Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro , however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b + F4/80 + MHC-II + cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS + inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.

The mucosal immune system represents the first line of defense against Brucella infection in nature. We used genetically deficient mice to identify the lymphocytes and signaling pathways implicated in the control of primary and secondary intranasal infection with B. melitensis. Our analysis of primary infection demonstrated that the effectors implicated differ at the early and late stages and are dependent on the organ. TCR-δ, TAP1, and IL-17RA deficiency specifically affects early control of Brucella in the lungs, whereas MHC class II (MHCII) and IFN-γR deficiency impairs late control in the lungs, spleen, and liver. Interestingly, IL-12p35−/− mice display enhanced Brucella growth in the spleen but not in the lungs or liver. Secondary intranasal infections are efficiently contained in the lung. In contrast to an i.p. infectious model, in which IL-12p35, MHCII, and B cells are strictly required for the control of secondary infection, we observed that only TCR-β deficiency or simultaneous neutralization of IL-12p35– and IL-17A–dependent pathways impairs the memory protective response against a secondary intranasal infection. Protection is not affected by TCR-δ, MHCII, TAP1, B cell, IL-17RA, or IL-12p35 deficiency, suggesting that CD4+ and CD8+ α/β+ T cells are sufficient to mount a protective immune response and that an IL-17A–mediated response can compensate for the partial deficiency of an IFN-γ–mediated response to control a Brucella challenge. These findings demonstrate that the nature of the protective memory response depends closely on the route of infection and highlights the role of IFN-γ–and IL-17RA–mediated responses in the control of mucosal infection by Brucella.

Brucella spp are intracellular bacteria that cause brucellosis, one of the most common zoonoses in the world. Given the serious medical consequences of this disease, a safe and effective human vaccine is urgently needed. Efforts to develop this vaccine have been hampered by our lack of understanding of what constitutes a protective memory response against Brucella. In this study, we characterize the cells and signaling pathways implicated in the generation of a protective immune memory response following priming by the injection of heat-killed or live Brucella melitensis 16M. Using a panel of gene-deficient mice, we demonstrated that during a secondary recall response, both the Brucella-specific humoral response and CD4+ Th1 cells must act together to confer protective immunity in the spleen to B. melitensis infection. Humoral protective immunity is induced by the inoculation of both heat-killed and live bacteria, and its development does not require T cells, MyD88/IL-12p35 signaling pathways, or an activation-induced deaminase–mediated isotype switch. In striking contrast, the presence of memory IFN-γ–producing CD4+ Th1 cells requires the administration of live bacteria and functional MyD88/IL-12p35 pathways. In summary, our work identifies several immune markers closely associated with protective immune memory and could help to define a rational strategy to obtain an effective human vaccine against brucellosis.

e Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. Brucella is a facultative intracellular alphaproteobacterium that causes abortion and infertility in mammals and leads to a debilitating febrile illness in humans. Although it is rarely fatal, human brucellosis can progress into a severe chronic disease if left untreated (1, 2). Despite significant progress, the incidence of human brucellosis remains very high in areas of endemicity (3). Complete eradication of Brucella would be impractical due to its presence in a large range of wild mammals (4, 5). Moreover, antibiotic treatment is costly, and patients frequently suffer from relapse of the illness (6, 7). Vaccination actually remains the only rational strategy to confer protection on populations living in countries of endemicity. Unfortunately, there is currently no available vaccine against human brucellosis, since all commercial animal vaccines are based on live attenuated strains of Brucella (8) that are still virulent in humans. Efforts to develop an effective vaccine are impaired by our poor knowledge of the Brucella life cycle in vivo and of the protective immune response induced by infection. In particular, the mechanisms of early Brucella dissemination in vivo remain largely unknown. A prominent characteristic of brucellosis is its long incubation period. The current hypothesis is that this period, corresponding to reduced activation of innate immunity, opens an "immunological window" and gives Brucella the chance to spread throughout the organism to establish a replication niche within phagocytic cells (9).Bacteremia is one major characteristic of human brucellosis (10) and is often associated with an increased risk of relapse (6, 11). Moreover, positive blood cultures are synonymous with secondary seeding and development of focal complications of the disease. Bacteremia in mice infected by intraperitoneal injection, the most frequent route of experimental infection, has been reported in some studies (12, 13). However, to our knowledge, the mechanism of Brucella persistence in the blood has never been investigated. In this study, we monitored bacteremia in mice for several weeks after the intraperito...

ObjectivesThe aim of this study was to characterize by classical biotyping and Multi-Locus variable number tandem repeats (VNTR) Analysis (MLVA) all Brucella spp. derived from human cases in Belgium from 1996 to 2015. Final goals were to determine the species and biovar, to trace-back on genetic grounds the origin of each strain when patient history and risk factors were missing, and to survey for particular trends at the national level.MethodsA total of 37 Brucella strains, isolated from 37 patients in Belgium, were analyzed by both classical biotyping and MLVA, and the genetic patterns compared to those of human strains isolated worldwide.ResultsClassical biotyping revealed that isolates were mainly Brucella melitensis. Most of them belonged to biovar 3, the most abundant biovar in the Mediterranean region. MLVA confirmed that Brucella melitensis is too diverse in VNTRs to be able to make clusters associated to each biovar, but it allowed retrieving precious epidemiological information. The analysis highlighted the imported nature of the strains from all over the world with a dominant part from the Mediterranean countries. Findings of the MLVA11 testing were in line with the travel history of patients coming from Italy, Turkey, Lebanon and Peru. The analysis was particularly useful because it suggested the geographical origin of the infection for 12/16 patients for whom no case history was available.ConclusionClassical biotyping and MLVA analysis are not exclusive but remain complementary tools for Brucella melitensis strain surveillance. MLVA11 is sufficient for Brucella-free countries such as Belgium to trace the geographical origin of infection, but complete MLVA16 is needed to search for links with endemic areas.