Blasco Canet, José María
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Blasco Canet
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José María
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Psicología y Pedagogía
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Publication Open Access Development of a selective culture medium for primary isolation of the main Brucella species(American Society for Microbiology, 2011) Miguel López, María Jesús de; Marín, Clara M.; Muñoz Álvaro, Pilar María; Dieste, L.; Grilló Dolset, María Jesús; Blasco Canet, José María; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaBacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA’s performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis.Publication Open Access Brucellosis vaccines: assessment of brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export(Public Library of Science, 2008) González, David; Grilló Dolset, María Jesús; Miguel, María Jesús de; Ali, Tara; Arce Gorvel, Vilma; Delrue, Rose May; Conde Álvarez, Raquel; Muñoz Álvaro, Pilar María; López Goñi, Ignacio; Iriarte, Maite; Marín, Clara M.; Weintraub, Andrej; Widmalm, Goeran; Zygmunt, Michel; Letesson, Jean-Jacques; Gorvel, Jean-Pierre; Blasco Canet, José María; Moriyón Uría, Ignacio; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaBackground: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.Publication Open Access Spatial distribution and risk factors of brucellosis in Iberian wild ungulates(BioMed Central, 2010) Muñoz Álvaro, Pilar María; Boadella, Mariana; Arnal, Maricruz; Miguel, María Jesús de; Revilla, Miguel; Martínez, David; Vicente, Joaquín; Acevedo, Pelayo; Oleaga, Álvaro; Ruiz Fons, Francisco; Marín, Clara M.; Prieto, José M.; Fuente, José de la; Barral, Marta; Barberán, Montserrat; Fernández de Luco, Daniel; Blasco Canet, José María; Gortázar, Christian; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaBackground: 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.