Bacterial host interaction of GFP-labelled Vibrio anguillarum HI-610 with gnotobiotic sea bass, Dicentrarchus labrax (L.), larvae (original) (raw)
Related papers
2011
27 The location and cell damage caused by Vibrio anguillarum, the causative agent of classical 28 vibriosis, within the developing gut of the newly hatched sea bass (Dicentrarchus labrax L.) 29 is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. 30 anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally 31 exposed to a V. anguillarum HI-610 pathogen labelled with the Green Fluorescent Protein 32 (GFP-HI-610) and sampled at regular intervals. Pathogenic colonisation of gut enterocytes 33 was observed 2 hrs post exposure (p.e.) and onwards, whereas bacteria within the swim 34 bladder were visualized 48 hrs p.e and onwards. Ultrastructural findings demonstrated direct 35 bacterial contact with the host cell in the oesophageal mucosa and putative attachment to 36 microvilli of midand hindgut enterocytes. The present findings form a starting point for 37 studies assessing the impact of potential candidates (p...
Diseases of Aquatic Organisms, 2014
Vibrio anguillarum is the major cause of haemorrhagic septicaemia, vibriosis, which is a severe disease affecting marine fish. In this work, it was found that the mortality of gnotobiotic sea bass larvae challenged with V. anguillarum was dependent on the number of dead fish in the vials at the moment of challenge. Based on this finding, the effect of dead hosts (homogenised sea bass larvae or brine shrimp) on the virulence of V. anguillarum towards sea bass larvae was further investigated. Addition of homogenised hosts led to significantly increased larval mortality of challenged larvae, and this was observed for 3 different V. anguillarum strains, i.e. 43, NB 10 and HI 610. In contrast, the addition of similar levels of tryptone had no effect on mortality. In line with this, the motility of all 3 V. anguillarum strains was significantly increased by the addition of homogenised hosts but not by tryptone. These results suggest that dead hosts increase infectivity of V. anguillarum, not merely by offering nutrients to the bacteria, but also by increasing virulence-associated activities such as motility.
Journal of Fish Diseases, 2013
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 pseudopode-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 witnessed 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.
Veterinary microbiology, 2015
In this study, we investigated the impact of the host factors mucin, bile salts and cholesterol on the virulence of the economically important aquatic pathogen Vibrio anguillarum towards sea bass larvae. Pretreatment of V. anguillarum with either one of the host factors (at 10 mg l(-1)) prior to inoculation into the sea bass rearing water increased virulence of the bacterium, although the effect of cholesterol was not significant. Each of the three host factors significantly increased several virulence-related phenotypes in V. anguillarum, i.e. protease activity, flagellar motility, biofilm formation and exopolysaccharide production, whereas there was no effect on growth of the bacterium under these conditions. Furthermore, the host factors increased the expression of genes involved in these phenotypes, i.e. the metalloprotease empA, the flagellar transcriptional regulator fleQ, the flagellin gene flaA, the chemotaxis methyltransferase gene cheR, the exopolysaccharide biosynthesis g...
Diseases of Aquatic Organisms
Induction of mild enterocolitis in zebrafish Danio rerio via ingestion of Vibrio anguillarum serovar O1 B. Randazzo, F. Abbate, F. Marino*, M. Mancuso, M. C. Guerrera, U. Muglia, M. Navarra, A. Germanà ABSTRACT: Vibrio anguillarum is the aetiological agent of a fatal haemorrhagic disease known as vibriosis that affects a wide range of fish species causing severe economic losses. Several investigations have been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. The aim of this paper was to evaluate the most effective way to induce a mild enteritis in a fish model, in order to allow further applications. The experiments were carried out using two methods of administration of V. anguillarum serotype O1 to adult zebrafish: via intra-peritoneal injection and via Artemia nauplii. The results showed that the intra-peritoneal administration often caused a massive fish death due to severe ...
World Journal of Microbiology and Biotechnology, 2012
Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.
Fish & Shellfish Immunology, 2017
The aim of this study was to assess the effects of low levels of dietary fish meal (FM) and fish oil (FO) on disease resistance and gut associated lymphoid tissue (GALT) response after an experimental intestinal infection with V. anguillarum in European sea bass (Dicentrarchus labrax) For that purpose, sea bass juveniles were fed one of four diets containing combined levels of FO and FM as follows: 20%FM/6%FO, 20%FM/3%FO, 5%FM/6%FO and 5%FM/3%FO during 153 days. At the end of the feeding trial, fish were subjected to either an in vivo exposure to a sub-lethal dose of V. anguillarum via anal inoculation or to an ex vivo exposure to V. anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) gut patterns of immunopositivity were studied. Growth performance was affected by dietary FM level, however ex vivo gut bacterial translocation rates and survival after the in vivo challenge test were affected by dietary FO level. After 5 months of feeding, low dietary FM levels led to a posterior gut up-regulation of interleukin-1 (IL-1) and TNF, major histocompatibility complex-II (MHCII) and cyclooxygenase-2 (COX2), which in turn reduced the gut associated lymphoid tissue (GALT) capacity of response after 24h post infection and conditioned European sea bass capacity to recover gut homeostasis 7 days post infection. Immunoreactivity to anti-iNOS and anti-TNF presented a gradient of increased immunopositivity towards the anus, regardless of the dietary FM/FO fed. Strong positive anti-TNFα isolated enterocytes were observed in the anterior gut in relation to low levels of dietary FM/FO. Submucosa and lamina propria immunoreactivity grade was related to the amount of leucocyte populations infiltrated and goblet cells presented immunopositivity to anti-iNOS but not to anti-TNFα. Thus, reducing FO content from 6% to a 3% by VO in European sea bass diets increases ex vivo and in vivo gut bacterial translocation rates, whereas reducing FM content from 20% down to 5% up-regulates the expression of several posterior gut inflammationrelated genes conditioning fish growth and GALT capacity of response after bacterial infection.
Journal of Aquaculture Research & Development, 2011
The aim of the present study was to observe the antagonistic relationship of the probiotic Pediococcus acidilactici and the pathogen Vibrio (Listonella) anguillarum in the intestine of rainbow trout (Oncorhynchus mykiss) by using the ex vivo intestinal sac method. Rainbow trout (240-250g) were fed either a control diet (commercial diet: 43% protein, 20% lipid) or a probiotic diet (control diet supplemented with P. acidilactici [Bactocell] at log 7 CFU g-1) for two weeks. The anterior intestine was then isolated for an ex vivo challenge study and intestinal sacs formed by shutting one end of the intestinal portion. The sacs were filled with PBS solutions containing either no bacteria (as a control), the probiotic (P. acidilactici), the pathogen Vibrio anguillarum, or probiotic+pathogen and incubated for one hour. At the end of the exposure bacterial levels in the lumen were determined by culture based approaches and colonisation of the mucosa assessed with PCR-DGGE and electron microscopy (EM). Intestinal morphology (observation of gross morphological damage, ultrstructural differences and the quantification of goblet cells and intra epithelial lecuocyte numbers) and bacterial infection/translocation was assessed using light microscopy (LM) and EM. Results revealed that V. anguillarum caused extensive histological damage to the gut but P. acidilactici did not. No signs of translocation of either the probiotic or pathogen were observed. Microbiological analyses indicated that P. acidilactici was able to outcompete V. anguillarum in the rainbow trout intestine and also to populate or colonise the mucosa. Additionally, elevated leucocyte levels and goblet cells in the epithelium of P. acidilactici fed fish, and intestines exposed to P. acidilactici, suggests that P. acidilactici might have potential use in controlling vibriosis. In vivo disease-challenge studies are warranted to ascertain if V. anguillarum infections can be controlled in rainbow trout using dietary applications of P. acidilactici.
FEMS Microbiology Letters, 2013
Vibrios, distributed in marine and brackish environments, can cause vibriosis in fish and shellfish under appropriate conditions. Previously, we clarified by thin-layer chromatography (TLC) overlay assay that 35 S-labeled Vibrio trachuri adhered to GM4 isolated from red sea bream intestine. However, whether GM4 actually functions on epithelial cells as an attachment site for vibrios still remains to be uncovered. We found that six isolates, classified as V. harveyi, V. campbellii, and V. splendidus, from intestinal microflora of red sea bream adhered to GM4 but not galactosylceramide (GalCer) by TLC-overlay assay. Tissue-overlay assays revealed that V. harveyi labeled with green fluorescent protein (GFP) adhered to epithelial cells of red sea bream intestine where GM4 and GalCer were found to be distributed on the top layer of actin filaments by immunohistochemical analysis using corresponding antibodies. The number of adhering vibrios was diminished by pretreatment with anti-GM4 antibody, but not anti-GalCer antibody. These results clearly indicate that vibrios adhere to epithelial cells of red sea bream intestine utilizing GM4 as an attachment site.