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alga Porphyridium cruentum. For this reason, this alga has been selected in this work to evaluate its potential immunostimulant effect on farmed fish. However, most studies performed to examine the immunostimulant ability of algae have been carried out by in vitro incubation of immune cells with algal extracts, and information on the in vivo effects of whole algal cells is still scarce [27]. In addition, algal extracts are inoculated intraperitoneally in theses studies. This route of administration, although very effective, is also very laborious, time-consuming, stressful for fish and difficult to apply to fingerlings [28, 29]. Oral administration of immunostimulants is a non-stressful method with minimum economic cost and effort and enables mass administration regardless of the fish size [7], but studies addressing this route of administration are scarce and usually include only algal extracts instead of whole cells [22]. In this study the potential immunostimulant effect of aqueous and ethanolic extracts obtained from P. cruentum on the respiratory burst activity of Senegalese sole phagocytes has been determined. In addition, the potential stimulation of the respiratory burst activity of phagocytes isolated from fish fed with a commercial diet supplemented with P. cruentum cells has been studied. In this case, potential synergetic or antagonic effects resulting from the alga diet and vaccination against P. damselae subsp. piscicida have been evaluated. Materials and Methods 2.1. Microorganisms The virulent strain Lg h41/01 of Photobacterium damselae subsp. piscicida isolated from diseased Senegalese sole [16] was selected to test the respiratory burst activity of sole phagocytes. The bacterial strain was cultured on tryptic soy agar (Oxoid) supplemented with 1.5% NaCl (TSAs) for 24 h at 22 ºC. Bacterial suspensions for respiratory burst assays were obtained from tubes containing tryptic soy broth (Oxoid) added with 1.5% NaCl (TSBs) inoculated with one colony from a TSAs plate and incubated at 22 ºC for 24 h. Then, the cultures were centrifuged at 6000 xg for 15 min at 4 ºC, and pellets were resuspended in L-15 medium at an optical density (600 nm) equal to 1(10 8 cells ml -1 ). 4
2.2. Alga culture The red microalga Porphyridium cruentum (S.F. Gray) Näegli was obtained from the collection at the Centro de Investigaciones Marinas de Cádiz, Cádiz, Spain. It was grown in Porphyridium medium [30] in batch culture at 25ºC, with a 12h photoperiod for 7 days. The algal biomass was centrifuged at 3000 xg, 15 min at 4ºC and the pellet was lyophilized. 2.3. Obtention of aqueous and ethanolic extracts The preparation of water-soluble extract, aqueous extract, was carried out as follows: 10 g of lyophilized alga was resuspended in 100 ml of HBSS (Hank’s Balanced Salt Solution) using a mortar and pestle. The extract was sonicated for 20 min and centrifuged at 3000 xg, 5 min. The supernatant was separated from the pellet and lyophilized and 10 mg of the lyophilized extract was resuspended in 1 ml of HBSS. Extraction of the non-soluble fraction of the alga, ethanolic extract, was carried out as described above, but instead of HBSS, ethanol was used. Dilutions from both extracts were prepared in HBSS to achieve concentrations of 10 mg ml -1 of lyophilized extract, 5 mg ml -1 , 2 mg ml -1 and 1 mg ml -1 . Commercial β-1,3-glucan from Euglena gracilis (BioChemika Fluka, Sigma) was used as a positive control of stimulation of respiratory burst activity. β-1,3-glucan (10 mg) was dissolved following commercial 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 . 2.4. Fish and experimental design Experiments to test the in vitro effect of P. cruentum on the respiratory burst activity of sole phagocytes were carried out. Sole of 200 g body weight, stocked in 250 l tanks with recirculating, aerated seawater at 20 ºC, 35‰ salinity, were used to isolate kidney phagocytes and determine the production of anion radicals in contact with aqueous and ethanolic extracts from P. cruentum and the commercial β-1,3-glucan. 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
Page 131 and 132: CONCLUSIONS Studies carried out on
Page 133: R R E F E R E N C I A S E F E R E N
Page 136 and 137: REFERENCIAS / REFEREN CES Aoki, T,
Page 138 and 139: REFERENCIAS / REFEREN CES Bell, MV,
Page 140 and 141: REFERENCIAS / REFEREN CES Dalmo, RA
Page 142 and 143: REFERENCIAS / REFEREN CES Hamaguchi
Page 144 and 145: REFERENCIAS / REFEREN CES Kono, Y &
Page 146 and 147: REFERENCIAS / REFEREN CES Magariño
Page 148 and 149: REFERENCIAS / REFEREN CES Ortuño,
Page 150 and 151: REFERENCIAS / REFEREN CES Salinas,
Page 152 and 153: REFERENCIAS / REFEREN CES Thompson,
Page 155: S E C C I Ó N D E A R T Í C U L O
Page 159: A RTÍCULO 1.1. A RTICLE 1.1.
Page 162 and 163: Journal of Fish Diseases 2003, 26,
Page 177: A RTÍCULO 2.1. A RTICLE 2.1.
Page 180 and 181: Abstract The stimulatory effect of
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 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
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Figure 2 Stimulation index 1.4 1.2
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Effect of dietary administration of
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1. Introduction Senegalese sole cul
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2.2. Fish and experimental design S
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2.5. Challenge with Photobacterium
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the densitometric curves of the pro
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most efficient probiotics for aquac
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acterial diversity only the dominan
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[9] Verschuere, L, Rombaut, G, Sorg
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[28] Burr, G, Gathin, D and Ricke,
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WB, editors. Techniques in Fish Imm
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and host-specific communities of ac
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Table 1 Primer Sequence (5’-3’)
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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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que perdieras parte de tu tiempo en
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