Fifteen different Rose Bengal antigens showed large differences with respect to pH, cell concentration and agglutination with the international standard anti-Brucella abortus serum, demonstrating the lack of international standardisation. Their sensitivity and specificity, compared with that of the complement fixation test, were evaluated for the diagnosis of B melitensis infection in culture-positive sheep, brucella-free ewes, and sheep and goats belonging to field flocks under different epidemiological conditions. All the Rose Bengal antigens and the complement fixation test had 100 per cent specificity when testing brucella-free sheep or animals belonging to flocks in unvaccinated brucellosis-free areas, but there were large differences in sensitivity between the Rose Bengal antigens with sera from culture-positive sheep or from animals belonging to infected flocks. When using the most sensitive antigen, no difference was observed in Rose Bengal sensitivity between animals infected with either biovar 1 or biovar 3 of B melitensis. The relationship between the sensitivity of the Rose Bengal antigens and cell concentration was unclear, but their sensitivity was related to the standardisation of the antigens with the international standard serum. The complement fixation test was less sensitive than the Rose Bengal test when testing culture-positive sheep. When testing sera from animals belonging to infected flocks with antigens standardised according to European Union rules, no great differences were observed in the sensitivities of the two tests. However, great differences in sensitivity between the Rose Bengal antigens were observed with sera from animals belonging to flocks with low levels of prevalence.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.

A mutant of Brucella suis bearing a Tn5 insertion in norD, the last gene of the operon norEFCBQD, encoding nitric oxide reductase, was unable to survive under anaerobic denitrifying conditions. The norD strain exhibited attenuated multiplication within nitric oxide-producing murine macrophages and rapid elimination in mice, hence demonstrating that norD is essential for Brucella virulence.The gram-negative intracellular pathogen Brucella sp. is the causative agent of brucellosis, which is transmissible to humans from domestic animal species that are infected with B. abortus, B. suis, or B. melitensis.Multiplication inside macrophages allows Brucella to be carried throughout the host organism and to colonize specific organs. The pathogenicity of brucellae and chronicity are based on the ability of the pathogen to adapt to the environmental conditions that it encounters in its replicative niche (18) and to avoid the killing mechanisms within macrophages (3,8,10,14).Intensive studies were performed at the genetic level to investigate the factors that are essential for the adaptation of Brucella to the intracellular conditions (5). Analysis of the intramacrophagic virulome (17) confirmed that the type IV secretion system encoded by virB (19) is, to date, the main virulence factor of B. suis. It also revealed that the B. suis replicative niche is characterized by low levels of nutrients and oxygen. The latter parameter was previously observed in the phagosomes of stimulated macrophages (13). Complete genome sequences (6,12,20) have revealed that Brucella possesses all of the genes that are necessary for a complete denitrification pathway resulting in the reduction of nitrate to nitrogen. Genes encoding the four reductases Nar (nitrate reductase), Nir (nitrite reductase), Nor (nitric oxide reductase), and Nos (nitrous oxide reductase) constitute a "denitrification island" in B. suis which shares numerous similarities with that of Sinorhizobium meliloti (21); both organisms belong to the ␣-subclass of the proteobacteria. The respiratory system could allow Brucella to survive under very low oxygen tension, using nitrogen oxides as terminal electron acceptors. Bacteria may also take advantage of denitrification to cope with nitric oxide (NO) production in the macrophages during the innate response against infection. NO production by infected macrophages is a major defense system in control of Brucella infection in mice (9, 16) and possibly, although more controversial, in human infection, as revealed by the use of human macrophages transfected with inducible NO synthase (7). Despite the low levels of NO that were released by human macrophages, the nitric oxide reductase of Neisseria meningitidis was found to confer intracellular resistance to NO and allowed its utilization, resulting in the optimal survival of this bacterium in nasopharyngeal mucosa (22). In fact, during an infection of murine macrophages producing high levels of NO, B. abortus displayed increased late survival (23). The authors of that work suggested th...

Expression of the high-oxygen-affinity cytochrome cbb3 and cytochrome bd ubiquinol oxidases of Brucella suis was studied in vitro and in the intramacrophagic niche, which was previously proposed to be oxygen limited. The cytochrome cbb3 oxidase was exclusively expressed in vitro, whereas the cytochrome bd oxidase was preferentially used inside macrophages and contributed to intracellular bacterial replication.Brucellosis, an anthropozoonosis encountered worldwide, is caused by the gram-negative intracellular pathogens Brucella spp., which utilize macrophages to multiply inside a specific niche (12) and to spread throughout the organism. Low levels of nutriments and oxygen, as revealed by the analysis of the intramacrophagic virulome (11), are major features of the Brucella suis replicative niche. Phagosomes of stimulated macrophages are known to have oxygen concentrations which are lower than those found in the extracellular environment (9). Furthermore, granulomatous structures generated by the immune system during localized infection within livers, spleens, or brains of patients are characterized by oxygen deficiency (2,20,21).Pathogenicity of brucellae and chronicity are due to the ability of the pathogen to adapt to the environmental conditions encountered in its replicative niche. To perform this task, Brucella has to modify its gene expression profile to rapidly adapt to the intracellular conditions. To this end, the bacteria induce a set of virulence genes, the main one being virB, encoding a type IV secretion system (17). Expression of genes involved in adaptation to oxygen-limited conditions appeared to be crucial for intramacrophagic survival of Brucella. A previous study identified a cydB mutant of Brucella abortus lacking the cytochrome bd oxidase with high affinity for oxygen as being highly attenuated in the mouse model of infection (6). Complete genome sequences (5,8,18) have revealed that Brucella possessed the locus ccoNOQP, potentially encoding another high-oxygen-affinity oxidase, the cytochrome cbb3-type terminal oxidase. We also identified a putative transcription regulator of the FixK/Fnr family in B. suis. The present study was undertaken to investigate (i) expression of the two operons encoding the cytochrome bd and cytochrome cbb3-type terminal oxidases in vitro as well as in bacteria obtained from infected cells and (ii) their respective roles in intracellular multiplication.

During bacterial sepsis, proinflammatory cytokines contribute to multiorgan failure and death in a process regulated in part by cytolytic cell granzymes. When challenged with a sublethal dose of the identified mouse pathogen Brucella microti, wild-type (WT) and granzyme A (gzmA)(-/-) mice eliminate the organism from liver and spleen in 2 or 3 weeks, whereas the bacteria persist in mice lacking perforin or granzyme B as well as in mice depleted of Tc cells. In comparison, after a fatal challenge, only gzmA(-/-) mice exhibit increased survival, which correlated with reduced proinflammatory cytokines. Depletion of natural killer (NK) cells protects WT mice from sepsis without influencing bacterial clearance and the transfer of WT, but not gzmA(-/-) NK, cells into gzmA(-/-) recipients restores the susceptibility to sepsis. Therefore, infection-related pathology, but not bacterial clearance, appears to require gzmA, suggesting the protease may be a therapeutic target for the prevention of bacterial sepsis without affecting immune control of the pathogen.

This work provides first evidence that the GAD system might play an essential role in the resistance of an environment-borne, pathogenic Brucella species to extreme acid shock and during passage through the host stomach following oral infection.

European rabbits (Oryctolagus cuniculus) are severely affected by rabbit haemorrhagic disease (RHD). Caused by a lagovirus, the disease leads to losses in the rabbit industry and has implications for wildlife conservation. Past RHD outbreaks have been caused by GI.1/RHDV genotype viruses. A new virus belonging to the GI.2/RHDV2/b genotype emerged in 2010, quickly spreading and replacing the former in several countries; however, limited data are available on its pathogenicity and epidemiological factors. The present work extends these issues and evaluates cross-protection between both genotypes. Ninety-four and 88 domestic rabbits were challenged with GI.2/RHDV2/b and GI.1b/RHDV variant isolates, respectively. Cross-protection was determined by a second challenge on survivors with the corresponding strain. Mortality by GI.2/RHDV2/b was highly variable due to unknown individual factors, whereas mortality by GI.1b/RHDV was associated with age. Mortality in rabbits < 4 weeks old was 84%, higher than previously reported. Cross-protection was not identical between the two viruses because the ratio of mortality rate ratios for the first and second challenges was 3.80 ± 2.68 times higher for GI.2/RHDV2/b than it was for GI.1b/RHDV. Rabbit susceptibility to GI.2/RHDV2/b varied greatly and appeared to be modulated by the innate functionality of the immune response and/or its prompt activation by other pathogens. GI.1b/RHDV pathogenicity appeared to be associated with undetermined age-related factors. These results suggest that GI.2/RHDV2/b may interact with other pathogens at the population level but does not satisfactorily explain the GI.1b/RHDV virus's quick replacement.

In experimental cellular and murine infections, B. microti exhibited a high pathogenic potential, compared with other Brucella species.