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Feeding assays were carried out with soles of 80 g mean body weight, which were randomly separated into six experimental groups, and stocked in six 250 l tanks (20 fish per tank) with similar culture conditions to those described above. The diet assayed was prepared in the laboratory from the commercial pellet diet routinely used in fish farms (Skreeting, Trouw España, Nutreco, Burgos, Spain). Briefly, the commercial pellet diet was crushed and mixed with tap water before adding the lyophilized alga Porphyridium cruentum at the desired concentration (10 g kg -1 ), and then made into pellets again. The re-made pellets were allowed to dry and stored at 4 ºC until use. The commercial pellet Sanostim (Skreeting, Trouw España, Nutreco, Burgos, Spain), containing β-glucans, was used to test the response of sole phagocytes. Two groups of fish received daily one of the different diets assayed: diet consisting of nonsupplemented commercial diet (control group); diet composed of the commercial diet containing immunostimulant Sanostim; and finally, a commercial diet supplemented with lyophilized alga (1%). Fish were fed at a rate of 20 g dry diet kg -1 biomass (2 %) per day for 4 weeks. The biomass of the fish in each aquarium was measured before the experiment and daily ration, being adjusted accordingly. No mortality was observed during the experiment. 2.5. Immunization assay Two weeks after beginning the feeding trial, fish from one tank per treatment were intraperitoneally inoculated with a bacterin of P. damselae subsp. piscicida. The formalin-killed aqueous vaccine was prepared with a virulent strain of P. damselae subsp. piscicida (Lg h411/01 ) isolated from diseased sole [16] according to the following protocol. Briefly, bacteria were cultured on TSAs for 24 h and one colony was inoculated in tubes containing 5 ml of TSBs. After 18 h incubation at 22 ºC, an aliquot of the culture, 50 μl, was inoculated in flasks with 50 ml TSBs and incubated at 22ºC with continuous shaking. After 18 h incubation the culture achieved O.D. 600 of 1.2. The total bacterial number was counted, obtaining a bacterial concentration of 6 x 10 8 bacteria ml -1 . Then bacterial cells were killed by addition of formaldehyde to achieve 1% final concentration, and overnight incubation. Sterility tests were performed by 6
spreading an aliquot of the bacterin on TSA plates and incubation for 2 days at 22 ºC. The vaccine was administered by intraperitoneal injection (0.1 ml per fish). Control fish were injected with phosphate buffer saline (PBS, pH 7.2). 2.6. Isolation of head kidney phagocytes Sole phagocytes were isolated from the kidney following the technique described by Secombes [31]. 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 xg for 30 min. Then the bands separated at the interface were resuspended in L-15 medium supplemented with P/S/G. The viable cell concentration was determined after staining with trypan blue and microscope counting. Aliquots of 100 μl containing 1x10 7 cells ml - 1 in L-15 medium supplemented with P/S/G were added to 96-well microtitre plates. After 3 h incubation at 22 ºC, non-adherent cells were removed and medium was substituted by L-15 and P/S/G supplemented with 2% FCS. Monolayers were incubated overnight at 22 ºC. 2.7. Respiratory burst activity The generation of intracellular superoxide radicals by sole phagocytes was determined by the reduction of nitro-blue tetrazolium (NBT) according to the technique described by Secombes [31] and Boesen et al. [32]. Phagocyte monolayers were washed with L-15 medium and HBSS (Hank´s Balanced Salt Solution) to remove any trace of the antibiotic. In order to test the in vitro effects of algal extracts on the production of superoxide radicals by sole phagocytes, a volume of 20 μl of the extracts (aqueous, ethanolic or β-glucan) were added to the wells (15 wells from 5 fish) containing phagocyte monolayers prepared as above. Then, 100 μl of NBT dissolved at 1 mg ml -1 in HBSS was added to the wells and the phagocytes incubated at 22 ºC for 30 min. Wells containing phagocytes were infected with 20 μl of P. damselae subsp. piscicida 7
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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
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 182 and 183: alga Porphyridium cruentum. For thi
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
Page 233: 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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