Deliverables Definition of specific protocols for colonisation of live prey organisms by potential probionts and their delivery to fish larvae The cultivation conditions of live feed organisms as rotifers and Artemia favour growth of opportunistic microorganisms. Disinfection may reduce the bacterial load of the live feed, but the treatment is not necessarily successful as the level of organic nutrients for bacterial growth is not affected and opportunistic microorganisms proliferate within short time after treatment. A change, rather than a total decimation, of the bacterial flora of live feed should be aimed at. This should be done in a way that reduce loading of detrimental bacteria into the larval rearing tanks through the live feed and that introduce bacteria with probiotic properties. To reduce the transfer of opportunistic bacteria from live feed cultures to the larvae, probiotic bacteria can be bioencapsulated by short term treatments of the cultures in suspensions, to exchange the flora of the live feed with the probiotics. If probiotic bacteria are unable to establish themselves on the mucosal surfaces of the larvae or on the tank walls, they will have to be added regularly, either to the water or via the live feed. Addition to water is most effective at stagnant rearing conditions, as water exchange normally will have stronger impact on the number of the added bacteria, than the growth rate of the bacteria will. The flora of live feed can be exchanged by incubating the organisms in water added the probionts. Dense suspensions are necessary to induce effective exchange of the flora, and load the organisms with high numbers of the probionts within 30 minutes. The probiotic bacteria may, however, have a short stability in rotifers and Artemia. As much as 90% of the probionts can be lost from the animals within one hour after bioencapsulation. When feeding with rotifers the loss may correspond to a load of >105CFU/ml to the water at each feeding. To reduce the total CFU of the organisms before they are fed to the larvae a decontamination step therefore should be included, and the project suggest that one hour decontamination in clean water reduce the bacterial content to normal or even lower levels. The project has shown that there are differences between bacterial strains in how they are ingested by live prey and how stable they are. Administration of single or mixed strains have impact on the uptake by live feed, and on the colonisation of larvae. Establishment of a culture collection of bacteria with inhibitory activity against turbot larval pathogenic bacteria A collection of over 3000 bacterial isolates from aquatic environments, including adult and larval fish, live feed organisms and rearing systems was available for screening to detect potential probiotic bacteria. Subsequently, the project used the targeted replica plating procedure to isolate antagonistic bacteria directly from turbot larval rearing units. This resulted in approx. 200 antagonistic strains (from screening more than 20,000 colonies). Development of a chemostat system for larval turbot rearing The lack of control over the microbiota in the larval rearing of marine fish is one of the main factors affecting variability of growth and, particularly survival in cultures Unpredictability of changes in the microflora of larval rearing can be diminished or avoided in axenic systems. Rearings in axenic chemostat systems have the advantage of their flexibility from the point of view of manipulation of the microflora and therefore the study of interactions between specific bacteria. A procedure has been developed for the larval rearing of turbot larvae in axenic chemostats. The rearing of axenic turbot larvae larval rearings in chemostats required the use of axenic microalgae, rotifers and newly hatched turbot larvae. For that, two protocols were proposed for the production of axenic rotifers and larvae, respectively. Protocols were based on baths of the organisms in mixtures of antibiotics, from which, mixture IIM resulted more appropriate. One of the advantages of sterile chemostats is that both microalgae and rotifers grow faster than in non-axenic systems. By applying the protocols proposed, turbot larvae could be successfully reared under axenic conditions until day 10 using initial densities of 105 cells of Isochrysis ml{-1}, 1 rotifer ml{-1} and 25 larvae l{-1}. Performances achieved with the methodology proposed were adequate for experimental purposes. Survivals at day 10 were about 40%, with a maximum of 80%. Identification of virulence mechanisms of pathogens of larval turbot and use of this knowledge in selection of probiotic bacteria Vibrio splendidus was identified as the cause of major mortalities in larval turbot rearing and in studies to identify the mechanism by which this occurs we have identified the gene for a cytotoxin closely related to an established toxin involved in enteric infections in humans. For the toxin we identified, inactivation of this gene renders the organism harmless to turbot larvae yet the bacteria still colonise the larval intestinal tract in high numbers. Expression of the toxin gene is subject to control by a regulatory gene related to a well-established group of virulence gene regulators but of a structure previously unknown. This knowledge allows a more rational selection of probiotic organisms to prevent intestinal tract infections. Development of a replica plating assay for the isolation and identification of novel probiotic organisms Samples were plated on standard media (marine agar) and when colonies are outgrown plates were replica-plated on agar seeded with Vibrio anguillarum. Subsequently, colonies causing clearing zones in the V. anguillarum agar layer are isolated from master plates and pure cultured. Antagonism against the fish pathogen is tested in a well diffusion assay to confirm sustained activity. Use of immunohistochemistry to characterise bacterial infections of marine fish larvae Immunolabelling methods may provide insights into the whereabout of infectious organisms in their hosts. Knowledge of the sites of entry, penetration of the hosts epithelium or epidermis, and the sites of proliferation of the pathogen, provide insights in the action of the pathogen. In addition, knowledge of the damages caused by the infection to the various tissues of the host may provide additional understanding fo the modes of action of a pathogen, and its interactions with its host. Immunohistochemical protocols have been developed using turbot larvae challenged with two different Vibrio spp. as model systems. Vibrio anguillarum strain 90-11-287 was generally detected in the epidermis of the larvae, despite added via water or rotifers. Only in a few cases, the bacterium provided any damages to the gastrointestinal tract. In contrast, Vibrio splendidus strain DMC-1 was dependent on oral challenge in order to induce mortality. In the latter case, damages to the host was concentrated in the gastrointestinal tract. In both cases clearly moribund larvae was characterised by systemic infections, with labelled bacterial cells detected in most organs. The project has demonstrated that immunohistochemistry is a powerful diagnostic tool applied to fish larvae. Development of model challenge systems to assess the pathogenic potential of suspected larval pathogens, and to assess adverse effects of candidate probionts A general limitation for testing the effectiveness of probiotic bacteria in in vivo trials is to have a reliable and reproducible challenge model with a pathogenic strain. For Roseobacter 27-4 a challenge model using the fish pathogen Vibrio anguillarum 90-11-287 has been developed. The pathogenic strain was selected due to the antagonism showed in vitro by Roseobacter 27-4 against V. anguillarum 90-11-287 and V. splendidus. V. anguillarum showed different pathogenicity depending on the administration mode. When added to the larvae via rotifer, the mortality obtained in 10 d old larvae was 28-44% with respect to mortalities in unchallenged larvae, which was adequate for testing the probiotic strain. This challenge model could be used to test the in vivo probiotic activity of other strains. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
