Identification of N-acyl homoserine lactone-degrading bacteria isolated from rainbow trout (Oncorhynchus mykiss).

AIMS: A variety of pathogens use quorum sensing (QS) to control the expression of their virulence factors. QS interference has hence been proposed as a promising antivirulence strategy. The specific aim of this study was to isolate bacteria from trout tissue able to degrade N-acyl homoserine lactones (AHL), a QS molecule family. METHODS AND RESULTS: In total 132 isolates were screened for AHL degradation using Chromobacterium violaceum CV026 as a biosensor. Twenty-four quorum-quenching (QQ) isolates were identified biochemically and characterized using 16S rDNA sequencing. They belong to Bacillus, Enterobacter, Citrobacter, Acinetobacter, Agrobacterium, Pseudomonas and Stentrophomonas genera. Four Bacillus spp. showed the highest and fastest QQ activity. AHL degradation proved to be enzymatic in most isolates (except for Stentrophomonas spp. and Pseudomonas sp.) as QQ activity could be destroyed by heat and/or proteinase K treatments. All QQ activity proved to be cell-bound except for Pseudomonas sp., where it could be detected in the supernatant. The results of aiiA gene homology analysis revealed the presence of aiiA gene encoding AHL lactonase in all examined isolates except Pseudomonas syringae and Enterobacter cloacae. The HXHXDH motif conserved in all AHL lactonases and considered to be essential for AHL degradation was detected in all AiiAs after sequence alignment. CONCLUSIONS: Some known and novel QQ bacteria were isolated from trouts and characterized in terms of enzymatic or nonenzymatic AHL degradation activity and their extracellular or intracellular location. In addition, an aiiA gene and its HXHXDH motif were detected in most isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: We could isolate and identify some novel QQ bacteria including Enterobacter hormaechei, Acinetobacter radioresistens and Citrobacter gillenii. The aiiA gene was detected for the first time in these strains as well as in Stenotrophomonas maltophilia. Our QQ isolates could be used for biocontrol of bacterial infections in aquaculture.

Characterization of phenotype variations of luminescent and non-luminescent variants of Vibrio harveyi wild type and quorum sensing mutants.

Vibrio harveyi, a luminescent Gram-negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non-luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence-related phenotypic traits of the wild-type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non-luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non-luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei (Boone), and the outcome for virus and cell.

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)-killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV-killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV-killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV-killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post-inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post-inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV-killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular-sensing molecules.

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge.

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35 g L-1 were subjected to salinities of 50 g L-1 , 35 g L-1 , 20 g L-1 , 10 g L-1 and 7 g L-1 or 5 g L-1 and simultaneously exposed to 105.5  SID50 mL-1 of WSSV for 5 h, after which the salinity was brought back to 35 g L-1 . Shrimp that were transferred from 35 g L-1 to 50 g L-1 , 35 g L-1 and 20 g L-1 did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35 g L-1 to 10 g L-1 and lower. The mortality in shrimp, subjected to a salinity change to 10 g L-1 , 7 g L-1 and 5 g L-1 , was 6.7%, 46.7% and 53.3%, respectively (P < 0.05). For testing the effect of moulting, shrimp in early premoult, moulting and post-moult were immersed in sea water containing 105.5  SID50 mL-1 of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post-moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P < 0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10 g L-1 and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures.

Differences in uptake and killing of pathogenic and non-pathogenic bacteria by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone).

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non-pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi-granulocytes) have the main function in phagocytosis of both pathogenic and non-pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non-virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post-inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non-virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.

Priming the immune system of Penaeid shrimp by bacterial HSP70 (DnaK).

This study was conducted to test the effect of DnaK on priming immune responses in Penaeid shrimp. Juvenile-specific pathogen-free (SPF) P. vannamei shrimp were injected with 0.05 µg recombinant DnaK. One hour post-DnaK priming, a non-lethal dose of Vibrio campbellii (10(5) CFU shrimp(-1)) was injected. Other treatments include only DnaK or V. campbellii injection or control with blank inocula. The haemolymph of three shrimp from each treatment was collected at 1.5, 6, 9 and 12 h post-DnaK priming (hpp). It was verified that injection with DnaK and V. campbellii challenge affected the transcription of 3 immune genes, transglutaminase-1 (TGase-1), prophenoloxidase-2 (proPO-2) and endogenous HSP70 (lvHSP70). In P. monodon, shrimp were first injected with DnaK at a dose of 10 µg shrimp(-1) and one hour later with 10(6) CFU of V. harveyi (BB120) shrimp(-1). Shrimp injected with DnaK showed a significant increase in proPO expression compared to the control (P < 0.05). Yet a double injection (DnaK and Vibrio) seemed to cause an antagonistic response at the level of expression, which was not equalled at the level of PO activity. Those results suggest that DnaK is able to modulate immune responses in P. vannamei and P. monodon.

Ingestion of food pellets containing Escherichia coli overexpressing the heat-shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection.

Feeding aquatic animals with bacterial encapsulated heat-shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK-DnaJ-GrpE, the prokaryotic equivalents of Hsp70-Hsp40-Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g(-1) pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.

Relation between virulence of Vibrio anguillarum strains and response to the host factors mucin, bile salts and cholesterol.

AIMS: In this study, we investigated the responsiveness of 15 Vibrio anguillarum strains to three host factors (mucin, bile salts and cholesterol). METHODS AND RESULTS: Three virulence-related phenotypes were investigated in this respect, i.e. motility, biofilm formation and exopolysaccharide production. Almost all V. anguillarum strains showed a significantly increased motility in the presence of either of the three host factors. Only five of the strains showed increased biofilm formation in the presence of host factors and only three strains showed increased exopolysaccharide production in the presence of the host factors. CONCLUSIONS: There were no significant correlations between the three putatively virulence-linked phenotypes (in the absence of host factors) and virulence to sea bass larvae. There was no correlation between responsiveness to the host factors (percentage increase in motility, biofilm formation or exopolysaccharide production in the presence of the three host factors) and virulence to sea bass larvae. However, the responses of these virulence-related phenotypes upon the addition of either of the three host factors were significantly correlated with each other. This result suggests that the mechanisms by which V. anguillarum responds to these three host factors is linked. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the mechanism by which V. anguillarum responds to the host factors mucin, bile salts and cholesterol seems to be linked, there is no correlation between host factor responsiveness and virulence towards sea bass larvae. This emphasizes that one should be careful when extrapolating results obtained for one particular strain to reach general conclusions on a species of pathogenic bacteria.

Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates.

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non-host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non-ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.

Protocol for quantitative shape analysis of deformities in early larval European seabass Dicentrarchus labrax.

This study established an optimized protocol for quantifying the shape variation of newly hatched larvae of European seabass Dicentrarchus labrax, focusing on the effect of fixatives and mounting on body shape from hatching until 14 days post hatching, while also minimizing the error introduced by handling. This assessment was performed based on both biometric and geometric shape data, with the latter relying on outline based elliptic Fourier analysis. The fixation and mounting effect on the total length and shape of newly hatched larvae of D. labrax was tested for four fixation treatments: (1) 8% formalin, (2) 70% ethanol, (3) 8% formalin for 48 h and then to 70% ethanol and (4) 3% phosphate-buffered glutaraldehyde. The analyses showed that no significant size and shape effect was observed on anaesthetized specimens 5 months post-fixation in glutaraldehyde, and that the glycerol mounting process of specimens fixed in glutaraldehyde provided the best results for further ontogenetic qualitative and quantitative analysis. The protocol proved to be sufficiently sensitive to even quantify and visualize subtle pre-fixation shape differences originating from a different egg batch.

Purification of white spot syndrome virus by iodixanol density gradient centrifugation.

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60,000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80,000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1,000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.

Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species.

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards - amongst others - ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes--a novel early defence strategy in larval fish.

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

Enhancement of Hsp70 synthesis protects common carp, Cyprinus carpio L., against lethal ammonia toxicity.

Exposure to TEX-OE®, a patented extract of the prickly pear cactus (Opuntia ficus indica) containing chaperone-stimulating factor, was shown to protect common carp, Cyprinus carpio L., fingerlings against acute ammonia stress. Survival was enhanced twofold from 50% to 95% after exposure to 5.92 mg L(-1) NH(3) , a level determined in the ammonia challenge bioassay as the 1-h LD50 concentration for this species. Survival of TEX-OE®-pre-exposed fish was enhanced by 20% over non-exposed controls during lethal ammonia challenge (14.21 mg L(-1) NH(3) ). Increase in the levels of gill and muscle Hsp70 was evident in TEX-OE®-pre-exposed fish but not in the unexposed controls, indicating that application of TEX-OE® accelerated carp endogenous Hsp70 synthesis during ammonia perturbation. Protection against ammonia was correlated with Hsp70 accretion.

Effect of poly-ß-hydroxybutyrate on Chinese mitten crab, Eriocheir sinensis, larvae challenged with pathogenic Vibrio anguillarum.

This study investigated the protective effect of poly-ß-hydroxybutyrate (PHB) on Chinese mitten crab, Eriocheir sinensis, zoea larvae challenged with pathogenic Vibrio anguillarum. PHB was delivered to the crab larvae through rotifer and Artemia bioencapsulation. Zoea 3 larvae were challenged with V. anguillarum at a final concentration of 10(5) CFU mL(-1). PHB-enriched rotifers and Artemia nauplii were added to the culture water 24 h prior to, upon and 24 h after challenge. The results confirmed that PHB could enhance the survival and growth of unexposed E. sinensis larvae. Moreover, PHB protected larvae from the pathogen as the larvae fed PHB-enriched live food showed the highest survival and development rate in all challenged groups (P < 0.05). Furthermore, larval performance was the best when PHB was delivered to the larvae 24 h before challenge (P < 0.05). In conclusion, our results indicate that PHB can be used as part of an effective strategy to protect E. sinensis larvae from V. anguillarum resulting in higher survival and better growth, especially when applied before the challenge.

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.

Expression of virulence genes in luminescent and nonluminescent isogenic vibrios and virulence towards gnotobiotic brine shrimp (Artemia franciscana).

AIMS: This study aimed to evaluate the expression levels of virulence gene regulators (luxR and toxR) and virulence factors (serine protease, metalloprotease and haemolysin) in luminescent and nonluminescent isogenic Vibrio harveyi and Vibrio campbellii. METHOD AND RESULTS: Nonluminescent variants have been reported before to become dominant in cultures of luminescent vibrios when grown under static conditions in the dark. Wild-type V. harveyi BB120, V. campbellii LMG 21363, quorum sensing mutants of V. harveyi BB120 and their previously reported nonluminescent isogenic counterparts were used in this study. The expression level of the virulence genes srp serine protease, vhp metalloprotease and vhh haemolysin, the quorum sensing master regulator gene luxR and the virulence regulator gene toxR in isogenic luminescent and nonluminescent strains were quantified using reverse transcriptase real-time PCR. These experiments revealed that the nonluminescent strains produced lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene (which is known to be regulated by quorum sensing). Finally, challenge tests with gnotobiotic brine shrimp (Artemia franciscana) larvae revealed that the nonluminescent strains are less virulent than their luminescent isogenic counterparts. CONCLUSION: Nonluminescent variants of V. harveyi and V. campbellii strains produce lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene and are less virulent to brine shrimp than their isogenic luminescent counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that adaptation of luminescent vibrios to specific growth conditions that result in a dominant nonluminescent phenotype is accompanied by a decreased adaptation to a host environment because of altered virulence gene regulation.

Stereology and computer assisted three-dimensional reconstruction as tools to study probiotic effects of Aeromonas hydrophila on the digestive tract of germ-free Artemia franciscana nauplii.

AIMS: Validation of stereology and three-dimensional reconstruction for monitoring the probiotic effect of Aeromonas hydrophila on the gut development of germ-free Artemia franciscana nauplii. METHODS AND RESULTS: Germ-free Artemia nauplii were cultured using Baker's yeast and dead Aer. hydrophila. Live Aer. hydrophila were added on the first day to the treatment group. The gut length and volume were monitored on days two and four using stereology and three-dimensional reconstruction. Both methods showed comparable results. Stereology was least labour intensive to estimate volumes, while three-dimensional reconstructions rendered architectural and topographical data of the gut. Moreover, a positive effect of probiotic bacterium, Aer. hydrophila is likely. CONCLUSION: Slight increment in the growth of the digestive tract of A. franciscana nauplii exerted by probiotic bacteria could be detected using stereology and three-dimensional reconstruction. SIGNIFICANCE AND IMPACT OF THE STUDY: The gnotobiotic Artemia rearing system is unique to investigate the effects of micro-organisms on the development of nauplii. However, in the base of this model system, only survival counts and length measurements exist as monitoring tools. Therefore, additional tools such as stereology and three-dimensional reconstruction are prerequisite to obtain more powerful analysis.

Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention.

Vibrio anguillarum, also known as Listonella anguillarum, is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. In both aquaculture and larviculture, this disease is responsible for severe economic losses worldwide. Because of its high morbidity and mortality rates, substantial research has been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. This review summarizes the current state of knowledge pertaining to V. anguillarum, focusing on pathogenesis, known virulence factors, diagnosis, prevention and treatment.

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi.

AIMS: To study the potential biocontrol activity of bioflocs technology. METHODS AND RESULTS: Glycerol-grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth-inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing-regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. CONCLUSIONS: Bioflocs grown on glycerol as carbon source inhibit quorum sensing-regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.