PhD Thesis MJProl .pdf - digital-csic Digital CSIC - Consejo Superior ...

ABSTRACT
Abstract
The purpose of this Ph.D. Thesis was to study the immobilization of the probiotic
strain Phaeobacter 27-4 in biofilters as a new strategy for the control of Vibrionaceae
infections in turbot (Psetta maxima) larval rearing. This new strategy guarantees the
permanence of the probiotic bacteria in the rearing system and protects turbot larvae
against the fish pathogen Listonella anguillarum 90-11-287 at the beginning of
exogenous feeding on the rotifer Brachionus plicatilis.
As a first step to accomplish the objectives, a reproducible and specific real-time
PCR method was developed for detection and quantification of the fish probiotic
Phaeobacter 27-4 and the target pathogens L. anguillarum 90-11-287 and V. splendidus
DMC-1 in presence of all the organisms involved in turbot larval rearing: microalgae
(Isochrysis galbana), rotifers (Brachionus plicatilis), Artemia nauplii and turbot larvae.
The developed real-time PCR protocol allowed monitoring and quantification of
probiotic and pathogenic bacteria during in vivo trials.
As rotifer is the usual way of entry for potential pathogenic Vibrionaceae into fish
larvae, in a second step in this Ph.D. Thesis, the colonization and residence time of two
pathogenic Vibrionaceae strains (L. anguillarum 90-11-287 and V. splendidus DMC-1)
in rotifers were studied. L. anguillarum colonized rotifers more efficiently than V.
splendidus and both pathogenic strains were released from rotifers to seawater, after
infected rotifers were transferred to rearing tanks. Residence time of L. anguillarum was
longer than for V. splendidus, being released slower to surrounding seawater. V.
splendidus grew and became predominant in the seawater of tanks. Both pathogens
remained in rotifer or seawater enough time to infect fish larvae, but their different
behaviour could determine different infection patterns, preferentially by ingestion of
prey or by active intake or contact with surrounding seawater.
The effect of L. anguillarum and V. splendidus on the bacterial community
associated with rotifers and seawater of rearing tanks was analysed by DGGE of PCRamplified
16S rDNA fragments. The bacterial community of rotifers did not present
marked species dominance, being composed by Gram negative bacteria belonging to α-
Proteobacteria, γ-Proteobacteria, Cytophaga-Flexibacter-Bacteroides group and a Gram
positive bacterium (Microbacterium sp). The incorporation of L. anguillarum or V.
splendidus did not reduce bacterial diversity and shifts in bacteria populations could be
explained by bacterial exchange between rotifers and seawater.
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