Zabalza Baranguá, Ana

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https://academica-e.unavarra.es/handle/2454/50194

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Zabalza Baranguá

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Ana

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Instituto de Agrobiotecnología (IdAB)

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810895

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Open Access
Simultaneous infections by different Salmonella strains in mesenteric lymph nodes of finishing pigs
(BioMed Central, 2014) Garrido González, Victoria; Sánchez Alarcón, Samanta Rita; San Román Aberasturi, Beatriz; Zabalza Baranguá, Ana; Díaz Tendero, Yasmín; Frutos, Cristina de; Mainar Jaime, Raúl Carlos; Grilló Dolset, María Jesús; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14064.RI1; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Background: Salmonellosis is a major worldwide zoonosis, and Salmonella-infected finishing pigs are considered one of the major sources of human infections in developed countries. Baseline studies on salmonellosis prevalence in fattening pigs in Europe are based on direct pathogen isolation from mesenteric lymph nodes (MLN). This procedure is considered the most reliable for diagnosing salmonellosis in apparently healthy pigs. The presence of simultaneous infections by different Salmonella strains in the same animal has never been reported and could have important epidemiological implications. Results: Fourteen finishing pigs belonging to 14 farms that showed high salmonellosis prevalence and a variety of circulating Salmonella strains, were found infected by Salmonella spp, and 7 of them were simultaneously infected with strains of 2 or 3 different serotypes. Typhimurium isolates showing resistance to several antimicrobials and carrying mobile integrons were the most frequently identified in the colonized MLN. Four animals were found infected by Salmonella spp. of a single serotype (Rissen or Derby) but showing 2 or 3 different antimicrobial resistance profiles, without evidence of mobile genetic element exchange in vivo. Conclusion: This is the first report clearly demonstrating that pigs naturally infected by Salmonella may harbour different Salmonella strains simultaneously. This may have implications in the interpretation of results from baseline studies, and also help to better understand human salmonellosis outbreaks and the horizontal transmission of antimicrobial resistance genes.
Open Access
Brucella melitensis wzm/wzt system: changes in the bacterial envelope lead to improved rev1Δwzm vaccine properties
(Frontiers Media, 2022) Mena Bueno, Sara; Poveda Urkixo, Irati; Irazoki, Oihane; Palacios Chaves, Leyre; Cava, Felipe; Zabalza Baranguá, Ana; Grilló Dolset, María Jesús; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Gobierno de Navarra / Nafarroako Gobernua
The lipopolysaccharide (LPS) O-polysaccharide (O-PS) is the main virulence factor in Brucella. After synthesis in the cytoplasmic membrane, O-PS is exported to the periplasm by the Wzm/Wzt system, where it is assembled into a LPS. This translocation also engages a bactoprenol carrier required for further biosynthesis pathways, such as cell wall biogenesis. Targeting O-PS export by blockage holds great potential for vaccine development, but little is known about the biological implications of each Wzm/Wzt moiety. To improve this knowledge and to elucidate its potential application as a vaccine, we constructed and studied wzm/wzt single- and double-deletion mutants, using the attenuated strain Brucella melitensis Rev1 as the parental strain. This allowed us to describe the composition of Brucella peptidoglycan for the first time. We observed that these mutants lack external O-PS yet trigger changes in genetic transcription and in phenotypic properties associated with the outer membrane and cell wall. The three mutants are highly attenuated; unexpectedly, Rev1Δwzm also excels as an immunogenic and effective vaccine against B. melitensis and Brucella ovis in mice, revealing that low persistence is not at odds with efficacy. Rev1Δwzm is attenuated in BeWo trophoblasts, does not infect mouse placentas, and is safe in pregnant ewes. Overall, these attributes and the minimal serological interference induced in sheep make Rev1Δwzm a highly promising vaccine candidate.
Open Access
Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine
(BioMed Central, 2013) Mancilla, Marcos; Grilló Dolset, María Jesús; Miguel López, María Jesús de; López Goñi, Ignacio; San Román Aberasturi, Beatriz; Zabalza Baranguá, Ana; Moriyón Uría, Ignacio; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.
Open Access
Desarrollo de vacunas marcadas con GFP frente a la brucelosis ovina y tests diagnósticos asociados
(2017) Zabalza Baranguá, Ana; Grilló Dolset, María Jesús; San Román Aberasturi, Beatriz; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
La brucelosis es una zoonosis extendida mundialmente con importantes repercusiones económicas y sanitarias, cuya principal fuente de infección para el ser humano son los pequeños rumiantes infectados por Brucella melitensis. En gran parte del mundo, el control de la brucelosis en estos animales debe basarse en la vacunación. B. melitensis Rev1 es la única vacuna disponible y recomendada internacionalmente para ovejas y cabras. Sin embargo, la vacunación con Rev1 genera una interferencia serológica que impide la Diferenciación entre Animales Infectados y Vacunados (DIVA), limitando el uso de esta cepa vacunal. Para tratar de solventar el problema DIVA, se ha desarrollado una técnica de marcaje xenogénico con la proteína de origen marino GFP. En el capítulo 1 de esta tesis, se obtuvieron dos isoformas de GFP recombinante y se desarrollaron tests serológicos utilizando dichas proteínas como antígeno diagnóstico. En el capítulo 2, se realizó el marcaje xenogénico de Rev1 con gfp mediante la inserción cromosómica dirigida de un mini-Tn7-gfp (Rev1::gfp) y un análisis serológico detallado, tanto en ratones BALB/c como en ganado ovino. La estrategia de vacunación con Rev1::gfp en mezcla con GFP y un booster posterior con GFP en hidróxido de aluminio, permitió identificar durante más de 6 meses post-booster a todos los animales que habían sido vacunados, utilizando las técnicas serológicas oficiales anti-LPS-S y un iELISA-GFP. Además, la vacuna clásica Rev1 posee otros inconvenientes como son su poder patógeno residual y resistencia a la estreptomicina, que recomiendan la búsqueda de nuevas vacunas frente a B. melitensis. Para ello, en el capítulo 3 se construyeron y caracterizaron los candidatos vacunales 16Δ.wzm y 16Δ.wzm::gfp, con LPS-R, a la vez que acumulan PS-0 en el interior bacteriano, y sensibles a estreptomicina. Estas propiedades confirieron una atenuación y eficacia frente a B. melitensis en ratones similares a las de Rev1. En corderos, la vacunación combinada de 16Δ.wzm::gfp con GFP generó una mínima interferencia en las pruebas convencionales con LPS-S y permitió identificar a los anímales vacunados mediante iELISA-GFP, sin necesidad de booster. Por otra parte, Rev1 ha permitido controlar la infección por Brucella ovis. En las zonas donde se ha abandonado la vacunación con Rev1, B. ovis es una patógeno emergente que causa graves pérdidas económicas. Puesto que no existe una vacuna específica frente a B. ovis, el capítulo 4 se centró en analizar las propiedades vacunales de una selección de cinco mutantes rugosos de B. melitensis obtenidos en un proyecto anterior, mediante inserción aleatoria del mini-Tn5. De ellos, se seleccionaron y construyeron por deleción en fase las cepas marcadas 16Δ.wzm::gfp (capítulo 3) y H38ΔwbkF::gfp (capítulo 4). Además, se seleccionó un mutante de B. ovis PA que había mostrado buenas propiedades en el modelo murino en un trabajo anterior, para su marcaje con el mini-Tn7-gfp, generando la cepa BoPAiΔomp10ΔugpBΔomp31::gfp. La vacunación con una de estas cepas marcadas, tanto por vía intraperitoneal como subcutánea, evidenció mejor protección con los mutantes de B. melitensis que con el triple mutante de B. avis en el modelo murino. Finalmente, se evaluó la inocuidad y respuesta serológica de 16MΔwzm::gfp y BoPAΔomp10iΔugpBfΔomp31::gfp en ganado ovino, tras la vacunación combinada del mutante con GFP y posterior booster con GFP en hidróxido de aluminio, siguiendo el protocolo descrito en el Capítulo 2. Al igual que con Rev1::gfp, esta estrategia permitió identificar por iELISA-GFP a todos los animales que habían sido vacunados, durante más de 4 meses post-booster. En conjunto, por un lado, el marcaje de Brucella con el mini-Tn7-gfp permitió conservar las propiedades microbiológicas y vacunales que poseían los correspondientes mutantes antes de ser marcados y, a su vez, permitió identificarlos fácilmente por visualización directa de la ftuorescencia y por una PCR-GFP múltiple específicamente diseñada. Por otro lado, Rev1::gfp y 16MΔwzm::gfp parecen buenas alternativas a Rev1 frente a infecciones por B. melitensis virulentas.
Open Access
Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice
(BioMed Central, 2014) Soler Lloréns, Pedro; Gil Ramírez, Yolanda; Zabalza Baranguá, Ana; Iriarte, Maite; Conde Álvarez, Raquel; Zúñiga Ripa, Amaia; San Román Aberasturi, Beatriz; Zygmunt, Michel; Vizcaíno, Nieves; Cloeckaert, Axel; Grilló Dolset, María Jesús; Moriyón Uría, Ignacio; López Goñi, Ignacio; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.