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Brucella ovis causes non-zoonotic ovine brucellosis of worldwide distribution and is responsible for important economic losses mainly derived from male genital lesions and reproductive fails. Studies about the virulence mechanisms of this rough species (lacking lipopolysaccharide O-chains) are underrepresented when compared to the main zoonotic Brucella species that are smooth (with O-chains). Zinc intoxication constitutes a defense mechanism of the host against bacterial pathogens, which have developed efflux systems to counterbalance toxicity. In this study, we have characterized three potential B. ovis zinc exporters, including the ZntA ortholog previously studied in B. abortus. Despite an in-frame deletion removing 100 amino acids from B. ovis ZntA, the protein retained strong zinc efflux properties. Only indirect evidence suggested a higher exporter activity for B. abortus ZntA, which, together with differences in ZntR-mediated regulation of zntA expression between B. ovis and B. abortus, could contribute to explaining why the ΔzntR mutant of B. abortus is attenuated while that of B. ovis is virulent. Additionally, B. ovis ZntA was revealed as a powerful cadmium exporter contributing to cobalt, copper, and nickel detoxification, properties not previously described for the B. abortus ortholog. Deletion mutants for BOV_0501 and BOV_A1100, also identified as potential zinc exporters and pseudogenes in B. abortus, behaved as the B. ovis parental strain in all tests performed. However, their overexpression in the ΔzntA mutant allowed the detection of discrete zinc and cobalt efflux activity for BOV_0501 and BOV_ A1100, respectively. Nevertheless, considering their low expression levels and the stronger activity of ZntA as a zinc and cobalt exporter, the biological role of BOV_0501 and BOV_A1100 is questionable. Results presented in this study evidence heterogeneity among pathogenic Brucellae regarding zinc export and, considering the virulence of B. ovis ΔzntA, suggest that hostmediated zinc intoxication is not a relevant mechanism to control B. ovis infection. [ABSTRACT FROM AUTHOR]

Brucella ovis is a non-zoonotic Brucella species lacking specific vaccine. It presents a narrow host range, a unique biology relative to other Brucella species, and important distinct surface properties. To increase our knowledge on its peculiar surface and virulence features, and seeking to develop a specific vaccine, multiple mutants for nine relevant cell-envelope-related genes were investigated. Mutants lacking Omp10 plus Omp19 could not be obtained, suggesting that at least one of these lipoproteins is required for viability. A similar result was obtained for the double deletion of omp31 and omp25 that encode two major surface proteins. Conversely, the absence of major Omp25c (proved essential for internalization in HeLa cells) together with Omp25 or Omp31 was tolerated by the bacterium. Although showing important in vitro and in vivo defects, the Δ omp10 Δ omp31 Δ omp25c mutant was obtained, demonstrating that B. ovis PA survives to the simultaneous absence of Omp10 and four out seven proteins of the Omp25/Omp31 family (i.e., Omp31, Omp25c, Omp25b, and Omp31b, the two latter naturally absent in B. ovis). Three multiple mutants were selected for a detailed analysis of virulence in the mouse model. The Δ omp31 Δ cgs and Δ omp10 Δ omp31 Δ omp25c mutants were highly attenuated when inoculated at 106 colony forming units/mouse but they established a persistent infection when the infection dose was increased 100-fold. The Δ omp10 Δ ugpB Δ omp31 mutant showed a similar behavior until week 3 post-infection but was then totally cleared from spleen. Accordingly, it was retained as vaccine candidate for mice protection assays. When compared to classical B. melitensis Rev1 heterologous vaccine, the triple mutant induced limited splenomegaly, a significantly higher antibody response against whole B. ovis PA cells, an equivalent memory cellular response and, according to spleen colonization measurements, better protection against a challenge with virulent B. ovis PA. Therefore, it would be a good candidate to be evaluated in the natural host as a specific vaccine against B. ovis that would avoid the drawbacks of B. melitensis Rev1. In addition, the lack in this attenuated strain of Omp31, recognized as a highly immunogenic protein during B. ovis infection, would favor the differentiation between infected and vaccinated animals using Omp31 as diagnostic target. [ABSTRACT FROM AUTHOR]

The factorial structure of the Dysexecutive Questionnaire (DEX) is an unresolved issue in scientific literature. One-to-five-factor solutions have been found in several studies by applying different research methods. Only a few of these studies used appropriate analysis procedures to suit a Likert scale-type of answer or investigated large enough samples to ensure the stability of factorial solutions. The present study examines a sample of 2151 subjects, 1482 from the general population and 669 from a clinical population. An unrestricted factorial analysis was carried out on both samples. The results unequivocally point to a single-factor solution in both samples. This means that only one latent variable is displayed in the DEX, which accounts for symptoms of oversight malfunction in activities of daily living. It is concluded that the diversity of results previously obtained in other studies may be due to using research methods that depict Likert-type scales on a continuum when they are actually ordinal categorical measures. In conclusion, the DEX should be considered a screening test that reports symptoms of prefrontal malfunction, although it is unable to specify what areas or functions have been affected, as previous studies have claimed. [ABSTRACT FROM AUTHOR]

Alfalfa ( Medicago sativa L.) is an important crop worldwide whose cropping in acid soils is hampered by the poor nodulation and yield commonly attributed to the sensitivity of its endosymbionts to acid pH. In this work, we isolated several acid-tolerant strains from alfalfa nodules in three acid soils in northwestern Spain. After grouping by RAPD fingerprinting, most strains were identified as Ensifer meliloti and only two strains as Ensifer medicae according to their 16S-23S intergenic spacer (ITS) sequences that allowed the differentiation of two groups within each one of these species. The two ITS groups of E. meliloti and the ITS group I of E. medicae have been previously found in Medicago nodules; however, the group II of E. medicae has been only found to date in Prosopis alba nodules. The analysis of the nodC gene showed that all strains isolated in this study belong to the symbiovar meliloti, grouping with the type strains of E. meliloti or E. medicae, but some harboured nodC gene alleles different from those found to date in alfalfa nodules. The strains of E. medicae belong to the symbiovar meliloti which should be also recognised in this species, although they harboured a nodC allele phylogenetically divergent to those from E. meliloti strains. Microcosm experiments showed that inoculation of alfalfa with selected acid-tolerant strains significantly increased yields in acid soils representing a suitable agricultural practice for alfalfa cropping in these soils. [ABSTRACT FROM AUTHOR]

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium– Vicia symbiosis. [ABSTRACT FROM AUTHOR]

The toxicity and mutagenecity of 1000-fold concentrates of 5 litre samples of wastewater from five different towns (containing 500, 600, 15000, 50000 and 190000 inhabitants) with different sewage treatment processes were determined. Two kinds of samples, including municipal wastewater entering the sewage treatment plants and the effluents discharged into the environment were analysed. After concentration through resins, overall toxicity was evaluated using the bioassay employing Photobacterium phosphoreum and samples were classified according to Vasseur et al. (1986) as 'Non-toxic', 'Slightly toxic', 'Toxic' or 'Highly toxic'. Mutagenecity was evaluated using the mutagenecity index determined by the Ames test, and samples were classified as 'Non-mutagenic', 'Slightly mutagenic', or 'Mutagenic', according to the 'two-fold' rule. The results obtained varied considerably; such that in some plants the toxicity values decreased during treatment whereas in others they remained unmodified or increased. None of the extracts analysed proved to be mutagenic. The data on toxicity and mutagenecity of the samples are apparently not correlated. [ABSTRACT FROM AUTHOR]