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The aim of this work is to evaluate the immunomodulatory capacity of the extracellular polysaccharidic fraction isolated from P. cruentum on respiratory burst activity of sole phagocytes against P. damselae subsp. piscicida. 2. Materials and Methods 2.1. Microorganisms The virulent strain of Photobacterium damselae subsp. piscicida Lg h41/01 isolated from diseased sole [17] was selected to test the respiratory burst activity of phagocytes from Senegalese sole treated with and without polysaccharidic fraction of P. cruentum. The bacterial strain was cultured on tryptic soy agar (Oxoid) supplemented with 1.5% NaCl (TSAs) for 24 h at 22 ºC. Bacterial suspensions were obtained from one colony of the previous culture on TSAs, inoculated in culture in tryptic soy broth (Oxoid) added with 1.5% NaCl (TSBs) and incubated at 22ºC for 24 h. Then, the cultures were centrifuged at 2000 xg for 20 min at 4ºC, and pellets resuspended in L-15 medium at an optical density (600 nm) equal to 1(10 8 cells ml -1 ). 2.2. Isolation of the extracellular polysaccharidic fraction from Porphyridium cruentum The red microalga P. cruentum (S.F. Gray) Nägeli obtained from the collection of Centro de Investigaciones Marinas de Cádiz, Cádiz, Spain was grown in Porphyridium medium [31] in batch culture at 25 ºC, with 12 h photoperiod for 7 days. Then, the algal culture was centrifuged at 10000 xg for 10 min. The polysaccharidic fraction was obtained by selective precipitation of the exocellular polysaccharides from the culture supernatant with N-cetylpyridinium bromide (Cetavlon) 2% (p/v), following the method described by Morris et al. [30]. The pellet was redissolved with 4 M NaCl, and the polysaccharide was flocculated again with ethanol (96%), centrifuged (10000 xg, 10 min), dialyzed against 2M NaCl and finally lyophilized. Dilutions from the lyophilised extract were prepared in HBSS to achieve concentrations of 10 mg of lyophilised per ml, 5 mg ml -1 , 2 mg ml -1 and 1 mg ml -1 . 4
On the other hand, β-1,3-glucan from Euglena gracilis (BioChemika Fluka, Sigma) was used as a positive control. Ten mg of this compound were dissolved according to the manufacturer instructions, and diluted in HBSS to achieve concentrations of 10 mg ml -1 , 5 mg ml -1 , 2 mg ml -1 and 1 mg ml -1 . 2.3. Inoculation with the polysaccharidic fraction and immunization assay Specimens of Senegalese sole of 50 g mean weight were randomly separated into groups of 20 fish each, and stocked into six 2500 l tanks with recirculating, aerated seawater at 22 ºC, 35‰ salinity and fed daily with a commercial pellet diet (Skreeting, Skreeting, Trouw España, Nutreco, Burgos, Spain). Two groups of 20 fish were intraperitoneally injected with a dose of 500 μg of the extracellular polysaccharidic fraction from P. cruentum per fish. Phagocytes from two groups of sole were sampled at 2 and 8 days from algal extract administration. One group of them was intraperitoneally inoculated with bacterin of P. damselae subsp. piscicida, 24 h after the administration by intraperitoneal injection of the polyssacharide. The formalin-killed aqueous vaccine was prepared according to the following description. Briefly, the selected strain of P. damselae subsp. piscicida isolated from diseased sole in Spain was cultured on TSAs, and one colony was transferred to one tube containing 5 ml of TSBs for 18 h at 22 ºC. After incubation at 22 ºC for 18 h, an aliquot of the culture, 50 μl, was inoculated in a flask containing 50 ml TSBs and incubated at 22 ºC for 18 h with continuous shaking. When the culture achieved O.D.600 of 1.2, corresponding to 6 x 10 8 bacteria ml -1 , the cells were killed by addition of formaldehyde (1% final concentration) and incubated overnight. Sterility trials were performed by spreading an aliquot of the vaccine preparation on TSAs plates and incubating for 2 days at 22 ºC. The vaccine was administered by intraperitoneal injection (0.1 ml per fish). The other two groups of fish were used as the controls and phosphate buffer saline (PBS, pH 7.2) was inoculated instead of the algal polysaccharide. One of these groups was vaccinated with a bacterin against P. damselae subsp. piscicida as described above. 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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Journal of Fish Diseases 2003, 26,
Page 167 and 168: Journal of Fish Diseases 2006, 29,
Page 177: A RTÍCULO 2.1. A RTICLE 2.1.
Page 180 and 181: Abstract The stimulatory effect of
Page 182 and 183: alga Porphyridium cruentum. For thi
Page 184 and 185: Feeding assays were carried out wit
Page 186 and 187: (10 8 bacterias ml -1 ) and used to
Page 188 and 189: 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: 1. Introduction Solea senegalensis
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 and 240: 1. Introduction Senegalese sole cul
Page 241 and 242: 2.2. Fish and experimental design S
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
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Table 2 Primer Control Pdp11 Pdp13
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Figure 3 Pearson correlation [0.0%-
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