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.

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.

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.

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.

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.

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.

Brucella neotomae Infection in Humans, Costa Rica.

Several species of Brucella are known to be zoonotic, but B. neotomae infection has been thought to be limited to wood rats. In 2008 and 2011, however, B. neotomae was isolated from cerebrospinal fluid of 2 men with neurobrucellosis. The nonzoonotic status of B. neotomae should be reassessed.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide.

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1ß by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

Genome-Wide Analysis Provides Evidence on the Genetic Relatedness of the Emergent Xylella fastidiosa Genotype in Italy to Isolates from Central America.

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp. pauca.

N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils.

Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.

Brucella canis is an intracellular pathogen that induces a lower proinflammatory response than smooth zoonotic counterparts.

Canine brucellosis caused by Brucella canis is a disease of dogs and a zoonotic risk. B. canis harbors most of the virulence determinants defined for the genus, but its pathogenic strategy remains unclear since it has not been demonstrated that this natural rough bacterium is an intracellular pathogen. Studies of B. canis outbreaks in kennel facilities indicated that infected dogs displaying clinical signs did not present hematological alterations. A virulent B. canis strain isolated from those outbreaks readily replicated in different organs of mice for a protracted period. However, the levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-12 in serum were close to background levels. Furthermore, B. canis induced lower levels of gamma interferon, less inflammation of the spleen, and a reduced number of granulomas in the liver in mice than did B. abortus. When the interaction of B. canis with cells was studied ex vivo, two patterns were observed, a predominant scattered cell-associated pattern of nonviable bacteria and an infrequent intracellular replicative pattern of viable bacteria in a perinuclear location. The second pattern, responsible for the increase in intracellular multiplication, was dependent on the type IV secretion system VirB and was seen only if the inoculum used for cell infections was in early exponential phase. Intracellular replicative B. canis followed an intracellular trafficking route undistinguishable from that of B. abortus. Although B. canis induces a lower proinflammatory response and has a stealthier replication cycle, it still displays the pathogenic properties of the genus and the ability to persist in infected organs based on the ability to multiply intracellularly.

The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition.

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

Brucella ceti infection in dolphins from the Western Mediterranean sea.

BACKGROUND: Brucella ceti infections have been increasingly reported in cetaceans. Brucellosis in these animals is associated with meningoencephalitis, abortion, discospondylitis', subcutaneous abscesses, endometritis and other pathological conditions B. ceti infections have been frequently described in dolphins from both, the Atlantic and Pacific Oceans. In the Mediterranean Sea, only two reports have been made: one from the Italian Tyrrhenian Sea and the other from the Adriatic Sea. RESULTS: We describe the clinical and pathological features of three cases of B. ceti infections in three dolphins stranded in the Mediterranean Catalonian coast. One striped dolphin had neurobrucellosis, showing lethargy, incoordination and lateral swimming due to meningoencephalitis, A B. ceti infected bottlenose dolphin had discospondylitis, and another striped dolphin did not show clinical signs or lesions related to Brucella infection. A detailed characterization of the three B. ceti isolates was performed by bacteriological, molecular, protein and fatty acid analyses. CONCLUSIONS: All the B. ceti strains originating from Mediterranean dolphins cluster together in a distinct phylogenetic clade, close to that formed by B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Our study confirms the severity of pathological signs in stranded dolphins and the relevance of B. ceti as a pathogen in the Mediterranean Sea.

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.

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.

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.

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.