Atlantic salmon, Salmo salar, were given 2-min bath immunization with Yersinia ruckeri 0-antigen bacterins at doses of 10, 100, and lo00 c(g mi-l . The uptake of the antigen was followed by light and electron microscopy of samples taken immediately and periodically after immunization, and the immune response monitored by the passive haemolytic plaque assay. The particulate antigen was observed in the gill mucus, adhering to and within the pavement cellscoveringthe gill filaments, and in mononuclear phagocytes below the epidermal gill cells. There was a direct doseresponse correlation in the observed immune response according to the numbers of splenic antibody-producing cells 14 days after immunization. The cells involved in the recognition and uptake of a bacterin are initial important steps in the immune response, and these studies may aid in the immunopotentiation of fish vaccines and bacterins.
Brucella abortus is a facultative intracellular pathogen that causes a zoonosis called brucellosis. This disease leads to abortion and infertility in cattle, and diverse complications in humans. B. abortus is a successful intracellular bacterium that has developed the ability to evade the host's immune system and it replicates in professional and non-professional phagocytic cells, persisting in the different tissues, and organs of its hosts. It has been described that Brucella expresses a polar flagellum under certain conditions, but its function is still unknown. In this study we evaluated the role of the FlgJ, a protein, presumably a peptidoglycan hydrolase involved in flagellum formation and in the virulence of B. abortus strain 2308. B. abortus 2308 flgJ mutant and complemented strains were constructed to study the function of the FlgJ protein in the context of the virulence of this pathogen in in vitro and in vivo assays. The results showed that the elimination of the flgJ gene delays the growth rate of B. abortus in culture, reduces its intracellular survival capacity in professional and non-professional phagocytic cells, rendering it unable to escape from the endocytic route and not reaching the endoplasmic reticulum. It also negatively affects their persistence in BALB/c mice. Functionally, the B. abortus 2308 flgJ gene restored motility to an E. coli flgJ mutant gene. Furthermore, it was discovered that the production of FlgJ protein is associated with the bacterial adherence by B. abortus. Therefore, although the specific function of the polar flagellum for Brucella is unknown, the data indicates that the flagellar flgJ gene and its product are required for full virulence of B. abortus 2308, since its deletion significantly reduces the fitness of this pathogen in vitro and in vivo.
Brucella abortus is a pathogen that survives in macrophages. Several virulence factors participate in this process, including the open reading frame (ORF) BAB1_0270 codifying for a zinc-dependent metalloproteinase (ZnMP). Here, its contribution in the intracellular adaptation of B. abortus was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus Δ270 strain. Results showed that this ZnMP did not participated in either the adherence or the initial intracellular traffic of B. abortus in macrophages. Nevertheless, its deletion significantly increased the co-localization of B. abortus Δ270 with phagolysosomal cathepsin D and reduced its co-localization with calnexin present in endoplasmic reticulum (RE)-derived vesicles. Although B. abortus Δ270 showed an upregulated expression of genes involved in virulence ( vjbR , hutC , bvrR , virB1 ), it was insufficient to reach a successful intracellular replication within macrophages. Furthermore, its attenuation favored in macrophages infected the production of high levels of cytokines (TNF-α and IL-6) and co-stimulatory proteins (CD80 and CD86), signals required in T cell activation. Finally, its deletion significantly reduced the ability of B. abortus Δ270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, and considering that this ZnMP has homology with ImmA/IrrE proteases, we discuss its role in the virulence of this pathogen, concluding that ZnMP is required in the intracellular adaptation of B. abortus 2308 during the infection of macrophages.
As an alternative brucellosis prevention method, we evaluated the immunogenicity induced by new multivalent DNA vaccines in BALB/c mice. We constructed the vaccines by fusion of BAB1_0273 and/or BAB1_0278 open reading frames (ORFs) from genomic island 3 (GI-3) and the Brucella abortus 2308 sodC gene with a link based on prolines and alanines (pV273-sod, pV278-sod, and pV273-278-sod, resp.). Results show that immunization with all tested multivalent DNA vaccines induced a specific humoral and cellular immune response. These novel multivalent vaccines significantly increased the production of IgM, IgG, and IgG2a antibodies as well as IFN-γ levels and the lymphoproliferative response of splenocytes. Although immunization with these multivalent vaccines induced a typical T-helper 1- (Th1-) dominated immune response, such immunogenicity conferred low protection levels in mice challenged with the B. abortus 2308 pathogenic strain. Our results demonstrated that the expression of BAB1_0273 and/or BABl_0278 antigens conjugated to SOD protein can polarize mice immunity to a Th1-type phenotype, conferring low levels of protection.
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