Comparación del test directo de anticuerpos fluorescentes y el cultivo bacteriológico para detección de Brucella suis

Methods available for detection of Brucella sp from different specimens include bacteriological culture or detection of specific DNA fragments by polymerase chain reaction. The use of fluorescein-labeled anti-Brucella globulin for demonstrating this antigen in animal tissues is a simple, easy, reproducible, cheap and fast technique. The aim of this work was to evaluate the gamma globulin fraction of polyclonal anti-Brucella abortus serum labeled with fluorescein iso-tio-cyanate (FITC-labeled antibody): 1) against different smooth and rough Brucella sp, 2) against bacterium of other genus, and 3) to compare direct fluorescent antibody test results with bacteriological culture for the detection of B. suis in different tissues from infected animals. This conjugate stained all Brucella sp with different intensities but it did not stain any heterologous bacterium tested. Background fluorescence associated with its use on smears from infected sources of different specimens was particularly low. Most of the infected tissues showed the presence of yellowish-green fluorescent organisms with brucella morphology. The tested FITC-labeled antibody allows a quick, effective and inexpensive diagnosis of brucellosis.El diagnóstico de brucelosis se apoya en el cultivo bacteriológico o en la detección de fragmentos de ADN de la bacteria mediante la reacción en cadena de la polimerasa. El empleo de una inmunoglobulina anti-Brucella conjugada a fluoresceína para la detección de este antígeno en tejidos constituye una técnica simple, fácil, reproducible, económica y rápida. El objetivo de este trabajo fue evaluar la fracción gammaglobulínica de un suero policlonal anti-Brucella abortus marcada con isotiocianato de fluoresceína (FITC), 1) contra distintas especies lisas y rugosas de Brucella sp, 2) contra bacterias de otros géneros, y 3) comparar los resultados obtenidos con la inmunofluorescencia directa y el cultivo bacteriológico para la detección de B. suis en distintos tejidos de porcinos infectados. Este conjugado detectó todas las brucelas con distinta intensidad de fluorescencia, pero no hubo fluorescencia inespecífica cuando se ensayaron las bacterias de otros géneros. La fluorescencia de fondo en muestras de los distintos tejidos infectados fue baja. La mayoría de los tejidos infectados mostraron la presencia de microorganismos verde-fluorescentes con la morfología de las brucelas. El anticuerpo conjugado a FITC permitió un diagnóstico de brucelosis rápido, efectivo y económico

An Oral Vaccine Based on U-Omp19 Induces Protection against B. abortus Mucosal Challenge by Inducing an Adaptive IL-17 Immune Response in Mice

As Brucella infections occur mainly through mucosal surfaces, the development of mucosal administered vaccines could be radical for the control of brucellosis. In this work we evaluated the potential of Brucella abortus 19 kDa outer membrane protein (U-Omp19) as an edible subunit vaccine against brucellosis. We investigated the protective immune response elicited against oral B. abortus infection after vaccination of mice with leaves from transgenic plants expressing U-Omp19; or with plant-made or E. coli-made purified U-Omp19. All tested U-Omp19 formulations induced protection against Brucella when orally administered without the need of adjuvants. U-Omp19 also induced protection against a systemic challenge when parenterally administered. This built-in adjuvant ability of U-Omp19 was independent of TLR4 and could be explained at least in part by its capability to activate dendritic cells in vivo. While unadjuvanted U-Omp19 intraperitoneally administered induced a specific Th1 response, following U-Omp19 oral delivery a mixed specific Th1-Th17 response was induced. Depletion of CD4+ T cells in mice orally vaccinated with U-Omp19 resulted in a loss of the elicited protection, indicating that this cell type mediates immune protection. The role of IL-17 against Brucella infection has never been explored. In this study, we determined that if IL-17A was neutralized in vivo during the challenge period, the mucosal U-Omp19 vaccine did not confer mucosal protection. On the contrary, IL-17A neutralization during the infection did not influence at all the subsistence and growth of this bacterium in PBS-immunized mice. All together, our results indicate that an oral unadjuvanted vaccine based on U-Omp19 induces protection against a mucosal challenge with Brucella abortus by inducing an adaptive IL-17 immune response. They also indicate different and important new aspects i) IL-17 does not contribute to reduce the bacterial burden in non vaccinated mice and ii) IL-17 plays a central role in vaccine mediated anti-Brucella mucosal immunity

Assisting Sustainability Through Achievement of an Integrated Health, Safety and Environmental Management System at Cardiff University

Universities use a multitude of methods to assist in driving sustainability forward. As large organisations, it can often be difficult to instigate change covering all aspects of the institution, without using a systematic method for implementation. This paper investigates the approach used by Cardiff University over a 5-year period to implement an integrated Health, Safety and Environmental Management System. It will show the approach used to ensure coverage across every academic school and professional service, and discuss this in terms of being one of the first UK Universities to achieve such integrated certification. It will then investigate how this approach has assisted in the development of wider sustainability issues and programmes across the entire institution, including “Green Impact”, through detailed investigation within three academic schools and a professional service. The problems and barriers encountered during the process will be discussed, along with approaches to mitigation of these. A brief analysis of Cardiff’s progression towards sustainable development compared to other Universities in Wales is also considered. Finally, some recommendations for further improvement at Cardiff will be discussed

The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis.

Knowing the inherent stimulatory properties of the lipid moiety of bacterial lipoproteins, we first hypothesized that Brucella abortus outer membrane protein (Omp)16 lipoprotein would be able to elicit a protective immune response without the need of external adjuvants. In this study, we demonstrate that Omp16 administered by the i.p. route confers significant protection against B. abortus infection and that the protective response evoked is independent of the protein lipidation. To date, Omp16 is the first Brucella protein that without the requirement of external adjuvants is able to induce similar protection levels to the control live vaccine S19. Moreover, the protein portion of Omp16 (unlipidated Omp16 [U-Omp16]) elicits a protective response when administered by the oral route. Either systemic or oral immunization with U-Omp16 elicits a Th1-specific response. These abilities of U-Omp16 indicate that it is endowed with self-adjuvanting properties. The adjuvanticity of U-Omp16 could be explained, at least in part, by its capacity to activate dendritic cells in vivo. U-Omp16 is also able to stimulate dendritic cells and macrophages in vitro. The latter property and its ability to induce a protective Th1 immune response against B. abortus infection have been found to be TLR4 dependent. The facts that U-Omp16 is an oral protective Ag and possesses a mucosal self-adjuvanting property led us to develop a plant-made vaccine expressing U-Omp16. Our results indicate that plant-expressed recombinant U-Omp16 is able to confer protective immunity, when given orally, indicating that a plant-based oral vaccine expressing U-Omp16 could be a valuable approach to controlling this disease

Oral administration of U-Omp19 expressing crude leaf material without adjuvant induces protection against oral <i>B. abortus</i> challenge in BALB/c mice.

a<p>The content of bacteria in spleens is represented as the mean log₁₀ CFU ± SD per group.</p>b<p>Significantly different from PBS-immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p>c<p>Significantly different from U-Omp19 immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p

Specific DTH response elicited after i.g. administration of adjuvant-free Omp19.

<p>Three weeks after the last i.g. immunization mice were intradermally challenged with U-Omp19 in their left footpad and an equal volume of saline into their right footpad. DTH response was measured at 72 h following injection of antigen. Each bar represents the mean increase in the footpad thickness ± SEM from 5 mice per group. (★) Significantly different from the mean increase thickness measured in PBS immunized mice (<i>P</i><0.05).These experiments were conducted three times with similar results.</p

Protection against oral <i>B. abortus</i> challenge in BALB/c mice immunized i.g. with <i>E. coli</i>- or <i>N. benthamiana</i>- derived purified U-Omp19 without adjuvants.

a<p>The content of bacteria in spleens is represented as the mean log₁₀ CFU ± SD per group.</p>b<p>Significantly different from PBS-immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p>c<p>Significantly different from <i>B. abortus</i> RB51 immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p