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Abstract The effects of the dietary administration of two bacterial probiotic strains from the Alteromonadaceae family, Pdp11 and Pdp13, on Senegalese sole intestinal microbiota were studied. The fish were fed four different diets: control (nonsupplemented), or diets supplemented with alginate alone, or as a carrier for Pdp11 (10 9 cfu g -1 ) or Pdp13 (10 9 cfu g -1 ) for sixty days. The effect of the different dietary treatments was assessed by measuring respiratory burst activity, protection against experimental infection with Photobacterium damselae subsp. piscicida and analysis of intestinal microbiota by 16S ribosomal RNA gene-targeted PCR-DGGE (denaturing gradient gel electrophoresis). We also evaluated, to which extend and how this technique could be effectively applied to examine bacterial diversity and dynamics of the sole (Solea senegalensis) intestinal microbiota, as influenced by the diet. Bacterial ribotypic diversity was determined using DGGE analysis of the V6 to V8 and V3 regions of 16S rRNA genes using two different sets of primers for PCR amplification. To our knowledge, this is the first report related to the effect of probiotics in the intestinal microbiota of sole, by cultivation-independent molecular ecological approaches. Keywords: Probiotics; Alteromonadaceae; Intestinal microbiota; DGGE; Sole (Solea senegalensis, Kaup 1858); Teleosts. 2
1. Introduction Senegalese sole culture (Solea senegalensis, Kaup 1858) is nowadays a very promising industry in the Mediterranean countries [1]. One of the most serious problems concerning sole production is the existence of infectious diseases, pseudotuberculosis caused by Photobacterium damselae subsp. piscicida, being the main limiting factor [2]. In fish farms, control of this bacterial pathogen is achieved by the administration of chemotherapeutic agents. However, over the last decade, drugresistant strains carrying a transferable R-plasmid have evolved, making treatment with antimicrobial chemotherapeutics less successful [3]. Therefore, immunoprophylaxis has become the best way to prevent pseudotuberculosis [4]. Throughout the last 20 years, there has been a variety of studies analyzing the effectiveness of immunization in preventing pseudotuberculosis [5]. A divalent vaccine against V. harveyi and P. damselae subsp. piscicida for sole has been reported [6]. This, however, only provides protection for a short period. Therefore, searching for new prophylactic methods, such as immunostimulants or probiotics seems a very promising alternative. Probiotics have been defined as live microbial preparations that improve the health and well-being of the host [7-10]. The research on probiotics for aquatic animals is increasing with the demand for environment-friendly aquaculture. Most of these studies have been related to challenge trials and suppression of pathogen growth by probiotic bacteria [11-21]. Recent works have been focused on the immunological enhancement of fish defence mechanisms by probiotic bacteria [22-27] and the effects that dietary administration of probiotics could exert on the intestinal microbiota of the host fish [28]. As immunomodulators, probiotics may enhance phagocytic activity and increase the production of reactive oxygen species by macrophages from species such as gilthead seabream [27, 29] and rainbow trout (Onchorynchus mykiss) [22]. The means of invasion and survival of P. damselae subsp. piscicida inside the host are still unknown, and while authors have reported the presence of intact bacteria inside fish cells, 3
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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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Page 190 and 191: unvaccinated fish. However, the ser
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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%-
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