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

The probiotic Shewanella putrefaciens PDP11 target virulence factors by modulating quorum sensing inhibition

Bacteria communicate with each other by producing signal molecules and regulating the production of virulence factors that have importance in pathogenicity. Quorum sensing (QS) is a bacterial communication mechanism based on the perception of population density and secretion of determining signal molecules called autoinducers (AI) such as the case of Acylhomoserine lactones (AHLs). AHLs-mediated QS processes seem to be common in the marine environment and among marine pathogenic bacteria, which pathogenesis could be mitigated by probiotics, among others. Probiotics are defined as live microbial cells that confer health benefits to the host and some of their mechanisms include the production of antagonistic compounds that are inhibitory toward pathogens. In this sense, Shewanella putrefaciens Pdp11, a strain described as a probiotic for use in aquaculture, has been analysed to mediate QS processes by quorum-quenching assays using synthetic AHLs. The enzymatic activity is estimated at around 80% and 30% for C8- and C10-HSL, respectively, while the rest of AHLs tested were not degraded by the Pdp11 strain. It would be an interesting feature of the probiotic Pdp11 strain since these AHLs are related to facilitating microbial adhesion by promoting biofilm formation among other virulence factors related to pathogens. On the other hand, a distinctive feature of AHL inactivated by lactonase is that it can be reactivated by acid treatment. In this way, little C8-AHL was recovered when it is extracted to pH2, which indicates the enzyme activity is not derived from the hydrolysis of the lactone ring derived from the action of lactonases, suggesting the enzyme activity in Pdp11 could be an AHL-acylase. The potential QQ activity of Pdp11 was unknown so, these preliminary studies led to a further as another promising probiotic QQ tool for aquaculture

Insights into the fecal microbiota of captive Iberian lynx (Lynx pardinus)

The Iberian lynx (Lynx pardinus) is an endangered species restricted to several areas of Spain and Portugal. Its low genetic diversity likely provokes immune depression and high susceptibility to infectious diseases. The intestinal microbiota is closely related to host health and nutrition. In order to contribute to the knowledge of the Iberian lynx intestinal microbiota, fecal microbiota of captive specimens from two breeding centers (“La Olivilla” and “El Acebuche”), located in Southern Spain, were studied by Denaturing Gradient Gel Electrophoresis (DGGE). Results grouped microbiota in two main clusters (I and III) which included DGGE patterns of 19 out of 36 specimens, cluster I being the most frequent in “La Olivilla” (50%) and cluster III in “El Acebuche” (55.55 %) specimens. Bacteroidetes, Firmicutes and Proteobacteria phyla were identified. Segregation of clusters I and III was attributed to different microorganism presence (Pseudomonas koreensis, Pseudomonas migulae, Carnobacterium sp., Arthrobacter, Robinsoniella peorensis and Ornithinibacillus sp.) and ability to use different carbon sources. Biolog EcoPlates® results indicate high functional diversity of fecal microbiota, it being higher in cluster III. The great impact of intestinal microbiota on host health supports the importance of its microbial composition understanding. This study is the first report of captive Iberian lynx fecal microbiota composition. [Int Microbiol 20(1): 31-41 (2017)]Keywords: Iberian lynx (Lynx pardinus) · fecal microbiota · biodiversit

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

Adsorption of salmonella in clay minerals and clay-based materials

A series of clay minerals and clay-based materials have been tested to eliminate one of the most dangerous bacteria we can find in the water: Salmonella. It has been proven that the use of clays and their PCH materials can be a suitable method for removing Salmonella from water. The results of this initial study show that all the materials analyzed have great salmonella adsorption capacities ranging from the lowest value observed in the mont-PCH sample (0.29 × 1010 CFU g−1 ) to the highest value observed in the natural palygorskite sample (1.52 × 1010 CFU g−1 ). Macroporosity, accessible external surface area, and the presence of silanol groups in the external surface of the particles appears to be the controlling factors for Salmonella adsorption capacity while it seems that the structural characteristics of the clay minerals and their respective PCH does not affect the adsorption capacityThe authors wish to thank the project BIA2017-82391-R for the financial suppor

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

Potential postbiotic activities of extracellular products of probiotic bacteria from gilthead seabream gastrointestinal tract

Probiotics have been established as a potential tool for improving gut health and environmental quality in aquaculture. However, recent evidence suggests that bacterial viability is not necessary to attain the beneficial-promoting effects. In this way, postbiotics have emerged providing a potential opportunity in the field of functional foods. They are soluble factors resulting from the metabolic activity of a probiotic or any released molecule, including short-chain fatty acids, enzymes, extracellular products, etc. that can have interesting properties inducing biological responses on health similar to probiotics while avoiding the administration of live microorganisms. Interestingly, its production can be affected by different factors such as the components of the culture media among others. Information on this type of postbiotic activities is very scarce, especially in the case of aquaculture, so the evaluation of the nutraceutical use of postbiotics to improve health management in fish and other cultivated aquatic organisms is an emerging area of research in aquaculture. In a previous work, we characterized four potential probiotics that were isolated from the gastrointestinal tract of Sparus aurata specimens fed with a diet containing a blend of microalgae. This diet involves a selection pressure on the intestinal microbiota of the fish that will be used to achieve enrichment in bacteria with a set of extracellular enzymatic activities capable of metabolizing and mobilizing the components of the diet enriched with microalgae. Here, we investigate the postbiotic potential of the extracellular products (ECPs) obtained from the four candidate probiotics grown on different microalgae-supplemented medium, and evaluate its enzymatic and antibacterial activity, and its cytotoxicity against the SAF-1 cell line. Our objective is to select different ECPs with a variety of activities that help the digestive process of seabream, with the aim to be included in aquafeeds.This work was funded by Junta de Andalucía (grant no. P20_00085) and co-financed by FEDER funds

Inactivation of simulated aquaculture stream bacteria at low temperature using advanced UVA- and solar-based oxidation methods

In this work the effect of water temperature (6 +/- 1 degrees C and 22 +/- 1 degrees C) on inactivation of bacteria (104 -106 CFU mL-1; Pseudomonas spp., Aeromonas spp. and Enterobacter spp.) in simulated aquaculture streams (SAS) using UVA based advanced oxidation processes (AOP) (H2O2-assisted UVA; photocatalysis; H2O2-assisted photocatalysis) and solar driven AOPs (H2O2-assisted solar disinfection, SODIS) was studied. Efficiency at 22 degrees C in terms of inactivation rate was higher using H2O2-assisted photocatalysis (H2O2/UVA-TiO2/polysiloxane) > H2O2-assisted UVA disinfection (UVA/H2O2 - 10 mg L-1) > photocatalysis (UVA-TiO2/polysiloxane) > UVA disinfection. At low temperature (6 degrees C) the inactivation rate increased with SODIS/H2O2 > SODIS > H2O2assisted UVA disinfection (UVA/H2O2 - 10 mg L-1) > H2O2-assisted photocatalysis (H2O2/UVA-TiO2/polysiloxane) > photocatalysis (UVA-TiO2/polysiloxane). The main results indicate that the inactivation rates increased when hydrogen peroxide (10 mg L-1) was used during H2O2-assisted UVA disinfection and photocatalysis. In addition, exposure of SAS to hydrogen peroxide for 24 h (in absence of light) at room temperature decreased the subsequent exposure UVA irradiation dose by almost four times. Drastic increase of inactivation rate was observed at low water temperature (6 +/- 1 degrees C) when UVA- and solarbased AOPs were employed compared to 22 +/- 1 degrees C. The treatment with SODIS proved to be more effective in Finland than in Spain. The effect of the low temperature (6 +/- 1 degrees C) was proposed as a critical factor during UVA disinfection (UVA/H2O2 and photocatalysis) that can increase the disinfection rate constant (kmax) by 1.3-5.2 times, leading to a reduction of the treatment costs ( euro m-3) by 1.3-3.3 times. The mechanism of observed enhanced disinfection at low water temperature (6 +/- 1 degrees C) when natural solar light and UVA are employed as irradiation sources for UVA/H2O2 and photocatalytic bacteria inactivation was proposed. No regrowth was observed in case of H2O2-assisted AOPs.This work was supported by a national research project (SUNRAS PROJECT, Project AGL2016-80507-R) funded by Ministerio de Economia y Competitividad (Plan Nacional de I + D + i (2013-2016). The research was also supported by a PIF contract (UCA/REC01VI/2017) funded by Vicerrectorado de Investigacion of Universidad de Cadiz. D. Sc. Juan Jose Rueda-Marquez is grateful for financial support from Academy of Finland within the project '' Combination of Advanced Oxidation Processes and Photobiotreatment for Sustainable Resource Recovery and Wastewater Reuse'' (application number 322339). T. Homola acknowledges financial support by Czech Science Foundation project 19-14770Y and project LM2018097 funded by the Ministry of Education, Youth and Sports of the Czech Republic. Documen

Relación entre la microbiota intestinal y la composición de ácidos grasos tras la administración del probiótico Shewanella putrefaciens Pdp11 bioencapsulado en Artemia en ejemplares larvarios de S. senegalensis

Estudios previos confirmaron que un pulso corto (10-30 dpe) del probiótico S. putrefaciens Pdp11 (2.5x10 7 cfu mL-1) bioencapsulado en Artemia y administrado a ejemplares larvarios de S. senegalensis incrementaron significativamente los niveles de lípidos totales y de ciertos ácidos grasos de los ejemplares. Dicha dieta suplementada produjo además cambios importantes en las especies microbianas intestinales de dichos organismos. La realización de un análisis de componentes principales (ACP) confirmó el efecto de la dieta probiótica en los ejemplares, sobre su microbiota y sobre a los niveles de ácidos grasos analizados