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: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.

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)

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Gene expression profiles of spleen, liver and head kidney in turbot (Scophthalmus maximus) along the infection process with Philasterides dicentrarchi using an immune-enriched oligo-microarray

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