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.
Genome-wide gene expression analysis of anguillid herpesvirus 1
Beurden, S.J. van; Peeters, B.P.H. ; Rottier, P.J.M. ; Davison, A.A. ; Engelsma, M.Y. - \ 2013
BMC Genomics 14 (2013). - ISSN 1471-2164 - 11 p.
channel catfish virus - time rt-pcr - dna microarray - european eel - murine gammaherpesvirus-68 - transcription - persistence - proteins - carp
Background Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR. Results Four immediate-early genes – open reading frames 1, 6A, 127 and 131 – were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins. Conclusions Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.
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
Brief communication: Soybean meal-induced uptake block in Atlantic salmon Salmo salar distal enterocytes
Urán, P. ; Aydin, R. ; Schrama, J.W. ; Verreth, J.A.J. ; Rombout, J.H.W.M. - \ 2008
Journal of Fish Biology 73 (2008)10. - ISSN 0022-1112 - p. 2571 - 2579.
ctenopharyngodon-idella val - trout oncorhynchus-mykiss - induced enteritis - rainbow-trout - intestinal epithelium - l. - transport - carp - gut - absorption
Soybean meal-induced enteritis was characterized by the disappearance of the supranuclear vacuoles (SNV) in the enterocytes of the distal intestine of Atlantic salmon Salmo salar. The loss of SNV was correlated with an endocytosis uptake block as shown with ferritin as a marker molecule.
A history of fish immunology and vaccination I. the early days
Muiswinkel, W.B. van - \ 2008
Fish and Shellfish Immunology 25 (2008)4. - ISSN 1050-4648 - p. 397 - 408.
trout salmo-gairdneri - dogfish mustelus canis - immune-response - immunoglobulin structure - oral immunization - antibody - carp - evolution - shark - homotransplantation
This historic review describes the people that were involved in studying some aspect of fish immunology and vaccination from as early as 1854. Between 1850 and 1940, most scientists were looking at fish from the angle of comparative anatomy, embryology, physiology, taxonomy and fish diseases. Most publications from this early period are describing the morphology of blood cells and hemopoietic or lymphoid organs. The first publications on specific immune responses and vaccination of fish were found in the period 1935-1938. However, the immune mechanisms behind protective immunization were largely unknown in those days. In the period after 1940, the first researchers can be found devoting their whole career to fish immunology. This paper has been organized largely by individuals and not so much by accomplishments. It is not the intent of this review to evaluate the scientific merit of the work discussed, but to provide the reader with information that was - at least in part - lost to the scientific community. Publications from before 1940 or in languages other than English (e.g. Russian) are usually not found by today's database searches on the Internet.
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
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
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
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
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.