Papel de las actividades superóxido dismutasa y catalasa en la ...

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separated at the interface were collected, centrifuged for 15 min at 500 × g and 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. 2.4. Respiratory burst activity Fish of the four experimental groups, fed with diets A, B, C or D, were assayed for 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 [45] and Boesen et al. [46]. Phagocyte monolayers were washed with L-15 medium and HBSS (Hank´s Balanced Salt Solution) to remove any trace of antibiotic. Then, NBT (100 μl) 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 P. damselae subsp. piscicida (10 8 bacterias ml -1 ) and used to determine the response of the phagocytes to the fish pathogen. As a positive control phorbol myristate acetate (PMA, Sigma) (1 μg ml -1 ), an activating agent of the respiratory burst, was used to stimulate the respiratory burst of non-infected phagocytes. The specificity of the reaction was tested by adding superoxide dismutase (SOD) (300 I.U. per well) to some wells containing PMA-stimulated phagocytes (data not shown). After incubation, cells were fixed in 70% methanol and reduced formazan within phagocytes was solubilised by adding 120 μl 2M KOH and 140 μl dimethyl sulfoxide (DMSO, Sigma). Finally, absorbance was read at 630 nm in a multiscan spectrophotometer (UV-1601 Spectrophotometer, Whitakker Bioproducts). 6
2.5. Challenge with Photobacterium damselae subsp. piscicida Groups of 10 fish (15-30 g weight) were challenged at the end of the feeding trial, after 60 days of beginning. For the challenge, three groups of fish were assayed, fed with diets A, B and C. Soles were intraperitoneally injected with a dose of P. damselae subsp. piscicida (Lg h41/01 ) of 5 x 10 4 cfu g -1 from a bacterial suspension in PBS (phosphate buffer saline) adjusted to 5 x 10 8 cfu ml -1 . As control, the same number of fish was inoculated with 0.1 ml PBS. Inoculated fish were followed daily for 10 days, and all mortalities were recorded, considering only those that could be linked to the bacterial challenge by reisolation in pure culture from internal organs of dead fish. 2.6. Analysis of the sole intestinal microbiota 2.6.1. Sample collection Three groups of fish were analyzed for intestinal microbiota composition, i.e. fish fed with diets A, B and C. Per treatment group, three specimens of Senegalese sole were sacrificed at the end of the feeding trial (60 days), and whole intestines were collected and stored at -80 ºC until further analysis. 2.6.2. DNA isolation from intestinal content and probiotic cultures The gut contents were homogenized in 4 ml PBS (phosphate buffer saline) pH 7.2, and a 1 ml aliquot was centrifuged at 1000 × g for 5 min. The supernatant was pretreated by enzymatic digestion with 40 μl proteinase K (20 mg/ml) and 50 μl dodecyl sulphate (SDS) 10% and incubated at 65 ºC for 30 min. Subsequently, DNA extraction from the suspension was performed with the Fast DNA Spin kit for soil (Qbiogene, Inc., Carlsbad, CA) according to manufacturer’s instructions. Agarose gel (1.5% [wt/vol]) electrophoresis in the presence of ethidium bromide was used to check visually for DNA quality and yield. Pure cultures of probiotic strains were grown until exponential phase in TSBs, and then centrifuged at 2500 × g for 15 min. Pellets were washed with PBS and centrifuged again. DNA was extracted from the resulting pellet resuspended in 500 μl PBS with the Fast DNA Spin kit (Qbiogene, Inc., Carlsbad, CA). 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
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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
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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 and 240: 1. Introduction Senegalese sole cul
Page 241: 2.2. Fish and experimental design S
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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