Project description:Here, we define the VanT-QS regulon and explore the diversity and trajectory of traits under QS regulation in the fish pathogen Vibrio anguillarum through comparative transcriptomics of two wildtype strains and their corresponding mutants artificially locked in QS-on (ΔvanO) or QS-off (ΔvanT) states. We reveal that populations of one strain primarily employ a QS-off response, while the other strain mainly maneuvers within the QS-on spectrum. Further examination of our V. anguillarum collection revealed that ~15 % of the strains were QS-negative and measurements of a total of 9 QS-positive strains suggested diverse QS responses, which underlines an extensive diversity of QS delegations. We show that QS control a plethora of genes involved in processes such as central metabolism, biofilm, competence, T6SS and virulence in V. anguillarum. Interestingly, the low QS response strain had an enlarged QS regulon compared to the high QS response strain, suggesting a potential trade-off between engagement in QS and output of the commitment. Moreover, the combination of present and previous virulence trials coupled with QS response measurements indicate that the QS state is an important driver of virulence toward fish larvae in V. anguillarum. We propose that infections by mixed-strain communities spanning diverse QS strategies might utilize the animal host more efficiently. Furthermore, we emphasize the importance of applying whole-cell spike-in normalization to study gene expression of cell populations with differential total RNA content per cell.
Project description:Total RNA was extracted from V. angularium susceptible and resistant rainbow trout, tissues (liver, spleen, gill), in different time points using GenEluteTM mammalian RNA kit (RTN350, Sigma-Aldrich, Denmark). After measuring quantity (NanoDrop 2000 spectrophotometer (Saveen & Werner, Denmark)) and quality (gel electrophoresis) of RNA, cDNA was synthetised in T100 thermocycler, Biorad, Denmark, using Oligo d(T)16 primer and TaqMan® Reverse Transcription Reagents (cat.no. N8080234, Thermo Fischer Scientific, Denmark). Primers and probes for total of 28 genes including three housekeeping genes were synthesized at TAG Copenhagen AS, Denmark. qPCR reactions were run by Brilliant III Ultra-Fast QPCR Master Mix (600881, AH Diagnostics AS, Denmark) for all samples. The fold changes analysed by the simplified 2-ΔΔCq method. Fingerlings of rainbow trout (mean body weight of 12 g) were exposed (2 h bathing, 18°C) to the pathogen V. anguillarum serotype O1 in a solution of 1.5x107 cfu/ml and observed for 14 d. Disease signs appeared three days post exposure (dpe) whereafter morbidity progressed exponentially until 6 dpe reaching a total morbidity/mortality of 55% within 11 days. we sampled fish for immune gene expression analysis when they first showed clinical signs, fish without clinical signs at the same time point and finally fish surviving the exposure to the pathogen. The different immune gene expression profiles in the different groups were addressed when discussing possible resistance mechanisms in rainbow trout.
Project description:Japanese flounder (Paralichthys olivaceus) is an economic important aquaculture fish that was susceptible to Vibrio anguillarum. To fully deciphered the molecular mechanisms underlying flounder host defense against V. anguillarum infection, we perform the micro-transcriptome analysis of founder spleen with and without V. anguillarum infection at 3 time points.
Project description:This data describes the clinical picture of natural Vibrio anguillarum infection in rainbow trout during an outbreak on a fish farm. Molecular mechanisms associated with the host immune response have been investigated using mass spectrometric analysis of trout plasma proteins. Three fish populations were identified among infected trout according to the severity of infection: fish with severe lesions (SL), with moderate infectious process (IP) and asymptomatic fish (AS). As expected, pro-inflammatory interleukins, complement components, acute phase proteins and antimicrobial peptides were implicated in the acute pathogenesis. Systemic coagulopathy was accompanied by increased antithrombotic reactions. Reconstruction of metabolic pathways also revealed a high energy requirement for the immune response in severely affected fish. An unexpected result was a small difference between fish with moderate symptoms and fish with no or minor external signs of pathology, proposed as resistant to infection. Increased production of antiproteases and enhanced blood coagulation cascade were observed in healthier fish, which may underlie the mechanisms of a controlled, non-self-damaging immune response to infection.
Project description:MVAV6203, an live attenuated vaccine showed a strong protection against V. anguillarum for Paralichthys olivaceus, Epinephelus coioides and zebrafish. However the mechanism of its protection, especially the mucosal-related response induced by immersion vaccination, is not fully understood. This study was conducted to reveal the changes of genes involved in both innative and adaptive immunity. For gene expression analysis, spleen of zebrafish from 3 vaccinated groups and 3 control groups were hybridized on a 4×44K aglient whole zebrafsih genome oligo microarray. Functional analysis of the microarray data was performed using KEGG and Gene Ontology (GO) analysis. Microarray gene expression analysis showed that the Th17-like immune response may play vital role in orchestrating the mucosal barrier against pathogen.
Project description:UnlabelledThe genetic heterogeneity of the close relatives Vibrio anguillarum and Vibrio ordalii, both serious pathogens of fish causing extensive losses in aquaculture, was studied. Eight housekeeping genes, i.e., atpA, ftsZ, gapA, gyrB, mreB, rpoA, topA, and pyrH, were partially sequenced in 116 isolates from diverse fish species and geographical areas. The eight genes appear to be under purifying selection, and the genetic diversity in the total data set was estimated to be 0.767 ± 0.026. Our multilocus sequence analysis (MLSA) scheme identified several widespread clonal complexes and resolved the isolates, for the most part, according to serotype. Serotype O2b isolates from diseased cod in Norway, Ireland, and Scotland were found to be extremely homogeneous. Horizontal gene transfer appears to be fairly common within and between clonal complexes. Taken together, MLSA and in silico DNA-DNA hybridization (DDH) calculations suggest that some isolates previously characterized as V ordalii, i.e., 12B09, FF93, FS144, and FS238, are in fact V. anguillarum isolates. The precise taxonomic situation for two isolates from Atlantic cod that display several traits consistent with V. ordalii, i.e., NVI 5286 and NVI 5918, and a single environmental strain that was previously considered to represent V. ordalii, i.e., FF167, is less clear.ImportanceIt is still being debated whether V. anguillarum and V ordalii represent separate bacterial species. Our study addresses this issue and elucidates the degree of genetic variability within this group of closely related bacteria, based on a substantial number of isolates. Our results clearly illustrate the existence of different populations among putative V ordalii isolates. On the basis of additional full-length genomic analysis, we conclude that most environmental isolates previously identified as V ordalii lie firmly within the species V. anguillarum While bona fide fish-pathogenic V ordalii isolates display a very close genetic relationship with V. anguillarum, they combine a clearly divergent evolutionary pattern with clear phenotypic differences. The study also highlights the need for further characterization of fish-pathogenic isolates from the northern Atlantic region that share phenotypic characteristics with V. ordalii but are genetically closer to V. anguillarum The retention of taxonomic distinctions between the phenotypically different groups of bacteria is of practical advantage to microbial ecologists and veterinarians.
Project description:The hemorrhagic septicemic disease vibriosis caused by Vibrio anguillarum shows noticeable similarities to invasive septicemia in humans, and in this case, the V. anguillarum-host system has the potential to serve as a model for understanding native eukaryotic host-pathogen interactions. Iron acquisition, as a fierce battle occurring between pathogenic V. anguillarum and the fish host, is a pivotal step for virulence. In this article, advances in defining the roles of iron uptake pathways in growth and virulence of V. anguillarum have been summarized, divided into five aspects, including siderophore biosynthesis and secretion, iron uptake, iron release, and regulation of iron uptake. Understanding the molecular mechanisms of iron acquisition will have important implications for the pathogenicity of this organism.
Project description:MVAV6203, an live attenuated vaccine showed a strong protection against V. anguillarum for Paralichthys olivaceus, Epinephelus coioides and zebrafish. However the mechanism of its protection, especially the mucosal-related response induced by immersion vaccination, is not fully understood. This study was conducted to reveal the changes of genes involved in both innative and adaptive immunity. For gene expression analysis, spleen of zebrafish from 3 vaccinated groups and 3 control groups were hybridized on a 4M-CM-^W44K aglient whole zebrafsih genome oligo microarray. Functional analysis of the microarray data was performed using KEGG and Gene Ontology (GO) analysis. Microarray gene expression analysis showed that the Th17-like immune response may play vital role in orchestrating the mucosal barrier against pathogen. Zebrafish were randomly divided into three vaccinated groups and three control groups. V.anguillarum MVAV6203 was cultured in high-salt Luria (LB) medium at 30 for 16h. The cells were harvested by centrifugation and rinsed twice in 2% saline. The desired number of cells was adjusted to 1.0E+08 CFU/mL with 2% saline. Six groups of 70 zebrafish were immersed in the aerated cell-resuspended saline or 2% saline for 10min at 24M-bM-^DM-^C. At 28 days post-vaccination pool of spleen tissue of 10 zebrafish from each group was harvested for microarray hybridization.