Luiz Felipe Caron

Avaliação microbiológica, histológica e imunológica de frangos de corte desafiados com Salmonella Enteritidis e Minnesota e tratados com ácidos orgânicos

Two experiments were carried out to evaluate effectiveness of organic acids against Salmonella enterica enterica serovars Enteritidis (SE) and Minnesota (SM) in broilers. In the first experiment three treatments were evaluated: T1 - feeding with organic acids, T2 - feeding with organic acids and organic acids in drink water, and T3 - control group. All animals were oral challenged with SE. Organic acids in diet (T1) and organic acids in diet and drink water (T2) reduced the shadding of Salmonella in crop and cecum 7 days post challenged with SE and reduced the CD3+ cells in jejunal mucosa of broilers. In the second experiment four treatments were evaluated, T1 - control group, T2 - control group oral challenged with Salmonella Minnesota (SM); T3 - oral challenged animals with SM and treated with organic acids in diet; T4 - oral challenged animals with SM and treated with organic acids in diet and in drink water. Organic acids in diet (T3) and organic acids in diet and in drink water (T4) reduced the shadding of SM in crop of challenged broilers, 7 days post inoculation The use of organic acids in diet and in water was more effective to control SE than SM.

Flow cytometric immune profiling of specific-pathogen-free chickens before and after infectious challenges

Broilers and layer chickens have been intensively selected for production parameters. This selection has affected immune capacity. Consequently, the fine-tuning of immune responses is becoming important for maximum productivity. Flow cytometry is a recurrent technology used for the immunophenotyping of birds. Studies, however, have focused on the mechanism of specific diseases or have used animals whose immunological condition could be biased-by vaccination or environmental stressors, for example. The aim of this study was to evaluate the immune status of specific-pathogen-free birds across different age ranges to characterize the natural changes that occur over time. Additionally, specific-pathogen-free chickens were challenged with four infectious agents, allowing identification of the subpopulations of peripheral blood immune cells that are consistently altered under various conditions. Several lymphocyte subsets vary naturally with aging, so the interpretation of results using animals of different age ranges must proceed with care. Parameters such as CD8(+)CD28(-), CD8αα(+), CD4(+)CD8(+), and CD8(+)TCRVβ1(+) have been shown to be valuable in understanding immune changes during disease. The use of these data allows a determination of the consistency of cytometric parameters under various conditions, which should ease the interpretation of immunophenotyping and the future application of cytometric analysis in the poultry industry.

Effect of Spray Escherichia coli Vaccine on the Immunity of Poultry

SUMMARY Avian pathogenic Escherichia coli is a current problem in the poultry industry, causing mortality and economic losses. This paper evaluates the dynamics in immune response after the use of spray vaccination against E. coli and, thereby, seeks to understand how the vaccine can provide protection. During the early stages of response to vaccination the presence of antigen-presenting cells is predominant, but these diminish within the first 7 days after vaccination. The immune correlate of protection of vaccination using the E. coli vaccine Poulvac E. coli (aroA-deficient mutant strain) probably does not depend on the production of circulating antibodies (as assessed through the presence of B lymphocytes) and is linked to the presence of CD4+TCRV&bgr;1+. These cells act on mucosa tissue stimulating the production of immunoglobulin A. Vaccination stimulated a high state of immunocompetence, as assessed by measurement of several cellular subsets. This state of “immune alertness,” however, may be associated with reduced weight gain. The high presence of naive and memory CD8 cells in the vaccinated group at 14 and 21 days postvaccination may indicate greater ability in the future to prevent tissue invasion by E. coli, based on the possibility that these cells will proliferate rapidly to a new stimulus. The simultaneous use of vaccine with the antibiotic ceftiofur sodium interferes with the immune response obtained through vaccination. In combination, the data obtained in this study indicate that the immune response produced by a spray vaccine against E. coli is mainly a cellular response, especially relevant to the sites in contact with the pathogen. It is suggested that there is a strong cell migration to the mucous membranes, where macrophages act first and then lymphocytes take part to protect the host. It is believed that recruited lymphocytes will act in the production of secreted IgA, which probably plays a greater role in the defense when compared with circulating immunoglobulins. The assessment of cellular dynamics by flow cytometry made it possible to elucidate the operation mechanism of the live E. coli vaccine. RESUMEN Nota de Investigación—Efecto de la vacuna contra E. coli en aerosol en la inmunidad de aves comerciales. La Escherichia coli patógena para las aves es un problema actual en la industria avícola, que es causa de mortalidad y pérdidas económicas. En este trabajo se evaluó la dinámica en la respuesta inmune después de la utilización de la vacunación en aerosol contra E. coli y de este modo, el objetivo de este trabajo es entender cómo esta vacuna puede proporcionar protección. Durante las primeras etapas de la respuesta a la vacunación, la presencia de células presentadoras de antígeno es predominante, pero esto disminuye dentro de los primeros siete días después de la vacunación. La correlación inmune de la protección por la vacunación utilizando la vacuna de E. coli Poulvac E. coli (cepa mutante deficiente del gene aroA) probablemente no depende de la producción de anticuerpos circulantes (como se evaluó a través de la presencia de linfocitos B) y está vinculada a la presencia de CD4+TCRV&bgr;1+. Estas células actúan sobre la mucosa estimulando la producción de inmunoglobulina A. La vacunación estimula un alto estado de inmunocompetencia, según lo evaluado por la medición de varios subconjuntos celulares. Sin embargo, este estado de “alerta inmune”, puede estar asociado con una reducción de la ganancia de peso. La alta presencia de células CD8 no estimuladas (naive) y de memoria en el grupo vacunado a los 14 y 21 días después de la vacunación, puede indicar una mayor capacidad en el futuro para prevenir la invasión de tejidos por E. coli, con base a la posibilidad de que estas células proliferan rápidamente ante un nuevo estímulo. El uso simultáneo de la vacuna con el ceftiofur sódico antibiótico interfiere con la respuesta inmune obtenida a través de la vacunación. En conjunto, los datos obtenidos en este estudio indican que la respuesta inmune producida por una vacuna en aerosol contra E. coli es principalmente una respuesta celular, que es especialmente importante en los sitios donde ocurre el contacto con el patógeno. Se sugiere que existe una fuerte migración de células hacia las membranas mucosas, donde los macrófagos actúan primero y luego los linfocitos toman parte para proteger al huésped. Se cree que los linfocitos reclutados actuarán en la producción de IgA secretada, que probablemente juega un papel más importante en la defensa cuando se compara con inmunoglobulinas circulantes. La evaluación de la dinámica celular por citometría de flujo hizo posible para dilucidar el mecanismo de operación de la vacuna viva de E. coli.

Physicochemical and immunological characterization of chitosan-coated bacteriophage nanoparticles for in vivo mycotoxin modeling

To propose a novel modeling of aflatoxin immunization and surrogate toxin conjugate from AFB1 vaccines, an immunogen based on the mimotope, (i.e. a peptide-displayed phage that mimics aflatoxins epitope without toxin hazards) was designed. The recombinant phage 3P30 was identified by phage display technology and exhibited the ability to bind, dose dependent, specifically to its cognate target - anti-AFB1 antibody. In immunization assay, the phage-displayed mimotope and its peptide chemically synthesized were able to induce specific anti-AFB1 antibodies, indicating the proof of concept for aflatoxin mimicry. Furthermore, the phage 3P30 was homogeneously coated with chitosan, which also provided a tridimensional matrix network for mucosal delivery. After intranasal immunization, chitosan coated phages improved specific immunogenicity compared to the free antigen. It can be concluded that affinity-selected phage may contribute to the rational design of epitope-based vaccines in a prospectus for the control of aflatoxins and possibly other mycotoxins, and that chitosan coating improved the vectorization of the vaccine by the mucosal route.

Effect of an Enterococcus faecium probiotic on specific IgA following live Salmonella Enteritidis vaccination of layer chickens

ABSTRACT Probiotics and immunization are being widely adopted by the poultry industry with the goal of controlling Salmonella enterica. However, the interaction between these two management protocols has been sparsely studied. The present study aimed to understand the role of an Enterococcus faecium probiotic in the production of salmonella-specific IgA in layers immunized with a live vaccine. Four groups were used: “Control” (no vaccine or probiotic); “Probiotic” (which received an E. faecium product); “Vaccine” (immunized with two doses of a live attenuated S. Enteritidis vaccine); and “Vaccine + probiotic”. Faecal salmonella-specific IgA was analysed 7 and 20 days post-vaccination (dpv) boost. At 7 dpv, the “Vaccine” and “Vaccine + probiotic” groups had similar IgA levels. However, at 20 dpv, IgA levels were two times higher in the “Vaccine + probiotic” group compared to the “Vaccine” group. To understand the role of the intestinal microbiota in this finding, bacterial diversity in faeces was analysed by 16S rRNA gene sequencing. The improvement in IgA production in probiotic-treated birds was accompanied by marked changes in the faecal microbiome. Some of the main differences between the “Vaccine” and “Vaccine + probiotic” groups included reduction of Escherichia-Shigella and increases in Blautia, Anaerotruncus and Lactobacillus in the latter group. Although no direct causal link can be established from this study design, it is possible that the E. faecium probiotic induces improved antibody production following vaccination via modulation of the intestinal microbiota.

Cecal Microbiota in Broilers Fed with Prebiotics

Prebiotics are typically fibrous compounds that pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the bowel and contribute to the well-being of their host (Gibson and Roberfroid, 1995). Some of the most widely used prebiotics in the poultry industry are fructo-oligosaccharides, mannan-oligosaccharides, galacto-oligosaccharides and beta-glucans (Huyghebaert et al., 2011). Selective fermentation of some prebiotics has been shown to induce changes in the composition and/or activity of the gastrointestinal microbiota, improving the health of the host (Gibson et al., 2004). Zhenping et al. (2013) showed increased growth performance, enhanced endocrine metabolism, and improved immune function in broiler chickens after in-feed supplementation with xylo-oligosaccharides prebiotics. Moreover, changes in enteric bacteria in the cecum (Spring et al., 2000) and improved intestinal morphology have been observed in broilers fed with dietary mannan-oligosaccharide (Baurhoo et al., 2009). Because the microbiota can be modified (Khoruts et al., 2010; Borody and Khoruts, 2012) it constitutes an attractive target for therapeutic manipulation. However, successful outcome of such manipulations require a better understanding of the interactions between the host and its microbiota (Hamilton et al., 2013; Van Nood et al., 2013). The composition and diversity of chicken intestinal microbiota were previously investigated using cultivation-based methodologies (Fernandez et al., 2002; Cross et al., 2007). However, the use of DNA-based molecular biology techniques, such as metagenomics and new generation DNA sequencing, allowed new opportunities to characterize uncultivable members of intestinal microbiota (Gong et al., 2002) shedding light on the composition and temporal spatial location of the microbial population in broilers intestine. Nevertheless, the knowledge of the structure, interactions and functions of the intestinal microbiota is still limited and fragmented (Oakley and Kogut, 2016). In this work the effect of dietary supplement with prebiotics derived from yeast wall (mannan-oligosaccharide and nucleotide) on the cecal microbiota of broilers was evaluated by the massive parallel sequencing of the 16S rRNA gene.