Selection of putative probiotics based on antigen-antibody cross-reaction with Photobacterium damselae subsp. piscicida and Vibrio harveyi for use in Senegalese sole (Solea senegalensis)

Probiotics can be used to reduce disease outbreaks in aquaculture. Some of them are characterised by their antagonistic activity against pathogens or the stimulation of the fish immune response, including the production of specific antibodies. If a probiotic has common antigens with a determined pathogen, it could produce antibodies with a cross-reaction to that pathogen. Thus, a probiotic with these characteristics could be used in a similar way to a live vaccine. The aim of this study was to select bacteria with antigenic similarity and antagonistic activity against the pathogens Photobacterium damselae subsp. piscicida and Vibrio harveyi, and to determine their ability to stimulate the production of antibodies in sole (Solea senegalensis, Kaup) with crossreaction against these pathogens. Dot blot was used to detect strains with cross-reaction using sera immunized against P. damselae subsp. piscicida Lg41.01 and V. harveyi Lg16.00. A total of 138 strains were selected from 718 strains, based on the intensity of the dot blot reaction. A second selection was performed to detect their ability to inhibit the pathogens growth. Five strains inhibited the growth of P. damselae subsp. piscicida, four strains inhibited the growth of V. harveyi, while two strains inhibited both pathogens. A Western blot confirmed the cross-reactions of the selected strains with the pathogens. Selected strains were subsequently inoculated into sole specimens by intraperitoneal injection. Four strains produced antibodies with cross-reaction against the pathogens. None mortality was observed in the inoculated fish. Further research demonstrated the storage capability of the selected strains in saline solution and feed, their growth at low pH, and identified their enzymatic characteristics. In conclusion, the selected strains showed antimicrobial activity and capacity to activate a specific immune response against fish pathogens

Effect of the potential probiotic Vibrio proteolyticus DCF12.2 on the immune system of Solea senegalensis and protection against Photobacterium damselae subsp. piscicida and Vibrio harveyi

Some of the characteristics of probiotics used in aquaculture are the antagonistic activity against pathogens and the activation of the immune response. Moreover, some probiotics with common antigens to pathogens can produce antibodies that react with these pathogens. One of those potential probiotics is Vibrio proteolyticus DCF12.2, a strain with the capacity to produce antibodies in soles (Solea senegalensis) that react with Photobacterium damselae subsp. piscicida and Vibrio harveyi antigens. Therefore, the objective of the work was to determine the capacity of DCF12.2 to activate the immune response in the sole, providing protection against both pathogens. For this purpose, DCF12.2 was administered to cultured soles by intraperitoneal injection, bath, and diet. The DCF12.2 strain activated the gene expression related to the fish immune response and increased the antibody production with cross-reaction to P. damselae subsp. piscicida and V. harveyi cells. Challenges with P. damselae subsp. piscicida resulted in a 30–40% relative percent survival (RPS) through DCF12.2 treatments. After challenge with V. harveyi, only intraperitoneal treatment protected the fish (67% RPS). In conclusion, the DCF12.2 strain showed great potential to be used to prevent diseases caused by both pathogens, and indicates a way to use probiotics as live vaccines.Ministry of Science of Spain (Plan Nacional de Investigación, AGL2010-17789

Detection of specific immune response in sole (Solea senegalensis) against Photobacterium damselae subsp. piscicida antigens.

Política de acceso abierto tomada de: https://v2.sherpa.ac.uk/id/publication/11293The pathogenic bacteria Photobacterium damselae subsp. piscicida affects the development of Solea senegalensis culture. Vaccines made with inactivated cells have produced a relative protection against the sickness, however the administration of subcellular and purified antigens as vaccine could increase the effectiveness of the immune response. Thus, the aim of this work was the determination of antigens of P. damselae subsp. piscicida involved in the specific immune response of S. senegalensis. Fish were immunized by intraperitoneal injection (i.p.) with inactivated extracellular polymeric substances (ECP) and whole cells of P. damselae subsp. piscicida, and Freund's incomplete adjuvant. Two months later fish were boosted with the same antigens. Serum from fish was collected to determine by ELISA the title of antibodies against subcellular fractions of bacteria (ECP, capsule, outer membrane proteins, O antigen and formalized whole cells). Significant differences were found between control and immunized fish, but differences between first immunization and booster were only found for O antigen and capsule. Western blots derived from 2D-PAGE of ECP and Outer Membrane Proteins (OMP), using sole immunized serum, detected two high reactive antigens from ECP. Proteins were identified, by mass spectrometry, as ATP-dependent metalloprotease and Telurite resistance proteins. In the case of OMP, three antigenic proteins were detected and identified as Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related

Characterization of Vibrio harveyi strains recovered from diseased farmed Senegalese sole (Solea senegalensis)

Aim: To characterize 16 Vibrio harveyi strains isolated from different epizootic outbreaks affecting farmed Senegalese sole. Materials and Results: The Vibrio harveyi strains tested have broad phenotypic diversity based on their biochemical and exoenzymatic patterns, outer membrane proteins (OMP), extracellular product (ECP) patterns and presence of prophages. Lethal dose 50 (LD50) of the strains and in vitro antagonism tests with two probiotic strains were also determined. The OMP analysis revealed three different patterns (A, M and V). The electrophoretic analysis of the ECP showed two different groups. All strains considered virulent based on their LD50 exhibited the same protein pattern in their ECP (pattern I), while all nonvirulent strains showed a different profile (pattern II). About 32% of the tested strains were positive for prophages, although a clear relationship between virulence and the presence of prophages has not been established. Conclusions: The results obtained have shown differences between virulent and avirulent strains isolated from diseased farmed Senegalese sole based on the protein patterns of their ECP. However, a clear relationship between virulence and presence of prophages has not been established. Significance and Impact of the Study: The differences observed between virulent and nonvirulent strains could be used to design prophylactic strategies against diseases caused by V. harveyi in farmed Senegalese sole

Procedimiento de preparación, conservación, y uso en peces, del probiótico SHEWANELLA PUTREFACIENS PDP 11 para el control de enfermedades y la mejora en el crecimiento

Número de solicitud 201100385Procedimiento de preparación, conservación, y uso en peces, del probiótico Shewanella putresfaciens Pdp11 para el control de enfermedades y la mejora en el crecimiento. Preferentemente, el probiótico, compuesto por células enteras de la cepa Pdp11, se cultiva en TSAs durante 24 h a 22ºC. La preparación de una suspensión del probiótico, preferentemente sin proceso previo de liofilización o de inactivación física o química, se realiza mediante su incorporación en una matriz de alginato, preferentemente alginato sádico al 0,5%. La suspensión preparada se puede conservar sin pérdida significativa de viabilidad durante 20 - 30 días a 4ºC. Los productos alimenticios para peces son preparados mediante la adición en agitación de la suspensión de probiótico y, adicionalmente, CaCl2 50 mM, preferentemente mediante atomización.Universidad de Málaga (50%). Universidad de Almería (50%

Preparado de bacterias probióticas para su administración oral a peces cultivados basado en la encapsulación en hidrogeles de alginato

Número de solicitud: 201100469La invención consiste en un preparado para la administración oral del probiótico Shewanella PDP11 a peces. El preparado se caracteriza por contener células bacterianas viables de la cepa CECT 7627 encapsuladas en un hidrogel basado en alginato cálcico en forma de partículas esféricas de morfología uniforme y tamaño modificable para adaptarse a peces de distinto tamaño. Las cápsulas contienen aditivos organolépticos y/o nutricionales que actúan como atrayentes para los animales. El preparado mantiene viables a las bacterias durante periodos prolongados, es estable en medios acuáticos, y soporta el paso por el tubo digestivo de los peces, sobre los que ejerce efectos biológicos favorables. Su administración por vía oral es independiente del alimento habitual, evitando así la inactivación del microorganismo durante la fabricación del pienso.Universidad de Almerí

An ex vivo Approach in European Seabass Leucocytes Supports the in vitro Regulation by Postbiotics of Aip56 Gene Expression of Photobacterium damselae subsp. piscicida

Shewanella putrefaciens Pdp11 (SpPdp11) is a probiotic strain assayed in aquaculture; however, its postbiotic potential is unknown. Postbiotics are bacterial metabolites, including extracellular products (ECPs) that improve host physiology and immunity. Their production and composition can be affected by different factors such as the growing conditions of the probiotics. Photobacterium damselae subsp. piscicida strain Lg 41/01 (Phdp) is one of the most important pathogens in marine aquaculture. The major virulent factor of this bacterium is the exotoxin aip56, responsible for inducing apoptosis of fish leucocytes. Viable SpPdp11 cells have been reported to increase resistance to challenges with Phdp. This work aimed to evaluate the effect of two ECPs, T2348-ECP and FM1548-ECP, obtained from SpPdp11 grown under different culture conditions that previously demonstrated to exert different degradative and non-cytotoxic activities, as well as the effect on pathogens biofilm formation. These SpPdp11-ECPs were then analyzed by their effect on the viability, phagocytosis, respiratory burst and apoptogenic activity against European sea bass leucocytes infected or not with Phdp supernatant. Both ECPs, T2348-ECP and FM1548-ECP, were not cytotoxic against leucocytes and significantly reduced their apoptosis. Phagocytosis and respiratory burst of leucocytes were significantly reduced by incubation with Phdp supernatant, and not influenced by incubation with T2348-ECP or FM1548-ECP. However, both activities were significantly increased after leucocyte incubation with combined T2348-ECP and FM1548-ECP with Phdp supernatant, compared to those incubated only with Phdp supernatant. Finally, both T2348-ECP and FM1548-ECP significantly reduced the relative in vitro expression of the Phdp aip56 encoding gene.Funding for open access publishing: Universidad Málaga/CBUA. This work was funded by the proyectos de investigación PID2020-113637RB-C21 and PID2020-113637RB-C22 funded by MCIN/AEI/10.13039/ 501100011033 and is part of the ThinkInAzul programme supported by MCIN with funding from European Union Next Generation EU (PRTR-C17. I1) and by the Comunidad Autónoma de la Región de Murcia-Fundación Séneca (Spain)

Gut microbiome specific changes in different behavioral profiles in a mouse social defeat stress model.

Comunicación de tipo PósterThe gut microbiome has arisen as one important modulator of general health, including brain function. In fact, disturbances in brain health are commonly mirrored in the microbiome, which could be contributing to pathology. One of the most common brain disorders is depression, which is tightly linked to environmental factors such as stress and drives alterations in regular behavior. However, not much is known about the role of the gut microbiome in response to stress and its relationship to behavior. In this study, the social defeat stress (SDS) paradigm was used as a depressive-like symptoms inducer in 8 w.o. male C57BL/6J mice for 10 days. Mice were segregated in stress resilient and sensitive according to behavior using K-means clustering and behavioral data was interpreted using principal component analysis. Then, the mice microbiome was extracted from fecal pellets after the stress protocol. DNA was extracted and purified followed by 16S (V3-V4) region amplification for sequencing. These data were analyzed to obtain diversity indexes and identify bacterial taxa within samples and groups. Data revealed that mice responded differently to the same stressor. Half the mice were found to have mild depressive-like symptoms whereas the other half showed profound alterations. Behavioral data was found to be explained in three factors: anhedonia, exploration, and motility. Stressed mice showed overall differences in their microbiome, being less diverse and populations associated with higher inflammation. Moreover, the healthy gut associated Verrucomicrobiae class was only identified in stress resilient mice, suggesting a possible relationship with their behavioral phenotype. Altogether, these results show a different behavioral response to stress in animals that reflects in their microbiome, which could be a key factor in determining stress resilience.This study was supported by Ministerio de Ciencia e Innovación - Plan Nacional I+D+I from Spain (PID2020-117464RB-I00) to CP and MP-M; FEDER/Junta de Andalucía - Proyectos I+D+I en el marco del Programa Operativo FEDER Andalucía 2014-2020 (UMA20-FEDERJA-112) to CP and MP-M; Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía (P20_00460) to CP. PC-P has been funded by the research project PID2020-117464RB-I00. Ministerio de Educación, Cultura y Deporte from Spain (FPU19/03629 to Infantes-López MI). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Shewanella putrefaciens Pdp11 extracts protect against betanodavirus infection.

Viral nervous necrosis is caused by the nervous necrosis virus (Betanodavirus genus), a naked virus with two positive-sense RNA segments. Betanodaviruses are classified into four species, being RGNNV predominant in the Mediterranean area. RGNNV causes high mortality in several fish species, including European seabass. There are two vaccines to protect seabass against RGNNV infection. In this regard, the development of strategies to protect different fish species against different viruses, such as the use of probiotics, is a key issue for the aquaculture industry. Shewanella putrefaciens Pdp11, SpPdp11, is a fish probiotic with proven positive effects on seabream and Senegalese sole, protecting those species against bacterial pathogens; however, its antiviral activity is unknown. This study evaluates the anti-RGNNV activity of sonicated-SpPdp11 extracts in vitro and in vivo. The in vitro evaluation was performed on E11 cells following 3 assays: (i) neutralization, (ii) 6-h pre-adsorption, (iii) post-adsorption, determining the inhibition of RGNNV-induced CPEs and quantifying viral replication. The immunostimulatory activity of SpPdp11 extracts was also examined, analysing the transcription of mx, hsp70, tnfα, e3 and tlr3 in E11 cells. For the in vivo evaluation, two European seabass groups were established: (i) control group, (ii) experimental group, fed with commercial pellet supplemented with SpPdp11 extracts. Animals were fed for 30 days and subsequently challenged by intramuscular injection. SpPdp11 extracts compromised RGNNV replication in E11 cells (67.3% and 55% CPE inhibition in pre-adsorption and post-adsorption assays, respectively), and modulated the transcription of all the immune-related genes examined. The highest induction was obtained for mx gene. Regarding the in vivo results, 82% of fish fed with the SpPdp11-supplemented diet survived to RGNNV infection, whereas the survival rate of fish fed with the control diet was 64%.P18-RT-1067 (Proyecto de Excelencia, Junta de Andalucia), PID2020-113637RB-C22 (MINECO/AEI/FEDER, UE). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Potential Effects of Microalgae-Supplemented Diets on the Growth, Blood Parameters, and the Activity of the Intestinal Microbiota in Sparus aurata and Mugil cephalus.

This work aimed to assess the suitability of a microalgal blend as a dietary ingredient for feeding juveniles of marine carnivorous and herbivorous teleost, as is the case of Sparus aurata and Mugil cephalus, respectively, and to isolate microorganisms from different media and characterize them on the base of their enzymatic activities and their antagonism against important fish pathogens. Thirty juveniles of each species (70 3.2 g S. aurata mean weight and 47 2.8 g M. cephalus mean weight) were distributed in four tanks (15 individuals each) corresponding to four independent dietary treatments (control and microalgae diets designed for each species). Fish were fed their corresponding diets ad libitum for 108 days. At the end of the trial, fish were weighed, and plasma, liver, perivisceral fat, and the entire intestines were obtained for the evaluation of growth performance and metabolic assessment. Furthermore, 117 bacterial strains were isolated in different culture media from the gastrointestinal tract of S. aurata fed the microalgae blend and further characterized for their potential use as probiotics in aquaculture. S. aurata fed the microalgae-supplemented diet (25% dietary inclusion) showed a significant increase in weight gain, specific growth rate, feed efficiency, hepatosomatic, and intestine length indices. However, growth performance and somatic indices in M. cephalus were not affected by the experimental diets. Plasma samples from S. aurata fed the microalgal diet revealed higher levels of glucose and triglycerides and a decrease in cortisol levels. No significant differences were found in any biochemical parameters among the experimental diets in M. cephalus. In conclusion, both species demonstrated a favorable adaptation to the nutritional formulation employed [...]Partial funding for open access charge: Universidad de Málag