Kristof Dierckens

A Review of the Functionality of Probiotics in the Larviculture Food Chain

During the past two decades, the use of probiotics as an alternative to the use of antibiotics has shown to be promising in aquaculture, particularly in fish and shellfish larviculture. This article reviews the studies on probiotics in larviculture, focusing on the current knowledge of their in vivo mechanisms of action. The article highlights that the in vivo mechanisms of action largely remain to be unravelled. Several methodologies are suggested for further in vivo research, including studies on gut microbiota composition, the use of gnotobiotic animals as test models, and the application of molecular techniques to study host–microbe and microbe–microbe interactions.

Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae.

The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae (Dicentrarchus labrax) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila, Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.

Toxicological evaluation of a bioadhesive nasal powder containing a starch and Carbopol® 974 P on rabbit nasal mucosa and slug mucosa

The purpose of this study is the investigation of possible adverse effects of a powder formulation containing drum-dried waxy maize (DDWM) starch and Carbopol 974 P (90/10) on the nasal mucosa of rabbits and the foot mucosa of slugs after multiple administrations. In the rabbit, the effect of the formulation was measured by the release of proteins and lactate dehydrogenase (LDH) from the nasal mucosa with a new non-invasive in vivo method and also by histopathology. The mucosal toxicity of the formulation was evaluated using slugs by measuring the effect on the body weight and the amount of mucus produced during a repeated contact period. Additionally, the release of proteins, lactate dehydrogenase and alkaline phosphatase from the body wall of the slugs after a repeated treatment was measured. Twenty four hours after the powder administration to the rabbits the release of the marker molecules was comparable with the negative controls. The histopathological study showed only a slight increase of granulocytes in the epithelium. The formulation induced a higher mucus production in the slugs but no additional effects were detected on the body weight and on the release of proteins. No enzymes were released from the body wall. The results indicate that the effect of the bioadhesive powder consisting of DDWM/Carbopol 974 P (90:10, w/w) on the mucosa was negligible.

Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis

Aims:  To evaluate the effect of Vibrio harveyi strains on the growth rate of the gnotobiotically cultured rotifer Brachionus plicatilis, and to establish whether quorum sensing is involved in the observed phenomena.

The Mucosal Toxicity of Different Benzalkonium Chloride Analogues Evaluated with an Alternative Test Using Slugs

AbstractPurpose. The objective of this study was to evaluate the mucosal toxicity of different benzalkonium chloride (BAC) analogues using slugs as the alternative test organism. Methods. The effect of different BAC analogues on the mucosal tissue of slugs was determined from the protein, lactate dehydrogenase, and alkaline phosphatase released from the foot mucosa after treatment. Additionally, mucus production and reduction in body weight of the slugs were measured. The eye irritation potency of the molecules was evaluated with the Bovine Corneal Opacity and Permeability (BCOP) assay. The antimicrobial activity of the different BAC analogues was also assessed. Results. All BAC analogues induced severe damage to the mucosal epithelium of the slugs, and the irritation increased with decreasing alkyl chain length: BAC-C16 < BAC-C14 < BAC-C12 ∼ BAC-mix. A similar ranking was obtained with the BCOP assay for eye irritation. The relative order of activities among the three BAC analogues was the same, i.e., BAC-C14 ≥ BAC-C16 > BAC-C12. The BAC-C14 exhibited higher activity than the BAC-mix. Conclusions. The toxicity and activity of BAC analogues depend on the alkyl chain length. The use of BAC-C14 as a conservative agent in pharmaceutical preparations instead of the BAC-mix should be considered.

Does Virulence Assessment of Vibrio anguillarum Using Sea Bass (Dicentrarchus labrax) Larvae Correspond with Genotypic and Phenotypic Characterization

Background Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. Methodology/Principal Findings We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog’s Phenotype MicroArray™ technology and some virulence factor assays. Conclusions/Significance Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum.

Convergent dynamics of the juvenile European sea bass gut microbiota induced by poly‐β‐hydroxybutyrate

Poly-β-hydroxybutyrate (PHB) is a bacterial energy and carbon storage compound which exhibits a controlling effect on the gastrointestinal microbiota. Its beneficial activities for aquaculture have already been shown in terms of increased disease resistance and growth performance in a number of studies. However, the action of PHB on the intestinal microbial community in the treated animals has not yet been studied in depth. In this research, the effects of PHB on the microbiota composition in the intestinal tract of juvenile sea bass were examined. It was found that fish cohabiting in the same tank were on average 87% similar regarding the intestinal microbiota. When subjected to the same treatment and environmental conditions but reared in different tanks, the compositions of the enteric communities diverged. The provision of PHB overruled this tank effect by sustaining a microbial core community in the gut that represented 60% of the total bacterial diversity at the highest PHB level of 10%. The microbial community compositions converged between replicate tanks upon supplementation of PHB and were characterized by high dynamics and increased evenness. The results are discussed in the framework of hypotheses that try to relate the intestinal microbial community composition to the health status of the host organisms.

RpoS and Indole Signaling Control the Virulence of Vibrio anguillarum towards Gnotobiotic Sea Bass (Dicentrarchus labrax) Larvae

Quorum sensing, bacterial cell-to-cell communication with small signal molecules, controls the virulence of many pathogens. In contrast to other vibrios, neither the VanI/VanR acylhomoserine lactone quorum sensing system, nor the three-channel quorum sensing system affects virulence of the economically important aquatic pathogen Vibrio anguillarum. Indole is another molecule that recently gained attention as a putative signal molecule. The data presented in this study indicate that indole signaling and the alternative sigma factor RpoS have a significant impact on the virulence of V. anguillarum. Deletion of rpoS resulted in increased expression of the indole biosynthesis gene tnaA and in increased production of indole. Both rpoS deletion and the addition of exogenous indole (50–100 µM) resulted in decreased biofilm formation, exopolysaccharide production (a phenotype that is required for pathogenicity) and expression of the exopolysaccharide synthesis gene wbfD. Further, indole inhibitors increased the virulence of the rpoS deletion mutant, suggesting that indole acts downstream of RpoS. Finally, in addition to the phenotypes found to be affected by indole, the rpoS deletion mutant also showed increased motility and decreased sensitivity to oxidative stress.

Bacterial host interaction of GFP-labelled Vibrio anguillarum HI-610 with gnotobiotic sea bass, Dicentrarchus labrax (L.), larvae

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.

Development of a bacterial challenge test for gnotobiotic Nile tilapia Oreochromis niloticus larvae.

Gastrointestinal microbiota have an important impact on fish health and disease, stimulating interest in a better understanding of how these gastrointestinal microbial communities are composed and consequently affect host fitness. In this respect, probiotic microorganisms have been extensively used in recent aquaculture production. To study the use of probiotics in the treatment of infectious diseases, the establishment of a method of experimental infection to obtain consistent results for mortality and infection in challenge tests is important. In pathogen-screening tests, 4 candidate pathogenic bacteria strains (Edwardsiella ictaluri gly09, E. ictaluri gly10, E. tarda LMG2793 and Streptococcus agalactiae LMG15977) were individually tested on xenic Nile tilapia larvae. Only Edwardsiella strains delivered via Artemia nauplii, with or without additional pathogen delivery via the culture water, led to increased mortality in fish larvae. A gnotobiotic Nile tilapia larvae model system was developed to provide a research tool to investigate the effects and modes-of-action of probiotics under controlled conditions. A double disinfection procedure using hydrogen peroxide and sodium hypochlorite solution was applied to the fish eggs, which were subsequently incubated in a cocktail of antibiotic and antifungal agents. In the gnotobiotic challenge test, E. ictaluri gly09R was added to the model system via Artemia nauplii and culture water, resulting in a significant mortality of the gnotobiotic fish larvae. The developed gnotobiotic Nile tilapia model can be used as a tool to extend understanding of the mechanisms involved in host-microbe interactions and to evaluate new methods of disease control.