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The need for species diversification in Mediterranean aquaculture has promoted the culture of omnivorous and herbivorous species. Within this context, it is worth noting the high potential presented by the species of the family Mugilidae (mullets), which is considered a priority within the current strategies of European aquaculture (Alexander et al. 2015). One of the mullets with potential for use in aquaculture is the thick-lipped grey mullet (Chelon labrosus), due to its great adaptability to different culture conditions, omnivorous profile, high osmoregulatory capacity, and resistance to environmental variations (Pujante et al., 2018). However, there is a lack of information regarding the composition and functions of its intestinal microbiota. Documenting the bacteria present in healthy individuals is the first step to understanding the impacts of microbial manipulation in aquaculture systems (Tarnecki et al., 2016). In this sense, the knowledge of bacterial diversity from healthy wild fish in their natural environment is essential. Furthermore, the study of the functionality of the intestinal microbiota of wild fish provides information on its capacity to adapt to different culture conditions. Thus, this study aimed to characterize the intestinal microbiota of wild C. labrosus and explore its potential functionality on the host. Predicted functions of intestinal microbiota showed the most abundant those related to amino acid metabolism, carbohydrate metabolism, energy metabolism, membrane transport, and cell replication and repair. Furthermore, the analysis revealed microbial functional genes related to the elimination of environmental toxins. These functions of intestinal microbiota might provide beneficial effects for the host. This work was funded by Project Agl-2017-83260R supported by the Agencia Estatal de Investigación (MINECO, Spanish Government), and by the Project Bluemaro (PID2020 116136 RB100) of the Ministry of Science and Innovation of Spain.

In recent years, a clear emphasis has been placed on replacing fishmeal and fish oil in aquafeeds with other alternative ingredients, including algae, particularly in low trophic omnivorous fish species. This work aimed at evaluating the effects of moderate dietary supplementation with the green microalga Chlorella fusca on growth, metabolism, and digestive functionality in juvenile thick-lipped grey mullet (Chelon labrosus). Fish were fed a control diet (CT) or a diet containing 15% C. fusca (C-15) biomass during 90 days. C. labrosus fed with the C-15 diet showed higher growth performance (in terms of final weight and length, weight gain, and specific growth rate) than the control group. Somatic indices and muscle proximate composition were similar at the end of the feeding trial. Regarding fatty acids profile, C. fusca-fed fish showed a selective retention of docosahexaenoic acid (DHA) in the liver, and arachidonic acid (ARA), eicosapentaenoic acid (EPA), and DHA in the muscle. Dietary inclusion of this microalga significantly increased intestinal total alkaline protease, leucine aminopeptidase, and alkaline phosphatase activities in specimens fed with C-15 diet. Furthermore, intestine histological analysis revealed the absence of damage signs on gut morphology in fish fed the microalgae supplemented diet. Thick-lipped grey mullets fed the C-15 diet increased plasma glucose and decreased plasma lactate. Overall, the effects observed on liver (lipid metabolism, glycolysis and glycogenolysis) enzyme activities, together with adequate fatty acid profile, metabolic response, and gut morphology, and a significant increase in the intestinal mucosa’s digestive and absorptive capacity, could explain the positive effects on growth performance obtained in fish fed the microalgae-supplemented diet. In conclusion, the results obtained showed that C. fusca is suitable as dietary ingredient for feeding thick-lipped grey mullet juveniles.

Made available in DSpace on 2020-12-12T02:01:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Aeromonas hydrophila is responsible for outbreaks of a severe infectious disease in fish farms around the world and is one of the major causes of economic losses to the neotropical fish farmers. This study assessed the induction of immune responses and protection against A. hydrophila in pacu, Piaractus mesopotamicus, vaccinated through intraperitoneal and immersion route with inactivated virulent strain. Fish were randomly distributed in three vaccinated groups: intraperitoneal (i.p.) route; immersion; and immersion + booster; and control group (unvaccinated). All vaccination protocols used the concentration of 1.7 × 108 CFU mL−1 of inactivated A. hydrophila., and an oil adjuvant was used for vaccine prepararion for i.p. route vaccination. Blood and skin mucus from 9 fishes per treatment were collected at 14, 28, 42 and 84 days post-vaccination (DPV) for determination of lysozyme concentration in skin mucus, as well as antibodies anti-A. hydrophila in blood serum and skin mucus. Fish were challenged at 84 DPV with homologous and virulent strain of A. hydrophila for evaluation of resistance against bacterial infection. The results demonstrated that vaccination with inactivated A. hydrophila suspension by i.p. or immersion resulted in significant increase of skin mucus lysozyme and specific antibody levels in serum and skin mucus, at 28 and 42 DPV, and this increase in innate and adaptive immunity remained significant in pacu vaccinated through i.p. route up to 84 DPV. Although no significant differences were observed in the survival study, pacu vaccinated through i.p. route presented 31,33% of relative percentage survival (RPS) in LD50–96h when compared unvaccinated fish challenged at 84 DPV. The results observed in this study indicate that vaccination programs with inactivated A. hydrophila, including booster doses by i.p. or immersion routes, could result in more effective protection in pacu against this bacteriosis, by increasing innate and adaptive mucosal and systemic immune responses. Laboratory of Aquatic Organisms Pathology (Lapoa) Aquaculture Center from UNESP (CAUNESP), Rodovia de Acesso Paulo Donato Castellane s/n Department of Microbiology Faculty of Sciences University of Malaga Department of Preventive Veterinary Medicine São Paulo State University (Unesp), Rodovia de Acesso Paulo Donato Castellane s/n, Zona Rural Department of Microbiology São Paulo State University (Unesp), Rodovia de Acesso Paulo Donato Castellane s/n, Zona Rural Laboratory of Animal Pharmacology and Toxicology Brazil University, Av. Hilário da Silva Passos, 950 Laboratory of Aquatic Organisms Pathology (Lapoa) Aquaculture Center from UNESP (CAUNESP), Rodovia de Acesso Paulo Donato Castellane s/n Department of Preventive Veterinary Medicine São Paulo State University (Unesp), Rodovia de Acesso Paulo Donato Castellane s/n, Zona Rural Department of Microbiology São Paulo State University (Unesp), Rodovia de Acesso Paulo Donato Castellane s/n, Zona Rural FAPESP: 2012/12256-7 CNPq: 432830/2016-2

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 cross-reaction 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.

Aquaculture procedures usually induce stress that affects the physiological status of fish. For this reason, the inclusion of additives in fish feeds to palliate stress might be a good alternative. This study aimed to assess the antimicrobial activity of citronella (Cymbopogon nardus) essential oil (CEO) against bacterial pathogens and to determine its dietary impact on the growth performance of Sparus aurata. In vitro tests confirmed that CEO possesses antimicrobial activity against several fish-specific pathogens. For the in vivo tests, three experimental groups were fed for 60 days with different concentrations of CEO: CTRL (0 mL kg−1 fish feed), CEO1 (1 mL kg−1 fish feed), and CEO2 (2 mL kg−1 fish feed). At the end of the experiment, the physiological status was characterized. Subsequently, the specimens of the CTRL and CEO2 groups were subjected to a challenge with an injection of Poly I:C for immune stimulation. Although S. aurata individuals tolerated CEO inclusion without compromising growth performance, it significantly reduced glycogen in the CEO2 group, concomitant to an increment of total peripheral leucocytes. Moreover, different hematological profiles’ responsive patterns against an inflammatory stimulus were observed. In conclusion, our results suggest that the use of CEO as a fish feed additive can prevent bacterial outbreaks and improve potential in vivo disease resistance in S. aurata without negatively affecting growth.

This study evaluated the control of streptococcosis outbreaks in Brazil, isolated from diseased sorubim and identified as Lactococcus garvieae by genetic sequencing. This report determined the potential for lactococcosis control in sorubim Pseudoplatystoma sp. with two vaccines: an aqueous-based, whole-cell inactivated vaccine (bacterin) and an oil-adjuvanted bacterin. Their efficacy was evaluated at 30 days post-vaccination (d.p.v.) by challenge with L. garvieae, and the antibody production response at 15, 30 and 60 d.p.v. and the non-specific immune response were compared amongst treatments. High protection levels (P

The 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.

Probiotics are currently being supplemented to cultured fish due to their benefits for fish performance. Herein, we tested the health protection and nutritional effects of probiotic Shewanella putrefaciens (Pdp11 strain), both fresh and lyophilized cells, on Senegalese sole (Solea senegalensis) juveniles. Pdp11 was incorporated into fish feed at concentration of 109 cells g−1, and then provided to juvenile sole for 2 months. Growth rates were significantly higher in fish fed the fresh probiotic compared with fish fed the control diet. Growth of fish receiving lyophilized bacteria was not improved when compared with controls. Body compositions (protein, total lipids and fatty acids profile) were similar for each dietary treatment. At the end of the study, fish were challenged by intraperitoneal inoculation with a pathogenic strain of Photobacterium damselae subsp. piscicida. Relative percentages of survival (RPS) ranged between 25% and 43.8% for fish receiving both probiotic supplemented diets. Both fresh and lyophilized Pdp11 cells conferred protection against P. damselae subsp. piscicida. However, only fresh Pdp11 enhanced Senegal sole performance. These findings should be taken into account for industry purposes in which lyophilized Pdp11 might be easier to manage.

The effects of dietary administration of probiotic Pdp11, a bacterial probiotic strain from the Alteromonadaceae family, on growth and stress tolerance to high stocking density was analyzed in juvenile gilthead sea bream Sparus auratus . In the first trial, juvenile specimens were fed during 116 days with control feed or diets supplemented with Pdp11, and growth, serum immunological parameters as well as plasmatic, hepatic and muscular metabolic parameters were assessed. Growth performance improved in group receiving probiotic compared to control group. No differences were observed for the assessed immunological or metabolic parameters. In the second trial, specimens from both groups from the first experiment were submitted to the following conditions for 15 days: i) group fed with control food and either under low density (3 kg/m 3 ), or high density (30 kg/m 3 ) and ii) group fed with probiotic supplemented feed and either under low density, or high density. Our results indicated that administration of probiotic Pdp11 in the diet promoted growth and improved stress tolerance under high stocking density, suggesting its use will be beneficial for the aquaculture industry of this species.

This study aimed to evaluate the influence of dietary administration of two probiotic strains belonging to Shewanella genus (Pdp11 and Pdp13), on juvenile Senegalese sole (Solea senegalensis, Kaup 1858). Fish were fed three experimental diets for 2 months: two diets supplemented with 109 cfu g−1 feed of Pdp11 and Pdp13 probiotics and one control group. At the end of the experiment, samples were collected to determine muscle and liver body composition and to study digestive tract and liver histology. A challenge with Photobacterium damselae subsp. piscicida (ip) was performed. Results showed a protection for fish receiving probiotic supplemented diets (Relative Percentage Survival 27.5 ± 2.5% for Pdp11; 32.5 ± 2.5% for Pdp13) and a better growth for control and Pdp11 diets. Higher total protein in muscle were detected for fish fed the probiotic diets and significant differences in muscle and liver fatty acid profiles were observed for Pdp11 and Pdp13 compared to the control. Light microscopy images from sole specimens fed the Pdp11 diet showed scarce presence of lipid droplets inside of enterocytes and hepatocytes while greater numbers were found in fish fed the control and Pdp13 diets. Finally, the DGGE technique results suggest that the addition of the probiotic strains of Shewanella genus highly influenced the intestinal microbiota of Senegalese sole specimens, although this effect may depend on the probiotic strain assayed.

The effects of dietary administration of two probiotics, Shewanella putrefaciens Pdp11 and Shewanella baltica Pdp13, on growth, respiratory burst activity of phagocytes of Senegalese sole (Solea senegalensis), and survival of fish challenged with Photobacterium damselae subsp. piscicida have been studied. Fish were fed for 60 days with three different diets: one control, and two diets supplemented with 109 cfu g− 1of probiotics Pdp11 (Pdp11 diet) and Pdp13 (Pdp13 diet). Respiratory burst activity of phagocytes from fish fed with diet Pdp11 significantly increased after 60 days of feeding, whilst this significant increase was not detected in phagocytes from fish fed control and Pdp13 diets. On the other hand, the cumulative percentage of mortality after the challenges with P. damselae subsp. piscicida, was 100% in the groups fed with control diet, whereas mortality rates observed in the groups fed with diets supplemented with Pdp11 and Pdp13 ranged from 75–100% and 65–80%, respectively. These results suggest that an increased respiratory burst activity of the phagocytes is not essential to increase the protection against the P. damselae subsp. piscicida. On the other hand, the use of both probiotics improved the growth, and the survival against the pathogen, in comparison with those fish receiving the control diet.

Senegalese sole cultures are frequently affected by Vibrio harveyi disease outbreaks. Vaccines in aquaculture are one of the most successful methods of preventing fish pathologies; however, these vaccines are usually composed of inactivated whole cells containing a wide pool of antigens, and some do not induce any protection against pathogens. Thus, the aim of this study was to identify immunogenic proteins of V. harveyi involved in the specific antibody production by Senegalese sole. S. senegalensis specimens were immunized, by intraperitoneal injection, with V. harveyi bacterin supplemented with inactivated extracellular polymeric substances (ECP) and Freund incomplete adjuvant to obtain polyclonal antiserum. One month later, specimens were re-inoculated with the same antigens. Sera from immunized fish were collected two months post first immunization. Strong specific immune response to V. harveyi antigens was detected by ELISA using bacterin (limit dilutions of sera were 1:64000), ECP (1:4000) and outer membrane proteins (OMP) (1:4000) as antigens. Presence of immunogenic proteins in V. harveyi ECP and OMP were determined by 2D-PAGE. For Western Blot analysis some gels were transferred onto nitrocellulose membranes and incubated with sera from S. senegalensis specimens immunized against V. harveyi. 2D-PAGE and Western Blot showed at least five reactive proteins in the ECP and two in the OMP fraction. The spots that clearly reacted with the sole antiserum were excised from stained gel, and analyzed by mass spectrometry (MALDI/TOFTOF). A database search was then performed, using MASCOT as the search method. According to the results, the five ECP spots were identified as Maltoporine, protein homologous to Metal dependent phosphohydrolase, two porins isoforms of V. harveyi and a protein homologous to the cell division protein FtsH. Reactive proteins in the OMP fraction were identified as the protein 3-hydroxyisobutyrate dehydrogenase and a protein homologous to acid phosphatase.

Four isolates obtained from gilthead seabream have been tested for their adhesion to the skin, gill and intestinal mucus of gilthead seabream, and for their ability to interfere with Listonella anguillarum ,a n important pathogen of farmed gilthead seabream. The ability to adhere to mucus was higher than 7% for all isolates. Three isolates showed an antagonistic eiect against some of the pathogenic strains tested. They were assayed to interfere with the attachment of L. anguillarum to the mucus of gilthead seabream. Only two isolates signi¢cantly reduced the adhesion of L. anguillarum to all of the mucus assayed under exclusion, competition and displacement conditions. According to the criteria applied, the isolate Pdp11 was selected and its in vivo probiotic potential was assessed by oral administration followed by challenge with the pathogen L. anguillarum. For the feeding trial, a group of 50 gilthead seabreams received a commercial diet supplemented with lyophilized10 8 CFU g 1 of this isolate for 15 days. An other group of similar characteristics received the non-supplemented commercial diet. After the challenge, the mortality of the ¢sh receiving the diet supplemented with the potential probiotic was signi¢cantly lower than that observed in the groups of ¢sh receiving the non-supplemented commercial diet.

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.

Bacteria isolated from an outbreak with moderate mortalities in farmed sole, Solea senegalensis (Kaup), in the south of Spain were identified as Vibrio harveyi and V. parahaemolyticus. Only bacterial strains showing swarming were virulent in sole and caused mortalities in experimentally inoculated fish. However, the signs of the disease were only reproduced with V. harveyi. The intramuscular inoculation of the extracellular products (ECPs) of both species produced mortalities in inoculated fish and the appearance of surface ulcers in the case of V. harveyi. However, the inoculation of sublethal doses of ECPs to fish showed a protective effect against V. harveyi.

This work concerns the case study of a Mediterranean Ramsar salt lake (Fuente de Piedra, southern Spain) that receives the treated wastewater of the local village treatment plant. The wastewater goes through a system of canals, water dams, and three semi-natural ponds that were built in 2005. This work aims to investigate the capacity of the system to assimilate the impact of wastewater effluents on Lake Fuente de Piedra. For this, four points were sampled on 27–29 April 2016, at the inlet and the outlet points of the first and the third semi-natural ponds, with three replicates each. Temperature, pH, and conductivity at the inlet were 19.62 °C, 7.99, and 3262.67 μS/cm, respectively, and increased through the pond system by 7.59%, 8.04%, and 37.34%, respectively. Phytoplankton concentration indicators decreased from the inlet point to the outlet point (chlorophyll a from >500 to 5 × 1010 to 4.3 × 109 μm3/mL). Zooplankton biovolume, in contrast, increased three orders of magnitude from the inlet (3.5 × 107 μm3/mL) to the outlet point (1.6 × 109 μm3/mL). Heterotrophic bacteria (1.29 × 105 cfu/mL) and faecal enterococci (1033 ± 351 cfu/100 mL) were high at the inlet point, but decreased at the outlet point by almost three orders of magnitude. Total phosphorous and total nitrogen decreased 40.3% and 23.1% through the pond system. The results showed an improvement in water quality in its passage through the built system. Additionally, as permanent wetlands with acceptable water quality, the water system attracts wild fauna during the dry summer, leading to the conclusion that these semi-natural or artificial wetlands should be extrapolated to other aquatic ecosystems (Mediterranean wetlands) that receive contributions of residual waters. Better functioning of the treatment plant is desirable to improve the conservation of the Ramsar and adjacent wetlands systems.

The role of the capsule inPhotobacterium damselasubsp.piscicida(formerlyPasteurella piscicida) as a virulence factor was evaluated by determining the phagocytic and bactericidal activities of macrophages of gilt-head seabream. Five capsulated strains ofP. damselasubsp.piscicidapathogenic for gilt-head seabream and one strain (EPOY-8803-II) described as noncapsulated and avirulent for this fish species were used in the study. Significant differences (P

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.

The effects of the dietary administration of two bacterial probiotic strains (Ppd11 and Pdp13) from the Alteromonadaceae family for 60 days, were assessed by measuring growth and feed efficiency, activities of leucine aminopeptidase and alkaline phosphatase and structural changes in the intestine of juvenile Senegalese sole. In addition, the profile of intestinal microbiota was studied by Denaturing Gradient Gel Electrophoresis. Growth and nutrient utilization were significantly higher in fish receiving probiotics than in those fed the control diet. No differences were observed in proximal composition between treatments, though higher lipid muscle content was measured in fish receiving Pdp13. Those fish also exhibited higher activities of AP when compared to Ppd11 and control groups. The profile of intestinal microbiota clearly separated those fish receiving probiotics from those of the control group. Microscopical examination revealed accumulation of lipid droplets in the enterocytes of fish receiving the control diet, but not in those fed on probiotics. Interactions between those structural changes and growth performance are discussed

Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (approximately 93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi. Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of approximately 93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens.

Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole.

The protection of cultured sole, Solea senegalensis, against Vibrio harveyi and Photobacterium damselae subsp. piscicida was evaluated following the use of a divalent vaccine prepared with formalized whole cells and extracellular products of virulent strains of both pathogenic microorganisms and administered by the immersion route. Two prolonged immersions of 5-10 g fish in the divalent bacterin at a 1-month interval gave high levels of protection similar to those obtained when the respective monovalent vaccines were administered by the intraperitoneal route [relative percentage of survival (RPS) values >70%], which indicates that the former procedure can be a useful strategy with small fish. The high protection afforded by the divalent vaccine in sole lasted for 4 months after which the RPS values against both pathogens decreased significantly.