Gene expression profiles after infection of rainbow trout with viral hemorrhagic septicemia virus (VHSV)
ABSTRACT: We describe here transcripts induced after intraperitoneal injection of rainbow trout with 2 different viruses, both belonging to strain 23.75 of viral hemorrhagic septicemia virus (VHSV): a deleted Nv gene (dNV) virus and a wild type (wt) virus. Two days after infection, differentially expressed transcript levels from selected immune-related trout genes were studied in internal organs (spleen and head kidney). Fishes were divided in two groups (3 fishes per group). The first group was intraperitoneally injected with 100000 pfu per trout of dNV VHSV, while the second group was injected with 100000 pfu/trout of wt VHSV. All fishes were sacrificed two days post infection.
Project description:The function of the non-virion protein (NV) of the Viral hemorrhagic septicemia virus (VHSV) has been long questioned but it still remains unknown. We report here the differences in gene expression profiles of trouts infected with deleted NV VHSV or wild type VHSV so that it could shed some light on the issue. Eight fingerlings of rainbow trout were used for the experiment. Four of them were injected with deleted NV VHSV and the rest were injected with wild type VHSV as a control. Head kidney and spleen of each trout were collected 2 days post-injection and total RNA was extracted.
Project description:Due to the common presence of a non-virion NV gene absent from other fish rhabdoviruses, NV-coding rhabdoviruses, such as the viral haemorrhagic septicemia (VHSV), were placed into a new Novirhabdovirus genus. Because conflicting results do exist on the importance of NV for VHSV trout pathogenicity and the NV function is still unclear, the effects of intraperitoneal injection of recombinant NV protein were studied by using home-designed immune-related microarrays from rainbow trout Oncorhynchus mykiss. Trouts were separated in two groups. One group was injected with PBS and it was used as control group. Second group was injected with soluble VHSV non-virion (NV) recombinant protein
Project description:Due to the common presence of a non-virion NV gene absent from other fish rhabdoviruses, NV-coding rhabdoviruses, such as the viral haemorrhagic septicemia (VHSV), were placed into a new Novirhabdovirus genus. Because conflicting results do exist on the importance of NV for VHSV trout pathogenicity and the NV function is still unclear, the effects of intraperitoneal injection of recombinant NV protein were studied by using home-designed immune-related microarrays from rainbow trout Oncorhynchus mykiss. The results were compared with parallel experiments using bacterial flagellins, a well know mammalian agonist of toll receptors (tlr5) with adjuvant activity recently described also in fish Four experimental groups were formed with four biological replicates each, 16 samples in total. First one was injected with NV recombinant protein in PBS, second one was a control group injected with PBS, the third group was injected with bacterial flagellins in PBS and the fourth group was injected with polihistidine peptide as a reference group because the recombinant proteins have a polihistidine tail. There is a technical replicate within each array.
Project description:Oral immunization of trout against infectious pancreatic necrosis virus (IPNV) have been recently reported by using a DNA vector coding the viral capsid VP2 gene encapsulated in alginate microspheres. We report here an study of the transcripts in head kidney and pyloric ceca 7 days after oral vaccination of rainbow trout by using a newly designed rainbow trout 60-mer oligo microarray focused in their immune-related genes. Trout were obtained from 4 different farms, one group of trout was defined as belonging to one of the farms. Each of the trout 4 groups were divided into two subgroups of 6 trout for each subgroup. Subgroup I was orally vaccinated with 10 µl of suspension of the vaccine microspheres each containing 10 µg of pcDNA-VP2 diluted in 10µl of PBS, while subgroup II received similar microspheres suspension but pcDNA. Head kidney and pyloric ceca were collected from each subgroup and RNA was extracted.
Project description:In order to explore the relative importance of adult zebrafish innate and adaptive immune responses to different kinds of infections, we compared the gene expression profile of VHSV-challenge-survivors, bacterial infection survivors and control non infected zebrafish. VHSV -infection-survivors zebrafish were divided in two groups of 12 individuals, first group was infected with VHSV (samples named A) and second group was mock-infected (samples named B). Two days after challenge, zebrafish of each group were divided in four subgroups (3 fishes per group) and head kidney and spleen of each individual were sampled. For each group, internal organs of three fishes were pooled, so four biological replicates were generated . Zebrafish surviving a natural infection caused by Aeromonas hydrophila and Vibrio fluvialis (samples named C) were divided in four groups (3 fishes per group) and head kidney and spleen were extracted. Finally, 12 healthy zebrafish were used as control group (samples named D) and processed in the same way as group C.
Project description:We describe here transcripts induced after infection of zebrafish with Spring Viremia Carp Virus (SVCV). Two days after infection, differentially expressed transcript levels from selected immune-related zebrafish genes were studied in internal organs (pooled spleen, head kidney). Also, transcripts from resistant fishes to viral infection one month after inoculation were studied. Three different experiments were performed to get three biological replicates. Fishes were divided into two groups in each experiment. First group was infected by immersion with SVCV 10^7 pfu/ml, second group was used as a control of non-infected fishes. 6 fishes per group were sacrificed two days post infection, whereas the rest of the infected fishes from the three experiments were maintained for 30 days in the aquariums and then survivors (six for experiment) were sacrificed. This submission includes three biological replicate groups for the non-infected fish and the two days post-infected fish, and two biological replicate groups for the 30 days post-infected fish.
Project description:In mammals, increasing data suggests that there exists a complex and bi-directional relationship between thyroid and immune systems. However the existence of such interactions in fish is unknown. Here, we administered the biologically active hormone 3.3′.5-triiodo-L-thyronine (T3) and the anti-thyroid drug, propilthiouracil (PTU) to juvenile rainbow trout and examined the head kidney expression profile with a custom-made microarray enriched in immune-related genes. A seven day-experiment was performed. Fish were divided in 3 groups. First group received 20 µg/g fish feed of T3; second group received 5000 µg/g fish feed of PTU; third group served as control. After 7 days orally administering T3 or PTU, differentially expressed transcript levels from selected immune-related rainbow trout genes were studied in head kidney. Three groups were studied (T3-treated, PTU treated and control), each group having 4 biological replicates (each replicate consisting of 2 pooled fish).
Project description:Because fin base is supposed to be the entry zone of some fish virus, we wanted to know which transcripts are induced after infection of zebrafish with Spring Viremia Carp Virus (SVCV). Two days after infection, differentially expressed transcript levels from selected immune-related zebrafish genes were studied in zebrafish fins. Also transcripts from resistant fishes to viral infection one month after inoculation were studied. Overall design: Three different experiments were performed to get three biological replicates. Fishes were divided in two groups in each experiment. First group was infected by immersion with SVCV 107 pfu/ml, second group was used as a control of non-infected fishes. 6 fishes per group were sacrified two days post infection, whereas the rest of infected fishes from the three experiments were maintained for 30 days in the aquariums and then survivors (six for experiment) were sacrified.
Project description:The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor α (TNFα) and other immune factors, are produced and act on the reproductive system. However, TNFα is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNFα in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). In control and recombinant trout TNFα (rtTNFα)-treated granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNFα on follicle contraction and testosterone production in preovulatory trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNFα-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.Treatment with rtTNFα induces ovarian cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNFα causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNFα induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. In view of these results, we propose that TNFα could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction. Reproductively mature female brown trout (Salmo trutta) from a cultured stock at the Piscifactoria de Bagà (Generalitat de Catalunya, Bagà, Spain) were kept under natural conditions of temperature and photoperiod. Fish at the preovulatory stage (according to the position of the germinal vesicle (GV) in oocytes that were cleared using a solution previously described), were anesthesized in 3-aminobenzoic acid ethyl ester (0.1 g/l; Sigma, Alcobendas, Spain) dissolved in fresh water, and the fish were sacrificed by concussion prior to the collection of the ovaries. The dissected ovaries were immediately used for the various in vitro assays. After dissection, brown trout preovulatory ovaries were placed in Hank´s balanced salt solution (HBSS) and individual ovarian follicles were manually separated with forceps from each ovary on ice, as previously described. To collect ovarian tissue for RNA extraction, preovulatory follicles from each of a total of three females were incubated (400 follicles/50 ml) in HBSS-BSA in the absence or presence of rtTNFα (100 ng/ ml, dissolved directly in HBSS-BSA), at 15ºC for 24 h with gentle shaking (100 rpm). At the end of the incubation follicles (previously de-yolked by gentle pressure) were removed, flash frozen in liquid nitrogen and stored at -80ºC until assayed.
Project description:Immune responses in higher vertebrates are classically separated into innate and adaptive (or specific) immunity. However, important gaps of knowledge about how adaptive responses are generated in lower vertebrates still remain unsolved. In order to explore the relative importance of adaptive and innate immune responses, we have studied zebrafish transcriptional responses to an infection with the Spring Viraemia of Carp virus (SVCV) in rag1-/- zebrafish mutants compared to wild type zebrafish by using both genome wide and immunological-targeted gene expression microarrays. Both wild type (wt) and mutant (rag1) zebrafish were divided in two groups with 3 individuals per group. First group was infected with 10^5 pfu per ml of SVCV, second group was mock-infected. Two days after challenge zebrafish were sampled, head kidney and spleen of each fish were extracted and pooled between each group. The experiment was repeated once to obtain two biological replicates.