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

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Journal of Fish Diseases 2006, 29, 355–364 PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida superoxide dismutases (SODs) and catalases. Production of SOD and catalase enzymes, which decompose superoxide and peroxide radicals, respectively, have been reported to contribute to the virulence of a great number of pathogens (Franzon, Arondel & Sansonetti 1990; Lynch & Kuramitsu 2000; Lefebre & Valvano 2001; Uzzau, Bossi & Figueroa-Bossi 2002). Superoxide dismutases are a family of metalloenzymes including four types depending on the metal cofactor, copper-zinc (Cu/Zn-SOD), manganese (Mn-SOD), iron (Fe-SOD) and nickel (Ni-SOD) (Lynch & Kuramitsu 2000). Three types of catalase have been described: monofunctional catalases, bifunctional catalases or catalase/ peroxidase and pseudocatalases or non-haeme catalases, with manganese as a metal cofactor (Loewen 1997). Microorganisms produce different SOD and catalase isozymes inducible under certain culture conditions such as high oxygen tension, low levels of iron or stationary growth phase (Crockford, Davis & Williams 1995; Schnell & Steinman 1995; Barnes, Horne & Ellis 1996; Polack, Dacheux, Delic-Attree, Toussaint & Vignais 1996; St John & Steinman 1996; Lynch & Kuramitsu 2000; Vattanaviboon & Mongkolsuk 2001). However, information on the SOD and catalase activities of P. damselae ssp. piscicida is scarce. The aim of this work was to determine whether P. damselae ssp. piscicida can express different SOD and catalase activities when cultured under different conditions, and whether these enzymatic activities may protect the bacterium in vitro from oxygen radicals generated during the macrophage respiratory burst. Materials and methods Bacteria Strains of P. damselae ssp. piscicida used in this study are listed in Table 1. Strains B180, D 26/98 , Pp8H, R45, R46, B51 and Lg h41/01 were isolated in our laboratory (Department of Microbiology, Faculty of Sciences, University of Málaga, Spain). Strains MT 1415, MT 1375, MT 1376 and MT 1379 were kindly provided by Dr A.C. Barnes (Marine Laboratory, Aberdeen, UK); strain DI-21S by Dr A.E. Toranzo (Department of Microbiology and Parasitology, Faculty of Chemistry, University of Santiago de Compostela, Spain) and EPOY- 8803-II by Dr K. Muroga (Faculty of Applied Biological Sciences, Hiroshima University, Hiroshima, Japan). Strains 17911 and 29690 were obtained from the American Type Culture Collection (ATCC). Virulence assays were carried out with two selected strains: Lg h41/01 and EPOY-8803-II. Assays to determine the lethal dose 50% (LD 50 ) for sole, Solea senegalensis (Kaup), were carried out following the methodology described by Santos (1991). Groups of five fish (10–15 g body weight) maintained in tanks at 24 °C, were intraperitoneally inoculated with 0.1 mL of serial bacterial dilutions containing 10 3 –10 8 cfu. The same number of fish was inoculated with phosphate-buffered saline (PBS) and used as a control. Inoculated fish were observed daily for 14 days, and all mortalities were recorded. Mortalities were considered to be due to the inoculation when the bacterial strain was isolated in pure culture from internal organs of dead fish. Lethal dose 50% (LD 50 ) represents the Strain Host Source 17911 Roccus americanus ATCC 29690 Seriola quinqueradiata ATCC B51 Dicentrarchus labrax UMA, Spain B180 Sparus aurata UMA, Spain D 26/98 S. aurata UMA, Spain Pp8H S. aurata UMA, Spain R45 S. aurata UMA, Spain R46 S. aurata UMA, Spain DI-21S S. aurata USC, Spain EPOY-8803-II Epinephelus akaara Japan Lg h41/01 Solea senegalensis UMA, Spain MT1415 D. labrax Marine Laboratory, Aberdeen, UK MT1375 D. labrax Marine Laboratory, Aberdeen, UK MT1376 S. aurata Marine Laboratory, Aberdeen, UK MT1379 S. aurata Marine Laboratory, Aberdeen, UK Table 1 Photobacterium damselae subsp. piscicida strains used in this study ATCC, American Type Culture Collection; UMA, University of Málaga; USC, University of Santiago de Compostela. Ó 2006 Blackwell Publishing Ltd 356
Journal of Fish Diseases 2006, 29, 355–364 PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida number of bacteria needed to kill 50% of the inoculated fish (Reed & Müench 1938). Strain Lg h41/01 with an LD 50 ¼ 2.8 · 10 4 cfu g )1 fish was considered virulent for sole and strain EPOY- 8803-II with LD 50 > 7.7 · 10 6 cfu g )1 fish was considered non-virulent. Bacterial growth conditions Bacteria were stored at )80 °C in tryptic soy broth (TSB; Oxoid Ltd., Basingstoke, UK) containing 2% NaCl and 20% glycerol. Bacteria were cultured on tryptic soy agar (TSA; Oxoid) containing 2% NaCl and incubated at 22 °C for 48 h. One colony was used to inoculate 5 mL TSBs and incubated for 18 h at 22 °C with shaking. Aliquots (25 lL) of these cultures were used to inoculate 250 mL TSBs which was incubated at 22 °C with shaking. The incubation time varied depending on the culture condition and strain to be assayed. Different growth conditions were assayed to determine the potential induction of SOD and catalase activities. Thus, 250-mL culture flasks were supplemented with an iron chelant, dipyridyl (100 lm), FeCl 3 Æ6H 2 O (100 lm) or MnSO 4 Æ2H 2 O (250 lm) to determine the influence of iron and manganese availability on enzymatic activity. In order to induce oxidative stress, methyl viologen (0.2 mm), which generates superoxide radicals, was added to mid-exponential cultures, which were then incubated for 8 h before centrifugation. The potential induction of enzymatic activities by hydrogen peroxide was tested in cultures after the addition of two pulses of hydrogen peroxide, one of 20 lm in the mid-exponential phase, and another of 2mm in the early stationary phase. Cells were harvested after 1-h incubation. The influence of the growth phase was investigated with bacteria harvested from mid-exponential (OD 600 ¼ 0.4–0.6) and early stationary (OD 600 ¼ 1–1.2) phase cultures. Preparation of crude extracts Bacteria were harvested from cultures grown as described above by centrifugation at 2000 g for 20 min at 4 °C and washing twice in 25 mm potassium phosphate buffer containing 1 mm disodium ethylene diamine tetraacetic acid (EDTA; Sigma-Aldrich, St. Louis, MO, USA), pH 7.2 and 0.5 mm phenyl methylsulphonyl fluoride (Sigma) followed by re-suspension in 1 mL of the same buffer. Suspensions were sonicated on ice for 120 s (four pulses of 30 s with 15 s cooling between bursts). Lysates were clarified twice by centrifugation at 10 000 g for 20 min at 4 °C. Supernatants were assayed for the detection of SOD and catalase and quantification of enzymatic activity on acrylamide gels. Total protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as standard. Polyacrylamide gel electrophoresis Electrophoresis was performed in non-denaturing discontinuous polyacrylamide mini-gels using the Bio-Rad Mini Protean II System (Bio-Rad Laboratories, Richmond, CA, USA) with a 10% acrylamide/bis separating gel (1.5 m Tris–HCl, pH 8.8) and a 4% acrylamide/bis stacking gel (0.5 m Tris– HCl, pH 8.3). The extracts in the sample buffer were applied to the gel at a concentration of 20–24 lg protein per lane. Gels were then stained for SOD or catalase and peroxidase activities. Detection and quantification of SOD activity Superoxide dismutase activity was visualized on gels by nitroblue tetrazolium (NBT; Sigma) negative staining (Beauchamp & Fridovich 1971). Briefly, gels were washed in distilled water, soaked in a solution of 2.45 mm NBT for 20 min, followed by 10-min incubation in darkness in a solution containing 50 mm potassium phosphate buffer (pH 7.2), 0.028 mm riboflavin (Sigma) and 28 mm tetramethylethylenediamine (TEMED; Sigma). Gels were illuminated on a light box to develop a dark background with achromatic bands corresponding to SOD activity, due to inhibition of the photochemical reduction of NBT to formazan blue. The method employed to quantify SOD activity is based on the ability of SOD to inhibit the reduction of NBT by superoxide (Winterbourn, Hawkins, Brian & Correll 1975; Worthington Enzyme Manual 1993). One unit is defined as the amount of enzyme causing half the maximum inhibition of NBT reduction. Different volumes of extracts were added to cuvettes containing 0.2 mL of a solution of 0.1 m EDTA, 0.3 mm sodium cyanide (NaCN; Sigma) and 0.1 mL of 1.5 mm NBT. Then, 0.05 mL of 0.12 mm riboflavin was added at zero time and at timed intervals. All cuvettes were incubated in a light box for 12 min and absorbance at 560 nm was read at timed Ó 2006 Blackwell Publishing Ltd 357
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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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Page 120 and 121: RESULTS AND DISCUSSION resist 100 m
Page 122 and 123: RESULTS AND DISCUSSION present work
Page 124 and 125: RESULTS AND DISCUSSION reason, once
Page 126 and 127: RESULTS AND DISCUSSION great number
Page 128 and 129: 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 167: 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 and 216: 1. Introduction Solea senegalensis
Page 217 and 218: On the other hand, β-1,3-glucan fr
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eaction was tested by adding supero
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Intraperitoneal inoculation of the
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defense mechanisms and protective i
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phagocytes from sole (Solea senegal
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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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