Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Modulation of the immune response of turbot (Scophthalmus maximus) after infection with viral haemorragic septicaemia virus

ABSTRACT: Turbot (Scophthalmus maximus) is a valuable resource for aquaculture in Galicia (NW Spain). Since it has been observed that viral hemorraghic septicaemia can affect turbot, among other finfish, increase of knowledge in molecular factors affected by the exposure to pathogen could help to develop strategies of VHSV prevention and treatment. In this study, it has been used a custom oligo-microarray by Agilent to identify genes differentially expressed in several turbot families showing different susceptibility to VHSV. Fishes from each family (n=30) were injected with either VHSV (Resistant) or control medium (Naive) and monitored for 30 days, when each group was splitted in two new groups and rechallenged with VHSV (Infected) or control medium (Control). Gene expression at the head kidney was evaluated, showing than an important proportion of the variation of the gene expression profiles is explained by the genetic background (family). After infection, fish showed an up-regulation of the interferon-induced Mx2 gene, the IL-8 gene and the VHSV-induced protein 5 gene compared with control groups. Familes with high mortality after VHSV infection showed lower levels of expression of molecules secreted in the mucus and, by contrast, higher expression of genes involved in viral entrance into target cells. 4 different families of turbot were subjected to challenged with VHSV and splitted after 30 days in 2

ORGANISM(S): Scophthalmus maximus

SUBMITTER: Pablo Balseiro

PROVIDER: E-GEOD-35804 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. A total number of 204 juvenile turbot were divided into 3 groups, two of them containing 72 fish and the last one 60 fish. Turbot were anaesthetized by immersion in 50 mg/ml buffered tricaine methanesulfonate (MS-222; Sigma) and then, fish from the first two groups were intramuscularly (i.m.) injected with 50 µl of PBS containing 2 µg of pMCV1.4 or pMCV1.4-G860. Turbot from the last batch were i.m. inoculated with 50 µl of PBS. At 8, 24 and 72 h after injection, 12 fish were removed from the first two tanks and, at 8 h after PBS inoculation, other 12 fish were taken from the last tank. These turbot were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for each treatment and time point (3 turbot/replicate). The remaining fish (36 in the plasmid-injected groups and 48 in the PBS-inoculated tank) were maintained during one month and then, 12 fish from the PBS injected group were separated to another tank. This new group of fish was intraperitoneally (i.p.) injected with 50 µl of MEM + penicillin and streptomycin + 2% FBS (PBS - MEM group), whereas the other turbot were i.p. infected with a dose of VHSV860 of 5 x 105 TCID50/fish (pMCV1.4 - VHSV and pMCV1.4-G860 - VHSV groups). At 8, 24 and 72 hours after infection, 12 fish were removed from the VHSV-infected tanks, and at 8 h after MEM injection the 12 fish were taken from the non-infected tank. The fish were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for treatment and time point (3 turbot/replicate)

Project description:We evaluated the expression profiles of turbot in spleen, liver and head kidney across five temporal points of the Philasterides dicentrarchi infection process using an 8x15K Agilent oligo-microarray. The microarray included 2,176 different 5-fold replicated gene probes designed from a turbot 3’ sequenced EST database. We were able to identify 221 differentially expressed (DE) genes (8.1% of the whole microarray), 113 in spleen, 83 in liver and 90 in head kidney, in at least one of the 5 temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using GO terms (69.1%) after the additional sequencing of DE genes from the 5’ end. Many DE genes were related to innate and acquired immune functions. A high proportion of DE genes were organ-specific (70.6%), although their associated GO functions showed notable similarities in the three organs. The most striking difference in functional distribution was observed between the up- and down-regulated gene groups. Up-regulated genes were mostly associated to immune functions, while down-regulated ones mainly involved metabolism-related genes. Genetic response appeared clustered in a few groups of genes with similar expression profiles along the temporal series. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to P. dicentrarchi to achieve more resistant broodstocks for turbot industry. We were interested in analyzing the response of turbot as species to P. dicentrarchi in three of the main immune organs (spleen, liver and head kidney) along a temporal series representing the main episodes of infection (1d, 3d, 7d, 15d, 25d). Accordingly, individual samples were pooled at each sampling point to average interindividual variation. A single control point (time 0; non-injected) was used since a very slight effect of injection on gene expression was previously reported in these three organs in turbot. Five fish were sacrificed on day 0 to be used as the unique control in the experiment and one hundred fifty were challenged with a highly virulent strain of P. dicentrarchi as previously described (Paramá et al., 2003). Groups of five fish were sacrificed at 1d, 3d, 7d, 15d and 25d post-challenging. Equal amounts of spleen, liver and head kidney were sampled from each fish, pooled per organ at each sampling point and immediately stored in liquid nitrogen.

Project description:Background: Enteromyxosis caused by the intestinal myxozoan parasite Enteromyxum scophthalmi is a serious threat for turbot (Scophthalmus maximus, L.) aquaculture, causing severe catarrhal enteritis leading to a cachectic syndrome, with no therapeutic options available. There are still many aspects of host-parasite interaction and disease pathogenesis that are yet to be elucidated, and to date, no analysis of the transcriptomic changes induced by E. scophthalmi in turbot organs has been conducted. In this study, RNA-seq technology was applied to head kidney, spleen and pyloric caeca of severely infected turbot with the aim of furthering our understanding of the pathogenetic mechanisms and turbot immune response against enteromyxosis. Results: A huge amount of information was generated with more than 23,000 identified genes in the three organs, amongst which 4,762 were differently expressed (DE) between infected and control fish. Associate gene functions were studied based on gene ontology terms and available literature, and the most interesting DE genes were classified into five categories: 1) immune and defence response; 2) apoptosis and cell proliferation; 3) iron metabolism and erythropoiesis; 4) cytoskeleton and extracellular matrix and 5) metabolism and digestive function. The analysis of down-regulated genes of the first category revealed evidences of a connexion failure between innate and adaptive immune response, especially represented by a high number of DE interferon-related genes in the three organs. Furthermore, we found an intense activation of local immune response at intestinal level that appeared exacerbated, whereas in kidney and spleen genes involved in adaptive immune response were mainly down-regulated. The apoptotic machinery was only clearly activated in pyloric caeca, while kidney and spleen showed a marked depression of genes related to erythropoiesis, probably related to disorders in iron homeostasis. The genetic signature of the causes and consequences of cachexia was also demonstrated by the down-regulation of the genes encoding structural proteins and those involved in the digestive metabolism. Conclusions: This transcriptomic study has enabled us to gain a better understanding of the pathogenesis of enteromyxosis and identify a large number of DE target genes that bring us closer to the development of strategies designed to effectively combat this pathogen. Four samples per organ (kidney, spleen and pyloric caeca) were sequenced by Illumina HiSeq 2000 as 100bp paired-end reads. For each organ, three samples were taken from Enteromyxum severely infected turbot and the remaining one was a pool of three control turbot.

Project description:An EST database from immune tissues was used to design the first high density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60mers) were successfully designed for 2716 out of 3482 unique sequences of the database. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida challenged fish at 3 days post-infection. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide and dye bias) factors using one-colour (1C) and two-colour (2C) -labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random effects variance revealed that technical variation was mostly negligible and biological variation represented the main factor, even using pooled samples. One-colour approach performed at least as well as 2C. A relevant proportion of genes turn out to be differentially labelled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defence response were detected at three days post-challenging. An unbalanced hierarchical design was planned to evaluate the sources of variation in microarray analysis. This design enabled us to consider most sources of noise in microarray analysis and evaluate their weight in the variability of the signal using an equilibrate number of replicates at each level and a not too large number of microarrays. According to this hierarchy, the following within slide sources of variation were assessed from top to bottom: biological (B), RNA extraction (E), labeling (L) and hybridization (H). Additionally, we studied variation between slides (S), the bias produced by labeling with different dyes (Cy3 and Cy5) and compared the performances of one-colour (1C) and two-colour (2C) -labeling approaches. A total of 24 microarrays grouped in three 8x15k slides were used for this design. One pool of five individuals was used as control and other two other pools of five individuals each (B1 and B2) were used as biological replicates. Three replicates were used for evaluating the variation associated with RNA extraction (E1, E2, E3), labeling (L1, L2, L3) and hybridization (H1, H2, H3) within slide. Some of these replicates were in turn hybridized in different slides to assess interslide variation. Three 2C hybridizations with dye swapping (6 microarrays) were performed in the same slide for comparison of 1C and 2C approaches and to analyze dye bias. Normalization within each microarray was carried out using the loess method. Normalization using all microarray data was done by performing the Aquantile method implemented in the limma package. To estimate the variation associated with replicates at the different steps of microarray analysis or to different slides we applied the Pearson correlation coefficient using log2 absolute treatment and treatment/control ratio values. Also an ANOVA of random effects was used to split the total variance into its components. For Dye bias evaluation and 1C vs. 2C comparisons we f estimated the proportion of common differentially expressed (DE) genes after Bonferroni correction at P <0.05 and P<0.01 at fold changes (FC) >1.5, 2 and 3. The degree of concordance between the different dyes and between 1C and 2C approaches was estimated as the proportion of common DE genes at each P and FC.

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Modulation of the immune response of turbot (Scophthalmus maximus) after infection with viral haemorragic septicaemia virus

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