BUSCADOR RECOLECTA

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., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21), Gobierno de Navarra (PT040-2018 and PT007-2019) projects. SM-B contracts were granted by the FEDER 2016–2018 program of Garantía Juvenil and by an UPNA pre-doctoral fellowship 2018–2022. IP-U Doctorados Industriales contract was cofounded by Gobierno de Navarra and CSIC. Research in the FC lab was supported by The Swedish Research Council (VR), The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS), and The Kempe Foundation.

La tutora de la tesis es Inmaculada Farrán Blanch, La brucelosis es una enfermedad zoonótica causada por bacterias del género Brucella que, además, conlleva grandes pérdidas económicas. La principal fuente de infección para el ser humano son los rumiantes, en los que la enfermedad cursa con aborto y otras alteraciones reproductivas. En la mayoría de las regiones afectadas, la vacunación de los animales es la única estrategia realista para controlar la infección. La cepa atenuada B. melitensis Rev1 es la única vacuna recomendada frente a la brucelosis ovina y caprina, pero posee ciertos inconvenientes, entre los que destacan la virulencia en ovejas gestantes, la interferencia serológica que generan, la posibilidad de infectar al ser humano y la resistencia a los tratamientos con estreptomicina, que instan la búsqueda de nuevas vacunas. El lipopolisacárido liso (S-LPS) de Brucella es un conocido factor de virulencia y también el antígeno inmunodominante en las pruebas de diagnóstico, por lo que sus distintas modificaciones representan una interesante aproximación en la búsqueda de una vacuna segura. Entre las rutas de biosíntesis del S-LPS, el sistema de dos componentes Wzm/Wzt es el responsable de transportar el O-PS sintetizado en la cara citoplasmática de la membrana interna hasta el espacio periplasmático, para su posterior ensamblaje al core-lípido A para formar el S-LPS. El bloqueo de esta ruta conlleva la creación de mutantes con LPS rugoso (R-LPS) capaces de acumular un O-PS dentro de las bacterias que puede ser decisivo para generar una respuesta inmune efectiva frente a la infección. Sin embargo, aún se desconocen las implicaciones biológicas de cada proteína del transportador. En particular, el bloqueo del O-PS podría acarrear modificaciones en la envuelta bacteriana, ya que su transporte requiere la participación del bactoprenol como molécula portadora, el cual también está involucrado en la biosíntesis de otras estructuras celulares. Con el objetivo de profundizar en el conocimiento de este sistema transportador de Brucella y su potencial aplicación en el desarrollo de vacunas, en el capítulo 1 de esta tesis se presenta la construcción y caracterización de mutantes Δwzm/Δwzt simples y doble derivados de Rev1. Además, con fines comparativos, se construyeron mutantes ∆wzm derivados de B. abortus 2308 y S19, y se utilizó un mutante 16M∆wzm derivado de la cepa virulenta B. melitensis 16M previamente desarrollado en el grupo de Sanidad Animal del Instituto de Agrobiotecnología (IdAB). Como resultado, los mutantes carentes de O-PS externo exhibieron cambios de expresión génica, propiedades antigénicas del O-PS interno y cambios fenotípicos asociados a modificaciones de la membrana externa y/o de la pared celular. Además, los mutantes Rev1 ∆wzm/∆wzt fueron muy susceptibles
tanto a polimixinas como a los antibióticos utilizados para el tratamiento de la brucelosis humana, en particular, a la estreptomicina; y mostraron una gran atenuación en ratón. Entre ellos, Rev1∆wzm destacó por inducir un pico de esplenomegalia transitoria, ser altamente inmunogénico y generar una protección eficaz frente a la infección virulenta por B. melitensis y por B. ovis en el modelo murino. En el capítulo 2, antes de estudiar la seguridad de Rev1∆wzm en su hospedador natural, se determinó la capacidad de este candidato vacunal para crecer en los medios de cultivo selectivos Farrell (FM) y CITA (CM), actualmente recomendados para el aislamiento primario de Brucella. La fuerte susceptibilidad del mutante a las polimixinas y la sinergia observada entre colistina y vancomicina conllevaron una severa inhibición de este candidato vacunal en ambos medios selectivos. Para resolverlo, se reformuló y evaluó el denominado Brucella Selective Medium (BSM) que, tras su evaluación con más de 1.700 muestras ovinas de campo, demostró ser capaz de mejorar el rendimiento de los medios anteriores. Además, profundizando en la productividad de BSM para otras brucellae, se observó que tanto BSM como CM inhibían el crecimiento de una colección de cepas B. abortus bv1. El análisis de las posibles causas llevó al desarrollo un segundo medio selectivo denominado Brucella Selective Improved Culture (BruSIC) que resultó permisivo para todas las cepas de Brucella analizadas y tan inhibidor como CM para las bacterias contaminantes habituales en muestras veterinarias. BruSIC ofrece la ventaja adicional de no precisar suero bovino para el aislamiento de cepas de Brucella suero-dependientes, lo que simplifica la preparación del medio, abarata costes y evita el uso de compuestos de origen animal. En el capítulo 3, se analizó la seguridad y respuesta inmune inducidas en corderos inoculados con Rev1∆wzm, demostrando que se activa la formación de anticuerpos sin interferir en las pruebas serológicas estándar para el diagnóstico de la brucelosis. Estos resultados, más otros logrados por el grupo de investigación en ganado adulto y en ovejas gestantes, hacen de este mutante un prometedor candidato vacunal para el control de la infección por B. melitensis y B. ovis en pequeños rumiantes. El contenido de estos tres capítulos de tesis ha sido publicado en las revistas científicas Frontiers in Microbiology (grupo Frontiers) y Microbiology Spectrum (ASM)., Brucellosis is a zoonotic disease caused by bacteria of the genus Brucella, that also entails large economic losses. The main infection source for humans are ruminants, in which the disease induces abortion and other reproductive disorders. In most affected regions, vaccinating animals is the only realistic strategy to control the infection. The attenuated strain B. melitensis Rev1 is the only recommended vaccine against ovine and caprine brucellosis, but it has several drawbacks, such as virulence when administered in pregnant animals, serological interference, possibility of infecting humans, and resistance to treatments including streptomycin, that urge the search for new vaccines. The smooth lipopolysaccharide (S-LPS) is a well-known virulence factor and the immunodominant antigen in diagnostic tests, thus, its modifications represent an interesting approach in the research for a safe vaccine. Among S-LPS biosynthesis pathways, the Wzm/Wzt two-component system is the responsible of transporting the O-PS synthesised onto the cytoplasmic side of the inner membrane to the periplasm, for its subsequent assembly to the core-lipid A to form the S-LPS. Blocking this pathway leads to the generation of rough LPS (R-LPS) mutants capable of accumulating O-PS within the bacteria, that may be decisive for an effective immune response against the infection. However, the biological implications of each transporter protein are still unknown. In particular, the O-PS blockage could lead to modifications in bacterial envelope, since the O-PS transport requires the participation of bactoprenol as a carrier molecule, that is also involved in the biosynthesis of other cellular structures. Aiming to deepen the knowledge about this system and its potential application in vaccines development, in Chapter 1 of this thesis, it is presented the construction and characterisation of single and double Δwzm/Δwzt mutants derived from Rev1. In addition, for comparative purposes, ∆wzm mutants were constructed from B. abortus 2308 and S19 and studied together with a 16M∆wzm mutant, derived from the B. melitensis 16M virulent strain previously developed in the Animal Health Group of the Institute of Agrobiotechnology (IdAB). As a result, the mutants lacking outer O-PS exhibited changes in gene expression, antigenic properties of inner O-PS, and phenotypic changes associated with outer membrane and/or cell wall modifications. In addition, Rev1 ∆wzm/∆wzt mutants were highly susceptible to polymyxins and other antibiotics used in human brucellosis treatment, particularly, to streptomycin; and they showed a large attenuation in mouse. Among them, Rev1∆wzm stood out for inducing a transient peak of splenomegaly, being highly immunogenic, and generating effective protection against B. melitensis and B. ovis virulent infections in murine model. In Chapter 2, prior to studying the safety of Rev1∆wzm in the natural host, it was determined the ability of this vaccine candidate to grow in Farrell (FM) and CITA (CM) selective culture media, currently recommended for Brucella primary isolation. The mutant strong susceptibility to polymyxins and the synergy between colistin and vancomycin observed led to the severe inhibition of the vaccine candidate in both selective media. To solve this, the named Brucella Selective Medium (BSM) was reformulated and evaluated with more than 1,700 ovine field samples, being able to improve the previous media performance. Furthermore, by exploring the productivity of BSM for other brucellae, it was observed that both BSM and CM inhibited the growth of a B. abortus bv1 strains collection. The analysis of the possible causes led to the development of a second selective medium called Brucella Selective Improved Culture (BruSIC), which was permissive for all the Brucella strains analysed, and as inhibitory as CM for the usual contaminant bacteria in veterinary samples. In addition, BruSIC offers the advantage of not requiring bovine serum to isolate serum-dependent Brucella strains, simplifying medium preparation, lowering costs and avoiding the use of compounds from animal sources. In Chapter 3, it was analysed the safety and immune response induced in lambs inoculated with Rev1∆wzm, demonstrating the activation of antibodies development without interfering in the standard serological tests for brucellosis diagnosis. These results, as well as others achieved by the research group in adult cattle and pregnant sheep, make this mutant a promising vaccine candidate to control B. melitensis and B. ovis infections in small ruminants. The content of these three thesis chapters has been published in the scientific journals Frontiers in Microbiology (Frontiers group) and Microbiology Spectrum (ASM)., La realización de la presente tesis doctoral ha sido posible gracias a la obtención de un contrato del Programa de Ayudas para la Formación de Personal Investigador Predoctoral de la UPNA 2018-2022, disfrutada en el Instituto de Agrobiotecnología (IdAB; CSIC-Gobierno de Navarra).Los trabajos realizados han sido financiados por los proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R y RTI2018-098658-B-C21) y del Gobierno de Navarra (PT040-2018 y PT007-2019)., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

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 (5×10 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., This work was partially supported by the FES-CONARE of Costa Rica (www.conare.ac.cr), projects UNA 0009-12, 0248-13, 0504-13, 0505-13 from Universidad Nacional (www.investigacion.una.ac.cr). Grant 803-C0-456, 803-B3-761 Red Temática de brucelosis, Vicerrectoría de Investigación of the University of Costa Rica (www.vinv.ucr.ac.cr). Grant B8762 from Espacio Estudios Avanzados (UCREA) from the University of Costa Rica (www.ucrea.ucr.ac.cr). Bilateral cooperation CSIC-CRUSA Foundation (ref. 2010CR0005)(www.crusa.cr); grants from the Spanish Minister of Economy and Competitiveness (MINECO refs. AGL2010-20247, AGL2011-30453-C04-00, AGL2014-58795-C4-2-R and RTC-2015-3618-1). Fellowship support for The doctoral contract of Ana Zabalza was funded by Public University of Navarra, and the postdoctoral contract of Beatriz San Román was funded by JAE-DOC program of CSIC co-funded by European Social Fund (FSE, European Commission) and MINECO (AGL2011-30453-C04-00) of Spain (www.mineco.gob.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21), Gobierno de Navarra (PT040-2018 and PT007-2019) projects. SM-B contracts were granted by the FEDER 2016–2018 program of Garantía Juvenil and by an UPNA pre-doctoral fellowship 2018–2022. IP-U Doctorados Industriales contract was cofounded by Gobierno de Navarra and CSIC. Research in the FC lab was supported by The Swedish Research Council (VR), The Knut and Alice Wallenberg Foundation (KAW), The Laboratory of Molecular Infection Medicine Sweden (MIMS), and The Kempe Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Brucellosis, a re-emerging zoonotic infection, threatens animal welfare and public health with serious economic consequences. A definitive diagnosis requires Brucella isolation by culturing field specimens in specific media. This study aimed to (i) assess the effectivity of recommended Farrell’s médium (FM) and CITA medium (CM) for the isolation of four Brucella melitensis strains (16M, Rev1, and the 16MDwzm and Rev1Dwzm in-frame deletion mutants) with variable susceptibility to polymyxins; (ii) develop a Brucella selective medium (BSM) suitable for these strains; (iii) test BSM, FM, and CM with other Brucella species; and (iv) develop an improved selective culture medium (BruSIC) for all brucellae, including B. abortus bv1. The four B. melitensis strains were strongly inhibited in FM and (except Rev1) CM. Since Rev1Dwzm’s CM inhibition was due to a synergistic effect of colistin and vancomycin, we formulated BSM with half the concentrations of both antibiotics, achieving a similar growth of B. melitensis to blood agar base (BAB) and an inhibition of contaminant microorganisms comparable to CM; CM performance was surpassed by BSM for the primary isolation of B. melitensis when tested in 1,789 real sheep samples. For other brucellae, BSM and CM were more inhibitory than FM for B. abortus bv1 when using plates immediately after preparation but not after $4 weeks of storage. To address this, we developed the improved solid medium BruSIC by replacing the calf serum in BSM with activated charcoal. BruSIC yielded faster colony growth than BSM and CM and similar CFU numbers than BAB (including for B. ovis in BAB-Serum) and inhibited accompanying microorganisms in sheep and cow samples as effectively as BSM., This study was funded by the Department of Industry of the Gobierno de Navarra (PT068-2018 and PT007-2019) and Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21). The contract of S.M.-B. was granted by a UPNA 2018-2022 predoctoral fellowship, and that of I.P.-U. was granted by the Doctorados Industriales program of the Gobierno de Navarra cofunded by CSIC. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We thank the staff of Laboratorio Central de Sanidad Animal (LCSA; Granada, Spain) for supplying some of the strains, and the staff of VISAVET (UCM, Madrid, Spain) and INTIA (Navarra Government, Spain) for their veterinary assistance and/or support in obtaining animal samples.

Trabajo presentado en la 2nd FEMS Conference on Microbiology, celebrada en Belgrado (Serbia), del 30 de junio al 2 de julio de 2022, Animal brucellosis is characterized by abortion and bacterial shedding in
livestock. Brucella isolation is the only denite conrmation of infections. For
this, the use of adequate selective culture media able to inhibit the growth
of overcoming contaminants is an essential tool. Currently, simultaneous
culture in Farrell’s (FM) and CITA (CM) media is the recommended method.
However, attenuated mutants susceptible to polymyxins can be inhibited in
these media, providing misled safety results in the natural host., This work was funded by Agencia Estatal de Investigación of the Ministerio de Ciencia of Spain (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21) and Gobierno de Navarra (PT040-2018 and PT007-2019) projects. S.M-B. contract was granted
by an UPNA predoctoral fellowship 2018-2022. I.P-U. “Doctorados Industriales” contract was cofounded by Gobierno de Navarra and CSIC. The funders had no role in study design, data collection and analysis or decision to publish.

Brucellosis, a worldwide zoonotic disease, is endemic in many developing countries. Besides causing significant economic losses for the livestock industry, it has severe consequences for human health. In endemic regions, small ruminants infected by Brucella melitensis are the main source of human brucellosis. Rev1, the only vaccine currently recommended to control the disease in sheep and goats, has several drawbacks. Rough lipopolysaccharide (R-LPS) mutants have been tested as alternatives, but most lack efficacy. Those in the Wzm/Wzt system responsible for O-polysaccharide export to the periplasm have been proposed as promising vaccine candidates, although to date they have been scarcely investigated in the natural host. In the present work, we studied the biological properties of a 16MΔwzm in-frame deletion mutant, including its safety in pregnant mice and sheep. In mice, 16MΔwzm prevented placental and fetal infections before parturition and protected against B. melitensis and Brucella ovis infections. In sheep, 16MΔwzm was equally safe in lambs, rams, and non-pregnant ewes, inducing some transient Rose Bengal reactions (<7 weeks). The serological reactions occurred earlier and more strongly in pregnant than in non-pregnant ewes and were significantly reduced when conjunctival rather than subcutaneous vaccination was used. In ewes vaccinated at mid-pregnancy, 16MΔwzm was not shed in vaginal discharges during the pregnancy and did not induce abortions/stillbirths. However, some ewes showed a transitory reactivation of infection in placentas and/or milk at parturition, accompanied by a seroconversion in smooth LPS (S-LPS) and/or R-LPS tests. Overall, 16MΔwzm can be considered as a safe vaccine for lambs, rams, and non-pregnant ewes, but its use at mid-pregnancy should be avoided to prevent vaccine dissemination at parturition. If the efficacy results against B. melitensis and B. ovis observed in mice are confirmed by further studies in the natural host, 16MΔwzm could constitute a useful vaccine., This work was funded by the Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades of Spain (AGL2014-58795-C4-2-R and RTI2018-098658-B-C21) and Dirección General de Industria, Energía Proyectos Estratégicos S3 of Gobierno de Navarra, Spain (projects PT068-2018 and PT007-2019). IPU was contracted in the context of the Doctorados Industriales program of Gobierno de Navarra, co-funded by CSIC (2017-2020). SMB contracts were funded by the “Garantía Juvenil” program of CSIC-FEDER 2016-2018 and a predoctoral (2018-2019) fellowship of the Public University of Navarra (UPNA, Spain). Also, we thank the scientific advice of Professor Jean-Jacques Letesson; and the technical support of Sagrario Pérez, Elena San Miguel and Manuel Barrón (LCA-Navarra, Spain) as well as the staff of the subcontracted companies Maeva Servet-Visavet and Animalia (Spain).

Trabajo presentado para lograr el título de Doctor por la Universidad Pública de Navarra, Departamento de Agronomía, Biotecnología y Alimentación, Programa de Doctorado en Biotecnología.--Calificación: Sobresaliente cum laude, La brucelosis es una enfermedad zoonótica causada por bacterias del género Brucella que, además, conlleva grandes pérdidas económicas. La principal fuente de infección para el ser humano son los rumiantes, en los que la enfermedad cursa con aborto y otras alteraciones reproductivas. En la mayoría de las regiones afectadas, la vacunación de los animales es la única estrategia realista para controlar la infección. La cepa atenuada B. melitensis Rev1 es la única vacuna recomendada frente a la brucelosis ovina y caprina, pero posee ciertos inconvenientes, entre los que destacan la virulencia en ovejas gestantes, la interferencia serológica que generan, la posibilidad de infectar al ser humano y la resistencia a los tratamientos con estreptomicina, que instan la búsqueda de nuevas vacunas., El lipopolisacárido liso (S-LPS) de Brucella es un conocido factor de virulencia y también el antígeno inmunodominante en las pruebas de diagnóstico, por lo que sus distintas modificaciones representan una interesante aproximación en la búsqueda de una vacuna segura. Entre las rutas de biosíntesis del S-LPS, el sistema de dos componentes Wzm/Wzt es el responsable de transportar el O-PS sintetizado en la cara citoplasmática de la membrana interna hasta el espacio periplasmático, para su posterior ensamblaje al core-lípido A para formar el S-LPS. El bloqueo de esta ruta conlleva la creación de mutantes con LPS rugoso (R-LPS) capaces de acumular un O-PS dentro de las bacterias que puede ser decisivo para generar una respuesta inmune efectiva frente a la infección. Sin embargo, aún se desconocen las implicaciones biológicas de cada proteína del transportador. En particular, el bloqueo del O-PS podría acarrear modificaciones en la envuelta bacteriana, ya que su transporte requiere la participación del bactoprenol como molécula portadora, el cual también está involucrado en la biosíntesis de otras estructuras celulares. Con el objetivo de profundizar en el conocimiento de este sistema transportador de Brucella y su potencial aplicación en el desarrollo de vacunas, en el capítulo 1 de esta tesis se presenta la construcción y caracterización de mutantes Δwzm/Δwzt simples y doble derivados de Rev1. Además, con fines comparativos, se construyeron mutantes ∆wzm derivados de B. abortus 2308 y S19, y se utilizó un mutante 16M∆wzm derivado de la cepa virulenta B. melitensis 16M previamente desarrollado en el grupo de Sanidad Animal del Instituto de Agrobiotecnología (IdAB). Como resultado, los mutantes carentes de O-PS externo exhibieron cambios de expresión génica, propiedades antigénicas del O-PS interno y cambios fenotípicos asociados a modificaciones de la membrana externa y/o de la pared celular. Además, los mutantes Rev1 ∆wzm/∆wzt fueron muy susceptibles tanto a polimixinas como a los antibióticos utilizados para el tratamiento de la brucelosis humana, en particular, a la estreptomicina; y mostraron una gran atenuación en ratón. Entre ellos, Rev1∆wzm destacó por inducir un pico de esplenomegalia transitoria, ser altamente inmunogénico y generar una protección eficaz frente a la infección virulenta por B. melitensis y por B. ovis en el modelo murino. En el capítulo 2, antes de estudiar la seguridad de Rev1∆wzm en su hospedador natural, se determinó la capacidad de este candidato vacunal para crecer en los medios de cultivo selectivos Farrell (FM) y CITA (CM), actualmente recomendados para el aislamiento primario de Brucella. La fuerte susceptibilidad del mutante a las polimixinas y la sinergia observada entre colistina y vancomicina conllevaron una severa inhibición de este candidato vacunal en ambos medios selectivos. Para resolverlo, se reformuló y evaluó el denominado Brucella Selective Medium (BSM) que, tras su evaluación con más de 1.700 muestras ovinas de campo, demostró ser capaz de mejorar el rendimiento de los medios anteriores. Además, profundizando en la productividad de BSM para otras brucellae, se observó que tanto BSM como CM inhibían el crecimiento de una colección de cepas B. abortus bv1. El análisis de las posibles causas llevó al desarrollo un segundo medio selectivo denominado Brucella Selective Improved Culture (BruSIC) que resultó permisivo para todas las cepas de Brucella analizadas y tan inhibidor como CM para las bacterias contaminantes habituales en muestras veterinarias. BruSIC ofrece la ventaja adicional de no precisar suero bovino para el aislamiento de cepas de Brucella suero-dependientes, lo que simplifica la preparación del medio, abarata costes y evita el uso de compuestos de origen animal. En el capítulo 3, se analizó la seguridad y respuesta inmune inducidas en corderos inoculados con Rev1∆wzm, demostrando que se activa la formación de anticuerpos sin interferir en las pruebas serológicas estándar para el diagnóstico de la brucelosis. Estos resultados, más otros logrados por el grupo de investigación en ganado adulto y en ovejas gestantes, hacen de este mutante un prometedor candidato vacunal para el control de la infección por B. melitensis y B. ovis en pequeños rumiantes. El contenido de estos tres capítulos de tesis ha sido publicado en las revistas científicas Frontiers in Microbiology (grupo Frontiers) y Microbiology Spectrum (ASM)., La realización de la presente tesis doctoral ha sido posible gracias a la obtención de un contrato del Programa de Ayudas para la Formación de Personal Investigador Predoctoral de la UPNA 2018-2022, disfrutada en el Instituto de Agrobiotecnología (IdAB; CSIC-Gobierno de Navarra).Los trabajos realizados han sido financiados por los proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades (AGL2014-58795-C4-2-R y RTI2018-098658-B-C21) y del Gobierno de Navarra (PT040-2018 y PT007-2019).