Efecto de la adición de dos probióticos (Shewanella putrefaciens y Shewanella baltica) en el engorde del lenguado senegalés (Solea senegalensis Kaup, 1858)

206 p.El conocimiento, prevención y tratamiento de las patologías existentes es posiblemente el factor más limitante en la actualidad para el engorde del lenguado (Solea senegalensis, Kaup 1858). Sin embargo, no hay evidencia de que ésta sea una especie más susceptible que otras a las infecciones bacterianas. Los actuales sistemas de producción intensiva suponen un estrés para los ejemplares en cultivo, ocasionando una menor eficiencia digestiva, una mayor susceptibilidad frente a patógenos potenciales y pérdidas económicas para la industria. Puesto que la producción en acuicultura depende, en buena medida, del estatus fisiológico de los peces, se hace preciso el empleo de piensos comerciales específicos y adecuados protocolos de cultivo que favorezcan el bienestar animal. La utilización de probióticos a nivel comercial en dieta (células microbianas con un efecto positivo para la salud del hospedador) es ya una herramienta eficaz para la mejora del metabolismo, reducción de deformidades y prevención de patologías en acuicultura de salmónido

Use of in-vivo induced antigen technology to identify bacterial genes expressed during Solea senegalensis infection with Photobacterium damselae subsp. piscicida

The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease outbreaks affecting several fish species including flatfish Solea senegalensis (Kaup). Phdp is able to avoid host defences by invasion and intracellular survival in non-phagocytic cells, mainly epithelial cells. Virulence factors reported in Phdp include restricting complement-mediated activity, apoptosis of phagocytes caused by exotoxins secretion, iron acquisition mechanisms such as siderophores that enable the pathogen to obtain iron from transferrin and ability to bind haemin and antioxidant enzymatic activities capable to counteract superoxide radicals (Do Vale et al., 2005; Andreoni and Magnano, 2014). Commonly, genes expressed during pathogen infection are important for pathogenicity. In vivo-induced antigen technology (IVIAT) (Handfield et al., 2000) has been used to identify in vivo-induced genes using pooled sera from fish that have experienced photobacteriosis. Materials and methods Sera were obtained from surviving S. senegalensis specimens after sublethal infection with Phdp (Lg41/01) and subsequently pooled and adsorbed against in vitro grown Phdp Lg41/01 and Escherichia coli BL21 (DE3) cells and lysates according to Handfield et al. (2000). The efficiency of sera adsorption was evaluated based on the immunoreactivity after each adsorption step with whole and lysed Phdp cells grown in vitro. A genomic expression library of Phdp Lg41/01 was generated in E. coli BL21 (DE3) using pET-30 expression system (Novagen, San Diego, CA, USA). The expression library was probed with adsorbed and non-absorbed sera using immunoblot technique. Reactive clones of in vivo-induced and in vitro antigens were obtained, purified and their inserted DNA sequenced (Macrogen Europe, Amsterdam, The Netherlands). Nucleotide sequences were compared against the NCBI protein database using BLASTx. Results A progressive reduction in sera immunoreactivity against in vitro grown Phdp cells was detected after the adsorption rounds, especially after the first adsorption step. Thus, following adsorption steps substantially removed antibodies against in vitro expressed antigens and resulted in relative enrichment in antibodies recognizing in vivo expressed antigens. The library from Phdp Lg14/01 constructed in E. coli BL21 (DE3) consisted of approximately 6500 recombinants. A total of 117 clones were selected for their reactivity with pooled adsorbed and non-adsorbed sera from convalescent S. senegalensis specimens after a first round of screening. In a second screening, 14 out of 117 candidate clones showed positive reaction, among which two clones were clearly positive and two gave weak reaction against adsorbed sera. Predicted proteins codified by inserted sequences have intracellular and membrane cell location and are involved in virulence, synthesis of intermediary products, energy metabolism and gene replication. Inosine-5’-monophosphate dehydrogenase (IMPDH) and alkyl hydroperoxide reductase (AhpC) have been identified as in vivo induced antigens expressed during S. senegalensis infection with Phdp. Iron/manganese superoxide dismutase (Fe/Mn-SOD) and alanyl-tRNA synthetase (AlaRS) proteins have also been identified, though with weak signal. Discussion and conclusion Identification of immunogenic bacterial proteins during Phdp infection is essential for understanding bacterial pathogenesis and development of effective vaccines. AhpC peroxidase activity has a protective role by reducing hydrogen peroxide, peroxynitrite and organic hydroperoxides. Immunization with AhpC conferred protection against Helicobacter pylori infection (O’Riordan et al., 2012). IMPDH catalyzes the conversion of products essential in de novo synthesis of guanine nucleotides. Adequate levels of purine nucleotides are critical for cell proliferation, nucleic acid replication, cell signaling and as a biochemical energy source. This gene is an important therapeutic target against bacterial diseases (Shu and Nair,2008). In conclusion, different genes expressed during Phdp infection in S. senegalensis have been identified. Among them, IMPDH and AhpC have been identified as in vivo induced antigens expressed during S. senegalensis infection with Phdp. Thus, they are likely to play a role in the virulence of Phdp. The antigenic character of these proteins makes them potential targets for the development of new vaccines. References Andreoni, F., and Magnani, M., 2014. Photobacteriosis: Prevention and Diagnosis. Journal of Immunology Research, 2014: 1-7. Do Vale, A., Silva, M.T., dos Santos, N.M., Nascimento, D.S., Reis Rodrigues, P., Costa Ramos, C., Ellis, A.E., and Azevedo, J.E., 2005. AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils. Molecular Microbiology, 58: 1025-1038. Handfield, M., Brady, L.J., Progulske-Fox, A., and Hillman, J.D., 2000. IVIAT: a novel method to identify microbial genes expressed specifically during human infections. Trends in Microbiology, 8: 336-339. O’Riordan A.A., Morales V.A., Mulligan L., Faheem N., Windle H.J., and Kelleher D.P., 2012. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine. Vaccine, 30:3876-3884. Shu, Q., and Nair, V., 2008. Inosine monophosphate dehydrogenase (IMPDH) as a target in drug discovery. Medicinal Research Reviews, 28:219-232.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Benefits of probiotic administration on growth and performance along metamorphosis and weaning of Senegalese sole (Solea senegalensis)

Suitable nutrition at first feeding in marine fish larvae is an important factor for successful larval and juvenile rearing. In this way probiotic supplementation may contribute to better face the habitual stress of metamorphosis and weaning at early sole stages. The aimof this study was to optimize Shewanella putrefaciens Pdp11 administration to Senegalese sole (Solea senegalensis) at early stages of development. For this purpose, we studied the influence of a short pulse of S. putrefaciens Pdp11 (10–30 dah) on growth performance, gut microbiota modulation, digestive enzymes activities and body composition. These probiotic bacteria were incorporated using Artemia as live vector (2.5 × 10 7 cfu mL −1 ) and supplied to sole specimens in a co-feeding regime (Gemma, Skretting) by triplicate. S. putrefaciens Pdp11 colonized larval gut being present from23 to 119 dah. Probiotic supply significantly modulated larval and fry gut microbiota. A PCA analysis including all the parameters analyzed strongly associated S. putrefaciens Pdp11 presence with a higher fish growth, a higher digestive proteolytic activity level and a fish body composition modulation along S. senegalensis rearing. In addition less size variability was obtained frommetamorphosis until the end of weaning. In conclusion a short pulse of S. putrefaciens Pdp11 (10– 30 dah) seems sufficient to obtain a suitable microbial modulation involved in a better growth performance and body composition that suggests a great potential for sole aquaculture production.En prens

Use of in vivo-induced antigen technology to identify bacterial genes expressed during solea senegalensis infection with Photobacterium damselae subsp. piscicida

The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important outbreaks affecting several fish species including flatfish Solea senegalensis. The aim of this work was to identify in vivo-induced expressed immunogenic proteins using pooled sera from fish that have experienced photobacteriosis. In vivo induced immunogenic proteins included inosine-5'-monophosphate dehydrogenase (Impdh) and alkyl hydroperoxide reductase (AhpC), two proteins involved in peptide synthesis: serine hydroxymethyl transferase (Shmt) and alanyl-tRNA synthetase (AlaRS) and the non-ribosomal peptide synthetase involved in the synthesis of the siderophore piscibactin (Irp2)

Modulation of the intestinal microbiota and expression of gene of senegalese sole (Solea senegalensis, Kaup 1858) after dietary administration of oxytetracycline.

Oxitetracycline and probiotics (Pdp11) administration to sole juveniles showed important changes in the species composition of intestinal microbiota that established a relationship between these species and gene expression

Proyecto de investigación: la motivación del profesorado en Castilla y León

En este estudio se pretende recopilar y analizar los diferentes factores que influyen en la motivación y desmotivación de los docentes. Partimos de la hipótesis de que cuanto mayor sea el tiempo trabajado mayor será la desmotivación del docente. Se trata de un estudio no experimental y descriptivo en el cual participarán 300 profesores de secundaria. Se evaluarán su motivación, de forma cuantitativa a través de la escala Maslach Burnout Inventory Maslach, C. (1997) y de forma cualitativa a través de grupos de discusión. Con los datos obtenidos se plantearán investigaciones análogas a nivel nacional y se extraerán las posibles soluciones que podrían mejorar la experiencia docente.The aim of this paper is to compile and analyse the different elements and factors which influence the motivation and demotivation of teachers. We start from the hypothesis that the longer is their teaching career, the greater will be their demotivation. This is a non-experimental and descriptive study in which 300 secondary school teachers will participate. Their motivation will be evaluated quantitatively using the Maslach Burnout Inventory scale Maslach, C. (1997) and qualitatively through discussion groups. Using the data obtained, analogous nationwide research will be carried out as well as possible solutions will be identified in order to improve the teaching experience

Isolation and characterization of a Bacillus velezensis D‑18 strain, as a potential probiotic in European seabass aquaculture

Author's accepted version (postprint)This is an Accepted Manuscript of an article published by Springer in Probiotics and Antimicrobial Proteins on 03/04/2021Available online: https://link.springer.com/content/pdf/10.1007/s12602-021-09782-8.pdfWithin the food-producing sectors, aquaculture is the one that has developed the greatest growth in recent decades, currently representing almost 50% of the world’s edible fish. The diseases can affect the final production in intensive aquaculture; in seabass, aquaculture vibriosis is one of the most important diseases producing huge economical losses in this industry. The usual methodology to solve the problems associated with the bacterial pathology has been the use of antibiotics, with known environmental consequences. This is why probiotic bacteria are proposed as an alternative fight against pathogenic bacteria. The aim of this study was to analyse a strain of Bacillus velezensis D-18 isolated from a wastewater sample collected from a fish farm, for use as probiotics in aquaculture. The strain was evaluated in vitro through various mechanisms of selection, obtaining as results for growth inhibition by co-culture a reduction of 30%; B. velezensis D-18 was able to survive at 1.5-h exposure to 10% seabass bile, and at pH 4, its survival is 5% and reducing by 60% the adhesion capacity of V. anguillarum 507 to the mucus of seabass and in vivo by performing a challenge. Therefore, in conclusion, we consider B. velezensis D-18 isolate from wastewater samples collected from the farms as a good candidate probiotic in the prevention of the infection by Vibrio anguillarum 507 in European seabass after in vitro and biosafety assays.acceptedVersio

Changes in intestinal microbiota and gene expression of immune system parameters caused by dietary administration of alginate and Shewanella putrefaciens Pdp11 in Senegalese sole (Solea senegalensis Kaup, 1858) specimens

Dietary administration of sodium alginate single or mixed with Pdp11 modulated the intestinal microbiota diversity of sole juveniles. Both treatments promoted a change in the species present in the gut in comparison with control group. In addition, oral administration of alginate promoted the appearance of an intestinal microbiota highly influenced by the prebiotic presence. Synbiotic administration of alginate with Pdp11 promoted a more pronounced decreasing in the similarity index and in the species richness. Both treatments modulated the expression of genes related with immune system functions