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type:RESEARCH ARTICLE, [Background] / The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. / [Results] / Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. / [Conclusions] / The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

Trypanosomes of the Trypanosoma genus are blood flagellates, and important causative agents of diseases of humans, livestock and cold-blooded species. Numerous in vitro studies and infection studies in mice contributed enormously to the insights into the biology of trypanosomes, their interaction with and evasion of the host immune system, as well as into various aspects related to vaccine failure and (uncontrolled) inflammation. A tight regulation of the early innate immune response to trypanosome infections was shown to be critical to obtain a balance between parasite control and inflammation-associated pathology. Trypanosome morphology was observed to be essential for their motility, the adaptation to their host’s environment and pathogenesis. One of the best-studied non-mammalian trypanosomes is Trypanosoma carassii, which presents many morphological similarities to mammalian trypanosomes. T. carassii is regularly observed co-infecting fish with Trypanoplasma spp such as T. borreli. Currently, few or no in vitro studies have been performed to unravel the swimming behaviour and host-pathogen interaction of Trypanoplasma species. For both trypanosomes and trypanoplasma, in vivo studies to visualise the parasite motility and host immune response have not been reported so far.    In this thesis we describe for the first time blood flagellate infections in vivo in the natural environment of a vertebrate host (zebrafish). We did this by studying the parasite motility in vitro and in vivo and the kinetics of innate immune responses in vivo. The T. carassii and T. borreli zebrafish infection models are promising complementary models to existing (mammalian) animal models, and can contribute to fundamental mechanistic insights into host-parasite interactions.

Rising demands for animal protein have caused an increase and further intensification of aquaculture over the last decades, which has subsequently led to increased disease pressure. Next to preventative vaccines as a solution, there is a drive to explore preventative approaches based on immunomodulation of innate immune responses. In either case, it isclear that a more detailed knowledge of innate immune responses is essential to help combat infectious diseases in aquaculture. Therefore, the overall aim of this thesis is to provide fundamental knowledge of the fish’ innate immune system and characterize polarized innate immune responses in carp with the emphasis on macrophages.In chapter 1 we touch upon the relevance of carp as an aquaculture species and explain why macrophages are considered essential players in innate immune responses, particularly in lower and cold-blooded vertebrates such as fish. We introduce macrophages as highly plastic cell types, introduce their activation signals and introduce the concept of macrophage polarization as it has been defined for mammalian macrophages. Then, following discussion of the framework that will help define macrophage polarization, we briefly summarize existing indications for the presence of polarized macrophages in carp. Finally, we shortly discuss how our findings can aid the development of immunomodulators that could help improve fish health in the context of aquaculture.We start by thoroughly reviewing the existing literature on macrophage polarization in fish in chapter 2. We review the stimuli frequently used to polarize macrophages in mammals, and the conservation of cytokines often associated with T helper 1 and T helper 2 subsets. We discuss approaching macrophage polarization in fish from a ‘macrophages first’ point of view and consider the plausibility that polarization in fish macrophages could rely primarily on sensing microbial infection or other innate danger signals. Furthermore, we discuss preliminar

In this thesis we report the development and initial testing of vaccines for common carp against Spring Vireamia of Carp Virus (SVCV) and Koi Herpes Virus (KHV), two important diseases of carp. We developed a DNA vaccine against SVCV, encoding the viral glycoprotein (G), and showed that intra-muscular injection of a very low dose (0.1 mg/g of fish) of the vaccine leads to full protection. Using multiple techniques we show that this vaccine induced a strong local (anti-viral) response as well as the induction of a B- and T- cell memory response. After this success we used the same DNA vaccine and in parallel developed recombinant baculoviruses expressing the G protein for oral vaccine delivery, allowing for stress-free vaccination without any local side-effects. The vaccine was encapsulated in alginate microspheres in order to protect it from intestinal degradation. Despite testing various vaccine doses, regimes and a mucosal adjuvant, no protection was obtained. Next to SVCV, we tested a DNA vaccine against KHV, encoding the ORF25 protein, but under the tested conditions the vaccine was not able to confer protection after intra-muscular injection or oral delivery. Given that understanding the adaptive immune response triggered by vaccination is of utmost importance to understand the underlining protective mechanisms, we developed multiple tools to characterize B cell and T cell responses in common carp. Using these new tools, we significantly contributed to the understanding of adaptive immune responses of common carp and especially, their role during infection and after vaccination.

Maybe most characteristic of the TargetFish1 project, which kicked off some five years ago with 30 partners from 10 EU member states, two associated countries (Norway, Israel) and one international cooperation partner country (Chile), has been the close cooperation between research groups and enterprises; more or less equally represented in this large consortium. In this respect, TargetFish has been revolutionary validating by this close cooperation fundamental knowledge for the development of next generation vaccines and different routes of vaccine administration. TargetFish had the ambition to demonstrate market applicability of improved vaccines or new prototype vaccines that would come forward from the project. Via frequent joint meetings of its partners, be it research group or enterprise, TargetFish aimed to drive vaccine development in an industrial applicable way. This could facilitate adoption of new intellectual property and stimulate the presentation of new fish vaccines on the market. The industry forum has been a platform for a continuing validation of the applied potential of the research outcomes. Workshops were organised at the different EAFP meetings to communicate the validation process to those not directly involved with the project but interested in the fish vaccine market. After a kick-off meeting during the EAFP in Tampere, Finland fours years ago and a second meeting at the EAFP in Las Palmas, Spain, two years ago, at the present EAFP in Belfast, Northern Ireland a final meeting was organised. This report is a summary of the 'Industrial Forum workshop' held at the EAFP in Belfast 2017 and provides a short overview of the highlights presented to, and discussed with, those present and interested in DNA vaccine development, policies and laws, production and delivery routes.

Background: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. Results: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. Conclusions: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

Coronaviruses from both the Alpha and Betacoronavirus genera, interfere with the type I interferon (IFN) response in various ways, ensuring limited activation of the IFN response in most cell types. Of Gammacoronaviruses that mainly infect birds, little is known about activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells and in a chicken cell line. Ifnß expression in fact, is delayed with respect to the peak of viral replication and accompanying accumulation of dsRNA. In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the IFN response at the transcriptional and translational level. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, signalling of non-self dsRNA through both MDA5 and TLR3 remains intact in IBV-infected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses.

In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8+ memory T cells while downregulating CD4+ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM+ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.

Larval zebrafish was subjected to a methodological exploration of the gastrointestinal microbiota and transcriptome. Assessed was the impact of two dietary inclusion levels of a novel protein meal (NPM) of animal origin (ragworm Nereis virens) on the gastrointestinal tract (GIT). Microbial development was assessed over the first 21 days post egg fertilisation (dpf) through 16S rRNA gene-based microbial composition profiling by pyrosequencing. Differentially expressed genes in the GIT were demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq). Larval zebrafish showed rapid temporal changes in microbial colonization but domination occurred by one to three bacterial species generally belonging to Proteobacteria and Firmicutes. The high iron content of NPM may have led to an increased relative abundance of bacteria that were related to potential pathogens and bacteria with an increased iron metabolism. Functional classification of the 328 differentially expressed genes indicated that the GIT of larvae fed at higher NPM level was more active in transmembrane ion transport and protein synthesis. mRNAseq analysis did not reveal a major activation of genes involved in the immune response or indicating differences in iron uptake and homeostasis in zebrafish fed at the high inclusion level of NPM

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

We have previously observed that in common carp (Cyprinus carpio), administration of β-glucan (MacroGard™) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through β-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by β-glucans in fish, MacroGardTM was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly I:C), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response and a set of immune related genes, including Mx, were analysed by real-time PCR in liver, spleen, head-kidney and mid-gut. Results obtained confirmed that treatment with β-glucan alone generally down-regulated the mRNA expression of pro-inflammatory cytokines when compared to untreated fish, while Mx gene expression remained stable. A similar expression pattern was observed for cytokines in samples obtained from β-glucan fed fish 24 h after injection with poly I:C. However, poly I:C injection markedly increased Mx gene expression but mainly in the group fed with β-glucan. Toll-like receptor 3 (TLR3) is the candidate pattern recognition receptor possibly responsible also in fish for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through a carp genome data mining, two sequences for carp TLR3 were retrieved (ccTLR3.1 and ccTLR3.2) and characterized. Constitutive gene expression of both genes was detected by rea

Sequencing of the common carp (Cyprinus carpio carpio Linnaeus, 1758) genome, with the objective of establishing carp as a model organism to supplement the closely related zebrafish (Danio rerio). The sequenced individual is a homozygous female (by gynogenesis) of R3 x R8 carp, the heterozygous offspring of a cross between fish of Hungarian origin (R8 strain) and of Polish origin (R3 strain).

Zebrafish has been explored as nutritional fish model with the purpose to assess the impact of two dietary inclusion levels of a novel iron-containing protein meal (IPM) of animal origin on the gastrointestinal tract (GIT) of the developing zebrafish. The development of the microbial community has been assessed over the first 21 days post egg fertilisation (dpf) through 16S rRNA gene-based microbial composition profiling by quantitative PCR (qPCR) and pyrosequencing using a Genome Sequencer FLX. The molecular regulation of physiological processes by differentially expressed genes in the GIT is demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq) using an Illumina HiSeq2000 focusing on genes that are functionally involved in iron uptake and homeostasis. Differential diet dependent phylogenetic diversity occurred. Larvae fed at high inclusion level of IPM differed from those fed at low level in early presence of Bacteroidetes, then an increase of Firmicutes and other phyla at the expense of the Actinobacteria. Finally with Firmicutes and Actinobacteria still present, Proteobacteria dominated. The abundance of Firmicutes in the larvae fed at high inclusion level of IPM at 14 and 21 dpf was much higher than those fed at low inclusion level which probably relates to their iron oxidizing capacity that may coincide with higher pH in the GIT. mRNAseq revealed that 328 genes were differentially expressed: expression of 214 genes was up-regulated and 114 genes down-regulated in larvae fed at high vs. low inclusion levels of IPM. Dominant gene groups representing ribosome components and activity and transport were up-regulated in the GIT of these larvae. 27 genes were identified as involved in iron homeostasis but were non-differentially expressed at a fold change 0.27 – 1.54. Functional classification of genes revealed that the GIT of larvae fed at higher IPM level are more active in transmembrane ion transport and protein synthesis. The marked differences in mi, Zebrafish has been explored as nutritional fish model with the purpose to assess the impact of two dietary inclusion levels of a novel iron-containing protein meal (IPM) of animal origin on the gastrointestinal tract (GIT) of the developing zebrafish. The development of the microbial community has been assessed over the first 21 days post egg fertilisation (dpf) through 16S rRNA gene-based microbial composition profiling by quantitative PCR (qPCR) and pyrosequencing using a Genome Sequencer FLX. The molecular regulation of physiological processes by differentially expressed genes in the GIT is demonstrated at 21 dpf by whole transcriptome sequencing (mRNAseq) using an Illumina HiSeq2000 focusing on genes that are functionally involved in iron uptake and homeostasis. Differential diet dependent phylogenetic diversity occurred. Larvae fed at high inclusion level of IPM differed from those fed at low level in early presence of Bacteroidetes, then an increase of Firmicutes and other phyla at the expense of the Actinobacteria. Finally with Firmicutes and Actinobacteria still present, Proteobacteria dominated. The abundance of Firmicutes in the larvae fed at high inclusion level of IPM at 14 and 21 dpf was much higher than those fed at low inclusion level which probably relates to their iron oxidizing capacity that may coincide with higher pH in the GIT. mRNAseq revealed that 328 genes were differentially expressed: expression of 214 genes was up-regulated and 114 genes down-regulated in larvae fed at high vs. low inclusion levels of IPM. Dominant gene groups representing ribosome components and activity and transport were up-regulated in the GIT of these larvae. 27 genes were identified as involved in iron homeostasis but were non-differentially expressed at a fold change 0.27 – 1.54. Functional classification of genes revealed that the GIT of larvae fed at higher IPM level are more active in transmembrane ion transport and protein synthesis. The marked differences in mi

cum laude graduation (with distinction)

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Item does not contain fulltext, We studied a predictive model of gene expression induced by mechanical injury of fish skin, to resolve the confounding effects on the immune system induced by injury and skin parasite-specific molecules. We applied real time quantitative PCR (RQ-PCR) to measure the expression of the pro-inflammatory cytokines CXCa, CXCb, interleukin (IL)1-beta, tumor necrosis factor alpha (TNFalpha), and the receptors IL1R1, CXCR1 and CXCR2 in skin of Cyprinus carpio after mechanical injury. We also studied the expression of the anti-inflammatory cytokine IL-10. Most obvious, specific up-regulation of the chemokine CXCa, the chemokine receptor CXCR1 and the pro-inflammatory cytokine IL-beta was detected at 2-3h after injury. In order to correlate gene expression patterns after injury with cell migration, we studied chemotaxis of head kidney leukocytes towards lysates of epithelioma papulosum cyprini (EPC) cells. Neutrophilic granulocytes were shown to migrate towards epithelial lysates. Using immunohistochemistry we observed that the early inflammatory response after injury involved an influx of cells, most probably neutrophilic granulocytes, into the injured area. This suggests that the increased expression of pro-inflammatory genes is related to a rapid influx of neutrophilic granulocytes.