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suggesting the ability of the bacterium to survive as an intracellular pathogen [30-33], others have observed that this pathogen is highly susceptible to oxidative radicals generated during the macrophage respiratory burst [34, 35]. For this reason, the stimulation of the respiratory burst activity of Senegalese sole phagocytes could facilitate a more effective destruction of P. damselae subsp. piscicida. Hence, the first objective of the present study was to evaluate the possible immunostimulatory effect of dietary administration of two probiotics [36-38] on respiratory burst activity of sole phagocytes. In addition, possible changes in the intestinal microbiota were evaluated by determining the genetic diversity of gut microbiota by DGGE (denaturing gradient gel electrophoresis) [39]. Several authors have reported on the composition of intestinal microbiota of different farmed fish [40- 44], although none of them have studied sole microbiota. On the other hand, the possible effect of probiotics on fish intestinal microbiota is still largely unknown [28]. Thus, this is the first report on sole gut microbiota and the possible changes in response to probiotic treatment. 2. Materials and Methods 2.1. Microorganisms Two bacteria strains isolated from gilthead seabream skin, Pdp11 and Pdp13, belonging to Alteromonadaceae family, Shewanella genus, were selected for this study because their in vitro characteristics suggested they could be considered as potential fish probiotics [36-38]. These strains were grown in tubes containing 5 ml of trypticase soy broth (Oxoid Ltd., Basingstoke, UK) supplemented with 1.5 % NaCl (TSBs) for 18 h at 22 ºC, with continuous shaking. Appropriate dilutions of the culture were spread onto plates of trypticase soy agar (Oxoid) supplemented with 1.5 % NaCl (TSAs). The number of culturable bacteria was determined by plate counting on TSAs. Bacteria were recovered from the plates, lyophilized and the concentration of lyophilized bacteria was adjusted per gram of pellet diet. 4
2.2. Fish and experimental design Senegalese sole specimens (Solea senegalensis, Kaup, 1838) of 15-30 g weight, were kept in four 300 l seawater tanks (75 fish per tank), 36 ‰ salinity, at 22 ºC and fed with a commercial pellet diet (Gemma 0.5, Skreeting, Trouw España, Nutreco, Burgos, Spain). Specimens were sacrificed by overdose of clave oil. Experimental diets were prepared in the laboratory from the commercial pellet diet. The diet preparation was carried out with alginate (0.5% kg -1 ) and 50 mM calcium chloride (0.4% kg -1 ) to facilitate the lyophilized bacteria incorporation to the pellet. Alginate and Ca Cl 2 were sprayed into the feed slowly, mixing with the required amount of lyophilized bacteria (10 9 cfu g -1 ). Thus, fish in each tank received one of four different diets: a commercial diet supplemented with Pdp11 (10 9 cfu g -1 ) (diet A); the same diet supplemented with Pdp13 (10 9 cfu g -1 ) (diet B); a diet consisting of the commercial diet (control without alginate) (diet C); and, finally, the fourth group of fish received a diet supplemented with alginate and calcium chloride (control with alginate) (diet D). Fish were fed at a rate of 15 g dry diet Kg -1 biomass (1.5 %) per day for 60 days. The biomass of the fish in each aquarium was measured at the beginning of the experiment and daily ratio was adjusted accordingly. No mortality was observed during the experiment. Sampling was carried out at the end of the feeding trial, day 60, for the challenge test and study of intestinal microbiota. Samples for measurement of the respiratory burst activity were taken at the middle of the trial and at the end, at day 30 and 60, respectively. 2.3. Isolation of head kidney phagocytes The influence of the treatment on respiratory burst activity was tested in phagocytes isolated from the kidney of soles following the technique described by Secombes [45]. Briefly, the kidney was removed aseptically and pushed through a 100 μm nylon mesh with Leibovitz medium (L-15) containing 2% foetal calf serum (FCS, Sigma), 1% penicillin-streptomycin (Sigma), 0.1 % (5 mg ml -1 ) gentamicine (Sigma) (P/S/G) and 10 U heparine ml -1 . This cell suspension was layered on a 30 to 51% Percoll (Amersham) gradient and centrifuged at 600 × g for 30 min. Then, the bands 5
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FACULTAD DE CIENCIAS DEPARTAMENTO D
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Los ensayos que constituyen esta Te
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- Díaz-Rosales, P, Arijo, S, Chabr
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Sabe esperar, aguarda que la marea
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ÍNDICE / INDEX Papel de las activi
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ÍNDICE / INDEX 2. Photobacterium d
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RESUMEN Photobacterium damselae sub
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INTRODUCCIÓN 1. LA ACUICULTURA. EL
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INTRODUCCIÓN saxatilis) (Hawke et
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INTRODUCCIÓN 2.2. SINTOMATOLOGÍA
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INTRODUCCIÓN las epidemias surgida
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INTRODUCCIÓN et al., 1997). En P.
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INTRODUCCIÓN 3.1. ESTALLIDO RESPIR
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INTRODUCCIÓN radicales generados e
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INTRODUCCIÓN Tabla 2 Ejemplos de m
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INTRODUCCIÓN de parasitismo en el
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INTRODUCCIÓN razón de la búsqued
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INTRODUCCIÓN araquidónico, están
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INTRODUCCIÓN simplificar la admini
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INTRODUCCIÓN sino también cuando
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INTRODUCCIÓN La necesidad de mejor
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OBJETIVOS El trabajo planteado en e
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MATERIA L Y MÉTODOS La metodologí
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RESULTADO S Y DISCUSIÓN El primer
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RESULTADO S Y DISCUSIÓN 1996; Lync
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RESULTADO S Y DISCUSIÓN Una vez de
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RESULTADO S Y DISCUSIÓN inmunoesti
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RESULTADO S Y DISCUSIÓN cruentum s
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RESULTADO S Y DISCUSIÓN permiten c
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RESULTADO S Y DISCUSIÓN harían fa
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CONCLUSIONES Tras los estudios real
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ABSTRACT Photobacterium damselae su
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INTRODUCTION 1. AQUACULTURE. THE CU
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INTRODUCTION With regard to Solea s
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INTRODUCTION 3.1. RESPIRATORY BURST
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INTRODUCTION Some catalases have al
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INTRODUCTION pathogens, study of th
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INTRODUCTION increase their bacteri
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INTRODUCTION 4.3.1. Porphyridium cr
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INTRODUCTION Different mechanisms h
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A I M S
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M A T E R I A L S A N D M E T H O D
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R E S U L T S A N D D I S C U S S I
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RESULTS AND DISCUSSION resist 100 m
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RESULTS AND DISCUSSION present work
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RESULTS AND DISCUSSION reason, once
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RESULTS AND DISCUSSION great number
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RESULTS AND DISCUSSION dominant spe
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CONCLUSIONS Studies carried out on
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R R E F E R E N C I A S E F E R E N
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REFERENCIAS / REFEREN CES Aoki, T,
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REFERENCIAS / REFEREN CES Bell, MV,
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REFERENCIAS / REFEREN CES Dalmo, RA
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REFERENCIAS / REFEREN CES Hamaguchi
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REFERENCIAS / REFEREN CES Kono, Y &
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REFERENCIAS / REFEREN CES Magariño
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REFERENCIAS / REFEREN CES Ortuño,
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REFERENCIAS / REFEREN CES Salinas,
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REFERENCIAS / REFEREN CES Thompson,
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S E C C I Ó N D E A R T Í C U L O
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A RTÍCULO 1.1. A RTICLE 1.1.
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Journal of Fish Diseases 2003, 26,
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A RTÍCULO 2.1. A RTICLE 2.1.
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Abstract The stimulatory effect of
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alga Porphyridium cruentum. For thi
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Feeding assays were carried out wit
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(10 8 bacterias ml -1 ) and used to
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are in agreement with the data repo
Page 190 and 191: unvaccinated fish. However, the ser
Page 192 and 193: immune response of gilthead seabrea
Page 194 and 195: [27] Duncan, PL and Klesius, PH. Ef
Page 196 and 197: (Macrogard) and alginic acid (Ergos
Page 198 and 199: [66] Salinas, I, Díaz-Rosales, P,
Page 201: Figure 1a 3 2.5 Stimulation index 2
Page 205: Figure 1c Stimulation index 3 2.5 2
Page 209: Figure 2b Stimulation index 3 2.5 2
Page 213 and 214: Effect of the extracellular polysac
Page 215 and 216: 1. Introduction Solea senegalensis
Page 217 and 218: On the other hand, β-1,3-glucan fr
Page 219 and 220: eaction was tested by adding supero
Page 221 and 222: Intraperitoneal inoculation of the
Page 223 and 224: defense mechanisms and protective i
Page 225: phagocytes from sole (Solea senegal
Page 229: Figure 1a Stimulation index 3 2.5 2
Page 233: Figure 2 Stimulation index 1.4 1.2
Page 237 and 238: Effect of dietary administration of
Page 239: 1. Introduction Senegalese sole cul
Page 243 and 244: 2.5. Challenge with Photobacterium
Page 245 and 246: the densitometric curves of the pro
Page 247 and 248: most efficient probiotics for aquac
Page 249 and 250: acterial diversity only the dominan
Page 251 and 252: [9] Verschuere, L, Rombaut, G, Sorg
Page 253 and 254: [28] Burr, G, Gathin, D and Ricke,
Page 255 and 256: WB, editors. Techniques in Fish Imm
Page 257: and host-specific communities of ac
Page 261: Table 1 Primer Sequence (5’-3’)
Page 265: Figure 1b Stimulation index 2.5 2 1
Page 269: Table 2 Primer Control Pdp11 Pdp13
Page 273: Figure 3 Pearson correlation [0.0%-
Page 276: que perdieras parte de tu tiempo en
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