Immobilization of Phaeobacter 27-4 in biofilters as a strategy for the control of Vibrionaceae infections in marine fish larval rearing

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.The developed real-time PCR protocol allowed monitoring and quantification of probiotic and pathogenic bacteria during in vivo trials.The developed real-time PCR protocol allowed monitoring and quantification of probiotic and pathogenic bacteria during in vivo trials.The developed real-time PCR protocol allowed monitoring and quantification of probiotic and pathogenic bacteria during in vivo trials.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.The third step consisted in the application of biofilters with the probiotic strain Phaeobacter 27-4 against Vibrionaceae infections in the rearing of turbot larvae. The growth and the antagonism (in vivo and in vitro) of the probiotic strain Phaeobacter 27- 4 was tested with different supports used in aquaculture biofilters (plastic balls, sintered glass pellets and ceramic cylinders) and under different culture conditions (soaked stagnant and submerged with or without agitation). The attachment and growth of Phaeobacter, as well as the formation of rosette-shaped microcolonies and the subsequent development of a biofilm, were different depending on the support and on the culture conditions. A multilayer biofilm was only detected on ceramic cylinders cultured in submerged stagnant conditions. The in vitro antagonistic activity of Phaeobacter 27-4 biofilters against the pathogenic Vibrionaceae L. anguillarum 90-11-287 and V. splendidus DMC-1 was different when immobilized on biofilters made of different materials. The degree of antagonism was affected by the support and culture conditions used for the growth of the probiotic strain. In absence of nutrients, the inactivation was similar for both pathogens, but the inhibition of growth promoted by the probiotic, in presence of nutrients, was higher for L. anguillarum than for V. splendidus. In presence of Phaeobacter, V. splendidus grew more than L. anguillarum and induced a lower growth of the probiotic in the culture medium. The presence of Phaeobacter also diminished the attachment of both pathogens to ceramic and sintered glass but not to plastic supports. The permanence and detachment kinetic of Phaeobacter 27-4 was tested in tanks with green seawater maintained under the conditions used in larval rearing. Phaeobacter showed a better permanence in porous supports, compared with the plastic one, being the lowest slope of detachment registered in ceramic biofilters. In DGGE profiles, ceramic biofilters showed the lowest number of bands, indicating that the presence of Phaeobacter in those biofilters avoids the colonization by other bacteria. Ceramic cylinders were selected for preparation of biofilters to be used in challenge trials at pilot-scale due to the capability of Phaeobacter 27-4 in that support to: i) develop a biofilm formed by rosette-shaped microcolonies, ii) antagonize two pathogens (L. anguillarum and V. splendidus), and iii) remain longer in the biofilters when maintained under larval rearing conditions. Additionally, ceramic is a resistant material, which would be advantageous for the scale-up in aquaculture facilities.In small scale trials conducted under larval rearing conditions and different nutrients levels, matured Phaeobacter 27-4 ceramic biofilters reduced the concentration of L. anguillarum 90-11-287 and total Vibrionaceae in green seawater with different nutrients levels. Phaeobacter 27-4 biofilters significantly reduced a 45 % the accumulated mortality in turbot larvae infected with the pathogen L. anguillarum 90-11-287 by diminishing pathogen levels and the concentration of total Vibrionaceae, mainly in seawater. Additionally, the probiotic biofilter reduced turbidity in the rearing tanks. DGGE analysis revealed that the incorporation of L. anguillarum or Phaeobacter matured biofilters did not displace or modify significantly the bacterial microbiota present on larvae, which showed low bacterial diversity. However, the presence of Phaeobacter biofilters diminished the carrying capacity in seawater of rearing tanks. The shifts occurring in the bacterial community of turbot larvae reflected the influence of rotifers on larvae bacterial communities, whereas in the seawater shifts were marked by disappearance of bands when Phaeobacter biofilters were present. Biofilters acted as a probiotic reservoir in the rearing system, maintaining the level of the probiotic in the seawater of tanks for at least ten days, period in which larvae are fed on rotifers and which is considered critical for larval survival. An important advantage of this biofilters is that their use does not require repeated additions of the probiotic, avoiding long-term cultures and bioencapsulation in rotifers. This feature simplifies the potential transference of this methodology to industrial hatcheries.Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria – Ministerio de Ciencia e Innovación (Project: Modificación de la microbiota bacteriana asociada a la utilización de bacterias probióticas en el cultivo de larvas de rodaballo. Reference: ACU03-003) Consejo Superior de Investigaciones Científicas (Project: Modo de acción y optimización del uso de Roseobacter 27-4 como probiótico en el cultivo de larvas de rodaballo. Reference: 2006 7 01 067)Peer reviewe