Use of water soluble extracts from ulva sp. by probiotics and fish bacterial pathogens

BACKGROUNDS The potential of seaweeds as dietary components is considered for a wide range of cultured fish species. In this context, Ulva is investigated as a good source of protein, minerals and vitamins. In addition, of probiotics are used to improve fish growth and modulate immune system and intestinal microbiota. To promote probiotics colonization and maintenance in the intestine, prebiotics are included in fish diets. Prebiotics are indigestible substrates used as energy sources for gastrointestinal microbiota, with a positive effect on the nutrition and health status of the host. In the present work, ability of selected probiotic and fish pathogen strains to use water soluble extracts from Ulva as nutrient source has been evaluated. MATERIALS AND METHODS Water-soluble extracts from Ulva sp. prepared by sonication of dehydrated samples were used to supplement minimum medium (M9). Probiotics and pathogens growth was evaluated based on the optical densities measured with a microplate reader. RESULTS AND CONCLUSIONS Probiotics were able to grow in minimum medium using water soluble extracts as nutrient source. On the other hand, P. damselae subsp. piscicida and V. harveyistrains were also able to grow with Ulvaextracts as nutrient source. However, incubation time to reach maximum growth was longer. Although Ulva extract may support growth of both probiotics and pathogen bacteria, faster growth of probiotics may help for the establishment of probiotic populations in the intestinal environment. In addition, beneficial effects on growth performance, gut microbiota, immunity and disease resistance of Ulva for Solea senegalensisare being studied. This work was funded by INIA, Ministerio de Economía y Competitividad and FEDER (RTA201400023 C0202).This work was funded by INIA, Ministerio de Economía y Competitividad and FEDER (RTA201400023 C0202).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

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)

Expresión de genes del sistema inmune innato del lenguado (Solea senegalensis) alimentados con Ulva ohnoi durante la infección con P. damselae subsp. piscicida

Las algas son fuente de proteínas y numerosas sustancias biológicamente activas. En el caso del género Ulva, son muy escasos los estudios sobre los efectos en el sistema inmune. En el presente trabajo se ha evaluado la respuesta a la infección por P. damselae subsp. piscicida de genes del sistema inmune innato en lenguados (Solea senegalensis) alimentados con una dieta suplementada con Ulva ohnoi. Los ejemplares de lenguado con un peso inicial de 10,7g se alimentaron durante 3 meses con una dieta control y otra conteniendo 5% de polvo seco de U. ohnoi. Transcurrido este periodo, los peces (54,17 g) se sometieron a infección frente a P. damselae subsp. piscicida y se determinó la expresión relativa de los genes que codifican para componentes del sistema del complemento, y citoquinas inflamatorias en hígado y riñón de los lenguados infectados. Los resultados obtenidos muestran, tras la infección, un incremento en la expresión de los genes que codifican para los componentes C1q4, C3, C4/1 y C9 así como TNF en el hígado de los peces que recibieron el alga. Por el contrario, se observaron niveles inferiores de expresión de los genes que codifican para las citoquinas IL1beta, IL6 e IL8b en el hígado de los ejemplares que recibieron el alga. A nivel del riñón, los lenguados alimentados con la dieta conteniendo Ulva sólo presentaron diferencias respecto al grupo control en la expresión de los genes que codifican para IL8b y C9. La activación del sistema del complemento da como resultado la formación del complejo de ataque de membrana, que tiene un papel esencial en la eliminación de microorganismos patógenos. Por otro lado, una respuesta inflamatoria controlada puede contribuir a limitar el daño derivado de la infección por P. damselae subsp. piscicida, en peces alimentados con la dieta conteniendo Ulva.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia de Innovación y FEDER (Ref. RTA2014 00023 C02

Respuesta inmune en mucosas de piel y branquias de Solea senegalensis tras la alimentación con una dieta conteniendo Ulva ohnoi (5%)

Abstract In the search of the improvement of the feeding of the fish in the aquaculture, macroalgae seem to be a promising source of nutrients and bioactive substances. In the case of the genus Ulva, the effect of its inclusion in feed on the growth, tissues composition and the stress response of some fish species has been evaluated. However, it is necessary to evaluate the effects on the immune system, as its inclusion could modify the resistance to pathogens. In the present work, we have studied the response of the immune system at lysozyme expression level, cellular immune response genes, and inflammatory response genes in mucous of skin and sole gills (Solea senegalensis) fed with a diet supplemented in Ulva ohnoi (5%). Results obtained indicate absence of significant changes in the expression of assayed immune genes. Resumen En la búsqueda de la mejora de la alimentación de los peces en la acuicultura, las algas se presentan como una prometedora fuente de nutrientes y sustancias bioactivas. En el caso del género Ulva, se ha publicado el efecto de su inclusión en piensos sobre el crecimiento, la composición de los tejidos y la respuesta al estrés de algunas especies de interés acuícola. Sin embargo, es necesario evaluar los efectos en el sistema inmune, puesto que su inclusión podría modificar la resistencia a patógenos. En el presente trabajo se ha estudiado la respuesta del sistema inmune a nivel de expresión de lisozimas, genes de respuesta inmune celular, y genes de la respuesta inflamatoria en piel y branquias de lenguados (Solea senegalensis) alimentados con una dieta suplementada en Ulva ohnoi (5%). Los resultados indican que no se produce un cambio significativo en la expresión de estos grupos de genes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia e Innovación (INIA) y FEDER (Ref. RTA2014 00023 C0202

Modulation of Intestinal Microbiota in Solea senegalensis Fed Low Dietary Level of Ulva ohnoi

Gastrointestinal (GI) microbiota has a relevant role in animal nutrition, modulation of the immune system and protection against pathogen invasion. Interest in algae as source of nutrients and functional ingredients for aquafeeds is increasing in order to substitute conventional feedstuffs by more sustainable resources. The diet is an important factor in the modulation of the microbiota composition, and functional ingredients have been proposed to shape the microbiota and contribute benefits to the host. However, fish microbiome research is still limited compared to other hosts. Solea senegalensis is a flat fish with high potential for aquaculture in South Europe. In this study, a characterization of the microbiome of S. senegalensis (GI) tract and the effects of feeding Ulva ohnoi supplemented diet has been carried out. Differences in the composition of the microbiota of anterior and posterior sections of S. senegalensis GI tract have been observed, Pseudomonas being more abundant in the anterior sections and Mycoplasmataceae the dominant taxa in the posterior GI tract sections. In addition, modulation of the GI microbiota of juvenile Senegalese sole fed for 45 days a diet containing low percentage of U. ohnoi has been observed in the present study. Microbiota of the anterior regions of the intestinal tract was mainly modulated, with higher abundance of Vibrio spp. in the GI tract of fish fed dietary U. ohnoi

Transcription of IVIAT and Virulence Genes in Photobacterium damselae Subsp. piscicida Infecting Solea senegalensis

Photobacterium damselae subsp. piscicida (Phdp) is responsible for disease outbreaks in marine aquaculture worldwide. Solea senegalensis, a valuable fish species for aquaculture in the south of Europe, is frequently affected by this pathogen. It is well established that bacteria respond to environmental signals and, in the case of pathogens, this ability may determine the outcome of their interaction with the host. Determination of gene expression under in vivo conditions constitutes a valuable tool in the assessment of microbial pathogenesis. Considering that different hosts may represent different environments for the pathogen, expression of Phdp virulence and in vivo induced antigen (IVIAT) genes during S. senegalensis infection has been determined in the present work. Increased transcription of genes encoding proteins involved in iron acquisition (Irp1, Irp2, HutB and HutD), oxidative stress defence (AhpC and Sod), adhesion (PDP_0080), toxins (AIP56) and metabolism (Impdh, Shmt and AlaRS) were detected in Phdp infecting S. senegalensis head kidney or liver. The highest increases corresponded to genes involved in survival under iron limiting conditions and oxidative stress, indicating their essential role during infection of sole. Results obtained give insight into Phdp virulence strategies and contribute to the identification of promising targets for the control of photobacteriosis