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. 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. 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.

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.

Brucella abortus is a facultative 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 post-infection) 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 bacteria become more adherent and penetrate more efficiently as compared to in vitro grown bacteria. At this late stage of infection, the bacterium locates within an autophagosome-like compartment, facing starving and acidic conditions. At this point, the two-component system BvrR/BvrS becomes activated, and the expression of the transcriptional regulator VjbR and the type IV secretion system VirB increased. Using bafilomycin to inhibit BvrR/BvrS activation and specific inhibitors for VjbR and VirB we showed that the BvrR/BvrS and VjbR systems correlate with the 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 participating 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.

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.

Brucellosis is a bacterial disease of animals and humans.Brucella abortusbarely activates the innate immune system at the onset of infection, and this bacterium is resistant to the microbicidal action of complement.

Brucellosis has therapeutic challenges due to 3%–15% relapses/therapeutic failures (R/TF) after antibiotic treatment. Therefore, determining the antibiotic concentration in tissues, the physiopathological parameters, and the R/TF after treatment is relevant. After exploring different antibiotic quantities, we found that a combined dose of 100 µg/g of doxycycline (for 45 days) and 7.5 µg/g of streptomycin (for 14 days), respectively, achieved therapeutic levels of more than fourfold minimum inhibitory concentrations (MICs) against Brucella abortus in the spleen, liver, bone marrow, and plasma of mice, causing minimal pathophysiological effects. After 30 days of infection, mice received antibiotics, and hematological, histopathological, biochemical, and immunological analyses were performed. After antibiotic therapy, the pathological, hematological, immunological, and physiological profiles paralleled those described in human brucellosis. Treatment lowered antibody titers, reduced proinflammatory cytokines, and reduced inflammation in the target organs for a protracted period. No bacteria were detected in tissues 8 weeks after treatment, suggesting complete recovery. However, despite high doxycycline and streptomycin concentrations in tissues, relapses appeared in 100% of the animals after 182 days post-infection, estimated by the bacterial counts and PCR from organs. This proportion contrasts with the 15% R/TF observed in humans after antibiotic treatments. None of the B. abortus isolated from relapses showed augmented MICs or mutations coding for antibiotic resistance in chromosomal-relevant regions. We discuss whether our findings constitute a general phenomenon or differences in the exhaustive screening method for bacteria detection related to the murine model. Along these lines, we envision likely mechanisms of bacterial persistence in tissues after antibiotic treatment.