M.C. Balebona

Bacteria recovered from diseased cultured gilthead sea bream (Sparus aurata L.) in southwestern Spain

A bacteriological study of 25 outbreaks affecting cultured gilthead sea bream (Sparus aurata L.) in southwestern Spain from 1997 to 2000 has been carried out. Each year, the highest number of outbreaks occurred during winter and affected fish with sizes ranging between 2 and 25 g. The most frequently isolated microorganisms were identified as Vibrio (69.90%). Other microorganisms belonging to Pseudomonas spp., Photobacterium damselae ssp. piscicida, Cytophaga/Flexibacterlike and Aeromonas spp. were isolated with a frequency lower than 10%. The outbreaks with the highest mortalities of cultured gilthead sea bream were due to P. damselae ssp. piscicida. An increase in the number of resistances to different antimicrobials has been observed in this study with respect to previous studies carried out in the same area. This increase was very high in the case of some species of Vibrio, Pseudomonas spp. and P. damselae ssp. piscicida. D 2003 Elsevier Science B.V. All rights reserved.

Intraspecific characterization of Vibrio alginolyticus isolates recovered from cultured fish in Spain

Aims: Intraspecific differentiation and characterization of Vibrio alginolyticus strains isolated from cultured fish in Spain.

The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease.

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.

Use of the probiotic Shewanella putrefaciens Pdp11 on the culture of Senegalese sole (Solea senegalensis, Kaup 1858) and gilthead seabream (Sparus aurata L.)

The application of probiotics on aquatic animals is increasing for a better fish welfare status as well as an environment-friendly activity which are actual demands of modern aquaculture industry. A bacterium from skin mucus of healthy gilthead seabream (Sparus aurata L.) has been isolated and identified as Shewanella putrefaciens Pdp11. Different studies have been done to know its application as probiotic in the Senegalese sole and gilthead seabream farming. This article reviews the studies carried out with this probiotic microorganism focusing on the current knowledge of its in vitro and in vivo mechanisms of action. The results suggested that the probiotic S. putrefaciens Pdp11, due to its beneficial effects, could be used in the aquaculture activity of both species.

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.

Dietary administration of the probiotic SpPdp11: Effects on the intestinal microbiota and immune-related gene expression of farmed Solea senegalensis treated with oxytetracycline

Few antimicrobials are currently authorised in the aquaculture industry to treat infectious diseases. Among them, oxytetracycline (OTC) is one of the first-choice drugs for nearly all bacterial diseases. The objective of this study was to evaluate the effect of the dietary administration of OTC both alone and jointly with the probiotic Shewanella putrefaciens Pdp11 (SpPdp11) on the intestinal microbiota and hepatic expression of genes related to immunity in Senegalese sole (Solea senegalensis) juveniles. The results demonstrated that the richness and diversity of the intestinal microbiota of fish treated with OTC decreased compared with those of the control group but that these effects were lessened by the simultaneous administration of SpPdp11. In addition, specimens that received OTC and SpPdp11 jointly showed a decreased intensity of the Denaturing Gradient Gel Electrophoresis (DGGE) bands related to Vibrio genus and the presence of DGGE bands related to Lactobacillus and Shewanella genera. The relationship among the intestinal microbiota of fish fed with control and OTC diets and the expression of the NADPH oxidase and CASPASE-6 genes was demonstrated by a Principal Components Analysis (PCA) carried out in this study. In contrast, a close relationship between the transcription of genes, such as NKEF, IGF-β, HSP70 and GP96, and the DGGE bands of fish treated jointly with OTC and SpPdp11 was observed in the PCA study. In summary, the results obtained in this study demonstrate that the administration of OTC results in the up-regulation of genes related to apoptosis but that the joint administration of OTC and S. putrefaciens Pdp11 increases the transcription of genes related to antiapoptotic effects and oxidative stress regulation. Further, a clear relationship between these changes and those detected in the intestinal microbiota is established.

Effect of dietary administration of Porphyridium cruentum on the respiratory burst activity of sole, Solea senegalensis (Kaup), phagocytes.

The stimulatory effect of the red microalga Porphyridium cruentum on respiratory burst activity of sole phagocytes was evaluated in vivo. Oral administration of a diet supplemented with lyophilized P. cruentum cells (10 g kg(-1)) stimulated respiratory burst activity after 4 weeks feeding in sole vaccinated with Photobacterium damselae subsp. piscicida bacterin.

Effects on intestinal microbiota and immune genes of Solea senegalensis after suspension of the administration of Shewanella putrefaciens Pdp11

The interaction host-intestinal microbiota is essential for the immunological homeostasis of the host. Probiotics, prebiotics and synbiotics are promising tools for the manipulation of the intestinal microbiota towards beneficial effects to the host. The objective of this study was to evaluate the modulation effect on the intestinal microbiota and the transcription of genes involved in the immune response in head kidney of Solea senegalensis after administration of diet supplemented with the prebiotic alginate and the probiotic Shewanella putrefaciens Pdp11 CECT 7627 (SpPdp11). The results showed higher adaptability to dietary changes in the intestinal microbiota of fish fed diet with alginate and SpPdp11 together compared to those fish that received an alginate-supplemented diet. The alginate-supplemented diet produced up-regulation of genes encoding proteins involved in immunological responses, such as complement, lysozyme G and transferrin, and oxidative stress, such as NADPH oxidase and glutation peroxidase. On the other hand, the administration of alginate combined with SpPdp11 resulted in a significant increase of the transcription of genes encoding for glutation peroxidase and HSP70, indicating a potential protective effect of SpPdp11 against oxidative stress. In addition, these effects were maintained after the suspension of the probiotic treatment. The relationship between the modulation of the intestinal microbiota and the expression of genes with protective effect against the oxidative stress was demonstrated by the Principal Components Analysis.

Two routes of infection with Photobacterium damselae subsp. piscicida are effective in the modulation of the transcription of immune related genes in Solea senegalensis

The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease outbreaks affecting cultured fish species including the flatfish Solea senegalensis. In the present work, transcription of iron metabolism related genes (TF, FERR-M, HP-1 and HAMP-1) as well as innate immune system components such as complement proteins (C3 and C7), lysozyme (LYS-G), TNF family (TNFα, TRAF-3), NCCRP-1 and heat shock protein encoding genes (HSP70, HSP90AA, HSP90AB and GP96) has been determined in the liver and kidney of S. senegalensis specimens after Phdp infection. Intraperitoneal injection (IP) and immersion (IM) routes have been used for infection. Fish developed specific antibodies in both cases, higher levels being detected in IP infected specimens. Both infection routes resulted in increased relative transcript levels of FERR-M, HP-1 and HAMP-1 genes and TF decreased relative transcription, conducting to lower iron availability for the pathogen. This response can be considered as a strategy to limit iron availability for Phdp, a pathogen capable to obtain iron from transferrin. Relative transcription of genes encoding lysozyme and complement factors C3 and C7 were also increased regardless the infection route; the liver was the main organ involved in the initial stages and the kidney in later stages of the infection. TNFα and TRAF-3 relative gene transcription increased 24h post-infection. TRAF-3 gene induction was detected 30 d post-infection, whilst no changes in TNFα were observed 72h or 30 d post-infection. NCCRP-1 changes were observed after IP infection in the liver and kidney; however, IM infection resulted only in slight changes in the kidney of infected fish. This different response observed maybe related to a lower number of invaded cells by the pathogen. Finally, changes in HSP90AB and GP96 have been detected after infection by both routes. Different late modulation has been observed in assayed genes depending on the route of infection. Thus, only LYS-G, TF, NCCRP-1, GP96 and HSP90AB gene transcription was modulated 30 d post-infection in the kidney of IM infected specimens; however, IP infected fish showed modulation in a higher number of genes both in liver and kidney tissues. The implications of these responses in resistance to infection by Phdp need to be elucidated.

Use of in vivo induced technology to identify antigens expressed by Photobacterium damselae subsp. piscicida during infection of Senegalese sole (Solea senegalensis)

ABSTRACT Photobacterium damselae subsp. piscicida (Phdp), the causative agent of photobacteriosis, is an important pathogen in marine aquaculture that affects many different fish species worldwide, including Solea senegalensis, an important fish species for aquaculture in the south of Europe. Bacteria express different repertoires of proteins in response to environmental conditions and when invading a host, sense in vivo environment and adapt by changing the expression of specific proteins. In the case of pathogens, identification of genes with up‐regulated expression in vivo compared to in vitro conditions might give an insight into the genes relevant to the bacterial virulence. In the present work, in vivo induced antigen technology (IVIAT) has been used to search for Phdp genes only expressed or up‐regulated in infected S. senegalensis. An expression library from Phdp was assayed against pooled sera from convalescent S. senegalensis specimens and 18 clones were positive, indicating that proteins encoded are expressed by Phdp during S. senegalensis infection and are immunogenic for this fish species. In addition, five proteins were reactive against adsorbed sera, indicating their in vivo induced character. Inosine‐5′‐monophosphate dehydrogenase, serine hydroxy methyltransferase and alanyl‐tRNA synthethase, involved in aminoacid and nucleotide metabolism, the protein with antioxidant activity alkyl hydroperoxide reductase and a non‐ribosomal peptide synthetase responsible for the synthesis of the siderophore piscibactin have been identified as antigens induced in Phdp during S. senegalensis infection. Proteins induced during in vivo growth of Phdp represent promising targets for the development of novel antimicrobial or prophylactic agents in the treatment and prevention of photobacteriosis. HIGHLIGHTSImmunogenic proteins expressed by Phdp during Solea senegalensis infection are identified.Iron and oxidative stress up‐regulate Phdp genes encoding immunogenic proteins.Genes encoding Phdp immunogenic proteins identified are up‐regulated in S. senegalensis kidney.