“Influencia de la alimentación en la presencia de Salmonella y composición del microbioma intestinal en porcino extensivo”

La salmonelosis es la segunda zoonosis más frecuente de la UE y el ganado porcino es, tras las aves, la segunda fuente de infección más importante para el ser humano. Por ello, la salmonelosis porcina constituye un serio problema de Salud Pública que requiere un especial control y vigilancia epidemiológica. Para preservar la salud de los consumidores, la UE recomienda realizar controles exhaustivos en todas las fases de la cadena alimentaria “de la granja a la mesa” con la intención de reducir progresivamente la carga bacteriana hasta llegar a los alimentos. Entre otras medidas, está previsto instaurar restricciones en el comercio internacional de cerdos y sus productos derivados para aquellos países que no cumplan los objetivos fijados de reducción de la prevalencia. Estas restricciones pueden tener importantes repercusiones económicas en nuestro país, puesto que el sector porcino es un pilar fundamental de los recursos ganaderos de España, que es el cuarto productor mundial tras EE.UU., China y Alemania. Asimismo, el porcino extensivo como el Basatxerri de País Vasco y norte de Navarra está cobrando importancia como producción de calidad y, a su vez, como estandarte de desarrollo rural sostenible y del bienestar animal. Sin embargo, se desconoce la situación epidemiológica y sanitaria de la salmonelosis en este tipo de animales. En este estudio, se analizó la presencia de Salmonella spp. en el contenido intestinal de una población representativa de cerdos Basatxerri y los factores de riesgo asociados a la misma. Asimismo, para evaluar la posible implicación de los factores alimentarios como fuente de exclusión directa o indirecta de Salmonella, se realizó un estudio del microbioma mediante secuenciación masiva del contenido intestinal. Como resultado, se aisló Salmonella en el 32,2% de los animales y en el 83,3% de las explotaciones analizadas, observando grandes diferencias de prevalencia entre explotaciones. Las cepas aisladas pertenecían mayoritariamente a la variante monofásica de S. Typhimurium (79,3%) y también a S. Bovismorbificans (10,3%), mostrando resistencia a al menos uno (89,7%) o a más de dos (86,2%) de los antimicrobianos analizados, siendo estreptomicina y sulfisoxazol los más involucrados. El análisis univariante detectó cuatro factores de riesgo directamente relacionados con la presencia de Salmonella en el contenido intestinal, que fueron: el número de animales/explotación; la composición del pienso administrado; el tipo de vegetación ingerida por los animales; y la limpieza y desinfección de los silos. Los tres últimos parámetros mostraron asociación entre sí, por lo que el análisis multivariante se realizó considerando la composición del pienso como el factor asociado más interesante, permitiendo identificar que la administración del Pienso A era un factor de riesgo (p=0,008) de salmonelosis en los animales analizados. Para determinar el peso relativo de las otras variables asociadas, sería necesario realizar un estudio complementario adecuado a este objetivo. El estudio del microbioma intestinal reveló claras diferencias entre las poblaciones bacterianas de los animales portadores y no portadores de Salmonella, detectando en los primeros un aumento de los phylum Firmicutes y Proteobacterias, acompañado de un descenso del phylum Bacteroides. La comparación de los OTUs indicó la existencia de diferencias en diez familias bacterianas entre los cerdos portadores y no portadores de Salmonella, con mayor peso estadístico de las Mycoplasmataceae, Enterobacteriaceae, Verrucomicrobiaceae y Pseudomonadaceae. En definitiva, todos los resultados indicaron que la alimentación animal (i.e. composición del pienso y/o la vegetación suplementaria) es un factor de riesgo de primera magnitud en la prevalencia de la salmonelosis porcina en explotaciones extensivas, por lo que puede ser utilizada como herramienta para el control sostenible de esta importante zoonosis desde la granja

Desarrollo de pruebas serológicas para la identificación de ovinos inmunizados con vacunas-GFP

Publishe

What have we learned from brucellosis in the mouse model?

Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-gamma, TLR9, Myd88, Tgammadelta and TNF-beta favor Brucella replication; whereas IL-1beta, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-gamma and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (greater than or equal to] 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model.This work was performed under agreement contract 2010020113, subscribed by UNA from Costa Rica and, CSIC, CITA, and UN from Spain. This work was funded by grants FIDA-2009 UNA, FS-CONARE UNA/UCR, NeTropica 8, and MICIT/CONICIT, CSIC-CRUSA (2010CR0005) from Costa Rica; and CICYT-MICINN (AGL2010-20247, AGL2008-04514-C03-00 and AGL2011-30453-C04-00) projects from Spain and grant ANR2010BLAN1308 Brutir., form France. This work was done as part of the UCR/DAAD Humboldt Fellow award 2012 to EM

In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [18F]fluoro-deoxyglucose–MicroPET in a mouse model

A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [18F]fluoro-deoxyglucose–MicroPET ([18F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [18F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [18F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches.This work was supported by grants from Gobierno de Navarra “IIM13002.RI1” and MICINN “CIT-010000-2009-32”

Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine

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.This work was funded by MINECO (reference project AGL2011-30453-C04) of Spain, the FIMA foundation and the European Union’s FP7/2007-2013 (grant agreement n° 221948, ICONZ - Integrated control of Neglected Zoonoses) and CSIC JAE-Doc program (FSE)

The extradomain a of fibronectin enhances the efficacy of lipopolysaccharide defective Salmonella bacterins as vaccines in mice

The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SE Delta waaL) or deep-defective (SE Delta gal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SE Delta waaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SE Delta waaL as non-live vaccine in the mouse model.This work was funded by Gobierno de Navarra and European Union (project EuroInnova-Navarra reference IIM10865.RI1-EP12). B.S.R., P.M.M. and X.D.A. post-doctoral contracts were granted by Gobierno de Navarra/JAE-doc CSIC, MICINN (Subprograma Juan de la Cierva) and Universidad Pública de Navarra, respectively