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

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“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

Multidrug resistant Salmonella enterica isolated from conventional pig farms using antimicrobial agents in preventative medicine programmes

A longitudinal study was conducted to investigate the presence of multidrug antimicrobial resistance (multi-AR) in Salmonella enterica in pigs reared under conventional preventative medicine programmes in Spain and the possible association of multi-AR with ceftiofur or tulathromycin treatment during the pre-weaning period. Groups of 7-day-old piglets were treated by intramuscular injection with ceftiofur on four farms (n = 40 piglets per farm) and with tulathromycin on another four farms (n = 40 piglets per farm). A control group of untreated piglets (n = 30 per farm) was present on each farm. Faecal swabs were collected for S. enterica culture prior to treatment, at 2, 7 and 180 days post-treatment, and at slaughter. Minimal inhibitory concentrations of 14 antimicrobial agents, pulsed-field gel electrophoresis and detection of resistance genes representing five families of antimicrobial agents were performed. Plasmids carrying cephalosporin resistant (CR) genes were characterised. Sixty-six S. enterica isolates were recovered from five of eight farms. Forty-seven isolates were multi-AR and four contained blaCTX-M genes harboured in conjugative plasmids of the IncI1 family; three of these isolates were recovered before treatment with ceftiofur. The most frequent AR genes detected were tet(A) (51/66, 77%), sul1 (17/66, 26%); tet(B) (15/66, 23%) and qnrB (10/66,15%). A direct relation between the use of ceftiofur in these conditions and the occurrence of CR S. enterica was not established. However, multi-AR was common, especially for ampicillin, streptomycin, sulphonamides and tetracycline. These antibiotics are used frequently in veterinary medicine in Spain and, therefore, should be used sparingly to minimise the spread of multi-ARThis work was supported by project AGL2011-28836 from the Ministerio de Economia y Competitividad of Spain, the Departamento de Innovación, Empresa y Empleo del Gobierno de Navarra (project reference IIQ14064.RI1) and Fundación Caja Navarra, Spain (project reference VATC 2014-0411 y VATC 2015-70628). Contract of LMG was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the European Social Fund. Contract of VG was supported by Fundación Caja Navarra, Spain (project reference VATC 2015-70628). We acknowledge the Centres de Recerca de Catalunya programme for financial support, and to John G. Wild for review of English

Salmonella infection in mesenteric lymph nodes of breeding sows

Salmonellosis is one of the main foodborne diseases worldwide. Breeding sows asymptomatically infected with Salmonella can transmit the pathogen to piglets and humans. The isolation of Salmonella from mesenteric lymph nodes (MLNs) is considered a demonstration of asymptomatic infection in swine. As previous breeding sow studies have been performed using feces, the aim of this work was to study the occurrence of Salmonella infections by sampling MLNs, in comparison to their serological status. First, Salmonella fecal shedding was studied in 12/16 large breeding farms to establish the framework of study. Then, MLN (n = 264) and blood (n = 237) samples were obtained at an abattoir from sows of 15 of these 16 breeding farms. Additionally, risk factors associated with Salmonella MLN infection were analyzed. A total of 6.1% (16/264) sows, distributed in 40% (6/15) of the farms, had the pathogen in MLN. Salmonella Typhimurium was the most frequent serovar isolated. Interestingly, 43.8% (7/16) of MLN isolates were susceptible to all the antimicrobials tested and were found distributed throughout all farms with at least one sow positive. As well, one isolate of the emerging DT195 clone was detected and found to be resistant to six antibiotic families (ASSuTNx-Cfx). The serovars and the resistance profiles of the Salmonella isolates from feces were completely different to those obtained from MLNs. The seroprevalence (41.8% of sows and 100% of farms) was higher than that of MLN infections, showing no concordance (k = 0.15) between these two diagnostic tests in sows. Strategies directed to correct two risk factors (i.e., administration of dry food and old premises) would most likely help to reduce Salmonella infections in breeding sows.The work was financed by Departamento de Industria, Energía e Innovación of the Navarra Government [reference IIQ14064.RI1] and Instituto Navarro de Tecnología e Infraestructuras Agroalimentarias S.A. -INTIA- [reference CAM2011030054]. VG, SS, BSR and LMG contracts were funded by UPNA postdoctoral fellowship, Erasmus Mundus EMUNDUS18 Program, CSIC JAE-Doc Program, and INIA-European Social Fund, respectively. We are grateful to farmers, veterinaries and slaughterhouse workers as well as to the temporal students Naroa Remondegui, June Landa (JAE-Intro CSIC-FEDER fellowship), Ruth Erro and Mirian Samblas

Simultaneous infections by different Salmonella strains in mesenteric lymph nodes of finishing pigs

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.The work was financed by Gobierno de Navarra (project reference IIQ14064.RI1) and INIA (project reference RTA2007-65). Contracts were funded by UPNA (VG postdoctoral contract, and AZB predoctoral fellowship), EMUNDUS18 program (SS) and CSIC in collaboration with the European Social Fund (BSR “Programa JAE-Doc” contract)

Prevalence of Salmonella in Free-Range Pigs: Risk Factors and Intestinal Microbiota Composition

Extensive pig systems are gaining importance as quality production systems and as the standard for sustainable rural development and animal welfare. However, the effects of natural foods on Salmonella epidemiology remain unknown. Herein, we assessed the presence of Salmonella and the composition of the gut microbiota in pigs from both Salmonella-free and high Salmonella prevalence farms. In addition, risk factors associated with the presence of Salmonella were investigated. The pathogen was found in 32.2% of animals and 83.3% of farms, showing large differences in prevalence between farms. Most isolates were serovars Typhimurium monophasic (79.3%) and Bovismorbificans (10.3%), and exhibited a multi-drug resistance profile (58.6%). Risk factor analysis identified feed composition, type/variety of vegetation available, and silos’ cleaning/disinfection as the main factors associated with Salmonella prevalence. Clear differences in the intestinal microbiota were found between Salmonella-positive and Salmonella-negative populations, showing the former with increasing Proteobacteria and decreasing Bacteroides populations. Butyrate and propionate producers including Clostridium, Turicibacter, Bacteroidaceae_uc, and Lactobacillus were more abundant in the Salmonella-negative group, whereas acetate producers like Sporobacter, Escherichia or Enterobacter were more abundant in the Salmonella-positive group. Overall, our results suggest that the presence of Salmonella in free-range pigs is directly related to the natural vegetation accessible, determining the composition of the intestinal microbiota.info:eu-repo/semantics/publishedVersio

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

Intestinal microbiota composition in free-range pigs is associated with the presence of Salmonella

Extensive pig systems are gaining importance as quality production systems and as stand ard for sustainable rural development and animal welfare. However, the effect of natural food on Salmonella epidemiology remains unknown. Here we assessed the presence of Salmonella in the in testinal content, the risks factors associated, and the gut microbiota composition in pigs selected from Salmonella-free and high prevalence farms. The pathogen was found in 32.2% of animals and 83.3% of farms, showing large differences in prevalence between farms. Most isolates were serovars Typhimurium monophasic (79.3%) and Bovismorbificans (10.3%), exhibiting multi-drug resistance (58.6%). Risk factor analysis identified feed composition, type/variety of vegetation available, and silos' cleaning/disinfection, as main factors associated with Salmonella prevalence. Clear differences in the intestinal microbiota were found between Salmonella-positive and Salmonella-negative popu- lations, showing the former increasing Proteobacteria and decreasing Bacteroides populations. Butyr ate and propionate producers, including Clostridium, Turicibacter, Bacteroidaceae_uc, and Lactoba- cillus were enriched in the Salmonella-negative group whereas acetate producers like Sporobacter, Escherichia or Enterobacter were more abundant in the Salmonella-positive group. Overall, our results suggest that the presence of Salmonella in free-range pigs' gut is directly related to the natural veg- etation accessible, determining the composition of the intestinal microbiota.This work was funded by the Caja Navarra Foundation (project reference 70628)

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”