Background: The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification.

-Pathogens that are transmitted between the environment, wildlife, livestock and humans represent major challenges for the protection of human and domestic animal health, the economic sustainability of agriculture, and the conservation of wildlife. Among such pathogens, the genus Mycobacterium is well represented by M. bovis, the etiological agent of bovine tuberculosis, M. avium ssp. paratuberculosis (Map) the etiological agent of Johne disease, M. avium ssp. avium (Maa) and in a few common cases by other emergent environmental mycobacteria. Epidemiologic surveys performed in Europe, North America and New Zealand have demonstrated the existence and importance of environmental and wildlife reservoirs of mycobacterial infections that limit the attempts of disease control programmes. The aim of this review is to examine the zoonotic aspects of mycobacteria transmitted from the environment and wildlife. This work is focused on the species of two main groups of mycobacteria classified as important pathogens for humans and animals: first, M. bovis, the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, captive wildlife, domestic livestock, non-human primates and humans; the second group examined, is the M. avium-intracellulare complex (MAC) which includes M. avium ssp. avium causing major health problems in AIDS patients and M. avium ssp. paratuberculosis the etiological agent of Johne disease in cattle and identified in patients with Crohn disease. MAC agents, in addition to a broad host range, are environmental mycobacteria found in numerous biotopes including the soil, water, aerosols, protozoa, deep litter and fresh tropical vegetation. This review examines the possible reservoirs of these pathogens in the environment and in wildlife, their role as sources of infection in humans and animals and their health impact on humans. The possibilities of control and management programmes for these mycobacterial infections are examined with regards to the importance of their natural reservoirs.

Dendritic cells (DC) are recognized as sentinels, which capture antigens in tissue and migrate to the lymph node, where they initiate immune responses. However, when a vaccine strain of green fluorescent protein-expressing Salmonella abortusovis (SAO) was inoculated into sheep oral mucosa, it induced accumulation of myeloid non-DC in the subcapsular sinus and paracortex of the draining lymph node, and SAO was mainly found associated with these cells (granulocytes and macrophages) but rarely with DC. To analyze how bacteria reached lymph nodes, we used cervical pseudo-afferent lymph duct catheterization. We showed that Salmonella administered in the oral mucosa were traveling free in lymph or associated with cells, largely with lymph monocytes and granulocytes but less with DC. SAO also induced a strong influx of these phagocytic cells in afferent lymph. Migrating DC presented a semi-mature phenotype, and SAO administration did not alter their expression of major histocompatibility complex type 2 and coactivation molecules. Compared with blood counterparts, lymph monocytes expressed lower levels of CD40, and granulocytes expressed higher levels of CD80. The data suggest that immunity to bacteria may result from the complex interplay between a mixture of phagocytic cell types, which transport antigens and are massively recruited via lymph to decisional lymph nodes.

Expression of the virB operon, encoding the type IV secretion system required for Brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suis VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suis from which chvE was deleted was fully virulent in a mouse model. Comparative analyses of various Brucella species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canis and B. suis, as well as the Brucella abortusand Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortus and B. melitensis strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virB expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virB operon are not uniformly regulated in different Brucella species. Since VirB proteins were shown to modulate Brucella phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.Bacteria belonging to the genus Brucella are gram-negative facultative intracellular pathogens of various wild and domestic mammals, and they also cause severe zoonotic infections in humans. Traditionally, three major species are distinguished by their preferences for certain animal hosts; Brucella abortus has a preference for cattle, Brucella melitensis has a preference for caprines, and Brucella suis has a preference for hogs. Whereas B. abortus is the livestock pathogen with the greatest economic impact, B. melitensis and B. suis account for most clinical cases in humans (15,42).In an attempt to unravel Brucella virulence factors by transposon mutagenesis, the crucial role of an operon similar to the virB operon of Agrobacterium tumefaciens encoding a type IV secretion system (T4SS) was revealed (35). The importance of the virB operon for Brucella virulence was further confirmed by signature-tagged mutagenesis both in vitro in a human macrophage infection model (24) and in vivo with mice (26). Further studies indicated that a complete Brucella virB operon was required for wild-type virulence in mice (47) or in macrophagelike cells (52,53). In nonphagocytic HeLa cells, the absence of some functional VirB proteins (B2, B4, and B9) did not...

The protein ClpA belongs to a diverse group of polypeptides named ClpATPases, which are highly conserved, and which include several molecular chaperones. In this study the gene encoding the 91 kDa protein b-ClpA of the facultative intracellular pathogen Brucella suis, which showed 70 % identity to ClpA of Rhodobacter blasticus, was identified and sequenced. Following heterologous expression in Escherichia coli strains SG1126 (∆clpA) and SG1127 (∆lon ∆clpA), b-ClpA replaced the function of E. coli ClpA, participating in the degradation of abnormal proteins. A b-clpA null mutant of B. suis was constructed, and growth experiments at 37 and 42 SC showed reduced growth rates for the null mutant, especially at the elevated temperature. The mutant complemented by b-clpA and overexpressing the gene was even more impaired at 37 and 42 SC. In intracellular infection of human THP-1 or murine J774 macrophage-like cells, the clpA null mutant and, to a lesser extent, the strain of B. suis overexpressing b-clpA behaved similarly to the wild-type strain. In a murine model of infection, however, the absence of ClpA significantly increased persistence of B. suis. These results showed that in B. suis the highly conserved protein ClpA by itself was dispensable for intramacrophagic growth, but was involved in temperature-dependent growth regulation, and in bacterial clearance from infected BALB/c mice.