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Brucellosis is a world-wide extended zoonosis that causes a grave problem in developing economies. Animal vaccination and diagnosis are essential to control brucellosis, and the need for accurate but also simple and low-cost tests that can be implemented in low-infrastructure laboratories has been emphasized. We evaluated bovine, sheep, goat and swine lateral flow immunochromatography assay kits (LFA), the Rose Bengal test (RBT) and a well-validated protein G indirect ELISA (iELISA) using sera of Brucella culture-positive and unvaccinated brucellosis free livestock. Sera from cattle vaccinated with S19 and RB51 brucellosis vaccines were also tested. Finally, we compared RBT and LFA using sera of white Fulani cattle of unknown bacteriological status from a brucellosis endemic area of Nigeria. Although differences were not statistically significant, RBT showed the highest values for diagnostic sensitivity/specificity in cattle (LFA, 96.6/98.8; RBT, 98.9/100; and iELISA, 96.6/100) and the iELISA yielded highest values in sheep (LFA, 94.0/100; RBT, 92.0/100; iELISA, 100/100), goats (LFA, 95.7/96.2; RBT, 97.8/100; iELISA, 100/100) and pigs (LFA, 92.3/100; RBT, 92.3/100; iELISA, 100/100). Vaccine S19 administered subcutaneously interfered in all tests but conjunctival application minimized the problem. Although designed not to interfere in serodiagnosis, vaccine RB51 interfered in LFA and iELISA but not in the RBT. We found closely similar apparent prevalence results when testing the Nigerian Fulani cattle by RBT and LFA. Although both RBT and LFA (showing similar diagnostic performance) are suitable for small laboratories in resource-limited areas, RBT has the advantage that a single reagent is useful in all animal species. Considering these advantages, its low cost and that it is also useful for human brucellosis diagnosis, RBT might be a good choice for resource-limited laboratories.

Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis . Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔ wadB (CO 2 -independent B. ovis with truncated LPS core oligosaccharide); Rev1:: wbdR Δ wbkC (carrying N-acetylated O-PS); and H38Δ wbkF ( B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38Δ wbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔ wadB and Rev1:: wbdR Δ wbkC conferred no or poor protection, respectively. All H38Δ wbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38Δ wbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis -free areas.

Brucellosis is a bacterial zoonosis caused by the genus Brucella, which mainly affects domestic animals. In these natural hosts, brucellae display a tropism towards the reproductive organs, such as the placenta, replicating in high numbers and leading to placentitis and abortion, an ability also exerted by the B. melitensis live-attenuated Rev1 strain, the only vaccine available for ovine brucellosis. It is broadly accepted that this tropism is mediated, at least in part, by the presence of certain preferred nutrients in the placenta, particularly erythritol, a polyol that is ultimately incorporated into the Brucella central carbon metabolism via two reactions dependent on transaldolase (Tal) or fructose-bisphosphate aldolase (Fba). In the light of these remarks, we propose that blocking the incorporation of erythritol into the central carbon metabolism of Rev1 by deleting the genes encoding Tal and Fba may impair the ability of the vaccine to proliferate massively in the placenta. Therefore, a Rev1ΔfbaΔtal double mutant was generated and confirmed to be unable to use erythritol. This mutant exhibited a reduced intracellular fitness both in BeWo trophoblasts and THP-1 macrophages. In the murine model, Rev1ΔfbaΔtal provided comparable protection to the Rev1 reference vaccine while inducing fewer adverse reproductive events in pregnant animals. Altogether, these results postulate the Rev1ΔfbaΔtal mutant as a reproductively safer Rev1-derived vaccine candidate to be studied in the natural host.

Brucellosis is one of the most common and widespread bacterial zoonoses and is caused by Gram-negative bacteria belonging to the genus Brucella. These organisms are able to infect and replicate within the placenta, resulting in abortion, one of the main clinical signs of brucellosis. Although the mouse model is widely used to study Brucella virulence and, more recently, to evaluate the protection of new vaccines, there is no clear consensus on the experimental conditions (e.g., mouse strains, doses, routes of inoculation, infection/pregnancy time) and the natural host reproducibility of the pregnant mouse model for reproductive brucellosis. This lack of consensus calls for a review that integrates the major findings regarding the effect of Brucella wild-type and vaccine strains infections on mouse pregnancy. We found sufficient evidence on the utility of the pregnant mouse model to study Brucella-induced placentitis and abortion and propose suitable experimental conditions (dose, time of infection) and pregnancy outcome readouts for B. abortus and B. melitensis studies. Finally, we discuss the utility and limitations of the pregnant mouse as a predictive model for the abortifacient effect of live Brucella vaccines.

Brucella is a genus of gram-negative bacteria that cause brucellosis . B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1–3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2Δ wbkF ) or only producing internal O-chain precursors (Bs2Δ wzm ) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2Δ wadB and Bs2Δ wadD ). We also investigated mutants in the pyruvate phosphate dikinase (Bs2Δ ppdK ) and phosphoenolpyruvate carboxykinase (Bs2Δ pckA ) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (10 8 and 10 5  CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2Δ wbkF , Bs2Δ wzm, Bs2Δ wadB and the Rev1 control (10 5  CFU). As described before for B. abortus , B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2Δ ppdK Δ pckA had the same metabolic phenotype as Bs2Δ ppdK and ppdK mutation was enough to generate attenuation. At 10 5  CFU, Bs2Δ ppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2Δ wbkF , the latter seems a better R vaccine candidate than Bs2Δ wzm . However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2Δ wbkF is advantageous over Bs2Δ wadB or Bs2Δ ppdK requires experiments in the natural host.

Sheep brucellosis is a worldwide extended disease caused by B. melitensis and B. ovis , two species respectively carrying smooth or rough lipopolysaccharide. Vaccine B. melitensis Rev1 is used against B. melitensis and B. ovis but induces an anti-smooth-lipopolysaccharide response interfering with B. melitensis serodiagnosis, which precludes its use against B. ovis where B. melitensis is absent. In mice, Rev1 deleted in wbkC ( Brucella lipopolysaccharide formyl-transferase) and carrying wbdR ( E. coli acetyl-transferase) triggered antibodies that could be differentiated from those evoked by wild-type strains, was comparatively attenuated and protected against B. ovis , suggesting its potential as a B. ovis vaccine.

Brucella bacteria cause brucellosis, a major zoonosis whose control requires efficient diagnosis and vaccines. Identification of classical Brucella spp. has traditionally relied on phenotypic characterization, including surface antigens and 5–10% CO 2 necessity for growth (CO 2 -dependence), a trait of Brucella ovis and most Brucella abortus biovars 1–4 strains. Although molecular tests are replacing phenotypic methods, CO 2 -dependence remains of interest as it conditions isolation and propagation and reflects Brucella metabolism, an area of active research. Here, we investigated the connection of CO 2 -dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO 2 to the bicarbonate used by anaplerotic and biosynthetic carboxylases. Based on the previous demonstration that B. suis carries two functional CAs (CAI and CAII), we analyzed the CA sequences of CO 2 -dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO 2 -dependent B. abortus and B. ovis , and that a modified CAII sequence explains the CO 2 -independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO 2 -independent growth of B. suis in rich media but not of B. abortus in rich media or B. suis in minimal media. Finally, we also found that insertion of a heterologous active CAII into B. ovis reverted the CO 2 -dependence but did not alter its virulence in the mouse model. These results allow a better understanding of central aspects of Brucella metabolism and, in the case of B. ovis , provide tools for large-scale production of diagnostic antigens and vaccines.

Background The role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates. Methods A multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar ( Sus scrofa ), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs. Results Mean apparent prevalence below 0.5% was identified in chamois ( Rupicapra pyrenaica ), Iberian wild goat ( Capra pyrenaica ), and red deer ( Cervus elaphus ). Roe deer ( Capreolus capreolus ), fallow deer ( Dama dama ), mouflon ( Ovis aries ) and Barbary sheep ( Ammotragus lervia ) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs. Conclusions In conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

In the original publication of this article [1], the corresponding author points out Pilar M. Muñoz and Raquel Conde‑Alvarez contributed equally to this work.In the original publication of this article [1], the corresponding author points out Pilar M. Muñoz and Raquel Conde‑Alvarez contributed equally to this work.