth inhibitory activity of the synthesized compounds against the H37Rv strain of Mycobacterium tuberculosis was determined to assess the viability of this concept. It was shown that all of the compounds designed as lipophilic precursors were more active than the unmodified polar isosteres of pyrazinoic and nicotinic acids.     

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Sixty two and 59 patients with tuberculous and nontuberculous salpingo-oophoritis, respectively, and 30 healthy females were examined. It was found that indirect solid-phase enzyme immunoassay (EIA) using ultrasonography of Mycobacterium tuberculosis H37Rv could detect antituberculosis antibodies in 66.7% of patients with tuberculous salpingo-oophoritis, and in 10% of healthy females. That using the antigen isolated from the cell wall extract of M. tuberculosis (whose molecular weight was 38 - 42 kD) could reveal them in 70.2, 4.3%, and 6.7%, respectively. After dissociation of immune complexes, EIA inhibition using affinity-purified antimycobacterial antisera displayed mycobacterial antigens in 75.0 and 4.3% of patients with tuberculous and nontuberculous salpingo-oophoritis, respectively, and in none healthy female. Thus, the detection of mycobacterial antigens and antituberculosis antibody may be successfully used in the complex diagnosis of tuberculosis of the female genitals.     

Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig

Two antigens from Mycobacterium tuberculosis, ESAT-6 and MPT64, elicited delayed-type hypersensitivity (DTH) skin responses in outbred guinea pigs infected with M. tuberculosis by the aerosol and intravenous routes but not those sensitized with M. bovis BCG or M. avium. The DTH epitope of ESAT-6 was mapped to the C terminus. Nonresponders to the individual antigens were found, but all animals responded to a combination of ESAT-6 and MPT64 or their respective minimal target peptides. Correspondingly, these molecules could form the basis of a new skin test for tuberculosis.     

Mycobacterium tuberculosis invades and replicates within type II alveolar cells

Although Mycobacterium tuberculosis is assumed to infect primarily alveolar macrophages after being aspirated into the lung in aerosol form, it is plausible to hypothesize that M. tuberculosis can come in contact with alveolar epithelial cells upon arrival into the alveolar space. Therefore, as a first step toward investigation of the interaction between M. tuberculosis and alveolar epithelial cells, we examined the ability of M. tuberculosis to bind to and invade alveolar epithelial cells in vitro. The H37Rv and H37Ra strains of M. tuberculosis were cultured to mid-log phase and used in both adherence and invasion assays. The A549 human type II alveolar cell line was cultured to confluence in RPMI 1640 supplemented with 5% fetal bovine serum, L-glutamine, and nonessential amino acids. H37Rv was more efficient in entering A549 cells than H37Ra, Mycobacterium avium, and Escherichia coli Hb101, and nonpiliated strain (4.7% +/- 1.0% of the initial inoculum in 2 h compared with 3.1% +/- 0.8%, 2.1% +/- 0.9%, and 0.03% +/- 0.0%, respectively). The invasion was more efficient at 37 degrees C than 30 degrees C (4.7% +/- 1.0% compared with 2.3% +/- 0.8%). H37Rv and H37Ra were both capable of multiplying intracellularly at a similar ration over 4 days. Binding was inhibited up to 55.7% by anti-CD51 antibody (antivitronectin receptor), up to 55% with anti-CD29 antibody (beta(1) integrin), and 79% with both antibodies used together. Update of M. tuberculosis H37Rv was microtubule and microfilament dependent. It was inhibited by 6l.4% in the presence of 10 micron colchicine and by 72.3% in the presence of 3 micron cytochalasin D, suggesting two separate pathways for uptake. Our results show that M. tuberculosis is capable of invading type II alveolar epithelial cells and raise the possibility that invasion of alveolar epithelial cells is associated with the pathogenesis of lung infection.     

Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.     

Fluctuating currents in puncture therapy

Antibodies to Mycobacterium tuberculosis antigens H37Rv and reverse strains previously isolated from patients with sarcoidosis with granular isolates were determined in 50 patients with sarcoidosis (including 16 patients isolating granular types) and 56 patients with tuberculosis, by using ELISA and immunoblotting. Serum antibodies from patients with sarcoidosis were ascertained to more commonly react in ELISA with the antigen (ultrasound disintegrant (USDs) obtained from reverse mycobacteria isolated (initially) from patients with sarcoidosis (AGS) than with the USD of the M. tuberculosis H37Rv (AGT) and, on the contrary, serum antibodies from patients with tuberculosis more frequently reacted with the M. tuberculosis H37Rv. The spectrum of serum antibodies from patients with sarcoidosis greatly differed at immunoblotting with AGS and AGT. There was most commonly a reaction with the antigenic determinants 79, 27, 30, and 50 kDa to AGS and that of the determinants 17, 35, 32 kDa to AGT.     

Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNF-alpha

Infection by Mycobacterium tuberculosis (MTB) induces human alveolar macrophage (AMphi) apoptosis by a TNF-alpha-dependent mechanism. The apoptotic response is postulated to be a defense mechanism, limiting the growth of this intracellular pathogen. Consistent with that model, recent studies showed that the virulent MTB strain H37Rv induces substantially less AMphi apoptosis than the attenuated strain H37Ra. We now report that AMphi infection with either H37Rv or H37Ra induces comparable levels of TNF-alpha measured by ELISA but that TNF-alpha bioactivity is reduced in supernatants of H37Rv-infected AMphi. Differential release of soluble TNFR2 (sTNFR2), with formation of inactive TNF-alpha-TNFR2 complexes accounted for the difference in TNF-alpha bioactivity in these cultures. Release of sTNFR2 by H37Rv-infected AMphi was IL-10 dependent since it was inhibited by neutralizing anti-IL-10 Ab. Thus, the effect of TNF-alpha produced by AMphi following infection can be modulated by virulent MTB, using IL-10 as an upstream mediator.     

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.     

Leishmania mexicana in C3H mice: BCG and levamisole treatment of established infections

The effect of BCG and levamisole on the course of established murine leishmaniasis was examined. C3H mice infected subcutaneously in the perinasal region with 10(5) L. mexicana promastigotes produced chronic non-ulcerating, non-healing lesions and demonstrated positive humoral and delayed hypersensitivity responses to leishmanial antigens. Infected animals were treated during months 3-5 of infection with either live BCG or with levamisole. Neither treatment resulted in resolution of lesions or in production of a hyperallergic form of infection; similarly, neither immune responses to leishmanial antigens nor histopathological features of lesions were significantly altered. BCG treatment resulted in accelerated growth of primary leishmanial lesions and in the appearance of metastases in some animals. Levamisole treatment of uninfected animals resulted in low levels of antibodies reacting with promastigote antigens, but not in positive delayed intradermal responses. BCG induced delayed intradermal sensitivity to PPD in both infected and control animals; significantly increased delayed reactions to leishmania, were observed in treated uninfected mice.     

Altered cytokine production and impaired antimycobacterial immunity in protein-malnourished guinea pigs

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), in response to PPD, despite the demonstration of higher serum levels of TNF-alpha and TGF-beta after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-beta 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.     

Superficial bladder tumors and increased reactivity against mycobacterial antigens before bacillus Calmette-Guerin therapy

PURPOSE: The precise mechanism of action of bacillus Calmette-Guerin (BCG) in bladder cancer treatment remains poorly understood. Whether bladder tumor cells are destroyed by nonspecific mechanisms or targeted by specifically activated lymphocytes recognizing cognate antigens is unclear. To investigate a possible cross-reactivity between BCG and bladder cell tumors, we tested before BCG treatment the lymphoproliferation of peripheral blood lymphocytes against several mycobacterial antigens, including the secreted fibronectin binding antigen 85 complex from BCG (AG 85) in patients with superficial bladder tumors compared to control matched patients. MATERIALS AND METHODS: Using a whole blood assay, T cell response against purified protein derivative, BCG extract, whole BCG, purified AG 85, and the nonspecific mitogens pokeweed and phytohemagglutinin was investigated in 79 patients with superficial bladder tumors before BCG and in 39 control subjects without malignancy matched for age and sex. Neither group had a history of tuberculosis. Lymphoproliferation was measured with a tritiated thymidine uptake assay on day 7 of culture. RESULTS: Of the 79 patients with superficial transitional cell carcinoma, a significant lymphoproliferative response before BCG against PPD, BCG extract, whole BCG and AG 85 was observed in 65 (82.2%), 67 (84.81%), 30 (37.97%) and 49 (62.02%) patients, respectively. Of the 39 controls only 26 (64.1%), 23 (58.9%), 3 (7.7%) and 3 (7.7%) patients, respectively, had a significant lymphoproliferation against PPD, BCG extract, BCG and AG 85 (p >0.05, p = 0.004, p = 0.00001 and p = 0.00001, respectively). In terms of lymphoproliferative levels, patients with superficial transitional cell carcinoma also showed a significantly higher response against PPD (p = 0.000012), BCG extract (p = 0.000001), AG 85 (p = 0.000001), whole BCG (p = 0.00001) and pokeweed (p = 0.01) than controls but not against phytohemagglutinin. CONCLUSIONS: Patients with superficial transitional cell carcinoma demonstrate an increased lymphoproliferation against mycobacterial antigens before BCG compared to control subjects. Although a nonspecific activation of the immune system cannot be excluded at this stage, our data may suggest the possible existence of bladder cancer antigens cross-reactive with mycobacterial antigens responsible for boosting precursor cells witnessing previous contacts with mycobacteria. The implication of these findings in the antitumoral mechanism of action of BCG are under investigation.     

Roger Brown

Salmonella abortusovis strain Rv6 (Sao Rv6) is a live attenuated vaccine used for a few years to protect ewes against abortive salmonellosis. As Salmonellae, particularly Salmonella aro mutants, have considerable potential as vehicles for the presentation of heterologous vaccine antigens, Sao Rv6 was tested in order to develop a vaccinal vehicle for small ruminants. Five vector plasmids were tested in Sao Rv6; these plasmids, which carry Maltose Binding Protein (MBP) expressed as protein, but differ in their promotors, had been previously tested in S. typhimurium strain SL3261, and were transferred into Sao Rv6. The five plasmids were stable in vitro, and the recombinant Sao Rv6 expressed MBP at various levels. Intraperitoneal infection of OF1 mice with the recombinant bacteria did not modify the characteristics of Sao Rv6; dissemination and infection levels were similar in all groups and all mice developed antibodies to Salmonella antigens as measured by ELISA. In contrast, only animals immunized with Sao Rv6 carrying the pNTE plasmid developed a serum antibody response to MBP. This plasmid was then tested in sheep; following subcutaneous immunization with Sao Rv6-pNTE, dissemination and infection levels were not modified in comparison with sheep immunized with Sao Rv6 lacking plasmid. Antibodies specific to MBP were detected in sera of sheep immunized with Sao Rv6-pNTE, purified MBP, and with S. typhimurium SL3261-pNTE as positive controls. These results demonstrate that Sao Rv6 can be used as a vehicle for heterologous antigens in sheep with pNTE as plasmid vector.