Comamonas testosteroni as the cause of mortality in embryonated chicken eggs of breeding broiler hens in Costa Rica.

Mortality of chicken embryos and first-week chickens was reported in a commercial incubator company in Costa Rica. Six 1-day-old Cobb chickens and twenty-four embryonated chicken eggs were examined in the Laboratory of Avian Pathology and the Laboratory of Bacteriology of the National University of Costa Rica. Twelve dead-in-shell embryos showed maceration and were immersed in a putrid, turbid, slightly thick brown liquid. Additionally, the other 12 embryonated eggs had milky yellow-orange content. The livers of those embryos had congestion, haemorrhages and multifocal cream foci of necrosis. Granulocytic infiltration was observed in the bursa of Fabricius, myocardium, liver, lung and kidney. Livers and egg yolks from six embryonated chickens and all 1-day-old chickens were aseptically collected and cultured. In addition, tissues from six better conserved embryos and all 1-day-old chickens were fixed in buffered formalin and embedded in paraffin. Biochemical and molecular tests identified Comamonas testosteroni as the cause of the early, middle and late embryo mortality. As all the eggshells from the sampled embryonated eggs were dirty with soiled a fecal matter, contamination after manipulating the eggs was considered the source of infection. C. testosteroni is an environmental microorganism that has rarely been reported to cause human disease. To our knowledge, this is the first report of C. testosteroni causing mortality in a hatchery. Cleaning and disinfection using ozone were implemented in the hatchery to eliminate the embryo mortality associated with C. testosteroni.

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

Virulent Brucella nosferati infecting Desmodus rotundus has emerging potential due to the broad foraging range of its bat host for humans and wild and domestic animals.

Desmodus rotundus, vampire bats, transmit dangerous infections, and brucellosis is a hazardous zoonotic disease, two adversities that coexist in the subtropical and tropical areas of the American continent. Here, we report a 47.89% Brucella infection prevalence in a colony of vampire bats inhabiting the tropical rainforest of Costa Rica. The bacterium induced placentitis and fetal death in bats. Wide-range phenotypic and genotypic characterization placed the Brucella organisms as a new pathogenic species named Brucella nosferati sp. nov., isolated from bat tissues, including the salivary glands, suggesting feeding behavior might favor transmission to their prey. Overall analyses placed B. nosferati as the etiological agent of a reported canine brucellosis case, demonstrating its potential for infecting other hosts. To assess the putative prey hosts, we analyzed the intestinal contents of 14 infected and 23 non-infected bats by proteomics. A total of 54,508 peptides sorted into 7,203 unique peptides corresponding to 1,521 proteins were identified. Twenty-three wildlife and domestic taxa, including humans, were foraged by B. nosferati-infected D. rotundus, suggesting contact of this bacterium with a broad range of hosts. Our approach is appropriate for detecting, in a single study, the prey preferences of vampire bats in a diverse area, demonstrating its suitability for control strategies where vampire bats thrive. IMPORTANCE The discovery that a high proportion of vampire bats in a tropical area is infected with pathogenic Brucella nosferati and that bats forage on humans and many wild and domestic animals is relevant from the perspective of emerging disease prevention. Indeed, bats harboring B. nosferati in their salivary glands may transmit this pathogenic bacterium to other hosts. This potential is not trivial since, besides the demonstrated pathogenicity, this bacterium possesses all the required virulent arsenal of dangerous Brucella organisms, including those that are zoonotic for humans. Our work has settled the basis for future surveillance actions in brucellosis control programs where these infected bats thrive. Moreover, our strategy to identify the foraging range of bats may be adapted for exploring the feeding habits of diverse animals, including arthropod vectors of infectious diseases, and therefore of interest to a broader audience besides experts on Brucella and bats.

Phenotypes controlled by the Brucella abortus two component system BvrR/BvrS are differentially impacted by BvrR phosphorylation.

Brucella abortus is a zoonotic pathogen whose virulence depends on its ability to survive intracellularly at the endoplasmic reticulum derived compartment. The two-component system BvrR/BvrS (BvrRS) is essential for intracellular survival due to the transcriptional control of the type IV secretion system VirB and its transcriptional regulator VjbR. It is a master regulator of several traits including membrane homeostasis by controlling gene expression of membrane components, such as Omp25. BvrR phosphorylation is related to DNA binding at target regions, thereby repressing or activating gene transcription. To understand the role of BvrR phosphorylation we generated dominant positive and negative versions of this response regulator, mimicking phosphorylated and non-phosphorylated BvrR states and, in addition to the wild-type version, these variants were introduced in a BvrR negative background. We then characterized BvrRS-controlled phenotypes and assessed the expression of proteins regulated by the system. We found two regulatory patterns exerted by BvrR. The first pattern was represented by resistance to polymyxin and expression of Omp25 (membrane conformation) which were restored to normal levels by the dominant positive and the wild-type version, but not the dominant negative BvrR. The second pattern was represented by intracellular survival and expression of VjbR and VirB (virulence) which were, again, complemented by the wild-type and the dominant positive variants of BvrR but were also significantly restored by complementation with the dominant negative BvrR. These results indicate a differential transcriptional response of the genes controlled to the phosphorylation status of BvrR and suggest that unphosphorylated BvrR binds and impacts the expression of a subset of genes. We confirmed this hypothesis by showing that the dominant negative BvrR did not interact with the omp25 promoter whereas it could interact with vjbR promoter. Furthermore, a global transcriptional analysis revealed that a subset of genes responds to the presence of the dominant negative BvrR. Thus, BvrR possesses diverse strategies to exert transcriptional control on the genes it regulates and, consequently, impacting on the phenotypes controlled by this response regulator.

Platelet depletion does not alter the course of Brucella abortus infection in vivo.

Brucellosis is a bacterial disease of animals and a zoonotic infection. Thrombocytopenia is a common outcome in long-lasting brucellosis in humans. Likewise, ex vivo experiments have shown that platelets may play a role in Brucella abortus infections. Following these reports, we explored the course of brucellosis in thrombocytopenic mice, using the non-toxic low-molecular-weight aspercetin protein that depletes platelets in vivo. Aspercetin does not induce systemic hemorrhage or inflammation, and when injected into mice, it generates a rapid dose-dependent drop in platelet counts without affecting central organs, disrupting hematological parameters, or the proinflammatory cytokine profile. Compared to the B. abortus infected control group, the infected thrombocytopenic mice did not show significant differences in the hematological profiles, pathological score, spleen, liver histopathology, or bacterial loads. Except for IL-6, which was higher in the infected thrombocytopenic mice, the TNF-α, IFN-γ and IL-10 did not significantly differ with the PBS-infected group. The results indicate that platelets do not play a significant role in modulating Brucella infection in vivo at the early stages of infection, which is commensurate with the stealthy strategy followed by Brucella organisms at the onset of the disease.

Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle.

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.

Canine brucellosis in Costa Rica reveals widespread Brucella canis infection and the recent introduction of foreign strains.

Brucellosis is a prevalent disease in Costa Rica (CR), with an increasing number of human infections. Close to half of homes in CR have one or more dogs, corresponding to ∼1.4 million canines, most of them in the Central Valley within or near the cities of San José, Heredia, and Alajuela. From 302 dog sera collected from this region, 19 were positive for Brucella canis antigens, and five had antibodies against smooth lipopolysaccharide, suggesting infections by both B. canis and other Brucella species. B. canis strains were isolated in the Central Valley from 26 kennel dogs and three pet dogs, all displaying clinical signs of canine brucellosis. We detected three recent introductions of different B. canis strains in kennels: two traced from Mexico and one from Panama. Multiple locus-variable number tandem repeats (MLVA-16) and whole-genome sequencing (WGSA) analyses showed that B. canis CR strains comprise three main lineages. The tree topologies obtained by WGSA and MLVA-16 just partially agreed, indicating that the latter analysis is not suitable for phylogenetic studies. The fatty acid methyl ester analysis resolved five different B. canis groups, showing less resolution power than the MLVA-16 and WGSA. Lactobacillic acid was absent in linages I and II but present in linage III, supporting the recent introductions of B. canis strains from Mexico. B. canis displaying putative functional cyclopropane synthase for the synthesis of lactobacillic acid are phylogenetically intertwined with B. canis with non-functional protein, indicating that mutations have occurred independently in the various lineages.

Intracellular Passage Triggers a Molecular Response in Brucella abortus That Increases Its Infectiousness.

Brucella abortus is a facultatively extracellular-intracellular pathogen that encounters a diversity of environments within the host cell. We report that bacteria extracted from infected cells at late stages (48 h postinfection) of the intracellular life cycle significantly increase their ability to multiply in new target cells. This increase depends on early interaction with the cell surface, since the bacteria become more adherent and penetrate more efficiently than in vitro-grown bacteria. At this late stage of infection, the bacterium locates within an autophagosome-like compartment, facing starvation and acidic conditions. At this point, the BvrR/BvrS two-component system becomes activated, and the expression of the transcriptional regulator VjbR and the type IV secretion system component VirB increases. Using bafilomycin to inhibit BvrR/BvrS activation and using specific inhibitors for VjbR and VirB, we showed that the BvrR/BvrS and VjbR systems correlate with increased interaction with new host cells, while the VirB system does not. Bacteria released from infected cells under natural conditions displayed the same phenotype as intracellular bacteria. We propose a model in which the B. abortus BvrR/BvrS system senses the transition from its replicative niche at the endoplasmic reticulum to the autophagosome-like exit compartment. This activation leads to the expression of VirB, which participates in the release of the bacterium from the cells, and an increase in VjbR expression that results in a more efficient interaction with new host cells.

Impacts of local population history and ecology on the evolution of a globally dispersed pathogen.

BACKGROUND: Pathogens with a global distribution face diverse biotic and abiotic conditions across populations. Moreover, the ecological and evolutionary history of each population is unique. Xylella fastidiosa is a xylem-dwelling bacterium infecting multiple plant hosts, often with detrimental effects. As a group, X. fastidiosa is divided into distinct subspecies with allopatric historical distributions and patterns of multiple introductions from numerous source populations. The capacity of X. fastidiosa to successfully colonize and cause disease in naïve plant hosts varies among subspecies, and potentially, among populations. Within Central America (i.e. Costa Rica) two X. fastidiosa subspecies coexist: the native subsp. fastidiosa and the introduced subsp. pauca. Using whole genome sequences, the patterns of gene gain/loss, genomic introgression, and genetic diversity were characterized within Costa Rica and contrasted to other X. fastidiosa populations. RESULTS: Within Costa Rica, accessory and core genome analyses showed a highly malleable genome with numerous intra- and inter-subspecific gain/loss events. Likewise, variable levels of inter-subspecific introgression were found within and between both coexisting subspecies; nonetheless, the direction of donor/recipient subspecies to the recombinant segments varied. Some strains appeared to recombine more frequently than others; however, no group of genes or gene functions were overrepresented within recombinant segments. Finally, the patterns of genetic diversity of subsp. fastidiosa in Costa Rica were consistent with those of other native populations (i.e. subsp. pauca in Brazil). CONCLUSIONS: Overall, this study shows the importance of characterizing local evolutionary and ecological history in the context of world-wide pathogen distribution.

Persistence of Brucella abortus lineages revealed by genomic characterization and phylodynamic analysis.

Brucellosis, caused by Brucella abortus, is a major disease of cattle and humans worldwide distributed. Eradication and control of the disease has been difficult in Central and South America, Central Asia, the Mediterranean and the Middle East. Epidemiological strategies combined with phylogenetic methods provide the high-resolution power needed to study relationships between surveillance data and pathogen population dynamics, using genetic diversity and spatiotemporal distributions. This information is crucial for prevention and control of disease spreading at a local and worldwide level. In Costa Rica (CR), the disease was first reported at the beginning of the 20th century and has not been controlled despite many efforts. We characterized 188 B. abortus isolates from CR recovered from cattle, humans and water buffalo, from 2003 to 2018, and whole genome sequencing (WGS) was performed in 95 of them. They were also assessed based on geographic origin, date of introduction, and phylogenetic associations in a worldwide and national context. Our results show circulation of five B. abortus lineages (I to V) in CR, phylogenetically related to isolates from the United States, United Kingdom, and South America. Lineage I was dominant and probably introduced at the end of the 19th century. Lineage II, represented by a single isolate from a water buffalo, clustered with a Colombian sample, and was likely introduced after 1845. Lineages III and IV were likely introduced during the early 2000s. Fourteen isolates from humans were found within the same lineage (lineage I) regardless of their geographic origin within the country. The main CR lineages, introduced more than 100 years ago, are widely spread throughout the country, in contrast to new introductions that seemed to be more geographically restricted. Following the brucellosis prevalence and the farming practices of several middle- and low-income countries, similar scenarios could be found in other regions worldwide.

Proteogenomic analysis of the Clostridium difficile exoproteome reveals a correlation between phylogenetic distribution and virulence potential.

C. difficile induces antibiotic-associated diarrhea due to the action of two secreted toxins, TcdA and TcdB. A considerable range of virulence among C. difficile strains has been widely reported. During a hospital outbreak, 46 isolates were collected that belonged to different genotypes. Of those, the majority corresponded to two virulent strains, the globally distributed Sequence Type 1 (ST1)_North American Pulsotype 1 (NAP1) and the endemic ST54_NAPCR1 genotypes, respectively. Whereas the virulence of the latter has been attributed to increased secretion of toxins and production of a highly cytotoxic TcdB, these characteristics do not explain the increased lethality of the former. We undertook a proteomic comparative approach of the isolates participating in the outbreak to look for proteins present in the exoproteome of the ST1_NAP1and ST54_NAPCR1 strains. We used a low virulent ST2_NAP4 strain isolated also in the outbreak as control. Dendrograms constructed using the exoproteomes of the strains were very similar to those created using genomic information, suggesting an association between secreted proteins and relative virulence of the strains. By 2D electrophoresis and mass spectrometry it was found that approximately half of the proteins are shared among strains of different genotypes. From the identified proteins, the surface-located SlpA draw our attention due to its detection in ST54_NAPCR1 exoproteomes. Biochemical analysis indicated that the processing of SlpA is different in the ST54_NAPCR1 strain and confirmed that this strain secretes more SlpA than its counterparts. Furthermore, SlpA from the ST54_NAPCR1 strain exerted an increased proinflammatory activity. Altogether, these results indicate that the exoproteome composition correlates with the C. difficile genotype and suggest that particular proteins secreted by some strains could synergize with the effects of TcdA and TcdB increasing their virulence.

Corrigendum: Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3.

[This corrects the article DOI: 10.3389/fvets.2019.00175.].

Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3.

Members of the genus Brucella cluster in two phylogenetic groups: classical and non-classical species. The former group is composed of Brucella species that cause disease in mammals, including humans. A Brucella species, labeled as Brucella sp. BCCN84.3, was isolated from the testes of a Saint Bernard dog suffering orchiepididymitis, in Costa Rica. Following standard microbiological methods, the bacterium was first defined as "Brucella melitensis biovar 2." Further molecular typing, identified the strain as an atypical "Brucella suis." Distinctive Brucella sp. BCCN84.3 markers, absent in other Brucella species and strains, were revealed by fatty acid methyl ester analysis, high resolution melting PCR and omp25 and omp2a/omp2b gene diversity. Analysis of multiple loci variable number of tandem repeats and whole genome sequencing demonstrated that this isolate was different from the currently described Brucella species. The smooth Brucella sp. BCCN84.3 clusters together with the classical Brucella clade and displays all the genes required for virulence. Brucella sp. BCCN84.3 is a species nova taxonomical entity displaying pathogenicity; therefore, relevant for differential diagnoses in the context of brucellosis. Considering the debate on the Brucella species concept, there is a need to describe the extant taxonomical entities of these pathogens in order to understand the dispersion and evolution.

Neutrophils as Trojan Horse Vehicles for Brucella abortus Macrophage Infection.

Brucella abortus is a stealthy intracellular bacterial pathogen of animals and humans. This bacterium promotes the premature cell death of neutrophils (PMN) and resists the killing action of these leukocytes. B. abortus-infected PMNs presented phosphatidylserine (PS) as "eat me" signal on the cell surface. This signal promoted direct contacts between PMNs and macrophages (Mϕs) and favored the phagocytosis of the infected dying PMNs. Once inside Mϕs, B. abortus replicated within Mϕs at significantly higher numbers than when Mϕs were infected with bacteria alone. The high levels of the regulatory IL-10 and the lower levels of proinflammatory TNF-α released by the B. abortus-PMN infected Mϕs, at the initial stages of the infection, suggested a non-phlogistic phagocytosis mechanism. Thereafter, the levels of proinflammatory cytokines increased in the B. abortus-PMN-infected Mϕs. Still, the efficient bacterial replication proceeded, regardless of the cytokine levels and Mϕ type. Blockage of PS with Annexin V on the surface of B. abortus-infected PMNs hindered their contact with Mϕs and hampered the association, internalization, and replication of B. abortus within these cells. We propose that B. abortus infected PMNs serve as "Trojan horse" vehicles for the efficient dispersion and replication of the bacterium within the host.

Neutrophils Dampen Adaptive Immunity in Brucellosis.

Brucella organisms are intracellular stealth pathogens of animals and humans. The bacteria overcome the assault of innate immunity at early stages of an infection. Removal of polymorphonuclear neutrophils (PMNs) at the onset of adaptive immunity against Brucella abortus favored bacterial elimination in mice. This was associated with higher levels of interferon gamma (IFN-γ) and a higher proportion of cells expressing interleukin 6 (IL-6) and inducible nitric oxide synthase (iNOS), compatible with M1 macrophages, in PMN-depleted B. abortus-infected (PMNd-Br) mice. At later times in the acute infection phase, the amounts of IFN-γ fell while IL-6, IL-10, and IL-12 became the predominant cytokines in PMNd-Br mice. IL-4, IL-1ß, and tumor necrosis factor alpha (TNF-α) remained at background levels at all times of the infection. Depletion of PMNs at the acute stages of infection promoted the premature resolution of spleen inflammation. The efficient removal of bacteria in the PMNd-Br mice was not due to an increase of antibodies, since the immunoglobulin isotype responses to Brucella antigens were dampened. Anti-Brucella antibodies abrogated the production of IL-6, IL-10, and IL-12 but did not affect the levels of IFN-γ at later stages of infection in PMNd-Br mice. These results demonstrate that PMNs have an active role in modulating the course of B. abortus infection after the adaptive immune response has already developed.

GFP tagging of Brucella melitensis Rev1 allows the identification of vaccinated sheep.

Brucellosis is a worldwide zoonosis causing important economic loss and a public health problem. Small ruminants are the preferred hosts of Brucella melitensis and thus the main source of human infections. Effective control of sheep and goat brucellosis has been achieved in several countries through vaccination with the live-attenuated B. melitensis Rev1 vaccine. However, Rev1 induces a long-lasting serological response that hinders the differentiation between infected and vaccinated animals. A Rev1::gfp strain expressing constitutively the Green Fluorescent Protein (GFP) was built by stable insertion of a mini-Tn7-gfp in the glmS-recG non-codifying chromosomal region. An associated indirect ELISA-GFP was developed to identify anti-GFP antibodies in vaccinated animals. The resulting Rev1::gfp kept the biological properties of the Rev1 reference strain, including residual virulence and efficacy in mice, and was readily distinguished from Rev1 and other Brucella field strains by direct visualization under ultraviolet illumination, fluorescence microscopy and a multiplex PCR-GFP. The Rev1::gfp strain did not elicit anti-GFP antibodies itself in lambs but when applied in combination with recombinant GFP induced an intense and long-lasting (>9 months) anti-GFP serological response readily detectable by the ELISA-GFP. Overall, our results confirm that Rev1 GFP-tagging can be a suitable alternative for identifying vaccinated sheep in infected contexts.

Depletion of Complement Enhances the Clearance of Brucella abortus in Mice.

Brucellosis is a bacterial disease of animals and humans. Brucella abortus barely activates the innate immune system at the onset of infection, and this bacterium is resistant to the microbicidal action of complement. Since complement stands as the first line of defense during bacterial invasions, we explored the role of complement in B. abortus infections. Brucella abortus-infected mice depleted of complement with cobra venom factor (CVF) showed the same survival rate as mice in the control group. The complement-depleted mice readily eliminated B. abortus from the spleen and did so more efficiently than the infected controls after 7 days of infection. The levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6) remained within background levels in complement-depleted B. abortus-infected mice. In contrast, the levels of the immune activator cytokine gamma interferon and the regulatory cytokine IL-10 were significantly increased. No significant histopathological changes in the liver and spleen were observed between the complement-depleted B. abortus-infected mice and the corresponding controls. The action exerted by Brucella on the immune system in the absence of complement may correspond to a broader phenomenon that involves several components of innate immunity.

Brucella abortus Senses the Intracellular Environment through the BvrR/BvrS Two-Component System, Which Allows B. abortus To Adapt to Its Replicative Niche.

Brucella abortus is a facultative extracellular-intracellular pathogen belonging to a group of Alphaproteobacteria that establishes close interactions with animal cells. This bacterium enters host cells in a membrane-bound compartment, avoiding the lysosomal route and reaching the endoplasmic reticulum through the action of the type IV secretion system, VirB. In this work, we demonstrate that the BvrR/BvrS two-component system senses the intracellular environment to mount the transcriptional response required for intracellular life adaptation. By combining a method to purify intracellularly extracted bacteria with a strategy that allows direct determination of BvrR phosphorylation, we showed that upon entrance to host cells, the regulatory protein BvrR was activated (BvrR-P) by phosphorylation at aspartate 58. This activation takes place in response to intracellular cues found in early compartments, such as low pH and nutrient deprivation. Furthermore, BvrR activation was followed by an increase in the expression of VjbR and VirB. The in vitro activation of this BvrR-P/VjbR/VirB virulence circuit rescued B. abortus from the inhibition of intracellular replication induced by bafilomycin treatment of cells, demonstrating the relevance of this mechanism for intracellular bacterial survival and replication. All together, our results indicate that B. abortus senses the transition from the extracellular to the intracellular milieu through BvrR/BvrS, allowing the bacterium to transit safely to its replicative niche. These results serve as a working model for understanding the role of this family of two-component systems in the adaptation to intracellular life of Alphaproteobacteria.

Brucellosis caused by the wood rat pathogen Brucella neotomae: two case reports.

BACKGROUND: Brucellosis is a chronic bacterial disease caused by members of the genus Brucella. Among the classical species stands Brucella neotomae, until now, a pathogen limited to wood rats. However, we have identified two brucellosis human cases caused by B. neotomae, demonstrating that this species has zoonotic potential. CASES PRESENTATION: Within almost 4 years of each other, a 64-year-old Costa Rican white Hispanic man and a 51-year-old Costa Rican white Hispanic man required medical care at public hospitals of Costa Rica. Their hematological and biochemical parameters were within normal limits. No adenopathies or visceral abnormalities were found. Both patients showed intermittent fever, disorientation, and general malaise and a positive Rose Bengal test compatible with Brucella infection. Blood and cerebrospinal fluid cultures rendered Gram-negative coccobacilli identified by genomic analysis as B. neotomae. After antibiotic treatment, the patients recovered with normal mental activities. CONCLUSIONS: This is the first report describing in detail the clinical disease caused by B. neotomae in two unrelated patients. In spite of previous claims, this bacterium keeps zoonotic potential. Proposals to generate vaccines by using B. neotomae as an immunogen must be reexamined and countries housing the natural reservoir must consider the zoonotic risk.