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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Innate immunity of carp : fishing for receptors
    Fink, Inge - \ 2017
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789463430753 - 240
    carp - cyprinus - immunity - platelets - macrophage activation - receptors - polarization - immunostimulation - immunology - karper - cyprinus - immuniteit - bloedplaatjes - macrofaag activering - receptoren - polarisatie - immunostimulatie - immunologie

    Recent decades have seen a significant intensification of aquaculture leading to increased risk of infections with several pathogenic organisms. On economical and ethical grounds it is more appropriate to improve general welfare conditions and prevent infections rather than treating disease outbreaks once they have occurred. Immunostimulation through feed can provide more efficient and sustainable control of diseases in aquaculture through enhancing the immunocompetence of fish; however, the underlying mechanisms are poorly characterized. The overall aim of this thesis was to perform a molecular and functional characterization of how pathogen-associated molecular patterns (PAMPs), such as β-glucans, affect the innate immune response of carp and which receptors on carp leukocytes are likely candidates to play a role in sensing such PAMPs.

    In chapter 1 we provide a framework for this thesis by introducing different classes of PAMPs, including β-glucans. These molecules were the centrepiece of an intra-European training network called NEMO (Protective immune modulation in warm water fish by feeding glucans), which this PhD project was part of. The scientific aim of the NEMO network was to develop a sustainable and cost-effective use of β-glucans as immunostimulants for aquaculture, using common carp as the model fish species, since on a global scale common carp is the most cultured fish species for food consumption. Our aims within the NEMO project entailed both the characterization of carp leukocytes and the characterization of candidate pattern recognition receptors (PRRs) that could play a role in sensing PAMPs and initiating immune responses. Chapter 1 therefore introduces the thrombocytes and macrophages pertinent to this thesis, as well as important classes of PRRs.

    In our first experimental study, described in chapter 2, we investigated the relevance of thrombocytes for the immune system of carp. We found that thrombocytes from healthy carp express a large number of immune-relevant genes, among which several cytokines and Toll-like receptors (Tlrs). Furthermore, we dissected the role of thrombocytes during infections with two different, albeit related, protozoan parasites, Trypanoplasma borreli and Trypanosoma carassii, and found thrombocytes were massively depleted from blood and spleen of fish infected with T. borreli. The pathology of this infection is associated with elevated levels of tissue nitration, prompting us to investigate, ex vivo, the effect of nitric oxide on thrombocytes. Our studies revealed that nitric oxide can induce a clear and rapid apoptosis of thrombocytes from healthy carp, supporting a role for nitric oxide-mediated control of immune-relevant thrombocytes during infection with T. borreli. Thereby, this particular study provided an excellent example of interplay between pathogen and the innate immune system of carp.

    We reviewed in chapter 3 another cell type central to innate immunity: the macrophage. We focused on the heterogeneity of macrophage activation states as these cells, at least in humans and mice, have the ability to polarize in several directions during an immune response. Based on the signals that lead to activation and the effector functions and cytokine profile as a result thereof, macrophages can be broadly divided into two types: classically activated macrophages induced in a T helper 1 (TH1) cytokine environment, and alternatively activated macrophages, induced in a TH2 cytokine environment. Mirroring the TH1–TH2 dichotomy, classically activated macrophages have also been termed M1, whereas alternatively activated macrophages have been termed M2. Classically activated macrophages are typically induced by stimulation with microbial ligands such as LPS in combination with pro-inflammatory cytokines such as IFNγ, and can be viewed as an extension of innate activated macrophages which are induced by microbial ligands only, thus are independent of cytokines. Alternatively activated macrophages are generated in the presence of IL4 and/or IL13. In addition to M1 and M2, one can distinguish regulatory macrophages, which are associated with the presence of the cytokine IL10. In this chapter, we reviewed the evidence of existence of polarized macrophages in teleost fish, among other things based on observations of the fundamentally different immune responses elicited by the parasites T. borreli and T. carassii.

    We further investigated the polarization of carp macrophages in chapter 4, where we obtained gene signature profiles of carp macrophages via a transcriptome approach. Independently of cytokines, carp macrophages showed the ability to differentiate into cells with functional characteristics highly comparable to those of mammalian M1 and M2, consistent with a conserved ability of macrophages to polarize into distinct subsets. In addition to obtaining a global view of gene expression, our transcriptome approach identified gene signatures for M1 and M2 macrophages which appear conserved from fish to mammals. We selected a number of these interesting genes that were differentially regulated between M1 and M2 macrophages and discussed in detail five potential M1 markers; il1β, ptx3a, saa, nos2b, and il12a – as well as five potential M2 markers; cyr61, inhba, timp2, tgm2, and arg2. These transcriptome studies may pave the way for future studies of polarized macrophages during immune responses in fish. Furthermore, additional analyses of the datasets described in this chapter will undoubtedly lead to the characterization of more genes relevant to macrophage polarization and recognition of immunostimulants.

    As part of the characterization of candidate PRRs that could play a role in sensing PAMPs and initiating immune responses, we studied the scavenger receptor Cd36 (chapter 5), which in mammals is expressed by many different (immune) cell types and plays a role in highly diverse processes, both homeostatic and pathologic. Among other things, it is often found associated with sensing of β-glucans and also with M2 macrophage activation, sparking our interest in this molecule in fish. We studied Cd36 in common carp as well as in zebrafish, a closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp was shown to have two paralogs of cd36. Although all genes show conserved synteny compared to mammalian CD36, unexpectedly we could not detect gene expression of cyprinid cd36 in macrophages or any other immune cell type or immune organ. Yet, because gene expression of cd36 was down-regulated during Mycobacterium marinum infection of zebrafish, and knockdown of cd36 in zebrafish embryos led to higher bacterial burden upon such infection, our data imply a role for Cd36 in immune responses of fish. Future studies are needed to clarify the exact mechanisms involved.

    As characterization of candidate PRRs we also examined the Toll-like receptors Tlr1 and Tlr2 (chapter 6). We identified a full-length, expressed tlr1 gene, a tlr1 pseudogene, and a second tlr2 gene next to the tlr2 which had been described previously. Sequence, phylogenetic and synteny analyses supported the conserved nature of these genes, and three-dimensional modelling showed a good fit with the mammalian TLR1/TLR2 heterodimer including the potential to bind to the prototypical ligand Pam3CSK4. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NFκB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. This prompted a discussion of methods available for studying ligand-binding properties of fish Tlrs.

    Finally, we discuss in chapter 7 the findings of this thesis in the context of the NEMO project. We present the concept of trained immunity, which could provide the conceptual framework within which the immune-stimulating ability of compounds such as β-glucans could be explained. We discuss recent advances in the field of TLR research as well as that of macrophage polarization, and highlight immunometabolism as a new area of interest which may help to illuminate the molecular events occurring in immune cells during health and disease. In conclusion, we found that carp leukocytes, along with their pattern recognition receptors, are central players of the innate immune system of carp. Our findings contribute to the understanding of mechanisms of immunostimulation, and expect this will enable the valorisation and use of immunostimulants for sustainable aquaculture and improvement of fish health.

    Herpesvirus kaapt afweersysteem karpers
    Sikkema, A. ; Forlenza, M. - \ 2015
    Resource: weekblad voor Wageningen UR 10 (2015)5. - ISSN 1874-3625 - p. 8 - 8.
    herpes - karper - dierenvirussen - immuunsysteem - diergezondheid - cyprinidae - herpes - carp - animal viruses - immune system - animal health - cyprinidae
    Een dodelijk virus bouwde meer dan 400 miljoen jaar geleden een molecuul van het afweersysteem van vissen in zijn genoom, tonen Wageningse celbiologen voor het eerst aan. Daardoor kan dit koiherpesvirus de afweer van karpers en sierkarpers (koi) omzeilen.
    Evaluatie rapportage over karperuitzet in Nederland
    Couperus, A.S. ; Keeken, O.A. van - \ 2015
    IJmuiden : IMARES (Rapport / IMARES Wageningen UR C020/15) - 39
    visstand - sportvissen - karper - visserijbeheer - waterkwaliteit - onderzoek - vergelijkingen - fish stocks - game fishes - carp - fishery management - water quality - research - comparisons
    De afgelopen jaren zijn vier rapporten verschenen over de ecologische impact van karper (Cyprinus carpio) in relatie tot de uitzet van deze soort in Nederlandse wateren. De vier rapporten geven verschillende weergaven van het effect van karper op de waterkwaliteit en ook hoeveel karper per hectare in Nederlandse wateren kan worden uitgezet. Het doel van deze desk-studie is om te bepalen waar verschillen tussen conclusies over het uitzetten van karper in de vier rapporten door kunnen worden verklaard. De vastgestelde verschillen in dit rapport zullen als uitgangspunt dienen voor een bijeenkomst met de betrokken partijen om tot een consensus te komen.
    Karperziekten in Nederland: toen en nu
    Haenen, O.L.M. - \ 2014
    Aquacultuur 29 (2014)5. - ISSN 1382-2764 - p. 36 - 41.
    visziekten - karper - cyprinidae - parasieten - bacterieziekten - virusziekten - fish diseases - carp - cyprinidae - parasites - bacterial diseases - viral diseases
    Ingegaan wordt op de belangrijkste ziekten van wilde en gehouden karper in Nederland. Daarbij gaat het zowel om al heel lang bekende als om één nieuwe ziekte, optredend in de diverse jaargetijden.
    "Koi Sleepy Disease" voor het eerst in Nederland aangetoond in koikarpers
    Haenen, O.L.M. ; Way, K. ; Stone, D. ; Engelsma, M.Y. - \ 2014
    Tijdschrift voor Diergeneeskunde 139 (2014)4. - ISSN 0040-7453 - p. 26 - 29.
    koi - cyprinidae - karper - cyprinus - virusziekten - diagnostiek - siervissen - visziekten - koi - cyprinidae - carp - cyprinus - viral diseases - diagnostics - ornamental fishes - fish diseases
    Eind september 2013 is de koikarperziekte 'Koi Sleepy Disease'(KSD) voor het eerst aangetoond in Nederland door het Vis-, schaal- en schelpdierziektelaboratorium van Central Veterinary Institute, onderdeel van Wageningen UR. De klinische verdenking werd bevestigd door het zusterlab CEFAS in Engeland. KSD wordt veroorzaakt door Carp Edema Virus (CEV), een pokkenvirus. Ziekteverschijnselen lijken erg op die van de al meerdere jaren aanwezige koi herpesvirusziekte (KHVD), veroorzaakt door koi herpesvirus (KHV). De klinische verschijnselen van beide ziekten zijn: enophthalmus, overmatige slijmproductie en kieuwnecrose. Een verschil vormt echter het onmiskenbare slaapgedrag waaraan de ziekte zijn naam dankt. Omdat Nederland vele hobbyvijvers telt en koikarpereigenaren hun dierenarts consulteren in verband met ziekte bij hun vissen is het belangrijk dat dierenartsen KSD en KHVD in het veld kunnen herkennen. Bij visziektelaboratorium kan vervolgens bevestigingsdiagnostiek plaatsvinden.
    Koi Sleepy Disease (KSD) door 'Carp Edema virus' : eerste detectie in Nederlandse Koi
    Haenen, O.L.M. ; Way, K. ; Stone, D. ; Engelsma, M.Y. - \ 2013
    Aquacultuur 2013 (2013)5. - ISSN 1382-2764 - p. 27 - 29.
    koi - cyprinidae - karper - cyprinus - siervissen - virusziekten - visteelt - diagnostiek - koi - cyprinidae - carp - cyprinus - ornamental fishes - viral diseases - fish culture - diagnostics
    De koi sector is bekend met verschillende ziekten van koi, zoals koi herpesvirus (KHV) en columnaris disease door Flavobacterium columnare. Dit najaar is echter een in Nederland nog niet eerder aangetoonde ziekte aangetroffen in koi: het koi sleepy disease (KSD). We gaan in dit artikel in op deze ziekte
    Stimulation of the innate immune system of carp: role of Toll-like receptors
    Pietretti, D. - \ 2013
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789461737878 - 213
    karper - cyprinus - immuunsysteem - verdedigingsmechanismen - immuniteitsreactie - immunostimulatie - bèta-glucaan - macrofagen - receptoren - immunologie - visteelt - aquacultuur - carp - cyprinus - immune system - defence mechanisms - immune response - immunostimulation - beta-glucan - macrophages - receptors - immunology - fish culture - aquaculture

    Toll-like receptors (TLRs), named after the Toll gene identified in fruit flies, are a family of evolutionary conserved proteins that play a key role in the innate immune system. TLRs are found inside or on the surface of immune cells of virtually all-living animals and recognize integral parts of microbes. Thereby, they are excellent candidate receptors for controlled stimulation of the innate immune system of, for example, fish in aquaculture. β-glucans are microbial compounds routinely added to fish feed for their health-promoting effects. They regulate innate immunity by stimulating fish cells to produce more oxygen and nitrogen radicals but are not recognized by TLRs.Instead, TLRs of cyprinid fish (zebrafish, carp) are stimulated by viral and/or parasitic infection. Although immunostimulation by β-glucans occurs via yet undefined receptors certainly, addition of integral but harmless parts of microbes to fish feed may help controlfish diseases in aquaculture.

    Transmission and control of Fish-borne Zoonotic Trematodes in aquaculture
    Boerlage, A.S. - \ 2013
    Wageningen University. Promotor(en): Mart de Jong; Johan Verreth, co-promotor(en): Lisette Graat. - S.l. : s.n. - ISBN 9789461736284 - 150
    trematoda - karper - vissen - zoönosen - ziekteoverdracht - ziektebestrijding - ziektemodellen - viskwekerijen - visteelt - aquacultuur - parasitologie - trematoda - carp - fishes - zoonoses - disease transmission - disease control - disease models - fish farms - fish culture - aquaculture - parasitology

    Fish-borne Zoonotic Trematodes (FZTs) affect the health of millions of humans worldwide. For persistence, the life cycle of FZTs depends on aquatic snails, fish, and definitive hosts like humans, pigs or chickens. Definitive hosts can become infected by eating raw or undercooked fish. Integrated Agriculture Aquaculture (IAA) systems improve the livelihood of small scale farmers, but may enhance transmission of FZTs because all types of hosts and all transmission routes can be present on a single farm. This thesiscombines experiments, statistical analyses and mathematical modelling to gain insight into transmission mechanisms of FZTs to fish in aquaculture and to use this insight to compare and discuss control measures against FZTs. Currently, medication of humans is the main strategy to control FZTs. Modelling indicated that this does not lead to elimination of FZTs because both humans and definitive hosts other than humans will maintain the life cycle of FZTs independently. Treatment of (a part of) these host types may eliminate FZTs, e.g. treating all humans and 54% of definitive hosts other than humans. Aquaculture may provide opportunities for control of FZTs by adapting management measures. Experiments showed that smaller fish get more often and more heavily infected with FZTs than larger fish; common carp (Cyprinus carpio) of more than 50 g rarely acquire new infections. Once carp are infected, FZTs persist for at least 27 weeks, implying that harvestable fish still contain FZTs and, therefore, are a risk to human health. In most IAA systems, fish are kept FZT free until 0.5 g before being stocked into a fish pond where they are very likely to be exposed to FZTs. Stocking fish at more than 25 g, or at more than 14 g in combination with treating all humans with anthelmintics, may lead to elimination of FZTs. Also, control of snails by either decreasing density or increasing mortality of snails may lead to elimination of FZTs in aquaculture. Farmers and policy makers should evaluate which combination of control measures is attractive to them.

    Karpersterfte in Nederland: Nederlands instituut onderzoekt karpersterftes
    Haenen, O.L.M. ; Tulden, P.W. van; Engelsma, M.Y. - \ 2012
    Visionair : het vakblad van sportvisserij Nederland 5 (2012)23. - ISSN 1569-7533 - p. 18 - 21.
    karper - visstand - sportvissen - hengelsport - virusziekten - spring viremia of carp virus - virussen - bacteriën - parasieten - carp - fish stocks - game fishes - angling - viral diseases - spring viremia of carp virus - viruses - bacteria - parasites
    Voor veel sportvissers is karper de favoriete vissoort. Dankzij een doordacht water- en visstandbeheer kent ons land een gezonde karperpopulatie. Tocht treedt er af en toe sterfte op bij deze vissen. Mede op verzoek van Sportvisserij Nederland onderzoekt het Centraal Veterinair Instituut in Lelystad de oorzaak van deze sterftes.
    Carp mucus and its role in mucosal defense
    Marel, M.C. van der - \ 2012
    Wageningen University. Promotor(en): Huub Savelkoul; D. Steinhagen, co-promotor(en): Jan Rombout. - S.l. : s.n. - ISBN 9789461734273 - 183
    karper - cyprinus - slijm - verdedigingsmechanismen - vissen - immunologie - glycoproteïnen - carp - cyprinus - mucus - defence mechanisms - fishes - immunology - glycoproteins
    Innate immune receptors in carp: recognition of protozoan parasites
    Ribeiro, C.M.S. - \ 2010
    Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Geert Wiegertjes. - [S.l. : S.n. - ISBN 9789085857747 - 217
    karper - immuniteitsreactie - receptoren - protozoa - parasieten - immuunsysteem - vaccins - hulpstoffen - immunologie - immuniteit - carp - immune response - receptors - protozoa - parasites - immune system - vaccines - adjuvants - immunology - immunity
    This PhD thesis reports on pattern recognition receptors involved in the immune responses of common carp (Cyprinus carpio) to two protozoan parasites Trypanoplasma borreli and Trypanosoma carassii. The immune responses of carp are fundamentally different when comparing these two extracellular blood parasites. T. borreli induces a characteristically high production of nitric oxide by macrophages, whereas T. carassii parasites seem to preferentially induce an alternative state of macrophage activation. These differences could be driven by differences in the initial engagement of pattern recognition receptors on carp macrophages with either of the two types of parasites. Based on known host-parasite interactions in mammalian vertebrates, Toll-like receptor 2 (TLR2) and TLR9 were selected as candidate receptors for parasite recognition by receptors carp macrophages. Transfection of human cell cultures with carp TLR2 and overexpression of TLR2 in carp macrophages, corroborated the ability of this receptor to bind peptidoglycan from Gram-positive bacteria and glycosylphosphatidylinositol anchors from protozoan parasites. The parasite T. carassii, in particular, induced a TLR2-mediated formation of the cytokine IL-23, leading to a Th17-like immune response in fish infected with T. carassii. Transfection of human cell cultures with carp TLR9 indicated this receptor recognizes bacterial DNA, but not protozoan DNA, and studies in carp macrophages indicated this recognition to be protease-dependent. A novel pattern recognition receptor of carp, named Soluble Immune-Type Receptor (SITR), was identified by investigating an enriched cDNA repertoire from macrophages stimulated by T. borreli. SITR is abundantly expressed in carp macrophages and seems to be secreted upon stimulation with T. borreli. Overexpression of SITR in mouse macrophages and knock-down of SITR in carp macrophages provided evidence for the involvement of this receptor in T. borreli-induced production of nitric oxide. The results presented in this PhD thesis have shed light on the evolution of innate immune receptors involved in the recognition of pathogens.
    Informatieblad Koi Herpesvirus (KHV) bij karper en koi
    Engelsma, M.Y. ; Haenen, O.L.M. - \ 2009
    Lelystad : Centraal Veterinair Instituut Wageningen UR
    visziekten - karper - koi - cyprinidae - vijverteelt - siervissen - herpes - herpesviridae - nefritis - fish diseases - carp - koi - cyprinidae - pond culture - ornamental fishes - herpes - herpesviridae - nephritis
    Beantwoording van de volgende vragen: Wat is Koi Herpesvirus (KHV)? Waar komt KHV voor? Hoe herken ik KHV? Hoe weet ik of mijn vis KHV heeft? Hoe komt vis aan KHV? Kunnen ook andere vissoorten in de vijver met KHV geïnfecteerd worden? Is er een behandeling voor KHV en hoe raak je het kwijt? Voorkomen van KHV en vaccinatie?
    Immune responses of carp : a molecular and cellular approach to infections
    Forlenza, M. - \ 2009
    Wageningen University. Promotor(en): Huub Savelkoul; Geert Wiegertjes. - S.l. : s.n. - ISBN 9789085855125 - 212
    karper - immuniteitsreactie - immuunsysteem - experimentele infecties - moleculaire biologie - celbiologie - immuniteit - immunologie - diermodellen - vergelijkend onderzoek - carp - immune response - immune system - experimental infections - molecular biology - cellular biology - immunity - immunology - animal models - comparative research - cum laude
    cum laude graduation (with distinction)
    Zieke koi gevaar voor wilde karpers
    Haenen, O.L.M. - \ 2009
    Kennis Online 6 (2009)juni. - p. 8 - 8.
    koi - siervissen - vissen - gezelschapsdieren - karper - cyprinus - koi - ornamental fishes - fishes - pets - carp - cyprinus
    Ontstoken, slijmerige kieuwen, ingevallen ogen en huidwondjes. Het zijn enkele van de ziekteverschijnselen van het koi herpesvirus (KHV). Sierkoi en gewone karpers kunnen er binnen een week aan sterven. “Er zijn veel ziektegevallen in achtertuinvijvers, bij sierkoi. In wilde karper hebben we het nog niet aangetoond, maar dat is een kwestie van tijd”, zegt Olga Haenen van het Vis- en chelpdierziektenlaboratorium van het Centraal Veterinair Instituut
    Improving the utilization of Silver carp (Hypopthalmichthys Molitrix) and other under-utilized fish species, especially Fresh water Bream (Abramis brama)
    Bartels, P.V. ; Kals, J. - \ 2008
    Wageningen : Agrotechnology & Food Sciences Group (Rapport / Agrotechnology and Food Sciences Group (AFSG) 767) - 71
    visverwerkende industrie - visverwerking - visproductie - visproducten - nieuwe producten - dierlijke eiwitten - snijden - voedselverwerking - karper - iran - fish industry - fish processing - fish production - fish products - new products - animal proteins - cutting - food processing - carp - iran
    Major histocompatibility (MH) polymorphism of common carp : link with disease resistance
    Rakus, K.L. - \ 2008
    Wageningen University. Promotor(en): Huub Savelkoul; A. Pilarczyk, co-promotor(en): Geert Wiegertjes; I. Irnazarow. - [S.l.] : S.n. - ISBN 9789085852445 - 169
    karper - major histocompatibility complex - polymorfisme - ziekteresistentie - genen - immuniteitsreactie - immunologie - visteelt - aquacultuur - carp - major histocompatibility complex - polymorphism - disease resistance - genes - immune response - immunology - fish culture - aquaculture
    The impact of diseases caused by a wide range of pathogens (viruses, bacteria
    and parasites) is the most important problem in aquaculture of common carp (Cyprinus
    carpio L.). Genetic selection aimed at obtaining population of more resistant common
    carp is potential and sustainable approach to disease control in semi-intensive carp pond
    farming. Genes of the major histocompatibility complex (MHC) are candidate marker
    genes for studies on association with disease resistance. The MHC contains some of the
    most polymorphic genes known to date and are considered crucial to adaptive
    immunity. MHC molecules bind both self and foreign peptides and present them to T
    lymphocytes (T cells). MHC class I molecules present endogenously derived peptides to
    CD8+ T cells, while MHC class II molecules present exogenously derived peptides to
    CD4+ T cells. Each MHC molecule has the ability to bind and present different groups
    of peptides in more or less successful ways. This can influence the immune response of
    an organism since the peptides derived from a certain pathogen may either not be
    presented by specific MHC molecules, which can result in higher susceptibility or, may
    be bound with a high affinity by specific MHC molecules which could lead to increased
    resistance to the pathogenic organism.
    In teleosts, unlike to humans, tetrapods and cartilaginous fish, class I and class II
    genes are not linked and segregate independently, which allows for association studies
    of only class I or only class II MH genes with disease resistance. MHC class II
    molecules generally have a broader spectrum of action in the immune system than the
    MHC class I genes. There are also observations that suggest a more intense selection
    pressure and a more rapid evolution of MH class II than class I alleles in fish. Although
    the expression of both MH class II chains is equivalent, the beta chain generally has a
    higher degree of polymorphism than the alpha chain. This thesis addressed possible
    implementations of MH class II B genes for selection aimed at improving of a common
    carp resistance in semi-intensive pond farming.
    In common carp there are two paralogous groups of MH class II B genes, Cyca-
    DAB1-like and Cyca-DAB3-like genes. In a preliminary study, we examined the
    polymorphism for the Cyca-DAB1-like and Cyca-DAB3-like genes in different
    European common carp lines (chapter 2). These carp lines were of various
    geographical origins and part of carp live gene bank, which is maintained at the Institute
    of Ichthyobiology and Aquaculture in Gołysz. Previous observations over a period of at least 15 years revealed significant differences between lines in survival rate and parasite
    load under natural conditions. Also, differences in resistance to atypical Aeromonas
    salmonicida in laboratory based challenge tests was observed, suggesting genetic
    differences in resistance between the carp lines. Analysis of polymorphism of MH class
    II B genes in selected carp lines revealed a ubiquitous presence and high polymorphism
    of Cyca-DAB1-like but not Cyca-DAB3-like genes. The observed allelic polymorphism
    for Cyca-DAB1-like genes rather than Cyca-DAB3-like genes stimulated further studies
    into the association of Cyca-DAB1-like allelic polymorphism and disease resistance of
    common carp.
    In order to study association between Cyca-DAB1-like gene polymorphism and
    resistance of common carp we optimized a technique designated polymerase chain
    reaction -restriction fragments- single strand conformation polymorphism (PCR-RFSSCP)
    to be able to screen and type large numbers of individual carp (chapter 3). The
    advantages of this technique are simplicity, high sensitivity and low costs. PCR-RFSSCP
    analysis of n = 79 carp individuals from 8 lines challenged with Aeromonas
    hydrophila revealed the presence of different genotypes consisting of unique
    combinations of Cyca-DAB1 and Cyca-DAB2 sequences. We found four alleles for the
    Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02). We noted that the
    Cyca-DAB2 gene was either homozygous or absent. The degree of heterozygosity of the
    Cyca-DAB1 and Cyca-DAB2 genes clearly correlated with the number of SSCP bands.
    Thus, we proved that PCR-RF-SSCP is a reliable technique that can be used for
    screening large number of individuals for investigating the Cyca-DAB1 and Cyca-DAB2
    genes polymorphism in common carp.
    Previously, we performed a long-term divergent selection of common carp for
    antibody production, which successfully resulted in carp lines with a different immune
    response. We studied the segregation of Cyca-DAB genes with the DNP-specific
    antibody response and we showed that the presence of Cyca-DAB1-like, but not Cyca-
    DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp
    (chapter 4). Background genes other than Cyca-DAB genes also influenced the level of
    antibody response. We also studied the transcription of both Cyca-DAB1-like and Cyca-
    DAB3-like genes in different organs of carp. The constitutive transcription for both
    Cyca-DAB1-like and Cyca-DAB3-like genes was high, although Cyca-DAB1-like genes consistently showed slightly higher mRNA transcription than Cyca-DAB3-like genes in
    some immunological relevant organs. Sequence information, constitutive transcription
    levels and co-segregation data indicated that both paralogous Cyca-DAB1-like and
    Cyca-DAB3-like groups represent functional MH class II B genes.
    We then proceeded to study association of Cyca-DAB1-like genotypes with
    resistance to four different pathogens; the bacterium Aeromonas hydrophila, the
    ectoparasite Argulus japonicus, and the blood parasite Trypanoplasma borreli (chapter
    5) and the viral pathogen Cyprinid herpesvirus-3 (CyHV-3) (chapter 6). We used a
    large number of individuals of different carp lines and revealed, using PCR-RF-SSCP,
    the presence of n = 9 unique Cyca-DAB1-like genotypes, of which three genotypes (B,
    D, and E) were most common (chapter 5). In general, Cyca-DAB2 was often
    homozygous or absent while allelic polymorphism was detected in Cyca-DAB1 gene.
    We could detect significant associations between genotype E and abundance of
    A. japonicus and between genotype D and higher level of parasitaemia after T. borreli
    infection. We also observed a significant association between Cyca-DAB1
    heterozygosity and lower level of parasitaemia after T. borreli infection. In chapter 6,
    we showed a strong association between Cyca-DAB1-like genotypes and resistance or
    susceptibility to CyHV-3. One genotype (E) performed significantly better, resulting in
    carp more resistant to CyHV-3, while three other genotypes (B, H and J) could be
    linked to higher susceptibility to the virus. Subsequent analysis of the alleles that
    compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05)
    to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to
    significantly decreased resistance to CyHV-3. The resistant genotype E did not
    comprise the Cyca-DAB2 gene and consisted of a homozygous Cyca-DAB1*05 allele.
    Phylogenetic analysis of all Cyca-DAB1 alleles showed that the Cyca-DAB1*05 allele
    represents the oldest allele in our study (chapter 7). We discussed the possibility for
    using Cyca-DAB1 allelic polymorphism as a potential genetic marker in future breeding
    programs of common carp (chapter 7). We expect that selection of carp for particular
    MH class II B genotypes or alleles could allow for an increased survival upon challenge
    with selected pathogens and possibly, increased survival rate under pond conditions.
    Transferrin polymorphism of common carp: link with disease resistance
    Jurecka, P.M. - \ 2008
    Wageningen University. Promotor(en): Huub Savelkoul; A. Pilarczyk, co-promotor(en): Geert Wiegertjes; I. Irnazarow. - [S.l.] : S.n. - ISBN 9789085852438 - 178
    karper - transferrine - polymorfisme - ziekteresistentie - genetisch bepaalde resistentie - trypanoplasma borreli - experimentele infectie - genexpressie - stikstofoxide - macrofagen - immunologie - carp - transferrin - polymorphism - disease resistance - genetic resistance - trypanoplasma borreli - experimental infection - gene expression - nitric oxide - macrophages - immunology
    Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many
    metabolic functions. Iron concentrations are tightly regulated, for example by ferritin,
    because excessive iron leads to tissue damage. Iron cannot cross cellular membranes
    directly and most cells acquire iron from the iron transporting protein transferrin (Tf),
    via transferrin receptors. During nutritional immunity the body reacts with a metabolic
    adjustment in order to render important nutrients unavailable to invading
    microorganisms. However, pathogens also have evolved a range of mechanisms to
    acquire iron from the host (chapter 1).
    In the study described in this thesis, we used a natural host-parasite model of common
    carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan
    kinetoplastid, extracellular blood parasite of carp to get more insight in the competition
    for iron between host and parasite. Transferrin of common carp is highly polymorphic
    with several alleles identified according to differences in electrophoretic mobility. We
    studied the implications of Tf polymorphisms for iron binding and modulation of
    immune function.
    We performed a series of challenge experiments infecting five genetically different,
    commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype
    may influence the susceptibility to pathogens. We observed a significant association of
    the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’
    and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We
    also showed that variation in resistance to T. borreli could be controlled by sex-related
    genetic factors. Examination of parasite growth in vitro, in culture media supplemented
    with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in
    number of parasites in media supplemented with serum from DD-typed animals
    (chapter 2).
    In general, pathogens also have mechanisms to acquire iron from the host. We
    developed a method for Tf depletion of carp serum using specific antibodies to carp Tf,
    and compared T. borreli multiplication and survival in the presence or absence of Tf in
    vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly
    showed that Tf is essential for parasite growth and multiplication (chapter 3). We
    isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol
    precipitation and ion-exchange chromatography (chapter 5). We showed that parasite
    growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in
    culture media containing different sera typed differently for Tf genotypes (chapter 3).
    We identified four complete coding sequences for common carp Tf alleles C, D, F and
    G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs
    of other species. We could show that carp Tf differs significantly in critical iron-binding
    sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species
    (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe
    indeed seems to affect the ability to bind iron, which may be compensated for by
    higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene
    expression of two Tf alleles D and G showed a comparably high gene expression level
    in liver and small but consistent differences in gene expression for allele D over allele G
    in other immunologically important organs (chapter 4). Our data suggest that the allelic
    polymorphism is not related to differences in iron binding and/or binding to the host Tf
    receptor but could be linked with other factors, such as competition for iron with
    pathogens (chapter 4).
    Transferrin itself may also exert effects that are not directly linked with maintaining
    iron levels and Tf cleavage products have been shown to stimulate macrophages to
    produce large amounts of nitric oxide (NO). To study the induction of NO in carp head
    kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G)
    to purity and showed that the level of activation of macrophages by Tf was different for
    the D and G allele (chapter 5). Differences in NO levels induced could be related to
    different cleavage forms of the two alleles D and G, as shown by Western blot,
    confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products
    induced significantly higher nitric oxide (NO) production than cleavage products of Gtype
    Tf. (chapter 5).
    Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The
    TfR-Tf complex then is internalised and transported to lysosomes, where Tf is
    proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like
    cysteine proteinase from T. borreli and production of a recombinant and biologically
    active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is
    able to digest host transferrin. Likely, Tf cleavage fragments are released from the
    trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages
    (chapter 7).
    Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in
    immunity of common carp and the influence of allelic polymorphism on competition for
    iron between host and pathogen (chapter 7). Further investigations should shed more
    light on the selective advantage of particular alleles to provide a basis for incorporating
    Tf as a genetic marker in marker-assisted selection programmes for increased resistance
    to diseases. This could contribute to improved survival of carp kept under semiintensive
    farming systems in ponds.
    A differential role for corticosteroid receptors in neuroendocrine-immune interactions in carp (Cyprinus carpio L.)
    Stolte, H.H. - \ 2008
    Wageningen University. Promotor(en): Huub Savelkoul; G. Flik, co-promotor(en): Lidy van Kemenade. - [S.l.] : S.n. - ISBN 9789085851998 - 231
    karper - corticoïden - hormoonreceptoren - immunologie - immuniteit - immuniteitsreactie - interacties - stress - cytokinen - visteelt - neuroendocrinologie - carp - corticoids - hormone receptors - immunology - immunity - immune response - interactions - stress - cytokines - fish culture - neuroendocrinology
    In this thesis we investigated the involvement of the receptors for the stress hormone cortisol in stress and immune regulation. We set out to characterise the pro-inflammatory cytokine interferon gamma (IFN-γ). Furthermore, we used a genome wide screen (microarray) to search for additional genes that might be involved in regulation of the stress or the immune response.

    In teleostean fishes cortisol can be bound by different receptors encoded by at least three different genes. An ancestral corticosteroid receptor (AncCR) is assumed to have been an effective receptor for cortisol in the ancestors of fishes. An early genomic duplication in the fish lineage, over 450 million years ago, led to separate glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) genes, both of which retained the ability to bind cortisol. A second major genomic duplication event took place only in teleostean fishes (not in other vertebrates), and gave rise to duplicate GR genes (GR1 and GR2). Even more variants developed as a result of alternative splicing of the GR1 gene which introduces a nine amino acid insert in the DNA-binding domain of GR1a, GR1b does not have this insert.

    To investigate how one ligand can regulate many and very diverse functions using multiple receptors, we describe the expression of GR1 (a and b), GR2 and MR and their sensitivity for cortisol in chapters 3 and 4. The three receptors are expressed in tissues that make up the neuroendocrine stress-axis (brain, hypothalamus and pituitary) and in cells that produce corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Decreased mRNA expression in brain after prolonged stress suggests an involvement in regulation of hypothalamo-pituitary-interrenal (HPI)-axis activity. In cells of the immune system MR expression is very low compared to GR expression and GR2 is preferentially expressed in lymphocytes. Transactivation assays shows that GR1 is a relatively ‘insensitive’ or ‘stress’ receptor, which can only become activated at stress levels of, whereas GR2 is a ‘sensitive’ receptor that will already be activated at basal levels of cortisol such as occur in non-stressed fish. MR sensitivity for cortisol is intermediate. We predict by tertiary protein modelling and confirmed by transfection assays, that the transactivation capacity of both splice variants (GR1a and GR1b) is similar. Based on the very low expression level in immune cells and the moderate transactivation capacity of MR we concluded that GRs rather then the MR primarily convey stress signals to the immune system. Next, we determined the expression profile of the duplicated GR genes in the immune system in chapters 4 and 5 to investigate the regulation of stress-induced immune modulation. Simultaneously we investigated the expression profile of (among others) heat shock protein 70 (Hsp70). This protein is required for binding of cortisol to the GR, but also has intrinsic immune modulatory functions, as it was shown to downregulate LPS-induced pro-inflammatory cytokine expression in vitro and in vivo. In head kidney phagocytes we found that only physiological stress levels of cortisol could reduce LPS-induced expression of pro-inflammatory cytokines, a response that appears mediated by the ‘stress’ receptor GR1. Moreover, we found that Hsp70 and GR1 (a and b) expression is increased after an immune stimulus in vitro and in vivo, whereas 24 hr restraint stress or 100nM cortisol-treatment hardly increases Hsp70 and GR1 expression levels. This suggests that an immune stimulus rather than increased cortisol levels increases the sensitivity for glucocorticoid regulation and thereby of the cytokine profiles in immune cells.

    To find additional genes involved in bidirectional neuroendocrine-immune communication we applied a genome wide screen of 9000 randomly picked cDNA clones. This has the advantage of an unprejudiced overview of regulated genes, but the sensitivity of the technique is limited. In chapter 6 we describe a microarray experiment in which we compared mild acute stress, to prolonged severe immune stimulation. We show that an immune response after parasite infection appears tightly regulated and comparable between individuals, whereas a mild acute stressor allows for more variable gene expression profiles. We found LOC406744 of the DUF727 protein family and nephrosin as new interesting candidate genes that may be involved in neuroendocrine-immune communication.

    The key pro-inflammatory cytokine IFN-γ, which is hypothesised to affect neurotransmitter and hormone release, had not been investigated in carp. In chapter 7 we show that carp have duplicate IFN-γ genes that are expressed in immune cells. IFN-γ-2 shows structural and functional characteristics simlar to those in other vertebrate IFN-γ genes and appears to be involved in T-lymphocyte function, whereas IFN-γ-1 is expressed in stimulated B-lymphocytes. Currently recombinant proteins are being produced which will enable us to further elucidate the role of both IFN-γ gene products in the immune system as well as in mediating the neuroendocrine stress response.

    Interestingly, as explained in chapter 8, both the glucocorticoid receptor and the IFN-γ genes are duplicated. The duplication-degeneration-complementation (DCC) model has been proposed as an explanation for the high retention of duplicate genes in fishes. The hypothesis assumes that following gene duplication, the two gene copies degenerate over time by random mutation to perform complementary functions that jointly match that of the single ancestral gene, termed ‘subfunctionalisation’. Indeed it appears that the duplicate GR genes have divided the general and ‘stress-related’ functions, reflected by their different sensitivity for cortisol. The duplicate IFN-γ genes appear to have divided B- and T-lymphocyte functions as targets suggested by their gene expression profiles upon selective stimulation.

    An important conclusion of this thesis is that duplicated glucocorticoid receptors and heat shock proteins are an integral part of the immune system. Immune stimuli rather than increased cortisol levels control GR and Hsp70 expression in immune cells. The differentially regulated expression of GR genes is at the basis of a balanced pro- and anti-inflammatory
    cytokine profile, immune cell viability and thus at the basis of the success of the fishes. This thesis illustrates the importance of extensive and effective bidirectional communication between the neuroendocrine and immune systems, which are at the basis of the successful evolution of the vertebrates.
    Etiology of soybean-induced enteritis in fish
    Urán Carmona, P.A. - \ 2008
    Wageningen University. Promotor(en): Johan Verreth, co-promotor(en): Johan Schrama; Jan Rombout. - [S.l.] : S.n. - ISBN 9789085049098 - 175
    Europese zalm - karper - sojabonen - visvoeding - enteritis - etiologie - dierpathologie - visteelt - aquacultuur - Atlantic salmon - carp - soyabeans - fish feeding - enteritis - aetiology - animal pathology - fish culture - aquaculture
    The inclusion of soybean meal (SBM), especially in the diet of Atlantic salmon, induces an inflammatory response of the distal intestinal mucosa, known as SBM-induced enteritis. A semi-quantitative scoring system was developed to assess the extent of the morphological changes observed in this study. The influence of SBM feeding has been investigated taking into account several dietary and non-dietary factors possibly involved in the induction of the disorder. It has been found that the severity of enteritis and its kinetics are dose-dependent. Electron microscopy studies indicated a block of the endocytosis process and a strong decrease of the microvilli length. Comparative studies were carried out in an omnivorous species and for the first time ever reported, the results suggested that the symptoms of enteritis also occur in common carp. Contrary to the observations in studies with Atlantic salmon, the common carp started to recover from week four onwards. Several cytokines were presumed to influence this process and they were correlated to the modulation of the inflammatory process triggered by the SBM-containing diet. The influence of different factors was measured according to the degree of enteritis developed. Low temperature (8 °C vs. 12 °C) seems to delay the onset of the symptoms. On the other hand, it was suggested that SBM-induced enteritis was not strongly influenced by either salinity or age. The extent of enteritis in Atlantic salmon depends on the origin and/or the processing of the soybeans. The morphological changes observed were induced when soyasaponins were fed to Atlantic salmon alone or in combination with other soybean components suggesting their possible role on the induction of enteritis. The actual causative components and its mechanisms of action need further research. It is concluded that the etiology and further development of SBM-induced enteritis is related to dietary factors rather than non-dietary factors. SBM inclusion levels and the commercial source used for the diet formulation have a great impact on the severity of the disorder, mainly affecting the endocytosis process. This thesis evidenced that the endocytosis block is directly related to the disappearance of the supranuclear vacuoles, which can be considered as the most striking feature in the onset of enteritis.
    Characterization of the stress response in 17a-hydroxylase deficient common carp (Cyprinus carpio L.)
    Nematollahi, M.A. - \ 2008
    Wageningen University. Promotor(en): Johan Verreth; Johan van Arendonk, co-promotor(en): Hans Komen; Hilde van Pelt-Heerschap. - [S.l.] : S.n. - ISBN 9789085049043 - 135
    karper - cyprinus - stressreactie - genexpressie - deficiëntie - stammen (biologisch) - dna-sequencing - polymerase-kettingreactie - nieren - carp - cyprinus - stress response - gene expression - deficiency - strains - dna sequencing - polymerase chain reaction - kidneys
    In this thesis we describe interrenal hyperplasia in a teleost fish, the common carp, Cyprinus carpio L, caused by 17α-hydroxylase deficiency. Two homozygous, XX male inbred strains, “E5” and “E7”, consistently show 5-10 fold less plasma cortisol in response to net confinement compared to normal male and female carp strains. Both E5 and E7 fish have enlarged head kidneys, which is caused by an increase in size and number of interrenal cells. The experiments and results described in this thesis aim to provide further insight into the genetic background of the 17α-hydroxylase deficiency, and the expression of genes involved in the stress response in P450c17 deficient common carp during and after stress. First, we describe the sequencing of the 17α-hydroxylase/17, 20 lyase gene, CYP17a1, in a normal, standard strain (“STD”) and one of the 17α-hydroxylase deficient strains (E5) of common carp. Two variants of the CYP17a1 gene were found. The nucleotide coding region of the CYP17a1.I variant contained 8 exons and 7 introns which is identical to the human CYP17. In total 3122 base pairs of the nucleotide sequence were determined. Variant I showed no differences between STD and E5, while variant II is missing exon 7 and part of exon 8 and contains a 3 bp insertion and 8 homozygous SNP’s downstream of exon 6. Next, we describe the stress response and gene expression in 17α-hydroxylase deficient E5 and normal STD common carp. The results show a significant increase of corticosterone levels in E5 carp in response to stress. Normal STD carp produce only small amounts of corticosterone. STD carp show a correlation between cortisol production and 11β-HSD2 mRNA expression during the stress response. In contrast, 11β-HSD2 expression is significantly lower in E5 fish and stays at a constant level during confinement. Real-time PCR analysis of StAR and 3β-HSD show a significantly higher expression of these enzymes in E5 during and after net confinement, but P450c21 not, suggesting that transcription of P450c21 is not a limiting step in corticosterone production. Messenger RNA levels in P450c17a1.I are quite variable with a trend towards lower expression levels in E5, suggesting a dysfunction at the transcriptional level. In the last part of this thesis, we investigate the inheritance of interrenal hyperplasia and low cortisol response using backcross and gynogenetic progeny of heterozygote carriers of common carp. Six-month old backcross (BC5 and BC7) and double haploid (DH) progenies were sacrificed after one hour net-confinement, blood sampled, and dissected to determine sex and head kidney-somatic index. Values for cortisol and head kidney index showed a continuous distribution in BC and DH progeny. Values for corticosterone, on the other hand showed a very clear segregation pattern in high and low responders, consistent with a single gene model. There was a significant difference in mean cortisol level between high (H) and low (L) corticosterone responders in BC and DH progeny groups. Surprisingely, H responders were predominantly male, while L responders were female or intersex. These results confirm that 17α-hydroxylase deficiency is inherited as a recessive mutation, and that 17α-deficiency and sex reversal are either caused by the same mutation (pleiotropy) or by closely linked genes.

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